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Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Blood creatine phosphokinase increased'. | Rapid and exceptional response to Sonidegib in a patient with multiple locally advanced basal cell carcinomas.
Locally advanced basal cell carcinoma (laBCC) represents a rare but possible occurrence in the vast scenario of dermatological diseases. It is well known that most BCC has a pathological activation of the hedgehog pathway, making them susceptible to targeted therapy with selective inhibitors. Sonidegib, approved for the treatment of laBCC on the basis of the results of the basal cell carcinoma outcomes with LDE225 treatment study, demonstrated rapid efficacy and a manageable safety profile. Here, we describe the case of a patient affected by multiple laBCC treated with Sonidegib. The patient experienced an important regression of tumors after only 2 months of therapy, with few side effects. This result confirms the role of Sonidegib as a valid and well-tolerated therapeutic option for laBCC.
Introduction
Basal cell carcinoma (BCC) is the most common form of skin cancer, making up approximately 80% of all nonmelanoma skin cancers [1]. The vast majority of BCCs can be effectively cured by complete histopathology controlled excision, which is the gold standard for BCC treatment. However, about 1–10% of lesions evolve towards advanced forms, including laBCC and Metastatic BCC [2]; in these cases, surgery may not represent a possible therapeutic option, because curing is unlikely and surgery might result in substantial deformity [3].
In order to avoid unreasonable postsurgical cosmetic changes, alternative approaches such as radiation, electrochemotherapy or systemic treatment [4] can be required, as specified by the European guidelines for the management of BCC [5]. Particularly, these approaches are indicated in patient affected by multiple comorbidities, numerous lesions or Gorlin–Goltz syndrome.
However, about 90% of BCCs have a pathologic activation of the Sonic hedgehog pathway, making them susceptible to targeted therapy with selective hedgehog pathway inhibitors (HPI) [6,7]. Notably, Sonidegib (Odomzo) demonstrated a sustained and clinically relevant efficacy and a manageable safety profile in phase II randomized, double-blind basal cell carcinoma outcomes with LDE225 treatment (BOLT) study [8,9].
Herein, we describe a rapid and exceptional response to Sonidegib in a patient affected by multiple disfiguring laBCC.
Case report
An otherwise healthy 71-year-old male patient comes to our attention for the sudden bleeding of an ulcerated abdominal lesion of 8 cm × 5 cm (Fig. 1a). Complete physical examination allowed us to identify about 30 other pink or reddish papules and plaques of different shapes and sizes (up to 15 cm) located on the face, trunk and limb; clinical and dermoscopic characteristics were compatible with BCCs (Fig. 1b–d).
Fig. 1 (a) Deep ulcerated abdominal lesion; (b) Basal cell carcinoma (BCC) of 15 cm × 8 cm located on the left shoulder; (c) multiple BCCs of different shapes and sizes on the anterior trunk, with a detail of the voluminous nodule at the internal cantus; (d) multiple BCC on the posterior trunk.
We collected the patient’s medical history, which was negative for comorbidity, chronic therapy or radiotherapy treatments; he reported a previous surgical treatment to remove a BCC on nasal dorsum without any dermatological follow-up for over 10 years. A skin biopsy was performed on the abdominal lesion, and the histological examination confirmed the diagnosis of BCC. The skull X-ray and the orthopantomography showed no evidence of calcification of the falx cerebri or odontogenic keratocysts; the patient did not appear to have skeletal anomalies or intellectual deficit; he also denies family history of skin cancers. All other criteria for a possible Gorlin–Goltz syndrome have been excluded.
Considering the extensive dimensions of the lesions on the abdomen and back, the numerousness of tumors, and the involvement of critical sites, we excluded the possibility of a surgical approach and decided, on the basis of the results of the BOLT study [8] and the evidence reported by Dummer et al. [10], to start systemic therapy with Sonidegib 200 mg, 1 cp per day.
Therapy was well-tolerated by the patient, except for a transient CTCAE grade I increase in the creatine phosphokinase, less than 2.5 × ULN, observed at the end of the first month of treatment, which did not require a dose adjustment. The other grades 1–2 side effects observed were dysgeusia and nocturnal muscle cramps resolving in a few minutes. Already at the end of the second month of treatment, the abdominal lesion appeared considerably reduced in size and depth (Fig. 2a). Furthermore, a similar clinical improvement was observed in other lesions; notably, the lesion on the back was less infiltrated and the size of the nodule at the internal cantus decreased significantly (Fig. 2b).
Fig. 2 (a) Abdominal basal cell carcinoma (BCC) considerably reduced in size and depth; (b) internal cantus BCC, significantly decreased.
At the end of the sixth month, there was no clinical or dermatoscopic evidence of BCC: the abdominal ulcerated lesion was healed (Fig. 3a), the nodule in the inner corner of the eye was not palpable and the lesions localized in the trunk assumed a scar-like appearance (Fig. 3b–d).
Fig. 3 (a) Ulcerated abdominal lesion healed; (b) scar appearance of the lesion localized in the left shoulder; (c) appearance of the anterior trunk and face lesions after 6 months of therapy; (d) appearance of the posterior trunk lesions after 6 months of therapy.
Currently, the treatment is ongoing, and the excellent clinical response persists.
Discussion
Although BCC is the most common skin cancer, the finding of laBCC is rather infrequent, often related to tumors with a long-term course, located in midface or on ears, with aggressive histopathologic subtype, perivascular or perineural infiltration, history of radiation exposure or previous surgical treatment failure. Patients with immunosuppressive status or multiple comorbidities are more affected [11]. In particular, neglected patients are one of the major contributing factors for the development of mutilating and aggressive BCC.
BCC occurs in the head area in 85–90% of cases [12] and can cause, when the tumor reaches a considerable size, social isolation in affected people. Here, the use of alternative therapeutic strategies is mandatory, to avoid cosmetically unacceptable postsurgical outcomes.
In phase II randomized double-blind BOLT study, which led to the approval of Sonidegib [8], the median time of response assessed by investigators was 1.9 months. Even in our patient, we observed a quick response to the treatment, with a significant improvement of all skin lesions after only 2 months. Already during the first weeks of therapy, in addition to an obvious reduction in the size of the tumors, the patient experienced a significative improvement of symptoms; in particular, the bleeding stopped and the patient reported a progressive reduction in the visual discomfort given by the BCC located at the inner corner of the eye. The rapidity of these events is very important as it promotes patient adherence to treatment, limiting the risk of spontaneous drop-out.
The good tolerability profile of Sonidegib detected by the BOLT study [8] was also confirmed in the long-term observations [10,13,14]. Also, in our experience, dysgeusia appeared as the first and main adverse event, whereas muscle cramps (reported as the first side effect in the patients received the 200 mg dose) arose later. However, also a slight spontaneously resolved increase in the CK value was observed.
Nevertheless, this value should be monitored, to evaluate any dose adjustment as required by the drug data sheet [8,9,13,14].
Even if the usefulness of HPI in the treatment of laBCC is widely confirmed [15], there are still few real-life experiences on Sonidegib [16,17] and anecdotal reports on multiple laBCC.
Our observations confirm the efficacy and safety of Sonidegib in this setting, suggesting it as an excellent therapeutic choice in neglected and laBCC.
Conclusion
In our real-life experience, Sonidegib has fulfilled its main goal, which is to obtain a rapid improvement of lesions and related symptoms, to avoid the risk of infection and anemia, to promote patient compliance towards the treatment and to reduce their social isolation improving his quality of life. The safety and effectiveness of the treatment will be confirmed by the long-term follow-up.
Acknowledgements
The authors are grateful to the patient for agreeing to share photos contributing to extend medical knowledge on this topic. The study received unrestricted grant from Sun Pharmaceutical Industries.
Conflicts of interest
We thank SunPharma for the non-conditioning contribution. There are no conflicts of interest. | SONIDEGIB | DrugsGivenReaction | CC BY-NC-ND | 33534224 | 20,359,494 | 2021-04-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Dysgeusia'. | Rapid and exceptional response to Sonidegib in a patient with multiple locally advanced basal cell carcinomas.
Locally advanced basal cell carcinoma (laBCC) represents a rare but possible occurrence in the vast scenario of dermatological diseases. It is well known that most BCC has a pathological activation of the hedgehog pathway, making them susceptible to targeted therapy with selective inhibitors. Sonidegib, approved for the treatment of laBCC on the basis of the results of the basal cell carcinoma outcomes with LDE225 treatment study, demonstrated rapid efficacy and a manageable safety profile. Here, we describe the case of a patient affected by multiple laBCC treated with Sonidegib. The patient experienced an important regression of tumors after only 2 months of therapy, with few side effects. This result confirms the role of Sonidegib as a valid and well-tolerated therapeutic option for laBCC.
Introduction
Basal cell carcinoma (BCC) is the most common form of skin cancer, making up approximately 80% of all nonmelanoma skin cancers [1]. The vast majority of BCCs can be effectively cured by complete histopathology controlled excision, which is the gold standard for BCC treatment. However, about 1–10% of lesions evolve towards advanced forms, including laBCC and Metastatic BCC [2]; in these cases, surgery may not represent a possible therapeutic option, because curing is unlikely and surgery might result in substantial deformity [3].
In order to avoid unreasonable postsurgical cosmetic changes, alternative approaches such as radiation, electrochemotherapy or systemic treatment [4] can be required, as specified by the European guidelines for the management of BCC [5]. Particularly, these approaches are indicated in patient affected by multiple comorbidities, numerous lesions or Gorlin–Goltz syndrome.
However, about 90% of BCCs have a pathologic activation of the Sonic hedgehog pathway, making them susceptible to targeted therapy with selective hedgehog pathway inhibitors (HPI) [6,7]. Notably, Sonidegib (Odomzo) demonstrated a sustained and clinically relevant efficacy and a manageable safety profile in phase II randomized, double-blind basal cell carcinoma outcomes with LDE225 treatment (BOLT) study [8,9].
Herein, we describe a rapid and exceptional response to Sonidegib in a patient affected by multiple disfiguring laBCC.
Case report
An otherwise healthy 71-year-old male patient comes to our attention for the sudden bleeding of an ulcerated abdominal lesion of 8 cm × 5 cm (Fig. 1a). Complete physical examination allowed us to identify about 30 other pink or reddish papules and plaques of different shapes and sizes (up to 15 cm) located on the face, trunk and limb; clinical and dermoscopic characteristics were compatible with BCCs (Fig. 1b–d).
Fig. 1 (a) Deep ulcerated abdominal lesion; (b) Basal cell carcinoma (BCC) of 15 cm × 8 cm located on the left shoulder; (c) multiple BCCs of different shapes and sizes on the anterior trunk, with a detail of the voluminous nodule at the internal cantus; (d) multiple BCC on the posterior trunk.
We collected the patient’s medical history, which was negative for comorbidity, chronic therapy or radiotherapy treatments; he reported a previous surgical treatment to remove a BCC on nasal dorsum without any dermatological follow-up for over 10 years. A skin biopsy was performed on the abdominal lesion, and the histological examination confirmed the diagnosis of BCC. The skull X-ray and the orthopantomography showed no evidence of calcification of the falx cerebri or odontogenic keratocysts; the patient did not appear to have skeletal anomalies or intellectual deficit; he also denies family history of skin cancers. All other criteria for a possible Gorlin–Goltz syndrome have been excluded.
Considering the extensive dimensions of the lesions on the abdomen and back, the numerousness of tumors, and the involvement of critical sites, we excluded the possibility of a surgical approach and decided, on the basis of the results of the BOLT study [8] and the evidence reported by Dummer et al. [10], to start systemic therapy with Sonidegib 200 mg, 1 cp per day.
Therapy was well-tolerated by the patient, except for a transient CTCAE grade I increase in the creatine phosphokinase, less than 2.5 × ULN, observed at the end of the first month of treatment, which did not require a dose adjustment. The other grades 1–2 side effects observed were dysgeusia and nocturnal muscle cramps resolving in a few minutes. Already at the end of the second month of treatment, the abdominal lesion appeared considerably reduced in size and depth (Fig. 2a). Furthermore, a similar clinical improvement was observed in other lesions; notably, the lesion on the back was less infiltrated and the size of the nodule at the internal cantus decreased significantly (Fig. 2b).
Fig. 2 (a) Abdominal basal cell carcinoma (BCC) considerably reduced in size and depth; (b) internal cantus BCC, significantly decreased.
At the end of the sixth month, there was no clinical or dermatoscopic evidence of BCC: the abdominal ulcerated lesion was healed (Fig. 3a), the nodule in the inner corner of the eye was not palpable and the lesions localized in the trunk assumed a scar-like appearance (Fig. 3b–d).
Fig. 3 (a) Ulcerated abdominal lesion healed; (b) scar appearance of the lesion localized in the left shoulder; (c) appearance of the anterior trunk and face lesions after 6 months of therapy; (d) appearance of the posterior trunk lesions after 6 months of therapy.
Currently, the treatment is ongoing, and the excellent clinical response persists.
Discussion
Although BCC is the most common skin cancer, the finding of laBCC is rather infrequent, often related to tumors with a long-term course, located in midface or on ears, with aggressive histopathologic subtype, perivascular or perineural infiltration, history of radiation exposure or previous surgical treatment failure. Patients with immunosuppressive status or multiple comorbidities are more affected [11]. In particular, neglected patients are one of the major contributing factors for the development of mutilating and aggressive BCC.
BCC occurs in the head area in 85–90% of cases [12] and can cause, when the tumor reaches a considerable size, social isolation in affected people. Here, the use of alternative therapeutic strategies is mandatory, to avoid cosmetically unacceptable postsurgical outcomes.
In phase II randomized double-blind BOLT study, which led to the approval of Sonidegib [8], the median time of response assessed by investigators was 1.9 months. Even in our patient, we observed a quick response to the treatment, with a significant improvement of all skin lesions after only 2 months. Already during the first weeks of therapy, in addition to an obvious reduction in the size of the tumors, the patient experienced a significative improvement of symptoms; in particular, the bleeding stopped and the patient reported a progressive reduction in the visual discomfort given by the BCC located at the inner corner of the eye. The rapidity of these events is very important as it promotes patient adherence to treatment, limiting the risk of spontaneous drop-out.
The good tolerability profile of Sonidegib detected by the BOLT study [8] was also confirmed in the long-term observations [10,13,14]. Also, in our experience, dysgeusia appeared as the first and main adverse event, whereas muscle cramps (reported as the first side effect in the patients received the 200 mg dose) arose later. However, also a slight spontaneously resolved increase in the CK value was observed.
Nevertheless, this value should be monitored, to evaluate any dose adjustment as required by the drug data sheet [8,9,13,14].
Even if the usefulness of HPI in the treatment of laBCC is widely confirmed [15], there are still few real-life experiences on Sonidegib [16,17] and anecdotal reports on multiple laBCC.
Our observations confirm the efficacy and safety of Sonidegib in this setting, suggesting it as an excellent therapeutic choice in neglected and laBCC.
Conclusion
In our real-life experience, Sonidegib has fulfilled its main goal, which is to obtain a rapid improvement of lesions and related symptoms, to avoid the risk of infection and anemia, to promote patient compliance towards the treatment and to reduce their social isolation improving his quality of life. The safety and effectiveness of the treatment will be confirmed by the long-term follow-up.
Acknowledgements
The authors are grateful to the patient for agreeing to share photos contributing to extend medical knowledge on this topic. The study received unrestricted grant from Sun Pharmaceutical Industries.
Conflicts of interest
We thank SunPharma for the non-conditioning contribution. There are no conflicts of interest. | SONIDEGIB | DrugsGivenReaction | CC BY-NC-ND | 33534224 | 20,359,494 | 2021-04-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Muscle spasms'. | Rapid and exceptional response to Sonidegib in a patient with multiple locally advanced basal cell carcinomas.
Locally advanced basal cell carcinoma (laBCC) represents a rare but possible occurrence in the vast scenario of dermatological diseases. It is well known that most BCC has a pathological activation of the hedgehog pathway, making them susceptible to targeted therapy with selective inhibitors. Sonidegib, approved for the treatment of laBCC on the basis of the results of the basal cell carcinoma outcomes with LDE225 treatment study, demonstrated rapid efficacy and a manageable safety profile. Here, we describe the case of a patient affected by multiple laBCC treated with Sonidegib. The patient experienced an important regression of tumors after only 2 months of therapy, with few side effects. This result confirms the role of Sonidegib as a valid and well-tolerated therapeutic option for laBCC.
Introduction
Basal cell carcinoma (BCC) is the most common form of skin cancer, making up approximately 80% of all nonmelanoma skin cancers [1]. The vast majority of BCCs can be effectively cured by complete histopathology controlled excision, which is the gold standard for BCC treatment. However, about 1–10% of lesions evolve towards advanced forms, including laBCC and Metastatic BCC [2]; in these cases, surgery may not represent a possible therapeutic option, because curing is unlikely and surgery might result in substantial deformity [3].
In order to avoid unreasonable postsurgical cosmetic changes, alternative approaches such as radiation, electrochemotherapy or systemic treatment [4] can be required, as specified by the European guidelines for the management of BCC [5]. Particularly, these approaches are indicated in patient affected by multiple comorbidities, numerous lesions or Gorlin–Goltz syndrome.
However, about 90% of BCCs have a pathologic activation of the Sonic hedgehog pathway, making them susceptible to targeted therapy with selective hedgehog pathway inhibitors (HPI) [6,7]. Notably, Sonidegib (Odomzo) demonstrated a sustained and clinically relevant efficacy and a manageable safety profile in phase II randomized, double-blind basal cell carcinoma outcomes with LDE225 treatment (BOLT) study [8,9].
Herein, we describe a rapid and exceptional response to Sonidegib in a patient affected by multiple disfiguring laBCC.
Case report
An otherwise healthy 71-year-old male patient comes to our attention for the sudden bleeding of an ulcerated abdominal lesion of 8 cm × 5 cm (Fig. 1a). Complete physical examination allowed us to identify about 30 other pink or reddish papules and plaques of different shapes and sizes (up to 15 cm) located on the face, trunk and limb; clinical and dermoscopic characteristics were compatible with BCCs (Fig. 1b–d).
Fig. 1 (a) Deep ulcerated abdominal lesion; (b) Basal cell carcinoma (BCC) of 15 cm × 8 cm located on the left shoulder; (c) multiple BCCs of different shapes and sizes on the anterior trunk, with a detail of the voluminous nodule at the internal cantus; (d) multiple BCC on the posterior trunk.
We collected the patient’s medical history, which was negative for comorbidity, chronic therapy or radiotherapy treatments; he reported a previous surgical treatment to remove a BCC on nasal dorsum without any dermatological follow-up for over 10 years. A skin biopsy was performed on the abdominal lesion, and the histological examination confirmed the diagnosis of BCC. The skull X-ray and the orthopantomography showed no evidence of calcification of the falx cerebri or odontogenic keratocysts; the patient did not appear to have skeletal anomalies or intellectual deficit; he also denies family history of skin cancers. All other criteria for a possible Gorlin–Goltz syndrome have been excluded.
Considering the extensive dimensions of the lesions on the abdomen and back, the numerousness of tumors, and the involvement of critical sites, we excluded the possibility of a surgical approach and decided, on the basis of the results of the BOLT study [8] and the evidence reported by Dummer et al. [10], to start systemic therapy with Sonidegib 200 mg, 1 cp per day.
Therapy was well-tolerated by the patient, except for a transient CTCAE grade I increase in the creatine phosphokinase, less than 2.5 × ULN, observed at the end of the first month of treatment, which did not require a dose adjustment. The other grades 1–2 side effects observed were dysgeusia and nocturnal muscle cramps resolving in a few minutes. Already at the end of the second month of treatment, the abdominal lesion appeared considerably reduced in size and depth (Fig. 2a). Furthermore, a similar clinical improvement was observed in other lesions; notably, the lesion on the back was less infiltrated and the size of the nodule at the internal cantus decreased significantly (Fig. 2b).
Fig. 2 (a) Abdominal basal cell carcinoma (BCC) considerably reduced in size and depth; (b) internal cantus BCC, significantly decreased.
At the end of the sixth month, there was no clinical or dermatoscopic evidence of BCC: the abdominal ulcerated lesion was healed (Fig. 3a), the nodule in the inner corner of the eye was not palpable and the lesions localized in the trunk assumed a scar-like appearance (Fig. 3b–d).
Fig. 3 (a) Ulcerated abdominal lesion healed; (b) scar appearance of the lesion localized in the left shoulder; (c) appearance of the anterior trunk and face lesions after 6 months of therapy; (d) appearance of the posterior trunk lesions after 6 months of therapy.
Currently, the treatment is ongoing, and the excellent clinical response persists.
Discussion
Although BCC is the most common skin cancer, the finding of laBCC is rather infrequent, often related to tumors with a long-term course, located in midface or on ears, with aggressive histopathologic subtype, perivascular or perineural infiltration, history of radiation exposure or previous surgical treatment failure. Patients with immunosuppressive status or multiple comorbidities are more affected [11]. In particular, neglected patients are one of the major contributing factors for the development of mutilating and aggressive BCC.
BCC occurs in the head area in 85–90% of cases [12] and can cause, when the tumor reaches a considerable size, social isolation in affected people. Here, the use of alternative therapeutic strategies is mandatory, to avoid cosmetically unacceptable postsurgical outcomes.
In phase II randomized double-blind BOLT study, which led to the approval of Sonidegib [8], the median time of response assessed by investigators was 1.9 months. Even in our patient, we observed a quick response to the treatment, with a significant improvement of all skin lesions after only 2 months. Already during the first weeks of therapy, in addition to an obvious reduction in the size of the tumors, the patient experienced a significative improvement of symptoms; in particular, the bleeding stopped and the patient reported a progressive reduction in the visual discomfort given by the BCC located at the inner corner of the eye. The rapidity of these events is very important as it promotes patient adherence to treatment, limiting the risk of spontaneous drop-out.
The good tolerability profile of Sonidegib detected by the BOLT study [8] was also confirmed in the long-term observations [10,13,14]. Also, in our experience, dysgeusia appeared as the first and main adverse event, whereas muscle cramps (reported as the first side effect in the patients received the 200 mg dose) arose later. However, also a slight spontaneously resolved increase in the CK value was observed.
Nevertheless, this value should be monitored, to evaluate any dose adjustment as required by the drug data sheet [8,9,13,14].
Even if the usefulness of HPI in the treatment of laBCC is widely confirmed [15], there are still few real-life experiences on Sonidegib [16,17] and anecdotal reports on multiple laBCC.
Our observations confirm the efficacy and safety of Sonidegib in this setting, suggesting it as an excellent therapeutic choice in neglected and laBCC.
Conclusion
In our real-life experience, Sonidegib has fulfilled its main goal, which is to obtain a rapid improvement of lesions and related symptoms, to avoid the risk of infection and anemia, to promote patient compliance towards the treatment and to reduce their social isolation improving his quality of life. The safety and effectiveness of the treatment will be confirmed by the long-term follow-up.
Acknowledgements
The authors are grateful to the patient for agreeing to share photos contributing to extend medical knowledge on this topic. The study received unrestricted grant from Sun Pharmaceutical Industries.
Conflicts of interest
We thank SunPharma for the non-conditioning contribution. There are no conflicts of interest. | SONIDEGIB | DrugsGivenReaction | CC BY-NC-ND | 33534224 | 20,359,494 | 2021-04-01 |
What was the administration route of drug 'SONIDEGIB'? | Rapid and exceptional response to Sonidegib in a patient with multiple locally advanced basal cell carcinomas.
Locally advanced basal cell carcinoma (laBCC) represents a rare but possible occurrence in the vast scenario of dermatological diseases. It is well known that most BCC has a pathological activation of the hedgehog pathway, making them susceptible to targeted therapy with selective inhibitors. Sonidegib, approved for the treatment of laBCC on the basis of the results of the basal cell carcinoma outcomes with LDE225 treatment study, demonstrated rapid efficacy and a manageable safety profile. Here, we describe the case of a patient affected by multiple laBCC treated with Sonidegib. The patient experienced an important regression of tumors after only 2 months of therapy, with few side effects. This result confirms the role of Sonidegib as a valid and well-tolerated therapeutic option for laBCC.
Introduction
Basal cell carcinoma (BCC) is the most common form of skin cancer, making up approximately 80% of all nonmelanoma skin cancers [1]. The vast majority of BCCs can be effectively cured by complete histopathology controlled excision, which is the gold standard for BCC treatment. However, about 1–10% of lesions evolve towards advanced forms, including laBCC and Metastatic BCC [2]; in these cases, surgery may not represent a possible therapeutic option, because curing is unlikely and surgery might result in substantial deformity [3].
In order to avoid unreasonable postsurgical cosmetic changes, alternative approaches such as radiation, electrochemotherapy or systemic treatment [4] can be required, as specified by the European guidelines for the management of BCC [5]. Particularly, these approaches are indicated in patient affected by multiple comorbidities, numerous lesions or Gorlin–Goltz syndrome.
However, about 90% of BCCs have a pathologic activation of the Sonic hedgehog pathway, making them susceptible to targeted therapy with selective hedgehog pathway inhibitors (HPI) [6,7]. Notably, Sonidegib (Odomzo) demonstrated a sustained and clinically relevant efficacy and a manageable safety profile in phase II randomized, double-blind basal cell carcinoma outcomes with LDE225 treatment (BOLT) study [8,9].
Herein, we describe a rapid and exceptional response to Sonidegib in a patient affected by multiple disfiguring laBCC.
Case report
An otherwise healthy 71-year-old male patient comes to our attention for the sudden bleeding of an ulcerated abdominal lesion of 8 cm × 5 cm (Fig. 1a). Complete physical examination allowed us to identify about 30 other pink or reddish papules and plaques of different shapes and sizes (up to 15 cm) located on the face, trunk and limb; clinical and dermoscopic characteristics were compatible with BCCs (Fig. 1b–d).
Fig. 1 (a) Deep ulcerated abdominal lesion; (b) Basal cell carcinoma (BCC) of 15 cm × 8 cm located on the left shoulder; (c) multiple BCCs of different shapes and sizes on the anterior trunk, with a detail of the voluminous nodule at the internal cantus; (d) multiple BCC on the posterior trunk.
We collected the patient’s medical history, which was negative for comorbidity, chronic therapy or radiotherapy treatments; he reported a previous surgical treatment to remove a BCC on nasal dorsum without any dermatological follow-up for over 10 years. A skin biopsy was performed on the abdominal lesion, and the histological examination confirmed the diagnosis of BCC. The skull X-ray and the orthopantomography showed no evidence of calcification of the falx cerebri or odontogenic keratocysts; the patient did not appear to have skeletal anomalies or intellectual deficit; he also denies family history of skin cancers. All other criteria for a possible Gorlin–Goltz syndrome have been excluded.
Considering the extensive dimensions of the lesions on the abdomen and back, the numerousness of tumors, and the involvement of critical sites, we excluded the possibility of a surgical approach and decided, on the basis of the results of the BOLT study [8] and the evidence reported by Dummer et al. [10], to start systemic therapy with Sonidegib 200 mg, 1 cp per day.
Therapy was well-tolerated by the patient, except for a transient CTCAE grade I increase in the creatine phosphokinase, less than 2.5 × ULN, observed at the end of the first month of treatment, which did not require a dose adjustment. The other grades 1–2 side effects observed were dysgeusia and nocturnal muscle cramps resolving in a few minutes. Already at the end of the second month of treatment, the abdominal lesion appeared considerably reduced in size and depth (Fig. 2a). Furthermore, a similar clinical improvement was observed in other lesions; notably, the lesion on the back was less infiltrated and the size of the nodule at the internal cantus decreased significantly (Fig. 2b).
Fig. 2 (a) Abdominal basal cell carcinoma (BCC) considerably reduced in size and depth; (b) internal cantus BCC, significantly decreased.
At the end of the sixth month, there was no clinical or dermatoscopic evidence of BCC: the abdominal ulcerated lesion was healed (Fig. 3a), the nodule in the inner corner of the eye was not palpable and the lesions localized in the trunk assumed a scar-like appearance (Fig. 3b–d).
Fig. 3 (a) Ulcerated abdominal lesion healed; (b) scar appearance of the lesion localized in the left shoulder; (c) appearance of the anterior trunk and face lesions after 6 months of therapy; (d) appearance of the posterior trunk lesions after 6 months of therapy.
Currently, the treatment is ongoing, and the excellent clinical response persists.
Discussion
Although BCC is the most common skin cancer, the finding of laBCC is rather infrequent, often related to tumors with a long-term course, located in midface or on ears, with aggressive histopathologic subtype, perivascular or perineural infiltration, history of radiation exposure or previous surgical treatment failure. Patients with immunosuppressive status or multiple comorbidities are more affected [11]. In particular, neglected patients are one of the major contributing factors for the development of mutilating and aggressive BCC.
BCC occurs in the head area in 85–90% of cases [12] and can cause, when the tumor reaches a considerable size, social isolation in affected people. Here, the use of alternative therapeutic strategies is mandatory, to avoid cosmetically unacceptable postsurgical outcomes.
In phase II randomized double-blind BOLT study, which led to the approval of Sonidegib [8], the median time of response assessed by investigators was 1.9 months. Even in our patient, we observed a quick response to the treatment, with a significant improvement of all skin lesions after only 2 months. Already during the first weeks of therapy, in addition to an obvious reduction in the size of the tumors, the patient experienced a significative improvement of symptoms; in particular, the bleeding stopped and the patient reported a progressive reduction in the visual discomfort given by the BCC located at the inner corner of the eye. The rapidity of these events is very important as it promotes patient adherence to treatment, limiting the risk of spontaneous drop-out.
The good tolerability profile of Sonidegib detected by the BOLT study [8] was also confirmed in the long-term observations [10,13,14]. Also, in our experience, dysgeusia appeared as the first and main adverse event, whereas muscle cramps (reported as the first side effect in the patients received the 200 mg dose) arose later. However, also a slight spontaneously resolved increase in the CK value was observed.
Nevertheless, this value should be monitored, to evaluate any dose adjustment as required by the drug data sheet [8,9,13,14].
Even if the usefulness of HPI in the treatment of laBCC is widely confirmed [15], there are still few real-life experiences on Sonidegib [16,17] and anecdotal reports on multiple laBCC.
Our observations confirm the efficacy and safety of Sonidegib in this setting, suggesting it as an excellent therapeutic choice in neglected and laBCC.
Conclusion
In our real-life experience, Sonidegib has fulfilled its main goal, which is to obtain a rapid improvement of lesions and related symptoms, to avoid the risk of infection and anemia, to promote patient compliance towards the treatment and to reduce their social isolation improving his quality of life. The safety and effectiveness of the treatment will be confirmed by the long-term follow-up.
Acknowledgements
The authors are grateful to the patient for agreeing to share photos contributing to extend medical knowledge on this topic. The study received unrestricted grant from Sun Pharmaceutical Industries.
Conflicts of interest
We thank SunPharma for the non-conditioning contribution. There are no conflicts of interest. | Oral | DrugAdministrationRoute | CC BY-NC-ND | 33534224 | 20,359,494 | 2021-04-01 |
What was the outcome of reaction 'Dysgeusia'? | Rapid and exceptional response to Sonidegib in a patient with multiple locally advanced basal cell carcinomas.
Locally advanced basal cell carcinoma (laBCC) represents a rare but possible occurrence in the vast scenario of dermatological diseases. It is well known that most BCC has a pathological activation of the hedgehog pathway, making them susceptible to targeted therapy with selective inhibitors. Sonidegib, approved for the treatment of laBCC on the basis of the results of the basal cell carcinoma outcomes with LDE225 treatment study, demonstrated rapid efficacy and a manageable safety profile. Here, we describe the case of a patient affected by multiple laBCC treated with Sonidegib. The patient experienced an important regression of tumors after only 2 months of therapy, with few side effects. This result confirms the role of Sonidegib as a valid and well-tolerated therapeutic option for laBCC.
Introduction
Basal cell carcinoma (BCC) is the most common form of skin cancer, making up approximately 80% of all nonmelanoma skin cancers [1]. The vast majority of BCCs can be effectively cured by complete histopathology controlled excision, which is the gold standard for BCC treatment. However, about 1–10% of lesions evolve towards advanced forms, including laBCC and Metastatic BCC [2]; in these cases, surgery may not represent a possible therapeutic option, because curing is unlikely and surgery might result in substantial deformity [3].
In order to avoid unreasonable postsurgical cosmetic changes, alternative approaches such as radiation, electrochemotherapy or systemic treatment [4] can be required, as specified by the European guidelines for the management of BCC [5]. Particularly, these approaches are indicated in patient affected by multiple comorbidities, numerous lesions or Gorlin–Goltz syndrome.
However, about 90% of BCCs have a pathologic activation of the Sonic hedgehog pathway, making them susceptible to targeted therapy with selective hedgehog pathway inhibitors (HPI) [6,7]. Notably, Sonidegib (Odomzo) demonstrated a sustained and clinically relevant efficacy and a manageable safety profile in phase II randomized, double-blind basal cell carcinoma outcomes with LDE225 treatment (BOLT) study [8,9].
Herein, we describe a rapid and exceptional response to Sonidegib in a patient affected by multiple disfiguring laBCC.
Case report
An otherwise healthy 71-year-old male patient comes to our attention for the sudden bleeding of an ulcerated abdominal lesion of 8 cm × 5 cm (Fig. 1a). Complete physical examination allowed us to identify about 30 other pink or reddish papules and plaques of different shapes and sizes (up to 15 cm) located on the face, trunk and limb; clinical and dermoscopic characteristics were compatible with BCCs (Fig. 1b–d).
Fig. 1 (a) Deep ulcerated abdominal lesion; (b) Basal cell carcinoma (BCC) of 15 cm × 8 cm located on the left shoulder; (c) multiple BCCs of different shapes and sizes on the anterior trunk, with a detail of the voluminous nodule at the internal cantus; (d) multiple BCC on the posterior trunk.
We collected the patient’s medical history, which was negative for comorbidity, chronic therapy or radiotherapy treatments; he reported a previous surgical treatment to remove a BCC on nasal dorsum without any dermatological follow-up for over 10 years. A skin biopsy was performed on the abdominal lesion, and the histological examination confirmed the diagnosis of BCC. The skull X-ray and the orthopantomography showed no evidence of calcification of the falx cerebri or odontogenic keratocysts; the patient did not appear to have skeletal anomalies or intellectual deficit; he also denies family history of skin cancers. All other criteria for a possible Gorlin–Goltz syndrome have been excluded.
Considering the extensive dimensions of the lesions on the abdomen and back, the numerousness of tumors, and the involvement of critical sites, we excluded the possibility of a surgical approach and decided, on the basis of the results of the BOLT study [8] and the evidence reported by Dummer et al. [10], to start systemic therapy with Sonidegib 200 mg, 1 cp per day.
Therapy was well-tolerated by the patient, except for a transient CTCAE grade I increase in the creatine phosphokinase, less than 2.5 × ULN, observed at the end of the first month of treatment, which did not require a dose adjustment. The other grades 1–2 side effects observed were dysgeusia and nocturnal muscle cramps resolving in a few minutes. Already at the end of the second month of treatment, the abdominal lesion appeared considerably reduced in size and depth (Fig. 2a). Furthermore, a similar clinical improvement was observed in other lesions; notably, the lesion on the back was less infiltrated and the size of the nodule at the internal cantus decreased significantly (Fig. 2b).
Fig. 2 (a) Abdominal basal cell carcinoma (BCC) considerably reduced in size and depth; (b) internal cantus BCC, significantly decreased.
At the end of the sixth month, there was no clinical or dermatoscopic evidence of BCC: the abdominal ulcerated lesion was healed (Fig. 3a), the nodule in the inner corner of the eye was not palpable and the lesions localized in the trunk assumed a scar-like appearance (Fig. 3b–d).
Fig. 3 (a) Ulcerated abdominal lesion healed; (b) scar appearance of the lesion localized in the left shoulder; (c) appearance of the anterior trunk and face lesions after 6 months of therapy; (d) appearance of the posterior trunk lesions after 6 months of therapy.
Currently, the treatment is ongoing, and the excellent clinical response persists.
Discussion
Although BCC is the most common skin cancer, the finding of laBCC is rather infrequent, often related to tumors with a long-term course, located in midface or on ears, with aggressive histopathologic subtype, perivascular or perineural infiltration, history of radiation exposure or previous surgical treatment failure. Patients with immunosuppressive status or multiple comorbidities are more affected [11]. In particular, neglected patients are one of the major contributing factors for the development of mutilating and aggressive BCC.
BCC occurs in the head area in 85–90% of cases [12] and can cause, when the tumor reaches a considerable size, social isolation in affected people. Here, the use of alternative therapeutic strategies is mandatory, to avoid cosmetically unacceptable postsurgical outcomes.
In phase II randomized double-blind BOLT study, which led to the approval of Sonidegib [8], the median time of response assessed by investigators was 1.9 months. Even in our patient, we observed a quick response to the treatment, with a significant improvement of all skin lesions after only 2 months. Already during the first weeks of therapy, in addition to an obvious reduction in the size of the tumors, the patient experienced a significative improvement of symptoms; in particular, the bleeding stopped and the patient reported a progressive reduction in the visual discomfort given by the BCC located at the inner corner of the eye. The rapidity of these events is very important as it promotes patient adherence to treatment, limiting the risk of spontaneous drop-out.
The good tolerability profile of Sonidegib detected by the BOLT study [8] was also confirmed in the long-term observations [10,13,14]. Also, in our experience, dysgeusia appeared as the first and main adverse event, whereas muscle cramps (reported as the first side effect in the patients received the 200 mg dose) arose later. However, also a slight spontaneously resolved increase in the CK value was observed.
Nevertheless, this value should be monitored, to evaluate any dose adjustment as required by the drug data sheet [8,9,13,14].
Even if the usefulness of HPI in the treatment of laBCC is widely confirmed [15], there are still few real-life experiences on Sonidegib [16,17] and anecdotal reports on multiple laBCC.
Our observations confirm the efficacy and safety of Sonidegib in this setting, suggesting it as an excellent therapeutic choice in neglected and laBCC.
Conclusion
In our real-life experience, Sonidegib has fulfilled its main goal, which is to obtain a rapid improvement of lesions and related symptoms, to avoid the risk of infection and anemia, to promote patient compliance towards the treatment and to reduce their social isolation improving his quality of life. The safety and effectiveness of the treatment will be confirmed by the long-term follow-up.
Acknowledgements
The authors are grateful to the patient for agreeing to share photos contributing to extend medical knowledge on this topic. The study received unrestricted grant from Sun Pharmaceutical Industries.
Conflicts of interest
We thank SunPharma for the non-conditioning contribution. There are no conflicts of interest. | Recovered | ReactionOutcome | CC BY-NC-ND | 33534224 | 20,359,494 | 2021-04-01 |
What was the outcome of reaction 'Muscle spasms'? | Rapid and exceptional response to Sonidegib in a patient with multiple locally advanced basal cell carcinomas.
Locally advanced basal cell carcinoma (laBCC) represents a rare but possible occurrence in the vast scenario of dermatological diseases. It is well known that most BCC has a pathological activation of the hedgehog pathway, making them susceptible to targeted therapy with selective inhibitors. Sonidegib, approved for the treatment of laBCC on the basis of the results of the basal cell carcinoma outcomes with LDE225 treatment study, demonstrated rapid efficacy and a manageable safety profile. Here, we describe the case of a patient affected by multiple laBCC treated with Sonidegib. The patient experienced an important regression of tumors after only 2 months of therapy, with few side effects. This result confirms the role of Sonidegib as a valid and well-tolerated therapeutic option for laBCC.
Introduction
Basal cell carcinoma (BCC) is the most common form of skin cancer, making up approximately 80% of all nonmelanoma skin cancers [1]. The vast majority of BCCs can be effectively cured by complete histopathology controlled excision, which is the gold standard for BCC treatment. However, about 1–10% of lesions evolve towards advanced forms, including laBCC and Metastatic BCC [2]; in these cases, surgery may not represent a possible therapeutic option, because curing is unlikely and surgery might result in substantial deformity [3].
In order to avoid unreasonable postsurgical cosmetic changes, alternative approaches such as radiation, electrochemotherapy or systemic treatment [4] can be required, as specified by the European guidelines for the management of BCC [5]. Particularly, these approaches are indicated in patient affected by multiple comorbidities, numerous lesions or Gorlin–Goltz syndrome.
However, about 90% of BCCs have a pathologic activation of the Sonic hedgehog pathway, making them susceptible to targeted therapy with selective hedgehog pathway inhibitors (HPI) [6,7]. Notably, Sonidegib (Odomzo) demonstrated a sustained and clinically relevant efficacy and a manageable safety profile in phase II randomized, double-blind basal cell carcinoma outcomes with LDE225 treatment (BOLT) study [8,9].
Herein, we describe a rapid and exceptional response to Sonidegib in a patient affected by multiple disfiguring laBCC.
Case report
An otherwise healthy 71-year-old male patient comes to our attention for the sudden bleeding of an ulcerated abdominal lesion of 8 cm × 5 cm (Fig. 1a). Complete physical examination allowed us to identify about 30 other pink or reddish papules and plaques of different shapes and sizes (up to 15 cm) located on the face, trunk and limb; clinical and dermoscopic characteristics were compatible with BCCs (Fig. 1b–d).
Fig. 1 (a) Deep ulcerated abdominal lesion; (b) Basal cell carcinoma (BCC) of 15 cm × 8 cm located on the left shoulder; (c) multiple BCCs of different shapes and sizes on the anterior trunk, with a detail of the voluminous nodule at the internal cantus; (d) multiple BCC on the posterior trunk.
We collected the patient’s medical history, which was negative for comorbidity, chronic therapy or radiotherapy treatments; he reported a previous surgical treatment to remove a BCC on nasal dorsum without any dermatological follow-up for over 10 years. A skin biopsy was performed on the abdominal lesion, and the histological examination confirmed the diagnosis of BCC. The skull X-ray and the orthopantomography showed no evidence of calcification of the falx cerebri or odontogenic keratocysts; the patient did not appear to have skeletal anomalies or intellectual deficit; he also denies family history of skin cancers. All other criteria for a possible Gorlin–Goltz syndrome have been excluded.
Considering the extensive dimensions of the lesions on the abdomen and back, the numerousness of tumors, and the involvement of critical sites, we excluded the possibility of a surgical approach and decided, on the basis of the results of the BOLT study [8] and the evidence reported by Dummer et al. [10], to start systemic therapy with Sonidegib 200 mg, 1 cp per day.
Therapy was well-tolerated by the patient, except for a transient CTCAE grade I increase in the creatine phosphokinase, less than 2.5 × ULN, observed at the end of the first month of treatment, which did not require a dose adjustment. The other grades 1–2 side effects observed were dysgeusia and nocturnal muscle cramps resolving in a few minutes. Already at the end of the second month of treatment, the abdominal lesion appeared considerably reduced in size and depth (Fig. 2a). Furthermore, a similar clinical improvement was observed in other lesions; notably, the lesion on the back was less infiltrated and the size of the nodule at the internal cantus decreased significantly (Fig. 2b).
Fig. 2 (a) Abdominal basal cell carcinoma (BCC) considerably reduced in size and depth; (b) internal cantus BCC, significantly decreased.
At the end of the sixth month, there was no clinical or dermatoscopic evidence of BCC: the abdominal ulcerated lesion was healed (Fig. 3a), the nodule in the inner corner of the eye was not palpable and the lesions localized in the trunk assumed a scar-like appearance (Fig. 3b–d).
Fig. 3 (a) Ulcerated abdominal lesion healed; (b) scar appearance of the lesion localized in the left shoulder; (c) appearance of the anterior trunk and face lesions after 6 months of therapy; (d) appearance of the posterior trunk lesions after 6 months of therapy.
Currently, the treatment is ongoing, and the excellent clinical response persists.
Discussion
Although BCC is the most common skin cancer, the finding of laBCC is rather infrequent, often related to tumors with a long-term course, located in midface or on ears, with aggressive histopathologic subtype, perivascular or perineural infiltration, history of radiation exposure or previous surgical treatment failure. Patients with immunosuppressive status or multiple comorbidities are more affected [11]. In particular, neglected patients are one of the major contributing factors for the development of mutilating and aggressive BCC.
BCC occurs in the head area in 85–90% of cases [12] and can cause, when the tumor reaches a considerable size, social isolation in affected people. Here, the use of alternative therapeutic strategies is mandatory, to avoid cosmetically unacceptable postsurgical outcomes.
In phase II randomized double-blind BOLT study, which led to the approval of Sonidegib [8], the median time of response assessed by investigators was 1.9 months. Even in our patient, we observed a quick response to the treatment, with a significant improvement of all skin lesions after only 2 months. Already during the first weeks of therapy, in addition to an obvious reduction in the size of the tumors, the patient experienced a significative improvement of symptoms; in particular, the bleeding stopped and the patient reported a progressive reduction in the visual discomfort given by the BCC located at the inner corner of the eye. The rapidity of these events is very important as it promotes patient adherence to treatment, limiting the risk of spontaneous drop-out.
The good tolerability profile of Sonidegib detected by the BOLT study [8] was also confirmed in the long-term observations [10,13,14]. Also, in our experience, dysgeusia appeared as the first and main adverse event, whereas muscle cramps (reported as the first side effect in the patients received the 200 mg dose) arose later. However, also a slight spontaneously resolved increase in the CK value was observed.
Nevertheless, this value should be monitored, to evaluate any dose adjustment as required by the drug data sheet [8,9,13,14].
Even if the usefulness of HPI in the treatment of laBCC is widely confirmed [15], there are still few real-life experiences on Sonidegib [16,17] and anecdotal reports on multiple laBCC.
Our observations confirm the efficacy and safety of Sonidegib in this setting, suggesting it as an excellent therapeutic choice in neglected and laBCC.
Conclusion
In our real-life experience, Sonidegib has fulfilled its main goal, which is to obtain a rapid improvement of lesions and related symptoms, to avoid the risk of infection and anemia, to promote patient compliance towards the treatment and to reduce their social isolation improving his quality of life. The safety and effectiveness of the treatment will be confirmed by the long-term follow-up.
Acknowledgements
The authors are grateful to the patient for agreeing to share photos contributing to extend medical knowledge on this topic. The study received unrestricted grant from Sun Pharmaceutical Industries.
Conflicts of interest
We thank SunPharma for the non-conditioning contribution. There are no conflicts of interest. | Recovered | ReactionOutcome | CC BY-NC-ND | 33534224 | 20,359,494 | 2021-04-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Condition aggravated'. | Case Report: Iliopsoas Hematoma during the Clinical Course of Severe COVID-19 in Two Male Patients.
Anticoagulation plays a major role in reducing the risk of systematic thrombosis in patients with severe COVID-19. Serious hemorrhagic complications, such as intracranial hemorrhage, have also been recognized. However, intra-abdominal hemorrhage is under-recognized because of its rare occurrence, despite high mortality. Here, we discuss two cases of spontaneous iliopsoas hematoma (IPH) likely caused by anticoagulants during the clinical course of COVID-19. We also explored published case reports to identify clinical characteristics of IPH in COVID-19 patients. The use of anticoagulants may increase the risk of lethal IPH among COVID-19 patients becsuse of scarce data on optimal dosage and adequate monitoring of anticoagulant effects. Rapid diagnosis and timely intervention are crucial to ensure good patient outcomes.
INTRODUCTION
As awareness of COVID-19-associated coagulopathy increases, thrombotic manifestations are implicated in mortality.1,2 A recent multicenter retrospective study from the United States reported a thrombotic complication rate was 9.5%, despite standard prophylactic anticoagulation dose.3 Furthermore, hemorrhagic events among COVID-19 patients have been recognized. The overall proportion of patients with bleeding complications range from 4.8% to 8%, of which approximately 3.5% had major bleeding.3,4
Herein, we describe two cases of life-threatening iliopsoas hematoma (IPH) during the clinical course of COVID-19. Both cases were recovering from acute respiratory distress syndrome owing to COVID-19 when they developed IPH. Moreover, they were receiving anticoagulants to prevent thrombosis. This report highlights IPH as a serious hemorrhagic complication in severe COVID-19 patients treated with anti-thrombotic agents.
CASE REPORT
Case 1.
A 62-year-old man was referred to our institution because of a 4-day history of fever, productive cough, and fatigue. Results of real-time PCR for SARS-CoV-2 performed for a nasal swab were positive.
On admission, his temperature was 37.2°C; blood pressure, 148/76 mmHg; heart rate, 100 beats/minute; respiratory rate, 24 breaths/minute; oxygen saturation (SpO2), 91–94% in ambient air; and ratio of arterial oxygen partial pressure to fractional inspired oxygen (P/F ratio), 316. Chest computed tomography (CT) on admission revealed ground-glass opacities in the bilateral peripheral lung fields, and laboratory findings were leukocyte count, 6.8 (normal, 3.3–8.6) × 109/L with 76.9% neutrophils; lymphocyte percentage, 18.3% (normal, 26–46%); platelet count, 178 (normal, 158–348) × 103/μL; interleukin-6 level, 89.1 pg/mL (cutoff < 4.0 pg/mL); C-reactive protein (CRP) level, 5.53 mg/dL (cutoff < 0.14 mg/dL); ferritin level, 537.1 (normal, 50–200) ng/mL; D-dimer level, 0.5 μg/mL (cutoff < 1.0 μg/mL); and fibrinogen level, 611 (normal, 180–350) mg/dL.
We prescribed favipiravir (loading dose of 1,800 mg on day 1, followed by 400 mg twice daily for 13 days), dexamethasone (6.6 mg/day intravenously administered for 10 days), and nafamostat (100 mg/day). Despite initial treatment, 10 L of oxygen supplementation was required to maintain SpO2 > 90% for the first 3 days after admission. Hence, tocilizumab (8 mg/kg) was intravenously administered on days 3 and 4. Hypoxia continued to worsen; thus, the patient was intubated and transferred to the intensive care unit (ICU). Recombinant human soluble thrombomodulin (rhsTM; 12,800 U twice daily) was administered to prevent thrombosis. After 5 days in the ICU, he fully recovered from acute respiratory failure, extubated, and returned to the ward on day 9. On the same day, rhsTM was replaced with daily subcutaneous enoxaparin (40 mg).
On day 14, the patient complained about severe and sudden pain in his left lower back after physiotherapy. His blood pressure and pulse rates were 118/86 mmHg and 115 beats/minute, respectively. Hemoglobin levels dropped from 15.7 g/dL to 13.2 g/dL with an elevated D-dimer level of 2.6 μg/mL. Contrast-enhanced abdominal and pelvic CT showed a hematoma (6.5 × 10.5 cm) in the left iliopsoas muscle spreading to the retroperitoneal space with extravasation (Figure 1A). Urgent transarterial embolization (TAE) was performed by interventional radiologists. Extravasation was identified from the left L3 and L4 lumbar arteries and common iliac arteries. Those arteries were selectively catheterized with a five French Mickelson catheter through which gelatin sponge particles were placed for occlusion. After the procedure, no active contrast extravasation was observed. He received six packed red blood cell (PRBC) units with intravenous fluids because his hemoglobin levels dropped to 7.5 g/dL. He was discharged from the hospital with improved hemoglobin levels (12.6 g/dL) on day 33.
Figure 1. Contrast-enhanced computed tomography of the abdomen and pelvis. (A) Case 1: A massive hematoma with extravasation (red arrow) in the left iliopsoas muscle spreading to the retroperitoneal space is observed. (B) Case 2: The swollen right iliopsoas muscle with retroperitoneal hematoma and extravasation is observed (red arrow). A small low-density area (blue arrow) in the left iliopsoas muscle suggests a hematoma.
Case 2.
A 79-year-old man presented with fever and general fatigue for 2 days and was transferred to our hospital owing to COVID-19–associated acute respiratory distress syndrome. A different general hospital administered favipiravir, dexamethasone, and subcutaneous unfractionated heparin (UFH) on referral day. Nevertheless, respiratory failure progressively worsened, and he was intubated.
Oxygen saturation was 95% under fraction of inspired oxygenation, 50%; P/F ratio 167; blood pressure, 132/65 mmHg; heart rate, 40 beats/minute; temperature, 38.4°C at the time of admission to our hospital. Initial laboratory findings were leukocyte count, 10.2 × 109/L with 89.7% neutrophils; lymphocyte percentage, 5.6%; platelet count, 129 × 103/μL; CRP level, 2.9 mg/dL; D-dimer level, 0.6 μg/mL; and fibrinogen level, 285 mg/dL. Chest CT revealed bilateral ground-glass opacities with a small amount of pleural effusion.
Remdesivir (loading dose of 200 mg on day 1, followed by 100 mg once daily for 10 days), methylprednisolone (1,000 mg for 3 days) followed by dexamethasone (6.6 mg for 7 days), nafamostat (100 mg/day), and intravenous tocilizumab (two consecutive doses, 8 mg/kg) were administered. Intravenous UFH was also administered, with the dosage adjusted to meet the target-activated partial thromboplastin time (aPTT) range of 1.5–2.5 times the control value. The P/F ratio increased from 167 to 290 on day 10, and he was extubated.
After extubation, blood pressure gradually decreased with tachycardia. Moreover, he complained of gradual worsening pain in the right lower back since extubation. His hemoglobin levels decreased from 12.2 g/dL to 9.7 g/dL, suggesting hemorrhagic hypovolemic shock. His platelet count was 158 × 103/μL; aPTT, 42.1 (normal, 23.5–35) seconds; D-dimer level, 2.2 μg/mL; and fibrinogen level, 167 mg/dL. Contrast-enhanced abdominal CT showed swelling of the right iliopsoas muscle with retroperitoneal hematoma, extravasation in the right psoas major muscle, and a small-density area in the left psoas major muscle (Figure 1B). Intravenous UFH infusion was immediately discontinued; TAE was urgently attempted with rapid volume replacement and four units of PRBC transfusion. However, he had cardiac arrest for 4 minutes during preoperative preparations. Although he responded to resuscitation, the second episode of cardiac arrest occurred within an hour. Because his family members did not want further resuscitation attempts, 18 hours after extubation, his death was confirmed.
DISCUSSION
The current case series showed the life-threatening IPH in two COVID-19 patients on either prophylactic or therapeutic dose of anticoagulants; whereas one patient was successfully treated with TAE, the other patient died because of hemorrhagic hypovolemic shock.
Iliopsoas hematoma is defined as retroperitoneal hemorrhage involving the iliopsoas muscle. The incidence of spontaneous IPH during hospitalization ranges from 0.3% to 0.4%.5,6 The precise frequency of spontaneous IPH in COVID-19 patients remains unclear; however, eight case series have been reported (Table 1).7–13 Several specific predisposing factors, including anticoagulation therapy, advanced age, and hemodialysis, are associated with developing spontaneous IPH.5 Previous reports in addition to the current case series were consistent with this evidence. Furthermore, concurrent off-level use of rhsTM or nafamostat mesylate may have contributed to major bleeding events in our cases. Despite insufficient data, those agents had been administered as compassionate therapy to alleviate hypercoagulation and for their antiviral effects.14,15
Table 1 Summary of case reports on iliopsoas hematoma in patients with COVID-19
Case no. Reference Age, years Sex Preexisting condition Severity of COVID-19* Anticoagulation at hemorrhage Intervention for hemorrhage Outcome Comments
1. Patel et al.7 69 M CAD, HTN, and type 2 DM Severe Therapeutic enoxaparin (1 mg/kg) TAE Alive –
2. Scialpi et al.8 57 M Non-Hodgkin’s lymphoma ND ND TAE Alive –
3. Erdinc and Raina9 58 F HTN, obesity (BMI, 62 kg/m2) Severe None ND Died Acute DVT coincided
4. Guo et al.10 71 M ND Critical ND, but presumably on anticoagulants because of ECMO TAE Alive On ECMO
5. Angileri et al.11 59 M ND Critical Prophylactic enoxaparin (60 mg/day) ND ND –
6. Conti et al.12 76 M CAD and HTN Severe Prophylactic LMWH (6,000 UI/day) TAE ND –
7. Conti et al.12 72 F HTN Severe Therapeutic LMWH (100 UI/kg/bid) TAE ND Acute DVT coincided
8. Bargellini et al.13 71 M Atrial fibrillation Prophylactic LMWH (6,000 UI/12 hours) TAE Alive
9. Case 1 62 M None Critical Prophylactic enoxaparin (40 mg bid subcutaneous injection) TAE Alive
10. Case 2 79 M HTN, obesity (BMI, 34.2 kg/m2), type 2 DM Critical Therapeutic unfractionated heparin TAE Died Died because of hypovolemic shock
bid = bis in die; BMI = body mass index; CAD = coronary artery disease; DM = diabetes; DVT = deep venous thrombosis; ECMO = extracorporeal membrane oxygenation; F = female; HTN = hypertension; LMWH = low-molecular-weight heparin; M = male; ND = not described; TAE = transarterial embolization.
* Severity of COVID-19 was defined as follows. Severe: individuals who have oxygen saturation < 94% on ambient air, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen < 300 mmHg, respiratory frequency > 30 breaths/minute, or lung infiltrates > 50%. Critical: individuals who have respiratory failure, septic shock, and/or multiple organ dysfunction.
Apart from anticoagulants, microvascular vulnerability derived from atherosclerosis and microtrauma, including cough, could lead to retroperitoneal bleeding; however, pathology has not confirmed the hypothesis.5 Whether direct effects of SARS-CoV-2 on vascular endothelial cells cause hemorrhage events is uncertain. However, several hypotheses have been raised: the affinity of SARS-CoV-2 for angiotensin-converting enzyme 2 receptors on endothelial cells can directly damage vessels, causing vessel wall rupture.16 By contrast, renin–angiotensin–aldosterone system dysregulation may contribute to increased blood pressure, causing hemorrhagic events.16 Erdinc and Raina9 reported a COVID-19 patient who developed IPH without anticoagulants.
The mortality rate of patients with spontaneous IPH, which occurred during hospitalization, is estimated to be between 30% and 50%.5,6 Given the high mortality rate, physicians should pay careful attention to the disease’s early signs. Thrombocytopenia (platelet count < 150 × 103 cells/μL) and elevated D-dimer levels (> 2.5 μg/mL) at initial presentation are predictive of hemorrhage-related complications during hospitalization.3 In fact, the initial platelet count in case 2 was 129 × 103 cells/μL.
One-third of patients with spontaneous IPH can present with Lenk’s triad: severe flank pain, hemodynamic shock, and palpable mass.17 Monitoring the effects of anticoagulants is imperative to minimize the risk of hemorrhagic events; however, there are some inherent issues. Artzer et al.6 reported that severe adverse events could occur among patients treated with UFH despite optimal aPTT values. For case 2, aPTT values were within the target range. Regarding low-molecular-weight heparin, anti-factor Xa activity and thromboelastography may not be reliable for monitoring.18 Contrast-enhanced CT plays an important role in diagnosis, providing a wide range of information on volume, spatial extent, and compressive complications or extravasation.5
An effective anticoagulation therapy with low risk of bleeding is urgently required. Despite the lack of data, some experts have suggested intensified anticoagulation strategy such as using enoxaparin (0.5 mg/kg twice daily) because standard-dose prophylaxis may not prevent thrombosis.19 A recent observational study showed that compared with a prophylactic anticoagulant, a therapeutic dose of an anticoagulant had the tendency to lower the risk of mortality among hospitalized patients with COVID-19, but it showed an insignificant difference.20 In addition, the therapeutic dose was associated with a higher risk of major hemorrhage than was prophylactic dose (3% versus 1.7%).
Although case reports are scarce for conclusion, our two cases, in addition to previous reports, suggest that COVID-19 patients treated with anticoagulants are at risk of IPH. The paucity of data on how to balance anticoagulation in COVID-19 patients may be attributed to hemorrhagic events. Until randomized trials prove the optimal anticoagulation treatment, there will be no clarity with respect to the ideal type and dose of anticoagulants for patients with COVID-19. If severe flank pain, along with anemia and hypovolemic signs, is present, CT should be used for evaluation.
ACKNOWLEDGMENTS
We thank the patients and their family members for permitting us to publish this report. The American Society of Tropical Medicine and Hygiene has waived the Open Access fee for this article due to the ongoing COVID-19 pandemic. | DEXAMETHASONE, FAVIPIRAVIR, HEPARIN SODIUM | DrugsGivenReaction | CC BY-NC | 33534775 | 19,826,864 | 2021-01-13 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Anal pruritus'. | Risk factors for ≥high-grade anal intraepithelial lesions in MSM living with HIV and the response to topical and surgical treatments.
The objective of this study in MSM living with HIV was to determine the incidence of HSIL and ASCC, related factors, and the response to treatment.
Data were gathered in 405 consecutive HIV-infected MSM (May 2010-December 2018) at baseline and annually on: sexual behavior, anal cytology, and HPV PCR and/or high-resolution anoscopy results. They could choose mucosectomy with electric scalpel (from May 2010) or self-administration of 5% imiquimod 3 times weekly for 16 weeks (from November 2013). A multivariate logistic regression model was developed for ≥HSIL-related factors using a step-wise approach to select variables, with a significance level of 0.05 for entry and 0.10 for exit, applying the Hosmer-Lemeshow test to assess the goodness of fit.
The study included 405 patients with a mean age of 36.2 years; 56.7% had bachelor´s degree, and 52.8% were smokers. They had a mean of 1 (IQR 1-7) sexual partner in the previous 12 months, median time since HIV diagnosis of 2 years, and mean CD4 nadir of 367.9 cells/uL; 86.7% were receiving ART, the mean CD4 level was 689.6 cells/uL, mean CD4/CD8 ratio was 0.77, and 85.9% of patients were undetectable. Incidence rates were 30.86/1,000 patient-years for ≥high squamous intraepithelial lesion (HSIL) and 81.22/100,000 for anal squamous cell carcinoma (ASCC). The ≥HSIL incidence significantly decreased from 42.9% (9/21) in 2010 to 4.1% (10/254) in 2018 (p = 0.034). ≥HSIL risk factors were infection with HPV 11 (OR 3.81; 95%CI 1.76-8.24), HPV 16 (OR 2.69, 95%CI 1.22-5.99), HPV 18 (OR 2.73, 95%CI 1.01-7.36), HPV 53 (OR 2.97, 95%CI 1.002-8.79); HPV 61 (OR 11.88, 95%CI 3.67-38.53); HPV 68 (OR 2.44, CI 95% 1.03-5.8); low CD4 nadir (OR1.002; 95%CI 1-1.004) and history of AIDS (OR 2.373, CI 95% 1.009-5.577). Among HSIL-positive patients, the response rate was higher after imiquimod than after surgical excision (96.7% vs 73.3%, p = 0.009) and there were fewer re-treatments (2.7% vs 23.4%, p = 0.02) and adverse events (2.7% vs 100%, p = 0.046); none developed ASCC.
HSIL screening and treatment programs reduce the incidence of HSIL, which is related to chronic HPV infection and poor immunological status. Self-administration of 5% imiquimod as first-line treatment of HSIL is more effective than surgery in HIV+ MSM.
Introduction
Anal squamous-cell carcinoma (ASCC) is frequent among individuals living with HIV (HIV+) and associated with a high mortality rate [1] and with an incidence of up to 131/100,000 people/year among HIV+ men who have sex with men (MSM) [2], with a similar survival rate to the general population [3]. The beginning of a decline in its incidence among MSM has been described by some observational studies but not by clinical trials, which may be explained by the participation of their study populations in an intraepithelial neoplasia screening and treatment program [4, 5], as recently reported by our group [6].
ASCC has been associated with persistent infection by high-risk oncogenic HPV (HR-HPV) genotypes [7]. Interaction between HIV and HPV, which have risk factors in common, has been reported to increase the risk of HPV and reduce the clearance rate among individuals living with HIV [8].
It remains controversial whether the treatment of high-grade anal intraepithelial lesions (HSILs) prevents the subsequent development of ASCC [9], and no standard approach has been established, so that treatments vary among centers according to their resources and experience. Therapeutic options include local topical treatments with trichloroacetic acid, imiquimod, or 5-fluoruracil and more invasive treatments such as excision, fulguration, or laser therapy [9]. Surgical excision used to be a treatment of choice but is associated with adverse effects, including anal stenosis, and with recurrences and incomplete resection, especially when the anal disease is extensive [10]. Fulguration is applied during high-resolution anoscopy (HRA) and has been associated with a recurrence rate of around 25% at 30 months in HIV+ patients, depending on the amount of fulguration [11]. In regard to infrared or laser coagulation, it can be applied in the consulting room and is well tolerated, but a recurrence rate of 61% at one year has been described in HIV+ patients [12]. Finally, topical therapies offer a non-invasive option with a good safety profile and few adverse effects [9]. In particular, imiquimod offers the advantage of self-administration, a low recurrence rate, and a high response rate in HIV+ patients, which was reported to be 61% in a clinical trial [13].
In this study of consecutive HIV+ MSM undergoing a screening/treatment program at our hospital, we previously reported that oncogenic genotype clearance in anal mucosa was associated with the length of ART but did not affect the incidence of HSILs or ASCC. In the present article, data from the same population [6] were analyzed with a different objective, which was to determine the incidence of HSIL and ASCC, the risk factors, and the response to imiquimod treatment and/or electrosurgical excision.
Patients and methods
Design
This longitudinal observational (naturalistic/naturalist) prospective study was conducted in a population of 405 adult HIV+ MSM patients with no history of ASCC, who were consecutively enrolled between May 2010 and December 2018 in a screening and follow-up program for dysplasic anal mucosa lesions. This population and the methodology applied were previously reported [6] in a previous study on HR-HPV clearance and acquisition rates and their relationship with the incidence of HSILs/ASCC in anal mucosa. At their baseline visit (V0) we obtained the written informed consent of the patients to participation in the study, which was approved by the ethics committee (CEIC) of the University Hospital “Virgen de las Nieves” and complied with Spanish data protection legislation (Law 15/1999, 13 December). This CEIC is integrated within the network of ethics Committees of Andalusian public health system (SSPA). CEIC is regulated by order/Decree 439/2010 December 14th of Andalusia. All data were treated in accordance with Spanish data protection legislation Law 15/1999, 13 December, on Personal Character Data Protection).
In brief, data were gathered at baseline (V0), 4–12 weeks, and subsequent follow-ups (at individualized intervals) on clinical-epidemiological and analytical variables and on the results of PCR (Gonorrhoeae, Mycoplasma spp, Chlamydia spp, Ureaplasma spp) and oral-anal-urethral exudate culture (Gonorrhoeae) studies in symptomatic patients or infected partners. In addition, two anal canal mucosa samples were taken at baseline for HPV detection and genotyping by qualitative PCR and for cytology study using the “thin-layer” technique (Processor Thin Prep 2000 (Hologic), and HRA was performed at 4–12 weeks taking samples of apparently normal mucosa and areas with Lugol-negative aceto-white lesions using an endoscopic retrograde cholangiopancreatography catheter. HRA results were obtained from 100% of patients, and findings of at least one follow-up anoscopy were available for 97.3% (394/405). HRA was performed by an infectious disease specialist specifically trained for one year by an expert in this technique from the Department of Digestive Disease of our hospital.
At one year, patients with a normal anoscopy result and LSIL (AIN1) were examined with cytology, HPV PCR, and anoscopy, whereas those with HSIL underwent electrosurgical mucosal resection (from May 2010) or self-administration of 5% imiquimod 3 times/week for 16 weeks (from November 2013), followed by another anoscopy. Surgery was the sole option from May 2010 until the introduction of imiquimod in 2013; since then, all patients have been offered imiquimod as first treatment option. If the imiquimod treatment failed, patients were offered extension of the course up to 18 weeks, a new 16-week cycle of imiquimod, or surgery. Between May 2012 and May 2014, quadrivalent HPV vaccine was received by patients with no HPV 16 or 18 infection and no presence or history of HSIL+-compatible lesions. Cytology results were categorized according to the Bethesda classification [14] and histological findings according to the LAST HPV standardization project [15].
Definition of variables
Abnormal cytology: Cytology findings of ASCUS, LSIL, or HSIL.
Histology with
≥HSILs: Histology findings from HSIL to ASCC.
Therapeutic failure: HSIL persistence after treatment.
Post-treatment HSIL recurrence: Re-appearance of previously treated lesion with normal post-treatment anoscopy.
Response to treatment: Disappearance of lesion in follow-up anoscopy after treatment with surgery or imiquimod.
Self-administration of 5% imiquimod. On Mondays, Wednesdays and Fridays, patients used a preloaded single-use insulin syringe (after cleaning drug remains from its surface) to apply imiquimod inside the anal canal, 3 cm from the anal verge, while in left lateral decubitus position, preferably before sleeping. Before starting this therapy, the patient received detailed instructions on the procedure from the attending physician and nurse.
Statistical analysis
SPSS 21.0 was used for data analyses. The descriptive statistics and tests used for bivariate analyses were previously reported in detail [6]. In the present study, a multivariate logistic regression model was developed for ≥HSIL-related factors, using Freeman’s formula [n = 10*(k+1)] [16], including significant variables in bivariate analysis (ART duration, infection with low-risk HPV genotypes, infection with HPV-6, 11, 16, 18, 53, 59, 61, or 68, and duration of infection with high-risk genotypes) and other variables deemed clinically relevant (smoking habit, history of AIDS, CD4 nadir, CD4/CD8 ratio, duration of infection with HR-HPV and mixed infection). A stepwise approach was used to select variables, with a significance level of 0.05 for entry and 0.10 for exit, applying the Hosmer-Lemeshow test to assess the goodness of fit. P≤0.05 was considered significant.
Results
Baseline characteristics of the cohort
Table 1 summarizes the baseline characteristics of the 405 HIV+MSM enrolled in the study, Table 2 exhibits the distribution of genotypes and Table 3 the cytology and anoscopy findings. In brief, the mean age was 36.2 years, and fewer than 10% were aged > 50 years. They had very good viro-immunological status, 85.6% were on ART at enrolment, and the mean interval since HIV diagnosis was around two years. There was a high prevalence of HPV infection: 76.9% with HR-genotypes, 73.1% with LR-genotypes and 58.1% with both. The prevalence of ≥HSILs was 21.7% (88/405), with an incidence of 30.86/1,000 patient-years, while the prevalence of ASCC was 0.74%, (4/405), with an incidence of 81.22/100,000 patient-years.
10.1371/journal.pone.0245870.t001Table 1 Characteristics of HIV-infected MSM patients.
Number of patients
n = 405
Age, mean (± SD) 36.2 (± 10.1)
<30 yrs, n (%) 123 (30.4)
30–50 yrs, n (%) 244 (60.2)
>50 yrs n (%) 38 (9.4)
Educational level
No studies 6 (1.5)
Primary studies 40 (9.9)
Secondary studies 129 (31.9)
University studies 230 (56.7)
Retired, n (%), 95% CI 22 (5.4) (3–7.5)
Origin
Europe 387 (95.6)
Central America 17 (4.2)
qHPV vaccine (2012–2014), n (%), 95% CI 66 (16.3)
Age at first sexual intercourse, median (IQR) 18 (16–20)
Number of lifetime male sex partners, median (IQR) 50 (15–150)
Number of male sex partners during previous 12 months, median (IQR) 1(1–7)
Habitual use of condoms, n (%), 95% CI 294 (72.6) (68.2–77.4)
Total number of sexual partners during follow-up, median (IQR) 54.5(20–154)
History of anal/genital warts, n (%), (95%CI) 128 (31.6), (27.1–36.1)
Anal/genital warts at baseline n (%), (95%CI) 93 (23), (18.6–26.8)
History of syphilis, n (%), IC95% 103 (25.4), (21.6–29.8)
History of other STI, n (%), IC 95% 110 (27.2), (23.1–31.6)
Time since HIV diagnosis (months), median (IQR) 25 (8–84)
CD4 at diagnosis of HIV (cel/uL), mean (± SD) 448± 298.17
HIV viral load at diagnosis of HIV (log), median (IQR) 4.61 (4.07–5.12)
CD4 nadir (cells/uL), mean (± SD) 367.93±233.85
CD4 nadir < 200 cells/uL, n (%), 95% CI 97 (23.9), (20–28.5)
CD4 cell count at baseline (cells/uL), mean (± SD) 689.64± 475.03
CD8 cell count at baseline (cells/uL), mean (± SD) 981.5±531.5
CD4 /CD8 ratio, mean (± SD) 0.77±0.70
HIV viral load at baseline (log), median (IQR) 0 (0–1.72)
Undetectable: < 50 HIV RNA copies/mL of plasma, n (%) 348 (85.9)
History of AIDS diagnosis, n (%), 95% CI 106 (26.2) (21.3–30.1)
HAART before inclusion, n (%), 95% CI 351 (86.7), (83.2–90)
Previous ART line, median (IQR) 1 (1–2)
Virological failure, n (%) 17 (4.8)
Median months of ART, median (IQR) 4 (16–56)
Chronic HCV infection, n (%) 14 (3.5)
Chronic HBV infection, n (%) 13 (3.2)
Smoker, pack/year, median (IQR) 1.5 (0–14)
Smoker, n (%), 95% CI 214 (52.8) (47.9–57.4)
Alcohol, SDU, median (IQR) 0 (0–4)
EX-IDU, n (%) 2 (0.5)
HCV, chronic infection by hepatitis C virus; HBV, chronic infection by hepatitis B virus; EX-IDU, ex-injecting drug users; STI: sexual transmitted infection; VL: viral load; IQR: interquartile range; SD, (standard deviation), SDU: standard drink unit.
10.1371/journal.pone.0245870.t002Table 2 Cytology, anoscopy, and HPV PCR results for the cohort.
Cohort of MSM-HIV patients
n = 405
Anal cytology, (n = 397), n (%), 95% CI
LSIL 190 (47.9), (43–52.7)
HSIL 13 (3.3), (1.8–5.1)
ASCUS 29 (7.3), (5.1–10.2)
Normal 165 (41.6), (37–46)
Anoscopy: Histology (n = 405), n (%), 95% CI
Normal 189 (46.7), (44.5–54)
LSIL 164 (40.4), (36.6–46)
HSIL 50 (12.3), (9.2–15.9)
ASCC 2 (0.5), (0–0.8)
n (%), 95% CI HPV PCR in anal mucosa
n = 394
HR-HPV 303 (76.9), (73–81.1)
LR-HPV 288 (73.1), (69–77)
HR and LR-HPV 229 (58.1), (53–63)
Median HR-HPV, IQR 1 (1–3)
Median LR-HPV, IQR 1 (0–2)
HPV 6 71 (18)
HPV 11 71 (18)
HPV 12 1 (0.3)
HPV 16 109 (27.7)
HPV 18 51 (12.9)
HPV 26 6 (1.5)
HPV 31 55 (14)
HPV 33 29 (7.4)
HPV 35 36 (9.1)
HPV 39 46 (11.7)
HPV-40 7 (1.8)
HPV-42 72 (18.3)
HPV-43 10 (2.5)
HPV 45 50 (12.7)
HPV 48 1 (0.3)
HPV 51 55 (14)
HPV 52 50 (12.7)
HPV 53 36 (9.1)
HPV 54 26 (6.6)
HPV 55 64 (16.2)
HPV 56 31 (7.9)
HPV 58 23 (5.8)
HPV 59 42 (10.7)
HPV 61 30 (7.6)
HPV 62 56 (14.2)
HPV 64 1 (0.3)
HPV 66 34 (8.6)
HPV 68 42 (10.7)
HPV 69 14 (3.6)
HPV 70 32 (8.1)
HPV 71 1 (0.3)
HPV 72 28 (7.1)
HPV 73 37 (9.4)
HPV 81 51 (12.9)
HPV 82 17 (4.3)
HPV 83 5 (1.3)
HPV 84 30 (7.6)
HPV 89 1 (0.3)
HPV 6108 13 (3.3)
HPV-AR subtype of HPV 18 (39,45,59,68) 164 (41.6)
HPV-AR subtype of HPV 16 (31,33,35,52,58,67) 207 (52.5)
LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance; ASCC, anal squamous cell cancer. HPV, human papillomavirus; HR-HPV: high-risk HPV, LR-HPV: low-risk HPV.
10.1371/journal.pone.0245870.t003Table 3 Risk factors associated with ≥HSILs in HIV+ MSM patients.
Bivariate and multivariate analysis.
≥HSIL NORMAL Bivariate Multivariate
N = 88 N = 317 p* OR 95% CI
Mean age (yrs), mean (± DS) 30.4 (± 7.6) 31.3 (± 8.3) 0.359 1.27 (0.71–2.29)
Retired, n (%) 6 (6.8) 16 (5) 0.594
Smoker, n (%) 50 (56.8) 164 (51.7) 0.398
Charlson Index, median (IQR) 0 (0–0) 0 (0–0) 0.178
Intercourse in previous 12 months, n (%) 78 (88.6) 286 (90.8) 0.545
qHPV Vaccine, n (%) 12 (13.6) 54(17) 0.445 1.72 (0.92–3.23)
Age at first sexual intercourse, (IQR) 18 (17–21) 18(16–20)
Genital/anal warts, n (%) 36(40.9) 92(29) 0.034
History of Syphilis, n (%) 24(27.3) 79(24.9) 0.654
HCV infection, n (%) 4 (4.5) 10 (3.2) 0.515
HBV infection, n (%) 2 (2.3) 11 (3.5) 0.742
Total NPS, baseline visit, median, (IQR) 50 (16–200) 50 (19.5–150) 0.543
NSP12m before last visit, median, (IQR) 1 (1–4) 1 (1–6.5) 0.078
Use of condom during study, n (%) 63 (71.6) 244 (76.9) 0.493
History of AIDS (A3, B3, C), n (%) 29 (33) 77(24.3) 0.102 2.37(1.009–5.58)
Time since HIV diagnosis (months), (IQR) 27 (9–83) 54(21–107) 0.386 1.002 (1.000–1.003)
CD4 nadir (cells/ul), mean (± SD) 366.8(±267.9) 368.2(±223.9) 0.959
CD4 nadir < 200 cells/uL, n (%) 24(27.6) 73(23.3) 0.412
Cd4 nadir <500 cells/uL, n (%) 39 (44.8) 161 (51.4) 0.275
Cd4 nadir >500 cells/uL, n (%) 24 (27.6) 77 (24.6) 0.571
CD4 cells/uL, mean (± SD) 674.3(± 347.3) 761.2(± 406.9) 0.069 0.99(0.99–1)
CD8 cells/uL, mean (± SD) 1017.3(± 474.1) 977.5(± 467.5) 0.487
CD4/CD8, mean (± SD) 0.75(± 0.43) 0.85(± 0.41) 0.042 0.53(0.2–1.38)
HIV VL (log), mean (± SD) 4.09 (± 3.35) 4.91(± 4.27) 0.042 1 (1–1)
ART during follow-up, n (%) 78 (88.6) 300 (94.6) 0.046 0.74 (0.22–2.56)
Median months of ART, median, (IQR) 0 (0–13) 24 (0–48) 0.003 0.99(0.99–1)
Virological failure, n (%) 1 (1.3) 4 (1.3) 1
N = 83 N = 291
Infection by Low-risk HPV genotype, n (%) 65 (78.3) 211 (72) 0.349
Infection by High-risk HPV genotype, n (%) 68 (83.9) 174 (59.8) 0.0001 2.72(0.87–8.54)
Infection by Low and High-risk HPV, n (%) 53 (63.9) 132 (45.4) 0.003 0.79 (0.25–2.46)
N° of HR-HPV genotypes, median (IQR) 2 (1–3) 1 (0–2) 0.0001 0.76 (0.58–1)
N° of LR-HPV genotypes, median (IQR) 1 (1–2) 1 (0–2) 0.082 0.83 (0.61–1.12)
Median months with VPH-AR (IQR) 11 (1–18) 1 (0–24) 0.07 0.98 (0.96–1.02)
Median months with VPH-BR (IQR) 8 (1–16) 11 (0–26) 0.487 1 (0.96–1.04)
Median months with mixed VPH infection (IQR) 1 (0–15) 1 (0–12) 0.102
HPV-6 21 (25.3) 47 (16.2) 0.057 1.61 (0.73–3.53)
HPV-11 21 (25.3) 37 (12.7) 0.005 3.81(1.76–8.24)
HPV-16 27 (32.5) 43 (14.8) 0.0001 2.69 (1.22–5.99)
HPV-18 16 (19.3) 26 (8.9) 0.008 2.73 (1.01–7.36)
HPV-31 10 (12) 29 (10) 0.584
HPV-33 4 (4.9) 17 (5.9) 0.733
HPV-35 6 (7.2) 18 (6.1) 0.781
HPV-39 9 (10.8) 24 (8.2) 0.462
HPV-42 12 (14.5) 53 (18.3) 0.419
HPV-45 9 (10.8) 31 (10.7) 0.961
HPV-51 12 (14.5) 26 (8.9) 0.142
HPV-52 10 (12) 40 (13.7) 0.689
HPV-53 12 (14.5) 13 (4.5) 0.001 2.97 (1.002–8.79)
HPV-54 6 (7.2) 24 (8.2) 0.763
HPV-55 11 (13.3) 50 (17.2) 0.393
HPV-59 11 (13.3) 19 (6.5) 0.047 1.8(0.66–4.83)
HPV-61 11 (13.3) 8 (2.7) 0.001 11.88 (3.67–38.53)
HPV-68 16 (19.3) 32 (11) 0.048 2.44(1.03–5.8)
HPV-70 8 (9.8) 24 (8.2) 0.667
HPV-81 10 (12) 56 (19.2) 0.129
P*: p-value
95% CI: 95% confidence interval
HIV+MSM, men who have sex with men living with HIV; LTI, Latent tuberculosis infection; HCV hepatitis C virus; HBV, hepatitis B virus; HPV, Human papillomavirus; EX-IDU, ex-injecting drug addict; VL, viral load. HR-HPV: high-risk HPV, LR-HPV: low-risk HPV; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance, NSPt, Total number of sexual partners; NSP12m: number of sexual partners in past 12 months
Factors associated with ≥HSIL
In the multivariate analysis, the presence of ≥HSILs was related to infection with HPV genotypes 11 (OR 3.81; CI95% 1.76–8.24), 16 (OR 2.69, 95%CI 1.22–5.99), 18 (OR 2.73, CI95% 1.01–7.36), 53 (OR 2.97, CI95% 1.002–8.79), 61 (OR 11.88, 95%CI 3.67–38.53) and 68 (OR 2.44, 95%CI 1.03–5.8), low CD4 nadir (OR1.002; CI95% 1–1.004), and history of AIDS (OR 2.37, 95%CI 1.009–5.58). The remaining results obtained are exhibited in Table 3.
HSIL patients and treatment outcomes
After excluding 11 patients who did not undergo two or more anoscopies, treatment outcomes were analyzed in a sample of 394 (87.2%) MSM, with a median follow-up of 36 months (IQR: 12–69), 1.215 patients-year. Table 4 exhibits the characteristic of the two HSIL treatment groups (imiquimod and surgery).
10.1371/journal.pone.0245870.t004Table 4 Baseline characteristics of HIV+MSM patients receiving imiquimod vs. surgery.
Imiquimod as first option Surgery as first option Bivariate
N = 32 N = 47 p*
Mean age (yrs), mean (± DS) 35.3.4 (± 11.48) 31.3 (± 8.3) 0.88
Retired, n (%) 2 (6.3) 3 (6.4) 1
Smoker, n (%) 19 (59.4) 29 (61.7) 0.68
Intercourse in previous 12 months, n(%) 28 (87.5) 47 (100) 0.72
qHPV Vaccine, n (%) 6 (18.7) 6 (12.8) 0.36
Age at first sexual intercourse, (IQR) 18 (16–21) 18(16–20) 0.53
Genital/anal warts, n (%) 12 (37.5) 23 (48.9) 0.53
History of Syphilis, n (%) 11(34.3) 13 (27.7) 0.33
HCV infection, n (%) 1 (3.1) 3 (6.4) 1
HBV infection, n (%) 0 (0) 2 (4.3) 0.53
Total NPS, baseline visit, median, (IQR) 55 (30–300) 36 (15–200) 0.14
NSP12m before last visit, median, (IQR) 2 (1–9) 1 (1–8) 0.55
Use of condom during study, n (%) 26 (81.3) 39 (82.9) 0.51
History of AIDS (A3, B3, C), n (%) 11 (34.4) 15 (31.9) 0.56
Time since HIV diagnosis (months), (IQR) 21 (7–111) 25 (9.5–64.3) 0.92
CD4 nadir (cells/ul), mean (± SD) 366.8(±267.9) 368.2(±223.9) 0.96
CD4 nadir < 200 cells/uL, n (%) 6(18.8) 15(31.9) 0.36
Cd4 nadir 200–500 cells/uL, n (%) 15 (46.9) 24 (51.1) 0.78
Cd4 nadir >500 cells/uL, n (%) 10 (31.3) 14 (29.8) 0.57
CD4 cells/uL, mean (± SD) 618.2(± 280.3) 675.7(± 334.1) 0.42
CD8 cells/uL, mean (± SD) 946.4(± 443.9) 1096(± 551.5) 0.17
CD4/CD8, mean (± SD) 0.78(± 0.46) 0.70(± 0.35) 0.42
HIV VL (log), mean (± SD) 3.9 (± 4.53) 3.55(± 3.98) 0.42
ART during follow-up, n (%) 28 (87.5) 45 (95.7) 0.6
Median months of ART, median, (IQR) 19.5 (6.5–44) 9 (2–64) 0.57
VL HIV < 50 copies/uL, n (%) 20(62.5) 39 (82.9) 0.28
Infection by Low-risk HPV genotype, n (%) 32 (100) 41 (87.2) 0.29
Infection by High-risk HPV genotype, n (%) 26 (81.3) 46 (97.8) 0.52
Infection by Low and High-risk HPV, n (%) 18 (56.3) 34 (72.3) 0.40
Sub-species HPV 18 (18, 39, 45, 59, 68) 16 (50) 29 (61.7) 0.61
Sub-species HPV 16 (16, 31, 33, 35, 52, 58, 67) 17 (53.1) 23 (48.9) 0.43
N° of HR-HPV genotypes, median (IQR) 2 (1–3.8) 2 (1–3) 0.28
N° of LR-HPV genotypes, median (IQR) 2 (0.3–3) 1 (1–2) 0.92
P*: p-value
95% CI: 95% confidence interval
HIV+MSM, men who have sex with men living with HIV; LTI, Latent tuberculosis infection; HCV hepatitis C virus; HBV, hepatitis B virus; HPV, Human papillomavirus; VL, viral load. HR-HPV: high-risk HPV, LR-HPV: low-risk HPV; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance, NSPt, Total number of sexual partners; NSP12m: number of sexual partners in past 12 months.
Mucosectomy was performed in 47 patients with HSIL, with a median follow-up of 60 months (IQR: 46–73 months) and median disease-free period of 48 months (IQR: 28–60 months).
Forty-one (87.2%) of these patients received surgery alone, five (10.6%) received surgery plus self-administered imiquimod, due to failure of surgery in two cases and recurrence in three, and one patient underwent successful surgery after the failure of imiquimod treatment. Surgical margins were disease-free in 35 patients (76.9%) after first surgery. The response rate was 73.3% to surgery as first-line treatment (33/45) versus 96.8% to imiquimod (31/32) (p = 0.009) as evaluated by post-treatment HRA. A median of one surgical intervention was performed (IQR:1–1), with 31 patients undergoing one, 9 needing two, and 1 needing four interventions. Recurrence was recorded in 7 patients (15.2%), and repeat excision of the same lesion in 11 (23.4%). All surgical patients reported adverse effects, with a median duration of 15 days post-surgery (IQR: 7–21 days), including bleeding with defecation in 32 (68%), pain requiring anti-analgesics in 39 (82.9%), rectal incontinence in 1 (2.1%), and transient anal stenosis in 3 (6.4%) versus 1 in the imiquimod group (2.7%, p = 0.046). Among the 43 patients with follow-up HPV PCR results, clearance of oncogenic VPH genotypes was observed in 19 (44.2%).
Thirty-seven patients with HSIL self-administered 5% imiquimod three times/week; the treatment lasted 16 weeks in 97.3% of these patients and 18 weeks in 2.7%. It was first-line treatment in 32 patients (86.4%) and administered after previous surgery in 5 (13.5%); all patients showed a complete response, except for one case of failure caused by intolerance to imiquimod. Only one patient (2.7%) needed retreatment of the same lesion, whereas 11 (23.4%) of the surgical group required repeat surgery (p = 0.02). The median number of affected quadrants was 1 (IQR: 1–2). Mean follow-up was 48 months (IQR: 35–57 months) and mean disease-free period 36 months (IQR: 12–48). Imiquimod was discontinued in one patient (2.7%) for adverse effects (anal itching, stinging, and/or pain) attributed to non-compliance with the treatment protocol. Among the 35 (94.6%) patients treated with imiquimod for whom follow-up HPV PCR results were available, clearance of oncogenic VPH genotypes was observed in 10 (28.6%) (p = 0.065). No patient treated with surgery or imiquimod progressed to ASCC.
We found significant reductions in ≥HSIL cases between 2010 and 2018 (42.9% (9/21) vs. 4.1% (10/245) p = 0.034), between 2010 and 2013 (42.9% (9/21) vs. 13.8% (22/159), p = 0.003), and between 2013 and 2016 (13.8% (22/159) vs. 4.8% (13/273), p = 0.0001), followed by a stabilization between 2016 and 2018 (4.8% (13/273) vs. 4.1% (10/245), p = 0.617). Four deaths were recorded during the follow-up: one patient with hepatic cirrhosis secondary to chronic HCV infection in 2012, one with Burkitt lymphoma in 2013, one with small-cell lung cancer in 2014, and one with metastatic ASCC in 2015. Further data on outcomes were previously reported in detail [6].
Discussion
Since the first HIV epidemic, the incidence of ASCC has increased in seropositive patients, mainly in MSM with AIDS [17]. After the initiation of our screening/treatment program in 2010, the ≥HSIL rate significantly decreased among HIV-infected MSM patients for six years and then subsequently stabilized, with no progression to ASCC in patients with treated HSIL. Our results support the proposition by the authors of the Swiss Cohort study that the incidence of ASCC among people living with HIV can be markedly reduced if they all receive ART and can be further diminished if they also undergo annual screening with anal cytology or anoscopy [18]. A recent study of 592 HIV patients, with a mean follow-up of 69 months, reported that the risk of progression from HSIL (AIN3) to ASCC was high and that ASCC screening was the only factor that reduced this risk [19]. Data from the Study for the Prevention of Anal Cancer (SPANC) [20] are expected to elucidate the natural evolution of HPV infection, allowing a more effective classification of patients at risk of ASCC. In the meantime, a program to screen, diagnose, treat and follow up anal mucosal dysplastic lesions appears recommendable, especially in HIV+ MSM.
In this prospective study of HIV+ MSM undergoing a screening/treatment program for anal mucosa dysplasic lesions, the presence of ≥high-grade anal intraepithelial lesions were related to infection by HPV genotypes 11 16, 18, 53, 61 and 68, a low CD4 nadir and a history of AIDS. This finding of a relationship between HSIL-positivity and poor immunological status is consistent with previous observations that prolonged antiretroviral treatment [21–23] and a high CD4 count, regardless of CD4 nadir [24], are protective factors against HSIL. A recent prospective study observed a similar incidence of HPV-16 and -18 genotypes in the anal mucosa of French HIV+MSM, but HPV-16 was more persistent and therefore more closely correlated with the presence of HSIL [25]. In a retrospective study of “alpha-human papillomavirus” in the anal mucosa of German HIV+ patients, the presence of HSIL and simultaneous infection was associated with high- and low-risk genotypes [26]. Currently, patients with a new diagnosis of HIV in Spain are usually MSM, and the diagnosis is late in 47.6% of these cases [27], with a CD4 count <200 cells/uL. The above data suggest that screening for anal dysplasia is essential in this type of patient.
Self-administration of 5% imiquimod was a highly effective therapeutic strategy against HSIL in this series of HIV+MSM. Most of them did not need to repeat the topical treatment, whereas around a quarter of the patients undergoing excision required another intervention. Furthermore, the therapeutic failure rate and dropout for adverse effects were lower in the imiquimod group than in the surgery group. Various studies have supported the efficacy of imiquimod to treat HSIL in HIV+ patients [13, 28, 29]. Thus, a double-blind randomized placebo-controlled clinical trial comparing between self-application of imiquimod (n = 28) versus placebo (n = 25) in the anal canal three times/week for 4 months found a significant association (P = 0.003) between imiquimod and a positive outcome [13]. In addition, a prospective, observational open study in 44 HIV+ patients with HSIL observed a response rate of 66% (29/44) for imiquimod [28]. Finally, a retrospective observational study in 28 HIV+ and HIV- patients observed a higher frequency of total or partial responses in those receiving anal tampon treatment with a 15 mg versus 6.25 mg dose of imiquimod, with no difference in CD4, HIV viral load, or serostatus [29].
Further advantages of 5% imiquimod in comparison to ablative therapies include its self-administration and its usefulness in cases of extensive disease. The surgical option was also effective in a large proportion of our patients, although some needed retreatment due to recurrence or incomplete excision. No cases of permanent stenosis or fecal incontinence were observed in the surgical group; however, surgery is not currently recommended due to its adverse effects, especially in patients with large lesions [30].
Limitations of this single-center study include its observational design, comparing the real-life clinical effectiveness of imiquimod and surgery rather than their efficacy (as in a clinical trial). In addition, it only included HIV+MSM, and these data cannot be extrapolated to other types of patient. Finally, 11 of the 405 enrolled patients did not undergo two or more anoscopies and were therefore lost to the follow-up. However, its strengths include the prospective design and long follow-up period, which was a mean of 36 months. In fact, the present cohort of HIV+ patients is one of the few published to date that was created to measure predetermined objectives.
In conclusion, HSIL screening and treatment programs reduce the incidence of this precursor of ASCC. Chronic mixed HPV infection and a history of poor immunological status are associated with the presence of HSILs. Self-administration of 5% imiquimod is more effective than surgery as first-line treatment of anal HSIL in HIV+MSM patients, with a lower recurrence rate and fewer adverse effects.
Supporting information
S1 File HIV MSM cohort database.
(SAV)
Click here for additional data file.
The authors are grateful to Mercedes Álvarez Romero for coordinating patients and drawing blood samples and to Marina Gutiérrez and Rodrigo López of the Pathology Department for processing samples. The authors are grateful to the participating patients. | IMIQUIMOD | DrugsGivenReaction | CC BY | 33534790 | 18,913,048 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug intolerance'. | Risk factors for ≥high-grade anal intraepithelial lesions in MSM living with HIV and the response to topical and surgical treatments.
The objective of this study in MSM living with HIV was to determine the incidence of HSIL and ASCC, related factors, and the response to treatment.
Data were gathered in 405 consecutive HIV-infected MSM (May 2010-December 2018) at baseline and annually on: sexual behavior, anal cytology, and HPV PCR and/or high-resolution anoscopy results. They could choose mucosectomy with electric scalpel (from May 2010) or self-administration of 5% imiquimod 3 times weekly for 16 weeks (from November 2013). A multivariate logistic regression model was developed for ≥HSIL-related factors using a step-wise approach to select variables, with a significance level of 0.05 for entry and 0.10 for exit, applying the Hosmer-Lemeshow test to assess the goodness of fit.
The study included 405 patients with a mean age of 36.2 years; 56.7% had bachelor´s degree, and 52.8% were smokers. They had a mean of 1 (IQR 1-7) sexual partner in the previous 12 months, median time since HIV diagnosis of 2 years, and mean CD4 nadir of 367.9 cells/uL; 86.7% were receiving ART, the mean CD4 level was 689.6 cells/uL, mean CD4/CD8 ratio was 0.77, and 85.9% of patients were undetectable. Incidence rates were 30.86/1,000 patient-years for ≥high squamous intraepithelial lesion (HSIL) and 81.22/100,000 for anal squamous cell carcinoma (ASCC). The ≥HSIL incidence significantly decreased from 42.9% (9/21) in 2010 to 4.1% (10/254) in 2018 (p = 0.034). ≥HSIL risk factors were infection with HPV 11 (OR 3.81; 95%CI 1.76-8.24), HPV 16 (OR 2.69, 95%CI 1.22-5.99), HPV 18 (OR 2.73, 95%CI 1.01-7.36), HPV 53 (OR 2.97, 95%CI 1.002-8.79); HPV 61 (OR 11.88, 95%CI 3.67-38.53); HPV 68 (OR 2.44, CI 95% 1.03-5.8); low CD4 nadir (OR1.002; 95%CI 1-1.004) and history of AIDS (OR 2.373, CI 95% 1.009-5.577). Among HSIL-positive patients, the response rate was higher after imiquimod than after surgical excision (96.7% vs 73.3%, p = 0.009) and there were fewer re-treatments (2.7% vs 23.4%, p = 0.02) and adverse events (2.7% vs 100%, p = 0.046); none developed ASCC.
HSIL screening and treatment programs reduce the incidence of HSIL, which is related to chronic HPV infection and poor immunological status. Self-administration of 5% imiquimod as first-line treatment of HSIL is more effective than surgery in HIV+ MSM.
Introduction
Anal squamous-cell carcinoma (ASCC) is frequent among individuals living with HIV (HIV+) and associated with a high mortality rate [1] and with an incidence of up to 131/100,000 people/year among HIV+ men who have sex with men (MSM) [2], with a similar survival rate to the general population [3]. The beginning of a decline in its incidence among MSM has been described by some observational studies but not by clinical trials, which may be explained by the participation of their study populations in an intraepithelial neoplasia screening and treatment program [4, 5], as recently reported by our group [6].
ASCC has been associated with persistent infection by high-risk oncogenic HPV (HR-HPV) genotypes [7]. Interaction between HIV and HPV, which have risk factors in common, has been reported to increase the risk of HPV and reduce the clearance rate among individuals living with HIV [8].
It remains controversial whether the treatment of high-grade anal intraepithelial lesions (HSILs) prevents the subsequent development of ASCC [9], and no standard approach has been established, so that treatments vary among centers according to their resources and experience. Therapeutic options include local topical treatments with trichloroacetic acid, imiquimod, or 5-fluoruracil and more invasive treatments such as excision, fulguration, or laser therapy [9]. Surgical excision used to be a treatment of choice but is associated with adverse effects, including anal stenosis, and with recurrences and incomplete resection, especially when the anal disease is extensive [10]. Fulguration is applied during high-resolution anoscopy (HRA) and has been associated with a recurrence rate of around 25% at 30 months in HIV+ patients, depending on the amount of fulguration [11]. In regard to infrared or laser coagulation, it can be applied in the consulting room and is well tolerated, but a recurrence rate of 61% at one year has been described in HIV+ patients [12]. Finally, topical therapies offer a non-invasive option with a good safety profile and few adverse effects [9]. In particular, imiquimod offers the advantage of self-administration, a low recurrence rate, and a high response rate in HIV+ patients, which was reported to be 61% in a clinical trial [13].
In this study of consecutive HIV+ MSM undergoing a screening/treatment program at our hospital, we previously reported that oncogenic genotype clearance in anal mucosa was associated with the length of ART but did not affect the incidence of HSILs or ASCC. In the present article, data from the same population [6] were analyzed with a different objective, which was to determine the incidence of HSIL and ASCC, the risk factors, and the response to imiquimod treatment and/or electrosurgical excision.
Patients and methods
Design
This longitudinal observational (naturalistic/naturalist) prospective study was conducted in a population of 405 adult HIV+ MSM patients with no history of ASCC, who were consecutively enrolled between May 2010 and December 2018 in a screening and follow-up program for dysplasic anal mucosa lesions. This population and the methodology applied were previously reported [6] in a previous study on HR-HPV clearance and acquisition rates and their relationship with the incidence of HSILs/ASCC in anal mucosa. At their baseline visit (V0) we obtained the written informed consent of the patients to participation in the study, which was approved by the ethics committee (CEIC) of the University Hospital “Virgen de las Nieves” and complied with Spanish data protection legislation (Law 15/1999, 13 December). This CEIC is integrated within the network of ethics Committees of Andalusian public health system (SSPA). CEIC is regulated by order/Decree 439/2010 December 14th of Andalusia. All data were treated in accordance with Spanish data protection legislation Law 15/1999, 13 December, on Personal Character Data Protection).
In brief, data were gathered at baseline (V0), 4–12 weeks, and subsequent follow-ups (at individualized intervals) on clinical-epidemiological and analytical variables and on the results of PCR (Gonorrhoeae, Mycoplasma spp, Chlamydia spp, Ureaplasma spp) and oral-anal-urethral exudate culture (Gonorrhoeae) studies in symptomatic patients or infected partners. In addition, two anal canal mucosa samples were taken at baseline for HPV detection and genotyping by qualitative PCR and for cytology study using the “thin-layer” technique (Processor Thin Prep 2000 (Hologic), and HRA was performed at 4–12 weeks taking samples of apparently normal mucosa and areas with Lugol-negative aceto-white lesions using an endoscopic retrograde cholangiopancreatography catheter. HRA results were obtained from 100% of patients, and findings of at least one follow-up anoscopy were available for 97.3% (394/405). HRA was performed by an infectious disease specialist specifically trained for one year by an expert in this technique from the Department of Digestive Disease of our hospital.
At one year, patients with a normal anoscopy result and LSIL (AIN1) were examined with cytology, HPV PCR, and anoscopy, whereas those with HSIL underwent electrosurgical mucosal resection (from May 2010) or self-administration of 5% imiquimod 3 times/week for 16 weeks (from November 2013), followed by another anoscopy. Surgery was the sole option from May 2010 until the introduction of imiquimod in 2013; since then, all patients have been offered imiquimod as first treatment option. If the imiquimod treatment failed, patients were offered extension of the course up to 18 weeks, a new 16-week cycle of imiquimod, or surgery. Between May 2012 and May 2014, quadrivalent HPV vaccine was received by patients with no HPV 16 or 18 infection and no presence or history of HSIL+-compatible lesions. Cytology results were categorized according to the Bethesda classification [14] and histological findings according to the LAST HPV standardization project [15].
Definition of variables
Abnormal cytology: Cytology findings of ASCUS, LSIL, or HSIL.
Histology with
≥HSILs: Histology findings from HSIL to ASCC.
Therapeutic failure: HSIL persistence after treatment.
Post-treatment HSIL recurrence: Re-appearance of previously treated lesion with normal post-treatment anoscopy.
Response to treatment: Disappearance of lesion in follow-up anoscopy after treatment with surgery or imiquimod.
Self-administration of 5% imiquimod. On Mondays, Wednesdays and Fridays, patients used a preloaded single-use insulin syringe (after cleaning drug remains from its surface) to apply imiquimod inside the anal canal, 3 cm from the anal verge, while in left lateral decubitus position, preferably before sleeping. Before starting this therapy, the patient received detailed instructions on the procedure from the attending physician and nurse.
Statistical analysis
SPSS 21.0 was used for data analyses. The descriptive statistics and tests used for bivariate analyses were previously reported in detail [6]. In the present study, a multivariate logistic regression model was developed for ≥HSIL-related factors, using Freeman’s formula [n = 10*(k+1)] [16], including significant variables in bivariate analysis (ART duration, infection with low-risk HPV genotypes, infection with HPV-6, 11, 16, 18, 53, 59, 61, or 68, and duration of infection with high-risk genotypes) and other variables deemed clinically relevant (smoking habit, history of AIDS, CD4 nadir, CD4/CD8 ratio, duration of infection with HR-HPV and mixed infection). A stepwise approach was used to select variables, with a significance level of 0.05 for entry and 0.10 for exit, applying the Hosmer-Lemeshow test to assess the goodness of fit. P≤0.05 was considered significant.
Results
Baseline characteristics of the cohort
Table 1 summarizes the baseline characteristics of the 405 HIV+MSM enrolled in the study, Table 2 exhibits the distribution of genotypes and Table 3 the cytology and anoscopy findings. In brief, the mean age was 36.2 years, and fewer than 10% were aged > 50 years. They had very good viro-immunological status, 85.6% were on ART at enrolment, and the mean interval since HIV diagnosis was around two years. There was a high prevalence of HPV infection: 76.9% with HR-genotypes, 73.1% with LR-genotypes and 58.1% with both. The prevalence of ≥HSILs was 21.7% (88/405), with an incidence of 30.86/1,000 patient-years, while the prevalence of ASCC was 0.74%, (4/405), with an incidence of 81.22/100,000 patient-years.
10.1371/journal.pone.0245870.t001Table 1 Characteristics of HIV-infected MSM patients.
Number of patients
n = 405
Age, mean (± SD) 36.2 (± 10.1)
<30 yrs, n (%) 123 (30.4)
30–50 yrs, n (%) 244 (60.2)
>50 yrs n (%) 38 (9.4)
Educational level
No studies 6 (1.5)
Primary studies 40 (9.9)
Secondary studies 129 (31.9)
University studies 230 (56.7)
Retired, n (%), 95% CI 22 (5.4) (3–7.5)
Origin
Europe 387 (95.6)
Central America 17 (4.2)
qHPV vaccine (2012–2014), n (%), 95% CI 66 (16.3)
Age at first sexual intercourse, median (IQR) 18 (16–20)
Number of lifetime male sex partners, median (IQR) 50 (15–150)
Number of male sex partners during previous 12 months, median (IQR) 1(1–7)
Habitual use of condoms, n (%), 95% CI 294 (72.6) (68.2–77.4)
Total number of sexual partners during follow-up, median (IQR) 54.5(20–154)
History of anal/genital warts, n (%), (95%CI) 128 (31.6), (27.1–36.1)
Anal/genital warts at baseline n (%), (95%CI) 93 (23), (18.6–26.8)
History of syphilis, n (%), IC95% 103 (25.4), (21.6–29.8)
History of other STI, n (%), IC 95% 110 (27.2), (23.1–31.6)
Time since HIV diagnosis (months), median (IQR) 25 (8–84)
CD4 at diagnosis of HIV (cel/uL), mean (± SD) 448± 298.17
HIV viral load at diagnosis of HIV (log), median (IQR) 4.61 (4.07–5.12)
CD4 nadir (cells/uL), mean (± SD) 367.93±233.85
CD4 nadir < 200 cells/uL, n (%), 95% CI 97 (23.9), (20–28.5)
CD4 cell count at baseline (cells/uL), mean (± SD) 689.64± 475.03
CD8 cell count at baseline (cells/uL), mean (± SD) 981.5±531.5
CD4 /CD8 ratio, mean (± SD) 0.77±0.70
HIV viral load at baseline (log), median (IQR) 0 (0–1.72)
Undetectable: < 50 HIV RNA copies/mL of plasma, n (%) 348 (85.9)
History of AIDS diagnosis, n (%), 95% CI 106 (26.2) (21.3–30.1)
HAART before inclusion, n (%), 95% CI 351 (86.7), (83.2–90)
Previous ART line, median (IQR) 1 (1–2)
Virological failure, n (%) 17 (4.8)
Median months of ART, median (IQR) 4 (16–56)
Chronic HCV infection, n (%) 14 (3.5)
Chronic HBV infection, n (%) 13 (3.2)
Smoker, pack/year, median (IQR) 1.5 (0–14)
Smoker, n (%), 95% CI 214 (52.8) (47.9–57.4)
Alcohol, SDU, median (IQR) 0 (0–4)
EX-IDU, n (%) 2 (0.5)
HCV, chronic infection by hepatitis C virus; HBV, chronic infection by hepatitis B virus; EX-IDU, ex-injecting drug users; STI: sexual transmitted infection; VL: viral load; IQR: interquartile range; SD, (standard deviation), SDU: standard drink unit.
10.1371/journal.pone.0245870.t002Table 2 Cytology, anoscopy, and HPV PCR results for the cohort.
Cohort of MSM-HIV patients
n = 405
Anal cytology, (n = 397), n (%), 95% CI
LSIL 190 (47.9), (43–52.7)
HSIL 13 (3.3), (1.8–5.1)
ASCUS 29 (7.3), (5.1–10.2)
Normal 165 (41.6), (37–46)
Anoscopy: Histology (n = 405), n (%), 95% CI
Normal 189 (46.7), (44.5–54)
LSIL 164 (40.4), (36.6–46)
HSIL 50 (12.3), (9.2–15.9)
ASCC 2 (0.5), (0–0.8)
n (%), 95% CI HPV PCR in anal mucosa
n = 394
HR-HPV 303 (76.9), (73–81.1)
LR-HPV 288 (73.1), (69–77)
HR and LR-HPV 229 (58.1), (53–63)
Median HR-HPV, IQR 1 (1–3)
Median LR-HPV, IQR 1 (0–2)
HPV 6 71 (18)
HPV 11 71 (18)
HPV 12 1 (0.3)
HPV 16 109 (27.7)
HPV 18 51 (12.9)
HPV 26 6 (1.5)
HPV 31 55 (14)
HPV 33 29 (7.4)
HPV 35 36 (9.1)
HPV 39 46 (11.7)
HPV-40 7 (1.8)
HPV-42 72 (18.3)
HPV-43 10 (2.5)
HPV 45 50 (12.7)
HPV 48 1 (0.3)
HPV 51 55 (14)
HPV 52 50 (12.7)
HPV 53 36 (9.1)
HPV 54 26 (6.6)
HPV 55 64 (16.2)
HPV 56 31 (7.9)
HPV 58 23 (5.8)
HPV 59 42 (10.7)
HPV 61 30 (7.6)
HPV 62 56 (14.2)
HPV 64 1 (0.3)
HPV 66 34 (8.6)
HPV 68 42 (10.7)
HPV 69 14 (3.6)
HPV 70 32 (8.1)
HPV 71 1 (0.3)
HPV 72 28 (7.1)
HPV 73 37 (9.4)
HPV 81 51 (12.9)
HPV 82 17 (4.3)
HPV 83 5 (1.3)
HPV 84 30 (7.6)
HPV 89 1 (0.3)
HPV 6108 13 (3.3)
HPV-AR subtype of HPV 18 (39,45,59,68) 164 (41.6)
HPV-AR subtype of HPV 16 (31,33,35,52,58,67) 207 (52.5)
LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance; ASCC, anal squamous cell cancer. HPV, human papillomavirus; HR-HPV: high-risk HPV, LR-HPV: low-risk HPV.
10.1371/journal.pone.0245870.t003Table 3 Risk factors associated with ≥HSILs in HIV+ MSM patients.
Bivariate and multivariate analysis.
≥HSIL NORMAL Bivariate Multivariate
N = 88 N = 317 p* OR 95% CI
Mean age (yrs), mean (± DS) 30.4 (± 7.6) 31.3 (± 8.3) 0.359 1.27 (0.71–2.29)
Retired, n (%) 6 (6.8) 16 (5) 0.594
Smoker, n (%) 50 (56.8) 164 (51.7) 0.398
Charlson Index, median (IQR) 0 (0–0) 0 (0–0) 0.178
Intercourse in previous 12 months, n (%) 78 (88.6) 286 (90.8) 0.545
qHPV Vaccine, n (%) 12 (13.6) 54(17) 0.445 1.72 (0.92–3.23)
Age at first sexual intercourse, (IQR) 18 (17–21) 18(16–20)
Genital/anal warts, n (%) 36(40.9) 92(29) 0.034
History of Syphilis, n (%) 24(27.3) 79(24.9) 0.654
HCV infection, n (%) 4 (4.5) 10 (3.2) 0.515
HBV infection, n (%) 2 (2.3) 11 (3.5) 0.742
Total NPS, baseline visit, median, (IQR) 50 (16–200) 50 (19.5–150) 0.543
NSP12m before last visit, median, (IQR) 1 (1–4) 1 (1–6.5) 0.078
Use of condom during study, n (%) 63 (71.6) 244 (76.9) 0.493
History of AIDS (A3, B3, C), n (%) 29 (33) 77(24.3) 0.102 2.37(1.009–5.58)
Time since HIV diagnosis (months), (IQR) 27 (9–83) 54(21–107) 0.386 1.002 (1.000–1.003)
CD4 nadir (cells/ul), mean (± SD) 366.8(±267.9) 368.2(±223.9) 0.959
CD4 nadir < 200 cells/uL, n (%) 24(27.6) 73(23.3) 0.412
Cd4 nadir <500 cells/uL, n (%) 39 (44.8) 161 (51.4) 0.275
Cd4 nadir >500 cells/uL, n (%) 24 (27.6) 77 (24.6) 0.571
CD4 cells/uL, mean (± SD) 674.3(± 347.3) 761.2(± 406.9) 0.069 0.99(0.99–1)
CD8 cells/uL, mean (± SD) 1017.3(± 474.1) 977.5(± 467.5) 0.487
CD4/CD8, mean (± SD) 0.75(± 0.43) 0.85(± 0.41) 0.042 0.53(0.2–1.38)
HIV VL (log), mean (± SD) 4.09 (± 3.35) 4.91(± 4.27) 0.042 1 (1–1)
ART during follow-up, n (%) 78 (88.6) 300 (94.6) 0.046 0.74 (0.22–2.56)
Median months of ART, median, (IQR) 0 (0–13) 24 (0–48) 0.003 0.99(0.99–1)
Virological failure, n (%) 1 (1.3) 4 (1.3) 1
N = 83 N = 291
Infection by Low-risk HPV genotype, n (%) 65 (78.3) 211 (72) 0.349
Infection by High-risk HPV genotype, n (%) 68 (83.9) 174 (59.8) 0.0001 2.72(0.87–8.54)
Infection by Low and High-risk HPV, n (%) 53 (63.9) 132 (45.4) 0.003 0.79 (0.25–2.46)
N° of HR-HPV genotypes, median (IQR) 2 (1–3) 1 (0–2) 0.0001 0.76 (0.58–1)
N° of LR-HPV genotypes, median (IQR) 1 (1–2) 1 (0–2) 0.082 0.83 (0.61–1.12)
Median months with VPH-AR (IQR) 11 (1–18) 1 (0–24) 0.07 0.98 (0.96–1.02)
Median months with VPH-BR (IQR) 8 (1–16) 11 (0–26) 0.487 1 (0.96–1.04)
Median months with mixed VPH infection (IQR) 1 (0–15) 1 (0–12) 0.102
HPV-6 21 (25.3) 47 (16.2) 0.057 1.61 (0.73–3.53)
HPV-11 21 (25.3) 37 (12.7) 0.005 3.81(1.76–8.24)
HPV-16 27 (32.5) 43 (14.8) 0.0001 2.69 (1.22–5.99)
HPV-18 16 (19.3) 26 (8.9) 0.008 2.73 (1.01–7.36)
HPV-31 10 (12) 29 (10) 0.584
HPV-33 4 (4.9) 17 (5.9) 0.733
HPV-35 6 (7.2) 18 (6.1) 0.781
HPV-39 9 (10.8) 24 (8.2) 0.462
HPV-42 12 (14.5) 53 (18.3) 0.419
HPV-45 9 (10.8) 31 (10.7) 0.961
HPV-51 12 (14.5) 26 (8.9) 0.142
HPV-52 10 (12) 40 (13.7) 0.689
HPV-53 12 (14.5) 13 (4.5) 0.001 2.97 (1.002–8.79)
HPV-54 6 (7.2) 24 (8.2) 0.763
HPV-55 11 (13.3) 50 (17.2) 0.393
HPV-59 11 (13.3) 19 (6.5) 0.047 1.8(0.66–4.83)
HPV-61 11 (13.3) 8 (2.7) 0.001 11.88 (3.67–38.53)
HPV-68 16 (19.3) 32 (11) 0.048 2.44(1.03–5.8)
HPV-70 8 (9.8) 24 (8.2) 0.667
HPV-81 10 (12) 56 (19.2) 0.129
P*: p-value
95% CI: 95% confidence interval
HIV+MSM, men who have sex with men living with HIV; LTI, Latent tuberculosis infection; HCV hepatitis C virus; HBV, hepatitis B virus; HPV, Human papillomavirus; EX-IDU, ex-injecting drug addict; VL, viral load. HR-HPV: high-risk HPV, LR-HPV: low-risk HPV; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance, NSPt, Total number of sexual partners; NSP12m: number of sexual partners in past 12 months
Factors associated with ≥HSIL
In the multivariate analysis, the presence of ≥HSILs was related to infection with HPV genotypes 11 (OR 3.81; CI95% 1.76–8.24), 16 (OR 2.69, 95%CI 1.22–5.99), 18 (OR 2.73, CI95% 1.01–7.36), 53 (OR 2.97, CI95% 1.002–8.79), 61 (OR 11.88, 95%CI 3.67–38.53) and 68 (OR 2.44, 95%CI 1.03–5.8), low CD4 nadir (OR1.002; CI95% 1–1.004), and history of AIDS (OR 2.37, 95%CI 1.009–5.58). The remaining results obtained are exhibited in Table 3.
HSIL patients and treatment outcomes
After excluding 11 patients who did not undergo two or more anoscopies, treatment outcomes were analyzed in a sample of 394 (87.2%) MSM, with a median follow-up of 36 months (IQR: 12–69), 1.215 patients-year. Table 4 exhibits the characteristic of the two HSIL treatment groups (imiquimod and surgery).
10.1371/journal.pone.0245870.t004Table 4 Baseline characteristics of HIV+MSM patients receiving imiquimod vs. surgery.
Imiquimod as first option Surgery as first option Bivariate
N = 32 N = 47 p*
Mean age (yrs), mean (± DS) 35.3.4 (± 11.48) 31.3 (± 8.3) 0.88
Retired, n (%) 2 (6.3) 3 (6.4) 1
Smoker, n (%) 19 (59.4) 29 (61.7) 0.68
Intercourse in previous 12 months, n(%) 28 (87.5) 47 (100) 0.72
qHPV Vaccine, n (%) 6 (18.7) 6 (12.8) 0.36
Age at first sexual intercourse, (IQR) 18 (16–21) 18(16–20) 0.53
Genital/anal warts, n (%) 12 (37.5) 23 (48.9) 0.53
History of Syphilis, n (%) 11(34.3) 13 (27.7) 0.33
HCV infection, n (%) 1 (3.1) 3 (6.4) 1
HBV infection, n (%) 0 (0) 2 (4.3) 0.53
Total NPS, baseline visit, median, (IQR) 55 (30–300) 36 (15–200) 0.14
NSP12m before last visit, median, (IQR) 2 (1–9) 1 (1–8) 0.55
Use of condom during study, n (%) 26 (81.3) 39 (82.9) 0.51
History of AIDS (A3, B3, C), n (%) 11 (34.4) 15 (31.9) 0.56
Time since HIV diagnosis (months), (IQR) 21 (7–111) 25 (9.5–64.3) 0.92
CD4 nadir (cells/ul), mean (± SD) 366.8(±267.9) 368.2(±223.9) 0.96
CD4 nadir < 200 cells/uL, n (%) 6(18.8) 15(31.9) 0.36
Cd4 nadir 200–500 cells/uL, n (%) 15 (46.9) 24 (51.1) 0.78
Cd4 nadir >500 cells/uL, n (%) 10 (31.3) 14 (29.8) 0.57
CD4 cells/uL, mean (± SD) 618.2(± 280.3) 675.7(± 334.1) 0.42
CD8 cells/uL, mean (± SD) 946.4(± 443.9) 1096(± 551.5) 0.17
CD4/CD8, mean (± SD) 0.78(± 0.46) 0.70(± 0.35) 0.42
HIV VL (log), mean (± SD) 3.9 (± 4.53) 3.55(± 3.98) 0.42
ART during follow-up, n (%) 28 (87.5) 45 (95.7) 0.6
Median months of ART, median, (IQR) 19.5 (6.5–44) 9 (2–64) 0.57
VL HIV < 50 copies/uL, n (%) 20(62.5) 39 (82.9) 0.28
Infection by Low-risk HPV genotype, n (%) 32 (100) 41 (87.2) 0.29
Infection by High-risk HPV genotype, n (%) 26 (81.3) 46 (97.8) 0.52
Infection by Low and High-risk HPV, n (%) 18 (56.3) 34 (72.3) 0.40
Sub-species HPV 18 (18, 39, 45, 59, 68) 16 (50) 29 (61.7) 0.61
Sub-species HPV 16 (16, 31, 33, 35, 52, 58, 67) 17 (53.1) 23 (48.9) 0.43
N° of HR-HPV genotypes, median (IQR) 2 (1–3.8) 2 (1–3) 0.28
N° of LR-HPV genotypes, median (IQR) 2 (0.3–3) 1 (1–2) 0.92
P*: p-value
95% CI: 95% confidence interval
HIV+MSM, men who have sex with men living with HIV; LTI, Latent tuberculosis infection; HCV hepatitis C virus; HBV, hepatitis B virus; HPV, Human papillomavirus; VL, viral load. HR-HPV: high-risk HPV, LR-HPV: low-risk HPV; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance, NSPt, Total number of sexual partners; NSP12m: number of sexual partners in past 12 months.
Mucosectomy was performed in 47 patients with HSIL, with a median follow-up of 60 months (IQR: 46–73 months) and median disease-free period of 48 months (IQR: 28–60 months).
Forty-one (87.2%) of these patients received surgery alone, five (10.6%) received surgery plus self-administered imiquimod, due to failure of surgery in two cases and recurrence in three, and one patient underwent successful surgery after the failure of imiquimod treatment. Surgical margins were disease-free in 35 patients (76.9%) after first surgery. The response rate was 73.3% to surgery as first-line treatment (33/45) versus 96.8% to imiquimod (31/32) (p = 0.009) as evaluated by post-treatment HRA. A median of one surgical intervention was performed (IQR:1–1), with 31 patients undergoing one, 9 needing two, and 1 needing four interventions. Recurrence was recorded in 7 patients (15.2%), and repeat excision of the same lesion in 11 (23.4%). All surgical patients reported adverse effects, with a median duration of 15 days post-surgery (IQR: 7–21 days), including bleeding with defecation in 32 (68%), pain requiring anti-analgesics in 39 (82.9%), rectal incontinence in 1 (2.1%), and transient anal stenosis in 3 (6.4%) versus 1 in the imiquimod group (2.7%, p = 0.046). Among the 43 patients with follow-up HPV PCR results, clearance of oncogenic VPH genotypes was observed in 19 (44.2%).
Thirty-seven patients with HSIL self-administered 5% imiquimod three times/week; the treatment lasted 16 weeks in 97.3% of these patients and 18 weeks in 2.7%. It was first-line treatment in 32 patients (86.4%) and administered after previous surgery in 5 (13.5%); all patients showed a complete response, except for one case of failure caused by intolerance to imiquimod. Only one patient (2.7%) needed retreatment of the same lesion, whereas 11 (23.4%) of the surgical group required repeat surgery (p = 0.02). The median number of affected quadrants was 1 (IQR: 1–2). Mean follow-up was 48 months (IQR: 35–57 months) and mean disease-free period 36 months (IQR: 12–48). Imiquimod was discontinued in one patient (2.7%) for adverse effects (anal itching, stinging, and/or pain) attributed to non-compliance with the treatment protocol. Among the 35 (94.6%) patients treated with imiquimod for whom follow-up HPV PCR results were available, clearance of oncogenic VPH genotypes was observed in 10 (28.6%) (p = 0.065). No patient treated with surgery or imiquimod progressed to ASCC.
We found significant reductions in ≥HSIL cases between 2010 and 2018 (42.9% (9/21) vs. 4.1% (10/245) p = 0.034), between 2010 and 2013 (42.9% (9/21) vs. 13.8% (22/159), p = 0.003), and between 2013 and 2016 (13.8% (22/159) vs. 4.8% (13/273), p = 0.0001), followed by a stabilization between 2016 and 2018 (4.8% (13/273) vs. 4.1% (10/245), p = 0.617). Four deaths were recorded during the follow-up: one patient with hepatic cirrhosis secondary to chronic HCV infection in 2012, one with Burkitt lymphoma in 2013, one with small-cell lung cancer in 2014, and one with metastatic ASCC in 2015. Further data on outcomes were previously reported in detail [6].
Discussion
Since the first HIV epidemic, the incidence of ASCC has increased in seropositive patients, mainly in MSM with AIDS [17]. After the initiation of our screening/treatment program in 2010, the ≥HSIL rate significantly decreased among HIV-infected MSM patients for six years and then subsequently stabilized, with no progression to ASCC in patients with treated HSIL. Our results support the proposition by the authors of the Swiss Cohort study that the incidence of ASCC among people living with HIV can be markedly reduced if they all receive ART and can be further diminished if they also undergo annual screening with anal cytology or anoscopy [18]. A recent study of 592 HIV patients, with a mean follow-up of 69 months, reported that the risk of progression from HSIL (AIN3) to ASCC was high and that ASCC screening was the only factor that reduced this risk [19]. Data from the Study for the Prevention of Anal Cancer (SPANC) [20] are expected to elucidate the natural evolution of HPV infection, allowing a more effective classification of patients at risk of ASCC. In the meantime, a program to screen, diagnose, treat and follow up anal mucosal dysplastic lesions appears recommendable, especially in HIV+ MSM.
In this prospective study of HIV+ MSM undergoing a screening/treatment program for anal mucosa dysplasic lesions, the presence of ≥high-grade anal intraepithelial lesions were related to infection by HPV genotypes 11 16, 18, 53, 61 and 68, a low CD4 nadir and a history of AIDS. This finding of a relationship between HSIL-positivity and poor immunological status is consistent with previous observations that prolonged antiretroviral treatment [21–23] and a high CD4 count, regardless of CD4 nadir [24], are protective factors against HSIL. A recent prospective study observed a similar incidence of HPV-16 and -18 genotypes in the anal mucosa of French HIV+MSM, but HPV-16 was more persistent and therefore more closely correlated with the presence of HSIL [25]. In a retrospective study of “alpha-human papillomavirus” in the anal mucosa of German HIV+ patients, the presence of HSIL and simultaneous infection was associated with high- and low-risk genotypes [26]. Currently, patients with a new diagnosis of HIV in Spain are usually MSM, and the diagnosis is late in 47.6% of these cases [27], with a CD4 count <200 cells/uL. The above data suggest that screening for anal dysplasia is essential in this type of patient.
Self-administration of 5% imiquimod was a highly effective therapeutic strategy against HSIL in this series of HIV+MSM. Most of them did not need to repeat the topical treatment, whereas around a quarter of the patients undergoing excision required another intervention. Furthermore, the therapeutic failure rate and dropout for adverse effects were lower in the imiquimod group than in the surgery group. Various studies have supported the efficacy of imiquimod to treat HSIL in HIV+ patients [13, 28, 29]. Thus, a double-blind randomized placebo-controlled clinical trial comparing between self-application of imiquimod (n = 28) versus placebo (n = 25) in the anal canal three times/week for 4 months found a significant association (P = 0.003) between imiquimod and a positive outcome [13]. In addition, a prospective, observational open study in 44 HIV+ patients with HSIL observed a response rate of 66% (29/44) for imiquimod [28]. Finally, a retrospective observational study in 28 HIV+ and HIV- patients observed a higher frequency of total or partial responses in those receiving anal tampon treatment with a 15 mg versus 6.25 mg dose of imiquimod, with no difference in CD4, HIV viral load, or serostatus [29].
Further advantages of 5% imiquimod in comparison to ablative therapies include its self-administration and its usefulness in cases of extensive disease. The surgical option was also effective in a large proportion of our patients, although some needed retreatment due to recurrence or incomplete excision. No cases of permanent stenosis or fecal incontinence were observed in the surgical group; however, surgery is not currently recommended due to its adverse effects, especially in patients with large lesions [30].
Limitations of this single-center study include its observational design, comparing the real-life clinical effectiveness of imiquimod and surgery rather than their efficacy (as in a clinical trial). In addition, it only included HIV+MSM, and these data cannot be extrapolated to other types of patient. Finally, 11 of the 405 enrolled patients did not undergo two or more anoscopies and were therefore lost to the follow-up. However, its strengths include the prospective design and long follow-up period, which was a mean of 36 months. In fact, the present cohort of HIV+ patients is one of the few published to date that was created to measure predetermined objectives.
In conclusion, HSIL screening and treatment programs reduce the incidence of this precursor of ASCC. Chronic mixed HPV infection and a history of poor immunological status are associated with the presence of HSILs. Self-administration of 5% imiquimod is more effective than surgery as first-line treatment of anal HSIL in HIV+MSM patients, with a lower recurrence rate and fewer adverse effects.
Supporting information
S1 File HIV MSM cohort database.
(SAV)
Click here for additional data file.
The authors are grateful to Mercedes Álvarez Romero for coordinating patients and drawing blood samples and to Marina Gutiérrez and Rodrigo López of the Pathology Department for processing samples. The authors are grateful to the participating patients. | IMIQUIMOD | DrugsGivenReaction | CC BY | 33534790 | 18,911,566 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Proctalgia'. | Risk factors for ≥high-grade anal intraepithelial lesions in MSM living with HIV and the response to topical and surgical treatments.
The objective of this study in MSM living with HIV was to determine the incidence of HSIL and ASCC, related factors, and the response to treatment.
Data were gathered in 405 consecutive HIV-infected MSM (May 2010-December 2018) at baseline and annually on: sexual behavior, anal cytology, and HPV PCR and/or high-resolution anoscopy results. They could choose mucosectomy with electric scalpel (from May 2010) or self-administration of 5% imiquimod 3 times weekly for 16 weeks (from November 2013). A multivariate logistic regression model was developed for ≥HSIL-related factors using a step-wise approach to select variables, with a significance level of 0.05 for entry and 0.10 for exit, applying the Hosmer-Lemeshow test to assess the goodness of fit.
The study included 405 patients with a mean age of 36.2 years; 56.7% had bachelor´s degree, and 52.8% were smokers. They had a mean of 1 (IQR 1-7) sexual partner in the previous 12 months, median time since HIV diagnosis of 2 years, and mean CD4 nadir of 367.9 cells/uL; 86.7% were receiving ART, the mean CD4 level was 689.6 cells/uL, mean CD4/CD8 ratio was 0.77, and 85.9% of patients were undetectable. Incidence rates were 30.86/1,000 patient-years for ≥high squamous intraepithelial lesion (HSIL) and 81.22/100,000 for anal squamous cell carcinoma (ASCC). The ≥HSIL incidence significantly decreased from 42.9% (9/21) in 2010 to 4.1% (10/254) in 2018 (p = 0.034). ≥HSIL risk factors were infection with HPV 11 (OR 3.81; 95%CI 1.76-8.24), HPV 16 (OR 2.69, 95%CI 1.22-5.99), HPV 18 (OR 2.73, 95%CI 1.01-7.36), HPV 53 (OR 2.97, 95%CI 1.002-8.79); HPV 61 (OR 11.88, 95%CI 3.67-38.53); HPV 68 (OR 2.44, CI 95% 1.03-5.8); low CD4 nadir (OR1.002; 95%CI 1-1.004) and history of AIDS (OR 2.373, CI 95% 1.009-5.577). Among HSIL-positive patients, the response rate was higher after imiquimod than after surgical excision (96.7% vs 73.3%, p = 0.009) and there were fewer re-treatments (2.7% vs 23.4%, p = 0.02) and adverse events (2.7% vs 100%, p = 0.046); none developed ASCC.
HSIL screening and treatment programs reduce the incidence of HSIL, which is related to chronic HPV infection and poor immunological status. Self-administration of 5% imiquimod as first-line treatment of HSIL is more effective than surgery in HIV+ MSM.
Introduction
Anal squamous-cell carcinoma (ASCC) is frequent among individuals living with HIV (HIV+) and associated with a high mortality rate [1] and with an incidence of up to 131/100,000 people/year among HIV+ men who have sex with men (MSM) [2], with a similar survival rate to the general population [3]. The beginning of a decline in its incidence among MSM has been described by some observational studies but not by clinical trials, which may be explained by the participation of their study populations in an intraepithelial neoplasia screening and treatment program [4, 5], as recently reported by our group [6].
ASCC has been associated with persistent infection by high-risk oncogenic HPV (HR-HPV) genotypes [7]. Interaction between HIV and HPV, which have risk factors in common, has been reported to increase the risk of HPV and reduce the clearance rate among individuals living with HIV [8].
It remains controversial whether the treatment of high-grade anal intraepithelial lesions (HSILs) prevents the subsequent development of ASCC [9], and no standard approach has been established, so that treatments vary among centers according to their resources and experience. Therapeutic options include local topical treatments with trichloroacetic acid, imiquimod, or 5-fluoruracil and more invasive treatments such as excision, fulguration, or laser therapy [9]. Surgical excision used to be a treatment of choice but is associated with adverse effects, including anal stenosis, and with recurrences and incomplete resection, especially when the anal disease is extensive [10]. Fulguration is applied during high-resolution anoscopy (HRA) and has been associated with a recurrence rate of around 25% at 30 months in HIV+ patients, depending on the amount of fulguration [11]. In regard to infrared or laser coagulation, it can be applied in the consulting room and is well tolerated, but a recurrence rate of 61% at one year has been described in HIV+ patients [12]. Finally, topical therapies offer a non-invasive option with a good safety profile and few adverse effects [9]. In particular, imiquimod offers the advantage of self-administration, a low recurrence rate, and a high response rate in HIV+ patients, which was reported to be 61% in a clinical trial [13].
In this study of consecutive HIV+ MSM undergoing a screening/treatment program at our hospital, we previously reported that oncogenic genotype clearance in anal mucosa was associated with the length of ART but did not affect the incidence of HSILs or ASCC. In the present article, data from the same population [6] were analyzed with a different objective, which was to determine the incidence of HSIL and ASCC, the risk factors, and the response to imiquimod treatment and/or electrosurgical excision.
Patients and methods
Design
This longitudinal observational (naturalistic/naturalist) prospective study was conducted in a population of 405 adult HIV+ MSM patients with no history of ASCC, who were consecutively enrolled between May 2010 and December 2018 in a screening and follow-up program for dysplasic anal mucosa lesions. This population and the methodology applied were previously reported [6] in a previous study on HR-HPV clearance and acquisition rates and their relationship with the incidence of HSILs/ASCC in anal mucosa. At their baseline visit (V0) we obtained the written informed consent of the patients to participation in the study, which was approved by the ethics committee (CEIC) of the University Hospital “Virgen de las Nieves” and complied with Spanish data protection legislation (Law 15/1999, 13 December). This CEIC is integrated within the network of ethics Committees of Andalusian public health system (SSPA). CEIC is regulated by order/Decree 439/2010 December 14th of Andalusia. All data were treated in accordance with Spanish data protection legislation Law 15/1999, 13 December, on Personal Character Data Protection).
In brief, data were gathered at baseline (V0), 4–12 weeks, and subsequent follow-ups (at individualized intervals) on clinical-epidemiological and analytical variables and on the results of PCR (Gonorrhoeae, Mycoplasma spp, Chlamydia spp, Ureaplasma spp) and oral-anal-urethral exudate culture (Gonorrhoeae) studies in symptomatic patients or infected partners. In addition, two anal canal mucosa samples were taken at baseline for HPV detection and genotyping by qualitative PCR and for cytology study using the “thin-layer” technique (Processor Thin Prep 2000 (Hologic), and HRA was performed at 4–12 weeks taking samples of apparently normal mucosa and areas with Lugol-negative aceto-white lesions using an endoscopic retrograde cholangiopancreatography catheter. HRA results were obtained from 100% of patients, and findings of at least one follow-up anoscopy were available for 97.3% (394/405). HRA was performed by an infectious disease specialist specifically trained for one year by an expert in this technique from the Department of Digestive Disease of our hospital.
At one year, patients with a normal anoscopy result and LSIL (AIN1) were examined with cytology, HPV PCR, and anoscopy, whereas those with HSIL underwent electrosurgical mucosal resection (from May 2010) or self-administration of 5% imiquimod 3 times/week for 16 weeks (from November 2013), followed by another anoscopy. Surgery was the sole option from May 2010 until the introduction of imiquimod in 2013; since then, all patients have been offered imiquimod as first treatment option. If the imiquimod treatment failed, patients were offered extension of the course up to 18 weeks, a new 16-week cycle of imiquimod, or surgery. Between May 2012 and May 2014, quadrivalent HPV vaccine was received by patients with no HPV 16 or 18 infection and no presence or history of HSIL+-compatible lesions. Cytology results were categorized according to the Bethesda classification [14] and histological findings according to the LAST HPV standardization project [15].
Definition of variables
Abnormal cytology: Cytology findings of ASCUS, LSIL, or HSIL.
Histology with
≥HSILs: Histology findings from HSIL to ASCC.
Therapeutic failure: HSIL persistence after treatment.
Post-treatment HSIL recurrence: Re-appearance of previously treated lesion with normal post-treatment anoscopy.
Response to treatment: Disappearance of lesion in follow-up anoscopy after treatment with surgery or imiquimod.
Self-administration of 5% imiquimod. On Mondays, Wednesdays and Fridays, patients used a preloaded single-use insulin syringe (after cleaning drug remains from its surface) to apply imiquimod inside the anal canal, 3 cm from the anal verge, while in left lateral decubitus position, preferably before sleeping. Before starting this therapy, the patient received detailed instructions on the procedure from the attending physician and nurse.
Statistical analysis
SPSS 21.0 was used for data analyses. The descriptive statistics and tests used for bivariate analyses were previously reported in detail [6]. In the present study, a multivariate logistic regression model was developed for ≥HSIL-related factors, using Freeman’s formula [n = 10*(k+1)] [16], including significant variables in bivariate analysis (ART duration, infection with low-risk HPV genotypes, infection with HPV-6, 11, 16, 18, 53, 59, 61, or 68, and duration of infection with high-risk genotypes) and other variables deemed clinically relevant (smoking habit, history of AIDS, CD4 nadir, CD4/CD8 ratio, duration of infection with HR-HPV and mixed infection). A stepwise approach was used to select variables, with a significance level of 0.05 for entry and 0.10 for exit, applying the Hosmer-Lemeshow test to assess the goodness of fit. P≤0.05 was considered significant.
Results
Baseline characteristics of the cohort
Table 1 summarizes the baseline characteristics of the 405 HIV+MSM enrolled in the study, Table 2 exhibits the distribution of genotypes and Table 3 the cytology and anoscopy findings. In brief, the mean age was 36.2 years, and fewer than 10% were aged > 50 years. They had very good viro-immunological status, 85.6% were on ART at enrolment, and the mean interval since HIV diagnosis was around two years. There was a high prevalence of HPV infection: 76.9% with HR-genotypes, 73.1% with LR-genotypes and 58.1% with both. The prevalence of ≥HSILs was 21.7% (88/405), with an incidence of 30.86/1,000 patient-years, while the prevalence of ASCC was 0.74%, (4/405), with an incidence of 81.22/100,000 patient-years.
10.1371/journal.pone.0245870.t001Table 1 Characteristics of HIV-infected MSM patients.
Number of patients
n = 405
Age, mean (± SD) 36.2 (± 10.1)
<30 yrs, n (%) 123 (30.4)
30–50 yrs, n (%) 244 (60.2)
>50 yrs n (%) 38 (9.4)
Educational level
No studies 6 (1.5)
Primary studies 40 (9.9)
Secondary studies 129 (31.9)
University studies 230 (56.7)
Retired, n (%), 95% CI 22 (5.4) (3–7.5)
Origin
Europe 387 (95.6)
Central America 17 (4.2)
qHPV vaccine (2012–2014), n (%), 95% CI 66 (16.3)
Age at first sexual intercourse, median (IQR) 18 (16–20)
Number of lifetime male sex partners, median (IQR) 50 (15–150)
Number of male sex partners during previous 12 months, median (IQR) 1(1–7)
Habitual use of condoms, n (%), 95% CI 294 (72.6) (68.2–77.4)
Total number of sexual partners during follow-up, median (IQR) 54.5(20–154)
History of anal/genital warts, n (%), (95%CI) 128 (31.6), (27.1–36.1)
Anal/genital warts at baseline n (%), (95%CI) 93 (23), (18.6–26.8)
History of syphilis, n (%), IC95% 103 (25.4), (21.6–29.8)
History of other STI, n (%), IC 95% 110 (27.2), (23.1–31.6)
Time since HIV diagnosis (months), median (IQR) 25 (8–84)
CD4 at diagnosis of HIV (cel/uL), mean (± SD) 448± 298.17
HIV viral load at diagnosis of HIV (log), median (IQR) 4.61 (4.07–5.12)
CD4 nadir (cells/uL), mean (± SD) 367.93±233.85
CD4 nadir < 200 cells/uL, n (%), 95% CI 97 (23.9), (20–28.5)
CD4 cell count at baseline (cells/uL), mean (± SD) 689.64± 475.03
CD8 cell count at baseline (cells/uL), mean (± SD) 981.5±531.5
CD4 /CD8 ratio, mean (± SD) 0.77±0.70
HIV viral load at baseline (log), median (IQR) 0 (0–1.72)
Undetectable: < 50 HIV RNA copies/mL of plasma, n (%) 348 (85.9)
History of AIDS diagnosis, n (%), 95% CI 106 (26.2) (21.3–30.1)
HAART before inclusion, n (%), 95% CI 351 (86.7), (83.2–90)
Previous ART line, median (IQR) 1 (1–2)
Virological failure, n (%) 17 (4.8)
Median months of ART, median (IQR) 4 (16–56)
Chronic HCV infection, n (%) 14 (3.5)
Chronic HBV infection, n (%) 13 (3.2)
Smoker, pack/year, median (IQR) 1.5 (0–14)
Smoker, n (%), 95% CI 214 (52.8) (47.9–57.4)
Alcohol, SDU, median (IQR) 0 (0–4)
EX-IDU, n (%) 2 (0.5)
HCV, chronic infection by hepatitis C virus; HBV, chronic infection by hepatitis B virus; EX-IDU, ex-injecting drug users; STI: sexual transmitted infection; VL: viral load; IQR: interquartile range; SD, (standard deviation), SDU: standard drink unit.
10.1371/journal.pone.0245870.t002Table 2 Cytology, anoscopy, and HPV PCR results for the cohort.
Cohort of MSM-HIV patients
n = 405
Anal cytology, (n = 397), n (%), 95% CI
LSIL 190 (47.9), (43–52.7)
HSIL 13 (3.3), (1.8–5.1)
ASCUS 29 (7.3), (5.1–10.2)
Normal 165 (41.6), (37–46)
Anoscopy: Histology (n = 405), n (%), 95% CI
Normal 189 (46.7), (44.5–54)
LSIL 164 (40.4), (36.6–46)
HSIL 50 (12.3), (9.2–15.9)
ASCC 2 (0.5), (0–0.8)
n (%), 95% CI HPV PCR in anal mucosa
n = 394
HR-HPV 303 (76.9), (73–81.1)
LR-HPV 288 (73.1), (69–77)
HR and LR-HPV 229 (58.1), (53–63)
Median HR-HPV, IQR 1 (1–3)
Median LR-HPV, IQR 1 (0–2)
HPV 6 71 (18)
HPV 11 71 (18)
HPV 12 1 (0.3)
HPV 16 109 (27.7)
HPV 18 51 (12.9)
HPV 26 6 (1.5)
HPV 31 55 (14)
HPV 33 29 (7.4)
HPV 35 36 (9.1)
HPV 39 46 (11.7)
HPV-40 7 (1.8)
HPV-42 72 (18.3)
HPV-43 10 (2.5)
HPV 45 50 (12.7)
HPV 48 1 (0.3)
HPV 51 55 (14)
HPV 52 50 (12.7)
HPV 53 36 (9.1)
HPV 54 26 (6.6)
HPV 55 64 (16.2)
HPV 56 31 (7.9)
HPV 58 23 (5.8)
HPV 59 42 (10.7)
HPV 61 30 (7.6)
HPV 62 56 (14.2)
HPV 64 1 (0.3)
HPV 66 34 (8.6)
HPV 68 42 (10.7)
HPV 69 14 (3.6)
HPV 70 32 (8.1)
HPV 71 1 (0.3)
HPV 72 28 (7.1)
HPV 73 37 (9.4)
HPV 81 51 (12.9)
HPV 82 17 (4.3)
HPV 83 5 (1.3)
HPV 84 30 (7.6)
HPV 89 1 (0.3)
HPV 6108 13 (3.3)
HPV-AR subtype of HPV 18 (39,45,59,68) 164 (41.6)
HPV-AR subtype of HPV 16 (31,33,35,52,58,67) 207 (52.5)
LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance; ASCC, anal squamous cell cancer. HPV, human papillomavirus; HR-HPV: high-risk HPV, LR-HPV: low-risk HPV.
10.1371/journal.pone.0245870.t003Table 3 Risk factors associated with ≥HSILs in HIV+ MSM patients.
Bivariate and multivariate analysis.
≥HSIL NORMAL Bivariate Multivariate
N = 88 N = 317 p* OR 95% CI
Mean age (yrs), mean (± DS) 30.4 (± 7.6) 31.3 (± 8.3) 0.359 1.27 (0.71–2.29)
Retired, n (%) 6 (6.8) 16 (5) 0.594
Smoker, n (%) 50 (56.8) 164 (51.7) 0.398
Charlson Index, median (IQR) 0 (0–0) 0 (0–0) 0.178
Intercourse in previous 12 months, n (%) 78 (88.6) 286 (90.8) 0.545
qHPV Vaccine, n (%) 12 (13.6) 54(17) 0.445 1.72 (0.92–3.23)
Age at first sexual intercourse, (IQR) 18 (17–21) 18(16–20)
Genital/anal warts, n (%) 36(40.9) 92(29) 0.034
History of Syphilis, n (%) 24(27.3) 79(24.9) 0.654
HCV infection, n (%) 4 (4.5) 10 (3.2) 0.515
HBV infection, n (%) 2 (2.3) 11 (3.5) 0.742
Total NPS, baseline visit, median, (IQR) 50 (16–200) 50 (19.5–150) 0.543
NSP12m before last visit, median, (IQR) 1 (1–4) 1 (1–6.5) 0.078
Use of condom during study, n (%) 63 (71.6) 244 (76.9) 0.493
History of AIDS (A3, B3, C), n (%) 29 (33) 77(24.3) 0.102 2.37(1.009–5.58)
Time since HIV diagnosis (months), (IQR) 27 (9–83) 54(21–107) 0.386 1.002 (1.000–1.003)
CD4 nadir (cells/ul), mean (± SD) 366.8(±267.9) 368.2(±223.9) 0.959
CD4 nadir < 200 cells/uL, n (%) 24(27.6) 73(23.3) 0.412
Cd4 nadir <500 cells/uL, n (%) 39 (44.8) 161 (51.4) 0.275
Cd4 nadir >500 cells/uL, n (%) 24 (27.6) 77 (24.6) 0.571
CD4 cells/uL, mean (± SD) 674.3(± 347.3) 761.2(± 406.9) 0.069 0.99(0.99–1)
CD8 cells/uL, mean (± SD) 1017.3(± 474.1) 977.5(± 467.5) 0.487
CD4/CD8, mean (± SD) 0.75(± 0.43) 0.85(± 0.41) 0.042 0.53(0.2–1.38)
HIV VL (log), mean (± SD) 4.09 (± 3.35) 4.91(± 4.27) 0.042 1 (1–1)
ART during follow-up, n (%) 78 (88.6) 300 (94.6) 0.046 0.74 (0.22–2.56)
Median months of ART, median, (IQR) 0 (0–13) 24 (0–48) 0.003 0.99(0.99–1)
Virological failure, n (%) 1 (1.3) 4 (1.3) 1
N = 83 N = 291
Infection by Low-risk HPV genotype, n (%) 65 (78.3) 211 (72) 0.349
Infection by High-risk HPV genotype, n (%) 68 (83.9) 174 (59.8) 0.0001 2.72(0.87–8.54)
Infection by Low and High-risk HPV, n (%) 53 (63.9) 132 (45.4) 0.003 0.79 (0.25–2.46)
N° of HR-HPV genotypes, median (IQR) 2 (1–3) 1 (0–2) 0.0001 0.76 (0.58–1)
N° of LR-HPV genotypes, median (IQR) 1 (1–2) 1 (0–2) 0.082 0.83 (0.61–1.12)
Median months with VPH-AR (IQR) 11 (1–18) 1 (0–24) 0.07 0.98 (0.96–1.02)
Median months with VPH-BR (IQR) 8 (1–16) 11 (0–26) 0.487 1 (0.96–1.04)
Median months with mixed VPH infection (IQR) 1 (0–15) 1 (0–12) 0.102
HPV-6 21 (25.3) 47 (16.2) 0.057 1.61 (0.73–3.53)
HPV-11 21 (25.3) 37 (12.7) 0.005 3.81(1.76–8.24)
HPV-16 27 (32.5) 43 (14.8) 0.0001 2.69 (1.22–5.99)
HPV-18 16 (19.3) 26 (8.9) 0.008 2.73 (1.01–7.36)
HPV-31 10 (12) 29 (10) 0.584
HPV-33 4 (4.9) 17 (5.9) 0.733
HPV-35 6 (7.2) 18 (6.1) 0.781
HPV-39 9 (10.8) 24 (8.2) 0.462
HPV-42 12 (14.5) 53 (18.3) 0.419
HPV-45 9 (10.8) 31 (10.7) 0.961
HPV-51 12 (14.5) 26 (8.9) 0.142
HPV-52 10 (12) 40 (13.7) 0.689
HPV-53 12 (14.5) 13 (4.5) 0.001 2.97 (1.002–8.79)
HPV-54 6 (7.2) 24 (8.2) 0.763
HPV-55 11 (13.3) 50 (17.2) 0.393
HPV-59 11 (13.3) 19 (6.5) 0.047 1.8(0.66–4.83)
HPV-61 11 (13.3) 8 (2.7) 0.001 11.88 (3.67–38.53)
HPV-68 16 (19.3) 32 (11) 0.048 2.44(1.03–5.8)
HPV-70 8 (9.8) 24 (8.2) 0.667
HPV-81 10 (12) 56 (19.2) 0.129
P*: p-value
95% CI: 95% confidence interval
HIV+MSM, men who have sex with men living with HIV; LTI, Latent tuberculosis infection; HCV hepatitis C virus; HBV, hepatitis B virus; HPV, Human papillomavirus; EX-IDU, ex-injecting drug addict; VL, viral load. HR-HPV: high-risk HPV, LR-HPV: low-risk HPV; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance, NSPt, Total number of sexual partners; NSP12m: number of sexual partners in past 12 months
Factors associated with ≥HSIL
In the multivariate analysis, the presence of ≥HSILs was related to infection with HPV genotypes 11 (OR 3.81; CI95% 1.76–8.24), 16 (OR 2.69, 95%CI 1.22–5.99), 18 (OR 2.73, CI95% 1.01–7.36), 53 (OR 2.97, CI95% 1.002–8.79), 61 (OR 11.88, 95%CI 3.67–38.53) and 68 (OR 2.44, 95%CI 1.03–5.8), low CD4 nadir (OR1.002; CI95% 1–1.004), and history of AIDS (OR 2.37, 95%CI 1.009–5.58). The remaining results obtained are exhibited in Table 3.
HSIL patients and treatment outcomes
After excluding 11 patients who did not undergo two or more anoscopies, treatment outcomes were analyzed in a sample of 394 (87.2%) MSM, with a median follow-up of 36 months (IQR: 12–69), 1.215 patients-year. Table 4 exhibits the characteristic of the two HSIL treatment groups (imiquimod and surgery).
10.1371/journal.pone.0245870.t004Table 4 Baseline characteristics of HIV+MSM patients receiving imiquimod vs. surgery.
Imiquimod as first option Surgery as first option Bivariate
N = 32 N = 47 p*
Mean age (yrs), mean (± DS) 35.3.4 (± 11.48) 31.3 (± 8.3) 0.88
Retired, n (%) 2 (6.3) 3 (6.4) 1
Smoker, n (%) 19 (59.4) 29 (61.7) 0.68
Intercourse in previous 12 months, n(%) 28 (87.5) 47 (100) 0.72
qHPV Vaccine, n (%) 6 (18.7) 6 (12.8) 0.36
Age at first sexual intercourse, (IQR) 18 (16–21) 18(16–20) 0.53
Genital/anal warts, n (%) 12 (37.5) 23 (48.9) 0.53
History of Syphilis, n (%) 11(34.3) 13 (27.7) 0.33
HCV infection, n (%) 1 (3.1) 3 (6.4) 1
HBV infection, n (%) 0 (0) 2 (4.3) 0.53
Total NPS, baseline visit, median, (IQR) 55 (30–300) 36 (15–200) 0.14
NSP12m before last visit, median, (IQR) 2 (1–9) 1 (1–8) 0.55
Use of condom during study, n (%) 26 (81.3) 39 (82.9) 0.51
History of AIDS (A3, B3, C), n (%) 11 (34.4) 15 (31.9) 0.56
Time since HIV diagnosis (months), (IQR) 21 (7–111) 25 (9.5–64.3) 0.92
CD4 nadir (cells/ul), mean (± SD) 366.8(±267.9) 368.2(±223.9) 0.96
CD4 nadir < 200 cells/uL, n (%) 6(18.8) 15(31.9) 0.36
Cd4 nadir 200–500 cells/uL, n (%) 15 (46.9) 24 (51.1) 0.78
Cd4 nadir >500 cells/uL, n (%) 10 (31.3) 14 (29.8) 0.57
CD4 cells/uL, mean (± SD) 618.2(± 280.3) 675.7(± 334.1) 0.42
CD8 cells/uL, mean (± SD) 946.4(± 443.9) 1096(± 551.5) 0.17
CD4/CD8, mean (± SD) 0.78(± 0.46) 0.70(± 0.35) 0.42
HIV VL (log), mean (± SD) 3.9 (± 4.53) 3.55(± 3.98) 0.42
ART during follow-up, n (%) 28 (87.5) 45 (95.7) 0.6
Median months of ART, median, (IQR) 19.5 (6.5–44) 9 (2–64) 0.57
VL HIV < 50 copies/uL, n (%) 20(62.5) 39 (82.9) 0.28
Infection by Low-risk HPV genotype, n (%) 32 (100) 41 (87.2) 0.29
Infection by High-risk HPV genotype, n (%) 26 (81.3) 46 (97.8) 0.52
Infection by Low and High-risk HPV, n (%) 18 (56.3) 34 (72.3) 0.40
Sub-species HPV 18 (18, 39, 45, 59, 68) 16 (50) 29 (61.7) 0.61
Sub-species HPV 16 (16, 31, 33, 35, 52, 58, 67) 17 (53.1) 23 (48.9) 0.43
N° of HR-HPV genotypes, median (IQR) 2 (1–3.8) 2 (1–3) 0.28
N° of LR-HPV genotypes, median (IQR) 2 (0.3–3) 1 (1–2) 0.92
P*: p-value
95% CI: 95% confidence interval
HIV+MSM, men who have sex with men living with HIV; LTI, Latent tuberculosis infection; HCV hepatitis C virus; HBV, hepatitis B virus; HPV, Human papillomavirus; VL, viral load. HR-HPV: high-risk HPV, LR-HPV: low-risk HPV; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance, NSPt, Total number of sexual partners; NSP12m: number of sexual partners in past 12 months.
Mucosectomy was performed in 47 patients with HSIL, with a median follow-up of 60 months (IQR: 46–73 months) and median disease-free period of 48 months (IQR: 28–60 months).
Forty-one (87.2%) of these patients received surgery alone, five (10.6%) received surgery plus self-administered imiquimod, due to failure of surgery in two cases and recurrence in three, and one patient underwent successful surgery after the failure of imiquimod treatment. Surgical margins were disease-free in 35 patients (76.9%) after first surgery. The response rate was 73.3% to surgery as first-line treatment (33/45) versus 96.8% to imiquimod (31/32) (p = 0.009) as evaluated by post-treatment HRA. A median of one surgical intervention was performed (IQR:1–1), with 31 patients undergoing one, 9 needing two, and 1 needing four interventions. Recurrence was recorded in 7 patients (15.2%), and repeat excision of the same lesion in 11 (23.4%). All surgical patients reported adverse effects, with a median duration of 15 days post-surgery (IQR: 7–21 days), including bleeding with defecation in 32 (68%), pain requiring anti-analgesics in 39 (82.9%), rectal incontinence in 1 (2.1%), and transient anal stenosis in 3 (6.4%) versus 1 in the imiquimod group (2.7%, p = 0.046). Among the 43 patients with follow-up HPV PCR results, clearance of oncogenic VPH genotypes was observed in 19 (44.2%).
Thirty-seven patients with HSIL self-administered 5% imiquimod three times/week; the treatment lasted 16 weeks in 97.3% of these patients and 18 weeks in 2.7%. It was first-line treatment in 32 patients (86.4%) and administered after previous surgery in 5 (13.5%); all patients showed a complete response, except for one case of failure caused by intolerance to imiquimod. Only one patient (2.7%) needed retreatment of the same lesion, whereas 11 (23.4%) of the surgical group required repeat surgery (p = 0.02). The median number of affected quadrants was 1 (IQR: 1–2). Mean follow-up was 48 months (IQR: 35–57 months) and mean disease-free period 36 months (IQR: 12–48). Imiquimod was discontinued in one patient (2.7%) for adverse effects (anal itching, stinging, and/or pain) attributed to non-compliance with the treatment protocol. Among the 35 (94.6%) patients treated with imiquimod for whom follow-up HPV PCR results were available, clearance of oncogenic VPH genotypes was observed in 10 (28.6%) (p = 0.065). No patient treated with surgery or imiquimod progressed to ASCC.
We found significant reductions in ≥HSIL cases between 2010 and 2018 (42.9% (9/21) vs. 4.1% (10/245) p = 0.034), between 2010 and 2013 (42.9% (9/21) vs. 13.8% (22/159), p = 0.003), and between 2013 and 2016 (13.8% (22/159) vs. 4.8% (13/273), p = 0.0001), followed by a stabilization between 2016 and 2018 (4.8% (13/273) vs. 4.1% (10/245), p = 0.617). Four deaths were recorded during the follow-up: one patient with hepatic cirrhosis secondary to chronic HCV infection in 2012, one with Burkitt lymphoma in 2013, one with small-cell lung cancer in 2014, and one with metastatic ASCC in 2015. Further data on outcomes were previously reported in detail [6].
Discussion
Since the first HIV epidemic, the incidence of ASCC has increased in seropositive patients, mainly in MSM with AIDS [17]. After the initiation of our screening/treatment program in 2010, the ≥HSIL rate significantly decreased among HIV-infected MSM patients for six years and then subsequently stabilized, with no progression to ASCC in patients with treated HSIL. Our results support the proposition by the authors of the Swiss Cohort study that the incidence of ASCC among people living with HIV can be markedly reduced if they all receive ART and can be further diminished if they also undergo annual screening with anal cytology or anoscopy [18]. A recent study of 592 HIV patients, with a mean follow-up of 69 months, reported that the risk of progression from HSIL (AIN3) to ASCC was high and that ASCC screening was the only factor that reduced this risk [19]. Data from the Study for the Prevention of Anal Cancer (SPANC) [20] are expected to elucidate the natural evolution of HPV infection, allowing a more effective classification of patients at risk of ASCC. In the meantime, a program to screen, diagnose, treat and follow up anal mucosal dysplastic lesions appears recommendable, especially in HIV+ MSM.
In this prospective study of HIV+ MSM undergoing a screening/treatment program for anal mucosa dysplasic lesions, the presence of ≥high-grade anal intraepithelial lesions were related to infection by HPV genotypes 11 16, 18, 53, 61 and 68, a low CD4 nadir and a history of AIDS. This finding of a relationship between HSIL-positivity and poor immunological status is consistent with previous observations that prolonged antiretroviral treatment [21–23] and a high CD4 count, regardless of CD4 nadir [24], are protective factors against HSIL. A recent prospective study observed a similar incidence of HPV-16 and -18 genotypes in the anal mucosa of French HIV+MSM, but HPV-16 was more persistent and therefore more closely correlated with the presence of HSIL [25]. In a retrospective study of “alpha-human papillomavirus” in the anal mucosa of German HIV+ patients, the presence of HSIL and simultaneous infection was associated with high- and low-risk genotypes [26]. Currently, patients with a new diagnosis of HIV in Spain are usually MSM, and the diagnosis is late in 47.6% of these cases [27], with a CD4 count <200 cells/uL. The above data suggest that screening for anal dysplasia is essential in this type of patient.
Self-administration of 5% imiquimod was a highly effective therapeutic strategy against HSIL in this series of HIV+MSM. Most of them did not need to repeat the topical treatment, whereas around a quarter of the patients undergoing excision required another intervention. Furthermore, the therapeutic failure rate and dropout for adverse effects were lower in the imiquimod group than in the surgery group. Various studies have supported the efficacy of imiquimod to treat HSIL in HIV+ patients [13, 28, 29]. Thus, a double-blind randomized placebo-controlled clinical trial comparing between self-application of imiquimod (n = 28) versus placebo (n = 25) in the anal canal three times/week for 4 months found a significant association (P = 0.003) between imiquimod and a positive outcome [13]. In addition, a prospective, observational open study in 44 HIV+ patients with HSIL observed a response rate of 66% (29/44) for imiquimod [28]. Finally, a retrospective observational study in 28 HIV+ and HIV- patients observed a higher frequency of total or partial responses in those receiving anal tampon treatment with a 15 mg versus 6.25 mg dose of imiquimod, with no difference in CD4, HIV viral load, or serostatus [29].
Further advantages of 5% imiquimod in comparison to ablative therapies include its self-administration and its usefulness in cases of extensive disease. The surgical option was also effective in a large proportion of our patients, although some needed retreatment due to recurrence or incomplete excision. No cases of permanent stenosis or fecal incontinence were observed in the surgical group; however, surgery is not currently recommended due to its adverse effects, especially in patients with large lesions [30].
Limitations of this single-center study include its observational design, comparing the real-life clinical effectiveness of imiquimod and surgery rather than their efficacy (as in a clinical trial). In addition, it only included HIV+MSM, and these data cannot be extrapolated to other types of patient. Finally, 11 of the 405 enrolled patients did not undergo two or more anoscopies and were therefore lost to the follow-up. However, its strengths include the prospective design and long follow-up period, which was a mean of 36 months. In fact, the present cohort of HIV+ patients is one of the few published to date that was created to measure predetermined objectives.
In conclusion, HSIL screening and treatment programs reduce the incidence of this precursor of ASCC. Chronic mixed HPV infection and a history of poor immunological status are associated with the presence of HSILs. Self-administration of 5% imiquimod is more effective than surgery as first-line treatment of anal HSIL in HIV+MSM patients, with a lower recurrence rate and fewer adverse effects.
Supporting information
S1 File HIV MSM cohort database.
(SAV)
Click here for additional data file.
The authors are grateful to Mercedes Álvarez Romero for coordinating patients and drawing blood samples and to Marina Gutiérrez and Rodrigo López of the Pathology Department for processing samples. The authors are grateful to the participating patients. | IMIQUIMOD | DrugsGivenReaction | CC BY | 33534790 | 18,913,048 | 2021 |
What was the administration route of drug 'IMIQUIMOD'? | Risk factors for ≥high-grade anal intraepithelial lesions in MSM living with HIV and the response to topical and surgical treatments.
The objective of this study in MSM living with HIV was to determine the incidence of HSIL and ASCC, related factors, and the response to treatment.
Data were gathered in 405 consecutive HIV-infected MSM (May 2010-December 2018) at baseline and annually on: sexual behavior, anal cytology, and HPV PCR and/or high-resolution anoscopy results. They could choose mucosectomy with electric scalpel (from May 2010) or self-administration of 5% imiquimod 3 times weekly for 16 weeks (from November 2013). A multivariate logistic regression model was developed for ≥HSIL-related factors using a step-wise approach to select variables, with a significance level of 0.05 for entry and 0.10 for exit, applying the Hosmer-Lemeshow test to assess the goodness of fit.
The study included 405 patients with a mean age of 36.2 years; 56.7% had bachelor´s degree, and 52.8% were smokers. They had a mean of 1 (IQR 1-7) sexual partner in the previous 12 months, median time since HIV diagnosis of 2 years, and mean CD4 nadir of 367.9 cells/uL; 86.7% were receiving ART, the mean CD4 level was 689.6 cells/uL, mean CD4/CD8 ratio was 0.77, and 85.9% of patients were undetectable. Incidence rates were 30.86/1,000 patient-years for ≥high squamous intraepithelial lesion (HSIL) and 81.22/100,000 for anal squamous cell carcinoma (ASCC). The ≥HSIL incidence significantly decreased from 42.9% (9/21) in 2010 to 4.1% (10/254) in 2018 (p = 0.034). ≥HSIL risk factors were infection with HPV 11 (OR 3.81; 95%CI 1.76-8.24), HPV 16 (OR 2.69, 95%CI 1.22-5.99), HPV 18 (OR 2.73, 95%CI 1.01-7.36), HPV 53 (OR 2.97, 95%CI 1.002-8.79); HPV 61 (OR 11.88, 95%CI 3.67-38.53); HPV 68 (OR 2.44, CI 95% 1.03-5.8); low CD4 nadir (OR1.002; 95%CI 1-1.004) and history of AIDS (OR 2.373, CI 95% 1.009-5.577). Among HSIL-positive patients, the response rate was higher after imiquimod than after surgical excision (96.7% vs 73.3%, p = 0.009) and there were fewer re-treatments (2.7% vs 23.4%, p = 0.02) and adverse events (2.7% vs 100%, p = 0.046); none developed ASCC.
HSIL screening and treatment programs reduce the incidence of HSIL, which is related to chronic HPV infection and poor immunological status. Self-administration of 5% imiquimod as first-line treatment of HSIL is more effective than surgery in HIV+ MSM.
Introduction
Anal squamous-cell carcinoma (ASCC) is frequent among individuals living with HIV (HIV+) and associated with a high mortality rate [1] and with an incidence of up to 131/100,000 people/year among HIV+ men who have sex with men (MSM) [2], with a similar survival rate to the general population [3]. The beginning of a decline in its incidence among MSM has been described by some observational studies but not by clinical trials, which may be explained by the participation of their study populations in an intraepithelial neoplasia screening and treatment program [4, 5], as recently reported by our group [6].
ASCC has been associated with persistent infection by high-risk oncogenic HPV (HR-HPV) genotypes [7]. Interaction between HIV and HPV, which have risk factors in common, has been reported to increase the risk of HPV and reduce the clearance rate among individuals living with HIV [8].
It remains controversial whether the treatment of high-grade anal intraepithelial lesions (HSILs) prevents the subsequent development of ASCC [9], and no standard approach has been established, so that treatments vary among centers according to their resources and experience. Therapeutic options include local topical treatments with trichloroacetic acid, imiquimod, or 5-fluoruracil and more invasive treatments such as excision, fulguration, or laser therapy [9]. Surgical excision used to be a treatment of choice but is associated with adverse effects, including anal stenosis, and with recurrences and incomplete resection, especially when the anal disease is extensive [10]. Fulguration is applied during high-resolution anoscopy (HRA) and has been associated with a recurrence rate of around 25% at 30 months in HIV+ patients, depending on the amount of fulguration [11]. In regard to infrared or laser coagulation, it can be applied in the consulting room and is well tolerated, but a recurrence rate of 61% at one year has been described in HIV+ patients [12]. Finally, topical therapies offer a non-invasive option with a good safety profile and few adverse effects [9]. In particular, imiquimod offers the advantage of self-administration, a low recurrence rate, and a high response rate in HIV+ patients, which was reported to be 61% in a clinical trial [13].
In this study of consecutive HIV+ MSM undergoing a screening/treatment program at our hospital, we previously reported that oncogenic genotype clearance in anal mucosa was associated with the length of ART but did not affect the incidence of HSILs or ASCC. In the present article, data from the same population [6] were analyzed with a different objective, which was to determine the incidence of HSIL and ASCC, the risk factors, and the response to imiquimod treatment and/or electrosurgical excision.
Patients and methods
Design
This longitudinal observational (naturalistic/naturalist) prospective study was conducted in a population of 405 adult HIV+ MSM patients with no history of ASCC, who were consecutively enrolled between May 2010 and December 2018 in a screening and follow-up program for dysplasic anal mucosa lesions. This population and the methodology applied were previously reported [6] in a previous study on HR-HPV clearance and acquisition rates and their relationship with the incidence of HSILs/ASCC in anal mucosa. At their baseline visit (V0) we obtained the written informed consent of the patients to participation in the study, which was approved by the ethics committee (CEIC) of the University Hospital “Virgen de las Nieves” and complied with Spanish data protection legislation (Law 15/1999, 13 December). This CEIC is integrated within the network of ethics Committees of Andalusian public health system (SSPA). CEIC is regulated by order/Decree 439/2010 December 14th of Andalusia. All data were treated in accordance with Spanish data protection legislation Law 15/1999, 13 December, on Personal Character Data Protection).
In brief, data were gathered at baseline (V0), 4–12 weeks, and subsequent follow-ups (at individualized intervals) on clinical-epidemiological and analytical variables and on the results of PCR (Gonorrhoeae, Mycoplasma spp, Chlamydia spp, Ureaplasma spp) and oral-anal-urethral exudate culture (Gonorrhoeae) studies in symptomatic patients or infected partners. In addition, two anal canal mucosa samples were taken at baseline for HPV detection and genotyping by qualitative PCR and for cytology study using the “thin-layer” technique (Processor Thin Prep 2000 (Hologic), and HRA was performed at 4–12 weeks taking samples of apparently normal mucosa and areas with Lugol-negative aceto-white lesions using an endoscopic retrograde cholangiopancreatography catheter. HRA results were obtained from 100% of patients, and findings of at least one follow-up anoscopy were available for 97.3% (394/405). HRA was performed by an infectious disease specialist specifically trained for one year by an expert in this technique from the Department of Digestive Disease of our hospital.
At one year, patients with a normal anoscopy result and LSIL (AIN1) were examined with cytology, HPV PCR, and anoscopy, whereas those with HSIL underwent electrosurgical mucosal resection (from May 2010) or self-administration of 5% imiquimod 3 times/week for 16 weeks (from November 2013), followed by another anoscopy. Surgery was the sole option from May 2010 until the introduction of imiquimod in 2013; since then, all patients have been offered imiquimod as first treatment option. If the imiquimod treatment failed, patients were offered extension of the course up to 18 weeks, a new 16-week cycle of imiquimod, or surgery. Between May 2012 and May 2014, quadrivalent HPV vaccine was received by patients with no HPV 16 or 18 infection and no presence or history of HSIL+-compatible lesions. Cytology results were categorized according to the Bethesda classification [14] and histological findings according to the LAST HPV standardization project [15].
Definition of variables
Abnormal cytology: Cytology findings of ASCUS, LSIL, or HSIL.
Histology with
≥HSILs: Histology findings from HSIL to ASCC.
Therapeutic failure: HSIL persistence after treatment.
Post-treatment HSIL recurrence: Re-appearance of previously treated lesion with normal post-treatment anoscopy.
Response to treatment: Disappearance of lesion in follow-up anoscopy after treatment with surgery or imiquimod.
Self-administration of 5% imiquimod. On Mondays, Wednesdays and Fridays, patients used a preloaded single-use insulin syringe (after cleaning drug remains from its surface) to apply imiquimod inside the anal canal, 3 cm from the anal verge, while in left lateral decubitus position, preferably before sleeping. Before starting this therapy, the patient received detailed instructions on the procedure from the attending physician and nurse.
Statistical analysis
SPSS 21.0 was used for data analyses. The descriptive statistics and tests used for bivariate analyses were previously reported in detail [6]. In the present study, a multivariate logistic regression model was developed for ≥HSIL-related factors, using Freeman’s formula [n = 10*(k+1)] [16], including significant variables in bivariate analysis (ART duration, infection with low-risk HPV genotypes, infection with HPV-6, 11, 16, 18, 53, 59, 61, or 68, and duration of infection with high-risk genotypes) and other variables deemed clinically relevant (smoking habit, history of AIDS, CD4 nadir, CD4/CD8 ratio, duration of infection with HR-HPV and mixed infection). A stepwise approach was used to select variables, with a significance level of 0.05 for entry and 0.10 for exit, applying the Hosmer-Lemeshow test to assess the goodness of fit. P≤0.05 was considered significant.
Results
Baseline characteristics of the cohort
Table 1 summarizes the baseline characteristics of the 405 HIV+MSM enrolled in the study, Table 2 exhibits the distribution of genotypes and Table 3 the cytology and anoscopy findings. In brief, the mean age was 36.2 years, and fewer than 10% were aged > 50 years. They had very good viro-immunological status, 85.6% were on ART at enrolment, and the mean interval since HIV diagnosis was around two years. There was a high prevalence of HPV infection: 76.9% with HR-genotypes, 73.1% with LR-genotypes and 58.1% with both. The prevalence of ≥HSILs was 21.7% (88/405), with an incidence of 30.86/1,000 patient-years, while the prevalence of ASCC was 0.74%, (4/405), with an incidence of 81.22/100,000 patient-years.
10.1371/journal.pone.0245870.t001Table 1 Characteristics of HIV-infected MSM patients.
Number of patients
n = 405
Age, mean (± SD) 36.2 (± 10.1)
<30 yrs, n (%) 123 (30.4)
30–50 yrs, n (%) 244 (60.2)
>50 yrs n (%) 38 (9.4)
Educational level
No studies 6 (1.5)
Primary studies 40 (9.9)
Secondary studies 129 (31.9)
University studies 230 (56.7)
Retired, n (%), 95% CI 22 (5.4) (3–7.5)
Origin
Europe 387 (95.6)
Central America 17 (4.2)
qHPV vaccine (2012–2014), n (%), 95% CI 66 (16.3)
Age at first sexual intercourse, median (IQR) 18 (16–20)
Number of lifetime male sex partners, median (IQR) 50 (15–150)
Number of male sex partners during previous 12 months, median (IQR) 1(1–7)
Habitual use of condoms, n (%), 95% CI 294 (72.6) (68.2–77.4)
Total number of sexual partners during follow-up, median (IQR) 54.5(20–154)
History of anal/genital warts, n (%), (95%CI) 128 (31.6), (27.1–36.1)
Anal/genital warts at baseline n (%), (95%CI) 93 (23), (18.6–26.8)
History of syphilis, n (%), IC95% 103 (25.4), (21.6–29.8)
History of other STI, n (%), IC 95% 110 (27.2), (23.1–31.6)
Time since HIV diagnosis (months), median (IQR) 25 (8–84)
CD4 at diagnosis of HIV (cel/uL), mean (± SD) 448± 298.17
HIV viral load at diagnosis of HIV (log), median (IQR) 4.61 (4.07–5.12)
CD4 nadir (cells/uL), mean (± SD) 367.93±233.85
CD4 nadir < 200 cells/uL, n (%), 95% CI 97 (23.9), (20–28.5)
CD4 cell count at baseline (cells/uL), mean (± SD) 689.64± 475.03
CD8 cell count at baseline (cells/uL), mean (± SD) 981.5±531.5
CD4 /CD8 ratio, mean (± SD) 0.77±0.70
HIV viral load at baseline (log), median (IQR) 0 (0–1.72)
Undetectable: < 50 HIV RNA copies/mL of plasma, n (%) 348 (85.9)
History of AIDS diagnosis, n (%), 95% CI 106 (26.2) (21.3–30.1)
HAART before inclusion, n (%), 95% CI 351 (86.7), (83.2–90)
Previous ART line, median (IQR) 1 (1–2)
Virological failure, n (%) 17 (4.8)
Median months of ART, median (IQR) 4 (16–56)
Chronic HCV infection, n (%) 14 (3.5)
Chronic HBV infection, n (%) 13 (3.2)
Smoker, pack/year, median (IQR) 1.5 (0–14)
Smoker, n (%), 95% CI 214 (52.8) (47.9–57.4)
Alcohol, SDU, median (IQR) 0 (0–4)
EX-IDU, n (%) 2 (0.5)
HCV, chronic infection by hepatitis C virus; HBV, chronic infection by hepatitis B virus; EX-IDU, ex-injecting drug users; STI: sexual transmitted infection; VL: viral load; IQR: interquartile range; SD, (standard deviation), SDU: standard drink unit.
10.1371/journal.pone.0245870.t002Table 2 Cytology, anoscopy, and HPV PCR results for the cohort.
Cohort of MSM-HIV patients
n = 405
Anal cytology, (n = 397), n (%), 95% CI
LSIL 190 (47.9), (43–52.7)
HSIL 13 (3.3), (1.8–5.1)
ASCUS 29 (7.3), (5.1–10.2)
Normal 165 (41.6), (37–46)
Anoscopy: Histology (n = 405), n (%), 95% CI
Normal 189 (46.7), (44.5–54)
LSIL 164 (40.4), (36.6–46)
HSIL 50 (12.3), (9.2–15.9)
ASCC 2 (0.5), (0–0.8)
n (%), 95% CI HPV PCR in anal mucosa
n = 394
HR-HPV 303 (76.9), (73–81.1)
LR-HPV 288 (73.1), (69–77)
HR and LR-HPV 229 (58.1), (53–63)
Median HR-HPV, IQR 1 (1–3)
Median LR-HPV, IQR 1 (0–2)
HPV 6 71 (18)
HPV 11 71 (18)
HPV 12 1 (0.3)
HPV 16 109 (27.7)
HPV 18 51 (12.9)
HPV 26 6 (1.5)
HPV 31 55 (14)
HPV 33 29 (7.4)
HPV 35 36 (9.1)
HPV 39 46 (11.7)
HPV-40 7 (1.8)
HPV-42 72 (18.3)
HPV-43 10 (2.5)
HPV 45 50 (12.7)
HPV 48 1 (0.3)
HPV 51 55 (14)
HPV 52 50 (12.7)
HPV 53 36 (9.1)
HPV 54 26 (6.6)
HPV 55 64 (16.2)
HPV 56 31 (7.9)
HPV 58 23 (5.8)
HPV 59 42 (10.7)
HPV 61 30 (7.6)
HPV 62 56 (14.2)
HPV 64 1 (0.3)
HPV 66 34 (8.6)
HPV 68 42 (10.7)
HPV 69 14 (3.6)
HPV 70 32 (8.1)
HPV 71 1 (0.3)
HPV 72 28 (7.1)
HPV 73 37 (9.4)
HPV 81 51 (12.9)
HPV 82 17 (4.3)
HPV 83 5 (1.3)
HPV 84 30 (7.6)
HPV 89 1 (0.3)
HPV 6108 13 (3.3)
HPV-AR subtype of HPV 18 (39,45,59,68) 164 (41.6)
HPV-AR subtype of HPV 16 (31,33,35,52,58,67) 207 (52.5)
LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance; ASCC, anal squamous cell cancer. HPV, human papillomavirus; HR-HPV: high-risk HPV, LR-HPV: low-risk HPV.
10.1371/journal.pone.0245870.t003Table 3 Risk factors associated with ≥HSILs in HIV+ MSM patients.
Bivariate and multivariate analysis.
≥HSIL NORMAL Bivariate Multivariate
N = 88 N = 317 p* OR 95% CI
Mean age (yrs), mean (± DS) 30.4 (± 7.6) 31.3 (± 8.3) 0.359 1.27 (0.71–2.29)
Retired, n (%) 6 (6.8) 16 (5) 0.594
Smoker, n (%) 50 (56.8) 164 (51.7) 0.398
Charlson Index, median (IQR) 0 (0–0) 0 (0–0) 0.178
Intercourse in previous 12 months, n (%) 78 (88.6) 286 (90.8) 0.545
qHPV Vaccine, n (%) 12 (13.6) 54(17) 0.445 1.72 (0.92–3.23)
Age at first sexual intercourse, (IQR) 18 (17–21) 18(16–20)
Genital/anal warts, n (%) 36(40.9) 92(29) 0.034
History of Syphilis, n (%) 24(27.3) 79(24.9) 0.654
HCV infection, n (%) 4 (4.5) 10 (3.2) 0.515
HBV infection, n (%) 2 (2.3) 11 (3.5) 0.742
Total NPS, baseline visit, median, (IQR) 50 (16–200) 50 (19.5–150) 0.543
NSP12m before last visit, median, (IQR) 1 (1–4) 1 (1–6.5) 0.078
Use of condom during study, n (%) 63 (71.6) 244 (76.9) 0.493
History of AIDS (A3, B3, C), n (%) 29 (33) 77(24.3) 0.102 2.37(1.009–5.58)
Time since HIV diagnosis (months), (IQR) 27 (9–83) 54(21–107) 0.386 1.002 (1.000–1.003)
CD4 nadir (cells/ul), mean (± SD) 366.8(±267.9) 368.2(±223.9) 0.959
CD4 nadir < 200 cells/uL, n (%) 24(27.6) 73(23.3) 0.412
Cd4 nadir <500 cells/uL, n (%) 39 (44.8) 161 (51.4) 0.275
Cd4 nadir >500 cells/uL, n (%) 24 (27.6) 77 (24.6) 0.571
CD4 cells/uL, mean (± SD) 674.3(± 347.3) 761.2(± 406.9) 0.069 0.99(0.99–1)
CD8 cells/uL, mean (± SD) 1017.3(± 474.1) 977.5(± 467.5) 0.487
CD4/CD8, mean (± SD) 0.75(± 0.43) 0.85(± 0.41) 0.042 0.53(0.2–1.38)
HIV VL (log), mean (± SD) 4.09 (± 3.35) 4.91(± 4.27) 0.042 1 (1–1)
ART during follow-up, n (%) 78 (88.6) 300 (94.6) 0.046 0.74 (0.22–2.56)
Median months of ART, median, (IQR) 0 (0–13) 24 (0–48) 0.003 0.99(0.99–1)
Virological failure, n (%) 1 (1.3) 4 (1.3) 1
N = 83 N = 291
Infection by Low-risk HPV genotype, n (%) 65 (78.3) 211 (72) 0.349
Infection by High-risk HPV genotype, n (%) 68 (83.9) 174 (59.8) 0.0001 2.72(0.87–8.54)
Infection by Low and High-risk HPV, n (%) 53 (63.9) 132 (45.4) 0.003 0.79 (0.25–2.46)
N° of HR-HPV genotypes, median (IQR) 2 (1–3) 1 (0–2) 0.0001 0.76 (0.58–1)
N° of LR-HPV genotypes, median (IQR) 1 (1–2) 1 (0–2) 0.082 0.83 (0.61–1.12)
Median months with VPH-AR (IQR) 11 (1–18) 1 (0–24) 0.07 0.98 (0.96–1.02)
Median months with VPH-BR (IQR) 8 (1–16) 11 (0–26) 0.487 1 (0.96–1.04)
Median months with mixed VPH infection (IQR) 1 (0–15) 1 (0–12) 0.102
HPV-6 21 (25.3) 47 (16.2) 0.057 1.61 (0.73–3.53)
HPV-11 21 (25.3) 37 (12.7) 0.005 3.81(1.76–8.24)
HPV-16 27 (32.5) 43 (14.8) 0.0001 2.69 (1.22–5.99)
HPV-18 16 (19.3) 26 (8.9) 0.008 2.73 (1.01–7.36)
HPV-31 10 (12) 29 (10) 0.584
HPV-33 4 (4.9) 17 (5.9) 0.733
HPV-35 6 (7.2) 18 (6.1) 0.781
HPV-39 9 (10.8) 24 (8.2) 0.462
HPV-42 12 (14.5) 53 (18.3) 0.419
HPV-45 9 (10.8) 31 (10.7) 0.961
HPV-51 12 (14.5) 26 (8.9) 0.142
HPV-52 10 (12) 40 (13.7) 0.689
HPV-53 12 (14.5) 13 (4.5) 0.001 2.97 (1.002–8.79)
HPV-54 6 (7.2) 24 (8.2) 0.763
HPV-55 11 (13.3) 50 (17.2) 0.393
HPV-59 11 (13.3) 19 (6.5) 0.047 1.8(0.66–4.83)
HPV-61 11 (13.3) 8 (2.7) 0.001 11.88 (3.67–38.53)
HPV-68 16 (19.3) 32 (11) 0.048 2.44(1.03–5.8)
HPV-70 8 (9.8) 24 (8.2) 0.667
HPV-81 10 (12) 56 (19.2) 0.129
P*: p-value
95% CI: 95% confidence interval
HIV+MSM, men who have sex with men living with HIV; LTI, Latent tuberculosis infection; HCV hepatitis C virus; HBV, hepatitis B virus; HPV, Human papillomavirus; EX-IDU, ex-injecting drug addict; VL, viral load. HR-HPV: high-risk HPV, LR-HPV: low-risk HPV; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance, NSPt, Total number of sexual partners; NSP12m: number of sexual partners in past 12 months
Factors associated with ≥HSIL
In the multivariate analysis, the presence of ≥HSILs was related to infection with HPV genotypes 11 (OR 3.81; CI95% 1.76–8.24), 16 (OR 2.69, 95%CI 1.22–5.99), 18 (OR 2.73, CI95% 1.01–7.36), 53 (OR 2.97, CI95% 1.002–8.79), 61 (OR 11.88, 95%CI 3.67–38.53) and 68 (OR 2.44, 95%CI 1.03–5.8), low CD4 nadir (OR1.002; CI95% 1–1.004), and history of AIDS (OR 2.37, 95%CI 1.009–5.58). The remaining results obtained are exhibited in Table 3.
HSIL patients and treatment outcomes
After excluding 11 patients who did not undergo two or more anoscopies, treatment outcomes were analyzed in a sample of 394 (87.2%) MSM, with a median follow-up of 36 months (IQR: 12–69), 1.215 patients-year. Table 4 exhibits the characteristic of the two HSIL treatment groups (imiquimod and surgery).
10.1371/journal.pone.0245870.t004Table 4 Baseline characteristics of HIV+MSM patients receiving imiquimod vs. surgery.
Imiquimod as first option Surgery as first option Bivariate
N = 32 N = 47 p*
Mean age (yrs), mean (± DS) 35.3.4 (± 11.48) 31.3 (± 8.3) 0.88
Retired, n (%) 2 (6.3) 3 (6.4) 1
Smoker, n (%) 19 (59.4) 29 (61.7) 0.68
Intercourse in previous 12 months, n(%) 28 (87.5) 47 (100) 0.72
qHPV Vaccine, n (%) 6 (18.7) 6 (12.8) 0.36
Age at first sexual intercourse, (IQR) 18 (16–21) 18(16–20) 0.53
Genital/anal warts, n (%) 12 (37.5) 23 (48.9) 0.53
History of Syphilis, n (%) 11(34.3) 13 (27.7) 0.33
HCV infection, n (%) 1 (3.1) 3 (6.4) 1
HBV infection, n (%) 0 (0) 2 (4.3) 0.53
Total NPS, baseline visit, median, (IQR) 55 (30–300) 36 (15–200) 0.14
NSP12m before last visit, median, (IQR) 2 (1–9) 1 (1–8) 0.55
Use of condom during study, n (%) 26 (81.3) 39 (82.9) 0.51
History of AIDS (A3, B3, C), n (%) 11 (34.4) 15 (31.9) 0.56
Time since HIV diagnosis (months), (IQR) 21 (7–111) 25 (9.5–64.3) 0.92
CD4 nadir (cells/ul), mean (± SD) 366.8(±267.9) 368.2(±223.9) 0.96
CD4 nadir < 200 cells/uL, n (%) 6(18.8) 15(31.9) 0.36
Cd4 nadir 200–500 cells/uL, n (%) 15 (46.9) 24 (51.1) 0.78
Cd4 nadir >500 cells/uL, n (%) 10 (31.3) 14 (29.8) 0.57
CD4 cells/uL, mean (± SD) 618.2(± 280.3) 675.7(± 334.1) 0.42
CD8 cells/uL, mean (± SD) 946.4(± 443.9) 1096(± 551.5) 0.17
CD4/CD8, mean (± SD) 0.78(± 0.46) 0.70(± 0.35) 0.42
HIV VL (log), mean (± SD) 3.9 (± 4.53) 3.55(± 3.98) 0.42
ART during follow-up, n (%) 28 (87.5) 45 (95.7) 0.6
Median months of ART, median, (IQR) 19.5 (6.5–44) 9 (2–64) 0.57
VL HIV < 50 copies/uL, n (%) 20(62.5) 39 (82.9) 0.28
Infection by Low-risk HPV genotype, n (%) 32 (100) 41 (87.2) 0.29
Infection by High-risk HPV genotype, n (%) 26 (81.3) 46 (97.8) 0.52
Infection by Low and High-risk HPV, n (%) 18 (56.3) 34 (72.3) 0.40
Sub-species HPV 18 (18, 39, 45, 59, 68) 16 (50) 29 (61.7) 0.61
Sub-species HPV 16 (16, 31, 33, 35, 52, 58, 67) 17 (53.1) 23 (48.9) 0.43
N° of HR-HPV genotypes, median (IQR) 2 (1–3.8) 2 (1–3) 0.28
N° of LR-HPV genotypes, median (IQR) 2 (0.3–3) 1 (1–2) 0.92
P*: p-value
95% CI: 95% confidence interval
HIV+MSM, men who have sex with men living with HIV; LTI, Latent tuberculosis infection; HCV hepatitis C virus; HBV, hepatitis B virus; HPV, Human papillomavirus; VL, viral load. HR-HPV: high-risk HPV, LR-HPV: low-risk HPV; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance, NSPt, Total number of sexual partners; NSP12m: number of sexual partners in past 12 months.
Mucosectomy was performed in 47 patients with HSIL, with a median follow-up of 60 months (IQR: 46–73 months) and median disease-free period of 48 months (IQR: 28–60 months).
Forty-one (87.2%) of these patients received surgery alone, five (10.6%) received surgery plus self-administered imiquimod, due to failure of surgery in two cases and recurrence in three, and one patient underwent successful surgery after the failure of imiquimod treatment. Surgical margins were disease-free in 35 patients (76.9%) after first surgery. The response rate was 73.3% to surgery as first-line treatment (33/45) versus 96.8% to imiquimod (31/32) (p = 0.009) as evaluated by post-treatment HRA. A median of one surgical intervention was performed (IQR:1–1), with 31 patients undergoing one, 9 needing two, and 1 needing four interventions. Recurrence was recorded in 7 patients (15.2%), and repeat excision of the same lesion in 11 (23.4%). All surgical patients reported adverse effects, with a median duration of 15 days post-surgery (IQR: 7–21 days), including bleeding with defecation in 32 (68%), pain requiring anti-analgesics in 39 (82.9%), rectal incontinence in 1 (2.1%), and transient anal stenosis in 3 (6.4%) versus 1 in the imiquimod group (2.7%, p = 0.046). Among the 43 patients with follow-up HPV PCR results, clearance of oncogenic VPH genotypes was observed in 19 (44.2%).
Thirty-seven patients with HSIL self-administered 5% imiquimod three times/week; the treatment lasted 16 weeks in 97.3% of these patients and 18 weeks in 2.7%. It was first-line treatment in 32 patients (86.4%) and administered after previous surgery in 5 (13.5%); all patients showed a complete response, except for one case of failure caused by intolerance to imiquimod. Only one patient (2.7%) needed retreatment of the same lesion, whereas 11 (23.4%) of the surgical group required repeat surgery (p = 0.02). The median number of affected quadrants was 1 (IQR: 1–2). Mean follow-up was 48 months (IQR: 35–57 months) and mean disease-free period 36 months (IQR: 12–48). Imiquimod was discontinued in one patient (2.7%) for adverse effects (anal itching, stinging, and/or pain) attributed to non-compliance with the treatment protocol. Among the 35 (94.6%) patients treated with imiquimod for whom follow-up HPV PCR results were available, clearance of oncogenic VPH genotypes was observed in 10 (28.6%) (p = 0.065). No patient treated with surgery or imiquimod progressed to ASCC.
We found significant reductions in ≥HSIL cases between 2010 and 2018 (42.9% (9/21) vs. 4.1% (10/245) p = 0.034), between 2010 and 2013 (42.9% (9/21) vs. 13.8% (22/159), p = 0.003), and between 2013 and 2016 (13.8% (22/159) vs. 4.8% (13/273), p = 0.0001), followed by a stabilization between 2016 and 2018 (4.8% (13/273) vs. 4.1% (10/245), p = 0.617). Four deaths were recorded during the follow-up: one patient with hepatic cirrhosis secondary to chronic HCV infection in 2012, one with Burkitt lymphoma in 2013, one with small-cell lung cancer in 2014, and one with metastatic ASCC in 2015. Further data on outcomes were previously reported in detail [6].
Discussion
Since the first HIV epidemic, the incidence of ASCC has increased in seropositive patients, mainly in MSM with AIDS [17]. After the initiation of our screening/treatment program in 2010, the ≥HSIL rate significantly decreased among HIV-infected MSM patients for six years and then subsequently stabilized, with no progression to ASCC in patients with treated HSIL. Our results support the proposition by the authors of the Swiss Cohort study that the incidence of ASCC among people living with HIV can be markedly reduced if they all receive ART and can be further diminished if they also undergo annual screening with anal cytology or anoscopy [18]. A recent study of 592 HIV patients, with a mean follow-up of 69 months, reported that the risk of progression from HSIL (AIN3) to ASCC was high and that ASCC screening was the only factor that reduced this risk [19]. Data from the Study for the Prevention of Anal Cancer (SPANC) [20] are expected to elucidate the natural evolution of HPV infection, allowing a more effective classification of patients at risk of ASCC. In the meantime, a program to screen, diagnose, treat and follow up anal mucosal dysplastic lesions appears recommendable, especially in HIV+ MSM.
In this prospective study of HIV+ MSM undergoing a screening/treatment program for anal mucosa dysplasic lesions, the presence of ≥high-grade anal intraepithelial lesions were related to infection by HPV genotypes 11 16, 18, 53, 61 and 68, a low CD4 nadir and a history of AIDS. This finding of a relationship between HSIL-positivity and poor immunological status is consistent with previous observations that prolonged antiretroviral treatment [21–23] and a high CD4 count, regardless of CD4 nadir [24], are protective factors against HSIL. A recent prospective study observed a similar incidence of HPV-16 and -18 genotypes in the anal mucosa of French HIV+MSM, but HPV-16 was more persistent and therefore more closely correlated with the presence of HSIL [25]. In a retrospective study of “alpha-human papillomavirus” in the anal mucosa of German HIV+ patients, the presence of HSIL and simultaneous infection was associated with high- and low-risk genotypes [26]. Currently, patients with a new diagnosis of HIV in Spain are usually MSM, and the diagnosis is late in 47.6% of these cases [27], with a CD4 count <200 cells/uL. The above data suggest that screening for anal dysplasia is essential in this type of patient.
Self-administration of 5% imiquimod was a highly effective therapeutic strategy against HSIL in this series of HIV+MSM. Most of them did not need to repeat the topical treatment, whereas around a quarter of the patients undergoing excision required another intervention. Furthermore, the therapeutic failure rate and dropout for adverse effects were lower in the imiquimod group than in the surgery group. Various studies have supported the efficacy of imiquimod to treat HSIL in HIV+ patients [13, 28, 29]. Thus, a double-blind randomized placebo-controlled clinical trial comparing between self-application of imiquimod (n = 28) versus placebo (n = 25) in the anal canal three times/week for 4 months found a significant association (P = 0.003) between imiquimod and a positive outcome [13]. In addition, a prospective, observational open study in 44 HIV+ patients with HSIL observed a response rate of 66% (29/44) for imiquimod [28]. Finally, a retrospective observational study in 28 HIV+ and HIV- patients observed a higher frequency of total or partial responses in those receiving anal tampon treatment with a 15 mg versus 6.25 mg dose of imiquimod, with no difference in CD4, HIV viral load, or serostatus [29].
Further advantages of 5% imiquimod in comparison to ablative therapies include its self-administration and its usefulness in cases of extensive disease. The surgical option was also effective in a large proportion of our patients, although some needed retreatment due to recurrence or incomplete excision. No cases of permanent stenosis or fecal incontinence were observed in the surgical group; however, surgery is not currently recommended due to its adverse effects, especially in patients with large lesions [30].
Limitations of this single-center study include its observational design, comparing the real-life clinical effectiveness of imiquimod and surgery rather than their efficacy (as in a clinical trial). In addition, it only included HIV+MSM, and these data cannot be extrapolated to other types of patient. Finally, 11 of the 405 enrolled patients did not undergo two or more anoscopies and were therefore lost to the follow-up. However, its strengths include the prospective design and long follow-up period, which was a mean of 36 months. In fact, the present cohort of HIV+ patients is one of the few published to date that was created to measure predetermined objectives.
In conclusion, HSIL screening and treatment programs reduce the incidence of this precursor of ASCC. Chronic mixed HPV infection and a history of poor immunological status are associated with the presence of HSILs. Self-administration of 5% imiquimod is more effective than surgery as first-line treatment of anal HSIL in HIV+MSM patients, with a lower recurrence rate and fewer adverse effects.
Supporting information
S1 File HIV MSM cohort database.
(SAV)
Click here for additional data file.
The authors are grateful to Mercedes Álvarez Romero for coordinating patients and drawing blood samples and to Marina Gutiérrez and Rodrigo López of the Pathology Department for processing samples. The authors are grateful to the participating patients. | Topical | DrugAdministrationRoute | CC BY | 33534790 | 18,913,048 | 2021 |
What was the dosage of drug 'IMIQUIMOD'? | Risk factors for ≥high-grade anal intraepithelial lesions in MSM living with HIV and the response to topical and surgical treatments.
The objective of this study in MSM living with HIV was to determine the incidence of HSIL and ASCC, related factors, and the response to treatment.
Data were gathered in 405 consecutive HIV-infected MSM (May 2010-December 2018) at baseline and annually on: sexual behavior, anal cytology, and HPV PCR and/or high-resolution anoscopy results. They could choose mucosectomy with electric scalpel (from May 2010) or self-administration of 5% imiquimod 3 times weekly for 16 weeks (from November 2013). A multivariate logistic regression model was developed for ≥HSIL-related factors using a step-wise approach to select variables, with a significance level of 0.05 for entry and 0.10 for exit, applying the Hosmer-Lemeshow test to assess the goodness of fit.
The study included 405 patients with a mean age of 36.2 years; 56.7% had bachelor´s degree, and 52.8% were smokers. They had a mean of 1 (IQR 1-7) sexual partner in the previous 12 months, median time since HIV diagnosis of 2 years, and mean CD4 nadir of 367.9 cells/uL; 86.7% were receiving ART, the mean CD4 level was 689.6 cells/uL, mean CD4/CD8 ratio was 0.77, and 85.9% of patients were undetectable. Incidence rates were 30.86/1,000 patient-years for ≥high squamous intraepithelial lesion (HSIL) and 81.22/100,000 for anal squamous cell carcinoma (ASCC). The ≥HSIL incidence significantly decreased from 42.9% (9/21) in 2010 to 4.1% (10/254) in 2018 (p = 0.034). ≥HSIL risk factors were infection with HPV 11 (OR 3.81; 95%CI 1.76-8.24), HPV 16 (OR 2.69, 95%CI 1.22-5.99), HPV 18 (OR 2.73, 95%CI 1.01-7.36), HPV 53 (OR 2.97, 95%CI 1.002-8.79); HPV 61 (OR 11.88, 95%CI 3.67-38.53); HPV 68 (OR 2.44, CI 95% 1.03-5.8); low CD4 nadir (OR1.002; 95%CI 1-1.004) and history of AIDS (OR 2.373, CI 95% 1.009-5.577). Among HSIL-positive patients, the response rate was higher after imiquimod than after surgical excision (96.7% vs 73.3%, p = 0.009) and there were fewer re-treatments (2.7% vs 23.4%, p = 0.02) and adverse events (2.7% vs 100%, p = 0.046); none developed ASCC.
HSIL screening and treatment programs reduce the incidence of HSIL, which is related to chronic HPV infection and poor immunological status. Self-administration of 5% imiquimod as first-line treatment of HSIL is more effective than surgery in HIV+ MSM.
Introduction
Anal squamous-cell carcinoma (ASCC) is frequent among individuals living with HIV (HIV+) and associated with a high mortality rate [1] and with an incidence of up to 131/100,000 people/year among HIV+ men who have sex with men (MSM) [2], with a similar survival rate to the general population [3]. The beginning of a decline in its incidence among MSM has been described by some observational studies but not by clinical trials, which may be explained by the participation of their study populations in an intraepithelial neoplasia screening and treatment program [4, 5], as recently reported by our group [6].
ASCC has been associated with persistent infection by high-risk oncogenic HPV (HR-HPV) genotypes [7]. Interaction between HIV and HPV, which have risk factors in common, has been reported to increase the risk of HPV and reduce the clearance rate among individuals living with HIV [8].
It remains controversial whether the treatment of high-grade anal intraepithelial lesions (HSILs) prevents the subsequent development of ASCC [9], and no standard approach has been established, so that treatments vary among centers according to their resources and experience. Therapeutic options include local topical treatments with trichloroacetic acid, imiquimod, or 5-fluoruracil and more invasive treatments such as excision, fulguration, or laser therapy [9]. Surgical excision used to be a treatment of choice but is associated with adverse effects, including anal stenosis, and with recurrences and incomplete resection, especially when the anal disease is extensive [10]. Fulguration is applied during high-resolution anoscopy (HRA) and has been associated with a recurrence rate of around 25% at 30 months in HIV+ patients, depending on the amount of fulguration [11]. In regard to infrared or laser coagulation, it can be applied in the consulting room and is well tolerated, but a recurrence rate of 61% at one year has been described in HIV+ patients [12]. Finally, topical therapies offer a non-invasive option with a good safety profile and few adverse effects [9]. In particular, imiquimod offers the advantage of self-administration, a low recurrence rate, and a high response rate in HIV+ patients, which was reported to be 61% in a clinical trial [13].
In this study of consecutive HIV+ MSM undergoing a screening/treatment program at our hospital, we previously reported that oncogenic genotype clearance in anal mucosa was associated with the length of ART but did not affect the incidence of HSILs or ASCC. In the present article, data from the same population [6] were analyzed with a different objective, which was to determine the incidence of HSIL and ASCC, the risk factors, and the response to imiquimod treatment and/or electrosurgical excision.
Patients and methods
Design
This longitudinal observational (naturalistic/naturalist) prospective study was conducted in a population of 405 adult HIV+ MSM patients with no history of ASCC, who were consecutively enrolled between May 2010 and December 2018 in a screening and follow-up program for dysplasic anal mucosa lesions. This population and the methodology applied were previously reported [6] in a previous study on HR-HPV clearance and acquisition rates and their relationship with the incidence of HSILs/ASCC in anal mucosa. At their baseline visit (V0) we obtained the written informed consent of the patients to participation in the study, which was approved by the ethics committee (CEIC) of the University Hospital “Virgen de las Nieves” and complied with Spanish data protection legislation (Law 15/1999, 13 December). This CEIC is integrated within the network of ethics Committees of Andalusian public health system (SSPA). CEIC is regulated by order/Decree 439/2010 December 14th of Andalusia. All data were treated in accordance with Spanish data protection legislation Law 15/1999, 13 December, on Personal Character Data Protection).
In brief, data were gathered at baseline (V0), 4–12 weeks, and subsequent follow-ups (at individualized intervals) on clinical-epidemiological and analytical variables and on the results of PCR (Gonorrhoeae, Mycoplasma spp, Chlamydia spp, Ureaplasma spp) and oral-anal-urethral exudate culture (Gonorrhoeae) studies in symptomatic patients or infected partners. In addition, two anal canal mucosa samples were taken at baseline for HPV detection and genotyping by qualitative PCR and for cytology study using the “thin-layer” technique (Processor Thin Prep 2000 (Hologic), and HRA was performed at 4–12 weeks taking samples of apparently normal mucosa and areas with Lugol-negative aceto-white lesions using an endoscopic retrograde cholangiopancreatography catheter. HRA results were obtained from 100% of patients, and findings of at least one follow-up anoscopy were available for 97.3% (394/405). HRA was performed by an infectious disease specialist specifically trained for one year by an expert in this technique from the Department of Digestive Disease of our hospital.
At one year, patients with a normal anoscopy result and LSIL (AIN1) were examined with cytology, HPV PCR, and anoscopy, whereas those with HSIL underwent electrosurgical mucosal resection (from May 2010) or self-administration of 5% imiquimod 3 times/week for 16 weeks (from November 2013), followed by another anoscopy. Surgery was the sole option from May 2010 until the introduction of imiquimod in 2013; since then, all patients have been offered imiquimod as first treatment option. If the imiquimod treatment failed, patients were offered extension of the course up to 18 weeks, a new 16-week cycle of imiquimod, or surgery. Between May 2012 and May 2014, quadrivalent HPV vaccine was received by patients with no HPV 16 or 18 infection and no presence or history of HSIL+-compatible lesions. Cytology results were categorized according to the Bethesda classification [14] and histological findings according to the LAST HPV standardization project [15].
Definition of variables
Abnormal cytology: Cytology findings of ASCUS, LSIL, or HSIL.
Histology with
≥HSILs: Histology findings from HSIL to ASCC.
Therapeutic failure: HSIL persistence after treatment.
Post-treatment HSIL recurrence: Re-appearance of previously treated lesion with normal post-treatment anoscopy.
Response to treatment: Disappearance of lesion in follow-up anoscopy after treatment with surgery or imiquimod.
Self-administration of 5% imiquimod. On Mondays, Wednesdays and Fridays, patients used a preloaded single-use insulin syringe (after cleaning drug remains from its surface) to apply imiquimod inside the anal canal, 3 cm from the anal verge, while in left lateral decubitus position, preferably before sleeping. Before starting this therapy, the patient received detailed instructions on the procedure from the attending physician and nurse.
Statistical analysis
SPSS 21.0 was used for data analyses. The descriptive statistics and tests used for bivariate analyses were previously reported in detail [6]. In the present study, a multivariate logistic regression model was developed for ≥HSIL-related factors, using Freeman’s formula [n = 10*(k+1)] [16], including significant variables in bivariate analysis (ART duration, infection with low-risk HPV genotypes, infection with HPV-6, 11, 16, 18, 53, 59, 61, or 68, and duration of infection with high-risk genotypes) and other variables deemed clinically relevant (smoking habit, history of AIDS, CD4 nadir, CD4/CD8 ratio, duration of infection with HR-HPV and mixed infection). A stepwise approach was used to select variables, with a significance level of 0.05 for entry and 0.10 for exit, applying the Hosmer-Lemeshow test to assess the goodness of fit. P≤0.05 was considered significant.
Results
Baseline characteristics of the cohort
Table 1 summarizes the baseline characteristics of the 405 HIV+MSM enrolled in the study, Table 2 exhibits the distribution of genotypes and Table 3 the cytology and anoscopy findings. In brief, the mean age was 36.2 years, and fewer than 10% were aged > 50 years. They had very good viro-immunological status, 85.6% were on ART at enrolment, and the mean interval since HIV diagnosis was around two years. There was a high prevalence of HPV infection: 76.9% with HR-genotypes, 73.1% with LR-genotypes and 58.1% with both. The prevalence of ≥HSILs was 21.7% (88/405), with an incidence of 30.86/1,000 patient-years, while the prevalence of ASCC was 0.74%, (4/405), with an incidence of 81.22/100,000 patient-years.
10.1371/journal.pone.0245870.t001Table 1 Characteristics of HIV-infected MSM patients.
Number of patients
n = 405
Age, mean (± SD) 36.2 (± 10.1)
<30 yrs, n (%) 123 (30.4)
30–50 yrs, n (%) 244 (60.2)
>50 yrs n (%) 38 (9.4)
Educational level
No studies 6 (1.5)
Primary studies 40 (9.9)
Secondary studies 129 (31.9)
University studies 230 (56.7)
Retired, n (%), 95% CI 22 (5.4) (3–7.5)
Origin
Europe 387 (95.6)
Central America 17 (4.2)
qHPV vaccine (2012–2014), n (%), 95% CI 66 (16.3)
Age at first sexual intercourse, median (IQR) 18 (16–20)
Number of lifetime male sex partners, median (IQR) 50 (15–150)
Number of male sex partners during previous 12 months, median (IQR) 1(1–7)
Habitual use of condoms, n (%), 95% CI 294 (72.6) (68.2–77.4)
Total number of sexual partners during follow-up, median (IQR) 54.5(20–154)
History of anal/genital warts, n (%), (95%CI) 128 (31.6), (27.1–36.1)
Anal/genital warts at baseline n (%), (95%CI) 93 (23), (18.6–26.8)
History of syphilis, n (%), IC95% 103 (25.4), (21.6–29.8)
History of other STI, n (%), IC 95% 110 (27.2), (23.1–31.6)
Time since HIV diagnosis (months), median (IQR) 25 (8–84)
CD4 at diagnosis of HIV (cel/uL), mean (± SD) 448± 298.17
HIV viral load at diagnosis of HIV (log), median (IQR) 4.61 (4.07–5.12)
CD4 nadir (cells/uL), mean (± SD) 367.93±233.85
CD4 nadir < 200 cells/uL, n (%), 95% CI 97 (23.9), (20–28.5)
CD4 cell count at baseline (cells/uL), mean (± SD) 689.64± 475.03
CD8 cell count at baseline (cells/uL), mean (± SD) 981.5±531.5
CD4 /CD8 ratio, mean (± SD) 0.77±0.70
HIV viral load at baseline (log), median (IQR) 0 (0–1.72)
Undetectable: < 50 HIV RNA copies/mL of plasma, n (%) 348 (85.9)
History of AIDS diagnosis, n (%), 95% CI 106 (26.2) (21.3–30.1)
HAART before inclusion, n (%), 95% CI 351 (86.7), (83.2–90)
Previous ART line, median (IQR) 1 (1–2)
Virological failure, n (%) 17 (4.8)
Median months of ART, median (IQR) 4 (16–56)
Chronic HCV infection, n (%) 14 (3.5)
Chronic HBV infection, n (%) 13 (3.2)
Smoker, pack/year, median (IQR) 1.5 (0–14)
Smoker, n (%), 95% CI 214 (52.8) (47.9–57.4)
Alcohol, SDU, median (IQR) 0 (0–4)
EX-IDU, n (%) 2 (0.5)
HCV, chronic infection by hepatitis C virus; HBV, chronic infection by hepatitis B virus; EX-IDU, ex-injecting drug users; STI: sexual transmitted infection; VL: viral load; IQR: interquartile range; SD, (standard deviation), SDU: standard drink unit.
10.1371/journal.pone.0245870.t002Table 2 Cytology, anoscopy, and HPV PCR results for the cohort.
Cohort of MSM-HIV patients
n = 405
Anal cytology, (n = 397), n (%), 95% CI
LSIL 190 (47.9), (43–52.7)
HSIL 13 (3.3), (1.8–5.1)
ASCUS 29 (7.3), (5.1–10.2)
Normal 165 (41.6), (37–46)
Anoscopy: Histology (n = 405), n (%), 95% CI
Normal 189 (46.7), (44.5–54)
LSIL 164 (40.4), (36.6–46)
HSIL 50 (12.3), (9.2–15.9)
ASCC 2 (0.5), (0–0.8)
n (%), 95% CI HPV PCR in anal mucosa
n = 394
HR-HPV 303 (76.9), (73–81.1)
LR-HPV 288 (73.1), (69–77)
HR and LR-HPV 229 (58.1), (53–63)
Median HR-HPV, IQR 1 (1–3)
Median LR-HPV, IQR 1 (0–2)
HPV 6 71 (18)
HPV 11 71 (18)
HPV 12 1 (0.3)
HPV 16 109 (27.7)
HPV 18 51 (12.9)
HPV 26 6 (1.5)
HPV 31 55 (14)
HPV 33 29 (7.4)
HPV 35 36 (9.1)
HPV 39 46 (11.7)
HPV-40 7 (1.8)
HPV-42 72 (18.3)
HPV-43 10 (2.5)
HPV 45 50 (12.7)
HPV 48 1 (0.3)
HPV 51 55 (14)
HPV 52 50 (12.7)
HPV 53 36 (9.1)
HPV 54 26 (6.6)
HPV 55 64 (16.2)
HPV 56 31 (7.9)
HPV 58 23 (5.8)
HPV 59 42 (10.7)
HPV 61 30 (7.6)
HPV 62 56 (14.2)
HPV 64 1 (0.3)
HPV 66 34 (8.6)
HPV 68 42 (10.7)
HPV 69 14 (3.6)
HPV 70 32 (8.1)
HPV 71 1 (0.3)
HPV 72 28 (7.1)
HPV 73 37 (9.4)
HPV 81 51 (12.9)
HPV 82 17 (4.3)
HPV 83 5 (1.3)
HPV 84 30 (7.6)
HPV 89 1 (0.3)
HPV 6108 13 (3.3)
HPV-AR subtype of HPV 18 (39,45,59,68) 164 (41.6)
HPV-AR subtype of HPV 16 (31,33,35,52,58,67) 207 (52.5)
LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance; ASCC, anal squamous cell cancer. HPV, human papillomavirus; HR-HPV: high-risk HPV, LR-HPV: low-risk HPV.
10.1371/journal.pone.0245870.t003Table 3 Risk factors associated with ≥HSILs in HIV+ MSM patients.
Bivariate and multivariate analysis.
≥HSIL NORMAL Bivariate Multivariate
N = 88 N = 317 p* OR 95% CI
Mean age (yrs), mean (± DS) 30.4 (± 7.6) 31.3 (± 8.3) 0.359 1.27 (0.71–2.29)
Retired, n (%) 6 (6.8) 16 (5) 0.594
Smoker, n (%) 50 (56.8) 164 (51.7) 0.398
Charlson Index, median (IQR) 0 (0–0) 0 (0–0) 0.178
Intercourse in previous 12 months, n (%) 78 (88.6) 286 (90.8) 0.545
qHPV Vaccine, n (%) 12 (13.6) 54(17) 0.445 1.72 (0.92–3.23)
Age at first sexual intercourse, (IQR) 18 (17–21) 18(16–20)
Genital/anal warts, n (%) 36(40.9) 92(29) 0.034
History of Syphilis, n (%) 24(27.3) 79(24.9) 0.654
HCV infection, n (%) 4 (4.5) 10 (3.2) 0.515
HBV infection, n (%) 2 (2.3) 11 (3.5) 0.742
Total NPS, baseline visit, median, (IQR) 50 (16–200) 50 (19.5–150) 0.543
NSP12m before last visit, median, (IQR) 1 (1–4) 1 (1–6.5) 0.078
Use of condom during study, n (%) 63 (71.6) 244 (76.9) 0.493
History of AIDS (A3, B3, C), n (%) 29 (33) 77(24.3) 0.102 2.37(1.009–5.58)
Time since HIV diagnosis (months), (IQR) 27 (9–83) 54(21–107) 0.386 1.002 (1.000–1.003)
CD4 nadir (cells/ul), mean (± SD) 366.8(±267.9) 368.2(±223.9) 0.959
CD4 nadir < 200 cells/uL, n (%) 24(27.6) 73(23.3) 0.412
Cd4 nadir <500 cells/uL, n (%) 39 (44.8) 161 (51.4) 0.275
Cd4 nadir >500 cells/uL, n (%) 24 (27.6) 77 (24.6) 0.571
CD4 cells/uL, mean (± SD) 674.3(± 347.3) 761.2(± 406.9) 0.069 0.99(0.99–1)
CD8 cells/uL, mean (± SD) 1017.3(± 474.1) 977.5(± 467.5) 0.487
CD4/CD8, mean (± SD) 0.75(± 0.43) 0.85(± 0.41) 0.042 0.53(0.2–1.38)
HIV VL (log), mean (± SD) 4.09 (± 3.35) 4.91(± 4.27) 0.042 1 (1–1)
ART during follow-up, n (%) 78 (88.6) 300 (94.6) 0.046 0.74 (0.22–2.56)
Median months of ART, median, (IQR) 0 (0–13) 24 (0–48) 0.003 0.99(0.99–1)
Virological failure, n (%) 1 (1.3) 4 (1.3) 1
N = 83 N = 291
Infection by Low-risk HPV genotype, n (%) 65 (78.3) 211 (72) 0.349
Infection by High-risk HPV genotype, n (%) 68 (83.9) 174 (59.8) 0.0001 2.72(0.87–8.54)
Infection by Low and High-risk HPV, n (%) 53 (63.9) 132 (45.4) 0.003 0.79 (0.25–2.46)
N° of HR-HPV genotypes, median (IQR) 2 (1–3) 1 (0–2) 0.0001 0.76 (0.58–1)
N° of LR-HPV genotypes, median (IQR) 1 (1–2) 1 (0–2) 0.082 0.83 (0.61–1.12)
Median months with VPH-AR (IQR) 11 (1–18) 1 (0–24) 0.07 0.98 (0.96–1.02)
Median months with VPH-BR (IQR) 8 (1–16) 11 (0–26) 0.487 1 (0.96–1.04)
Median months with mixed VPH infection (IQR) 1 (0–15) 1 (0–12) 0.102
HPV-6 21 (25.3) 47 (16.2) 0.057 1.61 (0.73–3.53)
HPV-11 21 (25.3) 37 (12.7) 0.005 3.81(1.76–8.24)
HPV-16 27 (32.5) 43 (14.8) 0.0001 2.69 (1.22–5.99)
HPV-18 16 (19.3) 26 (8.9) 0.008 2.73 (1.01–7.36)
HPV-31 10 (12) 29 (10) 0.584
HPV-33 4 (4.9) 17 (5.9) 0.733
HPV-35 6 (7.2) 18 (6.1) 0.781
HPV-39 9 (10.8) 24 (8.2) 0.462
HPV-42 12 (14.5) 53 (18.3) 0.419
HPV-45 9 (10.8) 31 (10.7) 0.961
HPV-51 12 (14.5) 26 (8.9) 0.142
HPV-52 10 (12) 40 (13.7) 0.689
HPV-53 12 (14.5) 13 (4.5) 0.001 2.97 (1.002–8.79)
HPV-54 6 (7.2) 24 (8.2) 0.763
HPV-55 11 (13.3) 50 (17.2) 0.393
HPV-59 11 (13.3) 19 (6.5) 0.047 1.8(0.66–4.83)
HPV-61 11 (13.3) 8 (2.7) 0.001 11.88 (3.67–38.53)
HPV-68 16 (19.3) 32 (11) 0.048 2.44(1.03–5.8)
HPV-70 8 (9.8) 24 (8.2) 0.667
HPV-81 10 (12) 56 (19.2) 0.129
P*: p-value
95% CI: 95% confidence interval
HIV+MSM, men who have sex with men living with HIV; LTI, Latent tuberculosis infection; HCV hepatitis C virus; HBV, hepatitis B virus; HPV, Human papillomavirus; EX-IDU, ex-injecting drug addict; VL, viral load. HR-HPV: high-risk HPV, LR-HPV: low-risk HPV; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance, NSPt, Total number of sexual partners; NSP12m: number of sexual partners in past 12 months
Factors associated with ≥HSIL
In the multivariate analysis, the presence of ≥HSILs was related to infection with HPV genotypes 11 (OR 3.81; CI95% 1.76–8.24), 16 (OR 2.69, 95%CI 1.22–5.99), 18 (OR 2.73, CI95% 1.01–7.36), 53 (OR 2.97, CI95% 1.002–8.79), 61 (OR 11.88, 95%CI 3.67–38.53) and 68 (OR 2.44, 95%CI 1.03–5.8), low CD4 nadir (OR1.002; CI95% 1–1.004), and history of AIDS (OR 2.37, 95%CI 1.009–5.58). The remaining results obtained are exhibited in Table 3.
HSIL patients and treatment outcomes
After excluding 11 patients who did not undergo two or more anoscopies, treatment outcomes were analyzed in a sample of 394 (87.2%) MSM, with a median follow-up of 36 months (IQR: 12–69), 1.215 patients-year. Table 4 exhibits the characteristic of the two HSIL treatment groups (imiquimod and surgery).
10.1371/journal.pone.0245870.t004Table 4 Baseline characteristics of HIV+MSM patients receiving imiquimod vs. surgery.
Imiquimod as first option Surgery as first option Bivariate
N = 32 N = 47 p*
Mean age (yrs), mean (± DS) 35.3.4 (± 11.48) 31.3 (± 8.3) 0.88
Retired, n (%) 2 (6.3) 3 (6.4) 1
Smoker, n (%) 19 (59.4) 29 (61.7) 0.68
Intercourse in previous 12 months, n(%) 28 (87.5) 47 (100) 0.72
qHPV Vaccine, n (%) 6 (18.7) 6 (12.8) 0.36
Age at first sexual intercourse, (IQR) 18 (16–21) 18(16–20) 0.53
Genital/anal warts, n (%) 12 (37.5) 23 (48.9) 0.53
History of Syphilis, n (%) 11(34.3) 13 (27.7) 0.33
HCV infection, n (%) 1 (3.1) 3 (6.4) 1
HBV infection, n (%) 0 (0) 2 (4.3) 0.53
Total NPS, baseline visit, median, (IQR) 55 (30–300) 36 (15–200) 0.14
NSP12m before last visit, median, (IQR) 2 (1–9) 1 (1–8) 0.55
Use of condom during study, n (%) 26 (81.3) 39 (82.9) 0.51
History of AIDS (A3, B3, C), n (%) 11 (34.4) 15 (31.9) 0.56
Time since HIV diagnosis (months), (IQR) 21 (7–111) 25 (9.5–64.3) 0.92
CD4 nadir (cells/ul), mean (± SD) 366.8(±267.9) 368.2(±223.9) 0.96
CD4 nadir < 200 cells/uL, n (%) 6(18.8) 15(31.9) 0.36
Cd4 nadir 200–500 cells/uL, n (%) 15 (46.9) 24 (51.1) 0.78
Cd4 nadir >500 cells/uL, n (%) 10 (31.3) 14 (29.8) 0.57
CD4 cells/uL, mean (± SD) 618.2(± 280.3) 675.7(± 334.1) 0.42
CD8 cells/uL, mean (± SD) 946.4(± 443.9) 1096(± 551.5) 0.17
CD4/CD8, mean (± SD) 0.78(± 0.46) 0.70(± 0.35) 0.42
HIV VL (log), mean (± SD) 3.9 (± 4.53) 3.55(± 3.98) 0.42
ART during follow-up, n (%) 28 (87.5) 45 (95.7) 0.6
Median months of ART, median, (IQR) 19.5 (6.5–44) 9 (2–64) 0.57
VL HIV < 50 copies/uL, n (%) 20(62.5) 39 (82.9) 0.28
Infection by Low-risk HPV genotype, n (%) 32 (100) 41 (87.2) 0.29
Infection by High-risk HPV genotype, n (%) 26 (81.3) 46 (97.8) 0.52
Infection by Low and High-risk HPV, n (%) 18 (56.3) 34 (72.3) 0.40
Sub-species HPV 18 (18, 39, 45, 59, 68) 16 (50) 29 (61.7) 0.61
Sub-species HPV 16 (16, 31, 33, 35, 52, 58, 67) 17 (53.1) 23 (48.9) 0.43
N° of HR-HPV genotypes, median (IQR) 2 (1–3.8) 2 (1–3) 0.28
N° of LR-HPV genotypes, median (IQR) 2 (0.3–3) 1 (1–2) 0.92
P*: p-value
95% CI: 95% confidence interval
HIV+MSM, men who have sex with men living with HIV; LTI, Latent tuberculosis infection; HCV hepatitis C virus; HBV, hepatitis B virus; HPV, Human papillomavirus; VL, viral load. HR-HPV: high-risk HPV, LR-HPV: low-risk HPV; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; ASC, atypical squamous cells of undetermined significance, NSPt, Total number of sexual partners; NSP12m: number of sexual partners in past 12 months.
Mucosectomy was performed in 47 patients with HSIL, with a median follow-up of 60 months (IQR: 46–73 months) and median disease-free period of 48 months (IQR: 28–60 months).
Forty-one (87.2%) of these patients received surgery alone, five (10.6%) received surgery plus self-administered imiquimod, due to failure of surgery in two cases and recurrence in three, and one patient underwent successful surgery after the failure of imiquimod treatment. Surgical margins were disease-free in 35 patients (76.9%) after first surgery. The response rate was 73.3% to surgery as first-line treatment (33/45) versus 96.8% to imiquimod (31/32) (p = 0.009) as evaluated by post-treatment HRA. A median of one surgical intervention was performed (IQR:1–1), with 31 patients undergoing one, 9 needing two, and 1 needing four interventions. Recurrence was recorded in 7 patients (15.2%), and repeat excision of the same lesion in 11 (23.4%). All surgical patients reported adverse effects, with a median duration of 15 days post-surgery (IQR: 7–21 days), including bleeding with defecation in 32 (68%), pain requiring anti-analgesics in 39 (82.9%), rectal incontinence in 1 (2.1%), and transient anal stenosis in 3 (6.4%) versus 1 in the imiquimod group (2.7%, p = 0.046). Among the 43 patients with follow-up HPV PCR results, clearance of oncogenic VPH genotypes was observed in 19 (44.2%).
Thirty-seven patients with HSIL self-administered 5% imiquimod three times/week; the treatment lasted 16 weeks in 97.3% of these patients and 18 weeks in 2.7%. It was first-line treatment in 32 patients (86.4%) and administered after previous surgery in 5 (13.5%); all patients showed a complete response, except for one case of failure caused by intolerance to imiquimod. Only one patient (2.7%) needed retreatment of the same lesion, whereas 11 (23.4%) of the surgical group required repeat surgery (p = 0.02). The median number of affected quadrants was 1 (IQR: 1–2). Mean follow-up was 48 months (IQR: 35–57 months) and mean disease-free period 36 months (IQR: 12–48). Imiquimod was discontinued in one patient (2.7%) for adverse effects (anal itching, stinging, and/or pain) attributed to non-compliance with the treatment protocol. Among the 35 (94.6%) patients treated with imiquimod for whom follow-up HPV PCR results were available, clearance of oncogenic VPH genotypes was observed in 10 (28.6%) (p = 0.065). No patient treated with surgery or imiquimod progressed to ASCC.
We found significant reductions in ≥HSIL cases between 2010 and 2018 (42.9% (9/21) vs. 4.1% (10/245) p = 0.034), between 2010 and 2013 (42.9% (9/21) vs. 13.8% (22/159), p = 0.003), and between 2013 and 2016 (13.8% (22/159) vs. 4.8% (13/273), p = 0.0001), followed by a stabilization between 2016 and 2018 (4.8% (13/273) vs. 4.1% (10/245), p = 0.617). Four deaths were recorded during the follow-up: one patient with hepatic cirrhosis secondary to chronic HCV infection in 2012, one with Burkitt lymphoma in 2013, one with small-cell lung cancer in 2014, and one with metastatic ASCC in 2015. Further data on outcomes were previously reported in detail [6].
Discussion
Since the first HIV epidemic, the incidence of ASCC has increased in seropositive patients, mainly in MSM with AIDS [17]. After the initiation of our screening/treatment program in 2010, the ≥HSIL rate significantly decreased among HIV-infected MSM patients for six years and then subsequently stabilized, with no progression to ASCC in patients with treated HSIL. Our results support the proposition by the authors of the Swiss Cohort study that the incidence of ASCC among people living with HIV can be markedly reduced if they all receive ART and can be further diminished if they also undergo annual screening with anal cytology or anoscopy [18]. A recent study of 592 HIV patients, with a mean follow-up of 69 months, reported that the risk of progression from HSIL (AIN3) to ASCC was high and that ASCC screening was the only factor that reduced this risk [19]. Data from the Study for the Prevention of Anal Cancer (SPANC) [20] are expected to elucidate the natural evolution of HPV infection, allowing a more effective classification of patients at risk of ASCC. In the meantime, a program to screen, diagnose, treat and follow up anal mucosal dysplastic lesions appears recommendable, especially in HIV+ MSM.
In this prospective study of HIV+ MSM undergoing a screening/treatment program for anal mucosa dysplasic lesions, the presence of ≥high-grade anal intraepithelial lesions were related to infection by HPV genotypes 11 16, 18, 53, 61 and 68, a low CD4 nadir and a history of AIDS. This finding of a relationship between HSIL-positivity and poor immunological status is consistent with previous observations that prolonged antiretroviral treatment [21–23] and a high CD4 count, regardless of CD4 nadir [24], are protective factors against HSIL. A recent prospective study observed a similar incidence of HPV-16 and -18 genotypes in the anal mucosa of French HIV+MSM, but HPV-16 was more persistent and therefore more closely correlated with the presence of HSIL [25]. In a retrospective study of “alpha-human papillomavirus” in the anal mucosa of German HIV+ patients, the presence of HSIL and simultaneous infection was associated with high- and low-risk genotypes [26]. Currently, patients with a new diagnosis of HIV in Spain are usually MSM, and the diagnosis is late in 47.6% of these cases [27], with a CD4 count <200 cells/uL. The above data suggest that screening for anal dysplasia is essential in this type of patient.
Self-administration of 5% imiquimod was a highly effective therapeutic strategy against HSIL in this series of HIV+MSM. Most of them did not need to repeat the topical treatment, whereas around a quarter of the patients undergoing excision required another intervention. Furthermore, the therapeutic failure rate and dropout for adverse effects were lower in the imiquimod group than in the surgery group. Various studies have supported the efficacy of imiquimod to treat HSIL in HIV+ patients [13, 28, 29]. Thus, a double-blind randomized placebo-controlled clinical trial comparing between self-application of imiquimod (n = 28) versus placebo (n = 25) in the anal canal three times/week for 4 months found a significant association (P = 0.003) between imiquimod and a positive outcome [13]. In addition, a prospective, observational open study in 44 HIV+ patients with HSIL observed a response rate of 66% (29/44) for imiquimod [28]. Finally, a retrospective observational study in 28 HIV+ and HIV- patients observed a higher frequency of total or partial responses in those receiving anal tampon treatment with a 15 mg versus 6.25 mg dose of imiquimod, with no difference in CD4, HIV viral load, or serostatus [29].
Further advantages of 5% imiquimod in comparison to ablative therapies include its self-administration and its usefulness in cases of extensive disease. The surgical option was also effective in a large proportion of our patients, although some needed retreatment due to recurrence or incomplete excision. No cases of permanent stenosis or fecal incontinence were observed in the surgical group; however, surgery is not currently recommended due to its adverse effects, especially in patients with large lesions [30].
Limitations of this single-center study include its observational design, comparing the real-life clinical effectiveness of imiquimod and surgery rather than their efficacy (as in a clinical trial). In addition, it only included HIV+MSM, and these data cannot be extrapolated to other types of patient. Finally, 11 of the 405 enrolled patients did not undergo two or more anoscopies and were therefore lost to the follow-up. However, its strengths include the prospective design and long follow-up period, which was a mean of 36 months. In fact, the present cohort of HIV+ patients is one of the few published to date that was created to measure predetermined objectives.
In conclusion, HSIL screening and treatment programs reduce the incidence of this precursor of ASCC. Chronic mixed HPV infection and a history of poor immunological status are associated with the presence of HSILs. Self-administration of 5% imiquimod is more effective than surgery as first-line treatment of anal HSIL in HIV+MSM patients, with a lower recurrence rate and fewer adverse effects.
Supporting information
S1 File HIV MSM cohort database.
(SAV)
Click here for additional data file.
The authors are grateful to Mercedes Álvarez Romero for coordinating patients and drawing blood samples and to Marina Gutiérrez and Rodrigo López of the Pathology Department for processing samples. The authors are grateful to the participating patients. | UNK, 3/WEEK | DrugDosageText | CC BY | 33534790 | 18,913,048 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Peptic ulcer haemorrhage'. | Long-Term Tacrolimus Blood Trough Level and Patient Survival in Adult Liver Transplantation.
Tacrolimus is the most widely used immunosuppressant in liver transplant (LT) patients. However, the ideal long-term target level for these patients is unknown. This retrospective study aimed to investigate the impact of tacrolimus blood concentration five years after LT on long-term patient survival outcomes in adult LT recipients. Patients who underwent LT between January 2004 and July 2014 at a tertiary medical center were included in this study (n = 189). The mean tacrolimus blood concentrations of each patient during the fifth year after LT were recorded and the overall survival rate was determined. A multivariate analysis of factors associated with long-term survival was conducted using a Cox's model. The median follow-up period was 9.63 years, and 144 patients (76.2%) underwent live donor LT. Sixteen patients died within 5 years of LT. In the Cox's model, patients with a mean tacrolimus blood trough level of 4.6-10.2 ng/mL had significantly better long-term survival than those with a mean tacrolimus blood trough level outside this range (estimated hazard ratio = 4.76; 95% confidence interval: 1.34-16.9, p = 0.016). Therefore, a tacrolimus level no lower than 4.6 ng/mL would be recommended in adult LT patients.
1. Introduction
Liver transplantation (LT) has become a mature treatment of end-stage liver disease in clinical practice [1]. The continuous improvement of effective immunosuppression treatments has led to a significant improvement in patient and graft survival in recent years [2]. Calcineurin inhibitors are the cornerstone of immunosuppression in LT [3], and tacrolimus is currently the mainstay of initial and maintenance immunosuppression therapies [4,5,6]. Tacrolimus reduces the incidence and severity of early and late T-cell mediated rejection by inhibiting T-cell production of interleukin-2 [5,6]. However, the long-term use of immunosuppressants leads to an increasing burden of toxicity. The reported toxic effects of calcineurin inhibitors include infections, chronic renal insufficiency, metabolic diseases (such as hyperlipidemia, hypertension, and diabetes mellitus), and malignancy [7,8,9], which, along with cardiovascular diseases, have been reported to be the major causes of morbidity and mortality after LT [3,10]. However, most LT patients cannot withdraw from lifelong immunosuppression therapy; the only exceptions are a few selected patients participating in experimental trials [11,12].
It is a clinical challenge to design a well-balanced immunosuppressive regimen for LT recipients. Tacrolimus has a narrow therapeutic dose range and its blood levels should be carefully monitored. The Advagraf (tacrolimus prolonged-release hard capsules) recommendation for adult LT patients is a blood trough level of 5–20 ng/mL in the early post-transplant period and 5–15 ng/mL during subsequent maintenance therapy. Nevertheless, the current recommendations from the clinical practice guidelines for the ideal tacrolimus level in adults after LT remain controversial. The American Association for the Study of Liver Diseases (AASLD) recommends a target blood trough level of 5–10 ng/mL for tacrolimus three months after LT [13]. The Consensus on Managing Modifiable Risk in Transplantation Group (COMMIT) recommends that the target tacrolimus blood trough levels be 6–10 ng/mL during the first month after LT and decrease to 4–8 ng/mL thereafter, except when used in combination with mammalian target of rapamycin (mTOR) inhibitors [14]. Moreover, the International Liver Transplant Society (ILTS) consensus statement on immunosuppression in LT recipients recommends the target blood trough levels of tacrolimus be 6–10 ng/mL three months after LT, lower than 5 ng/mL 12 months after LT, and decrease to 3 ng/mL thereafter, resulting in a blood trough level just above the lower limit of detection five years after LT [15]. However, the impact of the long-term tacrolimus blood trough level on the outcomes of the LT recipients remains unclear. This study aimed to suggest an appropriate tacrolimus blood trough level for adult patients five years after LT.
2. Materials and Methods
This study was approved by the Institutional Review Board of National Taiwan University Hospital and was conducted according to the Declaration of Helsinki. A total of 286 patients who underwent LT at 18 years or older at a tertiary medical center from January 2004 to July 2014 were recruited for this study. Patients who died within 5 years of LT, were lost to follow-up, or did not use tacrolimus-based calcineurin inhibitor for immunosuppressant therapy were excluded from this study (Figure 1). In addition, those who received mTOR inhibitor treatment were excluded, as mTOR inhibitors are typically used as a combination therapy to reduce the required dose of tacrolimus. The final analysis included 189 patients. All eligible patients were followed up for more than five years until August 2019. Patients who underwent a LT due to liver cancer met the Milan criteria (before 2006) or the criteria of the University of California, San Francisco (since 2006) at the time of LT. All patients received regular monthly or bi-monthly follow-ups at the outpatient clinic after LT. Routine blood examinations for the tacrolimus blood trough level, liver function, and renal function were conducted at each visit, and abdominal sonography was performed every 6 to 12 months.
The patients’ medical records were reviewed retrospectively to extract demographic and clinical data, including patient characteristics, laboratory tests, and survival outcomes. The serum bilirubin and creatinine data at the end of the fifth year after LT were used in this study. The tacrolimus level used in this study was the mean of the values obtained at the three follow-up visits during the fifth year after LT.
The immunosuppression protocol for adult LT patients consisted of tacrolimus, mycophenolate mofetil, and steroids. Tacrolimus was administered orally beginning on the first day after LT, and the dose was adjusted to achieve the desired therapeutic drug level. Basiliximab was administered immediately before graft reperfusion and on the fourth day after LT for induction therapy. A 500-mg intravenous bolus of methylprednisolone was administered immediately before reperfusion of the liver graft and was tapered to oral prednisolone over one week and reduced to withdrawal after six months.
All statistical analyses were performed using R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p ≤ 0.05. Continuous variables are presented as mean ± standard deviation (SD) and median (interquartile range, IQR), and categorical variables are presented as frequency (percentage, %). The survival curve was estimated by the Kaplan-Meier method. A univariate analysis was conducted to examine the differences in the distributions of continuous variables, categorical variables, and survival outcomes between the surviving and deceased liver recipients five years after LT using the Wilcoxon rank-sum test, Chi-square test, Fisher’s exact test, or log-rank test as appropriate for the data type. A multivariate analysis was performed to estimate the adjusted effects of risk factors or prognostic factors on the survival outcome using a multiple Cox’s proportional hazards model.
To ensure a good quality of regression analysis, the model-fitting techniques for variable selection, goodness-of-fit (GOF) assessment, and regression diagnostics and remedies were used in our regression analysis. Specifically, the stepwise variable selection procedure (with iterations between the forward and backward steps) was applied to obtain the best final regression model using the My.stepwise package of the R software [16]. All significant and non-significant relevant covariates from the univariate analysis (listed in Table 1) and some of the interaction terms were used in the multivariate analysis. The significance levels for entry and for stay were set to 0.15 for being conservative. With the aid of substantive knowledge, the best candidate final regression model was identified manually by dropping the covariates with p value > 0.05 one at a time until all regression coefficients were significantly different from 0. To assess the GOF of the fitted Cox’s model, the concordance and adjusted generalized R2 [17] were examined. A concordance ≥ 0.7 and an adjusted generalized R2 > 0.15 indicated an acceptable level of discrimination, power, and fitness.
Moreover, the smoothing option “pspline” (for the smoothing splines using a “p-spline” basis) was specified inside the coxph function of the survival package to smooth the effects of continuous covariates on the log-hazard rate of the simple and multiple Cox’s proportional hazards models in R. Then, the termplot function of the stats package was used to plot the smoothed effects of the continuous covariates on the log-hazard rate in R [18]. The regression diagnostics for the verification of proportional hazards assumption, residual analysis, detection of influential cases, and a multicollinearity check were applied to discover any model or data problems. A variance inflating factor (VIF) ≥ 10 in continuous covariates or VIF ≥ 2.5 in categorical covariates indicated the occurrence of the multicollinearity problem among some of the covariates in the fitted regression model.
3. Results
3.1. Patients’ Demographic and Clinical Characteristics
The median follow-up duration was 9.63 years (IQR: 7.2–11.4 years), and the mean follow-up was 9.58 ± 2.74 years. A total of 121 males (64.0%) and 68 females (36.0%) were included in this study (Table 1). The median age at LT was 54.26 years (IQR: 48.63–58.58 years, range: 18.3–73.1 years), and the mean age at LT was 52.7 ± 9.6 years. Among the 189 patients, 114 (76.2%) underwent living donor transplants and 45 (23.8%) underwent deceased donor transplants. The main indications for LT were hepatitis B virus (HBV) cirrhosis (52.9%), hepatocellular carcinoma (38.1%), hepatitis C virus (HCV) cirrhosis (22.2%), and fulminant hepatitis (12.2%). The median of the mean tacrolimus blood trough level during the fifth year after LT was 5.0 ng/mL (IQR: 4.12–6.33 ng/mL), and the mean tacrolimus blood trough level was 5.249 ± 1.71 ng/mL. The mean tacrolimus blood trough level was ≥ 5 ng/mL in 96 patients (50.8%) and < 5 ng/mL in 93 patients (49.2%), including 44 patients (23.3%) with a mean tacrolimus blood trough level < 4 ng/mL and 11 patients (5.8%) with a mean tacrolimus blood trough level < 3 ng/mL. No significant differences in gender, age at LT, body weight, blood type, graft type, etiology of LT, or serum total bilirubin were found between the deceased and surviving LT recipients (p > 0.05). However, serum creatinine levels > 1.5 mg/dL (23/173 vs. 8/16, p = 0.001), end-stage renal disease (2/173 vs. 3/16, p = 0.005), and mean tacrolimus blood trough levels < 4 ng/mL (36/173 vs. 8/16, p = 0.014) were significantly more common among deceased LT recipients compared to survivors. The causes of death included malignancies (n = 5), graft failures (n = 4), infection (n = 4), cerebrovascular accidents (n = 2), and duodenal ulcer bleeding (n = 1) (Table 2). The distribution of the tacrolimus trough level stratified by the etiology and cause of death among the 16 dead patients was shown in the box plot of Figure 2. There was no statistical significance in the tacrolimus trough level between the different causes of death (p = 0.3823).
3.2. Predictors of Patients’ Long-Term Survival
The Cox’s model fitted to the survival data for the multivariate analyses of the time to death after five years of LT is shown in Table 3. After adjusting for the effects of the other covariates, age at LT ≤ 27.011 years (estimated hazard ratio [HR] = 168.79, 95% confidence interval [C.I.]: 11.13–2559.51), pre-transplant autoimmune liver disease (HR = 8.12, 95% C.I.: 1.97–33.43), pre-transplant HCV infection × survival time in years (HR = 1.34, 95% C.I.: 1.12–1.60), serum creatinine level > 1.311 mg/dL × serum total bilirubin level > 1.411 mg/dL (HR = 921.69, 95% C.I.: 43.40–19,573.71), serum creatinine level > 1.311 mg/dL × serum total bilirubin level ≤ 0.792 mg/dL (HR = 105.68, 95% C.I.: 7.81–1430.79), serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level > 0.882 mg/dL (HR = 30.49, 95% C.I.: 2.98–312.34), and the mean tacrolimus trough level during the fifth year after LT ≤ 4.609 ng/mL or > 10.168 ng/mL (HR = 4.76, 95% C.I.: 1.34–16.94) were associated with a higher long-term mortality five years after LT. The time-dependent interaction term, HCV × survival time in years, was added to the Cox’s model to account for the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.292, indicates that the risk of mortality in patients with HCV increases with time five years post-LT. Moreover, the three second-order interaction terms between the serum creatinine and total bilirubin levels were compared to the other two possible combinations, serum creatinine level > 1.311 mg/dL × (0.792 mg/dL < serum total bilirubin level ≤ 1.411 mg/dL) and serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level ≤ 0.882 mg/dL, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and”.
All cut-off values of the continuous covariates (such as age at LT) were estimated by applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models. As shown in Figure 3, the optimal cut-off values of the mean tacrolimus trough level, 4.609 ng/mL and 10.168 ng/mL, were estimated directly in the “p-spline plot,” which allowed the visualization of the nonlinear effect of the averaged dosage of the tacrolimus-based immunosuppressant during the fifth year after LT on log(λ), where λ was the hazard rate of time to death five years after LT. Then, the Kaplan-Meier estimates of survival curves for time to death five years after LT were determined for the 66 patients with mean tacrolimus trough levels ≤ 4.609 ng/mL or > 10.168 ng/mL and the 123 patients with mean tacrolimus trough levels between 4.609 ng/mL and 10.168 ng/mL (log-rank test, p = 0.009) (Figure 4). In an additional subgroup analysis, we found that a mean tacrolimus trough level between 4.431 ng/mL and 6.332 ng/mL for patients with a serum creatinine level > 1.311 mg/dL improved survival. Finally, this Cox’s model had a concordance of 0.904 and an adjusted generalized R2 of 0.388, indicating that it fit the survival data very well.
4. Discussion
This is the first study to report an association between long-term tacrolimus blood trough level and long-term patient survival in adult LT recipients. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL in adult LT recipients is associated with a lower mortality rate. In patients with a serum creatinine > 1.3 mg/dL, a mean tacrolimus blood trough level of 4.4–6.3 ng/mL is associated with a lower mortality rate.
Decreasing tacrolimus use during long-term follow-up in LT recipients is a general concept in clinical practice. In this study, only one patient had the mean level > 10 ng/mL and a few patients had the mean level > 8 ng/mL (Figure 3). Patients with such high drug levels were most likely because of their poor compliance (not must because of they have truly such high trough level). Poor compliance indicates that a patient takes incorrect dosage of immunosuppressant that their physician prescribed, or takes immunosuppressant or blood exam at the wrong time, either of which might clearly be important factors that influence graft and survival outcome of LT patients during long-term follow-ups. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL is associated with lower mortality rate; however, the upper limit of 10.2 ng/mL suggested by the regression analysis was merely a value for caution, but not a recommended upper limit in real clinical practice. The long-term hazard of high level of tacrolimus is already widely known; therefore, we would focus more on the findings that it appears to increase the survival risks in LT recipients if their tacrolimus levels are maintained below 4.6 ng/mL during the long-term follow-ups.
Inadequate immunosuppression is associated with higher risks of graft rejection [19], while excess immunosuppression increases the risks of malignancy and infection and increases the adverse effects of drug toxicity. The tailored use of immunosuppressants should be considered based on the patient’s risks of graft rejection and infection and the patient’s medical comorbidities and liver disease status prior to LT [14]. Patients with autoimmune liver diseases may require more immunosuppression to prevent disease recurrence and graft rejection [20]. By contrast, lower doses of immunosuppression are recommended in patients who have undergone LT due to HCV, as high levels of immunosuppression are related to increased viral replication [21,22]. Rejection results in abnormal liver function and is one of the most important factors associated with poor long-term graft and patient outcomes [23,24,25]. Tacrolimus reduces the risk of T-cell mediated rejection to protect graft function and is the cornerstone of a successful LT. However, short and long-term adverse effects of tacrolimus such as infection, chronic renal insufficiency, metabolic diseases (hyperlipidemia, hypertension, and diabetes mellitus), and malignancy have been widely reported and influence patients’’ long-term outcomes [7,8,9]. The early causes of death after LT within one year are infection and graft loss, and the late causes of death three years post-LT are malignancy, cardiovascular disease, and renal failure [10]. As graft function typically stabilizes, factors associated with the long-term outcome are often patient-related factors (such as chronic medical diseases) that are usually associated with tacrolimus.
Previous studies have reported that minimizing tacrolimus use in the early post-LT period is associated with a lower risk of new-onset diabetes mellitus [26], a lower incidence of hyperlipidemia [27], and better long-term survival [19,28]. In recent years, studies have focused on the reduction or complete withdrawal of long-term immunosuppressants in LT recipients [12,29,30,31,32,33,34]. One study reported satisfactory outcomes with the combined use of tacrolimus and mycophenolate mofetil, which allowed for the tacrolimus dose to be reduced [35]. Other studies demonstrated that the concomitant use of everolimus may reduce the required dose of tacrolimus while having potential renal benefits [29,36]. However, only some LT recipients were able to discontinue the use of immunosuppressants, and these patients more frequently experienced biopsy-proven acute rejection [29]. The risk of chronic rejection during long-term follow-up remains in patients with inadequate immunosuppression [30,33]. The tacrolimus blood concentration in LT recipients may play a key role in long-term outcomes, as it is associated with long-term graft function and its adverse effects are related to several chronic medical diseases. However, to the best of our knowledge, this is the first report regarding the effect of long-term tacrolimus levels on the long-term survival outcomes of LT recipients.
Several factors are associated with the long-term outcomes of adult LT recipients, including pre-transplant primary sclerosing cholangitis, immunosuppression therapies, acute and chronic rejections, malignancy, and metabolic syndrome [37,38]. As listed in Table 3, we found several independent risk factors of long-term mortality in this study. Only four patients aged <27 years at the time of LT were included in this study, and one died due to pneumonia 6.3 years after LT. Patients who underwent LT due to autoimmune liver diseases had worse outcomes, as they were more likely to experience acute rejections [39] and suffer disease recurrence, leading to graft loss. Patients who underwent LT due to HCV infections had relatively poor post-LT outcomes due to the disease recurrence followed by graft dysfunction and failure [40]. As direct-acting antiviral agents have advanced [41], the survival outcomes of HCV patients should improve in the near future. Long-term renal and liver functions are reflected by serum creatinine and total bilirubin levels, respectively. We found that abnormal serum creatinine levels have a bigger impact than abnormal serum total bilirubin levels on the survival outcomes of LT recipients. The graft function of adult LT recipients is chronically stable five years after LT. While some patients may experience asymptomatic hyperbilirubinemia at this time, the long-term outcomes remain favorable. By contrast, the gradual deterioration of renal function may lead to chronic kidney disease or end-stage renal disease, affecting the long-term survival, especially in patients with long-term use of tacrolimus.
This study had some limitations. First, the data were obtained from a single medical center in Asia, which provided a relatively small number of eligible patients with single ethnicity and fewer death events. Second, the study spanned a long time period, and therefore the improvements in surgical and medical expertise and advances in immunosuppression therapies may have influenced the patient outcomes. Third, we did not collect or analyze the time-dependent tacrolimus blood trough level during the follow-up visits five years after LT.
In summary, we found an association between the long-term tacrolimus blood trough levels and the long-term survival five years after LT. A mean tacrolimus blood trough level outside the range of 4.6–10.2 ng/mL appeared to be an independent risk factor for long-term mortality. Further studies with larger sample sizes are needed to verify these results and to further identify an appropriate tacrolimus blood trough level for maintenance use.
Acknowledgments
We thank the coordinators (Hui-Ying Lin and Min-Heuy Lin) for their helpful efforts in data collection.
Author Contributions
C.-Y.H. drafted the manuscript and R.-H.H. designed the study. C.-Y.H., M.-C.H., and Y.-M.W. conducted data processing, and C.-Y.H. and C.-M.H. performed data analysis. P.-H.L. and R.-H.H. were the directors responsible for general organization and instruction. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of National Taiwan University Hospital.
Informed Consent Statement
Patient consent was waived due to retrospective design of the study.
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1 Patient flow diagram.
Figure 2 The box plot of tacrolimus mean trough level stratified by the etiology and cause of death among the 16 dead patients. There was no statistical difference of tacrolimus level between the different causes of death (p = 0.3823).
Figure 3 The p-spline plot for the nonlinear effect of the long-term tacrolimus blood trough levels on time to death. The tacrolimus blood trough level is shown on the X-axis, and the log(λ), where λ is the hazard rate of time to death five years after liver transplantation, is shown on the Y-axis. In this p-spline plot, the intersection between the horizontal green line (Y = 0) and the red curve yields the estimated optimal cut-off values for long-term tacrolimus blood trough levels at which the values of log(λ) will not change (4.609 ng/mL and 10.168 ng/mL). When the level is ≤4.609 ng/mL or >10.168 ng/mL, the value of log(λ) increases, indicating an increasing λ. When the level is >4.609 ng/mL and ≤10.168 ng/mL, the value of log(λ) decreases, indicating a decreasing λ. The vertical bars above the X-axis represent the patients’ actual mean tacrolimus blood trough levels obtained at the three follow-up visits during the fifth year after LT.
Figure 4 The Kaplan-Meier curve for time to death five years after liver transplantation. The survival curve of the 66 patients with a mean tacrolimus blood trough level ≤ 4.609 ng/mL or >10.168 ng/mL is shown in red while the survival curve of the 123 patients with a mean tacrolimus blood trough level > 4.609 ng/mL or ≤10.168 ng/mL is shown in green.
jpm-11-00090-t001_Table 1 Table 1 Univariate analysis for comparing the distributions of the demographic and clinical characteristics between the alive and dead adult liver recipients after five years of liver transplantations.
Variable All Patients
(n = 189) Alive
(n = 173) Dead
(n = 16) p Value
Gender 0.7899
Male 121 (64.0) 110 (90.9) 11 (9.1)
Female 68 (36.0) 63 (92.6) 5 (7.4)
Age at LT (years) 52.7 ± 9.6 52.6 ± 9.5 53.4 ± 10.7 0.6672
Body weight at LT (kg) 64.9 ± 12.2 65.0 ± 12.3 63.7 ± 12.1 0.4459
Blood type 0.8719
O 79 (41.8) 73 (92.4) 6 (7.6)
A 51 (27.0) 46 (90.2) 5 (9.8)
B 42 (22.2) 39 (92.9) 3 (7.1)
AB 17 (9.0) 15 (88.2) 2 (11.8)
Graft type 1.0000
Living donor 144 (76.2) 132 (91.7) 12 (8.3)
Deceased donor 45 (23.8) 41 (91.1) 4 (8.9)
Etiology for LT
Liver malignancy (HCC) 72 (38.1) 66 (91.7) 6 (8.3) 1.0000
Alcoholic cirrhosis 17 (9.0) 16 (94.1) 1 (5.9) 1.0000
HBV cirrhosis 100 (52.9) 92 (92.0) 8 (8.0) 1.0000
HCV cirrhosis 42 (22.2) 38 (90.5) 4 (9.5) 0.7577
Fulminant hepatitis 23 (12.2) 23 (100) 0 (0) 0.2259
Autoimmune disease 14 (7.4) 11 (78.6) 3 (21.4) 0.1017
Biliary atresia 5 (2.6) 5 (100) 0 (0) 1.0000
Other 10 (5.3) 9 (90) 1 (10) 0.5964
Total bilirubin (mg/dL) 0.973 ± 0.51 0.958 ± 1.53 1.133 ± 0.69 0.6260
Total bilirubin > 1 mg/dL 60 (31.7) 53 (88.3) 7 (11.7) 0.2770
Total bilirubin > 2 mg/dL 11 (5.8) 9 (81.8) 2 (18.2) 0.2360
Creatinine (mg/dL) 1.391 ± 1.25 1.279 ± 0.96 2.6 ± 2.74 0.0201 *
Creatinine > 1.5 mg/dL 31 (16.4) 23 (74.2) 8 (25.8) 0.0010 *
ESRD 5 (2.6) 2 (40.0) 3 (60.0) 0.0050 *
Tacrolimus mean level (ng/mL) 5.249 ± 1.71 5.263 ± 1.53 5.096 ± 3.12 0.9787
Tacrolimus level < 5 ng/mL 93 (49.2) 82 (88.2) 11 (11.8) 0.1216
Tacrolimus level < 4 ng/mL 44 (23.3) 36 (81.8) 8 (18.2) 0.0136 *
Tacrolimus level < 3 ng/mL 11 (5.8) 9 (81.8) 2 (18.2) 0.2356
Data are presented as mean ± standard deviation (SD) for continuous variables and frequency (percentage, %) for categorical variables. The p-values of statistical tests were calculated using the Wilcoxon rank-sum test for continuous variables and the Fisher’s exact test for categorical variables. * p value ≤ 0.05. Abbreviations: LT, liver transplantation; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; Total bilirubin, serum total bilirubin level; Creatinine, serum creatinine level; and ESRD, end-stage renal disease (defined by receiving hemodialysis regularly).
jpm-11-00090-t002_Table 2 Table 2 The causes of 16 deaths since 5 years after adult liver transplantations.
Causes of Deaths Number of Subjects
Malignancy 5 (31.25%)
De novo: Multiple myeloma, bladder cancer, colon cancer, prostate cancer 4
Recurrent: Hepatocellular carcinoma 1
Graft failure 4 (25.00%)
Chronic rejection 3
Autoimmune hepatitis 1
Infection 4 (25.00%)
Pneumonia 3
Urinary tract infection 1
Cerebral vascular event (intracerebral hemorrhage) 2 (12.50%)
Peptic ulcer bleeding 1 (6.25%)
jpm-11-00090-t003_Table 3 Table 3 Multivariate analysis for identifying the predictors of long-term overall survival after 5 years of liver transplantations by fitting a multiple Cox’s Model in the adult liver transplant recipients 1.
Covariate 2 Estimate Standard
Error Wald’s
z Test p Value Hazard
Ratio (HR) 95% Confidence
Interval (C.I.)
Age at LT ≤ 27.011 years 5.1286 1.3872 3.6970 0.0002 168.7851 11.130–2559.512
Autoimmune (including PBC) 2.0946 0.7219 2.9015 0.0037 8.1221 1.973–33.431
HCV × Overall survival years 0.2924 0.0914 3.1978 0.0014 1.3397 1.120–1.603
Cre > 1.311 × T-Bil > 1.411 mg/dL 6.8262 1.5591 4.3784 <0.0001 921.6940 43.401–19,573.712
Cre > 1.311 × T-Bil ≤ 0.792 mg/dL 4.6604 1.3294 3.5056 0.0005 105.6778 7.805–1430.790
Cre ≤ 1.311 × T-Bil > 0.882 mg/dL 3.4174 1.1871 2.8788 0.0040 30.4913 2.977–312.341
Tacrolimus mean ≤ 4.609 or > 10.168 ng/mL 1.5599 0.6479 2.4076 0.0161 4.7581 1.336–16.940
1 The above multiple Cox’s model was fitted to the 189 adult patients who underwent liver transplantations with 16 death events, for modeling the hazard rate of the right-censored overall survival time five years after liver transplantations. All the cut-off values of the continuous covariates (e.g., age at liver transplantations) were estimated by choosing the option of applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models (e.g., Figure 3). The time-dependent interaction term, HCV × Overall survival year, was added to the Cox’s model for handling the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.2924, indicated that the risk of dying in the patients with HCV would increase as time elapsed five years after liver transplantations. Moreover, the three second-order interaction terms, Cre > 1.311 × T-Bil > 1.411, Cre > 1.311 × T-Bil ≤ 0.792, and Cre ≤ 1.311 × T-Bil > 0.882, were compared to the other two possible combinations, Cre > 1.311 × (0.792 < T-Bil ≤ 1.411) and Cre ≤ 1.311 × T-Bil ≤ 0.882, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and.” Finally, both goodness-of-fit (GOF) measures, concordance = 0.9041 (se = 0.0265) > 0.7 and adjusted generalized R2 = 0.3878 > 0.15, indicated an excellent fit of this multiple Cox’s model to the observed survival data. 2 Abbreviations: LT, liver transplantation; PBC, primary biliary cirrhosis; HCV, hepatitis C viral infection; Cre, serum creatinine level (mg/dL); T-Bil, serum total bilirubin level (mg/dL); and Tacrolimus mean, the averaged dosage of the tacrolimus-based immunosuppressant (ng/mL).
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. | BASILIXIMAB, METHYLPREDNISOLONE, MYCOPHENOLATE MOFETIL, PREDNISOLONE, TACROLIMUS | DrugsGivenReaction | CC BY | 33535628 | 18,997,175 | 2021-02-01 |
What was the administration route of drug 'METHYLPREDNISOLONE'? | Long-Term Tacrolimus Blood Trough Level and Patient Survival in Adult Liver Transplantation.
Tacrolimus is the most widely used immunosuppressant in liver transplant (LT) patients. However, the ideal long-term target level for these patients is unknown. This retrospective study aimed to investigate the impact of tacrolimus blood concentration five years after LT on long-term patient survival outcomes in adult LT recipients. Patients who underwent LT between January 2004 and July 2014 at a tertiary medical center were included in this study (n = 189). The mean tacrolimus blood concentrations of each patient during the fifth year after LT were recorded and the overall survival rate was determined. A multivariate analysis of factors associated with long-term survival was conducted using a Cox's model. The median follow-up period was 9.63 years, and 144 patients (76.2%) underwent live donor LT. Sixteen patients died within 5 years of LT. In the Cox's model, patients with a mean tacrolimus blood trough level of 4.6-10.2 ng/mL had significantly better long-term survival than those with a mean tacrolimus blood trough level outside this range (estimated hazard ratio = 4.76; 95% confidence interval: 1.34-16.9, p = 0.016). Therefore, a tacrolimus level no lower than 4.6 ng/mL would be recommended in adult LT patients.
1. Introduction
Liver transplantation (LT) has become a mature treatment of end-stage liver disease in clinical practice [1]. The continuous improvement of effective immunosuppression treatments has led to a significant improvement in patient and graft survival in recent years [2]. Calcineurin inhibitors are the cornerstone of immunosuppression in LT [3], and tacrolimus is currently the mainstay of initial and maintenance immunosuppression therapies [4,5,6]. Tacrolimus reduces the incidence and severity of early and late T-cell mediated rejection by inhibiting T-cell production of interleukin-2 [5,6]. However, the long-term use of immunosuppressants leads to an increasing burden of toxicity. The reported toxic effects of calcineurin inhibitors include infections, chronic renal insufficiency, metabolic diseases (such as hyperlipidemia, hypertension, and diabetes mellitus), and malignancy [7,8,9], which, along with cardiovascular diseases, have been reported to be the major causes of morbidity and mortality after LT [3,10]. However, most LT patients cannot withdraw from lifelong immunosuppression therapy; the only exceptions are a few selected patients participating in experimental trials [11,12].
It is a clinical challenge to design a well-balanced immunosuppressive regimen for LT recipients. Tacrolimus has a narrow therapeutic dose range and its blood levels should be carefully monitored. The Advagraf (tacrolimus prolonged-release hard capsules) recommendation for adult LT patients is a blood trough level of 5–20 ng/mL in the early post-transplant period and 5–15 ng/mL during subsequent maintenance therapy. Nevertheless, the current recommendations from the clinical practice guidelines for the ideal tacrolimus level in adults after LT remain controversial. The American Association for the Study of Liver Diseases (AASLD) recommends a target blood trough level of 5–10 ng/mL for tacrolimus three months after LT [13]. The Consensus on Managing Modifiable Risk in Transplantation Group (COMMIT) recommends that the target tacrolimus blood trough levels be 6–10 ng/mL during the first month after LT and decrease to 4–8 ng/mL thereafter, except when used in combination with mammalian target of rapamycin (mTOR) inhibitors [14]. Moreover, the International Liver Transplant Society (ILTS) consensus statement on immunosuppression in LT recipients recommends the target blood trough levels of tacrolimus be 6–10 ng/mL three months after LT, lower than 5 ng/mL 12 months after LT, and decrease to 3 ng/mL thereafter, resulting in a blood trough level just above the lower limit of detection five years after LT [15]. However, the impact of the long-term tacrolimus blood trough level on the outcomes of the LT recipients remains unclear. This study aimed to suggest an appropriate tacrolimus blood trough level for adult patients five years after LT.
2. Materials and Methods
This study was approved by the Institutional Review Board of National Taiwan University Hospital and was conducted according to the Declaration of Helsinki. A total of 286 patients who underwent LT at 18 years or older at a tertiary medical center from January 2004 to July 2014 were recruited for this study. Patients who died within 5 years of LT, were lost to follow-up, or did not use tacrolimus-based calcineurin inhibitor for immunosuppressant therapy were excluded from this study (Figure 1). In addition, those who received mTOR inhibitor treatment were excluded, as mTOR inhibitors are typically used as a combination therapy to reduce the required dose of tacrolimus. The final analysis included 189 patients. All eligible patients were followed up for more than five years until August 2019. Patients who underwent a LT due to liver cancer met the Milan criteria (before 2006) or the criteria of the University of California, San Francisco (since 2006) at the time of LT. All patients received regular monthly or bi-monthly follow-ups at the outpatient clinic after LT. Routine blood examinations for the tacrolimus blood trough level, liver function, and renal function were conducted at each visit, and abdominal sonography was performed every 6 to 12 months.
The patients’ medical records were reviewed retrospectively to extract demographic and clinical data, including patient characteristics, laboratory tests, and survival outcomes. The serum bilirubin and creatinine data at the end of the fifth year after LT were used in this study. The tacrolimus level used in this study was the mean of the values obtained at the three follow-up visits during the fifth year after LT.
The immunosuppression protocol for adult LT patients consisted of tacrolimus, mycophenolate mofetil, and steroids. Tacrolimus was administered orally beginning on the first day after LT, and the dose was adjusted to achieve the desired therapeutic drug level. Basiliximab was administered immediately before graft reperfusion and on the fourth day after LT for induction therapy. A 500-mg intravenous bolus of methylprednisolone was administered immediately before reperfusion of the liver graft and was tapered to oral prednisolone over one week and reduced to withdrawal after six months.
All statistical analyses were performed using R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p ≤ 0.05. Continuous variables are presented as mean ± standard deviation (SD) and median (interquartile range, IQR), and categorical variables are presented as frequency (percentage, %). The survival curve was estimated by the Kaplan-Meier method. A univariate analysis was conducted to examine the differences in the distributions of continuous variables, categorical variables, and survival outcomes between the surviving and deceased liver recipients five years after LT using the Wilcoxon rank-sum test, Chi-square test, Fisher’s exact test, or log-rank test as appropriate for the data type. A multivariate analysis was performed to estimate the adjusted effects of risk factors or prognostic factors on the survival outcome using a multiple Cox’s proportional hazards model.
To ensure a good quality of regression analysis, the model-fitting techniques for variable selection, goodness-of-fit (GOF) assessment, and regression diagnostics and remedies were used in our regression analysis. Specifically, the stepwise variable selection procedure (with iterations between the forward and backward steps) was applied to obtain the best final regression model using the My.stepwise package of the R software [16]. All significant and non-significant relevant covariates from the univariate analysis (listed in Table 1) and some of the interaction terms were used in the multivariate analysis. The significance levels for entry and for stay were set to 0.15 for being conservative. With the aid of substantive knowledge, the best candidate final regression model was identified manually by dropping the covariates with p value > 0.05 one at a time until all regression coefficients were significantly different from 0. To assess the GOF of the fitted Cox’s model, the concordance and adjusted generalized R2 [17] were examined. A concordance ≥ 0.7 and an adjusted generalized R2 > 0.15 indicated an acceptable level of discrimination, power, and fitness.
Moreover, the smoothing option “pspline” (for the smoothing splines using a “p-spline” basis) was specified inside the coxph function of the survival package to smooth the effects of continuous covariates on the log-hazard rate of the simple and multiple Cox’s proportional hazards models in R. Then, the termplot function of the stats package was used to plot the smoothed effects of the continuous covariates on the log-hazard rate in R [18]. The regression diagnostics for the verification of proportional hazards assumption, residual analysis, detection of influential cases, and a multicollinearity check were applied to discover any model or data problems. A variance inflating factor (VIF) ≥ 10 in continuous covariates or VIF ≥ 2.5 in categorical covariates indicated the occurrence of the multicollinearity problem among some of the covariates in the fitted regression model.
3. Results
3.1. Patients’ Demographic and Clinical Characteristics
The median follow-up duration was 9.63 years (IQR: 7.2–11.4 years), and the mean follow-up was 9.58 ± 2.74 years. A total of 121 males (64.0%) and 68 females (36.0%) were included in this study (Table 1). The median age at LT was 54.26 years (IQR: 48.63–58.58 years, range: 18.3–73.1 years), and the mean age at LT was 52.7 ± 9.6 years. Among the 189 patients, 114 (76.2%) underwent living donor transplants and 45 (23.8%) underwent deceased donor transplants. The main indications for LT were hepatitis B virus (HBV) cirrhosis (52.9%), hepatocellular carcinoma (38.1%), hepatitis C virus (HCV) cirrhosis (22.2%), and fulminant hepatitis (12.2%). The median of the mean tacrolimus blood trough level during the fifth year after LT was 5.0 ng/mL (IQR: 4.12–6.33 ng/mL), and the mean tacrolimus blood trough level was 5.249 ± 1.71 ng/mL. The mean tacrolimus blood trough level was ≥ 5 ng/mL in 96 patients (50.8%) and < 5 ng/mL in 93 patients (49.2%), including 44 patients (23.3%) with a mean tacrolimus blood trough level < 4 ng/mL and 11 patients (5.8%) with a mean tacrolimus blood trough level < 3 ng/mL. No significant differences in gender, age at LT, body weight, blood type, graft type, etiology of LT, or serum total bilirubin were found between the deceased and surviving LT recipients (p > 0.05). However, serum creatinine levels > 1.5 mg/dL (23/173 vs. 8/16, p = 0.001), end-stage renal disease (2/173 vs. 3/16, p = 0.005), and mean tacrolimus blood trough levels < 4 ng/mL (36/173 vs. 8/16, p = 0.014) were significantly more common among deceased LT recipients compared to survivors. The causes of death included malignancies (n = 5), graft failures (n = 4), infection (n = 4), cerebrovascular accidents (n = 2), and duodenal ulcer bleeding (n = 1) (Table 2). The distribution of the tacrolimus trough level stratified by the etiology and cause of death among the 16 dead patients was shown in the box plot of Figure 2. There was no statistical significance in the tacrolimus trough level between the different causes of death (p = 0.3823).
3.2. Predictors of Patients’ Long-Term Survival
The Cox’s model fitted to the survival data for the multivariate analyses of the time to death after five years of LT is shown in Table 3. After adjusting for the effects of the other covariates, age at LT ≤ 27.011 years (estimated hazard ratio [HR] = 168.79, 95% confidence interval [C.I.]: 11.13–2559.51), pre-transplant autoimmune liver disease (HR = 8.12, 95% C.I.: 1.97–33.43), pre-transplant HCV infection × survival time in years (HR = 1.34, 95% C.I.: 1.12–1.60), serum creatinine level > 1.311 mg/dL × serum total bilirubin level > 1.411 mg/dL (HR = 921.69, 95% C.I.: 43.40–19,573.71), serum creatinine level > 1.311 mg/dL × serum total bilirubin level ≤ 0.792 mg/dL (HR = 105.68, 95% C.I.: 7.81–1430.79), serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level > 0.882 mg/dL (HR = 30.49, 95% C.I.: 2.98–312.34), and the mean tacrolimus trough level during the fifth year after LT ≤ 4.609 ng/mL or > 10.168 ng/mL (HR = 4.76, 95% C.I.: 1.34–16.94) were associated with a higher long-term mortality five years after LT. The time-dependent interaction term, HCV × survival time in years, was added to the Cox’s model to account for the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.292, indicates that the risk of mortality in patients with HCV increases with time five years post-LT. Moreover, the three second-order interaction terms between the serum creatinine and total bilirubin levels were compared to the other two possible combinations, serum creatinine level > 1.311 mg/dL × (0.792 mg/dL < serum total bilirubin level ≤ 1.411 mg/dL) and serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level ≤ 0.882 mg/dL, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and”.
All cut-off values of the continuous covariates (such as age at LT) were estimated by applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models. As shown in Figure 3, the optimal cut-off values of the mean tacrolimus trough level, 4.609 ng/mL and 10.168 ng/mL, were estimated directly in the “p-spline plot,” which allowed the visualization of the nonlinear effect of the averaged dosage of the tacrolimus-based immunosuppressant during the fifth year after LT on log(λ), where λ was the hazard rate of time to death five years after LT. Then, the Kaplan-Meier estimates of survival curves for time to death five years after LT were determined for the 66 patients with mean tacrolimus trough levels ≤ 4.609 ng/mL or > 10.168 ng/mL and the 123 patients with mean tacrolimus trough levels between 4.609 ng/mL and 10.168 ng/mL (log-rank test, p = 0.009) (Figure 4). In an additional subgroup analysis, we found that a mean tacrolimus trough level between 4.431 ng/mL and 6.332 ng/mL for patients with a serum creatinine level > 1.311 mg/dL improved survival. Finally, this Cox’s model had a concordance of 0.904 and an adjusted generalized R2 of 0.388, indicating that it fit the survival data very well.
4. Discussion
This is the first study to report an association between long-term tacrolimus blood trough level and long-term patient survival in adult LT recipients. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL in adult LT recipients is associated with a lower mortality rate. In patients with a serum creatinine > 1.3 mg/dL, a mean tacrolimus blood trough level of 4.4–6.3 ng/mL is associated with a lower mortality rate.
Decreasing tacrolimus use during long-term follow-up in LT recipients is a general concept in clinical practice. In this study, only one patient had the mean level > 10 ng/mL and a few patients had the mean level > 8 ng/mL (Figure 3). Patients with such high drug levels were most likely because of their poor compliance (not must because of they have truly such high trough level). Poor compliance indicates that a patient takes incorrect dosage of immunosuppressant that their physician prescribed, or takes immunosuppressant or blood exam at the wrong time, either of which might clearly be important factors that influence graft and survival outcome of LT patients during long-term follow-ups. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL is associated with lower mortality rate; however, the upper limit of 10.2 ng/mL suggested by the regression analysis was merely a value for caution, but not a recommended upper limit in real clinical practice. The long-term hazard of high level of tacrolimus is already widely known; therefore, we would focus more on the findings that it appears to increase the survival risks in LT recipients if their tacrolimus levels are maintained below 4.6 ng/mL during the long-term follow-ups.
Inadequate immunosuppression is associated with higher risks of graft rejection [19], while excess immunosuppression increases the risks of malignancy and infection and increases the adverse effects of drug toxicity. The tailored use of immunosuppressants should be considered based on the patient’s risks of graft rejection and infection and the patient’s medical comorbidities and liver disease status prior to LT [14]. Patients with autoimmune liver diseases may require more immunosuppression to prevent disease recurrence and graft rejection [20]. By contrast, lower doses of immunosuppression are recommended in patients who have undergone LT due to HCV, as high levels of immunosuppression are related to increased viral replication [21,22]. Rejection results in abnormal liver function and is one of the most important factors associated with poor long-term graft and patient outcomes [23,24,25]. Tacrolimus reduces the risk of T-cell mediated rejection to protect graft function and is the cornerstone of a successful LT. However, short and long-term adverse effects of tacrolimus such as infection, chronic renal insufficiency, metabolic diseases (hyperlipidemia, hypertension, and diabetes mellitus), and malignancy have been widely reported and influence patients’’ long-term outcomes [7,8,9]. The early causes of death after LT within one year are infection and graft loss, and the late causes of death three years post-LT are malignancy, cardiovascular disease, and renal failure [10]. As graft function typically stabilizes, factors associated with the long-term outcome are often patient-related factors (such as chronic medical diseases) that are usually associated with tacrolimus.
Previous studies have reported that minimizing tacrolimus use in the early post-LT period is associated with a lower risk of new-onset diabetes mellitus [26], a lower incidence of hyperlipidemia [27], and better long-term survival [19,28]. In recent years, studies have focused on the reduction or complete withdrawal of long-term immunosuppressants in LT recipients [12,29,30,31,32,33,34]. One study reported satisfactory outcomes with the combined use of tacrolimus and mycophenolate mofetil, which allowed for the tacrolimus dose to be reduced [35]. Other studies demonstrated that the concomitant use of everolimus may reduce the required dose of tacrolimus while having potential renal benefits [29,36]. However, only some LT recipients were able to discontinue the use of immunosuppressants, and these patients more frequently experienced biopsy-proven acute rejection [29]. The risk of chronic rejection during long-term follow-up remains in patients with inadequate immunosuppression [30,33]. The tacrolimus blood concentration in LT recipients may play a key role in long-term outcomes, as it is associated with long-term graft function and its adverse effects are related to several chronic medical diseases. However, to the best of our knowledge, this is the first report regarding the effect of long-term tacrolimus levels on the long-term survival outcomes of LT recipients.
Several factors are associated with the long-term outcomes of adult LT recipients, including pre-transplant primary sclerosing cholangitis, immunosuppression therapies, acute and chronic rejections, malignancy, and metabolic syndrome [37,38]. As listed in Table 3, we found several independent risk factors of long-term mortality in this study. Only four patients aged <27 years at the time of LT were included in this study, and one died due to pneumonia 6.3 years after LT. Patients who underwent LT due to autoimmune liver diseases had worse outcomes, as they were more likely to experience acute rejections [39] and suffer disease recurrence, leading to graft loss. Patients who underwent LT due to HCV infections had relatively poor post-LT outcomes due to the disease recurrence followed by graft dysfunction and failure [40]. As direct-acting antiviral agents have advanced [41], the survival outcomes of HCV patients should improve in the near future. Long-term renal and liver functions are reflected by serum creatinine and total bilirubin levels, respectively. We found that abnormal serum creatinine levels have a bigger impact than abnormal serum total bilirubin levels on the survival outcomes of LT recipients. The graft function of adult LT recipients is chronically stable five years after LT. While some patients may experience asymptomatic hyperbilirubinemia at this time, the long-term outcomes remain favorable. By contrast, the gradual deterioration of renal function may lead to chronic kidney disease or end-stage renal disease, affecting the long-term survival, especially in patients with long-term use of tacrolimus.
This study had some limitations. First, the data were obtained from a single medical center in Asia, which provided a relatively small number of eligible patients with single ethnicity and fewer death events. Second, the study spanned a long time period, and therefore the improvements in surgical and medical expertise and advances in immunosuppression therapies may have influenced the patient outcomes. Third, we did not collect or analyze the time-dependent tacrolimus blood trough level during the follow-up visits five years after LT.
In summary, we found an association between the long-term tacrolimus blood trough levels and the long-term survival five years after LT. A mean tacrolimus blood trough level outside the range of 4.6–10.2 ng/mL appeared to be an independent risk factor for long-term mortality. Further studies with larger sample sizes are needed to verify these results and to further identify an appropriate tacrolimus blood trough level for maintenance use.
Acknowledgments
We thank the coordinators (Hui-Ying Lin and Min-Heuy Lin) for their helpful efforts in data collection.
Author Contributions
C.-Y.H. drafted the manuscript and R.-H.H. designed the study. C.-Y.H., M.-C.H., and Y.-M.W. conducted data processing, and C.-Y.H. and C.-M.H. performed data analysis. P.-H.L. and R.-H.H. were the directors responsible for general organization and instruction. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of National Taiwan University Hospital.
Informed Consent Statement
Patient consent was waived due to retrospective design of the study.
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1 Patient flow diagram.
Figure 2 The box plot of tacrolimus mean trough level stratified by the etiology and cause of death among the 16 dead patients. There was no statistical difference of tacrolimus level between the different causes of death (p = 0.3823).
Figure 3 The p-spline plot for the nonlinear effect of the long-term tacrolimus blood trough levels on time to death. The tacrolimus blood trough level is shown on the X-axis, and the log(λ), where λ is the hazard rate of time to death five years after liver transplantation, is shown on the Y-axis. In this p-spline plot, the intersection between the horizontal green line (Y = 0) and the red curve yields the estimated optimal cut-off values for long-term tacrolimus blood trough levels at which the values of log(λ) will not change (4.609 ng/mL and 10.168 ng/mL). When the level is ≤4.609 ng/mL or >10.168 ng/mL, the value of log(λ) increases, indicating an increasing λ. When the level is >4.609 ng/mL and ≤10.168 ng/mL, the value of log(λ) decreases, indicating a decreasing λ. The vertical bars above the X-axis represent the patients’ actual mean tacrolimus blood trough levels obtained at the three follow-up visits during the fifth year after LT.
Figure 4 The Kaplan-Meier curve for time to death five years after liver transplantation. The survival curve of the 66 patients with a mean tacrolimus blood trough level ≤ 4.609 ng/mL or >10.168 ng/mL is shown in red while the survival curve of the 123 patients with a mean tacrolimus blood trough level > 4.609 ng/mL or ≤10.168 ng/mL is shown in green.
jpm-11-00090-t001_Table 1 Table 1 Univariate analysis for comparing the distributions of the demographic and clinical characteristics between the alive and dead adult liver recipients after five years of liver transplantations.
Variable All Patients
(n = 189) Alive
(n = 173) Dead
(n = 16) p Value
Gender 0.7899
Male 121 (64.0) 110 (90.9) 11 (9.1)
Female 68 (36.0) 63 (92.6) 5 (7.4)
Age at LT (years) 52.7 ± 9.6 52.6 ± 9.5 53.4 ± 10.7 0.6672
Body weight at LT (kg) 64.9 ± 12.2 65.0 ± 12.3 63.7 ± 12.1 0.4459
Blood type 0.8719
O 79 (41.8) 73 (92.4) 6 (7.6)
A 51 (27.0) 46 (90.2) 5 (9.8)
B 42 (22.2) 39 (92.9) 3 (7.1)
AB 17 (9.0) 15 (88.2) 2 (11.8)
Graft type 1.0000
Living donor 144 (76.2) 132 (91.7) 12 (8.3)
Deceased donor 45 (23.8) 41 (91.1) 4 (8.9)
Etiology for LT
Liver malignancy (HCC) 72 (38.1) 66 (91.7) 6 (8.3) 1.0000
Alcoholic cirrhosis 17 (9.0) 16 (94.1) 1 (5.9) 1.0000
HBV cirrhosis 100 (52.9) 92 (92.0) 8 (8.0) 1.0000
HCV cirrhosis 42 (22.2) 38 (90.5) 4 (9.5) 0.7577
Fulminant hepatitis 23 (12.2) 23 (100) 0 (0) 0.2259
Autoimmune disease 14 (7.4) 11 (78.6) 3 (21.4) 0.1017
Biliary atresia 5 (2.6) 5 (100) 0 (0) 1.0000
Other 10 (5.3) 9 (90) 1 (10) 0.5964
Total bilirubin (mg/dL) 0.973 ± 0.51 0.958 ± 1.53 1.133 ± 0.69 0.6260
Total bilirubin > 1 mg/dL 60 (31.7) 53 (88.3) 7 (11.7) 0.2770
Total bilirubin > 2 mg/dL 11 (5.8) 9 (81.8) 2 (18.2) 0.2360
Creatinine (mg/dL) 1.391 ± 1.25 1.279 ± 0.96 2.6 ± 2.74 0.0201 *
Creatinine > 1.5 mg/dL 31 (16.4) 23 (74.2) 8 (25.8) 0.0010 *
ESRD 5 (2.6) 2 (40.0) 3 (60.0) 0.0050 *
Tacrolimus mean level (ng/mL) 5.249 ± 1.71 5.263 ± 1.53 5.096 ± 3.12 0.9787
Tacrolimus level < 5 ng/mL 93 (49.2) 82 (88.2) 11 (11.8) 0.1216
Tacrolimus level < 4 ng/mL 44 (23.3) 36 (81.8) 8 (18.2) 0.0136 *
Tacrolimus level < 3 ng/mL 11 (5.8) 9 (81.8) 2 (18.2) 0.2356
Data are presented as mean ± standard deviation (SD) for continuous variables and frequency (percentage, %) for categorical variables. The p-values of statistical tests were calculated using the Wilcoxon rank-sum test for continuous variables and the Fisher’s exact test for categorical variables. * p value ≤ 0.05. Abbreviations: LT, liver transplantation; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; Total bilirubin, serum total bilirubin level; Creatinine, serum creatinine level; and ESRD, end-stage renal disease (defined by receiving hemodialysis regularly).
jpm-11-00090-t002_Table 2 Table 2 The causes of 16 deaths since 5 years after adult liver transplantations.
Causes of Deaths Number of Subjects
Malignancy 5 (31.25%)
De novo: Multiple myeloma, bladder cancer, colon cancer, prostate cancer 4
Recurrent: Hepatocellular carcinoma 1
Graft failure 4 (25.00%)
Chronic rejection 3
Autoimmune hepatitis 1
Infection 4 (25.00%)
Pneumonia 3
Urinary tract infection 1
Cerebral vascular event (intracerebral hemorrhage) 2 (12.50%)
Peptic ulcer bleeding 1 (6.25%)
jpm-11-00090-t003_Table 3 Table 3 Multivariate analysis for identifying the predictors of long-term overall survival after 5 years of liver transplantations by fitting a multiple Cox’s Model in the adult liver transplant recipients 1.
Covariate 2 Estimate Standard
Error Wald’s
z Test p Value Hazard
Ratio (HR) 95% Confidence
Interval (C.I.)
Age at LT ≤ 27.011 years 5.1286 1.3872 3.6970 0.0002 168.7851 11.130–2559.512
Autoimmune (including PBC) 2.0946 0.7219 2.9015 0.0037 8.1221 1.973–33.431
HCV × Overall survival years 0.2924 0.0914 3.1978 0.0014 1.3397 1.120–1.603
Cre > 1.311 × T-Bil > 1.411 mg/dL 6.8262 1.5591 4.3784 <0.0001 921.6940 43.401–19,573.712
Cre > 1.311 × T-Bil ≤ 0.792 mg/dL 4.6604 1.3294 3.5056 0.0005 105.6778 7.805–1430.790
Cre ≤ 1.311 × T-Bil > 0.882 mg/dL 3.4174 1.1871 2.8788 0.0040 30.4913 2.977–312.341
Tacrolimus mean ≤ 4.609 or > 10.168 ng/mL 1.5599 0.6479 2.4076 0.0161 4.7581 1.336–16.940
1 The above multiple Cox’s model was fitted to the 189 adult patients who underwent liver transplantations with 16 death events, for modeling the hazard rate of the right-censored overall survival time five years after liver transplantations. All the cut-off values of the continuous covariates (e.g., age at liver transplantations) were estimated by choosing the option of applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models (e.g., Figure 3). The time-dependent interaction term, HCV × Overall survival year, was added to the Cox’s model for handling the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.2924, indicated that the risk of dying in the patients with HCV would increase as time elapsed five years after liver transplantations. Moreover, the three second-order interaction terms, Cre > 1.311 × T-Bil > 1.411, Cre > 1.311 × T-Bil ≤ 0.792, and Cre ≤ 1.311 × T-Bil > 0.882, were compared to the other two possible combinations, Cre > 1.311 × (0.792 < T-Bil ≤ 1.411) and Cre ≤ 1.311 × T-Bil ≤ 0.882, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and.” Finally, both goodness-of-fit (GOF) measures, concordance = 0.9041 (se = 0.0265) > 0.7 and adjusted generalized R2 = 0.3878 > 0.15, indicated an excellent fit of this multiple Cox’s model to the observed survival data. 2 Abbreviations: LT, liver transplantation; PBC, primary biliary cirrhosis; HCV, hepatitis C viral infection; Cre, serum creatinine level (mg/dL); T-Bil, serum total bilirubin level (mg/dL); and Tacrolimus mean, the averaged dosage of the tacrolimus-based immunosuppressant (ng/mL).
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What was the administration route of drug 'PREDNISOLONE'? | Long-Term Tacrolimus Blood Trough Level and Patient Survival in Adult Liver Transplantation.
Tacrolimus is the most widely used immunosuppressant in liver transplant (LT) patients. However, the ideal long-term target level for these patients is unknown. This retrospective study aimed to investigate the impact of tacrolimus blood concentration five years after LT on long-term patient survival outcomes in adult LT recipients. Patients who underwent LT between January 2004 and July 2014 at a tertiary medical center were included in this study (n = 189). The mean tacrolimus blood concentrations of each patient during the fifth year after LT were recorded and the overall survival rate was determined. A multivariate analysis of factors associated with long-term survival was conducted using a Cox's model. The median follow-up period was 9.63 years, and 144 patients (76.2%) underwent live donor LT. Sixteen patients died within 5 years of LT. In the Cox's model, patients with a mean tacrolimus blood trough level of 4.6-10.2 ng/mL had significantly better long-term survival than those with a mean tacrolimus blood trough level outside this range (estimated hazard ratio = 4.76; 95% confidence interval: 1.34-16.9, p = 0.016). Therefore, a tacrolimus level no lower than 4.6 ng/mL would be recommended in adult LT patients.
1. Introduction
Liver transplantation (LT) has become a mature treatment of end-stage liver disease in clinical practice [1]. The continuous improvement of effective immunosuppression treatments has led to a significant improvement in patient and graft survival in recent years [2]. Calcineurin inhibitors are the cornerstone of immunosuppression in LT [3], and tacrolimus is currently the mainstay of initial and maintenance immunosuppression therapies [4,5,6]. Tacrolimus reduces the incidence and severity of early and late T-cell mediated rejection by inhibiting T-cell production of interleukin-2 [5,6]. However, the long-term use of immunosuppressants leads to an increasing burden of toxicity. The reported toxic effects of calcineurin inhibitors include infections, chronic renal insufficiency, metabolic diseases (such as hyperlipidemia, hypertension, and diabetes mellitus), and malignancy [7,8,9], which, along with cardiovascular diseases, have been reported to be the major causes of morbidity and mortality after LT [3,10]. However, most LT patients cannot withdraw from lifelong immunosuppression therapy; the only exceptions are a few selected patients participating in experimental trials [11,12].
It is a clinical challenge to design a well-balanced immunosuppressive regimen for LT recipients. Tacrolimus has a narrow therapeutic dose range and its blood levels should be carefully monitored. The Advagraf (tacrolimus prolonged-release hard capsules) recommendation for adult LT patients is a blood trough level of 5–20 ng/mL in the early post-transplant period and 5–15 ng/mL during subsequent maintenance therapy. Nevertheless, the current recommendations from the clinical practice guidelines for the ideal tacrolimus level in adults after LT remain controversial. The American Association for the Study of Liver Diseases (AASLD) recommends a target blood trough level of 5–10 ng/mL for tacrolimus three months after LT [13]. The Consensus on Managing Modifiable Risk in Transplantation Group (COMMIT) recommends that the target tacrolimus blood trough levels be 6–10 ng/mL during the first month after LT and decrease to 4–8 ng/mL thereafter, except when used in combination with mammalian target of rapamycin (mTOR) inhibitors [14]. Moreover, the International Liver Transplant Society (ILTS) consensus statement on immunosuppression in LT recipients recommends the target blood trough levels of tacrolimus be 6–10 ng/mL three months after LT, lower than 5 ng/mL 12 months after LT, and decrease to 3 ng/mL thereafter, resulting in a blood trough level just above the lower limit of detection five years after LT [15]. However, the impact of the long-term tacrolimus blood trough level on the outcomes of the LT recipients remains unclear. This study aimed to suggest an appropriate tacrolimus blood trough level for adult patients five years after LT.
2. Materials and Methods
This study was approved by the Institutional Review Board of National Taiwan University Hospital and was conducted according to the Declaration of Helsinki. A total of 286 patients who underwent LT at 18 years or older at a tertiary medical center from January 2004 to July 2014 were recruited for this study. Patients who died within 5 years of LT, were lost to follow-up, or did not use tacrolimus-based calcineurin inhibitor for immunosuppressant therapy were excluded from this study (Figure 1). In addition, those who received mTOR inhibitor treatment were excluded, as mTOR inhibitors are typically used as a combination therapy to reduce the required dose of tacrolimus. The final analysis included 189 patients. All eligible patients were followed up for more than five years until August 2019. Patients who underwent a LT due to liver cancer met the Milan criteria (before 2006) or the criteria of the University of California, San Francisco (since 2006) at the time of LT. All patients received regular monthly or bi-monthly follow-ups at the outpatient clinic after LT. Routine blood examinations for the tacrolimus blood trough level, liver function, and renal function were conducted at each visit, and abdominal sonography was performed every 6 to 12 months.
The patients’ medical records were reviewed retrospectively to extract demographic and clinical data, including patient characteristics, laboratory tests, and survival outcomes. The serum bilirubin and creatinine data at the end of the fifth year after LT were used in this study. The tacrolimus level used in this study was the mean of the values obtained at the three follow-up visits during the fifth year after LT.
The immunosuppression protocol for adult LT patients consisted of tacrolimus, mycophenolate mofetil, and steroids. Tacrolimus was administered orally beginning on the first day after LT, and the dose was adjusted to achieve the desired therapeutic drug level. Basiliximab was administered immediately before graft reperfusion and on the fourth day after LT for induction therapy. A 500-mg intravenous bolus of methylprednisolone was administered immediately before reperfusion of the liver graft and was tapered to oral prednisolone over one week and reduced to withdrawal after six months.
All statistical analyses were performed using R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p ≤ 0.05. Continuous variables are presented as mean ± standard deviation (SD) and median (interquartile range, IQR), and categorical variables are presented as frequency (percentage, %). The survival curve was estimated by the Kaplan-Meier method. A univariate analysis was conducted to examine the differences in the distributions of continuous variables, categorical variables, and survival outcomes between the surviving and deceased liver recipients five years after LT using the Wilcoxon rank-sum test, Chi-square test, Fisher’s exact test, or log-rank test as appropriate for the data type. A multivariate analysis was performed to estimate the adjusted effects of risk factors or prognostic factors on the survival outcome using a multiple Cox’s proportional hazards model.
To ensure a good quality of regression analysis, the model-fitting techniques for variable selection, goodness-of-fit (GOF) assessment, and regression diagnostics and remedies were used in our regression analysis. Specifically, the stepwise variable selection procedure (with iterations between the forward and backward steps) was applied to obtain the best final regression model using the My.stepwise package of the R software [16]. All significant and non-significant relevant covariates from the univariate analysis (listed in Table 1) and some of the interaction terms were used in the multivariate analysis. The significance levels for entry and for stay were set to 0.15 for being conservative. With the aid of substantive knowledge, the best candidate final regression model was identified manually by dropping the covariates with p value > 0.05 one at a time until all regression coefficients were significantly different from 0. To assess the GOF of the fitted Cox’s model, the concordance and adjusted generalized R2 [17] were examined. A concordance ≥ 0.7 and an adjusted generalized R2 > 0.15 indicated an acceptable level of discrimination, power, and fitness.
Moreover, the smoothing option “pspline” (for the smoothing splines using a “p-spline” basis) was specified inside the coxph function of the survival package to smooth the effects of continuous covariates on the log-hazard rate of the simple and multiple Cox’s proportional hazards models in R. Then, the termplot function of the stats package was used to plot the smoothed effects of the continuous covariates on the log-hazard rate in R [18]. The regression diagnostics for the verification of proportional hazards assumption, residual analysis, detection of influential cases, and a multicollinearity check were applied to discover any model or data problems. A variance inflating factor (VIF) ≥ 10 in continuous covariates or VIF ≥ 2.5 in categorical covariates indicated the occurrence of the multicollinearity problem among some of the covariates in the fitted regression model.
3. Results
3.1. Patients’ Demographic and Clinical Characteristics
The median follow-up duration was 9.63 years (IQR: 7.2–11.4 years), and the mean follow-up was 9.58 ± 2.74 years. A total of 121 males (64.0%) and 68 females (36.0%) were included in this study (Table 1). The median age at LT was 54.26 years (IQR: 48.63–58.58 years, range: 18.3–73.1 years), and the mean age at LT was 52.7 ± 9.6 years. Among the 189 patients, 114 (76.2%) underwent living donor transplants and 45 (23.8%) underwent deceased donor transplants. The main indications for LT were hepatitis B virus (HBV) cirrhosis (52.9%), hepatocellular carcinoma (38.1%), hepatitis C virus (HCV) cirrhosis (22.2%), and fulminant hepatitis (12.2%). The median of the mean tacrolimus blood trough level during the fifth year after LT was 5.0 ng/mL (IQR: 4.12–6.33 ng/mL), and the mean tacrolimus blood trough level was 5.249 ± 1.71 ng/mL. The mean tacrolimus blood trough level was ≥ 5 ng/mL in 96 patients (50.8%) and < 5 ng/mL in 93 patients (49.2%), including 44 patients (23.3%) with a mean tacrolimus blood trough level < 4 ng/mL and 11 patients (5.8%) with a mean tacrolimus blood trough level < 3 ng/mL. No significant differences in gender, age at LT, body weight, blood type, graft type, etiology of LT, or serum total bilirubin were found between the deceased and surviving LT recipients (p > 0.05). However, serum creatinine levels > 1.5 mg/dL (23/173 vs. 8/16, p = 0.001), end-stage renal disease (2/173 vs. 3/16, p = 0.005), and mean tacrolimus blood trough levels < 4 ng/mL (36/173 vs. 8/16, p = 0.014) were significantly more common among deceased LT recipients compared to survivors. The causes of death included malignancies (n = 5), graft failures (n = 4), infection (n = 4), cerebrovascular accidents (n = 2), and duodenal ulcer bleeding (n = 1) (Table 2). The distribution of the tacrolimus trough level stratified by the etiology and cause of death among the 16 dead patients was shown in the box plot of Figure 2. There was no statistical significance in the tacrolimus trough level between the different causes of death (p = 0.3823).
3.2. Predictors of Patients’ Long-Term Survival
The Cox’s model fitted to the survival data for the multivariate analyses of the time to death after five years of LT is shown in Table 3. After adjusting for the effects of the other covariates, age at LT ≤ 27.011 years (estimated hazard ratio [HR] = 168.79, 95% confidence interval [C.I.]: 11.13–2559.51), pre-transplant autoimmune liver disease (HR = 8.12, 95% C.I.: 1.97–33.43), pre-transplant HCV infection × survival time in years (HR = 1.34, 95% C.I.: 1.12–1.60), serum creatinine level > 1.311 mg/dL × serum total bilirubin level > 1.411 mg/dL (HR = 921.69, 95% C.I.: 43.40–19,573.71), serum creatinine level > 1.311 mg/dL × serum total bilirubin level ≤ 0.792 mg/dL (HR = 105.68, 95% C.I.: 7.81–1430.79), serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level > 0.882 mg/dL (HR = 30.49, 95% C.I.: 2.98–312.34), and the mean tacrolimus trough level during the fifth year after LT ≤ 4.609 ng/mL or > 10.168 ng/mL (HR = 4.76, 95% C.I.: 1.34–16.94) were associated with a higher long-term mortality five years after LT. The time-dependent interaction term, HCV × survival time in years, was added to the Cox’s model to account for the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.292, indicates that the risk of mortality in patients with HCV increases with time five years post-LT. Moreover, the three second-order interaction terms between the serum creatinine and total bilirubin levels were compared to the other two possible combinations, serum creatinine level > 1.311 mg/dL × (0.792 mg/dL < serum total bilirubin level ≤ 1.411 mg/dL) and serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level ≤ 0.882 mg/dL, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and”.
All cut-off values of the continuous covariates (such as age at LT) were estimated by applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models. As shown in Figure 3, the optimal cut-off values of the mean tacrolimus trough level, 4.609 ng/mL and 10.168 ng/mL, were estimated directly in the “p-spline plot,” which allowed the visualization of the nonlinear effect of the averaged dosage of the tacrolimus-based immunosuppressant during the fifth year after LT on log(λ), where λ was the hazard rate of time to death five years after LT. Then, the Kaplan-Meier estimates of survival curves for time to death five years after LT were determined for the 66 patients with mean tacrolimus trough levels ≤ 4.609 ng/mL or > 10.168 ng/mL and the 123 patients with mean tacrolimus trough levels between 4.609 ng/mL and 10.168 ng/mL (log-rank test, p = 0.009) (Figure 4). In an additional subgroup analysis, we found that a mean tacrolimus trough level between 4.431 ng/mL and 6.332 ng/mL for patients with a serum creatinine level > 1.311 mg/dL improved survival. Finally, this Cox’s model had a concordance of 0.904 and an adjusted generalized R2 of 0.388, indicating that it fit the survival data very well.
4. Discussion
This is the first study to report an association between long-term tacrolimus blood trough level and long-term patient survival in adult LT recipients. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL in adult LT recipients is associated with a lower mortality rate. In patients with a serum creatinine > 1.3 mg/dL, a mean tacrolimus blood trough level of 4.4–6.3 ng/mL is associated with a lower mortality rate.
Decreasing tacrolimus use during long-term follow-up in LT recipients is a general concept in clinical practice. In this study, only one patient had the mean level > 10 ng/mL and a few patients had the mean level > 8 ng/mL (Figure 3). Patients with such high drug levels were most likely because of their poor compliance (not must because of they have truly such high trough level). Poor compliance indicates that a patient takes incorrect dosage of immunosuppressant that their physician prescribed, or takes immunosuppressant or blood exam at the wrong time, either of which might clearly be important factors that influence graft and survival outcome of LT patients during long-term follow-ups. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL is associated with lower mortality rate; however, the upper limit of 10.2 ng/mL suggested by the regression analysis was merely a value for caution, but not a recommended upper limit in real clinical practice. The long-term hazard of high level of tacrolimus is already widely known; therefore, we would focus more on the findings that it appears to increase the survival risks in LT recipients if their tacrolimus levels are maintained below 4.6 ng/mL during the long-term follow-ups.
Inadequate immunosuppression is associated with higher risks of graft rejection [19], while excess immunosuppression increases the risks of malignancy and infection and increases the adverse effects of drug toxicity. The tailored use of immunosuppressants should be considered based on the patient’s risks of graft rejection and infection and the patient’s medical comorbidities and liver disease status prior to LT [14]. Patients with autoimmune liver diseases may require more immunosuppression to prevent disease recurrence and graft rejection [20]. By contrast, lower doses of immunosuppression are recommended in patients who have undergone LT due to HCV, as high levels of immunosuppression are related to increased viral replication [21,22]. Rejection results in abnormal liver function and is one of the most important factors associated with poor long-term graft and patient outcomes [23,24,25]. Tacrolimus reduces the risk of T-cell mediated rejection to protect graft function and is the cornerstone of a successful LT. However, short and long-term adverse effects of tacrolimus such as infection, chronic renal insufficiency, metabolic diseases (hyperlipidemia, hypertension, and diabetes mellitus), and malignancy have been widely reported and influence patients’’ long-term outcomes [7,8,9]. The early causes of death after LT within one year are infection and graft loss, and the late causes of death three years post-LT are malignancy, cardiovascular disease, and renal failure [10]. As graft function typically stabilizes, factors associated with the long-term outcome are often patient-related factors (such as chronic medical diseases) that are usually associated with tacrolimus.
Previous studies have reported that minimizing tacrolimus use in the early post-LT period is associated with a lower risk of new-onset diabetes mellitus [26], a lower incidence of hyperlipidemia [27], and better long-term survival [19,28]. In recent years, studies have focused on the reduction or complete withdrawal of long-term immunosuppressants in LT recipients [12,29,30,31,32,33,34]. One study reported satisfactory outcomes with the combined use of tacrolimus and mycophenolate mofetil, which allowed for the tacrolimus dose to be reduced [35]. Other studies demonstrated that the concomitant use of everolimus may reduce the required dose of tacrolimus while having potential renal benefits [29,36]. However, only some LT recipients were able to discontinue the use of immunosuppressants, and these patients more frequently experienced biopsy-proven acute rejection [29]. The risk of chronic rejection during long-term follow-up remains in patients with inadequate immunosuppression [30,33]. The tacrolimus blood concentration in LT recipients may play a key role in long-term outcomes, as it is associated with long-term graft function and its adverse effects are related to several chronic medical diseases. However, to the best of our knowledge, this is the first report regarding the effect of long-term tacrolimus levels on the long-term survival outcomes of LT recipients.
Several factors are associated with the long-term outcomes of adult LT recipients, including pre-transplant primary sclerosing cholangitis, immunosuppression therapies, acute and chronic rejections, malignancy, and metabolic syndrome [37,38]. As listed in Table 3, we found several independent risk factors of long-term mortality in this study. Only four patients aged <27 years at the time of LT were included in this study, and one died due to pneumonia 6.3 years after LT. Patients who underwent LT due to autoimmune liver diseases had worse outcomes, as they were more likely to experience acute rejections [39] and suffer disease recurrence, leading to graft loss. Patients who underwent LT due to HCV infections had relatively poor post-LT outcomes due to the disease recurrence followed by graft dysfunction and failure [40]. As direct-acting antiviral agents have advanced [41], the survival outcomes of HCV patients should improve in the near future. Long-term renal and liver functions are reflected by serum creatinine and total bilirubin levels, respectively. We found that abnormal serum creatinine levels have a bigger impact than abnormal serum total bilirubin levels on the survival outcomes of LT recipients. The graft function of adult LT recipients is chronically stable five years after LT. While some patients may experience asymptomatic hyperbilirubinemia at this time, the long-term outcomes remain favorable. By contrast, the gradual deterioration of renal function may lead to chronic kidney disease or end-stage renal disease, affecting the long-term survival, especially in patients with long-term use of tacrolimus.
This study had some limitations. First, the data were obtained from a single medical center in Asia, which provided a relatively small number of eligible patients with single ethnicity and fewer death events. Second, the study spanned a long time period, and therefore the improvements in surgical and medical expertise and advances in immunosuppression therapies may have influenced the patient outcomes. Third, we did not collect or analyze the time-dependent tacrolimus blood trough level during the follow-up visits five years after LT.
In summary, we found an association between the long-term tacrolimus blood trough levels and the long-term survival five years after LT. A mean tacrolimus blood trough level outside the range of 4.6–10.2 ng/mL appeared to be an independent risk factor for long-term mortality. Further studies with larger sample sizes are needed to verify these results and to further identify an appropriate tacrolimus blood trough level for maintenance use.
Acknowledgments
We thank the coordinators (Hui-Ying Lin and Min-Heuy Lin) for their helpful efforts in data collection.
Author Contributions
C.-Y.H. drafted the manuscript and R.-H.H. designed the study. C.-Y.H., M.-C.H., and Y.-M.W. conducted data processing, and C.-Y.H. and C.-M.H. performed data analysis. P.-H.L. and R.-H.H. were the directors responsible for general organization and instruction. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of National Taiwan University Hospital.
Informed Consent Statement
Patient consent was waived due to retrospective design of the study.
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1 Patient flow diagram.
Figure 2 The box plot of tacrolimus mean trough level stratified by the etiology and cause of death among the 16 dead patients. There was no statistical difference of tacrolimus level between the different causes of death (p = 0.3823).
Figure 3 The p-spline plot for the nonlinear effect of the long-term tacrolimus blood trough levels on time to death. The tacrolimus blood trough level is shown on the X-axis, and the log(λ), where λ is the hazard rate of time to death five years after liver transplantation, is shown on the Y-axis. In this p-spline plot, the intersection between the horizontal green line (Y = 0) and the red curve yields the estimated optimal cut-off values for long-term tacrolimus blood trough levels at which the values of log(λ) will not change (4.609 ng/mL and 10.168 ng/mL). When the level is ≤4.609 ng/mL or >10.168 ng/mL, the value of log(λ) increases, indicating an increasing λ. When the level is >4.609 ng/mL and ≤10.168 ng/mL, the value of log(λ) decreases, indicating a decreasing λ. The vertical bars above the X-axis represent the patients’ actual mean tacrolimus blood trough levels obtained at the three follow-up visits during the fifth year after LT.
Figure 4 The Kaplan-Meier curve for time to death five years after liver transplantation. The survival curve of the 66 patients with a mean tacrolimus blood trough level ≤ 4.609 ng/mL or >10.168 ng/mL is shown in red while the survival curve of the 123 patients with a mean tacrolimus blood trough level > 4.609 ng/mL or ≤10.168 ng/mL is shown in green.
jpm-11-00090-t001_Table 1 Table 1 Univariate analysis for comparing the distributions of the demographic and clinical characteristics between the alive and dead adult liver recipients after five years of liver transplantations.
Variable All Patients
(n = 189) Alive
(n = 173) Dead
(n = 16) p Value
Gender 0.7899
Male 121 (64.0) 110 (90.9) 11 (9.1)
Female 68 (36.0) 63 (92.6) 5 (7.4)
Age at LT (years) 52.7 ± 9.6 52.6 ± 9.5 53.4 ± 10.7 0.6672
Body weight at LT (kg) 64.9 ± 12.2 65.0 ± 12.3 63.7 ± 12.1 0.4459
Blood type 0.8719
O 79 (41.8) 73 (92.4) 6 (7.6)
A 51 (27.0) 46 (90.2) 5 (9.8)
B 42 (22.2) 39 (92.9) 3 (7.1)
AB 17 (9.0) 15 (88.2) 2 (11.8)
Graft type 1.0000
Living donor 144 (76.2) 132 (91.7) 12 (8.3)
Deceased donor 45 (23.8) 41 (91.1) 4 (8.9)
Etiology for LT
Liver malignancy (HCC) 72 (38.1) 66 (91.7) 6 (8.3) 1.0000
Alcoholic cirrhosis 17 (9.0) 16 (94.1) 1 (5.9) 1.0000
HBV cirrhosis 100 (52.9) 92 (92.0) 8 (8.0) 1.0000
HCV cirrhosis 42 (22.2) 38 (90.5) 4 (9.5) 0.7577
Fulminant hepatitis 23 (12.2) 23 (100) 0 (0) 0.2259
Autoimmune disease 14 (7.4) 11 (78.6) 3 (21.4) 0.1017
Biliary atresia 5 (2.6) 5 (100) 0 (0) 1.0000
Other 10 (5.3) 9 (90) 1 (10) 0.5964
Total bilirubin (mg/dL) 0.973 ± 0.51 0.958 ± 1.53 1.133 ± 0.69 0.6260
Total bilirubin > 1 mg/dL 60 (31.7) 53 (88.3) 7 (11.7) 0.2770
Total bilirubin > 2 mg/dL 11 (5.8) 9 (81.8) 2 (18.2) 0.2360
Creatinine (mg/dL) 1.391 ± 1.25 1.279 ± 0.96 2.6 ± 2.74 0.0201 *
Creatinine > 1.5 mg/dL 31 (16.4) 23 (74.2) 8 (25.8) 0.0010 *
ESRD 5 (2.6) 2 (40.0) 3 (60.0) 0.0050 *
Tacrolimus mean level (ng/mL) 5.249 ± 1.71 5.263 ± 1.53 5.096 ± 3.12 0.9787
Tacrolimus level < 5 ng/mL 93 (49.2) 82 (88.2) 11 (11.8) 0.1216
Tacrolimus level < 4 ng/mL 44 (23.3) 36 (81.8) 8 (18.2) 0.0136 *
Tacrolimus level < 3 ng/mL 11 (5.8) 9 (81.8) 2 (18.2) 0.2356
Data are presented as mean ± standard deviation (SD) for continuous variables and frequency (percentage, %) for categorical variables. The p-values of statistical tests were calculated using the Wilcoxon rank-sum test for continuous variables and the Fisher’s exact test for categorical variables. * p value ≤ 0.05. Abbreviations: LT, liver transplantation; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; Total bilirubin, serum total bilirubin level; Creatinine, serum creatinine level; and ESRD, end-stage renal disease (defined by receiving hemodialysis regularly).
jpm-11-00090-t002_Table 2 Table 2 The causes of 16 deaths since 5 years after adult liver transplantations.
Causes of Deaths Number of Subjects
Malignancy 5 (31.25%)
De novo: Multiple myeloma, bladder cancer, colon cancer, prostate cancer 4
Recurrent: Hepatocellular carcinoma 1
Graft failure 4 (25.00%)
Chronic rejection 3
Autoimmune hepatitis 1
Infection 4 (25.00%)
Pneumonia 3
Urinary tract infection 1
Cerebral vascular event (intracerebral hemorrhage) 2 (12.50%)
Peptic ulcer bleeding 1 (6.25%)
jpm-11-00090-t003_Table 3 Table 3 Multivariate analysis for identifying the predictors of long-term overall survival after 5 years of liver transplantations by fitting a multiple Cox’s Model in the adult liver transplant recipients 1.
Covariate 2 Estimate Standard
Error Wald’s
z Test p Value Hazard
Ratio (HR) 95% Confidence
Interval (C.I.)
Age at LT ≤ 27.011 years 5.1286 1.3872 3.6970 0.0002 168.7851 11.130–2559.512
Autoimmune (including PBC) 2.0946 0.7219 2.9015 0.0037 8.1221 1.973–33.431
HCV × Overall survival years 0.2924 0.0914 3.1978 0.0014 1.3397 1.120–1.603
Cre > 1.311 × T-Bil > 1.411 mg/dL 6.8262 1.5591 4.3784 <0.0001 921.6940 43.401–19,573.712
Cre > 1.311 × T-Bil ≤ 0.792 mg/dL 4.6604 1.3294 3.5056 0.0005 105.6778 7.805–1430.790
Cre ≤ 1.311 × T-Bil > 0.882 mg/dL 3.4174 1.1871 2.8788 0.0040 30.4913 2.977–312.341
Tacrolimus mean ≤ 4.609 or > 10.168 ng/mL 1.5599 0.6479 2.4076 0.0161 4.7581 1.336–16.940
1 The above multiple Cox’s model was fitted to the 189 adult patients who underwent liver transplantations with 16 death events, for modeling the hazard rate of the right-censored overall survival time five years after liver transplantations. All the cut-off values of the continuous covariates (e.g., age at liver transplantations) were estimated by choosing the option of applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models (e.g., Figure 3). The time-dependent interaction term, HCV × Overall survival year, was added to the Cox’s model for handling the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.2924, indicated that the risk of dying in the patients with HCV would increase as time elapsed five years after liver transplantations. Moreover, the three second-order interaction terms, Cre > 1.311 × T-Bil > 1.411, Cre > 1.311 × T-Bil ≤ 0.792, and Cre ≤ 1.311 × T-Bil > 0.882, were compared to the other two possible combinations, Cre > 1.311 × (0.792 < T-Bil ≤ 1.411) and Cre ≤ 1.311 × T-Bil ≤ 0.882, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and.” Finally, both goodness-of-fit (GOF) measures, concordance = 0.9041 (se = 0.0265) > 0.7 and adjusted generalized R2 = 0.3878 > 0.15, indicated an excellent fit of this multiple Cox’s model to the observed survival data. 2 Abbreviations: LT, liver transplantation; PBC, primary biliary cirrhosis; HCV, hepatitis C viral infection; Cre, serum creatinine level (mg/dL); T-Bil, serum total bilirubin level (mg/dL); and Tacrolimus mean, the averaged dosage of the tacrolimus-based immunosuppressant (ng/mL).
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What was the administration route of drug 'TACROLIMUS'? | Long-Term Tacrolimus Blood Trough Level and Patient Survival in Adult Liver Transplantation.
Tacrolimus is the most widely used immunosuppressant in liver transplant (LT) patients. However, the ideal long-term target level for these patients is unknown. This retrospective study aimed to investigate the impact of tacrolimus blood concentration five years after LT on long-term patient survival outcomes in adult LT recipients. Patients who underwent LT between January 2004 and July 2014 at a tertiary medical center were included in this study (n = 189). The mean tacrolimus blood concentrations of each patient during the fifth year after LT were recorded and the overall survival rate was determined. A multivariate analysis of factors associated with long-term survival was conducted using a Cox's model. The median follow-up period was 9.63 years, and 144 patients (76.2%) underwent live donor LT. Sixteen patients died within 5 years of LT. In the Cox's model, patients with a mean tacrolimus blood trough level of 4.6-10.2 ng/mL had significantly better long-term survival than those with a mean tacrolimus blood trough level outside this range (estimated hazard ratio = 4.76; 95% confidence interval: 1.34-16.9, p = 0.016). Therefore, a tacrolimus level no lower than 4.6 ng/mL would be recommended in adult LT patients.
1. Introduction
Liver transplantation (LT) has become a mature treatment of end-stage liver disease in clinical practice [1]. The continuous improvement of effective immunosuppression treatments has led to a significant improvement in patient and graft survival in recent years [2]. Calcineurin inhibitors are the cornerstone of immunosuppression in LT [3], and tacrolimus is currently the mainstay of initial and maintenance immunosuppression therapies [4,5,6]. Tacrolimus reduces the incidence and severity of early and late T-cell mediated rejection by inhibiting T-cell production of interleukin-2 [5,6]. However, the long-term use of immunosuppressants leads to an increasing burden of toxicity. The reported toxic effects of calcineurin inhibitors include infections, chronic renal insufficiency, metabolic diseases (such as hyperlipidemia, hypertension, and diabetes mellitus), and malignancy [7,8,9], which, along with cardiovascular diseases, have been reported to be the major causes of morbidity and mortality after LT [3,10]. However, most LT patients cannot withdraw from lifelong immunosuppression therapy; the only exceptions are a few selected patients participating in experimental trials [11,12].
It is a clinical challenge to design a well-balanced immunosuppressive regimen for LT recipients. Tacrolimus has a narrow therapeutic dose range and its blood levels should be carefully monitored. The Advagraf (tacrolimus prolonged-release hard capsules) recommendation for adult LT patients is a blood trough level of 5–20 ng/mL in the early post-transplant period and 5–15 ng/mL during subsequent maintenance therapy. Nevertheless, the current recommendations from the clinical practice guidelines for the ideal tacrolimus level in adults after LT remain controversial. The American Association for the Study of Liver Diseases (AASLD) recommends a target blood trough level of 5–10 ng/mL for tacrolimus three months after LT [13]. The Consensus on Managing Modifiable Risk in Transplantation Group (COMMIT) recommends that the target tacrolimus blood trough levels be 6–10 ng/mL during the first month after LT and decrease to 4–8 ng/mL thereafter, except when used in combination with mammalian target of rapamycin (mTOR) inhibitors [14]. Moreover, the International Liver Transplant Society (ILTS) consensus statement on immunosuppression in LT recipients recommends the target blood trough levels of tacrolimus be 6–10 ng/mL three months after LT, lower than 5 ng/mL 12 months after LT, and decrease to 3 ng/mL thereafter, resulting in a blood trough level just above the lower limit of detection five years after LT [15]. However, the impact of the long-term tacrolimus blood trough level on the outcomes of the LT recipients remains unclear. This study aimed to suggest an appropriate tacrolimus blood trough level for adult patients five years after LT.
2. Materials and Methods
This study was approved by the Institutional Review Board of National Taiwan University Hospital and was conducted according to the Declaration of Helsinki. A total of 286 patients who underwent LT at 18 years or older at a tertiary medical center from January 2004 to July 2014 were recruited for this study. Patients who died within 5 years of LT, were lost to follow-up, or did not use tacrolimus-based calcineurin inhibitor for immunosuppressant therapy were excluded from this study (Figure 1). In addition, those who received mTOR inhibitor treatment were excluded, as mTOR inhibitors are typically used as a combination therapy to reduce the required dose of tacrolimus. The final analysis included 189 patients. All eligible patients were followed up for more than five years until August 2019. Patients who underwent a LT due to liver cancer met the Milan criteria (before 2006) or the criteria of the University of California, San Francisco (since 2006) at the time of LT. All patients received regular monthly or bi-monthly follow-ups at the outpatient clinic after LT. Routine blood examinations for the tacrolimus blood trough level, liver function, and renal function were conducted at each visit, and abdominal sonography was performed every 6 to 12 months.
The patients’ medical records were reviewed retrospectively to extract demographic and clinical data, including patient characteristics, laboratory tests, and survival outcomes. The serum bilirubin and creatinine data at the end of the fifth year after LT were used in this study. The tacrolimus level used in this study was the mean of the values obtained at the three follow-up visits during the fifth year after LT.
The immunosuppression protocol for adult LT patients consisted of tacrolimus, mycophenolate mofetil, and steroids. Tacrolimus was administered orally beginning on the first day after LT, and the dose was adjusted to achieve the desired therapeutic drug level. Basiliximab was administered immediately before graft reperfusion and on the fourth day after LT for induction therapy. A 500-mg intravenous bolus of methylprednisolone was administered immediately before reperfusion of the liver graft and was tapered to oral prednisolone over one week and reduced to withdrawal after six months.
All statistical analyses were performed using R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p ≤ 0.05. Continuous variables are presented as mean ± standard deviation (SD) and median (interquartile range, IQR), and categorical variables are presented as frequency (percentage, %). The survival curve was estimated by the Kaplan-Meier method. A univariate analysis was conducted to examine the differences in the distributions of continuous variables, categorical variables, and survival outcomes between the surviving and deceased liver recipients five years after LT using the Wilcoxon rank-sum test, Chi-square test, Fisher’s exact test, or log-rank test as appropriate for the data type. A multivariate analysis was performed to estimate the adjusted effects of risk factors or prognostic factors on the survival outcome using a multiple Cox’s proportional hazards model.
To ensure a good quality of regression analysis, the model-fitting techniques for variable selection, goodness-of-fit (GOF) assessment, and regression diagnostics and remedies were used in our regression analysis. Specifically, the stepwise variable selection procedure (with iterations between the forward and backward steps) was applied to obtain the best final regression model using the My.stepwise package of the R software [16]. All significant and non-significant relevant covariates from the univariate analysis (listed in Table 1) and some of the interaction terms were used in the multivariate analysis. The significance levels for entry and for stay were set to 0.15 for being conservative. With the aid of substantive knowledge, the best candidate final regression model was identified manually by dropping the covariates with p value > 0.05 one at a time until all regression coefficients were significantly different from 0. To assess the GOF of the fitted Cox’s model, the concordance and adjusted generalized R2 [17] were examined. A concordance ≥ 0.7 and an adjusted generalized R2 > 0.15 indicated an acceptable level of discrimination, power, and fitness.
Moreover, the smoothing option “pspline” (for the smoothing splines using a “p-spline” basis) was specified inside the coxph function of the survival package to smooth the effects of continuous covariates on the log-hazard rate of the simple and multiple Cox’s proportional hazards models in R. Then, the termplot function of the stats package was used to plot the smoothed effects of the continuous covariates on the log-hazard rate in R [18]. The regression diagnostics for the verification of proportional hazards assumption, residual analysis, detection of influential cases, and a multicollinearity check were applied to discover any model or data problems. A variance inflating factor (VIF) ≥ 10 in continuous covariates or VIF ≥ 2.5 in categorical covariates indicated the occurrence of the multicollinearity problem among some of the covariates in the fitted regression model.
3. Results
3.1. Patients’ Demographic and Clinical Characteristics
The median follow-up duration was 9.63 years (IQR: 7.2–11.4 years), and the mean follow-up was 9.58 ± 2.74 years. A total of 121 males (64.0%) and 68 females (36.0%) were included in this study (Table 1). The median age at LT was 54.26 years (IQR: 48.63–58.58 years, range: 18.3–73.1 years), and the mean age at LT was 52.7 ± 9.6 years. Among the 189 patients, 114 (76.2%) underwent living donor transplants and 45 (23.8%) underwent deceased donor transplants. The main indications for LT were hepatitis B virus (HBV) cirrhosis (52.9%), hepatocellular carcinoma (38.1%), hepatitis C virus (HCV) cirrhosis (22.2%), and fulminant hepatitis (12.2%). The median of the mean tacrolimus blood trough level during the fifth year after LT was 5.0 ng/mL (IQR: 4.12–6.33 ng/mL), and the mean tacrolimus blood trough level was 5.249 ± 1.71 ng/mL. The mean tacrolimus blood trough level was ≥ 5 ng/mL in 96 patients (50.8%) and < 5 ng/mL in 93 patients (49.2%), including 44 patients (23.3%) with a mean tacrolimus blood trough level < 4 ng/mL and 11 patients (5.8%) with a mean tacrolimus blood trough level < 3 ng/mL. No significant differences in gender, age at LT, body weight, blood type, graft type, etiology of LT, or serum total bilirubin were found between the deceased and surviving LT recipients (p > 0.05). However, serum creatinine levels > 1.5 mg/dL (23/173 vs. 8/16, p = 0.001), end-stage renal disease (2/173 vs. 3/16, p = 0.005), and mean tacrolimus blood trough levels < 4 ng/mL (36/173 vs. 8/16, p = 0.014) were significantly more common among deceased LT recipients compared to survivors. The causes of death included malignancies (n = 5), graft failures (n = 4), infection (n = 4), cerebrovascular accidents (n = 2), and duodenal ulcer bleeding (n = 1) (Table 2). The distribution of the tacrolimus trough level stratified by the etiology and cause of death among the 16 dead patients was shown in the box plot of Figure 2. There was no statistical significance in the tacrolimus trough level between the different causes of death (p = 0.3823).
3.2. Predictors of Patients’ Long-Term Survival
The Cox’s model fitted to the survival data for the multivariate analyses of the time to death after five years of LT is shown in Table 3. After adjusting for the effects of the other covariates, age at LT ≤ 27.011 years (estimated hazard ratio [HR] = 168.79, 95% confidence interval [C.I.]: 11.13–2559.51), pre-transplant autoimmune liver disease (HR = 8.12, 95% C.I.: 1.97–33.43), pre-transplant HCV infection × survival time in years (HR = 1.34, 95% C.I.: 1.12–1.60), serum creatinine level > 1.311 mg/dL × serum total bilirubin level > 1.411 mg/dL (HR = 921.69, 95% C.I.: 43.40–19,573.71), serum creatinine level > 1.311 mg/dL × serum total bilirubin level ≤ 0.792 mg/dL (HR = 105.68, 95% C.I.: 7.81–1430.79), serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level > 0.882 mg/dL (HR = 30.49, 95% C.I.: 2.98–312.34), and the mean tacrolimus trough level during the fifth year after LT ≤ 4.609 ng/mL or > 10.168 ng/mL (HR = 4.76, 95% C.I.: 1.34–16.94) were associated with a higher long-term mortality five years after LT. The time-dependent interaction term, HCV × survival time in years, was added to the Cox’s model to account for the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.292, indicates that the risk of mortality in patients with HCV increases with time five years post-LT. Moreover, the three second-order interaction terms between the serum creatinine and total bilirubin levels were compared to the other two possible combinations, serum creatinine level > 1.311 mg/dL × (0.792 mg/dL < serum total bilirubin level ≤ 1.411 mg/dL) and serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level ≤ 0.882 mg/dL, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and”.
All cut-off values of the continuous covariates (such as age at LT) were estimated by applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models. As shown in Figure 3, the optimal cut-off values of the mean tacrolimus trough level, 4.609 ng/mL and 10.168 ng/mL, were estimated directly in the “p-spline plot,” which allowed the visualization of the nonlinear effect of the averaged dosage of the tacrolimus-based immunosuppressant during the fifth year after LT on log(λ), where λ was the hazard rate of time to death five years after LT. Then, the Kaplan-Meier estimates of survival curves for time to death five years after LT were determined for the 66 patients with mean tacrolimus trough levels ≤ 4.609 ng/mL or > 10.168 ng/mL and the 123 patients with mean tacrolimus trough levels between 4.609 ng/mL and 10.168 ng/mL (log-rank test, p = 0.009) (Figure 4). In an additional subgroup analysis, we found that a mean tacrolimus trough level between 4.431 ng/mL and 6.332 ng/mL for patients with a serum creatinine level > 1.311 mg/dL improved survival. Finally, this Cox’s model had a concordance of 0.904 and an adjusted generalized R2 of 0.388, indicating that it fit the survival data very well.
4. Discussion
This is the first study to report an association between long-term tacrolimus blood trough level and long-term patient survival in adult LT recipients. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL in adult LT recipients is associated with a lower mortality rate. In patients with a serum creatinine > 1.3 mg/dL, a mean tacrolimus blood trough level of 4.4–6.3 ng/mL is associated with a lower mortality rate.
Decreasing tacrolimus use during long-term follow-up in LT recipients is a general concept in clinical practice. In this study, only one patient had the mean level > 10 ng/mL and a few patients had the mean level > 8 ng/mL (Figure 3). Patients with such high drug levels were most likely because of their poor compliance (not must because of they have truly such high trough level). Poor compliance indicates that a patient takes incorrect dosage of immunosuppressant that their physician prescribed, or takes immunosuppressant or blood exam at the wrong time, either of which might clearly be important factors that influence graft and survival outcome of LT patients during long-term follow-ups. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL is associated with lower mortality rate; however, the upper limit of 10.2 ng/mL suggested by the regression analysis was merely a value for caution, but not a recommended upper limit in real clinical practice. The long-term hazard of high level of tacrolimus is already widely known; therefore, we would focus more on the findings that it appears to increase the survival risks in LT recipients if their tacrolimus levels are maintained below 4.6 ng/mL during the long-term follow-ups.
Inadequate immunosuppression is associated with higher risks of graft rejection [19], while excess immunosuppression increases the risks of malignancy and infection and increases the adverse effects of drug toxicity. The tailored use of immunosuppressants should be considered based on the patient’s risks of graft rejection and infection and the patient’s medical comorbidities and liver disease status prior to LT [14]. Patients with autoimmune liver diseases may require more immunosuppression to prevent disease recurrence and graft rejection [20]. By contrast, lower doses of immunosuppression are recommended in patients who have undergone LT due to HCV, as high levels of immunosuppression are related to increased viral replication [21,22]. Rejection results in abnormal liver function and is one of the most important factors associated with poor long-term graft and patient outcomes [23,24,25]. Tacrolimus reduces the risk of T-cell mediated rejection to protect graft function and is the cornerstone of a successful LT. However, short and long-term adverse effects of tacrolimus such as infection, chronic renal insufficiency, metabolic diseases (hyperlipidemia, hypertension, and diabetes mellitus), and malignancy have been widely reported and influence patients’’ long-term outcomes [7,8,9]. The early causes of death after LT within one year are infection and graft loss, and the late causes of death three years post-LT are malignancy, cardiovascular disease, and renal failure [10]. As graft function typically stabilizes, factors associated with the long-term outcome are often patient-related factors (such as chronic medical diseases) that are usually associated with tacrolimus.
Previous studies have reported that minimizing tacrolimus use in the early post-LT period is associated with a lower risk of new-onset diabetes mellitus [26], a lower incidence of hyperlipidemia [27], and better long-term survival [19,28]. In recent years, studies have focused on the reduction or complete withdrawal of long-term immunosuppressants in LT recipients [12,29,30,31,32,33,34]. One study reported satisfactory outcomes with the combined use of tacrolimus and mycophenolate mofetil, which allowed for the tacrolimus dose to be reduced [35]. Other studies demonstrated that the concomitant use of everolimus may reduce the required dose of tacrolimus while having potential renal benefits [29,36]. However, only some LT recipients were able to discontinue the use of immunosuppressants, and these patients more frequently experienced biopsy-proven acute rejection [29]. The risk of chronic rejection during long-term follow-up remains in patients with inadequate immunosuppression [30,33]. The tacrolimus blood concentration in LT recipients may play a key role in long-term outcomes, as it is associated with long-term graft function and its adverse effects are related to several chronic medical diseases. However, to the best of our knowledge, this is the first report regarding the effect of long-term tacrolimus levels on the long-term survival outcomes of LT recipients.
Several factors are associated with the long-term outcomes of adult LT recipients, including pre-transplant primary sclerosing cholangitis, immunosuppression therapies, acute and chronic rejections, malignancy, and metabolic syndrome [37,38]. As listed in Table 3, we found several independent risk factors of long-term mortality in this study. Only four patients aged <27 years at the time of LT were included in this study, and one died due to pneumonia 6.3 years after LT. Patients who underwent LT due to autoimmune liver diseases had worse outcomes, as they were more likely to experience acute rejections [39] and suffer disease recurrence, leading to graft loss. Patients who underwent LT due to HCV infections had relatively poor post-LT outcomes due to the disease recurrence followed by graft dysfunction and failure [40]. As direct-acting antiviral agents have advanced [41], the survival outcomes of HCV patients should improve in the near future. Long-term renal and liver functions are reflected by serum creatinine and total bilirubin levels, respectively. We found that abnormal serum creatinine levels have a bigger impact than abnormal serum total bilirubin levels on the survival outcomes of LT recipients. The graft function of adult LT recipients is chronically stable five years after LT. While some patients may experience asymptomatic hyperbilirubinemia at this time, the long-term outcomes remain favorable. By contrast, the gradual deterioration of renal function may lead to chronic kidney disease or end-stage renal disease, affecting the long-term survival, especially in patients with long-term use of tacrolimus.
This study had some limitations. First, the data were obtained from a single medical center in Asia, which provided a relatively small number of eligible patients with single ethnicity and fewer death events. Second, the study spanned a long time period, and therefore the improvements in surgical and medical expertise and advances in immunosuppression therapies may have influenced the patient outcomes. Third, we did not collect or analyze the time-dependent tacrolimus blood trough level during the follow-up visits five years after LT.
In summary, we found an association between the long-term tacrolimus blood trough levels and the long-term survival five years after LT. A mean tacrolimus blood trough level outside the range of 4.6–10.2 ng/mL appeared to be an independent risk factor for long-term mortality. Further studies with larger sample sizes are needed to verify these results and to further identify an appropriate tacrolimus blood trough level for maintenance use.
Acknowledgments
We thank the coordinators (Hui-Ying Lin and Min-Heuy Lin) for their helpful efforts in data collection.
Author Contributions
C.-Y.H. drafted the manuscript and R.-H.H. designed the study. C.-Y.H., M.-C.H., and Y.-M.W. conducted data processing, and C.-Y.H. and C.-M.H. performed data analysis. P.-H.L. and R.-H.H. were the directors responsible for general organization and instruction. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of National Taiwan University Hospital.
Informed Consent Statement
Patient consent was waived due to retrospective design of the study.
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1 Patient flow diagram.
Figure 2 The box plot of tacrolimus mean trough level stratified by the etiology and cause of death among the 16 dead patients. There was no statistical difference of tacrolimus level between the different causes of death (p = 0.3823).
Figure 3 The p-spline plot for the nonlinear effect of the long-term tacrolimus blood trough levels on time to death. The tacrolimus blood trough level is shown on the X-axis, and the log(λ), where λ is the hazard rate of time to death five years after liver transplantation, is shown on the Y-axis. In this p-spline plot, the intersection between the horizontal green line (Y = 0) and the red curve yields the estimated optimal cut-off values for long-term tacrolimus blood trough levels at which the values of log(λ) will not change (4.609 ng/mL and 10.168 ng/mL). When the level is ≤4.609 ng/mL or >10.168 ng/mL, the value of log(λ) increases, indicating an increasing λ. When the level is >4.609 ng/mL and ≤10.168 ng/mL, the value of log(λ) decreases, indicating a decreasing λ. The vertical bars above the X-axis represent the patients’ actual mean tacrolimus blood trough levels obtained at the three follow-up visits during the fifth year after LT.
Figure 4 The Kaplan-Meier curve for time to death five years after liver transplantation. The survival curve of the 66 patients with a mean tacrolimus blood trough level ≤ 4.609 ng/mL or >10.168 ng/mL is shown in red while the survival curve of the 123 patients with a mean tacrolimus blood trough level > 4.609 ng/mL or ≤10.168 ng/mL is shown in green.
jpm-11-00090-t001_Table 1 Table 1 Univariate analysis for comparing the distributions of the demographic and clinical characteristics between the alive and dead adult liver recipients after five years of liver transplantations.
Variable All Patients
(n = 189) Alive
(n = 173) Dead
(n = 16) p Value
Gender 0.7899
Male 121 (64.0) 110 (90.9) 11 (9.1)
Female 68 (36.0) 63 (92.6) 5 (7.4)
Age at LT (years) 52.7 ± 9.6 52.6 ± 9.5 53.4 ± 10.7 0.6672
Body weight at LT (kg) 64.9 ± 12.2 65.0 ± 12.3 63.7 ± 12.1 0.4459
Blood type 0.8719
O 79 (41.8) 73 (92.4) 6 (7.6)
A 51 (27.0) 46 (90.2) 5 (9.8)
B 42 (22.2) 39 (92.9) 3 (7.1)
AB 17 (9.0) 15 (88.2) 2 (11.8)
Graft type 1.0000
Living donor 144 (76.2) 132 (91.7) 12 (8.3)
Deceased donor 45 (23.8) 41 (91.1) 4 (8.9)
Etiology for LT
Liver malignancy (HCC) 72 (38.1) 66 (91.7) 6 (8.3) 1.0000
Alcoholic cirrhosis 17 (9.0) 16 (94.1) 1 (5.9) 1.0000
HBV cirrhosis 100 (52.9) 92 (92.0) 8 (8.0) 1.0000
HCV cirrhosis 42 (22.2) 38 (90.5) 4 (9.5) 0.7577
Fulminant hepatitis 23 (12.2) 23 (100) 0 (0) 0.2259
Autoimmune disease 14 (7.4) 11 (78.6) 3 (21.4) 0.1017
Biliary atresia 5 (2.6) 5 (100) 0 (0) 1.0000
Other 10 (5.3) 9 (90) 1 (10) 0.5964
Total bilirubin (mg/dL) 0.973 ± 0.51 0.958 ± 1.53 1.133 ± 0.69 0.6260
Total bilirubin > 1 mg/dL 60 (31.7) 53 (88.3) 7 (11.7) 0.2770
Total bilirubin > 2 mg/dL 11 (5.8) 9 (81.8) 2 (18.2) 0.2360
Creatinine (mg/dL) 1.391 ± 1.25 1.279 ± 0.96 2.6 ± 2.74 0.0201 *
Creatinine > 1.5 mg/dL 31 (16.4) 23 (74.2) 8 (25.8) 0.0010 *
ESRD 5 (2.6) 2 (40.0) 3 (60.0) 0.0050 *
Tacrolimus mean level (ng/mL) 5.249 ± 1.71 5.263 ± 1.53 5.096 ± 3.12 0.9787
Tacrolimus level < 5 ng/mL 93 (49.2) 82 (88.2) 11 (11.8) 0.1216
Tacrolimus level < 4 ng/mL 44 (23.3) 36 (81.8) 8 (18.2) 0.0136 *
Tacrolimus level < 3 ng/mL 11 (5.8) 9 (81.8) 2 (18.2) 0.2356
Data are presented as mean ± standard deviation (SD) for continuous variables and frequency (percentage, %) for categorical variables. The p-values of statistical tests were calculated using the Wilcoxon rank-sum test for continuous variables and the Fisher’s exact test for categorical variables. * p value ≤ 0.05. Abbreviations: LT, liver transplantation; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; Total bilirubin, serum total bilirubin level; Creatinine, serum creatinine level; and ESRD, end-stage renal disease (defined by receiving hemodialysis regularly).
jpm-11-00090-t002_Table 2 Table 2 The causes of 16 deaths since 5 years after adult liver transplantations.
Causes of Deaths Number of Subjects
Malignancy 5 (31.25%)
De novo: Multiple myeloma, bladder cancer, colon cancer, prostate cancer 4
Recurrent: Hepatocellular carcinoma 1
Graft failure 4 (25.00%)
Chronic rejection 3
Autoimmune hepatitis 1
Infection 4 (25.00%)
Pneumonia 3
Urinary tract infection 1
Cerebral vascular event (intracerebral hemorrhage) 2 (12.50%)
Peptic ulcer bleeding 1 (6.25%)
jpm-11-00090-t003_Table 3 Table 3 Multivariate analysis for identifying the predictors of long-term overall survival after 5 years of liver transplantations by fitting a multiple Cox’s Model in the adult liver transplant recipients 1.
Covariate 2 Estimate Standard
Error Wald’s
z Test p Value Hazard
Ratio (HR) 95% Confidence
Interval (C.I.)
Age at LT ≤ 27.011 years 5.1286 1.3872 3.6970 0.0002 168.7851 11.130–2559.512
Autoimmune (including PBC) 2.0946 0.7219 2.9015 0.0037 8.1221 1.973–33.431
HCV × Overall survival years 0.2924 0.0914 3.1978 0.0014 1.3397 1.120–1.603
Cre > 1.311 × T-Bil > 1.411 mg/dL 6.8262 1.5591 4.3784 <0.0001 921.6940 43.401–19,573.712
Cre > 1.311 × T-Bil ≤ 0.792 mg/dL 4.6604 1.3294 3.5056 0.0005 105.6778 7.805–1430.790
Cre ≤ 1.311 × T-Bil > 0.882 mg/dL 3.4174 1.1871 2.8788 0.0040 30.4913 2.977–312.341
Tacrolimus mean ≤ 4.609 or > 10.168 ng/mL 1.5599 0.6479 2.4076 0.0161 4.7581 1.336–16.940
1 The above multiple Cox’s model was fitted to the 189 adult patients who underwent liver transplantations with 16 death events, for modeling the hazard rate of the right-censored overall survival time five years after liver transplantations. All the cut-off values of the continuous covariates (e.g., age at liver transplantations) were estimated by choosing the option of applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models (e.g., Figure 3). The time-dependent interaction term, HCV × Overall survival year, was added to the Cox’s model for handling the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.2924, indicated that the risk of dying in the patients with HCV would increase as time elapsed five years after liver transplantations. Moreover, the three second-order interaction terms, Cre > 1.311 × T-Bil > 1.411, Cre > 1.311 × T-Bil ≤ 0.792, and Cre ≤ 1.311 × T-Bil > 0.882, were compared to the other two possible combinations, Cre > 1.311 × (0.792 < T-Bil ≤ 1.411) and Cre ≤ 1.311 × T-Bil ≤ 0.882, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and.” Finally, both goodness-of-fit (GOF) measures, concordance = 0.9041 (se = 0.0265) > 0.7 and adjusted generalized R2 = 0.3878 > 0.15, indicated an excellent fit of this multiple Cox’s model to the observed survival data. 2 Abbreviations: LT, liver transplantation; PBC, primary biliary cirrhosis; HCV, hepatitis C viral infection; Cre, serum creatinine level (mg/dL); T-Bil, serum total bilirubin level (mg/dL); and Tacrolimus mean, the averaged dosage of the tacrolimus-based immunosuppressant (ng/mL).
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. | Oral | DrugAdministrationRoute | CC BY | 33535628 | 18,997,175 | 2021-02-01 |
What was the dosage of drug 'BASILIXIMAB'? | Long-Term Tacrolimus Blood Trough Level and Patient Survival in Adult Liver Transplantation.
Tacrolimus is the most widely used immunosuppressant in liver transplant (LT) patients. However, the ideal long-term target level for these patients is unknown. This retrospective study aimed to investigate the impact of tacrolimus blood concentration five years after LT on long-term patient survival outcomes in adult LT recipients. Patients who underwent LT between January 2004 and July 2014 at a tertiary medical center were included in this study (n = 189). The mean tacrolimus blood concentrations of each patient during the fifth year after LT were recorded and the overall survival rate was determined. A multivariate analysis of factors associated with long-term survival was conducted using a Cox's model. The median follow-up period was 9.63 years, and 144 patients (76.2%) underwent live donor LT. Sixteen patients died within 5 years of LT. In the Cox's model, patients with a mean tacrolimus blood trough level of 4.6-10.2 ng/mL had significantly better long-term survival than those with a mean tacrolimus blood trough level outside this range (estimated hazard ratio = 4.76; 95% confidence interval: 1.34-16.9, p = 0.016). Therefore, a tacrolimus level no lower than 4.6 ng/mL would be recommended in adult LT patients.
1. Introduction
Liver transplantation (LT) has become a mature treatment of end-stage liver disease in clinical practice [1]. The continuous improvement of effective immunosuppression treatments has led to a significant improvement in patient and graft survival in recent years [2]. Calcineurin inhibitors are the cornerstone of immunosuppression in LT [3], and tacrolimus is currently the mainstay of initial and maintenance immunosuppression therapies [4,5,6]. Tacrolimus reduces the incidence and severity of early and late T-cell mediated rejection by inhibiting T-cell production of interleukin-2 [5,6]. However, the long-term use of immunosuppressants leads to an increasing burden of toxicity. The reported toxic effects of calcineurin inhibitors include infections, chronic renal insufficiency, metabolic diseases (such as hyperlipidemia, hypertension, and diabetes mellitus), and malignancy [7,8,9], which, along with cardiovascular diseases, have been reported to be the major causes of morbidity and mortality after LT [3,10]. However, most LT patients cannot withdraw from lifelong immunosuppression therapy; the only exceptions are a few selected patients participating in experimental trials [11,12].
It is a clinical challenge to design a well-balanced immunosuppressive regimen for LT recipients. Tacrolimus has a narrow therapeutic dose range and its blood levels should be carefully monitored. The Advagraf (tacrolimus prolonged-release hard capsules) recommendation for adult LT patients is a blood trough level of 5–20 ng/mL in the early post-transplant period and 5–15 ng/mL during subsequent maintenance therapy. Nevertheless, the current recommendations from the clinical practice guidelines for the ideal tacrolimus level in adults after LT remain controversial. The American Association for the Study of Liver Diseases (AASLD) recommends a target blood trough level of 5–10 ng/mL for tacrolimus three months after LT [13]. The Consensus on Managing Modifiable Risk in Transplantation Group (COMMIT) recommends that the target tacrolimus blood trough levels be 6–10 ng/mL during the first month after LT and decrease to 4–8 ng/mL thereafter, except when used in combination with mammalian target of rapamycin (mTOR) inhibitors [14]. Moreover, the International Liver Transplant Society (ILTS) consensus statement on immunosuppression in LT recipients recommends the target blood trough levels of tacrolimus be 6–10 ng/mL three months after LT, lower than 5 ng/mL 12 months after LT, and decrease to 3 ng/mL thereafter, resulting in a blood trough level just above the lower limit of detection five years after LT [15]. However, the impact of the long-term tacrolimus blood trough level on the outcomes of the LT recipients remains unclear. This study aimed to suggest an appropriate tacrolimus blood trough level for adult patients five years after LT.
2. Materials and Methods
This study was approved by the Institutional Review Board of National Taiwan University Hospital and was conducted according to the Declaration of Helsinki. A total of 286 patients who underwent LT at 18 years or older at a tertiary medical center from January 2004 to July 2014 were recruited for this study. Patients who died within 5 years of LT, were lost to follow-up, or did not use tacrolimus-based calcineurin inhibitor for immunosuppressant therapy were excluded from this study (Figure 1). In addition, those who received mTOR inhibitor treatment were excluded, as mTOR inhibitors are typically used as a combination therapy to reduce the required dose of tacrolimus. The final analysis included 189 patients. All eligible patients were followed up for more than five years until August 2019. Patients who underwent a LT due to liver cancer met the Milan criteria (before 2006) or the criteria of the University of California, San Francisco (since 2006) at the time of LT. All patients received regular monthly or bi-monthly follow-ups at the outpatient clinic after LT. Routine blood examinations for the tacrolimus blood trough level, liver function, and renal function were conducted at each visit, and abdominal sonography was performed every 6 to 12 months.
The patients’ medical records were reviewed retrospectively to extract demographic and clinical data, including patient characteristics, laboratory tests, and survival outcomes. The serum bilirubin and creatinine data at the end of the fifth year after LT were used in this study. The tacrolimus level used in this study was the mean of the values obtained at the three follow-up visits during the fifth year after LT.
The immunosuppression protocol for adult LT patients consisted of tacrolimus, mycophenolate mofetil, and steroids. Tacrolimus was administered orally beginning on the first day after LT, and the dose was adjusted to achieve the desired therapeutic drug level. Basiliximab was administered immediately before graft reperfusion and on the fourth day after LT for induction therapy. A 500-mg intravenous bolus of methylprednisolone was administered immediately before reperfusion of the liver graft and was tapered to oral prednisolone over one week and reduced to withdrawal after six months.
All statistical analyses were performed using R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p ≤ 0.05. Continuous variables are presented as mean ± standard deviation (SD) and median (interquartile range, IQR), and categorical variables are presented as frequency (percentage, %). The survival curve was estimated by the Kaplan-Meier method. A univariate analysis was conducted to examine the differences in the distributions of continuous variables, categorical variables, and survival outcomes between the surviving and deceased liver recipients five years after LT using the Wilcoxon rank-sum test, Chi-square test, Fisher’s exact test, or log-rank test as appropriate for the data type. A multivariate analysis was performed to estimate the adjusted effects of risk factors or prognostic factors on the survival outcome using a multiple Cox’s proportional hazards model.
To ensure a good quality of regression analysis, the model-fitting techniques for variable selection, goodness-of-fit (GOF) assessment, and regression diagnostics and remedies were used in our regression analysis. Specifically, the stepwise variable selection procedure (with iterations between the forward and backward steps) was applied to obtain the best final regression model using the My.stepwise package of the R software [16]. All significant and non-significant relevant covariates from the univariate analysis (listed in Table 1) and some of the interaction terms were used in the multivariate analysis. The significance levels for entry and for stay were set to 0.15 for being conservative. With the aid of substantive knowledge, the best candidate final regression model was identified manually by dropping the covariates with p value > 0.05 one at a time until all regression coefficients were significantly different from 0. To assess the GOF of the fitted Cox’s model, the concordance and adjusted generalized R2 [17] were examined. A concordance ≥ 0.7 and an adjusted generalized R2 > 0.15 indicated an acceptable level of discrimination, power, and fitness.
Moreover, the smoothing option “pspline” (for the smoothing splines using a “p-spline” basis) was specified inside the coxph function of the survival package to smooth the effects of continuous covariates on the log-hazard rate of the simple and multiple Cox’s proportional hazards models in R. Then, the termplot function of the stats package was used to plot the smoothed effects of the continuous covariates on the log-hazard rate in R [18]. The regression diagnostics for the verification of proportional hazards assumption, residual analysis, detection of influential cases, and a multicollinearity check were applied to discover any model or data problems. A variance inflating factor (VIF) ≥ 10 in continuous covariates or VIF ≥ 2.5 in categorical covariates indicated the occurrence of the multicollinearity problem among some of the covariates in the fitted regression model.
3. Results
3.1. Patients’ Demographic and Clinical Characteristics
The median follow-up duration was 9.63 years (IQR: 7.2–11.4 years), and the mean follow-up was 9.58 ± 2.74 years. A total of 121 males (64.0%) and 68 females (36.0%) were included in this study (Table 1). The median age at LT was 54.26 years (IQR: 48.63–58.58 years, range: 18.3–73.1 years), and the mean age at LT was 52.7 ± 9.6 years. Among the 189 patients, 114 (76.2%) underwent living donor transplants and 45 (23.8%) underwent deceased donor transplants. The main indications for LT were hepatitis B virus (HBV) cirrhosis (52.9%), hepatocellular carcinoma (38.1%), hepatitis C virus (HCV) cirrhosis (22.2%), and fulminant hepatitis (12.2%). The median of the mean tacrolimus blood trough level during the fifth year after LT was 5.0 ng/mL (IQR: 4.12–6.33 ng/mL), and the mean tacrolimus blood trough level was 5.249 ± 1.71 ng/mL. The mean tacrolimus blood trough level was ≥ 5 ng/mL in 96 patients (50.8%) and < 5 ng/mL in 93 patients (49.2%), including 44 patients (23.3%) with a mean tacrolimus blood trough level < 4 ng/mL and 11 patients (5.8%) with a mean tacrolimus blood trough level < 3 ng/mL. No significant differences in gender, age at LT, body weight, blood type, graft type, etiology of LT, or serum total bilirubin were found between the deceased and surviving LT recipients (p > 0.05). However, serum creatinine levels > 1.5 mg/dL (23/173 vs. 8/16, p = 0.001), end-stage renal disease (2/173 vs. 3/16, p = 0.005), and mean tacrolimus blood trough levels < 4 ng/mL (36/173 vs. 8/16, p = 0.014) were significantly more common among deceased LT recipients compared to survivors. The causes of death included malignancies (n = 5), graft failures (n = 4), infection (n = 4), cerebrovascular accidents (n = 2), and duodenal ulcer bleeding (n = 1) (Table 2). The distribution of the tacrolimus trough level stratified by the etiology and cause of death among the 16 dead patients was shown in the box plot of Figure 2. There was no statistical significance in the tacrolimus trough level between the different causes of death (p = 0.3823).
3.2. Predictors of Patients’ Long-Term Survival
The Cox’s model fitted to the survival data for the multivariate analyses of the time to death after five years of LT is shown in Table 3. After adjusting for the effects of the other covariates, age at LT ≤ 27.011 years (estimated hazard ratio [HR] = 168.79, 95% confidence interval [C.I.]: 11.13–2559.51), pre-transplant autoimmune liver disease (HR = 8.12, 95% C.I.: 1.97–33.43), pre-transplant HCV infection × survival time in years (HR = 1.34, 95% C.I.: 1.12–1.60), serum creatinine level > 1.311 mg/dL × serum total bilirubin level > 1.411 mg/dL (HR = 921.69, 95% C.I.: 43.40–19,573.71), serum creatinine level > 1.311 mg/dL × serum total bilirubin level ≤ 0.792 mg/dL (HR = 105.68, 95% C.I.: 7.81–1430.79), serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level > 0.882 mg/dL (HR = 30.49, 95% C.I.: 2.98–312.34), and the mean tacrolimus trough level during the fifth year after LT ≤ 4.609 ng/mL or > 10.168 ng/mL (HR = 4.76, 95% C.I.: 1.34–16.94) were associated with a higher long-term mortality five years after LT. The time-dependent interaction term, HCV × survival time in years, was added to the Cox’s model to account for the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.292, indicates that the risk of mortality in patients with HCV increases with time five years post-LT. Moreover, the three second-order interaction terms between the serum creatinine and total bilirubin levels were compared to the other two possible combinations, serum creatinine level > 1.311 mg/dL × (0.792 mg/dL < serum total bilirubin level ≤ 1.411 mg/dL) and serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level ≤ 0.882 mg/dL, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and”.
All cut-off values of the continuous covariates (such as age at LT) were estimated by applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models. As shown in Figure 3, the optimal cut-off values of the mean tacrolimus trough level, 4.609 ng/mL and 10.168 ng/mL, were estimated directly in the “p-spline plot,” which allowed the visualization of the nonlinear effect of the averaged dosage of the tacrolimus-based immunosuppressant during the fifth year after LT on log(λ), where λ was the hazard rate of time to death five years after LT. Then, the Kaplan-Meier estimates of survival curves for time to death five years after LT were determined for the 66 patients with mean tacrolimus trough levels ≤ 4.609 ng/mL or > 10.168 ng/mL and the 123 patients with mean tacrolimus trough levels between 4.609 ng/mL and 10.168 ng/mL (log-rank test, p = 0.009) (Figure 4). In an additional subgroup analysis, we found that a mean tacrolimus trough level between 4.431 ng/mL and 6.332 ng/mL for patients with a serum creatinine level > 1.311 mg/dL improved survival. Finally, this Cox’s model had a concordance of 0.904 and an adjusted generalized R2 of 0.388, indicating that it fit the survival data very well.
4. Discussion
This is the first study to report an association between long-term tacrolimus blood trough level and long-term patient survival in adult LT recipients. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL in adult LT recipients is associated with a lower mortality rate. In patients with a serum creatinine > 1.3 mg/dL, a mean tacrolimus blood trough level of 4.4–6.3 ng/mL is associated with a lower mortality rate.
Decreasing tacrolimus use during long-term follow-up in LT recipients is a general concept in clinical practice. In this study, only one patient had the mean level > 10 ng/mL and a few patients had the mean level > 8 ng/mL (Figure 3). Patients with such high drug levels were most likely because of their poor compliance (not must because of they have truly such high trough level). Poor compliance indicates that a patient takes incorrect dosage of immunosuppressant that their physician prescribed, or takes immunosuppressant or blood exam at the wrong time, either of which might clearly be important factors that influence graft and survival outcome of LT patients during long-term follow-ups. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL is associated with lower mortality rate; however, the upper limit of 10.2 ng/mL suggested by the regression analysis was merely a value for caution, but not a recommended upper limit in real clinical practice. The long-term hazard of high level of tacrolimus is already widely known; therefore, we would focus more on the findings that it appears to increase the survival risks in LT recipients if their tacrolimus levels are maintained below 4.6 ng/mL during the long-term follow-ups.
Inadequate immunosuppression is associated with higher risks of graft rejection [19], while excess immunosuppression increases the risks of malignancy and infection and increases the adverse effects of drug toxicity. The tailored use of immunosuppressants should be considered based on the patient’s risks of graft rejection and infection and the patient’s medical comorbidities and liver disease status prior to LT [14]. Patients with autoimmune liver diseases may require more immunosuppression to prevent disease recurrence and graft rejection [20]. By contrast, lower doses of immunosuppression are recommended in patients who have undergone LT due to HCV, as high levels of immunosuppression are related to increased viral replication [21,22]. Rejection results in abnormal liver function and is one of the most important factors associated with poor long-term graft and patient outcomes [23,24,25]. Tacrolimus reduces the risk of T-cell mediated rejection to protect graft function and is the cornerstone of a successful LT. However, short and long-term adverse effects of tacrolimus such as infection, chronic renal insufficiency, metabolic diseases (hyperlipidemia, hypertension, and diabetes mellitus), and malignancy have been widely reported and influence patients’’ long-term outcomes [7,8,9]. The early causes of death after LT within one year are infection and graft loss, and the late causes of death three years post-LT are malignancy, cardiovascular disease, and renal failure [10]. As graft function typically stabilizes, factors associated with the long-term outcome are often patient-related factors (such as chronic medical diseases) that are usually associated with tacrolimus.
Previous studies have reported that minimizing tacrolimus use in the early post-LT period is associated with a lower risk of new-onset diabetes mellitus [26], a lower incidence of hyperlipidemia [27], and better long-term survival [19,28]. In recent years, studies have focused on the reduction or complete withdrawal of long-term immunosuppressants in LT recipients [12,29,30,31,32,33,34]. One study reported satisfactory outcomes with the combined use of tacrolimus and mycophenolate mofetil, which allowed for the tacrolimus dose to be reduced [35]. Other studies demonstrated that the concomitant use of everolimus may reduce the required dose of tacrolimus while having potential renal benefits [29,36]. However, only some LT recipients were able to discontinue the use of immunosuppressants, and these patients more frequently experienced biopsy-proven acute rejection [29]. The risk of chronic rejection during long-term follow-up remains in patients with inadequate immunosuppression [30,33]. The tacrolimus blood concentration in LT recipients may play a key role in long-term outcomes, as it is associated with long-term graft function and its adverse effects are related to several chronic medical diseases. However, to the best of our knowledge, this is the first report regarding the effect of long-term tacrolimus levels on the long-term survival outcomes of LT recipients.
Several factors are associated with the long-term outcomes of adult LT recipients, including pre-transplant primary sclerosing cholangitis, immunosuppression therapies, acute and chronic rejections, malignancy, and metabolic syndrome [37,38]. As listed in Table 3, we found several independent risk factors of long-term mortality in this study. Only four patients aged <27 years at the time of LT were included in this study, and one died due to pneumonia 6.3 years after LT. Patients who underwent LT due to autoimmune liver diseases had worse outcomes, as they were more likely to experience acute rejections [39] and suffer disease recurrence, leading to graft loss. Patients who underwent LT due to HCV infections had relatively poor post-LT outcomes due to the disease recurrence followed by graft dysfunction and failure [40]. As direct-acting antiviral agents have advanced [41], the survival outcomes of HCV patients should improve in the near future. Long-term renal and liver functions are reflected by serum creatinine and total bilirubin levels, respectively. We found that abnormal serum creatinine levels have a bigger impact than abnormal serum total bilirubin levels on the survival outcomes of LT recipients. The graft function of adult LT recipients is chronically stable five years after LT. While some patients may experience asymptomatic hyperbilirubinemia at this time, the long-term outcomes remain favorable. By contrast, the gradual deterioration of renal function may lead to chronic kidney disease or end-stage renal disease, affecting the long-term survival, especially in patients with long-term use of tacrolimus.
This study had some limitations. First, the data were obtained from a single medical center in Asia, which provided a relatively small number of eligible patients with single ethnicity and fewer death events. Second, the study spanned a long time period, and therefore the improvements in surgical and medical expertise and advances in immunosuppression therapies may have influenced the patient outcomes. Third, we did not collect or analyze the time-dependent tacrolimus blood trough level during the follow-up visits five years after LT.
In summary, we found an association between the long-term tacrolimus blood trough levels and the long-term survival five years after LT. A mean tacrolimus blood trough level outside the range of 4.6–10.2 ng/mL appeared to be an independent risk factor for long-term mortality. Further studies with larger sample sizes are needed to verify these results and to further identify an appropriate tacrolimus blood trough level for maintenance use.
Acknowledgments
We thank the coordinators (Hui-Ying Lin and Min-Heuy Lin) for their helpful efforts in data collection.
Author Contributions
C.-Y.H. drafted the manuscript and R.-H.H. designed the study. C.-Y.H., M.-C.H., and Y.-M.W. conducted data processing, and C.-Y.H. and C.-M.H. performed data analysis. P.-H.L. and R.-H.H. were the directors responsible for general organization and instruction. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of National Taiwan University Hospital.
Informed Consent Statement
Patient consent was waived due to retrospective design of the study.
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1 Patient flow diagram.
Figure 2 The box plot of tacrolimus mean trough level stratified by the etiology and cause of death among the 16 dead patients. There was no statistical difference of tacrolimus level between the different causes of death (p = 0.3823).
Figure 3 The p-spline plot for the nonlinear effect of the long-term tacrolimus blood trough levels on time to death. The tacrolimus blood trough level is shown on the X-axis, and the log(λ), where λ is the hazard rate of time to death five years after liver transplantation, is shown on the Y-axis. In this p-spline plot, the intersection between the horizontal green line (Y = 0) and the red curve yields the estimated optimal cut-off values for long-term tacrolimus blood trough levels at which the values of log(λ) will not change (4.609 ng/mL and 10.168 ng/mL). When the level is ≤4.609 ng/mL or >10.168 ng/mL, the value of log(λ) increases, indicating an increasing λ. When the level is >4.609 ng/mL and ≤10.168 ng/mL, the value of log(λ) decreases, indicating a decreasing λ. The vertical bars above the X-axis represent the patients’ actual mean tacrolimus blood trough levels obtained at the three follow-up visits during the fifth year after LT.
Figure 4 The Kaplan-Meier curve for time to death five years after liver transplantation. The survival curve of the 66 patients with a mean tacrolimus blood trough level ≤ 4.609 ng/mL or >10.168 ng/mL is shown in red while the survival curve of the 123 patients with a mean tacrolimus blood trough level > 4.609 ng/mL or ≤10.168 ng/mL is shown in green.
jpm-11-00090-t001_Table 1 Table 1 Univariate analysis for comparing the distributions of the demographic and clinical characteristics between the alive and dead adult liver recipients after five years of liver transplantations.
Variable All Patients
(n = 189) Alive
(n = 173) Dead
(n = 16) p Value
Gender 0.7899
Male 121 (64.0) 110 (90.9) 11 (9.1)
Female 68 (36.0) 63 (92.6) 5 (7.4)
Age at LT (years) 52.7 ± 9.6 52.6 ± 9.5 53.4 ± 10.7 0.6672
Body weight at LT (kg) 64.9 ± 12.2 65.0 ± 12.3 63.7 ± 12.1 0.4459
Blood type 0.8719
O 79 (41.8) 73 (92.4) 6 (7.6)
A 51 (27.0) 46 (90.2) 5 (9.8)
B 42 (22.2) 39 (92.9) 3 (7.1)
AB 17 (9.0) 15 (88.2) 2 (11.8)
Graft type 1.0000
Living donor 144 (76.2) 132 (91.7) 12 (8.3)
Deceased donor 45 (23.8) 41 (91.1) 4 (8.9)
Etiology for LT
Liver malignancy (HCC) 72 (38.1) 66 (91.7) 6 (8.3) 1.0000
Alcoholic cirrhosis 17 (9.0) 16 (94.1) 1 (5.9) 1.0000
HBV cirrhosis 100 (52.9) 92 (92.0) 8 (8.0) 1.0000
HCV cirrhosis 42 (22.2) 38 (90.5) 4 (9.5) 0.7577
Fulminant hepatitis 23 (12.2) 23 (100) 0 (0) 0.2259
Autoimmune disease 14 (7.4) 11 (78.6) 3 (21.4) 0.1017
Biliary atresia 5 (2.6) 5 (100) 0 (0) 1.0000
Other 10 (5.3) 9 (90) 1 (10) 0.5964
Total bilirubin (mg/dL) 0.973 ± 0.51 0.958 ± 1.53 1.133 ± 0.69 0.6260
Total bilirubin > 1 mg/dL 60 (31.7) 53 (88.3) 7 (11.7) 0.2770
Total bilirubin > 2 mg/dL 11 (5.8) 9 (81.8) 2 (18.2) 0.2360
Creatinine (mg/dL) 1.391 ± 1.25 1.279 ± 0.96 2.6 ± 2.74 0.0201 *
Creatinine > 1.5 mg/dL 31 (16.4) 23 (74.2) 8 (25.8) 0.0010 *
ESRD 5 (2.6) 2 (40.0) 3 (60.0) 0.0050 *
Tacrolimus mean level (ng/mL) 5.249 ± 1.71 5.263 ± 1.53 5.096 ± 3.12 0.9787
Tacrolimus level < 5 ng/mL 93 (49.2) 82 (88.2) 11 (11.8) 0.1216
Tacrolimus level < 4 ng/mL 44 (23.3) 36 (81.8) 8 (18.2) 0.0136 *
Tacrolimus level < 3 ng/mL 11 (5.8) 9 (81.8) 2 (18.2) 0.2356
Data are presented as mean ± standard deviation (SD) for continuous variables and frequency (percentage, %) for categorical variables. The p-values of statistical tests were calculated using the Wilcoxon rank-sum test for continuous variables and the Fisher’s exact test for categorical variables. * p value ≤ 0.05. Abbreviations: LT, liver transplantation; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; Total bilirubin, serum total bilirubin level; Creatinine, serum creatinine level; and ESRD, end-stage renal disease (defined by receiving hemodialysis regularly).
jpm-11-00090-t002_Table 2 Table 2 The causes of 16 deaths since 5 years after adult liver transplantations.
Causes of Deaths Number of Subjects
Malignancy 5 (31.25%)
De novo: Multiple myeloma, bladder cancer, colon cancer, prostate cancer 4
Recurrent: Hepatocellular carcinoma 1
Graft failure 4 (25.00%)
Chronic rejection 3
Autoimmune hepatitis 1
Infection 4 (25.00%)
Pneumonia 3
Urinary tract infection 1
Cerebral vascular event (intracerebral hemorrhage) 2 (12.50%)
Peptic ulcer bleeding 1 (6.25%)
jpm-11-00090-t003_Table 3 Table 3 Multivariate analysis for identifying the predictors of long-term overall survival after 5 years of liver transplantations by fitting a multiple Cox’s Model in the adult liver transplant recipients 1.
Covariate 2 Estimate Standard
Error Wald’s
z Test p Value Hazard
Ratio (HR) 95% Confidence
Interval (C.I.)
Age at LT ≤ 27.011 years 5.1286 1.3872 3.6970 0.0002 168.7851 11.130–2559.512
Autoimmune (including PBC) 2.0946 0.7219 2.9015 0.0037 8.1221 1.973–33.431
HCV × Overall survival years 0.2924 0.0914 3.1978 0.0014 1.3397 1.120–1.603
Cre > 1.311 × T-Bil > 1.411 mg/dL 6.8262 1.5591 4.3784 <0.0001 921.6940 43.401–19,573.712
Cre > 1.311 × T-Bil ≤ 0.792 mg/dL 4.6604 1.3294 3.5056 0.0005 105.6778 7.805–1430.790
Cre ≤ 1.311 × T-Bil > 0.882 mg/dL 3.4174 1.1871 2.8788 0.0040 30.4913 2.977–312.341
Tacrolimus mean ≤ 4.609 or > 10.168 ng/mL 1.5599 0.6479 2.4076 0.0161 4.7581 1.336–16.940
1 The above multiple Cox’s model was fitted to the 189 adult patients who underwent liver transplantations with 16 death events, for modeling the hazard rate of the right-censored overall survival time five years after liver transplantations. All the cut-off values of the continuous covariates (e.g., age at liver transplantations) were estimated by choosing the option of applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models (e.g., Figure 3). The time-dependent interaction term, HCV × Overall survival year, was added to the Cox’s model for handling the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.2924, indicated that the risk of dying in the patients with HCV would increase as time elapsed five years after liver transplantations. Moreover, the three second-order interaction terms, Cre > 1.311 × T-Bil > 1.411, Cre > 1.311 × T-Bil ≤ 0.792, and Cre ≤ 1.311 × T-Bil > 0.882, were compared to the other two possible combinations, Cre > 1.311 × (0.792 < T-Bil ≤ 1.411) and Cre ≤ 1.311 × T-Bil ≤ 0.882, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and.” Finally, both goodness-of-fit (GOF) measures, concordance = 0.9041 (se = 0.0265) > 0.7 and adjusted generalized R2 = 0.3878 > 0.15, indicated an excellent fit of this multiple Cox’s model to the observed survival data. 2 Abbreviations: LT, liver transplantation; PBC, primary biliary cirrhosis; HCV, hepatitis C viral infection; Cre, serum creatinine level (mg/dL); T-Bil, serum total bilirubin level (mg/dL); and Tacrolimus mean, the averaged dosage of the tacrolimus-based immunosuppressant (ng/mL).
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What was the dosage of drug 'METHYLPREDNISOLONE'? | Long-Term Tacrolimus Blood Trough Level and Patient Survival in Adult Liver Transplantation.
Tacrolimus is the most widely used immunosuppressant in liver transplant (LT) patients. However, the ideal long-term target level for these patients is unknown. This retrospective study aimed to investigate the impact of tacrolimus blood concentration five years after LT on long-term patient survival outcomes in adult LT recipients. Patients who underwent LT between January 2004 and July 2014 at a tertiary medical center were included in this study (n = 189). The mean tacrolimus blood concentrations of each patient during the fifth year after LT were recorded and the overall survival rate was determined. A multivariate analysis of factors associated with long-term survival was conducted using a Cox's model. The median follow-up period was 9.63 years, and 144 patients (76.2%) underwent live donor LT. Sixteen patients died within 5 years of LT. In the Cox's model, patients with a mean tacrolimus blood trough level of 4.6-10.2 ng/mL had significantly better long-term survival than those with a mean tacrolimus blood trough level outside this range (estimated hazard ratio = 4.76; 95% confidence interval: 1.34-16.9, p = 0.016). Therefore, a tacrolimus level no lower than 4.6 ng/mL would be recommended in adult LT patients.
1. Introduction
Liver transplantation (LT) has become a mature treatment of end-stage liver disease in clinical practice [1]. The continuous improvement of effective immunosuppression treatments has led to a significant improvement in patient and graft survival in recent years [2]. Calcineurin inhibitors are the cornerstone of immunosuppression in LT [3], and tacrolimus is currently the mainstay of initial and maintenance immunosuppression therapies [4,5,6]. Tacrolimus reduces the incidence and severity of early and late T-cell mediated rejection by inhibiting T-cell production of interleukin-2 [5,6]. However, the long-term use of immunosuppressants leads to an increasing burden of toxicity. The reported toxic effects of calcineurin inhibitors include infections, chronic renal insufficiency, metabolic diseases (such as hyperlipidemia, hypertension, and diabetes mellitus), and malignancy [7,8,9], which, along with cardiovascular diseases, have been reported to be the major causes of morbidity and mortality after LT [3,10]. However, most LT patients cannot withdraw from lifelong immunosuppression therapy; the only exceptions are a few selected patients participating in experimental trials [11,12].
It is a clinical challenge to design a well-balanced immunosuppressive regimen for LT recipients. Tacrolimus has a narrow therapeutic dose range and its blood levels should be carefully monitored. The Advagraf (tacrolimus prolonged-release hard capsules) recommendation for adult LT patients is a blood trough level of 5–20 ng/mL in the early post-transplant period and 5–15 ng/mL during subsequent maintenance therapy. Nevertheless, the current recommendations from the clinical practice guidelines for the ideal tacrolimus level in adults after LT remain controversial. The American Association for the Study of Liver Diseases (AASLD) recommends a target blood trough level of 5–10 ng/mL for tacrolimus three months after LT [13]. The Consensus on Managing Modifiable Risk in Transplantation Group (COMMIT) recommends that the target tacrolimus blood trough levels be 6–10 ng/mL during the first month after LT and decrease to 4–8 ng/mL thereafter, except when used in combination with mammalian target of rapamycin (mTOR) inhibitors [14]. Moreover, the International Liver Transplant Society (ILTS) consensus statement on immunosuppression in LT recipients recommends the target blood trough levels of tacrolimus be 6–10 ng/mL three months after LT, lower than 5 ng/mL 12 months after LT, and decrease to 3 ng/mL thereafter, resulting in a blood trough level just above the lower limit of detection five years after LT [15]. However, the impact of the long-term tacrolimus blood trough level on the outcomes of the LT recipients remains unclear. This study aimed to suggest an appropriate tacrolimus blood trough level for adult patients five years after LT.
2. Materials and Methods
This study was approved by the Institutional Review Board of National Taiwan University Hospital and was conducted according to the Declaration of Helsinki. A total of 286 patients who underwent LT at 18 years or older at a tertiary medical center from January 2004 to July 2014 were recruited for this study. Patients who died within 5 years of LT, were lost to follow-up, or did not use tacrolimus-based calcineurin inhibitor for immunosuppressant therapy were excluded from this study (Figure 1). In addition, those who received mTOR inhibitor treatment were excluded, as mTOR inhibitors are typically used as a combination therapy to reduce the required dose of tacrolimus. The final analysis included 189 patients. All eligible patients were followed up for more than five years until August 2019. Patients who underwent a LT due to liver cancer met the Milan criteria (before 2006) or the criteria of the University of California, San Francisco (since 2006) at the time of LT. All patients received regular monthly or bi-monthly follow-ups at the outpatient clinic after LT. Routine blood examinations for the tacrolimus blood trough level, liver function, and renal function were conducted at each visit, and abdominal sonography was performed every 6 to 12 months.
The patients’ medical records were reviewed retrospectively to extract demographic and clinical data, including patient characteristics, laboratory tests, and survival outcomes. The serum bilirubin and creatinine data at the end of the fifth year after LT were used in this study. The tacrolimus level used in this study was the mean of the values obtained at the three follow-up visits during the fifth year after LT.
The immunosuppression protocol for adult LT patients consisted of tacrolimus, mycophenolate mofetil, and steroids. Tacrolimus was administered orally beginning on the first day after LT, and the dose was adjusted to achieve the desired therapeutic drug level. Basiliximab was administered immediately before graft reperfusion and on the fourth day after LT for induction therapy. A 500-mg intravenous bolus of methylprednisolone was administered immediately before reperfusion of the liver graft and was tapered to oral prednisolone over one week and reduced to withdrawal after six months.
All statistical analyses were performed using R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p ≤ 0.05. Continuous variables are presented as mean ± standard deviation (SD) and median (interquartile range, IQR), and categorical variables are presented as frequency (percentage, %). The survival curve was estimated by the Kaplan-Meier method. A univariate analysis was conducted to examine the differences in the distributions of continuous variables, categorical variables, and survival outcomes between the surviving and deceased liver recipients five years after LT using the Wilcoxon rank-sum test, Chi-square test, Fisher’s exact test, or log-rank test as appropriate for the data type. A multivariate analysis was performed to estimate the adjusted effects of risk factors or prognostic factors on the survival outcome using a multiple Cox’s proportional hazards model.
To ensure a good quality of regression analysis, the model-fitting techniques for variable selection, goodness-of-fit (GOF) assessment, and regression diagnostics and remedies were used in our regression analysis. Specifically, the stepwise variable selection procedure (with iterations between the forward and backward steps) was applied to obtain the best final regression model using the My.stepwise package of the R software [16]. All significant and non-significant relevant covariates from the univariate analysis (listed in Table 1) and some of the interaction terms were used in the multivariate analysis. The significance levels for entry and for stay were set to 0.15 for being conservative. With the aid of substantive knowledge, the best candidate final regression model was identified manually by dropping the covariates with p value > 0.05 one at a time until all regression coefficients were significantly different from 0. To assess the GOF of the fitted Cox’s model, the concordance and adjusted generalized R2 [17] were examined. A concordance ≥ 0.7 and an adjusted generalized R2 > 0.15 indicated an acceptable level of discrimination, power, and fitness.
Moreover, the smoothing option “pspline” (for the smoothing splines using a “p-spline” basis) was specified inside the coxph function of the survival package to smooth the effects of continuous covariates on the log-hazard rate of the simple and multiple Cox’s proportional hazards models in R. Then, the termplot function of the stats package was used to plot the smoothed effects of the continuous covariates on the log-hazard rate in R [18]. The regression diagnostics for the verification of proportional hazards assumption, residual analysis, detection of influential cases, and a multicollinearity check were applied to discover any model or data problems. A variance inflating factor (VIF) ≥ 10 in continuous covariates or VIF ≥ 2.5 in categorical covariates indicated the occurrence of the multicollinearity problem among some of the covariates in the fitted regression model.
3. Results
3.1. Patients’ Demographic and Clinical Characteristics
The median follow-up duration was 9.63 years (IQR: 7.2–11.4 years), and the mean follow-up was 9.58 ± 2.74 years. A total of 121 males (64.0%) and 68 females (36.0%) were included in this study (Table 1). The median age at LT was 54.26 years (IQR: 48.63–58.58 years, range: 18.3–73.1 years), and the mean age at LT was 52.7 ± 9.6 years. Among the 189 patients, 114 (76.2%) underwent living donor transplants and 45 (23.8%) underwent deceased donor transplants. The main indications for LT were hepatitis B virus (HBV) cirrhosis (52.9%), hepatocellular carcinoma (38.1%), hepatitis C virus (HCV) cirrhosis (22.2%), and fulminant hepatitis (12.2%). The median of the mean tacrolimus blood trough level during the fifth year after LT was 5.0 ng/mL (IQR: 4.12–6.33 ng/mL), and the mean tacrolimus blood trough level was 5.249 ± 1.71 ng/mL. The mean tacrolimus blood trough level was ≥ 5 ng/mL in 96 patients (50.8%) and < 5 ng/mL in 93 patients (49.2%), including 44 patients (23.3%) with a mean tacrolimus blood trough level < 4 ng/mL and 11 patients (5.8%) with a mean tacrolimus blood trough level < 3 ng/mL. No significant differences in gender, age at LT, body weight, blood type, graft type, etiology of LT, or serum total bilirubin were found between the deceased and surviving LT recipients (p > 0.05). However, serum creatinine levels > 1.5 mg/dL (23/173 vs. 8/16, p = 0.001), end-stage renal disease (2/173 vs. 3/16, p = 0.005), and mean tacrolimus blood trough levels < 4 ng/mL (36/173 vs. 8/16, p = 0.014) were significantly more common among deceased LT recipients compared to survivors. The causes of death included malignancies (n = 5), graft failures (n = 4), infection (n = 4), cerebrovascular accidents (n = 2), and duodenal ulcer bleeding (n = 1) (Table 2). The distribution of the tacrolimus trough level stratified by the etiology and cause of death among the 16 dead patients was shown in the box plot of Figure 2. There was no statistical significance in the tacrolimus trough level between the different causes of death (p = 0.3823).
3.2. Predictors of Patients’ Long-Term Survival
The Cox’s model fitted to the survival data for the multivariate analyses of the time to death after five years of LT is shown in Table 3. After adjusting for the effects of the other covariates, age at LT ≤ 27.011 years (estimated hazard ratio [HR] = 168.79, 95% confidence interval [C.I.]: 11.13–2559.51), pre-transplant autoimmune liver disease (HR = 8.12, 95% C.I.: 1.97–33.43), pre-transplant HCV infection × survival time in years (HR = 1.34, 95% C.I.: 1.12–1.60), serum creatinine level > 1.311 mg/dL × serum total bilirubin level > 1.411 mg/dL (HR = 921.69, 95% C.I.: 43.40–19,573.71), serum creatinine level > 1.311 mg/dL × serum total bilirubin level ≤ 0.792 mg/dL (HR = 105.68, 95% C.I.: 7.81–1430.79), serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level > 0.882 mg/dL (HR = 30.49, 95% C.I.: 2.98–312.34), and the mean tacrolimus trough level during the fifth year after LT ≤ 4.609 ng/mL or > 10.168 ng/mL (HR = 4.76, 95% C.I.: 1.34–16.94) were associated with a higher long-term mortality five years after LT. The time-dependent interaction term, HCV × survival time in years, was added to the Cox’s model to account for the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.292, indicates that the risk of mortality in patients with HCV increases with time five years post-LT. Moreover, the three second-order interaction terms between the serum creatinine and total bilirubin levels were compared to the other two possible combinations, serum creatinine level > 1.311 mg/dL × (0.792 mg/dL < serum total bilirubin level ≤ 1.411 mg/dL) and serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level ≤ 0.882 mg/dL, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and”.
All cut-off values of the continuous covariates (such as age at LT) were estimated by applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models. As shown in Figure 3, the optimal cut-off values of the mean tacrolimus trough level, 4.609 ng/mL and 10.168 ng/mL, were estimated directly in the “p-spline plot,” which allowed the visualization of the nonlinear effect of the averaged dosage of the tacrolimus-based immunosuppressant during the fifth year after LT on log(λ), where λ was the hazard rate of time to death five years after LT. Then, the Kaplan-Meier estimates of survival curves for time to death five years after LT were determined for the 66 patients with mean tacrolimus trough levels ≤ 4.609 ng/mL or > 10.168 ng/mL and the 123 patients with mean tacrolimus trough levels between 4.609 ng/mL and 10.168 ng/mL (log-rank test, p = 0.009) (Figure 4). In an additional subgroup analysis, we found that a mean tacrolimus trough level between 4.431 ng/mL and 6.332 ng/mL for patients with a serum creatinine level > 1.311 mg/dL improved survival. Finally, this Cox’s model had a concordance of 0.904 and an adjusted generalized R2 of 0.388, indicating that it fit the survival data very well.
4. Discussion
This is the first study to report an association between long-term tacrolimus blood trough level and long-term patient survival in adult LT recipients. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL in adult LT recipients is associated with a lower mortality rate. In patients with a serum creatinine > 1.3 mg/dL, a mean tacrolimus blood trough level of 4.4–6.3 ng/mL is associated with a lower mortality rate.
Decreasing tacrolimus use during long-term follow-up in LT recipients is a general concept in clinical practice. In this study, only one patient had the mean level > 10 ng/mL and a few patients had the mean level > 8 ng/mL (Figure 3). Patients with such high drug levels were most likely because of their poor compliance (not must because of they have truly such high trough level). Poor compliance indicates that a patient takes incorrect dosage of immunosuppressant that their physician prescribed, or takes immunosuppressant or blood exam at the wrong time, either of which might clearly be important factors that influence graft and survival outcome of LT patients during long-term follow-ups. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL is associated with lower mortality rate; however, the upper limit of 10.2 ng/mL suggested by the regression analysis was merely a value for caution, but not a recommended upper limit in real clinical practice. The long-term hazard of high level of tacrolimus is already widely known; therefore, we would focus more on the findings that it appears to increase the survival risks in LT recipients if their tacrolimus levels are maintained below 4.6 ng/mL during the long-term follow-ups.
Inadequate immunosuppression is associated with higher risks of graft rejection [19], while excess immunosuppression increases the risks of malignancy and infection and increases the adverse effects of drug toxicity. The tailored use of immunosuppressants should be considered based on the patient’s risks of graft rejection and infection and the patient’s medical comorbidities and liver disease status prior to LT [14]. Patients with autoimmune liver diseases may require more immunosuppression to prevent disease recurrence and graft rejection [20]. By contrast, lower doses of immunosuppression are recommended in patients who have undergone LT due to HCV, as high levels of immunosuppression are related to increased viral replication [21,22]. Rejection results in abnormal liver function and is one of the most important factors associated with poor long-term graft and patient outcomes [23,24,25]. Tacrolimus reduces the risk of T-cell mediated rejection to protect graft function and is the cornerstone of a successful LT. However, short and long-term adverse effects of tacrolimus such as infection, chronic renal insufficiency, metabolic diseases (hyperlipidemia, hypertension, and diabetes mellitus), and malignancy have been widely reported and influence patients’’ long-term outcomes [7,8,9]. The early causes of death after LT within one year are infection and graft loss, and the late causes of death three years post-LT are malignancy, cardiovascular disease, and renal failure [10]. As graft function typically stabilizes, factors associated with the long-term outcome are often patient-related factors (such as chronic medical diseases) that are usually associated with tacrolimus.
Previous studies have reported that minimizing tacrolimus use in the early post-LT period is associated with a lower risk of new-onset diabetes mellitus [26], a lower incidence of hyperlipidemia [27], and better long-term survival [19,28]. In recent years, studies have focused on the reduction or complete withdrawal of long-term immunosuppressants in LT recipients [12,29,30,31,32,33,34]. One study reported satisfactory outcomes with the combined use of tacrolimus and mycophenolate mofetil, which allowed for the tacrolimus dose to be reduced [35]. Other studies demonstrated that the concomitant use of everolimus may reduce the required dose of tacrolimus while having potential renal benefits [29,36]. However, only some LT recipients were able to discontinue the use of immunosuppressants, and these patients more frequently experienced biopsy-proven acute rejection [29]. The risk of chronic rejection during long-term follow-up remains in patients with inadequate immunosuppression [30,33]. The tacrolimus blood concentration in LT recipients may play a key role in long-term outcomes, as it is associated with long-term graft function and its adverse effects are related to several chronic medical diseases. However, to the best of our knowledge, this is the first report regarding the effect of long-term tacrolimus levels on the long-term survival outcomes of LT recipients.
Several factors are associated with the long-term outcomes of adult LT recipients, including pre-transplant primary sclerosing cholangitis, immunosuppression therapies, acute and chronic rejections, malignancy, and metabolic syndrome [37,38]. As listed in Table 3, we found several independent risk factors of long-term mortality in this study. Only four patients aged <27 years at the time of LT were included in this study, and one died due to pneumonia 6.3 years after LT. Patients who underwent LT due to autoimmune liver diseases had worse outcomes, as they were more likely to experience acute rejections [39] and suffer disease recurrence, leading to graft loss. Patients who underwent LT due to HCV infections had relatively poor post-LT outcomes due to the disease recurrence followed by graft dysfunction and failure [40]. As direct-acting antiviral agents have advanced [41], the survival outcomes of HCV patients should improve in the near future. Long-term renal and liver functions are reflected by serum creatinine and total bilirubin levels, respectively. We found that abnormal serum creatinine levels have a bigger impact than abnormal serum total bilirubin levels on the survival outcomes of LT recipients. The graft function of adult LT recipients is chronically stable five years after LT. While some patients may experience asymptomatic hyperbilirubinemia at this time, the long-term outcomes remain favorable. By contrast, the gradual deterioration of renal function may lead to chronic kidney disease or end-stage renal disease, affecting the long-term survival, especially in patients with long-term use of tacrolimus.
This study had some limitations. First, the data were obtained from a single medical center in Asia, which provided a relatively small number of eligible patients with single ethnicity and fewer death events. Second, the study spanned a long time period, and therefore the improvements in surgical and medical expertise and advances in immunosuppression therapies may have influenced the patient outcomes. Third, we did not collect or analyze the time-dependent tacrolimus blood trough level during the follow-up visits five years after LT.
In summary, we found an association between the long-term tacrolimus blood trough levels and the long-term survival five years after LT. A mean tacrolimus blood trough level outside the range of 4.6–10.2 ng/mL appeared to be an independent risk factor for long-term mortality. Further studies with larger sample sizes are needed to verify these results and to further identify an appropriate tacrolimus blood trough level for maintenance use.
Acknowledgments
We thank the coordinators (Hui-Ying Lin and Min-Heuy Lin) for their helpful efforts in data collection.
Author Contributions
C.-Y.H. drafted the manuscript and R.-H.H. designed the study. C.-Y.H., M.-C.H., and Y.-M.W. conducted data processing, and C.-Y.H. and C.-M.H. performed data analysis. P.-H.L. and R.-H.H. were the directors responsible for general organization and instruction. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of National Taiwan University Hospital.
Informed Consent Statement
Patient consent was waived due to retrospective design of the study.
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1 Patient flow diagram.
Figure 2 The box plot of tacrolimus mean trough level stratified by the etiology and cause of death among the 16 dead patients. There was no statistical difference of tacrolimus level between the different causes of death (p = 0.3823).
Figure 3 The p-spline plot for the nonlinear effect of the long-term tacrolimus blood trough levels on time to death. The tacrolimus blood trough level is shown on the X-axis, and the log(λ), where λ is the hazard rate of time to death five years after liver transplantation, is shown on the Y-axis. In this p-spline plot, the intersection between the horizontal green line (Y = 0) and the red curve yields the estimated optimal cut-off values for long-term tacrolimus blood trough levels at which the values of log(λ) will not change (4.609 ng/mL and 10.168 ng/mL). When the level is ≤4.609 ng/mL or >10.168 ng/mL, the value of log(λ) increases, indicating an increasing λ. When the level is >4.609 ng/mL and ≤10.168 ng/mL, the value of log(λ) decreases, indicating a decreasing λ. The vertical bars above the X-axis represent the patients’ actual mean tacrolimus blood trough levels obtained at the three follow-up visits during the fifth year after LT.
Figure 4 The Kaplan-Meier curve for time to death five years after liver transplantation. The survival curve of the 66 patients with a mean tacrolimus blood trough level ≤ 4.609 ng/mL or >10.168 ng/mL is shown in red while the survival curve of the 123 patients with a mean tacrolimus blood trough level > 4.609 ng/mL or ≤10.168 ng/mL is shown in green.
jpm-11-00090-t001_Table 1 Table 1 Univariate analysis for comparing the distributions of the demographic and clinical characteristics between the alive and dead adult liver recipients after five years of liver transplantations.
Variable All Patients
(n = 189) Alive
(n = 173) Dead
(n = 16) p Value
Gender 0.7899
Male 121 (64.0) 110 (90.9) 11 (9.1)
Female 68 (36.0) 63 (92.6) 5 (7.4)
Age at LT (years) 52.7 ± 9.6 52.6 ± 9.5 53.4 ± 10.7 0.6672
Body weight at LT (kg) 64.9 ± 12.2 65.0 ± 12.3 63.7 ± 12.1 0.4459
Blood type 0.8719
O 79 (41.8) 73 (92.4) 6 (7.6)
A 51 (27.0) 46 (90.2) 5 (9.8)
B 42 (22.2) 39 (92.9) 3 (7.1)
AB 17 (9.0) 15 (88.2) 2 (11.8)
Graft type 1.0000
Living donor 144 (76.2) 132 (91.7) 12 (8.3)
Deceased donor 45 (23.8) 41 (91.1) 4 (8.9)
Etiology for LT
Liver malignancy (HCC) 72 (38.1) 66 (91.7) 6 (8.3) 1.0000
Alcoholic cirrhosis 17 (9.0) 16 (94.1) 1 (5.9) 1.0000
HBV cirrhosis 100 (52.9) 92 (92.0) 8 (8.0) 1.0000
HCV cirrhosis 42 (22.2) 38 (90.5) 4 (9.5) 0.7577
Fulminant hepatitis 23 (12.2) 23 (100) 0 (0) 0.2259
Autoimmune disease 14 (7.4) 11 (78.6) 3 (21.4) 0.1017
Biliary atresia 5 (2.6) 5 (100) 0 (0) 1.0000
Other 10 (5.3) 9 (90) 1 (10) 0.5964
Total bilirubin (mg/dL) 0.973 ± 0.51 0.958 ± 1.53 1.133 ± 0.69 0.6260
Total bilirubin > 1 mg/dL 60 (31.7) 53 (88.3) 7 (11.7) 0.2770
Total bilirubin > 2 mg/dL 11 (5.8) 9 (81.8) 2 (18.2) 0.2360
Creatinine (mg/dL) 1.391 ± 1.25 1.279 ± 0.96 2.6 ± 2.74 0.0201 *
Creatinine > 1.5 mg/dL 31 (16.4) 23 (74.2) 8 (25.8) 0.0010 *
ESRD 5 (2.6) 2 (40.0) 3 (60.0) 0.0050 *
Tacrolimus mean level (ng/mL) 5.249 ± 1.71 5.263 ± 1.53 5.096 ± 3.12 0.9787
Tacrolimus level < 5 ng/mL 93 (49.2) 82 (88.2) 11 (11.8) 0.1216
Tacrolimus level < 4 ng/mL 44 (23.3) 36 (81.8) 8 (18.2) 0.0136 *
Tacrolimus level < 3 ng/mL 11 (5.8) 9 (81.8) 2 (18.2) 0.2356
Data are presented as mean ± standard deviation (SD) for continuous variables and frequency (percentage, %) for categorical variables. The p-values of statistical tests were calculated using the Wilcoxon rank-sum test for continuous variables and the Fisher’s exact test for categorical variables. * p value ≤ 0.05. Abbreviations: LT, liver transplantation; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; Total bilirubin, serum total bilirubin level; Creatinine, serum creatinine level; and ESRD, end-stage renal disease (defined by receiving hemodialysis regularly).
jpm-11-00090-t002_Table 2 Table 2 The causes of 16 deaths since 5 years after adult liver transplantations.
Causes of Deaths Number of Subjects
Malignancy 5 (31.25%)
De novo: Multiple myeloma, bladder cancer, colon cancer, prostate cancer 4
Recurrent: Hepatocellular carcinoma 1
Graft failure 4 (25.00%)
Chronic rejection 3
Autoimmune hepatitis 1
Infection 4 (25.00%)
Pneumonia 3
Urinary tract infection 1
Cerebral vascular event (intracerebral hemorrhage) 2 (12.50%)
Peptic ulcer bleeding 1 (6.25%)
jpm-11-00090-t003_Table 3 Table 3 Multivariate analysis for identifying the predictors of long-term overall survival after 5 years of liver transplantations by fitting a multiple Cox’s Model in the adult liver transplant recipients 1.
Covariate 2 Estimate Standard
Error Wald’s
z Test p Value Hazard
Ratio (HR) 95% Confidence
Interval (C.I.)
Age at LT ≤ 27.011 years 5.1286 1.3872 3.6970 0.0002 168.7851 11.130–2559.512
Autoimmune (including PBC) 2.0946 0.7219 2.9015 0.0037 8.1221 1.973–33.431
HCV × Overall survival years 0.2924 0.0914 3.1978 0.0014 1.3397 1.120–1.603
Cre > 1.311 × T-Bil > 1.411 mg/dL 6.8262 1.5591 4.3784 <0.0001 921.6940 43.401–19,573.712
Cre > 1.311 × T-Bil ≤ 0.792 mg/dL 4.6604 1.3294 3.5056 0.0005 105.6778 7.805–1430.790
Cre ≤ 1.311 × T-Bil > 0.882 mg/dL 3.4174 1.1871 2.8788 0.0040 30.4913 2.977–312.341
Tacrolimus mean ≤ 4.609 or > 10.168 ng/mL 1.5599 0.6479 2.4076 0.0161 4.7581 1.336–16.940
1 The above multiple Cox’s model was fitted to the 189 adult patients who underwent liver transplantations with 16 death events, for modeling the hazard rate of the right-censored overall survival time five years after liver transplantations. All the cut-off values of the continuous covariates (e.g., age at liver transplantations) were estimated by choosing the option of applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models (e.g., Figure 3). The time-dependent interaction term, HCV × Overall survival year, was added to the Cox’s model for handling the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.2924, indicated that the risk of dying in the patients with HCV would increase as time elapsed five years after liver transplantations. Moreover, the three second-order interaction terms, Cre > 1.311 × T-Bil > 1.411, Cre > 1.311 × T-Bil ≤ 0.792, and Cre ≤ 1.311 × T-Bil > 0.882, were compared to the other two possible combinations, Cre > 1.311 × (0.792 < T-Bil ≤ 1.411) and Cre ≤ 1.311 × T-Bil ≤ 0.882, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and.” Finally, both goodness-of-fit (GOF) measures, concordance = 0.9041 (se = 0.0265) > 0.7 and adjusted generalized R2 = 0.3878 > 0.15, indicated an excellent fit of this multiple Cox’s model to the observed survival data. 2 Abbreviations: LT, liver transplantation; PBC, primary biliary cirrhosis; HCV, hepatitis C viral infection; Cre, serum creatinine level (mg/dL); T-Bil, serum total bilirubin level (mg/dL); and Tacrolimus mean, the averaged dosage of the tacrolimus-based immunosuppressant (ng/mL).
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What was the dosage of drug 'MYCOPHENOLATE MOFETIL'? | Long-Term Tacrolimus Blood Trough Level and Patient Survival in Adult Liver Transplantation.
Tacrolimus is the most widely used immunosuppressant in liver transplant (LT) patients. However, the ideal long-term target level for these patients is unknown. This retrospective study aimed to investigate the impact of tacrolimus blood concentration five years after LT on long-term patient survival outcomes in adult LT recipients. Patients who underwent LT between January 2004 and July 2014 at a tertiary medical center were included in this study (n = 189). The mean tacrolimus blood concentrations of each patient during the fifth year after LT were recorded and the overall survival rate was determined. A multivariate analysis of factors associated with long-term survival was conducted using a Cox's model. The median follow-up period was 9.63 years, and 144 patients (76.2%) underwent live donor LT. Sixteen patients died within 5 years of LT. In the Cox's model, patients with a mean tacrolimus blood trough level of 4.6-10.2 ng/mL had significantly better long-term survival than those with a mean tacrolimus blood trough level outside this range (estimated hazard ratio = 4.76; 95% confidence interval: 1.34-16.9, p = 0.016). Therefore, a tacrolimus level no lower than 4.6 ng/mL would be recommended in adult LT patients.
1. Introduction
Liver transplantation (LT) has become a mature treatment of end-stage liver disease in clinical practice [1]. The continuous improvement of effective immunosuppression treatments has led to a significant improvement in patient and graft survival in recent years [2]. Calcineurin inhibitors are the cornerstone of immunosuppression in LT [3], and tacrolimus is currently the mainstay of initial and maintenance immunosuppression therapies [4,5,6]. Tacrolimus reduces the incidence and severity of early and late T-cell mediated rejection by inhibiting T-cell production of interleukin-2 [5,6]. However, the long-term use of immunosuppressants leads to an increasing burden of toxicity. The reported toxic effects of calcineurin inhibitors include infections, chronic renal insufficiency, metabolic diseases (such as hyperlipidemia, hypertension, and diabetes mellitus), and malignancy [7,8,9], which, along with cardiovascular diseases, have been reported to be the major causes of morbidity and mortality after LT [3,10]. However, most LT patients cannot withdraw from lifelong immunosuppression therapy; the only exceptions are a few selected patients participating in experimental trials [11,12].
It is a clinical challenge to design a well-balanced immunosuppressive regimen for LT recipients. Tacrolimus has a narrow therapeutic dose range and its blood levels should be carefully monitored. The Advagraf (tacrolimus prolonged-release hard capsules) recommendation for adult LT patients is a blood trough level of 5–20 ng/mL in the early post-transplant period and 5–15 ng/mL during subsequent maintenance therapy. Nevertheless, the current recommendations from the clinical practice guidelines for the ideal tacrolimus level in adults after LT remain controversial. The American Association for the Study of Liver Diseases (AASLD) recommends a target blood trough level of 5–10 ng/mL for tacrolimus three months after LT [13]. The Consensus on Managing Modifiable Risk in Transplantation Group (COMMIT) recommends that the target tacrolimus blood trough levels be 6–10 ng/mL during the first month after LT and decrease to 4–8 ng/mL thereafter, except when used in combination with mammalian target of rapamycin (mTOR) inhibitors [14]. Moreover, the International Liver Transplant Society (ILTS) consensus statement on immunosuppression in LT recipients recommends the target blood trough levels of tacrolimus be 6–10 ng/mL three months after LT, lower than 5 ng/mL 12 months after LT, and decrease to 3 ng/mL thereafter, resulting in a blood trough level just above the lower limit of detection five years after LT [15]. However, the impact of the long-term tacrolimus blood trough level on the outcomes of the LT recipients remains unclear. This study aimed to suggest an appropriate tacrolimus blood trough level for adult patients five years after LT.
2. Materials and Methods
This study was approved by the Institutional Review Board of National Taiwan University Hospital and was conducted according to the Declaration of Helsinki. A total of 286 patients who underwent LT at 18 years or older at a tertiary medical center from January 2004 to July 2014 were recruited for this study. Patients who died within 5 years of LT, were lost to follow-up, or did not use tacrolimus-based calcineurin inhibitor for immunosuppressant therapy were excluded from this study (Figure 1). In addition, those who received mTOR inhibitor treatment were excluded, as mTOR inhibitors are typically used as a combination therapy to reduce the required dose of tacrolimus. The final analysis included 189 patients. All eligible patients were followed up for more than five years until August 2019. Patients who underwent a LT due to liver cancer met the Milan criteria (before 2006) or the criteria of the University of California, San Francisco (since 2006) at the time of LT. All patients received regular monthly or bi-monthly follow-ups at the outpatient clinic after LT. Routine blood examinations for the tacrolimus blood trough level, liver function, and renal function were conducted at each visit, and abdominal sonography was performed every 6 to 12 months.
The patients’ medical records were reviewed retrospectively to extract demographic and clinical data, including patient characteristics, laboratory tests, and survival outcomes. The serum bilirubin and creatinine data at the end of the fifth year after LT were used in this study. The tacrolimus level used in this study was the mean of the values obtained at the three follow-up visits during the fifth year after LT.
The immunosuppression protocol for adult LT patients consisted of tacrolimus, mycophenolate mofetil, and steroids. Tacrolimus was administered orally beginning on the first day after LT, and the dose was adjusted to achieve the desired therapeutic drug level. Basiliximab was administered immediately before graft reperfusion and on the fourth day after LT for induction therapy. A 500-mg intravenous bolus of methylprednisolone was administered immediately before reperfusion of the liver graft and was tapered to oral prednisolone over one week and reduced to withdrawal after six months.
All statistical analyses were performed using R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p ≤ 0.05. Continuous variables are presented as mean ± standard deviation (SD) and median (interquartile range, IQR), and categorical variables are presented as frequency (percentage, %). The survival curve was estimated by the Kaplan-Meier method. A univariate analysis was conducted to examine the differences in the distributions of continuous variables, categorical variables, and survival outcomes between the surviving and deceased liver recipients five years after LT using the Wilcoxon rank-sum test, Chi-square test, Fisher’s exact test, or log-rank test as appropriate for the data type. A multivariate analysis was performed to estimate the adjusted effects of risk factors or prognostic factors on the survival outcome using a multiple Cox’s proportional hazards model.
To ensure a good quality of regression analysis, the model-fitting techniques for variable selection, goodness-of-fit (GOF) assessment, and regression diagnostics and remedies were used in our regression analysis. Specifically, the stepwise variable selection procedure (with iterations between the forward and backward steps) was applied to obtain the best final regression model using the My.stepwise package of the R software [16]. All significant and non-significant relevant covariates from the univariate analysis (listed in Table 1) and some of the interaction terms were used in the multivariate analysis. The significance levels for entry and for stay were set to 0.15 for being conservative. With the aid of substantive knowledge, the best candidate final regression model was identified manually by dropping the covariates with p value > 0.05 one at a time until all regression coefficients were significantly different from 0. To assess the GOF of the fitted Cox’s model, the concordance and adjusted generalized R2 [17] were examined. A concordance ≥ 0.7 and an adjusted generalized R2 > 0.15 indicated an acceptable level of discrimination, power, and fitness.
Moreover, the smoothing option “pspline” (for the smoothing splines using a “p-spline” basis) was specified inside the coxph function of the survival package to smooth the effects of continuous covariates on the log-hazard rate of the simple and multiple Cox’s proportional hazards models in R. Then, the termplot function of the stats package was used to plot the smoothed effects of the continuous covariates on the log-hazard rate in R [18]. The regression diagnostics for the verification of proportional hazards assumption, residual analysis, detection of influential cases, and a multicollinearity check were applied to discover any model or data problems. A variance inflating factor (VIF) ≥ 10 in continuous covariates or VIF ≥ 2.5 in categorical covariates indicated the occurrence of the multicollinearity problem among some of the covariates in the fitted regression model.
3. Results
3.1. Patients’ Demographic and Clinical Characteristics
The median follow-up duration was 9.63 years (IQR: 7.2–11.4 years), and the mean follow-up was 9.58 ± 2.74 years. A total of 121 males (64.0%) and 68 females (36.0%) were included in this study (Table 1). The median age at LT was 54.26 years (IQR: 48.63–58.58 years, range: 18.3–73.1 years), and the mean age at LT was 52.7 ± 9.6 years. Among the 189 patients, 114 (76.2%) underwent living donor transplants and 45 (23.8%) underwent deceased donor transplants. The main indications for LT were hepatitis B virus (HBV) cirrhosis (52.9%), hepatocellular carcinoma (38.1%), hepatitis C virus (HCV) cirrhosis (22.2%), and fulminant hepatitis (12.2%). The median of the mean tacrolimus blood trough level during the fifth year after LT was 5.0 ng/mL (IQR: 4.12–6.33 ng/mL), and the mean tacrolimus blood trough level was 5.249 ± 1.71 ng/mL. The mean tacrolimus blood trough level was ≥ 5 ng/mL in 96 patients (50.8%) and < 5 ng/mL in 93 patients (49.2%), including 44 patients (23.3%) with a mean tacrolimus blood trough level < 4 ng/mL and 11 patients (5.8%) with a mean tacrolimus blood trough level < 3 ng/mL. No significant differences in gender, age at LT, body weight, blood type, graft type, etiology of LT, or serum total bilirubin were found between the deceased and surviving LT recipients (p > 0.05). However, serum creatinine levels > 1.5 mg/dL (23/173 vs. 8/16, p = 0.001), end-stage renal disease (2/173 vs. 3/16, p = 0.005), and mean tacrolimus blood trough levels < 4 ng/mL (36/173 vs. 8/16, p = 0.014) were significantly more common among deceased LT recipients compared to survivors. The causes of death included malignancies (n = 5), graft failures (n = 4), infection (n = 4), cerebrovascular accidents (n = 2), and duodenal ulcer bleeding (n = 1) (Table 2). The distribution of the tacrolimus trough level stratified by the etiology and cause of death among the 16 dead patients was shown in the box plot of Figure 2. There was no statistical significance in the tacrolimus trough level between the different causes of death (p = 0.3823).
3.2. Predictors of Patients’ Long-Term Survival
The Cox’s model fitted to the survival data for the multivariate analyses of the time to death after five years of LT is shown in Table 3. After adjusting for the effects of the other covariates, age at LT ≤ 27.011 years (estimated hazard ratio [HR] = 168.79, 95% confidence interval [C.I.]: 11.13–2559.51), pre-transplant autoimmune liver disease (HR = 8.12, 95% C.I.: 1.97–33.43), pre-transplant HCV infection × survival time in years (HR = 1.34, 95% C.I.: 1.12–1.60), serum creatinine level > 1.311 mg/dL × serum total bilirubin level > 1.411 mg/dL (HR = 921.69, 95% C.I.: 43.40–19,573.71), serum creatinine level > 1.311 mg/dL × serum total bilirubin level ≤ 0.792 mg/dL (HR = 105.68, 95% C.I.: 7.81–1430.79), serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level > 0.882 mg/dL (HR = 30.49, 95% C.I.: 2.98–312.34), and the mean tacrolimus trough level during the fifth year after LT ≤ 4.609 ng/mL or > 10.168 ng/mL (HR = 4.76, 95% C.I.: 1.34–16.94) were associated with a higher long-term mortality five years after LT. The time-dependent interaction term, HCV × survival time in years, was added to the Cox’s model to account for the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.292, indicates that the risk of mortality in patients with HCV increases with time five years post-LT. Moreover, the three second-order interaction terms between the serum creatinine and total bilirubin levels were compared to the other two possible combinations, serum creatinine level > 1.311 mg/dL × (0.792 mg/dL < serum total bilirubin level ≤ 1.411 mg/dL) and serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level ≤ 0.882 mg/dL, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and”.
All cut-off values of the continuous covariates (such as age at LT) were estimated by applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models. As shown in Figure 3, the optimal cut-off values of the mean tacrolimus trough level, 4.609 ng/mL and 10.168 ng/mL, were estimated directly in the “p-spline plot,” which allowed the visualization of the nonlinear effect of the averaged dosage of the tacrolimus-based immunosuppressant during the fifth year after LT on log(λ), where λ was the hazard rate of time to death five years after LT. Then, the Kaplan-Meier estimates of survival curves for time to death five years after LT were determined for the 66 patients with mean tacrolimus trough levels ≤ 4.609 ng/mL or > 10.168 ng/mL and the 123 patients with mean tacrolimus trough levels between 4.609 ng/mL and 10.168 ng/mL (log-rank test, p = 0.009) (Figure 4). In an additional subgroup analysis, we found that a mean tacrolimus trough level between 4.431 ng/mL and 6.332 ng/mL for patients with a serum creatinine level > 1.311 mg/dL improved survival. Finally, this Cox’s model had a concordance of 0.904 and an adjusted generalized R2 of 0.388, indicating that it fit the survival data very well.
4. Discussion
This is the first study to report an association between long-term tacrolimus blood trough level and long-term patient survival in adult LT recipients. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL in adult LT recipients is associated with a lower mortality rate. In patients with a serum creatinine > 1.3 mg/dL, a mean tacrolimus blood trough level of 4.4–6.3 ng/mL is associated with a lower mortality rate.
Decreasing tacrolimus use during long-term follow-up in LT recipients is a general concept in clinical practice. In this study, only one patient had the mean level > 10 ng/mL and a few patients had the mean level > 8 ng/mL (Figure 3). Patients with such high drug levels were most likely because of their poor compliance (not must because of they have truly such high trough level). Poor compliance indicates that a patient takes incorrect dosage of immunosuppressant that their physician prescribed, or takes immunosuppressant or blood exam at the wrong time, either of which might clearly be important factors that influence graft and survival outcome of LT patients during long-term follow-ups. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL is associated with lower mortality rate; however, the upper limit of 10.2 ng/mL suggested by the regression analysis was merely a value for caution, but not a recommended upper limit in real clinical practice. The long-term hazard of high level of tacrolimus is already widely known; therefore, we would focus more on the findings that it appears to increase the survival risks in LT recipients if their tacrolimus levels are maintained below 4.6 ng/mL during the long-term follow-ups.
Inadequate immunosuppression is associated with higher risks of graft rejection [19], while excess immunosuppression increases the risks of malignancy and infection and increases the adverse effects of drug toxicity. The tailored use of immunosuppressants should be considered based on the patient’s risks of graft rejection and infection and the patient’s medical comorbidities and liver disease status prior to LT [14]. Patients with autoimmune liver diseases may require more immunosuppression to prevent disease recurrence and graft rejection [20]. By contrast, lower doses of immunosuppression are recommended in patients who have undergone LT due to HCV, as high levels of immunosuppression are related to increased viral replication [21,22]. Rejection results in abnormal liver function and is one of the most important factors associated with poor long-term graft and patient outcomes [23,24,25]. Tacrolimus reduces the risk of T-cell mediated rejection to protect graft function and is the cornerstone of a successful LT. However, short and long-term adverse effects of tacrolimus such as infection, chronic renal insufficiency, metabolic diseases (hyperlipidemia, hypertension, and diabetes mellitus), and malignancy have been widely reported and influence patients’’ long-term outcomes [7,8,9]. The early causes of death after LT within one year are infection and graft loss, and the late causes of death three years post-LT are malignancy, cardiovascular disease, and renal failure [10]. As graft function typically stabilizes, factors associated with the long-term outcome are often patient-related factors (such as chronic medical diseases) that are usually associated with tacrolimus.
Previous studies have reported that minimizing tacrolimus use in the early post-LT period is associated with a lower risk of new-onset diabetes mellitus [26], a lower incidence of hyperlipidemia [27], and better long-term survival [19,28]. In recent years, studies have focused on the reduction or complete withdrawal of long-term immunosuppressants in LT recipients [12,29,30,31,32,33,34]. One study reported satisfactory outcomes with the combined use of tacrolimus and mycophenolate mofetil, which allowed for the tacrolimus dose to be reduced [35]. Other studies demonstrated that the concomitant use of everolimus may reduce the required dose of tacrolimus while having potential renal benefits [29,36]. However, only some LT recipients were able to discontinue the use of immunosuppressants, and these patients more frequently experienced biopsy-proven acute rejection [29]. The risk of chronic rejection during long-term follow-up remains in patients with inadequate immunosuppression [30,33]. The tacrolimus blood concentration in LT recipients may play a key role in long-term outcomes, as it is associated with long-term graft function and its adverse effects are related to several chronic medical diseases. However, to the best of our knowledge, this is the first report regarding the effect of long-term tacrolimus levels on the long-term survival outcomes of LT recipients.
Several factors are associated with the long-term outcomes of adult LT recipients, including pre-transplant primary sclerosing cholangitis, immunosuppression therapies, acute and chronic rejections, malignancy, and metabolic syndrome [37,38]. As listed in Table 3, we found several independent risk factors of long-term mortality in this study. Only four patients aged <27 years at the time of LT were included in this study, and one died due to pneumonia 6.3 years after LT. Patients who underwent LT due to autoimmune liver diseases had worse outcomes, as they were more likely to experience acute rejections [39] and suffer disease recurrence, leading to graft loss. Patients who underwent LT due to HCV infections had relatively poor post-LT outcomes due to the disease recurrence followed by graft dysfunction and failure [40]. As direct-acting antiviral agents have advanced [41], the survival outcomes of HCV patients should improve in the near future. Long-term renal and liver functions are reflected by serum creatinine and total bilirubin levels, respectively. We found that abnormal serum creatinine levels have a bigger impact than abnormal serum total bilirubin levels on the survival outcomes of LT recipients. The graft function of adult LT recipients is chronically stable five years after LT. While some patients may experience asymptomatic hyperbilirubinemia at this time, the long-term outcomes remain favorable. By contrast, the gradual deterioration of renal function may lead to chronic kidney disease or end-stage renal disease, affecting the long-term survival, especially in patients with long-term use of tacrolimus.
This study had some limitations. First, the data were obtained from a single medical center in Asia, which provided a relatively small number of eligible patients with single ethnicity and fewer death events. Second, the study spanned a long time period, and therefore the improvements in surgical and medical expertise and advances in immunosuppression therapies may have influenced the patient outcomes. Third, we did not collect or analyze the time-dependent tacrolimus blood trough level during the follow-up visits five years after LT.
In summary, we found an association between the long-term tacrolimus blood trough levels and the long-term survival five years after LT. A mean tacrolimus blood trough level outside the range of 4.6–10.2 ng/mL appeared to be an independent risk factor for long-term mortality. Further studies with larger sample sizes are needed to verify these results and to further identify an appropriate tacrolimus blood trough level for maintenance use.
Acknowledgments
We thank the coordinators (Hui-Ying Lin and Min-Heuy Lin) for their helpful efforts in data collection.
Author Contributions
C.-Y.H. drafted the manuscript and R.-H.H. designed the study. C.-Y.H., M.-C.H., and Y.-M.W. conducted data processing, and C.-Y.H. and C.-M.H. performed data analysis. P.-H.L. and R.-H.H. were the directors responsible for general organization and instruction. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of National Taiwan University Hospital.
Informed Consent Statement
Patient consent was waived due to retrospective design of the study.
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1 Patient flow diagram.
Figure 2 The box plot of tacrolimus mean trough level stratified by the etiology and cause of death among the 16 dead patients. There was no statistical difference of tacrolimus level between the different causes of death (p = 0.3823).
Figure 3 The p-spline plot for the nonlinear effect of the long-term tacrolimus blood trough levels on time to death. The tacrolimus blood trough level is shown on the X-axis, and the log(λ), where λ is the hazard rate of time to death five years after liver transplantation, is shown on the Y-axis. In this p-spline plot, the intersection between the horizontal green line (Y = 0) and the red curve yields the estimated optimal cut-off values for long-term tacrolimus blood trough levels at which the values of log(λ) will not change (4.609 ng/mL and 10.168 ng/mL). When the level is ≤4.609 ng/mL or >10.168 ng/mL, the value of log(λ) increases, indicating an increasing λ. When the level is >4.609 ng/mL and ≤10.168 ng/mL, the value of log(λ) decreases, indicating a decreasing λ. The vertical bars above the X-axis represent the patients’ actual mean tacrolimus blood trough levels obtained at the three follow-up visits during the fifth year after LT.
Figure 4 The Kaplan-Meier curve for time to death five years after liver transplantation. The survival curve of the 66 patients with a mean tacrolimus blood trough level ≤ 4.609 ng/mL or >10.168 ng/mL is shown in red while the survival curve of the 123 patients with a mean tacrolimus blood trough level > 4.609 ng/mL or ≤10.168 ng/mL is shown in green.
jpm-11-00090-t001_Table 1 Table 1 Univariate analysis for comparing the distributions of the demographic and clinical characteristics between the alive and dead adult liver recipients after five years of liver transplantations.
Variable All Patients
(n = 189) Alive
(n = 173) Dead
(n = 16) p Value
Gender 0.7899
Male 121 (64.0) 110 (90.9) 11 (9.1)
Female 68 (36.0) 63 (92.6) 5 (7.4)
Age at LT (years) 52.7 ± 9.6 52.6 ± 9.5 53.4 ± 10.7 0.6672
Body weight at LT (kg) 64.9 ± 12.2 65.0 ± 12.3 63.7 ± 12.1 0.4459
Blood type 0.8719
O 79 (41.8) 73 (92.4) 6 (7.6)
A 51 (27.0) 46 (90.2) 5 (9.8)
B 42 (22.2) 39 (92.9) 3 (7.1)
AB 17 (9.0) 15 (88.2) 2 (11.8)
Graft type 1.0000
Living donor 144 (76.2) 132 (91.7) 12 (8.3)
Deceased donor 45 (23.8) 41 (91.1) 4 (8.9)
Etiology for LT
Liver malignancy (HCC) 72 (38.1) 66 (91.7) 6 (8.3) 1.0000
Alcoholic cirrhosis 17 (9.0) 16 (94.1) 1 (5.9) 1.0000
HBV cirrhosis 100 (52.9) 92 (92.0) 8 (8.0) 1.0000
HCV cirrhosis 42 (22.2) 38 (90.5) 4 (9.5) 0.7577
Fulminant hepatitis 23 (12.2) 23 (100) 0 (0) 0.2259
Autoimmune disease 14 (7.4) 11 (78.6) 3 (21.4) 0.1017
Biliary atresia 5 (2.6) 5 (100) 0 (0) 1.0000
Other 10 (5.3) 9 (90) 1 (10) 0.5964
Total bilirubin (mg/dL) 0.973 ± 0.51 0.958 ± 1.53 1.133 ± 0.69 0.6260
Total bilirubin > 1 mg/dL 60 (31.7) 53 (88.3) 7 (11.7) 0.2770
Total bilirubin > 2 mg/dL 11 (5.8) 9 (81.8) 2 (18.2) 0.2360
Creatinine (mg/dL) 1.391 ± 1.25 1.279 ± 0.96 2.6 ± 2.74 0.0201 *
Creatinine > 1.5 mg/dL 31 (16.4) 23 (74.2) 8 (25.8) 0.0010 *
ESRD 5 (2.6) 2 (40.0) 3 (60.0) 0.0050 *
Tacrolimus mean level (ng/mL) 5.249 ± 1.71 5.263 ± 1.53 5.096 ± 3.12 0.9787
Tacrolimus level < 5 ng/mL 93 (49.2) 82 (88.2) 11 (11.8) 0.1216
Tacrolimus level < 4 ng/mL 44 (23.3) 36 (81.8) 8 (18.2) 0.0136 *
Tacrolimus level < 3 ng/mL 11 (5.8) 9 (81.8) 2 (18.2) 0.2356
Data are presented as mean ± standard deviation (SD) for continuous variables and frequency (percentage, %) for categorical variables. The p-values of statistical tests were calculated using the Wilcoxon rank-sum test for continuous variables and the Fisher’s exact test for categorical variables. * p value ≤ 0.05. Abbreviations: LT, liver transplantation; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; Total bilirubin, serum total bilirubin level; Creatinine, serum creatinine level; and ESRD, end-stage renal disease (defined by receiving hemodialysis regularly).
jpm-11-00090-t002_Table 2 Table 2 The causes of 16 deaths since 5 years after adult liver transplantations.
Causes of Deaths Number of Subjects
Malignancy 5 (31.25%)
De novo: Multiple myeloma, bladder cancer, colon cancer, prostate cancer 4
Recurrent: Hepatocellular carcinoma 1
Graft failure 4 (25.00%)
Chronic rejection 3
Autoimmune hepatitis 1
Infection 4 (25.00%)
Pneumonia 3
Urinary tract infection 1
Cerebral vascular event (intracerebral hemorrhage) 2 (12.50%)
Peptic ulcer bleeding 1 (6.25%)
jpm-11-00090-t003_Table 3 Table 3 Multivariate analysis for identifying the predictors of long-term overall survival after 5 years of liver transplantations by fitting a multiple Cox’s Model in the adult liver transplant recipients 1.
Covariate 2 Estimate Standard
Error Wald’s
z Test p Value Hazard
Ratio (HR) 95% Confidence
Interval (C.I.)
Age at LT ≤ 27.011 years 5.1286 1.3872 3.6970 0.0002 168.7851 11.130–2559.512
Autoimmune (including PBC) 2.0946 0.7219 2.9015 0.0037 8.1221 1.973–33.431
HCV × Overall survival years 0.2924 0.0914 3.1978 0.0014 1.3397 1.120–1.603
Cre > 1.311 × T-Bil > 1.411 mg/dL 6.8262 1.5591 4.3784 <0.0001 921.6940 43.401–19,573.712
Cre > 1.311 × T-Bil ≤ 0.792 mg/dL 4.6604 1.3294 3.5056 0.0005 105.6778 7.805–1430.790
Cre ≤ 1.311 × T-Bil > 0.882 mg/dL 3.4174 1.1871 2.8788 0.0040 30.4913 2.977–312.341
Tacrolimus mean ≤ 4.609 or > 10.168 ng/mL 1.5599 0.6479 2.4076 0.0161 4.7581 1.336–16.940
1 The above multiple Cox’s model was fitted to the 189 adult patients who underwent liver transplantations with 16 death events, for modeling the hazard rate of the right-censored overall survival time five years after liver transplantations. All the cut-off values of the continuous covariates (e.g., age at liver transplantations) were estimated by choosing the option of applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models (e.g., Figure 3). The time-dependent interaction term, HCV × Overall survival year, was added to the Cox’s model for handling the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.2924, indicated that the risk of dying in the patients with HCV would increase as time elapsed five years after liver transplantations. Moreover, the three second-order interaction terms, Cre > 1.311 × T-Bil > 1.411, Cre > 1.311 × T-Bil ≤ 0.792, and Cre ≤ 1.311 × T-Bil > 0.882, were compared to the other two possible combinations, Cre > 1.311 × (0.792 < T-Bil ≤ 1.411) and Cre ≤ 1.311 × T-Bil ≤ 0.882, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and.” Finally, both goodness-of-fit (GOF) measures, concordance = 0.9041 (se = 0.0265) > 0.7 and adjusted generalized R2 = 0.3878 > 0.15, indicated an excellent fit of this multiple Cox’s model to the observed survival data. 2 Abbreviations: LT, liver transplantation; PBC, primary biliary cirrhosis; HCV, hepatitis C viral infection; Cre, serum creatinine level (mg/dL); T-Bil, serum total bilirubin level (mg/dL); and Tacrolimus mean, the averaged dosage of the tacrolimus-based immunosuppressant (ng/mL).
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What was the dosage of drug 'PREDNISOLONE'? | Long-Term Tacrolimus Blood Trough Level and Patient Survival in Adult Liver Transplantation.
Tacrolimus is the most widely used immunosuppressant in liver transplant (LT) patients. However, the ideal long-term target level for these patients is unknown. This retrospective study aimed to investigate the impact of tacrolimus blood concentration five years after LT on long-term patient survival outcomes in adult LT recipients. Patients who underwent LT between January 2004 and July 2014 at a tertiary medical center were included in this study (n = 189). The mean tacrolimus blood concentrations of each patient during the fifth year after LT were recorded and the overall survival rate was determined. A multivariate analysis of factors associated with long-term survival was conducted using a Cox's model. The median follow-up period was 9.63 years, and 144 patients (76.2%) underwent live donor LT. Sixteen patients died within 5 years of LT. In the Cox's model, patients with a mean tacrolimus blood trough level of 4.6-10.2 ng/mL had significantly better long-term survival than those with a mean tacrolimus blood trough level outside this range (estimated hazard ratio = 4.76; 95% confidence interval: 1.34-16.9, p = 0.016). Therefore, a tacrolimus level no lower than 4.6 ng/mL would be recommended in adult LT patients.
1. Introduction
Liver transplantation (LT) has become a mature treatment of end-stage liver disease in clinical practice [1]. The continuous improvement of effective immunosuppression treatments has led to a significant improvement in patient and graft survival in recent years [2]. Calcineurin inhibitors are the cornerstone of immunosuppression in LT [3], and tacrolimus is currently the mainstay of initial and maintenance immunosuppression therapies [4,5,6]. Tacrolimus reduces the incidence and severity of early and late T-cell mediated rejection by inhibiting T-cell production of interleukin-2 [5,6]. However, the long-term use of immunosuppressants leads to an increasing burden of toxicity. The reported toxic effects of calcineurin inhibitors include infections, chronic renal insufficiency, metabolic diseases (such as hyperlipidemia, hypertension, and diabetes mellitus), and malignancy [7,8,9], which, along with cardiovascular diseases, have been reported to be the major causes of morbidity and mortality after LT [3,10]. However, most LT patients cannot withdraw from lifelong immunosuppression therapy; the only exceptions are a few selected patients participating in experimental trials [11,12].
It is a clinical challenge to design a well-balanced immunosuppressive regimen for LT recipients. Tacrolimus has a narrow therapeutic dose range and its blood levels should be carefully monitored. The Advagraf (tacrolimus prolonged-release hard capsules) recommendation for adult LT patients is a blood trough level of 5–20 ng/mL in the early post-transplant period and 5–15 ng/mL during subsequent maintenance therapy. Nevertheless, the current recommendations from the clinical practice guidelines for the ideal tacrolimus level in adults after LT remain controversial. The American Association for the Study of Liver Diseases (AASLD) recommends a target blood trough level of 5–10 ng/mL for tacrolimus three months after LT [13]. The Consensus on Managing Modifiable Risk in Transplantation Group (COMMIT) recommends that the target tacrolimus blood trough levels be 6–10 ng/mL during the first month after LT and decrease to 4–8 ng/mL thereafter, except when used in combination with mammalian target of rapamycin (mTOR) inhibitors [14]. Moreover, the International Liver Transplant Society (ILTS) consensus statement on immunosuppression in LT recipients recommends the target blood trough levels of tacrolimus be 6–10 ng/mL three months after LT, lower than 5 ng/mL 12 months after LT, and decrease to 3 ng/mL thereafter, resulting in a blood trough level just above the lower limit of detection five years after LT [15]. However, the impact of the long-term tacrolimus blood trough level on the outcomes of the LT recipients remains unclear. This study aimed to suggest an appropriate tacrolimus blood trough level for adult patients five years after LT.
2. Materials and Methods
This study was approved by the Institutional Review Board of National Taiwan University Hospital and was conducted according to the Declaration of Helsinki. A total of 286 patients who underwent LT at 18 years or older at a tertiary medical center from January 2004 to July 2014 were recruited for this study. Patients who died within 5 years of LT, were lost to follow-up, or did not use tacrolimus-based calcineurin inhibitor for immunosuppressant therapy were excluded from this study (Figure 1). In addition, those who received mTOR inhibitor treatment were excluded, as mTOR inhibitors are typically used as a combination therapy to reduce the required dose of tacrolimus. The final analysis included 189 patients. All eligible patients were followed up for more than five years until August 2019. Patients who underwent a LT due to liver cancer met the Milan criteria (before 2006) or the criteria of the University of California, San Francisco (since 2006) at the time of LT. All patients received regular monthly or bi-monthly follow-ups at the outpatient clinic after LT. Routine blood examinations for the tacrolimus blood trough level, liver function, and renal function were conducted at each visit, and abdominal sonography was performed every 6 to 12 months.
The patients’ medical records were reviewed retrospectively to extract demographic and clinical data, including patient characteristics, laboratory tests, and survival outcomes. The serum bilirubin and creatinine data at the end of the fifth year after LT were used in this study. The tacrolimus level used in this study was the mean of the values obtained at the three follow-up visits during the fifth year after LT.
The immunosuppression protocol for adult LT patients consisted of tacrolimus, mycophenolate mofetil, and steroids. Tacrolimus was administered orally beginning on the first day after LT, and the dose was adjusted to achieve the desired therapeutic drug level. Basiliximab was administered immediately before graft reperfusion and on the fourth day after LT for induction therapy. A 500-mg intravenous bolus of methylprednisolone was administered immediately before reperfusion of the liver graft and was tapered to oral prednisolone over one week and reduced to withdrawal after six months.
All statistical analyses were performed using R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p ≤ 0.05. Continuous variables are presented as mean ± standard deviation (SD) and median (interquartile range, IQR), and categorical variables are presented as frequency (percentage, %). The survival curve was estimated by the Kaplan-Meier method. A univariate analysis was conducted to examine the differences in the distributions of continuous variables, categorical variables, and survival outcomes between the surviving and deceased liver recipients five years after LT using the Wilcoxon rank-sum test, Chi-square test, Fisher’s exact test, or log-rank test as appropriate for the data type. A multivariate analysis was performed to estimate the adjusted effects of risk factors or prognostic factors on the survival outcome using a multiple Cox’s proportional hazards model.
To ensure a good quality of regression analysis, the model-fitting techniques for variable selection, goodness-of-fit (GOF) assessment, and regression diagnostics and remedies were used in our regression analysis. Specifically, the stepwise variable selection procedure (with iterations between the forward and backward steps) was applied to obtain the best final regression model using the My.stepwise package of the R software [16]. All significant and non-significant relevant covariates from the univariate analysis (listed in Table 1) and some of the interaction terms were used in the multivariate analysis. The significance levels for entry and for stay were set to 0.15 for being conservative. With the aid of substantive knowledge, the best candidate final regression model was identified manually by dropping the covariates with p value > 0.05 one at a time until all regression coefficients were significantly different from 0. To assess the GOF of the fitted Cox’s model, the concordance and adjusted generalized R2 [17] were examined. A concordance ≥ 0.7 and an adjusted generalized R2 > 0.15 indicated an acceptable level of discrimination, power, and fitness.
Moreover, the smoothing option “pspline” (for the smoothing splines using a “p-spline” basis) was specified inside the coxph function of the survival package to smooth the effects of continuous covariates on the log-hazard rate of the simple and multiple Cox’s proportional hazards models in R. Then, the termplot function of the stats package was used to plot the smoothed effects of the continuous covariates on the log-hazard rate in R [18]. The regression diagnostics for the verification of proportional hazards assumption, residual analysis, detection of influential cases, and a multicollinearity check were applied to discover any model or data problems. A variance inflating factor (VIF) ≥ 10 in continuous covariates or VIF ≥ 2.5 in categorical covariates indicated the occurrence of the multicollinearity problem among some of the covariates in the fitted regression model.
3. Results
3.1. Patients’ Demographic and Clinical Characteristics
The median follow-up duration was 9.63 years (IQR: 7.2–11.4 years), and the mean follow-up was 9.58 ± 2.74 years. A total of 121 males (64.0%) and 68 females (36.0%) were included in this study (Table 1). The median age at LT was 54.26 years (IQR: 48.63–58.58 years, range: 18.3–73.1 years), and the mean age at LT was 52.7 ± 9.6 years. Among the 189 patients, 114 (76.2%) underwent living donor transplants and 45 (23.8%) underwent deceased donor transplants. The main indications for LT were hepatitis B virus (HBV) cirrhosis (52.9%), hepatocellular carcinoma (38.1%), hepatitis C virus (HCV) cirrhosis (22.2%), and fulminant hepatitis (12.2%). The median of the mean tacrolimus blood trough level during the fifth year after LT was 5.0 ng/mL (IQR: 4.12–6.33 ng/mL), and the mean tacrolimus blood trough level was 5.249 ± 1.71 ng/mL. The mean tacrolimus blood trough level was ≥ 5 ng/mL in 96 patients (50.8%) and < 5 ng/mL in 93 patients (49.2%), including 44 patients (23.3%) with a mean tacrolimus blood trough level < 4 ng/mL and 11 patients (5.8%) with a mean tacrolimus blood trough level < 3 ng/mL. No significant differences in gender, age at LT, body weight, blood type, graft type, etiology of LT, or serum total bilirubin were found between the deceased and surviving LT recipients (p > 0.05). However, serum creatinine levels > 1.5 mg/dL (23/173 vs. 8/16, p = 0.001), end-stage renal disease (2/173 vs. 3/16, p = 0.005), and mean tacrolimus blood trough levels < 4 ng/mL (36/173 vs. 8/16, p = 0.014) were significantly more common among deceased LT recipients compared to survivors. The causes of death included malignancies (n = 5), graft failures (n = 4), infection (n = 4), cerebrovascular accidents (n = 2), and duodenal ulcer bleeding (n = 1) (Table 2). The distribution of the tacrolimus trough level stratified by the etiology and cause of death among the 16 dead patients was shown in the box plot of Figure 2. There was no statistical significance in the tacrolimus trough level between the different causes of death (p = 0.3823).
3.2. Predictors of Patients’ Long-Term Survival
The Cox’s model fitted to the survival data for the multivariate analyses of the time to death after five years of LT is shown in Table 3. After adjusting for the effects of the other covariates, age at LT ≤ 27.011 years (estimated hazard ratio [HR] = 168.79, 95% confidence interval [C.I.]: 11.13–2559.51), pre-transplant autoimmune liver disease (HR = 8.12, 95% C.I.: 1.97–33.43), pre-transplant HCV infection × survival time in years (HR = 1.34, 95% C.I.: 1.12–1.60), serum creatinine level > 1.311 mg/dL × serum total bilirubin level > 1.411 mg/dL (HR = 921.69, 95% C.I.: 43.40–19,573.71), serum creatinine level > 1.311 mg/dL × serum total bilirubin level ≤ 0.792 mg/dL (HR = 105.68, 95% C.I.: 7.81–1430.79), serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level > 0.882 mg/dL (HR = 30.49, 95% C.I.: 2.98–312.34), and the mean tacrolimus trough level during the fifth year after LT ≤ 4.609 ng/mL or > 10.168 ng/mL (HR = 4.76, 95% C.I.: 1.34–16.94) were associated with a higher long-term mortality five years after LT. The time-dependent interaction term, HCV × survival time in years, was added to the Cox’s model to account for the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.292, indicates that the risk of mortality in patients with HCV increases with time five years post-LT. Moreover, the three second-order interaction terms between the serum creatinine and total bilirubin levels were compared to the other two possible combinations, serum creatinine level > 1.311 mg/dL × (0.792 mg/dL < serum total bilirubin level ≤ 1.411 mg/dL) and serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level ≤ 0.882 mg/dL, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and”.
All cut-off values of the continuous covariates (such as age at LT) were estimated by applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models. As shown in Figure 3, the optimal cut-off values of the mean tacrolimus trough level, 4.609 ng/mL and 10.168 ng/mL, were estimated directly in the “p-spline plot,” which allowed the visualization of the nonlinear effect of the averaged dosage of the tacrolimus-based immunosuppressant during the fifth year after LT on log(λ), where λ was the hazard rate of time to death five years after LT. Then, the Kaplan-Meier estimates of survival curves for time to death five years after LT were determined for the 66 patients with mean tacrolimus trough levels ≤ 4.609 ng/mL or > 10.168 ng/mL and the 123 patients with mean tacrolimus trough levels between 4.609 ng/mL and 10.168 ng/mL (log-rank test, p = 0.009) (Figure 4). In an additional subgroup analysis, we found that a mean tacrolimus trough level between 4.431 ng/mL and 6.332 ng/mL for patients with a serum creatinine level > 1.311 mg/dL improved survival. Finally, this Cox’s model had a concordance of 0.904 and an adjusted generalized R2 of 0.388, indicating that it fit the survival data very well.
4. Discussion
This is the first study to report an association between long-term tacrolimus blood trough level and long-term patient survival in adult LT recipients. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL in adult LT recipients is associated with a lower mortality rate. In patients with a serum creatinine > 1.3 mg/dL, a mean tacrolimus blood trough level of 4.4–6.3 ng/mL is associated with a lower mortality rate.
Decreasing tacrolimus use during long-term follow-up in LT recipients is a general concept in clinical practice. In this study, only one patient had the mean level > 10 ng/mL and a few patients had the mean level > 8 ng/mL (Figure 3). Patients with such high drug levels were most likely because of their poor compliance (not must because of they have truly such high trough level). Poor compliance indicates that a patient takes incorrect dosage of immunosuppressant that their physician prescribed, or takes immunosuppressant or blood exam at the wrong time, either of which might clearly be important factors that influence graft and survival outcome of LT patients during long-term follow-ups. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL is associated with lower mortality rate; however, the upper limit of 10.2 ng/mL suggested by the regression analysis was merely a value for caution, but not a recommended upper limit in real clinical practice. The long-term hazard of high level of tacrolimus is already widely known; therefore, we would focus more on the findings that it appears to increase the survival risks in LT recipients if their tacrolimus levels are maintained below 4.6 ng/mL during the long-term follow-ups.
Inadequate immunosuppression is associated with higher risks of graft rejection [19], while excess immunosuppression increases the risks of malignancy and infection and increases the adverse effects of drug toxicity. The tailored use of immunosuppressants should be considered based on the patient’s risks of graft rejection and infection and the patient’s medical comorbidities and liver disease status prior to LT [14]. Patients with autoimmune liver diseases may require more immunosuppression to prevent disease recurrence and graft rejection [20]. By contrast, lower doses of immunosuppression are recommended in patients who have undergone LT due to HCV, as high levels of immunosuppression are related to increased viral replication [21,22]. Rejection results in abnormal liver function and is one of the most important factors associated with poor long-term graft and patient outcomes [23,24,25]. Tacrolimus reduces the risk of T-cell mediated rejection to protect graft function and is the cornerstone of a successful LT. However, short and long-term adverse effects of tacrolimus such as infection, chronic renal insufficiency, metabolic diseases (hyperlipidemia, hypertension, and diabetes mellitus), and malignancy have been widely reported and influence patients’’ long-term outcomes [7,8,9]. The early causes of death after LT within one year are infection and graft loss, and the late causes of death three years post-LT are malignancy, cardiovascular disease, and renal failure [10]. As graft function typically stabilizes, factors associated with the long-term outcome are often patient-related factors (such as chronic medical diseases) that are usually associated with tacrolimus.
Previous studies have reported that minimizing tacrolimus use in the early post-LT period is associated with a lower risk of new-onset diabetes mellitus [26], a lower incidence of hyperlipidemia [27], and better long-term survival [19,28]. In recent years, studies have focused on the reduction or complete withdrawal of long-term immunosuppressants in LT recipients [12,29,30,31,32,33,34]. One study reported satisfactory outcomes with the combined use of tacrolimus and mycophenolate mofetil, which allowed for the tacrolimus dose to be reduced [35]. Other studies demonstrated that the concomitant use of everolimus may reduce the required dose of tacrolimus while having potential renal benefits [29,36]. However, only some LT recipients were able to discontinue the use of immunosuppressants, and these patients more frequently experienced biopsy-proven acute rejection [29]. The risk of chronic rejection during long-term follow-up remains in patients with inadequate immunosuppression [30,33]. The tacrolimus blood concentration in LT recipients may play a key role in long-term outcomes, as it is associated with long-term graft function and its adverse effects are related to several chronic medical diseases. However, to the best of our knowledge, this is the first report regarding the effect of long-term tacrolimus levels on the long-term survival outcomes of LT recipients.
Several factors are associated with the long-term outcomes of adult LT recipients, including pre-transplant primary sclerosing cholangitis, immunosuppression therapies, acute and chronic rejections, malignancy, and metabolic syndrome [37,38]. As listed in Table 3, we found several independent risk factors of long-term mortality in this study. Only four patients aged <27 years at the time of LT were included in this study, and one died due to pneumonia 6.3 years after LT. Patients who underwent LT due to autoimmune liver diseases had worse outcomes, as they were more likely to experience acute rejections [39] and suffer disease recurrence, leading to graft loss. Patients who underwent LT due to HCV infections had relatively poor post-LT outcomes due to the disease recurrence followed by graft dysfunction and failure [40]. As direct-acting antiviral agents have advanced [41], the survival outcomes of HCV patients should improve in the near future. Long-term renal and liver functions are reflected by serum creatinine and total bilirubin levels, respectively. We found that abnormal serum creatinine levels have a bigger impact than abnormal serum total bilirubin levels on the survival outcomes of LT recipients. The graft function of adult LT recipients is chronically stable five years after LT. While some patients may experience asymptomatic hyperbilirubinemia at this time, the long-term outcomes remain favorable. By contrast, the gradual deterioration of renal function may lead to chronic kidney disease or end-stage renal disease, affecting the long-term survival, especially in patients with long-term use of tacrolimus.
This study had some limitations. First, the data were obtained from a single medical center in Asia, which provided a relatively small number of eligible patients with single ethnicity and fewer death events. Second, the study spanned a long time period, and therefore the improvements in surgical and medical expertise and advances in immunosuppression therapies may have influenced the patient outcomes. Third, we did not collect or analyze the time-dependent tacrolimus blood trough level during the follow-up visits five years after LT.
In summary, we found an association between the long-term tacrolimus blood trough levels and the long-term survival five years after LT. A mean tacrolimus blood trough level outside the range of 4.6–10.2 ng/mL appeared to be an independent risk factor for long-term mortality. Further studies with larger sample sizes are needed to verify these results and to further identify an appropriate tacrolimus blood trough level for maintenance use.
Acknowledgments
We thank the coordinators (Hui-Ying Lin and Min-Heuy Lin) for their helpful efforts in data collection.
Author Contributions
C.-Y.H. drafted the manuscript and R.-H.H. designed the study. C.-Y.H., M.-C.H., and Y.-M.W. conducted data processing, and C.-Y.H. and C.-M.H. performed data analysis. P.-H.L. and R.-H.H. were the directors responsible for general organization and instruction. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of National Taiwan University Hospital.
Informed Consent Statement
Patient consent was waived due to retrospective design of the study.
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1 Patient flow diagram.
Figure 2 The box plot of tacrolimus mean trough level stratified by the etiology and cause of death among the 16 dead patients. There was no statistical difference of tacrolimus level between the different causes of death (p = 0.3823).
Figure 3 The p-spline plot for the nonlinear effect of the long-term tacrolimus blood trough levels on time to death. The tacrolimus blood trough level is shown on the X-axis, and the log(λ), where λ is the hazard rate of time to death five years after liver transplantation, is shown on the Y-axis. In this p-spline plot, the intersection between the horizontal green line (Y = 0) and the red curve yields the estimated optimal cut-off values for long-term tacrolimus blood trough levels at which the values of log(λ) will not change (4.609 ng/mL and 10.168 ng/mL). When the level is ≤4.609 ng/mL or >10.168 ng/mL, the value of log(λ) increases, indicating an increasing λ. When the level is >4.609 ng/mL and ≤10.168 ng/mL, the value of log(λ) decreases, indicating a decreasing λ. The vertical bars above the X-axis represent the patients’ actual mean tacrolimus blood trough levels obtained at the three follow-up visits during the fifth year after LT.
Figure 4 The Kaplan-Meier curve for time to death five years after liver transplantation. The survival curve of the 66 patients with a mean tacrolimus blood trough level ≤ 4.609 ng/mL or >10.168 ng/mL is shown in red while the survival curve of the 123 patients with a mean tacrolimus blood trough level > 4.609 ng/mL or ≤10.168 ng/mL is shown in green.
jpm-11-00090-t001_Table 1 Table 1 Univariate analysis for comparing the distributions of the demographic and clinical characteristics between the alive and dead adult liver recipients after five years of liver transplantations.
Variable All Patients
(n = 189) Alive
(n = 173) Dead
(n = 16) p Value
Gender 0.7899
Male 121 (64.0) 110 (90.9) 11 (9.1)
Female 68 (36.0) 63 (92.6) 5 (7.4)
Age at LT (years) 52.7 ± 9.6 52.6 ± 9.5 53.4 ± 10.7 0.6672
Body weight at LT (kg) 64.9 ± 12.2 65.0 ± 12.3 63.7 ± 12.1 0.4459
Blood type 0.8719
O 79 (41.8) 73 (92.4) 6 (7.6)
A 51 (27.0) 46 (90.2) 5 (9.8)
B 42 (22.2) 39 (92.9) 3 (7.1)
AB 17 (9.0) 15 (88.2) 2 (11.8)
Graft type 1.0000
Living donor 144 (76.2) 132 (91.7) 12 (8.3)
Deceased donor 45 (23.8) 41 (91.1) 4 (8.9)
Etiology for LT
Liver malignancy (HCC) 72 (38.1) 66 (91.7) 6 (8.3) 1.0000
Alcoholic cirrhosis 17 (9.0) 16 (94.1) 1 (5.9) 1.0000
HBV cirrhosis 100 (52.9) 92 (92.0) 8 (8.0) 1.0000
HCV cirrhosis 42 (22.2) 38 (90.5) 4 (9.5) 0.7577
Fulminant hepatitis 23 (12.2) 23 (100) 0 (0) 0.2259
Autoimmune disease 14 (7.4) 11 (78.6) 3 (21.4) 0.1017
Biliary atresia 5 (2.6) 5 (100) 0 (0) 1.0000
Other 10 (5.3) 9 (90) 1 (10) 0.5964
Total bilirubin (mg/dL) 0.973 ± 0.51 0.958 ± 1.53 1.133 ± 0.69 0.6260
Total bilirubin > 1 mg/dL 60 (31.7) 53 (88.3) 7 (11.7) 0.2770
Total bilirubin > 2 mg/dL 11 (5.8) 9 (81.8) 2 (18.2) 0.2360
Creatinine (mg/dL) 1.391 ± 1.25 1.279 ± 0.96 2.6 ± 2.74 0.0201 *
Creatinine > 1.5 mg/dL 31 (16.4) 23 (74.2) 8 (25.8) 0.0010 *
ESRD 5 (2.6) 2 (40.0) 3 (60.0) 0.0050 *
Tacrolimus mean level (ng/mL) 5.249 ± 1.71 5.263 ± 1.53 5.096 ± 3.12 0.9787
Tacrolimus level < 5 ng/mL 93 (49.2) 82 (88.2) 11 (11.8) 0.1216
Tacrolimus level < 4 ng/mL 44 (23.3) 36 (81.8) 8 (18.2) 0.0136 *
Tacrolimus level < 3 ng/mL 11 (5.8) 9 (81.8) 2 (18.2) 0.2356
Data are presented as mean ± standard deviation (SD) for continuous variables and frequency (percentage, %) for categorical variables. The p-values of statistical tests were calculated using the Wilcoxon rank-sum test for continuous variables and the Fisher’s exact test for categorical variables. * p value ≤ 0.05. Abbreviations: LT, liver transplantation; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; Total bilirubin, serum total bilirubin level; Creatinine, serum creatinine level; and ESRD, end-stage renal disease (defined by receiving hemodialysis regularly).
jpm-11-00090-t002_Table 2 Table 2 The causes of 16 deaths since 5 years after adult liver transplantations.
Causes of Deaths Number of Subjects
Malignancy 5 (31.25%)
De novo: Multiple myeloma, bladder cancer, colon cancer, prostate cancer 4
Recurrent: Hepatocellular carcinoma 1
Graft failure 4 (25.00%)
Chronic rejection 3
Autoimmune hepatitis 1
Infection 4 (25.00%)
Pneumonia 3
Urinary tract infection 1
Cerebral vascular event (intracerebral hemorrhage) 2 (12.50%)
Peptic ulcer bleeding 1 (6.25%)
jpm-11-00090-t003_Table 3 Table 3 Multivariate analysis for identifying the predictors of long-term overall survival after 5 years of liver transplantations by fitting a multiple Cox’s Model in the adult liver transplant recipients 1.
Covariate 2 Estimate Standard
Error Wald’s
z Test p Value Hazard
Ratio (HR) 95% Confidence
Interval (C.I.)
Age at LT ≤ 27.011 years 5.1286 1.3872 3.6970 0.0002 168.7851 11.130–2559.512
Autoimmune (including PBC) 2.0946 0.7219 2.9015 0.0037 8.1221 1.973–33.431
HCV × Overall survival years 0.2924 0.0914 3.1978 0.0014 1.3397 1.120–1.603
Cre > 1.311 × T-Bil > 1.411 mg/dL 6.8262 1.5591 4.3784 <0.0001 921.6940 43.401–19,573.712
Cre > 1.311 × T-Bil ≤ 0.792 mg/dL 4.6604 1.3294 3.5056 0.0005 105.6778 7.805–1430.790
Cre ≤ 1.311 × T-Bil > 0.882 mg/dL 3.4174 1.1871 2.8788 0.0040 30.4913 2.977–312.341
Tacrolimus mean ≤ 4.609 or > 10.168 ng/mL 1.5599 0.6479 2.4076 0.0161 4.7581 1.336–16.940
1 The above multiple Cox’s model was fitted to the 189 adult patients who underwent liver transplantations with 16 death events, for modeling the hazard rate of the right-censored overall survival time five years after liver transplantations. All the cut-off values of the continuous covariates (e.g., age at liver transplantations) were estimated by choosing the option of applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models (e.g., Figure 3). The time-dependent interaction term, HCV × Overall survival year, was added to the Cox’s model for handling the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.2924, indicated that the risk of dying in the patients with HCV would increase as time elapsed five years after liver transplantations. Moreover, the three second-order interaction terms, Cre > 1.311 × T-Bil > 1.411, Cre > 1.311 × T-Bil ≤ 0.792, and Cre ≤ 1.311 × T-Bil > 0.882, were compared to the other two possible combinations, Cre > 1.311 × (0.792 < T-Bil ≤ 1.411) and Cre ≤ 1.311 × T-Bil ≤ 0.882, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and.” Finally, both goodness-of-fit (GOF) measures, concordance = 0.9041 (se = 0.0265) > 0.7 and adjusted generalized R2 = 0.3878 > 0.15, indicated an excellent fit of this multiple Cox’s model to the observed survival data. 2 Abbreviations: LT, liver transplantation; PBC, primary biliary cirrhosis; HCV, hepatitis C viral infection; Cre, serum creatinine level (mg/dL); T-Bil, serum total bilirubin level (mg/dL); and Tacrolimus mean, the averaged dosage of the tacrolimus-based immunosuppressant (ng/mL).
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What was the dosage of drug 'TACROLIMUS'? | Long-Term Tacrolimus Blood Trough Level and Patient Survival in Adult Liver Transplantation.
Tacrolimus is the most widely used immunosuppressant in liver transplant (LT) patients. However, the ideal long-term target level for these patients is unknown. This retrospective study aimed to investigate the impact of tacrolimus blood concentration five years after LT on long-term patient survival outcomes in adult LT recipients. Patients who underwent LT between January 2004 and July 2014 at a tertiary medical center were included in this study (n = 189). The mean tacrolimus blood concentrations of each patient during the fifth year after LT were recorded and the overall survival rate was determined. A multivariate analysis of factors associated with long-term survival was conducted using a Cox's model. The median follow-up period was 9.63 years, and 144 patients (76.2%) underwent live donor LT. Sixteen patients died within 5 years of LT. In the Cox's model, patients with a mean tacrolimus blood trough level of 4.6-10.2 ng/mL had significantly better long-term survival than those with a mean tacrolimus blood trough level outside this range (estimated hazard ratio = 4.76; 95% confidence interval: 1.34-16.9, p = 0.016). Therefore, a tacrolimus level no lower than 4.6 ng/mL would be recommended in adult LT patients.
1. Introduction
Liver transplantation (LT) has become a mature treatment of end-stage liver disease in clinical practice [1]. The continuous improvement of effective immunosuppression treatments has led to a significant improvement in patient and graft survival in recent years [2]. Calcineurin inhibitors are the cornerstone of immunosuppression in LT [3], and tacrolimus is currently the mainstay of initial and maintenance immunosuppression therapies [4,5,6]. Tacrolimus reduces the incidence and severity of early and late T-cell mediated rejection by inhibiting T-cell production of interleukin-2 [5,6]. However, the long-term use of immunosuppressants leads to an increasing burden of toxicity. The reported toxic effects of calcineurin inhibitors include infections, chronic renal insufficiency, metabolic diseases (such as hyperlipidemia, hypertension, and diabetes mellitus), and malignancy [7,8,9], which, along with cardiovascular diseases, have been reported to be the major causes of morbidity and mortality after LT [3,10]. However, most LT patients cannot withdraw from lifelong immunosuppression therapy; the only exceptions are a few selected patients participating in experimental trials [11,12].
It is a clinical challenge to design a well-balanced immunosuppressive regimen for LT recipients. Tacrolimus has a narrow therapeutic dose range and its blood levels should be carefully monitored. The Advagraf (tacrolimus prolonged-release hard capsules) recommendation for adult LT patients is a blood trough level of 5–20 ng/mL in the early post-transplant period and 5–15 ng/mL during subsequent maintenance therapy. Nevertheless, the current recommendations from the clinical practice guidelines for the ideal tacrolimus level in adults after LT remain controversial. The American Association for the Study of Liver Diseases (AASLD) recommends a target blood trough level of 5–10 ng/mL for tacrolimus three months after LT [13]. The Consensus on Managing Modifiable Risk in Transplantation Group (COMMIT) recommends that the target tacrolimus blood trough levels be 6–10 ng/mL during the first month after LT and decrease to 4–8 ng/mL thereafter, except when used in combination with mammalian target of rapamycin (mTOR) inhibitors [14]. Moreover, the International Liver Transplant Society (ILTS) consensus statement on immunosuppression in LT recipients recommends the target blood trough levels of tacrolimus be 6–10 ng/mL three months after LT, lower than 5 ng/mL 12 months after LT, and decrease to 3 ng/mL thereafter, resulting in a blood trough level just above the lower limit of detection five years after LT [15]. However, the impact of the long-term tacrolimus blood trough level on the outcomes of the LT recipients remains unclear. This study aimed to suggest an appropriate tacrolimus blood trough level for adult patients five years after LT.
2. Materials and Methods
This study was approved by the Institutional Review Board of National Taiwan University Hospital and was conducted according to the Declaration of Helsinki. A total of 286 patients who underwent LT at 18 years or older at a tertiary medical center from January 2004 to July 2014 were recruited for this study. Patients who died within 5 years of LT, were lost to follow-up, or did not use tacrolimus-based calcineurin inhibitor for immunosuppressant therapy were excluded from this study (Figure 1). In addition, those who received mTOR inhibitor treatment were excluded, as mTOR inhibitors are typically used as a combination therapy to reduce the required dose of tacrolimus. The final analysis included 189 patients. All eligible patients were followed up for more than five years until August 2019. Patients who underwent a LT due to liver cancer met the Milan criteria (before 2006) or the criteria of the University of California, San Francisco (since 2006) at the time of LT. All patients received regular monthly or bi-monthly follow-ups at the outpatient clinic after LT. Routine blood examinations for the tacrolimus blood trough level, liver function, and renal function were conducted at each visit, and abdominal sonography was performed every 6 to 12 months.
The patients’ medical records were reviewed retrospectively to extract demographic and clinical data, including patient characteristics, laboratory tests, and survival outcomes. The serum bilirubin and creatinine data at the end of the fifth year after LT were used in this study. The tacrolimus level used in this study was the mean of the values obtained at the three follow-up visits during the fifth year after LT.
The immunosuppression protocol for adult LT patients consisted of tacrolimus, mycophenolate mofetil, and steroids. Tacrolimus was administered orally beginning on the first day after LT, and the dose was adjusted to achieve the desired therapeutic drug level. Basiliximab was administered immediately before graft reperfusion and on the fourth day after LT for induction therapy. A 500-mg intravenous bolus of methylprednisolone was administered immediately before reperfusion of the liver graft and was tapered to oral prednisolone over one week and reduced to withdrawal after six months.
All statistical analyses were performed using R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p ≤ 0.05. Continuous variables are presented as mean ± standard deviation (SD) and median (interquartile range, IQR), and categorical variables are presented as frequency (percentage, %). The survival curve was estimated by the Kaplan-Meier method. A univariate analysis was conducted to examine the differences in the distributions of continuous variables, categorical variables, and survival outcomes between the surviving and deceased liver recipients five years after LT using the Wilcoxon rank-sum test, Chi-square test, Fisher’s exact test, or log-rank test as appropriate for the data type. A multivariate analysis was performed to estimate the adjusted effects of risk factors or prognostic factors on the survival outcome using a multiple Cox’s proportional hazards model.
To ensure a good quality of regression analysis, the model-fitting techniques for variable selection, goodness-of-fit (GOF) assessment, and regression diagnostics and remedies were used in our regression analysis. Specifically, the stepwise variable selection procedure (with iterations between the forward and backward steps) was applied to obtain the best final regression model using the My.stepwise package of the R software [16]. All significant and non-significant relevant covariates from the univariate analysis (listed in Table 1) and some of the interaction terms were used in the multivariate analysis. The significance levels for entry and for stay were set to 0.15 for being conservative. With the aid of substantive knowledge, the best candidate final regression model was identified manually by dropping the covariates with p value > 0.05 one at a time until all regression coefficients were significantly different from 0. To assess the GOF of the fitted Cox’s model, the concordance and adjusted generalized R2 [17] were examined. A concordance ≥ 0.7 and an adjusted generalized R2 > 0.15 indicated an acceptable level of discrimination, power, and fitness.
Moreover, the smoothing option “pspline” (for the smoothing splines using a “p-spline” basis) was specified inside the coxph function of the survival package to smooth the effects of continuous covariates on the log-hazard rate of the simple and multiple Cox’s proportional hazards models in R. Then, the termplot function of the stats package was used to plot the smoothed effects of the continuous covariates on the log-hazard rate in R [18]. The regression diagnostics for the verification of proportional hazards assumption, residual analysis, detection of influential cases, and a multicollinearity check were applied to discover any model or data problems. A variance inflating factor (VIF) ≥ 10 in continuous covariates or VIF ≥ 2.5 in categorical covariates indicated the occurrence of the multicollinearity problem among some of the covariates in the fitted regression model.
3. Results
3.1. Patients’ Demographic and Clinical Characteristics
The median follow-up duration was 9.63 years (IQR: 7.2–11.4 years), and the mean follow-up was 9.58 ± 2.74 years. A total of 121 males (64.0%) and 68 females (36.0%) were included in this study (Table 1). The median age at LT was 54.26 years (IQR: 48.63–58.58 years, range: 18.3–73.1 years), and the mean age at LT was 52.7 ± 9.6 years. Among the 189 patients, 114 (76.2%) underwent living donor transplants and 45 (23.8%) underwent deceased donor transplants. The main indications for LT were hepatitis B virus (HBV) cirrhosis (52.9%), hepatocellular carcinoma (38.1%), hepatitis C virus (HCV) cirrhosis (22.2%), and fulminant hepatitis (12.2%). The median of the mean tacrolimus blood trough level during the fifth year after LT was 5.0 ng/mL (IQR: 4.12–6.33 ng/mL), and the mean tacrolimus blood trough level was 5.249 ± 1.71 ng/mL. The mean tacrolimus blood trough level was ≥ 5 ng/mL in 96 patients (50.8%) and < 5 ng/mL in 93 patients (49.2%), including 44 patients (23.3%) with a mean tacrolimus blood trough level < 4 ng/mL and 11 patients (5.8%) with a mean tacrolimus blood trough level < 3 ng/mL. No significant differences in gender, age at LT, body weight, blood type, graft type, etiology of LT, or serum total bilirubin were found between the deceased and surviving LT recipients (p > 0.05). However, serum creatinine levels > 1.5 mg/dL (23/173 vs. 8/16, p = 0.001), end-stage renal disease (2/173 vs. 3/16, p = 0.005), and mean tacrolimus blood trough levels < 4 ng/mL (36/173 vs. 8/16, p = 0.014) were significantly more common among deceased LT recipients compared to survivors. The causes of death included malignancies (n = 5), graft failures (n = 4), infection (n = 4), cerebrovascular accidents (n = 2), and duodenal ulcer bleeding (n = 1) (Table 2). The distribution of the tacrolimus trough level stratified by the etiology and cause of death among the 16 dead patients was shown in the box plot of Figure 2. There was no statistical significance in the tacrolimus trough level between the different causes of death (p = 0.3823).
3.2. Predictors of Patients’ Long-Term Survival
The Cox’s model fitted to the survival data for the multivariate analyses of the time to death after five years of LT is shown in Table 3. After adjusting for the effects of the other covariates, age at LT ≤ 27.011 years (estimated hazard ratio [HR] = 168.79, 95% confidence interval [C.I.]: 11.13–2559.51), pre-transplant autoimmune liver disease (HR = 8.12, 95% C.I.: 1.97–33.43), pre-transplant HCV infection × survival time in years (HR = 1.34, 95% C.I.: 1.12–1.60), serum creatinine level > 1.311 mg/dL × serum total bilirubin level > 1.411 mg/dL (HR = 921.69, 95% C.I.: 43.40–19,573.71), serum creatinine level > 1.311 mg/dL × serum total bilirubin level ≤ 0.792 mg/dL (HR = 105.68, 95% C.I.: 7.81–1430.79), serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level > 0.882 mg/dL (HR = 30.49, 95% C.I.: 2.98–312.34), and the mean tacrolimus trough level during the fifth year after LT ≤ 4.609 ng/mL or > 10.168 ng/mL (HR = 4.76, 95% C.I.: 1.34–16.94) were associated with a higher long-term mortality five years after LT. The time-dependent interaction term, HCV × survival time in years, was added to the Cox’s model to account for the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.292, indicates that the risk of mortality in patients with HCV increases with time five years post-LT. Moreover, the three second-order interaction terms between the serum creatinine and total bilirubin levels were compared to the other two possible combinations, serum creatinine level > 1.311 mg/dL × (0.792 mg/dL < serum total bilirubin level ≤ 1.411 mg/dL) and serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level ≤ 0.882 mg/dL, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and”.
All cut-off values of the continuous covariates (such as age at LT) were estimated by applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models. As shown in Figure 3, the optimal cut-off values of the mean tacrolimus trough level, 4.609 ng/mL and 10.168 ng/mL, were estimated directly in the “p-spline plot,” which allowed the visualization of the nonlinear effect of the averaged dosage of the tacrolimus-based immunosuppressant during the fifth year after LT on log(λ), where λ was the hazard rate of time to death five years after LT. Then, the Kaplan-Meier estimates of survival curves for time to death five years after LT were determined for the 66 patients with mean tacrolimus trough levels ≤ 4.609 ng/mL or > 10.168 ng/mL and the 123 patients with mean tacrolimus trough levels between 4.609 ng/mL and 10.168 ng/mL (log-rank test, p = 0.009) (Figure 4). In an additional subgroup analysis, we found that a mean tacrolimus trough level between 4.431 ng/mL and 6.332 ng/mL for patients with a serum creatinine level > 1.311 mg/dL improved survival. Finally, this Cox’s model had a concordance of 0.904 and an adjusted generalized R2 of 0.388, indicating that it fit the survival data very well.
4. Discussion
This is the first study to report an association between long-term tacrolimus blood trough level and long-term patient survival in adult LT recipients. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL in adult LT recipients is associated with a lower mortality rate. In patients with a serum creatinine > 1.3 mg/dL, a mean tacrolimus blood trough level of 4.4–6.3 ng/mL is associated with a lower mortality rate.
Decreasing tacrolimus use during long-term follow-up in LT recipients is a general concept in clinical practice. In this study, only one patient had the mean level > 10 ng/mL and a few patients had the mean level > 8 ng/mL (Figure 3). Patients with such high drug levels were most likely because of their poor compliance (not must because of they have truly such high trough level). Poor compliance indicates that a patient takes incorrect dosage of immunosuppressant that their physician prescribed, or takes immunosuppressant or blood exam at the wrong time, either of which might clearly be important factors that influence graft and survival outcome of LT patients during long-term follow-ups. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL is associated with lower mortality rate; however, the upper limit of 10.2 ng/mL suggested by the regression analysis was merely a value for caution, but not a recommended upper limit in real clinical practice. The long-term hazard of high level of tacrolimus is already widely known; therefore, we would focus more on the findings that it appears to increase the survival risks in LT recipients if their tacrolimus levels are maintained below 4.6 ng/mL during the long-term follow-ups.
Inadequate immunosuppression is associated with higher risks of graft rejection [19], while excess immunosuppression increases the risks of malignancy and infection and increases the adverse effects of drug toxicity. The tailored use of immunosuppressants should be considered based on the patient’s risks of graft rejection and infection and the patient’s medical comorbidities and liver disease status prior to LT [14]. Patients with autoimmune liver diseases may require more immunosuppression to prevent disease recurrence and graft rejection [20]. By contrast, lower doses of immunosuppression are recommended in patients who have undergone LT due to HCV, as high levels of immunosuppression are related to increased viral replication [21,22]. Rejection results in abnormal liver function and is one of the most important factors associated with poor long-term graft and patient outcomes [23,24,25]. Tacrolimus reduces the risk of T-cell mediated rejection to protect graft function and is the cornerstone of a successful LT. However, short and long-term adverse effects of tacrolimus such as infection, chronic renal insufficiency, metabolic diseases (hyperlipidemia, hypertension, and diabetes mellitus), and malignancy have been widely reported and influence patients’’ long-term outcomes [7,8,9]. The early causes of death after LT within one year are infection and graft loss, and the late causes of death three years post-LT are malignancy, cardiovascular disease, and renal failure [10]. As graft function typically stabilizes, factors associated with the long-term outcome are often patient-related factors (such as chronic medical diseases) that are usually associated with tacrolimus.
Previous studies have reported that minimizing tacrolimus use in the early post-LT period is associated with a lower risk of new-onset diabetes mellitus [26], a lower incidence of hyperlipidemia [27], and better long-term survival [19,28]. In recent years, studies have focused on the reduction or complete withdrawal of long-term immunosuppressants in LT recipients [12,29,30,31,32,33,34]. One study reported satisfactory outcomes with the combined use of tacrolimus and mycophenolate mofetil, which allowed for the tacrolimus dose to be reduced [35]. Other studies demonstrated that the concomitant use of everolimus may reduce the required dose of tacrolimus while having potential renal benefits [29,36]. However, only some LT recipients were able to discontinue the use of immunosuppressants, and these patients more frequently experienced biopsy-proven acute rejection [29]. The risk of chronic rejection during long-term follow-up remains in patients with inadequate immunosuppression [30,33]. The tacrolimus blood concentration in LT recipients may play a key role in long-term outcomes, as it is associated with long-term graft function and its adverse effects are related to several chronic medical diseases. However, to the best of our knowledge, this is the first report regarding the effect of long-term tacrolimus levels on the long-term survival outcomes of LT recipients.
Several factors are associated with the long-term outcomes of adult LT recipients, including pre-transplant primary sclerosing cholangitis, immunosuppression therapies, acute and chronic rejections, malignancy, and metabolic syndrome [37,38]. As listed in Table 3, we found several independent risk factors of long-term mortality in this study. Only four patients aged <27 years at the time of LT were included in this study, and one died due to pneumonia 6.3 years after LT. Patients who underwent LT due to autoimmune liver diseases had worse outcomes, as they were more likely to experience acute rejections [39] and suffer disease recurrence, leading to graft loss. Patients who underwent LT due to HCV infections had relatively poor post-LT outcomes due to the disease recurrence followed by graft dysfunction and failure [40]. As direct-acting antiviral agents have advanced [41], the survival outcomes of HCV patients should improve in the near future. Long-term renal and liver functions are reflected by serum creatinine and total bilirubin levels, respectively. We found that abnormal serum creatinine levels have a bigger impact than abnormal serum total bilirubin levels on the survival outcomes of LT recipients. The graft function of adult LT recipients is chronically stable five years after LT. While some patients may experience asymptomatic hyperbilirubinemia at this time, the long-term outcomes remain favorable. By contrast, the gradual deterioration of renal function may lead to chronic kidney disease or end-stage renal disease, affecting the long-term survival, especially in patients with long-term use of tacrolimus.
This study had some limitations. First, the data were obtained from a single medical center in Asia, which provided a relatively small number of eligible patients with single ethnicity and fewer death events. Second, the study spanned a long time period, and therefore the improvements in surgical and medical expertise and advances in immunosuppression therapies may have influenced the patient outcomes. Third, we did not collect or analyze the time-dependent tacrolimus blood trough level during the follow-up visits five years after LT.
In summary, we found an association between the long-term tacrolimus blood trough levels and the long-term survival five years after LT. A mean tacrolimus blood trough level outside the range of 4.6–10.2 ng/mL appeared to be an independent risk factor for long-term mortality. Further studies with larger sample sizes are needed to verify these results and to further identify an appropriate tacrolimus blood trough level for maintenance use.
Acknowledgments
We thank the coordinators (Hui-Ying Lin and Min-Heuy Lin) for their helpful efforts in data collection.
Author Contributions
C.-Y.H. drafted the manuscript and R.-H.H. designed the study. C.-Y.H., M.-C.H., and Y.-M.W. conducted data processing, and C.-Y.H. and C.-M.H. performed data analysis. P.-H.L. and R.-H.H. were the directors responsible for general organization and instruction. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of National Taiwan University Hospital.
Informed Consent Statement
Patient consent was waived due to retrospective design of the study.
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1 Patient flow diagram.
Figure 2 The box plot of tacrolimus mean trough level stratified by the etiology and cause of death among the 16 dead patients. There was no statistical difference of tacrolimus level between the different causes of death (p = 0.3823).
Figure 3 The p-spline plot for the nonlinear effect of the long-term tacrolimus blood trough levels on time to death. The tacrolimus blood trough level is shown on the X-axis, and the log(λ), where λ is the hazard rate of time to death five years after liver transplantation, is shown on the Y-axis. In this p-spline plot, the intersection between the horizontal green line (Y = 0) and the red curve yields the estimated optimal cut-off values for long-term tacrolimus blood trough levels at which the values of log(λ) will not change (4.609 ng/mL and 10.168 ng/mL). When the level is ≤4.609 ng/mL or >10.168 ng/mL, the value of log(λ) increases, indicating an increasing λ. When the level is >4.609 ng/mL and ≤10.168 ng/mL, the value of log(λ) decreases, indicating a decreasing λ. The vertical bars above the X-axis represent the patients’ actual mean tacrolimus blood trough levels obtained at the three follow-up visits during the fifth year after LT.
Figure 4 The Kaplan-Meier curve for time to death five years after liver transplantation. The survival curve of the 66 patients with a mean tacrolimus blood trough level ≤ 4.609 ng/mL or >10.168 ng/mL is shown in red while the survival curve of the 123 patients with a mean tacrolimus blood trough level > 4.609 ng/mL or ≤10.168 ng/mL is shown in green.
jpm-11-00090-t001_Table 1 Table 1 Univariate analysis for comparing the distributions of the demographic and clinical characteristics between the alive and dead adult liver recipients after five years of liver transplantations.
Variable All Patients
(n = 189) Alive
(n = 173) Dead
(n = 16) p Value
Gender 0.7899
Male 121 (64.0) 110 (90.9) 11 (9.1)
Female 68 (36.0) 63 (92.6) 5 (7.4)
Age at LT (years) 52.7 ± 9.6 52.6 ± 9.5 53.4 ± 10.7 0.6672
Body weight at LT (kg) 64.9 ± 12.2 65.0 ± 12.3 63.7 ± 12.1 0.4459
Blood type 0.8719
O 79 (41.8) 73 (92.4) 6 (7.6)
A 51 (27.0) 46 (90.2) 5 (9.8)
B 42 (22.2) 39 (92.9) 3 (7.1)
AB 17 (9.0) 15 (88.2) 2 (11.8)
Graft type 1.0000
Living donor 144 (76.2) 132 (91.7) 12 (8.3)
Deceased donor 45 (23.8) 41 (91.1) 4 (8.9)
Etiology for LT
Liver malignancy (HCC) 72 (38.1) 66 (91.7) 6 (8.3) 1.0000
Alcoholic cirrhosis 17 (9.0) 16 (94.1) 1 (5.9) 1.0000
HBV cirrhosis 100 (52.9) 92 (92.0) 8 (8.0) 1.0000
HCV cirrhosis 42 (22.2) 38 (90.5) 4 (9.5) 0.7577
Fulminant hepatitis 23 (12.2) 23 (100) 0 (0) 0.2259
Autoimmune disease 14 (7.4) 11 (78.6) 3 (21.4) 0.1017
Biliary atresia 5 (2.6) 5 (100) 0 (0) 1.0000
Other 10 (5.3) 9 (90) 1 (10) 0.5964
Total bilirubin (mg/dL) 0.973 ± 0.51 0.958 ± 1.53 1.133 ± 0.69 0.6260
Total bilirubin > 1 mg/dL 60 (31.7) 53 (88.3) 7 (11.7) 0.2770
Total bilirubin > 2 mg/dL 11 (5.8) 9 (81.8) 2 (18.2) 0.2360
Creatinine (mg/dL) 1.391 ± 1.25 1.279 ± 0.96 2.6 ± 2.74 0.0201 *
Creatinine > 1.5 mg/dL 31 (16.4) 23 (74.2) 8 (25.8) 0.0010 *
ESRD 5 (2.6) 2 (40.0) 3 (60.0) 0.0050 *
Tacrolimus mean level (ng/mL) 5.249 ± 1.71 5.263 ± 1.53 5.096 ± 3.12 0.9787
Tacrolimus level < 5 ng/mL 93 (49.2) 82 (88.2) 11 (11.8) 0.1216
Tacrolimus level < 4 ng/mL 44 (23.3) 36 (81.8) 8 (18.2) 0.0136 *
Tacrolimus level < 3 ng/mL 11 (5.8) 9 (81.8) 2 (18.2) 0.2356
Data are presented as mean ± standard deviation (SD) for continuous variables and frequency (percentage, %) for categorical variables. The p-values of statistical tests were calculated using the Wilcoxon rank-sum test for continuous variables and the Fisher’s exact test for categorical variables. * p value ≤ 0.05. Abbreviations: LT, liver transplantation; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; Total bilirubin, serum total bilirubin level; Creatinine, serum creatinine level; and ESRD, end-stage renal disease (defined by receiving hemodialysis regularly).
jpm-11-00090-t002_Table 2 Table 2 The causes of 16 deaths since 5 years after adult liver transplantations.
Causes of Deaths Number of Subjects
Malignancy 5 (31.25%)
De novo: Multiple myeloma, bladder cancer, colon cancer, prostate cancer 4
Recurrent: Hepatocellular carcinoma 1
Graft failure 4 (25.00%)
Chronic rejection 3
Autoimmune hepatitis 1
Infection 4 (25.00%)
Pneumonia 3
Urinary tract infection 1
Cerebral vascular event (intracerebral hemorrhage) 2 (12.50%)
Peptic ulcer bleeding 1 (6.25%)
jpm-11-00090-t003_Table 3 Table 3 Multivariate analysis for identifying the predictors of long-term overall survival after 5 years of liver transplantations by fitting a multiple Cox’s Model in the adult liver transplant recipients 1.
Covariate 2 Estimate Standard
Error Wald’s
z Test p Value Hazard
Ratio (HR) 95% Confidence
Interval (C.I.)
Age at LT ≤ 27.011 years 5.1286 1.3872 3.6970 0.0002 168.7851 11.130–2559.512
Autoimmune (including PBC) 2.0946 0.7219 2.9015 0.0037 8.1221 1.973–33.431
HCV × Overall survival years 0.2924 0.0914 3.1978 0.0014 1.3397 1.120–1.603
Cre > 1.311 × T-Bil > 1.411 mg/dL 6.8262 1.5591 4.3784 <0.0001 921.6940 43.401–19,573.712
Cre > 1.311 × T-Bil ≤ 0.792 mg/dL 4.6604 1.3294 3.5056 0.0005 105.6778 7.805–1430.790
Cre ≤ 1.311 × T-Bil > 0.882 mg/dL 3.4174 1.1871 2.8788 0.0040 30.4913 2.977–312.341
Tacrolimus mean ≤ 4.609 or > 10.168 ng/mL 1.5599 0.6479 2.4076 0.0161 4.7581 1.336–16.940
1 The above multiple Cox’s model was fitted to the 189 adult patients who underwent liver transplantations with 16 death events, for modeling the hazard rate of the right-censored overall survival time five years after liver transplantations. All the cut-off values of the continuous covariates (e.g., age at liver transplantations) were estimated by choosing the option of applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models (e.g., Figure 3). The time-dependent interaction term, HCV × Overall survival year, was added to the Cox’s model for handling the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.2924, indicated that the risk of dying in the patients with HCV would increase as time elapsed five years after liver transplantations. Moreover, the three second-order interaction terms, Cre > 1.311 × T-Bil > 1.411, Cre > 1.311 × T-Bil ≤ 0.792, and Cre ≤ 1.311 × T-Bil > 0.882, were compared to the other two possible combinations, Cre > 1.311 × (0.792 < T-Bil ≤ 1.411) and Cre ≤ 1.311 × T-Bil ≤ 0.882, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and.” Finally, both goodness-of-fit (GOF) measures, concordance = 0.9041 (se = 0.0265) > 0.7 and adjusted generalized R2 = 0.3878 > 0.15, indicated an excellent fit of this multiple Cox’s model to the observed survival data. 2 Abbreviations: LT, liver transplantation; PBC, primary biliary cirrhosis; HCV, hepatitis C viral infection; Cre, serum creatinine level (mg/dL); T-Bil, serum total bilirubin level (mg/dL); and Tacrolimus mean, the averaged dosage of the tacrolimus-based immunosuppressant (ng/mL).
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What was the outcome of reaction 'Peptic ulcer haemorrhage'? | Long-Term Tacrolimus Blood Trough Level and Patient Survival in Adult Liver Transplantation.
Tacrolimus is the most widely used immunosuppressant in liver transplant (LT) patients. However, the ideal long-term target level for these patients is unknown. This retrospective study aimed to investigate the impact of tacrolimus blood concentration five years after LT on long-term patient survival outcomes in adult LT recipients. Patients who underwent LT between January 2004 and July 2014 at a tertiary medical center were included in this study (n = 189). The mean tacrolimus blood concentrations of each patient during the fifth year after LT were recorded and the overall survival rate was determined. A multivariate analysis of factors associated with long-term survival was conducted using a Cox's model. The median follow-up period was 9.63 years, and 144 patients (76.2%) underwent live donor LT. Sixteen patients died within 5 years of LT. In the Cox's model, patients with a mean tacrolimus blood trough level of 4.6-10.2 ng/mL had significantly better long-term survival than those with a mean tacrolimus blood trough level outside this range (estimated hazard ratio = 4.76; 95% confidence interval: 1.34-16.9, p = 0.016). Therefore, a tacrolimus level no lower than 4.6 ng/mL would be recommended in adult LT patients.
1. Introduction
Liver transplantation (LT) has become a mature treatment of end-stage liver disease in clinical practice [1]. The continuous improvement of effective immunosuppression treatments has led to a significant improvement in patient and graft survival in recent years [2]. Calcineurin inhibitors are the cornerstone of immunosuppression in LT [3], and tacrolimus is currently the mainstay of initial and maintenance immunosuppression therapies [4,5,6]. Tacrolimus reduces the incidence and severity of early and late T-cell mediated rejection by inhibiting T-cell production of interleukin-2 [5,6]. However, the long-term use of immunosuppressants leads to an increasing burden of toxicity. The reported toxic effects of calcineurin inhibitors include infections, chronic renal insufficiency, metabolic diseases (such as hyperlipidemia, hypertension, and diabetes mellitus), and malignancy [7,8,9], which, along with cardiovascular diseases, have been reported to be the major causes of morbidity and mortality after LT [3,10]. However, most LT patients cannot withdraw from lifelong immunosuppression therapy; the only exceptions are a few selected patients participating in experimental trials [11,12].
It is a clinical challenge to design a well-balanced immunosuppressive regimen for LT recipients. Tacrolimus has a narrow therapeutic dose range and its blood levels should be carefully monitored. The Advagraf (tacrolimus prolonged-release hard capsules) recommendation for adult LT patients is a blood trough level of 5–20 ng/mL in the early post-transplant period and 5–15 ng/mL during subsequent maintenance therapy. Nevertheless, the current recommendations from the clinical practice guidelines for the ideal tacrolimus level in adults after LT remain controversial. The American Association for the Study of Liver Diseases (AASLD) recommends a target blood trough level of 5–10 ng/mL for tacrolimus three months after LT [13]. The Consensus on Managing Modifiable Risk in Transplantation Group (COMMIT) recommends that the target tacrolimus blood trough levels be 6–10 ng/mL during the first month after LT and decrease to 4–8 ng/mL thereafter, except when used in combination with mammalian target of rapamycin (mTOR) inhibitors [14]. Moreover, the International Liver Transplant Society (ILTS) consensus statement on immunosuppression in LT recipients recommends the target blood trough levels of tacrolimus be 6–10 ng/mL three months after LT, lower than 5 ng/mL 12 months after LT, and decrease to 3 ng/mL thereafter, resulting in a blood trough level just above the lower limit of detection five years after LT [15]. However, the impact of the long-term tacrolimus blood trough level on the outcomes of the LT recipients remains unclear. This study aimed to suggest an appropriate tacrolimus blood trough level for adult patients five years after LT.
2. Materials and Methods
This study was approved by the Institutional Review Board of National Taiwan University Hospital and was conducted according to the Declaration of Helsinki. A total of 286 patients who underwent LT at 18 years or older at a tertiary medical center from January 2004 to July 2014 were recruited for this study. Patients who died within 5 years of LT, were lost to follow-up, or did not use tacrolimus-based calcineurin inhibitor for immunosuppressant therapy were excluded from this study (Figure 1). In addition, those who received mTOR inhibitor treatment were excluded, as mTOR inhibitors are typically used as a combination therapy to reduce the required dose of tacrolimus. The final analysis included 189 patients. All eligible patients were followed up for more than five years until August 2019. Patients who underwent a LT due to liver cancer met the Milan criteria (before 2006) or the criteria of the University of California, San Francisco (since 2006) at the time of LT. All patients received regular monthly or bi-monthly follow-ups at the outpatient clinic after LT. Routine blood examinations for the tacrolimus blood trough level, liver function, and renal function were conducted at each visit, and abdominal sonography was performed every 6 to 12 months.
The patients’ medical records were reviewed retrospectively to extract demographic and clinical data, including patient characteristics, laboratory tests, and survival outcomes. The serum bilirubin and creatinine data at the end of the fifth year after LT were used in this study. The tacrolimus level used in this study was the mean of the values obtained at the three follow-up visits during the fifth year after LT.
The immunosuppression protocol for adult LT patients consisted of tacrolimus, mycophenolate mofetil, and steroids. Tacrolimus was administered orally beginning on the first day after LT, and the dose was adjusted to achieve the desired therapeutic drug level. Basiliximab was administered immediately before graft reperfusion and on the fourth day after LT for induction therapy. A 500-mg intravenous bolus of methylprednisolone was administered immediately before reperfusion of the liver graft and was tapered to oral prednisolone over one week and reduced to withdrawal after six months.
All statistical analyses were performed using R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p ≤ 0.05. Continuous variables are presented as mean ± standard deviation (SD) and median (interquartile range, IQR), and categorical variables are presented as frequency (percentage, %). The survival curve was estimated by the Kaplan-Meier method. A univariate analysis was conducted to examine the differences in the distributions of continuous variables, categorical variables, and survival outcomes between the surviving and deceased liver recipients five years after LT using the Wilcoxon rank-sum test, Chi-square test, Fisher’s exact test, or log-rank test as appropriate for the data type. A multivariate analysis was performed to estimate the adjusted effects of risk factors or prognostic factors on the survival outcome using a multiple Cox’s proportional hazards model.
To ensure a good quality of regression analysis, the model-fitting techniques for variable selection, goodness-of-fit (GOF) assessment, and regression diagnostics and remedies were used in our regression analysis. Specifically, the stepwise variable selection procedure (with iterations between the forward and backward steps) was applied to obtain the best final regression model using the My.stepwise package of the R software [16]. All significant and non-significant relevant covariates from the univariate analysis (listed in Table 1) and some of the interaction terms were used in the multivariate analysis. The significance levels for entry and for stay were set to 0.15 for being conservative. With the aid of substantive knowledge, the best candidate final regression model was identified manually by dropping the covariates with p value > 0.05 one at a time until all regression coefficients were significantly different from 0. To assess the GOF of the fitted Cox’s model, the concordance and adjusted generalized R2 [17] were examined. A concordance ≥ 0.7 and an adjusted generalized R2 > 0.15 indicated an acceptable level of discrimination, power, and fitness.
Moreover, the smoothing option “pspline” (for the smoothing splines using a “p-spline” basis) was specified inside the coxph function of the survival package to smooth the effects of continuous covariates on the log-hazard rate of the simple and multiple Cox’s proportional hazards models in R. Then, the termplot function of the stats package was used to plot the smoothed effects of the continuous covariates on the log-hazard rate in R [18]. The regression diagnostics for the verification of proportional hazards assumption, residual analysis, detection of influential cases, and a multicollinearity check were applied to discover any model or data problems. A variance inflating factor (VIF) ≥ 10 in continuous covariates or VIF ≥ 2.5 in categorical covariates indicated the occurrence of the multicollinearity problem among some of the covariates in the fitted regression model.
3. Results
3.1. Patients’ Demographic and Clinical Characteristics
The median follow-up duration was 9.63 years (IQR: 7.2–11.4 years), and the mean follow-up was 9.58 ± 2.74 years. A total of 121 males (64.0%) and 68 females (36.0%) were included in this study (Table 1). The median age at LT was 54.26 years (IQR: 48.63–58.58 years, range: 18.3–73.1 years), and the mean age at LT was 52.7 ± 9.6 years. Among the 189 patients, 114 (76.2%) underwent living donor transplants and 45 (23.8%) underwent deceased donor transplants. The main indications for LT were hepatitis B virus (HBV) cirrhosis (52.9%), hepatocellular carcinoma (38.1%), hepatitis C virus (HCV) cirrhosis (22.2%), and fulminant hepatitis (12.2%). The median of the mean tacrolimus blood trough level during the fifth year after LT was 5.0 ng/mL (IQR: 4.12–6.33 ng/mL), and the mean tacrolimus blood trough level was 5.249 ± 1.71 ng/mL. The mean tacrolimus blood trough level was ≥ 5 ng/mL in 96 patients (50.8%) and < 5 ng/mL in 93 patients (49.2%), including 44 patients (23.3%) with a mean tacrolimus blood trough level < 4 ng/mL and 11 patients (5.8%) with a mean tacrolimus blood trough level < 3 ng/mL. No significant differences in gender, age at LT, body weight, blood type, graft type, etiology of LT, or serum total bilirubin were found between the deceased and surviving LT recipients (p > 0.05). However, serum creatinine levels > 1.5 mg/dL (23/173 vs. 8/16, p = 0.001), end-stage renal disease (2/173 vs. 3/16, p = 0.005), and mean tacrolimus blood trough levels < 4 ng/mL (36/173 vs. 8/16, p = 0.014) were significantly more common among deceased LT recipients compared to survivors. The causes of death included malignancies (n = 5), graft failures (n = 4), infection (n = 4), cerebrovascular accidents (n = 2), and duodenal ulcer bleeding (n = 1) (Table 2). The distribution of the tacrolimus trough level stratified by the etiology and cause of death among the 16 dead patients was shown in the box plot of Figure 2. There was no statistical significance in the tacrolimus trough level between the different causes of death (p = 0.3823).
3.2. Predictors of Patients’ Long-Term Survival
The Cox’s model fitted to the survival data for the multivariate analyses of the time to death after five years of LT is shown in Table 3. After adjusting for the effects of the other covariates, age at LT ≤ 27.011 years (estimated hazard ratio [HR] = 168.79, 95% confidence interval [C.I.]: 11.13–2559.51), pre-transplant autoimmune liver disease (HR = 8.12, 95% C.I.: 1.97–33.43), pre-transplant HCV infection × survival time in years (HR = 1.34, 95% C.I.: 1.12–1.60), serum creatinine level > 1.311 mg/dL × serum total bilirubin level > 1.411 mg/dL (HR = 921.69, 95% C.I.: 43.40–19,573.71), serum creatinine level > 1.311 mg/dL × serum total bilirubin level ≤ 0.792 mg/dL (HR = 105.68, 95% C.I.: 7.81–1430.79), serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level > 0.882 mg/dL (HR = 30.49, 95% C.I.: 2.98–312.34), and the mean tacrolimus trough level during the fifth year after LT ≤ 4.609 ng/mL or > 10.168 ng/mL (HR = 4.76, 95% C.I.: 1.34–16.94) were associated with a higher long-term mortality five years after LT. The time-dependent interaction term, HCV × survival time in years, was added to the Cox’s model to account for the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.292, indicates that the risk of mortality in patients with HCV increases with time five years post-LT. Moreover, the three second-order interaction terms between the serum creatinine and total bilirubin levels were compared to the other two possible combinations, serum creatinine level > 1.311 mg/dL × (0.792 mg/dL < serum total bilirubin level ≤ 1.411 mg/dL) and serum creatinine level ≤ 1.311 mg/dL × serum total bilirubin level ≤ 0.882 mg/dL, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and”.
All cut-off values of the continuous covariates (such as age at LT) were estimated by applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models. As shown in Figure 3, the optimal cut-off values of the mean tacrolimus trough level, 4.609 ng/mL and 10.168 ng/mL, were estimated directly in the “p-spline plot,” which allowed the visualization of the nonlinear effect of the averaged dosage of the tacrolimus-based immunosuppressant during the fifth year after LT on log(λ), where λ was the hazard rate of time to death five years after LT. Then, the Kaplan-Meier estimates of survival curves for time to death five years after LT were determined for the 66 patients with mean tacrolimus trough levels ≤ 4.609 ng/mL or > 10.168 ng/mL and the 123 patients with mean tacrolimus trough levels between 4.609 ng/mL and 10.168 ng/mL (log-rank test, p = 0.009) (Figure 4). In an additional subgroup analysis, we found that a mean tacrolimus trough level between 4.431 ng/mL and 6.332 ng/mL for patients with a serum creatinine level > 1.311 mg/dL improved survival. Finally, this Cox’s model had a concordance of 0.904 and an adjusted generalized R2 of 0.388, indicating that it fit the survival data very well.
4. Discussion
This is the first study to report an association between long-term tacrolimus blood trough level and long-term patient survival in adult LT recipients. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL in adult LT recipients is associated with a lower mortality rate. In patients with a serum creatinine > 1.3 mg/dL, a mean tacrolimus blood trough level of 4.4–6.3 ng/mL is associated with a lower mortality rate.
Decreasing tacrolimus use during long-term follow-up in LT recipients is a general concept in clinical practice. In this study, only one patient had the mean level > 10 ng/mL and a few patients had the mean level > 8 ng/mL (Figure 3). Patients with such high drug levels were most likely because of their poor compliance (not must because of they have truly such high trough level). Poor compliance indicates that a patient takes incorrect dosage of immunosuppressant that their physician prescribed, or takes immunosuppressant or blood exam at the wrong time, either of which might clearly be important factors that influence graft and survival outcome of LT patients during long-term follow-ups. We found that a long-term tacrolimus blood trough level of 4.6–10.2 ng/mL is associated with lower mortality rate; however, the upper limit of 10.2 ng/mL suggested by the regression analysis was merely a value for caution, but not a recommended upper limit in real clinical practice. The long-term hazard of high level of tacrolimus is already widely known; therefore, we would focus more on the findings that it appears to increase the survival risks in LT recipients if their tacrolimus levels are maintained below 4.6 ng/mL during the long-term follow-ups.
Inadequate immunosuppression is associated with higher risks of graft rejection [19], while excess immunosuppression increases the risks of malignancy and infection and increases the adverse effects of drug toxicity. The tailored use of immunosuppressants should be considered based on the patient’s risks of graft rejection and infection and the patient’s medical comorbidities and liver disease status prior to LT [14]. Patients with autoimmune liver diseases may require more immunosuppression to prevent disease recurrence and graft rejection [20]. By contrast, lower doses of immunosuppression are recommended in patients who have undergone LT due to HCV, as high levels of immunosuppression are related to increased viral replication [21,22]. Rejection results in abnormal liver function and is one of the most important factors associated with poor long-term graft and patient outcomes [23,24,25]. Tacrolimus reduces the risk of T-cell mediated rejection to protect graft function and is the cornerstone of a successful LT. However, short and long-term adverse effects of tacrolimus such as infection, chronic renal insufficiency, metabolic diseases (hyperlipidemia, hypertension, and diabetes mellitus), and malignancy have been widely reported and influence patients’’ long-term outcomes [7,8,9]. The early causes of death after LT within one year are infection and graft loss, and the late causes of death three years post-LT are malignancy, cardiovascular disease, and renal failure [10]. As graft function typically stabilizes, factors associated with the long-term outcome are often patient-related factors (such as chronic medical diseases) that are usually associated with tacrolimus.
Previous studies have reported that minimizing tacrolimus use in the early post-LT period is associated with a lower risk of new-onset diabetes mellitus [26], a lower incidence of hyperlipidemia [27], and better long-term survival [19,28]. In recent years, studies have focused on the reduction or complete withdrawal of long-term immunosuppressants in LT recipients [12,29,30,31,32,33,34]. One study reported satisfactory outcomes with the combined use of tacrolimus and mycophenolate mofetil, which allowed for the tacrolimus dose to be reduced [35]. Other studies demonstrated that the concomitant use of everolimus may reduce the required dose of tacrolimus while having potential renal benefits [29,36]. However, only some LT recipients were able to discontinue the use of immunosuppressants, and these patients more frequently experienced biopsy-proven acute rejection [29]. The risk of chronic rejection during long-term follow-up remains in patients with inadequate immunosuppression [30,33]. The tacrolimus blood concentration in LT recipients may play a key role in long-term outcomes, as it is associated with long-term graft function and its adverse effects are related to several chronic medical diseases. However, to the best of our knowledge, this is the first report regarding the effect of long-term tacrolimus levels on the long-term survival outcomes of LT recipients.
Several factors are associated with the long-term outcomes of adult LT recipients, including pre-transplant primary sclerosing cholangitis, immunosuppression therapies, acute and chronic rejections, malignancy, and metabolic syndrome [37,38]. As listed in Table 3, we found several independent risk factors of long-term mortality in this study. Only four patients aged <27 years at the time of LT were included in this study, and one died due to pneumonia 6.3 years after LT. Patients who underwent LT due to autoimmune liver diseases had worse outcomes, as they were more likely to experience acute rejections [39] and suffer disease recurrence, leading to graft loss. Patients who underwent LT due to HCV infections had relatively poor post-LT outcomes due to the disease recurrence followed by graft dysfunction and failure [40]. As direct-acting antiviral agents have advanced [41], the survival outcomes of HCV patients should improve in the near future. Long-term renal and liver functions are reflected by serum creatinine and total bilirubin levels, respectively. We found that abnormal serum creatinine levels have a bigger impact than abnormal serum total bilirubin levels on the survival outcomes of LT recipients. The graft function of adult LT recipients is chronically stable five years after LT. While some patients may experience asymptomatic hyperbilirubinemia at this time, the long-term outcomes remain favorable. By contrast, the gradual deterioration of renal function may lead to chronic kidney disease or end-stage renal disease, affecting the long-term survival, especially in patients with long-term use of tacrolimus.
This study had some limitations. First, the data were obtained from a single medical center in Asia, which provided a relatively small number of eligible patients with single ethnicity and fewer death events. Second, the study spanned a long time period, and therefore the improvements in surgical and medical expertise and advances in immunosuppression therapies may have influenced the patient outcomes. Third, we did not collect or analyze the time-dependent tacrolimus blood trough level during the follow-up visits five years after LT.
In summary, we found an association between the long-term tacrolimus blood trough levels and the long-term survival five years after LT. A mean tacrolimus blood trough level outside the range of 4.6–10.2 ng/mL appeared to be an independent risk factor for long-term mortality. Further studies with larger sample sizes are needed to verify these results and to further identify an appropriate tacrolimus blood trough level for maintenance use.
Acknowledgments
We thank the coordinators (Hui-Ying Lin and Min-Heuy Lin) for their helpful efforts in data collection.
Author Contributions
C.-Y.H. drafted the manuscript and R.-H.H. designed the study. C.-Y.H., M.-C.H., and Y.-M.W. conducted data processing, and C.-Y.H. and C.-M.H. performed data analysis. P.-H.L. and R.-H.H. were the directors responsible for general organization and instruction. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of National Taiwan University Hospital.
Informed Consent Statement
Patient consent was waived due to retrospective design of the study.
Data Availability Statement
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1 Patient flow diagram.
Figure 2 The box plot of tacrolimus mean trough level stratified by the etiology and cause of death among the 16 dead patients. There was no statistical difference of tacrolimus level between the different causes of death (p = 0.3823).
Figure 3 The p-spline plot for the nonlinear effect of the long-term tacrolimus blood trough levels on time to death. The tacrolimus blood trough level is shown on the X-axis, and the log(λ), where λ is the hazard rate of time to death five years after liver transplantation, is shown on the Y-axis. In this p-spline plot, the intersection between the horizontal green line (Y = 0) and the red curve yields the estimated optimal cut-off values for long-term tacrolimus blood trough levels at which the values of log(λ) will not change (4.609 ng/mL and 10.168 ng/mL). When the level is ≤4.609 ng/mL or >10.168 ng/mL, the value of log(λ) increases, indicating an increasing λ. When the level is >4.609 ng/mL and ≤10.168 ng/mL, the value of log(λ) decreases, indicating a decreasing λ. The vertical bars above the X-axis represent the patients’ actual mean tacrolimus blood trough levels obtained at the three follow-up visits during the fifth year after LT.
Figure 4 The Kaplan-Meier curve for time to death five years after liver transplantation. The survival curve of the 66 patients with a mean tacrolimus blood trough level ≤ 4.609 ng/mL or >10.168 ng/mL is shown in red while the survival curve of the 123 patients with a mean tacrolimus blood trough level > 4.609 ng/mL or ≤10.168 ng/mL is shown in green.
jpm-11-00090-t001_Table 1 Table 1 Univariate analysis for comparing the distributions of the demographic and clinical characteristics between the alive and dead adult liver recipients after five years of liver transplantations.
Variable All Patients
(n = 189) Alive
(n = 173) Dead
(n = 16) p Value
Gender 0.7899
Male 121 (64.0) 110 (90.9) 11 (9.1)
Female 68 (36.0) 63 (92.6) 5 (7.4)
Age at LT (years) 52.7 ± 9.6 52.6 ± 9.5 53.4 ± 10.7 0.6672
Body weight at LT (kg) 64.9 ± 12.2 65.0 ± 12.3 63.7 ± 12.1 0.4459
Blood type 0.8719
O 79 (41.8) 73 (92.4) 6 (7.6)
A 51 (27.0) 46 (90.2) 5 (9.8)
B 42 (22.2) 39 (92.9) 3 (7.1)
AB 17 (9.0) 15 (88.2) 2 (11.8)
Graft type 1.0000
Living donor 144 (76.2) 132 (91.7) 12 (8.3)
Deceased donor 45 (23.8) 41 (91.1) 4 (8.9)
Etiology for LT
Liver malignancy (HCC) 72 (38.1) 66 (91.7) 6 (8.3) 1.0000
Alcoholic cirrhosis 17 (9.0) 16 (94.1) 1 (5.9) 1.0000
HBV cirrhosis 100 (52.9) 92 (92.0) 8 (8.0) 1.0000
HCV cirrhosis 42 (22.2) 38 (90.5) 4 (9.5) 0.7577
Fulminant hepatitis 23 (12.2) 23 (100) 0 (0) 0.2259
Autoimmune disease 14 (7.4) 11 (78.6) 3 (21.4) 0.1017
Biliary atresia 5 (2.6) 5 (100) 0 (0) 1.0000
Other 10 (5.3) 9 (90) 1 (10) 0.5964
Total bilirubin (mg/dL) 0.973 ± 0.51 0.958 ± 1.53 1.133 ± 0.69 0.6260
Total bilirubin > 1 mg/dL 60 (31.7) 53 (88.3) 7 (11.7) 0.2770
Total bilirubin > 2 mg/dL 11 (5.8) 9 (81.8) 2 (18.2) 0.2360
Creatinine (mg/dL) 1.391 ± 1.25 1.279 ± 0.96 2.6 ± 2.74 0.0201 *
Creatinine > 1.5 mg/dL 31 (16.4) 23 (74.2) 8 (25.8) 0.0010 *
ESRD 5 (2.6) 2 (40.0) 3 (60.0) 0.0050 *
Tacrolimus mean level (ng/mL) 5.249 ± 1.71 5.263 ± 1.53 5.096 ± 3.12 0.9787
Tacrolimus level < 5 ng/mL 93 (49.2) 82 (88.2) 11 (11.8) 0.1216
Tacrolimus level < 4 ng/mL 44 (23.3) 36 (81.8) 8 (18.2) 0.0136 *
Tacrolimus level < 3 ng/mL 11 (5.8) 9 (81.8) 2 (18.2) 0.2356
Data are presented as mean ± standard deviation (SD) for continuous variables and frequency (percentage, %) for categorical variables. The p-values of statistical tests were calculated using the Wilcoxon rank-sum test for continuous variables and the Fisher’s exact test for categorical variables. * p value ≤ 0.05. Abbreviations: LT, liver transplantation; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; Total bilirubin, serum total bilirubin level; Creatinine, serum creatinine level; and ESRD, end-stage renal disease (defined by receiving hemodialysis regularly).
jpm-11-00090-t002_Table 2 Table 2 The causes of 16 deaths since 5 years after adult liver transplantations.
Causes of Deaths Number of Subjects
Malignancy 5 (31.25%)
De novo: Multiple myeloma, bladder cancer, colon cancer, prostate cancer 4
Recurrent: Hepatocellular carcinoma 1
Graft failure 4 (25.00%)
Chronic rejection 3
Autoimmune hepatitis 1
Infection 4 (25.00%)
Pneumonia 3
Urinary tract infection 1
Cerebral vascular event (intracerebral hemorrhage) 2 (12.50%)
Peptic ulcer bleeding 1 (6.25%)
jpm-11-00090-t003_Table 3 Table 3 Multivariate analysis for identifying the predictors of long-term overall survival after 5 years of liver transplantations by fitting a multiple Cox’s Model in the adult liver transplant recipients 1.
Covariate 2 Estimate Standard
Error Wald’s
z Test p Value Hazard
Ratio (HR) 95% Confidence
Interval (C.I.)
Age at LT ≤ 27.011 years 5.1286 1.3872 3.6970 0.0002 168.7851 11.130–2559.512
Autoimmune (including PBC) 2.0946 0.7219 2.9015 0.0037 8.1221 1.973–33.431
HCV × Overall survival years 0.2924 0.0914 3.1978 0.0014 1.3397 1.120–1.603
Cre > 1.311 × T-Bil > 1.411 mg/dL 6.8262 1.5591 4.3784 <0.0001 921.6940 43.401–19,573.712
Cre > 1.311 × T-Bil ≤ 0.792 mg/dL 4.6604 1.3294 3.5056 0.0005 105.6778 7.805–1430.790
Cre ≤ 1.311 × T-Bil > 0.882 mg/dL 3.4174 1.1871 2.8788 0.0040 30.4913 2.977–312.341
Tacrolimus mean ≤ 4.609 or > 10.168 ng/mL 1.5599 0.6479 2.4076 0.0161 4.7581 1.336–16.940
1 The above multiple Cox’s model was fitted to the 189 adult patients who underwent liver transplantations with 16 death events, for modeling the hazard rate of the right-censored overall survival time five years after liver transplantations. All the cut-off values of the continuous covariates (e.g., age at liver transplantations) were estimated by choosing the option of applying the p-spline smoothing techniques in fitting simple and multiple Cox’s proportional hazards models (e.g., Figure 3). The time-dependent interaction term, HCV × Overall survival year, was added to the Cox’s model for handling the non-proportional hazards problem between the patients with and without HCV, and its positive-valued regression coefficient estimate, 0.2924, indicated that the risk of dying in the patients with HCV would increase as time elapsed five years after liver transplantations. Moreover, the three second-order interaction terms, Cre > 1.311 × T-Bil > 1.411, Cre > 1.311 × T-Bil ≤ 0.792, and Cre ≤ 1.311 × T-Bil > 0.882, were compared to the other two possible combinations, Cre > 1.311 × (0.792 < T-Bil ≤ 1.411) and Cre ≤ 1.311 × T-Bil ≤ 0.882, as the reference group (i.e., HR = 1.0), where the cross sign × can be literally interpreted as “and.” Finally, both goodness-of-fit (GOF) measures, concordance = 0.9041 (se = 0.0265) > 0.7 and adjusted generalized R2 = 0.3878 > 0.15, indicated an excellent fit of this multiple Cox’s model to the observed survival data. 2 Abbreviations: LT, liver transplantation; PBC, primary biliary cirrhosis; HCV, hepatitis C viral infection; Cre, serum creatinine level (mg/dL); T-Bil, serum total bilirubin level (mg/dL); and Tacrolimus mean, the averaged dosage of the tacrolimus-based immunosuppressant (ng/mL).
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Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Anaemia'. | Neoadjuvant therapy of BRCA1-driven ovarian cancer by combination of cisplatin, mitomycin C and doxorubicin.
BACKGROUND
Cisplatin, mitomycin C and anthracyclines demonstrate high activity in BRCA1-deficient tumors. This study aimed to evaluate the efficacy of the triplet combination of these drugs in BRCA1-driven high-grade serous ovarian carcinomas (HGSOCs).
METHODS
Ten HGSOC patients with germ-line BRCA1 mutation received neoadjuvant chemotherapy (NACT) consisting of mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks (MAP regimen). The comparator group included 16 women, who received standard NACT combination of paclitaxel 175 mg/m2 and carboplatin (6 AUC), given every 3 weeks (TCbP scheme).
RESULTS
None of the patients treated by the MAP scheme demonstrated complete pathologic response in ovaries, while 4 women showed absence of tumor cells in surgically excised omental specimens. When chemotherapy response scores (CRS) were considered, poor responsiveness (CRS 1) was not observed in the MAP group, but was common for the TCbP regimen (6/16 (38 %) for ovaries and 5/16 (31 %) for omentum; p = 0.05 and 0.12, respectively). Median treatment-free interval (TFI) was not reached in women treated by the MAP, but was 9.5 months for the TCbP scheme (p = 0.1). The rate of the recurrence within 1 year after the completion of the treatment was 4/10 (40 %) for the MAP and 10/13 (77 %) for the TCbP (p = 0.1).
CONCLUSIONS
The attempt to intensify NACT by administering combination of 3 drugs did not result in high rate of complete pathologic responses. However, there was a trend towards higher efficacy of the MAP regimen versus conventional TCbP scheme with regard to CRS and clinical outcomes.
Background
Ovarian cancer (OC) is a common malignancy, which holds the leading position in the mortality caused by gynecological tumors [1]. The worldwide incidence of OC approaches approximately three hundred thousand new cases per year, with almost two-thirds of affected patients dying from this disease [2]. High-grade serous ovarian cancer (HGSOC) is the most frequent OC histological type. A significant portion of HGSOCs is attributed to germ-line mutations in BRCA1 or BRCA2 genes. BRCA1/2-driven ovarian tumors usually develop via inactivation of the remaining allele of the involved gene. Consequently, these cancers demonstrate a tumor-selective deficiency in DNA repair by homologous recombination and pronounced sensitivity to platinum compounds, PARP inhibitors and mitomycin C [3, 4].
Ovarian tumors often do not cause symptoms at early stages; therefore, most HGSOC patients are diagnosed with already inoperable disease. These women are often subjected to neoadjuvant chemotherapy (NACT), which is aimed to reduce tumor burden and allow surgical intervention [5]. BRCA1-associated ovarian malignancies demonstrate significantly better responses to the NACT as compared to sporadic neoplasms [6]. Although these patients usually undergo complete cytoreductive surgery followed by adjuvant therapy, most BRCA1-driven HGSOCs eventually relapse [7]. These relapses are attributed to the acquisition of the resistance of tumor clones to systemic therapy. The most known mechanism of acquired platinum resistance is the emergence of mutations, which restore the open reading frame in the BRCA1 gene [4]. This route is mainly applicable to heavily pretreated patients but appears to be less characteristic for the initial phases of OC therapy [8]. On the other hand, NACT often results in the selection of BRCA1-proficient cells, which exist in small amounts in chemonaive tumors and repopulate tumor mass during platinum exposure [9].
Intensification of the therapy is a common approach aimed to prevent the emergence of resistant clones. We have previously reported promising results of applying cisplatin plus mitomycin C combination for the NACT of BRCA1-driven carcinomas. This therapy resulted in a significant reduction of the tumor burden in all analyzed patients and in complete pathologic responses observed in 2/12 (17 %) treated women [10]. We reasoned that combining this regimen with an additional drug may further improve the outcomes of NACT. Previous studies suggested that BRCA1-driven tumors are particularly sensitive to anthracyclines, while their responsiveness to taxanes is under the question [10, 11]. Consequently, we decided to add doxorubicin to cisplatin plus mitomycin C as a third drug. Here we present the results of the trial involving this 3-drug combination.
Methods
The design of the study was discussed on the council involving medical oncologists, cancer gynecologists and hereditary cancer specialists. It was decided that the pilot trial would include 10 patients with initially inoperable BRCA1-driven HGSOC, and the main end-point will be the rate of pathologic complete responses. While all patients received the same neoadjuvant regimen (MAP: mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8), cisplatin 80 mg/m2 (day 1), given every 4 weeks), the physicians were permitted to administer the therapy of their choice after the surgery. This approach provided some flexibility given that the combination of carboplatin and paclitaxel (TCbP) is a standard option for the treatment of ovarian cancer [1] and that some post-NACT tumor samples have restored BRCA1 function and therefore may not be potentially responsive to platinum drugs [8, 9]. The recruitment of patients was performed from August 2017 to December 2018 based on the results of the PCR-based test for Slavic recurrent germ-line mutations [12, 13]. According to the study protocol, all tumor samples were subjected to the loss-of-heterozygosity (LOH) analysis before the NACT and after the surgery. LOH test was performed as described in [9]. All tumors were also analyzed for the TP53 somatic mutations, given that TP53 inactivation is a ubiquitous feature of BRCA1-driven carcinomas [14]. The study was approved by the local Ethics Committee. All patients included in the study provided informed consent.
Although this study was not randomized, we considered for the comparison of treatment outcomes 16 consecutive patients with germ-line BRCA1 mutations, who were referred to the N.N. Petrov Institute of Oncology (St.-Petersburg, Russia) between February 2017 and December 2019 and were subjected to a standard NACT combination of paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks. Most of these patients were negative for PCR-detectable recurrent BRCA1 mutations; however, they were found to carry a germ-line pathogenic allele upon the analysis of the entire BRCA1 and BRCA2 coding sequence, i.e., after the start of NACT [13].
All women receiving MAP or TCbP were managed by the same surgical team. Tumor responses were evaluated according to RECIST criteria using computed tomography and magnetic resonance imaging. None of the patients treated by MAP or TCbP received bevacizumab. Three patients in the MAP arm but none in the TCbP group were subjected to the hyperthermic intraperitoneal chemotherapy (HIPEC) during surgery. None of the included patients described in this report received maintenance by PARP inhibitors after completion of the first-line therapy, as this indication was not approved in Russia at the time of the study.
The statistical analysis was performed using SPSS 13.0 software package. Age distribution and the duration of the follow-up were compared by the Mann-Whitney U-test. Median treatment-free interval (TFI) was evaluated using Kaplan-Meyer curves. Other comparisons were performed with the Fisher’s exact test.
Results
Five patients included in the study of the neoadjuvant combination of mitomycin C, cisplatin and doxorubicin had stage IIIC HGSOC and another 5 women were diagnosed with stage IV disease (Table 1). Partial response to this therapy was observed in all 10 cases considered. Seven women had toxicities of grades 1 or 2; 2 patients had toxicity grade 3 and 1 subject experienced grade IV thrombocytopenia. None of the HGSOCs showed a complete pathological response in the ovaries, and only one woman demonstrated chemotherapy response score (CRS) 3, according to Böhm et al. [15]. Omental tumor response, which is more predictive for the disease outcome than adnexal CRS [15], showed considerably better values: 4 women had no residual tumor cells in the omentum, 1 patient had CRS 3 and 5 cases demonstrated CRS 2. There were no instances of poor responsiveness to the therapy categorized as CRS 1. The median TFI and progression-free survival (PFS) were not reached.
Table 1 BRCA1-mutated HGSOC patients receiving neoadjuvant therapy consisting of mitomycin C, doxorubicin and cisplatin
ID Age Stage BRCA1 mutation Somatic BRCA1 status before NACT MAP cycles
(NACT) Surgical debulking Response by RECIST CRS (ovary / omentum) Somatic BRCA1 status after NACT ACT
(cycles) TFI, months PFS, months OS, months Toxicities and grades TP53 mutation
MAP1 64 IIIC c.5266dupC LOH 3 Complete + HIPEC PR (-44 %) CRS 3 / no tumor cells in omentum LOH MAP (2) 25.4+ 32.2+ 32.2+ Anemia 1; nephrotoxicity 1 C135W
MAP2 35 IVA (pleuritis) c.68_69delAG LOH 3 Complete + HIPEC PR (-73 %) CRS 2 / no tumor cells in omentum na MAP (1), AT (2) 6.9 14.4 26.9 Anemia 1 R175H
MAP3 50 IIIC c.4034delA LOH 4 Complete PR (-35 %) CRS 2 / CRS 2 LOH MAP (3) 23.6+ 31.6+ 31.6+ Anemia 1 Y234H
MAP4 57 IVB (lymph nodes) c.5266dupC LOH 3 Complete + HIPEC PR (-90 %) CRS 2 / CRS 3 Retention of the wild-type allele AT (3) 29.2+ 36.6+ 36.6+ Diarrhea 1; emesis 1; gastritis 1; nausea 1; leukopenia 2; nephrotoxicity 2; thrombocytopenia 4 R213X
MAP5 50 IIIC c.5266dupC na 3 Complete PR (-33 %) CRS 2 / no tumor cells in omentum LOH MAP (3) 22.2+ 29.5+ 29.5+ Anemia 1; leukopenia 1; nausea 1; thrombocytopenia 1 c.97-1G > A
MAP6 45 IVB (spleen, lymph nodes) c.1961delA LOH 4 Suboptimal PR (-47 %) CRS 2 / CRS 2 LOH TCbP (6) 21+ 30.4+ 30.4+ Anemia 1; nausea 1; thrombosis R213X
MAP7 56 IVB (pleuritis, lymph nodes) c.5266dupC na 3 Complete PR (-47 %) CRS 2 / CRS 2 LOH MAP (1) 4.1 8.3 29.8+ Anemia 2; nephrotoxicity 3 c.314delG
MAP8 58 IIIC c.4034delA LOH 5 Complete PR (-46 %) CRS 2 / CRS 2 LOH TCbP (4) 11.5 20.7 28.2+ Anemia 1; neutropenia 2 R248Q
MAP9 46 IIIC c.4034delA LOH 4 Complete PR (-64 %) CRS 2 / CRS 2 LOH MAP (2) 3.1 11.2 15.8 Thrombocytopenia 2 I255S
MAP10 40 IVB (pleuritis, lymph nodes) c.5266dupC LOH 4 Complete PR (-36 %) na / no tumor cells in omentum na None 28.5+ 37.6+ 37.6+ Anemia 1; hepatotoxicity 2; thrombocytopenia 2; nephrotoxicity 3 M133R
ACT Adjuvant chemotherapy, AT Doxorubicin 60 mg/m2 and paclitaxel 175 mg/m2, every 3 weeks, CRS Chemotherapy response score, HIPEC hyperthermic intraperitoneal chemotherapy, LOH Loss of heterozygosity, na Not analyzed, MAP Mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks, NACT Neoadjuvant chemotherapy, OS Overall survival, PFS Progression-free survival, PR Partial response, TCbP Paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks, TFI Treatment-free interval.
Notes: Tumor responses presented in the table describe the status of patients observed after the completion of the NACT, i.e. straight before the surgery. Patient MAP10 was diagnosed with ovarian cancer upon surgery, which was performed in another hospital and was limited to the excision of ovaries. She was considered eligible for the NACT study, as she had a significant tumor burden and could not be subjected to primary debulking surgery; she received no adjuvant therapy, as no residual tumor cells was seen in the surgical material. Patient MAP4 demonstrated the restoration of heterozygosity in a post-NACT tumor sample, suggesting that the tumor may no longer be platinum-sensitive [8, 9]; based on this finding, combination of paclitaxel and doxorubicin was given after the surgery; similarly, this combination was incorporated in the adjuvant treatment for patient MAP2, where the molecular analysis of post-NACT tumor tissue failed to establish somatic BRCA1 status. Patients MAP6 and MAP8 received TCbP combination after the surgery due to preference of their primary physicians.
Sixteen patients receiving paclitaxel plus carboplatin had slightly more favorable stage distribution, as 12 subjects had HGSOC of stage IIIC and 4 patients demonstrated stage IV disease (Table 2). While all patients treated by MAP showed partial response, 4/16 (25 %) women subjected to the TCbP combination produced only the disease stabilization and there was one HGSOC with the progression on this therapy. There was a remarkable difference from MAP regimen with regard to pathological responses, as minimal response score was observed in 6/16 (38 %) cases for ovarian tumor masses and 5/16 (31 %) HGSOCs for omental metastases (p = 0.05 and 0.12, respectively). While the median follow-up for the TCbP group was shorter than for MAP patients, median TFI was already achieved and reached 9.5 months (Fig. 1). Thirteen patients had sufficient follow-up to evaluate 1-year outcomes; the recurrence rate at 1 year after the completion of the treatment was 10/13 (77 %) for the TCbP, while the same value was 4/10 (40 %) for the MAP regimen (p = 0.1).
Table 2 Comparative characteristics of patients with BRCA1-mutated HGSOC receiving NACT combination of mitomycin C, doxorubicin and cisplatin versus women treated by paclitaxel plus carboplatin
Clinical characteristics MAP group (N = 10) TCbP group (N = 16) Statistical comparison
Median age of onset (range) 50 (35–64) 49 (37–72) Not significant
Pattern of BRCA1 mutations c.5266dupC (n = 5), c.4034delA (n = 3), c.68_69delAG (n = 1), c.1961delA (n = 1) c.5266dupC (n = 2), c.4034delA (n = 2), c.68_69delAG (n = 1), C61G (n = 1), c.1510delC (n = 1), Q563X (n = 1), c.2076dupT (n = 1), c.2983_2984delAA (n = 1), c.3247del5 (n = 1), c.3601_3602delGG (n = 1), c.3718_3719delCA (n = 1), Y1509X (n = 1), G1706E (n = 1), c.5152 + 1G > T (n = 1) p = 0.005 (founder vs. non-founder mutations; Fisher’s exact test)
FIGO stage
IIIC 5 (50 %) 12 (75 %) p = 0.23 (stage III vs. IV; Fisher’s exact test)
IVA 1 (10 %) 2 (13 %)
IVB 4 (40 %) 2 (13 %)
NACT cycles (range) 3–5 3–8
Cytoreduction
Optimal 9 (90 %) 14 (88 %) p = 1.0 (Fisher’s exact test)
Suboptimal 1 (10 %) 1 (6 %)
None 0 1 (6 %)
Response by RECIST
CR 0 0 p = 0.12 (objective response vs. lack of objective response; Fisher’s exact test)
PR 10 (100 %) 11 (69 %)
SD 0 4 (25 %)
PD 0 1 (6 %)
Chemotherapy response score (CRS) in the ovaries
CRS 1 0 6 (38 %) p = 0.05 (CRS 1 vs. other; Fisher’s exact test)
CRS 2 8 (80 %) 9 (56 %)
CRS 3 1 (10 %) 0
Tissue not available for evaluation 1 (10 %) 1 (10 %)
Chemotherapy response score (CRS) in the omentum
CRS 1 0 5 (31 %) p = 0.12 (CRS 1 vs. other; Fisher’s exact test)
CRS 2 5 (40 %) 8 (50 %)
CRS 3 1 (10 %) 0
No tumor cells 4 (40 %) 2 (13 %)
Tissue not available for evaluation 0 1 (6 %)
ACT cycles (range) 1–6 1–6
Median follow-up, months (range) 30.1 (15.8–36.6) 23.4 (10.7–45.2) p = 0.28 (Mann-Whitney Test)
Median treatment-free interval (95 % CI) Not reached 9.5 (7.8–11.2) p = 0.109 (Log Rank [Mantel-Cox])
Recurrence within one year after completion of treatment 4 (40 %) 10/13a (77 %) p = 0.1 (Fisher’s exact test)
ACT adjuvant chemotherapy, CRS chemotherapy response score, MAP mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8), cisplatin 80 mg/m2 (day 1), given every 4 weeks, NACT neoadjuvant chemotherapy, TCbP paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks
a13 out of 16 patients had sufficient follow-up for the estimation of 1-year recurrence rate
Fig. 1 Treatment-free interval for patients treated with the combination of mitomycin C, cisplatin and doxorubicin and for women treated by the paclitaxel plus carboplatin doublet
Discussion
Our previous study involving 12 BRCA1-mutated HGSOCs treated with the combination of cisplatin and mitomycin C revealed complete pathologic responses in 2 out of 12 patients [10]. We anticipated that the addition of doxorubicin to this combination may increase the rate of elimination of all tumor cells detectable in surgically excised tissues. The obtained data are sufficient to state that the applied triplet does not significantly increase the rate of complete pathologic responses as compared to the previously applied combination of two drugs.
At the same time, short-term results of MAP therapy look encouraging. In addition to a reasonably good rate of objective responses, half of the included cases demonstrated complete or nearly-complete absence of tumor cells in the omentum. Omental response score is the main predictor of the long-term outcomes of NACT, so it is a valuable marker allowing robust evaluation of various chemotherapy regimens [15].
Previous studies suggested that the TCbP regimen may be less efficient in BRCA1-mutated HGSOCs as compared to other NACT schemes [10]. These data sets compared prospective and retrospective patients treated by different surgeons. The quality of surgical debulking is critical for the outcome of HGSOC treatment, therefore these comparisons are prone to biases. Within the present study, we analyzed groups of patients who were managed at the same time interval by the same group of surgeons. However, the MAP and TCbP groups of patients were not balanced with regard to the pattern of mutations. The selection of patients for the MAP therapy involved rapid PCR tests for recurrent Slavic mutations [13]. The TCbP HGSOC group is significantly enriched by subjects with “rare” BRCA1 pathogenic alleles, which were detected by the next-generation sequencing analysis after the start of NACT. There are some data suggesting that distinct BRCA1 and BRCA2 mutations may exert distinct sensitivity to platinum compounds and PARP inhibitors [4]. Although we acknowledge differences in the pattern of BRCA1 mutations as a limitation of the study, it should be noted that all published trials on PARP inhibitors did not consider the type of mutation as a confounding factor [4, 16].
The landscape of the treatment BRCA1/2-mutated HGSOC is rapidly evolving. In particular, PARP inhibitors have been recently included in the standards for the first-line maintenance therapy, as they significantly delay the time to tumor recurrence [16]. None of the patients considered in this report received PARP inhibitors because they were not locally approved for early lines of HGSOC treatment at the time of the study. Consequently, it is unclear whether the differences observed between distinct NACT regimens will be maintained upon the incorporation of PARP-targeted drugs.
Conclusions
In summary, this study suggests that BRCA1-associated HGSOCs may require distinct therapeutic NACT regimens as compared to conventional TCbP doublet. If this is the case, the fast turn-around time for BRCA1/2 testing could become a critical factor for appropriate treatment decisions. Recent data indicate that BRCA1/2-associated HGSOCs do not show inferior outcomes when treated by NACT before the surgery, while primary surgical intervention is clearly the best approach in sporadic ovarian tumors [7, 17, 18]. These findings are likely to increase the acceptance of NACT for BRCA1/2 germline mutation carriers and, therefore, stimulate large neoadjuvant clinical trials for this category of HGSOC patients.
Abbreviations
CRSChemotherapy response score
HGSOCHigh-grade serous ovarian cancer
LOHLoss of heterozygosity
MAPMitomycin C, anthracycline, platinum
NACTNeoadjuvant chemotherapy
OCOvarian cancer
PFSProgression-free survival
TCbPTaxanes and carboplatin
TFITreatment-free interval
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Authors’ contributions
TG, KK, IB, KG, EN and OS managed study patients and analyzed clinical data; TS performed the molecular analysis; AI carried out morphological analysis; AS integrated and analyzed the obtained data and contributed to the manuscript preparation; EI designed the study and wrote the first draft of the manuscript. All authors have read and approved the final version of the manuscript.
Funding
This work has been supported by the Ministry of Science and Higher Education of the Russian Federation (grant № 075-15-2020-789).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was performed in full accordance with ethics guidelines. A written informed consent from study participants was obtained.
Consent for publication
All living patients provided the consent for publication.
Competing interests
The authors declare that they have no competing interests. | CISPLATIN, DOXORUBICIN HYDROCHLORIDE, MITOMYCIN | DrugsGivenReaction | CC BY | 33536037 | 18,976,042 | 2021-02-03 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Nephropathy toxic'. | Neoadjuvant therapy of BRCA1-driven ovarian cancer by combination of cisplatin, mitomycin C and doxorubicin.
BACKGROUND
Cisplatin, mitomycin C and anthracyclines demonstrate high activity in BRCA1-deficient tumors. This study aimed to evaluate the efficacy of the triplet combination of these drugs in BRCA1-driven high-grade serous ovarian carcinomas (HGSOCs).
METHODS
Ten HGSOC patients with germ-line BRCA1 mutation received neoadjuvant chemotherapy (NACT) consisting of mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks (MAP regimen). The comparator group included 16 women, who received standard NACT combination of paclitaxel 175 mg/m2 and carboplatin (6 AUC), given every 3 weeks (TCbP scheme).
RESULTS
None of the patients treated by the MAP scheme demonstrated complete pathologic response in ovaries, while 4 women showed absence of tumor cells in surgically excised omental specimens. When chemotherapy response scores (CRS) were considered, poor responsiveness (CRS 1) was not observed in the MAP group, but was common for the TCbP regimen (6/16 (38 %) for ovaries and 5/16 (31 %) for omentum; p = 0.05 and 0.12, respectively). Median treatment-free interval (TFI) was not reached in women treated by the MAP, but was 9.5 months for the TCbP scheme (p = 0.1). The rate of the recurrence within 1 year after the completion of the treatment was 4/10 (40 %) for the MAP and 10/13 (77 %) for the TCbP (p = 0.1).
CONCLUSIONS
The attempt to intensify NACT by administering combination of 3 drugs did not result in high rate of complete pathologic responses. However, there was a trend towards higher efficacy of the MAP regimen versus conventional TCbP scheme with regard to CRS and clinical outcomes.
Background
Ovarian cancer (OC) is a common malignancy, which holds the leading position in the mortality caused by gynecological tumors [1]. The worldwide incidence of OC approaches approximately three hundred thousand new cases per year, with almost two-thirds of affected patients dying from this disease [2]. High-grade serous ovarian cancer (HGSOC) is the most frequent OC histological type. A significant portion of HGSOCs is attributed to germ-line mutations in BRCA1 or BRCA2 genes. BRCA1/2-driven ovarian tumors usually develop via inactivation of the remaining allele of the involved gene. Consequently, these cancers demonstrate a tumor-selective deficiency in DNA repair by homologous recombination and pronounced sensitivity to platinum compounds, PARP inhibitors and mitomycin C [3, 4].
Ovarian tumors often do not cause symptoms at early stages; therefore, most HGSOC patients are diagnosed with already inoperable disease. These women are often subjected to neoadjuvant chemotherapy (NACT), which is aimed to reduce tumor burden and allow surgical intervention [5]. BRCA1-associated ovarian malignancies demonstrate significantly better responses to the NACT as compared to sporadic neoplasms [6]. Although these patients usually undergo complete cytoreductive surgery followed by adjuvant therapy, most BRCA1-driven HGSOCs eventually relapse [7]. These relapses are attributed to the acquisition of the resistance of tumor clones to systemic therapy. The most known mechanism of acquired platinum resistance is the emergence of mutations, which restore the open reading frame in the BRCA1 gene [4]. This route is mainly applicable to heavily pretreated patients but appears to be less characteristic for the initial phases of OC therapy [8]. On the other hand, NACT often results in the selection of BRCA1-proficient cells, which exist in small amounts in chemonaive tumors and repopulate tumor mass during platinum exposure [9].
Intensification of the therapy is a common approach aimed to prevent the emergence of resistant clones. We have previously reported promising results of applying cisplatin plus mitomycin C combination for the NACT of BRCA1-driven carcinomas. This therapy resulted in a significant reduction of the tumor burden in all analyzed patients and in complete pathologic responses observed in 2/12 (17 %) treated women [10]. We reasoned that combining this regimen with an additional drug may further improve the outcomes of NACT. Previous studies suggested that BRCA1-driven tumors are particularly sensitive to anthracyclines, while their responsiveness to taxanes is under the question [10, 11]. Consequently, we decided to add doxorubicin to cisplatin plus mitomycin C as a third drug. Here we present the results of the trial involving this 3-drug combination.
Methods
The design of the study was discussed on the council involving medical oncologists, cancer gynecologists and hereditary cancer specialists. It was decided that the pilot trial would include 10 patients with initially inoperable BRCA1-driven HGSOC, and the main end-point will be the rate of pathologic complete responses. While all patients received the same neoadjuvant regimen (MAP: mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8), cisplatin 80 mg/m2 (day 1), given every 4 weeks), the physicians were permitted to administer the therapy of their choice after the surgery. This approach provided some flexibility given that the combination of carboplatin and paclitaxel (TCbP) is a standard option for the treatment of ovarian cancer [1] and that some post-NACT tumor samples have restored BRCA1 function and therefore may not be potentially responsive to platinum drugs [8, 9]. The recruitment of patients was performed from August 2017 to December 2018 based on the results of the PCR-based test for Slavic recurrent germ-line mutations [12, 13]. According to the study protocol, all tumor samples were subjected to the loss-of-heterozygosity (LOH) analysis before the NACT and after the surgery. LOH test was performed as described in [9]. All tumors were also analyzed for the TP53 somatic mutations, given that TP53 inactivation is a ubiquitous feature of BRCA1-driven carcinomas [14]. The study was approved by the local Ethics Committee. All patients included in the study provided informed consent.
Although this study was not randomized, we considered for the comparison of treatment outcomes 16 consecutive patients with germ-line BRCA1 mutations, who were referred to the N.N. Petrov Institute of Oncology (St.-Petersburg, Russia) between February 2017 and December 2019 and were subjected to a standard NACT combination of paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks. Most of these patients were negative for PCR-detectable recurrent BRCA1 mutations; however, they were found to carry a germ-line pathogenic allele upon the analysis of the entire BRCA1 and BRCA2 coding sequence, i.e., after the start of NACT [13].
All women receiving MAP or TCbP were managed by the same surgical team. Tumor responses were evaluated according to RECIST criteria using computed tomography and magnetic resonance imaging. None of the patients treated by MAP or TCbP received bevacizumab. Three patients in the MAP arm but none in the TCbP group were subjected to the hyperthermic intraperitoneal chemotherapy (HIPEC) during surgery. None of the included patients described in this report received maintenance by PARP inhibitors after completion of the first-line therapy, as this indication was not approved in Russia at the time of the study.
The statistical analysis was performed using SPSS 13.0 software package. Age distribution and the duration of the follow-up were compared by the Mann-Whitney U-test. Median treatment-free interval (TFI) was evaluated using Kaplan-Meyer curves. Other comparisons were performed with the Fisher’s exact test.
Results
Five patients included in the study of the neoadjuvant combination of mitomycin C, cisplatin and doxorubicin had stage IIIC HGSOC and another 5 women were diagnosed with stage IV disease (Table 1). Partial response to this therapy was observed in all 10 cases considered. Seven women had toxicities of grades 1 or 2; 2 patients had toxicity grade 3 and 1 subject experienced grade IV thrombocytopenia. None of the HGSOCs showed a complete pathological response in the ovaries, and only one woman demonstrated chemotherapy response score (CRS) 3, according to Böhm et al. [15]. Omental tumor response, which is more predictive for the disease outcome than adnexal CRS [15], showed considerably better values: 4 women had no residual tumor cells in the omentum, 1 patient had CRS 3 and 5 cases demonstrated CRS 2. There were no instances of poor responsiveness to the therapy categorized as CRS 1. The median TFI and progression-free survival (PFS) were not reached.
Table 1 BRCA1-mutated HGSOC patients receiving neoadjuvant therapy consisting of mitomycin C, doxorubicin and cisplatin
ID Age Stage BRCA1 mutation Somatic BRCA1 status before NACT MAP cycles
(NACT) Surgical debulking Response by RECIST CRS (ovary / omentum) Somatic BRCA1 status after NACT ACT
(cycles) TFI, months PFS, months OS, months Toxicities and grades TP53 mutation
MAP1 64 IIIC c.5266dupC LOH 3 Complete + HIPEC PR (-44 %) CRS 3 / no tumor cells in omentum LOH MAP (2) 25.4+ 32.2+ 32.2+ Anemia 1; nephrotoxicity 1 C135W
MAP2 35 IVA (pleuritis) c.68_69delAG LOH 3 Complete + HIPEC PR (-73 %) CRS 2 / no tumor cells in omentum na MAP (1), AT (2) 6.9 14.4 26.9 Anemia 1 R175H
MAP3 50 IIIC c.4034delA LOH 4 Complete PR (-35 %) CRS 2 / CRS 2 LOH MAP (3) 23.6+ 31.6+ 31.6+ Anemia 1 Y234H
MAP4 57 IVB (lymph nodes) c.5266dupC LOH 3 Complete + HIPEC PR (-90 %) CRS 2 / CRS 3 Retention of the wild-type allele AT (3) 29.2+ 36.6+ 36.6+ Diarrhea 1; emesis 1; gastritis 1; nausea 1; leukopenia 2; nephrotoxicity 2; thrombocytopenia 4 R213X
MAP5 50 IIIC c.5266dupC na 3 Complete PR (-33 %) CRS 2 / no tumor cells in omentum LOH MAP (3) 22.2+ 29.5+ 29.5+ Anemia 1; leukopenia 1; nausea 1; thrombocytopenia 1 c.97-1G > A
MAP6 45 IVB (spleen, lymph nodes) c.1961delA LOH 4 Suboptimal PR (-47 %) CRS 2 / CRS 2 LOH TCbP (6) 21+ 30.4+ 30.4+ Anemia 1; nausea 1; thrombosis R213X
MAP7 56 IVB (pleuritis, lymph nodes) c.5266dupC na 3 Complete PR (-47 %) CRS 2 / CRS 2 LOH MAP (1) 4.1 8.3 29.8+ Anemia 2; nephrotoxicity 3 c.314delG
MAP8 58 IIIC c.4034delA LOH 5 Complete PR (-46 %) CRS 2 / CRS 2 LOH TCbP (4) 11.5 20.7 28.2+ Anemia 1; neutropenia 2 R248Q
MAP9 46 IIIC c.4034delA LOH 4 Complete PR (-64 %) CRS 2 / CRS 2 LOH MAP (2) 3.1 11.2 15.8 Thrombocytopenia 2 I255S
MAP10 40 IVB (pleuritis, lymph nodes) c.5266dupC LOH 4 Complete PR (-36 %) na / no tumor cells in omentum na None 28.5+ 37.6+ 37.6+ Anemia 1; hepatotoxicity 2; thrombocytopenia 2; nephrotoxicity 3 M133R
ACT Adjuvant chemotherapy, AT Doxorubicin 60 mg/m2 and paclitaxel 175 mg/m2, every 3 weeks, CRS Chemotherapy response score, HIPEC hyperthermic intraperitoneal chemotherapy, LOH Loss of heterozygosity, na Not analyzed, MAP Mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks, NACT Neoadjuvant chemotherapy, OS Overall survival, PFS Progression-free survival, PR Partial response, TCbP Paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks, TFI Treatment-free interval.
Notes: Tumor responses presented in the table describe the status of patients observed after the completion of the NACT, i.e. straight before the surgery. Patient MAP10 was diagnosed with ovarian cancer upon surgery, which was performed in another hospital and was limited to the excision of ovaries. She was considered eligible for the NACT study, as she had a significant tumor burden and could not be subjected to primary debulking surgery; she received no adjuvant therapy, as no residual tumor cells was seen in the surgical material. Patient MAP4 demonstrated the restoration of heterozygosity in a post-NACT tumor sample, suggesting that the tumor may no longer be platinum-sensitive [8, 9]; based on this finding, combination of paclitaxel and doxorubicin was given after the surgery; similarly, this combination was incorporated in the adjuvant treatment for patient MAP2, where the molecular analysis of post-NACT tumor tissue failed to establish somatic BRCA1 status. Patients MAP6 and MAP8 received TCbP combination after the surgery due to preference of their primary physicians.
Sixteen patients receiving paclitaxel plus carboplatin had slightly more favorable stage distribution, as 12 subjects had HGSOC of stage IIIC and 4 patients demonstrated stage IV disease (Table 2). While all patients treated by MAP showed partial response, 4/16 (25 %) women subjected to the TCbP combination produced only the disease stabilization and there was one HGSOC with the progression on this therapy. There was a remarkable difference from MAP regimen with regard to pathological responses, as minimal response score was observed in 6/16 (38 %) cases for ovarian tumor masses and 5/16 (31 %) HGSOCs for omental metastases (p = 0.05 and 0.12, respectively). While the median follow-up for the TCbP group was shorter than for MAP patients, median TFI was already achieved and reached 9.5 months (Fig. 1). Thirteen patients had sufficient follow-up to evaluate 1-year outcomes; the recurrence rate at 1 year after the completion of the treatment was 10/13 (77 %) for the TCbP, while the same value was 4/10 (40 %) for the MAP regimen (p = 0.1).
Table 2 Comparative characteristics of patients with BRCA1-mutated HGSOC receiving NACT combination of mitomycin C, doxorubicin and cisplatin versus women treated by paclitaxel plus carboplatin
Clinical characteristics MAP group (N = 10) TCbP group (N = 16) Statistical comparison
Median age of onset (range) 50 (35–64) 49 (37–72) Not significant
Pattern of BRCA1 mutations c.5266dupC (n = 5), c.4034delA (n = 3), c.68_69delAG (n = 1), c.1961delA (n = 1) c.5266dupC (n = 2), c.4034delA (n = 2), c.68_69delAG (n = 1), C61G (n = 1), c.1510delC (n = 1), Q563X (n = 1), c.2076dupT (n = 1), c.2983_2984delAA (n = 1), c.3247del5 (n = 1), c.3601_3602delGG (n = 1), c.3718_3719delCA (n = 1), Y1509X (n = 1), G1706E (n = 1), c.5152 + 1G > T (n = 1) p = 0.005 (founder vs. non-founder mutations; Fisher’s exact test)
FIGO stage
IIIC 5 (50 %) 12 (75 %) p = 0.23 (stage III vs. IV; Fisher’s exact test)
IVA 1 (10 %) 2 (13 %)
IVB 4 (40 %) 2 (13 %)
NACT cycles (range) 3–5 3–8
Cytoreduction
Optimal 9 (90 %) 14 (88 %) p = 1.0 (Fisher’s exact test)
Suboptimal 1 (10 %) 1 (6 %)
None 0 1 (6 %)
Response by RECIST
CR 0 0 p = 0.12 (objective response vs. lack of objective response; Fisher’s exact test)
PR 10 (100 %) 11 (69 %)
SD 0 4 (25 %)
PD 0 1 (6 %)
Chemotherapy response score (CRS) in the ovaries
CRS 1 0 6 (38 %) p = 0.05 (CRS 1 vs. other; Fisher’s exact test)
CRS 2 8 (80 %) 9 (56 %)
CRS 3 1 (10 %) 0
Tissue not available for evaluation 1 (10 %) 1 (10 %)
Chemotherapy response score (CRS) in the omentum
CRS 1 0 5 (31 %) p = 0.12 (CRS 1 vs. other; Fisher’s exact test)
CRS 2 5 (40 %) 8 (50 %)
CRS 3 1 (10 %) 0
No tumor cells 4 (40 %) 2 (13 %)
Tissue not available for evaluation 0 1 (6 %)
ACT cycles (range) 1–6 1–6
Median follow-up, months (range) 30.1 (15.8–36.6) 23.4 (10.7–45.2) p = 0.28 (Mann-Whitney Test)
Median treatment-free interval (95 % CI) Not reached 9.5 (7.8–11.2) p = 0.109 (Log Rank [Mantel-Cox])
Recurrence within one year after completion of treatment 4 (40 %) 10/13a (77 %) p = 0.1 (Fisher’s exact test)
ACT adjuvant chemotherapy, CRS chemotherapy response score, MAP mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8), cisplatin 80 mg/m2 (day 1), given every 4 weeks, NACT neoadjuvant chemotherapy, TCbP paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks
a13 out of 16 patients had sufficient follow-up for the estimation of 1-year recurrence rate
Fig. 1 Treatment-free interval for patients treated with the combination of mitomycin C, cisplatin and doxorubicin and for women treated by the paclitaxel plus carboplatin doublet
Discussion
Our previous study involving 12 BRCA1-mutated HGSOCs treated with the combination of cisplatin and mitomycin C revealed complete pathologic responses in 2 out of 12 patients [10]. We anticipated that the addition of doxorubicin to this combination may increase the rate of elimination of all tumor cells detectable in surgically excised tissues. The obtained data are sufficient to state that the applied triplet does not significantly increase the rate of complete pathologic responses as compared to the previously applied combination of two drugs.
At the same time, short-term results of MAP therapy look encouraging. In addition to a reasonably good rate of objective responses, half of the included cases demonstrated complete or nearly-complete absence of tumor cells in the omentum. Omental response score is the main predictor of the long-term outcomes of NACT, so it is a valuable marker allowing robust evaluation of various chemotherapy regimens [15].
Previous studies suggested that the TCbP regimen may be less efficient in BRCA1-mutated HGSOCs as compared to other NACT schemes [10]. These data sets compared prospective and retrospective patients treated by different surgeons. The quality of surgical debulking is critical for the outcome of HGSOC treatment, therefore these comparisons are prone to biases. Within the present study, we analyzed groups of patients who were managed at the same time interval by the same group of surgeons. However, the MAP and TCbP groups of patients were not balanced with regard to the pattern of mutations. The selection of patients for the MAP therapy involved rapid PCR tests for recurrent Slavic mutations [13]. The TCbP HGSOC group is significantly enriched by subjects with “rare” BRCA1 pathogenic alleles, which were detected by the next-generation sequencing analysis after the start of NACT. There are some data suggesting that distinct BRCA1 and BRCA2 mutations may exert distinct sensitivity to platinum compounds and PARP inhibitors [4]. Although we acknowledge differences in the pattern of BRCA1 mutations as a limitation of the study, it should be noted that all published trials on PARP inhibitors did not consider the type of mutation as a confounding factor [4, 16].
The landscape of the treatment BRCA1/2-mutated HGSOC is rapidly evolving. In particular, PARP inhibitors have been recently included in the standards for the first-line maintenance therapy, as they significantly delay the time to tumor recurrence [16]. None of the patients considered in this report received PARP inhibitors because they were not locally approved for early lines of HGSOC treatment at the time of the study. Consequently, it is unclear whether the differences observed between distinct NACT regimens will be maintained upon the incorporation of PARP-targeted drugs.
Conclusions
In summary, this study suggests that BRCA1-associated HGSOCs may require distinct therapeutic NACT regimens as compared to conventional TCbP doublet. If this is the case, the fast turn-around time for BRCA1/2 testing could become a critical factor for appropriate treatment decisions. Recent data indicate that BRCA1/2-associated HGSOCs do not show inferior outcomes when treated by NACT before the surgery, while primary surgical intervention is clearly the best approach in sporadic ovarian tumors [7, 17, 18]. These findings are likely to increase the acceptance of NACT for BRCA1/2 germline mutation carriers and, therefore, stimulate large neoadjuvant clinical trials for this category of HGSOC patients.
Abbreviations
CRSChemotherapy response score
HGSOCHigh-grade serous ovarian cancer
LOHLoss of heterozygosity
MAPMitomycin C, anthracycline, platinum
NACTNeoadjuvant chemotherapy
OCOvarian cancer
PFSProgression-free survival
TCbPTaxanes and carboplatin
TFITreatment-free interval
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Authors’ contributions
TG, KK, IB, KG, EN and OS managed study patients and analyzed clinical data; TS performed the molecular analysis; AI carried out morphological analysis; AS integrated and analyzed the obtained data and contributed to the manuscript preparation; EI designed the study and wrote the first draft of the manuscript. All authors have read and approved the final version of the manuscript.
Funding
This work has been supported by the Ministry of Science and Higher Education of the Russian Federation (grant № 075-15-2020-789).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was performed in full accordance with ethics guidelines. A written informed consent from study participants was obtained.
Consent for publication
All living patients provided the consent for publication.
Competing interests
The authors declare that they have no competing interests. | CISPLATIN, DOXORUBICIN HYDROCHLORIDE, MITOMYCIN | DrugsGivenReaction | CC BY | 33536037 | 18,976,042 | 2021-02-03 |
What was the dosage of drug 'CISPLATIN'? | Neoadjuvant therapy of BRCA1-driven ovarian cancer by combination of cisplatin, mitomycin C and doxorubicin.
BACKGROUND
Cisplatin, mitomycin C and anthracyclines demonstrate high activity in BRCA1-deficient tumors. This study aimed to evaluate the efficacy of the triplet combination of these drugs in BRCA1-driven high-grade serous ovarian carcinomas (HGSOCs).
METHODS
Ten HGSOC patients with germ-line BRCA1 mutation received neoadjuvant chemotherapy (NACT) consisting of mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks (MAP regimen). The comparator group included 16 women, who received standard NACT combination of paclitaxel 175 mg/m2 and carboplatin (6 AUC), given every 3 weeks (TCbP scheme).
RESULTS
None of the patients treated by the MAP scheme demonstrated complete pathologic response in ovaries, while 4 women showed absence of tumor cells in surgically excised omental specimens. When chemotherapy response scores (CRS) were considered, poor responsiveness (CRS 1) was not observed in the MAP group, but was common for the TCbP regimen (6/16 (38 %) for ovaries and 5/16 (31 %) for omentum; p = 0.05 and 0.12, respectively). Median treatment-free interval (TFI) was not reached in women treated by the MAP, but was 9.5 months for the TCbP scheme (p = 0.1). The rate of the recurrence within 1 year after the completion of the treatment was 4/10 (40 %) for the MAP and 10/13 (77 %) for the TCbP (p = 0.1).
CONCLUSIONS
The attempt to intensify NACT by administering combination of 3 drugs did not result in high rate of complete pathologic responses. However, there was a trend towards higher efficacy of the MAP regimen versus conventional TCbP scheme with regard to CRS and clinical outcomes.
Background
Ovarian cancer (OC) is a common malignancy, which holds the leading position in the mortality caused by gynecological tumors [1]. The worldwide incidence of OC approaches approximately three hundred thousand new cases per year, with almost two-thirds of affected patients dying from this disease [2]. High-grade serous ovarian cancer (HGSOC) is the most frequent OC histological type. A significant portion of HGSOCs is attributed to germ-line mutations in BRCA1 or BRCA2 genes. BRCA1/2-driven ovarian tumors usually develop via inactivation of the remaining allele of the involved gene. Consequently, these cancers demonstrate a tumor-selective deficiency in DNA repair by homologous recombination and pronounced sensitivity to platinum compounds, PARP inhibitors and mitomycin C [3, 4].
Ovarian tumors often do not cause symptoms at early stages; therefore, most HGSOC patients are diagnosed with already inoperable disease. These women are often subjected to neoadjuvant chemotherapy (NACT), which is aimed to reduce tumor burden and allow surgical intervention [5]. BRCA1-associated ovarian malignancies demonstrate significantly better responses to the NACT as compared to sporadic neoplasms [6]. Although these patients usually undergo complete cytoreductive surgery followed by adjuvant therapy, most BRCA1-driven HGSOCs eventually relapse [7]. These relapses are attributed to the acquisition of the resistance of tumor clones to systemic therapy. The most known mechanism of acquired platinum resistance is the emergence of mutations, which restore the open reading frame in the BRCA1 gene [4]. This route is mainly applicable to heavily pretreated patients but appears to be less characteristic for the initial phases of OC therapy [8]. On the other hand, NACT often results in the selection of BRCA1-proficient cells, which exist in small amounts in chemonaive tumors and repopulate tumor mass during platinum exposure [9].
Intensification of the therapy is a common approach aimed to prevent the emergence of resistant clones. We have previously reported promising results of applying cisplatin plus mitomycin C combination for the NACT of BRCA1-driven carcinomas. This therapy resulted in a significant reduction of the tumor burden in all analyzed patients and in complete pathologic responses observed in 2/12 (17 %) treated women [10]. We reasoned that combining this regimen with an additional drug may further improve the outcomes of NACT. Previous studies suggested that BRCA1-driven tumors are particularly sensitive to anthracyclines, while their responsiveness to taxanes is under the question [10, 11]. Consequently, we decided to add doxorubicin to cisplatin plus mitomycin C as a third drug. Here we present the results of the trial involving this 3-drug combination.
Methods
The design of the study was discussed on the council involving medical oncologists, cancer gynecologists and hereditary cancer specialists. It was decided that the pilot trial would include 10 patients with initially inoperable BRCA1-driven HGSOC, and the main end-point will be the rate of pathologic complete responses. While all patients received the same neoadjuvant regimen (MAP: mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8), cisplatin 80 mg/m2 (day 1), given every 4 weeks), the physicians were permitted to administer the therapy of their choice after the surgery. This approach provided some flexibility given that the combination of carboplatin and paclitaxel (TCbP) is a standard option for the treatment of ovarian cancer [1] and that some post-NACT tumor samples have restored BRCA1 function and therefore may not be potentially responsive to platinum drugs [8, 9]. The recruitment of patients was performed from August 2017 to December 2018 based on the results of the PCR-based test for Slavic recurrent germ-line mutations [12, 13]. According to the study protocol, all tumor samples were subjected to the loss-of-heterozygosity (LOH) analysis before the NACT and after the surgery. LOH test was performed as described in [9]. All tumors were also analyzed for the TP53 somatic mutations, given that TP53 inactivation is a ubiquitous feature of BRCA1-driven carcinomas [14]. The study was approved by the local Ethics Committee. All patients included in the study provided informed consent.
Although this study was not randomized, we considered for the comparison of treatment outcomes 16 consecutive patients with germ-line BRCA1 mutations, who were referred to the N.N. Petrov Institute of Oncology (St.-Petersburg, Russia) between February 2017 and December 2019 and were subjected to a standard NACT combination of paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks. Most of these patients were negative for PCR-detectable recurrent BRCA1 mutations; however, they were found to carry a germ-line pathogenic allele upon the analysis of the entire BRCA1 and BRCA2 coding sequence, i.e., after the start of NACT [13].
All women receiving MAP or TCbP were managed by the same surgical team. Tumor responses were evaluated according to RECIST criteria using computed tomography and magnetic resonance imaging. None of the patients treated by MAP or TCbP received bevacizumab. Three patients in the MAP arm but none in the TCbP group were subjected to the hyperthermic intraperitoneal chemotherapy (HIPEC) during surgery. None of the included patients described in this report received maintenance by PARP inhibitors after completion of the first-line therapy, as this indication was not approved in Russia at the time of the study.
The statistical analysis was performed using SPSS 13.0 software package. Age distribution and the duration of the follow-up were compared by the Mann-Whitney U-test. Median treatment-free interval (TFI) was evaluated using Kaplan-Meyer curves. Other comparisons were performed with the Fisher’s exact test.
Results
Five patients included in the study of the neoadjuvant combination of mitomycin C, cisplatin and doxorubicin had stage IIIC HGSOC and another 5 women were diagnosed with stage IV disease (Table 1). Partial response to this therapy was observed in all 10 cases considered. Seven women had toxicities of grades 1 or 2; 2 patients had toxicity grade 3 and 1 subject experienced grade IV thrombocytopenia. None of the HGSOCs showed a complete pathological response in the ovaries, and only one woman demonstrated chemotherapy response score (CRS) 3, according to Böhm et al. [15]. Omental tumor response, which is more predictive for the disease outcome than adnexal CRS [15], showed considerably better values: 4 women had no residual tumor cells in the omentum, 1 patient had CRS 3 and 5 cases demonstrated CRS 2. There were no instances of poor responsiveness to the therapy categorized as CRS 1. The median TFI and progression-free survival (PFS) were not reached.
Table 1 BRCA1-mutated HGSOC patients receiving neoadjuvant therapy consisting of mitomycin C, doxorubicin and cisplatin
ID Age Stage BRCA1 mutation Somatic BRCA1 status before NACT MAP cycles
(NACT) Surgical debulking Response by RECIST CRS (ovary / omentum) Somatic BRCA1 status after NACT ACT
(cycles) TFI, months PFS, months OS, months Toxicities and grades TP53 mutation
MAP1 64 IIIC c.5266dupC LOH 3 Complete + HIPEC PR (-44 %) CRS 3 / no tumor cells in omentum LOH MAP (2) 25.4+ 32.2+ 32.2+ Anemia 1; nephrotoxicity 1 C135W
MAP2 35 IVA (pleuritis) c.68_69delAG LOH 3 Complete + HIPEC PR (-73 %) CRS 2 / no tumor cells in omentum na MAP (1), AT (2) 6.9 14.4 26.9 Anemia 1 R175H
MAP3 50 IIIC c.4034delA LOH 4 Complete PR (-35 %) CRS 2 / CRS 2 LOH MAP (3) 23.6+ 31.6+ 31.6+ Anemia 1 Y234H
MAP4 57 IVB (lymph nodes) c.5266dupC LOH 3 Complete + HIPEC PR (-90 %) CRS 2 / CRS 3 Retention of the wild-type allele AT (3) 29.2+ 36.6+ 36.6+ Diarrhea 1; emesis 1; gastritis 1; nausea 1; leukopenia 2; nephrotoxicity 2; thrombocytopenia 4 R213X
MAP5 50 IIIC c.5266dupC na 3 Complete PR (-33 %) CRS 2 / no tumor cells in omentum LOH MAP (3) 22.2+ 29.5+ 29.5+ Anemia 1; leukopenia 1; nausea 1; thrombocytopenia 1 c.97-1G > A
MAP6 45 IVB (spleen, lymph nodes) c.1961delA LOH 4 Suboptimal PR (-47 %) CRS 2 / CRS 2 LOH TCbP (6) 21+ 30.4+ 30.4+ Anemia 1; nausea 1; thrombosis R213X
MAP7 56 IVB (pleuritis, lymph nodes) c.5266dupC na 3 Complete PR (-47 %) CRS 2 / CRS 2 LOH MAP (1) 4.1 8.3 29.8+ Anemia 2; nephrotoxicity 3 c.314delG
MAP8 58 IIIC c.4034delA LOH 5 Complete PR (-46 %) CRS 2 / CRS 2 LOH TCbP (4) 11.5 20.7 28.2+ Anemia 1; neutropenia 2 R248Q
MAP9 46 IIIC c.4034delA LOH 4 Complete PR (-64 %) CRS 2 / CRS 2 LOH MAP (2) 3.1 11.2 15.8 Thrombocytopenia 2 I255S
MAP10 40 IVB (pleuritis, lymph nodes) c.5266dupC LOH 4 Complete PR (-36 %) na / no tumor cells in omentum na None 28.5+ 37.6+ 37.6+ Anemia 1; hepatotoxicity 2; thrombocytopenia 2; nephrotoxicity 3 M133R
ACT Adjuvant chemotherapy, AT Doxorubicin 60 mg/m2 and paclitaxel 175 mg/m2, every 3 weeks, CRS Chemotherapy response score, HIPEC hyperthermic intraperitoneal chemotherapy, LOH Loss of heterozygosity, na Not analyzed, MAP Mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks, NACT Neoadjuvant chemotherapy, OS Overall survival, PFS Progression-free survival, PR Partial response, TCbP Paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks, TFI Treatment-free interval.
Notes: Tumor responses presented in the table describe the status of patients observed after the completion of the NACT, i.e. straight before the surgery. Patient MAP10 was diagnosed with ovarian cancer upon surgery, which was performed in another hospital and was limited to the excision of ovaries. She was considered eligible for the NACT study, as she had a significant tumor burden and could not be subjected to primary debulking surgery; she received no adjuvant therapy, as no residual tumor cells was seen in the surgical material. Patient MAP4 demonstrated the restoration of heterozygosity in a post-NACT tumor sample, suggesting that the tumor may no longer be platinum-sensitive [8, 9]; based on this finding, combination of paclitaxel and doxorubicin was given after the surgery; similarly, this combination was incorporated in the adjuvant treatment for patient MAP2, where the molecular analysis of post-NACT tumor tissue failed to establish somatic BRCA1 status. Patients MAP6 and MAP8 received TCbP combination after the surgery due to preference of their primary physicians.
Sixteen patients receiving paclitaxel plus carboplatin had slightly more favorable stage distribution, as 12 subjects had HGSOC of stage IIIC and 4 patients demonstrated stage IV disease (Table 2). While all patients treated by MAP showed partial response, 4/16 (25 %) women subjected to the TCbP combination produced only the disease stabilization and there was one HGSOC with the progression on this therapy. There was a remarkable difference from MAP regimen with regard to pathological responses, as minimal response score was observed in 6/16 (38 %) cases for ovarian tumor masses and 5/16 (31 %) HGSOCs for omental metastases (p = 0.05 and 0.12, respectively). While the median follow-up for the TCbP group was shorter than for MAP patients, median TFI was already achieved and reached 9.5 months (Fig. 1). Thirteen patients had sufficient follow-up to evaluate 1-year outcomes; the recurrence rate at 1 year after the completion of the treatment was 10/13 (77 %) for the TCbP, while the same value was 4/10 (40 %) for the MAP regimen (p = 0.1).
Table 2 Comparative characteristics of patients with BRCA1-mutated HGSOC receiving NACT combination of mitomycin C, doxorubicin and cisplatin versus women treated by paclitaxel plus carboplatin
Clinical characteristics MAP group (N = 10) TCbP group (N = 16) Statistical comparison
Median age of onset (range) 50 (35–64) 49 (37–72) Not significant
Pattern of BRCA1 mutations c.5266dupC (n = 5), c.4034delA (n = 3), c.68_69delAG (n = 1), c.1961delA (n = 1) c.5266dupC (n = 2), c.4034delA (n = 2), c.68_69delAG (n = 1), C61G (n = 1), c.1510delC (n = 1), Q563X (n = 1), c.2076dupT (n = 1), c.2983_2984delAA (n = 1), c.3247del5 (n = 1), c.3601_3602delGG (n = 1), c.3718_3719delCA (n = 1), Y1509X (n = 1), G1706E (n = 1), c.5152 + 1G > T (n = 1) p = 0.005 (founder vs. non-founder mutations; Fisher’s exact test)
FIGO stage
IIIC 5 (50 %) 12 (75 %) p = 0.23 (stage III vs. IV; Fisher’s exact test)
IVA 1 (10 %) 2 (13 %)
IVB 4 (40 %) 2 (13 %)
NACT cycles (range) 3–5 3–8
Cytoreduction
Optimal 9 (90 %) 14 (88 %) p = 1.0 (Fisher’s exact test)
Suboptimal 1 (10 %) 1 (6 %)
None 0 1 (6 %)
Response by RECIST
CR 0 0 p = 0.12 (objective response vs. lack of objective response; Fisher’s exact test)
PR 10 (100 %) 11 (69 %)
SD 0 4 (25 %)
PD 0 1 (6 %)
Chemotherapy response score (CRS) in the ovaries
CRS 1 0 6 (38 %) p = 0.05 (CRS 1 vs. other; Fisher’s exact test)
CRS 2 8 (80 %) 9 (56 %)
CRS 3 1 (10 %) 0
Tissue not available for evaluation 1 (10 %) 1 (10 %)
Chemotherapy response score (CRS) in the omentum
CRS 1 0 5 (31 %) p = 0.12 (CRS 1 vs. other; Fisher’s exact test)
CRS 2 5 (40 %) 8 (50 %)
CRS 3 1 (10 %) 0
No tumor cells 4 (40 %) 2 (13 %)
Tissue not available for evaluation 0 1 (6 %)
ACT cycles (range) 1–6 1–6
Median follow-up, months (range) 30.1 (15.8–36.6) 23.4 (10.7–45.2) p = 0.28 (Mann-Whitney Test)
Median treatment-free interval (95 % CI) Not reached 9.5 (7.8–11.2) p = 0.109 (Log Rank [Mantel-Cox])
Recurrence within one year after completion of treatment 4 (40 %) 10/13a (77 %) p = 0.1 (Fisher’s exact test)
ACT adjuvant chemotherapy, CRS chemotherapy response score, MAP mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8), cisplatin 80 mg/m2 (day 1), given every 4 weeks, NACT neoadjuvant chemotherapy, TCbP paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks
a13 out of 16 patients had sufficient follow-up for the estimation of 1-year recurrence rate
Fig. 1 Treatment-free interval for patients treated with the combination of mitomycin C, cisplatin and doxorubicin and for women treated by the paclitaxel plus carboplatin doublet
Discussion
Our previous study involving 12 BRCA1-mutated HGSOCs treated with the combination of cisplatin and mitomycin C revealed complete pathologic responses in 2 out of 12 patients [10]. We anticipated that the addition of doxorubicin to this combination may increase the rate of elimination of all tumor cells detectable in surgically excised tissues. The obtained data are sufficient to state that the applied triplet does not significantly increase the rate of complete pathologic responses as compared to the previously applied combination of two drugs.
At the same time, short-term results of MAP therapy look encouraging. In addition to a reasonably good rate of objective responses, half of the included cases demonstrated complete or nearly-complete absence of tumor cells in the omentum. Omental response score is the main predictor of the long-term outcomes of NACT, so it is a valuable marker allowing robust evaluation of various chemotherapy regimens [15].
Previous studies suggested that the TCbP regimen may be less efficient in BRCA1-mutated HGSOCs as compared to other NACT schemes [10]. These data sets compared prospective and retrospective patients treated by different surgeons. The quality of surgical debulking is critical for the outcome of HGSOC treatment, therefore these comparisons are prone to biases. Within the present study, we analyzed groups of patients who were managed at the same time interval by the same group of surgeons. However, the MAP and TCbP groups of patients were not balanced with regard to the pattern of mutations. The selection of patients for the MAP therapy involved rapid PCR tests for recurrent Slavic mutations [13]. The TCbP HGSOC group is significantly enriched by subjects with “rare” BRCA1 pathogenic alleles, which were detected by the next-generation sequencing analysis after the start of NACT. There are some data suggesting that distinct BRCA1 and BRCA2 mutations may exert distinct sensitivity to platinum compounds and PARP inhibitors [4]. Although we acknowledge differences in the pattern of BRCA1 mutations as a limitation of the study, it should be noted that all published trials on PARP inhibitors did not consider the type of mutation as a confounding factor [4, 16].
The landscape of the treatment BRCA1/2-mutated HGSOC is rapidly evolving. In particular, PARP inhibitors have been recently included in the standards for the first-line maintenance therapy, as they significantly delay the time to tumor recurrence [16]. None of the patients considered in this report received PARP inhibitors because they were not locally approved for early lines of HGSOC treatment at the time of the study. Consequently, it is unclear whether the differences observed between distinct NACT regimens will be maintained upon the incorporation of PARP-targeted drugs.
Conclusions
In summary, this study suggests that BRCA1-associated HGSOCs may require distinct therapeutic NACT regimens as compared to conventional TCbP doublet. If this is the case, the fast turn-around time for BRCA1/2 testing could become a critical factor for appropriate treatment decisions. Recent data indicate that BRCA1/2-associated HGSOCs do not show inferior outcomes when treated by NACT before the surgery, while primary surgical intervention is clearly the best approach in sporadic ovarian tumors [7, 17, 18]. These findings are likely to increase the acceptance of NACT for BRCA1/2 germline mutation carriers and, therefore, stimulate large neoadjuvant clinical trials for this category of HGSOC patients.
Abbreviations
CRSChemotherapy response score
HGSOCHigh-grade serous ovarian cancer
LOHLoss of heterozygosity
MAPMitomycin C, anthracycline, platinum
NACTNeoadjuvant chemotherapy
OCOvarian cancer
PFSProgression-free survival
TCbPTaxanes and carboplatin
TFITreatment-free interval
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Authors’ contributions
TG, KK, IB, KG, EN and OS managed study patients and analyzed clinical data; TS performed the molecular analysis; AI carried out morphological analysis; AS integrated and analyzed the obtained data and contributed to the manuscript preparation; EI designed the study and wrote the first draft of the manuscript. All authors have read and approved the final version of the manuscript.
Funding
This work has been supported by the Ministry of Science and Higher Education of the Russian Federation (grant № 075-15-2020-789).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was performed in full accordance with ethics guidelines. A written informed consent from study participants was obtained.
Consent for publication
All living patients provided the consent for publication.
Competing interests
The authors declare that they have no competing interests. | DAY 1 | DrugDosageText | CC BY | 33536037 | 18,976,042 | 2021-02-03 |
What was the dosage of drug 'DOXORUBICIN HYDROCHLORIDE'? | Neoadjuvant therapy of BRCA1-driven ovarian cancer by combination of cisplatin, mitomycin C and doxorubicin.
BACKGROUND
Cisplatin, mitomycin C and anthracyclines demonstrate high activity in BRCA1-deficient tumors. This study aimed to evaluate the efficacy of the triplet combination of these drugs in BRCA1-driven high-grade serous ovarian carcinomas (HGSOCs).
METHODS
Ten HGSOC patients with germ-line BRCA1 mutation received neoadjuvant chemotherapy (NACT) consisting of mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks (MAP regimen). The comparator group included 16 women, who received standard NACT combination of paclitaxel 175 mg/m2 and carboplatin (6 AUC), given every 3 weeks (TCbP scheme).
RESULTS
None of the patients treated by the MAP scheme demonstrated complete pathologic response in ovaries, while 4 women showed absence of tumor cells in surgically excised omental specimens. When chemotherapy response scores (CRS) were considered, poor responsiveness (CRS 1) was not observed in the MAP group, but was common for the TCbP regimen (6/16 (38 %) for ovaries and 5/16 (31 %) for omentum; p = 0.05 and 0.12, respectively). Median treatment-free interval (TFI) was not reached in women treated by the MAP, but was 9.5 months for the TCbP scheme (p = 0.1). The rate of the recurrence within 1 year after the completion of the treatment was 4/10 (40 %) for the MAP and 10/13 (77 %) for the TCbP (p = 0.1).
CONCLUSIONS
The attempt to intensify NACT by administering combination of 3 drugs did not result in high rate of complete pathologic responses. However, there was a trend towards higher efficacy of the MAP regimen versus conventional TCbP scheme with regard to CRS and clinical outcomes.
Background
Ovarian cancer (OC) is a common malignancy, which holds the leading position in the mortality caused by gynecological tumors [1]. The worldwide incidence of OC approaches approximately three hundred thousand new cases per year, with almost two-thirds of affected patients dying from this disease [2]. High-grade serous ovarian cancer (HGSOC) is the most frequent OC histological type. A significant portion of HGSOCs is attributed to germ-line mutations in BRCA1 or BRCA2 genes. BRCA1/2-driven ovarian tumors usually develop via inactivation of the remaining allele of the involved gene. Consequently, these cancers demonstrate a tumor-selective deficiency in DNA repair by homologous recombination and pronounced sensitivity to platinum compounds, PARP inhibitors and mitomycin C [3, 4].
Ovarian tumors often do not cause symptoms at early stages; therefore, most HGSOC patients are diagnosed with already inoperable disease. These women are often subjected to neoadjuvant chemotherapy (NACT), which is aimed to reduce tumor burden and allow surgical intervention [5]. BRCA1-associated ovarian malignancies demonstrate significantly better responses to the NACT as compared to sporadic neoplasms [6]. Although these patients usually undergo complete cytoreductive surgery followed by adjuvant therapy, most BRCA1-driven HGSOCs eventually relapse [7]. These relapses are attributed to the acquisition of the resistance of tumor clones to systemic therapy. The most known mechanism of acquired platinum resistance is the emergence of mutations, which restore the open reading frame in the BRCA1 gene [4]. This route is mainly applicable to heavily pretreated patients but appears to be less characteristic for the initial phases of OC therapy [8]. On the other hand, NACT often results in the selection of BRCA1-proficient cells, which exist in small amounts in chemonaive tumors and repopulate tumor mass during platinum exposure [9].
Intensification of the therapy is a common approach aimed to prevent the emergence of resistant clones. We have previously reported promising results of applying cisplatin plus mitomycin C combination for the NACT of BRCA1-driven carcinomas. This therapy resulted in a significant reduction of the tumor burden in all analyzed patients and in complete pathologic responses observed in 2/12 (17 %) treated women [10]. We reasoned that combining this regimen with an additional drug may further improve the outcomes of NACT. Previous studies suggested that BRCA1-driven tumors are particularly sensitive to anthracyclines, while their responsiveness to taxanes is under the question [10, 11]. Consequently, we decided to add doxorubicin to cisplatin plus mitomycin C as a third drug. Here we present the results of the trial involving this 3-drug combination.
Methods
The design of the study was discussed on the council involving medical oncologists, cancer gynecologists and hereditary cancer specialists. It was decided that the pilot trial would include 10 patients with initially inoperable BRCA1-driven HGSOC, and the main end-point will be the rate of pathologic complete responses. While all patients received the same neoadjuvant regimen (MAP: mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8), cisplatin 80 mg/m2 (day 1), given every 4 weeks), the physicians were permitted to administer the therapy of their choice after the surgery. This approach provided some flexibility given that the combination of carboplatin and paclitaxel (TCbP) is a standard option for the treatment of ovarian cancer [1] and that some post-NACT tumor samples have restored BRCA1 function and therefore may not be potentially responsive to platinum drugs [8, 9]. The recruitment of patients was performed from August 2017 to December 2018 based on the results of the PCR-based test for Slavic recurrent germ-line mutations [12, 13]. According to the study protocol, all tumor samples were subjected to the loss-of-heterozygosity (LOH) analysis before the NACT and after the surgery. LOH test was performed as described in [9]. All tumors were also analyzed for the TP53 somatic mutations, given that TP53 inactivation is a ubiquitous feature of BRCA1-driven carcinomas [14]. The study was approved by the local Ethics Committee. All patients included in the study provided informed consent.
Although this study was not randomized, we considered for the comparison of treatment outcomes 16 consecutive patients with germ-line BRCA1 mutations, who were referred to the N.N. Petrov Institute of Oncology (St.-Petersburg, Russia) between February 2017 and December 2019 and were subjected to a standard NACT combination of paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks. Most of these patients were negative for PCR-detectable recurrent BRCA1 mutations; however, they were found to carry a germ-line pathogenic allele upon the analysis of the entire BRCA1 and BRCA2 coding sequence, i.e., after the start of NACT [13].
All women receiving MAP or TCbP were managed by the same surgical team. Tumor responses were evaluated according to RECIST criteria using computed tomography and magnetic resonance imaging. None of the patients treated by MAP or TCbP received bevacizumab. Three patients in the MAP arm but none in the TCbP group were subjected to the hyperthermic intraperitoneal chemotherapy (HIPEC) during surgery. None of the included patients described in this report received maintenance by PARP inhibitors after completion of the first-line therapy, as this indication was not approved in Russia at the time of the study.
The statistical analysis was performed using SPSS 13.0 software package. Age distribution and the duration of the follow-up were compared by the Mann-Whitney U-test. Median treatment-free interval (TFI) was evaluated using Kaplan-Meyer curves. Other comparisons were performed with the Fisher’s exact test.
Results
Five patients included in the study of the neoadjuvant combination of mitomycin C, cisplatin and doxorubicin had stage IIIC HGSOC and another 5 women were diagnosed with stage IV disease (Table 1). Partial response to this therapy was observed in all 10 cases considered. Seven women had toxicities of grades 1 or 2; 2 patients had toxicity grade 3 and 1 subject experienced grade IV thrombocytopenia. None of the HGSOCs showed a complete pathological response in the ovaries, and only one woman demonstrated chemotherapy response score (CRS) 3, according to Böhm et al. [15]. Omental tumor response, which is more predictive for the disease outcome than adnexal CRS [15], showed considerably better values: 4 women had no residual tumor cells in the omentum, 1 patient had CRS 3 and 5 cases demonstrated CRS 2. There were no instances of poor responsiveness to the therapy categorized as CRS 1. The median TFI and progression-free survival (PFS) were not reached.
Table 1 BRCA1-mutated HGSOC patients receiving neoadjuvant therapy consisting of mitomycin C, doxorubicin and cisplatin
ID Age Stage BRCA1 mutation Somatic BRCA1 status before NACT MAP cycles
(NACT) Surgical debulking Response by RECIST CRS (ovary / omentum) Somatic BRCA1 status after NACT ACT
(cycles) TFI, months PFS, months OS, months Toxicities and grades TP53 mutation
MAP1 64 IIIC c.5266dupC LOH 3 Complete + HIPEC PR (-44 %) CRS 3 / no tumor cells in omentum LOH MAP (2) 25.4+ 32.2+ 32.2+ Anemia 1; nephrotoxicity 1 C135W
MAP2 35 IVA (pleuritis) c.68_69delAG LOH 3 Complete + HIPEC PR (-73 %) CRS 2 / no tumor cells in omentum na MAP (1), AT (2) 6.9 14.4 26.9 Anemia 1 R175H
MAP3 50 IIIC c.4034delA LOH 4 Complete PR (-35 %) CRS 2 / CRS 2 LOH MAP (3) 23.6+ 31.6+ 31.6+ Anemia 1 Y234H
MAP4 57 IVB (lymph nodes) c.5266dupC LOH 3 Complete + HIPEC PR (-90 %) CRS 2 / CRS 3 Retention of the wild-type allele AT (3) 29.2+ 36.6+ 36.6+ Diarrhea 1; emesis 1; gastritis 1; nausea 1; leukopenia 2; nephrotoxicity 2; thrombocytopenia 4 R213X
MAP5 50 IIIC c.5266dupC na 3 Complete PR (-33 %) CRS 2 / no tumor cells in omentum LOH MAP (3) 22.2+ 29.5+ 29.5+ Anemia 1; leukopenia 1; nausea 1; thrombocytopenia 1 c.97-1G > A
MAP6 45 IVB (spleen, lymph nodes) c.1961delA LOH 4 Suboptimal PR (-47 %) CRS 2 / CRS 2 LOH TCbP (6) 21+ 30.4+ 30.4+ Anemia 1; nausea 1; thrombosis R213X
MAP7 56 IVB (pleuritis, lymph nodes) c.5266dupC na 3 Complete PR (-47 %) CRS 2 / CRS 2 LOH MAP (1) 4.1 8.3 29.8+ Anemia 2; nephrotoxicity 3 c.314delG
MAP8 58 IIIC c.4034delA LOH 5 Complete PR (-46 %) CRS 2 / CRS 2 LOH TCbP (4) 11.5 20.7 28.2+ Anemia 1; neutropenia 2 R248Q
MAP9 46 IIIC c.4034delA LOH 4 Complete PR (-64 %) CRS 2 / CRS 2 LOH MAP (2) 3.1 11.2 15.8 Thrombocytopenia 2 I255S
MAP10 40 IVB (pleuritis, lymph nodes) c.5266dupC LOH 4 Complete PR (-36 %) na / no tumor cells in omentum na None 28.5+ 37.6+ 37.6+ Anemia 1; hepatotoxicity 2; thrombocytopenia 2; nephrotoxicity 3 M133R
ACT Adjuvant chemotherapy, AT Doxorubicin 60 mg/m2 and paclitaxel 175 mg/m2, every 3 weeks, CRS Chemotherapy response score, HIPEC hyperthermic intraperitoneal chemotherapy, LOH Loss of heterozygosity, na Not analyzed, MAP Mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks, NACT Neoadjuvant chemotherapy, OS Overall survival, PFS Progression-free survival, PR Partial response, TCbP Paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks, TFI Treatment-free interval.
Notes: Tumor responses presented in the table describe the status of patients observed after the completion of the NACT, i.e. straight before the surgery. Patient MAP10 was diagnosed with ovarian cancer upon surgery, which was performed in another hospital and was limited to the excision of ovaries. She was considered eligible for the NACT study, as she had a significant tumor burden and could not be subjected to primary debulking surgery; she received no adjuvant therapy, as no residual tumor cells was seen in the surgical material. Patient MAP4 demonstrated the restoration of heterozygosity in a post-NACT tumor sample, suggesting that the tumor may no longer be platinum-sensitive [8, 9]; based on this finding, combination of paclitaxel and doxorubicin was given after the surgery; similarly, this combination was incorporated in the adjuvant treatment for patient MAP2, where the molecular analysis of post-NACT tumor tissue failed to establish somatic BRCA1 status. Patients MAP6 and MAP8 received TCbP combination after the surgery due to preference of their primary physicians.
Sixteen patients receiving paclitaxel plus carboplatin had slightly more favorable stage distribution, as 12 subjects had HGSOC of stage IIIC and 4 patients demonstrated stage IV disease (Table 2). While all patients treated by MAP showed partial response, 4/16 (25 %) women subjected to the TCbP combination produced only the disease stabilization and there was one HGSOC with the progression on this therapy. There was a remarkable difference from MAP regimen with regard to pathological responses, as minimal response score was observed in 6/16 (38 %) cases for ovarian tumor masses and 5/16 (31 %) HGSOCs for omental metastases (p = 0.05 and 0.12, respectively). While the median follow-up for the TCbP group was shorter than for MAP patients, median TFI was already achieved and reached 9.5 months (Fig. 1). Thirteen patients had sufficient follow-up to evaluate 1-year outcomes; the recurrence rate at 1 year after the completion of the treatment was 10/13 (77 %) for the TCbP, while the same value was 4/10 (40 %) for the MAP regimen (p = 0.1).
Table 2 Comparative characteristics of patients with BRCA1-mutated HGSOC receiving NACT combination of mitomycin C, doxorubicin and cisplatin versus women treated by paclitaxel plus carboplatin
Clinical characteristics MAP group (N = 10) TCbP group (N = 16) Statistical comparison
Median age of onset (range) 50 (35–64) 49 (37–72) Not significant
Pattern of BRCA1 mutations c.5266dupC (n = 5), c.4034delA (n = 3), c.68_69delAG (n = 1), c.1961delA (n = 1) c.5266dupC (n = 2), c.4034delA (n = 2), c.68_69delAG (n = 1), C61G (n = 1), c.1510delC (n = 1), Q563X (n = 1), c.2076dupT (n = 1), c.2983_2984delAA (n = 1), c.3247del5 (n = 1), c.3601_3602delGG (n = 1), c.3718_3719delCA (n = 1), Y1509X (n = 1), G1706E (n = 1), c.5152 + 1G > T (n = 1) p = 0.005 (founder vs. non-founder mutations; Fisher’s exact test)
FIGO stage
IIIC 5 (50 %) 12 (75 %) p = 0.23 (stage III vs. IV; Fisher’s exact test)
IVA 1 (10 %) 2 (13 %)
IVB 4 (40 %) 2 (13 %)
NACT cycles (range) 3–5 3–8
Cytoreduction
Optimal 9 (90 %) 14 (88 %) p = 1.0 (Fisher’s exact test)
Suboptimal 1 (10 %) 1 (6 %)
None 0 1 (6 %)
Response by RECIST
CR 0 0 p = 0.12 (objective response vs. lack of objective response; Fisher’s exact test)
PR 10 (100 %) 11 (69 %)
SD 0 4 (25 %)
PD 0 1 (6 %)
Chemotherapy response score (CRS) in the ovaries
CRS 1 0 6 (38 %) p = 0.05 (CRS 1 vs. other; Fisher’s exact test)
CRS 2 8 (80 %) 9 (56 %)
CRS 3 1 (10 %) 0
Tissue not available for evaluation 1 (10 %) 1 (10 %)
Chemotherapy response score (CRS) in the omentum
CRS 1 0 5 (31 %) p = 0.12 (CRS 1 vs. other; Fisher’s exact test)
CRS 2 5 (40 %) 8 (50 %)
CRS 3 1 (10 %) 0
No tumor cells 4 (40 %) 2 (13 %)
Tissue not available for evaluation 0 1 (6 %)
ACT cycles (range) 1–6 1–6
Median follow-up, months (range) 30.1 (15.8–36.6) 23.4 (10.7–45.2) p = 0.28 (Mann-Whitney Test)
Median treatment-free interval (95 % CI) Not reached 9.5 (7.8–11.2) p = 0.109 (Log Rank [Mantel-Cox])
Recurrence within one year after completion of treatment 4 (40 %) 10/13a (77 %) p = 0.1 (Fisher’s exact test)
ACT adjuvant chemotherapy, CRS chemotherapy response score, MAP mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8), cisplatin 80 mg/m2 (day 1), given every 4 weeks, NACT neoadjuvant chemotherapy, TCbP paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks
a13 out of 16 patients had sufficient follow-up for the estimation of 1-year recurrence rate
Fig. 1 Treatment-free interval for patients treated with the combination of mitomycin C, cisplatin and doxorubicin and for women treated by the paclitaxel plus carboplatin doublet
Discussion
Our previous study involving 12 BRCA1-mutated HGSOCs treated with the combination of cisplatin and mitomycin C revealed complete pathologic responses in 2 out of 12 patients [10]. We anticipated that the addition of doxorubicin to this combination may increase the rate of elimination of all tumor cells detectable in surgically excised tissues. The obtained data are sufficient to state that the applied triplet does not significantly increase the rate of complete pathologic responses as compared to the previously applied combination of two drugs.
At the same time, short-term results of MAP therapy look encouraging. In addition to a reasonably good rate of objective responses, half of the included cases demonstrated complete or nearly-complete absence of tumor cells in the omentum. Omental response score is the main predictor of the long-term outcomes of NACT, so it is a valuable marker allowing robust evaluation of various chemotherapy regimens [15].
Previous studies suggested that the TCbP regimen may be less efficient in BRCA1-mutated HGSOCs as compared to other NACT schemes [10]. These data sets compared prospective and retrospective patients treated by different surgeons. The quality of surgical debulking is critical for the outcome of HGSOC treatment, therefore these comparisons are prone to biases. Within the present study, we analyzed groups of patients who were managed at the same time interval by the same group of surgeons. However, the MAP and TCbP groups of patients were not balanced with regard to the pattern of mutations. The selection of patients for the MAP therapy involved rapid PCR tests for recurrent Slavic mutations [13]. The TCbP HGSOC group is significantly enriched by subjects with “rare” BRCA1 pathogenic alleles, which were detected by the next-generation sequencing analysis after the start of NACT. There are some data suggesting that distinct BRCA1 and BRCA2 mutations may exert distinct sensitivity to platinum compounds and PARP inhibitors [4]. Although we acknowledge differences in the pattern of BRCA1 mutations as a limitation of the study, it should be noted that all published trials on PARP inhibitors did not consider the type of mutation as a confounding factor [4, 16].
The landscape of the treatment BRCA1/2-mutated HGSOC is rapidly evolving. In particular, PARP inhibitors have been recently included in the standards for the first-line maintenance therapy, as they significantly delay the time to tumor recurrence [16]. None of the patients considered in this report received PARP inhibitors because they were not locally approved for early lines of HGSOC treatment at the time of the study. Consequently, it is unclear whether the differences observed between distinct NACT regimens will be maintained upon the incorporation of PARP-targeted drugs.
Conclusions
In summary, this study suggests that BRCA1-associated HGSOCs may require distinct therapeutic NACT regimens as compared to conventional TCbP doublet. If this is the case, the fast turn-around time for BRCA1/2 testing could become a critical factor for appropriate treatment decisions. Recent data indicate that BRCA1/2-associated HGSOCs do not show inferior outcomes when treated by NACT before the surgery, while primary surgical intervention is clearly the best approach in sporadic ovarian tumors [7, 17, 18]. These findings are likely to increase the acceptance of NACT for BRCA1/2 germline mutation carriers and, therefore, stimulate large neoadjuvant clinical trials for this category of HGSOC patients.
Abbreviations
CRSChemotherapy response score
HGSOCHigh-grade serous ovarian cancer
LOHLoss of heterozygosity
MAPMitomycin C, anthracycline, platinum
NACTNeoadjuvant chemotherapy
OCOvarian cancer
PFSProgression-free survival
TCbPTaxanes and carboplatin
TFITreatment-free interval
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Authors’ contributions
TG, KK, IB, KG, EN and OS managed study patients and analyzed clinical data; TS performed the molecular analysis; AI carried out morphological analysis; AS integrated and analyzed the obtained data and contributed to the manuscript preparation; EI designed the study and wrote the first draft of the manuscript. All authors have read and approved the final version of the manuscript.
Funding
This work has been supported by the Ministry of Science and Higher Education of the Russian Federation (grant № 075-15-2020-789).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was performed in full accordance with ethics guidelines. A written informed consent from study participants was obtained.
Consent for publication
All living patients provided the consent for publication.
Competing interests
The authors declare that they have no competing interests. | DAYS 1 AND 8 | DrugDosageText | CC BY | 33536037 | 18,976,042 | 2021-02-03 |
What was the dosage of drug 'MITOMYCIN'? | Neoadjuvant therapy of BRCA1-driven ovarian cancer by combination of cisplatin, mitomycin C and doxorubicin.
BACKGROUND
Cisplatin, mitomycin C and anthracyclines demonstrate high activity in BRCA1-deficient tumors. This study aimed to evaluate the efficacy of the triplet combination of these drugs in BRCA1-driven high-grade serous ovarian carcinomas (HGSOCs).
METHODS
Ten HGSOC patients with germ-line BRCA1 mutation received neoadjuvant chemotherapy (NACT) consisting of mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks (MAP regimen). The comparator group included 16 women, who received standard NACT combination of paclitaxel 175 mg/m2 and carboplatin (6 AUC), given every 3 weeks (TCbP scheme).
RESULTS
None of the patients treated by the MAP scheme demonstrated complete pathologic response in ovaries, while 4 women showed absence of tumor cells in surgically excised omental specimens. When chemotherapy response scores (CRS) were considered, poor responsiveness (CRS 1) was not observed in the MAP group, but was common for the TCbP regimen (6/16 (38 %) for ovaries and 5/16 (31 %) for omentum; p = 0.05 and 0.12, respectively). Median treatment-free interval (TFI) was not reached in women treated by the MAP, but was 9.5 months for the TCbP scheme (p = 0.1). The rate of the recurrence within 1 year after the completion of the treatment was 4/10 (40 %) for the MAP and 10/13 (77 %) for the TCbP (p = 0.1).
CONCLUSIONS
The attempt to intensify NACT by administering combination of 3 drugs did not result in high rate of complete pathologic responses. However, there was a trend towards higher efficacy of the MAP regimen versus conventional TCbP scheme with regard to CRS and clinical outcomes.
Background
Ovarian cancer (OC) is a common malignancy, which holds the leading position in the mortality caused by gynecological tumors [1]. The worldwide incidence of OC approaches approximately three hundred thousand new cases per year, with almost two-thirds of affected patients dying from this disease [2]. High-grade serous ovarian cancer (HGSOC) is the most frequent OC histological type. A significant portion of HGSOCs is attributed to germ-line mutations in BRCA1 or BRCA2 genes. BRCA1/2-driven ovarian tumors usually develop via inactivation of the remaining allele of the involved gene. Consequently, these cancers demonstrate a tumor-selective deficiency in DNA repair by homologous recombination and pronounced sensitivity to platinum compounds, PARP inhibitors and mitomycin C [3, 4].
Ovarian tumors often do not cause symptoms at early stages; therefore, most HGSOC patients are diagnosed with already inoperable disease. These women are often subjected to neoadjuvant chemotherapy (NACT), which is aimed to reduce tumor burden and allow surgical intervention [5]. BRCA1-associated ovarian malignancies demonstrate significantly better responses to the NACT as compared to sporadic neoplasms [6]. Although these patients usually undergo complete cytoreductive surgery followed by adjuvant therapy, most BRCA1-driven HGSOCs eventually relapse [7]. These relapses are attributed to the acquisition of the resistance of tumor clones to systemic therapy. The most known mechanism of acquired platinum resistance is the emergence of mutations, which restore the open reading frame in the BRCA1 gene [4]. This route is mainly applicable to heavily pretreated patients but appears to be less characteristic for the initial phases of OC therapy [8]. On the other hand, NACT often results in the selection of BRCA1-proficient cells, which exist in small amounts in chemonaive tumors and repopulate tumor mass during platinum exposure [9].
Intensification of the therapy is a common approach aimed to prevent the emergence of resistant clones. We have previously reported promising results of applying cisplatin plus mitomycin C combination for the NACT of BRCA1-driven carcinomas. This therapy resulted in a significant reduction of the tumor burden in all analyzed patients and in complete pathologic responses observed in 2/12 (17 %) treated women [10]. We reasoned that combining this regimen with an additional drug may further improve the outcomes of NACT. Previous studies suggested that BRCA1-driven tumors are particularly sensitive to anthracyclines, while their responsiveness to taxanes is under the question [10, 11]. Consequently, we decided to add doxorubicin to cisplatin plus mitomycin C as a third drug. Here we present the results of the trial involving this 3-drug combination.
Methods
The design of the study was discussed on the council involving medical oncologists, cancer gynecologists and hereditary cancer specialists. It was decided that the pilot trial would include 10 patients with initially inoperable BRCA1-driven HGSOC, and the main end-point will be the rate of pathologic complete responses. While all patients received the same neoadjuvant regimen (MAP: mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8), cisplatin 80 mg/m2 (day 1), given every 4 weeks), the physicians were permitted to administer the therapy of their choice after the surgery. This approach provided some flexibility given that the combination of carboplatin and paclitaxel (TCbP) is a standard option for the treatment of ovarian cancer [1] and that some post-NACT tumor samples have restored BRCA1 function and therefore may not be potentially responsive to platinum drugs [8, 9]. The recruitment of patients was performed from August 2017 to December 2018 based on the results of the PCR-based test for Slavic recurrent germ-line mutations [12, 13]. According to the study protocol, all tumor samples were subjected to the loss-of-heterozygosity (LOH) analysis before the NACT and after the surgery. LOH test was performed as described in [9]. All tumors were also analyzed for the TP53 somatic mutations, given that TP53 inactivation is a ubiquitous feature of BRCA1-driven carcinomas [14]. The study was approved by the local Ethics Committee. All patients included in the study provided informed consent.
Although this study was not randomized, we considered for the comparison of treatment outcomes 16 consecutive patients with germ-line BRCA1 mutations, who were referred to the N.N. Petrov Institute of Oncology (St.-Petersburg, Russia) between February 2017 and December 2019 and were subjected to a standard NACT combination of paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks. Most of these patients were negative for PCR-detectable recurrent BRCA1 mutations; however, they were found to carry a germ-line pathogenic allele upon the analysis of the entire BRCA1 and BRCA2 coding sequence, i.e., after the start of NACT [13].
All women receiving MAP or TCbP were managed by the same surgical team. Tumor responses were evaluated according to RECIST criteria using computed tomography and magnetic resonance imaging. None of the patients treated by MAP or TCbP received bevacizumab. Three patients in the MAP arm but none in the TCbP group were subjected to the hyperthermic intraperitoneal chemotherapy (HIPEC) during surgery. None of the included patients described in this report received maintenance by PARP inhibitors after completion of the first-line therapy, as this indication was not approved in Russia at the time of the study.
The statistical analysis was performed using SPSS 13.0 software package. Age distribution and the duration of the follow-up were compared by the Mann-Whitney U-test. Median treatment-free interval (TFI) was evaluated using Kaplan-Meyer curves. Other comparisons were performed with the Fisher’s exact test.
Results
Five patients included in the study of the neoadjuvant combination of mitomycin C, cisplatin and doxorubicin had stage IIIC HGSOC and another 5 women were diagnosed with stage IV disease (Table 1). Partial response to this therapy was observed in all 10 cases considered. Seven women had toxicities of grades 1 or 2; 2 patients had toxicity grade 3 and 1 subject experienced grade IV thrombocytopenia. None of the HGSOCs showed a complete pathological response in the ovaries, and only one woman demonstrated chemotherapy response score (CRS) 3, according to Böhm et al. [15]. Omental tumor response, which is more predictive for the disease outcome than adnexal CRS [15], showed considerably better values: 4 women had no residual tumor cells in the omentum, 1 patient had CRS 3 and 5 cases demonstrated CRS 2. There were no instances of poor responsiveness to the therapy categorized as CRS 1. The median TFI and progression-free survival (PFS) were not reached.
Table 1 BRCA1-mutated HGSOC patients receiving neoadjuvant therapy consisting of mitomycin C, doxorubicin and cisplatin
ID Age Stage BRCA1 mutation Somatic BRCA1 status before NACT MAP cycles
(NACT) Surgical debulking Response by RECIST CRS (ovary / omentum) Somatic BRCA1 status after NACT ACT
(cycles) TFI, months PFS, months OS, months Toxicities and grades TP53 mutation
MAP1 64 IIIC c.5266dupC LOH 3 Complete + HIPEC PR (-44 %) CRS 3 / no tumor cells in omentum LOH MAP (2) 25.4+ 32.2+ 32.2+ Anemia 1; nephrotoxicity 1 C135W
MAP2 35 IVA (pleuritis) c.68_69delAG LOH 3 Complete + HIPEC PR (-73 %) CRS 2 / no tumor cells in omentum na MAP (1), AT (2) 6.9 14.4 26.9 Anemia 1 R175H
MAP3 50 IIIC c.4034delA LOH 4 Complete PR (-35 %) CRS 2 / CRS 2 LOH MAP (3) 23.6+ 31.6+ 31.6+ Anemia 1 Y234H
MAP4 57 IVB (lymph nodes) c.5266dupC LOH 3 Complete + HIPEC PR (-90 %) CRS 2 / CRS 3 Retention of the wild-type allele AT (3) 29.2+ 36.6+ 36.6+ Diarrhea 1; emesis 1; gastritis 1; nausea 1; leukopenia 2; nephrotoxicity 2; thrombocytopenia 4 R213X
MAP5 50 IIIC c.5266dupC na 3 Complete PR (-33 %) CRS 2 / no tumor cells in omentum LOH MAP (3) 22.2+ 29.5+ 29.5+ Anemia 1; leukopenia 1; nausea 1; thrombocytopenia 1 c.97-1G > A
MAP6 45 IVB (spleen, lymph nodes) c.1961delA LOH 4 Suboptimal PR (-47 %) CRS 2 / CRS 2 LOH TCbP (6) 21+ 30.4+ 30.4+ Anemia 1; nausea 1; thrombosis R213X
MAP7 56 IVB (pleuritis, lymph nodes) c.5266dupC na 3 Complete PR (-47 %) CRS 2 / CRS 2 LOH MAP (1) 4.1 8.3 29.8+ Anemia 2; nephrotoxicity 3 c.314delG
MAP8 58 IIIC c.4034delA LOH 5 Complete PR (-46 %) CRS 2 / CRS 2 LOH TCbP (4) 11.5 20.7 28.2+ Anemia 1; neutropenia 2 R248Q
MAP9 46 IIIC c.4034delA LOH 4 Complete PR (-64 %) CRS 2 / CRS 2 LOH MAP (2) 3.1 11.2 15.8 Thrombocytopenia 2 I255S
MAP10 40 IVB (pleuritis, lymph nodes) c.5266dupC LOH 4 Complete PR (-36 %) na / no tumor cells in omentum na None 28.5+ 37.6+ 37.6+ Anemia 1; hepatotoxicity 2; thrombocytopenia 2; nephrotoxicity 3 M133R
ACT Adjuvant chemotherapy, AT Doxorubicin 60 mg/m2 and paclitaxel 175 mg/m2, every 3 weeks, CRS Chemotherapy response score, HIPEC hyperthermic intraperitoneal chemotherapy, LOH Loss of heterozygosity, na Not analyzed, MAP Mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8) and cisplatin 80 mg/m2 (day 1), given every 4 weeks, NACT Neoadjuvant chemotherapy, OS Overall survival, PFS Progression-free survival, PR Partial response, TCbP Paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks, TFI Treatment-free interval.
Notes: Tumor responses presented in the table describe the status of patients observed after the completion of the NACT, i.e. straight before the surgery. Patient MAP10 was diagnosed with ovarian cancer upon surgery, which was performed in another hospital and was limited to the excision of ovaries. She was considered eligible for the NACT study, as she had a significant tumor burden and could not be subjected to primary debulking surgery; she received no adjuvant therapy, as no residual tumor cells was seen in the surgical material. Patient MAP4 demonstrated the restoration of heterozygosity in a post-NACT tumor sample, suggesting that the tumor may no longer be platinum-sensitive [8, 9]; based on this finding, combination of paclitaxel and doxorubicin was given after the surgery; similarly, this combination was incorporated in the adjuvant treatment for patient MAP2, where the molecular analysis of post-NACT tumor tissue failed to establish somatic BRCA1 status. Patients MAP6 and MAP8 received TCbP combination after the surgery due to preference of their primary physicians.
Sixteen patients receiving paclitaxel plus carboplatin had slightly more favorable stage distribution, as 12 subjects had HGSOC of stage IIIC and 4 patients demonstrated stage IV disease (Table 2). While all patients treated by MAP showed partial response, 4/16 (25 %) women subjected to the TCbP combination produced only the disease stabilization and there was one HGSOC with the progression on this therapy. There was a remarkable difference from MAP regimen with regard to pathological responses, as minimal response score was observed in 6/16 (38 %) cases for ovarian tumor masses and 5/16 (31 %) HGSOCs for omental metastases (p = 0.05 and 0.12, respectively). While the median follow-up for the TCbP group was shorter than for MAP patients, median TFI was already achieved and reached 9.5 months (Fig. 1). Thirteen patients had sufficient follow-up to evaluate 1-year outcomes; the recurrence rate at 1 year after the completion of the treatment was 10/13 (77 %) for the TCbP, while the same value was 4/10 (40 %) for the MAP regimen (p = 0.1).
Table 2 Comparative characteristics of patients with BRCA1-mutated HGSOC receiving NACT combination of mitomycin C, doxorubicin and cisplatin versus women treated by paclitaxel plus carboplatin
Clinical characteristics MAP group (N = 10) TCbP group (N = 16) Statistical comparison
Median age of onset (range) 50 (35–64) 49 (37–72) Not significant
Pattern of BRCA1 mutations c.5266dupC (n = 5), c.4034delA (n = 3), c.68_69delAG (n = 1), c.1961delA (n = 1) c.5266dupC (n = 2), c.4034delA (n = 2), c.68_69delAG (n = 1), C61G (n = 1), c.1510delC (n = 1), Q563X (n = 1), c.2076dupT (n = 1), c.2983_2984delAA (n = 1), c.3247del5 (n = 1), c.3601_3602delGG (n = 1), c.3718_3719delCA (n = 1), Y1509X (n = 1), G1706E (n = 1), c.5152 + 1G > T (n = 1) p = 0.005 (founder vs. non-founder mutations; Fisher’s exact test)
FIGO stage
IIIC 5 (50 %) 12 (75 %) p = 0.23 (stage III vs. IV; Fisher’s exact test)
IVA 1 (10 %) 2 (13 %)
IVB 4 (40 %) 2 (13 %)
NACT cycles (range) 3–5 3–8
Cytoreduction
Optimal 9 (90 %) 14 (88 %) p = 1.0 (Fisher’s exact test)
Suboptimal 1 (10 %) 1 (6 %)
None 0 1 (6 %)
Response by RECIST
CR 0 0 p = 0.12 (objective response vs. lack of objective response; Fisher’s exact test)
PR 10 (100 %) 11 (69 %)
SD 0 4 (25 %)
PD 0 1 (6 %)
Chemotherapy response score (CRS) in the ovaries
CRS 1 0 6 (38 %) p = 0.05 (CRS 1 vs. other; Fisher’s exact test)
CRS 2 8 (80 %) 9 (56 %)
CRS 3 1 (10 %) 0
Tissue not available for evaluation 1 (10 %) 1 (10 %)
Chemotherapy response score (CRS) in the omentum
CRS 1 0 5 (31 %) p = 0.12 (CRS 1 vs. other; Fisher’s exact test)
CRS 2 5 (40 %) 8 (50 %)
CRS 3 1 (10 %) 0
No tumor cells 4 (40 %) 2 (13 %)
Tissue not available for evaluation 0 1 (6 %)
ACT cycles (range) 1–6 1–6
Median follow-up, months (range) 30.1 (15.8–36.6) 23.4 (10.7–45.2) p = 0.28 (Mann-Whitney Test)
Median treatment-free interval (95 % CI) Not reached 9.5 (7.8–11.2) p = 0.109 (Log Rank [Mantel-Cox])
Recurrence within one year after completion of treatment 4 (40 %) 10/13a (77 %) p = 0.1 (Fisher’s exact test)
ACT adjuvant chemotherapy, CRS chemotherapy response score, MAP mitomycin C 10 mg/m2 (day 1), doxorubicin 30 mg/m2 (days 1 and 8), cisplatin 80 mg/m2 (day 1), given every 4 weeks, NACT neoadjuvant chemotherapy, TCbP paclitaxel 175 mg/m2 plus carboplatin (6 AUC), given every 3 weeks
a13 out of 16 patients had sufficient follow-up for the estimation of 1-year recurrence rate
Fig. 1 Treatment-free interval for patients treated with the combination of mitomycin C, cisplatin and doxorubicin and for women treated by the paclitaxel plus carboplatin doublet
Discussion
Our previous study involving 12 BRCA1-mutated HGSOCs treated with the combination of cisplatin and mitomycin C revealed complete pathologic responses in 2 out of 12 patients [10]. We anticipated that the addition of doxorubicin to this combination may increase the rate of elimination of all tumor cells detectable in surgically excised tissues. The obtained data are sufficient to state that the applied triplet does not significantly increase the rate of complete pathologic responses as compared to the previously applied combination of two drugs.
At the same time, short-term results of MAP therapy look encouraging. In addition to a reasonably good rate of objective responses, half of the included cases demonstrated complete or nearly-complete absence of tumor cells in the omentum. Omental response score is the main predictor of the long-term outcomes of NACT, so it is a valuable marker allowing robust evaluation of various chemotherapy regimens [15].
Previous studies suggested that the TCbP regimen may be less efficient in BRCA1-mutated HGSOCs as compared to other NACT schemes [10]. These data sets compared prospective and retrospective patients treated by different surgeons. The quality of surgical debulking is critical for the outcome of HGSOC treatment, therefore these comparisons are prone to biases. Within the present study, we analyzed groups of patients who were managed at the same time interval by the same group of surgeons. However, the MAP and TCbP groups of patients were not balanced with regard to the pattern of mutations. The selection of patients for the MAP therapy involved rapid PCR tests for recurrent Slavic mutations [13]. The TCbP HGSOC group is significantly enriched by subjects with “rare” BRCA1 pathogenic alleles, which were detected by the next-generation sequencing analysis after the start of NACT. There are some data suggesting that distinct BRCA1 and BRCA2 mutations may exert distinct sensitivity to platinum compounds and PARP inhibitors [4]. Although we acknowledge differences in the pattern of BRCA1 mutations as a limitation of the study, it should be noted that all published trials on PARP inhibitors did not consider the type of mutation as a confounding factor [4, 16].
The landscape of the treatment BRCA1/2-mutated HGSOC is rapidly evolving. In particular, PARP inhibitors have been recently included in the standards for the first-line maintenance therapy, as they significantly delay the time to tumor recurrence [16]. None of the patients considered in this report received PARP inhibitors because they were not locally approved for early lines of HGSOC treatment at the time of the study. Consequently, it is unclear whether the differences observed between distinct NACT regimens will be maintained upon the incorporation of PARP-targeted drugs.
Conclusions
In summary, this study suggests that BRCA1-associated HGSOCs may require distinct therapeutic NACT regimens as compared to conventional TCbP doublet. If this is the case, the fast turn-around time for BRCA1/2 testing could become a critical factor for appropriate treatment decisions. Recent data indicate that BRCA1/2-associated HGSOCs do not show inferior outcomes when treated by NACT before the surgery, while primary surgical intervention is clearly the best approach in sporadic ovarian tumors [7, 17, 18]. These findings are likely to increase the acceptance of NACT for BRCA1/2 germline mutation carriers and, therefore, stimulate large neoadjuvant clinical trials for this category of HGSOC patients.
Abbreviations
CRSChemotherapy response score
HGSOCHigh-grade serous ovarian cancer
LOHLoss of heterozygosity
MAPMitomycin C, anthracycline, platinum
NACTNeoadjuvant chemotherapy
OCOvarian cancer
PFSProgression-free survival
TCbPTaxanes and carboplatin
TFITreatment-free interval
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Authors’ contributions
TG, KK, IB, KG, EN and OS managed study patients and analyzed clinical data; TS performed the molecular analysis; AI carried out morphological analysis; AS integrated and analyzed the obtained data and contributed to the manuscript preparation; EI designed the study and wrote the first draft of the manuscript. All authors have read and approved the final version of the manuscript.
Funding
This work has been supported by the Ministry of Science and Higher Education of the Russian Federation (grant № 075-15-2020-789).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was performed in full accordance with ethics guidelines. A written informed consent from study participants was obtained.
Consent for publication
All living patients provided the consent for publication.
Competing interests
The authors declare that they have no competing interests. | DAY 1 | DrugDosageText | CC BY | 33536037 | 18,976,042 | 2021-02-03 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'No adverse event'. | Cannabinoid treatment for autism: a proof-of-concept randomized trial.
Endocannabinoid dysfunction in animal models of autism spectrum disorder (ASD) and accumulating, albeit anecdotal, evidence for efficacy in humans motivated this placebo-controlled double-blind comparison of two oral cannabinoid solutions in 150 participants (age 5-21 years) with ASD.
We tested (1) BOL-DP-O-01-W, a whole-plant cannabis extract containing cannabidiol and Δ9-tetrahydrocannabinol at a 20:1 ratio and (2) BOL-DP-O-01, purified cannabidiol and Δ9-tetrahydrocannabinol at the same ratio. Participants (N = 150) received either placebo or cannabinoids for 12-weeks (testing efficacy) followed by a 4-week washout and predetermined cross-over for another 12 weeks to further assess tolerability. Registered primary efficacy outcome measures were improvement in behavioral problems (differences between whole-plant extract and placebo) on the Home Situation Questionnaire-ASD (HSQ-ASD) and the Clinical Global Impression-Improvement scale with disruptive behavior anchor points (CGI-I). Secondary measures were Social Responsiveness Scale (SRS-2) and Autism Parenting Stress Index (APSI).
Changes in Total Scores of HSQ-ASD (primary-outcome) and APSI (secondary-outcome) did not differ among groups. Disruptive behavior on the CGI-I (co-primary outcome) was either much or very much improved in 49% on whole-plant extract (n = 45) versus 21% on placebo (n = 47; p = 0.005). Median SRS Total Score (secondary-outcome) improved by 14.9 on whole-plant extract (n = 34) versus 3.6 points after placebo (n = 36); p = 0.009). There were no treatment-related serious adverse events. Common adverse events included somnolence and decreased appetite, reported for 28% and 25% on whole-plant extract, respectively (n = 95); 23% and 21% on pure-cannabinoids (n = 93), and 8% and 15% on placebo (n = 94). Limitations Lack of pharmacokinetic data and a wide range of ages and functional levels among participants warrant caution when interpreting the results.
This interventional study provides evidence that BOL-DP-O-01-W and BOL-DP-O-01, administrated for 3 months, are well tolerated. Evidence for efficacy of these interventions are mixed and insufficient. Further testing of cannabinoids in ASD is recommended. Trial registration ClinicalTrials.gov: NCT02956226. Registered 06 November 2016, https://clinicaltrials.gov/ct2/show/NCT02956226.
Background
There is no established pharmacological treatment for the core symptoms of autism spectrum disorder (ASD), persistent deficits in social communication, and repetitive, restrictive patterns of behavior [1]; the efficacy and tolerability of pharmacotherapies addressing comorbid disruptive behaviors are relatively low [2].
Consumption of cannabis is reported to enhance interpersonal communication [3] and decrease hostile feelings [4]. The main components of the cannabis plant (phytocannabinoids) are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). THC activates the type-1 cannabinoid receptor (CB1R) in the brain; it is psychoactive and can lead to anxiety and psychosis [5]. CBD, on the other hand, is an allosteric modulator of the CB1R and might decrease the effects of CB1R agonists such as THC. It is not psychoactive and has a relatively high toxicity threshold [5]. While THC consumption, especially at a young age, can lead to addiction, cognitive decline, motivational loss, and psychosis, co-consumption of CBD might reduce these risks [6].
CBD also appears to have anxiolytic, antipsychotic, antiepileptic, and neuroprotective properties that may be mediated through receptors such as serotonin 5-HT1A, glycine α3 and α1, TRPV1, GPR55, GABAA, and PPARγ, and by inhibiting adenosine reuptake [7–11]. A single oral administration of 600 mg CBD to 34 men (17 neurotypicals and 17 with ASD) increased prefrontal GABA activity in neurotypicals and decreased GABA activity in those with ASD [12].
Epidiolex is a cannabis-derived pure CBD compound which was approved by the U.S. FDA in 2018 for the treatment of two severe forms of epilepsy [13]. This may be relevant for patients with ASD, as 10–30% also have epilepsy, and several pathophysiological pathways are implicated in both disorders [11, 14].
The endocannabinoid system is a cell-signaling system composed of the cannabinoid receptors, their endogenous ligands (endocannabinoids, mainly anandamide and 2-AG), transporters, and enzymes which produce and degrade the endocannabinoids [15].
Studies in animal models suggest a reduced endocannabinoid tone in ASD [16–19]. Stimulation of the endocannabinoid system [16–19] and administration of CBD [17] have improved social deficits in some models. Additionally, children with ASD have been found to have lower peripheral endocannabinoid levels [20, 21].
These preclinical data and case-series, reporting treatment with artisanal CBD-rich, cannabis strains [22–26] have triggered widespread use of various cannabis strains in children with ASD, despite a lack of controlled studies. Furthermore, the cannabis plant contains a wide range of minor cannabinoids, terpenes, and flavonoids which differ by strain. These components have also been reported to impact human behaviour [27, 28]. Various combinations of these components have been proposed to have a synergistic pharmacological effect ('the entourage effect') [29]. Whether presumed effects of cannabis in ASD should be attributed to CBD or THC, or whether minor cannabinoids, terpenes, and flavonoids also contribute therapeutically remains unclear. Accordingly, we performed a proof-of-concept, placebo-controlled trial of whole-plant extract and pure cannabinoids in children and adolescents with ASD. We hypothesized that whole-plant extract, per the entourage effect, would be more effective than placebo for disruptive behaviors; assessing this hypothesis was our primary objective. A secondary objective was to assess the efficacy of pure cannabinoids which are more standardized and repeatable than whole-plant extracts and hence more suitable for pharmacotherapy.
Methods
Standard protocol approvals, registrations, and patient consents
NCT02956226 was approved by the Institutional Review Board at Shaare Zedek Medical Center and the Israeli Ministry of Health prior to participant enrollment. Participants’ parents provided written informed consent and written assent was obtained from participants when appropriate.
Study design
This proof-of-concept, randomized, double-blind, placebo-controlled trial was conducted in a single referral center—Shaare Zedek Medical Center, Jerusalem, Israel. Eligible participants were children and adolescents (5–21 years old) with an ASD diagnosis per DSM-5 criteria, confirmed by Autism Diagnostic Observation Schedule (ADOS-2), and moderate or greater behavioral problems (rating ≥ 4) on the Clinical Global Impression (CGI)-Severity scale (Table 1). Anchoring instructions (provided in the Additional file 1) were used so that the CGI-S would quantify behavioral difficulties rather than overall ASD severity.Table 1 Inclusion and exclusion criteria for study participation
Inclusion criteria 1. Male or female outpatients aged 5–21 years olda
2. Diagnosis of ASD according to Diagnostic and Statistical Manual of Mental Disorders [Fifth Edition; DSM-5]
3. Moderate or greater behavioral problems as measured by a Clinical Global Impression Scale—Severity (CGI-S) score of 4 or higher at screeningb
4. Involvement of a parent or caregiver able to consistently complete assessments throughout the study
Exclusion criteria 1. Lifetime history of psychotic disorder
2. Current or former treatment with cannabinoids
3. A medical condition (such as heart, liver, renal or hematological disorder) that impacts the subject's ability to participate in the study or makes the subject predisposed to severe adverse events
4. Subjects who have had changes in pharmacological, educational, or behavioral treatments for 4 weeks prior to randomization or planned changes in existing interventions for the duration of the trial
aIn Israel, special education programs for individuals with ASD and neuropediatric clinics continue to follow patients with ASD until they are 21 years old
bTo assign CGI-S scores, structured criteria were used to rate behavioral difficulties on the CGI-S, rather than overall ASD severity
Participants were randomly assigned (1:1:1 ratio) to 1 of 3 treatments for 12-weeks. Treatments were: (1) oral placebo, (2) whole-plant cannabis extract containing CBD and THC at a 20:1 ratio, and (3) pure CBD and pure THC at the same ratio and concentration. Randomization and blinding processes are described in the Additional file 1.
The primary objective was to evaluate whether whole-plant cannabis extract would induce a significant improvement in behavioral assessments compared to placebo. We used the same CBD: THC ratio as in previous open-label case series [22–24]. We did not use a ‘CBD only’ arm in this initial study, as we hypothesized that the CBD-THC combination would be more efficacious because of direct effects of THC on the endocannabinoid system.
For ethical reasons, we used a crossover design in which all participants would receive cannabinoids at least once: after 12-weeks of treatment (‘Period-1’) and a 4-week washout period, participants crossed-over to a predetermined second 12-week treatment (‘Period-2’; Fig. 1). The cross-over design was intended to allow within-participant analyses, comparing the two treatments that each participant received. As we had noted a substantial improvement in our open observational study with whole-plant extract [22], we ordered treatments a priori to minimize the likelihood of substantial improvement of severe disruptive behaviors in the first period and deterioration in the second period. As we hypothesized that whole-plant extract would be more effective than pure cannabinoids, we excluded the sequence of whole-plant extract followed by placebo.Fig. 1 Study design
Preliminary analyses revealed a treatment order effect: change from baseline was greater in the first period than in the second, suggesting a greater initial placebo effect. As a treatment order effect impairs the validity of within-participant analyses, we decided to evaluate between-group efficacy only during the first period. Data from both periods were examined for safety and tolerability. For transparency, we present within-participant analyses and between-participant analyses of period-2 (Additional file 1).
Intervention
Cannabis plants (Topaz strain; BOL Pharma, Israel) were subjected to CO2 extraction. The extract was either immediately dissolved in olive oil (BOL-DP-O-01-W) or underwent further purification to 99% pure CBD and then was dissolved in olive oil (BOL-DP-O-01). The final concentrations of CBD and THC in both solutions were 167 mg/ml CBD and 8.35 mg/ml THC. Flavorings were added to all three solutions to make taste and scent uniform.
In each treatment period, starting dose was 1 mg/kg/d CBD (and 0.05 mg/kg/d THC). The dose was increased by 1 mg/kg/d CBD (and 0.05 mg/kg/d THC) every other day up to 10 mg/kg body weight per day CBD (and 0.5 mg/kg/d THC) for children weighing 20–40 kg or 7.5 mg/kg/d CBD (and 0.375 mg/kg/d THC) for weight > 40 kg (to a maximum of 420 mg CBD and 21 mg THC per day) divided into 3 daily doses. Treatments were given orally (sublingual whenever possible) as an add-on to any ongoing stable medication. At the end of each treatment period, the study treatment was gradually decreased over 2 weeks.
Baseline evaluations
Baseline assessments included: ADOS-2 [30], a standardized assessment of communication, social interaction, play, and imaginary use of materials; Vineland Adaptive Behavior Scales (VABS) [31], a caregiver interview assessing Communication, Socialization, and Daily Living Skills; and Childhood Autism Rating Scale-Second edition (CARS2-ST) [32], a quantitative measure of direct behavior observation.
Outcomes
Primary outcomes: We designated two co-primary outcome measures to assess ASD associated disruptive behaviors: Home Situations Questionnaire-ASD (HSQ-ASD) and CGI-Improvement (CGI-I) targeting behavioral problems.
HSQ-ASD [33] is a 24-item parent-rated measure of noncompliant behavior in children with ASD. The scale yields per-item mean scores of 0 to 9 (higher is worse) [33].
CGI-I [34] was used to measure improvement in disruptive behaviors from baseline by incorporating anchoring instructions related to behavioral difficulties (Anchors appear in the Additional file 1). As in the standard CGI-I, scores ranged from 1 (very much improved) through 4 (unchanged) to 7 (very much worse). Scores of 1 or 2 (much improved) were defined as a positive response; all others indicated a negative response [34]. CGI-I was assessed at the end of each treatment period. The same clinician (AA) assessed and rated the CGI-S and CGI-I of all participants.
Secondary outcomes included the Social Responsiveness Scale-2nd edition (SRS-2), the Autism Parenting Stress Index (APSI), and adverse events.
SRS-2: [35] this 65-item, caregiver questionnaire quantifies autism symptom severity (total scores range from 0 to 195; higher is worse).
APSI: [36] this 13-item parent-rated measure assesses parenting stress in three categories: core social disability, difficult-to-manage behavior, and physical issues.
Adverse events were assessed using a modified Liverpool Adverse Events Profile (LAEP) including the 19 original LAEP [37] items plus 15 items covering all significant adverse effects of CBD and THC reported in prior pediatric studies.
Statistical analyses
The primary aim of this study was to test the superiority of whole-plant-extract over placebo in treating ASD associated behavioral problems, using the HSQ-ASD and the CGI-I for disruptive behaviors. The comparison between pure-cannabinoids and placebo was registered as a secondary outcome. Sample size calculation was based on an effect size of f = 0.67 (in total HSQ-ASD score) [38] and standard deviation of 3 points in the within-participant difference between placebo and whole-plant extract conditions. To achieve 80% power with 2.5% alpha (adjusted for two co-primary endpoints) requires a sample of 43 patients per group. To account for attrition, an additional 15% were enrolled. A total of 50 participants per arm was set to test primary study endpoints. Analyses were performed using JMP version 14 (SAS Institute, Cary, NC, USA). All P values were two-sided. Specific statistical tests used and corrections applied for multiple comparisons are indicated in figure/table legends.
For details on the cannabinoid preparations, randomization process, important changes to methods after trial commencement, anchoring instructions for rating the CGI-S and CGI-I, and the CONSORT checklist, see Additional file 2.
Results
Between 11 January 2017 and 12 April 2018, 150 children and adolescents (mean age 11.8 ± 4.1 years, median 11.25, range 5.1–20.8; 80% boys) entered the trial. ASD symptoms were ‘severe’ in 78.7% per ADOS-2 (Comparison Score = 8–10) [30] and adaptive level was ‘low’ (Standard Score ≤ 70) in 88% per Vineland Behavior Scales [31].
Screening, randomization and attrition are shown in Fig. 2 and participant characteristics are provided in Table 2. Fifty participants were randomly assigned to each of the 3 treatments in Period-1 and 44 per group completed the study (12% overall attrition).Fig. 2 Trial profile: screening, randomization and treatment periods
Table 2 Participant characteristics
All Placebo in 1st period; whole- plant in the 2nd Pure cannabinoids in 1st period; placebo in the 2nd Whole-plant in 1st period; pure cannabinoids in 2nd P-valuea
Age: mean ± SD
[median, range]
11.8 ± 4.1
[11.3, 5.1–20.8]
11.7 ± 3.8
[10.7, 5.8–20]
11.6 ± 4.3
[10.3, 5.1–20.4]
12.1 ± 4.3
[12.6, 5.1–20.8]
0.79
Sex (% girls) 20% 16% 16% 28% 0.22
ADOS-2 Total Score
mean ± SD [median, range]
21.8 ± 6.0
[23, 7–32]
22.1 ± 6.5
[23.5, 7–32]
22.5 ± 5.8
[24, 11–32]
20.9 ± 5.8
[21, 9–30]
0.41
VABS Standard Score
mean ± SD [median, range]
52.3 ± 14.5
[51, 20–102]
52.0 ± 15.0
[49, 26–102]
52.4 ± 15.2
[54, 25–89]
52.3 ± 13.6
[52, 20–78]
0.27
CARS Total Score
mean ± SD [median, range]
45.4 ± 8.4
[47.5, 29.5–59]
46.0 ± 8.5
[47.5, 30.5–59]
45.5 ± 8.9
[48.5, 29.5–57.5]
44.6 ± 7.8
[46.5, 31–56.5]
0.55
CGI-S maladaptive behavior
mean ± SD [median, range]
5.6 ± 0.7
[6, 4–7]
5.5 ± 0.7
[6, 4–7]
5.6 ± 0.7
[6, 4–7]
5.6 ± 0.7
[6, 4–7]
0.78
HSQ Total Score (baseline)
mean ± SD [median, range]
3.5 ± 1.7
[3.3, 0.3–8.5]
3.7 ± 1.5
[3.7, 0.7–6.0]
3.2 ± 1.5
[3.1, 0.7–6.6]
3.7 ± 2.1
[3.6, 0.3–8.5]
0.33
SRS-2 Total Score (baseline)
mean ± SD [median, range]
119 ± 27
[121, 53–180]
122 ± 23
[124, 53–159]
118 ± 31
[118, 64–178]
117 ± 27
[117, 66–180]
0.37
APSI Total Score (baseline)
mean ± SD [median, range]
27.1 ± 10.4
[26, 7–54]
28.3 ± 10.3
[27, 11–50]
25.8 ± 10.4
[25, 8–54]
27.4 ± 10.7
[25, 7–48]
0.67
BMI (baseline)
mean ± SD [median, range]
20.8 ± 5.7
[19.0, 12.3–39.6]
20.5 ± 5.2
[19.1, 12.8–34]
20.5 ± 6.0
[19.1, 12.3–39.6]
21.3 ± 6.1
[19.0, 13.9–39.6]
0.67
Epilepsy 9% 8% 8% 10% 0.92
Concomitant medications
Any medication 72% 72% 68% 76% 0.67
Antipsychotics 54% 58% 44% 60% 0.22
SSRIs 15% 12% 16% 16% 0.80
Antiepileptics (also given as mood stabilizers) 12% 12% 12% 12% 1.0
Stimulants 12% 8% 22% 6% 0.033
Benzodiazepines 7% 2% 8% 10% 0.19
Alpha-2 agonists 4% 4% 2% 6% 0.58
ADOS-2 Autism Diagnostic Observation Schedule-2nd edition, (Modules 1, 2 and 3 were used for 55%, 17%, and 28% of the participants, respectively, without significant differences among the 3 study arms); VABS Vineland Adaptive Behavior Scales; CARS Childhood Autism Rating Scale; CGI-S Clinical Global Impression–Severity [5 = markedly ill, 6 = severely ill, 7 = among the most extremely ill patients; all referencing disruptive behaviors]; HSQ Home Situations Questionnaire; SRS-2 Social Responsiveness Scale-2nd edition; APSI Autism Parenting Stress Index; SSRIs Selective serotonin reuptake inhibitors
aCategorical parameters (sex, epilepsy and medications) were compared using likelihood ratio chi-square tests. Continuous parameters were compared using the Kruskal–Wallis test if data distribution was non-normal but similar across groups (BMI) and using median tests if data distribution was non-normal and different across groups (age, assessment scores)
Safety and tolerability of cannabinoid treatment with BOL-DP-O-01-W (whole-plant extract) and BOL-DP-O-01 (pure cannabinoids)
Adverse events (AEs) were reported whenever they occurred, and caregivers were proactively asked about them at each study visit, and every 4 weeks using a structured questionnaire. AEs were documented whether considered related to study treatments or not. Reports of new adverse events or worsening of previously reported events were rated mild (present, but not problematic), moderate (problematic and leading to study drug dose decrease), or severe (posing a problem requiring medical intervention). Serious AEs were possibly life-threatening events or any requiring hospitalization. Overall, 95 participants received a whole-plant extract, 93 received pure cannabinoids, and 94 received a placebo.
There were no treatment-related severe or serious AEs. Six participants had an unrelated serious event (Additional file 1: Table S1). Overall, mild AEs were not significantly more frequent during cannabinoid treatment (mild AEs were reported 383, 388, and 353 times, in 89, 79, and 78 participants during treatment with whole-plant extract, pure cannabinoids, and placebo, respectively). Moderate AEs were reported 80, 78, and 57 times, in 44, 45, and 26 participants during treatment with whole-plant extract, pure cannabinoids, and placebo, respectively. AEs that were more common during cannabinoid treatment are presented in Table 3. The full list of adverse events and correlations with age, sex, treatment dose, and concomitant medications appears in Additional file 1: Table S2.Table 3 Common adverse events reported during either 12-week treatment period
Whole-plant extract
CBD 5.5 mg/kg/d; n = 95 (%) Pure cannabinoids
CBD 5.5 mg/kg/d; n = 93 (%) Placebo
n = 94 (%) P value (placebo vs cannabinoids)
Somnolence 27 24 7.5 < 0.001
Mild 20 18.5 7.5
Moderate 7 5.5 0
Severe 0 0 0
Decreased appetite 24 22 15 0.157
Mild 21 16.5 13
Moderate 3 5.5 2
Severe 0 0 0
Weight loss 12 13 4 0.053
Mild 9 12 3
Moderate 3 1 1
Severe 0 0 0
Tiredness 25 34 19 0.077
Mild 21 28.5 18
Moderate 4 5.5 1
Severe 0 0 0
Euphoria 20 19 13 0.201
Mild 15 16 12
Moderate 5 3 1
Severe 0 0 0
Anxiety 20 27 14 0.084
Mild 17 25 11
Moderate 3 2 3
CBD: cannabidiol (CBD:THC ratio was 20:1 for both cannabinoids tested; the average daily dose per kg was lower than the target dose as many participants weighted over 42 kg and reached the maximal daily dose)
Bold: sum of mild + moderate + severe for each adverse event
Impact of cannabinoid treatment with BOL-DP-O-01-W (whole-plant extract) and BOL-DP-O-01 (pure cannabinoids) on behavior
The impact of cannabinoid treatment on behavioral problems was assessed using the HSQ-ASD [33], and the CGI-I [34] (co-primary outcome measures). The APSI [36] (secondary outcome measure) also reflects the child’s behavior. HSQ-ASD total scores and APSI total scores did not differ significantly between participants who received cannabinoids and participants who received placebo (Table 4). On the CGI-I, 49% of 45 participants who received whole-plant cannabinoids responded (either much or very much improved) [34] compared with 21% of 47 on placebo (p = 0.005, Fig. 3). Of the 45 participants who received pure cannabinoids, 38% responded, which was not significantly higher than placebo (p = 0.08).Table 4 Impact of cannabinoid treatment, as reflected by change from baseline to end of treatment period 1 in total scores of HSQ-ASD, SRS-2, and APSI
Median (range) [n] Pairwise P
Assessment Whole-plant extract Pure cannabinoids Placebo Whole-plant versus placebo Pure C. versus placebo Whole-plant versus pure C
HSQ-ASD − 1.1 (− 3.8 to 1.6) [40] − 0.7 (− 4.4 to 3.8) [42] − 0.5 (− 3.7 to 2.5) [39] 0.575 0.915 0.508
SRS-2 − 14.9 (− 45 to 15) [34] − 8.2 (− 69 to 45) [28] − 3.6 (− 63 to 35) [36] 0.009 0.801 0.202
APSI − 5.4 (− 39 to 13) [38] − 4.9 (− 19 to 22) [42] − 1.5 (− 26 to 20) [42] 0.502 0.513 0.991
HSQ Home Situations Questionnaire–ASD; SRS-2 Social Responsiveness Scale-2nd edition; APSI Autism Parenting Stress Index
Median tests were used, as distributions were non-normal
P-values are based on Mood’s Median Test of each pairwise comparison
Fig. 3 Participants (%) whose behavioral problems either much improved or very much improved on the CGI-I scale following treatment. Response to 12-week treatment using the Clinical Global Impression-Improvement (CGI-I). Positive response in this scale is defined as a rating of ‘much improved' or 'very much improved' [34]. Outcome was analyzed using Likelihood ratio chi-square test. P value is unadjusted. *Remains significant after Bonferroni-correction for multiple comparisons
None of these 3 measures (HSQ-ASD, CGI-I and APSI) differed significantly between participants who received whole-plant extract versus pure cannabinoids (Table 4).
Second treatment period results are presented in Additional file 1: Table S3 and Additional file 1: Figure S2 for transparency but not further discussed because of a significant order effect.
Impact of BOL-DP-O-01-W (whole-plant extract) and BOL-DP-O-01 (pure cannabinoids) on Social Responsiveness Scale scores
ASD symptoms (secondary outcome) were assessed with the SRS-2 [35]. Improvement in SRS-2 total score was significantly higher following treatment with whole-plant extract compared with placebo (Table 4). Median total score improved by 3.6 points after placebo (n = 36) versus 14.9 on whole-plant extract (n = 34; p = 0.009) and 8.2 on pure cannabinoids (n = 28; p = 0.80). Results of the second treatment period are presented in Additional file 1: Table S3 and Additional file 1: Figure S3 for transparency.
Exploratory analyses: impact of BOL-DP-O-01-W (whole-plant extract) and BOL-DP-O-01 (pure cannabinoids) treatment on Body Mass Index (BMI)
Baseline BMIs were equivalent across treatment groups (Table 2). The BMI of participants who received cannabinoids decreased during active treatment [Median {25%, 75%}] by − 0.45 {− 1.15, 0.18} in Period-1 (n = 44) and − 0.12 {− 0.77, 0.18} in Period-2 (n = 40)] following treatment with whole-plant extract; BMI decreased by − 0.36 {− 1.09, 0.24} in Period-1 (n = 44) and − 0.01 {− 0.61, 0.48} in Period-2 (n = 43) following treatment with pure cannabinoids. Changes in BMI following cannabinoid treatment (either whole-plant extract or pure cannabinoids) were − 0.36 {− 1.14, 0.2} in Period-1 (n = 88) and − 0.01 {− 0.7, 0.38} in Period-2 (n = 83). During treatment with placebo, changes in BMI were 0.16 {− 0.25, 0.56} in Period-1 (n = 43; p < 0.0001 versus cannabinoids) and 0.30, {0, 0.96} in Period-2 (n = 43; p = 0.002 versus cannabinoids).
Notably, participants with higher BMI at baseline had a more prominent decrease in BMI following cannabinoid treatment [The decrease in BMI was positively correlated with baseline BMI (F = 4.3, p = 0.042 in Period-1, F = 8.6, p = 0.005 in Period-2)]. Change in BMI following placebo was not significantly correlated with baseline BMI (Fig. 4).Fig. 4 Impact of cannabinoid treatment on BMI. Change in BMI during 12-week treatment with either cannabinoids or placebo. a Whole-plant extract versus pure cannabinoids; b Cannabinoid treatment (either whole-plant extract or pure cannabinoids) versus placebo; c Distribution of data, bars represent 10%, 25%, Median, 75% and 90%, differences between placebo and cannabinoids were analyzed using median test; d, e Change in BMI as function of baseline level, stratified by treatment (cannabinoids or placebo) and treatment period (d first period, e second period). Correlations were analyzed using linear regression
Exploratory analyses: possible moderators of treatment effects
Additional file 1: Table S4 presents possible moderators of treatment response. Severity of ASD core symptoms at baseline (as assessed by ADOS-2) and concomitant use of medications were not significantly associated with response to either pure cannabinoids or whole-plant extract, on any assessment.
Males were more likely to improve on the HSQ-ASD and SRS-2. Younger children were more likely to improve on the CGI-I and APSI. Participants who had somnolence during cannabinoid treatment were more likely to respond per the CGI-I assessment. However, treatment with the whole-plant extract remained significantly associated with improvement on the CGI-I and SRS-2 after controlling for somnolence and for concomitant use of medications during treatment [Odds Ratio {95% confidence interval} of 6.08 {1.91, 21.82} (p = 0.003) and 3.56 {1.31, 10.28} (p = 0.015), respectively].
Correlations between treatment dose (per Kg of body weight) and treatment response are presented in Additional file 1: Table S5. The average treatment dose during the first period was 5.7 ± 2.6 mg/kg/d of CBD in the whole-plant extract arm and 5.9 ± 2.7 mg/kg/d of CBD in the pure cannabinoids arm. A higher dose of whole-plant extract correlated with higher behavioral improvement on the CGI-I (rs = − 0.29, n = 45, p = 0.050). Cannabinoid dose did not correlate significantly with any other endpoints for either whole-plant extract or pure cannabinoids.
Concomitant medications
Study treatments were added to ongoing behavioral or pharmacological treatments. Planned changes in such treatments or a change in the 4 weeks prior to randomization were exclusionary.
Concomitant medications were taken by 72% of participants (Table 2). Adverse events or response were not significantly associated with concomitant medication use (Additional file 1: Table S2 and S3), except for somnolence which was higher in those on chronic medications (p = 0.001).
Discussion
Currently, there are no established medications for the core autistic symptoms. Risperidone and aripiprazole have been approved by the U.S. Food and Drug Administration (FDA) to treat comorbid irritability [2] but these medications often cause obesity and metabolic syndrome [2, 39].
In this study, we have demonstrated for the first time in a placebo-controlled trial that cannabinoid treatment has the potential to decrease disruptive behaviors associated with ASD, with acceptable tolerability. This is specifically important for the many individuals with ASD who are overweight, as cannabinoid treatment was associated with net weight-loss (Fig. 4) in contrast to the substantial weight gain usually produced by antipsychotics.
Two co-primary outcomes were designated to assess improvement in disruptive behaviors following cannabinoid treatment: a parent questionnaire (HSQ-ASD) and an interview-based clinician assessment (CGI-I).
HSQ-ASD scores did not differ significantly between participants who received cannabinoids and participants who received placebo. However, as our cohort included children and adolescents with a wide range of function levels, many participants had 4 or more items which were not applicable on the HSQ-ASD, limiting sample size on this scale (Table 4).
The clinician assessment was based on a detailed description of the most bothersome behavioral problems at baseline and an extensive interview at the end of each treatment period focused on those problems. Using this patient- and family-centered tool customized for each participant, we found that 49% of participants receiving the whole-plant extract treatment responded versus 21% on placebo (p = 0.005).
Intriguingly, one of our secondary outcomes, the SRS-2, provided preliminary evidence that cannabinoid treatment might improve core symptoms of ASD (Table 4). This finding could be of high importance if confirmed in future studies, as studies exploring pharmacological interventions for the ASD core symptoms are scarce.
Although not reportable as evidence of efficacy due to crossover effects, Additional file 1: Figures S2 and S3 show that results in the second treatment period were similar to those in the first.
Other possible implications of this preliminary study for future studies and selected clinical use include feasibility of sublingual administration in children with low adaptive level, and feasibility of a starting dose of 1 mg/kg/d of CBD and a gradual increase over 2–3 weeks to a target of 5–10 mg/kg/d divided into 2–3 daily doses.
The study explored two cannabinoid compounds, differing by the absence of terpenes, flavonoids, and minor cannabinoids in the pure-cannabinoid compound. While additive and even synergistic therapeutic effects of these additional components have been suggested ('entourage' effect) [28, 29], we did not find clear advantages for the whole-plant extract over pure cannabinoids, suggesting that attempts to search for the optimal 'entourage' effect across cannabis strains with the same CBD:THC ratio are likely to be challenging. As previously reported in studies of children with refractory epilepsy [40, 41], we also found relatively high placebo effects, emphasizing the importance of placebo in studies of medical cannabis.
Similar to these studies we also found somnolence to be the most prevalent adverse event but importantly, cannabinoid treatment remained significantly associated with a positive response on the CGI-I and SRS-2 assessments after controlling for somnolence during treatment [Odds ratio of 6.08, p = 0.003].
Cannabinoids might affect behavior and communication through several mechanisms. THC activates CB1R and has been associated with enhanced social behavior in multiple studies [42, 43]. CBD is a 5-HT1A receptor agonist, which might facilitate anxiolytic effects. Its presumed antipsychotic effect is attributed to partial agonism at dopamine D2 receptors, similar to the antipsychotic action of aripiprazole [44].
Limitations
Our study had several limitations. Although it was designed as a cross-over study, preliminary analyses revealed a treatment order effect which prevented the use of data from the second treatment period and limited sample size. As this was the first clinical study in the ASD field, we included a wide range of levels of function. Unfortunately, the standardized questionnaires contained many items that were inapplicable for some low-functioning participants, resulting in numerous invalid scores and decreased statistical power on those measures. We did not perform genetic or intelligence quotient evaluations and could not assess the effects of genetic background or cognitive level on treatment response. We did collect data on concomitant medications but were not powered to detect effects on treatment response or on adverse events. We did not obtain data on pharmacokinetics of the interventions and concomitant medications nor tests of liver enzymes and complete blood count, although we detected no clinical evidence of hepatic or hematologic dysfunction.
Conclusions
Novel pharmacological treatments for the core and comorbid symptoms of ASD are urgently needed. Preclinical studies implicate the endocannabinoid system in the pathophysiology of ASD. In a controlled study of 150 participants, we found that BOL-DP-O-01-W, a whole-plant extract which contains CBD and THC in a 20:1 ratio, improved disruptive behaviors on one of two primary outcome measures and on a secondary outcome, an index of ASD core symptoms, with acceptable adverse events. These data suggest that cannabinoids should be further investigated in ASD.
Future studies should consider recruiting participants within narrower ranges of age and functional levels, assess the long-term tolerability and safety of cannabinoid treatments, and identify target populations within the autism spectrum that might benefit most from these treatments.
Supplementary Information
Additional file 1. Data Supplement.
Additional file 2. CONSORT checklist.
Abbreviations
APSIAutism Parenting Stress Index
ASDAutism spectrum disorder
CB1RType-1 cannabinoid receptor
CBDCannabidiol
CGI-S/CGI-IClinical Global Impression–Severity/Improvement
HSQHome Situations Questionnaire
SRSSocial Responsiveness Scale
THCTetrahydrocannabinol
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13229-021-00420-2.
Acknowledgements
The statistical analysis was conducted by: Elliot Sprecher, PhD, Technion Faculty of Medicine, Haifa, Israel and Yishai Friedlander, M.P.H, Public Heath, Ben-Gurion University, Israel. Anchoring instructions for rating the CGI-S and CGI-I were developed in consultation with Dr. Elizabeth Berry-Kravis.
Authors’ contributions
Dr. Aran conceptualized and designed the study, recruited participants, carried out the initial analyses, drafted the initial manuscript, and reviewed and revised the manuscript. Ms. Harel collected data, carried out the initial analyses, and reviewed and revised the manuscript. Drs. Cassuto, Schnapp, Watted, Shmueli and Golan recruited participants, collected data, and reviewed and revised the manuscript. Ms. Polyansky designed the data collection instruments, collected data, carried out the initial analyses, and reviewed and revised the manuscrip. Dr. Castellanos interpreted data, drafted the initial manuscript, and critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. All authors read and approved the final manuscript.
Funding
The study was funded by BOL Pharma, Revadim, Israel and the National Institute for Psychobiology in Israel (#203-17-18). The funding bodies were not involved in any way in the study design, collection, analysis and interpretation of data or in the writing of the manuscript.
Availability of data and materials
The authors declare that the data supporting study findings are available within the paper and its Additional file. The remaining data are available from the corresponding author upon reasonable request.
Ethics approval and consent to participate
All research procedures were approved by the Shaare Zedek Medical Center Review Board and Israeli Ministry of Health prior to participant enrollment. Participants’ parents provided written consent prior to initiation of any experimental procedures, and written assent was obtained from participants when appropriate.
Consent for publication
Not applicable.
Competing interests
Adi Aran and F. Xavier Castellanos report receiving personal fees and stock options for advisory roles at BOL Pharma. The remaining authors have no conflicts of interest to disclose. | ARIPIPRAZOLE | DrugsGivenReaction | CC BY | 33536055 | 19,917,757 | 2021-02-03 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Angle closure glaucoma'. | Pseudophakic Angle Closure Due to Vitreous Block Following Ureteroscopic Lithotripsy.
Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye following ureteroscopic lithotripsy under general anesthesia. The patient had a history of coconut hit into his left eye which resulted in traumatic anterior lens subluxation, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. Prior scleral fixation procedure, anterior vitrectomy was not performed. Clinical examination showed mushroom-shaped vitreous in the anterior chamber with absolute pupillary block, which had resulted in acute angle closure. Thus, topical and oral antiglaucoma medications were administered to achieve normal intraocular pressure in the left eye, followed by laser peripheral iridotomy in that eye. The anterior chamber depth was successfully increased. Limited anterior vitrectomy by a pars plana approach was performed to prevent recurrent angle closure. The patient's vision improved and his intraocular pressure remained controlled without any antiglaucoma medication.
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. Cautious intraoperative anterior vitrectomy and surgical iridectomy are warranted. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Introduction
Acute angle closure is less common in patients with pseudophakia, compared with patients with phakia; this type of closure can have various causes.1 The underlying mechanism can involve force originating at the level of the iris; examples include absolute pupillary block due to iris–intraocular lens contact (eg, during anterior chamber intraocular lens implantation), iris–air contact, iris–silicone oil contact, iris–residual posterior capsule contact, iris–postoperative membrane contact, and iris–vitreous contact. In addition to pupillary block, angle closure in patients with pseudophakia can also result from factors such as Soemmering ring,2 ciliochoroidal detachment, and malignant glaucoma.3 Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
Case Report
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye. The patient had a history of traumatic anterior lens subluxation four years prior, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. A few hours prior to onset of his symptoms, he had undergone ureteroscopic lithotripsy under general anesthesia. During the operation, the patient was placed in a lithotomy position. Eight hundred milliliters of isotonic saline were used during the operation. Both intravenous anesthetic agents and inhaled anesthetic agents were used during the procedure (ie, propofol, atropine, nitrous oxide, and sevoflurane).
Upon presentation in our clinic, the patient’s best-corrected visual acuity was 20/70 in the right eye and 20/200 in the left eye. His IOP was 12 mmHg in the right eye and 57 mmHg in the left eye. Slit-lamp biomicroscopy examination showed ciliary injection and generalized bedewing of the cornea in the left eye. Grade 1-shallow anterior chamber and protrusion of the anterior hyaloid membrane were noted (Figure 1). The vitreous was pushed forward by pressure that originated from behind the iris, resulting in vitreous–corneal apposition. The scleral-fixated intraocular lens was pushed backward into an abnormally posterior position. Slit-lamp biomicroscopy findings in the right eye were unremarkable. The anterior chamber in the right eye was deep with a posterior chamber intraocular lens; one year prior, the patient had undergone uneventful phacoemulsification in the right eye. Cup to disc ratios were 0.3 in both eyes. Gonioscopy revealed 360-degree angle closure in the left eye. The patient then underwent ultrasound biomicroscopy. The findings demonstrated asymmetrical pupillary block in the left eye, with an obviously shallow anterior chamber. The scleral-fixated intraocular lens was pushed away from the iris plane (Figure 2). However, vitreous mass could not be determined by ultrasound biomicroscopy because of poor resolution. Pentacam imaging revealed a vitreous mass in the anterior chamber, which appeared as a faint curvilinear line around the mid-iris zone.Figure 1 Anterior segment photograph demonstrated vitreous protrusion into anterior chamber.
Figure 2 Ultrasound biomicroscopy (UBM) showed shallow anterior chamber with abnormal position of scleral-fixated intraocular lens.
The patient was treated with two topical antiglaucoma medications and oral acetazolamide for reduction of IOP. Following normalization of IOP and clearing of the cornea, laser iridotomy was performed in the patient’s left eye. The anterior chamber underwent a dramatic change to the normal deep configuration following treatment with laser peripheral iridotomy. The vitreous–corneal apposition no longer persisted and the iris returned to a flat configuration. Subsequent anterior segment optical coherence tomography showed a clear relationship among the vitreous, cornea, and iris (Figure 3). Four weeks after the acute angle closure event, the patient underwent anterior vitrectomy using a pars plana approach in the left eye; this removed the vitreous and was expected to prevent recurrent vitreous block-induced angle closure. The patient’s vision returned to normal (best-corrected visual acuity of 20/20) in the left eye. The IOP remained at approximately 10–15 mmHg without any antiglaucoma medication. Gonioscopy revealed a normal open angle without any residual peripheral anterior synechia.Figure 3 Anterior segment optical coherence tomography showed mushroom-shaped vitreous after laser peripheral iridotomy.
Discussion
Our patient presented with acute angle closure in the left eye, a few hours after a ureteroscopic procedure. The patient had a notable risk factor: vitreous prolapse due to prior trauma with inadequate vitrectomy. Certain actions such as the Valsalva maneuver are presumed to increase IOP and may facilitate vitreous movement from the posterior segment to the anterior segment in patients with a ruptured anterior hyaloid membrane.4 Both intubation and extubation can trigger the gag reflex and severe coughing, which increase intrathoracic pressure and IOP.5 Some types of anesthetic agents and depths of anesthesia may also interfere with pupillary size,6 thereby facilitating iris–vitreous blockage. Furthermore, in some stages of anesthesia, a mydriatic pupil may allow the vitreous to move from the posterior segment to the pupillary plane, which results in vitreous block during return to the normal pupillary size condition. The lithotomy position might not have affected the occurrence of angle closure in our patient because his head and body were at the same level. However, the lithotomy position can increase venous return, thereby increasing cardiac output. The clinical manifestation in this patient was acute angle closure due to an abrupt increase in IOP. Slit-lamp biomicroscopy revealed absolute pupillary block. Ancillary assessment by ultrasound biomicroscopy confirmed vitreous-induced pupillary block. Initial management comprised reduction of pressure to limit optic nerve damage, using topical and oral aqueous suppressants. Subsequently, laser peripheral iridotomy was performed to eliminate angle closure at the pupillary plane. In our patient, angle closure mainly occurred at the pupillary plane, but was not due to the mass effect of the vitreous behind the iris. Therefore, this event was completely resolved by laser peripheral iridotomy. However, definite treatment with anterior vitrectomy should be performed when possible because vitreous behind the iris can cause recurrent blockage if it occludes the laser peripheral iridotomy and pupil simultaneously.
Conclusion
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The Institutional Review Board, Faculty of Medicine, Ramathibodi hospital granted approval to publish the case details.
Author Contributions
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work. | ATROPINE, NITROUS OXIDE, PROPOFOL, SEVOFLURANE, SODIUM CHLORIDE | DrugsGivenReaction | CC BY-NC | 33536795 | 19,031,650 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Vitreous disorder'. | Pseudophakic Angle Closure Due to Vitreous Block Following Ureteroscopic Lithotripsy.
Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye following ureteroscopic lithotripsy under general anesthesia. The patient had a history of coconut hit into his left eye which resulted in traumatic anterior lens subluxation, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. Prior scleral fixation procedure, anterior vitrectomy was not performed. Clinical examination showed mushroom-shaped vitreous in the anterior chamber with absolute pupillary block, which had resulted in acute angle closure. Thus, topical and oral antiglaucoma medications were administered to achieve normal intraocular pressure in the left eye, followed by laser peripheral iridotomy in that eye. The anterior chamber depth was successfully increased. Limited anterior vitrectomy by a pars plana approach was performed to prevent recurrent angle closure. The patient's vision improved and his intraocular pressure remained controlled without any antiglaucoma medication.
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. Cautious intraoperative anterior vitrectomy and surgical iridectomy are warranted. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Introduction
Acute angle closure is less common in patients with pseudophakia, compared with patients with phakia; this type of closure can have various causes.1 The underlying mechanism can involve force originating at the level of the iris; examples include absolute pupillary block due to iris–intraocular lens contact (eg, during anterior chamber intraocular lens implantation), iris–air contact, iris–silicone oil contact, iris–residual posterior capsule contact, iris–postoperative membrane contact, and iris–vitreous contact. In addition to pupillary block, angle closure in patients with pseudophakia can also result from factors such as Soemmering ring,2 ciliochoroidal detachment, and malignant glaucoma.3 Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
Case Report
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye. The patient had a history of traumatic anterior lens subluxation four years prior, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. A few hours prior to onset of his symptoms, he had undergone ureteroscopic lithotripsy under general anesthesia. During the operation, the patient was placed in a lithotomy position. Eight hundred milliliters of isotonic saline were used during the operation. Both intravenous anesthetic agents and inhaled anesthetic agents were used during the procedure (ie, propofol, atropine, nitrous oxide, and sevoflurane).
Upon presentation in our clinic, the patient’s best-corrected visual acuity was 20/70 in the right eye and 20/200 in the left eye. His IOP was 12 mmHg in the right eye and 57 mmHg in the left eye. Slit-lamp biomicroscopy examination showed ciliary injection and generalized bedewing of the cornea in the left eye. Grade 1-shallow anterior chamber and protrusion of the anterior hyaloid membrane were noted (Figure 1). The vitreous was pushed forward by pressure that originated from behind the iris, resulting in vitreous–corneal apposition. The scleral-fixated intraocular lens was pushed backward into an abnormally posterior position. Slit-lamp biomicroscopy findings in the right eye were unremarkable. The anterior chamber in the right eye was deep with a posterior chamber intraocular lens; one year prior, the patient had undergone uneventful phacoemulsification in the right eye. Cup to disc ratios were 0.3 in both eyes. Gonioscopy revealed 360-degree angle closure in the left eye. The patient then underwent ultrasound biomicroscopy. The findings demonstrated asymmetrical pupillary block in the left eye, with an obviously shallow anterior chamber. The scleral-fixated intraocular lens was pushed away from the iris plane (Figure 2). However, vitreous mass could not be determined by ultrasound biomicroscopy because of poor resolution. Pentacam imaging revealed a vitreous mass in the anterior chamber, which appeared as a faint curvilinear line around the mid-iris zone.Figure 1 Anterior segment photograph demonstrated vitreous protrusion into anterior chamber.
Figure 2 Ultrasound biomicroscopy (UBM) showed shallow anterior chamber with abnormal position of scleral-fixated intraocular lens.
The patient was treated with two topical antiglaucoma medications and oral acetazolamide for reduction of IOP. Following normalization of IOP and clearing of the cornea, laser iridotomy was performed in the patient’s left eye. The anterior chamber underwent a dramatic change to the normal deep configuration following treatment with laser peripheral iridotomy. The vitreous–corneal apposition no longer persisted and the iris returned to a flat configuration. Subsequent anterior segment optical coherence tomography showed a clear relationship among the vitreous, cornea, and iris (Figure 3). Four weeks after the acute angle closure event, the patient underwent anterior vitrectomy using a pars plana approach in the left eye; this removed the vitreous and was expected to prevent recurrent vitreous block-induced angle closure. The patient’s vision returned to normal (best-corrected visual acuity of 20/20) in the left eye. The IOP remained at approximately 10–15 mmHg without any antiglaucoma medication. Gonioscopy revealed a normal open angle without any residual peripheral anterior synechia.Figure 3 Anterior segment optical coherence tomography showed mushroom-shaped vitreous after laser peripheral iridotomy.
Discussion
Our patient presented with acute angle closure in the left eye, a few hours after a ureteroscopic procedure. The patient had a notable risk factor: vitreous prolapse due to prior trauma with inadequate vitrectomy. Certain actions such as the Valsalva maneuver are presumed to increase IOP and may facilitate vitreous movement from the posterior segment to the anterior segment in patients with a ruptured anterior hyaloid membrane.4 Both intubation and extubation can trigger the gag reflex and severe coughing, which increase intrathoracic pressure and IOP.5 Some types of anesthetic agents and depths of anesthesia may also interfere with pupillary size,6 thereby facilitating iris–vitreous blockage. Furthermore, in some stages of anesthesia, a mydriatic pupil may allow the vitreous to move from the posterior segment to the pupillary plane, which results in vitreous block during return to the normal pupillary size condition. The lithotomy position might not have affected the occurrence of angle closure in our patient because his head and body were at the same level. However, the lithotomy position can increase venous return, thereby increasing cardiac output. The clinical manifestation in this patient was acute angle closure due to an abrupt increase in IOP. Slit-lamp biomicroscopy revealed absolute pupillary block. Ancillary assessment by ultrasound biomicroscopy confirmed vitreous-induced pupillary block. Initial management comprised reduction of pressure to limit optic nerve damage, using topical and oral aqueous suppressants. Subsequently, laser peripheral iridotomy was performed to eliminate angle closure at the pupillary plane. In our patient, angle closure mainly occurred at the pupillary plane, but was not due to the mass effect of the vitreous behind the iris. Therefore, this event was completely resolved by laser peripheral iridotomy. However, definite treatment with anterior vitrectomy should be performed when possible because vitreous behind the iris can cause recurrent blockage if it occludes the laser peripheral iridotomy and pupil simultaneously.
Conclusion
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The Institutional Review Board, Faculty of Medicine, Ramathibodi hospital granted approval to publish the case details.
Author Contributions
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work. | ATROPINE, NITROUS OXIDE, PROPOFOL, SEVOFLURANE, SODIUM CHLORIDE | DrugsGivenReaction | CC BY-NC | 33536795 | 19,031,650 | 2021 |
What was the dosage of drug 'ATROPINE'? | Pseudophakic Angle Closure Due to Vitreous Block Following Ureteroscopic Lithotripsy.
Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye following ureteroscopic lithotripsy under general anesthesia. The patient had a history of coconut hit into his left eye which resulted in traumatic anterior lens subluxation, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. Prior scleral fixation procedure, anterior vitrectomy was not performed. Clinical examination showed mushroom-shaped vitreous in the anterior chamber with absolute pupillary block, which had resulted in acute angle closure. Thus, topical and oral antiglaucoma medications were administered to achieve normal intraocular pressure in the left eye, followed by laser peripheral iridotomy in that eye. The anterior chamber depth was successfully increased. Limited anterior vitrectomy by a pars plana approach was performed to prevent recurrent angle closure. The patient's vision improved and his intraocular pressure remained controlled without any antiglaucoma medication.
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. Cautious intraoperative anterior vitrectomy and surgical iridectomy are warranted. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Introduction
Acute angle closure is less common in patients with pseudophakia, compared with patients with phakia; this type of closure can have various causes.1 The underlying mechanism can involve force originating at the level of the iris; examples include absolute pupillary block due to iris–intraocular lens contact (eg, during anterior chamber intraocular lens implantation), iris–air contact, iris–silicone oil contact, iris–residual posterior capsule contact, iris–postoperative membrane contact, and iris–vitreous contact. In addition to pupillary block, angle closure in patients with pseudophakia can also result from factors such as Soemmering ring,2 ciliochoroidal detachment, and malignant glaucoma.3 Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
Case Report
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye. The patient had a history of traumatic anterior lens subluxation four years prior, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. A few hours prior to onset of his symptoms, he had undergone ureteroscopic lithotripsy under general anesthesia. During the operation, the patient was placed in a lithotomy position. Eight hundred milliliters of isotonic saline were used during the operation. Both intravenous anesthetic agents and inhaled anesthetic agents were used during the procedure (ie, propofol, atropine, nitrous oxide, and sevoflurane).
Upon presentation in our clinic, the patient’s best-corrected visual acuity was 20/70 in the right eye and 20/200 in the left eye. His IOP was 12 mmHg in the right eye and 57 mmHg in the left eye. Slit-lamp biomicroscopy examination showed ciliary injection and generalized bedewing of the cornea in the left eye. Grade 1-shallow anterior chamber and protrusion of the anterior hyaloid membrane were noted (Figure 1). The vitreous was pushed forward by pressure that originated from behind the iris, resulting in vitreous–corneal apposition. The scleral-fixated intraocular lens was pushed backward into an abnormally posterior position. Slit-lamp biomicroscopy findings in the right eye were unremarkable. The anterior chamber in the right eye was deep with a posterior chamber intraocular lens; one year prior, the patient had undergone uneventful phacoemulsification in the right eye. Cup to disc ratios were 0.3 in both eyes. Gonioscopy revealed 360-degree angle closure in the left eye. The patient then underwent ultrasound biomicroscopy. The findings demonstrated asymmetrical pupillary block in the left eye, with an obviously shallow anterior chamber. The scleral-fixated intraocular lens was pushed away from the iris plane (Figure 2). However, vitreous mass could not be determined by ultrasound biomicroscopy because of poor resolution. Pentacam imaging revealed a vitreous mass in the anterior chamber, which appeared as a faint curvilinear line around the mid-iris zone.Figure 1 Anterior segment photograph demonstrated vitreous protrusion into anterior chamber.
Figure 2 Ultrasound biomicroscopy (UBM) showed shallow anterior chamber with abnormal position of scleral-fixated intraocular lens.
The patient was treated with two topical antiglaucoma medications and oral acetazolamide for reduction of IOP. Following normalization of IOP and clearing of the cornea, laser iridotomy was performed in the patient’s left eye. The anterior chamber underwent a dramatic change to the normal deep configuration following treatment with laser peripheral iridotomy. The vitreous–corneal apposition no longer persisted and the iris returned to a flat configuration. Subsequent anterior segment optical coherence tomography showed a clear relationship among the vitreous, cornea, and iris (Figure 3). Four weeks after the acute angle closure event, the patient underwent anterior vitrectomy using a pars plana approach in the left eye; this removed the vitreous and was expected to prevent recurrent vitreous block-induced angle closure. The patient’s vision returned to normal (best-corrected visual acuity of 20/20) in the left eye. The IOP remained at approximately 10–15 mmHg without any antiglaucoma medication. Gonioscopy revealed a normal open angle without any residual peripheral anterior synechia.Figure 3 Anterior segment optical coherence tomography showed mushroom-shaped vitreous after laser peripheral iridotomy.
Discussion
Our patient presented with acute angle closure in the left eye, a few hours after a ureteroscopic procedure. The patient had a notable risk factor: vitreous prolapse due to prior trauma with inadequate vitrectomy. Certain actions such as the Valsalva maneuver are presumed to increase IOP and may facilitate vitreous movement from the posterior segment to the anterior segment in patients with a ruptured anterior hyaloid membrane.4 Both intubation and extubation can trigger the gag reflex and severe coughing, which increase intrathoracic pressure and IOP.5 Some types of anesthetic agents and depths of anesthesia may also interfere with pupillary size,6 thereby facilitating iris–vitreous blockage. Furthermore, in some stages of anesthesia, a mydriatic pupil may allow the vitreous to move from the posterior segment to the pupillary plane, which results in vitreous block during return to the normal pupillary size condition. The lithotomy position might not have affected the occurrence of angle closure in our patient because his head and body were at the same level. However, the lithotomy position can increase venous return, thereby increasing cardiac output. The clinical manifestation in this patient was acute angle closure due to an abrupt increase in IOP. Slit-lamp biomicroscopy revealed absolute pupillary block. Ancillary assessment by ultrasound biomicroscopy confirmed vitreous-induced pupillary block. Initial management comprised reduction of pressure to limit optic nerve damage, using topical and oral aqueous suppressants. Subsequently, laser peripheral iridotomy was performed to eliminate angle closure at the pupillary plane. In our patient, angle closure mainly occurred at the pupillary plane, but was not due to the mass effect of the vitreous behind the iris. Therefore, this event was completely resolved by laser peripheral iridotomy. However, definite treatment with anterior vitrectomy should be performed when possible because vitreous behind the iris can cause recurrent blockage if it occludes the laser peripheral iridotomy and pupil simultaneously.
Conclusion
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The Institutional Review Board, Faculty of Medicine, Ramathibodi hospital granted approval to publish the case details.
Author Contributions
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work. | NOT STATED | DrugDosageText | CC BY-NC | 33536795 | 19,031,650 | 2021 |
What was the dosage of drug 'NITROUS OXIDE'? | Pseudophakic Angle Closure Due to Vitreous Block Following Ureteroscopic Lithotripsy.
Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye following ureteroscopic lithotripsy under general anesthesia. The patient had a history of coconut hit into his left eye which resulted in traumatic anterior lens subluxation, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. Prior scleral fixation procedure, anterior vitrectomy was not performed. Clinical examination showed mushroom-shaped vitreous in the anterior chamber with absolute pupillary block, which had resulted in acute angle closure. Thus, topical and oral antiglaucoma medications were administered to achieve normal intraocular pressure in the left eye, followed by laser peripheral iridotomy in that eye. The anterior chamber depth was successfully increased. Limited anterior vitrectomy by a pars plana approach was performed to prevent recurrent angle closure. The patient's vision improved and his intraocular pressure remained controlled without any antiglaucoma medication.
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. Cautious intraoperative anterior vitrectomy and surgical iridectomy are warranted. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Introduction
Acute angle closure is less common in patients with pseudophakia, compared with patients with phakia; this type of closure can have various causes.1 The underlying mechanism can involve force originating at the level of the iris; examples include absolute pupillary block due to iris–intraocular lens contact (eg, during anterior chamber intraocular lens implantation), iris–air contact, iris–silicone oil contact, iris–residual posterior capsule contact, iris–postoperative membrane contact, and iris–vitreous contact. In addition to pupillary block, angle closure in patients with pseudophakia can also result from factors such as Soemmering ring,2 ciliochoroidal detachment, and malignant glaucoma.3 Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
Case Report
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye. The patient had a history of traumatic anterior lens subluxation four years prior, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. A few hours prior to onset of his symptoms, he had undergone ureteroscopic lithotripsy under general anesthesia. During the operation, the patient was placed in a lithotomy position. Eight hundred milliliters of isotonic saline were used during the operation. Both intravenous anesthetic agents and inhaled anesthetic agents were used during the procedure (ie, propofol, atropine, nitrous oxide, and sevoflurane).
Upon presentation in our clinic, the patient’s best-corrected visual acuity was 20/70 in the right eye and 20/200 in the left eye. His IOP was 12 mmHg in the right eye and 57 mmHg in the left eye. Slit-lamp biomicroscopy examination showed ciliary injection and generalized bedewing of the cornea in the left eye. Grade 1-shallow anterior chamber and protrusion of the anterior hyaloid membrane were noted (Figure 1). The vitreous was pushed forward by pressure that originated from behind the iris, resulting in vitreous–corneal apposition. The scleral-fixated intraocular lens was pushed backward into an abnormally posterior position. Slit-lamp biomicroscopy findings in the right eye were unremarkable. The anterior chamber in the right eye was deep with a posterior chamber intraocular lens; one year prior, the patient had undergone uneventful phacoemulsification in the right eye. Cup to disc ratios were 0.3 in both eyes. Gonioscopy revealed 360-degree angle closure in the left eye. The patient then underwent ultrasound biomicroscopy. The findings demonstrated asymmetrical pupillary block in the left eye, with an obviously shallow anterior chamber. The scleral-fixated intraocular lens was pushed away from the iris plane (Figure 2). However, vitreous mass could not be determined by ultrasound biomicroscopy because of poor resolution. Pentacam imaging revealed a vitreous mass in the anterior chamber, which appeared as a faint curvilinear line around the mid-iris zone.Figure 1 Anterior segment photograph demonstrated vitreous protrusion into anterior chamber.
Figure 2 Ultrasound biomicroscopy (UBM) showed shallow anterior chamber with abnormal position of scleral-fixated intraocular lens.
The patient was treated with two topical antiglaucoma medications and oral acetazolamide for reduction of IOP. Following normalization of IOP and clearing of the cornea, laser iridotomy was performed in the patient’s left eye. The anterior chamber underwent a dramatic change to the normal deep configuration following treatment with laser peripheral iridotomy. The vitreous–corneal apposition no longer persisted and the iris returned to a flat configuration. Subsequent anterior segment optical coherence tomography showed a clear relationship among the vitreous, cornea, and iris (Figure 3). Four weeks after the acute angle closure event, the patient underwent anterior vitrectomy using a pars plana approach in the left eye; this removed the vitreous and was expected to prevent recurrent vitreous block-induced angle closure. The patient’s vision returned to normal (best-corrected visual acuity of 20/20) in the left eye. The IOP remained at approximately 10–15 mmHg without any antiglaucoma medication. Gonioscopy revealed a normal open angle without any residual peripheral anterior synechia.Figure 3 Anterior segment optical coherence tomography showed mushroom-shaped vitreous after laser peripheral iridotomy.
Discussion
Our patient presented with acute angle closure in the left eye, a few hours after a ureteroscopic procedure. The patient had a notable risk factor: vitreous prolapse due to prior trauma with inadequate vitrectomy. Certain actions such as the Valsalva maneuver are presumed to increase IOP and may facilitate vitreous movement from the posterior segment to the anterior segment in patients with a ruptured anterior hyaloid membrane.4 Both intubation and extubation can trigger the gag reflex and severe coughing, which increase intrathoracic pressure and IOP.5 Some types of anesthetic agents and depths of anesthesia may also interfere with pupillary size,6 thereby facilitating iris–vitreous blockage. Furthermore, in some stages of anesthesia, a mydriatic pupil may allow the vitreous to move from the posterior segment to the pupillary plane, which results in vitreous block during return to the normal pupillary size condition. The lithotomy position might not have affected the occurrence of angle closure in our patient because his head and body were at the same level. However, the lithotomy position can increase venous return, thereby increasing cardiac output. The clinical manifestation in this patient was acute angle closure due to an abrupt increase in IOP. Slit-lamp biomicroscopy revealed absolute pupillary block. Ancillary assessment by ultrasound biomicroscopy confirmed vitreous-induced pupillary block. Initial management comprised reduction of pressure to limit optic nerve damage, using topical and oral aqueous suppressants. Subsequently, laser peripheral iridotomy was performed to eliminate angle closure at the pupillary plane. In our patient, angle closure mainly occurred at the pupillary plane, but was not due to the mass effect of the vitreous behind the iris. Therefore, this event was completely resolved by laser peripheral iridotomy. However, definite treatment with anterior vitrectomy should be performed when possible because vitreous behind the iris can cause recurrent blockage if it occludes the laser peripheral iridotomy and pupil simultaneously.
Conclusion
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The Institutional Review Board, Faculty of Medicine, Ramathibodi hospital granted approval to publish the case details.
Author Contributions
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work. | NOT STATED | DrugDosageText | CC BY-NC | 33536795 | 19,031,650 | 2021 |
What was the dosage of drug 'PROPOFOL'? | Pseudophakic Angle Closure Due to Vitreous Block Following Ureteroscopic Lithotripsy.
Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye following ureteroscopic lithotripsy under general anesthesia. The patient had a history of coconut hit into his left eye which resulted in traumatic anterior lens subluxation, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. Prior scleral fixation procedure, anterior vitrectomy was not performed. Clinical examination showed mushroom-shaped vitreous in the anterior chamber with absolute pupillary block, which had resulted in acute angle closure. Thus, topical and oral antiglaucoma medications were administered to achieve normal intraocular pressure in the left eye, followed by laser peripheral iridotomy in that eye. The anterior chamber depth was successfully increased. Limited anterior vitrectomy by a pars plana approach was performed to prevent recurrent angle closure. The patient's vision improved and his intraocular pressure remained controlled without any antiglaucoma medication.
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. Cautious intraoperative anterior vitrectomy and surgical iridectomy are warranted. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Introduction
Acute angle closure is less common in patients with pseudophakia, compared with patients with phakia; this type of closure can have various causes.1 The underlying mechanism can involve force originating at the level of the iris; examples include absolute pupillary block due to iris–intraocular lens contact (eg, during anterior chamber intraocular lens implantation), iris–air contact, iris–silicone oil contact, iris–residual posterior capsule contact, iris–postoperative membrane contact, and iris–vitreous contact. In addition to pupillary block, angle closure in patients with pseudophakia can also result from factors such as Soemmering ring,2 ciliochoroidal detachment, and malignant glaucoma.3 Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
Case Report
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye. The patient had a history of traumatic anterior lens subluxation four years prior, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. A few hours prior to onset of his symptoms, he had undergone ureteroscopic lithotripsy under general anesthesia. During the operation, the patient was placed in a lithotomy position. Eight hundred milliliters of isotonic saline were used during the operation. Both intravenous anesthetic agents and inhaled anesthetic agents were used during the procedure (ie, propofol, atropine, nitrous oxide, and sevoflurane).
Upon presentation in our clinic, the patient’s best-corrected visual acuity was 20/70 in the right eye and 20/200 in the left eye. His IOP was 12 mmHg in the right eye and 57 mmHg in the left eye. Slit-lamp biomicroscopy examination showed ciliary injection and generalized bedewing of the cornea in the left eye. Grade 1-shallow anterior chamber and protrusion of the anterior hyaloid membrane were noted (Figure 1). The vitreous was pushed forward by pressure that originated from behind the iris, resulting in vitreous–corneal apposition. The scleral-fixated intraocular lens was pushed backward into an abnormally posterior position. Slit-lamp biomicroscopy findings in the right eye were unremarkable. The anterior chamber in the right eye was deep with a posterior chamber intraocular lens; one year prior, the patient had undergone uneventful phacoemulsification in the right eye. Cup to disc ratios were 0.3 in both eyes. Gonioscopy revealed 360-degree angle closure in the left eye. The patient then underwent ultrasound biomicroscopy. The findings demonstrated asymmetrical pupillary block in the left eye, with an obviously shallow anterior chamber. The scleral-fixated intraocular lens was pushed away from the iris plane (Figure 2). However, vitreous mass could not be determined by ultrasound biomicroscopy because of poor resolution. Pentacam imaging revealed a vitreous mass in the anterior chamber, which appeared as a faint curvilinear line around the mid-iris zone.Figure 1 Anterior segment photograph demonstrated vitreous protrusion into anterior chamber.
Figure 2 Ultrasound biomicroscopy (UBM) showed shallow anterior chamber with abnormal position of scleral-fixated intraocular lens.
The patient was treated with two topical antiglaucoma medications and oral acetazolamide for reduction of IOP. Following normalization of IOP and clearing of the cornea, laser iridotomy was performed in the patient’s left eye. The anterior chamber underwent a dramatic change to the normal deep configuration following treatment with laser peripheral iridotomy. The vitreous–corneal apposition no longer persisted and the iris returned to a flat configuration. Subsequent anterior segment optical coherence tomography showed a clear relationship among the vitreous, cornea, and iris (Figure 3). Four weeks after the acute angle closure event, the patient underwent anterior vitrectomy using a pars plana approach in the left eye; this removed the vitreous and was expected to prevent recurrent vitreous block-induced angle closure. The patient’s vision returned to normal (best-corrected visual acuity of 20/20) in the left eye. The IOP remained at approximately 10–15 mmHg without any antiglaucoma medication. Gonioscopy revealed a normal open angle without any residual peripheral anterior synechia.Figure 3 Anterior segment optical coherence tomography showed mushroom-shaped vitreous after laser peripheral iridotomy.
Discussion
Our patient presented with acute angle closure in the left eye, a few hours after a ureteroscopic procedure. The patient had a notable risk factor: vitreous prolapse due to prior trauma with inadequate vitrectomy. Certain actions such as the Valsalva maneuver are presumed to increase IOP and may facilitate vitreous movement from the posterior segment to the anterior segment in patients with a ruptured anterior hyaloid membrane.4 Both intubation and extubation can trigger the gag reflex and severe coughing, which increase intrathoracic pressure and IOP.5 Some types of anesthetic agents and depths of anesthesia may also interfere with pupillary size,6 thereby facilitating iris–vitreous blockage. Furthermore, in some stages of anesthesia, a mydriatic pupil may allow the vitreous to move from the posterior segment to the pupillary plane, which results in vitreous block during return to the normal pupillary size condition. The lithotomy position might not have affected the occurrence of angle closure in our patient because his head and body were at the same level. However, the lithotomy position can increase venous return, thereby increasing cardiac output. The clinical manifestation in this patient was acute angle closure due to an abrupt increase in IOP. Slit-lamp biomicroscopy revealed absolute pupillary block. Ancillary assessment by ultrasound biomicroscopy confirmed vitreous-induced pupillary block. Initial management comprised reduction of pressure to limit optic nerve damage, using topical and oral aqueous suppressants. Subsequently, laser peripheral iridotomy was performed to eliminate angle closure at the pupillary plane. In our patient, angle closure mainly occurred at the pupillary plane, but was not due to the mass effect of the vitreous behind the iris. Therefore, this event was completely resolved by laser peripheral iridotomy. However, definite treatment with anterior vitrectomy should be performed when possible because vitreous behind the iris can cause recurrent blockage if it occludes the laser peripheral iridotomy and pupil simultaneously.
Conclusion
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The Institutional Review Board, Faculty of Medicine, Ramathibodi hospital granted approval to publish the case details.
Author Contributions
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work. | NOT STATED | DrugDosageText | CC BY-NC | 33536795 | 19,031,650 | 2021 |
What was the dosage of drug 'SEVOFLURANE'? | Pseudophakic Angle Closure Due to Vitreous Block Following Ureteroscopic Lithotripsy.
Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye following ureteroscopic lithotripsy under general anesthesia. The patient had a history of coconut hit into his left eye which resulted in traumatic anterior lens subluxation, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. Prior scleral fixation procedure, anterior vitrectomy was not performed. Clinical examination showed mushroom-shaped vitreous in the anterior chamber with absolute pupillary block, which had resulted in acute angle closure. Thus, topical and oral antiglaucoma medications were administered to achieve normal intraocular pressure in the left eye, followed by laser peripheral iridotomy in that eye. The anterior chamber depth was successfully increased. Limited anterior vitrectomy by a pars plana approach was performed to prevent recurrent angle closure. The patient's vision improved and his intraocular pressure remained controlled without any antiglaucoma medication.
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. Cautious intraoperative anterior vitrectomy and surgical iridectomy are warranted. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Introduction
Acute angle closure is less common in patients with pseudophakia, compared with patients with phakia; this type of closure can have various causes.1 The underlying mechanism can involve force originating at the level of the iris; examples include absolute pupillary block due to iris–intraocular lens contact (eg, during anterior chamber intraocular lens implantation), iris–air contact, iris–silicone oil contact, iris–residual posterior capsule contact, iris–postoperative membrane contact, and iris–vitreous contact. In addition to pupillary block, angle closure in patients with pseudophakia can also result from factors such as Soemmering ring,2 ciliochoroidal detachment, and malignant glaucoma.3 Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
Case Report
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye. The patient had a history of traumatic anterior lens subluxation four years prior, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. A few hours prior to onset of his symptoms, he had undergone ureteroscopic lithotripsy under general anesthesia. During the operation, the patient was placed in a lithotomy position. Eight hundred milliliters of isotonic saline were used during the operation. Both intravenous anesthetic agents and inhaled anesthetic agents were used during the procedure (ie, propofol, atropine, nitrous oxide, and sevoflurane).
Upon presentation in our clinic, the patient’s best-corrected visual acuity was 20/70 in the right eye and 20/200 in the left eye. His IOP was 12 mmHg in the right eye and 57 mmHg in the left eye. Slit-lamp biomicroscopy examination showed ciliary injection and generalized bedewing of the cornea in the left eye. Grade 1-shallow anterior chamber and protrusion of the anterior hyaloid membrane were noted (Figure 1). The vitreous was pushed forward by pressure that originated from behind the iris, resulting in vitreous–corneal apposition. The scleral-fixated intraocular lens was pushed backward into an abnormally posterior position. Slit-lamp biomicroscopy findings in the right eye were unremarkable. The anterior chamber in the right eye was deep with a posterior chamber intraocular lens; one year prior, the patient had undergone uneventful phacoemulsification in the right eye. Cup to disc ratios were 0.3 in both eyes. Gonioscopy revealed 360-degree angle closure in the left eye. The patient then underwent ultrasound biomicroscopy. The findings demonstrated asymmetrical pupillary block in the left eye, with an obviously shallow anterior chamber. The scleral-fixated intraocular lens was pushed away from the iris plane (Figure 2). However, vitreous mass could not be determined by ultrasound biomicroscopy because of poor resolution. Pentacam imaging revealed a vitreous mass in the anterior chamber, which appeared as a faint curvilinear line around the mid-iris zone.Figure 1 Anterior segment photograph demonstrated vitreous protrusion into anterior chamber.
Figure 2 Ultrasound biomicroscopy (UBM) showed shallow anterior chamber with abnormal position of scleral-fixated intraocular lens.
The patient was treated with two topical antiglaucoma medications and oral acetazolamide for reduction of IOP. Following normalization of IOP and clearing of the cornea, laser iridotomy was performed in the patient’s left eye. The anterior chamber underwent a dramatic change to the normal deep configuration following treatment with laser peripheral iridotomy. The vitreous–corneal apposition no longer persisted and the iris returned to a flat configuration. Subsequent anterior segment optical coherence tomography showed a clear relationship among the vitreous, cornea, and iris (Figure 3). Four weeks after the acute angle closure event, the patient underwent anterior vitrectomy using a pars plana approach in the left eye; this removed the vitreous and was expected to prevent recurrent vitreous block-induced angle closure. The patient’s vision returned to normal (best-corrected visual acuity of 20/20) in the left eye. The IOP remained at approximately 10–15 mmHg without any antiglaucoma medication. Gonioscopy revealed a normal open angle without any residual peripheral anterior synechia.Figure 3 Anterior segment optical coherence tomography showed mushroom-shaped vitreous after laser peripheral iridotomy.
Discussion
Our patient presented with acute angle closure in the left eye, a few hours after a ureteroscopic procedure. The patient had a notable risk factor: vitreous prolapse due to prior trauma with inadequate vitrectomy. Certain actions such as the Valsalva maneuver are presumed to increase IOP and may facilitate vitreous movement from the posterior segment to the anterior segment in patients with a ruptured anterior hyaloid membrane.4 Both intubation and extubation can trigger the gag reflex and severe coughing, which increase intrathoracic pressure and IOP.5 Some types of anesthetic agents and depths of anesthesia may also interfere with pupillary size,6 thereby facilitating iris–vitreous blockage. Furthermore, in some stages of anesthesia, a mydriatic pupil may allow the vitreous to move from the posterior segment to the pupillary plane, which results in vitreous block during return to the normal pupillary size condition. The lithotomy position might not have affected the occurrence of angle closure in our patient because his head and body were at the same level. However, the lithotomy position can increase venous return, thereby increasing cardiac output. The clinical manifestation in this patient was acute angle closure due to an abrupt increase in IOP. Slit-lamp biomicroscopy revealed absolute pupillary block. Ancillary assessment by ultrasound biomicroscopy confirmed vitreous-induced pupillary block. Initial management comprised reduction of pressure to limit optic nerve damage, using topical and oral aqueous suppressants. Subsequently, laser peripheral iridotomy was performed to eliminate angle closure at the pupillary plane. In our patient, angle closure mainly occurred at the pupillary plane, but was not due to the mass effect of the vitreous behind the iris. Therefore, this event was completely resolved by laser peripheral iridotomy. However, definite treatment with anterior vitrectomy should be performed when possible because vitreous behind the iris can cause recurrent blockage if it occludes the laser peripheral iridotomy and pupil simultaneously.
Conclusion
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The Institutional Review Board, Faculty of Medicine, Ramathibodi hospital granted approval to publish the case details.
Author Contributions
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work. | NOT STATED | DrugDosageText | CC BY-NC | 33536795 | 19,031,650 | 2021 |
What was the dosage of drug 'SODIUM CHLORIDE'? | Pseudophakic Angle Closure Due to Vitreous Block Following Ureteroscopic Lithotripsy.
Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye following ureteroscopic lithotripsy under general anesthesia. The patient had a history of coconut hit into his left eye which resulted in traumatic anterior lens subluxation, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. Prior scleral fixation procedure, anterior vitrectomy was not performed. Clinical examination showed mushroom-shaped vitreous in the anterior chamber with absolute pupillary block, which had resulted in acute angle closure. Thus, topical and oral antiglaucoma medications were administered to achieve normal intraocular pressure in the left eye, followed by laser peripheral iridotomy in that eye. The anterior chamber depth was successfully increased. Limited anterior vitrectomy by a pars plana approach was performed to prevent recurrent angle closure. The patient's vision improved and his intraocular pressure remained controlled without any antiglaucoma medication.
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. Cautious intraoperative anterior vitrectomy and surgical iridectomy are warranted. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Introduction
Acute angle closure is less common in patients with pseudophakia, compared with patients with phakia; this type of closure can have various causes.1 The underlying mechanism can involve force originating at the level of the iris; examples include absolute pupillary block due to iris–intraocular lens contact (eg, during anterior chamber intraocular lens implantation), iris–air contact, iris–silicone oil contact, iris–residual posterior capsule contact, iris–postoperative membrane contact, and iris–vitreous contact. In addition to pupillary block, angle closure in patients with pseudophakia can also result from factors such as Soemmering ring,2 ciliochoroidal detachment, and malignant glaucoma.3 Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
Case Report
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye. The patient had a history of traumatic anterior lens subluxation four years prior, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. A few hours prior to onset of his symptoms, he had undergone ureteroscopic lithotripsy under general anesthesia. During the operation, the patient was placed in a lithotomy position. Eight hundred milliliters of isotonic saline were used during the operation. Both intravenous anesthetic agents and inhaled anesthetic agents were used during the procedure (ie, propofol, atropine, nitrous oxide, and sevoflurane).
Upon presentation in our clinic, the patient’s best-corrected visual acuity was 20/70 in the right eye and 20/200 in the left eye. His IOP was 12 mmHg in the right eye and 57 mmHg in the left eye. Slit-lamp biomicroscopy examination showed ciliary injection and generalized bedewing of the cornea in the left eye. Grade 1-shallow anterior chamber and protrusion of the anterior hyaloid membrane were noted (Figure 1). The vitreous was pushed forward by pressure that originated from behind the iris, resulting in vitreous–corneal apposition. The scleral-fixated intraocular lens was pushed backward into an abnormally posterior position. Slit-lamp biomicroscopy findings in the right eye were unremarkable. The anterior chamber in the right eye was deep with a posterior chamber intraocular lens; one year prior, the patient had undergone uneventful phacoemulsification in the right eye. Cup to disc ratios were 0.3 in both eyes. Gonioscopy revealed 360-degree angle closure in the left eye. The patient then underwent ultrasound biomicroscopy. The findings demonstrated asymmetrical pupillary block in the left eye, with an obviously shallow anterior chamber. The scleral-fixated intraocular lens was pushed away from the iris plane (Figure 2). However, vitreous mass could not be determined by ultrasound biomicroscopy because of poor resolution. Pentacam imaging revealed a vitreous mass in the anterior chamber, which appeared as a faint curvilinear line around the mid-iris zone.Figure 1 Anterior segment photograph demonstrated vitreous protrusion into anterior chamber.
Figure 2 Ultrasound biomicroscopy (UBM) showed shallow anterior chamber with abnormal position of scleral-fixated intraocular lens.
The patient was treated with two topical antiglaucoma medications and oral acetazolamide for reduction of IOP. Following normalization of IOP and clearing of the cornea, laser iridotomy was performed in the patient’s left eye. The anterior chamber underwent a dramatic change to the normal deep configuration following treatment with laser peripheral iridotomy. The vitreous–corneal apposition no longer persisted and the iris returned to a flat configuration. Subsequent anterior segment optical coherence tomography showed a clear relationship among the vitreous, cornea, and iris (Figure 3). Four weeks after the acute angle closure event, the patient underwent anterior vitrectomy using a pars plana approach in the left eye; this removed the vitreous and was expected to prevent recurrent vitreous block-induced angle closure. The patient’s vision returned to normal (best-corrected visual acuity of 20/20) in the left eye. The IOP remained at approximately 10–15 mmHg without any antiglaucoma medication. Gonioscopy revealed a normal open angle without any residual peripheral anterior synechia.Figure 3 Anterior segment optical coherence tomography showed mushroom-shaped vitreous after laser peripheral iridotomy.
Discussion
Our patient presented with acute angle closure in the left eye, a few hours after a ureteroscopic procedure. The patient had a notable risk factor: vitreous prolapse due to prior trauma with inadequate vitrectomy. Certain actions such as the Valsalva maneuver are presumed to increase IOP and may facilitate vitreous movement from the posterior segment to the anterior segment in patients with a ruptured anterior hyaloid membrane.4 Both intubation and extubation can trigger the gag reflex and severe coughing, which increase intrathoracic pressure and IOP.5 Some types of anesthetic agents and depths of anesthesia may also interfere with pupillary size,6 thereby facilitating iris–vitreous blockage. Furthermore, in some stages of anesthesia, a mydriatic pupil may allow the vitreous to move from the posterior segment to the pupillary plane, which results in vitreous block during return to the normal pupillary size condition. The lithotomy position might not have affected the occurrence of angle closure in our patient because his head and body were at the same level. However, the lithotomy position can increase venous return, thereby increasing cardiac output. The clinical manifestation in this patient was acute angle closure due to an abrupt increase in IOP. Slit-lamp biomicroscopy revealed absolute pupillary block. Ancillary assessment by ultrasound biomicroscopy confirmed vitreous-induced pupillary block. Initial management comprised reduction of pressure to limit optic nerve damage, using topical and oral aqueous suppressants. Subsequently, laser peripheral iridotomy was performed to eliminate angle closure at the pupillary plane. In our patient, angle closure mainly occurred at the pupillary plane, but was not due to the mass effect of the vitreous behind the iris. Therefore, this event was completely resolved by laser peripheral iridotomy. However, definite treatment with anterior vitrectomy should be performed when possible because vitreous behind the iris can cause recurrent blockage if it occludes the laser peripheral iridotomy and pupil simultaneously.
Conclusion
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The Institutional Review Board, Faculty of Medicine, Ramathibodi hospital granted approval to publish the case details.
Author Contributions
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work. | 800 MILLILITERS | DrugDosageText | CC BY-NC | 33536795 | 19,031,650 | 2021 |
What was the outcome of reaction 'Angle closure glaucoma'? | Pseudophakic Angle Closure Due to Vitreous Block Following Ureteroscopic Lithotripsy.
Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye following ureteroscopic lithotripsy under general anesthesia. The patient had a history of coconut hit into his left eye which resulted in traumatic anterior lens subluxation, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. Prior scleral fixation procedure, anterior vitrectomy was not performed. Clinical examination showed mushroom-shaped vitreous in the anterior chamber with absolute pupillary block, which had resulted in acute angle closure. Thus, topical and oral antiglaucoma medications were administered to achieve normal intraocular pressure in the left eye, followed by laser peripheral iridotomy in that eye. The anterior chamber depth was successfully increased. Limited anterior vitrectomy by a pars plana approach was performed to prevent recurrent angle closure. The patient's vision improved and his intraocular pressure remained controlled without any antiglaucoma medication.
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. Cautious intraoperative anterior vitrectomy and surgical iridectomy are warranted. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Introduction
Acute angle closure is less common in patients with pseudophakia, compared with patients with phakia; this type of closure can have various causes.1 The underlying mechanism can involve force originating at the level of the iris; examples include absolute pupillary block due to iris–intraocular lens contact (eg, during anterior chamber intraocular lens implantation), iris–air contact, iris–silicone oil contact, iris–residual posterior capsule contact, iris–postoperative membrane contact, and iris–vitreous contact. In addition to pupillary block, angle closure in patients with pseudophakia can also result from factors such as Soemmering ring,2 ciliochoroidal detachment, and malignant glaucoma.3 Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
Case Report
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye. The patient had a history of traumatic anterior lens subluxation four years prior, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. A few hours prior to onset of his symptoms, he had undergone ureteroscopic lithotripsy under general anesthesia. During the operation, the patient was placed in a lithotomy position. Eight hundred milliliters of isotonic saline were used during the operation. Both intravenous anesthetic agents and inhaled anesthetic agents were used during the procedure (ie, propofol, atropine, nitrous oxide, and sevoflurane).
Upon presentation in our clinic, the patient’s best-corrected visual acuity was 20/70 in the right eye and 20/200 in the left eye. His IOP was 12 mmHg in the right eye and 57 mmHg in the left eye. Slit-lamp biomicroscopy examination showed ciliary injection and generalized bedewing of the cornea in the left eye. Grade 1-shallow anterior chamber and protrusion of the anterior hyaloid membrane were noted (Figure 1). The vitreous was pushed forward by pressure that originated from behind the iris, resulting in vitreous–corneal apposition. The scleral-fixated intraocular lens was pushed backward into an abnormally posterior position. Slit-lamp biomicroscopy findings in the right eye were unremarkable. The anterior chamber in the right eye was deep with a posterior chamber intraocular lens; one year prior, the patient had undergone uneventful phacoemulsification in the right eye. Cup to disc ratios were 0.3 in both eyes. Gonioscopy revealed 360-degree angle closure in the left eye. The patient then underwent ultrasound biomicroscopy. The findings demonstrated asymmetrical pupillary block in the left eye, with an obviously shallow anterior chamber. The scleral-fixated intraocular lens was pushed away from the iris plane (Figure 2). However, vitreous mass could not be determined by ultrasound biomicroscopy because of poor resolution. Pentacam imaging revealed a vitreous mass in the anterior chamber, which appeared as a faint curvilinear line around the mid-iris zone.Figure 1 Anterior segment photograph demonstrated vitreous protrusion into anterior chamber.
Figure 2 Ultrasound biomicroscopy (UBM) showed shallow anterior chamber with abnormal position of scleral-fixated intraocular lens.
The patient was treated with two topical antiglaucoma medications and oral acetazolamide for reduction of IOP. Following normalization of IOP and clearing of the cornea, laser iridotomy was performed in the patient’s left eye. The anterior chamber underwent a dramatic change to the normal deep configuration following treatment with laser peripheral iridotomy. The vitreous–corneal apposition no longer persisted and the iris returned to a flat configuration. Subsequent anterior segment optical coherence tomography showed a clear relationship among the vitreous, cornea, and iris (Figure 3). Four weeks after the acute angle closure event, the patient underwent anterior vitrectomy using a pars plana approach in the left eye; this removed the vitreous and was expected to prevent recurrent vitreous block-induced angle closure. The patient’s vision returned to normal (best-corrected visual acuity of 20/20) in the left eye. The IOP remained at approximately 10–15 mmHg without any antiglaucoma medication. Gonioscopy revealed a normal open angle without any residual peripheral anterior synechia.Figure 3 Anterior segment optical coherence tomography showed mushroom-shaped vitreous after laser peripheral iridotomy.
Discussion
Our patient presented with acute angle closure in the left eye, a few hours after a ureteroscopic procedure. The patient had a notable risk factor: vitreous prolapse due to prior trauma with inadequate vitrectomy. Certain actions such as the Valsalva maneuver are presumed to increase IOP and may facilitate vitreous movement from the posterior segment to the anterior segment in patients with a ruptured anterior hyaloid membrane.4 Both intubation and extubation can trigger the gag reflex and severe coughing, which increase intrathoracic pressure and IOP.5 Some types of anesthetic agents and depths of anesthesia may also interfere with pupillary size,6 thereby facilitating iris–vitreous blockage. Furthermore, in some stages of anesthesia, a mydriatic pupil may allow the vitreous to move from the posterior segment to the pupillary plane, which results in vitreous block during return to the normal pupillary size condition. The lithotomy position might not have affected the occurrence of angle closure in our patient because his head and body were at the same level. However, the lithotomy position can increase venous return, thereby increasing cardiac output. The clinical manifestation in this patient was acute angle closure due to an abrupt increase in IOP. Slit-lamp biomicroscopy revealed absolute pupillary block. Ancillary assessment by ultrasound biomicroscopy confirmed vitreous-induced pupillary block. Initial management comprised reduction of pressure to limit optic nerve damage, using topical and oral aqueous suppressants. Subsequently, laser peripheral iridotomy was performed to eliminate angle closure at the pupillary plane. In our patient, angle closure mainly occurred at the pupillary plane, but was not due to the mass effect of the vitreous behind the iris. Therefore, this event was completely resolved by laser peripheral iridotomy. However, definite treatment with anterior vitrectomy should be performed when possible because vitreous behind the iris can cause recurrent blockage if it occludes the laser peripheral iridotomy and pupil simultaneously.
Conclusion
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The Institutional Review Board, Faculty of Medicine, Ramathibodi hospital granted approval to publish the case details.
Author Contributions
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work. | Recovered | ReactionOutcome | CC BY-NC | 33536795 | 19,031,650 | 2021 |
What was the outcome of reaction 'Vitreous disorder'? | Pseudophakic Angle Closure Due to Vitreous Block Following Ureteroscopic Lithotripsy.
Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye following ureteroscopic lithotripsy under general anesthesia. The patient had a history of coconut hit into his left eye which resulted in traumatic anterior lens subluxation, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. Prior scleral fixation procedure, anterior vitrectomy was not performed. Clinical examination showed mushroom-shaped vitreous in the anterior chamber with absolute pupillary block, which had resulted in acute angle closure. Thus, topical and oral antiglaucoma medications were administered to achieve normal intraocular pressure in the left eye, followed by laser peripheral iridotomy in that eye. The anterior chamber depth was successfully increased. Limited anterior vitrectomy by a pars plana approach was performed to prevent recurrent angle closure. The patient's vision improved and his intraocular pressure remained controlled without any antiglaucoma medication.
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. Cautious intraoperative anterior vitrectomy and surgical iridectomy are warranted. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Introduction
Acute angle closure is less common in patients with pseudophakia, compared with patients with phakia; this type of closure can have various causes.1 The underlying mechanism can involve force originating at the level of the iris; examples include absolute pupillary block due to iris–intraocular lens contact (eg, during anterior chamber intraocular lens implantation), iris–air contact, iris–silicone oil contact, iris–residual posterior capsule contact, iris–postoperative membrane contact, and iris–vitreous contact. In addition to pupillary block, angle closure in patients with pseudophakia can also result from factors such as Soemmering ring,2 ciliochoroidal detachment, and malignant glaucoma.3 Here, we describe a patient who exhibited pseudophakic angle closure due to vitreous block following ureteroscopic lithotripsy under general anesthesia.
Case Report
A 57-year-old Thai man presented with sudden eye pain and blurring of vision in the left eye. The patient had a history of traumatic anterior lens subluxation four years prior, for which he had undergone phacoemulsification and scleral-fixated intraocular lens implantation in the left eye. A few hours prior to onset of his symptoms, he had undergone ureteroscopic lithotripsy under general anesthesia. During the operation, the patient was placed in a lithotomy position. Eight hundred milliliters of isotonic saline were used during the operation. Both intravenous anesthetic agents and inhaled anesthetic agents were used during the procedure (ie, propofol, atropine, nitrous oxide, and sevoflurane).
Upon presentation in our clinic, the patient’s best-corrected visual acuity was 20/70 in the right eye and 20/200 in the left eye. His IOP was 12 mmHg in the right eye and 57 mmHg in the left eye. Slit-lamp biomicroscopy examination showed ciliary injection and generalized bedewing of the cornea in the left eye. Grade 1-shallow anterior chamber and protrusion of the anterior hyaloid membrane were noted (Figure 1). The vitreous was pushed forward by pressure that originated from behind the iris, resulting in vitreous–corneal apposition. The scleral-fixated intraocular lens was pushed backward into an abnormally posterior position. Slit-lamp biomicroscopy findings in the right eye were unremarkable. The anterior chamber in the right eye was deep with a posterior chamber intraocular lens; one year prior, the patient had undergone uneventful phacoemulsification in the right eye. Cup to disc ratios were 0.3 in both eyes. Gonioscopy revealed 360-degree angle closure in the left eye. The patient then underwent ultrasound biomicroscopy. The findings demonstrated asymmetrical pupillary block in the left eye, with an obviously shallow anterior chamber. The scleral-fixated intraocular lens was pushed away from the iris plane (Figure 2). However, vitreous mass could not be determined by ultrasound biomicroscopy because of poor resolution. Pentacam imaging revealed a vitreous mass in the anterior chamber, which appeared as a faint curvilinear line around the mid-iris zone.Figure 1 Anterior segment photograph demonstrated vitreous protrusion into anterior chamber.
Figure 2 Ultrasound biomicroscopy (UBM) showed shallow anterior chamber with abnormal position of scleral-fixated intraocular lens.
The patient was treated with two topical antiglaucoma medications and oral acetazolamide for reduction of IOP. Following normalization of IOP and clearing of the cornea, laser iridotomy was performed in the patient’s left eye. The anterior chamber underwent a dramatic change to the normal deep configuration following treatment with laser peripheral iridotomy. The vitreous–corneal apposition no longer persisted and the iris returned to a flat configuration. Subsequent anterior segment optical coherence tomography showed a clear relationship among the vitreous, cornea, and iris (Figure 3). Four weeks after the acute angle closure event, the patient underwent anterior vitrectomy using a pars plana approach in the left eye; this removed the vitreous and was expected to prevent recurrent vitreous block-induced angle closure. The patient’s vision returned to normal (best-corrected visual acuity of 20/20) in the left eye. The IOP remained at approximately 10–15 mmHg without any antiglaucoma medication. Gonioscopy revealed a normal open angle without any residual peripheral anterior synechia.Figure 3 Anterior segment optical coherence tomography showed mushroom-shaped vitreous after laser peripheral iridotomy.
Discussion
Our patient presented with acute angle closure in the left eye, a few hours after a ureteroscopic procedure. The patient had a notable risk factor: vitreous prolapse due to prior trauma with inadequate vitrectomy. Certain actions such as the Valsalva maneuver are presumed to increase IOP and may facilitate vitreous movement from the posterior segment to the anterior segment in patients with a ruptured anterior hyaloid membrane.4 Both intubation and extubation can trigger the gag reflex and severe coughing, which increase intrathoracic pressure and IOP.5 Some types of anesthetic agents and depths of anesthesia may also interfere with pupillary size,6 thereby facilitating iris–vitreous blockage. Furthermore, in some stages of anesthesia, a mydriatic pupil may allow the vitreous to move from the posterior segment to the pupillary plane, which results in vitreous block during return to the normal pupillary size condition. The lithotomy position might not have affected the occurrence of angle closure in our patient because his head and body were at the same level. However, the lithotomy position can increase venous return, thereby increasing cardiac output. The clinical manifestation in this patient was acute angle closure due to an abrupt increase in IOP. Slit-lamp biomicroscopy revealed absolute pupillary block. Ancillary assessment by ultrasound biomicroscopy confirmed vitreous-induced pupillary block. Initial management comprised reduction of pressure to limit optic nerve damage, using topical and oral aqueous suppressants. Subsequently, laser peripheral iridotomy was performed to eliminate angle closure at the pupillary plane. In our patient, angle closure mainly occurred at the pupillary plane, but was not due to the mass effect of the vitreous behind the iris. Therefore, this event was completely resolved by laser peripheral iridotomy. However, definite treatment with anterior vitrectomy should be performed when possible because vitreous behind the iris can cause recurrent blockage if it occludes the laser peripheral iridotomy and pupil simultaneously.
Conclusion
Vitreous block can occur in patients with pseudophakia, especially in the presence of a ruptured posterior capsule. General anesthesia may contribute to the onset of vitreous block in susceptible patients.
Consent
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. The Institutional Review Board, Faculty of Medicine, Ramathibodi hospital granted approval to publish the case details.
Author Contributions
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work. | Recovered | ReactionOutcome | CC BY-NC | 33536795 | 19,031,650 | 2021 |
What was the dosage of drug 'OLANZAPINE'? | A very rare case of priapism under aripiprazole in a patient followed for bipolar disorder: A CARE-compliant report.
Priapism is a urological emergency characterized by abnormally prolonged, painful and irreducible erection. It occurs without a sexual stimulation and habitually exceeds 6 h. About a half of iatrogenic priapisms are believed to be associated with antipsychotics. Until to date, very few cases of aripiprazole-associated priapism were reported.
In this case report, we present the clinical findings of a 40-year-old patient that developed priapism after treatment with aripiprazole after his hospitalization for an episode of clinical mania following treatment discontinuation for bipolar I disorder. The management was successful and priapism was resolved spontaneously.
Despite its low affinity to alpha-1 adrenergic receptors, aripiprazole may be associated with priapism. Several potential factors involved in the pathogenesis of this adverse event have been reported in the literature including history of priapism in a different class of neuroleptics and consumption of psychoactive drugs which are the principal factors found in our case.
Priapism may occur even during treatment with antipsychotics that have a low affinity to alpha1-adrenergic receptors. All patients on antipsychotics should be informed about the risk of this rare but serious adverse event.
1 Introduction
Priapism is a rare condition in which abnormally prolonged (>6 hours), painful, and irreducible erection without any sexual stimulations nor ejaculation can be observed [1]. Despite its rareness, it remains an extremely serious condition that requires appropriate management. Therefore, priapism is a medical and surgical emergency that may lead to severe sequelae, particularly, erectile dysfunction after fibrosis of the cavernous bodies [2]. Iatrogenic origins of priapism, especially associated with pharmacological treatments, are found implicated in 25–40% of the cases in which antipsychotics occupy 50% [3,4]. However, there are few published cases in the literature that reported aripiprazole-induced priapism. In this paper, we report a new case of this association in a patient treated for bipolar I disorder based on CARE-guidelines [5].
1.1 Presentation of case
Our patient is a 40 year-old male, married, father of a child, and working as a nurse. He has been followed for 18 years for bipolar I disorder diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders V (DSM-V) criteria. He is a chronic user of tobacco at a rate of 30 packets/years and cannabis at a rate of 1 g per day. Cocaine was occasionally used. Moreover, he has a history of priapism induced by chlorpromazine during his hospitalization in 2016 for manic access. In that time, the management of his case required surgical intervention by puncture-washing of the cavernous bodies. After this adverse event, sedative neuroleptics were proscribed for him. Of note, our patient has no other personal or family history including psychiatric disorders. The disease history of our patient dates back to 2005, with the onset of clinical mania that required hospitalization and treatment with escalating doses of sodium valproate to 1500 mg/day. During follow up, the occurrence of several relapses of manic and depressive symptoms were noticed which required his hospitalization with free intervals between episodes. The patient was treated using single agents several including olanzapine (20 mg/day), risperidone (8 mg/day), and sertraline (50 mg/day). The treatments changes were proposed to the patient because of the marked adverse events reported principally sedation and priapism that needed his hospitalization in 2016.
In 2019, the patient was admitted to our psychiatric hospital for psychomotor excitement, tachypsychia with logorrhea, mood lability, ideas of grandeur and multiple projects, which were associated with insomnia and a tendency to hetero aggressiveness. According to DSM-V criteria, a mania in the context of bipolar I disorder following discontinuation of treatment for negative insight was retained as a diagnosis. Notably, there were no linguistic, cultural or financial issues during the management of our patient. Initially, our patient was treated with aripiprazole at a dose of 15 mg/day in combination with diazepam (30 mg/day). On day 10 of his hospitalization, the patient presented with a persistent and painful erection. He was referred to the urology department for management. A preliminary clinical assessment found no urological abnormalities. The diagnosis of priapism induced by aripiprazole was therefore retained. Fortunately, the erection resolved spontaneously, and the patient started benzodiazepines alone (diazepam 30 mg/day) with surveillance of his clinical and mental status. Subsequently, sodium valproate (1500 mg/day) combined with psychoeducation were proposed to the patient. The choice of this molecule was based on the good improvement and tolerance seen in the previous episodes as reported by the patient. The evolution was marked by the disappearance of manic symptoms, good tolerance, as well as non-recurrence of priapism.
2 Discussion
Despite the occurrence of aripiprazole-induced priapism is rare, its severity and difficult management should alert the attention of clinicians. Remarkably, a significant strength of our clinical case is the absence of any drug interactions that might justify the association of other molecules with priapism. In fact, our patient was treated with aripiprazole and diazepam only. Importantly, the recognition of this adverse event by the patient allowed us to intervene quickly. This is not always possible especially in patients with non-stabilized mental disorders. The selection of an appropriate treatment for the patient (sodium valproate) was guided by the nature of his psychiatric disorder (bipolar disorder type 1), the previous response to this treatment and the absence of affinity for alpha-1-adrenergic receptors. Therefore, this medication for this indication does not seem to increase the risk for developing priapism.
Several lines of evidence on the occurrence of priapism when using antipsychotics have supported the neuromuscular hypothesis that remained the most adopted worldwide. It suggests that antipsychotics-associated priapism depends on the ability of alpha-1-adrenergic receptors blockade of the cavernous bodies [6]. The current published literature reported several clinical cases of priapism associated with the two classes of antipsychotics including classical and atypical molecules [7]. In this perspective, haloperidol, chlorpromazine, levomepromazine and thioridazine are among the classical neuroleptics that have the greatest affinity for alpha-1 adrenergic receptors. Clozapine, quetiapine, risperidone and olanzapine are the atypical antipsychotics with high affinity for these receptors [[8], [9], [10]]. However, aripiprazole has the lowest affinity for this receptor among all atypical antipsychotics [11] (Table 1). Besides, despite this characteristic, cases of priapism induced by aripiprazole have been reported (Table 2). Two previous reports suggested an association between the dose of aripiprazole and priapism [12,13]. In 2006, Mago et al. discussed a case of recurrent priapism that was associated with the administration of aripiprazole [14]. In addition, priapism has been reported when aripiprazole was combined with oxcarbazepine and lithium [15]. Other authors reported a case of priapism with 10 mg of aripiprazole within a few hours of its first administration to patients with schizophrenia [16,17].Table 1 Affinity of antipsychotics to alpha-1 adrenergic receptors.
Table 1Antipsychotics Affinity
Amisulpride -
Aripiprazole ±
Olanzapine ++
Clozapine +++
Haloperidol +++
Quetiapine +++
Risperidone ++++
Chlorpromazine ++++
Table 2 Summary of published cases and reports on aripiprazole-associated priapism.
Table 2Author/year Article type Country of origin Patient’ gender and age Psychiatric disorder Treatments and outcomes
Negin et al., 2005 [15] Letter to the editor USA -Male
−16 Pervasive developmental disorder and bipolar disorder -Oxcarbazepine combined to lithium and aripiprazole
-After two days of this treatment, two discrete episodes of prolonged penile erection were reported
-No further prolonged erections were recorded after maintenance treatment with lithium and aripiprazole only
Mago et al., 2006 [14] Letter to the editor USA -Male
−47 Chronic paranoid schizophrenia -Aripiprazole (dose not reported)
-Recurrent priapism treated by cavernal irrigation and phenylephrine each time combined to pseudoephedrine
-Lost to follow-up after discharge
Aguilar et al., 2009 [13] Letter to the editor Spain -Male
−23 Schizophrenic disorder -Aripiprazole (20 mg/day) and dipotassium clorazepate.
-Increase in the dose to 30 mg/day following the activation of the disease
-Painful erection after two days of treatment for more than 24 hours
-No similar episodes were noticed after urological intervention and dose reduction of aripiprazole to 20mg/day
Hsu et al., 2011 [12] Letter to the editor Taiwan -Male
−24 Psychotic disorder (not specified) -Initial aripiprazole at a dose of 10mg/day with gradual increase to up to 25mg
-Priapism was observed following this monotherapy
-Switching to olanzapine (10 mg/day) was effective for priapism disappearance
Togul et al., 2012 [16] Conference abstract Turkey -Male
−30 Schizophrenia -Aripiprazole at a dose of 10 mg/day.
-The patient reported a painful erection 8 hours later.
-Priapism disappeared after switching to olanzapine (20 mg/day).
Trivedi et al., 2016 [17] Case report India -Male
-An adolescent (age non specified) Paranoid schizophrenia -Aripiprazole 10 mg/day and lorazepam 2 mg.
-Priapism after 7 hours treated successfully with blood aspiration with saline in combination to local adrenaline
Our patient case differs from the rare reports published mainly by the nature of the underlying psychiatric disease. On the other hand, other common points are present. The repeated occurrence of priapism under different antipsychotics could refer to a predisposition to this adverse event [18,19]. The use of psychoactive substances would also increase the risk of occurrence of priapism [20]. This was the case of our patient who is a chronic user of tobacco, cannabis and occasional cocaine. So far, the occurrence of priapism in our case does not appear to be dose related. In fact, our clinical case must attract the attention of practitioners to several key points. First, the systematic search for a history of priapism before administration of an antipsychotic should be performed because the literature raises cases of repeated priapism in these patients. Furthermore, a careful follow up of patients treated with antipsychotics especially those who have unstable psychiatric diseases should be implemented. And finally, the use of antipsychotics with weak or absent alpha1-adrenergic affinity should be recommended along with psychoeducation. During follow up, our patient was very satisfied with our management despite the stress experienced during his hospitalization for priapism.
3 Conclusion
Priapism is a serious urological emergency that requires a rapid and an adequate management. The factors predisposing to its occurrence after aripiprazole use or other antipsychotics need to be evaluated in well conducted robust studies. This will be essential to identify patients at risk to develop priapism. Patients under treatments using antipsychotics should be informed of the risk of this adverse event.
Conflict of interest
None declared.
Sources of funding
None.
Ethical approval
N/a.
Consent
Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.
Author contribution
Dr Salah-Eddine El Jabiry collected data and wrote the manuscript. Dr Atif Mansour participated in the patient management. Professors Mohammed Barrimi, Bouchra Oneib, and Fatima El Ghazouani supervised the article writing. All the authors approved the final version.
Research registration
N/a.
Guarantor
Dr Salah-Eddine El Jabiry.
Provenance and peer review
Not commissioned, externally peer-reviewed.
Appendix A Supplementary data
The following is the supplementary data related to this article:Multimedia component 1
Multimedia component 1
Appendix A Supplementary data related to this article can be found at https://doi.org/10.1016/j.amsu.2021.01.015. | 20 MILLIGRAM PER DAY | DrugDosageText | CC BY | 33537133 | 18,891,426 | 2021-02 |
What was the dosage of drug 'RISPERIDONE'? | A very rare case of priapism under aripiprazole in a patient followed for bipolar disorder: A CARE-compliant report.
Priapism is a urological emergency characterized by abnormally prolonged, painful and irreducible erection. It occurs without a sexual stimulation and habitually exceeds 6 h. About a half of iatrogenic priapisms are believed to be associated with antipsychotics. Until to date, very few cases of aripiprazole-associated priapism were reported.
In this case report, we present the clinical findings of a 40-year-old patient that developed priapism after treatment with aripiprazole after his hospitalization for an episode of clinical mania following treatment discontinuation for bipolar I disorder. The management was successful and priapism was resolved spontaneously.
Despite its low affinity to alpha-1 adrenergic receptors, aripiprazole may be associated with priapism. Several potential factors involved in the pathogenesis of this adverse event have been reported in the literature including history of priapism in a different class of neuroleptics and consumption of psychoactive drugs which are the principal factors found in our case.
Priapism may occur even during treatment with antipsychotics that have a low affinity to alpha1-adrenergic receptors. All patients on antipsychotics should be informed about the risk of this rare but serious adverse event.
1 Introduction
Priapism is a rare condition in which abnormally prolonged (>6 hours), painful, and irreducible erection without any sexual stimulations nor ejaculation can be observed [1]. Despite its rareness, it remains an extremely serious condition that requires appropriate management. Therefore, priapism is a medical and surgical emergency that may lead to severe sequelae, particularly, erectile dysfunction after fibrosis of the cavernous bodies [2]. Iatrogenic origins of priapism, especially associated with pharmacological treatments, are found implicated in 25–40% of the cases in which antipsychotics occupy 50% [3,4]. However, there are few published cases in the literature that reported aripiprazole-induced priapism. In this paper, we report a new case of this association in a patient treated for bipolar I disorder based on CARE-guidelines [5].
1.1 Presentation of case
Our patient is a 40 year-old male, married, father of a child, and working as a nurse. He has been followed for 18 years for bipolar I disorder diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders V (DSM-V) criteria. He is a chronic user of tobacco at a rate of 30 packets/years and cannabis at a rate of 1 g per day. Cocaine was occasionally used. Moreover, he has a history of priapism induced by chlorpromazine during his hospitalization in 2016 for manic access. In that time, the management of his case required surgical intervention by puncture-washing of the cavernous bodies. After this adverse event, sedative neuroleptics were proscribed for him. Of note, our patient has no other personal or family history including psychiatric disorders. The disease history of our patient dates back to 2005, with the onset of clinical mania that required hospitalization and treatment with escalating doses of sodium valproate to 1500 mg/day. During follow up, the occurrence of several relapses of manic and depressive symptoms were noticed which required his hospitalization with free intervals between episodes. The patient was treated using single agents several including olanzapine (20 mg/day), risperidone (8 mg/day), and sertraline (50 mg/day). The treatments changes were proposed to the patient because of the marked adverse events reported principally sedation and priapism that needed his hospitalization in 2016.
In 2019, the patient was admitted to our psychiatric hospital for psychomotor excitement, tachypsychia with logorrhea, mood lability, ideas of grandeur and multiple projects, which were associated with insomnia and a tendency to hetero aggressiveness. According to DSM-V criteria, a mania in the context of bipolar I disorder following discontinuation of treatment for negative insight was retained as a diagnosis. Notably, there were no linguistic, cultural or financial issues during the management of our patient. Initially, our patient was treated with aripiprazole at a dose of 15 mg/day in combination with diazepam (30 mg/day). On day 10 of his hospitalization, the patient presented with a persistent and painful erection. He was referred to the urology department for management. A preliminary clinical assessment found no urological abnormalities. The diagnosis of priapism induced by aripiprazole was therefore retained. Fortunately, the erection resolved spontaneously, and the patient started benzodiazepines alone (diazepam 30 mg/day) with surveillance of his clinical and mental status. Subsequently, sodium valproate (1500 mg/day) combined with psychoeducation were proposed to the patient. The choice of this molecule was based on the good improvement and tolerance seen in the previous episodes as reported by the patient. The evolution was marked by the disappearance of manic symptoms, good tolerance, as well as non-recurrence of priapism.
2 Discussion
Despite the occurrence of aripiprazole-induced priapism is rare, its severity and difficult management should alert the attention of clinicians. Remarkably, a significant strength of our clinical case is the absence of any drug interactions that might justify the association of other molecules with priapism. In fact, our patient was treated with aripiprazole and diazepam only. Importantly, the recognition of this adverse event by the patient allowed us to intervene quickly. This is not always possible especially in patients with non-stabilized mental disorders. The selection of an appropriate treatment for the patient (sodium valproate) was guided by the nature of his psychiatric disorder (bipolar disorder type 1), the previous response to this treatment and the absence of affinity for alpha-1-adrenergic receptors. Therefore, this medication for this indication does not seem to increase the risk for developing priapism.
Several lines of evidence on the occurrence of priapism when using antipsychotics have supported the neuromuscular hypothesis that remained the most adopted worldwide. It suggests that antipsychotics-associated priapism depends on the ability of alpha-1-adrenergic receptors blockade of the cavernous bodies [6]. The current published literature reported several clinical cases of priapism associated with the two classes of antipsychotics including classical and atypical molecules [7]. In this perspective, haloperidol, chlorpromazine, levomepromazine and thioridazine are among the classical neuroleptics that have the greatest affinity for alpha-1 adrenergic receptors. Clozapine, quetiapine, risperidone and olanzapine are the atypical antipsychotics with high affinity for these receptors [[8], [9], [10]]. However, aripiprazole has the lowest affinity for this receptor among all atypical antipsychotics [11] (Table 1). Besides, despite this characteristic, cases of priapism induced by aripiprazole have been reported (Table 2). Two previous reports suggested an association between the dose of aripiprazole and priapism [12,13]. In 2006, Mago et al. discussed a case of recurrent priapism that was associated with the administration of aripiprazole [14]. In addition, priapism has been reported when aripiprazole was combined with oxcarbazepine and lithium [15]. Other authors reported a case of priapism with 10 mg of aripiprazole within a few hours of its first administration to patients with schizophrenia [16,17].Table 1 Affinity of antipsychotics to alpha-1 adrenergic receptors.
Table 1Antipsychotics Affinity
Amisulpride -
Aripiprazole ±
Olanzapine ++
Clozapine +++
Haloperidol +++
Quetiapine +++
Risperidone ++++
Chlorpromazine ++++
Table 2 Summary of published cases and reports on aripiprazole-associated priapism.
Table 2Author/year Article type Country of origin Patient’ gender and age Psychiatric disorder Treatments and outcomes
Negin et al., 2005 [15] Letter to the editor USA -Male
−16 Pervasive developmental disorder and bipolar disorder -Oxcarbazepine combined to lithium and aripiprazole
-After two days of this treatment, two discrete episodes of prolonged penile erection were reported
-No further prolonged erections were recorded after maintenance treatment with lithium and aripiprazole only
Mago et al., 2006 [14] Letter to the editor USA -Male
−47 Chronic paranoid schizophrenia -Aripiprazole (dose not reported)
-Recurrent priapism treated by cavernal irrigation and phenylephrine each time combined to pseudoephedrine
-Lost to follow-up after discharge
Aguilar et al., 2009 [13] Letter to the editor Spain -Male
−23 Schizophrenic disorder -Aripiprazole (20 mg/day) and dipotassium clorazepate.
-Increase in the dose to 30 mg/day following the activation of the disease
-Painful erection after two days of treatment for more than 24 hours
-No similar episodes were noticed after urological intervention and dose reduction of aripiprazole to 20mg/day
Hsu et al., 2011 [12] Letter to the editor Taiwan -Male
−24 Psychotic disorder (not specified) -Initial aripiprazole at a dose of 10mg/day with gradual increase to up to 25mg
-Priapism was observed following this monotherapy
-Switching to olanzapine (10 mg/day) was effective for priapism disappearance
Togul et al., 2012 [16] Conference abstract Turkey -Male
−30 Schizophrenia -Aripiprazole at a dose of 10 mg/day.
-The patient reported a painful erection 8 hours later.
-Priapism disappeared after switching to olanzapine (20 mg/day).
Trivedi et al., 2016 [17] Case report India -Male
-An adolescent (age non specified) Paranoid schizophrenia -Aripiprazole 10 mg/day and lorazepam 2 mg.
-Priapism after 7 hours treated successfully with blood aspiration with saline in combination to local adrenaline
Our patient case differs from the rare reports published mainly by the nature of the underlying psychiatric disease. On the other hand, other common points are present. The repeated occurrence of priapism under different antipsychotics could refer to a predisposition to this adverse event [18,19]. The use of psychoactive substances would also increase the risk of occurrence of priapism [20]. This was the case of our patient who is a chronic user of tobacco, cannabis and occasional cocaine. So far, the occurrence of priapism in our case does not appear to be dose related. In fact, our clinical case must attract the attention of practitioners to several key points. First, the systematic search for a history of priapism before administration of an antipsychotic should be performed because the literature raises cases of repeated priapism in these patients. Furthermore, a careful follow up of patients treated with antipsychotics especially those who have unstable psychiatric diseases should be implemented. And finally, the use of antipsychotics with weak or absent alpha1-adrenergic affinity should be recommended along with psychoeducation. During follow up, our patient was very satisfied with our management despite the stress experienced during his hospitalization for priapism.
3 Conclusion
Priapism is a serious urological emergency that requires a rapid and an adequate management. The factors predisposing to its occurrence after aripiprazole use or other antipsychotics need to be evaluated in well conducted robust studies. This will be essential to identify patients at risk to develop priapism. Patients under treatments using antipsychotics should be informed of the risk of this adverse event.
Conflict of interest
None declared.
Sources of funding
None.
Ethical approval
N/a.
Consent
Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.
Author contribution
Dr Salah-Eddine El Jabiry collected data and wrote the manuscript. Dr Atif Mansour participated in the patient management. Professors Mohammed Barrimi, Bouchra Oneib, and Fatima El Ghazouani supervised the article writing. All the authors approved the final version.
Research registration
N/a.
Guarantor
Dr Salah-Eddine El Jabiry.
Provenance and peer review
Not commissioned, externally peer-reviewed.
Appendix A Supplementary data
The following is the supplementary data related to this article:Multimedia component 1
Multimedia component 1
Appendix A Supplementary data related to this article can be found at https://doi.org/10.1016/j.amsu.2021.01.015. | 8 MILLIGRAM PER DAY | DrugDosageText | CC BY | 33537133 | 18,891,426 | 2021-02 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Aspergillus infection'. | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | AZATHIOPRINE, CYCLOPHOSPHAMIDE, CYCLOSPORINE, METHYLPREDNISOLONE, MYCOPHENOLATE MOFETIL, PREDNISOLONE, RITUXIMAB, TACROLIMUS, THYMOCYTE IMMUNE GLOBULIN NOS | DrugsGivenReaction | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Cytomegalovirus viraemia'. | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | AZATHIOPRINE, CYCLOPHOSPHAMIDE, CYCLOSPORINE, METHYLPREDNISOLONE, MYCOPHENOLATE MOFETIL, PREDNISOLONE, RITUXIMAB, TACROLIMUS, THYMOCYTE IMMUNE GLOBULIN NOS | DrugsGivenReaction | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Influenza'. | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | AZATHIOPRINE, CYCLOPHOSPHAMIDE, CYCLOSPORINE, METHYLPREDNISOLONE, MYCOPHENOLATE MOFETIL, PREDNISOLONE, RITUXIMAB, TACROLIMUS, THYMOCYTE IMMUNE GLOBULIN NOS | DrugsGivenReaction | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Leukopenia'. | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | AZATHIOPRINE, CYCLOPHOSPHAMIDE, CYCLOSPORINE, METHYLPREDNISOLONE, MYCOPHENOLATE MOFETIL, PREDNISOLONE, RITUXIMAB, TACROLIMUS, THYMOCYTE IMMUNE GLOBULIN NOS | DrugsGivenReaction | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Off label use'. | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | AZATHIOPRINE, CYCLOPHOSPHAMIDE, CYCLOSPORINE, METHYLPREDNISOLONE, MYCOPHENOLATE MOFETIL, PREDNISOLONE, RITUXIMAB, TACROLIMUS, THYMOCYTE IMMUNE GLOBULIN NOS | DrugsGivenReaction | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Therapy partial responder'. | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | AZATHIOPRINE, CYCLOPHOSPHAMIDE, CYCLOSPORINE, METHYLPREDNISOLONE, MYCOPHENOLATE MOFETIL, PREDNISOLONE, RITUXIMAB, TACROLIMUS, THYMOCYTE IMMUNE GLOBULIN NOS | DrugsGivenReaction | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
What was the administration route of drug 'CYCLOSPORINE'? | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | Oral | DrugAdministrationRoute | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
What was the administration route of drug 'METHYLPREDNISOLONE'? | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | Intravenous (not otherwise specified) | DrugAdministrationRoute | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
What was the administration route of drug 'MYCOPHENOLATE MOFETIL'? | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | Oral | DrugAdministrationRoute | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
What was the administration route of drug 'PREDNISOLONE'? | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | Oral | DrugAdministrationRoute | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
What was the dosage of drug 'METHYLPREDNISOLONE'? | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | 1 MILLIGRAM/KILOGRAM | DrugDosageText | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
What was the dosage of drug 'MYCOPHENOLATE MOFETIL'? | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | UNK (DOSE DECREASED) | DrugDosageText | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
What was the dosage of drug 'THYMOCYTE IMMUNE GLOBULIN NOS'? | IL-6 receptor blockade for allograft dysfunction after lung transplantation in a patient with COPA syndrome.
COPA syndrome is a genetic disorder of retrograde cis-Golgi vesicle transport that leads to upregulation of pro-inflammatory cytokines (mainly IL-1β and IL-6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post-lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA-ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy.
Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre- and post-LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre- and post-LTx were stimulated with PMA, LPS and anti-CD3/CD28 antibodies. Post-LTx endobronchial biopsies underwent microarray-based gene expression analysis. Results were compared to non-COPA LTx recipients and non-LTx healthy controls.
Multiplexed cytokine analysis showed rising type I/II IFNs, and IL-6 in BAL post-LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL-6 signalling pathways. Tocilizumab (IL-6 receptor antibody) administration for 3 months (4 mg kg-1, monthly) effectively suppressed IL-6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal.
Clinical effectiveness of IL-6 receptor blockade was not observed. However, we identified IL-6 upregulation associated with graft injury, effective IL-6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL-6 blockade in post-LTx care that should be investigated further.
Introduction
COPA syndrome is a monogenic disorder of immune dysregulation associated with mutations in the COPA gene on chromosome 1 encoding the coatomer‐associated protein subunit alpha. The syndrome was first described in 2015 based on whole exome sequencing.
1
The pathogenesis is of dysfunctional retrograde Golgi to endoplasmic reticulum (ER) protein transport, leading to the accumulation of unfolded proteins and increased ER stress. This causes an upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and skews the T‐helper (Th) response towards a Th17 phenotype, associated with autoimmune disease. Clinical manifestations reported with this syndrome include inflammatory arthropathies, glomerulonephritis, interstitial lung disease (ILD) and pulmonary haemorrhage.
2
High autoantibody titres are also common.
The impact of the innate immune dysfunction associated with COPA syndrome on post‐lung transplant (LTx) outcomes is unknown. The potential risk is that impaired regulation of pro‐inflammatory pathways will exacerbate immune‐mediated allograft injury and impair outcome. The recent identification of COPA syndrome
1
means that experience of performing LTx for this indication is limited. Encouragingly, a recent case report described stable lung function with no episodes of acute cellular rejection (ACR) or antibody‐mediated rejection (AMR) after 15 months of follow‐up.
3
The immunosuppressive approach was reported to included peri‐operative plasmapheresis (PLEX) and rituximab, induction anti‐thymocyte globulin (ATG), and long‐term maintenance intravenous immunoglobulin (IVIg).
In this case report, we describe our experience with lung transplantation (LTx) for ILD associated with COPA syndrome. We report progressive allograft dysfunction that occurred early post‐LTx and provide detailed immunologic analysis that informed therapeutic approach. Although the outcome was not successful, we hope that the description of the challenges faced will provide mechanistic insights into disease pathways that may be investigated in the management of COPA syndrome and post‐LTx allograft dysfunction.
Case report
We report the case of a 38‐year‐old Asian woman who underwent LTx for progressive ILD associated with COPA syndrome. Prior to LTx, COPA syndrome management had included immunosuppression (cyclophosphamide, azathioprine and prednisone) in a similar approach taken by other groups.
4
In addition to ILD, COPA syndrome had manifested as glomerulonephritis and arthropathy. Other relevant medical history included hypertension, osteoporosis and a pulmonary embolus. At the time of referral for LTx, she presented with advanced restrictive lung disease [TLC 3.2 L or 63% predicted; DLCO unrecordable; cellular and fibrotic NSIP pattern with emphysematous/cystic changes (Figure 1)], secondary pulmonary hypertension and hypoxic respiratory failure. Pre‐LTx assessment revealed normal coronary arteries, no evidence of oesophageal dysfunction and preserved renal function with stable, non‐nephrotic range proteinuria. Pre‐LTx human leucocyte antigen (HLA) testing revealed a high level of sensitisation, with a calculated panel reactive antibody (cPRA) of 28% for class I and 75% for class II HLA.
Figure 1 Pre‐transplant CT chest and explanted lung pathology. (a) Thoracic CT scan performed 1 week prior to lung transplant showed extensive cystic lung disease and fibrosis. (b) Explant pathology showed emphysematous/cystic changes, bronchiectasis, and a cellular and fibrotic nonspecific interstitial pneumonia pattern of interstitial lung disease. The upper image shows 20× magnification and the lower image 40× magnification.
At the time of LTx, a positive antibody crossmatch – identifying both auto and donor specific‐antibodies (DSA) – necessitated antibody desensitisation [intra‐ and post‐operative PLEX, IVIg (1 g kg−1) and ATG (5 mg kg−1)].
5
Bilateral LTx surgery was performed on central venoarterial extracorporeal membrane oxygenation support due to high pulmonary pressures and low lung volumes. Surgery was uneventful, post‐operative recovery was routine, and grade 1 (mild) primary graft dysfunction was noted at 72 h. She was discharged from hospital on the 22nd post‐operative day (POD), mobilising independently on room air. Maintenance oral immunosuppression at discharge composed of cyclosporin, mycophenolate and prednisone.
Despite this good early outcome, acute graft dysfunction (decline in FEV1 of 0.5 L from baseline; ill‐defined, inflammatory nodules on CT chest; BAL neutrophilia with Enterococcus cloacae on BAL culture) was observed 2 months post‐transplant. This was associated with grade 1 acute cellular rejection (ACR) and probable antibody‐mediated rejection (AMR)
6
(de novo HLA DQ DSA but C4d stain negative) on transbronchial biopsies. Intravenous antibiotics were administered for infection, guided by culture result. Despite this antibiotic course, allograft function did not recover. Treatment of ACR and AMR was commenced, including high‐dose methylprednisolone, PLEX, IVIg and rituximab. Cyclosporin was also changed to tacrolimus at this time. Although a small improvement in FEV1 was seen with this treatment, allograft function did not return to baseline (Figure 2) and an ongoing requirement for supplementary oxygen was noted. Immunosuppressive strategy was complicated by leucopenia, low‐grade CMV viraemia, Influenza A and the identification of Aspergillus fumigatus in BAL necessitating mycophenolate dose reduction.
Figure 2 Summary of the post‐transplant clinical course. This figure highlights the post‐transplant forced expiratory volume in one second (FEV1), with arrows indicating infection incidences, diagnostic sample collections and immunosuppressive treatment.
Investigations
As described above, COPA syndrome results in the upregulation of pro‐inflammatory cytokines leading to interstitial lung disease amongst other disorders. In this case, we hypothesised that persistent immune dysregulation might underlie the pathogenesis of progressive graft injury, as IL‐6, IL‐1β and Th17 upregulation have all been associated with COPA syndrome. If so, these processes may augment or exacerbate the alloimmune response. Therefore, to evaluate whether pro‐inflammatory cytokines presented potential therapeutic targets in this unique case, we obtained peripheral blood mononuclear cells (PBMC), serum, bronchoalveolar lavage (BAL) and lung mucosal tissue for analysis from the patient, non‐COPA LTx recipients with or without acute rejection (n = 3 for each), and two healthy volunteers.
Multiplexed cytokine analysis of BAL at 45, 62, 91 and 102 POD showed rising type I and II IFNs, IL‐17, and IL‐6 in BAL that decreased after treatment of ACR and AMR, but subsequently rebounded following clinical deterioration (Figure 3a). In our patient, plasma IL‐6 and TNFα exhibited a particularly sharp rise post‐LTx in comparison with non‐COPA LTx recipients with acute rejection (Figure 3b). PBMCs from the patient at 3 months post‐LTx exhibited marked LPS‐recruitable IL‐6 production but minimal responsiveness to anti‐CD3, suggesting that T‐cell responses were controlled and that cells of the myeloid lineage were driving clinical deterioration through an IL‐6 signalling pathway. Interestingly, analysis of cytokine production of PBMCs in response to different stimuli showed that this patient's post‐LTx management effectively controlled type I interferon production in her T cells and myeloid cells (Figure 3f). Microarray‐based gene expression profiling of endobronchial biopsies
7
at POD 102 revealed a very unusual pattern of gene expression distinct from other biopsies in the reference set (Figure 3c).
Figure 3 Post‐transplant cytokine and gene expression analysis. (a) Multiplex cytokine analysis of BAL, from three non‐COPA LTx patients without acute rejection (NAR), three non‐COPA LTx patients with spirometrically significant acute rejection (SSAR, with decrease in FEV1), and in the patient with COPA syndrome over time. Blue arrow indicates time of PLEX/IVIg treatment. (b) Cytokine analysis of plasma samples from three non‐COPA LTx patients with SSAR (grey circles) and the patient with COPA syndrome (black triangles) pre‐ and 3 months post‐transplant. One healthy volunteer is included (grey square) as a baseline comparator. (c) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 102 (prior to tocilizumab) compared to a reference set of biopsies from other LTx patients (small circles). Biopsies are classified according to their similarity to one of four archetypes (large circles: R1, normal; R2, T‐cell‐mediated rejection; R3, sampling heterogeneity; R4, late/fibrotic). The patient's biopsy was highly dissimilar from the reference set at POD 102. (d) IL‐6 level in BAL of the patient with COPA syndrome pre‐ and post‐tocilizumab therapy (green arrow), and from three non‐COPA LTx recipients without acute rejection (NoAR) and three non‐COPA LTx recipients with SSAR. IL‐6 level in plasma from 3 non‐COPA LTx patients with SSAR (grey circles) pre‐ and 3 months post‐transplant and the patient with COPA syndrome (black triangles) before and after tocilizumab therapy. (e) Orthogonal principal component analysis of the gene expression profile from an endobronchial biopsy of the patient (yellow triangle) at POD 226 (after tocilizumab) compared to a reference set of other biopsies (small circles). Results indicate that biopsy gene expression moved closer to R1 (normal) after tocilizumab at POD 226. (f) PBMCs from the patient with COPA syndrome pre‐transplant (open triangles) and 3 months post‐transplant (closed triangles) and from two healthy volunteers (grey squares) were stimulated with phorbol 12‐myristate 13‐acetate (PMA), anti‐CD3 and anti‐CD28 antibodies (CD3), or lipopolysaccharide (LPS). (g) Autopsy revealed widespread obliterative bronchiolitis and thickened interlobular septae, with no evidence of cellular or antibody‐mediated rejection. The left figure is H&E stain, and the right figure is elastic‐trichrome stain (20× magnification). (h) Thoracic CT performed 8 months post‐LTx showed diffuse bronchiectasis, scattered ground‐glass opacities and diffuse air‐trapping suggestive of CLAD.
Treatment
In view of the results of the cytokine analysis seen in this case, in addition to progressive allograft dysfunction, we administered tocilizumab 4 mg kg−1 monthly for three doses. Tocilizumab was well tolerated by the patient and effectively suppressed IL‐6 in BAL (Figure 3d). Mucosal gene expression profile following treatment suggested improvement in features of T‐cell‐mediated rejection and greater similarity to normal biopsies (Figure 3e). Tocilizumab was discontinued after three doses, due to a lack of substantial clinical improvement. After tocilizumab discontinuation, an increase in serum IL‐6 suggested that ongoing IL‐6 suppression may have been efficacious (Figure 3d).
Differential diagnosis
Baseline lung allograft dysfunction
The failure to achieve ‘normal’ pulmonary function following transplant (based on non‐LTx population reference ranges) has been termed baseline lung allograft dysfunction (BLAD).
8
The presence, and severity, of FEV1 impairment compared to ‘population norms’ has been associated with increasing mortality risk. Factors associated with BLAD include ILD as a LTx indication and donor smoking history (> 20 packs per years). Conceptually, one might also expect other factors to contribute to a failure to achieve a ‘normal’ FEV1, such as size‐mismatching, primary graft dysfunction (PGD), and early or ongoing lung injury (infection, aspiration, rejection). In this case, whilst the diagnosis of ILD may have been a risk factor for BLAD, the donor did not have a smoking history and the donor lung was well size‐matched (donor–recipient predicted TLC ratio 1.02). Peri‐operative donor and recipient BAL cultures were negative, and mild PGD (grade 1) was experienced at 72 h.
T‐ or B‐ cell‐mediated acute rejection
Bronchoscopy at POD 46 revealed minimal ACR (grade A1Bx) and a positive BAL culture for E. cloacae. Despite appropriate intravenous antibiotics, a decline in lung function occurred and treatment of ACR with IV methylprednisolone (1 mg kg−1 for 3 days) was indicated. The diagnosis of ‘probable AMR’ based on graft injury, histology and de novo DSA (DQ4 and DQ6) was managed with PLEX, IVIg and rituximab. No further TBBx were performed due to concerns associated with low lung function. Following AMR treatment, DQ6 DSA resolved but DQ4 DSA persisted. Autopsy histology did not identify evidence of ACR or AMR (Figure 3g), suggesting that the immunosuppressive approach taken in this case was successful in treating acute rejection but that other immune processes contributed to progressive allograft dysfunction.
Infection as a cause of progressive graft dysfunction
During post‐LTx follow‐up, this patient underwent frequent screening for infection, including regular bronchoscopy (including BAL with bacterial, fungal and mycobacterial cultures), sputum C+S and nasopharyngeal swabs for viral PCR. In view of the augmented immunosuppressive strategy, infection was carefully considered as the aetiology for ongoing graft dysfunction. Notably, bacterial organisms were only identified on two occasions post‐LTx and treated appropriately. Influenza A was identified on two separate occasions and treated with antivirals. Aside from pneumonitis, respiratory viral infection has been associated with the potentiation of an alloimmune response in LTx recipients.
9
Whilst we are unable to confirm whether influenza contributed to the potentiation of immune‐mediated injury in this case, we did not identify an expansion of either class 1 or 2 HLA antibodies following these infections. Finally, the identification of A. fumigatus in BAL was managed with pre‐emptive antifungal therapy, in view of the risk of invasive aspergillosis associated with further immunosuppression. With this strategy, we did not identify radiographic evidence of aspergillosis on CT imaging and serum galactomannan remained negative.
Allograft injury related to a dysregulation of the immune response associated with COPA syndrome
Throughout the post‐LTx course, this patient underwent a CT thorax approximately monthly. At 3 months post‐LTx, there was radiological evidence of bronchiolitis obliterans and this progressed on interval scans. The development of BOS at this early time‐point is uncommon and likely driven by allo‐immune responses to noxious stimuli. The lack of clinical benefit to ACR/AMR treatment, in addition to the pro‐inflammatory cytokine profile seen, raises the question as to whether the progressive graft injury in this case was a result of, or exacerbated by, the immunological dysfunction intrinsic to COPA syndrome.
Discussion
The likely pathophysiology underpinning this case is of a dysfunctional immune response causing early and ongoing graft injury leading to progressive chronic lung allograft dysfunction (CLAD). Whether the trigger for this injurious immune response was infection, alloimmunity and/or COPA‐associated immune dysregulation is unclear. However, the pro‐inflammatory cytokine response described with an over‐expression of IL‐6 presented a potential therapeutic target following the failure of standard immunosuppression.
Tocilizumab – a monoclonal anti‐IL‐6 receptor antibody – is FDA approved for use in rheumatoid arthritis, juvenile idiopathic arthritis and giant cell arteritis. IL‐6 is thought to play an important role in the progression of autoimmune disease and has been implicated in the expansion and activation of both B and T cells, as well as in the initiation of the acute phase inflammatory response. In organ transplant animal models, improved survival has been reported in IL‐6‐deficient heart
10
and kidney
11
allografts. In renal transplant medicine, tocilizumab has been investigated in pre‐transplant HLA desensitisation
12
and in the treatment of chronic AMR.
13
,
14
In lung transplantation, raised IL‐6 levels in BAL have been reported to be associated with primary graft dysfunction,
15
ACR
16
and CLAD.
17
Unfortunately, the administration of tocilizumab did not provide a clinically meaningful benefit in this case. This patient experienced progressive allograft dysfunction and death at 9 months post‐LTx. However, we hypothesise that if IL‐6 has a role in ongoing graft injury, the commencement of tocilizumab at an earlier phase of alloimmune injury may be more beneficial than that seen in this case [when radiographic evidence of BOS was present (Figure 3h)].
Lessons to be learnt
We identify IL‐6 as a potentially important cytokine in the development of graft dysfunction and report effective suppression of IL‐6 in BAL with tocilizumab. In addition, we report the improvement of injury as measured by mucosal gene transcription, suggesting reduced cellular injury and rejection with this treatment. This insight has value to the LTx community in general, as there is a growing evidence for IL‐6 receptor blockade in promoting immune tolerance.
18
We further support this theory by identifying elevated BAL IL‐6 concentrations in non‐COPA LTx patients experiencing acute rejection (Figure 3a). Further research is required to determine whether IL‐6 suppression leads to clinically meaningful endpoints.
Conflict of interest
Peter Riddell, Sajad Moshkelgosha, Liran Levy, Prodipto Pal, Kieran Halloran, Lianne Singer and Shaf Keshavjee declare no conflict of interest. Phil Halloran is Owner/founder of Transcriptome Sciences Inc. and received honoraria for lectures from Thermo Fisher and Astellas. Michael Parkes is Employee of Transcriptome Sciences Inc. Tereza Martinu and Stephen Juvet received research grant support from Sanofi.
Author contributions
Peter Riddell: Conceptualization; Formal analysis; Visualization; Writing‐original draft; Writing‐review & editing. Sajad Moshkelgosh: Data curation; Investigation; Methodology; Visualization; Writing‐review & editing. Liran Levy: Data curation; Formal analysis; Writing‐review & editing. Nina Chang: Data curation; Visualization; Writing‐review & editing. Prodipto Pal: Data curation; Visualization; Writing‐review & editing. Kieran Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Phil Halloran: Data curation; Formal analysis; Visualization; Writing‐review & editing. Michael Parkes: Data curation; Formal analysis; Visualization; Writing‐review & editing. Lianne Singer: Conceptualization; Formal analysis; Writing‐review & editing. Shaf Keshavjee: Conceptualization; Methodology; Writing‐review & editing. Tereza Martinu: Conceptualization; Data curation; Formal analysis; Methodology; Visualization; Writing‐review & editing. Stephen Juvet: Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Supervision; Writing‐review & editing.
Acknowledgments
The authors acknowledge Dr Ronald Laxer (SickKids Hospital, Toronto), Dr Anthony Shum (University of California, San Francisco), Dr Marco Gattorno (Gaslini Institute, Genoa) and Dr Simon Helfgott (Harvard Medical School, Boston) for helpful discussions regarding clinical management and interpretation of data.
Ethics board approval
Studies on samples from the healthy volunteers and LTx recipients were approved by the institutional review board at the University Health Network, Toronto. | 5 MILLIGRAM/KILOGRAM | DrugDosageText | CC BY-NC-ND | 33537146 | 20,950,102 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Accidental exposure to product by child'. | Pediatric Toxidrome Simulation Curriculum: Lidocaine-Induced Methemoglobinemia.
Lidocaine is a common local anesthetic used during minor procedures performed on pediatric patients. A rare but toxic and life-threatening side effect of lidocaine is methemoglobinemia. It should be considered in children who are hypoxic after exposure to an oxidizing agent.
We developed this simulation case for pediatric emergency medicine (PEM) fellows, but it can be adapted for interprofessional simulation. The case involved a 1-month-old male with hypoxia and resulting central cyanosis after exposure to lidocaine. The team performed an initial evaluation and intervention, collected a history, and developed a differential diagnosis for hypoxia and central cyanosis in an infant. Methemoglobinemia was confirmed by CO-oximetry. Preparatory materials, a debriefing guide, and scenario evaluation forms assisted with facilitation.
Fifty-six participants (including 18 PEM fellows) completed this simulation across four institutions. Participants rated the scenario on a 5-point Likert scale (1 = strongly disagree, 5 = strongly agree), finding it to be relevant to their work (median = 5) and realistic (median = 5). After participation in the simulation, learners felt confident in their ability to recognize methemoglobinemia (median = 4) and implement a plan to stabilize an infant with hypoxia (median = 4).
This simulation represents a resource for learners in the pediatric emergency department. It teaches the recognition and management of an infant with lidocaine toxicity and resultant methemoglobinemia. It uses experiential learning to teach and reinforce a systematic approach to the evaluation and management of a critically ill infant with acquired methemoglobinemia.
Educational Objectives
After participation in this simulation session, learners will be able to:
1. Perform a primary survey of a critically ill pediatric patient.
2. Implement a plan to stabilize a hypoxic and cyanotic neonate.
3. Develop a systematic approach for the evaluation of hypoxia and central cyanosis in a pediatric patient.
4. Describe the signs and symptoms of acquired methemoglobinemia in a pediatric patient.
5. Manage a pediatric patient with acquired methemoglobinemia.
6. Demonstrate teamwork and communication skills in a resuscitation setting.
Introduction
Methemoglobinemia is a rare but life-threatening disorder and can present at any age after exposure to an oxidizing agent. It occurs when hemoglobin becomes altered, causing the irreversible binding of oxygen through oxidation of ferrous ions associated with heme to the ferric state. The ferric ions of heme (Fe+++) cannot dissociate from oxygen, causing a left shift in the oxyhemoglobin dissociation curve and subsequent hypoxia.1 The impaired oxygen delivery to the tissues causes central cyanosis even with typical supportive measures to improve tissue oxygenation.1 The resultant hypoxia may lead to respiratory failure, end-organ damage, and death in severe cases.1
Methemoglobinemia develops as the result of a congenital condition or toxin exposure. Congenital methemoglobinemia is rare and is due to an abnormal hemoglobin structure or a deficiency in a reducing enzyme. These mutations can be autosomal recessive or dominant and include disease entities such as cytochrome b5 reductase deficiency and hemoglobin M disease.2 Acquired cases of methemoglobinemia are more prevalent, although the incidence is not well defined. Typically, medications and other chemical substances with oxidative potential are the cause. Common medications with oxidative potential include local anesthetics (lidocaine, benzocaine, and prilocaine), sulfa-containing medications, and dapsone.3 Infants are particularly susceptible to methemoglobinemia when facing an oxidative stress due to the immaturity of their enzymatic systems and increased potential for accidental overdose.3
Methemoglobinemia presents with hypoxia causing cyanosis, which can be a diagnostic challenge in a neonate. The possible causes of hypoxia in a neonate are extensive and include, but are not limited to, cyanotic congenital heart disease, sepsis, pneumonia, congenital airway or pulmonary anatomic abnormalities, pulmonary interstitial abnormalities, neurologic conditions, toxidromes, and hemoglobinopathies. While pulmonary etiologies are the most common cause of hypoxia and central cyanosis in an otherwise healthy child, methemoglobinemia should be considered in patients with exposure to oxidizing agents or a family history of congenital methemoglobinemia.
Infants experiencing acute methemoglobinemia may develop a physical exam finding of central cyanosis, hypoxia, increased work of breathing, altered mental status, respiratory depression, and seizures.3 The end result may be death.3 Central cyanosis appears differently depending on the pigmentation of a child's skin. Children with more skin pigmentation have bluish-grey color changes most noticeable on the tongue and mucosal membranes. Children with less skin pigmentation have blue color changes most noticeable on the tongue, mucosal membranes, and perioral area. A key indication of possible methemoglobinemia is a peripheral oxygen saturation detected by pulse oximetry that does not improve with oxygen administration. Upon phlebotomy, the blood remains dark brown in color after exposure to oxygen.3 The arterial blood gas is typically within normal limits. A peripheral capillary oxygen saturation (SpO2) monitor often underestimates hypoxia in the setting of high levels of methemoglobin, so this should not be relied upon for clinical monitoring.4 CO-oximetry should be used to detect the percentage of methemoglobin. It does this by measuring specific wavelengths of light corresponding to methemoglobin (660 nm and 940 nm).4
A majority of cases of methemoglobinemia resolve with supportive care only. More severe cases, however, can be treated with methylene blue to reduce the ferric ions of the heme molecules back to their ferrous state. This allows for appropriate oxygen dissociation from hemoglobin.5 The most commonly accepted methemoglobin level requiring treatment is a level greater than 20% of hemoglobin.6 If the patient is significantly symptomatic, however, methylene blue may be administered with lower levels. It is typically dosed at one to two milligrams per kilogram.6 The overall efficacy of methylene blue in the treatment of methemoglobinemia varies. The primary contraindication to methylene blue use is glucose-6-phophate dehydrogenase deficiency due to the risk of hemolysis.6
This simulation case presents a rare but potentially lethal toxic exposure of topical lidocaine, which induces methemoglobinemia. Content experts in pediatric emergency medicine (PEM) and toxicology created this simulation and originally designed it for PEM fellow education. It is also relevant to other health professionals who care for acutely ill children, including pediatric residents, emergency medicine residents, family medicine residents, medical students, advanced practice providers, and nurses. Learners must evaluate and stabilize an infant presenting with central cyanosis and respiratory distress of unknown etiology. This simulation serves as an educational tool to accommodate this need. Importantly, toxidromes due to medication are a content domain for the PEM certification examination.7
This scenario encourages active learning and integration of previously acquired knowledge, skills, and behaviors as learners must evaluate and stabilize an infant presenting with central cyanosis and respiratory distress of unknown etiology. While the underlying etiology is lidocaine-induced methemoglobinemia, participants must work as a team to use a systematic approach to care for an acutely ill pediatric patient, a theme common to the emergent care of children. Through this scenario, learners must evaluate a cyanotic and hypoxic infant, implement initial resuscitation interventions, develop a broad differential diagnosis, and implement a diagnostic plan leading them to the eventual diagnosis of methemoglobinemia. While other simulation cases addressing methemoglobinemia exist, this scenario is unique in its focus on a neonate necessitating a broad differential diagnosis.8,9 This simulation-based curriculum can be used independently or in series with other sessions from the Pediatric Toxidrome Simulation Curriculum.10–15
Methods
Development
We created this simulation to teach learners a systemic approach to PEM care and promote interpersonal dialogue and communication. The primary goals of the case included working as a team evaluating a neonatal patient with hypoxia and central cyanosis, implementing initial steps of stabilization, thinking through a differential diagnosis, and identifying signs and symptoms of methemoglobinemia.
This simulation scenario was created by PEM attendings with expertise in simulation and curriculum design. A toxicologist provided additional support in the case creation as a content expert. This case was initially created for PEM fellows as part of their recurring simulation-based education. It was also implemented with other members of the emergency department (ED) clinical team who provide medical care to pediatric patients in an ED setting. No specific preparation was required by the learners prior to participation in the simulation. Participants had to have prerequisite knowledge about rapid evaluation and management of neonatal respiratory distress and skills in airway management, which were expected skills for medical personnel working in a pediatric ED. The instructors were provided with the simulation scenario (Appendix A), simulation environment preparation (Appendix B), diagnostic images including electrocardiogram and chest radiograph (Appendix C), teamwork and communication glossary (Appendix D), debriefing guide (Appendix E), and participant evaluation form (Appendix F). Educational slides (Appendix G) were reviewed with participants after the simulation to augment learning and reinforce important concepts. Assessment of the curriculum focused on levels 1 and 2 of Kirkpatrick's model.16
Equipment/Environment
All sites conducted this simulation in a pediatric ED or simulation laboratory with a high-fidelity, infant-sized manikin. The case could also be run in other clinical locations and/or using a low-fidelity manikin. Specific environmental preparation was available in Appendix B. We began the simulation with the manikin on the hospital bed after being brought to the ED by parents. Participants were told that he was in respiratory distress. There was no intravenous access. Equipment and medications commonly encountered in the ED environment were available as outlined in Appendix B. Printouts of an electrocardiogram and chest radiograph were available upon request, all of which showed normal findings (Appendix C).
If using a low-fidelity manikin, facilitators can report vital signs verbally or write them in an area easily seen by all participants. Physical exam findings can be verbally described during the evaluation. The central characteristic of this simulation, cyanosis, can be verbally communicated to the participants. Additionally, moulage can be considered to make the patient appear to be cyanotic.
Personnel
This simulation scenario was performed with seven to 16 learners during each session. Participants were oriented to the simulation, including the manikin and safe learning environment principles. Available roles included three physician/medical provider roles, four nursing roles, and two instructor roles. For sessions in which interdisciplinary team members were not present, physicians filled these roles. The optimal number of participants was seven (three physician/medical provider roles and four nursing roles). The optimal number of instructors was two. Roles for pharmacists and respiratory therapists could be incorporated into the simulation if personnel were available. If the scenario was completed with an interdisciplinary team, personnel maintained the roles of their employment; for example, a fellow would be in the role of a physician, and a nurse would be in the role of a nurse. If the participant group was smaller than the typical clinical team, the simulation would be completed with a minimum of three participants fulfilling physician roles and one facilitator fulfilling an instructor role. This would, however, decrease the opportunity to practice teamwork and communication skills. For larger groups, such as the group of sixteen participants, learners who did not have a designated role were recommended to observe and participate in the didactics and debrief. Instructors were all PEM-trained physicians with experience in medical simulation.
Implementation
This scenario was implemented with a total of 56 participants (including 18 PEM fellows) at four sites across the United States. Each site performed the scenario between one and three times with different groups of learners. The scenario was described in Appendix A. The scenario started with the infant in an ED exam room after having been rushed there from triage. The ED team was called to bedside to evaluate the child with central cyanosis and respiratory distress. The team member assigned to the role of nurse applied the monitors, which showed an oxygen saturation of 85% via the simulation monitor. This alerted the team to make attempts to support the child's breathing during the primary survey. A facilitator or embedded participant played the role of the parent and, upon request, gave the team history, which helped narrow the differential diagnosis to methemoglobinemia induced by lidocaine exposure. Labs as requested in addition to use of CO-oximetry confirmed the diagnosis and appropriate treatment. At participant request, electrocardiogram and chest radiograph (Appendix C) were available and unremarkable. Appropriate sign-out to the neonatal intensive care unit was expected at the conclusion of the scenario. Facilitators were provided with teamwork and communication tools prior to each session, found in Appendix D. This allowed for standardized terminology to debrief the scenario in order to optimize the educational experience. The debriefing guide was provided in Appendix E to allow facilitators to provide effective feedback and participants to obtain optimal learning from the experience. A slide-based didactic to aid in the debriefing was provided in Appendix G. An evaluation form for participants to complete after case participation was provided in Appendix F and could be used to adjust the case in the future.
Assessment
The scenario was facilitated and debriefed by PEM physicians experienced in simulation. The participants were PEM fellows, emergency medicine residents, pediatric and emergency medicine interns, PEM RNs, PEM attendings, a respiratory therapist, and a physician assistant. Facilitators provided pediatric resuscitation and content expertise as it related to managing a cyanotic infant and methemoglobinemia. Facilitators also provided participant performance feedback in accordance with the learning objectives. Following the scenario debriefing, participants completed the evaluation form (Appendix F) to give facilitators feedback on the relevance, realism, and overall learning experience of the simulation scenario. The evaluation form included statements rated on a 5-point Likert scale (1 = strongly disagree, 2 = disagree, 3 = neutral, 4 = agree, 5 = strongly agree) to assess the success of the simulation in addressing the learning objectives and to report confidence managing a similar scenario after participation. Respondents had the opportunity to provide additional feedback on the clinical impact and ideas for scenario improvement through free text and open-ended questions, including “Can you list/describe one or more ways this simulation session will change how you do your job?”, “How can we improve this scenario?”, and “Additional comments.” Median Likert scores were calculated for each item on the survey.
Debriefing
We used the tools in Appendices D and E to assist the facilitation debrief sessions after the simulations. These guides provided an outline for leading a debriefing session. We recommend that these sessions begin by encouraging participants to share their overall reflection of the scenario, followed by a structured discussion outlined in Appendix E. The discussion included topics of teamwork and communication (Appendix D) as well as reiteration of diagnostic and management skills.
Results
The scenario was facilitated by experienced PEM simulation faculty across four institutions who provided content expertise and constructive feedback to learners with regard to the learning objectives. A total of 56 individuals participated in the simulations and completed the postsimulation survey. Participants included PEM fellows, emergency medicine residents, pediatric and emergency medicine interns, a physician assistant, a respiratory therapist, PEM nurses, and PEM attendings. Numbers of participants from each discipline and level of training are described in Table 1. The largest portion was PEM fellows, who made up 32% of participants. Emergency medicine residents composed the second largest group of 25% of the total participants. The smallest simulation was completed with seven participants, and the largest was completed with 16 participants. In groups with more participants than available roles, learners without assigned roles observed the simulation and participated in the didactic and debrief. Table 2 summarizes participants' experience related to the simulation case and session facilitation. Overall, participants strongly agreed that the simulation was relevant to their work, realistic, and effective in teaching the objectives and that it promoted team reflection. Table 3 summarizes participant self-appraisal after participation related to the learning objectives. Overall, participants were confident in their ability to perform the designated learning objectives on subsequent patients.
Table 1. Simulation Participants (N = 56)
Table 2. Participant Experience During the Simulation Session (N = 56)
Table 3. Participant Clinical Confidence After Participating in the Session (N = 56)
Each participant was given the opportunity to respond to the following question: “Can you list/describe one or more ways this session will change how you do your job?” Responses primarily discussed improved differential diagnosis building with respect to a hypoxic and cyanotic pediatric patient and further understanding of methemoglobinemia pathophysiology. Responses included the following:
• “Critically think about cyanosis in a patient with a normal venous blood gas.”
• “Recognize that seemingly benign medications (to parents) can have true complications.”
• “Thinking of methemoglobinemia as part of my differential diagnosis for cyanosis.”
• “Understanding of indications for methylene blue treatment.”
• “Encourage me and widen differential diagnosis and gather more extensive history.”
• “Knowing availability of CO-oximetry within my practice.”
Of note, one PEM fellow, who had participated in the first iteration of this case, encountered a child with cyanosis and desaturations unresponsive to supplemental oxygenation several months after completing the simulation. He attributed his quick consideration of and evaluation for methemoglobinemia to having participated in this simulation session.
Participants' suggestions for improvement to the session included making the SpO2 reading 85%-88%, keeping learner groups to fewer than 10 participants, and allowing more time for the session and debrief.
Discussion
Due to widespread availability of topical anesthetics and the severity of the potential complication of methemoglobinemia, health care providers caring for children in ED settings must be able to recognize and treat this rare but fatal complication. This simulation provides an opportunity for PEM fellows to review the differential diagnosis of a hypoxic and cyanotic infant; identify history, symptoms, and physical exam findings consistent with lidocaine toxicity and resultant methemoglobinemia; and strengthen team communication skills. While other curricula related to lidocaine toxicity have appeared in MedEdPORTAL, none are specific to the evaluation of a hypoxic and cyanotic infant.8,9 This curriculum provides a modality for learners to expand their differential diagnosis to include lidocaine toxicity in hypoxic infants. For many pediatric and emergency medicine providers, this scenario is rare but life-threatening. Prompt recognition and appropriate management are important. Additionally, the scenario allows learners to enhance teamwork and communication skills in a controlled environment while also providing a dedicated space to debrief team dynamics.
Multiple learners have participated in the simulation, which is necessary to refine and adapt the material. The simulation case was adjusted after the initial iteration to include an oxygenation saturation typical of methemoglobinemia. Larger groups of learners identified a benefit to completing the simulation in smaller groups. While that was not always possible due to time constraints and predetermined learner group size, subsequent sessions should be held with groups small enough to allow ample opportunity for participation and engagement. If group sizes must be larger than the number of personnel required to run the case, some participants should be assigned an observer role with a special focus on providing observations and feedback to the other participants. This allows the simulation to maintain the realism of a resuscitation. There should be sufficient participants and facilitators to ensure the crucial roles are filled.
A primary limitation of this activity is the difficulty of incorporating all providers typically present on a resuscitation team due to scheduling constraints of nurses, respiratory therapists, and pharmacists. While the simulation was created for PEM fellows, the interdisciplinary teamwork and debrief are enhanced by the incorporation of health care providers from various disciplines. In the future, this simulation should prioritize PEM fellow involvement with incorporation of disciplines who typically care for children in the ED. While PEM fellows make up the largest group of participants for this simulation, they constitute only 32% of participants. This is due to availability of PEM fellows at the institutions included in the evaluation of the simulation and the simultaneous desire to include all health care professionals working in the ED. A limitation of the evaluation process is the reliance of the evaluations on participant perception of improvement of skills after completing the session. Teach-back methods and testing/retesting knowledge, behaviors, and skills through additional simulations have not been evaluated. The immediate feedback provided by learners, however, has been reviewed and assessed for integration in future iterations, as described in preceding paragraphs. Additionally, to better define the time frame within which these knowledge and skills are obtained and reevaluated, future iterations of this simulation should include a time frame associated with each objective.
Appendices
Simulation Case.docx
Environment Preparation.docx
Images.pptx
Teamwork and Communication Glossary.docx
Debriefing Guide.docx
Evaluation Form.docx
Didactics.pptx
All appendices are peer reviewed as integral parts of the Original Publication.
Acknowledgments
The authors would like to thank Jennifer Reid, MD, and Kimberly Stone, MD, for their contributions to the development and execution of these simulations.
Disclosures
None to report.
Funding/Support
None to report.
Ethical Approval
Reported as not applicable. | LIDOCAINE | DrugsGivenReaction | CC BY | 33537407 | 19,389,785 | 2021-01-28 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Cyanosis neonatal'. | Pediatric Toxidrome Simulation Curriculum: Lidocaine-Induced Methemoglobinemia.
Lidocaine is a common local anesthetic used during minor procedures performed on pediatric patients. A rare but toxic and life-threatening side effect of lidocaine is methemoglobinemia. It should be considered in children who are hypoxic after exposure to an oxidizing agent.
We developed this simulation case for pediatric emergency medicine (PEM) fellows, but it can be adapted for interprofessional simulation. The case involved a 1-month-old male with hypoxia and resulting central cyanosis after exposure to lidocaine. The team performed an initial evaluation and intervention, collected a history, and developed a differential diagnosis for hypoxia and central cyanosis in an infant. Methemoglobinemia was confirmed by CO-oximetry. Preparatory materials, a debriefing guide, and scenario evaluation forms assisted with facilitation.
Fifty-six participants (including 18 PEM fellows) completed this simulation across four institutions. Participants rated the scenario on a 5-point Likert scale (1 = strongly disagree, 5 = strongly agree), finding it to be relevant to their work (median = 5) and realistic (median = 5). After participation in the simulation, learners felt confident in their ability to recognize methemoglobinemia (median = 4) and implement a plan to stabilize an infant with hypoxia (median = 4).
This simulation represents a resource for learners in the pediatric emergency department. It teaches the recognition and management of an infant with lidocaine toxicity and resultant methemoglobinemia. It uses experiential learning to teach and reinforce a systematic approach to the evaluation and management of a critically ill infant with acquired methemoglobinemia.
Educational Objectives
After participation in this simulation session, learners will be able to:
1. Perform a primary survey of a critically ill pediatric patient.
2. Implement a plan to stabilize a hypoxic and cyanotic neonate.
3. Develop a systematic approach for the evaluation of hypoxia and central cyanosis in a pediatric patient.
4. Describe the signs and symptoms of acquired methemoglobinemia in a pediatric patient.
5. Manage a pediatric patient with acquired methemoglobinemia.
6. Demonstrate teamwork and communication skills in a resuscitation setting.
Introduction
Methemoglobinemia is a rare but life-threatening disorder and can present at any age after exposure to an oxidizing agent. It occurs when hemoglobin becomes altered, causing the irreversible binding of oxygen through oxidation of ferrous ions associated with heme to the ferric state. The ferric ions of heme (Fe+++) cannot dissociate from oxygen, causing a left shift in the oxyhemoglobin dissociation curve and subsequent hypoxia.1 The impaired oxygen delivery to the tissues causes central cyanosis even with typical supportive measures to improve tissue oxygenation.1 The resultant hypoxia may lead to respiratory failure, end-organ damage, and death in severe cases.1
Methemoglobinemia develops as the result of a congenital condition or toxin exposure. Congenital methemoglobinemia is rare and is due to an abnormal hemoglobin structure or a deficiency in a reducing enzyme. These mutations can be autosomal recessive or dominant and include disease entities such as cytochrome b5 reductase deficiency and hemoglobin M disease.2 Acquired cases of methemoglobinemia are more prevalent, although the incidence is not well defined. Typically, medications and other chemical substances with oxidative potential are the cause. Common medications with oxidative potential include local anesthetics (lidocaine, benzocaine, and prilocaine), sulfa-containing medications, and dapsone.3 Infants are particularly susceptible to methemoglobinemia when facing an oxidative stress due to the immaturity of their enzymatic systems and increased potential for accidental overdose.3
Methemoglobinemia presents with hypoxia causing cyanosis, which can be a diagnostic challenge in a neonate. The possible causes of hypoxia in a neonate are extensive and include, but are not limited to, cyanotic congenital heart disease, sepsis, pneumonia, congenital airway or pulmonary anatomic abnormalities, pulmonary interstitial abnormalities, neurologic conditions, toxidromes, and hemoglobinopathies. While pulmonary etiologies are the most common cause of hypoxia and central cyanosis in an otherwise healthy child, methemoglobinemia should be considered in patients with exposure to oxidizing agents or a family history of congenital methemoglobinemia.
Infants experiencing acute methemoglobinemia may develop a physical exam finding of central cyanosis, hypoxia, increased work of breathing, altered mental status, respiratory depression, and seizures.3 The end result may be death.3 Central cyanosis appears differently depending on the pigmentation of a child's skin. Children with more skin pigmentation have bluish-grey color changes most noticeable on the tongue and mucosal membranes. Children with less skin pigmentation have blue color changes most noticeable on the tongue, mucosal membranes, and perioral area. A key indication of possible methemoglobinemia is a peripheral oxygen saturation detected by pulse oximetry that does not improve with oxygen administration. Upon phlebotomy, the blood remains dark brown in color after exposure to oxygen.3 The arterial blood gas is typically within normal limits. A peripheral capillary oxygen saturation (SpO2) monitor often underestimates hypoxia in the setting of high levels of methemoglobin, so this should not be relied upon for clinical monitoring.4 CO-oximetry should be used to detect the percentage of methemoglobin. It does this by measuring specific wavelengths of light corresponding to methemoglobin (660 nm and 940 nm).4
A majority of cases of methemoglobinemia resolve with supportive care only. More severe cases, however, can be treated with methylene blue to reduce the ferric ions of the heme molecules back to their ferrous state. This allows for appropriate oxygen dissociation from hemoglobin.5 The most commonly accepted methemoglobin level requiring treatment is a level greater than 20% of hemoglobin.6 If the patient is significantly symptomatic, however, methylene blue may be administered with lower levels. It is typically dosed at one to two milligrams per kilogram.6 The overall efficacy of methylene blue in the treatment of methemoglobinemia varies. The primary contraindication to methylene blue use is glucose-6-phophate dehydrogenase deficiency due to the risk of hemolysis.6
This simulation case presents a rare but potentially lethal toxic exposure of topical lidocaine, which induces methemoglobinemia. Content experts in pediatric emergency medicine (PEM) and toxicology created this simulation and originally designed it for PEM fellow education. It is also relevant to other health professionals who care for acutely ill children, including pediatric residents, emergency medicine residents, family medicine residents, medical students, advanced practice providers, and nurses. Learners must evaluate and stabilize an infant presenting with central cyanosis and respiratory distress of unknown etiology. This simulation serves as an educational tool to accommodate this need. Importantly, toxidromes due to medication are a content domain for the PEM certification examination.7
This scenario encourages active learning and integration of previously acquired knowledge, skills, and behaviors as learners must evaluate and stabilize an infant presenting with central cyanosis and respiratory distress of unknown etiology. While the underlying etiology is lidocaine-induced methemoglobinemia, participants must work as a team to use a systematic approach to care for an acutely ill pediatric patient, a theme common to the emergent care of children. Through this scenario, learners must evaluate a cyanotic and hypoxic infant, implement initial resuscitation interventions, develop a broad differential diagnosis, and implement a diagnostic plan leading them to the eventual diagnosis of methemoglobinemia. While other simulation cases addressing methemoglobinemia exist, this scenario is unique in its focus on a neonate necessitating a broad differential diagnosis.8,9 This simulation-based curriculum can be used independently or in series with other sessions from the Pediatric Toxidrome Simulation Curriculum.10–15
Methods
Development
We created this simulation to teach learners a systemic approach to PEM care and promote interpersonal dialogue and communication. The primary goals of the case included working as a team evaluating a neonatal patient with hypoxia and central cyanosis, implementing initial steps of stabilization, thinking through a differential diagnosis, and identifying signs and symptoms of methemoglobinemia.
This simulation scenario was created by PEM attendings with expertise in simulation and curriculum design. A toxicologist provided additional support in the case creation as a content expert. This case was initially created for PEM fellows as part of their recurring simulation-based education. It was also implemented with other members of the emergency department (ED) clinical team who provide medical care to pediatric patients in an ED setting. No specific preparation was required by the learners prior to participation in the simulation. Participants had to have prerequisite knowledge about rapid evaluation and management of neonatal respiratory distress and skills in airway management, which were expected skills for medical personnel working in a pediatric ED. The instructors were provided with the simulation scenario (Appendix A), simulation environment preparation (Appendix B), diagnostic images including electrocardiogram and chest radiograph (Appendix C), teamwork and communication glossary (Appendix D), debriefing guide (Appendix E), and participant evaluation form (Appendix F). Educational slides (Appendix G) were reviewed with participants after the simulation to augment learning and reinforce important concepts. Assessment of the curriculum focused on levels 1 and 2 of Kirkpatrick's model.16
Equipment/Environment
All sites conducted this simulation in a pediatric ED or simulation laboratory with a high-fidelity, infant-sized manikin. The case could also be run in other clinical locations and/or using a low-fidelity manikin. Specific environmental preparation was available in Appendix B. We began the simulation with the manikin on the hospital bed after being brought to the ED by parents. Participants were told that he was in respiratory distress. There was no intravenous access. Equipment and medications commonly encountered in the ED environment were available as outlined in Appendix B. Printouts of an electrocardiogram and chest radiograph were available upon request, all of which showed normal findings (Appendix C).
If using a low-fidelity manikin, facilitators can report vital signs verbally or write them in an area easily seen by all participants. Physical exam findings can be verbally described during the evaluation. The central characteristic of this simulation, cyanosis, can be verbally communicated to the participants. Additionally, moulage can be considered to make the patient appear to be cyanotic.
Personnel
This simulation scenario was performed with seven to 16 learners during each session. Participants were oriented to the simulation, including the manikin and safe learning environment principles. Available roles included three physician/medical provider roles, four nursing roles, and two instructor roles. For sessions in which interdisciplinary team members were not present, physicians filled these roles. The optimal number of participants was seven (three physician/medical provider roles and four nursing roles). The optimal number of instructors was two. Roles for pharmacists and respiratory therapists could be incorporated into the simulation if personnel were available. If the scenario was completed with an interdisciplinary team, personnel maintained the roles of their employment; for example, a fellow would be in the role of a physician, and a nurse would be in the role of a nurse. If the participant group was smaller than the typical clinical team, the simulation would be completed with a minimum of three participants fulfilling physician roles and one facilitator fulfilling an instructor role. This would, however, decrease the opportunity to practice teamwork and communication skills. For larger groups, such as the group of sixteen participants, learners who did not have a designated role were recommended to observe and participate in the didactics and debrief. Instructors were all PEM-trained physicians with experience in medical simulation.
Implementation
This scenario was implemented with a total of 56 participants (including 18 PEM fellows) at four sites across the United States. Each site performed the scenario between one and three times with different groups of learners. The scenario was described in Appendix A. The scenario started with the infant in an ED exam room after having been rushed there from triage. The ED team was called to bedside to evaluate the child with central cyanosis and respiratory distress. The team member assigned to the role of nurse applied the monitors, which showed an oxygen saturation of 85% via the simulation monitor. This alerted the team to make attempts to support the child's breathing during the primary survey. A facilitator or embedded participant played the role of the parent and, upon request, gave the team history, which helped narrow the differential diagnosis to methemoglobinemia induced by lidocaine exposure. Labs as requested in addition to use of CO-oximetry confirmed the diagnosis and appropriate treatment. At participant request, electrocardiogram and chest radiograph (Appendix C) were available and unremarkable. Appropriate sign-out to the neonatal intensive care unit was expected at the conclusion of the scenario. Facilitators were provided with teamwork and communication tools prior to each session, found in Appendix D. This allowed for standardized terminology to debrief the scenario in order to optimize the educational experience. The debriefing guide was provided in Appendix E to allow facilitators to provide effective feedback and participants to obtain optimal learning from the experience. A slide-based didactic to aid in the debriefing was provided in Appendix G. An evaluation form for participants to complete after case participation was provided in Appendix F and could be used to adjust the case in the future.
Assessment
The scenario was facilitated and debriefed by PEM physicians experienced in simulation. The participants were PEM fellows, emergency medicine residents, pediatric and emergency medicine interns, PEM RNs, PEM attendings, a respiratory therapist, and a physician assistant. Facilitators provided pediatric resuscitation and content expertise as it related to managing a cyanotic infant and methemoglobinemia. Facilitators also provided participant performance feedback in accordance with the learning objectives. Following the scenario debriefing, participants completed the evaluation form (Appendix F) to give facilitators feedback on the relevance, realism, and overall learning experience of the simulation scenario. The evaluation form included statements rated on a 5-point Likert scale (1 = strongly disagree, 2 = disagree, 3 = neutral, 4 = agree, 5 = strongly agree) to assess the success of the simulation in addressing the learning objectives and to report confidence managing a similar scenario after participation. Respondents had the opportunity to provide additional feedback on the clinical impact and ideas for scenario improvement through free text and open-ended questions, including “Can you list/describe one or more ways this simulation session will change how you do your job?”, “How can we improve this scenario?”, and “Additional comments.” Median Likert scores were calculated for each item on the survey.
Debriefing
We used the tools in Appendices D and E to assist the facilitation debrief sessions after the simulations. These guides provided an outline for leading a debriefing session. We recommend that these sessions begin by encouraging participants to share their overall reflection of the scenario, followed by a structured discussion outlined in Appendix E. The discussion included topics of teamwork and communication (Appendix D) as well as reiteration of diagnostic and management skills.
Results
The scenario was facilitated by experienced PEM simulation faculty across four institutions who provided content expertise and constructive feedback to learners with regard to the learning objectives. A total of 56 individuals participated in the simulations and completed the postsimulation survey. Participants included PEM fellows, emergency medicine residents, pediatric and emergency medicine interns, a physician assistant, a respiratory therapist, PEM nurses, and PEM attendings. Numbers of participants from each discipline and level of training are described in Table 1. The largest portion was PEM fellows, who made up 32% of participants. Emergency medicine residents composed the second largest group of 25% of the total participants. The smallest simulation was completed with seven participants, and the largest was completed with 16 participants. In groups with more participants than available roles, learners without assigned roles observed the simulation and participated in the didactic and debrief. Table 2 summarizes participants' experience related to the simulation case and session facilitation. Overall, participants strongly agreed that the simulation was relevant to their work, realistic, and effective in teaching the objectives and that it promoted team reflection. Table 3 summarizes participant self-appraisal after participation related to the learning objectives. Overall, participants were confident in their ability to perform the designated learning objectives on subsequent patients.
Table 1. Simulation Participants (N = 56)
Table 2. Participant Experience During the Simulation Session (N = 56)
Table 3. Participant Clinical Confidence After Participating in the Session (N = 56)
Each participant was given the opportunity to respond to the following question: “Can you list/describe one or more ways this session will change how you do your job?” Responses primarily discussed improved differential diagnosis building with respect to a hypoxic and cyanotic pediatric patient and further understanding of methemoglobinemia pathophysiology. Responses included the following:
• “Critically think about cyanosis in a patient with a normal venous blood gas.”
• “Recognize that seemingly benign medications (to parents) can have true complications.”
• “Thinking of methemoglobinemia as part of my differential diagnosis for cyanosis.”
• “Understanding of indications for methylene blue treatment.”
• “Encourage me and widen differential diagnosis and gather more extensive history.”
• “Knowing availability of CO-oximetry within my practice.”
Of note, one PEM fellow, who had participated in the first iteration of this case, encountered a child with cyanosis and desaturations unresponsive to supplemental oxygenation several months after completing the simulation. He attributed his quick consideration of and evaluation for methemoglobinemia to having participated in this simulation session.
Participants' suggestions for improvement to the session included making the SpO2 reading 85%-88%, keeping learner groups to fewer than 10 participants, and allowing more time for the session and debrief.
Discussion
Due to widespread availability of topical anesthetics and the severity of the potential complication of methemoglobinemia, health care providers caring for children in ED settings must be able to recognize and treat this rare but fatal complication. This simulation provides an opportunity for PEM fellows to review the differential diagnosis of a hypoxic and cyanotic infant; identify history, symptoms, and physical exam findings consistent with lidocaine toxicity and resultant methemoglobinemia; and strengthen team communication skills. While other curricula related to lidocaine toxicity have appeared in MedEdPORTAL, none are specific to the evaluation of a hypoxic and cyanotic infant.8,9 This curriculum provides a modality for learners to expand their differential diagnosis to include lidocaine toxicity in hypoxic infants. For many pediatric and emergency medicine providers, this scenario is rare but life-threatening. Prompt recognition and appropriate management are important. Additionally, the scenario allows learners to enhance teamwork and communication skills in a controlled environment while also providing a dedicated space to debrief team dynamics.
Multiple learners have participated in the simulation, which is necessary to refine and adapt the material. The simulation case was adjusted after the initial iteration to include an oxygenation saturation typical of methemoglobinemia. Larger groups of learners identified a benefit to completing the simulation in smaller groups. While that was not always possible due to time constraints and predetermined learner group size, subsequent sessions should be held with groups small enough to allow ample opportunity for participation and engagement. If group sizes must be larger than the number of personnel required to run the case, some participants should be assigned an observer role with a special focus on providing observations and feedback to the other participants. This allows the simulation to maintain the realism of a resuscitation. There should be sufficient participants and facilitators to ensure the crucial roles are filled.
A primary limitation of this activity is the difficulty of incorporating all providers typically present on a resuscitation team due to scheduling constraints of nurses, respiratory therapists, and pharmacists. While the simulation was created for PEM fellows, the interdisciplinary teamwork and debrief are enhanced by the incorporation of health care providers from various disciplines. In the future, this simulation should prioritize PEM fellow involvement with incorporation of disciplines who typically care for children in the ED. While PEM fellows make up the largest group of participants for this simulation, they constitute only 32% of participants. This is due to availability of PEM fellows at the institutions included in the evaluation of the simulation and the simultaneous desire to include all health care professionals working in the ED. A limitation of the evaluation process is the reliance of the evaluations on participant perception of improvement of skills after completing the session. Teach-back methods and testing/retesting knowledge, behaviors, and skills through additional simulations have not been evaluated. The immediate feedback provided by learners, however, has been reviewed and assessed for integration in future iterations, as described in preceding paragraphs. Additionally, to better define the time frame within which these knowledge and skills are obtained and reevaluated, future iterations of this simulation should include a time frame associated with each objective.
Appendices
Simulation Case.docx
Environment Preparation.docx
Images.pptx
Teamwork and Communication Glossary.docx
Debriefing Guide.docx
Evaluation Form.docx
Didactics.pptx
All appendices are peer reviewed as integral parts of the Original Publication.
Acknowledgments
The authors would like to thank Jennifer Reid, MD, and Kimberly Stone, MD, for their contributions to the development and execution of these simulations.
Disclosures
None to report.
Funding/Support
None to report.
Ethical Approval
Reported as not applicable. | LIDOCAINE | DrugsGivenReaction | CC BY | 33537407 | 19,389,785 | 2021-01-28 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Methaemoglobinaemia'. | Pediatric Toxidrome Simulation Curriculum: Lidocaine-Induced Methemoglobinemia.
Lidocaine is a common local anesthetic used during minor procedures performed on pediatric patients. A rare but toxic and life-threatening side effect of lidocaine is methemoglobinemia. It should be considered in children who are hypoxic after exposure to an oxidizing agent.
We developed this simulation case for pediatric emergency medicine (PEM) fellows, but it can be adapted for interprofessional simulation. The case involved a 1-month-old male with hypoxia and resulting central cyanosis after exposure to lidocaine. The team performed an initial evaluation and intervention, collected a history, and developed a differential diagnosis for hypoxia and central cyanosis in an infant. Methemoglobinemia was confirmed by CO-oximetry. Preparatory materials, a debriefing guide, and scenario evaluation forms assisted with facilitation.
Fifty-six participants (including 18 PEM fellows) completed this simulation across four institutions. Participants rated the scenario on a 5-point Likert scale (1 = strongly disagree, 5 = strongly agree), finding it to be relevant to their work (median = 5) and realistic (median = 5). After participation in the simulation, learners felt confident in their ability to recognize methemoglobinemia (median = 4) and implement a plan to stabilize an infant with hypoxia (median = 4).
This simulation represents a resource for learners in the pediatric emergency department. It teaches the recognition and management of an infant with lidocaine toxicity and resultant methemoglobinemia. It uses experiential learning to teach and reinforce a systematic approach to the evaluation and management of a critically ill infant with acquired methemoglobinemia.
Educational Objectives
After participation in this simulation session, learners will be able to:
1. Perform a primary survey of a critically ill pediatric patient.
2. Implement a plan to stabilize a hypoxic and cyanotic neonate.
3. Develop a systematic approach for the evaluation of hypoxia and central cyanosis in a pediatric patient.
4. Describe the signs and symptoms of acquired methemoglobinemia in a pediatric patient.
5. Manage a pediatric patient with acquired methemoglobinemia.
6. Demonstrate teamwork and communication skills in a resuscitation setting.
Introduction
Methemoglobinemia is a rare but life-threatening disorder and can present at any age after exposure to an oxidizing agent. It occurs when hemoglobin becomes altered, causing the irreversible binding of oxygen through oxidation of ferrous ions associated with heme to the ferric state. The ferric ions of heme (Fe+++) cannot dissociate from oxygen, causing a left shift in the oxyhemoglobin dissociation curve and subsequent hypoxia.1 The impaired oxygen delivery to the tissues causes central cyanosis even with typical supportive measures to improve tissue oxygenation.1 The resultant hypoxia may lead to respiratory failure, end-organ damage, and death in severe cases.1
Methemoglobinemia develops as the result of a congenital condition or toxin exposure. Congenital methemoglobinemia is rare and is due to an abnormal hemoglobin structure or a deficiency in a reducing enzyme. These mutations can be autosomal recessive or dominant and include disease entities such as cytochrome b5 reductase deficiency and hemoglobin M disease.2 Acquired cases of methemoglobinemia are more prevalent, although the incidence is not well defined. Typically, medications and other chemical substances with oxidative potential are the cause. Common medications with oxidative potential include local anesthetics (lidocaine, benzocaine, and prilocaine), sulfa-containing medications, and dapsone.3 Infants are particularly susceptible to methemoglobinemia when facing an oxidative stress due to the immaturity of their enzymatic systems and increased potential for accidental overdose.3
Methemoglobinemia presents with hypoxia causing cyanosis, which can be a diagnostic challenge in a neonate. The possible causes of hypoxia in a neonate are extensive and include, but are not limited to, cyanotic congenital heart disease, sepsis, pneumonia, congenital airway or pulmonary anatomic abnormalities, pulmonary interstitial abnormalities, neurologic conditions, toxidromes, and hemoglobinopathies. While pulmonary etiologies are the most common cause of hypoxia and central cyanosis in an otherwise healthy child, methemoglobinemia should be considered in patients with exposure to oxidizing agents or a family history of congenital methemoglobinemia.
Infants experiencing acute methemoglobinemia may develop a physical exam finding of central cyanosis, hypoxia, increased work of breathing, altered mental status, respiratory depression, and seizures.3 The end result may be death.3 Central cyanosis appears differently depending on the pigmentation of a child's skin. Children with more skin pigmentation have bluish-grey color changes most noticeable on the tongue and mucosal membranes. Children with less skin pigmentation have blue color changes most noticeable on the tongue, mucosal membranes, and perioral area. A key indication of possible methemoglobinemia is a peripheral oxygen saturation detected by pulse oximetry that does not improve with oxygen administration. Upon phlebotomy, the blood remains dark brown in color after exposure to oxygen.3 The arterial blood gas is typically within normal limits. A peripheral capillary oxygen saturation (SpO2) monitor often underestimates hypoxia in the setting of high levels of methemoglobin, so this should not be relied upon for clinical monitoring.4 CO-oximetry should be used to detect the percentage of methemoglobin. It does this by measuring specific wavelengths of light corresponding to methemoglobin (660 nm and 940 nm).4
A majority of cases of methemoglobinemia resolve with supportive care only. More severe cases, however, can be treated with methylene blue to reduce the ferric ions of the heme molecules back to their ferrous state. This allows for appropriate oxygen dissociation from hemoglobin.5 The most commonly accepted methemoglobin level requiring treatment is a level greater than 20% of hemoglobin.6 If the patient is significantly symptomatic, however, methylene blue may be administered with lower levels. It is typically dosed at one to two milligrams per kilogram.6 The overall efficacy of methylene blue in the treatment of methemoglobinemia varies. The primary contraindication to methylene blue use is glucose-6-phophate dehydrogenase deficiency due to the risk of hemolysis.6
This simulation case presents a rare but potentially lethal toxic exposure of topical lidocaine, which induces methemoglobinemia. Content experts in pediatric emergency medicine (PEM) and toxicology created this simulation and originally designed it for PEM fellow education. It is also relevant to other health professionals who care for acutely ill children, including pediatric residents, emergency medicine residents, family medicine residents, medical students, advanced practice providers, and nurses. Learners must evaluate and stabilize an infant presenting with central cyanosis and respiratory distress of unknown etiology. This simulation serves as an educational tool to accommodate this need. Importantly, toxidromes due to medication are a content domain for the PEM certification examination.7
This scenario encourages active learning and integration of previously acquired knowledge, skills, and behaviors as learners must evaluate and stabilize an infant presenting with central cyanosis and respiratory distress of unknown etiology. While the underlying etiology is lidocaine-induced methemoglobinemia, participants must work as a team to use a systematic approach to care for an acutely ill pediatric patient, a theme common to the emergent care of children. Through this scenario, learners must evaluate a cyanotic and hypoxic infant, implement initial resuscitation interventions, develop a broad differential diagnosis, and implement a diagnostic plan leading them to the eventual diagnosis of methemoglobinemia. While other simulation cases addressing methemoglobinemia exist, this scenario is unique in its focus on a neonate necessitating a broad differential diagnosis.8,9 This simulation-based curriculum can be used independently or in series with other sessions from the Pediatric Toxidrome Simulation Curriculum.10–15
Methods
Development
We created this simulation to teach learners a systemic approach to PEM care and promote interpersonal dialogue and communication. The primary goals of the case included working as a team evaluating a neonatal patient with hypoxia and central cyanosis, implementing initial steps of stabilization, thinking through a differential diagnosis, and identifying signs and symptoms of methemoglobinemia.
This simulation scenario was created by PEM attendings with expertise in simulation and curriculum design. A toxicologist provided additional support in the case creation as a content expert. This case was initially created for PEM fellows as part of their recurring simulation-based education. It was also implemented with other members of the emergency department (ED) clinical team who provide medical care to pediatric patients in an ED setting. No specific preparation was required by the learners prior to participation in the simulation. Participants had to have prerequisite knowledge about rapid evaluation and management of neonatal respiratory distress and skills in airway management, which were expected skills for medical personnel working in a pediatric ED. The instructors were provided with the simulation scenario (Appendix A), simulation environment preparation (Appendix B), diagnostic images including electrocardiogram and chest radiograph (Appendix C), teamwork and communication glossary (Appendix D), debriefing guide (Appendix E), and participant evaluation form (Appendix F). Educational slides (Appendix G) were reviewed with participants after the simulation to augment learning and reinforce important concepts. Assessment of the curriculum focused on levels 1 and 2 of Kirkpatrick's model.16
Equipment/Environment
All sites conducted this simulation in a pediatric ED or simulation laboratory with a high-fidelity, infant-sized manikin. The case could also be run in other clinical locations and/or using a low-fidelity manikin. Specific environmental preparation was available in Appendix B. We began the simulation with the manikin on the hospital bed after being brought to the ED by parents. Participants were told that he was in respiratory distress. There was no intravenous access. Equipment and medications commonly encountered in the ED environment were available as outlined in Appendix B. Printouts of an electrocardiogram and chest radiograph were available upon request, all of which showed normal findings (Appendix C).
If using a low-fidelity manikin, facilitators can report vital signs verbally or write them in an area easily seen by all participants. Physical exam findings can be verbally described during the evaluation. The central characteristic of this simulation, cyanosis, can be verbally communicated to the participants. Additionally, moulage can be considered to make the patient appear to be cyanotic.
Personnel
This simulation scenario was performed with seven to 16 learners during each session. Participants were oriented to the simulation, including the manikin and safe learning environment principles. Available roles included three physician/medical provider roles, four nursing roles, and two instructor roles. For sessions in which interdisciplinary team members were not present, physicians filled these roles. The optimal number of participants was seven (three physician/medical provider roles and four nursing roles). The optimal number of instructors was two. Roles for pharmacists and respiratory therapists could be incorporated into the simulation if personnel were available. If the scenario was completed with an interdisciplinary team, personnel maintained the roles of their employment; for example, a fellow would be in the role of a physician, and a nurse would be in the role of a nurse. If the participant group was smaller than the typical clinical team, the simulation would be completed with a minimum of three participants fulfilling physician roles and one facilitator fulfilling an instructor role. This would, however, decrease the opportunity to practice teamwork and communication skills. For larger groups, such as the group of sixteen participants, learners who did not have a designated role were recommended to observe and participate in the didactics and debrief. Instructors were all PEM-trained physicians with experience in medical simulation.
Implementation
This scenario was implemented with a total of 56 participants (including 18 PEM fellows) at four sites across the United States. Each site performed the scenario between one and three times with different groups of learners. The scenario was described in Appendix A. The scenario started with the infant in an ED exam room after having been rushed there from triage. The ED team was called to bedside to evaluate the child with central cyanosis and respiratory distress. The team member assigned to the role of nurse applied the monitors, which showed an oxygen saturation of 85% via the simulation monitor. This alerted the team to make attempts to support the child's breathing during the primary survey. A facilitator or embedded participant played the role of the parent and, upon request, gave the team history, which helped narrow the differential diagnosis to methemoglobinemia induced by lidocaine exposure. Labs as requested in addition to use of CO-oximetry confirmed the diagnosis and appropriate treatment. At participant request, electrocardiogram and chest radiograph (Appendix C) were available and unremarkable. Appropriate sign-out to the neonatal intensive care unit was expected at the conclusion of the scenario. Facilitators were provided with teamwork and communication tools prior to each session, found in Appendix D. This allowed for standardized terminology to debrief the scenario in order to optimize the educational experience. The debriefing guide was provided in Appendix E to allow facilitators to provide effective feedback and participants to obtain optimal learning from the experience. A slide-based didactic to aid in the debriefing was provided in Appendix G. An evaluation form for participants to complete after case participation was provided in Appendix F and could be used to adjust the case in the future.
Assessment
The scenario was facilitated and debriefed by PEM physicians experienced in simulation. The participants were PEM fellows, emergency medicine residents, pediatric and emergency medicine interns, PEM RNs, PEM attendings, a respiratory therapist, and a physician assistant. Facilitators provided pediatric resuscitation and content expertise as it related to managing a cyanotic infant and methemoglobinemia. Facilitators also provided participant performance feedback in accordance with the learning objectives. Following the scenario debriefing, participants completed the evaluation form (Appendix F) to give facilitators feedback on the relevance, realism, and overall learning experience of the simulation scenario. The evaluation form included statements rated on a 5-point Likert scale (1 = strongly disagree, 2 = disagree, 3 = neutral, 4 = agree, 5 = strongly agree) to assess the success of the simulation in addressing the learning objectives and to report confidence managing a similar scenario after participation. Respondents had the opportunity to provide additional feedback on the clinical impact and ideas for scenario improvement through free text and open-ended questions, including “Can you list/describe one or more ways this simulation session will change how you do your job?”, “How can we improve this scenario?”, and “Additional comments.” Median Likert scores were calculated for each item on the survey.
Debriefing
We used the tools in Appendices D and E to assist the facilitation debrief sessions after the simulations. These guides provided an outline for leading a debriefing session. We recommend that these sessions begin by encouraging participants to share their overall reflection of the scenario, followed by a structured discussion outlined in Appendix E. The discussion included topics of teamwork and communication (Appendix D) as well as reiteration of diagnostic and management skills.
Results
The scenario was facilitated by experienced PEM simulation faculty across four institutions who provided content expertise and constructive feedback to learners with regard to the learning objectives. A total of 56 individuals participated in the simulations and completed the postsimulation survey. Participants included PEM fellows, emergency medicine residents, pediatric and emergency medicine interns, a physician assistant, a respiratory therapist, PEM nurses, and PEM attendings. Numbers of participants from each discipline and level of training are described in Table 1. The largest portion was PEM fellows, who made up 32% of participants. Emergency medicine residents composed the second largest group of 25% of the total participants. The smallest simulation was completed with seven participants, and the largest was completed with 16 participants. In groups with more participants than available roles, learners without assigned roles observed the simulation and participated in the didactic and debrief. Table 2 summarizes participants' experience related to the simulation case and session facilitation. Overall, participants strongly agreed that the simulation was relevant to their work, realistic, and effective in teaching the objectives and that it promoted team reflection. Table 3 summarizes participant self-appraisal after participation related to the learning objectives. Overall, participants were confident in their ability to perform the designated learning objectives on subsequent patients.
Table 1. Simulation Participants (N = 56)
Table 2. Participant Experience During the Simulation Session (N = 56)
Table 3. Participant Clinical Confidence After Participating in the Session (N = 56)
Each participant was given the opportunity to respond to the following question: “Can you list/describe one or more ways this session will change how you do your job?” Responses primarily discussed improved differential diagnosis building with respect to a hypoxic and cyanotic pediatric patient and further understanding of methemoglobinemia pathophysiology. Responses included the following:
• “Critically think about cyanosis in a patient with a normal venous blood gas.”
• “Recognize that seemingly benign medications (to parents) can have true complications.”
• “Thinking of methemoglobinemia as part of my differential diagnosis for cyanosis.”
• “Understanding of indications for methylene blue treatment.”
• “Encourage me and widen differential diagnosis and gather more extensive history.”
• “Knowing availability of CO-oximetry within my practice.”
Of note, one PEM fellow, who had participated in the first iteration of this case, encountered a child with cyanosis and desaturations unresponsive to supplemental oxygenation several months after completing the simulation. He attributed his quick consideration of and evaluation for methemoglobinemia to having participated in this simulation session.
Participants' suggestions for improvement to the session included making the SpO2 reading 85%-88%, keeping learner groups to fewer than 10 participants, and allowing more time for the session and debrief.
Discussion
Due to widespread availability of topical anesthetics and the severity of the potential complication of methemoglobinemia, health care providers caring for children in ED settings must be able to recognize and treat this rare but fatal complication. This simulation provides an opportunity for PEM fellows to review the differential diagnosis of a hypoxic and cyanotic infant; identify history, symptoms, and physical exam findings consistent with lidocaine toxicity and resultant methemoglobinemia; and strengthen team communication skills. While other curricula related to lidocaine toxicity have appeared in MedEdPORTAL, none are specific to the evaluation of a hypoxic and cyanotic infant.8,9 This curriculum provides a modality for learners to expand their differential diagnosis to include lidocaine toxicity in hypoxic infants. For many pediatric and emergency medicine providers, this scenario is rare but life-threatening. Prompt recognition and appropriate management are important. Additionally, the scenario allows learners to enhance teamwork and communication skills in a controlled environment while also providing a dedicated space to debrief team dynamics.
Multiple learners have participated in the simulation, which is necessary to refine and adapt the material. The simulation case was adjusted after the initial iteration to include an oxygenation saturation typical of methemoglobinemia. Larger groups of learners identified a benefit to completing the simulation in smaller groups. While that was not always possible due to time constraints and predetermined learner group size, subsequent sessions should be held with groups small enough to allow ample opportunity for participation and engagement. If group sizes must be larger than the number of personnel required to run the case, some participants should be assigned an observer role with a special focus on providing observations and feedback to the other participants. This allows the simulation to maintain the realism of a resuscitation. There should be sufficient participants and facilitators to ensure the crucial roles are filled.
A primary limitation of this activity is the difficulty of incorporating all providers typically present on a resuscitation team due to scheduling constraints of nurses, respiratory therapists, and pharmacists. While the simulation was created for PEM fellows, the interdisciplinary teamwork and debrief are enhanced by the incorporation of health care providers from various disciplines. In the future, this simulation should prioritize PEM fellow involvement with incorporation of disciplines who typically care for children in the ED. While PEM fellows make up the largest group of participants for this simulation, they constitute only 32% of participants. This is due to availability of PEM fellows at the institutions included in the evaluation of the simulation and the simultaneous desire to include all health care professionals working in the ED. A limitation of the evaluation process is the reliance of the evaluations on participant perception of improvement of skills after completing the session. Teach-back methods and testing/retesting knowledge, behaviors, and skills through additional simulations have not been evaluated. The immediate feedback provided by learners, however, has been reviewed and assessed for integration in future iterations, as described in preceding paragraphs. Additionally, to better define the time frame within which these knowledge and skills are obtained and reevaluated, future iterations of this simulation should include a time frame associated with each objective.
Appendices
Simulation Case.docx
Environment Preparation.docx
Images.pptx
Teamwork and Communication Glossary.docx
Debriefing Guide.docx
Evaluation Form.docx
Didactics.pptx
All appendices are peer reviewed as integral parts of the Original Publication.
Acknowledgments
The authors would like to thank Jennifer Reid, MD, and Kimberly Stone, MD, for their contributions to the development and execution of these simulations.
Disclosures
None to report.
Funding/Support
None to report.
Ethical Approval
Reported as not applicable. | LIDOCAINE | DrugsGivenReaction | CC BY | 33537407 | 19,389,785 | 2021-01-28 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Neonatal hypoxia'. | Pediatric Toxidrome Simulation Curriculum: Lidocaine-Induced Methemoglobinemia.
Lidocaine is a common local anesthetic used during minor procedures performed on pediatric patients. A rare but toxic and life-threatening side effect of lidocaine is methemoglobinemia. It should be considered in children who are hypoxic after exposure to an oxidizing agent.
We developed this simulation case for pediatric emergency medicine (PEM) fellows, but it can be adapted for interprofessional simulation. The case involved a 1-month-old male with hypoxia and resulting central cyanosis after exposure to lidocaine. The team performed an initial evaluation and intervention, collected a history, and developed a differential diagnosis for hypoxia and central cyanosis in an infant. Methemoglobinemia was confirmed by CO-oximetry. Preparatory materials, a debriefing guide, and scenario evaluation forms assisted with facilitation.
Fifty-six participants (including 18 PEM fellows) completed this simulation across four institutions. Participants rated the scenario on a 5-point Likert scale (1 = strongly disagree, 5 = strongly agree), finding it to be relevant to their work (median = 5) and realistic (median = 5). After participation in the simulation, learners felt confident in their ability to recognize methemoglobinemia (median = 4) and implement a plan to stabilize an infant with hypoxia (median = 4).
This simulation represents a resource for learners in the pediatric emergency department. It teaches the recognition and management of an infant with lidocaine toxicity and resultant methemoglobinemia. It uses experiential learning to teach and reinforce a systematic approach to the evaluation and management of a critically ill infant with acquired methemoglobinemia.
Educational Objectives
After participation in this simulation session, learners will be able to:
1. Perform a primary survey of a critically ill pediatric patient.
2. Implement a plan to stabilize a hypoxic and cyanotic neonate.
3. Develop a systematic approach for the evaluation of hypoxia and central cyanosis in a pediatric patient.
4. Describe the signs and symptoms of acquired methemoglobinemia in a pediatric patient.
5. Manage a pediatric patient with acquired methemoglobinemia.
6. Demonstrate teamwork and communication skills in a resuscitation setting.
Introduction
Methemoglobinemia is a rare but life-threatening disorder and can present at any age after exposure to an oxidizing agent. It occurs when hemoglobin becomes altered, causing the irreversible binding of oxygen through oxidation of ferrous ions associated with heme to the ferric state. The ferric ions of heme (Fe+++) cannot dissociate from oxygen, causing a left shift in the oxyhemoglobin dissociation curve and subsequent hypoxia.1 The impaired oxygen delivery to the tissues causes central cyanosis even with typical supportive measures to improve tissue oxygenation.1 The resultant hypoxia may lead to respiratory failure, end-organ damage, and death in severe cases.1
Methemoglobinemia develops as the result of a congenital condition or toxin exposure. Congenital methemoglobinemia is rare and is due to an abnormal hemoglobin structure or a deficiency in a reducing enzyme. These mutations can be autosomal recessive or dominant and include disease entities such as cytochrome b5 reductase deficiency and hemoglobin M disease.2 Acquired cases of methemoglobinemia are more prevalent, although the incidence is not well defined. Typically, medications and other chemical substances with oxidative potential are the cause. Common medications with oxidative potential include local anesthetics (lidocaine, benzocaine, and prilocaine), sulfa-containing medications, and dapsone.3 Infants are particularly susceptible to methemoglobinemia when facing an oxidative stress due to the immaturity of their enzymatic systems and increased potential for accidental overdose.3
Methemoglobinemia presents with hypoxia causing cyanosis, which can be a diagnostic challenge in a neonate. The possible causes of hypoxia in a neonate are extensive and include, but are not limited to, cyanotic congenital heart disease, sepsis, pneumonia, congenital airway or pulmonary anatomic abnormalities, pulmonary interstitial abnormalities, neurologic conditions, toxidromes, and hemoglobinopathies. While pulmonary etiologies are the most common cause of hypoxia and central cyanosis in an otherwise healthy child, methemoglobinemia should be considered in patients with exposure to oxidizing agents or a family history of congenital methemoglobinemia.
Infants experiencing acute methemoglobinemia may develop a physical exam finding of central cyanosis, hypoxia, increased work of breathing, altered mental status, respiratory depression, and seizures.3 The end result may be death.3 Central cyanosis appears differently depending on the pigmentation of a child's skin. Children with more skin pigmentation have bluish-grey color changes most noticeable on the tongue and mucosal membranes. Children with less skin pigmentation have blue color changes most noticeable on the tongue, mucosal membranes, and perioral area. A key indication of possible methemoglobinemia is a peripheral oxygen saturation detected by pulse oximetry that does not improve with oxygen administration. Upon phlebotomy, the blood remains dark brown in color after exposure to oxygen.3 The arterial blood gas is typically within normal limits. A peripheral capillary oxygen saturation (SpO2) monitor often underestimates hypoxia in the setting of high levels of methemoglobin, so this should not be relied upon for clinical monitoring.4 CO-oximetry should be used to detect the percentage of methemoglobin. It does this by measuring specific wavelengths of light corresponding to methemoglobin (660 nm and 940 nm).4
A majority of cases of methemoglobinemia resolve with supportive care only. More severe cases, however, can be treated with methylene blue to reduce the ferric ions of the heme molecules back to their ferrous state. This allows for appropriate oxygen dissociation from hemoglobin.5 The most commonly accepted methemoglobin level requiring treatment is a level greater than 20% of hemoglobin.6 If the patient is significantly symptomatic, however, methylene blue may be administered with lower levels. It is typically dosed at one to two milligrams per kilogram.6 The overall efficacy of methylene blue in the treatment of methemoglobinemia varies. The primary contraindication to methylene blue use is glucose-6-phophate dehydrogenase deficiency due to the risk of hemolysis.6
This simulation case presents a rare but potentially lethal toxic exposure of topical lidocaine, which induces methemoglobinemia. Content experts in pediatric emergency medicine (PEM) and toxicology created this simulation and originally designed it for PEM fellow education. It is also relevant to other health professionals who care for acutely ill children, including pediatric residents, emergency medicine residents, family medicine residents, medical students, advanced practice providers, and nurses. Learners must evaluate and stabilize an infant presenting with central cyanosis and respiratory distress of unknown etiology. This simulation serves as an educational tool to accommodate this need. Importantly, toxidromes due to medication are a content domain for the PEM certification examination.7
This scenario encourages active learning and integration of previously acquired knowledge, skills, and behaviors as learners must evaluate and stabilize an infant presenting with central cyanosis and respiratory distress of unknown etiology. While the underlying etiology is lidocaine-induced methemoglobinemia, participants must work as a team to use a systematic approach to care for an acutely ill pediatric patient, a theme common to the emergent care of children. Through this scenario, learners must evaluate a cyanotic and hypoxic infant, implement initial resuscitation interventions, develop a broad differential diagnosis, and implement a diagnostic plan leading them to the eventual diagnosis of methemoglobinemia. While other simulation cases addressing methemoglobinemia exist, this scenario is unique in its focus on a neonate necessitating a broad differential diagnosis.8,9 This simulation-based curriculum can be used independently or in series with other sessions from the Pediatric Toxidrome Simulation Curriculum.10–15
Methods
Development
We created this simulation to teach learners a systemic approach to PEM care and promote interpersonal dialogue and communication. The primary goals of the case included working as a team evaluating a neonatal patient with hypoxia and central cyanosis, implementing initial steps of stabilization, thinking through a differential diagnosis, and identifying signs and symptoms of methemoglobinemia.
This simulation scenario was created by PEM attendings with expertise in simulation and curriculum design. A toxicologist provided additional support in the case creation as a content expert. This case was initially created for PEM fellows as part of their recurring simulation-based education. It was also implemented with other members of the emergency department (ED) clinical team who provide medical care to pediatric patients in an ED setting. No specific preparation was required by the learners prior to participation in the simulation. Participants had to have prerequisite knowledge about rapid evaluation and management of neonatal respiratory distress and skills in airway management, which were expected skills for medical personnel working in a pediatric ED. The instructors were provided with the simulation scenario (Appendix A), simulation environment preparation (Appendix B), diagnostic images including electrocardiogram and chest radiograph (Appendix C), teamwork and communication glossary (Appendix D), debriefing guide (Appendix E), and participant evaluation form (Appendix F). Educational slides (Appendix G) were reviewed with participants after the simulation to augment learning and reinforce important concepts. Assessment of the curriculum focused on levels 1 and 2 of Kirkpatrick's model.16
Equipment/Environment
All sites conducted this simulation in a pediatric ED or simulation laboratory with a high-fidelity, infant-sized manikin. The case could also be run in other clinical locations and/or using a low-fidelity manikin. Specific environmental preparation was available in Appendix B. We began the simulation with the manikin on the hospital bed after being brought to the ED by parents. Participants were told that he was in respiratory distress. There was no intravenous access. Equipment and medications commonly encountered in the ED environment were available as outlined in Appendix B. Printouts of an electrocardiogram and chest radiograph were available upon request, all of which showed normal findings (Appendix C).
If using a low-fidelity manikin, facilitators can report vital signs verbally or write them in an area easily seen by all participants. Physical exam findings can be verbally described during the evaluation. The central characteristic of this simulation, cyanosis, can be verbally communicated to the participants. Additionally, moulage can be considered to make the patient appear to be cyanotic.
Personnel
This simulation scenario was performed with seven to 16 learners during each session. Participants were oriented to the simulation, including the manikin and safe learning environment principles. Available roles included three physician/medical provider roles, four nursing roles, and two instructor roles. For sessions in which interdisciplinary team members were not present, physicians filled these roles. The optimal number of participants was seven (three physician/medical provider roles and four nursing roles). The optimal number of instructors was two. Roles for pharmacists and respiratory therapists could be incorporated into the simulation if personnel were available. If the scenario was completed with an interdisciplinary team, personnel maintained the roles of their employment; for example, a fellow would be in the role of a physician, and a nurse would be in the role of a nurse. If the participant group was smaller than the typical clinical team, the simulation would be completed with a minimum of three participants fulfilling physician roles and one facilitator fulfilling an instructor role. This would, however, decrease the opportunity to practice teamwork and communication skills. For larger groups, such as the group of sixteen participants, learners who did not have a designated role were recommended to observe and participate in the didactics and debrief. Instructors were all PEM-trained physicians with experience in medical simulation.
Implementation
This scenario was implemented with a total of 56 participants (including 18 PEM fellows) at four sites across the United States. Each site performed the scenario between one and three times with different groups of learners. The scenario was described in Appendix A. The scenario started with the infant in an ED exam room after having been rushed there from triage. The ED team was called to bedside to evaluate the child with central cyanosis and respiratory distress. The team member assigned to the role of nurse applied the monitors, which showed an oxygen saturation of 85% via the simulation monitor. This alerted the team to make attempts to support the child's breathing during the primary survey. A facilitator or embedded participant played the role of the parent and, upon request, gave the team history, which helped narrow the differential diagnosis to methemoglobinemia induced by lidocaine exposure. Labs as requested in addition to use of CO-oximetry confirmed the diagnosis and appropriate treatment. At participant request, electrocardiogram and chest radiograph (Appendix C) were available and unremarkable. Appropriate sign-out to the neonatal intensive care unit was expected at the conclusion of the scenario. Facilitators were provided with teamwork and communication tools prior to each session, found in Appendix D. This allowed for standardized terminology to debrief the scenario in order to optimize the educational experience. The debriefing guide was provided in Appendix E to allow facilitators to provide effective feedback and participants to obtain optimal learning from the experience. A slide-based didactic to aid in the debriefing was provided in Appendix G. An evaluation form for participants to complete after case participation was provided in Appendix F and could be used to adjust the case in the future.
Assessment
The scenario was facilitated and debriefed by PEM physicians experienced in simulation. The participants were PEM fellows, emergency medicine residents, pediatric and emergency medicine interns, PEM RNs, PEM attendings, a respiratory therapist, and a physician assistant. Facilitators provided pediatric resuscitation and content expertise as it related to managing a cyanotic infant and methemoglobinemia. Facilitators also provided participant performance feedback in accordance with the learning objectives. Following the scenario debriefing, participants completed the evaluation form (Appendix F) to give facilitators feedback on the relevance, realism, and overall learning experience of the simulation scenario. The evaluation form included statements rated on a 5-point Likert scale (1 = strongly disagree, 2 = disagree, 3 = neutral, 4 = agree, 5 = strongly agree) to assess the success of the simulation in addressing the learning objectives and to report confidence managing a similar scenario after participation. Respondents had the opportunity to provide additional feedback on the clinical impact and ideas for scenario improvement through free text and open-ended questions, including “Can you list/describe one or more ways this simulation session will change how you do your job?”, “How can we improve this scenario?”, and “Additional comments.” Median Likert scores were calculated for each item on the survey.
Debriefing
We used the tools in Appendices D and E to assist the facilitation debrief sessions after the simulations. These guides provided an outline for leading a debriefing session. We recommend that these sessions begin by encouraging participants to share their overall reflection of the scenario, followed by a structured discussion outlined in Appendix E. The discussion included topics of teamwork and communication (Appendix D) as well as reiteration of diagnostic and management skills.
Results
The scenario was facilitated by experienced PEM simulation faculty across four institutions who provided content expertise and constructive feedback to learners with regard to the learning objectives. A total of 56 individuals participated in the simulations and completed the postsimulation survey. Participants included PEM fellows, emergency medicine residents, pediatric and emergency medicine interns, a physician assistant, a respiratory therapist, PEM nurses, and PEM attendings. Numbers of participants from each discipline and level of training are described in Table 1. The largest portion was PEM fellows, who made up 32% of participants. Emergency medicine residents composed the second largest group of 25% of the total participants. The smallest simulation was completed with seven participants, and the largest was completed with 16 participants. In groups with more participants than available roles, learners without assigned roles observed the simulation and participated in the didactic and debrief. Table 2 summarizes participants' experience related to the simulation case and session facilitation. Overall, participants strongly agreed that the simulation was relevant to their work, realistic, and effective in teaching the objectives and that it promoted team reflection. Table 3 summarizes participant self-appraisal after participation related to the learning objectives. Overall, participants were confident in their ability to perform the designated learning objectives on subsequent patients.
Table 1. Simulation Participants (N = 56)
Table 2. Participant Experience During the Simulation Session (N = 56)
Table 3. Participant Clinical Confidence After Participating in the Session (N = 56)
Each participant was given the opportunity to respond to the following question: “Can you list/describe one or more ways this session will change how you do your job?” Responses primarily discussed improved differential diagnosis building with respect to a hypoxic and cyanotic pediatric patient and further understanding of methemoglobinemia pathophysiology. Responses included the following:
• “Critically think about cyanosis in a patient with a normal venous blood gas.”
• “Recognize that seemingly benign medications (to parents) can have true complications.”
• “Thinking of methemoglobinemia as part of my differential diagnosis for cyanosis.”
• “Understanding of indications for methylene blue treatment.”
• “Encourage me and widen differential diagnosis and gather more extensive history.”
• “Knowing availability of CO-oximetry within my practice.”
Of note, one PEM fellow, who had participated in the first iteration of this case, encountered a child with cyanosis and desaturations unresponsive to supplemental oxygenation several months after completing the simulation. He attributed his quick consideration of and evaluation for methemoglobinemia to having participated in this simulation session.
Participants' suggestions for improvement to the session included making the SpO2 reading 85%-88%, keeping learner groups to fewer than 10 participants, and allowing more time for the session and debrief.
Discussion
Due to widespread availability of topical anesthetics and the severity of the potential complication of methemoglobinemia, health care providers caring for children in ED settings must be able to recognize and treat this rare but fatal complication. This simulation provides an opportunity for PEM fellows to review the differential diagnosis of a hypoxic and cyanotic infant; identify history, symptoms, and physical exam findings consistent with lidocaine toxicity and resultant methemoglobinemia; and strengthen team communication skills. While other curricula related to lidocaine toxicity have appeared in MedEdPORTAL, none are specific to the evaluation of a hypoxic and cyanotic infant.8,9 This curriculum provides a modality for learners to expand their differential diagnosis to include lidocaine toxicity in hypoxic infants. For many pediatric and emergency medicine providers, this scenario is rare but life-threatening. Prompt recognition and appropriate management are important. Additionally, the scenario allows learners to enhance teamwork and communication skills in a controlled environment while also providing a dedicated space to debrief team dynamics.
Multiple learners have participated in the simulation, which is necessary to refine and adapt the material. The simulation case was adjusted after the initial iteration to include an oxygenation saturation typical of methemoglobinemia. Larger groups of learners identified a benefit to completing the simulation in smaller groups. While that was not always possible due to time constraints and predetermined learner group size, subsequent sessions should be held with groups small enough to allow ample opportunity for participation and engagement. If group sizes must be larger than the number of personnel required to run the case, some participants should be assigned an observer role with a special focus on providing observations and feedback to the other participants. This allows the simulation to maintain the realism of a resuscitation. There should be sufficient participants and facilitators to ensure the crucial roles are filled.
A primary limitation of this activity is the difficulty of incorporating all providers typically present on a resuscitation team due to scheduling constraints of nurses, respiratory therapists, and pharmacists. While the simulation was created for PEM fellows, the interdisciplinary teamwork and debrief are enhanced by the incorporation of health care providers from various disciplines. In the future, this simulation should prioritize PEM fellow involvement with incorporation of disciplines who typically care for children in the ED. While PEM fellows make up the largest group of participants for this simulation, they constitute only 32% of participants. This is due to availability of PEM fellows at the institutions included in the evaluation of the simulation and the simultaneous desire to include all health care professionals working in the ED. A limitation of the evaluation process is the reliance of the evaluations on participant perception of improvement of skills after completing the session. Teach-back methods and testing/retesting knowledge, behaviors, and skills through additional simulations have not been evaluated. The immediate feedback provided by learners, however, has been reviewed and assessed for integration in future iterations, as described in preceding paragraphs. Additionally, to better define the time frame within which these knowledge and skills are obtained and reevaluated, future iterations of this simulation should include a time frame associated with each objective.
Appendices
Simulation Case.docx
Environment Preparation.docx
Images.pptx
Teamwork and Communication Glossary.docx
Debriefing Guide.docx
Evaluation Form.docx
Didactics.pptx
All appendices are peer reviewed as integral parts of the Original Publication.
Acknowledgments
The authors would like to thank Jennifer Reid, MD, and Kimberly Stone, MD, for their contributions to the development and execution of these simulations.
Disclosures
None to report.
Funding/Support
None to report.
Ethical Approval
Reported as not applicable. | LIDOCAINE | DrugsGivenReaction | CC BY | 33537407 | 19,389,785 | 2021-01-28 |
What was the administration route of drug 'RITUXIMAB'? | Cyclophosphamide as a Treatment for Focal Segmental Glomerular Sclerosis Recurrence in a Kidney Transplant Patient.
BACKGROUND Primary focal segmental glomerular sclerosis (FSGS) frequently causes recurrence after kidney transplantation, leading to graft loss in half of the patients. Conservative treatment of FSGS is the main acceptable method due to the lack of randomized clinical trials. A few strategies are known to treat FSGS recurrence, such as plasmapheresis and intravenous immunoglobulin (IVIG), but failure to achieve remission may occur. In addition, some of these treatment strategies are more established in pediatric patients and lack evidence in adult patients. CASE REPORT We describe the case of a 24-year-old woman who had a kidney transplant due to FSGS and was admitted to the hospital for an evaluation of lower-limb and facial swelling. Her kidney biopsy showed segmental glomerulosclerosis compatible with recurrence of FSGS. Her FSGS relapses were further confirmed by increase in serum creatinine and proteinuria. The patient had several FSGS relapses that were treated by different combinations of plasmapheresis, pulse steroid, mycophenolic acid, tacrolimus, prednisolone, IVIG, and IV rituximab. She did not respond to conventional therapy and was eventually treated successfully using cyclophosphamide and remained in remission afterward. CONCLUSIONS FSGS has a high recurrence rate after kidney transplantation. A few options to achieve remission have been investigated. In this report, we present the case of a young woman with FSGS recurrence after a kidney transplant, achieving remission successfully with cyclophosphamide. Cyclophosphamide can be used a treatment of FSGS recurrence in a transplanted kidney when all other options have been exhausted. Additional research is needed to assess the efficacy and safety profile of cyclophosphamide in such cases.
Background
Focal segmental glomerulosclerosis (FSGS) is defined as segmental obliteration of the capillary lumina by the hyaline matrix component involving some of the nephrons [1]. It is considered the most frequent pathological lesions in adults with nephrotic syndrome [2]. FSGS is divided into 2 types based on the etiology of the disease. An idiopathic origin is regarded as primary FSGS. Causes of secondary FSGS include adaptive response of glomerular hypertension resulting from glomerulonephritis and diabetes mellitus, as well as hereditary/genetic glomeropathies (viral or drug-induced) and adaptive responses [3]. Moreover, primary FSGS is the only type related to recurrence in a transplanted kidney.
Treating FSGS conservatively is generally the main acceptable method due to the dearth of randomized clinical trials; however, kidney survival in non-nephrotic patients is significantly better than in nephrotic patients [4]. Angiotensin-converting enzyme inhibitors (ACEI) or angiotensin-II receptor blockers (ARBs) are beneficial in reducing proteinuria and improving long-term kidney function by controlling blood pressure [5]. Nonetheless, possible adverse effects such as hyperkalemia and acute kidney injury must be monitored. In addition to rennin-angiotensin system inhibition, lifestyle modifications are essential to achieve a goal of blood pressure less than 130/80 mmHg [5].
About 40–70% of patients with FSGS need dialysis within 10–20 years after diagnosis [3,6,7]. For patients reaching end-stage renal disease, kidney transplantation is the most suitable treatment option. However, primary FSGS has a recurrence rate of up to 30% [9]. Treatment of FSGS recurrence is challenging because of the limited treatment options. Plasmapheresis is the most common treatment and is reported to achieve remission in up to 63% of adults with recurrent FSGS following kidney transplantation [10]. Other medical treatment options include drugs such as calcineurin inhibitors, rituximab, and cyclophosphamide [1]. However, some of these options lack evidence in adult patients.
Herein, we present a case of an adult patient with FSGS treated by renal transplant who then developed a recurrence and was subsequently managed successfully with cyclophosphamide.
Case Report
A 24-year-old woman had been diagnosed with primary FSGS at age 10 years; nephrotic syndrome was noted on biopsy as primary FSGS. The patient was found to have primary steroid-resistant nephrotic syndrome and was started on peritoneal dialysis (PD). Three years later, she received a single-kidney transplant from a living non-related donor; her post-transplant kidney function was normal. She was started on mycophenolic acid (MMF), tacrolimus, and prednisolone after the transplant. The patient was compliant with her medications, but 2 years later she presented with bilateral lower-limb edema and facial puffiness. Her serum creatinine (SCr) had an almost 3-fold increase from her baseline of 60–70 umol/L. The initial kidney ultrasound (US) revealed mild increased size and echogenicity of the transplanted kidney (Figure 1A), as well as an increase in resistive index of the main renal artery (Figure 1B). A biopsy showed borderline changes for acute T cell-mediated rejection and features consistent with recurrent/de novo FSGS (Figure 2A). The pathological features included only 1 out 5 glomeruli with global glomerulosclerosis, 6 glomeruli with segmental sclerosis, mild interstitial fibrosis, and tubular atrophy. After excluding secondary causes of FSGS, the patient was treated by plasmapheresis in addition to pulse steroid and responded well, with SCr level returning to baseline. Three years later, she presented with symptoms similar to the first episode of recurrence. Her SCr was within her normal baseline, but urine analysis was positive for proteinuria. On admission, laboratory findings included decreased albumin (21 g/L) and 24-h urine collection with a protein level of 5.2 g/day. Renal US images revealed a mild degree of hydronephrosis of the transplanted kidney, without stones, focal masses, or perinephric collection.
The kidney size was estimated to be 12×5.1 cm with cortical thickness of 1.3 cm (Figure 3). A biopsy was planned immediately, which revealed segmental glomerulosclerosis in 4 out of 8 glomeruli, mild arteriolar hyalinosis, minimal interstitial fibrosis, and tubular atrophy, showing a recurrence of FSGS without evidence of acute active cellular rejection (Figure 2B–2D). She was treated initially with 9 sessions of plasmapheresis in combination with intravenous immunoglobulin (IVIG) and pulse steroid. The patient did not respond, and proteinuria persisted, exceeding 5 g/day; therefore, she was started on IV rituximab 375 mg. After the first dose, the patient responded well, with complete remission of FSGS noted by absence of proteinuria, improvement of albumin level (46 g/L), and reduction of 24-h urine protein to 145 mg/day. The patient was discharged on tacrolimus, and another dose of IV rituximab 375 mg was administered 1 week later. One month later, she experienced bilateral optic atrophy as an adverse effect of tacrolimus; thus, she was switched to cyclosporine. Two months after the second recurrence, repeated 24-h urine protein was 12.8 g/day, but the renal profile was normal. The patient was admitted as a case of FSGS recurrence and started on plasmapheresis and received 5 consecutive sessions, which reduced proteinuria to 6.2 g/day. She was discharged to continue 2 session per week of plasmapheresis for 3 months. During the second month of the planned plasmapheresis, the patient developed anasarca, and repeated 24-h urine protein was more than 20 g/day. Since her condition did not improve after plasmapheresis and administration of rituximab, cyclophosphamide was added to the plasmapheresis that she already was receiving, after discussion and informed consent. She was started on daily oral cyclophosphamide 100 mg, in addition to discontinuation of MMF. At 17 days after starting cyclophosphamide, her symptoms improved, and proteinuria fell to 0.7 g/day (Figure 4 demonstrates the timeline of FSGS disease, relapses, and medication regimen). She was discharged on the same dose of cyclophosphamide, prednisolone 10 mg, and cyclosporine 75 mg. Three months later, she had completed a 3-month course of cyclophosphamide, and MMF was resumed. Subsequently, on regular follow-up, she remained in partial remission, with proteinuria ranging from 0.5 g/day to 0.8 g/day.
Discussion
There are few options to treat FSGS recurrence after kidney transplantation. Many studies have noted evidence to support plasma exchange in treating primary recurrent FSGS. Plasma exchange has been shown to ameliorate proteinuria, improve kidney function, and prevent allograft loss [11]. Rituximab’s role in preventing and treating the recurrence of FSGS was not fully established; however, rituximab achieved a significant decrease in proteinuria and recurrence rate as prophylactic therapy in high-risk patients [12].
Our patient received multiple sessions of plasmapheresis, 2 doses of rituximab, and steroids, but she had another FSGS recurrence within 3 months. Moreover, a meta-analysis demonstrated that FSGS recurrence after kidney transplant can be treated with the combination of rituximab and plasmapheresis, which helped achieve remission in 72.7% of the cases [13]. Nonetheless, rituximab has shown beneficial outcomes in adults with FSGS recurrence after transplant, but there is no consensus on the exact dose or duration. Some patients have been reported to achieve complete remission after 2 doses [14], but others may require 3 or 4 doses [15]. Due to a lack of substantial evidence of the efficacy and safety of repetitive rituximab use, a trial of cyclophosphamide to save the graft improved our patient’s symptoms and resolved her proteinuria.
Cyclophosphamide is a nitrogen mustard drug; its main effect is DNA alkylation [16]. The drug affects Th2 cytokines like IL-4 and IL-10 by increasing the secretion in the blood and decreases interferon-gamma secretion [17]. However, the exact mechanism by which cyclophosphamide achieves its immunomodulatory effects is unclear. Theories include type I interferons induction, regulatory T cell elimination, and reducing rejection risk by decreasing the sensitivity of host cells to donor T cells [17].
When used in patients with FSGS with native kidneys, cyclophosphamide has shown beneficial outcomes in achieving partial or complete remission [18]. Experts differ regarding use of cyclophosphamide in cases of FSGS recurrence after kidney. In the pediatric population, a case series of cyclophosphamide use in 11 children showed that 9 of them achieved remission, with 7 becoming free of disease [19]. Moreover, another case series on 6 children with recurrent FSGS treated with a combination of cyclophosphamide and plasma exchange resulted in complete remission in 3 patients, while the rest had partial remission [20]. On the other hand, there is limited data in adult patients treated with cyclophosphamide for FSGS recurrence after kidney transplantation. A case report by Hristea et al describes a case similar to ours, in which the patient received plasmapheresis and rituximab, while cyclophosphamide was used to consolidate the outcomes rather than solely treating recurrence [21].
Our patient received plasmapheresis, rituximab, and steroids, without significant remission. Cyclophosphamide was initiated, which resulted in partial remission. The effect of cyclophosphamide alone was not studied before; nonetheless, our patient received cyclophosphamide after exhausting the other options, but FSGS recurrence was eventually treated successfully.
A limitation of this case report is that the patient’s improvement started almost 4 months after starting rituximab, which may be the reason for achieving remission.
Conclusions
FSGS has a high recurrence rate after kidney transplantation. A few options to gain remission are investigated. In this report, we present the case of a young woman with FSGS recurrence after kidney transplant, achieving remission successfully with cyclophosphamide. Further randomized controlled trials are needed to evaluate the efficacy and safety of this method.
Conflict of Interest
None.
Figure 1. Renal ultrasound showing: (A) Increased size and echogenicity of transplanted kidney. (B) Increase in resistive index of the main renal artery.
Figure 2. (A) In August 2016, an image representative of a glomerulus involvement by segmental sclerosis: segmental obliteration of a capillary loop segment with associated expansion of mesangial matrix by hyaline matrix, in a non-characteristic localization appearing red, PAS stain, ×400. A capsular adhesion of the corresponding segment is present. (B) In May 2019, the image demonstrates glomeruli involvement, in a non-characteristic localization, by segmental sclerosis and capsular adhesion, H&E stain, ×400. (C) In May 2019, the image demonstrates a corticomedullary glomerulus involvement by segmental sclerosis and capsular adhesion, H&E stain, ×400. (D) An electron microscopic image demonstrates effacement and fusion of podocyte foot processes over long segments (>90%) of the glomerular basement membranes, EM, ×2000.
Figure 3. Renal ultrasound the transplanted kidney with a size of 12×5.1 cm and cortical thickness of 1.3 cm.
Figure 4. Timeline of FSGS disease, relapses, and medication regimen. FSGS: focal segmental glomerulosclerosis. PD – peritoneal dialysis; MMF – mycophenolic acid; IVIG – intravenous immunoglobulin. | Intravenous (not otherwise specified) | DrugAdministrationRoute | CC BY-NC-ND | 33539328 | 19,002,074 | 2021-02-04 |
What was the dosage of drug 'MYCOPHENOLIC ACID'? | Cyclophosphamide as a Treatment for Focal Segmental Glomerular Sclerosis Recurrence in a Kidney Transplant Patient.
BACKGROUND Primary focal segmental glomerular sclerosis (FSGS) frequently causes recurrence after kidney transplantation, leading to graft loss in half of the patients. Conservative treatment of FSGS is the main acceptable method due to the lack of randomized clinical trials. A few strategies are known to treat FSGS recurrence, such as plasmapheresis and intravenous immunoglobulin (IVIG), but failure to achieve remission may occur. In addition, some of these treatment strategies are more established in pediatric patients and lack evidence in adult patients. CASE REPORT We describe the case of a 24-year-old woman who had a kidney transplant due to FSGS and was admitted to the hospital for an evaluation of lower-limb and facial swelling. Her kidney biopsy showed segmental glomerulosclerosis compatible with recurrence of FSGS. Her FSGS relapses were further confirmed by increase in serum creatinine and proteinuria. The patient had several FSGS relapses that were treated by different combinations of plasmapheresis, pulse steroid, mycophenolic acid, tacrolimus, prednisolone, IVIG, and IV rituximab. She did not respond to conventional therapy and was eventually treated successfully using cyclophosphamide and remained in remission afterward. CONCLUSIONS FSGS has a high recurrence rate after kidney transplantation. A few options to achieve remission have been investigated. In this report, we present the case of a young woman with FSGS recurrence after a kidney transplant, achieving remission successfully with cyclophosphamide. Cyclophosphamide can be used a treatment of FSGS recurrence in a transplanted kidney when all other options have been exhausted. Additional research is needed to assess the efficacy and safety profile of cyclophosphamide in such cases.
Background
Focal segmental glomerulosclerosis (FSGS) is defined as segmental obliteration of the capillary lumina by the hyaline matrix component involving some of the nephrons [1]. It is considered the most frequent pathological lesions in adults with nephrotic syndrome [2]. FSGS is divided into 2 types based on the etiology of the disease. An idiopathic origin is regarded as primary FSGS. Causes of secondary FSGS include adaptive response of glomerular hypertension resulting from glomerulonephritis and diabetes mellitus, as well as hereditary/genetic glomeropathies (viral or drug-induced) and adaptive responses [3]. Moreover, primary FSGS is the only type related to recurrence in a transplanted kidney.
Treating FSGS conservatively is generally the main acceptable method due to the dearth of randomized clinical trials; however, kidney survival in non-nephrotic patients is significantly better than in nephrotic patients [4]. Angiotensin-converting enzyme inhibitors (ACEI) or angiotensin-II receptor blockers (ARBs) are beneficial in reducing proteinuria and improving long-term kidney function by controlling blood pressure [5]. Nonetheless, possible adverse effects such as hyperkalemia and acute kidney injury must be monitored. In addition to rennin-angiotensin system inhibition, lifestyle modifications are essential to achieve a goal of blood pressure less than 130/80 mmHg [5].
About 40–70% of patients with FSGS need dialysis within 10–20 years after diagnosis [3,6,7]. For patients reaching end-stage renal disease, kidney transplantation is the most suitable treatment option. However, primary FSGS has a recurrence rate of up to 30% [9]. Treatment of FSGS recurrence is challenging because of the limited treatment options. Plasmapheresis is the most common treatment and is reported to achieve remission in up to 63% of adults with recurrent FSGS following kidney transplantation [10]. Other medical treatment options include drugs such as calcineurin inhibitors, rituximab, and cyclophosphamide [1]. However, some of these options lack evidence in adult patients.
Herein, we present a case of an adult patient with FSGS treated by renal transplant who then developed a recurrence and was subsequently managed successfully with cyclophosphamide.
Case Report
A 24-year-old woman had been diagnosed with primary FSGS at age 10 years; nephrotic syndrome was noted on biopsy as primary FSGS. The patient was found to have primary steroid-resistant nephrotic syndrome and was started on peritoneal dialysis (PD). Three years later, she received a single-kidney transplant from a living non-related donor; her post-transplant kidney function was normal. She was started on mycophenolic acid (MMF), tacrolimus, and prednisolone after the transplant. The patient was compliant with her medications, but 2 years later she presented with bilateral lower-limb edema and facial puffiness. Her serum creatinine (SCr) had an almost 3-fold increase from her baseline of 60–70 umol/L. The initial kidney ultrasound (US) revealed mild increased size and echogenicity of the transplanted kidney (Figure 1A), as well as an increase in resistive index of the main renal artery (Figure 1B). A biopsy showed borderline changes for acute T cell-mediated rejection and features consistent with recurrent/de novo FSGS (Figure 2A). The pathological features included only 1 out 5 glomeruli with global glomerulosclerosis, 6 glomeruli with segmental sclerosis, mild interstitial fibrosis, and tubular atrophy. After excluding secondary causes of FSGS, the patient was treated by plasmapheresis in addition to pulse steroid and responded well, with SCr level returning to baseline. Three years later, she presented with symptoms similar to the first episode of recurrence. Her SCr was within her normal baseline, but urine analysis was positive for proteinuria. On admission, laboratory findings included decreased albumin (21 g/L) and 24-h urine collection with a protein level of 5.2 g/day. Renal US images revealed a mild degree of hydronephrosis of the transplanted kidney, without stones, focal masses, or perinephric collection.
The kidney size was estimated to be 12×5.1 cm with cortical thickness of 1.3 cm (Figure 3). A biopsy was planned immediately, which revealed segmental glomerulosclerosis in 4 out of 8 glomeruli, mild arteriolar hyalinosis, minimal interstitial fibrosis, and tubular atrophy, showing a recurrence of FSGS without evidence of acute active cellular rejection (Figure 2B–2D). She was treated initially with 9 sessions of plasmapheresis in combination with intravenous immunoglobulin (IVIG) and pulse steroid. The patient did not respond, and proteinuria persisted, exceeding 5 g/day; therefore, she was started on IV rituximab 375 mg. After the first dose, the patient responded well, with complete remission of FSGS noted by absence of proteinuria, improvement of albumin level (46 g/L), and reduction of 24-h urine protein to 145 mg/day. The patient was discharged on tacrolimus, and another dose of IV rituximab 375 mg was administered 1 week later. One month later, she experienced bilateral optic atrophy as an adverse effect of tacrolimus; thus, she was switched to cyclosporine. Two months after the second recurrence, repeated 24-h urine protein was 12.8 g/day, but the renal profile was normal. The patient was admitted as a case of FSGS recurrence and started on plasmapheresis and received 5 consecutive sessions, which reduced proteinuria to 6.2 g/day. She was discharged to continue 2 session per week of plasmapheresis for 3 months. During the second month of the planned plasmapheresis, the patient developed anasarca, and repeated 24-h urine protein was more than 20 g/day. Since her condition did not improve after plasmapheresis and administration of rituximab, cyclophosphamide was added to the plasmapheresis that she already was receiving, after discussion and informed consent. She was started on daily oral cyclophosphamide 100 mg, in addition to discontinuation of MMF. At 17 days after starting cyclophosphamide, her symptoms improved, and proteinuria fell to 0.7 g/day (Figure 4 demonstrates the timeline of FSGS disease, relapses, and medication regimen). She was discharged on the same dose of cyclophosphamide, prednisolone 10 mg, and cyclosporine 75 mg. Three months later, she had completed a 3-month course of cyclophosphamide, and MMF was resumed. Subsequently, on regular follow-up, she remained in partial remission, with proteinuria ranging from 0.5 g/day to 0.8 g/day.
Discussion
There are few options to treat FSGS recurrence after kidney transplantation. Many studies have noted evidence to support plasma exchange in treating primary recurrent FSGS. Plasma exchange has been shown to ameliorate proteinuria, improve kidney function, and prevent allograft loss [11]. Rituximab’s role in preventing and treating the recurrence of FSGS was not fully established; however, rituximab achieved a significant decrease in proteinuria and recurrence rate as prophylactic therapy in high-risk patients [12].
Our patient received multiple sessions of plasmapheresis, 2 doses of rituximab, and steroids, but she had another FSGS recurrence within 3 months. Moreover, a meta-analysis demonstrated that FSGS recurrence after kidney transplant can be treated with the combination of rituximab and plasmapheresis, which helped achieve remission in 72.7% of the cases [13]. Nonetheless, rituximab has shown beneficial outcomes in adults with FSGS recurrence after transplant, but there is no consensus on the exact dose or duration. Some patients have been reported to achieve complete remission after 2 doses [14], but others may require 3 or 4 doses [15]. Due to a lack of substantial evidence of the efficacy and safety of repetitive rituximab use, a trial of cyclophosphamide to save the graft improved our patient’s symptoms and resolved her proteinuria.
Cyclophosphamide is a nitrogen mustard drug; its main effect is DNA alkylation [16]. The drug affects Th2 cytokines like IL-4 and IL-10 by increasing the secretion in the blood and decreases interferon-gamma secretion [17]. However, the exact mechanism by which cyclophosphamide achieves its immunomodulatory effects is unclear. Theories include type I interferons induction, regulatory T cell elimination, and reducing rejection risk by decreasing the sensitivity of host cells to donor T cells [17].
When used in patients with FSGS with native kidneys, cyclophosphamide has shown beneficial outcomes in achieving partial or complete remission [18]. Experts differ regarding use of cyclophosphamide in cases of FSGS recurrence after kidney. In the pediatric population, a case series of cyclophosphamide use in 11 children showed that 9 of them achieved remission, with 7 becoming free of disease [19]. Moreover, another case series on 6 children with recurrent FSGS treated with a combination of cyclophosphamide and plasma exchange resulted in complete remission in 3 patients, while the rest had partial remission [20]. On the other hand, there is limited data in adult patients treated with cyclophosphamide for FSGS recurrence after kidney transplantation. A case report by Hristea et al describes a case similar to ours, in which the patient received plasmapheresis and rituximab, while cyclophosphamide was used to consolidate the outcomes rather than solely treating recurrence [21].
Our patient received plasmapheresis, rituximab, and steroids, without significant remission. Cyclophosphamide was initiated, which resulted in partial remission. The effect of cyclophosphamide alone was not studied before; nonetheless, our patient received cyclophosphamide after exhausting the other options, but FSGS recurrence was eventually treated successfully.
A limitation of this case report is that the patient’s improvement started almost 4 months after starting rituximab, which may be the reason for achieving remission.
Conclusions
FSGS has a high recurrence rate after kidney transplantation. A few options to gain remission are investigated. In this report, we present the case of a young woman with FSGS recurrence after kidney transplant, achieving remission successfully with cyclophosphamide. Further randomized controlled trials are needed to evaluate the efficacy and safety of this method.
Conflict of Interest
None.
Figure 1. Renal ultrasound showing: (A) Increased size and echogenicity of transplanted kidney. (B) Increase in resistive index of the main renal artery.
Figure 2. (A) In August 2016, an image representative of a glomerulus involvement by segmental sclerosis: segmental obliteration of a capillary loop segment with associated expansion of mesangial matrix by hyaline matrix, in a non-characteristic localization appearing red, PAS stain, ×400. A capsular adhesion of the corresponding segment is present. (B) In May 2019, the image demonstrates glomeruli involvement, in a non-characteristic localization, by segmental sclerosis and capsular adhesion, H&E stain, ×400. (C) In May 2019, the image demonstrates a corticomedullary glomerulus involvement by segmental sclerosis and capsular adhesion, H&E stain, ×400. (D) An electron microscopic image demonstrates effacement and fusion of podocyte foot processes over long segments (>90%) of the glomerular basement membranes, EM, ×2000.
Figure 3. Renal ultrasound the transplanted kidney with a size of 12×5.1 cm and cortical thickness of 1.3 cm.
Figure 4. Timeline of FSGS disease, relapses, and medication regimen. FSGS: focal segmental glomerulosclerosis. PD – peritoneal dialysis; MMF – mycophenolic acid; IVIG – intravenous immunoglobulin. | UNK UNK, UNKNOWN FREQ. | DrugDosageText | CC BY-NC-ND | 33539328 | 18,877,825 | 2021-02-04 |
What was the dosage of drug 'TACROLIMUS'? | Cyclophosphamide as a Treatment for Focal Segmental Glomerular Sclerosis Recurrence in a Kidney Transplant Patient.
BACKGROUND Primary focal segmental glomerular sclerosis (FSGS) frequently causes recurrence after kidney transplantation, leading to graft loss in half of the patients. Conservative treatment of FSGS is the main acceptable method due to the lack of randomized clinical trials. A few strategies are known to treat FSGS recurrence, such as plasmapheresis and intravenous immunoglobulin (IVIG), but failure to achieve remission may occur. In addition, some of these treatment strategies are more established in pediatric patients and lack evidence in adult patients. CASE REPORT We describe the case of a 24-year-old woman who had a kidney transplant due to FSGS and was admitted to the hospital for an evaluation of lower-limb and facial swelling. Her kidney biopsy showed segmental glomerulosclerosis compatible with recurrence of FSGS. Her FSGS relapses were further confirmed by increase in serum creatinine and proteinuria. The patient had several FSGS relapses that were treated by different combinations of plasmapheresis, pulse steroid, mycophenolic acid, tacrolimus, prednisolone, IVIG, and IV rituximab. She did not respond to conventional therapy and was eventually treated successfully using cyclophosphamide and remained in remission afterward. CONCLUSIONS FSGS has a high recurrence rate after kidney transplantation. A few options to achieve remission have been investigated. In this report, we present the case of a young woman with FSGS recurrence after a kidney transplant, achieving remission successfully with cyclophosphamide. Cyclophosphamide can be used a treatment of FSGS recurrence in a transplanted kidney when all other options have been exhausted. Additional research is needed to assess the efficacy and safety profile of cyclophosphamide in such cases.
Background
Focal segmental glomerulosclerosis (FSGS) is defined as segmental obliteration of the capillary lumina by the hyaline matrix component involving some of the nephrons [1]. It is considered the most frequent pathological lesions in adults with nephrotic syndrome [2]. FSGS is divided into 2 types based on the etiology of the disease. An idiopathic origin is regarded as primary FSGS. Causes of secondary FSGS include adaptive response of glomerular hypertension resulting from glomerulonephritis and diabetes mellitus, as well as hereditary/genetic glomeropathies (viral or drug-induced) and adaptive responses [3]. Moreover, primary FSGS is the only type related to recurrence in a transplanted kidney.
Treating FSGS conservatively is generally the main acceptable method due to the dearth of randomized clinical trials; however, kidney survival in non-nephrotic patients is significantly better than in nephrotic patients [4]. Angiotensin-converting enzyme inhibitors (ACEI) or angiotensin-II receptor blockers (ARBs) are beneficial in reducing proteinuria and improving long-term kidney function by controlling blood pressure [5]. Nonetheless, possible adverse effects such as hyperkalemia and acute kidney injury must be monitored. In addition to rennin-angiotensin system inhibition, lifestyle modifications are essential to achieve a goal of blood pressure less than 130/80 mmHg [5].
About 40–70% of patients with FSGS need dialysis within 10–20 years after diagnosis [3,6,7]. For patients reaching end-stage renal disease, kidney transplantation is the most suitable treatment option. However, primary FSGS has a recurrence rate of up to 30% [9]. Treatment of FSGS recurrence is challenging because of the limited treatment options. Plasmapheresis is the most common treatment and is reported to achieve remission in up to 63% of adults with recurrent FSGS following kidney transplantation [10]. Other medical treatment options include drugs such as calcineurin inhibitors, rituximab, and cyclophosphamide [1]. However, some of these options lack evidence in adult patients.
Herein, we present a case of an adult patient with FSGS treated by renal transplant who then developed a recurrence and was subsequently managed successfully with cyclophosphamide.
Case Report
A 24-year-old woman had been diagnosed with primary FSGS at age 10 years; nephrotic syndrome was noted on biopsy as primary FSGS. The patient was found to have primary steroid-resistant nephrotic syndrome and was started on peritoneal dialysis (PD). Three years later, she received a single-kidney transplant from a living non-related donor; her post-transplant kidney function was normal. She was started on mycophenolic acid (MMF), tacrolimus, and prednisolone after the transplant. The patient was compliant with her medications, but 2 years later she presented with bilateral lower-limb edema and facial puffiness. Her serum creatinine (SCr) had an almost 3-fold increase from her baseline of 60–70 umol/L. The initial kidney ultrasound (US) revealed mild increased size and echogenicity of the transplanted kidney (Figure 1A), as well as an increase in resistive index of the main renal artery (Figure 1B). A biopsy showed borderline changes for acute T cell-mediated rejection and features consistent with recurrent/de novo FSGS (Figure 2A). The pathological features included only 1 out 5 glomeruli with global glomerulosclerosis, 6 glomeruli with segmental sclerosis, mild interstitial fibrosis, and tubular atrophy. After excluding secondary causes of FSGS, the patient was treated by plasmapheresis in addition to pulse steroid and responded well, with SCr level returning to baseline. Three years later, she presented with symptoms similar to the first episode of recurrence. Her SCr was within her normal baseline, but urine analysis was positive for proteinuria. On admission, laboratory findings included decreased albumin (21 g/L) and 24-h urine collection with a protein level of 5.2 g/day. Renal US images revealed a mild degree of hydronephrosis of the transplanted kidney, without stones, focal masses, or perinephric collection.
The kidney size was estimated to be 12×5.1 cm with cortical thickness of 1.3 cm (Figure 3). A biopsy was planned immediately, which revealed segmental glomerulosclerosis in 4 out of 8 glomeruli, mild arteriolar hyalinosis, minimal interstitial fibrosis, and tubular atrophy, showing a recurrence of FSGS without evidence of acute active cellular rejection (Figure 2B–2D). She was treated initially with 9 sessions of plasmapheresis in combination with intravenous immunoglobulin (IVIG) and pulse steroid. The patient did not respond, and proteinuria persisted, exceeding 5 g/day; therefore, she was started on IV rituximab 375 mg. After the first dose, the patient responded well, with complete remission of FSGS noted by absence of proteinuria, improvement of albumin level (46 g/L), and reduction of 24-h urine protein to 145 mg/day. The patient was discharged on tacrolimus, and another dose of IV rituximab 375 mg was administered 1 week later. One month later, she experienced bilateral optic atrophy as an adverse effect of tacrolimus; thus, she was switched to cyclosporine. Two months after the second recurrence, repeated 24-h urine protein was 12.8 g/day, but the renal profile was normal. The patient was admitted as a case of FSGS recurrence and started on plasmapheresis and received 5 consecutive sessions, which reduced proteinuria to 6.2 g/day. She was discharged to continue 2 session per week of plasmapheresis for 3 months. During the second month of the planned plasmapheresis, the patient developed anasarca, and repeated 24-h urine protein was more than 20 g/day. Since her condition did not improve after plasmapheresis and administration of rituximab, cyclophosphamide was added to the plasmapheresis that she already was receiving, after discussion and informed consent. She was started on daily oral cyclophosphamide 100 mg, in addition to discontinuation of MMF. At 17 days after starting cyclophosphamide, her symptoms improved, and proteinuria fell to 0.7 g/day (Figure 4 demonstrates the timeline of FSGS disease, relapses, and medication regimen). She was discharged on the same dose of cyclophosphamide, prednisolone 10 mg, and cyclosporine 75 mg. Three months later, she had completed a 3-month course of cyclophosphamide, and MMF was resumed. Subsequently, on regular follow-up, she remained in partial remission, with proteinuria ranging from 0.5 g/day to 0.8 g/day.
Discussion
There are few options to treat FSGS recurrence after kidney transplantation. Many studies have noted evidence to support plasma exchange in treating primary recurrent FSGS. Plasma exchange has been shown to ameliorate proteinuria, improve kidney function, and prevent allograft loss [11]. Rituximab’s role in preventing and treating the recurrence of FSGS was not fully established; however, rituximab achieved a significant decrease in proteinuria and recurrence rate as prophylactic therapy in high-risk patients [12].
Our patient received multiple sessions of plasmapheresis, 2 doses of rituximab, and steroids, but she had another FSGS recurrence within 3 months. Moreover, a meta-analysis demonstrated that FSGS recurrence after kidney transplant can be treated with the combination of rituximab and plasmapheresis, which helped achieve remission in 72.7% of the cases [13]. Nonetheless, rituximab has shown beneficial outcomes in adults with FSGS recurrence after transplant, but there is no consensus on the exact dose or duration. Some patients have been reported to achieve complete remission after 2 doses [14], but others may require 3 or 4 doses [15]. Due to a lack of substantial evidence of the efficacy and safety of repetitive rituximab use, a trial of cyclophosphamide to save the graft improved our patient’s symptoms and resolved her proteinuria.
Cyclophosphamide is a nitrogen mustard drug; its main effect is DNA alkylation [16]. The drug affects Th2 cytokines like IL-4 and IL-10 by increasing the secretion in the blood and decreases interferon-gamma secretion [17]. However, the exact mechanism by which cyclophosphamide achieves its immunomodulatory effects is unclear. Theories include type I interferons induction, regulatory T cell elimination, and reducing rejection risk by decreasing the sensitivity of host cells to donor T cells [17].
When used in patients with FSGS with native kidneys, cyclophosphamide has shown beneficial outcomes in achieving partial or complete remission [18]. Experts differ regarding use of cyclophosphamide in cases of FSGS recurrence after kidney. In the pediatric population, a case series of cyclophosphamide use in 11 children showed that 9 of them achieved remission, with 7 becoming free of disease [19]. Moreover, another case series on 6 children with recurrent FSGS treated with a combination of cyclophosphamide and plasma exchange resulted in complete remission in 3 patients, while the rest had partial remission [20]. On the other hand, there is limited data in adult patients treated with cyclophosphamide for FSGS recurrence after kidney transplantation. A case report by Hristea et al describes a case similar to ours, in which the patient received plasmapheresis and rituximab, while cyclophosphamide was used to consolidate the outcomes rather than solely treating recurrence [21].
Our patient received plasmapheresis, rituximab, and steroids, without significant remission. Cyclophosphamide was initiated, which resulted in partial remission. The effect of cyclophosphamide alone was not studied before; nonetheless, our patient received cyclophosphamide after exhausting the other options, but FSGS recurrence was eventually treated successfully.
A limitation of this case report is that the patient’s improvement started almost 4 months after starting rituximab, which may be the reason for achieving remission.
Conclusions
FSGS has a high recurrence rate after kidney transplantation. A few options to gain remission are investigated. In this report, we present the case of a young woman with FSGS recurrence after kidney transplant, achieving remission successfully with cyclophosphamide. Further randomized controlled trials are needed to evaluate the efficacy and safety of this method.
Conflict of Interest
None.
Figure 1. Renal ultrasound showing: (A) Increased size and echogenicity of transplanted kidney. (B) Increase in resistive index of the main renal artery.
Figure 2. (A) In August 2016, an image representative of a glomerulus involvement by segmental sclerosis: segmental obliteration of a capillary loop segment with associated expansion of mesangial matrix by hyaline matrix, in a non-characteristic localization appearing red, PAS stain, ×400. A capsular adhesion of the corresponding segment is present. (B) In May 2019, the image demonstrates glomeruli involvement, in a non-characteristic localization, by segmental sclerosis and capsular adhesion, H&E stain, ×400. (C) In May 2019, the image demonstrates a corticomedullary glomerulus involvement by segmental sclerosis and capsular adhesion, H&E stain, ×400. (D) An electron microscopic image demonstrates effacement and fusion of podocyte foot processes over long segments (>90%) of the glomerular basement membranes, EM, ×2000.
Figure 3. Renal ultrasound the transplanted kidney with a size of 12×5.1 cm and cortical thickness of 1.3 cm.
Figure 4. Timeline of FSGS disease, relapses, and medication regimen. FSGS: focal segmental glomerulosclerosis. PD – peritoneal dialysis; MMF – mycophenolic acid; IVIG – intravenous immunoglobulin. | UNK UNK, UNKNOWN FREQ. | DrugDosageText | CC BY-NC-ND | 33539328 | 18,877,825 | 2021-02-04 |
What was the outcome of reaction 'Optic atrophy'? | Cyclophosphamide as a Treatment for Focal Segmental Glomerular Sclerosis Recurrence in a Kidney Transplant Patient.
BACKGROUND Primary focal segmental glomerular sclerosis (FSGS) frequently causes recurrence after kidney transplantation, leading to graft loss in half of the patients. Conservative treatment of FSGS is the main acceptable method due to the lack of randomized clinical trials. A few strategies are known to treat FSGS recurrence, such as plasmapheresis and intravenous immunoglobulin (IVIG), but failure to achieve remission may occur. In addition, some of these treatment strategies are more established in pediatric patients and lack evidence in adult patients. CASE REPORT We describe the case of a 24-year-old woman who had a kidney transplant due to FSGS and was admitted to the hospital for an evaluation of lower-limb and facial swelling. Her kidney biopsy showed segmental glomerulosclerosis compatible with recurrence of FSGS. Her FSGS relapses were further confirmed by increase in serum creatinine and proteinuria. The patient had several FSGS relapses that were treated by different combinations of plasmapheresis, pulse steroid, mycophenolic acid, tacrolimus, prednisolone, IVIG, and IV rituximab. She did not respond to conventional therapy and was eventually treated successfully using cyclophosphamide and remained in remission afterward. CONCLUSIONS FSGS has a high recurrence rate after kidney transplantation. A few options to achieve remission have been investigated. In this report, we present the case of a young woman with FSGS recurrence after a kidney transplant, achieving remission successfully with cyclophosphamide. Cyclophosphamide can be used a treatment of FSGS recurrence in a transplanted kidney when all other options have been exhausted. Additional research is needed to assess the efficacy and safety profile of cyclophosphamide in such cases.
Background
Focal segmental glomerulosclerosis (FSGS) is defined as segmental obliteration of the capillary lumina by the hyaline matrix component involving some of the nephrons [1]. It is considered the most frequent pathological lesions in adults with nephrotic syndrome [2]. FSGS is divided into 2 types based on the etiology of the disease. An idiopathic origin is regarded as primary FSGS. Causes of secondary FSGS include adaptive response of glomerular hypertension resulting from glomerulonephritis and diabetes mellitus, as well as hereditary/genetic glomeropathies (viral or drug-induced) and adaptive responses [3]. Moreover, primary FSGS is the only type related to recurrence in a transplanted kidney.
Treating FSGS conservatively is generally the main acceptable method due to the dearth of randomized clinical trials; however, kidney survival in non-nephrotic patients is significantly better than in nephrotic patients [4]. Angiotensin-converting enzyme inhibitors (ACEI) or angiotensin-II receptor blockers (ARBs) are beneficial in reducing proteinuria and improving long-term kidney function by controlling blood pressure [5]. Nonetheless, possible adverse effects such as hyperkalemia and acute kidney injury must be monitored. In addition to rennin-angiotensin system inhibition, lifestyle modifications are essential to achieve a goal of blood pressure less than 130/80 mmHg [5].
About 40–70% of patients with FSGS need dialysis within 10–20 years after diagnosis [3,6,7]. For patients reaching end-stage renal disease, kidney transplantation is the most suitable treatment option. However, primary FSGS has a recurrence rate of up to 30% [9]. Treatment of FSGS recurrence is challenging because of the limited treatment options. Plasmapheresis is the most common treatment and is reported to achieve remission in up to 63% of adults with recurrent FSGS following kidney transplantation [10]. Other medical treatment options include drugs such as calcineurin inhibitors, rituximab, and cyclophosphamide [1]. However, some of these options lack evidence in adult patients.
Herein, we present a case of an adult patient with FSGS treated by renal transplant who then developed a recurrence and was subsequently managed successfully with cyclophosphamide.
Case Report
A 24-year-old woman had been diagnosed with primary FSGS at age 10 years; nephrotic syndrome was noted on biopsy as primary FSGS. The patient was found to have primary steroid-resistant nephrotic syndrome and was started on peritoneal dialysis (PD). Three years later, she received a single-kidney transplant from a living non-related donor; her post-transplant kidney function was normal. She was started on mycophenolic acid (MMF), tacrolimus, and prednisolone after the transplant. The patient was compliant with her medications, but 2 years later she presented with bilateral lower-limb edema and facial puffiness. Her serum creatinine (SCr) had an almost 3-fold increase from her baseline of 60–70 umol/L. The initial kidney ultrasound (US) revealed mild increased size and echogenicity of the transplanted kidney (Figure 1A), as well as an increase in resistive index of the main renal artery (Figure 1B). A biopsy showed borderline changes for acute T cell-mediated rejection and features consistent with recurrent/de novo FSGS (Figure 2A). The pathological features included only 1 out 5 glomeruli with global glomerulosclerosis, 6 glomeruli with segmental sclerosis, mild interstitial fibrosis, and tubular atrophy. After excluding secondary causes of FSGS, the patient was treated by plasmapheresis in addition to pulse steroid and responded well, with SCr level returning to baseline. Three years later, she presented with symptoms similar to the first episode of recurrence. Her SCr was within her normal baseline, but urine analysis was positive for proteinuria. On admission, laboratory findings included decreased albumin (21 g/L) and 24-h urine collection with a protein level of 5.2 g/day. Renal US images revealed a mild degree of hydronephrosis of the transplanted kidney, without stones, focal masses, or perinephric collection.
The kidney size was estimated to be 12×5.1 cm with cortical thickness of 1.3 cm (Figure 3). A biopsy was planned immediately, which revealed segmental glomerulosclerosis in 4 out of 8 glomeruli, mild arteriolar hyalinosis, minimal interstitial fibrosis, and tubular atrophy, showing a recurrence of FSGS without evidence of acute active cellular rejection (Figure 2B–2D). She was treated initially with 9 sessions of plasmapheresis in combination with intravenous immunoglobulin (IVIG) and pulse steroid. The patient did not respond, and proteinuria persisted, exceeding 5 g/day; therefore, she was started on IV rituximab 375 mg. After the first dose, the patient responded well, with complete remission of FSGS noted by absence of proteinuria, improvement of albumin level (46 g/L), and reduction of 24-h urine protein to 145 mg/day. The patient was discharged on tacrolimus, and another dose of IV rituximab 375 mg was administered 1 week later. One month later, she experienced bilateral optic atrophy as an adverse effect of tacrolimus; thus, she was switched to cyclosporine. Two months after the second recurrence, repeated 24-h urine protein was 12.8 g/day, but the renal profile was normal. The patient was admitted as a case of FSGS recurrence and started on plasmapheresis and received 5 consecutive sessions, which reduced proteinuria to 6.2 g/day. She was discharged to continue 2 session per week of plasmapheresis for 3 months. During the second month of the planned plasmapheresis, the patient developed anasarca, and repeated 24-h urine protein was more than 20 g/day. Since her condition did not improve after plasmapheresis and administration of rituximab, cyclophosphamide was added to the plasmapheresis that she already was receiving, after discussion and informed consent. She was started on daily oral cyclophosphamide 100 mg, in addition to discontinuation of MMF. At 17 days after starting cyclophosphamide, her symptoms improved, and proteinuria fell to 0.7 g/day (Figure 4 demonstrates the timeline of FSGS disease, relapses, and medication regimen). She was discharged on the same dose of cyclophosphamide, prednisolone 10 mg, and cyclosporine 75 mg. Three months later, she had completed a 3-month course of cyclophosphamide, and MMF was resumed. Subsequently, on regular follow-up, she remained in partial remission, with proteinuria ranging from 0.5 g/day to 0.8 g/day.
Discussion
There are few options to treat FSGS recurrence after kidney transplantation. Many studies have noted evidence to support plasma exchange in treating primary recurrent FSGS. Plasma exchange has been shown to ameliorate proteinuria, improve kidney function, and prevent allograft loss [11]. Rituximab’s role in preventing and treating the recurrence of FSGS was not fully established; however, rituximab achieved a significant decrease in proteinuria and recurrence rate as prophylactic therapy in high-risk patients [12].
Our patient received multiple sessions of plasmapheresis, 2 doses of rituximab, and steroids, but she had another FSGS recurrence within 3 months. Moreover, a meta-analysis demonstrated that FSGS recurrence after kidney transplant can be treated with the combination of rituximab and plasmapheresis, which helped achieve remission in 72.7% of the cases [13]. Nonetheless, rituximab has shown beneficial outcomes in adults with FSGS recurrence after transplant, but there is no consensus on the exact dose or duration. Some patients have been reported to achieve complete remission after 2 doses [14], but others may require 3 or 4 doses [15]. Due to a lack of substantial evidence of the efficacy and safety of repetitive rituximab use, a trial of cyclophosphamide to save the graft improved our patient’s symptoms and resolved her proteinuria.
Cyclophosphamide is a nitrogen mustard drug; its main effect is DNA alkylation [16]. The drug affects Th2 cytokines like IL-4 and IL-10 by increasing the secretion in the blood and decreases interferon-gamma secretion [17]. However, the exact mechanism by which cyclophosphamide achieves its immunomodulatory effects is unclear. Theories include type I interferons induction, regulatory T cell elimination, and reducing rejection risk by decreasing the sensitivity of host cells to donor T cells [17].
When used in patients with FSGS with native kidneys, cyclophosphamide has shown beneficial outcomes in achieving partial or complete remission [18]. Experts differ regarding use of cyclophosphamide in cases of FSGS recurrence after kidney. In the pediatric population, a case series of cyclophosphamide use in 11 children showed that 9 of them achieved remission, with 7 becoming free of disease [19]. Moreover, another case series on 6 children with recurrent FSGS treated with a combination of cyclophosphamide and plasma exchange resulted in complete remission in 3 patients, while the rest had partial remission [20]. On the other hand, there is limited data in adult patients treated with cyclophosphamide for FSGS recurrence after kidney transplantation. A case report by Hristea et al describes a case similar to ours, in which the patient received plasmapheresis and rituximab, while cyclophosphamide was used to consolidate the outcomes rather than solely treating recurrence [21].
Our patient received plasmapheresis, rituximab, and steroids, without significant remission. Cyclophosphamide was initiated, which resulted in partial remission. The effect of cyclophosphamide alone was not studied before; nonetheless, our patient received cyclophosphamide after exhausting the other options, but FSGS recurrence was eventually treated successfully.
A limitation of this case report is that the patient’s improvement started almost 4 months after starting rituximab, which may be the reason for achieving remission.
Conclusions
FSGS has a high recurrence rate after kidney transplantation. A few options to gain remission are investigated. In this report, we present the case of a young woman with FSGS recurrence after kidney transplant, achieving remission successfully with cyclophosphamide. Further randomized controlled trials are needed to evaluate the efficacy and safety of this method.
Conflict of Interest
None.
Figure 1. Renal ultrasound showing: (A) Increased size and echogenicity of transplanted kidney. (B) Increase in resistive index of the main renal artery.
Figure 2. (A) In August 2016, an image representative of a glomerulus involvement by segmental sclerosis: segmental obliteration of a capillary loop segment with associated expansion of mesangial matrix by hyaline matrix, in a non-characteristic localization appearing red, PAS stain, ×400. A capsular adhesion of the corresponding segment is present. (B) In May 2019, the image demonstrates glomeruli involvement, in a non-characteristic localization, by segmental sclerosis and capsular adhesion, H&E stain, ×400. (C) In May 2019, the image demonstrates a corticomedullary glomerulus involvement by segmental sclerosis and capsular adhesion, H&E stain, ×400. (D) An electron microscopic image demonstrates effacement and fusion of podocyte foot processes over long segments (>90%) of the glomerular basement membranes, EM, ×2000.
Figure 3. Renal ultrasound the transplanted kidney with a size of 12×5.1 cm and cortical thickness of 1.3 cm.
Figure 4. Timeline of FSGS disease, relapses, and medication regimen. FSGS: focal segmental glomerulosclerosis. PD – peritoneal dialysis; MMF – mycophenolic acid; IVIG – intravenous immunoglobulin. | Recovered | ReactionOutcome | CC BY-NC-ND | 33539328 | 19,002,074 | 2021-02-04 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Abnormal dreams'. | Higher plasma drug levels in elderly people living with HIV treated with darunavir.
The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age.
Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases.
Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV).
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.
Introduction
Antiretroviral therapy (ART) has dramatically changed the life expectancy of people living with HIV (PLHIV). HIV can now be considered a chronic infection, and the expected life span of PLHIV who receive efficient treatment is comparable to HIV-negative individuals [1, 2]. As a consequence, an increasing number of PLHIV are of older age. For example, in 2018 51% of PLHIV in the US were 50 years of age or older [3].
The risk of developing age-related and lifestyle-related diseases increases with age. PLHIV are, in addition, at higher risk of non-infectious comorbidities compared to the general population [4–6]. Furthermore, elderly people are, in general, at higher risk of adverse events to medications and may require lower doses of medications than recommended for younger individuals [7, 8]. It has been reported that PLHIV above 50 years of age have more concomitant medications and a higher risk of potential drug-drug interactions (PDDIs) compared to PLHIV below 50 years of age [9, 10].
Since the mid 1990s the standard regime for HIV treatment is two nucleoside reverse-transcriptase inhibitors (NRTIs) combined with a third agent from another drug class (most commonly a nucleoside reverse-transcriptase inhibitor (NNRTI), a protease inhibitor (PI) or an integrase inhibitor (INSTI). A dolutegravir (INSTI) or Efavirenz (NNRTI) containing regimen is recommended by WHO as first line treatment today [11]. In the Swedish setting a dolutegravir or darunavir containing regimen is recommended by the Swedish Reference Group for Antiviral Therapy [12]. Neither WHO nor Sweden have specific treatment recommendations for elderly PLHIV.
Scientific data on the pharmacokinetics of PIs and NNRTIs in individuals 65 years of age and older are scarce. The primary objective of this study was to investigate differences in steady-state plasma drug levels of ATV, DRV and EFV in PLHIV ≥ 65 years of age as compared to PLHIV ≤ 49 years of age. Secondary objectives were to study differences in self-reported side-effects, concomitant chronic diseases and medications, and PDDIs.
Methods
PLHIV who were followed at four HIV centers in Sweden: the Department of Infectious Diseases at Sahlgrenska University Hospital in Gothenburg; the Department of Infectious Diseases at South Älvsborg Hospital in Borås; the Department of Infectious Diseases at Karolinska University Hospital Huddinge in Stockholm; and the Department of Infectious Diseases at Stockholm South General Hospital in Stockholm, and met the inclusion criteria (age, 65 years of age or older for the study group or 49 years of age or younger for the control group; and on stable ART containing atazanavir (ATV), darunavir (DRV) or efavirenz (EFV) for more than 6 months) were eligible for inclusion in this cross-sectional study. On the day of inclusion, a blood sample for analysis of plasma drug level was taken, and concomitant medications (including non-prescription drugs and herbal supplements) and any side-effects related to ART were recorded in a standardized questionnaire, S1 File. Blood samples drawn between 6 to 36 hours after last dose of medication was included in the analysis of steady-state drug levels and adjusted for time with ANCOVA analysis. Adherence was recorded using a modified ACTG adherence questionnaire [13]. Any missed dose during the preceding 4 days was considered as non-adherence.
Comorbidities were registered by structured medical record reviews. PDDIs were analyzed using the Liverpool University HIV drug interactions [14] and Janusmed [15] webtools. The Liverpool University HIV drug interactions database definitions for PDDIs were used and red flag (drugs should not be co-administered) and orange flag (a potential interaction that may require dose monitoring, alteration of drug dosage or timing of administration) interactions were included in the analysis. If there was an interaction between a comedication and both the PI and the booster, the interaction was counted as one interaction in the analysis. Individuals taking DRV b.i.d. or ATV without ritonavir booster were excluded from the analysis of plasma drug levels.
All study participants gave their written informed consent and ethics approval for the study was granted by the Research Ethics Committee at Gothenburg University.
Laboratory analyses
Plasma samples were frozen at –70°C immediately after sampling until analysis. Drug levels were analyzed using a reverse-phase High Pressure Liquid Chromatography (HPLC) with ultraviolet (UV) detection at the routine pharmacology analytical laboratory at Karolinska University Hospital, Huddinge in Stockholm, Sweden. The method was CAP (College of American Pathologists) and Swedac accredited and has been described elsewhere [16]. Routine clinical methods were used to analyze CD4 cell count, liver enzymes and creatinine according to local laboratory standards.
Statistical analyses
Differences in plasma drug levels were analyzed with ANCOVA (adjusting for time) with log-transformed concentrations of ATV, DRV, and EFV. Chi-square test and Fisher’s exact test were used to compare the frequencies of side-effects, CD4/CD8 ratios, and AIDS diagnosis as appropriate. Mann Whitney U-test and Kruskal Wallis test (with Bonferroni correction for multiple tests) were used to compare frequencies of concomitant medications and PDDIs. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics, Armonk, NY, USA) or Prism version 8.0 (Graphpad Software Inc., La Jolla, CA, USA).
Results
One hundred and seventy-two individuals 65 years of age or older were eligible for inclusion and were asked to participate in the study at the four sites. Between November 2013 and August 2015, 100 individuals were enrolled in the study group (ATV n = 19; DRV n = 35; EFV n = 46) and 99 individuals in the control group (ATV n = 18; DRV n = 37; EFV n = 44). Baseline characteristics are listed in Table 1. Three individuals had HIV RNA blips (HIV RNA 59–156 copies/mL) at inclusion; all other patients had HIV RNA levels < 50 copies/mL at inclusion. Twenty-seven patients were excluded from the plasma drug level analysis: 15 individuals received DRV b.i.d. and 9 were treated with ATV, either unboosted or with dosing not according to clinical standards. Three were excluded from the plasma drug level analysis due to sample management (elapsed time since last dose less than 6 hours, or elapsed time since last dose unknown). Patients included in the plasma drug level analysis received DRV/r 800/100 mg, ATV/r 300/100 mg, or EFV 600 mg q.d. There was a significant difference in ALT levels between study and control group in the ATV arm, however the majority of subjects had ALT within the normal range. The study group had a lower glomerular filtration rate (GFR) in all arms compared to controls, although within the normal range.
10.1371/journal.pone.0246171.t001 Table 1 Baseline characteristics.
ATV DRV EFV
Study n = 19 Control n = 18 p Study n = 35 Control n = 37 p Study n = 46 Control n = 44 p
Age (median [IQR]) 68 (66–70) 46 (40.75–47.5) 68 (67–72) 45 (37.5–47) 69 (67–72) 43 (37–46)
Gender (M/F) (n) 13/6 17/1 0.09 33/2 32/5 0.43 41/5 38/6 0.76
BMI (median [IQR]) 24.3 (22.3–27.2) 25.3 (23.9–27.9) 0.28 25.7 (23.0–26.9) 23.5 (22.5–26.0) 0.17 24.4 (21.9–27.8) 24.2 (22.1–28.5) 0.65
GFR (ml/min) (median [IQR]) 83.7 (71.0–96.8) 118.0 (109.9–134.4) <0.001 86.1 (55.4–95.3) 110.4 (97.0–135.8) <0.001 79.9 (67.6–98.5) 124.3 (116.0–145.2) <0.001
ALT (μkat/L) (median [IQR]) 0.40 (0.30–0.55) 0.63 (0.52–0.75) 0.017 0.32 (0.24–0.48) 0.41 (0.31–0.55) 0.14 0.48 (0.33–0.56) 0.55 (0.38–0.85) 0.056
CD4 cell count (median [IQR]) 650 (370–730) 730 (490–912.5) 0.13 560 (420–650) 600 (465–830) 0.54 535 (380–687.5) 575 (407.5–750) 0.39
CD4 cell count Nadir (median [IQR]) 130 (69–223) 241 (187–337) 0.001 185 (90–281) 218 (40–310) 0.97 208 (158–263) 219 (167–303) 0.49
Backbone 3TC/ABC 9 14 0.09 11 14 0.63 23 12 0.03
Backbone FTC/TDF 10 4 0.09 5 12 0.03 23 30 0.09
Backbone other** 0 0 NA 19 9 0.02 0 2 0.24
Comorbidities (n) (median [IQR]) 2 (1–4) 2 (0.25–3) 0.31 3 (2–5) 1 (1–3) 0.001 3 (2–4.25) 1 (0–2) <0.001
** Other backbones: RAL, ETV, RAL + maraviroc, RAL + EFV, RAL + DRV, LPV/r, RAL + 3TC, 3TC, DTG + 3TC, RPV, no backbone.
The steady-state DRV concentrations were significantly higher in the study group (n = 25) compared to the control group (n = 30) (p = 0.047), Fig 1. The geometric mean was 48% higher in the study group than in the control group. The analysis of the ATV arm (study group n = 19, control group n = 18) showed a difference in steady state-levels (geometric mean 69% higher in the study group), with a trend towards statistical significance (p = 0.056). No statistically significant difference between the groups was found in the EFV (p = 0.87) arm. There were no differences in self-reported adherence between the study group (96% adherent) and control group (93% adherent) (p = 0.537), or between different treatment arms either in the study or the control group.
10.1371/journal.pone.0246171.g001 Fig 1 Steady-state levels of ART.
Steady-state levels in plasma of A) Darunavir (p = 0.047), B) Atazanavir (p = 0.056), and C) Efavirenz (p = 0.87) in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group).
There were no statistically significant differences in reported side-effects between the study group (23%) and the control group (34%) (p = 0.146), Fig 2. When dividing the groups according to drug regimen (taking the study group together with the controls), the DRV group had a higher rate of reported side-effects (ATV: 16.7%; DRV: 39.4%; EFV: 25.9%; p = 0.038), which was significantly different compared to the ATV arm. The difference remained when PLHIV ≥ 65 years of age were analysed separately (ATV: 0%; DRV: 36.7%; EFV: 23.8%; p = 0.014), with significant differences between DRV and ATV, and EFV and ATV. In the DRV arm there were no significant difference in reported side effects between the study group and the control group, (p = 0.80). The most commonly-reported side-effect in the DRV groups was diarrhea.
10.1371/journal.pone.0246171.g002 Fig 2 Frequency of self-reported side-effects.
A: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) (ns). B: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) divided into treatment arms (p = 0.038). C: Frequency of self-reported side-effects divided into groups according to treatment arm and study group vs. control group.
As expected, the study group had a significantly higher mean (range) number of concomitant medications, 3.7 (0–12), compared to the control group, 1.1 (0–10) (p < 0.001). Accordingly, the study group had significantly more PDDIs (mean (range)) 1.1 (0–6) compared to the control group, 0.3 (0–3) (p < 0.001). The most common PDDIs for ATV were statins and beta-blocking agents, and statins and antidepressants for DRV and EFV.
Analysis of the study group showed that the DRV arm had significantly more PDDIs (mean [range]) 1.4 (0–6) than the EFV arm 0.7 (0–3) (p = 0.03). The ATV arm had a mean (range) of 1.2 (0–4) PDDIs, not significantly different compared to either the DRV arm or the EFV arm (see Table 2). Notably, the use of concomitant medications was not higher in the DRV arm. Eight individuals in the study group had red flag PDDIs: DRV/r and alfuzosin (risk for severe hypotension, n = 2); DRV/r and clopidogrel (reduced effect of clopidogrel, n = 3); DRV/r and alfuzosin + clopidogrel (n = 1); ATV and budesonide (increased risk of steroid side-effects, n = 1); and ATV and lansoprazole (reduced ATV uptake, n = 1). Whereas no one in the control group had a red flag interaction.
10.1371/journal.pone.0246171.t002 Table 2 Potential drug-drug interactions.
PDDIs (n) (mean [range]) p
Atazanavir 1.2 (0–4)
Darunavir 1.4 (1–6) 0.026
Efavirenz 0.7 (0–3)
Number of potential drug-drug interactions (PDDIs) in the study group presented by treatment regime.
There were no differences in CD4/CD8 ratios (≥ 1 or < 1) between the study group (≥ 1 n = 35 (35%)) and control group (≥ 1 n = 40 (40%)) (p = 0.43), the ATV/DRV/EFV arms, or between arms in the different groups. No differences were found in the history of AIDS defining events in the study group vs. control group. In total 38 individuals had one or more AIDS defining diagnoses, S1 Table.
Discussion
We found a difference in the steady-state plasma drug levels of DRV in PLHIV who were ≥ 65 years of age, as compared to PLHIV < 50 years of age. To our knowledge, only one previous study has addressed the question of plasma DRV levels in elderly PLHIV [17]. In agreement with our results, the authors reported higher DRV levels in individuals > 60 years of age compared to those ≤ 40 years of age.
The difference in plasma drug levels is also consistent with earlier findings regarding other PIs, including ATV [18–20]. We noted a difference in ATV levels between elderly and younger PLHIV, with a trend towards statistical significance. The lack of significance is probably due to the small sample size. In a previous report by Avihingsanon et al., higher trough levels and higher exposure to ATV in PLHIV > 42 years of age was found compared to individuals ≤ 42 years of age, consistent with our results. This difference was more pronounced in PLHIV > 50 years of age [18]. Winston et al. also found a significant association between age and plasma drug levels of PIs [19]. We did not find any significant difference in EFV plasma drug levels in the study group compared to the control group. This is in agreement with the findings in other reports [19, 21].
There is only very limited pharmacokinetic data on ART in PLHIV older than 65 years of age. There are, however, several general age-related biological changes that may affect the metabolism of ART, e.g. decrease of liver and renal function and changes in body composition that influence the volume of distribution [22]. ATV, DRV and EFV are metabolized in the liver (ATV and DRV mainly through CYP3A4 and EFV through CYP 3A4 and CYP2B6). Only a minor portion of these drugs is eliminated through the kidneys. Therefore, the difference in drug levels in the DRV and ATV arm cannot be explained by differences in GFR. We found no clinically measurable difference in liver function measured by ALT but other changes in liver function related to age may have affected the drug levels.
Other aspects not related to metabolism may also affect the efficacy of ART. Older PLHIV have been shown to be more adherent to their treatment regimen than younger individuals [23, 24]. However, with increasing age there is a higher risk of cognitive impairment that may affect the adherence in the oldest. No difference in adherence was found in our study to support or reject either higher or lower adherence in elderly PLHIV.
Overall, we found a higher frequency of self-reported side-effects in the DRV arm, in comparison to the ATV and the EFV arms (although not significantly different from the EFV arm). The higher frequency was also present in the study group (although not statistically significant).
To the best of our knowledge this is the only study that compared the frequency of side-effects between different PIs/NNRTIs in PLHIV older than 65 years of age. A possible reason for the DRV arm having a higher frequency of side-effects might be that DRV was chosen because of extensive ART history and viral resistance, resulting in few available alternative regimes at the time of inclusion in the study and as a consequence there was a higher tolerance of side-effects. On the other hand, no difference in self-reported side effects was noted between the DRV study group and control group, even though we found a difference in steady state plasma drug levels. This observation may reflect under reporting in the study group. While it is not possible in the present study to establish a causal link, further studies are needed illuminate this issue.
PDDIs are common among PLHIV [25, 26], and the risk increases with age due to increasing frequencies of comorbidities and concomitant medications [9, 10]. Our result is in line with these earlier studies. Red flag interactions has been reported in 2% to 5.6% of PLHIV and 7.1% to 8.7% in PLHIV ≥ 65 years of age in earlier studies, similar to our findings [26–29].
ATV and DRV are both PIs and therefore they have, in general, the same PDDIs. However, they differ in regard to some frequently used drug classes e.g. beta blockers and PPIs. Since EFV is a NNRTI it has another drug interaction profile. DRV accounted for the majority of the red flag interactions found in our study, consistent with earlier findings of PLHIV in all ages [27]. Other studies have reported a higher probability of an orange or red flag PDDI in individuals treated with a PI (not restricted to those ≥ 65) [9, 28]. This is consistent with our finding that the DRV arm in the study group had a higher mean of PDDIs than the EFV arm. We did not find a significant difference between the ATV and EFV arms, however this may be due to the small sample size. The majority of the red flag interactions found were related to the concomitant use of alfuzosin, for treatment of benign prostatic hyperplasia, and/or clopidogrel, for treatment of vascular disease, which both are conditions that have a higher prevalence in older ages.
Our study has several limitations. Since we analysed steady-state plasma drug levels and not trough levels of ART, it is difficult to compare our results with trough levels in other studies. Thus, we were unable to evaluate potentially toxic plasma drug levels of ART drugs or levels below the proposed minimal effective concentrations. Patients were sampled from 6 to 36 hours after their last dose of ART. This was adjusted for in the statistical model, but there remains a risk that, because of this approach, we were not able to detect minor differences in drug levels between the study and control arms for ATV and EFV. Also, there was a difference in back-bone between PLHIV on DRV compared to ATV and EFV that may have affected self-reported side-effects. Concomitant medications may also have influenced the self-reported side-effects, though the participants were asked specifically to report side-effects related to ART. The PDDIs were calculated only for ATV, DRV or EFV regimens and therefore differences in back-bone likely did not affect the results. In addition, interactions between NRTIs and other medications are uncommon. The participants in the study were included consecutively at four sites in Sweden and it is possible that this introduced a selection bias, favoring PLHIV with frequent visits.
Conclusion
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV who were 65 years of age or older, as compared to controls who were 49 years of age and younger. Our findings are important for the management of elderly PLHIV and raise the question of whether regular monitoring of plasma levels and dose adjustment of DRV and other PIs is warranted in the elderly.
Supporting information
S1 Table Aids defining diagnoses.
(DOCX)
Click here for additional data file.
S2 Table Data set.
(XLSX)
Click here for additional data file.
S1 File Questionnaire/case report form.
(DOCX)
Click here for additional data file.
We would like to thank all the participants in the study, study nurses and technical staff. | CETIRIZINE HYDROCHLORIDE, EFAVIRENZ, EMTRICITABINE\TENOFOVIR DISOPROXIL, VALACYCLOVIR HYDROCHLORIDE | DrugsGivenReaction | CC BY | 33539440 | 19,781,519 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Coma'. | Higher plasma drug levels in elderly people living with HIV treated with darunavir.
The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age.
Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases.
Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV).
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.
Introduction
Antiretroviral therapy (ART) has dramatically changed the life expectancy of people living with HIV (PLHIV). HIV can now be considered a chronic infection, and the expected life span of PLHIV who receive efficient treatment is comparable to HIV-negative individuals [1, 2]. As a consequence, an increasing number of PLHIV are of older age. For example, in 2018 51% of PLHIV in the US were 50 years of age or older [3].
The risk of developing age-related and lifestyle-related diseases increases with age. PLHIV are, in addition, at higher risk of non-infectious comorbidities compared to the general population [4–6]. Furthermore, elderly people are, in general, at higher risk of adverse events to medications and may require lower doses of medications than recommended for younger individuals [7, 8]. It has been reported that PLHIV above 50 years of age have more concomitant medications and a higher risk of potential drug-drug interactions (PDDIs) compared to PLHIV below 50 years of age [9, 10].
Since the mid 1990s the standard regime for HIV treatment is two nucleoside reverse-transcriptase inhibitors (NRTIs) combined with a third agent from another drug class (most commonly a nucleoside reverse-transcriptase inhibitor (NNRTI), a protease inhibitor (PI) or an integrase inhibitor (INSTI). A dolutegravir (INSTI) or Efavirenz (NNRTI) containing regimen is recommended by WHO as first line treatment today [11]. In the Swedish setting a dolutegravir or darunavir containing regimen is recommended by the Swedish Reference Group for Antiviral Therapy [12]. Neither WHO nor Sweden have specific treatment recommendations for elderly PLHIV.
Scientific data on the pharmacokinetics of PIs and NNRTIs in individuals 65 years of age and older are scarce. The primary objective of this study was to investigate differences in steady-state plasma drug levels of ATV, DRV and EFV in PLHIV ≥ 65 years of age as compared to PLHIV ≤ 49 years of age. Secondary objectives were to study differences in self-reported side-effects, concomitant chronic diseases and medications, and PDDIs.
Methods
PLHIV who were followed at four HIV centers in Sweden: the Department of Infectious Diseases at Sahlgrenska University Hospital in Gothenburg; the Department of Infectious Diseases at South Älvsborg Hospital in Borås; the Department of Infectious Diseases at Karolinska University Hospital Huddinge in Stockholm; and the Department of Infectious Diseases at Stockholm South General Hospital in Stockholm, and met the inclusion criteria (age, 65 years of age or older for the study group or 49 years of age or younger for the control group; and on stable ART containing atazanavir (ATV), darunavir (DRV) or efavirenz (EFV) for more than 6 months) were eligible for inclusion in this cross-sectional study. On the day of inclusion, a blood sample for analysis of plasma drug level was taken, and concomitant medications (including non-prescription drugs and herbal supplements) and any side-effects related to ART were recorded in a standardized questionnaire, S1 File. Blood samples drawn between 6 to 36 hours after last dose of medication was included in the analysis of steady-state drug levels and adjusted for time with ANCOVA analysis. Adherence was recorded using a modified ACTG adherence questionnaire [13]. Any missed dose during the preceding 4 days was considered as non-adherence.
Comorbidities were registered by structured medical record reviews. PDDIs were analyzed using the Liverpool University HIV drug interactions [14] and Janusmed [15] webtools. The Liverpool University HIV drug interactions database definitions for PDDIs were used and red flag (drugs should not be co-administered) and orange flag (a potential interaction that may require dose monitoring, alteration of drug dosage or timing of administration) interactions were included in the analysis. If there was an interaction between a comedication and both the PI and the booster, the interaction was counted as one interaction in the analysis. Individuals taking DRV b.i.d. or ATV without ritonavir booster were excluded from the analysis of plasma drug levels.
All study participants gave their written informed consent and ethics approval for the study was granted by the Research Ethics Committee at Gothenburg University.
Laboratory analyses
Plasma samples were frozen at –70°C immediately after sampling until analysis. Drug levels were analyzed using a reverse-phase High Pressure Liquid Chromatography (HPLC) with ultraviolet (UV) detection at the routine pharmacology analytical laboratory at Karolinska University Hospital, Huddinge in Stockholm, Sweden. The method was CAP (College of American Pathologists) and Swedac accredited and has been described elsewhere [16]. Routine clinical methods were used to analyze CD4 cell count, liver enzymes and creatinine according to local laboratory standards.
Statistical analyses
Differences in plasma drug levels were analyzed with ANCOVA (adjusting for time) with log-transformed concentrations of ATV, DRV, and EFV. Chi-square test and Fisher’s exact test were used to compare the frequencies of side-effects, CD4/CD8 ratios, and AIDS diagnosis as appropriate. Mann Whitney U-test and Kruskal Wallis test (with Bonferroni correction for multiple tests) were used to compare frequencies of concomitant medications and PDDIs. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics, Armonk, NY, USA) or Prism version 8.0 (Graphpad Software Inc., La Jolla, CA, USA).
Results
One hundred and seventy-two individuals 65 years of age or older were eligible for inclusion and were asked to participate in the study at the four sites. Between November 2013 and August 2015, 100 individuals were enrolled in the study group (ATV n = 19; DRV n = 35; EFV n = 46) and 99 individuals in the control group (ATV n = 18; DRV n = 37; EFV n = 44). Baseline characteristics are listed in Table 1. Three individuals had HIV RNA blips (HIV RNA 59–156 copies/mL) at inclusion; all other patients had HIV RNA levels < 50 copies/mL at inclusion. Twenty-seven patients were excluded from the plasma drug level analysis: 15 individuals received DRV b.i.d. and 9 were treated with ATV, either unboosted or with dosing not according to clinical standards. Three were excluded from the plasma drug level analysis due to sample management (elapsed time since last dose less than 6 hours, or elapsed time since last dose unknown). Patients included in the plasma drug level analysis received DRV/r 800/100 mg, ATV/r 300/100 mg, or EFV 600 mg q.d. There was a significant difference in ALT levels between study and control group in the ATV arm, however the majority of subjects had ALT within the normal range. The study group had a lower glomerular filtration rate (GFR) in all arms compared to controls, although within the normal range.
10.1371/journal.pone.0246171.t001 Table 1 Baseline characteristics.
ATV DRV EFV
Study n = 19 Control n = 18 p Study n = 35 Control n = 37 p Study n = 46 Control n = 44 p
Age (median [IQR]) 68 (66–70) 46 (40.75–47.5) 68 (67–72) 45 (37.5–47) 69 (67–72) 43 (37–46)
Gender (M/F) (n) 13/6 17/1 0.09 33/2 32/5 0.43 41/5 38/6 0.76
BMI (median [IQR]) 24.3 (22.3–27.2) 25.3 (23.9–27.9) 0.28 25.7 (23.0–26.9) 23.5 (22.5–26.0) 0.17 24.4 (21.9–27.8) 24.2 (22.1–28.5) 0.65
GFR (ml/min) (median [IQR]) 83.7 (71.0–96.8) 118.0 (109.9–134.4) <0.001 86.1 (55.4–95.3) 110.4 (97.0–135.8) <0.001 79.9 (67.6–98.5) 124.3 (116.0–145.2) <0.001
ALT (μkat/L) (median [IQR]) 0.40 (0.30–0.55) 0.63 (0.52–0.75) 0.017 0.32 (0.24–0.48) 0.41 (0.31–0.55) 0.14 0.48 (0.33–0.56) 0.55 (0.38–0.85) 0.056
CD4 cell count (median [IQR]) 650 (370–730) 730 (490–912.5) 0.13 560 (420–650) 600 (465–830) 0.54 535 (380–687.5) 575 (407.5–750) 0.39
CD4 cell count Nadir (median [IQR]) 130 (69–223) 241 (187–337) 0.001 185 (90–281) 218 (40–310) 0.97 208 (158–263) 219 (167–303) 0.49
Backbone 3TC/ABC 9 14 0.09 11 14 0.63 23 12 0.03
Backbone FTC/TDF 10 4 0.09 5 12 0.03 23 30 0.09
Backbone other** 0 0 NA 19 9 0.02 0 2 0.24
Comorbidities (n) (median [IQR]) 2 (1–4) 2 (0.25–3) 0.31 3 (2–5) 1 (1–3) 0.001 3 (2–4.25) 1 (0–2) <0.001
** Other backbones: RAL, ETV, RAL + maraviroc, RAL + EFV, RAL + DRV, LPV/r, RAL + 3TC, 3TC, DTG + 3TC, RPV, no backbone.
The steady-state DRV concentrations were significantly higher in the study group (n = 25) compared to the control group (n = 30) (p = 0.047), Fig 1. The geometric mean was 48% higher in the study group than in the control group. The analysis of the ATV arm (study group n = 19, control group n = 18) showed a difference in steady state-levels (geometric mean 69% higher in the study group), with a trend towards statistical significance (p = 0.056). No statistically significant difference between the groups was found in the EFV (p = 0.87) arm. There were no differences in self-reported adherence between the study group (96% adherent) and control group (93% adherent) (p = 0.537), or between different treatment arms either in the study or the control group.
10.1371/journal.pone.0246171.g001 Fig 1 Steady-state levels of ART.
Steady-state levels in plasma of A) Darunavir (p = 0.047), B) Atazanavir (p = 0.056), and C) Efavirenz (p = 0.87) in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group).
There were no statistically significant differences in reported side-effects between the study group (23%) and the control group (34%) (p = 0.146), Fig 2. When dividing the groups according to drug regimen (taking the study group together with the controls), the DRV group had a higher rate of reported side-effects (ATV: 16.7%; DRV: 39.4%; EFV: 25.9%; p = 0.038), which was significantly different compared to the ATV arm. The difference remained when PLHIV ≥ 65 years of age were analysed separately (ATV: 0%; DRV: 36.7%; EFV: 23.8%; p = 0.014), with significant differences between DRV and ATV, and EFV and ATV. In the DRV arm there were no significant difference in reported side effects between the study group and the control group, (p = 0.80). The most commonly-reported side-effect in the DRV groups was diarrhea.
10.1371/journal.pone.0246171.g002 Fig 2 Frequency of self-reported side-effects.
A: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) (ns). B: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) divided into treatment arms (p = 0.038). C: Frequency of self-reported side-effects divided into groups according to treatment arm and study group vs. control group.
As expected, the study group had a significantly higher mean (range) number of concomitant medications, 3.7 (0–12), compared to the control group, 1.1 (0–10) (p < 0.001). Accordingly, the study group had significantly more PDDIs (mean (range)) 1.1 (0–6) compared to the control group, 0.3 (0–3) (p < 0.001). The most common PDDIs for ATV were statins and beta-blocking agents, and statins and antidepressants for DRV and EFV.
Analysis of the study group showed that the DRV arm had significantly more PDDIs (mean [range]) 1.4 (0–6) than the EFV arm 0.7 (0–3) (p = 0.03). The ATV arm had a mean (range) of 1.2 (0–4) PDDIs, not significantly different compared to either the DRV arm or the EFV arm (see Table 2). Notably, the use of concomitant medications was not higher in the DRV arm. Eight individuals in the study group had red flag PDDIs: DRV/r and alfuzosin (risk for severe hypotension, n = 2); DRV/r and clopidogrel (reduced effect of clopidogrel, n = 3); DRV/r and alfuzosin + clopidogrel (n = 1); ATV and budesonide (increased risk of steroid side-effects, n = 1); and ATV and lansoprazole (reduced ATV uptake, n = 1). Whereas no one in the control group had a red flag interaction.
10.1371/journal.pone.0246171.t002 Table 2 Potential drug-drug interactions.
PDDIs (n) (mean [range]) p
Atazanavir 1.2 (0–4)
Darunavir 1.4 (1–6) 0.026
Efavirenz 0.7 (0–3)
Number of potential drug-drug interactions (PDDIs) in the study group presented by treatment regime.
There were no differences in CD4/CD8 ratios (≥ 1 or < 1) between the study group (≥ 1 n = 35 (35%)) and control group (≥ 1 n = 40 (40%)) (p = 0.43), the ATV/DRV/EFV arms, or between arms in the different groups. No differences were found in the history of AIDS defining events in the study group vs. control group. In total 38 individuals had one or more AIDS defining diagnoses, S1 Table.
Discussion
We found a difference in the steady-state plasma drug levels of DRV in PLHIV who were ≥ 65 years of age, as compared to PLHIV < 50 years of age. To our knowledge, only one previous study has addressed the question of plasma DRV levels in elderly PLHIV [17]. In agreement with our results, the authors reported higher DRV levels in individuals > 60 years of age compared to those ≤ 40 years of age.
The difference in plasma drug levels is also consistent with earlier findings regarding other PIs, including ATV [18–20]. We noted a difference in ATV levels between elderly and younger PLHIV, with a trend towards statistical significance. The lack of significance is probably due to the small sample size. In a previous report by Avihingsanon et al., higher trough levels and higher exposure to ATV in PLHIV > 42 years of age was found compared to individuals ≤ 42 years of age, consistent with our results. This difference was more pronounced in PLHIV > 50 years of age [18]. Winston et al. also found a significant association between age and plasma drug levels of PIs [19]. We did not find any significant difference in EFV plasma drug levels in the study group compared to the control group. This is in agreement with the findings in other reports [19, 21].
There is only very limited pharmacokinetic data on ART in PLHIV older than 65 years of age. There are, however, several general age-related biological changes that may affect the metabolism of ART, e.g. decrease of liver and renal function and changes in body composition that influence the volume of distribution [22]. ATV, DRV and EFV are metabolized in the liver (ATV and DRV mainly through CYP3A4 and EFV through CYP 3A4 and CYP2B6). Only a minor portion of these drugs is eliminated through the kidneys. Therefore, the difference in drug levels in the DRV and ATV arm cannot be explained by differences in GFR. We found no clinically measurable difference in liver function measured by ALT but other changes in liver function related to age may have affected the drug levels.
Other aspects not related to metabolism may also affect the efficacy of ART. Older PLHIV have been shown to be more adherent to their treatment regimen than younger individuals [23, 24]. However, with increasing age there is a higher risk of cognitive impairment that may affect the adherence in the oldest. No difference in adherence was found in our study to support or reject either higher or lower adherence in elderly PLHIV.
Overall, we found a higher frequency of self-reported side-effects in the DRV arm, in comparison to the ATV and the EFV arms (although not significantly different from the EFV arm). The higher frequency was also present in the study group (although not statistically significant).
To the best of our knowledge this is the only study that compared the frequency of side-effects between different PIs/NNRTIs in PLHIV older than 65 years of age. A possible reason for the DRV arm having a higher frequency of side-effects might be that DRV was chosen because of extensive ART history and viral resistance, resulting in few available alternative regimes at the time of inclusion in the study and as a consequence there was a higher tolerance of side-effects. On the other hand, no difference in self-reported side effects was noted between the DRV study group and control group, even though we found a difference in steady state plasma drug levels. This observation may reflect under reporting in the study group. While it is not possible in the present study to establish a causal link, further studies are needed illuminate this issue.
PDDIs are common among PLHIV [25, 26], and the risk increases with age due to increasing frequencies of comorbidities and concomitant medications [9, 10]. Our result is in line with these earlier studies. Red flag interactions has been reported in 2% to 5.6% of PLHIV and 7.1% to 8.7% in PLHIV ≥ 65 years of age in earlier studies, similar to our findings [26–29].
ATV and DRV are both PIs and therefore they have, in general, the same PDDIs. However, they differ in regard to some frequently used drug classes e.g. beta blockers and PPIs. Since EFV is a NNRTI it has another drug interaction profile. DRV accounted for the majority of the red flag interactions found in our study, consistent with earlier findings of PLHIV in all ages [27]. Other studies have reported a higher probability of an orange or red flag PDDI in individuals treated with a PI (not restricted to those ≥ 65) [9, 28]. This is consistent with our finding that the DRV arm in the study group had a higher mean of PDDIs than the EFV arm. We did not find a significant difference between the ATV and EFV arms, however this may be due to the small sample size. The majority of the red flag interactions found were related to the concomitant use of alfuzosin, for treatment of benign prostatic hyperplasia, and/or clopidogrel, for treatment of vascular disease, which both are conditions that have a higher prevalence in older ages.
Our study has several limitations. Since we analysed steady-state plasma drug levels and not trough levels of ART, it is difficult to compare our results with trough levels in other studies. Thus, we were unable to evaluate potentially toxic plasma drug levels of ART drugs or levels below the proposed minimal effective concentrations. Patients were sampled from 6 to 36 hours after their last dose of ART. This was adjusted for in the statistical model, but there remains a risk that, because of this approach, we were not able to detect minor differences in drug levels between the study and control arms for ATV and EFV. Also, there was a difference in back-bone between PLHIV on DRV compared to ATV and EFV that may have affected self-reported side-effects. Concomitant medications may also have influenced the self-reported side-effects, though the participants were asked specifically to report side-effects related to ART. The PDDIs were calculated only for ATV, DRV or EFV regimens and therefore differences in back-bone likely did not affect the results. In addition, interactions between NRTIs and other medications are uncommon. The participants in the study were included consecutively at four sites in Sweden and it is possible that this introduced a selection bias, favoring PLHIV with frequent visits.
Conclusion
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV who were 65 years of age or older, as compared to controls who were 49 years of age and younger. Our findings are important for the management of elderly PLHIV and raise the question of whether regular monitoring of plasma levels and dose adjustment of DRV and other PIs is warranted in the elderly.
Supporting information
S1 Table Aids defining diagnoses.
(DOCX)
Click here for additional data file.
S2 Table Data set.
(XLSX)
Click here for additional data file.
S1 File Questionnaire/case report form.
(DOCX)
Click here for additional data file.
We would like to thank all the participants in the study, study nurses and technical staff. | CETIRIZINE HYDROCHLORIDE, EFAVIRENZ, EMTRICITABINE\TENOFOVIR DISOPROXIL, VALACYCLOVIR HYDROCHLORIDE | DrugsGivenReaction | CC BY | 33539440 | 19,781,519 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Diabetes mellitus'. | Higher plasma drug levels in elderly people living with HIV treated with darunavir.
The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age.
Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases.
Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV).
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.
Introduction
Antiretroviral therapy (ART) has dramatically changed the life expectancy of people living with HIV (PLHIV). HIV can now be considered a chronic infection, and the expected life span of PLHIV who receive efficient treatment is comparable to HIV-negative individuals [1, 2]. As a consequence, an increasing number of PLHIV are of older age. For example, in 2018 51% of PLHIV in the US were 50 years of age or older [3].
The risk of developing age-related and lifestyle-related diseases increases with age. PLHIV are, in addition, at higher risk of non-infectious comorbidities compared to the general population [4–6]. Furthermore, elderly people are, in general, at higher risk of adverse events to medications and may require lower doses of medications than recommended for younger individuals [7, 8]. It has been reported that PLHIV above 50 years of age have more concomitant medications and a higher risk of potential drug-drug interactions (PDDIs) compared to PLHIV below 50 years of age [9, 10].
Since the mid 1990s the standard regime for HIV treatment is two nucleoside reverse-transcriptase inhibitors (NRTIs) combined with a third agent from another drug class (most commonly a nucleoside reverse-transcriptase inhibitor (NNRTI), a protease inhibitor (PI) or an integrase inhibitor (INSTI). A dolutegravir (INSTI) or Efavirenz (NNRTI) containing regimen is recommended by WHO as first line treatment today [11]. In the Swedish setting a dolutegravir or darunavir containing regimen is recommended by the Swedish Reference Group for Antiviral Therapy [12]. Neither WHO nor Sweden have specific treatment recommendations for elderly PLHIV.
Scientific data on the pharmacokinetics of PIs and NNRTIs in individuals 65 years of age and older are scarce. The primary objective of this study was to investigate differences in steady-state plasma drug levels of ATV, DRV and EFV in PLHIV ≥ 65 years of age as compared to PLHIV ≤ 49 years of age. Secondary objectives were to study differences in self-reported side-effects, concomitant chronic diseases and medications, and PDDIs.
Methods
PLHIV who were followed at four HIV centers in Sweden: the Department of Infectious Diseases at Sahlgrenska University Hospital in Gothenburg; the Department of Infectious Diseases at South Älvsborg Hospital in Borås; the Department of Infectious Diseases at Karolinska University Hospital Huddinge in Stockholm; and the Department of Infectious Diseases at Stockholm South General Hospital in Stockholm, and met the inclusion criteria (age, 65 years of age or older for the study group or 49 years of age or younger for the control group; and on stable ART containing atazanavir (ATV), darunavir (DRV) or efavirenz (EFV) for more than 6 months) were eligible for inclusion in this cross-sectional study. On the day of inclusion, a blood sample for analysis of plasma drug level was taken, and concomitant medications (including non-prescription drugs and herbal supplements) and any side-effects related to ART were recorded in a standardized questionnaire, S1 File. Blood samples drawn between 6 to 36 hours after last dose of medication was included in the analysis of steady-state drug levels and adjusted for time with ANCOVA analysis. Adherence was recorded using a modified ACTG adherence questionnaire [13]. Any missed dose during the preceding 4 days was considered as non-adherence.
Comorbidities were registered by structured medical record reviews. PDDIs were analyzed using the Liverpool University HIV drug interactions [14] and Janusmed [15] webtools. The Liverpool University HIV drug interactions database definitions for PDDIs were used and red flag (drugs should not be co-administered) and orange flag (a potential interaction that may require dose monitoring, alteration of drug dosage or timing of administration) interactions were included in the analysis. If there was an interaction between a comedication and both the PI and the booster, the interaction was counted as one interaction in the analysis. Individuals taking DRV b.i.d. or ATV without ritonavir booster were excluded from the analysis of plasma drug levels.
All study participants gave their written informed consent and ethics approval for the study was granted by the Research Ethics Committee at Gothenburg University.
Laboratory analyses
Plasma samples were frozen at –70°C immediately after sampling until analysis. Drug levels were analyzed using a reverse-phase High Pressure Liquid Chromatography (HPLC) with ultraviolet (UV) detection at the routine pharmacology analytical laboratory at Karolinska University Hospital, Huddinge in Stockholm, Sweden. The method was CAP (College of American Pathologists) and Swedac accredited and has been described elsewhere [16]. Routine clinical methods were used to analyze CD4 cell count, liver enzymes and creatinine according to local laboratory standards.
Statistical analyses
Differences in plasma drug levels were analyzed with ANCOVA (adjusting for time) with log-transformed concentrations of ATV, DRV, and EFV. Chi-square test and Fisher’s exact test were used to compare the frequencies of side-effects, CD4/CD8 ratios, and AIDS diagnosis as appropriate. Mann Whitney U-test and Kruskal Wallis test (with Bonferroni correction for multiple tests) were used to compare frequencies of concomitant medications and PDDIs. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics, Armonk, NY, USA) or Prism version 8.0 (Graphpad Software Inc., La Jolla, CA, USA).
Results
One hundred and seventy-two individuals 65 years of age or older were eligible for inclusion and were asked to participate in the study at the four sites. Between November 2013 and August 2015, 100 individuals were enrolled in the study group (ATV n = 19; DRV n = 35; EFV n = 46) and 99 individuals in the control group (ATV n = 18; DRV n = 37; EFV n = 44). Baseline characteristics are listed in Table 1. Three individuals had HIV RNA blips (HIV RNA 59–156 copies/mL) at inclusion; all other patients had HIV RNA levels < 50 copies/mL at inclusion. Twenty-seven patients were excluded from the plasma drug level analysis: 15 individuals received DRV b.i.d. and 9 were treated with ATV, either unboosted or with dosing not according to clinical standards. Three were excluded from the plasma drug level analysis due to sample management (elapsed time since last dose less than 6 hours, or elapsed time since last dose unknown). Patients included in the plasma drug level analysis received DRV/r 800/100 mg, ATV/r 300/100 mg, or EFV 600 mg q.d. There was a significant difference in ALT levels between study and control group in the ATV arm, however the majority of subjects had ALT within the normal range. The study group had a lower glomerular filtration rate (GFR) in all arms compared to controls, although within the normal range.
10.1371/journal.pone.0246171.t001 Table 1 Baseline characteristics.
ATV DRV EFV
Study n = 19 Control n = 18 p Study n = 35 Control n = 37 p Study n = 46 Control n = 44 p
Age (median [IQR]) 68 (66–70) 46 (40.75–47.5) 68 (67–72) 45 (37.5–47) 69 (67–72) 43 (37–46)
Gender (M/F) (n) 13/6 17/1 0.09 33/2 32/5 0.43 41/5 38/6 0.76
BMI (median [IQR]) 24.3 (22.3–27.2) 25.3 (23.9–27.9) 0.28 25.7 (23.0–26.9) 23.5 (22.5–26.0) 0.17 24.4 (21.9–27.8) 24.2 (22.1–28.5) 0.65
GFR (ml/min) (median [IQR]) 83.7 (71.0–96.8) 118.0 (109.9–134.4) <0.001 86.1 (55.4–95.3) 110.4 (97.0–135.8) <0.001 79.9 (67.6–98.5) 124.3 (116.0–145.2) <0.001
ALT (μkat/L) (median [IQR]) 0.40 (0.30–0.55) 0.63 (0.52–0.75) 0.017 0.32 (0.24–0.48) 0.41 (0.31–0.55) 0.14 0.48 (0.33–0.56) 0.55 (0.38–0.85) 0.056
CD4 cell count (median [IQR]) 650 (370–730) 730 (490–912.5) 0.13 560 (420–650) 600 (465–830) 0.54 535 (380–687.5) 575 (407.5–750) 0.39
CD4 cell count Nadir (median [IQR]) 130 (69–223) 241 (187–337) 0.001 185 (90–281) 218 (40–310) 0.97 208 (158–263) 219 (167–303) 0.49
Backbone 3TC/ABC 9 14 0.09 11 14 0.63 23 12 0.03
Backbone FTC/TDF 10 4 0.09 5 12 0.03 23 30 0.09
Backbone other** 0 0 NA 19 9 0.02 0 2 0.24
Comorbidities (n) (median [IQR]) 2 (1–4) 2 (0.25–3) 0.31 3 (2–5) 1 (1–3) 0.001 3 (2–4.25) 1 (0–2) <0.001
** Other backbones: RAL, ETV, RAL + maraviroc, RAL + EFV, RAL + DRV, LPV/r, RAL + 3TC, 3TC, DTG + 3TC, RPV, no backbone.
The steady-state DRV concentrations were significantly higher in the study group (n = 25) compared to the control group (n = 30) (p = 0.047), Fig 1. The geometric mean was 48% higher in the study group than in the control group. The analysis of the ATV arm (study group n = 19, control group n = 18) showed a difference in steady state-levels (geometric mean 69% higher in the study group), with a trend towards statistical significance (p = 0.056). No statistically significant difference between the groups was found in the EFV (p = 0.87) arm. There were no differences in self-reported adherence between the study group (96% adherent) and control group (93% adherent) (p = 0.537), or between different treatment arms either in the study or the control group.
10.1371/journal.pone.0246171.g001 Fig 1 Steady-state levels of ART.
Steady-state levels in plasma of A) Darunavir (p = 0.047), B) Atazanavir (p = 0.056), and C) Efavirenz (p = 0.87) in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group).
There were no statistically significant differences in reported side-effects between the study group (23%) and the control group (34%) (p = 0.146), Fig 2. When dividing the groups according to drug regimen (taking the study group together with the controls), the DRV group had a higher rate of reported side-effects (ATV: 16.7%; DRV: 39.4%; EFV: 25.9%; p = 0.038), which was significantly different compared to the ATV arm. The difference remained when PLHIV ≥ 65 years of age were analysed separately (ATV: 0%; DRV: 36.7%; EFV: 23.8%; p = 0.014), with significant differences between DRV and ATV, and EFV and ATV. In the DRV arm there were no significant difference in reported side effects between the study group and the control group, (p = 0.80). The most commonly-reported side-effect in the DRV groups was diarrhea.
10.1371/journal.pone.0246171.g002 Fig 2 Frequency of self-reported side-effects.
A: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) (ns). B: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) divided into treatment arms (p = 0.038). C: Frequency of self-reported side-effects divided into groups according to treatment arm and study group vs. control group.
As expected, the study group had a significantly higher mean (range) number of concomitant medications, 3.7 (0–12), compared to the control group, 1.1 (0–10) (p < 0.001). Accordingly, the study group had significantly more PDDIs (mean (range)) 1.1 (0–6) compared to the control group, 0.3 (0–3) (p < 0.001). The most common PDDIs for ATV were statins and beta-blocking agents, and statins and antidepressants for DRV and EFV.
Analysis of the study group showed that the DRV arm had significantly more PDDIs (mean [range]) 1.4 (0–6) than the EFV arm 0.7 (0–3) (p = 0.03). The ATV arm had a mean (range) of 1.2 (0–4) PDDIs, not significantly different compared to either the DRV arm or the EFV arm (see Table 2). Notably, the use of concomitant medications was not higher in the DRV arm. Eight individuals in the study group had red flag PDDIs: DRV/r and alfuzosin (risk for severe hypotension, n = 2); DRV/r and clopidogrel (reduced effect of clopidogrel, n = 3); DRV/r and alfuzosin + clopidogrel (n = 1); ATV and budesonide (increased risk of steroid side-effects, n = 1); and ATV and lansoprazole (reduced ATV uptake, n = 1). Whereas no one in the control group had a red flag interaction.
10.1371/journal.pone.0246171.t002 Table 2 Potential drug-drug interactions.
PDDIs (n) (mean [range]) p
Atazanavir 1.2 (0–4)
Darunavir 1.4 (1–6) 0.026
Efavirenz 0.7 (0–3)
Number of potential drug-drug interactions (PDDIs) in the study group presented by treatment regime.
There were no differences in CD4/CD8 ratios (≥ 1 or < 1) between the study group (≥ 1 n = 35 (35%)) and control group (≥ 1 n = 40 (40%)) (p = 0.43), the ATV/DRV/EFV arms, or between arms in the different groups. No differences were found in the history of AIDS defining events in the study group vs. control group. In total 38 individuals had one or more AIDS defining diagnoses, S1 Table.
Discussion
We found a difference in the steady-state plasma drug levels of DRV in PLHIV who were ≥ 65 years of age, as compared to PLHIV < 50 years of age. To our knowledge, only one previous study has addressed the question of plasma DRV levels in elderly PLHIV [17]. In agreement with our results, the authors reported higher DRV levels in individuals > 60 years of age compared to those ≤ 40 years of age.
The difference in plasma drug levels is also consistent with earlier findings regarding other PIs, including ATV [18–20]. We noted a difference in ATV levels between elderly and younger PLHIV, with a trend towards statistical significance. The lack of significance is probably due to the small sample size. In a previous report by Avihingsanon et al., higher trough levels and higher exposure to ATV in PLHIV > 42 years of age was found compared to individuals ≤ 42 years of age, consistent with our results. This difference was more pronounced in PLHIV > 50 years of age [18]. Winston et al. also found a significant association between age and plasma drug levels of PIs [19]. We did not find any significant difference in EFV plasma drug levels in the study group compared to the control group. This is in agreement with the findings in other reports [19, 21].
There is only very limited pharmacokinetic data on ART in PLHIV older than 65 years of age. There are, however, several general age-related biological changes that may affect the metabolism of ART, e.g. decrease of liver and renal function and changes in body composition that influence the volume of distribution [22]. ATV, DRV and EFV are metabolized in the liver (ATV and DRV mainly through CYP3A4 and EFV through CYP 3A4 and CYP2B6). Only a minor portion of these drugs is eliminated through the kidneys. Therefore, the difference in drug levels in the DRV and ATV arm cannot be explained by differences in GFR. We found no clinically measurable difference in liver function measured by ALT but other changes in liver function related to age may have affected the drug levels.
Other aspects not related to metabolism may also affect the efficacy of ART. Older PLHIV have been shown to be more adherent to their treatment regimen than younger individuals [23, 24]. However, with increasing age there is a higher risk of cognitive impairment that may affect the adherence in the oldest. No difference in adherence was found in our study to support or reject either higher or lower adherence in elderly PLHIV.
Overall, we found a higher frequency of self-reported side-effects in the DRV arm, in comparison to the ATV and the EFV arms (although not significantly different from the EFV arm). The higher frequency was also present in the study group (although not statistically significant).
To the best of our knowledge this is the only study that compared the frequency of side-effects between different PIs/NNRTIs in PLHIV older than 65 years of age. A possible reason for the DRV arm having a higher frequency of side-effects might be that DRV was chosen because of extensive ART history and viral resistance, resulting in few available alternative regimes at the time of inclusion in the study and as a consequence there was a higher tolerance of side-effects. On the other hand, no difference in self-reported side effects was noted between the DRV study group and control group, even though we found a difference in steady state plasma drug levels. This observation may reflect under reporting in the study group. While it is not possible in the present study to establish a causal link, further studies are needed illuminate this issue.
PDDIs are common among PLHIV [25, 26], and the risk increases with age due to increasing frequencies of comorbidities and concomitant medications [9, 10]. Our result is in line with these earlier studies. Red flag interactions has been reported in 2% to 5.6% of PLHIV and 7.1% to 8.7% in PLHIV ≥ 65 years of age in earlier studies, similar to our findings [26–29].
ATV and DRV are both PIs and therefore they have, in general, the same PDDIs. However, they differ in regard to some frequently used drug classes e.g. beta blockers and PPIs. Since EFV is a NNRTI it has another drug interaction profile. DRV accounted for the majority of the red flag interactions found in our study, consistent with earlier findings of PLHIV in all ages [27]. Other studies have reported a higher probability of an orange or red flag PDDI in individuals treated with a PI (not restricted to those ≥ 65) [9, 28]. This is consistent with our finding that the DRV arm in the study group had a higher mean of PDDIs than the EFV arm. We did not find a significant difference between the ATV and EFV arms, however this may be due to the small sample size. The majority of the red flag interactions found were related to the concomitant use of alfuzosin, for treatment of benign prostatic hyperplasia, and/or clopidogrel, for treatment of vascular disease, which both are conditions that have a higher prevalence in older ages.
Our study has several limitations. Since we analysed steady-state plasma drug levels and not trough levels of ART, it is difficult to compare our results with trough levels in other studies. Thus, we were unable to evaluate potentially toxic plasma drug levels of ART drugs or levels below the proposed minimal effective concentrations. Patients were sampled from 6 to 36 hours after their last dose of ART. This was adjusted for in the statistical model, but there remains a risk that, because of this approach, we were not able to detect minor differences in drug levels between the study and control arms for ATV and EFV. Also, there was a difference in back-bone between PLHIV on DRV compared to ATV and EFV that may have affected self-reported side-effects. Concomitant medications may also have influenced the self-reported side-effects, though the participants were asked specifically to report side-effects related to ART. The PDDIs were calculated only for ATV, DRV or EFV regimens and therefore differences in back-bone likely did not affect the results. In addition, interactions between NRTIs and other medications are uncommon. The participants in the study were included consecutively at four sites in Sweden and it is possible that this introduced a selection bias, favoring PLHIV with frequent visits.
Conclusion
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV who were 65 years of age or older, as compared to controls who were 49 years of age and younger. Our findings are important for the management of elderly PLHIV and raise the question of whether regular monitoring of plasma levels and dose adjustment of DRV and other PIs is warranted in the elderly.
Supporting information
S1 Table Aids defining diagnoses.
(DOCX)
Click here for additional data file.
S2 Table Data set.
(XLSX)
Click here for additional data file.
S1 File Questionnaire/case report form.
(DOCX)
Click here for additional data file.
We would like to thank all the participants in the study, study nurses and technical staff. | ABACAVIR SULFATE\LAMIVUDINE, ACETAMINOPHEN, EFAVIRENZ, FOSAMPRENAVIR CALCIUM, INSULIN HUMAN, METOPROLOL, RAMIPRIL, REPAGLINIDE, RITONAVIR, TRAMADOL HYDROCHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY | 33539440 | 19,781,509 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Erectile dysfunction'. | Higher plasma drug levels in elderly people living with HIV treated with darunavir.
The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age.
Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases.
Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV).
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.
Introduction
Antiretroviral therapy (ART) has dramatically changed the life expectancy of people living with HIV (PLHIV). HIV can now be considered a chronic infection, and the expected life span of PLHIV who receive efficient treatment is comparable to HIV-negative individuals [1, 2]. As a consequence, an increasing number of PLHIV are of older age. For example, in 2018 51% of PLHIV in the US were 50 years of age or older [3].
The risk of developing age-related and lifestyle-related diseases increases with age. PLHIV are, in addition, at higher risk of non-infectious comorbidities compared to the general population [4–6]. Furthermore, elderly people are, in general, at higher risk of adverse events to medications and may require lower doses of medications than recommended for younger individuals [7, 8]. It has been reported that PLHIV above 50 years of age have more concomitant medications and a higher risk of potential drug-drug interactions (PDDIs) compared to PLHIV below 50 years of age [9, 10].
Since the mid 1990s the standard regime for HIV treatment is two nucleoside reverse-transcriptase inhibitors (NRTIs) combined with a third agent from another drug class (most commonly a nucleoside reverse-transcriptase inhibitor (NNRTI), a protease inhibitor (PI) or an integrase inhibitor (INSTI). A dolutegravir (INSTI) or Efavirenz (NNRTI) containing regimen is recommended by WHO as first line treatment today [11]. In the Swedish setting a dolutegravir or darunavir containing regimen is recommended by the Swedish Reference Group for Antiviral Therapy [12]. Neither WHO nor Sweden have specific treatment recommendations for elderly PLHIV.
Scientific data on the pharmacokinetics of PIs and NNRTIs in individuals 65 years of age and older are scarce. The primary objective of this study was to investigate differences in steady-state plasma drug levels of ATV, DRV and EFV in PLHIV ≥ 65 years of age as compared to PLHIV ≤ 49 years of age. Secondary objectives were to study differences in self-reported side-effects, concomitant chronic diseases and medications, and PDDIs.
Methods
PLHIV who were followed at four HIV centers in Sweden: the Department of Infectious Diseases at Sahlgrenska University Hospital in Gothenburg; the Department of Infectious Diseases at South Älvsborg Hospital in Borås; the Department of Infectious Diseases at Karolinska University Hospital Huddinge in Stockholm; and the Department of Infectious Diseases at Stockholm South General Hospital in Stockholm, and met the inclusion criteria (age, 65 years of age or older for the study group or 49 years of age or younger for the control group; and on stable ART containing atazanavir (ATV), darunavir (DRV) or efavirenz (EFV) for more than 6 months) were eligible for inclusion in this cross-sectional study. On the day of inclusion, a blood sample for analysis of plasma drug level was taken, and concomitant medications (including non-prescription drugs and herbal supplements) and any side-effects related to ART were recorded in a standardized questionnaire, S1 File. Blood samples drawn between 6 to 36 hours after last dose of medication was included in the analysis of steady-state drug levels and adjusted for time with ANCOVA analysis. Adherence was recorded using a modified ACTG adherence questionnaire [13]. Any missed dose during the preceding 4 days was considered as non-adherence.
Comorbidities were registered by structured medical record reviews. PDDIs were analyzed using the Liverpool University HIV drug interactions [14] and Janusmed [15] webtools. The Liverpool University HIV drug interactions database definitions for PDDIs were used and red flag (drugs should not be co-administered) and orange flag (a potential interaction that may require dose monitoring, alteration of drug dosage or timing of administration) interactions were included in the analysis. If there was an interaction between a comedication and both the PI and the booster, the interaction was counted as one interaction in the analysis. Individuals taking DRV b.i.d. or ATV without ritonavir booster were excluded from the analysis of plasma drug levels.
All study participants gave their written informed consent and ethics approval for the study was granted by the Research Ethics Committee at Gothenburg University.
Laboratory analyses
Plasma samples were frozen at –70°C immediately after sampling until analysis. Drug levels were analyzed using a reverse-phase High Pressure Liquid Chromatography (HPLC) with ultraviolet (UV) detection at the routine pharmacology analytical laboratory at Karolinska University Hospital, Huddinge in Stockholm, Sweden. The method was CAP (College of American Pathologists) and Swedac accredited and has been described elsewhere [16]. Routine clinical methods were used to analyze CD4 cell count, liver enzymes and creatinine according to local laboratory standards.
Statistical analyses
Differences in plasma drug levels were analyzed with ANCOVA (adjusting for time) with log-transformed concentrations of ATV, DRV, and EFV. Chi-square test and Fisher’s exact test were used to compare the frequencies of side-effects, CD4/CD8 ratios, and AIDS diagnosis as appropriate. Mann Whitney U-test and Kruskal Wallis test (with Bonferroni correction for multiple tests) were used to compare frequencies of concomitant medications and PDDIs. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics, Armonk, NY, USA) or Prism version 8.0 (Graphpad Software Inc., La Jolla, CA, USA).
Results
One hundred and seventy-two individuals 65 years of age or older were eligible for inclusion and were asked to participate in the study at the four sites. Between November 2013 and August 2015, 100 individuals were enrolled in the study group (ATV n = 19; DRV n = 35; EFV n = 46) and 99 individuals in the control group (ATV n = 18; DRV n = 37; EFV n = 44). Baseline characteristics are listed in Table 1. Three individuals had HIV RNA blips (HIV RNA 59–156 copies/mL) at inclusion; all other patients had HIV RNA levels < 50 copies/mL at inclusion. Twenty-seven patients were excluded from the plasma drug level analysis: 15 individuals received DRV b.i.d. and 9 were treated with ATV, either unboosted or with dosing not according to clinical standards. Three were excluded from the plasma drug level analysis due to sample management (elapsed time since last dose less than 6 hours, or elapsed time since last dose unknown). Patients included in the plasma drug level analysis received DRV/r 800/100 mg, ATV/r 300/100 mg, or EFV 600 mg q.d. There was a significant difference in ALT levels between study and control group in the ATV arm, however the majority of subjects had ALT within the normal range. The study group had a lower glomerular filtration rate (GFR) in all arms compared to controls, although within the normal range.
10.1371/journal.pone.0246171.t001 Table 1 Baseline characteristics.
ATV DRV EFV
Study n = 19 Control n = 18 p Study n = 35 Control n = 37 p Study n = 46 Control n = 44 p
Age (median [IQR]) 68 (66–70) 46 (40.75–47.5) 68 (67–72) 45 (37.5–47) 69 (67–72) 43 (37–46)
Gender (M/F) (n) 13/6 17/1 0.09 33/2 32/5 0.43 41/5 38/6 0.76
BMI (median [IQR]) 24.3 (22.3–27.2) 25.3 (23.9–27.9) 0.28 25.7 (23.0–26.9) 23.5 (22.5–26.0) 0.17 24.4 (21.9–27.8) 24.2 (22.1–28.5) 0.65
GFR (ml/min) (median [IQR]) 83.7 (71.0–96.8) 118.0 (109.9–134.4) <0.001 86.1 (55.4–95.3) 110.4 (97.0–135.8) <0.001 79.9 (67.6–98.5) 124.3 (116.0–145.2) <0.001
ALT (μkat/L) (median [IQR]) 0.40 (0.30–0.55) 0.63 (0.52–0.75) 0.017 0.32 (0.24–0.48) 0.41 (0.31–0.55) 0.14 0.48 (0.33–0.56) 0.55 (0.38–0.85) 0.056
CD4 cell count (median [IQR]) 650 (370–730) 730 (490–912.5) 0.13 560 (420–650) 600 (465–830) 0.54 535 (380–687.5) 575 (407.5–750) 0.39
CD4 cell count Nadir (median [IQR]) 130 (69–223) 241 (187–337) 0.001 185 (90–281) 218 (40–310) 0.97 208 (158–263) 219 (167–303) 0.49
Backbone 3TC/ABC 9 14 0.09 11 14 0.63 23 12 0.03
Backbone FTC/TDF 10 4 0.09 5 12 0.03 23 30 0.09
Backbone other** 0 0 NA 19 9 0.02 0 2 0.24
Comorbidities (n) (median [IQR]) 2 (1–4) 2 (0.25–3) 0.31 3 (2–5) 1 (1–3) 0.001 3 (2–4.25) 1 (0–2) <0.001
** Other backbones: RAL, ETV, RAL + maraviroc, RAL + EFV, RAL + DRV, LPV/r, RAL + 3TC, 3TC, DTG + 3TC, RPV, no backbone.
The steady-state DRV concentrations were significantly higher in the study group (n = 25) compared to the control group (n = 30) (p = 0.047), Fig 1. The geometric mean was 48% higher in the study group than in the control group. The analysis of the ATV arm (study group n = 19, control group n = 18) showed a difference in steady state-levels (geometric mean 69% higher in the study group), with a trend towards statistical significance (p = 0.056). No statistically significant difference between the groups was found in the EFV (p = 0.87) arm. There were no differences in self-reported adherence between the study group (96% adherent) and control group (93% adherent) (p = 0.537), or between different treatment arms either in the study or the control group.
10.1371/journal.pone.0246171.g001 Fig 1 Steady-state levels of ART.
Steady-state levels in plasma of A) Darunavir (p = 0.047), B) Atazanavir (p = 0.056), and C) Efavirenz (p = 0.87) in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group).
There were no statistically significant differences in reported side-effects between the study group (23%) and the control group (34%) (p = 0.146), Fig 2. When dividing the groups according to drug regimen (taking the study group together with the controls), the DRV group had a higher rate of reported side-effects (ATV: 16.7%; DRV: 39.4%; EFV: 25.9%; p = 0.038), which was significantly different compared to the ATV arm. The difference remained when PLHIV ≥ 65 years of age were analysed separately (ATV: 0%; DRV: 36.7%; EFV: 23.8%; p = 0.014), with significant differences between DRV and ATV, and EFV and ATV. In the DRV arm there were no significant difference in reported side effects between the study group and the control group, (p = 0.80). The most commonly-reported side-effect in the DRV groups was diarrhea.
10.1371/journal.pone.0246171.g002 Fig 2 Frequency of self-reported side-effects.
A: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) (ns). B: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) divided into treatment arms (p = 0.038). C: Frequency of self-reported side-effects divided into groups according to treatment arm and study group vs. control group.
As expected, the study group had a significantly higher mean (range) number of concomitant medications, 3.7 (0–12), compared to the control group, 1.1 (0–10) (p < 0.001). Accordingly, the study group had significantly more PDDIs (mean (range)) 1.1 (0–6) compared to the control group, 0.3 (0–3) (p < 0.001). The most common PDDIs for ATV were statins and beta-blocking agents, and statins and antidepressants for DRV and EFV.
Analysis of the study group showed that the DRV arm had significantly more PDDIs (mean [range]) 1.4 (0–6) than the EFV arm 0.7 (0–3) (p = 0.03). The ATV arm had a mean (range) of 1.2 (0–4) PDDIs, not significantly different compared to either the DRV arm or the EFV arm (see Table 2). Notably, the use of concomitant medications was not higher in the DRV arm. Eight individuals in the study group had red flag PDDIs: DRV/r and alfuzosin (risk for severe hypotension, n = 2); DRV/r and clopidogrel (reduced effect of clopidogrel, n = 3); DRV/r and alfuzosin + clopidogrel (n = 1); ATV and budesonide (increased risk of steroid side-effects, n = 1); and ATV and lansoprazole (reduced ATV uptake, n = 1). Whereas no one in the control group had a red flag interaction.
10.1371/journal.pone.0246171.t002 Table 2 Potential drug-drug interactions.
PDDIs (n) (mean [range]) p
Atazanavir 1.2 (0–4)
Darunavir 1.4 (1–6) 0.026
Efavirenz 0.7 (0–3)
Number of potential drug-drug interactions (PDDIs) in the study group presented by treatment regime.
There were no differences in CD4/CD8 ratios (≥ 1 or < 1) between the study group (≥ 1 n = 35 (35%)) and control group (≥ 1 n = 40 (40%)) (p = 0.43), the ATV/DRV/EFV arms, or between arms in the different groups. No differences were found in the history of AIDS defining events in the study group vs. control group. In total 38 individuals had one or more AIDS defining diagnoses, S1 Table.
Discussion
We found a difference in the steady-state plasma drug levels of DRV in PLHIV who were ≥ 65 years of age, as compared to PLHIV < 50 years of age. To our knowledge, only one previous study has addressed the question of plasma DRV levels in elderly PLHIV [17]. In agreement with our results, the authors reported higher DRV levels in individuals > 60 years of age compared to those ≤ 40 years of age.
The difference in plasma drug levels is also consistent with earlier findings regarding other PIs, including ATV [18–20]. We noted a difference in ATV levels between elderly and younger PLHIV, with a trend towards statistical significance. The lack of significance is probably due to the small sample size. In a previous report by Avihingsanon et al., higher trough levels and higher exposure to ATV in PLHIV > 42 years of age was found compared to individuals ≤ 42 years of age, consistent with our results. This difference was more pronounced in PLHIV > 50 years of age [18]. Winston et al. also found a significant association between age and plasma drug levels of PIs [19]. We did not find any significant difference in EFV plasma drug levels in the study group compared to the control group. This is in agreement with the findings in other reports [19, 21].
There is only very limited pharmacokinetic data on ART in PLHIV older than 65 years of age. There are, however, several general age-related biological changes that may affect the metabolism of ART, e.g. decrease of liver and renal function and changes in body composition that influence the volume of distribution [22]. ATV, DRV and EFV are metabolized in the liver (ATV and DRV mainly through CYP3A4 and EFV through CYP 3A4 and CYP2B6). Only a minor portion of these drugs is eliminated through the kidneys. Therefore, the difference in drug levels in the DRV and ATV arm cannot be explained by differences in GFR. We found no clinically measurable difference in liver function measured by ALT but other changes in liver function related to age may have affected the drug levels.
Other aspects not related to metabolism may also affect the efficacy of ART. Older PLHIV have been shown to be more adherent to their treatment regimen than younger individuals [23, 24]. However, with increasing age there is a higher risk of cognitive impairment that may affect the adherence in the oldest. No difference in adherence was found in our study to support or reject either higher or lower adherence in elderly PLHIV.
Overall, we found a higher frequency of self-reported side-effects in the DRV arm, in comparison to the ATV and the EFV arms (although not significantly different from the EFV arm). The higher frequency was also present in the study group (although not statistically significant).
To the best of our knowledge this is the only study that compared the frequency of side-effects between different PIs/NNRTIs in PLHIV older than 65 years of age. A possible reason for the DRV arm having a higher frequency of side-effects might be that DRV was chosen because of extensive ART history and viral resistance, resulting in few available alternative regimes at the time of inclusion in the study and as a consequence there was a higher tolerance of side-effects. On the other hand, no difference in self-reported side effects was noted between the DRV study group and control group, even though we found a difference in steady state plasma drug levels. This observation may reflect under reporting in the study group. While it is not possible in the present study to establish a causal link, further studies are needed illuminate this issue.
PDDIs are common among PLHIV [25, 26], and the risk increases with age due to increasing frequencies of comorbidities and concomitant medications [9, 10]. Our result is in line with these earlier studies. Red flag interactions has been reported in 2% to 5.6% of PLHIV and 7.1% to 8.7% in PLHIV ≥ 65 years of age in earlier studies, similar to our findings [26–29].
ATV and DRV are both PIs and therefore they have, in general, the same PDDIs. However, they differ in regard to some frequently used drug classes e.g. beta blockers and PPIs. Since EFV is a NNRTI it has another drug interaction profile. DRV accounted for the majority of the red flag interactions found in our study, consistent with earlier findings of PLHIV in all ages [27]. Other studies have reported a higher probability of an orange or red flag PDDI in individuals treated with a PI (not restricted to those ≥ 65) [9, 28]. This is consistent with our finding that the DRV arm in the study group had a higher mean of PDDIs than the EFV arm. We did not find a significant difference between the ATV and EFV arms, however this may be due to the small sample size. The majority of the red flag interactions found were related to the concomitant use of alfuzosin, for treatment of benign prostatic hyperplasia, and/or clopidogrel, for treatment of vascular disease, which both are conditions that have a higher prevalence in older ages.
Our study has several limitations. Since we analysed steady-state plasma drug levels and not trough levels of ART, it is difficult to compare our results with trough levels in other studies. Thus, we were unable to evaluate potentially toxic plasma drug levels of ART drugs or levels below the proposed minimal effective concentrations. Patients were sampled from 6 to 36 hours after their last dose of ART. This was adjusted for in the statistical model, but there remains a risk that, because of this approach, we were not able to detect minor differences in drug levels between the study and control arms for ATV and EFV. Also, there was a difference in back-bone between PLHIV on DRV compared to ATV and EFV that may have affected self-reported side-effects. Concomitant medications may also have influenced the self-reported side-effects, though the participants were asked specifically to report side-effects related to ART. The PDDIs were calculated only for ATV, DRV or EFV regimens and therefore differences in back-bone likely did not affect the results. In addition, interactions between NRTIs and other medications are uncommon. The participants in the study were included consecutively at four sites in Sweden and it is possible that this introduced a selection bias, favoring PLHIV with frequent visits.
Conclusion
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV who were 65 years of age or older, as compared to controls who were 49 years of age and younger. Our findings are important for the management of elderly PLHIV and raise the question of whether regular monitoring of plasma levels and dose adjustment of DRV and other PIs is warranted in the elderly.
Supporting information
S1 Table Aids defining diagnoses.
(DOCX)
Click here for additional data file.
S2 Table Data set.
(XLSX)
Click here for additional data file.
S1 File Questionnaire/case report form.
(DOCX)
Click here for additional data file.
We would like to thank all the participants in the study, study nurses and technical staff. | CETIRIZINE HYDROCHLORIDE, EFAVIRENZ, EMTRICITABINE\TENOFOVIR DISOPROXIL, VALACYCLOVIR HYDROCHLORIDE | DrugsGivenReaction | CC BY | 33539440 | 19,781,519 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Fat redistribution'. | Higher plasma drug levels in elderly people living with HIV treated with darunavir.
The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age.
Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases.
Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV).
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.
Introduction
Antiretroviral therapy (ART) has dramatically changed the life expectancy of people living with HIV (PLHIV). HIV can now be considered a chronic infection, and the expected life span of PLHIV who receive efficient treatment is comparable to HIV-negative individuals [1, 2]. As a consequence, an increasing number of PLHIV are of older age. For example, in 2018 51% of PLHIV in the US were 50 years of age or older [3].
The risk of developing age-related and lifestyle-related diseases increases with age. PLHIV are, in addition, at higher risk of non-infectious comorbidities compared to the general population [4–6]. Furthermore, elderly people are, in general, at higher risk of adverse events to medications and may require lower doses of medications than recommended for younger individuals [7, 8]. It has been reported that PLHIV above 50 years of age have more concomitant medications and a higher risk of potential drug-drug interactions (PDDIs) compared to PLHIV below 50 years of age [9, 10].
Since the mid 1990s the standard regime for HIV treatment is two nucleoside reverse-transcriptase inhibitors (NRTIs) combined with a third agent from another drug class (most commonly a nucleoside reverse-transcriptase inhibitor (NNRTI), a protease inhibitor (PI) or an integrase inhibitor (INSTI). A dolutegravir (INSTI) or Efavirenz (NNRTI) containing regimen is recommended by WHO as first line treatment today [11]. In the Swedish setting a dolutegravir or darunavir containing regimen is recommended by the Swedish Reference Group for Antiviral Therapy [12]. Neither WHO nor Sweden have specific treatment recommendations for elderly PLHIV.
Scientific data on the pharmacokinetics of PIs and NNRTIs in individuals 65 years of age and older are scarce. The primary objective of this study was to investigate differences in steady-state plasma drug levels of ATV, DRV and EFV in PLHIV ≥ 65 years of age as compared to PLHIV ≤ 49 years of age. Secondary objectives were to study differences in self-reported side-effects, concomitant chronic diseases and medications, and PDDIs.
Methods
PLHIV who were followed at four HIV centers in Sweden: the Department of Infectious Diseases at Sahlgrenska University Hospital in Gothenburg; the Department of Infectious Diseases at South Älvsborg Hospital in Borås; the Department of Infectious Diseases at Karolinska University Hospital Huddinge in Stockholm; and the Department of Infectious Diseases at Stockholm South General Hospital in Stockholm, and met the inclusion criteria (age, 65 years of age or older for the study group or 49 years of age or younger for the control group; and on stable ART containing atazanavir (ATV), darunavir (DRV) or efavirenz (EFV) for more than 6 months) were eligible for inclusion in this cross-sectional study. On the day of inclusion, a blood sample for analysis of plasma drug level was taken, and concomitant medications (including non-prescription drugs and herbal supplements) and any side-effects related to ART were recorded in a standardized questionnaire, S1 File. Blood samples drawn between 6 to 36 hours after last dose of medication was included in the analysis of steady-state drug levels and adjusted for time with ANCOVA analysis. Adherence was recorded using a modified ACTG adherence questionnaire [13]. Any missed dose during the preceding 4 days was considered as non-adherence.
Comorbidities were registered by structured medical record reviews. PDDIs were analyzed using the Liverpool University HIV drug interactions [14] and Janusmed [15] webtools. The Liverpool University HIV drug interactions database definitions for PDDIs were used and red flag (drugs should not be co-administered) and orange flag (a potential interaction that may require dose monitoring, alteration of drug dosage or timing of administration) interactions were included in the analysis. If there was an interaction between a comedication and both the PI and the booster, the interaction was counted as one interaction in the analysis. Individuals taking DRV b.i.d. or ATV without ritonavir booster were excluded from the analysis of plasma drug levels.
All study participants gave their written informed consent and ethics approval for the study was granted by the Research Ethics Committee at Gothenburg University.
Laboratory analyses
Plasma samples were frozen at –70°C immediately after sampling until analysis. Drug levels were analyzed using a reverse-phase High Pressure Liquid Chromatography (HPLC) with ultraviolet (UV) detection at the routine pharmacology analytical laboratory at Karolinska University Hospital, Huddinge in Stockholm, Sweden. The method was CAP (College of American Pathologists) and Swedac accredited and has been described elsewhere [16]. Routine clinical methods were used to analyze CD4 cell count, liver enzymes and creatinine according to local laboratory standards.
Statistical analyses
Differences in plasma drug levels were analyzed with ANCOVA (adjusting for time) with log-transformed concentrations of ATV, DRV, and EFV. Chi-square test and Fisher’s exact test were used to compare the frequencies of side-effects, CD4/CD8 ratios, and AIDS diagnosis as appropriate. Mann Whitney U-test and Kruskal Wallis test (with Bonferroni correction for multiple tests) were used to compare frequencies of concomitant medications and PDDIs. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics, Armonk, NY, USA) or Prism version 8.0 (Graphpad Software Inc., La Jolla, CA, USA).
Results
One hundred and seventy-two individuals 65 years of age or older were eligible for inclusion and were asked to participate in the study at the four sites. Between November 2013 and August 2015, 100 individuals were enrolled in the study group (ATV n = 19; DRV n = 35; EFV n = 46) and 99 individuals in the control group (ATV n = 18; DRV n = 37; EFV n = 44). Baseline characteristics are listed in Table 1. Three individuals had HIV RNA blips (HIV RNA 59–156 copies/mL) at inclusion; all other patients had HIV RNA levels < 50 copies/mL at inclusion. Twenty-seven patients were excluded from the plasma drug level analysis: 15 individuals received DRV b.i.d. and 9 were treated with ATV, either unboosted or with dosing not according to clinical standards. Three were excluded from the plasma drug level analysis due to sample management (elapsed time since last dose less than 6 hours, or elapsed time since last dose unknown). Patients included in the plasma drug level analysis received DRV/r 800/100 mg, ATV/r 300/100 mg, or EFV 600 mg q.d. There was a significant difference in ALT levels between study and control group in the ATV arm, however the majority of subjects had ALT within the normal range. The study group had a lower glomerular filtration rate (GFR) in all arms compared to controls, although within the normal range.
10.1371/journal.pone.0246171.t001 Table 1 Baseline characteristics.
ATV DRV EFV
Study n = 19 Control n = 18 p Study n = 35 Control n = 37 p Study n = 46 Control n = 44 p
Age (median [IQR]) 68 (66–70) 46 (40.75–47.5) 68 (67–72) 45 (37.5–47) 69 (67–72) 43 (37–46)
Gender (M/F) (n) 13/6 17/1 0.09 33/2 32/5 0.43 41/5 38/6 0.76
BMI (median [IQR]) 24.3 (22.3–27.2) 25.3 (23.9–27.9) 0.28 25.7 (23.0–26.9) 23.5 (22.5–26.0) 0.17 24.4 (21.9–27.8) 24.2 (22.1–28.5) 0.65
GFR (ml/min) (median [IQR]) 83.7 (71.0–96.8) 118.0 (109.9–134.4) <0.001 86.1 (55.4–95.3) 110.4 (97.0–135.8) <0.001 79.9 (67.6–98.5) 124.3 (116.0–145.2) <0.001
ALT (μkat/L) (median [IQR]) 0.40 (0.30–0.55) 0.63 (0.52–0.75) 0.017 0.32 (0.24–0.48) 0.41 (0.31–0.55) 0.14 0.48 (0.33–0.56) 0.55 (0.38–0.85) 0.056
CD4 cell count (median [IQR]) 650 (370–730) 730 (490–912.5) 0.13 560 (420–650) 600 (465–830) 0.54 535 (380–687.5) 575 (407.5–750) 0.39
CD4 cell count Nadir (median [IQR]) 130 (69–223) 241 (187–337) 0.001 185 (90–281) 218 (40–310) 0.97 208 (158–263) 219 (167–303) 0.49
Backbone 3TC/ABC 9 14 0.09 11 14 0.63 23 12 0.03
Backbone FTC/TDF 10 4 0.09 5 12 0.03 23 30 0.09
Backbone other** 0 0 NA 19 9 0.02 0 2 0.24
Comorbidities (n) (median [IQR]) 2 (1–4) 2 (0.25–3) 0.31 3 (2–5) 1 (1–3) 0.001 3 (2–4.25) 1 (0–2) <0.001
** Other backbones: RAL, ETV, RAL + maraviroc, RAL + EFV, RAL + DRV, LPV/r, RAL + 3TC, 3TC, DTG + 3TC, RPV, no backbone.
The steady-state DRV concentrations were significantly higher in the study group (n = 25) compared to the control group (n = 30) (p = 0.047), Fig 1. The geometric mean was 48% higher in the study group than in the control group. The analysis of the ATV arm (study group n = 19, control group n = 18) showed a difference in steady state-levels (geometric mean 69% higher in the study group), with a trend towards statistical significance (p = 0.056). No statistically significant difference between the groups was found in the EFV (p = 0.87) arm. There were no differences in self-reported adherence between the study group (96% adherent) and control group (93% adherent) (p = 0.537), or between different treatment arms either in the study or the control group.
10.1371/journal.pone.0246171.g001 Fig 1 Steady-state levels of ART.
Steady-state levels in plasma of A) Darunavir (p = 0.047), B) Atazanavir (p = 0.056), and C) Efavirenz (p = 0.87) in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group).
There were no statistically significant differences in reported side-effects between the study group (23%) and the control group (34%) (p = 0.146), Fig 2. When dividing the groups according to drug regimen (taking the study group together with the controls), the DRV group had a higher rate of reported side-effects (ATV: 16.7%; DRV: 39.4%; EFV: 25.9%; p = 0.038), which was significantly different compared to the ATV arm. The difference remained when PLHIV ≥ 65 years of age were analysed separately (ATV: 0%; DRV: 36.7%; EFV: 23.8%; p = 0.014), with significant differences between DRV and ATV, and EFV and ATV. In the DRV arm there were no significant difference in reported side effects between the study group and the control group, (p = 0.80). The most commonly-reported side-effect in the DRV groups was diarrhea.
10.1371/journal.pone.0246171.g002 Fig 2 Frequency of self-reported side-effects.
A: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) (ns). B: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) divided into treatment arms (p = 0.038). C: Frequency of self-reported side-effects divided into groups according to treatment arm and study group vs. control group.
As expected, the study group had a significantly higher mean (range) number of concomitant medications, 3.7 (0–12), compared to the control group, 1.1 (0–10) (p < 0.001). Accordingly, the study group had significantly more PDDIs (mean (range)) 1.1 (0–6) compared to the control group, 0.3 (0–3) (p < 0.001). The most common PDDIs for ATV were statins and beta-blocking agents, and statins and antidepressants for DRV and EFV.
Analysis of the study group showed that the DRV arm had significantly more PDDIs (mean [range]) 1.4 (0–6) than the EFV arm 0.7 (0–3) (p = 0.03). The ATV arm had a mean (range) of 1.2 (0–4) PDDIs, not significantly different compared to either the DRV arm or the EFV arm (see Table 2). Notably, the use of concomitant medications was not higher in the DRV arm. Eight individuals in the study group had red flag PDDIs: DRV/r and alfuzosin (risk for severe hypotension, n = 2); DRV/r and clopidogrel (reduced effect of clopidogrel, n = 3); DRV/r and alfuzosin + clopidogrel (n = 1); ATV and budesonide (increased risk of steroid side-effects, n = 1); and ATV and lansoprazole (reduced ATV uptake, n = 1). Whereas no one in the control group had a red flag interaction.
10.1371/journal.pone.0246171.t002 Table 2 Potential drug-drug interactions.
PDDIs (n) (mean [range]) p
Atazanavir 1.2 (0–4)
Darunavir 1.4 (1–6) 0.026
Efavirenz 0.7 (0–3)
Number of potential drug-drug interactions (PDDIs) in the study group presented by treatment regime.
There were no differences in CD4/CD8 ratios (≥ 1 or < 1) between the study group (≥ 1 n = 35 (35%)) and control group (≥ 1 n = 40 (40%)) (p = 0.43), the ATV/DRV/EFV arms, or between arms in the different groups. No differences were found in the history of AIDS defining events in the study group vs. control group. In total 38 individuals had one or more AIDS defining diagnoses, S1 Table.
Discussion
We found a difference in the steady-state plasma drug levels of DRV in PLHIV who were ≥ 65 years of age, as compared to PLHIV < 50 years of age. To our knowledge, only one previous study has addressed the question of plasma DRV levels in elderly PLHIV [17]. In agreement with our results, the authors reported higher DRV levels in individuals > 60 years of age compared to those ≤ 40 years of age.
The difference in plasma drug levels is also consistent with earlier findings regarding other PIs, including ATV [18–20]. We noted a difference in ATV levels between elderly and younger PLHIV, with a trend towards statistical significance. The lack of significance is probably due to the small sample size. In a previous report by Avihingsanon et al., higher trough levels and higher exposure to ATV in PLHIV > 42 years of age was found compared to individuals ≤ 42 years of age, consistent with our results. This difference was more pronounced in PLHIV > 50 years of age [18]. Winston et al. also found a significant association between age and plasma drug levels of PIs [19]. We did not find any significant difference in EFV plasma drug levels in the study group compared to the control group. This is in agreement with the findings in other reports [19, 21].
There is only very limited pharmacokinetic data on ART in PLHIV older than 65 years of age. There are, however, several general age-related biological changes that may affect the metabolism of ART, e.g. decrease of liver and renal function and changes in body composition that influence the volume of distribution [22]. ATV, DRV and EFV are metabolized in the liver (ATV and DRV mainly through CYP3A4 and EFV through CYP 3A4 and CYP2B6). Only a minor portion of these drugs is eliminated through the kidneys. Therefore, the difference in drug levels in the DRV and ATV arm cannot be explained by differences in GFR. We found no clinically measurable difference in liver function measured by ALT but other changes in liver function related to age may have affected the drug levels.
Other aspects not related to metabolism may also affect the efficacy of ART. Older PLHIV have been shown to be more adherent to their treatment regimen than younger individuals [23, 24]. However, with increasing age there is a higher risk of cognitive impairment that may affect the adherence in the oldest. No difference in adherence was found in our study to support or reject either higher or lower adherence in elderly PLHIV.
Overall, we found a higher frequency of self-reported side-effects in the DRV arm, in comparison to the ATV and the EFV arms (although not significantly different from the EFV arm). The higher frequency was also present in the study group (although not statistically significant).
To the best of our knowledge this is the only study that compared the frequency of side-effects between different PIs/NNRTIs in PLHIV older than 65 years of age. A possible reason for the DRV arm having a higher frequency of side-effects might be that DRV was chosen because of extensive ART history and viral resistance, resulting in few available alternative regimes at the time of inclusion in the study and as a consequence there was a higher tolerance of side-effects. On the other hand, no difference in self-reported side effects was noted between the DRV study group and control group, even though we found a difference in steady state plasma drug levels. This observation may reflect under reporting in the study group. While it is not possible in the present study to establish a causal link, further studies are needed illuminate this issue.
PDDIs are common among PLHIV [25, 26], and the risk increases with age due to increasing frequencies of comorbidities and concomitant medications [9, 10]. Our result is in line with these earlier studies. Red flag interactions has been reported in 2% to 5.6% of PLHIV and 7.1% to 8.7% in PLHIV ≥ 65 years of age in earlier studies, similar to our findings [26–29].
ATV and DRV are both PIs and therefore they have, in general, the same PDDIs. However, they differ in regard to some frequently used drug classes e.g. beta blockers and PPIs. Since EFV is a NNRTI it has another drug interaction profile. DRV accounted for the majority of the red flag interactions found in our study, consistent with earlier findings of PLHIV in all ages [27]. Other studies have reported a higher probability of an orange or red flag PDDI in individuals treated with a PI (not restricted to those ≥ 65) [9, 28]. This is consistent with our finding that the DRV arm in the study group had a higher mean of PDDIs than the EFV arm. We did not find a significant difference between the ATV and EFV arms, however this may be due to the small sample size. The majority of the red flag interactions found were related to the concomitant use of alfuzosin, for treatment of benign prostatic hyperplasia, and/or clopidogrel, for treatment of vascular disease, which both are conditions that have a higher prevalence in older ages.
Our study has several limitations. Since we analysed steady-state plasma drug levels and not trough levels of ART, it is difficult to compare our results with trough levels in other studies. Thus, we were unable to evaluate potentially toxic plasma drug levels of ART drugs or levels below the proposed minimal effective concentrations. Patients were sampled from 6 to 36 hours after their last dose of ART. This was adjusted for in the statistical model, but there remains a risk that, because of this approach, we were not able to detect minor differences in drug levels between the study and control arms for ATV and EFV. Also, there was a difference in back-bone between PLHIV on DRV compared to ATV and EFV that may have affected self-reported side-effects. Concomitant medications may also have influenced the self-reported side-effects, though the participants were asked specifically to report side-effects related to ART. The PDDIs were calculated only for ATV, DRV or EFV regimens and therefore differences in back-bone likely did not affect the results. In addition, interactions between NRTIs and other medications are uncommon. The participants in the study were included consecutively at four sites in Sweden and it is possible that this introduced a selection bias, favoring PLHIV with frequent visits.
Conclusion
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV who were 65 years of age or older, as compared to controls who were 49 years of age and younger. Our findings are important for the management of elderly PLHIV and raise the question of whether regular monitoring of plasma levels and dose adjustment of DRV and other PIs is warranted in the elderly.
Supporting information
S1 Table Aids defining diagnoses.
(DOCX)
Click here for additional data file.
S2 Table Data set.
(XLSX)
Click here for additional data file.
S1 File Questionnaire/case report form.
(DOCX)
Click here for additional data file.
We would like to thank all the participants in the study, study nurses and technical staff. | ABACAVIR SULFATE\LAMIVUDINE, ACETAMINOPHEN, EFAVIRENZ, FOSAMPRENAVIR CALCIUM, INSULIN HUMAN, METOPROLOL, RAMIPRIL, REPAGLINIDE, RITONAVIR, TRAMADOL HYDROCHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY | 33539440 | 19,781,509 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Labelled drug-drug interaction issue'. | Higher plasma drug levels in elderly people living with HIV treated with darunavir.
The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age.
Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases.
Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV).
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.
Introduction
Antiretroviral therapy (ART) has dramatically changed the life expectancy of people living with HIV (PLHIV). HIV can now be considered a chronic infection, and the expected life span of PLHIV who receive efficient treatment is comparable to HIV-negative individuals [1, 2]. As a consequence, an increasing number of PLHIV are of older age. For example, in 2018 51% of PLHIV in the US were 50 years of age or older [3].
The risk of developing age-related and lifestyle-related diseases increases with age. PLHIV are, in addition, at higher risk of non-infectious comorbidities compared to the general population [4–6]. Furthermore, elderly people are, in general, at higher risk of adverse events to medications and may require lower doses of medications than recommended for younger individuals [7, 8]. It has been reported that PLHIV above 50 years of age have more concomitant medications and a higher risk of potential drug-drug interactions (PDDIs) compared to PLHIV below 50 years of age [9, 10].
Since the mid 1990s the standard regime for HIV treatment is two nucleoside reverse-transcriptase inhibitors (NRTIs) combined with a third agent from another drug class (most commonly a nucleoside reverse-transcriptase inhibitor (NNRTI), a protease inhibitor (PI) or an integrase inhibitor (INSTI). A dolutegravir (INSTI) or Efavirenz (NNRTI) containing regimen is recommended by WHO as first line treatment today [11]. In the Swedish setting a dolutegravir or darunavir containing regimen is recommended by the Swedish Reference Group for Antiviral Therapy [12]. Neither WHO nor Sweden have specific treatment recommendations for elderly PLHIV.
Scientific data on the pharmacokinetics of PIs and NNRTIs in individuals 65 years of age and older are scarce. The primary objective of this study was to investigate differences in steady-state plasma drug levels of ATV, DRV and EFV in PLHIV ≥ 65 years of age as compared to PLHIV ≤ 49 years of age. Secondary objectives were to study differences in self-reported side-effects, concomitant chronic diseases and medications, and PDDIs.
Methods
PLHIV who were followed at four HIV centers in Sweden: the Department of Infectious Diseases at Sahlgrenska University Hospital in Gothenburg; the Department of Infectious Diseases at South Älvsborg Hospital in Borås; the Department of Infectious Diseases at Karolinska University Hospital Huddinge in Stockholm; and the Department of Infectious Diseases at Stockholm South General Hospital in Stockholm, and met the inclusion criteria (age, 65 years of age or older for the study group or 49 years of age or younger for the control group; and on stable ART containing atazanavir (ATV), darunavir (DRV) or efavirenz (EFV) for more than 6 months) were eligible for inclusion in this cross-sectional study. On the day of inclusion, a blood sample for analysis of plasma drug level was taken, and concomitant medications (including non-prescription drugs and herbal supplements) and any side-effects related to ART were recorded in a standardized questionnaire, S1 File. Blood samples drawn between 6 to 36 hours after last dose of medication was included in the analysis of steady-state drug levels and adjusted for time with ANCOVA analysis. Adherence was recorded using a modified ACTG adherence questionnaire [13]. Any missed dose during the preceding 4 days was considered as non-adherence.
Comorbidities were registered by structured medical record reviews. PDDIs were analyzed using the Liverpool University HIV drug interactions [14] and Janusmed [15] webtools. The Liverpool University HIV drug interactions database definitions for PDDIs were used and red flag (drugs should not be co-administered) and orange flag (a potential interaction that may require dose monitoring, alteration of drug dosage or timing of administration) interactions were included in the analysis. If there was an interaction between a comedication and both the PI and the booster, the interaction was counted as one interaction in the analysis. Individuals taking DRV b.i.d. or ATV without ritonavir booster were excluded from the analysis of plasma drug levels.
All study participants gave their written informed consent and ethics approval for the study was granted by the Research Ethics Committee at Gothenburg University.
Laboratory analyses
Plasma samples were frozen at –70°C immediately after sampling until analysis. Drug levels were analyzed using a reverse-phase High Pressure Liquid Chromatography (HPLC) with ultraviolet (UV) detection at the routine pharmacology analytical laboratory at Karolinska University Hospital, Huddinge in Stockholm, Sweden. The method was CAP (College of American Pathologists) and Swedac accredited and has been described elsewhere [16]. Routine clinical methods were used to analyze CD4 cell count, liver enzymes and creatinine according to local laboratory standards.
Statistical analyses
Differences in plasma drug levels were analyzed with ANCOVA (adjusting for time) with log-transformed concentrations of ATV, DRV, and EFV. Chi-square test and Fisher’s exact test were used to compare the frequencies of side-effects, CD4/CD8 ratios, and AIDS diagnosis as appropriate. Mann Whitney U-test and Kruskal Wallis test (with Bonferroni correction for multiple tests) were used to compare frequencies of concomitant medications and PDDIs. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics, Armonk, NY, USA) or Prism version 8.0 (Graphpad Software Inc., La Jolla, CA, USA).
Results
One hundred and seventy-two individuals 65 years of age or older were eligible for inclusion and were asked to participate in the study at the four sites. Between November 2013 and August 2015, 100 individuals were enrolled in the study group (ATV n = 19; DRV n = 35; EFV n = 46) and 99 individuals in the control group (ATV n = 18; DRV n = 37; EFV n = 44). Baseline characteristics are listed in Table 1. Three individuals had HIV RNA blips (HIV RNA 59–156 copies/mL) at inclusion; all other patients had HIV RNA levels < 50 copies/mL at inclusion. Twenty-seven patients were excluded from the plasma drug level analysis: 15 individuals received DRV b.i.d. and 9 were treated with ATV, either unboosted or with dosing not according to clinical standards. Three were excluded from the plasma drug level analysis due to sample management (elapsed time since last dose less than 6 hours, or elapsed time since last dose unknown). Patients included in the plasma drug level analysis received DRV/r 800/100 mg, ATV/r 300/100 mg, or EFV 600 mg q.d. There was a significant difference in ALT levels between study and control group in the ATV arm, however the majority of subjects had ALT within the normal range. The study group had a lower glomerular filtration rate (GFR) in all arms compared to controls, although within the normal range.
10.1371/journal.pone.0246171.t001 Table 1 Baseline characteristics.
ATV DRV EFV
Study n = 19 Control n = 18 p Study n = 35 Control n = 37 p Study n = 46 Control n = 44 p
Age (median [IQR]) 68 (66–70) 46 (40.75–47.5) 68 (67–72) 45 (37.5–47) 69 (67–72) 43 (37–46)
Gender (M/F) (n) 13/6 17/1 0.09 33/2 32/5 0.43 41/5 38/6 0.76
BMI (median [IQR]) 24.3 (22.3–27.2) 25.3 (23.9–27.9) 0.28 25.7 (23.0–26.9) 23.5 (22.5–26.0) 0.17 24.4 (21.9–27.8) 24.2 (22.1–28.5) 0.65
GFR (ml/min) (median [IQR]) 83.7 (71.0–96.8) 118.0 (109.9–134.4) <0.001 86.1 (55.4–95.3) 110.4 (97.0–135.8) <0.001 79.9 (67.6–98.5) 124.3 (116.0–145.2) <0.001
ALT (μkat/L) (median [IQR]) 0.40 (0.30–0.55) 0.63 (0.52–0.75) 0.017 0.32 (0.24–0.48) 0.41 (0.31–0.55) 0.14 0.48 (0.33–0.56) 0.55 (0.38–0.85) 0.056
CD4 cell count (median [IQR]) 650 (370–730) 730 (490–912.5) 0.13 560 (420–650) 600 (465–830) 0.54 535 (380–687.5) 575 (407.5–750) 0.39
CD4 cell count Nadir (median [IQR]) 130 (69–223) 241 (187–337) 0.001 185 (90–281) 218 (40–310) 0.97 208 (158–263) 219 (167–303) 0.49
Backbone 3TC/ABC 9 14 0.09 11 14 0.63 23 12 0.03
Backbone FTC/TDF 10 4 0.09 5 12 0.03 23 30 0.09
Backbone other** 0 0 NA 19 9 0.02 0 2 0.24
Comorbidities (n) (median [IQR]) 2 (1–4) 2 (0.25–3) 0.31 3 (2–5) 1 (1–3) 0.001 3 (2–4.25) 1 (0–2) <0.001
** Other backbones: RAL, ETV, RAL + maraviroc, RAL + EFV, RAL + DRV, LPV/r, RAL + 3TC, 3TC, DTG + 3TC, RPV, no backbone.
The steady-state DRV concentrations were significantly higher in the study group (n = 25) compared to the control group (n = 30) (p = 0.047), Fig 1. The geometric mean was 48% higher in the study group than in the control group. The analysis of the ATV arm (study group n = 19, control group n = 18) showed a difference in steady state-levels (geometric mean 69% higher in the study group), with a trend towards statistical significance (p = 0.056). No statistically significant difference between the groups was found in the EFV (p = 0.87) arm. There were no differences in self-reported adherence between the study group (96% adherent) and control group (93% adherent) (p = 0.537), or between different treatment arms either in the study or the control group.
10.1371/journal.pone.0246171.g001 Fig 1 Steady-state levels of ART.
Steady-state levels in plasma of A) Darunavir (p = 0.047), B) Atazanavir (p = 0.056), and C) Efavirenz (p = 0.87) in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group).
There were no statistically significant differences in reported side-effects between the study group (23%) and the control group (34%) (p = 0.146), Fig 2. When dividing the groups according to drug regimen (taking the study group together with the controls), the DRV group had a higher rate of reported side-effects (ATV: 16.7%; DRV: 39.4%; EFV: 25.9%; p = 0.038), which was significantly different compared to the ATV arm. The difference remained when PLHIV ≥ 65 years of age were analysed separately (ATV: 0%; DRV: 36.7%; EFV: 23.8%; p = 0.014), with significant differences between DRV and ATV, and EFV and ATV. In the DRV arm there were no significant difference in reported side effects between the study group and the control group, (p = 0.80). The most commonly-reported side-effect in the DRV groups was diarrhea.
10.1371/journal.pone.0246171.g002 Fig 2 Frequency of self-reported side-effects.
A: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) (ns). B: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) divided into treatment arms (p = 0.038). C: Frequency of self-reported side-effects divided into groups according to treatment arm and study group vs. control group.
As expected, the study group had a significantly higher mean (range) number of concomitant medications, 3.7 (0–12), compared to the control group, 1.1 (0–10) (p < 0.001). Accordingly, the study group had significantly more PDDIs (mean (range)) 1.1 (0–6) compared to the control group, 0.3 (0–3) (p < 0.001). The most common PDDIs for ATV were statins and beta-blocking agents, and statins and antidepressants for DRV and EFV.
Analysis of the study group showed that the DRV arm had significantly more PDDIs (mean [range]) 1.4 (0–6) than the EFV arm 0.7 (0–3) (p = 0.03). The ATV arm had a mean (range) of 1.2 (0–4) PDDIs, not significantly different compared to either the DRV arm or the EFV arm (see Table 2). Notably, the use of concomitant medications was not higher in the DRV arm. Eight individuals in the study group had red flag PDDIs: DRV/r and alfuzosin (risk for severe hypotension, n = 2); DRV/r and clopidogrel (reduced effect of clopidogrel, n = 3); DRV/r and alfuzosin + clopidogrel (n = 1); ATV and budesonide (increased risk of steroid side-effects, n = 1); and ATV and lansoprazole (reduced ATV uptake, n = 1). Whereas no one in the control group had a red flag interaction.
10.1371/journal.pone.0246171.t002 Table 2 Potential drug-drug interactions.
PDDIs (n) (mean [range]) p
Atazanavir 1.2 (0–4)
Darunavir 1.4 (1–6) 0.026
Efavirenz 0.7 (0–3)
Number of potential drug-drug interactions (PDDIs) in the study group presented by treatment regime.
There were no differences in CD4/CD8 ratios (≥ 1 or < 1) between the study group (≥ 1 n = 35 (35%)) and control group (≥ 1 n = 40 (40%)) (p = 0.43), the ATV/DRV/EFV arms, or between arms in the different groups. No differences were found in the history of AIDS defining events in the study group vs. control group. In total 38 individuals had one or more AIDS defining diagnoses, S1 Table.
Discussion
We found a difference in the steady-state plasma drug levels of DRV in PLHIV who were ≥ 65 years of age, as compared to PLHIV < 50 years of age. To our knowledge, only one previous study has addressed the question of plasma DRV levels in elderly PLHIV [17]. In agreement with our results, the authors reported higher DRV levels in individuals > 60 years of age compared to those ≤ 40 years of age.
The difference in plasma drug levels is also consistent with earlier findings regarding other PIs, including ATV [18–20]. We noted a difference in ATV levels between elderly and younger PLHIV, with a trend towards statistical significance. The lack of significance is probably due to the small sample size. In a previous report by Avihingsanon et al., higher trough levels and higher exposure to ATV in PLHIV > 42 years of age was found compared to individuals ≤ 42 years of age, consistent with our results. This difference was more pronounced in PLHIV > 50 years of age [18]. Winston et al. also found a significant association between age and plasma drug levels of PIs [19]. We did not find any significant difference in EFV plasma drug levels in the study group compared to the control group. This is in agreement with the findings in other reports [19, 21].
There is only very limited pharmacokinetic data on ART in PLHIV older than 65 years of age. There are, however, several general age-related biological changes that may affect the metabolism of ART, e.g. decrease of liver and renal function and changes in body composition that influence the volume of distribution [22]. ATV, DRV and EFV are metabolized in the liver (ATV and DRV mainly through CYP3A4 and EFV through CYP 3A4 and CYP2B6). Only a minor portion of these drugs is eliminated through the kidneys. Therefore, the difference in drug levels in the DRV and ATV arm cannot be explained by differences in GFR. We found no clinically measurable difference in liver function measured by ALT but other changes in liver function related to age may have affected the drug levels.
Other aspects not related to metabolism may also affect the efficacy of ART. Older PLHIV have been shown to be more adherent to their treatment regimen than younger individuals [23, 24]. However, with increasing age there is a higher risk of cognitive impairment that may affect the adherence in the oldest. No difference in adherence was found in our study to support or reject either higher or lower adherence in elderly PLHIV.
Overall, we found a higher frequency of self-reported side-effects in the DRV arm, in comparison to the ATV and the EFV arms (although not significantly different from the EFV arm). The higher frequency was also present in the study group (although not statistically significant).
To the best of our knowledge this is the only study that compared the frequency of side-effects between different PIs/NNRTIs in PLHIV older than 65 years of age. A possible reason for the DRV arm having a higher frequency of side-effects might be that DRV was chosen because of extensive ART history and viral resistance, resulting in few available alternative regimes at the time of inclusion in the study and as a consequence there was a higher tolerance of side-effects. On the other hand, no difference in self-reported side effects was noted between the DRV study group and control group, even though we found a difference in steady state plasma drug levels. This observation may reflect under reporting in the study group. While it is not possible in the present study to establish a causal link, further studies are needed illuminate this issue.
PDDIs are common among PLHIV [25, 26], and the risk increases with age due to increasing frequencies of comorbidities and concomitant medications [9, 10]. Our result is in line with these earlier studies. Red flag interactions has been reported in 2% to 5.6% of PLHIV and 7.1% to 8.7% in PLHIV ≥ 65 years of age in earlier studies, similar to our findings [26–29].
ATV and DRV are both PIs and therefore they have, in general, the same PDDIs. However, they differ in regard to some frequently used drug classes e.g. beta blockers and PPIs. Since EFV is a NNRTI it has another drug interaction profile. DRV accounted for the majority of the red flag interactions found in our study, consistent with earlier findings of PLHIV in all ages [27]. Other studies have reported a higher probability of an orange or red flag PDDI in individuals treated with a PI (not restricted to those ≥ 65) [9, 28]. This is consistent with our finding that the DRV arm in the study group had a higher mean of PDDIs than the EFV arm. We did not find a significant difference between the ATV and EFV arms, however this may be due to the small sample size. The majority of the red flag interactions found were related to the concomitant use of alfuzosin, for treatment of benign prostatic hyperplasia, and/or clopidogrel, for treatment of vascular disease, which both are conditions that have a higher prevalence in older ages.
Our study has several limitations. Since we analysed steady-state plasma drug levels and not trough levels of ART, it is difficult to compare our results with trough levels in other studies. Thus, we were unable to evaluate potentially toxic plasma drug levels of ART drugs or levels below the proposed minimal effective concentrations. Patients were sampled from 6 to 36 hours after their last dose of ART. This was adjusted for in the statistical model, but there remains a risk that, because of this approach, we were not able to detect minor differences in drug levels between the study and control arms for ATV and EFV. Also, there was a difference in back-bone between PLHIV on DRV compared to ATV and EFV that may have affected self-reported side-effects. Concomitant medications may also have influenced the self-reported side-effects, though the participants were asked specifically to report side-effects related to ART. The PDDIs were calculated only for ATV, DRV or EFV regimens and therefore differences in back-bone likely did not affect the results. In addition, interactions between NRTIs and other medications are uncommon. The participants in the study were included consecutively at four sites in Sweden and it is possible that this introduced a selection bias, favoring PLHIV with frequent visits.
Conclusion
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV who were 65 years of age or older, as compared to controls who were 49 years of age and younger. Our findings are important for the management of elderly PLHIV and raise the question of whether regular monitoring of plasma levels and dose adjustment of DRV and other PIs is warranted in the elderly.
Supporting information
S1 Table Aids defining diagnoses.
(DOCX)
Click here for additional data file.
S2 Table Data set.
(XLSX)
Click here for additional data file.
S1 File Questionnaire/case report form.
(DOCX)
Click here for additional data file.
We would like to thank all the participants in the study, study nurses and technical staff. | ABACAVIR SULFATE\LAMIVUDINE, ACETAMINOPHEN, EFAVIRENZ, FOSAMPRENAVIR CALCIUM, INSULIN HUMAN, METOPROLOL, RAMIPRIL, REPAGLINIDE, RITONAVIR, TRAMADOL HYDROCHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY | 33539440 | 19,781,509 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Sensory loss'. | Higher plasma drug levels in elderly people living with HIV treated with darunavir.
The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age.
Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases.
Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV).
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.
Introduction
Antiretroviral therapy (ART) has dramatically changed the life expectancy of people living with HIV (PLHIV). HIV can now be considered a chronic infection, and the expected life span of PLHIV who receive efficient treatment is comparable to HIV-negative individuals [1, 2]. As a consequence, an increasing number of PLHIV are of older age. For example, in 2018 51% of PLHIV in the US were 50 years of age or older [3].
The risk of developing age-related and lifestyle-related diseases increases with age. PLHIV are, in addition, at higher risk of non-infectious comorbidities compared to the general population [4–6]. Furthermore, elderly people are, in general, at higher risk of adverse events to medications and may require lower doses of medications than recommended for younger individuals [7, 8]. It has been reported that PLHIV above 50 years of age have more concomitant medications and a higher risk of potential drug-drug interactions (PDDIs) compared to PLHIV below 50 years of age [9, 10].
Since the mid 1990s the standard regime for HIV treatment is two nucleoside reverse-transcriptase inhibitors (NRTIs) combined with a third agent from another drug class (most commonly a nucleoside reverse-transcriptase inhibitor (NNRTI), a protease inhibitor (PI) or an integrase inhibitor (INSTI). A dolutegravir (INSTI) or Efavirenz (NNRTI) containing regimen is recommended by WHO as first line treatment today [11]. In the Swedish setting a dolutegravir or darunavir containing regimen is recommended by the Swedish Reference Group for Antiviral Therapy [12]. Neither WHO nor Sweden have specific treatment recommendations for elderly PLHIV.
Scientific data on the pharmacokinetics of PIs and NNRTIs in individuals 65 years of age and older are scarce. The primary objective of this study was to investigate differences in steady-state plasma drug levels of ATV, DRV and EFV in PLHIV ≥ 65 years of age as compared to PLHIV ≤ 49 years of age. Secondary objectives were to study differences in self-reported side-effects, concomitant chronic diseases and medications, and PDDIs.
Methods
PLHIV who were followed at four HIV centers in Sweden: the Department of Infectious Diseases at Sahlgrenska University Hospital in Gothenburg; the Department of Infectious Diseases at South Älvsborg Hospital in Borås; the Department of Infectious Diseases at Karolinska University Hospital Huddinge in Stockholm; and the Department of Infectious Diseases at Stockholm South General Hospital in Stockholm, and met the inclusion criteria (age, 65 years of age or older for the study group or 49 years of age or younger for the control group; and on stable ART containing atazanavir (ATV), darunavir (DRV) or efavirenz (EFV) for more than 6 months) were eligible for inclusion in this cross-sectional study. On the day of inclusion, a blood sample for analysis of plasma drug level was taken, and concomitant medications (including non-prescription drugs and herbal supplements) and any side-effects related to ART were recorded in a standardized questionnaire, S1 File. Blood samples drawn between 6 to 36 hours after last dose of medication was included in the analysis of steady-state drug levels and adjusted for time with ANCOVA analysis. Adherence was recorded using a modified ACTG adherence questionnaire [13]. Any missed dose during the preceding 4 days was considered as non-adherence.
Comorbidities were registered by structured medical record reviews. PDDIs were analyzed using the Liverpool University HIV drug interactions [14] and Janusmed [15] webtools. The Liverpool University HIV drug interactions database definitions for PDDIs were used and red flag (drugs should not be co-administered) and orange flag (a potential interaction that may require dose monitoring, alteration of drug dosage or timing of administration) interactions were included in the analysis. If there was an interaction between a comedication and both the PI and the booster, the interaction was counted as one interaction in the analysis. Individuals taking DRV b.i.d. or ATV without ritonavir booster were excluded from the analysis of plasma drug levels.
All study participants gave their written informed consent and ethics approval for the study was granted by the Research Ethics Committee at Gothenburg University.
Laboratory analyses
Plasma samples were frozen at –70°C immediately after sampling until analysis. Drug levels were analyzed using a reverse-phase High Pressure Liquid Chromatography (HPLC) with ultraviolet (UV) detection at the routine pharmacology analytical laboratory at Karolinska University Hospital, Huddinge in Stockholm, Sweden. The method was CAP (College of American Pathologists) and Swedac accredited and has been described elsewhere [16]. Routine clinical methods were used to analyze CD4 cell count, liver enzymes and creatinine according to local laboratory standards.
Statistical analyses
Differences in plasma drug levels were analyzed with ANCOVA (adjusting for time) with log-transformed concentrations of ATV, DRV, and EFV. Chi-square test and Fisher’s exact test were used to compare the frequencies of side-effects, CD4/CD8 ratios, and AIDS diagnosis as appropriate. Mann Whitney U-test and Kruskal Wallis test (with Bonferroni correction for multiple tests) were used to compare frequencies of concomitant medications and PDDIs. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics, Armonk, NY, USA) or Prism version 8.0 (Graphpad Software Inc., La Jolla, CA, USA).
Results
One hundred and seventy-two individuals 65 years of age or older were eligible for inclusion and were asked to participate in the study at the four sites. Between November 2013 and August 2015, 100 individuals were enrolled in the study group (ATV n = 19; DRV n = 35; EFV n = 46) and 99 individuals in the control group (ATV n = 18; DRV n = 37; EFV n = 44). Baseline characteristics are listed in Table 1. Three individuals had HIV RNA blips (HIV RNA 59–156 copies/mL) at inclusion; all other patients had HIV RNA levels < 50 copies/mL at inclusion. Twenty-seven patients were excluded from the plasma drug level analysis: 15 individuals received DRV b.i.d. and 9 were treated with ATV, either unboosted or with dosing not according to clinical standards. Three were excluded from the plasma drug level analysis due to sample management (elapsed time since last dose less than 6 hours, or elapsed time since last dose unknown). Patients included in the plasma drug level analysis received DRV/r 800/100 mg, ATV/r 300/100 mg, or EFV 600 mg q.d. There was a significant difference in ALT levels between study and control group in the ATV arm, however the majority of subjects had ALT within the normal range. The study group had a lower glomerular filtration rate (GFR) in all arms compared to controls, although within the normal range.
10.1371/journal.pone.0246171.t001 Table 1 Baseline characteristics.
ATV DRV EFV
Study n = 19 Control n = 18 p Study n = 35 Control n = 37 p Study n = 46 Control n = 44 p
Age (median [IQR]) 68 (66–70) 46 (40.75–47.5) 68 (67–72) 45 (37.5–47) 69 (67–72) 43 (37–46)
Gender (M/F) (n) 13/6 17/1 0.09 33/2 32/5 0.43 41/5 38/6 0.76
BMI (median [IQR]) 24.3 (22.3–27.2) 25.3 (23.9–27.9) 0.28 25.7 (23.0–26.9) 23.5 (22.5–26.0) 0.17 24.4 (21.9–27.8) 24.2 (22.1–28.5) 0.65
GFR (ml/min) (median [IQR]) 83.7 (71.0–96.8) 118.0 (109.9–134.4) <0.001 86.1 (55.4–95.3) 110.4 (97.0–135.8) <0.001 79.9 (67.6–98.5) 124.3 (116.0–145.2) <0.001
ALT (μkat/L) (median [IQR]) 0.40 (0.30–0.55) 0.63 (0.52–0.75) 0.017 0.32 (0.24–0.48) 0.41 (0.31–0.55) 0.14 0.48 (0.33–0.56) 0.55 (0.38–0.85) 0.056
CD4 cell count (median [IQR]) 650 (370–730) 730 (490–912.5) 0.13 560 (420–650) 600 (465–830) 0.54 535 (380–687.5) 575 (407.5–750) 0.39
CD4 cell count Nadir (median [IQR]) 130 (69–223) 241 (187–337) 0.001 185 (90–281) 218 (40–310) 0.97 208 (158–263) 219 (167–303) 0.49
Backbone 3TC/ABC 9 14 0.09 11 14 0.63 23 12 0.03
Backbone FTC/TDF 10 4 0.09 5 12 0.03 23 30 0.09
Backbone other** 0 0 NA 19 9 0.02 0 2 0.24
Comorbidities (n) (median [IQR]) 2 (1–4) 2 (0.25–3) 0.31 3 (2–5) 1 (1–3) 0.001 3 (2–4.25) 1 (0–2) <0.001
** Other backbones: RAL, ETV, RAL + maraviroc, RAL + EFV, RAL + DRV, LPV/r, RAL + 3TC, 3TC, DTG + 3TC, RPV, no backbone.
The steady-state DRV concentrations were significantly higher in the study group (n = 25) compared to the control group (n = 30) (p = 0.047), Fig 1. The geometric mean was 48% higher in the study group than in the control group. The analysis of the ATV arm (study group n = 19, control group n = 18) showed a difference in steady state-levels (geometric mean 69% higher in the study group), with a trend towards statistical significance (p = 0.056). No statistically significant difference between the groups was found in the EFV (p = 0.87) arm. There were no differences in self-reported adherence between the study group (96% adherent) and control group (93% adherent) (p = 0.537), or between different treatment arms either in the study or the control group.
10.1371/journal.pone.0246171.g001 Fig 1 Steady-state levels of ART.
Steady-state levels in plasma of A) Darunavir (p = 0.047), B) Atazanavir (p = 0.056), and C) Efavirenz (p = 0.87) in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group).
There were no statistically significant differences in reported side-effects between the study group (23%) and the control group (34%) (p = 0.146), Fig 2. When dividing the groups according to drug regimen (taking the study group together with the controls), the DRV group had a higher rate of reported side-effects (ATV: 16.7%; DRV: 39.4%; EFV: 25.9%; p = 0.038), which was significantly different compared to the ATV arm. The difference remained when PLHIV ≥ 65 years of age were analysed separately (ATV: 0%; DRV: 36.7%; EFV: 23.8%; p = 0.014), with significant differences between DRV and ATV, and EFV and ATV. In the DRV arm there were no significant difference in reported side effects between the study group and the control group, (p = 0.80). The most commonly-reported side-effect in the DRV groups was diarrhea.
10.1371/journal.pone.0246171.g002 Fig 2 Frequency of self-reported side-effects.
A: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) (ns). B: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) divided into treatment arms (p = 0.038). C: Frequency of self-reported side-effects divided into groups according to treatment arm and study group vs. control group.
As expected, the study group had a significantly higher mean (range) number of concomitant medications, 3.7 (0–12), compared to the control group, 1.1 (0–10) (p < 0.001). Accordingly, the study group had significantly more PDDIs (mean (range)) 1.1 (0–6) compared to the control group, 0.3 (0–3) (p < 0.001). The most common PDDIs for ATV were statins and beta-blocking agents, and statins and antidepressants for DRV and EFV.
Analysis of the study group showed that the DRV arm had significantly more PDDIs (mean [range]) 1.4 (0–6) than the EFV arm 0.7 (0–3) (p = 0.03). The ATV arm had a mean (range) of 1.2 (0–4) PDDIs, not significantly different compared to either the DRV arm or the EFV arm (see Table 2). Notably, the use of concomitant medications was not higher in the DRV arm. Eight individuals in the study group had red flag PDDIs: DRV/r and alfuzosin (risk for severe hypotension, n = 2); DRV/r and clopidogrel (reduced effect of clopidogrel, n = 3); DRV/r and alfuzosin + clopidogrel (n = 1); ATV and budesonide (increased risk of steroid side-effects, n = 1); and ATV and lansoprazole (reduced ATV uptake, n = 1). Whereas no one in the control group had a red flag interaction.
10.1371/journal.pone.0246171.t002 Table 2 Potential drug-drug interactions.
PDDIs (n) (mean [range]) p
Atazanavir 1.2 (0–4)
Darunavir 1.4 (1–6) 0.026
Efavirenz 0.7 (0–3)
Number of potential drug-drug interactions (PDDIs) in the study group presented by treatment regime.
There were no differences in CD4/CD8 ratios (≥ 1 or < 1) between the study group (≥ 1 n = 35 (35%)) and control group (≥ 1 n = 40 (40%)) (p = 0.43), the ATV/DRV/EFV arms, or between arms in the different groups. No differences were found in the history of AIDS defining events in the study group vs. control group. In total 38 individuals had one or more AIDS defining diagnoses, S1 Table.
Discussion
We found a difference in the steady-state plasma drug levels of DRV in PLHIV who were ≥ 65 years of age, as compared to PLHIV < 50 years of age. To our knowledge, only one previous study has addressed the question of plasma DRV levels in elderly PLHIV [17]. In agreement with our results, the authors reported higher DRV levels in individuals > 60 years of age compared to those ≤ 40 years of age.
The difference in plasma drug levels is also consistent with earlier findings regarding other PIs, including ATV [18–20]. We noted a difference in ATV levels between elderly and younger PLHIV, with a trend towards statistical significance. The lack of significance is probably due to the small sample size. In a previous report by Avihingsanon et al., higher trough levels and higher exposure to ATV in PLHIV > 42 years of age was found compared to individuals ≤ 42 years of age, consistent with our results. This difference was more pronounced in PLHIV > 50 years of age [18]. Winston et al. also found a significant association between age and plasma drug levels of PIs [19]. We did not find any significant difference in EFV plasma drug levels in the study group compared to the control group. This is in agreement with the findings in other reports [19, 21].
There is only very limited pharmacokinetic data on ART in PLHIV older than 65 years of age. There are, however, several general age-related biological changes that may affect the metabolism of ART, e.g. decrease of liver and renal function and changes in body composition that influence the volume of distribution [22]. ATV, DRV and EFV are metabolized in the liver (ATV and DRV mainly through CYP3A4 and EFV through CYP 3A4 and CYP2B6). Only a minor portion of these drugs is eliminated through the kidneys. Therefore, the difference in drug levels in the DRV and ATV arm cannot be explained by differences in GFR. We found no clinically measurable difference in liver function measured by ALT but other changes in liver function related to age may have affected the drug levels.
Other aspects not related to metabolism may also affect the efficacy of ART. Older PLHIV have been shown to be more adherent to their treatment regimen than younger individuals [23, 24]. However, with increasing age there is a higher risk of cognitive impairment that may affect the adherence in the oldest. No difference in adherence was found in our study to support or reject either higher or lower adherence in elderly PLHIV.
Overall, we found a higher frequency of self-reported side-effects in the DRV arm, in comparison to the ATV and the EFV arms (although not significantly different from the EFV arm). The higher frequency was also present in the study group (although not statistically significant).
To the best of our knowledge this is the only study that compared the frequency of side-effects between different PIs/NNRTIs in PLHIV older than 65 years of age. A possible reason for the DRV arm having a higher frequency of side-effects might be that DRV was chosen because of extensive ART history and viral resistance, resulting in few available alternative regimes at the time of inclusion in the study and as a consequence there was a higher tolerance of side-effects. On the other hand, no difference in self-reported side effects was noted between the DRV study group and control group, even though we found a difference in steady state plasma drug levels. This observation may reflect under reporting in the study group. While it is not possible in the present study to establish a causal link, further studies are needed illuminate this issue.
PDDIs are common among PLHIV [25, 26], and the risk increases with age due to increasing frequencies of comorbidities and concomitant medications [9, 10]. Our result is in line with these earlier studies. Red flag interactions has been reported in 2% to 5.6% of PLHIV and 7.1% to 8.7% in PLHIV ≥ 65 years of age in earlier studies, similar to our findings [26–29].
ATV and DRV are both PIs and therefore they have, in general, the same PDDIs. However, they differ in regard to some frequently used drug classes e.g. beta blockers and PPIs. Since EFV is a NNRTI it has another drug interaction profile. DRV accounted for the majority of the red flag interactions found in our study, consistent with earlier findings of PLHIV in all ages [27]. Other studies have reported a higher probability of an orange or red flag PDDI in individuals treated with a PI (not restricted to those ≥ 65) [9, 28]. This is consistent with our finding that the DRV arm in the study group had a higher mean of PDDIs than the EFV arm. We did not find a significant difference between the ATV and EFV arms, however this may be due to the small sample size. The majority of the red flag interactions found were related to the concomitant use of alfuzosin, for treatment of benign prostatic hyperplasia, and/or clopidogrel, for treatment of vascular disease, which both are conditions that have a higher prevalence in older ages.
Our study has several limitations. Since we analysed steady-state plasma drug levels and not trough levels of ART, it is difficult to compare our results with trough levels in other studies. Thus, we were unable to evaluate potentially toxic plasma drug levels of ART drugs or levels below the proposed minimal effective concentrations. Patients were sampled from 6 to 36 hours after their last dose of ART. This was adjusted for in the statistical model, but there remains a risk that, because of this approach, we were not able to detect minor differences in drug levels between the study and control arms for ATV and EFV. Also, there was a difference in back-bone between PLHIV on DRV compared to ATV and EFV that may have affected self-reported side-effects. Concomitant medications may also have influenced the self-reported side-effects, though the participants were asked specifically to report side-effects related to ART. The PDDIs were calculated only for ATV, DRV or EFV regimens and therefore differences in back-bone likely did not affect the results. In addition, interactions between NRTIs and other medications are uncommon. The participants in the study were included consecutively at four sites in Sweden and it is possible that this introduced a selection bias, favoring PLHIV with frequent visits.
Conclusion
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV who were 65 years of age or older, as compared to controls who were 49 years of age and younger. Our findings are important for the management of elderly PLHIV and raise the question of whether regular monitoring of plasma levels and dose adjustment of DRV and other PIs is warranted in the elderly.
Supporting information
S1 Table Aids defining diagnoses.
(DOCX)
Click here for additional data file.
S2 Table Data set.
(XLSX)
Click here for additional data file.
S1 File Questionnaire/case report form.
(DOCX)
Click here for additional data file.
We would like to thank all the participants in the study, study nurses and technical staff. | ABACAVIR SULFATE\LAMIVUDINE, ACETAMINOPHEN, EFAVIRENZ, FOSAMPRENAVIR CALCIUM, INSULIN HUMAN, METOPROLOL, RAMIPRIL, REPAGLINIDE, RITONAVIR, TRAMADOL HYDROCHLORIDE, TRANEXAMIC ACID | DrugsGivenReaction | CC BY | 33539440 | 19,781,509 | 2021 |
What was the administration route of drug 'EFAVIRENZ'? | Higher plasma drug levels in elderly people living with HIV treated with darunavir.
The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age.
Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases.
Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV).
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.
Introduction
Antiretroviral therapy (ART) has dramatically changed the life expectancy of people living with HIV (PLHIV). HIV can now be considered a chronic infection, and the expected life span of PLHIV who receive efficient treatment is comparable to HIV-negative individuals [1, 2]. As a consequence, an increasing number of PLHIV are of older age. For example, in 2018 51% of PLHIV in the US were 50 years of age or older [3].
The risk of developing age-related and lifestyle-related diseases increases with age. PLHIV are, in addition, at higher risk of non-infectious comorbidities compared to the general population [4–6]. Furthermore, elderly people are, in general, at higher risk of adverse events to medications and may require lower doses of medications than recommended for younger individuals [7, 8]. It has been reported that PLHIV above 50 years of age have more concomitant medications and a higher risk of potential drug-drug interactions (PDDIs) compared to PLHIV below 50 years of age [9, 10].
Since the mid 1990s the standard regime for HIV treatment is two nucleoside reverse-transcriptase inhibitors (NRTIs) combined with a third agent from another drug class (most commonly a nucleoside reverse-transcriptase inhibitor (NNRTI), a protease inhibitor (PI) or an integrase inhibitor (INSTI). A dolutegravir (INSTI) or Efavirenz (NNRTI) containing regimen is recommended by WHO as first line treatment today [11]. In the Swedish setting a dolutegravir or darunavir containing regimen is recommended by the Swedish Reference Group for Antiviral Therapy [12]. Neither WHO nor Sweden have specific treatment recommendations for elderly PLHIV.
Scientific data on the pharmacokinetics of PIs and NNRTIs in individuals 65 years of age and older are scarce. The primary objective of this study was to investigate differences in steady-state plasma drug levels of ATV, DRV and EFV in PLHIV ≥ 65 years of age as compared to PLHIV ≤ 49 years of age. Secondary objectives were to study differences in self-reported side-effects, concomitant chronic diseases and medications, and PDDIs.
Methods
PLHIV who were followed at four HIV centers in Sweden: the Department of Infectious Diseases at Sahlgrenska University Hospital in Gothenburg; the Department of Infectious Diseases at South Älvsborg Hospital in Borås; the Department of Infectious Diseases at Karolinska University Hospital Huddinge in Stockholm; and the Department of Infectious Diseases at Stockholm South General Hospital in Stockholm, and met the inclusion criteria (age, 65 years of age or older for the study group or 49 years of age or younger for the control group; and on stable ART containing atazanavir (ATV), darunavir (DRV) or efavirenz (EFV) for more than 6 months) were eligible for inclusion in this cross-sectional study. On the day of inclusion, a blood sample for analysis of plasma drug level was taken, and concomitant medications (including non-prescription drugs and herbal supplements) and any side-effects related to ART were recorded in a standardized questionnaire, S1 File. Blood samples drawn between 6 to 36 hours after last dose of medication was included in the analysis of steady-state drug levels and adjusted for time with ANCOVA analysis. Adherence was recorded using a modified ACTG adherence questionnaire [13]. Any missed dose during the preceding 4 days was considered as non-adherence.
Comorbidities were registered by structured medical record reviews. PDDIs were analyzed using the Liverpool University HIV drug interactions [14] and Janusmed [15] webtools. The Liverpool University HIV drug interactions database definitions for PDDIs were used and red flag (drugs should not be co-administered) and orange flag (a potential interaction that may require dose monitoring, alteration of drug dosage or timing of administration) interactions were included in the analysis. If there was an interaction between a comedication and both the PI and the booster, the interaction was counted as one interaction in the analysis. Individuals taking DRV b.i.d. or ATV without ritonavir booster were excluded from the analysis of plasma drug levels.
All study participants gave their written informed consent and ethics approval for the study was granted by the Research Ethics Committee at Gothenburg University.
Laboratory analyses
Plasma samples were frozen at –70°C immediately after sampling until analysis. Drug levels were analyzed using a reverse-phase High Pressure Liquid Chromatography (HPLC) with ultraviolet (UV) detection at the routine pharmacology analytical laboratory at Karolinska University Hospital, Huddinge in Stockholm, Sweden. The method was CAP (College of American Pathologists) and Swedac accredited and has been described elsewhere [16]. Routine clinical methods were used to analyze CD4 cell count, liver enzymes and creatinine according to local laboratory standards.
Statistical analyses
Differences in plasma drug levels were analyzed with ANCOVA (adjusting for time) with log-transformed concentrations of ATV, DRV, and EFV. Chi-square test and Fisher’s exact test were used to compare the frequencies of side-effects, CD4/CD8 ratios, and AIDS diagnosis as appropriate. Mann Whitney U-test and Kruskal Wallis test (with Bonferroni correction for multiple tests) were used to compare frequencies of concomitant medications and PDDIs. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics, Armonk, NY, USA) or Prism version 8.0 (Graphpad Software Inc., La Jolla, CA, USA).
Results
One hundred and seventy-two individuals 65 years of age or older were eligible for inclusion and were asked to participate in the study at the four sites. Between November 2013 and August 2015, 100 individuals were enrolled in the study group (ATV n = 19; DRV n = 35; EFV n = 46) and 99 individuals in the control group (ATV n = 18; DRV n = 37; EFV n = 44). Baseline characteristics are listed in Table 1. Three individuals had HIV RNA blips (HIV RNA 59–156 copies/mL) at inclusion; all other patients had HIV RNA levels < 50 copies/mL at inclusion. Twenty-seven patients were excluded from the plasma drug level analysis: 15 individuals received DRV b.i.d. and 9 were treated with ATV, either unboosted or with dosing not according to clinical standards. Three were excluded from the plasma drug level analysis due to sample management (elapsed time since last dose less than 6 hours, or elapsed time since last dose unknown). Patients included in the plasma drug level analysis received DRV/r 800/100 mg, ATV/r 300/100 mg, or EFV 600 mg q.d. There was a significant difference in ALT levels between study and control group in the ATV arm, however the majority of subjects had ALT within the normal range. The study group had a lower glomerular filtration rate (GFR) in all arms compared to controls, although within the normal range.
10.1371/journal.pone.0246171.t001 Table 1 Baseline characteristics.
ATV DRV EFV
Study n = 19 Control n = 18 p Study n = 35 Control n = 37 p Study n = 46 Control n = 44 p
Age (median [IQR]) 68 (66–70) 46 (40.75–47.5) 68 (67–72) 45 (37.5–47) 69 (67–72) 43 (37–46)
Gender (M/F) (n) 13/6 17/1 0.09 33/2 32/5 0.43 41/5 38/6 0.76
BMI (median [IQR]) 24.3 (22.3–27.2) 25.3 (23.9–27.9) 0.28 25.7 (23.0–26.9) 23.5 (22.5–26.0) 0.17 24.4 (21.9–27.8) 24.2 (22.1–28.5) 0.65
GFR (ml/min) (median [IQR]) 83.7 (71.0–96.8) 118.0 (109.9–134.4) <0.001 86.1 (55.4–95.3) 110.4 (97.0–135.8) <0.001 79.9 (67.6–98.5) 124.3 (116.0–145.2) <0.001
ALT (μkat/L) (median [IQR]) 0.40 (0.30–0.55) 0.63 (0.52–0.75) 0.017 0.32 (0.24–0.48) 0.41 (0.31–0.55) 0.14 0.48 (0.33–0.56) 0.55 (0.38–0.85) 0.056
CD4 cell count (median [IQR]) 650 (370–730) 730 (490–912.5) 0.13 560 (420–650) 600 (465–830) 0.54 535 (380–687.5) 575 (407.5–750) 0.39
CD4 cell count Nadir (median [IQR]) 130 (69–223) 241 (187–337) 0.001 185 (90–281) 218 (40–310) 0.97 208 (158–263) 219 (167–303) 0.49
Backbone 3TC/ABC 9 14 0.09 11 14 0.63 23 12 0.03
Backbone FTC/TDF 10 4 0.09 5 12 0.03 23 30 0.09
Backbone other** 0 0 NA 19 9 0.02 0 2 0.24
Comorbidities (n) (median [IQR]) 2 (1–4) 2 (0.25–3) 0.31 3 (2–5) 1 (1–3) 0.001 3 (2–4.25) 1 (0–2) <0.001
** Other backbones: RAL, ETV, RAL + maraviroc, RAL + EFV, RAL + DRV, LPV/r, RAL + 3TC, 3TC, DTG + 3TC, RPV, no backbone.
The steady-state DRV concentrations were significantly higher in the study group (n = 25) compared to the control group (n = 30) (p = 0.047), Fig 1. The geometric mean was 48% higher in the study group than in the control group. The analysis of the ATV arm (study group n = 19, control group n = 18) showed a difference in steady state-levels (geometric mean 69% higher in the study group), with a trend towards statistical significance (p = 0.056). No statistically significant difference between the groups was found in the EFV (p = 0.87) arm. There were no differences in self-reported adherence between the study group (96% adherent) and control group (93% adherent) (p = 0.537), or between different treatment arms either in the study or the control group.
10.1371/journal.pone.0246171.g001 Fig 1 Steady-state levels of ART.
Steady-state levels in plasma of A) Darunavir (p = 0.047), B) Atazanavir (p = 0.056), and C) Efavirenz (p = 0.87) in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group).
There were no statistically significant differences in reported side-effects between the study group (23%) and the control group (34%) (p = 0.146), Fig 2. When dividing the groups according to drug regimen (taking the study group together with the controls), the DRV group had a higher rate of reported side-effects (ATV: 16.7%; DRV: 39.4%; EFV: 25.9%; p = 0.038), which was significantly different compared to the ATV arm. The difference remained when PLHIV ≥ 65 years of age were analysed separately (ATV: 0%; DRV: 36.7%; EFV: 23.8%; p = 0.014), with significant differences between DRV and ATV, and EFV and ATV. In the DRV arm there were no significant difference in reported side effects between the study group and the control group, (p = 0.80). The most commonly-reported side-effect in the DRV groups was diarrhea.
10.1371/journal.pone.0246171.g002 Fig 2 Frequency of self-reported side-effects.
A: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) (ns). B: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) divided into treatment arms (p = 0.038). C: Frequency of self-reported side-effects divided into groups according to treatment arm and study group vs. control group.
As expected, the study group had a significantly higher mean (range) number of concomitant medications, 3.7 (0–12), compared to the control group, 1.1 (0–10) (p < 0.001). Accordingly, the study group had significantly more PDDIs (mean (range)) 1.1 (0–6) compared to the control group, 0.3 (0–3) (p < 0.001). The most common PDDIs for ATV were statins and beta-blocking agents, and statins and antidepressants for DRV and EFV.
Analysis of the study group showed that the DRV arm had significantly more PDDIs (mean [range]) 1.4 (0–6) than the EFV arm 0.7 (0–3) (p = 0.03). The ATV arm had a mean (range) of 1.2 (0–4) PDDIs, not significantly different compared to either the DRV arm or the EFV arm (see Table 2). Notably, the use of concomitant medications was not higher in the DRV arm. Eight individuals in the study group had red flag PDDIs: DRV/r and alfuzosin (risk for severe hypotension, n = 2); DRV/r and clopidogrel (reduced effect of clopidogrel, n = 3); DRV/r and alfuzosin + clopidogrel (n = 1); ATV and budesonide (increased risk of steroid side-effects, n = 1); and ATV and lansoprazole (reduced ATV uptake, n = 1). Whereas no one in the control group had a red flag interaction.
10.1371/journal.pone.0246171.t002 Table 2 Potential drug-drug interactions.
PDDIs (n) (mean [range]) p
Atazanavir 1.2 (0–4)
Darunavir 1.4 (1–6) 0.026
Efavirenz 0.7 (0–3)
Number of potential drug-drug interactions (PDDIs) in the study group presented by treatment regime.
There were no differences in CD4/CD8 ratios (≥ 1 or < 1) between the study group (≥ 1 n = 35 (35%)) and control group (≥ 1 n = 40 (40%)) (p = 0.43), the ATV/DRV/EFV arms, or between arms in the different groups. No differences were found in the history of AIDS defining events in the study group vs. control group. In total 38 individuals had one or more AIDS defining diagnoses, S1 Table.
Discussion
We found a difference in the steady-state plasma drug levels of DRV in PLHIV who were ≥ 65 years of age, as compared to PLHIV < 50 years of age. To our knowledge, only one previous study has addressed the question of plasma DRV levels in elderly PLHIV [17]. In agreement with our results, the authors reported higher DRV levels in individuals > 60 years of age compared to those ≤ 40 years of age.
The difference in plasma drug levels is also consistent with earlier findings regarding other PIs, including ATV [18–20]. We noted a difference in ATV levels between elderly and younger PLHIV, with a trend towards statistical significance. The lack of significance is probably due to the small sample size. In a previous report by Avihingsanon et al., higher trough levels and higher exposure to ATV in PLHIV > 42 years of age was found compared to individuals ≤ 42 years of age, consistent with our results. This difference was more pronounced in PLHIV > 50 years of age [18]. Winston et al. also found a significant association between age and plasma drug levels of PIs [19]. We did not find any significant difference in EFV plasma drug levels in the study group compared to the control group. This is in agreement with the findings in other reports [19, 21].
There is only very limited pharmacokinetic data on ART in PLHIV older than 65 years of age. There are, however, several general age-related biological changes that may affect the metabolism of ART, e.g. decrease of liver and renal function and changes in body composition that influence the volume of distribution [22]. ATV, DRV and EFV are metabolized in the liver (ATV and DRV mainly through CYP3A4 and EFV through CYP 3A4 and CYP2B6). Only a minor portion of these drugs is eliminated through the kidneys. Therefore, the difference in drug levels in the DRV and ATV arm cannot be explained by differences in GFR. We found no clinically measurable difference in liver function measured by ALT but other changes in liver function related to age may have affected the drug levels.
Other aspects not related to metabolism may also affect the efficacy of ART. Older PLHIV have been shown to be more adherent to their treatment regimen than younger individuals [23, 24]. However, with increasing age there is a higher risk of cognitive impairment that may affect the adherence in the oldest. No difference in adherence was found in our study to support or reject either higher or lower adherence in elderly PLHIV.
Overall, we found a higher frequency of self-reported side-effects in the DRV arm, in comparison to the ATV and the EFV arms (although not significantly different from the EFV arm). The higher frequency was also present in the study group (although not statistically significant).
To the best of our knowledge this is the only study that compared the frequency of side-effects between different PIs/NNRTIs in PLHIV older than 65 years of age. A possible reason for the DRV arm having a higher frequency of side-effects might be that DRV was chosen because of extensive ART history and viral resistance, resulting in few available alternative regimes at the time of inclusion in the study and as a consequence there was a higher tolerance of side-effects. On the other hand, no difference in self-reported side effects was noted between the DRV study group and control group, even though we found a difference in steady state plasma drug levels. This observation may reflect under reporting in the study group. While it is not possible in the present study to establish a causal link, further studies are needed illuminate this issue.
PDDIs are common among PLHIV [25, 26], and the risk increases with age due to increasing frequencies of comorbidities and concomitant medications [9, 10]. Our result is in line with these earlier studies. Red flag interactions has been reported in 2% to 5.6% of PLHIV and 7.1% to 8.7% in PLHIV ≥ 65 years of age in earlier studies, similar to our findings [26–29].
ATV and DRV are both PIs and therefore they have, in general, the same PDDIs. However, they differ in regard to some frequently used drug classes e.g. beta blockers and PPIs. Since EFV is a NNRTI it has another drug interaction profile. DRV accounted for the majority of the red flag interactions found in our study, consistent with earlier findings of PLHIV in all ages [27]. Other studies have reported a higher probability of an orange or red flag PDDI in individuals treated with a PI (not restricted to those ≥ 65) [9, 28]. This is consistent with our finding that the DRV arm in the study group had a higher mean of PDDIs than the EFV arm. We did not find a significant difference between the ATV and EFV arms, however this may be due to the small sample size. The majority of the red flag interactions found were related to the concomitant use of alfuzosin, for treatment of benign prostatic hyperplasia, and/or clopidogrel, for treatment of vascular disease, which both are conditions that have a higher prevalence in older ages.
Our study has several limitations. Since we analysed steady-state plasma drug levels and not trough levels of ART, it is difficult to compare our results with trough levels in other studies. Thus, we were unable to evaluate potentially toxic plasma drug levels of ART drugs or levels below the proposed minimal effective concentrations. Patients were sampled from 6 to 36 hours after their last dose of ART. This was adjusted for in the statistical model, but there remains a risk that, because of this approach, we were not able to detect minor differences in drug levels between the study and control arms for ATV and EFV. Also, there was a difference in back-bone between PLHIV on DRV compared to ATV and EFV that may have affected self-reported side-effects. Concomitant medications may also have influenced the self-reported side-effects, though the participants were asked specifically to report side-effects related to ART. The PDDIs were calculated only for ATV, DRV or EFV regimens and therefore differences in back-bone likely did not affect the results. In addition, interactions between NRTIs and other medications are uncommon. The participants in the study were included consecutively at four sites in Sweden and it is possible that this introduced a selection bias, favoring PLHIV with frequent visits.
Conclusion
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV who were 65 years of age or older, as compared to controls who were 49 years of age and younger. Our findings are important for the management of elderly PLHIV and raise the question of whether regular monitoring of plasma levels and dose adjustment of DRV and other PIs is warranted in the elderly.
Supporting information
S1 Table Aids defining diagnoses.
(DOCX)
Click here for additional data file.
S2 Table Data set.
(XLSX)
Click here for additional data file.
S1 File Questionnaire/case report form.
(DOCX)
Click here for additional data file.
We would like to thank all the participants in the study, study nurses and technical staff. | Oral | DrugAdministrationRoute | CC BY | 33539440 | 19,781,509 | 2021 |
What was the dosage of drug 'ABACAVIR SULFATE\LAMIVUDINE'? | Higher plasma drug levels in elderly people living with HIV treated with darunavir.
The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age.
Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases.
Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV).
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.
Introduction
Antiretroviral therapy (ART) has dramatically changed the life expectancy of people living with HIV (PLHIV). HIV can now be considered a chronic infection, and the expected life span of PLHIV who receive efficient treatment is comparable to HIV-negative individuals [1, 2]. As a consequence, an increasing number of PLHIV are of older age. For example, in 2018 51% of PLHIV in the US were 50 years of age or older [3].
The risk of developing age-related and lifestyle-related diseases increases with age. PLHIV are, in addition, at higher risk of non-infectious comorbidities compared to the general population [4–6]. Furthermore, elderly people are, in general, at higher risk of adverse events to medications and may require lower doses of medications than recommended for younger individuals [7, 8]. It has been reported that PLHIV above 50 years of age have more concomitant medications and a higher risk of potential drug-drug interactions (PDDIs) compared to PLHIV below 50 years of age [9, 10].
Since the mid 1990s the standard regime for HIV treatment is two nucleoside reverse-transcriptase inhibitors (NRTIs) combined with a third agent from another drug class (most commonly a nucleoside reverse-transcriptase inhibitor (NNRTI), a protease inhibitor (PI) or an integrase inhibitor (INSTI). A dolutegravir (INSTI) or Efavirenz (NNRTI) containing regimen is recommended by WHO as first line treatment today [11]. In the Swedish setting a dolutegravir or darunavir containing regimen is recommended by the Swedish Reference Group for Antiviral Therapy [12]. Neither WHO nor Sweden have specific treatment recommendations for elderly PLHIV.
Scientific data on the pharmacokinetics of PIs and NNRTIs in individuals 65 years of age and older are scarce. The primary objective of this study was to investigate differences in steady-state plasma drug levels of ATV, DRV and EFV in PLHIV ≥ 65 years of age as compared to PLHIV ≤ 49 years of age. Secondary objectives were to study differences in self-reported side-effects, concomitant chronic diseases and medications, and PDDIs.
Methods
PLHIV who were followed at four HIV centers in Sweden: the Department of Infectious Diseases at Sahlgrenska University Hospital in Gothenburg; the Department of Infectious Diseases at South Älvsborg Hospital in Borås; the Department of Infectious Diseases at Karolinska University Hospital Huddinge in Stockholm; and the Department of Infectious Diseases at Stockholm South General Hospital in Stockholm, and met the inclusion criteria (age, 65 years of age or older for the study group or 49 years of age or younger for the control group; and on stable ART containing atazanavir (ATV), darunavir (DRV) or efavirenz (EFV) for more than 6 months) were eligible for inclusion in this cross-sectional study. On the day of inclusion, a blood sample for analysis of plasma drug level was taken, and concomitant medications (including non-prescription drugs and herbal supplements) and any side-effects related to ART were recorded in a standardized questionnaire, S1 File. Blood samples drawn between 6 to 36 hours after last dose of medication was included in the analysis of steady-state drug levels and adjusted for time with ANCOVA analysis. Adherence was recorded using a modified ACTG adherence questionnaire [13]. Any missed dose during the preceding 4 days was considered as non-adherence.
Comorbidities were registered by structured medical record reviews. PDDIs were analyzed using the Liverpool University HIV drug interactions [14] and Janusmed [15] webtools. The Liverpool University HIV drug interactions database definitions for PDDIs were used and red flag (drugs should not be co-administered) and orange flag (a potential interaction that may require dose monitoring, alteration of drug dosage or timing of administration) interactions were included in the analysis. If there was an interaction between a comedication and both the PI and the booster, the interaction was counted as one interaction in the analysis. Individuals taking DRV b.i.d. or ATV without ritonavir booster were excluded from the analysis of plasma drug levels.
All study participants gave their written informed consent and ethics approval for the study was granted by the Research Ethics Committee at Gothenburg University.
Laboratory analyses
Plasma samples were frozen at –70°C immediately after sampling until analysis. Drug levels were analyzed using a reverse-phase High Pressure Liquid Chromatography (HPLC) with ultraviolet (UV) detection at the routine pharmacology analytical laboratory at Karolinska University Hospital, Huddinge in Stockholm, Sweden. The method was CAP (College of American Pathologists) and Swedac accredited and has been described elsewhere [16]. Routine clinical methods were used to analyze CD4 cell count, liver enzymes and creatinine according to local laboratory standards.
Statistical analyses
Differences in plasma drug levels were analyzed with ANCOVA (adjusting for time) with log-transformed concentrations of ATV, DRV, and EFV. Chi-square test and Fisher’s exact test were used to compare the frequencies of side-effects, CD4/CD8 ratios, and AIDS diagnosis as appropriate. Mann Whitney U-test and Kruskal Wallis test (with Bonferroni correction for multiple tests) were used to compare frequencies of concomitant medications and PDDIs. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics, Armonk, NY, USA) or Prism version 8.0 (Graphpad Software Inc., La Jolla, CA, USA).
Results
One hundred and seventy-two individuals 65 years of age or older were eligible for inclusion and were asked to participate in the study at the four sites. Between November 2013 and August 2015, 100 individuals were enrolled in the study group (ATV n = 19; DRV n = 35; EFV n = 46) and 99 individuals in the control group (ATV n = 18; DRV n = 37; EFV n = 44). Baseline characteristics are listed in Table 1. Three individuals had HIV RNA blips (HIV RNA 59–156 copies/mL) at inclusion; all other patients had HIV RNA levels < 50 copies/mL at inclusion. Twenty-seven patients were excluded from the plasma drug level analysis: 15 individuals received DRV b.i.d. and 9 were treated with ATV, either unboosted or with dosing not according to clinical standards. Three were excluded from the plasma drug level analysis due to sample management (elapsed time since last dose less than 6 hours, or elapsed time since last dose unknown). Patients included in the plasma drug level analysis received DRV/r 800/100 mg, ATV/r 300/100 mg, or EFV 600 mg q.d. There was a significant difference in ALT levels between study and control group in the ATV arm, however the majority of subjects had ALT within the normal range. The study group had a lower glomerular filtration rate (GFR) in all arms compared to controls, although within the normal range.
10.1371/journal.pone.0246171.t001 Table 1 Baseline characteristics.
ATV DRV EFV
Study n = 19 Control n = 18 p Study n = 35 Control n = 37 p Study n = 46 Control n = 44 p
Age (median [IQR]) 68 (66–70) 46 (40.75–47.5) 68 (67–72) 45 (37.5–47) 69 (67–72) 43 (37–46)
Gender (M/F) (n) 13/6 17/1 0.09 33/2 32/5 0.43 41/5 38/6 0.76
BMI (median [IQR]) 24.3 (22.3–27.2) 25.3 (23.9–27.9) 0.28 25.7 (23.0–26.9) 23.5 (22.5–26.0) 0.17 24.4 (21.9–27.8) 24.2 (22.1–28.5) 0.65
GFR (ml/min) (median [IQR]) 83.7 (71.0–96.8) 118.0 (109.9–134.4) <0.001 86.1 (55.4–95.3) 110.4 (97.0–135.8) <0.001 79.9 (67.6–98.5) 124.3 (116.0–145.2) <0.001
ALT (μkat/L) (median [IQR]) 0.40 (0.30–0.55) 0.63 (0.52–0.75) 0.017 0.32 (0.24–0.48) 0.41 (0.31–0.55) 0.14 0.48 (0.33–0.56) 0.55 (0.38–0.85) 0.056
CD4 cell count (median [IQR]) 650 (370–730) 730 (490–912.5) 0.13 560 (420–650) 600 (465–830) 0.54 535 (380–687.5) 575 (407.5–750) 0.39
CD4 cell count Nadir (median [IQR]) 130 (69–223) 241 (187–337) 0.001 185 (90–281) 218 (40–310) 0.97 208 (158–263) 219 (167–303) 0.49
Backbone 3TC/ABC 9 14 0.09 11 14 0.63 23 12 0.03
Backbone FTC/TDF 10 4 0.09 5 12 0.03 23 30 0.09
Backbone other** 0 0 NA 19 9 0.02 0 2 0.24
Comorbidities (n) (median [IQR]) 2 (1–4) 2 (0.25–3) 0.31 3 (2–5) 1 (1–3) 0.001 3 (2–4.25) 1 (0–2) <0.001
** Other backbones: RAL, ETV, RAL + maraviroc, RAL + EFV, RAL + DRV, LPV/r, RAL + 3TC, 3TC, DTG + 3TC, RPV, no backbone.
The steady-state DRV concentrations were significantly higher in the study group (n = 25) compared to the control group (n = 30) (p = 0.047), Fig 1. The geometric mean was 48% higher in the study group than in the control group. The analysis of the ATV arm (study group n = 19, control group n = 18) showed a difference in steady state-levels (geometric mean 69% higher in the study group), with a trend towards statistical significance (p = 0.056). No statistically significant difference between the groups was found in the EFV (p = 0.87) arm. There were no differences in self-reported adherence between the study group (96% adherent) and control group (93% adherent) (p = 0.537), or between different treatment arms either in the study or the control group.
10.1371/journal.pone.0246171.g001 Fig 1 Steady-state levels of ART.
Steady-state levels in plasma of A) Darunavir (p = 0.047), B) Atazanavir (p = 0.056), and C) Efavirenz (p = 0.87) in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group).
There were no statistically significant differences in reported side-effects between the study group (23%) and the control group (34%) (p = 0.146), Fig 2. When dividing the groups according to drug regimen (taking the study group together with the controls), the DRV group had a higher rate of reported side-effects (ATV: 16.7%; DRV: 39.4%; EFV: 25.9%; p = 0.038), which was significantly different compared to the ATV arm. The difference remained when PLHIV ≥ 65 years of age were analysed separately (ATV: 0%; DRV: 36.7%; EFV: 23.8%; p = 0.014), with significant differences between DRV and ATV, and EFV and ATV. In the DRV arm there were no significant difference in reported side effects between the study group and the control group, (p = 0.80). The most commonly-reported side-effect in the DRV groups was diarrhea.
10.1371/journal.pone.0246171.g002 Fig 2 Frequency of self-reported side-effects.
A: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) (ns). B: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) divided into treatment arms (p = 0.038). C: Frequency of self-reported side-effects divided into groups according to treatment arm and study group vs. control group.
As expected, the study group had a significantly higher mean (range) number of concomitant medications, 3.7 (0–12), compared to the control group, 1.1 (0–10) (p < 0.001). Accordingly, the study group had significantly more PDDIs (mean (range)) 1.1 (0–6) compared to the control group, 0.3 (0–3) (p < 0.001). The most common PDDIs for ATV were statins and beta-blocking agents, and statins and antidepressants for DRV and EFV.
Analysis of the study group showed that the DRV arm had significantly more PDDIs (mean [range]) 1.4 (0–6) than the EFV arm 0.7 (0–3) (p = 0.03). The ATV arm had a mean (range) of 1.2 (0–4) PDDIs, not significantly different compared to either the DRV arm or the EFV arm (see Table 2). Notably, the use of concomitant medications was not higher in the DRV arm. Eight individuals in the study group had red flag PDDIs: DRV/r and alfuzosin (risk for severe hypotension, n = 2); DRV/r and clopidogrel (reduced effect of clopidogrel, n = 3); DRV/r and alfuzosin + clopidogrel (n = 1); ATV and budesonide (increased risk of steroid side-effects, n = 1); and ATV and lansoprazole (reduced ATV uptake, n = 1). Whereas no one in the control group had a red flag interaction.
10.1371/journal.pone.0246171.t002 Table 2 Potential drug-drug interactions.
PDDIs (n) (mean [range]) p
Atazanavir 1.2 (0–4)
Darunavir 1.4 (1–6) 0.026
Efavirenz 0.7 (0–3)
Number of potential drug-drug interactions (PDDIs) in the study group presented by treatment regime.
There were no differences in CD4/CD8 ratios (≥ 1 or < 1) between the study group (≥ 1 n = 35 (35%)) and control group (≥ 1 n = 40 (40%)) (p = 0.43), the ATV/DRV/EFV arms, or between arms in the different groups. No differences were found in the history of AIDS defining events in the study group vs. control group. In total 38 individuals had one or more AIDS defining diagnoses, S1 Table.
Discussion
We found a difference in the steady-state plasma drug levels of DRV in PLHIV who were ≥ 65 years of age, as compared to PLHIV < 50 years of age. To our knowledge, only one previous study has addressed the question of plasma DRV levels in elderly PLHIV [17]. In agreement with our results, the authors reported higher DRV levels in individuals > 60 years of age compared to those ≤ 40 years of age.
The difference in plasma drug levels is also consistent with earlier findings regarding other PIs, including ATV [18–20]. We noted a difference in ATV levels between elderly and younger PLHIV, with a trend towards statistical significance. The lack of significance is probably due to the small sample size. In a previous report by Avihingsanon et al., higher trough levels and higher exposure to ATV in PLHIV > 42 years of age was found compared to individuals ≤ 42 years of age, consistent with our results. This difference was more pronounced in PLHIV > 50 years of age [18]. Winston et al. also found a significant association between age and plasma drug levels of PIs [19]. We did not find any significant difference in EFV plasma drug levels in the study group compared to the control group. This is in agreement with the findings in other reports [19, 21].
There is only very limited pharmacokinetic data on ART in PLHIV older than 65 years of age. There are, however, several general age-related biological changes that may affect the metabolism of ART, e.g. decrease of liver and renal function and changes in body composition that influence the volume of distribution [22]. ATV, DRV and EFV are metabolized in the liver (ATV and DRV mainly through CYP3A4 and EFV through CYP 3A4 and CYP2B6). Only a minor portion of these drugs is eliminated through the kidneys. Therefore, the difference in drug levels in the DRV and ATV arm cannot be explained by differences in GFR. We found no clinically measurable difference in liver function measured by ALT but other changes in liver function related to age may have affected the drug levels.
Other aspects not related to metabolism may also affect the efficacy of ART. Older PLHIV have been shown to be more adherent to their treatment regimen than younger individuals [23, 24]. However, with increasing age there is a higher risk of cognitive impairment that may affect the adherence in the oldest. No difference in adherence was found in our study to support or reject either higher or lower adherence in elderly PLHIV.
Overall, we found a higher frequency of self-reported side-effects in the DRV arm, in comparison to the ATV and the EFV arms (although not significantly different from the EFV arm). The higher frequency was also present in the study group (although not statistically significant).
To the best of our knowledge this is the only study that compared the frequency of side-effects between different PIs/NNRTIs in PLHIV older than 65 years of age. A possible reason for the DRV arm having a higher frequency of side-effects might be that DRV was chosen because of extensive ART history and viral resistance, resulting in few available alternative regimes at the time of inclusion in the study and as a consequence there was a higher tolerance of side-effects. On the other hand, no difference in self-reported side effects was noted between the DRV study group and control group, even though we found a difference in steady state plasma drug levels. This observation may reflect under reporting in the study group. While it is not possible in the present study to establish a causal link, further studies are needed illuminate this issue.
PDDIs are common among PLHIV [25, 26], and the risk increases with age due to increasing frequencies of comorbidities and concomitant medications [9, 10]. Our result is in line with these earlier studies. Red flag interactions has been reported in 2% to 5.6% of PLHIV and 7.1% to 8.7% in PLHIV ≥ 65 years of age in earlier studies, similar to our findings [26–29].
ATV and DRV are both PIs and therefore they have, in general, the same PDDIs. However, they differ in regard to some frequently used drug classes e.g. beta blockers and PPIs. Since EFV is a NNRTI it has another drug interaction profile. DRV accounted for the majority of the red flag interactions found in our study, consistent with earlier findings of PLHIV in all ages [27]. Other studies have reported a higher probability of an orange or red flag PDDI in individuals treated with a PI (not restricted to those ≥ 65) [9, 28]. This is consistent with our finding that the DRV arm in the study group had a higher mean of PDDIs than the EFV arm. We did not find a significant difference between the ATV and EFV arms, however this may be due to the small sample size. The majority of the red flag interactions found were related to the concomitant use of alfuzosin, for treatment of benign prostatic hyperplasia, and/or clopidogrel, for treatment of vascular disease, which both are conditions that have a higher prevalence in older ages.
Our study has several limitations. Since we analysed steady-state plasma drug levels and not trough levels of ART, it is difficult to compare our results with trough levels in other studies. Thus, we were unable to evaluate potentially toxic plasma drug levels of ART drugs or levels below the proposed minimal effective concentrations. Patients were sampled from 6 to 36 hours after their last dose of ART. This was adjusted for in the statistical model, but there remains a risk that, because of this approach, we were not able to detect minor differences in drug levels between the study and control arms for ATV and EFV. Also, there was a difference in back-bone between PLHIV on DRV compared to ATV and EFV that may have affected self-reported side-effects. Concomitant medications may also have influenced the self-reported side-effects, though the participants were asked specifically to report side-effects related to ART. The PDDIs were calculated only for ATV, DRV or EFV regimens and therefore differences in back-bone likely did not affect the results. In addition, interactions between NRTIs and other medications are uncommon. The participants in the study were included consecutively at four sites in Sweden and it is possible that this introduced a selection bias, favoring PLHIV with frequent visits.
Conclusion
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV who were 65 years of age or older, as compared to controls who were 49 years of age and younger. Our findings are important for the management of elderly PLHIV and raise the question of whether regular monitoring of plasma levels and dose adjustment of DRV and other PIs is warranted in the elderly.
Supporting information
S1 Table Aids defining diagnoses.
(DOCX)
Click here for additional data file.
S2 Table Data set.
(XLSX)
Click here for additional data file.
S1 File Questionnaire/case report form.
(DOCX)
Click here for additional data file.
We would like to thank all the participants in the study, study nurses and technical staff. | 300/600 MG 1X1 | DrugDosageText | CC BY | 33539440 | 19,781,509 | 2021 |
What was the dosage of drug 'EMTRICITABINE\TENOFOVIR DISOPROXIL'? | Higher plasma drug levels in elderly people living with HIV treated with darunavir.
The proportion of elderly people living with HIV-1 (PLHIV) is rising. In older patients, comorbidities and concomitant medications are more frequent, increasing the risk of potential drug-drug interactions (PDDIs). Data on the pharmacokinetics of ART in individuals aged ≥ 65 years of age are scarce. We compared plasma drug levels of ART, PDDIs, and side-effects in PLHIV aged ≥ 65 years of age, with controls ≤ 49 years of age.
Patients ≥ 65 years of age and controls ≤ 49 years of age, all of whom were on stable treatment with atazanavir (ATV), darunavir (DRV), or efavirenz (EFV) were included cross-sectionally. Plasma drug levels of ART were analyzed, comorbidities, concomitant medication, adherence, and side-effects recorded, and PDDIs analyzed using drug interactions databases.
Between 2013 and 2015, we included 100 individuals ≥ 65 years of age (study group) and 99 controls (≤ 49 years of age). Steady-state DRV concentrations were significantly higher in the study group than in the control group (p = 0.047). In the ATV group there was a trend towards a significant difference (p = 0.056). No significant differences were found in the EFV arm. The DRV arm had a higher frequency of reported side-effects than the ATV and EFV arms in the study group (36.7% vs. 0% and 23.8% respectively (p = 0.014), with significant differences between DRV vs. ATV, and EFV vs. ATV).
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV aged ≥ 65 years of age, compared to controls ≤ 49 years of age.
Introduction
Antiretroviral therapy (ART) has dramatically changed the life expectancy of people living with HIV (PLHIV). HIV can now be considered a chronic infection, and the expected life span of PLHIV who receive efficient treatment is comparable to HIV-negative individuals [1, 2]. As a consequence, an increasing number of PLHIV are of older age. For example, in 2018 51% of PLHIV in the US were 50 years of age or older [3].
The risk of developing age-related and lifestyle-related diseases increases with age. PLHIV are, in addition, at higher risk of non-infectious comorbidities compared to the general population [4–6]. Furthermore, elderly people are, in general, at higher risk of adverse events to medications and may require lower doses of medications than recommended for younger individuals [7, 8]. It has been reported that PLHIV above 50 years of age have more concomitant medications and a higher risk of potential drug-drug interactions (PDDIs) compared to PLHIV below 50 years of age [9, 10].
Since the mid 1990s the standard regime for HIV treatment is two nucleoside reverse-transcriptase inhibitors (NRTIs) combined with a third agent from another drug class (most commonly a nucleoside reverse-transcriptase inhibitor (NNRTI), a protease inhibitor (PI) or an integrase inhibitor (INSTI). A dolutegravir (INSTI) or Efavirenz (NNRTI) containing regimen is recommended by WHO as first line treatment today [11]. In the Swedish setting a dolutegravir or darunavir containing regimen is recommended by the Swedish Reference Group for Antiviral Therapy [12]. Neither WHO nor Sweden have specific treatment recommendations for elderly PLHIV.
Scientific data on the pharmacokinetics of PIs and NNRTIs in individuals 65 years of age and older are scarce. The primary objective of this study was to investigate differences in steady-state plasma drug levels of ATV, DRV and EFV in PLHIV ≥ 65 years of age as compared to PLHIV ≤ 49 years of age. Secondary objectives were to study differences in self-reported side-effects, concomitant chronic diseases and medications, and PDDIs.
Methods
PLHIV who were followed at four HIV centers in Sweden: the Department of Infectious Diseases at Sahlgrenska University Hospital in Gothenburg; the Department of Infectious Diseases at South Älvsborg Hospital in Borås; the Department of Infectious Diseases at Karolinska University Hospital Huddinge in Stockholm; and the Department of Infectious Diseases at Stockholm South General Hospital in Stockholm, and met the inclusion criteria (age, 65 years of age or older for the study group or 49 years of age or younger for the control group; and on stable ART containing atazanavir (ATV), darunavir (DRV) or efavirenz (EFV) for more than 6 months) were eligible for inclusion in this cross-sectional study. On the day of inclusion, a blood sample for analysis of plasma drug level was taken, and concomitant medications (including non-prescription drugs and herbal supplements) and any side-effects related to ART were recorded in a standardized questionnaire, S1 File. Blood samples drawn between 6 to 36 hours after last dose of medication was included in the analysis of steady-state drug levels and adjusted for time with ANCOVA analysis. Adherence was recorded using a modified ACTG adherence questionnaire [13]. Any missed dose during the preceding 4 days was considered as non-adherence.
Comorbidities were registered by structured medical record reviews. PDDIs were analyzed using the Liverpool University HIV drug interactions [14] and Janusmed [15] webtools. The Liverpool University HIV drug interactions database definitions for PDDIs were used and red flag (drugs should not be co-administered) and orange flag (a potential interaction that may require dose monitoring, alteration of drug dosage or timing of administration) interactions were included in the analysis. If there was an interaction between a comedication and both the PI and the booster, the interaction was counted as one interaction in the analysis. Individuals taking DRV b.i.d. or ATV without ritonavir booster were excluded from the analysis of plasma drug levels.
All study participants gave their written informed consent and ethics approval for the study was granted by the Research Ethics Committee at Gothenburg University.
Laboratory analyses
Plasma samples were frozen at –70°C immediately after sampling until analysis. Drug levels were analyzed using a reverse-phase High Pressure Liquid Chromatography (HPLC) with ultraviolet (UV) detection at the routine pharmacology analytical laboratory at Karolinska University Hospital, Huddinge in Stockholm, Sweden. The method was CAP (College of American Pathologists) and Swedac accredited and has been described elsewhere [16]. Routine clinical methods were used to analyze CD4 cell count, liver enzymes and creatinine according to local laboratory standards.
Statistical analyses
Differences in plasma drug levels were analyzed with ANCOVA (adjusting for time) with log-transformed concentrations of ATV, DRV, and EFV. Chi-square test and Fisher’s exact test were used to compare the frequencies of side-effects, CD4/CD8 ratios, and AIDS diagnosis as appropriate. Mann Whitney U-test and Kruskal Wallis test (with Bonferroni correction for multiple tests) were used to compare frequencies of concomitant medications and PDDIs. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 25 (IBM SPSS Statistics, Armonk, NY, USA) or Prism version 8.0 (Graphpad Software Inc., La Jolla, CA, USA).
Results
One hundred and seventy-two individuals 65 years of age or older were eligible for inclusion and were asked to participate in the study at the four sites. Between November 2013 and August 2015, 100 individuals were enrolled in the study group (ATV n = 19; DRV n = 35; EFV n = 46) and 99 individuals in the control group (ATV n = 18; DRV n = 37; EFV n = 44). Baseline characteristics are listed in Table 1. Three individuals had HIV RNA blips (HIV RNA 59–156 copies/mL) at inclusion; all other patients had HIV RNA levels < 50 copies/mL at inclusion. Twenty-seven patients were excluded from the plasma drug level analysis: 15 individuals received DRV b.i.d. and 9 were treated with ATV, either unboosted or with dosing not according to clinical standards. Three were excluded from the plasma drug level analysis due to sample management (elapsed time since last dose less than 6 hours, or elapsed time since last dose unknown). Patients included in the plasma drug level analysis received DRV/r 800/100 mg, ATV/r 300/100 mg, or EFV 600 mg q.d. There was a significant difference in ALT levels between study and control group in the ATV arm, however the majority of subjects had ALT within the normal range. The study group had a lower glomerular filtration rate (GFR) in all arms compared to controls, although within the normal range.
10.1371/journal.pone.0246171.t001 Table 1 Baseline characteristics.
ATV DRV EFV
Study n = 19 Control n = 18 p Study n = 35 Control n = 37 p Study n = 46 Control n = 44 p
Age (median [IQR]) 68 (66–70) 46 (40.75–47.5) 68 (67–72) 45 (37.5–47) 69 (67–72) 43 (37–46)
Gender (M/F) (n) 13/6 17/1 0.09 33/2 32/5 0.43 41/5 38/6 0.76
BMI (median [IQR]) 24.3 (22.3–27.2) 25.3 (23.9–27.9) 0.28 25.7 (23.0–26.9) 23.5 (22.5–26.0) 0.17 24.4 (21.9–27.8) 24.2 (22.1–28.5) 0.65
GFR (ml/min) (median [IQR]) 83.7 (71.0–96.8) 118.0 (109.9–134.4) <0.001 86.1 (55.4–95.3) 110.4 (97.0–135.8) <0.001 79.9 (67.6–98.5) 124.3 (116.0–145.2) <0.001
ALT (μkat/L) (median [IQR]) 0.40 (0.30–0.55) 0.63 (0.52–0.75) 0.017 0.32 (0.24–0.48) 0.41 (0.31–0.55) 0.14 0.48 (0.33–0.56) 0.55 (0.38–0.85) 0.056
CD4 cell count (median [IQR]) 650 (370–730) 730 (490–912.5) 0.13 560 (420–650) 600 (465–830) 0.54 535 (380–687.5) 575 (407.5–750) 0.39
CD4 cell count Nadir (median [IQR]) 130 (69–223) 241 (187–337) 0.001 185 (90–281) 218 (40–310) 0.97 208 (158–263) 219 (167–303) 0.49
Backbone 3TC/ABC 9 14 0.09 11 14 0.63 23 12 0.03
Backbone FTC/TDF 10 4 0.09 5 12 0.03 23 30 0.09
Backbone other** 0 0 NA 19 9 0.02 0 2 0.24
Comorbidities (n) (median [IQR]) 2 (1–4) 2 (0.25–3) 0.31 3 (2–5) 1 (1–3) 0.001 3 (2–4.25) 1 (0–2) <0.001
** Other backbones: RAL, ETV, RAL + maraviroc, RAL + EFV, RAL + DRV, LPV/r, RAL + 3TC, 3TC, DTG + 3TC, RPV, no backbone.
The steady-state DRV concentrations were significantly higher in the study group (n = 25) compared to the control group (n = 30) (p = 0.047), Fig 1. The geometric mean was 48% higher in the study group than in the control group. The analysis of the ATV arm (study group n = 19, control group n = 18) showed a difference in steady state-levels (geometric mean 69% higher in the study group), with a trend towards statistical significance (p = 0.056). No statistically significant difference between the groups was found in the EFV (p = 0.87) arm. There were no differences in self-reported adherence between the study group (96% adherent) and control group (93% adherent) (p = 0.537), or between different treatment arms either in the study or the control group.
10.1371/journal.pone.0246171.g001 Fig 1 Steady-state levels of ART.
Steady-state levels in plasma of A) Darunavir (p = 0.047), B) Atazanavir (p = 0.056), and C) Efavirenz (p = 0.87) in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group).
There were no statistically significant differences in reported side-effects between the study group (23%) and the control group (34%) (p = 0.146), Fig 2. When dividing the groups according to drug regimen (taking the study group together with the controls), the DRV group had a higher rate of reported side-effects (ATV: 16.7%; DRV: 39.4%; EFV: 25.9%; p = 0.038), which was significantly different compared to the ATV arm. The difference remained when PLHIV ≥ 65 years of age were analysed separately (ATV: 0%; DRV: 36.7%; EFV: 23.8%; p = 0.014), with significant differences between DRV and ATV, and EFV and ATV. In the DRV arm there were no significant difference in reported side effects between the study group and the control group, (p = 0.80). The most commonly-reported side-effect in the DRV groups was diarrhea.
10.1371/journal.pone.0246171.g002 Fig 2 Frequency of self-reported side-effects.
A: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) (ns). B: Frequency of self-reported side-effects in individuals 65 years of age or older (study group) and individuals 49 years of age or younger (control group) divided into treatment arms (p = 0.038). C: Frequency of self-reported side-effects divided into groups according to treatment arm and study group vs. control group.
As expected, the study group had a significantly higher mean (range) number of concomitant medications, 3.7 (0–12), compared to the control group, 1.1 (0–10) (p < 0.001). Accordingly, the study group had significantly more PDDIs (mean (range)) 1.1 (0–6) compared to the control group, 0.3 (0–3) (p < 0.001). The most common PDDIs for ATV were statins and beta-blocking agents, and statins and antidepressants for DRV and EFV.
Analysis of the study group showed that the DRV arm had significantly more PDDIs (mean [range]) 1.4 (0–6) than the EFV arm 0.7 (0–3) (p = 0.03). The ATV arm had a mean (range) of 1.2 (0–4) PDDIs, not significantly different compared to either the DRV arm or the EFV arm (see Table 2). Notably, the use of concomitant medications was not higher in the DRV arm. Eight individuals in the study group had red flag PDDIs: DRV/r and alfuzosin (risk for severe hypotension, n = 2); DRV/r and clopidogrel (reduced effect of clopidogrel, n = 3); DRV/r and alfuzosin + clopidogrel (n = 1); ATV and budesonide (increased risk of steroid side-effects, n = 1); and ATV and lansoprazole (reduced ATV uptake, n = 1). Whereas no one in the control group had a red flag interaction.
10.1371/journal.pone.0246171.t002 Table 2 Potential drug-drug interactions.
PDDIs (n) (mean [range]) p
Atazanavir 1.2 (0–4)
Darunavir 1.4 (1–6) 0.026
Efavirenz 0.7 (0–3)
Number of potential drug-drug interactions (PDDIs) in the study group presented by treatment regime.
There were no differences in CD4/CD8 ratios (≥ 1 or < 1) between the study group (≥ 1 n = 35 (35%)) and control group (≥ 1 n = 40 (40%)) (p = 0.43), the ATV/DRV/EFV arms, or between arms in the different groups. No differences were found in the history of AIDS defining events in the study group vs. control group. In total 38 individuals had one or more AIDS defining diagnoses, S1 Table.
Discussion
We found a difference in the steady-state plasma drug levels of DRV in PLHIV who were ≥ 65 years of age, as compared to PLHIV < 50 years of age. To our knowledge, only one previous study has addressed the question of plasma DRV levels in elderly PLHIV [17]. In agreement with our results, the authors reported higher DRV levels in individuals > 60 years of age compared to those ≤ 40 years of age.
The difference in plasma drug levels is also consistent with earlier findings regarding other PIs, including ATV [18–20]. We noted a difference in ATV levels between elderly and younger PLHIV, with a trend towards statistical significance. The lack of significance is probably due to the small sample size. In a previous report by Avihingsanon et al., higher trough levels and higher exposure to ATV in PLHIV > 42 years of age was found compared to individuals ≤ 42 years of age, consistent with our results. This difference was more pronounced in PLHIV > 50 years of age [18]. Winston et al. also found a significant association between age and plasma drug levels of PIs [19]. We did not find any significant difference in EFV plasma drug levels in the study group compared to the control group. This is in agreement with the findings in other reports [19, 21].
There is only very limited pharmacokinetic data on ART in PLHIV older than 65 years of age. There are, however, several general age-related biological changes that may affect the metabolism of ART, e.g. decrease of liver and renal function and changes in body composition that influence the volume of distribution [22]. ATV, DRV and EFV are metabolized in the liver (ATV and DRV mainly through CYP3A4 and EFV through CYP 3A4 and CYP2B6). Only a minor portion of these drugs is eliminated through the kidneys. Therefore, the difference in drug levels in the DRV and ATV arm cannot be explained by differences in GFR. We found no clinically measurable difference in liver function measured by ALT but other changes in liver function related to age may have affected the drug levels.
Other aspects not related to metabolism may also affect the efficacy of ART. Older PLHIV have been shown to be more adherent to their treatment regimen than younger individuals [23, 24]. However, with increasing age there is a higher risk of cognitive impairment that may affect the adherence in the oldest. No difference in adherence was found in our study to support or reject either higher or lower adherence in elderly PLHIV.
Overall, we found a higher frequency of self-reported side-effects in the DRV arm, in comparison to the ATV and the EFV arms (although not significantly different from the EFV arm). The higher frequency was also present in the study group (although not statistically significant).
To the best of our knowledge this is the only study that compared the frequency of side-effects between different PIs/NNRTIs in PLHIV older than 65 years of age. A possible reason for the DRV arm having a higher frequency of side-effects might be that DRV was chosen because of extensive ART history and viral resistance, resulting in few available alternative regimes at the time of inclusion in the study and as a consequence there was a higher tolerance of side-effects. On the other hand, no difference in self-reported side effects was noted between the DRV study group and control group, even though we found a difference in steady state plasma drug levels. This observation may reflect under reporting in the study group. While it is not possible in the present study to establish a causal link, further studies are needed illuminate this issue.
PDDIs are common among PLHIV [25, 26], and the risk increases with age due to increasing frequencies of comorbidities and concomitant medications [9, 10]. Our result is in line with these earlier studies. Red flag interactions has been reported in 2% to 5.6% of PLHIV and 7.1% to 8.7% in PLHIV ≥ 65 years of age in earlier studies, similar to our findings [26–29].
ATV and DRV are both PIs and therefore they have, in general, the same PDDIs. However, they differ in regard to some frequently used drug classes e.g. beta blockers and PPIs. Since EFV is a NNRTI it has another drug interaction profile. DRV accounted for the majority of the red flag interactions found in our study, consistent with earlier findings of PLHIV in all ages [27]. Other studies have reported a higher probability of an orange or red flag PDDI in individuals treated with a PI (not restricted to those ≥ 65) [9, 28]. This is consistent with our finding that the DRV arm in the study group had a higher mean of PDDIs than the EFV arm. We did not find a significant difference between the ATV and EFV arms, however this may be due to the small sample size. The majority of the red flag interactions found were related to the concomitant use of alfuzosin, for treatment of benign prostatic hyperplasia, and/or clopidogrel, for treatment of vascular disease, which both are conditions that have a higher prevalence in older ages.
Our study has several limitations. Since we analysed steady-state plasma drug levels and not trough levels of ART, it is difficult to compare our results with trough levels in other studies. Thus, we were unable to evaluate potentially toxic plasma drug levels of ART drugs or levels below the proposed minimal effective concentrations. Patients were sampled from 6 to 36 hours after their last dose of ART. This was adjusted for in the statistical model, but there remains a risk that, because of this approach, we were not able to detect minor differences in drug levels between the study and control arms for ATV and EFV. Also, there was a difference in back-bone between PLHIV on DRV compared to ATV and EFV that may have affected self-reported side-effects. Concomitant medications may also have influenced the self-reported side-effects, though the participants were asked specifically to report side-effects related to ART. The PDDIs were calculated only for ATV, DRV or EFV regimens and therefore differences in back-bone likely did not affect the results. In addition, interactions between NRTIs and other medications are uncommon. The participants in the study were included consecutively at four sites in Sweden and it is possible that this introduced a selection bias, favoring PLHIV with frequent visits.
Conclusion
Higher steady-state plasma levels of DRV and ATV (but not EFV) were found in PLHIV who were 65 years of age or older, as compared to controls who were 49 years of age and younger. Our findings are important for the management of elderly PLHIV and raise the question of whether regular monitoring of plasma levels and dose adjustment of DRV and other PIs is warranted in the elderly.
Supporting information
S1 Table Aids defining diagnoses.
(DOCX)
Click here for additional data file.
S2 Table Data set.
(XLSX)
Click here for additional data file.
S1 File Questionnaire/case report form.
(DOCX)
Click here for additional data file.
We would like to thank all the participants in the study, study nurses and technical staff. | 1, QD (200/245 MG 1X1) | DrugDosageText | CC BY | 33539440 | 19,781,519 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Septic shock'. | Real-World Clinical Outcomes and Prognostic Factors for Patients with Advanced Angiosarcoma who Received Systemic Treatment.
OBJECTIVE
Angiosarcoma is a highly aggressive mesenchymal tumor. Although systemic chemotherapy is often considered for the inoperable or metastatic angiosarcoma, the outcome of such treatment is unsatisfactory and poorly delineated.
We reviewed electronic medical records of 75 patients with angiosarcoma who were treated with systemic chemotherapy for inoperable or metastatic disease. Patients were classified as having liver involvement if they had either primary or metastatic hepatic lesions.
RESULTS
Among the patients evaluated, 51 patients (68%) were male and 24 patients (32%) had primary cutaneous angiosarcoma. Liver involvement was present in 28 patients (37.3%). A total of 59 patients received first-line weekly paclitaxel (wPac) and showed an objective response rate (ORR) of 23.7% (n=14), a median progression-free survival (mPFS) of 4.0 months (95% confidence interval [CI], 3.0 to 6.1), and a median overall survival (mOS) of 10.2 months (95% CI, 7.0 to 14.6). Among patients without liver involvement, patients receiving wPac (n=35) had significantly prolonged mPFS (5.8 months vs. 3.2 months, respectively; p=0.014) with a tendency for prolonged mOS (13.8 months vs. 11.6 months, respectively; p=0.13) than those receiving other regimens (n=12). A total of 24 patients received second- or later-line pazopanib monotherapy and showed an ORR of 16.7% (n=4), a mPFS of 2.4 months (95% CI, 1.8 to 4.3) and a mOS of 5.4 months (95% CI, 3.5 to not available).
CONCLUSIONS
Treatment with first-line wPac and later-line pazopanib seems to provide survival benefit, especially for patients with advanced angiosarcoma without liver involvement.
pmcIntroduction
Angiosarcoma is a rare and highly aggressive sarcoma subtype and occurs throughout the body at any age [1]. The prognosis of angiosarcoma is generally poor even in localized disease with 5-year overall survival (OS) up to only 60% with a median survival of around 3–10 months for metastatic disease [1–4].
In case of localized disease, standard treatment is surgical resection, sometimes combined with preoperative and postoperative radiation. However, negative surgical margins could be often difficult to achieve especially in the head and neck or in the presence of multifocal disease. These inoperable or metastatic diseases are treated with systemic chemotherapy [5]. Doxorubicin and weekly paclitaxel (wPac) are currently regarded as a preferred option for first- or second-line therapy with a median OS of approximately 8 to 10 months [6,7]. However due to the rarity of the disease, there are only limited efficacy data for such therapies.
Meanwhile, several components involved in angiogenesis have been investigated as potential targets to treat angiosarcoma [8]. These include vascular endothelial growth factor (VEGF) and multiple VEGF receptors, which are the key regulators of angiogenesis that are overexpressed in angiosarcoma [9,10]. Bevacizumab is a recombinant human antibody against VEGF and has been tested in clinical trials both as a monotherapy and in combination with other drugs [11]. Multi-target tyrosine kinase inhibitors (sunitinib, sorafenib, and pazopanib) have also been applied, but the efficacy of such treatment was inconclusive as the numbers of angiosarcoma patients involved were low [1,12].
As consensus treatment strategy for angiosarcoma is not well established, it is necessary to better understand the clinicopathological features of angiosarcoma and correlate them with the treatment response and clinical outcomes. In this retrospective study, we reviewed the patients with advanced angiosarcoma who undergone systemic chemotherapy and evaluated clinical outcomes and their prognostic significance. In addition, we tried to delineate the efficacy of systemic therapies currently available for advanced angiosarcoma.
Materials and Methods
1. Patients
We retrospectively reviewed data of 96 adult patients (age > 18 years) with metastatic or advanced angiosarcoma from 2005 to 2018 at Seoul National University Hospital and Seoul National University Bundang Hospital. The diagnosis of metastatic or advanced angiosarcoma was based on the histopathological analysis according to 2013 World Health Organization Classification of Tumors of Soft Tissue and Bone [13]. Exclusion criteria were: patients who had mixed histology with other soft tissue sarcoma (STS) subtypes (five patients); those who did not receive systemic chemotherapy in locally advanced or metastatic setting (six patients); and those who had no available treatment administration record (10 patients). Patient records were accessed for age, sex, date of last follow-up or deaths, disease site (primary or metastatic), Eastern Cooperative Oncology Group performance status scale (ECOG PS), prior therapy (surgery, radiation, or chemotherapy), and treatment outcome. Tumor grade was evaluated using Federation Nationale des Centres de Lutte le Cancer (FNCLCC) grade.
2. Data collection
We assessed tumor responses according to Response Evaluation Criteria in Solid Tumors guideline, ver. 1.1 [14]. Objective response rate (ORR) was defined as percentage of patients who experienced partial response (PR) or complete response (CR). The grade of adverse events was assigned according to the National Cancer Institute Common Terminology Criteria for Adverse Events ver. 5.0 [15].
3. Data analysis
The endpoint for the prognostic factor analysis was OS, which was defined as the time from date of treatment initiation to date of death or last contact. The impact of covariates on OS was estimated using Cox models (hazard ratio [HR], 95% confidence interval [CI]). The factors analyzed for univariate analysis were age, sex, ECOG PS, primary sites, presence of liver involvement, lung involvement, bone involvement, lymph node involvement, FNCLCC grade, previous operation intent, pre-chemotherapy laboratory results of albumin and bilirubin. For multivariate analysis, the factors that were significant in univariate analyses were used. Progression-free survival (PFS) was defined as time to progression or death from the initiation of chemotherapy. Kaplan-Meier estimates were used for both OS and PFS analysis. Fisher’s exact test was used to compare ORR. Logistic regression analysis was performed to evaluate the association of factors with response. Continues variables are shown as medians and ranges and categorical variables as percentages. Wilcoxon rank-sum test was used to compare continuous variables. All p-values were two-sided, with p < 0.05 indicating statistical significance. R ver. 3.6.1 software (R Development Core Team, https://www.r-project.org/) was used for statistical analyses.
Results
1. Patient demographics
A total of 75 patients who received palliative chemotherapy for advanced angiosarcoma were enrolled. Among these, 24 had primary cutaneous angiosarcoma. The most common primary sites were the scalp (n=19, 25.3%), liver (n=17, 22.7%), and bone (n=7, 9.3%). Two patients had radiation-induced angiosarcoma of the cervix and nasopharynx, respectively, both of whom were involved in previous radiotherapy more than 10 years ago. Other demographic features are summarized in Table 1.
At the time of the first-line palliative chemotherapy, liver involvement either by primary mass or metastatic mass was present in 28 patients (37.3%). Lung, lymph node, and bone involvement were present in 24 (32%), 24 (32%), and 26 (34.7%) of patients, respectively.
2. Clinical outcomes and prognostic factors
The median OS of the 75 patients from the first-line palliative chemotherapy initiation was 10.2 months (95% CI, 8.6 to 13.8) (Fig. 1A). OS did not differ between patients with primary cutaneous and non-cutaneous angiosarcoma (8.7 months; 95% CI, 6.6 to 21.6 vs. 10.3 months; 95% CI, 7.2 to 14.5; p=0.73) (S1A Fig.). None of primary scalp, primary pulmonary, primary cardiac angiosarcoma, and FNCLCC grade were significantly associated with OS in our analysis (Table 2, S1B Fig.).
Patients with primary hepatic angiosarcoma tended to have poorer OS than those without liver involvement (4.0 months; 95% CI, 2.7 to not available [NA] vs. 12.5 months; 95% CI, 9.6 to 16.7; p=0.05). Patients with liver metastasis that originated from other organs tended to have poorer OS than those without liver involvement (8.7 months; 95% CI, 6.4 to NA for patients with liver metastasis; p=0.06) (Fig. 1B). When patients with primary and metastatic sites of liver were combined as having liver involvement, presence of liver involvement was significantly associated with poor OS (7.0 months; 95% CI, 4.0 to 10.3 vs. 12.5 months; 95% CI, 9.6 to 16.7; p=0.02) (Fig. 1C).
In the univariate Cox proportional hazard regression analysis, ECOG PS and presence of liver involvement were significantly associated with poor OS (HR, 2.54; 95% CI, 1.26 to 5.10; p=0.009 for ECOG PS; HR, 1.96; 95% CI, 1.12 to 3.43; p=0.018 for presence of liver involvement) (Table 2). In addition, higher pre-chemotherapy bilirubin and lower pre-chemotherapy albumin were associated with poor OS (HR, 1.37; 95% CI, 1.14 to 1.63; p < 0.001 for bilirubin; and HR, 0.58; 95% CI, 0.40 to 0.87; p=0.007 for albumin, respectively) (Table 2). In the multivariate Cox proportional hazard regression analysis, presence of liver involvement and pre-chemotherapy bilirubin level were associated with poor OS (HR, 2.27; 95% CI, 1.13 to 4.57; p=0.022 for presence of liver involvement; and HR, 1.27; 95% CI, 1.04 to 1.53; p=0.017 for bilirubin, respectively) (Table 2).
3. First-line weekly paclitaxel
A total of 59 patients received first-line wPac. ORR was 23.7% (n=14) including one CR and 13 PR patients (S2 Table 1). ORR in patients with liver involvement was 12.5% (3 of 24 patients) and ORR in patients without liver involvement was 31.4% (11 of 24 patients, p=0.12 compared to patients with liver involvements). Median PFS was 4.0 months (95% CI, 3.0 to 6.1) and OS was 10.2 months (95% CI, 7.0 to 14.6). Response to wPac was associated with prolonged PFS and OS (PFS: 7.0 months; 95% CI, 6.1 to NA for responders vs. 3.0 months; 95% CI, 2.5 to 4.0 for non-responders; p=0.018, Fig. 2A; OS: 21.6 months; 95% CI, 13.8 to NA for responders vs. 7.0 months; 95% CI, 4.0 to 12.1 for non-responders; p=0.012) (Fig. 2B). An example case of a patient who showed favorable response to wPac is shown in S3 Fig. The patient had recurred scalp angiosarcoma with lymph nodes and right parotid gland metastasis which showed complete remission with wPac and PFS of 33.4 months.
ECOG PS, liver involvement, high pre-chemotherapy bilirubin and low pre-chemotherapy albumin level were associated with poor PFS in univariate Cox proportional hazard regression analysis (S4 Table). In multivariate analysis, only pre-chemotherapy albumin level was significantly associated with PFS (HR, 0.56; 95% CI, 0.32 to 0.96; p=0.036). None of the factors were associated with response to first-line wPac in logistic regression analysis (S4 Table). For the adverse events of interest, neuropathy of any grade was observed in 39.0% (n=23) of patients, and most of them (n=19) were tolerable as grade 1. Febrile neutropenia of any grade was observed in 10.2% (n=6) of patients and one patient died of septic shock.
Remaining 16 patients received either one of doxorubicin-based chemotherapy (n=7) or non-doxorubicin-based chemotherapy (n=9) other than wPac as first-line treatment (S5 Table). ORR in these patients was 25% (n=4) including one CR and three PR patients (S2 Table). Compared with these 16 patients, patients who received first-line wPac did not show significant PFS and OS differences (S6 Fig.). However, when patients with liver involvement were excluded, those who received wPac (n=35) showed significantly prolonged PFS (5.8 months; 95% CI, 3.2 to 7.9 vs. 3.15 months; 95% CI, 2.0 to NA, respectively; p=0.014) (Fig. 2C) and tendency to prolonged OS (13.8 months; 95% CI, 10.0 to NA vs. 11.6 months; 95% CI, 9.4 to NA, respectively; p=0.13) (Fig. 2D) compared with patients who received other regimens (n=12).
4. Later-line pazopanib
A total of 24 patients received pazopanib monotherapy for advanced angiosarcoma. Most of these patients received pazopanib as second-line treatment (n=18, 75%), and none of the patients received pazopanib as first-line treatment. ORR was 16.7% (n=4) including four PR patients. The median PFS was 2.4 months (95% CI, 1.8 to 4.3 months) (Fig. 3A) and OS was 5.4 months (95% CI, 3.5 to NA months) (Fig. 3B). Responders showed significantly prolonged PFS compared to non-responders (6.4 months; 95% CI, 3.5 to NA vs. 1.9 months; 95% CI, 1.6 to 3.2, respectively; p=0.02) (Fig. 3A) while OS did not significantly differ between responders and non-responders (9.5 months; 95% CI, 3.5 to NA vs. 5.0 months; 95% CI, 3.5 to NA; p=0.58) (Fig. 3B). The most common side effect was hypertension (n=4, 16.7%), followed by diarrhea (n=3, 12.5%) and hand-foot syndrome (n=2, 8.3%).
Discussion
Here we described the clinical outcomes and prognostic factors of advanced angiosarcoma patients treated with systemic chemotherapy. ECOG PS was a significant prognostic factor in our study, which is consistent with previous literature [4]. Primary hepatic angiosarcoma had been associated with poor prognosis [4], and we found that liver involvement by primary or metastatic mass was associated with poor prognosis. Although previous study involving angiosarcoma patients some of whom received radical surgery described that histologic grade is associated with prognosis [16], we did not find any association of histologic grade with prognosis in patients with advanced stages.
We observed that some of the patients responded to first-line wPac or later-line pazopanib who showed significantly prolonged survival. Although there had been no clinical trials comparing the outcome of palliative systemic chemotherapy versus best supportive care, these results indicate that there are subsets of patients who may benefit from the palliative first-line wPac and second-line pazopanib in terms of survival gain. The same has also been suggested in previous retrospective analysis [17]. We also demonstrated that patients without liver involvement might benefit from wPac compared with other regimens.
Our results on first-line wPac showed seemingly inferior efficacy compared with previous studies. A prospective phase II clinical trial on wPac produced ORR of 45.8%, PFS of 6.6 months, and OS of 19.5 months [18]. Retrospective studies on first-line wPac demonstrated ORR of 45.5%–52%, PFS of 5.6–5.7 months, and OS of 13.1–18.6 months [17,19]. However, these results could be partly due to relatively lower number of patients with poor ECOG PS and liver involvement enrolled in other studies. Indeed, we showed in our study that both of these clinical features were poor prognostic factors. Another previous phase II clinical trial on wPac for angiosarcoma patients showed ORR of 18%, PFS of 4 months and OS of 8 months, which were comparable with our results [7].
Efficacy of second- or later-line of pazopanib in our study showed inferior results compared with the previous study of phase II clinical trial of pazopanib for metastatic STS, which showed PFS of 4.6 months and OS of 12.5 months [12]. However, this result may be attributed to the higher portion of other STSs in the enrolled patients such as leiomyosarcoma and synovial sarcoma, which have generally better prognosis than angiosarcoma [1]. Anti-angiogenesis agents for angiosarcoma generally produced ORR up to 14% and PFS around 3.8–4.7 months, which are similar to our study results [1]. A retrospective study on pazopanib for advanced vascular sarcomas also exhibited comparable ORR of 20%, and PFS and OS of 3 and 9.9 months, respectively, in angiosarcoma [20]. A multicenter phase II prospective trial of pazopanib is ongoing to confirm these results (Clinical trial information: NCT01462630).
Angiosarcoma is an endothelial tumor in which all three subtypes of VEGF receptors can be overexpressed [9,10,21]. In addition, recurrent mutations in PTPRB and PLCG1 genes, which are intimately linked to angiogenesis, have also been identified in angiosarcoma [22]. Therefore, there has been great interest in targeting angiogenesis for angiosarcoma [1]. Both wPac and pazopanib have anti-angiogenesis effects. wPac exerts anti-angiogenesis effects by multiple mechanisms including inhibition of the release of VEGF and angiopoietin-1 by tumor cells [23], while pazopanib directly targets VEGFR1/2/3, platelet-derived growth factor receptor and several other key proteins responsible for angiogenesis [24]. Despite some efficacy of using anti-angiogenesis drugs in angiosarcoma patients, there seems to be more factors involved in resistance to such treatments in angiosarcoma [25].
In this study, we showed that liver involvement, either by primary mass or metastatic mass, was potentially associated with poor treatment outcome. Poor survival of hepatic angiosarcoma has been reported in association with various clinical features such as intraabdominal hemorrhage due to tumor rupture and acute liver failure [26,27]. We showed that poor survival associated with liver involvement may be partly due to poor liver function represented as pre-chemotherapy bilirubin and albumin level, which would have affected drug tolerability and patient’s performance status. However, tendency to poor PFS and ORR in patients with liver involvement implicate additional mechanisms on tumor biology. Such resistance mechanism of liver involvement by tumors has been also implicated in other cancers and other antitumor agents, such as lung cancer to epidermal growth factor receptor tyrosine kinase inhibitors and melanoma to immune checkpoint inhibitors, which exhibited reduced CD8+ T cell density at the invasive tumor margin at pretreatment biopsies [28,29]. In addition, previous study showed that vessel co-option, a process whereby cancer incorporates pre-existing vessels from surrounding tissue, is a clinically relevant mechanism of resistance to anti-angiogenic therapy in liver metastases of colorectal and breast cancers [30]. Whether these mechanisms are also involved in angiosarcoma resistance would require further studies.
The limitations of this study include its retrospective nature; therefore, the results presented should be interpreted carefully. Also, as angiosarcoma can have diverse patterns of the clinical course according to involved sites, the ORR, the PFS, and the OS presented in this study would have been affected by the heterogeneity. However, due to the rarity of this disease, designing a clinical trial for advanced angiosarcoma is difficult, as observed in the previous phase II clinical trial, which enrolled 30 patients with heterogeneous primary sites [7]. In addition, most of the clinical trials involving angiosarcoma include other sarcomas, which results in even more heterogeneous study population with angiosarcoma being a small portion of the entire population. In this study, we collected data from homogeneous and relatively large number of patients in advanced setting who received palliative systemic chemotherapy. Therefore, our results provide clinicians with fair information of real-world treatment outcomes in advanced angiosarcoma patients. Secondly, whether liver function represented as more relevant scoring systems such as Child-Pugh or Model for End-Stage Liver Disease scores is correlated with clinical outcome should be further evaluated. Nevertheless, the baseline serum total bilirubin and albumin levels may provide clinical insights to physicians and researchers in deciding on treatments and designing stratifications for clinical trials. Lastly, our study implicates the liver involvement as a prognostic marker, not a predictive marker. Therefore, the presence or absence of liver involvement should not be used as a factor that could influence the decision on the initiation of chemotherapy.
In conclusion, treatment with first-line chemotherapy with wPac regimen and later-line pazopanib showed comparable efficacy to that reported in previous studies and may provide survival benefit for subset of patients, especially those without liver involvement. Clinicians would need to be aware that patients with presence of liver involvement may have poor prognosis. Further investigations with large number in multicenters or prospective design considering these results are warranted.
We thank Juyoun Kim, a data manager at Seoul National University Hospital, who managed the database. We also thank Seonah Ha, PhD, who assisted in medical writing.
Electronic Supplementary Material
Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).
Fig. 1 Overall survival of advanced angiosarcoma patients treated with systemic chemotherapy and the associated prognostic factors. Kaplan-Meier survival curves represent overall survival of advanced angiosarcoma patients. (A) Overall survival of patients included in the study. (B) Blue line represents patients without any liver involvement; red line represents patients with primary hepatic angiosarcoma; green line represents patients with liver metastasis. (C) Blue line represents patients without any liver involvement; red line represents patients with liver involvement. Censored data are marked with vertical segments and numbers at risk are demonstrated on the table at the bottom of each plot.
Fig. 2 Progression-free survival and overall survival of advanced angiosarcoma patients treated with first-line weekly paclitaxel (wT) or other regimens. Kaplan-Meier survival curves represent progression-free survival and overall survival of advanced angiosarcoma patients. (A, B) Survival curves show progression-free survival and overall survival of patients according to response to first-line wT. Blue line represents the progression-free survival of all patients who received first-line wT. Red dashed line represents non-responder and green dashed line represents responder. (C, D) Survival curves show progression-free survival and overall survival of patients without liver involvements according to the chemotherapy regimens. Blue line represents patients who received first-line wT and red line represents patients who received other regimens. Censored data are marked with cross segments and numbers at risk are demonstrated on the table at the bottom of each plot.
Fig. 3 Progression-free survival and overall survival of advanced angiosarcoma patients treated with second- or later-line pazopanib. Kaplan-Meier survival curves represent progression-free survival and overall survival of advanced angiosarcoma patients. (A, B) Survival curves show progression-free survival and overall survival of patients according to response to second- or later-line pazopanib. Blue lines represent the progression-free survival and overall survival of all patients who received first-line weekly paclitaxel. Red lines represent non-responder and green lines represent responder. Censored data are marked with cross segments and numbers at risk are demonstrated on the table at the bottom of each plot.
Table 1 Demographic features of patients
No. (%) (n=75)
Age at first chemotherapy, median (range, yr) 63 (30–81)
Sex
Male 51 (68.0)
Female 24 (32.0)
ECOG PS at first chemotherapy
0–1 62 (82.7)
2–3 11 (14.7)
NA 2 (2.7)
Primary site
Scalp 19 (25.3)
Liver 17 (22.7)
Bone 7 (9.3)
Heart 6 (8.0)
Lung 6 (8.0)
Skin of extremities 5 (6.7)
Spleen 4 (5.3)
Othersa) 11 (14.7)
FNCLCC grade
1 5 (6.7)
2 33 (44.0)
3 15 (20.0)
NA 22 (29.3)
Previous operation intent
None 38 (50.1)
Curative 21 (28.0)
Palliative 16 (21.3)
ECOG PS, Eastern Cooperative Oncology Group performance status; FNCLCC, Federation Nationale des Centres de Lutte Contre Le Cancer; NA, not available.
a) Locations of others include the breast (2 cases), large artery, kidney, spinal nerve root, deep soft tissue of thigh, soft tissue of mediastinum, soft tissue of retroperitoneum, pleura, uterine cervix, and nasopharynx.
Table 2 Cox proportional hazard analysis of the prognostic factors of overall survival in patients with advanced angiosarcoma at the time of the first chemotherapy
Univariate analysis Multivariate analysis
HR (95% CI) p-value HR (95% CI) p-value
Age 1.01 (0.99–1.03) 0.438 -
Sex -
Male Reference
Female 1.01 (0.57–1.78) 0.981
ECOG PS
0, 1 Reference Reference
2, 3 2.54 (1.26–5.10) 0.009 1.50 (0.60–3.78) 0.386
Primary cutaneous angiosarcoma -
Yes Reference
No 0.90 (0.51–1.59) 0.726
Primary scalp angiosarcoma -
Yes Reference
No 0.96 (0.53–1.74) 0.883
Primary site: liver -
No Reference
Yes 1.87 (0.94–3.72) 0.073
Primary site: lung -
No Reference
Yes 0.88 (0.35–2.22) 0.787
Primary site: heart -
No Reference
Yes 1.16 (0.46–2.93) 0.761
Presence of liver involvement
No Reference Reference
Yes 1.96 (1.12–3.43) 0.018 2.27 (1.13–4.57) 0.022
Presence of lung involvement -
No Reference
Yes 1.41 (0.80–2.46) 0.233
Presence of bone involvement -
No Reference
Yes 1.01 (0.59–1.75) 0.966
Presence of lymph node involvement -
No Reference
Yes 0.74 (0.41–1.34) 0.314
FNCLCC grade -
1 Reference
2 1.53 (0.53–4.45) 0.432
3 1.03 (0.31–3.44) 0.956
Previous operation intent -
None Reference
Curative 0.60 (0.32–1.14) 0.121
Palliative 0.62 (0.31–1.23) 0.173
Pre-chemotherapy bilirubin 1.37 (1.14–1.63) < 0.001 1.26 (1.04–1.53) 0.017
Pre-chemotherapy albumin 0.58 (0.40–0.87) 0.007 0.82 (0.50–1.34) 0.421
CI, confidence interval; ECOG PS, Eastern Cooperative Oncology Group performance status; FNCLCC, Federation Nationale des Centres de Lutte Contre Le Cancer; HR, hazard ratio; -, not done.
Ethical Statement
All data collection and analyses were conducted after the review and approval of the institutional review board (IRB approval number: H-1807-198-967) and were done in compliance with the Declaration of Helsinki. Informed consent was not required for this study because of its retrospective nature.
Author Contributions
Conceived and designed the analysis: Park C, Kim M.
Collected the data: Park C, Kim M, Kwak Y, Moon KC, Kim SH, Keam B, Kim YJ, Kim TM, Kim DW.
Contributed data or analysis tools: Park C, Kim M, Kwak Y, Moon KC, Kim SH, Keam B, Kim YJ, Kim TM, Kim DW.
Performed the analysis: Park C, Kim M.
Wrote the paper: Park C, Kim M.
Conflict of interest
Conflict of interest relevant to this article was not reported. | PACLITAXEL | DrugsGivenReaction | CC BY-NC | 33540493 | 18,914,526 | 2021-10 |
What was the dosage of drug 'PACLITAXEL'? | Real-World Clinical Outcomes and Prognostic Factors for Patients with Advanced Angiosarcoma who Received Systemic Treatment.
OBJECTIVE
Angiosarcoma is a highly aggressive mesenchymal tumor. Although systemic chemotherapy is often considered for the inoperable or metastatic angiosarcoma, the outcome of such treatment is unsatisfactory and poorly delineated.
We reviewed electronic medical records of 75 patients with angiosarcoma who were treated with systemic chemotherapy for inoperable or metastatic disease. Patients were classified as having liver involvement if they had either primary or metastatic hepatic lesions.
RESULTS
Among the patients evaluated, 51 patients (68%) were male and 24 patients (32%) had primary cutaneous angiosarcoma. Liver involvement was present in 28 patients (37.3%). A total of 59 patients received first-line weekly paclitaxel (wPac) and showed an objective response rate (ORR) of 23.7% (n=14), a median progression-free survival (mPFS) of 4.0 months (95% confidence interval [CI], 3.0 to 6.1), and a median overall survival (mOS) of 10.2 months (95% CI, 7.0 to 14.6). Among patients without liver involvement, patients receiving wPac (n=35) had significantly prolonged mPFS (5.8 months vs. 3.2 months, respectively; p=0.014) with a tendency for prolonged mOS (13.8 months vs. 11.6 months, respectively; p=0.13) than those receiving other regimens (n=12). A total of 24 patients received second- or later-line pazopanib monotherapy and showed an ORR of 16.7% (n=4), a mPFS of 2.4 months (95% CI, 1.8 to 4.3) and a mOS of 5.4 months (95% CI, 3.5 to not available).
CONCLUSIONS
Treatment with first-line wPac and later-line pazopanib seems to provide survival benefit, especially for patients with advanced angiosarcoma without liver involvement.
pmcIntroduction
Angiosarcoma is a rare and highly aggressive sarcoma subtype and occurs throughout the body at any age [1]. The prognosis of angiosarcoma is generally poor even in localized disease with 5-year overall survival (OS) up to only 60% with a median survival of around 3–10 months for metastatic disease [1–4].
In case of localized disease, standard treatment is surgical resection, sometimes combined with preoperative and postoperative radiation. However, negative surgical margins could be often difficult to achieve especially in the head and neck or in the presence of multifocal disease. These inoperable or metastatic diseases are treated with systemic chemotherapy [5]. Doxorubicin and weekly paclitaxel (wPac) are currently regarded as a preferred option for first- or second-line therapy with a median OS of approximately 8 to 10 months [6,7]. However due to the rarity of the disease, there are only limited efficacy data for such therapies.
Meanwhile, several components involved in angiogenesis have been investigated as potential targets to treat angiosarcoma [8]. These include vascular endothelial growth factor (VEGF) and multiple VEGF receptors, which are the key regulators of angiogenesis that are overexpressed in angiosarcoma [9,10]. Bevacizumab is a recombinant human antibody against VEGF and has been tested in clinical trials both as a monotherapy and in combination with other drugs [11]. Multi-target tyrosine kinase inhibitors (sunitinib, sorafenib, and pazopanib) have also been applied, but the efficacy of such treatment was inconclusive as the numbers of angiosarcoma patients involved were low [1,12].
As consensus treatment strategy for angiosarcoma is not well established, it is necessary to better understand the clinicopathological features of angiosarcoma and correlate them with the treatment response and clinical outcomes. In this retrospective study, we reviewed the patients with advanced angiosarcoma who undergone systemic chemotherapy and evaluated clinical outcomes and their prognostic significance. In addition, we tried to delineate the efficacy of systemic therapies currently available for advanced angiosarcoma.
Materials and Methods
1. Patients
We retrospectively reviewed data of 96 adult patients (age > 18 years) with metastatic or advanced angiosarcoma from 2005 to 2018 at Seoul National University Hospital and Seoul National University Bundang Hospital. The diagnosis of metastatic or advanced angiosarcoma was based on the histopathological analysis according to 2013 World Health Organization Classification of Tumors of Soft Tissue and Bone [13]. Exclusion criteria were: patients who had mixed histology with other soft tissue sarcoma (STS) subtypes (five patients); those who did not receive systemic chemotherapy in locally advanced or metastatic setting (six patients); and those who had no available treatment administration record (10 patients). Patient records were accessed for age, sex, date of last follow-up or deaths, disease site (primary or metastatic), Eastern Cooperative Oncology Group performance status scale (ECOG PS), prior therapy (surgery, radiation, or chemotherapy), and treatment outcome. Tumor grade was evaluated using Federation Nationale des Centres de Lutte le Cancer (FNCLCC) grade.
2. Data collection
We assessed tumor responses according to Response Evaluation Criteria in Solid Tumors guideline, ver. 1.1 [14]. Objective response rate (ORR) was defined as percentage of patients who experienced partial response (PR) or complete response (CR). The grade of adverse events was assigned according to the National Cancer Institute Common Terminology Criteria for Adverse Events ver. 5.0 [15].
3. Data analysis
The endpoint for the prognostic factor analysis was OS, which was defined as the time from date of treatment initiation to date of death or last contact. The impact of covariates on OS was estimated using Cox models (hazard ratio [HR], 95% confidence interval [CI]). The factors analyzed for univariate analysis were age, sex, ECOG PS, primary sites, presence of liver involvement, lung involvement, bone involvement, lymph node involvement, FNCLCC grade, previous operation intent, pre-chemotherapy laboratory results of albumin and bilirubin. For multivariate analysis, the factors that were significant in univariate analyses were used. Progression-free survival (PFS) was defined as time to progression or death from the initiation of chemotherapy. Kaplan-Meier estimates were used for both OS and PFS analysis. Fisher’s exact test was used to compare ORR. Logistic regression analysis was performed to evaluate the association of factors with response. Continues variables are shown as medians and ranges and categorical variables as percentages. Wilcoxon rank-sum test was used to compare continuous variables. All p-values were two-sided, with p < 0.05 indicating statistical significance. R ver. 3.6.1 software (R Development Core Team, https://www.r-project.org/) was used for statistical analyses.
Results
1. Patient demographics
A total of 75 patients who received palliative chemotherapy for advanced angiosarcoma were enrolled. Among these, 24 had primary cutaneous angiosarcoma. The most common primary sites were the scalp (n=19, 25.3%), liver (n=17, 22.7%), and bone (n=7, 9.3%). Two patients had radiation-induced angiosarcoma of the cervix and nasopharynx, respectively, both of whom were involved in previous radiotherapy more than 10 years ago. Other demographic features are summarized in Table 1.
At the time of the first-line palliative chemotherapy, liver involvement either by primary mass or metastatic mass was present in 28 patients (37.3%). Lung, lymph node, and bone involvement were present in 24 (32%), 24 (32%), and 26 (34.7%) of patients, respectively.
2. Clinical outcomes and prognostic factors
The median OS of the 75 patients from the first-line palliative chemotherapy initiation was 10.2 months (95% CI, 8.6 to 13.8) (Fig. 1A). OS did not differ between patients with primary cutaneous and non-cutaneous angiosarcoma (8.7 months; 95% CI, 6.6 to 21.6 vs. 10.3 months; 95% CI, 7.2 to 14.5; p=0.73) (S1A Fig.). None of primary scalp, primary pulmonary, primary cardiac angiosarcoma, and FNCLCC grade were significantly associated with OS in our analysis (Table 2, S1B Fig.).
Patients with primary hepatic angiosarcoma tended to have poorer OS than those without liver involvement (4.0 months; 95% CI, 2.7 to not available [NA] vs. 12.5 months; 95% CI, 9.6 to 16.7; p=0.05). Patients with liver metastasis that originated from other organs tended to have poorer OS than those without liver involvement (8.7 months; 95% CI, 6.4 to NA for patients with liver metastasis; p=0.06) (Fig. 1B). When patients with primary and metastatic sites of liver were combined as having liver involvement, presence of liver involvement was significantly associated with poor OS (7.0 months; 95% CI, 4.0 to 10.3 vs. 12.5 months; 95% CI, 9.6 to 16.7; p=0.02) (Fig. 1C).
In the univariate Cox proportional hazard regression analysis, ECOG PS and presence of liver involvement were significantly associated with poor OS (HR, 2.54; 95% CI, 1.26 to 5.10; p=0.009 for ECOG PS; HR, 1.96; 95% CI, 1.12 to 3.43; p=0.018 for presence of liver involvement) (Table 2). In addition, higher pre-chemotherapy bilirubin and lower pre-chemotherapy albumin were associated with poor OS (HR, 1.37; 95% CI, 1.14 to 1.63; p < 0.001 for bilirubin; and HR, 0.58; 95% CI, 0.40 to 0.87; p=0.007 for albumin, respectively) (Table 2). In the multivariate Cox proportional hazard regression analysis, presence of liver involvement and pre-chemotherapy bilirubin level were associated with poor OS (HR, 2.27; 95% CI, 1.13 to 4.57; p=0.022 for presence of liver involvement; and HR, 1.27; 95% CI, 1.04 to 1.53; p=0.017 for bilirubin, respectively) (Table 2).
3. First-line weekly paclitaxel
A total of 59 patients received first-line wPac. ORR was 23.7% (n=14) including one CR and 13 PR patients (S2 Table 1). ORR in patients with liver involvement was 12.5% (3 of 24 patients) and ORR in patients without liver involvement was 31.4% (11 of 24 patients, p=0.12 compared to patients with liver involvements). Median PFS was 4.0 months (95% CI, 3.0 to 6.1) and OS was 10.2 months (95% CI, 7.0 to 14.6). Response to wPac was associated with prolonged PFS and OS (PFS: 7.0 months; 95% CI, 6.1 to NA for responders vs. 3.0 months; 95% CI, 2.5 to 4.0 for non-responders; p=0.018, Fig. 2A; OS: 21.6 months; 95% CI, 13.8 to NA for responders vs. 7.0 months; 95% CI, 4.0 to 12.1 for non-responders; p=0.012) (Fig. 2B). An example case of a patient who showed favorable response to wPac is shown in S3 Fig. The patient had recurred scalp angiosarcoma with lymph nodes and right parotid gland metastasis which showed complete remission with wPac and PFS of 33.4 months.
ECOG PS, liver involvement, high pre-chemotherapy bilirubin and low pre-chemotherapy albumin level were associated with poor PFS in univariate Cox proportional hazard regression analysis (S4 Table). In multivariate analysis, only pre-chemotherapy albumin level was significantly associated with PFS (HR, 0.56; 95% CI, 0.32 to 0.96; p=0.036). None of the factors were associated with response to first-line wPac in logistic regression analysis (S4 Table). For the adverse events of interest, neuropathy of any grade was observed in 39.0% (n=23) of patients, and most of them (n=19) were tolerable as grade 1. Febrile neutropenia of any grade was observed in 10.2% (n=6) of patients and one patient died of septic shock.
Remaining 16 patients received either one of doxorubicin-based chemotherapy (n=7) or non-doxorubicin-based chemotherapy (n=9) other than wPac as first-line treatment (S5 Table). ORR in these patients was 25% (n=4) including one CR and three PR patients (S2 Table). Compared with these 16 patients, patients who received first-line wPac did not show significant PFS and OS differences (S6 Fig.). However, when patients with liver involvement were excluded, those who received wPac (n=35) showed significantly prolonged PFS (5.8 months; 95% CI, 3.2 to 7.9 vs. 3.15 months; 95% CI, 2.0 to NA, respectively; p=0.014) (Fig. 2C) and tendency to prolonged OS (13.8 months; 95% CI, 10.0 to NA vs. 11.6 months; 95% CI, 9.4 to NA, respectively; p=0.13) (Fig. 2D) compared with patients who received other regimens (n=12).
4. Later-line pazopanib
A total of 24 patients received pazopanib monotherapy for advanced angiosarcoma. Most of these patients received pazopanib as second-line treatment (n=18, 75%), and none of the patients received pazopanib as first-line treatment. ORR was 16.7% (n=4) including four PR patients. The median PFS was 2.4 months (95% CI, 1.8 to 4.3 months) (Fig. 3A) and OS was 5.4 months (95% CI, 3.5 to NA months) (Fig. 3B). Responders showed significantly prolonged PFS compared to non-responders (6.4 months; 95% CI, 3.5 to NA vs. 1.9 months; 95% CI, 1.6 to 3.2, respectively; p=0.02) (Fig. 3A) while OS did not significantly differ between responders and non-responders (9.5 months; 95% CI, 3.5 to NA vs. 5.0 months; 95% CI, 3.5 to NA; p=0.58) (Fig. 3B). The most common side effect was hypertension (n=4, 16.7%), followed by diarrhea (n=3, 12.5%) and hand-foot syndrome (n=2, 8.3%).
Discussion
Here we described the clinical outcomes and prognostic factors of advanced angiosarcoma patients treated with systemic chemotherapy. ECOG PS was a significant prognostic factor in our study, which is consistent with previous literature [4]. Primary hepatic angiosarcoma had been associated with poor prognosis [4], and we found that liver involvement by primary or metastatic mass was associated with poor prognosis. Although previous study involving angiosarcoma patients some of whom received radical surgery described that histologic grade is associated with prognosis [16], we did not find any association of histologic grade with prognosis in patients with advanced stages.
We observed that some of the patients responded to first-line wPac or later-line pazopanib who showed significantly prolonged survival. Although there had been no clinical trials comparing the outcome of palliative systemic chemotherapy versus best supportive care, these results indicate that there are subsets of patients who may benefit from the palliative first-line wPac and second-line pazopanib in terms of survival gain. The same has also been suggested in previous retrospective analysis [17]. We also demonstrated that patients without liver involvement might benefit from wPac compared with other regimens.
Our results on first-line wPac showed seemingly inferior efficacy compared with previous studies. A prospective phase II clinical trial on wPac produced ORR of 45.8%, PFS of 6.6 months, and OS of 19.5 months [18]. Retrospective studies on first-line wPac demonstrated ORR of 45.5%–52%, PFS of 5.6–5.7 months, and OS of 13.1–18.6 months [17,19]. However, these results could be partly due to relatively lower number of patients with poor ECOG PS and liver involvement enrolled in other studies. Indeed, we showed in our study that both of these clinical features were poor prognostic factors. Another previous phase II clinical trial on wPac for angiosarcoma patients showed ORR of 18%, PFS of 4 months and OS of 8 months, which were comparable with our results [7].
Efficacy of second- or later-line of pazopanib in our study showed inferior results compared with the previous study of phase II clinical trial of pazopanib for metastatic STS, which showed PFS of 4.6 months and OS of 12.5 months [12]. However, this result may be attributed to the higher portion of other STSs in the enrolled patients such as leiomyosarcoma and synovial sarcoma, which have generally better prognosis than angiosarcoma [1]. Anti-angiogenesis agents for angiosarcoma generally produced ORR up to 14% and PFS around 3.8–4.7 months, which are similar to our study results [1]. A retrospective study on pazopanib for advanced vascular sarcomas also exhibited comparable ORR of 20%, and PFS and OS of 3 and 9.9 months, respectively, in angiosarcoma [20]. A multicenter phase II prospective trial of pazopanib is ongoing to confirm these results (Clinical trial information: NCT01462630).
Angiosarcoma is an endothelial tumor in which all three subtypes of VEGF receptors can be overexpressed [9,10,21]. In addition, recurrent mutations in PTPRB and PLCG1 genes, which are intimately linked to angiogenesis, have also been identified in angiosarcoma [22]. Therefore, there has been great interest in targeting angiogenesis for angiosarcoma [1]. Both wPac and pazopanib have anti-angiogenesis effects. wPac exerts anti-angiogenesis effects by multiple mechanisms including inhibition of the release of VEGF and angiopoietin-1 by tumor cells [23], while pazopanib directly targets VEGFR1/2/3, platelet-derived growth factor receptor and several other key proteins responsible for angiogenesis [24]. Despite some efficacy of using anti-angiogenesis drugs in angiosarcoma patients, there seems to be more factors involved in resistance to such treatments in angiosarcoma [25].
In this study, we showed that liver involvement, either by primary mass or metastatic mass, was potentially associated with poor treatment outcome. Poor survival of hepatic angiosarcoma has been reported in association with various clinical features such as intraabdominal hemorrhage due to tumor rupture and acute liver failure [26,27]. We showed that poor survival associated with liver involvement may be partly due to poor liver function represented as pre-chemotherapy bilirubin and albumin level, which would have affected drug tolerability and patient’s performance status. However, tendency to poor PFS and ORR in patients with liver involvement implicate additional mechanisms on tumor biology. Such resistance mechanism of liver involvement by tumors has been also implicated in other cancers and other antitumor agents, such as lung cancer to epidermal growth factor receptor tyrosine kinase inhibitors and melanoma to immune checkpoint inhibitors, which exhibited reduced CD8+ T cell density at the invasive tumor margin at pretreatment biopsies [28,29]. In addition, previous study showed that vessel co-option, a process whereby cancer incorporates pre-existing vessels from surrounding tissue, is a clinically relevant mechanism of resistance to anti-angiogenic therapy in liver metastases of colorectal and breast cancers [30]. Whether these mechanisms are also involved in angiosarcoma resistance would require further studies.
The limitations of this study include its retrospective nature; therefore, the results presented should be interpreted carefully. Also, as angiosarcoma can have diverse patterns of the clinical course according to involved sites, the ORR, the PFS, and the OS presented in this study would have been affected by the heterogeneity. However, due to the rarity of this disease, designing a clinical trial for advanced angiosarcoma is difficult, as observed in the previous phase II clinical trial, which enrolled 30 patients with heterogeneous primary sites [7]. In addition, most of the clinical trials involving angiosarcoma include other sarcomas, which results in even more heterogeneous study population with angiosarcoma being a small portion of the entire population. In this study, we collected data from homogeneous and relatively large number of patients in advanced setting who received palliative systemic chemotherapy. Therefore, our results provide clinicians with fair information of real-world treatment outcomes in advanced angiosarcoma patients. Secondly, whether liver function represented as more relevant scoring systems such as Child-Pugh or Model for End-Stage Liver Disease scores is correlated with clinical outcome should be further evaluated. Nevertheless, the baseline serum total bilirubin and albumin levels may provide clinical insights to physicians and researchers in deciding on treatments and designing stratifications for clinical trials. Lastly, our study implicates the liver involvement as a prognostic marker, not a predictive marker. Therefore, the presence or absence of liver involvement should not be used as a factor that could influence the decision on the initiation of chemotherapy.
In conclusion, treatment with first-line chemotherapy with wPac regimen and later-line pazopanib showed comparable efficacy to that reported in previous studies and may provide survival benefit for subset of patients, especially those without liver involvement. Clinicians would need to be aware that patients with presence of liver involvement may have poor prognosis. Further investigations with large number in multicenters or prospective design considering these results are warranted.
We thank Juyoun Kim, a data manager at Seoul National University Hospital, who managed the database. We also thank Seonah Ha, PhD, who assisted in medical writing.
Electronic Supplementary Material
Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).
Fig. 1 Overall survival of advanced angiosarcoma patients treated with systemic chemotherapy and the associated prognostic factors. Kaplan-Meier survival curves represent overall survival of advanced angiosarcoma patients. (A) Overall survival of patients included in the study. (B) Blue line represents patients without any liver involvement; red line represents patients with primary hepatic angiosarcoma; green line represents patients with liver metastasis. (C) Blue line represents patients without any liver involvement; red line represents patients with liver involvement. Censored data are marked with vertical segments and numbers at risk are demonstrated on the table at the bottom of each plot.
Fig. 2 Progression-free survival and overall survival of advanced angiosarcoma patients treated with first-line weekly paclitaxel (wT) or other regimens. Kaplan-Meier survival curves represent progression-free survival and overall survival of advanced angiosarcoma patients. (A, B) Survival curves show progression-free survival and overall survival of patients according to response to first-line wT. Blue line represents the progression-free survival of all patients who received first-line wT. Red dashed line represents non-responder and green dashed line represents responder. (C, D) Survival curves show progression-free survival and overall survival of patients without liver involvements according to the chemotherapy regimens. Blue line represents patients who received first-line wT and red line represents patients who received other regimens. Censored data are marked with cross segments and numbers at risk are demonstrated on the table at the bottom of each plot.
Fig. 3 Progression-free survival and overall survival of advanced angiosarcoma patients treated with second- or later-line pazopanib. Kaplan-Meier survival curves represent progression-free survival and overall survival of advanced angiosarcoma patients. (A, B) Survival curves show progression-free survival and overall survival of patients according to response to second- or later-line pazopanib. Blue lines represent the progression-free survival and overall survival of all patients who received first-line weekly paclitaxel. Red lines represent non-responder and green lines represent responder. Censored data are marked with cross segments and numbers at risk are demonstrated on the table at the bottom of each plot.
Table 1 Demographic features of patients
No. (%) (n=75)
Age at first chemotherapy, median (range, yr) 63 (30–81)
Sex
Male 51 (68.0)
Female 24 (32.0)
ECOG PS at first chemotherapy
0–1 62 (82.7)
2–3 11 (14.7)
NA 2 (2.7)
Primary site
Scalp 19 (25.3)
Liver 17 (22.7)
Bone 7 (9.3)
Heart 6 (8.0)
Lung 6 (8.0)
Skin of extremities 5 (6.7)
Spleen 4 (5.3)
Othersa) 11 (14.7)
FNCLCC grade
1 5 (6.7)
2 33 (44.0)
3 15 (20.0)
NA 22 (29.3)
Previous operation intent
None 38 (50.1)
Curative 21 (28.0)
Palliative 16 (21.3)
ECOG PS, Eastern Cooperative Oncology Group performance status; FNCLCC, Federation Nationale des Centres de Lutte Contre Le Cancer; NA, not available.
a) Locations of others include the breast (2 cases), large artery, kidney, spinal nerve root, deep soft tissue of thigh, soft tissue of mediastinum, soft tissue of retroperitoneum, pleura, uterine cervix, and nasopharynx.
Table 2 Cox proportional hazard analysis of the prognostic factors of overall survival in patients with advanced angiosarcoma at the time of the first chemotherapy
Univariate analysis Multivariate analysis
HR (95% CI) p-value HR (95% CI) p-value
Age 1.01 (0.99–1.03) 0.438 -
Sex -
Male Reference
Female 1.01 (0.57–1.78) 0.981
ECOG PS
0, 1 Reference Reference
2, 3 2.54 (1.26–5.10) 0.009 1.50 (0.60–3.78) 0.386
Primary cutaneous angiosarcoma -
Yes Reference
No 0.90 (0.51–1.59) 0.726
Primary scalp angiosarcoma -
Yes Reference
No 0.96 (0.53–1.74) 0.883
Primary site: liver -
No Reference
Yes 1.87 (0.94–3.72) 0.073
Primary site: lung -
No Reference
Yes 0.88 (0.35–2.22) 0.787
Primary site: heart -
No Reference
Yes 1.16 (0.46–2.93) 0.761
Presence of liver involvement
No Reference Reference
Yes 1.96 (1.12–3.43) 0.018 2.27 (1.13–4.57) 0.022
Presence of lung involvement -
No Reference
Yes 1.41 (0.80–2.46) 0.233
Presence of bone involvement -
No Reference
Yes 1.01 (0.59–1.75) 0.966
Presence of lymph node involvement -
No Reference
Yes 0.74 (0.41–1.34) 0.314
FNCLCC grade -
1 Reference
2 1.53 (0.53–4.45) 0.432
3 1.03 (0.31–3.44) 0.956
Previous operation intent -
None Reference
Curative 0.60 (0.32–1.14) 0.121
Palliative 0.62 (0.31–1.23) 0.173
Pre-chemotherapy bilirubin 1.37 (1.14–1.63) < 0.001 1.26 (1.04–1.53) 0.017
Pre-chemotherapy albumin 0.58 (0.40–0.87) 0.007 0.82 (0.50–1.34) 0.421
CI, confidence interval; ECOG PS, Eastern Cooperative Oncology Group performance status; FNCLCC, Federation Nationale des Centres de Lutte Contre Le Cancer; HR, hazard ratio; -, not done.
Ethical Statement
All data collection and analyses were conducted after the review and approval of the institutional review board (IRB approval number: H-1807-198-967) and were done in compliance with the Declaration of Helsinki. Informed consent was not required for this study because of its retrospective nature.
Author Contributions
Conceived and designed the analysis: Park C, Kim M.
Collected the data: Park C, Kim M, Kwak Y, Moon KC, Kim SH, Keam B, Kim YJ, Kim TM, Kim DW.
Contributed data or analysis tools: Park C, Kim M, Kwak Y, Moon KC, Kim SH, Keam B, Kim YJ, Kim TM, Kim DW.
Performed the analysis: Park C, Kim M.
Wrote the paper: Park C, Kim M.
Conflict of interest
Conflict of interest relevant to this article was not reported. | WEEKLY | DrugDosageText | CC BY-NC | 33540493 | 18,914,526 | 2021-10 |
What was the outcome of reaction 'Septic shock'? | Real-World Clinical Outcomes and Prognostic Factors for Patients with Advanced Angiosarcoma who Received Systemic Treatment.
OBJECTIVE
Angiosarcoma is a highly aggressive mesenchymal tumor. Although systemic chemotherapy is often considered for the inoperable or metastatic angiosarcoma, the outcome of such treatment is unsatisfactory and poorly delineated.
We reviewed electronic medical records of 75 patients with angiosarcoma who were treated with systemic chemotherapy for inoperable or metastatic disease. Patients were classified as having liver involvement if they had either primary or metastatic hepatic lesions.
RESULTS
Among the patients evaluated, 51 patients (68%) were male and 24 patients (32%) had primary cutaneous angiosarcoma. Liver involvement was present in 28 patients (37.3%). A total of 59 patients received first-line weekly paclitaxel (wPac) and showed an objective response rate (ORR) of 23.7% (n=14), a median progression-free survival (mPFS) of 4.0 months (95% confidence interval [CI], 3.0 to 6.1), and a median overall survival (mOS) of 10.2 months (95% CI, 7.0 to 14.6). Among patients without liver involvement, patients receiving wPac (n=35) had significantly prolonged mPFS (5.8 months vs. 3.2 months, respectively; p=0.014) with a tendency for prolonged mOS (13.8 months vs. 11.6 months, respectively; p=0.13) than those receiving other regimens (n=12). A total of 24 patients received second- or later-line pazopanib monotherapy and showed an ORR of 16.7% (n=4), a mPFS of 2.4 months (95% CI, 1.8 to 4.3) and a mOS of 5.4 months (95% CI, 3.5 to not available).
CONCLUSIONS
Treatment with first-line wPac and later-line pazopanib seems to provide survival benefit, especially for patients with advanced angiosarcoma without liver involvement.
pmcIntroduction
Angiosarcoma is a rare and highly aggressive sarcoma subtype and occurs throughout the body at any age [1]. The prognosis of angiosarcoma is generally poor even in localized disease with 5-year overall survival (OS) up to only 60% with a median survival of around 3–10 months for metastatic disease [1–4].
In case of localized disease, standard treatment is surgical resection, sometimes combined with preoperative and postoperative radiation. However, negative surgical margins could be often difficult to achieve especially in the head and neck or in the presence of multifocal disease. These inoperable or metastatic diseases are treated with systemic chemotherapy [5]. Doxorubicin and weekly paclitaxel (wPac) are currently regarded as a preferred option for first- or second-line therapy with a median OS of approximately 8 to 10 months [6,7]. However due to the rarity of the disease, there are only limited efficacy data for such therapies.
Meanwhile, several components involved in angiogenesis have been investigated as potential targets to treat angiosarcoma [8]. These include vascular endothelial growth factor (VEGF) and multiple VEGF receptors, which are the key regulators of angiogenesis that are overexpressed in angiosarcoma [9,10]. Bevacizumab is a recombinant human antibody against VEGF and has been tested in clinical trials both as a monotherapy and in combination with other drugs [11]. Multi-target tyrosine kinase inhibitors (sunitinib, sorafenib, and pazopanib) have also been applied, but the efficacy of such treatment was inconclusive as the numbers of angiosarcoma patients involved were low [1,12].
As consensus treatment strategy for angiosarcoma is not well established, it is necessary to better understand the clinicopathological features of angiosarcoma and correlate them with the treatment response and clinical outcomes. In this retrospective study, we reviewed the patients with advanced angiosarcoma who undergone systemic chemotherapy and evaluated clinical outcomes and their prognostic significance. In addition, we tried to delineate the efficacy of systemic therapies currently available for advanced angiosarcoma.
Materials and Methods
1. Patients
We retrospectively reviewed data of 96 adult patients (age > 18 years) with metastatic or advanced angiosarcoma from 2005 to 2018 at Seoul National University Hospital and Seoul National University Bundang Hospital. The diagnosis of metastatic or advanced angiosarcoma was based on the histopathological analysis according to 2013 World Health Organization Classification of Tumors of Soft Tissue and Bone [13]. Exclusion criteria were: patients who had mixed histology with other soft tissue sarcoma (STS) subtypes (five patients); those who did not receive systemic chemotherapy in locally advanced or metastatic setting (six patients); and those who had no available treatment administration record (10 patients). Patient records were accessed for age, sex, date of last follow-up or deaths, disease site (primary or metastatic), Eastern Cooperative Oncology Group performance status scale (ECOG PS), prior therapy (surgery, radiation, or chemotherapy), and treatment outcome. Tumor grade was evaluated using Federation Nationale des Centres de Lutte le Cancer (FNCLCC) grade.
2. Data collection
We assessed tumor responses according to Response Evaluation Criteria in Solid Tumors guideline, ver. 1.1 [14]. Objective response rate (ORR) was defined as percentage of patients who experienced partial response (PR) or complete response (CR). The grade of adverse events was assigned according to the National Cancer Institute Common Terminology Criteria for Adverse Events ver. 5.0 [15].
3. Data analysis
The endpoint for the prognostic factor analysis was OS, which was defined as the time from date of treatment initiation to date of death or last contact. The impact of covariates on OS was estimated using Cox models (hazard ratio [HR], 95% confidence interval [CI]). The factors analyzed for univariate analysis were age, sex, ECOG PS, primary sites, presence of liver involvement, lung involvement, bone involvement, lymph node involvement, FNCLCC grade, previous operation intent, pre-chemotherapy laboratory results of albumin and bilirubin. For multivariate analysis, the factors that were significant in univariate analyses were used. Progression-free survival (PFS) was defined as time to progression or death from the initiation of chemotherapy. Kaplan-Meier estimates were used for both OS and PFS analysis. Fisher’s exact test was used to compare ORR. Logistic regression analysis was performed to evaluate the association of factors with response. Continues variables are shown as medians and ranges and categorical variables as percentages. Wilcoxon rank-sum test was used to compare continuous variables. All p-values were two-sided, with p < 0.05 indicating statistical significance. R ver. 3.6.1 software (R Development Core Team, https://www.r-project.org/) was used for statistical analyses.
Results
1. Patient demographics
A total of 75 patients who received palliative chemotherapy for advanced angiosarcoma were enrolled. Among these, 24 had primary cutaneous angiosarcoma. The most common primary sites were the scalp (n=19, 25.3%), liver (n=17, 22.7%), and bone (n=7, 9.3%). Two patients had radiation-induced angiosarcoma of the cervix and nasopharynx, respectively, both of whom were involved in previous radiotherapy more than 10 years ago. Other demographic features are summarized in Table 1.
At the time of the first-line palliative chemotherapy, liver involvement either by primary mass or metastatic mass was present in 28 patients (37.3%). Lung, lymph node, and bone involvement were present in 24 (32%), 24 (32%), and 26 (34.7%) of patients, respectively.
2. Clinical outcomes and prognostic factors
The median OS of the 75 patients from the first-line palliative chemotherapy initiation was 10.2 months (95% CI, 8.6 to 13.8) (Fig. 1A). OS did not differ between patients with primary cutaneous and non-cutaneous angiosarcoma (8.7 months; 95% CI, 6.6 to 21.6 vs. 10.3 months; 95% CI, 7.2 to 14.5; p=0.73) (S1A Fig.). None of primary scalp, primary pulmonary, primary cardiac angiosarcoma, and FNCLCC grade were significantly associated with OS in our analysis (Table 2, S1B Fig.).
Patients with primary hepatic angiosarcoma tended to have poorer OS than those without liver involvement (4.0 months; 95% CI, 2.7 to not available [NA] vs. 12.5 months; 95% CI, 9.6 to 16.7; p=0.05). Patients with liver metastasis that originated from other organs tended to have poorer OS than those without liver involvement (8.7 months; 95% CI, 6.4 to NA for patients with liver metastasis; p=0.06) (Fig. 1B). When patients with primary and metastatic sites of liver were combined as having liver involvement, presence of liver involvement was significantly associated with poor OS (7.0 months; 95% CI, 4.0 to 10.3 vs. 12.5 months; 95% CI, 9.6 to 16.7; p=0.02) (Fig. 1C).
In the univariate Cox proportional hazard regression analysis, ECOG PS and presence of liver involvement were significantly associated with poor OS (HR, 2.54; 95% CI, 1.26 to 5.10; p=0.009 for ECOG PS; HR, 1.96; 95% CI, 1.12 to 3.43; p=0.018 for presence of liver involvement) (Table 2). In addition, higher pre-chemotherapy bilirubin and lower pre-chemotherapy albumin were associated with poor OS (HR, 1.37; 95% CI, 1.14 to 1.63; p < 0.001 for bilirubin; and HR, 0.58; 95% CI, 0.40 to 0.87; p=0.007 for albumin, respectively) (Table 2). In the multivariate Cox proportional hazard regression analysis, presence of liver involvement and pre-chemotherapy bilirubin level were associated with poor OS (HR, 2.27; 95% CI, 1.13 to 4.57; p=0.022 for presence of liver involvement; and HR, 1.27; 95% CI, 1.04 to 1.53; p=0.017 for bilirubin, respectively) (Table 2).
3. First-line weekly paclitaxel
A total of 59 patients received first-line wPac. ORR was 23.7% (n=14) including one CR and 13 PR patients (S2 Table 1). ORR in patients with liver involvement was 12.5% (3 of 24 patients) and ORR in patients without liver involvement was 31.4% (11 of 24 patients, p=0.12 compared to patients with liver involvements). Median PFS was 4.0 months (95% CI, 3.0 to 6.1) and OS was 10.2 months (95% CI, 7.0 to 14.6). Response to wPac was associated with prolonged PFS and OS (PFS: 7.0 months; 95% CI, 6.1 to NA for responders vs. 3.0 months; 95% CI, 2.5 to 4.0 for non-responders; p=0.018, Fig. 2A; OS: 21.6 months; 95% CI, 13.8 to NA for responders vs. 7.0 months; 95% CI, 4.0 to 12.1 for non-responders; p=0.012) (Fig. 2B). An example case of a patient who showed favorable response to wPac is shown in S3 Fig. The patient had recurred scalp angiosarcoma with lymph nodes and right parotid gland metastasis which showed complete remission with wPac and PFS of 33.4 months.
ECOG PS, liver involvement, high pre-chemotherapy bilirubin and low pre-chemotherapy albumin level were associated with poor PFS in univariate Cox proportional hazard regression analysis (S4 Table). In multivariate analysis, only pre-chemotherapy albumin level was significantly associated with PFS (HR, 0.56; 95% CI, 0.32 to 0.96; p=0.036). None of the factors were associated with response to first-line wPac in logistic regression analysis (S4 Table). For the adverse events of interest, neuropathy of any grade was observed in 39.0% (n=23) of patients, and most of them (n=19) were tolerable as grade 1. Febrile neutropenia of any grade was observed in 10.2% (n=6) of patients and one patient died of septic shock.
Remaining 16 patients received either one of doxorubicin-based chemotherapy (n=7) or non-doxorubicin-based chemotherapy (n=9) other than wPac as first-line treatment (S5 Table). ORR in these patients was 25% (n=4) including one CR and three PR patients (S2 Table). Compared with these 16 patients, patients who received first-line wPac did not show significant PFS and OS differences (S6 Fig.). However, when patients with liver involvement were excluded, those who received wPac (n=35) showed significantly prolonged PFS (5.8 months; 95% CI, 3.2 to 7.9 vs. 3.15 months; 95% CI, 2.0 to NA, respectively; p=0.014) (Fig. 2C) and tendency to prolonged OS (13.8 months; 95% CI, 10.0 to NA vs. 11.6 months; 95% CI, 9.4 to NA, respectively; p=0.13) (Fig. 2D) compared with patients who received other regimens (n=12).
4. Later-line pazopanib
A total of 24 patients received pazopanib monotherapy for advanced angiosarcoma. Most of these patients received pazopanib as second-line treatment (n=18, 75%), and none of the patients received pazopanib as first-line treatment. ORR was 16.7% (n=4) including four PR patients. The median PFS was 2.4 months (95% CI, 1.8 to 4.3 months) (Fig. 3A) and OS was 5.4 months (95% CI, 3.5 to NA months) (Fig. 3B). Responders showed significantly prolonged PFS compared to non-responders (6.4 months; 95% CI, 3.5 to NA vs. 1.9 months; 95% CI, 1.6 to 3.2, respectively; p=0.02) (Fig. 3A) while OS did not significantly differ between responders and non-responders (9.5 months; 95% CI, 3.5 to NA vs. 5.0 months; 95% CI, 3.5 to NA; p=0.58) (Fig. 3B). The most common side effect was hypertension (n=4, 16.7%), followed by diarrhea (n=3, 12.5%) and hand-foot syndrome (n=2, 8.3%).
Discussion
Here we described the clinical outcomes and prognostic factors of advanced angiosarcoma patients treated with systemic chemotherapy. ECOG PS was a significant prognostic factor in our study, which is consistent with previous literature [4]. Primary hepatic angiosarcoma had been associated with poor prognosis [4], and we found that liver involvement by primary or metastatic mass was associated with poor prognosis. Although previous study involving angiosarcoma patients some of whom received radical surgery described that histologic grade is associated with prognosis [16], we did not find any association of histologic grade with prognosis in patients with advanced stages.
We observed that some of the patients responded to first-line wPac or later-line pazopanib who showed significantly prolonged survival. Although there had been no clinical trials comparing the outcome of palliative systemic chemotherapy versus best supportive care, these results indicate that there are subsets of patients who may benefit from the palliative first-line wPac and second-line pazopanib in terms of survival gain. The same has also been suggested in previous retrospective analysis [17]. We also demonstrated that patients without liver involvement might benefit from wPac compared with other regimens.
Our results on first-line wPac showed seemingly inferior efficacy compared with previous studies. A prospective phase II clinical trial on wPac produced ORR of 45.8%, PFS of 6.6 months, and OS of 19.5 months [18]. Retrospective studies on first-line wPac demonstrated ORR of 45.5%–52%, PFS of 5.6–5.7 months, and OS of 13.1–18.6 months [17,19]. However, these results could be partly due to relatively lower number of patients with poor ECOG PS and liver involvement enrolled in other studies. Indeed, we showed in our study that both of these clinical features were poor prognostic factors. Another previous phase II clinical trial on wPac for angiosarcoma patients showed ORR of 18%, PFS of 4 months and OS of 8 months, which were comparable with our results [7].
Efficacy of second- or later-line of pazopanib in our study showed inferior results compared with the previous study of phase II clinical trial of pazopanib for metastatic STS, which showed PFS of 4.6 months and OS of 12.5 months [12]. However, this result may be attributed to the higher portion of other STSs in the enrolled patients such as leiomyosarcoma and synovial sarcoma, which have generally better prognosis than angiosarcoma [1]. Anti-angiogenesis agents for angiosarcoma generally produced ORR up to 14% and PFS around 3.8–4.7 months, which are similar to our study results [1]. A retrospective study on pazopanib for advanced vascular sarcomas also exhibited comparable ORR of 20%, and PFS and OS of 3 and 9.9 months, respectively, in angiosarcoma [20]. A multicenter phase II prospective trial of pazopanib is ongoing to confirm these results (Clinical trial information: NCT01462630).
Angiosarcoma is an endothelial tumor in which all three subtypes of VEGF receptors can be overexpressed [9,10,21]. In addition, recurrent mutations in PTPRB and PLCG1 genes, which are intimately linked to angiogenesis, have also been identified in angiosarcoma [22]. Therefore, there has been great interest in targeting angiogenesis for angiosarcoma [1]. Both wPac and pazopanib have anti-angiogenesis effects. wPac exerts anti-angiogenesis effects by multiple mechanisms including inhibition of the release of VEGF and angiopoietin-1 by tumor cells [23], while pazopanib directly targets VEGFR1/2/3, platelet-derived growth factor receptor and several other key proteins responsible for angiogenesis [24]. Despite some efficacy of using anti-angiogenesis drugs in angiosarcoma patients, there seems to be more factors involved in resistance to such treatments in angiosarcoma [25].
In this study, we showed that liver involvement, either by primary mass or metastatic mass, was potentially associated with poor treatment outcome. Poor survival of hepatic angiosarcoma has been reported in association with various clinical features such as intraabdominal hemorrhage due to tumor rupture and acute liver failure [26,27]. We showed that poor survival associated with liver involvement may be partly due to poor liver function represented as pre-chemotherapy bilirubin and albumin level, which would have affected drug tolerability and patient’s performance status. However, tendency to poor PFS and ORR in patients with liver involvement implicate additional mechanisms on tumor biology. Such resistance mechanism of liver involvement by tumors has been also implicated in other cancers and other antitumor agents, such as lung cancer to epidermal growth factor receptor tyrosine kinase inhibitors and melanoma to immune checkpoint inhibitors, which exhibited reduced CD8+ T cell density at the invasive tumor margin at pretreatment biopsies [28,29]. In addition, previous study showed that vessel co-option, a process whereby cancer incorporates pre-existing vessels from surrounding tissue, is a clinically relevant mechanism of resistance to anti-angiogenic therapy in liver metastases of colorectal and breast cancers [30]. Whether these mechanisms are also involved in angiosarcoma resistance would require further studies.
The limitations of this study include its retrospective nature; therefore, the results presented should be interpreted carefully. Also, as angiosarcoma can have diverse patterns of the clinical course according to involved sites, the ORR, the PFS, and the OS presented in this study would have been affected by the heterogeneity. However, due to the rarity of this disease, designing a clinical trial for advanced angiosarcoma is difficult, as observed in the previous phase II clinical trial, which enrolled 30 patients with heterogeneous primary sites [7]. In addition, most of the clinical trials involving angiosarcoma include other sarcomas, which results in even more heterogeneous study population with angiosarcoma being a small portion of the entire population. In this study, we collected data from homogeneous and relatively large number of patients in advanced setting who received palliative systemic chemotherapy. Therefore, our results provide clinicians with fair information of real-world treatment outcomes in advanced angiosarcoma patients. Secondly, whether liver function represented as more relevant scoring systems such as Child-Pugh or Model for End-Stage Liver Disease scores is correlated with clinical outcome should be further evaluated. Nevertheless, the baseline serum total bilirubin and albumin levels may provide clinical insights to physicians and researchers in deciding on treatments and designing stratifications for clinical trials. Lastly, our study implicates the liver involvement as a prognostic marker, not a predictive marker. Therefore, the presence or absence of liver involvement should not be used as a factor that could influence the decision on the initiation of chemotherapy.
In conclusion, treatment with first-line chemotherapy with wPac regimen and later-line pazopanib showed comparable efficacy to that reported in previous studies and may provide survival benefit for subset of patients, especially those without liver involvement. Clinicians would need to be aware that patients with presence of liver involvement may have poor prognosis. Further investigations with large number in multicenters or prospective design considering these results are warranted.
We thank Juyoun Kim, a data manager at Seoul National University Hospital, who managed the database. We also thank Seonah Ha, PhD, who assisted in medical writing.
Electronic Supplementary Material
Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).
Fig. 1 Overall survival of advanced angiosarcoma patients treated with systemic chemotherapy and the associated prognostic factors. Kaplan-Meier survival curves represent overall survival of advanced angiosarcoma patients. (A) Overall survival of patients included in the study. (B) Blue line represents patients without any liver involvement; red line represents patients with primary hepatic angiosarcoma; green line represents patients with liver metastasis. (C) Blue line represents patients without any liver involvement; red line represents patients with liver involvement. Censored data are marked with vertical segments and numbers at risk are demonstrated on the table at the bottom of each plot.
Fig. 2 Progression-free survival and overall survival of advanced angiosarcoma patients treated with first-line weekly paclitaxel (wT) or other regimens. Kaplan-Meier survival curves represent progression-free survival and overall survival of advanced angiosarcoma patients. (A, B) Survival curves show progression-free survival and overall survival of patients according to response to first-line wT. Blue line represents the progression-free survival of all patients who received first-line wT. Red dashed line represents non-responder and green dashed line represents responder. (C, D) Survival curves show progression-free survival and overall survival of patients without liver involvements according to the chemotherapy regimens. Blue line represents patients who received first-line wT and red line represents patients who received other regimens. Censored data are marked with cross segments and numbers at risk are demonstrated on the table at the bottom of each plot.
Fig. 3 Progression-free survival and overall survival of advanced angiosarcoma patients treated with second- or later-line pazopanib. Kaplan-Meier survival curves represent progression-free survival and overall survival of advanced angiosarcoma patients. (A, B) Survival curves show progression-free survival and overall survival of patients according to response to second- or later-line pazopanib. Blue lines represent the progression-free survival and overall survival of all patients who received first-line weekly paclitaxel. Red lines represent non-responder and green lines represent responder. Censored data are marked with cross segments and numbers at risk are demonstrated on the table at the bottom of each plot.
Table 1 Demographic features of patients
No. (%) (n=75)
Age at first chemotherapy, median (range, yr) 63 (30–81)
Sex
Male 51 (68.0)
Female 24 (32.0)
ECOG PS at first chemotherapy
0–1 62 (82.7)
2–3 11 (14.7)
NA 2 (2.7)
Primary site
Scalp 19 (25.3)
Liver 17 (22.7)
Bone 7 (9.3)
Heart 6 (8.0)
Lung 6 (8.0)
Skin of extremities 5 (6.7)
Spleen 4 (5.3)
Othersa) 11 (14.7)
FNCLCC grade
1 5 (6.7)
2 33 (44.0)
3 15 (20.0)
NA 22 (29.3)
Previous operation intent
None 38 (50.1)
Curative 21 (28.0)
Palliative 16 (21.3)
ECOG PS, Eastern Cooperative Oncology Group performance status; FNCLCC, Federation Nationale des Centres de Lutte Contre Le Cancer; NA, not available.
a) Locations of others include the breast (2 cases), large artery, kidney, spinal nerve root, deep soft tissue of thigh, soft tissue of mediastinum, soft tissue of retroperitoneum, pleura, uterine cervix, and nasopharynx.
Table 2 Cox proportional hazard analysis of the prognostic factors of overall survival in patients with advanced angiosarcoma at the time of the first chemotherapy
Univariate analysis Multivariate analysis
HR (95% CI) p-value HR (95% CI) p-value
Age 1.01 (0.99–1.03) 0.438 -
Sex -
Male Reference
Female 1.01 (0.57–1.78) 0.981
ECOG PS
0, 1 Reference Reference
2, 3 2.54 (1.26–5.10) 0.009 1.50 (0.60–3.78) 0.386
Primary cutaneous angiosarcoma -
Yes Reference
No 0.90 (0.51–1.59) 0.726
Primary scalp angiosarcoma -
Yes Reference
No 0.96 (0.53–1.74) 0.883
Primary site: liver -
No Reference
Yes 1.87 (0.94–3.72) 0.073
Primary site: lung -
No Reference
Yes 0.88 (0.35–2.22) 0.787
Primary site: heart -
No Reference
Yes 1.16 (0.46–2.93) 0.761
Presence of liver involvement
No Reference Reference
Yes 1.96 (1.12–3.43) 0.018 2.27 (1.13–4.57) 0.022
Presence of lung involvement -
No Reference
Yes 1.41 (0.80–2.46) 0.233
Presence of bone involvement -
No Reference
Yes 1.01 (0.59–1.75) 0.966
Presence of lymph node involvement -
No Reference
Yes 0.74 (0.41–1.34) 0.314
FNCLCC grade -
1 Reference
2 1.53 (0.53–4.45) 0.432
3 1.03 (0.31–3.44) 0.956
Previous operation intent -
None Reference
Curative 0.60 (0.32–1.14) 0.121
Palliative 0.62 (0.31–1.23) 0.173
Pre-chemotherapy bilirubin 1.37 (1.14–1.63) < 0.001 1.26 (1.04–1.53) 0.017
Pre-chemotherapy albumin 0.58 (0.40–0.87) 0.007 0.82 (0.50–1.34) 0.421
CI, confidence interval; ECOG PS, Eastern Cooperative Oncology Group performance status; FNCLCC, Federation Nationale des Centres de Lutte Contre Le Cancer; HR, hazard ratio; -, not done.
Ethical Statement
All data collection and analyses were conducted after the review and approval of the institutional review board (IRB approval number: H-1807-198-967) and were done in compliance with the Declaration of Helsinki. Informed consent was not required for this study because of its retrospective nature.
Author Contributions
Conceived and designed the analysis: Park C, Kim M.
Collected the data: Park C, Kim M, Kwak Y, Moon KC, Kim SH, Keam B, Kim YJ, Kim TM, Kim DW.
Contributed data or analysis tools: Park C, Kim M, Kwak Y, Moon KC, Kim SH, Keam B, Kim YJ, Kim TM, Kim DW.
Performed the analysis: Park C, Kim M.
Wrote the paper: Park C, Kim M.
Conflict of interest
Conflict of interest relevant to this article was not reported. | Fatal | ReactionOutcome | CC BY-NC | 33540493 | 18,914,526 | 2021-10 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug interaction'. | Genetic Polymorphism Effect on Warfarin-Rifampin Interaction: A Case Report and Review of Literature.
Warfarin-rifampin interaction has been reported since the 1970s. Due to rifampin's strong induction of CYP2C9, most cases could not attain the target international normalized ratio (INR) despite warfarin dose escalation. Genetic polymorphisms determine up to 50% of warfarin dose variability. A 38-year-old woman was started on warfarin and rifampin for cerebral venous sinus thrombosis and pulmonary tuberculosis. Over six weeks, the daily warfarin dose was increased from 3 to 10 mg to attain three consecutive in-clinic therapeutic INRs. She completed three complications-free months of warfarin treatment with time in therapeutic range (TTR) of 46%. We performed retrospective genetic testing to determine the patient's CYP2C9, CYP4F2, and VKORC1 genotypes and whether they had affected the interaction outcome. The analysis revealed that the subject carries CYP2C9*3*3 and VKORC1-1639 (GA) mutations, classifying her as a slow metabolizer and, hence, highly warfarin-sensitive. This was reflected on how the case responded to a relatively lower dose than previously reported cases that did not achieve the target on warfarin daily doses up to 35 mg. This is the first report addressing the genotype effect on this interaction. Patients with genetic variants requiring low warfarin doses are more likely to respond at a feasible dose while on rifampin. Future studies to evaluate warfarin-rifampin-gene interaction are warranted.
Introduction
Warfarin, a vitamin K antagonist, remains the preferred oral anticoagulation for atrial fibrillation with prosthetic cardiac valves or rheumatic heart disease with more than mild mitral stenosis, and venous thromboembolism (VTE) at unusual sites.1,2 It is a mixture of two racemic isomers, R and S-warfarin. Both impair the vitamin k-dependent proteins production via inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).3 Cytochrome P450 2C9 (CYP2C9) extensively metabolizes S-warfarin, the stereoisomer of predominant potency, to the inactive 7-hydroxywarfarin.3
VKORC1 and CYP2C9 genetic polymorphisms, with other genetic variants, determine up to 50% of warfarin dose variance.3,4 The most studied and common variant alleles of CYP2C9 are CYP2C9*2 (rs1799853) and *3 (rs1057910), which result from missense mutations associated with diminished catalytic activity, poor warfarin metabolism, and decreased dose requirements.5
VKORC1–1639G>A (rs9923231) genotype variants (GA and AA) contribute majorly to sensitizing warfarin.5 On the other hand, CYP4F2*3, a nonsynonymous variant of the gene coding for the primary liver vitamin K oxidase, CYP4F2, has been associated in some studies with a modest increase of warfarin dose requirements (8–11%).3 These mutations were incorporated, with other clinical factors, into dosing algorithms which were shown to provide better warfarin dose prediction.3 Gage and International Warfarin Pharmacogenetics Consortium (IWPC), are among the most widely studied algorithms and are contained in the website (www.warfarindosing.org), which calculates the initial dose with the ability to adjust for CYP2C9*5, *6, CYP4F2, and GGCX genotypes.3 Genetic-based dosing of warfarin upon initiation was shown in some studies to improve target international normalized ratio (INR) attainment and time in therapeutic range (TTR) during the initial month.6 This was mainly mediated by CYP2C9 and VKORC1 polymorphisms which significantly impact the maintenance dose requirement.3,4,7 The Food and Drug Administration (FDA) has also approved warfarin label modifications with dosing guidance based on the CYP2C9 and VKORC1 genotypes.8 Apart from genetics, numerous warfarin drug interactions have been reported requiring dose adjustments and frequent INR monitoring to avoid bleeding or anticoagulation failure.9
Antituberculous management includes rifampin, isoniazid, ethambutol, and pyrazinamide. While ethambutol and pyrazinamide are neither cytochrome P450s inhibitors nor reported to affect warfarin, isoniazid is a week inhibitor of CYP3A4, which is not FDA classified as a clinical index inhibitor.10 While it has been reported to increase warfarin’s hypoprothrombinemic effect in two cases,11,12 the interaction magnitude is considered minimal, with no action recommended.13
Rifampin, a life-saving antimicrobial for tuberculosis, endocarditis, and meningitis,14 is a clinically significant inducer of CYP2B6, CYP2C19, CYP2C8, CYP2C9, and CYP3A4, as well as P glycoprotein.10 It induces CYP2C9 transcription by binding to its main De-novo synthesis regulatory nuclear receptor, pregnane X receptor (PXR).5,15 The binding increases the CYP2C9 mRNA expression by up to six-folds.15 That leads to a higher amount of the enzyme, and extensive metabolism of the substrate/victim drug.15 While the onset of induction can be few days with rifampin,16 the time required to reach maximal enzyme abundance and new steady state is more than two weeks based on the CYP turnover and degradation half-life.16–19 FDA classifies rifampin as a moderate inducer of CYP2C9, defined as a decrease in the substrates area under the concentration-time curve (AUC) by 50% to less than 80%.10 That was based on two healthy-volunteers studies with probe substrates, S-warfarin and tolbutamide.20,21 However, the first study’s duration was only four days,20 and 12–15 days in the latter,21 which, yet, showed high variability of CYP2C9 activity (1–7.4-fold).22 These short durations may imply that rifampin is rather a strong CYP2C9 inducer as AUC ratio were measured before reaching maximal induction.16–19
Rifampin almost eliminates warfarin’s therapeutic effect, which required extensive dose escalation in all cases and is typically associated with the inability to maintain therapeutic range. The interaction has been described since the 1970s.20,23,24 Till the late 1980s, multiple reports showed a significant increase in warfarin dose requirements with rifampin.25–27 After the INR test was universally adopted,28 several reports demonstrated that most patients who required the anti-infective along with anticoagulation were unable to maintain target INR.14,29–36
This report aims to describe a case who received warfarin and rifampin concomitantly and the interaction outcome, and to perform genetic testing to determine the patient’s CYP2C9, VKORC1, CALU, and CYP4F2 genotypes and whether they could explain the response to warfarin dose escalation.
Case Description
A 38-year-old Ethiopian woman with a weight of 60 kg, a height of 150 cm, and a history of diabetes and immune thrombocytopenic purpura (ITP) on metformin and chronic eltrombopag presented to the emergency of Hamad General Hospital in Qatar on January 13, 2020, with dizziness, severe diffuse headache, photophobia, and multiple vomiting for three days. Intracranial computed tomography (CT) venogram showed cerebral venous sinus thrombosis (CVST). Eltrombopag was stopped. Since the Glasgow Coma Score (GCS) dropped to 11, the patient was admitted to the medical ICU with sedation, analgesia, and close neurologic observation. Because of low platelet count (PC), 32X109/L, she was started on 0.5 gm/kg intravenous immunoglobulin (IVIG) plus steroids for three days to raise the PC above 50X109/L in order to initiate anticoagulation. Two days later, PC reached 75X109/L, and heparin continuous IV infusion was initiated with platelets transfusion as the patient was neurologically deteriorating, and repeated CT showed extension of thrombosis with intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). On January 19, although PC was maintained above 200X109/L, heparin was suspended due to a sharp drop of hemoglobin to 5 gm/dL with no identified source of bleeding. The patient was transfused immediately. The next day hemoglobin increased to 8 then maintained at 9–10 gm/dL. Heparin was resumed on January 23. Hematology planned to start rituximab for ITP, yet the Quantiferone test for tuberculosis (TB) was positive. CT chest on January 26 revealed consolidation patches in the right upper and middle lung lobes suggesting active TB. Since bronchoscopy was not feasible due to the high bleeding risk, the pulmonary, MICU, and infectious diseases teams decided to start empiric anti-TB medications based on radiology and follow response by imaging. The rituximab plan was aborted.
On January 29, the patient was started on daily rifampin 600 mg, isoniazid 300 mg, ethambutol 1100 mg, and pyrazinamide 1600 mg. She was transferred to the medicine ward after sedation withdrawal. On January 31, anticoagulation was shifted to daily oral warfarin 5 mg with twice-daily enoxaparin 60 mg as bridging. Three days later, warfarin was held for one day then resumed with dose reduction to 3 mg due to a sharp INR increase to 3.7. The next day, INR dropped to 1.0, then increased gradually after three days to 1.7, 1.9, and reached 2.0 on February 9. Enoxaparin was stopped, and the patient was discharged on warfarin 3 mg, anti-TB medications, pyridoxine 40 mg, metformin 500 mg twice daily, and metoprolol 50 mg twice daily. Following in anticoagulation clinic, on February 12, after 14 days of rifampin, INR was 2.6; therefore, the same warfarin dose continued. Although decreased to 1.7 on February 16, the warfarin dose remained. A week later, INR dropped to 1.3, so warfarin was escalated to 4 mg/day, and enoxaparin resumed. Over the next three weeks, the dose was gradually escalated up to 10 mg, after four days of which, INR reached 2.3 on March 15. Enoxaparin was stopped, and the patient was maintained on daily warfarin 10 mg. The anti-TB medications were switched to (Rifampin 600 mg/Isoniazid 300 mg) on March 24. INR was maintained in the next two clinic visits at 3 and 2.9 on March 23 and April 6, respectively. On May 4, the INR was 1.0 in the last anticoagulation clinic visit. That could not be explained by non-compliance as per the patient interview. The planned three-month duration of anticoagulation ended, so warfarin was stopped. Rifampin/isoniazid continued till July 14, 2020. The patient was interviewed on each visit and excluded any adverse effects. She had normal follow up laboratory values throughout the treatment. Details of warfarin daily dose and INR are shown in [Figure 1].Figure 1 This graph represents the daily warfarin dose, rifampin days, and INR overtime. The bottom x-axis represents dates. The top x-axis represents rifampin days. The left y-axis represents the daily warfarin dose in milligrams and is shown by the vertical bars. The right Y-axis represents the INR and is shown by the black diamond points. The therapeutic range is indicated between the two dotted lines (2.0–3.0).
Methods
The patient was approached by one of the study investigators and explained the reasons and expectations of the research. The patient confirmed her understanding, and agreement to provide saliva sample for genetic testing as well as to have the case published by signing an Institutional Review Board (IRB) approved informed consent form. She was asked to provide a saliva sample using Oragene•DNA (OG-500) self-collection kit (DNA genotek, USA). Hereafter, the kit was kept in a water bath at 50 C° overnight for DNA extraction. The prepIT®•L2P standard protocol for the purification of DNA was used for DNA extraction.37 The purified DNA’s quality and quantity were evaluated by Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific). Finally, the sample was genotyped for detecting the following genetic variants: CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), CYP4F2*3 (rs2108622),VKORC1 (rs9923231), VKORC1 Asp36Tyr (rs61742245), and CALU (rs339097). This was performed using the QuantStudio™ 5 Real-Time Polymerase Chain Reaction (PCR) system for Human Identification, 96-well, 0.2 mL, desktop manufactured by Applied BiosystemsTM.
Results
The genotyping revealed that the patient is a carrier of CYP2C9*3*3 homozygous, VKORC1-1639 (GA) heterozygous, and CYP4F2 (CC) wild-type homozygous. Based on this genetic profile, the subject is considered a slow metabolizer which indicates high warfarin sensitivity. On the other hand, both VKORC1 Asp36Tyr (CT) and CALU (AG) genotyping were heterozygous indicating partial warfarin resistance.
As it is shown in Figure 1, the sudden rise in INR with a moderate standard warfarin dose of 5 mg is very well explained by the CYP2C9 loss of function genetic variant carried by this patient (CYP2C9*3/*3). A few weeks later and with the interacting effect of rifampin reaching its peak, the daily warfarin dose requirement for the patient increased reaching 10 mg.
Discussion
In this case report, we observed warfarin–rifampin drug interaction in a patient with CVST and pulmonary TB. The interaction management required warfarin dose-escalation, frequent INR monitoring, and low molecular weight heparin (LMWH) bridging over six weeks. On a daily warfarin dose of 10 mg, our patient attained three consecutive therapeutic INR levels in three clinic visits. That is considered a “stable warfarin dose,” as defined in most studies,38 despite the one subtherapeutic INR in the last treatment day. The TTR, calculated by the Rosendaal method,39 was 46.2%. The three-month warfarin anticoagulation treatment, combined with rifampin, was completed with difficulty, yet no complications.
Since the World Health Organization (WHO) adopted INR in the 1980s,28 more than nineteen cases of warfarin–rifampin interaction have been reported.14,29–36 Despite extensive warfarin dose escalation over a prolonged time, the majority could not attain target INR while on the combination.14,29–32,34–36 Cases are summarized in [Table 1]. Excessive warfarin exposure and hemorrhage after rifampin discontinuation have been reported, implying the importance of close monitoring and careful dose de-escalation after rifampin stoppage.33,40 Due to the lack of laboratory monitorable parameters like INR, labels of direct oral anticoagulants (DOACs), that are substrates of CYP3A4 and/or P glycoprotein, recommend avoiding concomitant use with rifampin to avoid unmanageable therapeutic failures.41Table 1 Summary of Studies of Warfarin–Rifampicin Interaction with INR Monitoring
Study Patient’s Data Sequence and Duration During Concomitant Use After Rifampin Stopped
Case No. Age Sex Warfarin Indication Rifampin Start in Relation to Warfarin Start (Days) Concomitant Duration (Days) Average Initial Warfarin Dose (mg/day)a,b Average Last Warfarin Dose (mg/day)a,b Warfarin Dose Increase Target INR Target Attained Time to Stable Warfarin Dosec (Days) TTR Stable Warfarin Dosec (mg/day) Time to Stable Warfarin Dosec (Days)
Casner, 199629 1 36 M PE −2 20 7.8b 20b 169% 2.0–3.0 No NA NA 10 10
Lee & Trasher, 200130 1 58 M LVT −120 113 7.5b 25b 233% 2.0–3.0 No NA NA 7.5 35
Kim et al, 200631 1 79 M DVT −11 30 5a 30b 500% 2.0–3.0 No NA NA 6 60
Krajewski, 201032 1 71 M AF +14 45 5.7a 25b 340% 2.0–3.0 No NA NA 5 120
Martins et al, 201333 1 59 F AF +300 203 6.4a 11.4a 78% 2.0–3.0 Yes 104 50% 5.4 60
Maina et al, 201336 1 17 F DVT −7 UNK 10b 27.7a 177.3% 2.0–3.0 Yes 63 52% UNK UNK
2 24 F RHD/LAT −42 UNK 5b 5.8a 16% 2.0–3.0 Yesd 66 67% UNK UNK
3 36 M DVT −44 UNK 12b 11.4b −4.8% 2.0–3.0 No NA 24% UNK UNK
4 64 F DVT −45 UNK 10b 11.5b 15.3% 2.0–3.0 No NA 47% UNK UNK
5 22 F DVT −88 UNK 10b 4.8a −37% 2.0–3.0 Yese 12 54% UNK UNK
6 9 M DVT 0 UNK 5b 5.3b 5.8% 2.0–3.0 No NA 53% UNK UNK
7 49 M DVT −3 UNK 5b 9.5a 89.3% 2.0–3.0 Yes 49 42% UNK UNK
8 30 F PE −35 UNK 5b 27a 440.9% 2.0–3.0 Yes 67 30% UNK UNK
9 29 F DVT −31 UNK 5b 11.8a 135.8% 2.0–3.0 Yes 7 40% UNK UNK
10 41 M Stroke & DVT −46 UNK 6b 6.5a 8.3% 2.0–3.0 Yes 63 66% UNK UNK
Dawson et al, 201634 1 60 M MV replaced +UNK 42 8a 35b 340% 2.5–3.5 No NA NA 8 28
Fahmi et al, 201614 1 34 F MV replaced +19 years 42 7.5a 30b 300% 2.5–3.5 No NA NA 11.4 35
Shibata et al, 201735 1 70 F Stroke +UNK 365 4a 15a 275% 2.0–3.0 Yes UNK UNK 4 30
2 80 F AF +UNK 330 2.5a 10b 300% 2.0–3.0 No NA NA 3 60
Notes: Maina et al did not report rifampin cessation times or confirmed adherence to medications other than warfarin,36 which may have affected response. aTherapeutic INR attained on the dose. bTherapeutic INR not attained on the dose. cDefined as three consecutive therapeutic INR levels on the same warfarin dose (In Maina et al case series, defined as two consecutive therapeutic INRs). dOccasional warfarin overdoses. eMissed warfarin doses.
Abbreviations: AF, atrial fibrillation; DVT, deep venous thrombosis; INR, international normalized ratio; LAT, left arterial thrombosis; LVT, left ventricular thrombus; MV, mitral valve; NA, not applicable; PE, pulmonary embolism; RHD, rheumatic heart disease; TTR, time in therapeutic range; UNK, unknown.
Our case had one supratherapeutic INR after three warfarin doses of 5 mg and five days of rifampin commencement explained by her CYP2C9 poor metabolizer phenotype. She had two consecutive therapeutic INR readings after 12 and 14 days of rifampin on daily warfarin 3 mg. However, INR dropped below therapeutic after 17 days of rifampin, likely due to the latter’s liver enzyme induction. Comparable patterns have been observed in other cases when the commencement sequence was a few days apart or when rifampin was added to chronic warfarin. In 1996, Casner29 reported a patient who had one therapeutic INR after 13 rifampin days, which declined to subtherapeutic until ten days after the rifampin stoppage. The case reported by Kim et al31 had one in-target INR after warfarin 20 mg, which dropped then increased back to target for a week after escalation to 25 mg on rifampin days 25 and 33. Then, the INR declined steadily even after warfarin dose was raised to 30 mg.31 An INR of 4.4 after three days of rifampin in a mechanical valve replacement case, reported by Fahmi et al14, necessitated the holding of warfarin for five days. Then INR dropped to below the desired 7–14 days from the combination. In the case reported by Dawson et al34, it took 17 days for the interaction to become apparent. These reports, aligned with our report, indicate that the time-course to rifampin CYP2C9 induction is more than 14 days. Studies of this rifampin duration or less are inadequate to evaluate the full impact of such interaction. It is vital to monitor INR frequently during the first two weeks of rifampin and shortly after to avoid exposing the patient to subtherapeutic levels with no bridging.
Our patient’s CYP2C9 genotyping revealed that she is a slow metabolizer with a homozygous CYP2C9*3*3, which indicates high warfarin sensitivity and the lowest dose requirements. Additionally, VKORC1 –1639G>A genotyping showed upstream variant (AG) requiring lower warfarin doses.3 The patient reached three consecutive therapeutic INRs on a daily warfarin dose of 10 mg, which is generally considered a high dose. However, compared with most cases that received rifampin with warfarin doses up to 35 mg and no target attainment, 10 mg represents a relatively reasonable dose. The estimated therapeutic warfarin dose for our case, calculated using clinical factors via www.warfarindosing.org, was 5.8 mg/day. However, when the genotyping results were added to the calculation, the estimated therapeutic dose was 2 mg/day. The FDA warfarin label expected maintenance dosing for CYP2C9*3/*3 combined with VKORC1 (AG) is ranged from 0.5 to 2 mg/day.8 That implies that 10 mg is at least a 400% increase from the therapeutic dose without rifampin. Interestingly, most reported cases who reached therapeutic INR during the concomitant warfarin–rifampin use received relatively low warfarin doses around 10 to 15 mg/day.33,35,36 Since the reported cases were not tested for genotype variations; genetic polymorphism might represent an unrevealed explanation of the variable warfarin responses among patients during rifampin use.
Up to our knowledge, this is the first warfarin–rifampin interaction case report to address genetic polymorphism as a contributing factor in the interaction outcome since it was first described in the 1970s. Nevertheless, there are studies of rifampin induction effect on CYP2C9 variants. Vormfelde et al42 used tolbutamide as a probe substrate to evaluate rifampin effect on CYP2C9 activity in 128 healthy volunteers with different genotypes. While the pre-rifampin enzyme activity difference between *1/*1 and *3/*3 was six-folds, the induction effect was around two-fold regardless of the genotype.42 It is important to emphasize that the study duration was only four days,42 which may not represent the subsequent enzyme induction phases. George et al43, using phenytoin as a probe drug, studied the total effect of one month of rifampin therapy on CYP2C9 various genotypes in forty-eight new TB patients. Joined data from twelve mutant patients showed that rifampin’s induction potential was statistically significant regardless of the genotype.43 While these studies suggest that rifampin increases the CYP2C9 concentration with the same ratio,42,43 the catalytic activity would be genotype-dependent.5
Our case may represent an example of phenoconversion, a phenomenon of genotype-phenotype mismatch, in which an individual’s metabolizing enzyme is functionally converted from a poor metabolizer to an intermediate or extensive metabolizer or vice versa due to the use of an enzyme inducer or inhibitor, respectively.44–46 Rifampin shifted the patient from her genotype-based poor metabolizer status to a functional rapid metabolizer status that required warfarin daily dose escalation.46 However, because the patient’s gene-based estimated warfarin dose was 2 mg, escalation to 10 mg, a feasible dose compared with other interaction reports, was sufficient to attain therapeutic INR.
While the patient confirmed complete adherence, the INR dropped to 1.0 on the last day of warfarin therapy and day 97 of rifampin. One explanation may be a late CYP2C9 induction phase by rifampin. CYP2C9 half-life has been reported to be much longer than other CYPs as CYP3A4. Shibata et al35 monitored CYP2C9 and CYP3A activities in two cases who were receiving rifampin and warfarin concomitantly after rifampin discontinuation. The CYP2C9 estimated half-lives were 25.7 and 16.8 days, compared with CYP3A half-lives of 2.4 and 11.5 days, in the first and second case, respectively.35 Indicating that the CYP2C9 turnover can take up to months. Moreover, having a CYP2C9*3*3 genotype might have prolonged the time-course to maximal induction. Since warfarin was stopped at that point, verifying these explanations is not possible.
Conclusion
This case report demonstrated the highly significant effect of rifampin metabolic induction and genetic polymorphism on warfarin dose requirements. Our findings reveal a genetic explanation of the variable patients’ responses to different warfarin doses while on rifampin. While wild-type patients are not likely to respond to extreme warfarin doses due to the drug interaction with rifampin, patients with loss-of-function genetic variants of CYP2C9 and VKORC1 are more likely to respond at a feasible dose level. Future studies are warranted to evaluate the genotype variants’ effect on the interaction, which might benefit in selecting likely warfarin responders when rifampin therapy is needed.
Ethical Approval
Ethical approval for this case report was provided by the Medical Research Committee (MRC) of Hamad Medical Corporation (HMC) (#MRC-04-20-428).
Author Contributions
All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that they have no conflicts of interest in this work. | ELTROMBOPAG, ENOXAPARIN, ETHAMBUTOL HYDROCHLORIDE, HEPARIN SODIUM, HUMAN IMMUNOGLOBULIN G, HUMAN PLATELET, ALLOGENIC, ISONIAZID, METFORMIN HYDROCHLORIDE, PYRAZINAMIDE, RIFAMPIN, WARFARIN SODIUM | DrugsGivenReaction | CC BY | 33542643 | 18,944,693 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'International normalised ratio increased'. | Genetic Polymorphism Effect on Warfarin-Rifampin Interaction: A Case Report and Review of Literature.
Warfarin-rifampin interaction has been reported since the 1970s. Due to rifampin's strong induction of CYP2C9, most cases could not attain the target international normalized ratio (INR) despite warfarin dose escalation. Genetic polymorphisms determine up to 50% of warfarin dose variability. A 38-year-old woman was started on warfarin and rifampin for cerebral venous sinus thrombosis and pulmonary tuberculosis. Over six weeks, the daily warfarin dose was increased from 3 to 10 mg to attain three consecutive in-clinic therapeutic INRs. She completed three complications-free months of warfarin treatment with time in therapeutic range (TTR) of 46%. We performed retrospective genetic testing to determine the patient's CYP2C9, CYP4F2, and VKORC1 genotypes and whether they had affected the interaction outcome. The analysis revealed that the subject carries CYP2C9*3*3 and VKORC1-1639 (GA) mutations, classifying her as a slow metabolizer and, hence, highly warfarin-sensitive. This was reflected on how the case responded to a relatively lower dose than previously reported cases that did not achieve the target on warfarin daily doses up to 35 mg. This is the first report addressing the genotype effect on this interaction. Patients with genetic variants requiring low warfarin doses are more likely to respond at a feasible dose while on rifampin. Future studies to evaluate warfarin-rifampin-gene interaction are warranted.
Introduction
Warfarin, a vitamin K antagonist, remains the preferred oral anticoagulation for atrial fibrillation with prosthetic cardiac valves or rheumatic heart disease with more than mild mitral stenosis, and venous thromboembolism (VTE) at unusual sites.1,2 It is a mixture of two racemic isomers, R and S-warfarin. Both impair the vitamin k-dependent proteins production via inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).3 Cytochrome P450 2C9 (CYP2C9) extensively metabolizes S-warfarin, the stereoisomer of predominant potency, to the inactive 7-hydroxywarfarin.3
VKORC1 and CYP2C9 genetic polymorphisms, with other genetic variants, determine up to 50% of warfarin dose variance.3,4 The most studied and common variant alleles of CYP2C9 are CYP2C9*2 (rs1799853) and *3 (rs1057910), which result from missense mutations associated with diminished catalytic activity, poor warfarin metabolism, and decreased dose requirements.5
VKORC1–1639G>A (rs9923231) genotype variants (GA and AA) contribute majorly to sensitizing warfarin.5 On the other hand, CYP4F2*3, a nonsynonymous variant of the gene coding for the primary liver vitamin K oxidase, CYP4F2, has been associated in some studies with a modest increase of warfarin dose requirements (8–11%).3 These mutations were incorporated, with other clinical factors, into dosing algorithms which were shown to provide better warfarin dose prediction.3 Gage and International Warfarin Pharmacogenetics Consortium (IWPC), are among the most widely studied algorithms and are contained in the website (www.warfarindosing.org), which calculates the initial dose with the ability to adjust for CYP2C9*5, *6, CYP4F2, and GGCX genotypes.3 Genetic-based dosing of warfarin upon initiation was shown in some studies to improve target international normalized ratio (INR) attainment and time in therapeutic range (TTR) during the initial month.6 This was mainly mediated by CYP2C9 and VKORC1 polymorphisms which significantly impact the maintenance dose requirement.3,4,7 The Food and Drug Administration (FDA) has also approved warfarin label modifications with dosing guidance based on the CYP2C9 and VKORC1 genotypes.8 Apart from genetics, numerous warfarin drug interactions have been reported requiring dose adjustments and frequent INR monitoring to avoid bleeding or anticoagulation failure.9
Antituberculous management includes rifampin, isoniazid, ethambutol, and pyrazinamide. While ethambutol and pyrazinamide are neither cytochrome P450s inhibitors nor reported to affect warfarin, isoniazid is a week inhibitor of CYP3A4, which is not FDA classified as a clinical index inhibitor.10 While it has been reported to increase warfarin’s hypoprothrombinemic effect in two cases,11,12 the interaction magnitude is considered minimal, with no action recommended.13
Rifampin, a life-saving antimicrobial for tuberculosis, endocarditis, and meningitis,14 is a clinically significant inducer of CYP2B6, CYP2C19, CYP2C8, CYP2C9, and CYP3A4, as well as P glycoprotein.10 It induces CYP2C9 transcription by binding to its main De-novo synthesis regulatory nuclear receptor, pregnane X receptor (PXR).5,15 The binding increases the CYP2C9 mRNA expression by up to six-folds.15 That leads to a higher amount of the enzyme, and extensive metabolism of the substrate/victim drug.15 While the onset of induction can be few days with rifampin,16 the time required to reach maximal enzyme abundance and new steady state is more than two weeks based on the CYP turnover and degradation half-life.16–19 FDA classifies rifampin as a moderate inducer of CYP2C9, defined as a decrease in the substrates area under the concentration-time curve (AUC) by 50% to less than 80%.10 That was based on two healthy-volunteers studies with probe substrates, S-warfarin and tolbutamide.20,21 However, the first study’s duration was only four days,20 and 12–15 days in the latter,21 which, yet, showed high variability of CYP2C9 activity (1–7.4-fold).22 These short durations may imply that rifampin is rather a strong CYP2C9 inducer as AUC ratio were measured before reaching maximal induction.16–19
Rifampin almost eliminates warfarin’s therapeutic effect, which required extensive dose escalation in all cases and is typically associated with the inability to maintain therapeutic range. The interaction has been described since the 1970s.20,23,24 Till the late 1980s, multiple reports showed a significant increase in warfarin dose requirements with rifampin.25–27 After the INR test was universally adopted,28 several reports demonstrated that most patients who required the anti-infective along with anticoagulation were unable to maintain target INR.14,29–36
This report aims to describe a case who received warfarin and rifampin concomitantly and the interaction outcome, and to perform genetic testing to determine the patient’s CYP2C9, VKORC1, CALU, and CYP4F2 genotypes and whether they could explain the response to warfarin dose escalation.
Case Description
A 38-year-old Ethiopian woman with a weight of 60 kg, a height of 150 cm, and a history of diabetes and immune thrombocytopenic purpura (ITP) on metformin and chronic eltrombopag presented to the emergency of Hamad General Hospital in Qatar on January 13, 2020, with dizziness, severe diffuse headache, photophobia, and multiple vomiting for three days. Intracranial computed tomography (CT) venogram showed cerebral venous sinus thrombosis (CVST). Eltrombopag was stopped. Since the Glasgow Coma Score (GCS) dropped to 11, the patient was admitted to the medical ICU with sedation, analgesia, and close neurologic observation. Because of low platelet count (PC), 32X109/L, she was started on 0.5 gm/kg intravenous immunoglobulin (IVIG) plus steroids for three days to raise the PC above 50X109/L in order to initiate anticoagulation. Two days later, PC reached 75X109/L, and heparin continuous IV infusion was initiated with platelets transfusion as the patient was neurologically deteriorating, and repeated CT showed extension of thrombosis with intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). On January 19, although PC was maintained above 200X109/L, heparin was suspended due to a sharp drop of hemoglobin to 5 gm/dL with no identified source of bleeding. The patient was transfused immediately. The next day hemoglobin increased to 8 then maintained at 9–10 gm/dL. Heparin was resumed on January 23. Hematology planned to start rituximab for ITP, yet the Quantiferone test for tuberculosis (TB) was positive. CT chest on January 26 revealed consolidation patches in the right upper and middle lung lobes suggesting active TB. Since bronchoscopy was not feasible due to the high bleeding risk, the pulmonary, MICU, and infectious diseases teams decided to start empiric anti-TB medications based on radiology and follow response by imaging. The rituximab plan was aborted.
On January 29, the patient was started on daily rifampin 600 mg, isoniazid 300 mg, ethambutol 1100 mg, and pyrazinamide 1600 mg. She was transferred to the medicine ward after sedation withdrawal. On January 31, anticoagulation was shifted to daily oral warfarin 5 mg with twice-daily enoxaparin 60 mg as bridging. Three days later, warfarin was held for one day then resumed with dose reduction to 3 mg due to a sharp INR increase to 3.7. The next day, INR dropped to 1.0, then increased gradually after three days to 1.7, 1.9, and reached 2.0 on February 9. Enoxaparin was stopped, and the patient was discharged on warfarin 3 mg, anti-TB medications, pyridoxine 40 mg, metformin 500 mg twice daily, and metoprolol 50 mg twice daily. Following in anticoagulation clinic, on February 12, after 14 days of rifampin, INR was 2.6; therefore, the same warfarin dose continued. Although decreased to 1.7 on February 16, the warfarin dose remained. A week later, INR dropped to 1.3, so warfarin was escalated to 4 mg/day, and enoxaparin resumed. Over the next three weeks, the dose was gradually escalated up to 10 mg, after four days of which, INR reached 2.3 on March 15. Enoxaparin was stopped, and the patient was maintained on daily warfarin 10 mg. The anti-TB medications were switched to (Rifampin 600 mg/Isoniazid 300 mg) on March 24. INR was maintained in the next two clinic visits at 3 and 2.9 on March 23 and April 6, respectively. On May 4, the INR was 1.0 in the last anticoagulation clinic visit. That could not be explained by non-compliance as per the patient interview. The planned three-month duration of anticoagulation ended, so warfarin was stopped. Rifampin/isoniazid continued till July 14, 2020. The patient was interviewed on each visit and excluded any adverse effects. She had normal follow up laboratory values throughout the treatment. Details of warfarin daily dose and INR are shown in [Figure 1].Figure 1 This graph represents the daily warfarin dose, rifampin days, and INR overtime. The bottom x-axis represents dates. The top x-axis represents rifampin days. The left y-axis represents the daily warfarin dose in milligrams and is shown by the vertical bars. The right Y-axis represents the INR and is shown by the black diamond points. The therapeutic range is indicated between the two dotted lines (2.0–3.0).
Methods
The patient was approached by one of the study investigators and explained the reasons and expectations of the research. The patient confirmed her understanding, and agreement to provide saliva sample for genetic testing as well as to have the case published by signing an Institutional Review Board (IRB) approved informed consent form. She was asked to provide a saliva sample using Oragene•DNA (OG-500) self-collection kit (DNA genotek, USA). Hereafter, the kit was kept in a water bath at 50 C° overnight for DNA extraction. The prepIT®•L2P standard protocol for the purification of DNA was used for DNA extraction.37 The purified DNA’s quality and quantity were evaluated by Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific). Finally, the sample was genotyped for detecting the following genetic variants: CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), CYP4F2*3 (rs2108622),VKORC1 (rs9923231), VKORC1 Asp36Tyr (rs61742245), and CALU (rs339097). This was performed using the QuantStudio™ 5 Real-Time Polymerase Chain Reaction (PCR) system for Human Identification, 96-well, 0.2 mL, desktop manufactured by Applied BiosystemsTM.
Results
The genotyping revealed that the patient is a carrier of CYP2C9*3*3 homozygous, VKORC1-1639 (GA) heterozygous, and CYP4F2 (CC) wild-type homozygous. Based on this genetic profile, the subject is considered a slow metabolizer which indicates high warfarin sensitivity. On the other hand, both VKORC1 Asp36Tyr (CT) and CALU (AG) genotyping were heterozygous indicating partial warfarin resistance.
As it is shown in Figure 1, the sudden rise in INR with a moderate standard warfarin dose of 5 mg is very well explained by the CYP2C9 loss of function genetic variant carried by this patient (CYP2C9*3/*3). A few weeks later and with the interacting effect of rifampin reaching its peak, the daily warfarin dose requirement for the patient increased reaching 10 mg.
Discussion
In this case report, we observed warfarin–rifampin drug interaction in a patient with CVST and pulmonary TB. The interaction management required warfarin dose-escalation, frequent INR monitoring, and low molecular weight heparin (LMWH) bridging over six weeks. On a daily warfarin dose of 10 mg, our patient attained three consecutive therapeutic INR levels in three clinic visits. That is considered a “stable warfarin dose,” as defined in most studies,38 despite the one subtherapeutic INR in the last treatment day. The TTR, calculated by the Rosendaal method,39 was 46.2%. The three-month warfarin anticoagulation treatment, combined with rifampin, was completed with difficulty, yet no complications.
Since the World Health Organization (WHO) adopted INR in the 1980s,28 more than nineteen cases of warfarin–rifampin interaction have been reported.14,29–36 Despite extensive warfarin dose escalation over a prolonged time, the majority could not attain target INR while on the combination.14,29–32,34–36 Cases are summarized in [Table 1]. Excessive warfarin exposure and hemorrhage after rifampin discontinuation have been reported, implying the importance of close monitoring and careful dose de-escalation after rifampin stoppage.33,40 Due to the lack of laboratory monitorable parameters like INR, labels of direct oral anticoagulants (DOACs), that are substrates of CYP3A4 and/or P glycoprotein, recommend avoiding concomitant use with rifampin to avoid unmanageable therapeutic failures.41Table 1 Summary of Studies of Warfarin–Rifampicin Interaction with INR Monitoring
Study Patient’s Data Sequence and Duration During Concomitant Use After Rifampin Stopped
Case No. Age Sex Warfarin Indication Rifampin Start in Relation to Warfarin Start (Days) Concomitant Duration (Days) Average Initial Warfarin Dose (mg/day)a,b Average Last Warfarin Dose (mg/day)a,b Warfarin Dose Increase Target INR Target Attained Time to Stable Warfarin Dosec (Days) TTR Stable Warfarin Dosec (mg/day) Time to Stable Warfarin Dosec (Days)
Casner, 199629 1 36 M PE −2 20 7.8b 20b 169% 2.0–3.0 No NA NA 10 10
Lee & Trasher, 200130 1 58 M LVT −120 113 7.5b 25b 233% 2.0–3.0 No NA NA 7.5 35
Kim et al, 200631 1 79 M DVT −11 30 5a 30b 500% 2.0–3.0 No NA NA 6 60
Krajewski, 201032 1 71 M AF +14 45 5.7a 25b 340% 2.0–3.0 No NA NA 5 120
Martins et al, 201333 1 59 F AF +300 203 6.4a 11.4a 78% 2.0–3.0 Yes 104 50% 5.4 60
Maina et al, 201336 1 17 F DVT −7 UNK 10b 27.7a 177.3% 2.0–3.0 Yes 63 52% UNK UNK
2 24 F RHD/LAT −42 UNK 5b 5.8a 16% 2.0–3.0 Yesd 66 67% UNK UNK
3 36 M DVT −44 UNK 12b 11.4b −4.8% 2.0–3.0 No NA 24% UNK UNK
4 64 F DVT −45 UNK 10b 11.5b 15.3% 2.0–3.0 No NA 47% UNK UNK
5 22 F DVT −88 UNK 10b 4.8a −37% 2.0–3.0 Yese 12 54% UNK UNK
6 9 M DVT 0 UNK 5b 5.3b 5.8% 2.0–3.0 No NA 53% UNK UNK
7 49 M DVT −3 UNK 5b 9.5a 89.3% 2.0–3.0 Yes 49 42% UNK UNK
8 30 F PE −35 UNK 5b 27a 440.9% 2.0–3.0 Yes 67 30% UNK UNK
9 29 F DVT −31 UNK 5b 11.8a 135.8% 2.0–3.0 Yes 7 40% UNK UNK
10 41 M Stroke & DVT −46 UNK 6b 6.5a 8.3% 2.0–3.0 Yes 63 66% UNK UNK
Dawson et al, 201634 1 60 M MV replaced +UNK 42 8a 35b 340% 2.5–3.5 No NA NA 8 28
Fahmi et al, 201614 1 34 F MV replaced +19 years 42 7.5a 30b 300% 2.5–3.5 No NA NA 11.4 35
Shibata et al, 201735 1 70 F Stroke +UNK 365 4a 15a 275% 2.0–3.0 Yes UNK UNK 4 30
2 80 F AF +UNK 330 2.5a 10b 300% 2.0–3.0 No NA NA 3 60
Notes: Maina et al did not report rifampin cessation times or confirmed adherence to medications other than warfarin,36 which may have affected response. aTherapeutic INR attained on the dose. bTherapeutic INR not attained on the dose. cDefined as three consecutive therapeutic INR levels on the same warfarin dose (In Maina et al case series, defined as two consecutive therapeutic INRs). dOccasional warfarin overdoses. eMissed warfarin doses.
Abbreviations: AF, atrial fibrillation; DVT, deep venous thrombosis; INR, international normalized ratio; LAT, left arterial thrombosis; LVT, left ventricular thrombus; MV, mitral valve; NA, not applicable; PE, pulmonary embolism; RHD, rheumatic heart disease; TTR, time in therapeutic range; UNK, unknown.
Our case had one supratherapeutic INR after three warfarin doses of 5 mg and five days of rifampin commencement explained by her CYP2C9 poor metabolizer phenotype. She had two consecutive therapeutic INR readings after 12 and 14 days of rifampin on daily warfarin 3 mg. However, INR dropped below therapeutic after 17 days of rifampin, likely due to the latter’s liver enzyme induction. Comparable patterns have been observed in other cases when the commencement sequence was a few days apart or when rifampin was added to chronic warfarin. In 1996, Casner29 reported a patient who had one therapeutic INR after 13 rifampin days, which declined to subtherapeutic until ten days after the rifampin stoppage. The case reported by Kim et al31 had one in-target INR after warfarin 20 mg, which dropped then increased back to target for a week after escalation to 25 mg on rifampin days 25 and 33. Then, the INR declined steadily even after warfarin dose was raised to 30 mg.31 An INR of 4.4 after three days of rifampin in a mechanical valve replacement case, reported by Fahmi et al14, necessitated the holding of warfarin for five days. Then INR dropped to below the desired 7–14 days from the combination. In the case reported by Dawson et al34, it took 17 days for the interaction to become apparent. These reports, aligned with our report, indicate that the time-course to rifampin CYP2C9 induction is more than 14 days. Studies of this rifampin duration or less are inadequate to evaluate the full impact of such interaction. It is vital to monitor INR frequently during the first two weeks of rifampin and shortly after to avoid exposing the patient to subtherapeutic levels with no bridging.
Our patient’s CYP2C9 genotyping revealed that she is a slow metabolizer with a homozygous CYP2C9*3*3, which indicates high warfarin sensitivity and the lowest dose requirements. Additionally, VKORC1 –1639G>A genotyping showed upstream variant (AG) requiring lower warfarin doses.3 The patient reached three consecutive therapeutic INRs on a daily warfarin dose of 10 mg, which is generally considered a high dose. However, compared with most cases that received rifampin with warfarin doses up to 35 mg and no target attainment, 10 mg represents a relatively reasonable dose. The estimated therapeutic warfarin dose for our case, calculated using clinical factors via www.warfarindosing.org, was 5.8 mg/day. However, when the genotyping results were added to the calculation, the estimated therapeutic dose was 2 mg/day. The FDA warfarin label expected maintenance dosing for CYP2C9*3/*3 combined with VKORC1 (AG) is ranged from 0.5 to 2 mg/day.8 That implies that 10 mg is at least a 400% increase from the therapeutic dose without rifampin. Interestingly, most reported cases who reached therapeutic INR during the concomitant warfarin–rifampin use received relatively low warfarin doses around 10 to 15 mg/day.33,35,36 Since the reported cases were not tested for genotype variations; genetic polymorphism might represent an unrevealed explanation of the variable warfarin responses among patients during rifampin use.
Up to our knowledge, this is the first warfarin–rifampin interaction case report to address genetic polymorphism as a contributing factor in the interaction outcome since it was first described in the 1970s. Nevertheless, there are studies of rifampin induction effect on CYP2C9 variants. Vormfelde et al42 used tolbutamide as a probe substrate to evaluate rifampin effect on CYP2C9 activity in 128 healthy volunteers with different genotypes. While the pre-rifampin enzyme activity difference between *1/*1 and *3/*3 was six-folds, the induction effect was around two-fold regardless of the genotype.42 It is important to emphasize that the study duration was only four days,42 which may not represent the subsequent enzyme induction phases. George et al43, using phenytoin as a probe drug, studied the total effect of one month of rifampin therapy on CYP2C9 various genotypes in forty-eight new TB patients. Joined data from twelve mutant patients showed that rifampin’s induction potential was statistically significant regardless of the genotype.43 While these studies suggest that rifampin increases the CYP2C9 concentration with the same ratio,42,43 the catalytic activity would be genotype-dependent.5
Our case may represent an example of phenoconversion, a phenomenon of genotype-phenotype mismatch, in which an individual’s metabolizing enzyme is functionally converted from a poor metabolizer to an intermediate or extensive metabolizer or vice versa due to the use of an enzyme inducer or inhibitor, respectively.44–46 Rifampin shifted the patient from her genotype-based poor metabolizer status to a functional rapid metabolizer status that required warfarin daily dose escalation.46 However, because the patient’s gene-based estimated warfarin dose was 2 mg, escalation to 10 mg, a feasible dose compared with other interaction reports, was sufficient to attain therapeutic INR.
While the patient confirmed complete adherence, the INR dropped to 1.0 on the last day of warfarin therapy and day 97 of rifampin. One explanation may be a late CYP2C9 induction phase by rifampin. CYP2C9 half-life has been reported to be much longer than other CYPs as CYP3A4. Shibata et al35 monitored CYP2C9 and CYP3A activities in two cases who were receiving rifampin and warfarin concomitantly after rifampin discontinuation. The CYP2C9 estimated half-lives were 25.7 and 16.8 days, compared with CYP3A half-lives of 2.4 and 11.5 days, in the first and second case, respectively.35 Indicating that the CYP2C9 turnover can take up to months. Moreover, having a CYP2C9*3*3 genotype might have prolonged the time-course to maximal induction. Since warfarin was stopped at that point, verifying these explanations is not possible.
Conclusion
This case report demonstrated the highly significant effect of rifampin metabolic induction and genetic polymorphism on warfarin dose requirements. Our findings reveal a genetic explanation of the variable patients’ responses to different warfarin doses while on rifampin. While wild-type patients are not likely to respond to extreme warfarin doses due to the drug interaction with rifampin, patients with loss-of-function genetic variants of CYP2C9 and VKORC1 are more likely to respond at a feasible dose level. Future studies are warranted to evaluate the genotype variants’ effect on the interaction, which might benefit in selecting likely warfarin responders when rifampin therapy is needed.
Ethical Approval
Ethical approval for this case report was provided by the Medical Research Committee (MRC) of Hamad Medical Corporation (HMC) (#MRC-04-20-428).
Author Contributions
All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that they have no conflicts of interest in this work. | ELTROMBOPAG, ENOXAPARIN, ETHAMBUTOL HYDROCHLORIDE, HEPARIN SODIUM, HUMAN IMMUNOGLOBULIN G, HUMAN PLATELET, ALLOGENIC, ISONIAZID, METFORMIN HYDROCHLORIDE, PYRAZINAMIDE, RIFAMPIN, WARFARIN SODIUM | DrugsGivenReaction | CC BY | 33542643 | 18,944,693 | 2021 |
What is the weight of the patient? | Genetic Polymorphism Effect on Warfarin-Rifampin Interaction: A Case Report and Review of Literature.
Warfarin-rifampin interaction has been reported since the 1970s. Due to rifampin's strong induction of CYP2C9, most cases could not attain the target international normalized ratio (INR) despite warfarin dose escalation. Genetic polymorphisms determine up to 50% of warfarin dose variability. A 38-year-old woman was started on warfarin and rifampin for cerebral venous sinus thrombosis and pulmonary tuberculosis. Over six weeks, the daily warfarin dose was increased from 3 to 10 mg to attain three consecutive in-clinic therapeutic INRs. She completed three complications-free months of warfarin treatment with time in therapeutic range (TTR) of 46%. We performed retrospective genetic testing to determine the patient's CYP2C9, CYP4F2, and VKORC1 genotypes and whether they had affected the interaction outcome. The analysis revealed that the subject carries CYP2C9*3*3 and VKORC1-1639 (GA) mutations, classifying her as a slow metabolizer and, hence, highly warfarin-sensitive. This was reflected on how the case responded to a relatively lower dose than previously reported cases that did not achieve the target on warfarin daily doses up to 35 mg. This is the first report addressing the genotype effect on this interaction. Patients with genetic variants requiring low warfarin doses are more likely to respond at a feasible dose while on rifampin. Future studies to evaluate warfarin-rifampin-gene interaction are warranted.
Introduction
Warfarin, a vitamin K antagonist, remains the preferred oral anticoagulation for atrial fibrillation with prosthetic cardiac valves or rheumatic heart disease with more than mild mitral stenosis, and venous thromboembolism (VTE) at unusual sites.1,2 It is a mixture of two racemic isomers, R and S-warfarin. Both impair the vitamin k-dependent proteins production via inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).3 Cytochrome P450 2C9 (CYP2C9) extensively metabolizes S-warfarin, the stereoisomer of predominant potency, to the inactive 7-hydroxywarfarin.3
VKORC1 and CYP2C9 genetic polymorphisms, with other genetic variants, determine up to 50% of warfarin dose variance.3,4 The most studied and common variant alleles of CYP2C9 are CYP2C9*2 (rs1799853) and *3 (rs1057910), which result from missense mutations associated with diminished catalytic activity, poor warfarin metabolism, and decreased dose requirements.5
VKORC1–1639G>A (rs9923231) genotype variants (GA and AA) contribute majorly to sensitizing warfarin.5 On the other hand, CYP4F2*3, a nonsynonymous variant of the gene coding for the primary liver vitamin K oxidase, CYP4F2, has been associated in some studies with a modest increase of warfarin dose requirements (8–11%).3 These mutations were incorporated, with other clinical factors, into dosing algorithms which were shown to provide better warfarin dose prediction.3 Gage and International Warfarin Pharmacogenetics Consortium (IWPC), are among the most widely studied algorithms and are contained in the website (www.warfarindosing.org), which calculates the initial dose with the ability to adjust for CYP2C9*5, *6, CYP4F2, and GGCX genotypes.3 Genetic-based dosing of warfarin upon initiation was shown in some studies to improve target international normalized ratio (INR) attainment and time in therapeutic range (TTR) during the initial month.6 This was mainly mediated by CYP2C9 and VKORC1 polymorphisms which significantly impact the maintenance dose requirement.3,4,7 The Food and Drug Administration (FDA) has also approved warfarin label modifications with dosing guidance based on the CYP2C9 and VKORC1 genotypes.8 Apart from genetics, numerous warfarin drug interactions have been reported requiring dose adjustments and frequent INR monitoring to avoid bleeding or anticoagulation failure.9
Antituberculous management includes rifampin, isoniazid, ethambutol, and pyrazinamide. While ethambutol and pyrazinamide are neither cytochrome P450s inhibitors nor reported to affect warfarin, isoniazid is a week inhibitor of CYP3A4, which is not FDA classified as a clinical index inhibitor.10 While it has been reported to increase warfarin’s hypoprothrombinemic effect in two cases,11,12 the interaction magnitude is considered minimal, with no action recommended.13
Rifampin, a life-saving antimicrobial for tuberculosis, endocarditis, and meningitis,14 is a clinically significant inducer of CYP2B6, CYP2C19, CYP2C8, CYP2C9, and CYP3A4, as well as P glycoprotein.10 It induces CYP2C9 transcription by binding to its main De-novo synthesis regulatory nuclear receptor, pregnane X receptor (PXR).5,15 The binding increases the CYP2C9 mRNA expression by up to six-folds.15 That leads to a higher amount of the enzyme, and extensive metabolism of the substrate/victim drug.15 While the onset of induction can be few days with rifampin,16 the time required to reach maximal enzyme abundance and new steady state is more than two weeks based on the CYP turnover and degradation half-life.16–19 FDA classifies rifampin as a moderate inducer of CYP2C9, defined as a decrease in the substrates area under the concentration-time curve (AUC) by 50% to less than 80%.10 That was based on two healthy-volunteers studies with probe substrates, S-warfarin and tolbutamide.20,21 However, the first study’s duration was only four days,20 and 12–15 days in the latter,21 which, yet, showed high variability of CYP2C9 activity (1–7.4-fold).22 These short durations may imply that rifampin is rather a strong CYP2C9 inducer as AUC ratio were measured before reaching maximal induction.16–19
Rifampin almost eliminates warfarin’s therapeutic effect, which required extensive dose escalation in all cases and is typically associated with the inability to maintain therapeutic range. The interaction has been described since the 1970s.20,23,24 Till the late 1980s, multiple reports showed a significant increase in warfarin dose requirements with rifampin.25–27 After the INR test was universally adopted,28 several reports demonstrated that most patients who required the anti-infective along with anticoagulation were unable to maintain target INR.14,29–36
This report aims to describe a case who received warfarin and rifampin concomitantly and the interaction outcome, and to perform genetic testing to determine the patient’s CYP2C9, VKORC1, CALU, and CYP4F2 genotypes and whether they could explain the response to warfarin dose escalation.
Case Description
A 38-year-old Ethiopian woman with a weight of 60 kg, a height of 150 cm, and a history of diabetes and immune thrombocytopenic purpura (ITP) on metformin and chronic eltrombopag presented to the emergency of Hamad General Hospital in Qatar on January 13, 2020, with dizziness, severe diffuse headache, photophobia, and multiple vomiting for three days. Intracranial computed tomography (CT) venogram showed cerebral venous sinus thrombosis (CVST). Eltrombopag was stopped. Since the Glasgow Coma Score (GCS) dropped to 11, the patient was admitted to the medical ICU with sedation, analgesia, and close neurologic observation. Because of low platelet count (PC), 32X109/L, she was started on 0.5 gm/kg intravenous immunoglobulin (IVIG) plus steroids for three days to raise the PC above 50X109/L in order to initiate anticoagulation. Two days later, PC reached 75X109/L, and heparin continuous IV infusion was initiated with platelets transfusion as the patient was neurologically deteriorating, and repeated CT showed extension of thrombosis with intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). On January 19, although PC was maintained above 200X109/L, heparin was suspended due to a sharp drop of hemoglobin to 5 gm/dL with no identified source of bleeding. The patient was transfused immediately. The next day hemoglobin increased to 8 then maintained at 9–10 gm/dL. Heparin was resumed on January 23. Hematology planned to start rituximab for ITP, yet the Quantiferone test for tuberculosis (TB) was positive. CT chest on January 26 revealed consolidation patches in the right upper and middle lung lobes suggesting active TB. Since bronchoscopy was not feasible due to the high bleeding risk, the pulmonary, MICU, and infectious diseases teams decided to start empiric anti-TB medications based on radiology and follow response by imaging. The rituximab plan was aborted.
On January 29, the patient was started on daily rifampin 600 mg, isoniazid 300 mg, ethambutol 1100 mg, and pyrazinamide 1600 mg. She was transferred to the medicine ward after sedation withdrawal. On January 31, anticoagulation was shifted to daily oral warfarin 5 mg with twice-daily enoxaparin 60 mg as bridging. Three days later, warfarin was held for one day then resumed with dose reduction to 3 mg due to a sharp INR increase to 3.7. The next day, INR dropped to 1.0, then increased gradually after three days to 1.7, 1.9, and reached 2.0 on February 9. Enoxaparin was stopped, and the patient was discharged on warfarin 3 mg, anti-TB medications, pyridoxine 40 mg, metformin 500 mg twice daily, and metoprolol 50 mg twice daily. Following in anticoagulation clinic, on February 12, after 14 days of rifampin, INR was 2.6; therefore, the same warfarin dose continued. Although decreased to 1.7 on February 16, the warfarin dose remained. A week later, INR dropped to 1.3, so warfarin was escalated to 4 mg/day, and enoxaparin resumed. Over the next three weeks, the dose was gradually escalated up to 10 mg, after four days of which, INR reached 2.3 on March 15. Enoxaparin was stopped, and the patient was maintained on daily warfarin 10 mg. The anti-TB medications were switched to (Rifampin 600 mg/Isoniazid 300 mg) on March 24. INR was maintained in the next two clinic visits at 3 and 2.9 on March 23 and April 6, respectively. On May 4, the INR was 1.0 in the last anticoagulation clinic visit. That could not be explained by non-compliance as per the patient interview. The planned three-month duration of anticoagulation ended, so warfarin was stopped. Rifampin/isoniazid continued till July 14, 2020. The patient was interviewed on each visit and excluded any adverse effects. She had normal follow up laboratory values throughout the treatment. Details of warfarin daily dose and INR are shown in [Figure 1].Figure 1 This graph represents the daily warfarin dose, rifampin days, and INR overtime. The bottom x-axis represents dates. The top x-axis represents rifampin days. The left y-axis represents the daily warfarin dose in milligrams and is shown by the vertical bars. The right Y-axis represents the INR and is shown by the black diamond points. The therapeutic range is indicated between the two dotted lines (2.0–3.0).
Methods
The patient was approached by one of the study investigators and explained the reasons and expectations of the research. The patient confirmed her understanding, and agreement to provide saliva sample for genetic testing as well as to have the case published by signing an Institutional Review Board (IRB) approved informed consent form. She was asked to provide a saliva sample using Oragene•DNA (OG-500) self-collection kit (DNA genotek, USA). Hereafter, the kit was kept in a water bath at 50 C° overnight for DNA extraction. The prepIT®•L2P standard protocol for the purification of DNA was used for DNA extraction.37 The purified DNA’s quality and quantity were evaluated by Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific). Finally, the sample was genotyped for detecting the following genetic variants: CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), CYP4F2*3 (rs2108622),VKORC1 (rs9923231), VKORC1 Asp36Tyr (rs61742245), and CALU (rs339097). This was performed using the QuantStudio™ 5 Real-Time Polymerase Chain Reaction (PCR) system for Human Identification, 96-well, 0.2 mL, desktop manufactured by Applied BiosystemsTM.
Results
The genotyping revealed that the patient is a carrier of CYP2C9*3*3 homozygous, VKORC1-1639 (GA) heterozygous, and CYP4F2 (CC) wild-type homozygous. Based on this genetic profile, the subject is considered a slow metabolizer which indicates high warfarin sensitivity. On the other hand, both VKORC1 Asp36Tyr (CT) and CALU (AG) genotyping were heterozygous indicating partial warfarin resistance.
As it is shown in Figure 1, the sudden rise in INR with a moderate standard warfarin dose of 5 mg is very well explained by the CYP2C9 loss of function genetic variant carried by this patient (CYP2C9*3/*3). A few weeks later and with the interacting effect of rifampin reaching its peak, the daily warfarin dose requirement for the patient increased reaching 10 mg.
Discussion
In this case report, we observed warfarin–rifampin drug interaction in a patient with CVST and pulmonary TB. The interaction management required warfarin dose-escalation, frequent INR monitoring, and low molecular weight heparin (LMWH) bridging over six weeks. On a daily warfarin dose of 10 mg, our patient attained three consecutive therapeutic INR levels in three clinic visits. That is considered a “stable warfarin dose,” as defined in most studies,38 despite the one subtherapeutic INR in the last treatment day. The TTR, calculated by the Rosendaal method,39 was 46.2%. The three-month warfarin anticoagulation treatment, combined with rifampin, was completed with difficulty, yet no complications.
Since the World Health Organization (WHO) adopted INR in the 1980s,28 more than nineteen cases of warfarin–rifampin interaction have been reported.14,29–36 Despite extensive warfarin dose escalation over a prolonged time, the majority could not attain target INR while on the combination.14,29–32,34–36 Cases are summarized in [Table 1]. Excessive warfarin exposure and hemorrhage after rifampin discontinuation have been reported, implying the importance of close monitoring and careful dose de-escalation after rifampin stoppage.33,40 Due to the lack of laboratory monitorable parameters like INR, labels of direct oral anticoagulants (DOACs), that are substrates of CYP3A4 and/or P glycoprotein, recommend avoiding concomitant use with rifampin to avoid unmanageable therapeutic failures.41Table 1 Summary of Studies of Warfarin–Rifampicin Interaction with INR Monitoring
Study Patient’s Data Sequence and Duration During Concomitant Use After Rifampin Stopped
Case No. Age Sex Warfarin Indication Rifampin Start in Relation to Warfarin Start (Days) Concomitant Duration (Days) Average Initial Warfarin Dose (mg/day)a,b Average Last Warfarin Dose (mg/day)a,b Warfarin Dose Increase Target INR Target Attained Time to Stable Warfarin Dosec (Days) TTR Stable Warfarin Dosec (mg/day) Time to Stable Warfarin Dosec (Days)
Casner, 199629 1 36 M PE −2 20 7.8b 20b 169% 2.0–3.0 No NA NA 10 10
Lee & Trasher, 200130 1 58 M LVT −120 113 7.5b 25b 233% 2.0–3.0 No NA NA 7.5 35
Kim et al, 200631 1 79 M DVT −11 30 5a 30b 500% 2.0–3.0 No NA NA 6 60
Krajewski, 201032 1 71 M AF +14 45 5.7a 25b 340% 2.0–3.0 No NA NA 5 120
Martins et al, 201333 1 59 F AF +300 203 6.4a 11.4a 78% 2.0–3.0 Yes 104 50% 5.4 60
Maina et al, 201336 1 17 F DVT −7 UNK 10b 27.7a 177.3% 2.0–3.0 Yes 63 52% UNK UNK
2 24 F RHD/LAT −42 UNK 5b 5.8a 16% 2.0–3.0 Yesd 66 67% UNK UNK
3 36 M DVT −44 UNK 12b 11.4b −4.8% 2.0–3.0 No NA 24% UNK UNK
4 64 F DVT −45 UNK 10b 11.5b 15.3% 2.0–3.0 No NA 47% UNK UNK
5 22 F DVT −88 UNK 10b 4.8a −37% 2.0–3.0 Yese 12 54% UNK UNK
6 9 M DVT 0 UNK 5b 5.3b 5.8% 2.0–3.0 No NA 53% UNK UNK
7 49 M DVT −3 UNK 5b 9.5a 89.3% 2.0–3.0 Yes 49 42% UNK UNK
8 30 F PE −35 UNK 5b 27a 440.9% 2.0–3.0 Yes 67 30% UNK UNK
9 29 F DVT −31 UNK 5b 11.8a 135.8% 2.0–3.0 Yes 7 40% UNK UNK
10 41 M Stroke & DVT −46 UNK 6b 6.5a 8.3% 2.0–3.0 Yes 63 66% UNK UNK
Dawson et al, 201634 1 60 M MV replaced +UNK 42 8a 35b 340% 2.5–3.5 No NA NA 8 28
Fahmi et al, 201614 1 34 F MV replaced +19 years 42 7.5a 30b 300% 2.5–3.5 No NA NA 11.4 35
Shibata et al, 201735 1 70 F Stroke +UNK 365 4a 15a 275% 2.0–3.0 Yes UNK UNK 4 30
2 80 F AF +UNK 330 2.5a 10b 300% 2.0–3.0 No NA NA 3 60
Notes: Maina et al did not report rifampin cessation times or confirmed adherence to medications other than warfarin,36 which may have affected response. aTherapeutic INR attained on the dose. bTherapeutic INR not attained on the dose. cDefined as three consecutive therapeutic INR levels on the same warfarin dose (In Maina et al case series, defined as two consecutive therapeutic INRs). dOccasional warfarin overdoses. eMissed warfarin doses.
Abbreviations: AF, atrial fibrillation; DVT, deep venous thrombosis; INR, international normalized ratio; LAT, left arterial thrombosis; LVT, left ventricular thrombus; MV, mitral valve; NA, not applicable; PE, pulmonary embolism; RHD, rheumatic heart disease; TTR, time in therapeutic range; UNK, unknown.
Our case had one supratherapeutic INR after three warfarin doses of 5 mg and five days of rifampin commencement explained by her CYP2C9 poor metabolizer phenotype. She had two consecutive therapeutic INR readings after 12 and 14 days of rifampin on daily warfarin 3 mg. However, INR dropped below therapeutic after 17 days of rifampin, likely due to the latter’s liver enzyme induction. Comparable patterns have been observed in other cases when the commencement sequence was a few days apart or when rifampin was added to chronic warfarin. In 1996, Casner29 reported a patient who had one therapeutic INR after 13 rifampin days, which declined to subtherapeutic until ten days after the rifampin stoppage. The case reported by Kim et al31 had one in-target INR after warfarin 20 mg, which dropped then increased back to target for a week after escalation to 25 mg on rifampin days 25 and 33. Then, the INR declined steadily even after warfarin dose was raised to 30 mg.31 An INR of 4.4 after three days of rifampin in a mechanical valve replacement case, reported by Fahmi et al14, necessitated the holding of warfarin for five days. Then INR dropped to below the desired 7–14 days from the combination. In the case reported by Dawson et al34, it took 17 days for the interaction to become apparent. These reports, aligned with our report, indicate that the time-course to rifampin CYP2C9 induction is more than 14 days. Studies of this rifampin duration or less are inadequate to evaluate the full impact of such interaction. It is vital to monitor INR frequently during the first two weeks of rifampin and shortly after to avoid exposing the patient to subtherapeutic levels with no bridging.
Our patient’s CYP2C9 genotyping revealed that she is a slow metabolizer with a homozygous CYP2C9*3*3, which indicates high warfarin sensitivity and the lowest dose requirements. Additionally, VKORC1 –1639G>A genotyping showed upstream variant (AG) requiring lower warfarin doses.3 The patient reached three consecutive therapeutic INRs on a daily warfarin dose of 10 mg, which is generally considered a high dose. However, compared with most cases that received rifampin with warfarin doses up to 35 mg and no target attainment, 10 mg represents a relatively reasonable dose. The estimated therapeutic warfarin dose for our case, calculated using clinical factors via www.warfarindosing.org, was 5.8 mg/day. However, when the genotyping results were added to the calculation, the estimated therapeutic dose was 2 mg/day. The FDA warfarin label expected maintenance dosing for CYP2C9*3/*3 combined with VKORC1 (AG) is ranged from 0.5 to 2 mg/day.8 That implies that 10 mg is at least a 400% increase from the therapeutic dose without rifampin. Interestingly, most reported cases who reached therapeutic INR during the concomitant warfarin–rifampin use received relatively low warfarin doses around 10 to 15 mg/day.33,35,36 Since the reported cases were not tested for genotype variations; genetic polymorphism might represent an unrevealed explanation of the variable warfarin responses among patients during rifampin use.
Up to our knowledge, this is the first warfarin–rifampin interaction case report to address genetic polymorphism as a contributing factor in the interaction outcome since it was first described in the 1970s. Nevertheless, there are studies of rifampin induction effect on CYP2C9 variants. Vormfelde et al42 used tolbutamide as a probe substrate to evaluate rifampin effect on CYP2C9 activity in 128 healthy volunteers with different genotypes. While the pre-rifampin enzyme activity difference between *1/*1 and *3/*3 was six-folds, the induction effect was around two-fold regardless of the genotype.42 It is important to emphasize that the study duration was only four days,42 which may not represent the subsequent enzyme induction phases. George et al43, using phenytoin as a probe drug, studied the total effect of one month of rifampin therapy on CYP2C9 various genotypes in forty-eight new TB patients. Joined data from twelve mutant patients showed that rifampin’s induction potential was statistically significant regardless of the genotype.43 While these studies suggest that rifampin increases the CYP2C9 concentration with the same ratio,42,43 the catalytic activity would be genotype-dependent.5
Our case may represent an example of phenoconversion, a phenomenon of genotype-phenotype mismatch, in which an individual’s metabolizing enzyme is functionally converted from a poor metabolizer to an intermediate or extensive metabolizer or vice versa due to the use of an enzyme inducer or inhibitor, respectively.44–46 Rifampin shifted the patient from her genotype-based poor metabolizer status to a functional rapid metabolizer status that required warfarin daily dose escalation.46 However, because the patient’s gene-based estimated warfarin dose was 2 mg, escalation to 10 mg, a feasible dose compared with other interaction reports, was sufficient to attain therapeutic INR.
While the patient confirmed complete adherence, the INR dropped to 1.0 on the last day of warfarin therapy and day 97 of rifampin. One explanation may be a late CYP2C9 induction phase by rifampin. CYP2C9 half-life has been reported to be much longer than other CYPs as CYP3A4. Shibata et al35 monitored CYP2C9 and CYP3A activities in two cases who were receiving rifampin and warfarin concomitantly after rifampin discontinuation. The CYP2C9 estimated half-lives were 25.7 and 16.8 days, compared with CYP3A half-lives of 2.4 and 11.5 days, in the first and second case, respectively.35 Indicating that the CYP2C9 turnover can take up to months. Moreover, having a CYP2C9*3*3 genotype might have prolonged the time-course to maximal induction. Since warfarin was stopped at that point, verifying these explanations is not possible.
Conclusion
This case report demonstrated the highly significant effect of rifampin metabolic induction and genetic polymorphism on warfarin dose requirements. Our findings reveal a genetic explanation of the variable patients’ responses to different warfarin doses while on rifampin. While wild-type patients are not likely to respond to extreme warfarin doses due to the drug interaction with rifampin, patients with loss-of-function genetic variants of CYP2C9 and VKORC1 are more likely to respond at a feasible dose level. Future studies are warranted to evaluate the genotype variants’ effect on the interaction, which might benefit in selecting likely warfarin responders when rifampin therapy is needed.
Ethical Approval
Ethical approval for this case report was provided by the Medical Research Committee (MRC) of Hamad Medical Corporation (HMC) (#MRC-04-20-428).
Author Contributions
All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that they have no conflicts of interest in this work. | 60 kg. | Weight | CC BY | 33542643 | 18,944,693 | 2021 |
What was the administration route of drug 'HEPARIN SODIUM'? | Genetic Polymorphism Effect on Warfarin-Rifampin Interaction: A Case Report and Review of Literature.
Warfarin-rifampin interaction has been reported since the 1970s. Due to rifampin's strong induction of CYP2C9, most cases could not attain the target international normalized ratio (INR) despite warfarin dose escalation. Genetic polymorphisms determine up to 50% of warfarin dose variability. A 38-year-old woman was started on warfarin and rifampin for cerebral venous sinus thrombosis and pulmonary tuberculosis. Over six weeks, the daily warfarin dose was increased from 3 to 10 mg to attain three consecutive in-clinic therapeutic INRs. She completed three complications-free months of warfarin treatment with time in therapeutic range (TTR) of 46%. We performed retrospective genetic testing to determine the patient's CYP2C9, CYP4F2, and VKORC1 genotypes and whether they had affected the interaction outcome. The analysis revealed that the subject carries CYP2C9*3*3 and VKORC1-1639 (GA) mutations, classifying her as a slow metabolizer and, hence, highly warfarin-sensitive. This was reflected on how the case responded to a relatively lower dose than previously reported cases that did not achieve the target on warfarin daily doses up to 35 mg. This is the first report addressing the genotype effect on this interaction. Patients with genetic variants requiring low warfarin doses are more likely to respond at a feasible dose while on rifampin. Future studies to evaluate warfarin-rifampin-gene interaction are warranted.
Introduction
Warfarin, a vitamin K antagonist, remains the preferred oral anticoagulation for atrial fibrillation with prosthetic cardiac valves or rheumatic heart disease with more than mild mitral stenosis, and venous thromboembolism (VTE) at unusual sites.1,2 It is a mixture of two racemic isomers, R and S-warfarin. Both impair the vitamin k-dependent proteins production via inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).3 Cytochrome P450 2C9 (CYP2C9) extensively metabolizes S-warfarin, the stereoisomer of predominant potency, to the inactive 7-hydroxywarfarin.3
VKORC1 and CYP2C9 genetic polymorphisms, with other genetic variants, determine up to 50% of warfarin dose variance.3,4 The most studied and common variant alleles of CYP2C9 are CYP2C9*2 (rs1799853) and *3 (rs1057910), which result from missense mutations associated with diminished catalytic activity, poor warfarin metabolism, and decreased dose requirements.5
VKORC1–1639G>A (rs9923231) genotype variants (GA and AA) contribute majorly to sensitizing warfarin.5 On the other hand, CYP4F2*3, a nonsynonymous variant of the gene coding for the primary liver vitamin K oxidase, CYP4F2, has been associated in some studies with a modest increase of warfarin dose requirements (8–11%).3 These mutations were incorporated, with other clinical factors, into dosing algorithms which were shown to provide better warfarin dose prediction.3 Gage and International Warfarin Pharmacogenetics Consortium (IWPC), are among the most widely studied algorithms and are contained in the website (www.warfarindosing.org), which calculates the initial dose with the ability to adjust for CYP2C9*5, *6, CYP4F2, and GGCX genotypes.3 Genetic-based dosing of warfarin upon initiation was shown in some studies to improve target international normalized ratio (INR) attainment and time in therapeutic range (TTR) during the initial month.6 This was mainly mediated by CYP2C9 and VKORC1 polymorphisms which significantly impact the maintenance dose requirement.3,4,7 The Food and Drug Administration (FDA) has also approved warfarin label modifications with dosing guidance based on the CYP2C9 and VKORC1 genotypes.8 Apart from genetics, numerous warfarin drug interactions have been reported requiring dose adjustments and frequent INR monitoring to avoid bleeding or anticoagulation failure.9
Antituberculous management includes rifampin, isoniazid, ethambutol, and pyrazinamide. While ethambutol and pyrazinamide are neither cytochrome P450s inhibitors nor reported to affect warfarin, isoniazid is a week inhibitor of CYP3A4, which is not FDA classified as a clinical index inhibitor.10 While it has been reported to increase warfarin’s hypoprothrombinemic effect in two cases,11,12 the interaction magnitude is considered minimal, with no action recommended.13
Rifampin, a life-saving antimicrobial for tuberculosis, endocarditis, and meningitis,14 is a clinically significant inducer of CYP2B6, CYP2C19, CYP2C8, CYP2C9, and CYP3A4, as well as P glycoprotein.10 It induces CYP2C9 transcription by binding to its main De-novo synthesis regulatory nuclear receptor, pregnane X receptor (PXR).5,15 The binding increases the CYP2C9 mRNA expression by up to six-folds.15 That leads to a higher amount of the enzyme, and extensive metabolism of the substrate/victim drug.15 While the onset of induction can be few days with rifampin,16 the time required to reach maximal enzyme abundance and new steady state is more than two weeks based on the CYP turnover and degradation half-life.16–19 FDA classifies rifampin as a moderate inducer of CYP2C9, defined as a decrease in the substrates area under the concentration-time curve (AUC) by 50% to less than 80%.10 That was based on two healthy-volunteers studies with probe substrates, S-warfarin and tolbutamide.20,21 However, the first study’s duration was only four days,20 and 12–15 days in the latter,21 which, yet, showed high variability of CYP2C9 activity (1–7.4-fold).22 These short durations may imply that rifampin is rather a strong CYP2C9 inducer as AUC ratio were measured before reaching maximal induction.16–19
Rifampin almost eliminates warfarin’s therapeutic effect, which required extensive dose escalation in all cases and is typically associated with the inability to maintain therapeutic range. The interaction has been described since the 1970s.20,23,24 Till the late 1980s, multiple reports showed a significant increase in warfarin dose requirements with rifampin.25–27 After the INR test was universally adopted,28 several reports demonstrated that most patients who required the anti-infective along with anticoagulation were unable to maintain target INR.14,29–36
This report aims to describe a case who received warfarin and rifampin concomitantly and the interaction outcome, and to perform genetic testing to determine the patient’s CYP2C9, VKORC1, CALU, and CYP4F2 genotypes and whether they could explain the response to warfarin dose escalation.
Case Description
A 38-year-old Ethiopian woman with a weight of 60 kg, a height of 150 cm, and a history of diabetes and immune thrombocytopenic purpura (ITP) on metformin and chronic eltrombopag presented to the emergency of Hamad General Hospital in Qatar on January 13, 2020, with dizziness, severe diffuse headache, photophobia, and multiple vomiting for three days. Intracranial computed tomography (CT) venogram showed cerebral venous sinus thrombosis (CVST). Eltrombopag was stopped. Since the Glasgow Coma Score (GCS) dropped to 11, the patient was admitted to the medical ICU with sedation, analgesia, and close neurologic observation. Because of low platelet count (PC), 32X109/L, she was started on 0.5 gm/kg intravenous immunoglobulin (IVIG) plus steroids for three days to raise the PC above 50X109/L in order to initiate anticoagulation. Two days later, PC reached 75X109/L, and heparin continuous IV infusion was initiated with platelets transfusion as the patient was neurologically deteriorating, and repeated CT showed extension of thrombosis with intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). On January 19, although PC was maintained above 200X109/L, heparin was suspended due to a sharp drop of hemoglobin to 5 gm/dL with no identified source of bleeding. The patient was transfused immediately. The next day hemoglobin increased to 8 then maintained at 9–10 gm/dL. Heparin was resumed on January 23. Hematology planned to start rituximab for ITP, yet the Quantiferone test for tuberculosis (TB) was positive. CT chest on January 26 revealed consolidation patches in the right upper and middle lung lobes suggesting active TB. Since bronchoscopy was not feasible due to the high bleeding risk, the pulmonary, MICU, and infectious diseases teams decided to start empiric anti-TB medications based on radiology and follow response by imaging. The rituximab plan was aborted.
On January 29, the patient was started on daily rifampin 600 mg, isoniazid 300 mg, ethambutol 1100 mg, and pyrazinamide 1600 mg. She was transferred to the medicine ward after sedation withdrawal. On January 31, anticoagulation was shifted to daily oral warfarin 5 mg with twice-daily enoxaparin 60 mg as bridging. Three days later, warfarin was held for one day then resumed with dose reduction to 3 mg due to a sharp INR increase to 3.7. The next day, INR dropped to 1.0, then increased gradually after three days to 1.7, 1.9, and reached 2.0 on February 9. Enoxaparin was stopped, and the patient was discharged on warfarin 3 mg, anti-TB medications, pyridoxine 40 mg, metformin 500 mg twice daily, and metoprolol 50 mg twice daily. Following in anticoagulation clinic, on February 12, after 14 days of rifampin, INR was 2.6; therefore, the same warfarin dose continued. Although decreased to 1.7 on February 16, the warfarin dose remained. A week later, INR dropped to 1.3, so warfarin was escalated to 4 mg/day, and enoxaparin resumed. Over the next three weeks, the dose was gradually escalated up to 10 mg, after four days of which, INR reached 2.3 on March 15. Enoxaparin was stopped, and the patient was maintained on daily warfarin 10 mg. The anti-TB medications were switched to (Rifampin 600 mg/Isoniazid 300 mg) on March 24. INR was maintained in the next two clinic visits at 3 and 2.9 on March 23 and April 6, respectively. On May 4, the INR was 1.0 in the last anticoagulation clinic visit. That could not be explained by non-compliance as per the patient interview. The planned three-month duration of anticoagulation ended, so warfarin was stopped. Rifampin/isoniazid continued till July 14, 2020. The patient was interviewed on each visit and excluded any adverse effects. She had normal follow up laboratory values throughout the treatment. Details of warfarin daily dose and INR are shown in [Figure 1].Figure 1 This graph represents the daily warfarin dose, rifampin days, and INR overtime. The bottom x-axis represents dates. The top x-axis represents rifampin days. The left y-axis represents the daily warfarin dose in milligrams and is shown by the vertical bars. The right Y-axis represents the INR and is shown by the black diamond points. The therapeutic range is indicated between the two dotted lines (2.0–3.0).
Methods
The patient was approached by one of the study investigators and explained the reasons and expectations of the research. The patient confirmed her understanding, and agreement to provide saliva sample for genetic testing as well as to have the case published by signing an Institutional Review Board (IRB) approved informed consent form. She was asked to provide a saliva sample using Oragene•DNA (OG-500) self-collection kit (DNA genotek, USA). Hereafter, the kit was kept in a water bath at 50 C° overnight for DNA extraction. The prepIT®•L2P standard protocol for the purification of DNA was used for DNA extraction.37 The purified DNA’s quality and quantity were evaluated by Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific). Finally, the sample was genotyped for detecting the following genetic variants: CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), CYP4F2*3 (rs2108622),VKORC1 (rs9923231), VKORC1 Asp36Tyr (rs61742245), and CALU (rs339097). This was performed using the QuantStudio™ 5 Real-Time Polymerase Chain Reaction (PCR) system for Human Identification, 96-well, 0.2 mL, desktop manufactured by Applied BiosystemsTM.
Results
The genotyping revealed that the patient is a carrier of CYP2C9*3*3 homozygous, VKORC1-1639 (GA) heterozygous, and CYP4F2 (CC) wild-type homozygous. Based on this genetic profile, the subject is considered a slow metabolizer which indicates high warfarin sensitivity. On the other hand, both VKORC1 Asp36Tyr (CT) and CALU (AG) genotyping were heterozygous indicating partial warfarin resistance.
As it is shown in Figure 1, the sudden rise in INR with a moderate standard warfarin dose of 5 mg is very well explained by the CYP2C9 loss of function genetic variant carried by this patient (CYP2C9*3/*3). A few weeks later and with the interacting effect of rifampin reaching its peak, the daily warfarin dose requirement for the patient increased reaching 10 mg.
Discussion
In this case report, we observed warfarin–rifampin drug interaction in a patient with CVST and pulmonary TB. The interaction management required warfarin dose-escalation, frequent INR monitoring, and low molecular weight heparin (LMWH) bridging over six weeks. On a daily warfarin dose of 10 mg, our patient attained three consecutive therapeutic INR levels in three clinic visits. That is considered a “stable warfarin dose,” as defined in most studies,38 despite the one subtherapeutic INR in the last treatment day. The TTR, calculated by the Rosendaal method,39 was 46.2%. The three-month warfarin anticoagulation treatment, combined with rifampin, was completed with difficulty, yet no complications.
Since the World Health Organization (WHO) adopted INR in the 1980s,28 more than nineteen cases of warfarin–rifampin interaction have been reported.14,29–36 Despite extensive warfarin dose escalation over a prolonged time, the majority could not attain target INR while on the combination.14,29–32,34–36 Cases are summarized in [Table 1]. Excessive warfarin exposure and hemorrhage after rifampin discontinuation have been reported, implying the importance of close monitoring and careful dose de-escalation after rifampin stoppage.33,40 Due to the lack of laboratory monitorable parameters like INR, labels of direct oral anticoagulants (DOACs), that are substrates of CYP3A4 and/or P glycoprotein, recommend avoiding concomitant use with rifampin to avoid unmanageable therapeutic failures.41Table 1 Summary of Studies of Warfarin–Rifampicin Interaction with INR Monitoring
Study Patient’s Data Sequence and Duration During Concomitant Use After Rifampin Stopped
Case No. Age Sex Warfarin Indication Rifampin Start in Relation to Warfarin Start (Days) Concomitant Duration (Days) Average Initial Warfarin Dose (mg/day)a,b Average Last Warfarin Dose (mg/day)a,b Warfarin Dose Increase Target INR Target Attained Time to Stable Warfarin Dosec (Days) TTR Stable Warfarin Dosec (mg/day) Time to Stable Warfarin Dosec (Days)
Casner, 199629 1 36 M PE −2 20 7.8b 20b 169% 2.0–3.0 No NA NA 10 10
Lee & Trasher, 200130 1 58 M LVT −120 113 7.5b 25b 233% 2.0–3.0 No NA NA 7.5 35
Kim et al, 200631 1 79 M DVT −11 30 5a 30b 500% 2.0–3.0 No NA NA 6 60
Krajewski, 201032 1 71 M AF +14 45 5.7a 25b 340% 2.0–3.0 No NA NA 5 120
Martins et al, 201333 1 59 F AF +300 203 6.4a 11.4a 78% 2.0–3.0 Yes 104 50% 5.4 60
Maina et al, 201336 1 17 F DVT −7 UNK 10b 27.7a 177.3% 2.0–3.0 Yes 63 52% UNK UNK
2 24 F RHD/LAT −42 UNK 5b 5.8a 16% 2.0–3.0 Yesd 66 67% UNK UNK
3 36 M DVT −44 UNK 12b 11.4b −4.8% 2.0–3.0 No NA 24% UNK UNK
4 64 F DVT −45 UNK 10b 11.5b 15.3% 2.0–3.0 No NA 47% UNK UNK
5 22 F DVT −88 UNK 10b 4.8a −37% 2.0–3.0 Yese 12 54% UNK UNK
6 9 M DVT 0 UNK 5b 5.3b 5.8% 2.0–3.0 No NA 53% UNK UNK
7 49 M DVT −3 UNK 5b 9.5a 89.3% 2.0–3.0 Yes 49 42% UNK UNK
8 30 F PE −35 UNK 5b 27a 440.9% 2.0–3.0 Yes 67 30% UNK UNK
9 29 F DVT −31 UNK 5b 11.8a 135.8% 2.0–3.0 Yes 7 40% UNK UNK
10 41 M Stroke & DVT −46 UNK 6b 6.5a 8.3% 2.0–3.0 Yes 63 66% UNK UNK
Dawson et al, 201634 1 60 M MV replaced +UNK 42 8a 35b 340% 2.5–3.5 No NA NA 8 28
Fahmi et al, 201614 1 34 F MV replaced +19 years 42 7.5a 30b 300% 2.5–3.5 No NA NA 11.4 35
Shibata et al, 201735 1 70 F Stroke +UNK 365 4a 15a 275% 2.0–3.0 Yes UNK UNK 4 30
2 80 F AF +UNK 330 2.5a 10b 300% 2.0–3.0 No NA NA 3 60
Notes: Maina et al did not report rifampin cessation times or confirmed adherence to medications other than warfarin,36 which may have affected response. aTherapeutic INR attained on the dose. bTherapeutic INR not attained on the dose. cDefined as three consecutive therapeutic INR levels on the same warfarin dose (In Maina et al case series, defined as two consecutive therapeutic INRs). dOccasional warfarin overdoses. eMissed warfarin doses.
Abbreviations: AF, atrial fibrillation; DVT, deep venous thrombosis; INR, international normalized ratio; LAT, left arterial thrombosis; LVT, left ventricular thrombus; MV, mitral valve; NA, not applicable; PE, pulmonary embolism; RHD, rheumatic heart disease; TTR, time in therapeutic range; UNK, unknown.
Our case had one supratherapeutic INR after three warfarin doses of 5 mg and five days of rifampin commencement explained by her CYP2C9 poor metabolizer phenotype. She had two consecutive therapeutic INR readings after 12 and 14 days of rifampin on daily warfarin 3 mg. However, INR dropped below therapeutic after 17 days of rifampin, likely due to the latter’s liver enzyme induction. Comparable patterns have been observed in other cases when the commencement sequence was a few days apart or when rifampin was added to chronic warfarin. In 1996, Casner29 reported a patient who had one therapeutic INR after 13 rifampin days, which declined to subtherapeutic until ten days after the rifampin stoppage. The case reported by Kim et al31 had one in-target INR after warfarin 20 mg, which dropped then increased back to target for a week after escalation to 25 mg on rifampin days 25 and 33. Then, the INR declined steadily even after warfarin dose was raised to 30 mg.31 An INR of 4.4 after three days of rifampin in a mechanical valve replacement case, reported by Fahmi et al14, necessitated the holding of warfarin for five days. Then INR dropped to below the desired 7–14 days from the combination. In the case reported by Dawson et al34, it took 17 days for the interaction to become apparent. These reports, aligned with our report, indicate that the time-course to rifampin CYP2C9 induction is more than 14 days. Studies of this rifampin duration or less are inadequate to evaluate the full impact of such interaction. It is vital to monitor INR frequently during the first two weeks of rifampin and shortly after to avoid exposing the patient to subtherapeutic levels with no bridging.
Our patient’s CYP2C9 genotyping revealed that she is a slow metabolizer with a homozygous CYP2C9*3*3, which indicates high warfarin sensitivity and the lowest dose requirements. Additionally, VKORC1 –1639G>A genotyping showed upstream variant (AG) requiring lower warfarin doses.3 The patient reached three consecutive therapeutic INRs on a daily warfarin dose of 10 mg, which is generally considered a high dose. However, compared with most cases that received rifampin with warfarin doses up to 35 mg and no target attainment, 10 mg represents a relatively reasonable dose. The estimated therapeutic warfarin dose for our case, calculated using clinical factors via www.warfarindosing.org, was 5.8 mg/day. However, when the genotyping results were added to the calculation, the estimated therapeutic dose was 2 mg/day. The FDA warfarin label expected maintenance dosing for CYP2C9*3/*3 combined with VKORC1 (AG) is ranged from 0.5 to 2 mg/day.8 That implies that 10 mg is at least a 400% increase from the therapeutic dose without rifampin. Interestingly, most reported cases who reached therapeutic INR during the concomitant warfarin–rifampin use received relatively low warfarin doses around 10 to 15 mg/day.33,35,36 Since the reported cases were not tested for genotype variations; genetic polymorphism might represent an unrevealed explanation of the variable warfarin responses among patients during rifampin use.
Up to our knowledge, this is the first warfarin–rifampin interaction case report to address genetic polymorphism as a contributing factor in the interaction outcome since it was first described in the 1970s. Nevertheless, there are studies of rifampin induction effect on CYP2C9 variants. Vormfelde et al42 used tolbutamide as a probe substrate to evaluate rifampin effect on CYP2C9 activity in 128 healthy volunteers with different genotypes. While the pre-rifampin enzyme activity difference between *1/*1 and *3/*3 was six-folds, the induction effect was around two-fold regardless of the genotype.42 It is important to emphasize that the study duration was only four days,42 which may not represent the subsequent enzyme induction phases. George et al43, using phenytoin as a probe drug, studied the total effect of one month of rifampin therapy on CYP2C9 various genotypes in forty-eight new TB patients. Joined data from twelve mutant patients showed that rifampin’s induction potential was statistically significant regardless of the genotype.43 While these studies suggest that rifampin increases the CYP2C9 concentration with the same ratio,42,43 the catalytic activity would be genotype-dependent.5
Our case may represent an example of phenoconversion, a phenomenon of genotype-phenotype mismatch, in which an individual’s metabolizing enzyme is functionally converted from a poor metabolizer to an intermediate or extensive metabolizer or vice versa due to the use of an enzyme inducer or inhibitor, respectively.44–46 Rifampin shifted the patient from her genotype-based poor metabolizer status to a functional rapid metabolizer status that required warfarin daily dose escalation.46 However, because the patient’s gene-based estimated warfarin dose was 2 mg, escalation to 10 mg, a feasible dose compared with other interaction reports, was sufficient to attain therapeutic INR.
While the patient confirmed complete adherence, the INR dropped to 1.0 on the last day of warfarin therapy and day 97 of rifampin. One explanation may be a late CYP2C9 induction phase by rifampin. CYP2C9 half-life has been reported to be much longer than other CYPs as CYP3A4. Shibata et al35 monitored CYP2C9 and CYP3A activities in two cases who were receiving rifampin and warfarin concomitantly after rifampin discontinuation. The CYP2C9 estimated half-lives were 25.7 and 16.8 days, compared with CYP3A half-lives of 2.4 and 11.5 days, in the first and second case, respectively.35 Indicating that the CYP2C9 turnover can take up to months. Moreover, having a CYP2C9*3*3 genotype might have prolonged the time-course to maximal induction. Since warfarin was stopped at that point, verifying these explanations is not possible.
Conclusion
This case report demonstrated the highly significant effect of rifampin metabolic induction and genetic polymorphism on warfarin dose requirements. Our findings reveal a genetic explanation of the variable patients’ responses to different warfarin doses while on rifampin. While wild-type patients are not likely to respond to extreme warfarin doses due to the drug interaction with rifampin, patients with loss-of-function genetic variants of CYP2C9 and VKORC1 are more likely to respond at a feasible dose level. Future studies are warranted to evaluate the genotype variants’ effect on the interaction, which might benefit in selecting likely warfarin responders when rifampin therapy is needed.
Ethical Approval
Ethical approval for this case report was provided by the Medical Research Committee (MRC) of Hamad Medical Corporation (HMC) (#MRC-04-20-428).
Author Contributions
All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that they have no conflicts of interest in this work. | Intravenous drip | DrugAdministrationRoute | CC BY | 33542643 | 18,944,693 | 2021 |
What was the administration route of drug 'HUMAN IMMUNOGLOBULIN G'? | Genetic Polymorphism Effect on Warfarin-Rifampin Interaction: A Case Report and Review of Literature.
Warfarin-rifampin interaction has been reported since the 1970s. Due to rifampin's strong induction of CYP2C9, most cases could not attain the target international normalized ratio (INR) despite warfarin dose escalation. Genetic polymorphisms determine up to 50% of warfarin dose variability. A 38-year-old woman was started on warfarin and rifampin for cerebral venous sinus thrombosis and pulmonary tuberculosis. Over six weeks, the daily warfarin dose was increased from 3 to 10 mg to attain three consecutive in-clinic therapeutic INRs. She completed three complications-free months of warfarin treatment with time in therapeutic range (TTR) of 46%. We performed retrospective genetic testing to determine the patient's CYP2C9, CYP4F2, and VKORC1 genotypes and whether they had affected the interaction outcome. The analysis revealed that the subject carries CYP2C9*3*3 and VKORC1-1639 (GA) mutations, classifying her as a slow metabolizer and, hence, highly warfarin-sensitive. This was reflected on how the case responded to a relatively lower dose than previously reported cases that did not achieve the target on warfarin daily doses up to 35 mg. This is the first report addressing the genotype effect on this interaction. Patients with genetic variants requiring low warfarin doses are more likely to respond at a feasible dose while on rifampin. Future studies to evaluate warfarin-rifampin-gene interaction are warranted.
Introduction
Warfarin, a vitamin K antagonist, remains the preferred oral anticoagulation for atrial fibrillation with prosthetic cardiac valves or rheumatic heart disease with more than mild mitral stenosis, and venous thromboembolism (VTE) at unusual sites.1,2 It is a mixture of two racemic isomers, R and S-warfarin. Both impair the vitamin k-dependent proteins production via inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).3 Cytochrome P450 2C9 (CYP2C9) extensively metabolizes S-warfarin, the stereoisomer of predominant potency, to the inactive 7-hydroxywarfarin.3
VKORC1 and CYP2C9 genetic polymorphisms, with other genetic variants, determine up to 50% of warfarin dose variance.3,4 The most studied and common variant alleles of CYP2C9 are CYP2C9*2 (rs1799853) and *3 (rs1057910), which result from missense mutations associated with diminished catalytic activity, poor warfarin metabolism, and decreased dose requirements.5
VKORC1–1639G>A (rs9923231) genotype variants (GA and AA) contribute majorly to sensitizing warfarin.5 On the other hand, CYP4F2*3, a nonsynonymous variant of the gene coding for the primary liver vitamin K oxidase, CYP4F2, has been associated in some studies with a modest increase of warfarin dose requirements (8–11%).3 These mutations were incorporated, with other clinical factors, into dosing algorithms which were shown to provide better warfarin dose prediction.3 Gage and International Warfarin Pharmacogenetics Consortium (IWPC), are among the most widely studied algorithms and are contained in the website (www.warfarindosing.org), which calculates the initial dose with the ability to adjust for CYP2C9*5, *6, CYP4F2, and GGCX genotypes.3 Genetic-based dosing of warfarin upon initiation was shown in some studies to improve target international normalized ratio (INR) attainment and time in therapeutic range (TTR) during the initial month.6 This was mainly mediated by CYP2C9 and VKORC1 polymorphisms which significantly impact the maintenance dose requirement.3,4,7 The Food and Drug Administration (FDA) has also approved warfarin label modifications with dosing guidance based on the CYP2C9 and VKORC1 genotypes.8 Apart from genetics, numerous warfarin drug interactions have been reported requiring dose adjustments and frequent INR monitoring to avoid bleeding or anticoagulation failure.9
Antituberculous management includes rifampin, isoniazid, ethambutol, and pyrazinamide. While ethambutol and pyrazinamide are neither cytochrome P450s inhibitors nor reported to affect warfarin, isoniazid is a week inhibitor of CYP3A4, which is not FDA classified as a clinical index inhibitor.10 While it has been reported to increase warfarin’s hypoprothrombinemic effect in two cases,11,12 the interaction magnitude is considered minimal, with no action recommended.13
Rifampin, a life-saving antimicrobial for tuberculosis, endocarditis, and meningitis,14 is a clinically significant inducer of CYP2B6, CYP2C19, CYP2C8, CYP2C9, and CYP3A4, as well as P glycoprotein.10 It induces CYP2C9 transcription by binding to its main De-novo synthesis regulatory nuclear receptor, pregnane X receptor (PXR).5,15 The binding increases the CYP2C9 mRNA expression by up to six-folds.15 That leads to a higher amount of the enzyme, and extensive metabolism of the substrate/victim drug.15 While the onset of induction can be few days with rifampin,16 the time required to reach maximal enzyme abundance and new steady state is more than two weeks based on the CYP turnover and degradation half-life.16–19 FDA classifies rifampin as a moderate inducer of CYP2C9, defined as a decrease in the substrates area under the concentration-time curve (AUC) by 50% to less than 80%.10 That was based on two healthy-volunteers studies with probe substrates, S-warfarin and tolbutamide.20,21 However, the first study’s duration was only four days,20 and 12–15 days in the latter,21 which, yet, showed high variability of CYP2C9 activity (1–7.4-fold).22 These short durations may imply that rifampin is rather a strong CYP2C9 inducer as AUC ratio were measured before reaching maximal induction.16–19
Rifampin almost eliminates warfarin’s therapeutic effect, which required extensive dose escalation in all cases and is typically associated with the inability to maintain therapeutic range. The interaction has been described since the 1970s.20,23,24 Till the late 1980s, multiple reports showed a significant increase in warfarin dose requirements with rifampin.25–27 After the INR test was universally adopted,28 several reports demonstrated that most patients who required the anti-infective along with anticoagulation were unable to maintain target INR.14,29–36
This report aims to describe a case who received warfarin and rifampin concomitantly and the interaction outcome, and to perform genetic testing to determine the patient’s CYP2C9, VKORC1, CALU, and CYP4F2 genotypes and whether they could explain the response to warfarin dose escalation.
Case Description
A 38-year-old Ethiopian woman with a weight of 60 kg, a height of 150 cm, and a history of diabetes and immune thrombocytopenic purpura (ITP) on metformin and chronic eltrombopag presented to the emergency of Hamad General Hospital in Qatar on January 13, 2020, with dizziness, severe diffuse headache, photophobia, and multiple vomiting for three days. Intracranial computed tomography (CT) venogram showed cerebral venous sinus thrombosis (CVST). Eltrombopag was stopped. Since the Glasgow Coma Score (GCS) dropped to 11, the patient was admitted to the medical ICU with sedation, analgesia, and close neurologic observation. Because of low platelet count (PC), 32X109/L, she was started on 0.5 gm/kg intravenous immunoglobulin (IVIG) plus steroids for three days to raise the PC above 50X109/L in order to initiate anticoagulation. Two days later, PC reached 75X109/L, and heparin continuous IV infusion was initiated with platelets transfusion as the patient was neurologically deteriorating, and repeated CT showed extension of thrombosis with intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). On January 19, although PC was maintained above 200X109/L, heparin was suspended due to a sharp drop of hemoglobin to 5 gm/dL with no identified source of bleeding. The patient was transfused immediately. The next day hemoglobin increased to 8 then maintained at 9–10 gm/dL. Heparin was resumed on January 23. Hematology planned to start rituximab for ITP, yet the Quantiferone test for tuberculosis (TB) was positive. CT chest on January 26 revealed consolidation patches in the right upper and middle lung lobes suggesting active TB. Since bronchoscopy was not feasible due to the high bleeding risk, the pulmonary, MICU, and infectious diseases teams decided to start empiric anti-TB medications based on radiology and follow response by imaging. The rituximab plan was aborted.
On January 29, the patient was started on daily rifampin 600 mg, isoniazid 300 mg, ethambutol 1100 mg, and pyrazinamide 1600 mg. She was transferred to the medicine ward after sedation withdrawal. On January 31, anticoagulation was shifted to daily oral warfarin 5 mg with twice-daily enoxaparin 60 mg as bridging. Three days later, warfarin was held for one day then resumed with dose reduction to 3 mg due to a sharp INR increase to 3.7. The next day, INR dropped to 1.0, then increased gradually after three days to 1.7, 1.9, and reached 2.0 on February 9. Enoxaparin was stopped, and the patient was discharged on warfarin 3 mg, anti-TB medications, pyridoxine 40 mg, metformin 500 mg twice daily, and metoprolol 50 mg twice daily. Following in anticoagulation clinic, on February 12, after 14 days of rifampin, INR was 2.6; therefore, the same warfarin dose continued. Although decreased to 1.7 on February 16, the warfarin dose remained. A week later, INR dropped to 1.3, so warfarin was escalated to 4 mg/day, and enoxaparin resumed. Over the next three weeks, the dose was gradually escalated up to 10 mg, after four days of which, INR reached 2.3 on March 15. Enoxaparin was stopped, and the patient was maintained on daily warfarin 10 mg. The anti-TB medications were switched to (Rifampin 600 mg/Isoniazid 300 mg) on March 24. INR was maintained in the next two clinic visits at 3 and 2.9 on March 23 and April 6, respectively. On May 4, the INR was 1.0 in the last anticoagulation clinic visit. That could not be explained by non-compliance as per the patient interview. The planned three-month duration of anticoagulation ended, so warfarin was stopped. Rifampin/isoniazid continued till July 14, 2020. The patient was interviewed on each visit and excluded any adverse effects. She had normal follow up laboratory values throughout the treatment. Details of warfarin daily dose and INR are shown in [Figure 1].Figure 1 This graph represents the daily warfarin dose, rifampin days, and INR overtime. The bottom x-axis represents dates. The top x-axis represents rifampin days. The left y-axis represents the daily warfarin dose in milligrams and is shown by the vertical bars. The right Y-axis represents the INR and is shown by the black diamond points. The therapeutic range is indicated between the two dotted lines (2.0–3.0).
Methods
The patient was approached by one of the study investigators and explained the reasons and expectations of the research. The patient confirmed her understanding, and agreement to provide saliva sample for genetic testing as well as to have the case published by signing an Institutional Review Board (IRB) approved informed consent form. She was asked to provide a saliva sample using Oragene•DNA (OG-500) self-collection kit (DNA genotek, USA). Hereafter, the kit was kept in a water bath at 50 C° overnight for DNA extraction. The prepIT®•L2P standard protocol for the purification of DNA was used for DNA extraction.37 The purified DNA’s quality and quantity were evaluated by Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific). Finally, the sample was genotyped for detecting the following genetic variants: CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), CYP4F2*3 (rs2108622),VKORC1 (rs9923231), VKORC1 Asp36Tyr (rs61742245), and CALU (rs339097). This was performed using the QuantStudio™ 5 Real-Time Polymerase Chain Reaction (PCR) system for Human Identification, 96-well, 0.2 mL, desktop manufactured by Applied BiosystemsTM.
Results
The genotyping revealed that the patient is a carrier of CYP2C9*3*3 homozygous, VKORC1-1639 (GA) heterozygous, and CYP4F2 (CC) wild-type homozygous. Based on this genetic profile, the subject is considered a slow metabolizer which indicates high warfarin sensitivity. On the other hand, both VKORC1 Asp36Tyr (CT) and CALU (AG) genotyping were heterozygous indicating partial warfarin resistance.
As it is shown in Figure 1, the sudden rise in INR with a moderate standard warfarin dose of 5 mg is very well explained by the CYP2C9 loss of function genetic variant carried by this patient (CYP2C9*3/*3). A few weeks later and with the interacting effect of rifampin reaching its peak, the daily warfarin dose requirement for the patient increased reaching 10 mg.
Discussion
In this case report, we observed warfarin–rifampin drug interaction in a patient with CVST and pulmonary TB. The interaction management required warfarin dose-escalation, frequent INR monitoring, and low molecular weight heparin (LMWH) bridging over six weeks. On a daily warfarin dose of 10 mg, our patient attained three consecutive therapeutic INR levels in three clinic visits. That is considered a “stable warfarin dose,” as defined in most studies,38 despite the one subtherapeutic INR in the last treatment day. The TTR, calculated by the Rosendaal method,39 was 46.2%. The three-month warfarin anticoagulation treatment, combined with rifampin, was completed with difficulty, yet no complications.
Since the World Health Organization (WHO) adopted INR in the 1980s,28 more than nineteen cases of warfarin–rifampin interaction have been reported.14,29–36 Despite extensive warfarin dose escalation over a prolonged time, the majority could not attain target INR while on the combination.14,29–32,34–36 Cases are summarized in [Table 1]. Excessive warfarin exposure and hemorrhage after rifampin discontinuation have been reported, implying the importance of close monitoring and careful dose de-escalation after rifampin stoppage.33,40 Due to the lack of laboratory monitorable parameters like INR, labels of direct oral anticoagulants (DOACs), that are substrates of CYP3A4 and/or P glycoprotein, recommend avoiding concomitant use with rifampin to avoid unmanageable therapeutic failures.41Table 1 Summary of Studies of Warfarin–Rifampicin Interaction with INR Monitoring
Study Patient’s Data Sequence and Duration During Concomitant Use After Rifampin Stopped
Case No. Age Sex Warfarin Indication Rifampin Start in Relation to Warfarin Start (Days) Concomitant Duration (Days) Average Initial Warfarin Dose (mg/day)a,b Average Last Warfarin Dose (mg/day)a,b Warfarin Dose Increase Target INR Target Attained Time to Stable Warfarin Dosec (Days) TTR Stable Warfarin Dosec (mg/day) Time to Stable Warfarin Dosec (Days)
Casner, 199629 1 36 M PE −2 20 7.8b 20b 169% 2.0–3.0 No NA NA 10 10
Lee & Trasher, 200130 1 58 M LVT −120 113 7.5b 25b 233% 2.0–3.0 No NA NA 7.5 35
Kim et al, 200631 1 79 M DVT −11 30 5a 30b 500% 2.0–3.0 No NA NA 6 60
Krajewski, 201032 1 71 M AF +14 45 5.7a 25b 340% 2.0–3.0 No NA NA 5 120
Martins et al, 201333 1 59 F AF +300 203 6.4a 11.4a 78% 2.0–3.0 Yes 104 50% 5.4 60
Maina et al, 201336 1 17 F DVT −7 UNK 10b 27.7a 177.3% 2.0–3.0 Yes 63 52% UNK UNK
2 24 F RHD/LAT −42 UNK 5b 5.8a 16% 2.0–3.0 Yesd 66 67% UNK UNK
3 36 M DVT −44 UNK 12b 11.4b −4.8% 2.0–3.0 No NA 24% UNK UNK
4 64 F DVT −45 UNK 10b 11.5b 15.3% 2.0–3.0 No NA 47% UNK UNK
5 22 F DVT −88 UNK 10b 4.8a −37% 2.0–3.0 Yese 12 54% UNK UNK
6 9 M DVT 0 UNK 5b 5.3b 5.8% 2.0–3.0 No NA 53% UNK UNK
7 49 M DVT −3 UNK 5b 9.5a 89.3% 2.0–3.0 Yes 49 42% UNK UNK
8 30 F PE −35 UNK 5b 27a 440.9% 2.0–3.0 Yes 67 30% UNK UNK
9 29 F DVT −31 UNK 5b 11.8a 135.8% 2.0–3.0 Yes 7 40% UNK UNK
10 41 M Stroke & DVT −46 UNK 6b 6.5a 8.3% 2.0–3.0 Yes 63 66% UNK UNK
Dawson et al, 201634 1 60 M MV replaced +UNK 42 8a 35b 340% 2.5–3.5 No NA NA 8 28
Fahmi et al, 201614 1 34 F MV replaced +19 years 42 7.5a 30b 300% 2.5–3.5 No NA NA 11.4 35
Shibata et al, 201735 1 70 F Stroke +UNK 365 4a 15a 275% 2.0–3.0 Yes UNK UNK 4 30
2 80 F AF +UNK 330 2.5a 10b 300% 2.0–3.0 No NA NA 3 60
Notes: Maina et al did not report rifampin cessation times or confirmed adherence to medications other than warfarin,36 which may have affected response. aTherapeutic INR attained on the dose. bTherapeutic INR not attained on the dose. cDefined as three consecutive therapeutic INR levels on the same warfarin dose (In Maina et al case series, defined as two consecutive therapeutic INRs). dOccasional warfarin overdoses. eMissed warfarin doses.
Abbreviations: AF, atrial fibrillation; DVT, deep venous thrombosis; INR, international normalized ratio; LAT, left arterial thrombosis; LVT, left ventricular thrombus; MV, mitral valve; NA, not applicable; PE, pulmonary embolism; RHD, rheumatic heart disease; TTR, time in therapeutic range; UNK, unknown.
Our case had one supratherapeutic INR after three warfarin doses of 5 mg and five days of rifampin commencement explained by her CYP2C9 poor metabolizer phenotype. She had two consecutive therapeutic INR readings after 12 and 14 days of rifampin on daily warfarin 3 mg. However, INR dropped below therapeutic after 17 days of rifampin, likely due to the latter’s liver enzyme induction. Comparable patterns have been observed in other cases when the commencement sequence was a few days apart or when rifampin was added to chronic warfarin. In 1996, Casner29 reported a patient who had one therapeutic INR after 13 rifampin days, which declined to subtherapeutic until ten days after the rifampin stoppage. The case reported by Kim et al31 had one in-target INR after warfarin 20 mg, which dropped then increased back to target for a week after escalation to 25 mg on rifampin days 25 and 33. Then, the INR declined steadily even after warfarin dose was raised to 30 mg.31 An INR of 4.4 after three days of rifampin in a mechanical valve replacement case, reported by Fahmi et al14, necessitated the holding of warfarin for five days. Then INR dropped to below the desired 7–14 days from the combination. In the case reported by Dawson et al34, it took 17 days for the interaction to become apparent. These reports, aligned with our report, indicate that the time-course to rifampin CYP2C9 induction is more than 14 days. Studies of this rifampin duration or less are inadequate to evaluate the full impact of such interaction. It is vital to monitor INR frequently during the first two weeks of rifampin and shortly after to avoid exposing the patient to subtherapeutic levels with no bridging.
Our patient’s CYP2C9 genotyping revealed that she is a slow metabolizer with a homozygous CYP2C9*3*3, which indicates high warfarin sensitivity and the lowest dose requirements. Additionally, VKORC1 –1639G>A genotyping showed upstream variant (AG) requiring lower warfarin doses.3 The patient reached three consecutive therapeutic INRs on a daily warfarin dose of 10 mg, which is generally considered a high dose. However, compared with most cases that received rifampin with warfarin doses up to 35 mg and no target attainment, 10 mg represents a relatively reasonable dose. The estimated therapeutic warfarin dose for our case, calculated using clinical factors via www.warfarindosing.org, was 5.8 mg/day. However, when the genotyping results were added to the calculation, the estimated therapeutic dose was 2 mg/day. The FDA warfarin label expected maintenance dosing for CYP2C9*3/*3 combined with VKORC1 (AG) is ranged from 0.5 to 2 mg/day.8 That implies that 10 mg is at least a 400% increase from the therapeutic dose without rifampin. Interestingly, most reported cases who reached therapeutic INR during the concomitant warfarin–rifampin use received relatively low warfarin doses around 10 to 15 mg/day.33,35,36 Since the reported cases were not tested for genotype variations; genetic polymorphism might represent an unrevealed explanation of the variable warfarin responses among patients during rifampin use.
Up to our knowledge, this is the first warfarin–rifampin interaction case report to address genetic polymorphism as a contributing factor in the interaction outcome since it was first described in the 1970s. Nevertheless, there are studies of rifampin induction effect on CYP2C9 variants. Vormfelde et al42 used tolbutamide as a probe substrate to evaluate rifampin effect on CYP2C9 activity in 128 healthy volunteers with different genotypes. While the pre-rifampin enzyme activity difference between *1/*1 and *3/*3 was six-folds, the induction effect was around two-fold regardless of the genotype.42 It is important to emphasize that the study duration was only four days,42 which may not represent the subsequent enzyme induction phases. George et al43, using phenytoin as a probe drug, studied the total effect of one month of rifampin therapy on CYP2C9 various genotypes in forty-eight new TB patients. Joined data from twelve mutant patients showed that rifampin’s induction potential was statistically significant regardless of the genotype.43 While these studies suggest that rifampin increases the CYP2C9 concentration with the same ratio,42,43 the catalytic activity would be genotype-dependent.5
Our case may represent an example of phenoconversion, a phenomenon of genotype-phenotype mismatch, in which an individual’s metabolizing enzyme is functionally converted from a poor metabolizer to an intermediate or extensive metabolizer or vice versa due to the use of an enzyme inducer or inhibitor, respectively.44–46 Rifampin shifted the patient from her genotype-based poor metabolizer status to a functional rapid metabolizer status that required warfarin daily dose escalation.46 However, because the patient’s gene-based estimated warfarin dose was 2 mg, escalation to 10 mg, a feasible dose compared with other interaction reports, was sufficient to attain therapeutic INR.
While the patient confirmed complete adherence, the INR dropped to 1.0 on the last day of warfarin therapy and day 97 of rifampin. One explanation may be a late CYP2C9 induction phase by rifampin. CYP2C9 half-life has been reported to be much longer than other CYPs as CYP3A4. Shibata et al35 monitored CYP2C9 and CYP3A activities in two cases who were receiving rifampin and warfarin concomitantly after rifampin discontinuation. The CYP2C9 estimated half-lives were 25.7 and 16.8 days, compared with CYP3A half-lives of 2.4 and 11.5 days, in the first and second case, respectively.35 Indicating that the CYP2C9 turnover can take up to months. Moreover, having a CYP2C9*3*3 genotype might have prolonged the time-course to maximal induction. Since warfarin was stopped at that point, verifying these explanations is not possible.
Conclusion
This case report demonstrated the highly significant effect of rifampin metabolic induction and genetic polymorphism on warfarin dose requirements. Our findings reveal a genetic explanation of the variable patients’ responses to different warfarin doses while on rifampin. While wild-type patients are not likely to respond to extreme warfarin doses due to the drug interaction with rifampin, patients with loss-of-function genetic variants of CYP2C9 and VKORC1 are more likely to respond at a feasible dose level. Future studies are warranted to evaluate the genotype variants’ effect on the interaction, which might benefit in selecting likely warfarin responders when rifampin therapy is needed.
Ethical Approval
Ethical approval for this case report was provided by the Medical Research Committee (MRC) of Hamad Medical Corporation (HMC) (#MRC-04-20-428).
Author Contributions
All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that they have no conflicts of interest in this work. | Intravenous (not otherwise specified) | DrugAdministrationRoute | CC BY | 33542643 | 18,944,693 | 2021 |
What was the administration route of drug 'WARFARIN SODIUM'? | Genetic Polymorphism Effect on Warfarin-Rifampin Interaction: A Case Report and Review of Literature.
Warfarin-rifampin interaction has been reported since the 1970s. Due to rifampin's strong induction of CYP2C9, most cases could not attain the target international normalized ratio (INR) despite warfarin dose escalation. Genetic polymorphisms determine up to 50% of warfarin dose variability. A 38-year-old woman was started on warfarin and rifampin for cerebral venous sinus thrombosis and pulmonary tuberculosis. Over six weeks, the daily warfarin dose was increased from 3 to 10 mg to attain three consecutive in-clinic therapeutic INRs. She completed three complications-free months of warfarin treatment with time in therapeutic range (TTR) of 46%. We performed retrospective genetic testing to determine the patient's CYP2C9, CYP4F2, and VKORC1 genotypes and whether they had affected the interaction outcome. The analysis revealed that the subject carries CYP2C9*3*3 and VKORC1-1639 (GA) mutations, classifying her as a slow metabolizer and, hence, highly warfarin-sensitive. This was reflected on how the case responded to a relatively lower dose than previously reported cases that did not achieve the target on warfarin daily doses up to 35 mg. This is the first report addressing the genotype effect on this interaction. Patients with genetic variants requiring low warfarin doses are more likely to respond at a feasible dose while on rifampin. Future studies to evaluate warfarin-rifampin-gene interaction are warranted.
Introduction
Warfarin, a vitamin K antagonist, remains the preferred oral anticoagulation for atrial fibrillation with prosthetic cardiac valves or rheumatic heart disease with more than mild mitral stenosis, and venous thromboembolism (VTE) at unusual sites.1,2 It is a mixture of two racemic isomers, R and S-warfarin. Both impair the vitamin k-dependent proteins production via inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).3 Cytochrome P450 2C9 (CYP2C9) extensively metabolizes S-warfarin, the stereoisomer of predominant potency, to the inactive 7-hydroxywarfarin.3
VKORC1 and CYP2C9 genetic polymorphisms, with other genetic variants, determine up to 50% of warfarin dose variance.3,4 The most studied and common variant alleles of CYP2C9 are CYP2C9*2 (rs1799853) and *3 (rs1057910), which result from missense mutations associated with diminished catalytic activity, poor warfarin metabolism, and decreased dose requirements.5
VKORC1–1639G>A (rs9923231) genotype variants (GA and AA) contribute majorly to sensitizing warfarin.5 On the other hand, CYP4F2*3, a nonsynonymous variant of the gene coding for the primary liver vitamin K oxidase, CYP4F2, has been associated in some studies with a modest increase of warfarin dose requirements (8–11%).3 These mutations were incorporated, with other clinical factors, into dosing algorithms which were shown to provide better warfarin dose prediction.3 Gage and International Warfarin Pharmacogenetics Consortium (IWPC), are among the most widely studied algorithms and are contained in the website (www.warfarindosing.org), which calculates the initial dose with the ability to adjust for CYP2C9*5, *6, CYP4F2, and GGCX genotypes.3 Genetic-based dosing of warfarin upon initiation was shown in some studies to improve target international normalized ratio (INR) attainment and time in therapeutic range (TTR) during the initial month.6 This was mainly mediated by CYP2C9 and VKORC1 polymorphisms which significantly impact the maintenance dose requirement.3,4,7 The Food and Drug Administration (FDA) has also approved warfarin label modifications with dosing guidance based on the CYP2C9 and VKORC1 genotypes.8 Apart from genetics, numerous warfarin drug interactions have been reported requiring dose adjustments and frequent INR monitoring to avoid bleeding or anticoagulation failure.9
Antituberculous management includes rifampin, isoniazid, ethambutol, and pyrazinamide. While ethambutol and pyrazinamide are neither cytochrome P450s inhibitors nor reported to affect warfarin, isoniazid is a week inhibitor of CYP3A4, which is not FDA classified as a clinical index inhibitor.10 While it has been reported to increase warfarin’s hypoprothrombinemic effect in two cases,11,12 the interaction magnitude is considered minimal, with no action recommended.13
Rifampin, a life-saving antimicrobial for tuberculosis, endocarditis, and meningitis,14 is a clinically significant inducer of CYP2B6, CYP2C19, CYP2C8, CYP2C9, and CYP3A4, as well as P glycoprotein.10 It induces CYP2C9 transcription by binding to its main De-novo synthesis regulatory nuclear receptor, pregnane X receptor (PXR).5,15 The binding increases the CYP2C9 mRNA expression by up to six-folds.15 That leads to a higher amount of the enzyme, and extensive metabolism of the substrate/victim drug.15 While the onset of induction can be few days with rifampin,16 the time required to reach maximal enzyme abundance and new steady state is more than two weeks based on the CYP turnover and degradation half-life.16–19 FDA classifies rifampin as a moderate inducer of CYP2C9, defined as a decrease in the substrates area under the concentration-time curve (AUC) by 50% to less than 80%.10 That was based on two healthy-volunteers studies with probe substrates, S-warfarin and tolbutamide.20,21 However, the first study’s duration was only four days,20 and 12–15 days in the latter,21 which, yet, showed high variability of CYP2C9 activity (1–7.4-fold).22 These short durations may imply that rifampin is rather a strong CYP2C9 inducer as AUC ratio were measured before reaching maximal induction.16–19
Rifampin almost eliminates warfarin’s therapeutic effect, which required extensive dose escalation in all cases and is typically associated with the inability to maintain therapeutic range. The interaction has been described since the 1970s.20,23,24 Till the late 1980s, multiple reports showed a significant increase in warfarin dose requirements with rifampin.25–27 After the INR test was universally adopted,28 several reports demonstrated that most patients who required the anti-infective along with anticoagulation were unable to maintain target INR.14,29–36
This report aims to describe a case who received warfarin and rifampin concomitantly and the interaction outcome, and to perform genetic testing to determine the patient’s CYP2C9, VKORC1, CALU, and CYP4F2 genotypes and whether they could explain the response to warfarin dose escalation.
Case Description
A 38-year-old Ethiopian woman with a weight of 60 kg, a height of 150 cm, and a history of diabetes and immune thrombocytopenic purpura (ITP) on metformin and chronic eltrombopag presented to the emergency of Hamad General Hospital in Qatar on January 13, 2020, with dizziness, severe diffuse headache, photophobia, and multiple vomiting for three days. Intracranial computed tomography (CT) venogram showed cerebral venous sinus thrombosis (CVST). Eltrombopag was stopped. Since the Glasgow Coma Score (GCS) dropped to 11, the patient was admitted to the medical ICU with sedation, analgesia, and close neurologic observation. Because of low platelet count (PC), 32X109/L, she was started on 0.5 gm/kg intravenous immunoglobulin (IVIG) plus steroids for three days to raise the PC above 50X109/L in order to initiate anticoagulation. Two days later, PC reached 75X109/L, and heparin continuous IV infusion was initiated with platelets transfusion as the patient was neurologically deteriorating, and repeated CT showed extension of thrombosis with intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). On January 19, although PC was maintained above 200X109/L, heparin was suspended due to a sharp drop of hemoglobin to 5 gm/dL with no identified source of bleeding. The patient was transfused immediately. The next day hemoglobin increased to 8 then maintained at 9–10 gm/dL. Heparin was resumed on January 23. Hematology planned to start rituximab for ITP, yet the Quantiferone test for tuberculosis (TB) was positive. CT chest on January 26 revealed consolidation patches in the right upper and middle lung lobes suggesting active TB. Since bronchoscopy was not feasible due to the high bleeding risk, the pulmonary, MICU, and infectious diseases teams decided to start empiric anti-TB medications based on radiology and follow response by imaging. The rituximab plan was aborted.
On January 29, the patient was started on daily rifampin 600 mg, isoniazid 300 mg, ethambutol 1100 mg, and pyrazinamide 1600 mg. She was transferred to the medicine ward after sedation withdrawal. On January 31, anticoagulation was shifted to daily oral warfarin 5 mg with twice-daily enoxaparin 60 mg as bridging. Three days later, warfarin was held for one day then resumed with dose reduction to 3 mg due to a sharp INR increase to 3.7. The next day, INR dropped to 1.0, then increased gradually after three days to 1.7, 1.9, and reached 2.0 on February 9. Enoxaparin was stopped, and the patient was discharged on warfarin 3 mg, anti-TB medications, pyridoxine 40 mg, metformin 500 mg twice daily, and metoprolol 50 mg twice daily. Following in anticoagulation clinic, on February 12, after 14 days of rifampin, INR was 2.6; therefore, the same warfarin dose continued. Although decreased to 1.7 on February 16, the warfarin dose remained. A week later, INR dropped to 1.3, so warfarin was escalated to 4 mg/day, and enoxaparin resumed. Over the next three weeks, the dose was gradually escalated up to 10 mg, after four days of which, INR reached 2.3 on March 15. Enoxaparin was stopped, and the patient was maintained on daily warfarin 10 mg. The anti-TB medications were switched to (Rifampin 600 mg/Isoniazid 300 mg) on March 24. INR was maintained in the next two clinic visits at 3 and 2.9 on March 23 and April 6, respectively. On May 4, the INR was 1.0 in the last anticoagulation clinic visit. That could not be explained by non-compliance as per the patient interview. The planned three-month duration of anticoagulation ended, so warfarin was stopped. Rifampin/isoniazid continued till July 14, 2020. The patient was interviewed on each visit and excluded any adverse effects. She had normal follow up laboratory values throughout the treatment. Details of warfarin daily dose and INR are shown in [Figure 1].Figure 1 This graph represents the daily warfarin dose, rifampin days, and INR overtime. The bottom x-axis represents dates. The top x-axis represents rifampin days. The left y-axis represents the daily warfarin dose in milligrams and is shown by the vertical bars. The right Y-axis represents the INR and is shown by the black diamond points. The therapeutic range is indicated between the two dotted lines (2.0–3.0).
Methods
The patient was approached by one of the study investigators and explained the reasons and expectations of the research. The patient confirmed her understanding, and agreement to provide saliva sample for genetic testing as well as to have the case published by signing an Institutional Review Board (IRB) approved informed consent form. She was asked to provide a saliva sample using Oragene•DNA (OG-500) self-collection kit (DNA genotek, USA). Hereafter, the kit was kept in a water bath at 50 C° overnight for DNA extraction. The prepIT®•L2P standard protocol for the purification of DNA was used for DNA extraction.37 The purified DNA’s quality and quantity were evaluated by Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific). Finally, the sample was genotyped for detecting the following genetic variants: CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), CYP4F2*3 (rs2108622),VKORC1 (rs9923231), VKORC1 Asp36Tyr (rs61742245), and CALU (rs339097). This was performed using the QuantStudio™ 5 Real-Time Polymerase Chain Reaction (PCR) system for Human Identification, 96-well, 0.2 mL, desktop manufactured by Applied BiosystemsTM.
Results
The genotyping revealed that the patient is a carrier of CYP2C9*3*3 homozygous, VKORC1-1639 (GA) heterozygous, and CYP4F2 (CC) wild-type homozygous. Based on this genetic profile, the subject is considered a slow metabolizer which indicates high warfarin sensitivity. On the other hand, both VKORC1 Asp36Tyr (CT) and CALU (AG) genotyping were heterozygous indicating partial warfarin resistance.
As it is shown in Figure 1, the sudden rise in INR with a moderate standard warfarin dose of 5 mg is very well explained by the CYP2C9 loss of function genetic variant carried by this patient (CYP2C9*3/*3). A few weeks later and with the interacting effect of rifampin reaching its peak, the daily warfarin dose requirement for the patient increased reaching 10 mg.
Discussion
In this case report, we observed warfarin–rifampin drug interaction in a patient with CVST and pulmonary TB. The interaction management required warfarin dose-escalation, frequent INR monitoring, and low molecular weight heparin (LMWH) bridging over six weeks. On a daily warfarin dose of 10 mg, our patient attained three consecutive therapeutic INR levels in three clinic visits. That is considered a “stable warfarin dose,” as defined in most studies,38 despite the one subtherapeutic INR in the last treatment day. The TTR, calculated by the Rosendaal method,39 was 46.2%. The three-month warfarin anticoagulation treatment, combined with rifampin, was completed with difficulty, yet no complications.
Since the World Health Organization (WHO) adopted INR in the 1980s,28 more than nineteen cases of warfarin–rifampin interaction have been reported.14,29–36 Despite extensive warfarin dose escalation over a prolonged time, the majority could not attain target INR while on the combination.14,29–32,34–36 Cases are summarized in [Table 1]. Excessive warfarin exposure and hemorrhage after rifampin discontinuation have been reported, implying the importance of close monitoring and careful dose de-escalation after rifampin stoppage.33,40 Due to the lack of laboratory monitorable parameters like INR, labels of direct oral anticoagulants (DOACs), that are substrates of CYP3A4 and/or P glycoprotein, recommend avoiding concomitant use with rifampin to avoid unmanageable therapeutic failures.41Table 1 Summary of Studies of Warfarin–Rifampicin Interaction with INR Monitoring
Study Patient’s Data Sequence and Duration During Concomitant Use After Rifampin Stopped
Case No. Age Sex Warfarin Indication Rifampin Start in Relation to Warfarin Start (Days) Concomitant Duration (Days) Average Initial Warfarin Dose (mg/day)a,b Average Last Warfarin Dose (mg/day)a,b Warfarin Dose Increase Target INR Target Attained Time to Stable Warfarin Dosec (Days) TTR Stable Warfarin Dosec (mg/day) Time to Stable Warfarin Dosec (Days)
Casner, 199629 1 36 M PE −2 20 7.8b 20b 169% 2.0–3.0 No NA NA 10 10
Lee & Trasher, 200130 1 58 M LVT −120 113 7.5b 25b 233% 2.0–3.0 No NA NA 7.5 35
Kim et al, 200631 1 79 M DVT −11 30 5a 30b 500% 2.0–3.0 No NA NA 6 60
Krajewski, 201032 1 71 M AF +14 45 5.7a 25b 340% 2.0–3.0 No NA NA 5 120
Martins et al, 201333 1 59 F AF +300 203 6.4a 11.4a 78% 2.0–3.0 Yes 104 50% 5.4 60
Maina et al, 201336 1 17 F DVT −7 UNK 10b 27.7a 177.3% 2.0–3.0 Yes 63 52% UNK UNK
2 24 F RHD/LAT −42 UNK 5b 5.8a 16% 2.0–3.0 Yesd 66 67% UNK UNK
3 36 M DVT −44 UNK 12b 11.4b −4.8% 2.0–3.0 No NA 24% UNK UNK
4 64 F DVT −45 UNK 10b 11.5b 15.3% 2.0–3.0 No NA 47% UNK UNK
5 22 F DVT −88 UNK 10b 4.8a −37% 2.0–3.0 Yese 12 54% UNK UNK
6 9 M DVT 0 UNK 5b 5.3b 5.8% 2.0–3.0 No NA 53% UNK UNK
7 49 M DVT −3 UNK 5b 9.5a 89.3% 2.0–3.0 Yes 49 42% UNK UNK
8 30 F PE −35 UNK 5b 27a 440.9% 2.0–3.0 Yes 67 30% UNK UNK
9 29 F DVT −31 UNK 5b 11.8a 135.8% 2.0–3.0 Yes 7 40% UNK UNK
10 41 M Stroke & DVT −46 UNK 6b 6.5a 8.3% 2.0–3.0 Yes 63 66% UNK UNK
Dawson et al, 201634 1 60 M MV replaced +UNK 42 8a 35b 340% 2.5–3.5 No NA NA 8 28
Fahmi et al, 201614 1 34 F MV replaced +19 years 42 7.5a 30b 300% 2.5–3.5 No NA NA 11.4 35
Shibata et al, 201735 1 70 F Stroke +UNK 365 4a 15a 275% 2.0–3.0 Yes UNK UNK 4 30
2 80 F AF +UNK 330 2.5a 10b 300% 2.0–3.0 No NA NA 3 60
Notes: Maina et al did not report rifampin cessation times or confirmed adherence to medications other than warfarin,36 which may have affected response. aTherapeutic INR attained on the dose. bTherapeutic INR not attained on the dose. cDefined as three consecutive therapeutic INR levels on the same warfarin dose (In Maina et al case series, defined as two consecutive therapeutic INRs). dOccasional warfarin overdoses. eMissed warfarin doses.
Abbreviations: AF, atrial fibrillation; DVT, deep venous thrombosis; INR, international normalized ratio; LAT, left arterial thrombosis; LVT, left ventricular thrombus; MV, mitral valve; NA, not applicable; PE, pulmonary embolism; RHD, rheumatic heart disease; TTR, time in therapeutic range; UNK, unknown.
Our case had one supratherapeutic INR after three warfarin doses of 5 mg and five days of rifampin commencement explained by her CYP2C9 poor metabolizer phenotype. She had two consecutive therapeutic INR readings after 12 and 14 days of rifampin on daily warfarin 3 mg. However, INR dropped below therapeutic after 17 days of rifampin, likely due to the latter’s liver enzyme induction. Comparable patterns have been observed in other cases when the commencement sequence was a few days apart or when rifampin was added to chronic warfarin. In 1996, Casner29 reported a patient who had one therapeutic INR after 13 rifampin days, which declined to subtherapeutic until ten days after the rifampin stoppage. The case reported by Kim et al31 had one in-target INR after warfarin 20 mg, which dropped then increased back to target for a week after escalation to 25 mg on rifampin days 25 and 33. Then, the INR declined steadily even after warfarin dose was raised to 30 mg.31 An INR of 4.4 after three days of rifampin in a mechanical valve replacement case, reported by Fahmi et al14, necessitated the holding of warfarin for five days. Then INR dropped to below the desired 7–14 days from the combination. In the case reported by Dawson et al34, it took 17 days for the interaction to become apparent. These reports, aligned with our report, indicate that the time-course to rifampin CYP2C9 induction is more than 14 days. Studies of this rifampin duration or less are inadequate to evaluate the full impact of such interaction. It is vital to monitor INR frequently during the first two weeks of rifampin and shortly after to avoid exposing the patient to subtherapeutic levels with no bridging.
Our patient’s CYP2C9 genotyping revealed that she is a slow metabolizer with a homozygous CYP2C9*3*3, which indicates high warfarin sensitivity and the lowest dose requirements. Additionally, VKORC1 –1639G>A genotyping showed upstream variant (AG) requiring lower warfarin doses.3 The patient reached three consecutive therapeutic INRs on a daily warfarin dose of 10 mg, which is generally considered a high dose. However, compared with most cases that received rifampin with warfarin doses up to 35 mg and no target attainment, 10 mg represents a relatively reasonable dose. The estimated therapeutic warfarin dose for our case, calculated using clinical factors via www.warfarindosing.org, was 5.8 mg/day. However, when the genotyping results were added to the calculation, the estimated therapeutic dose was 2 mg/day. The FDA warfarin label expected maintenance dosing for CYP2C9*3/*3 combined with VKORC1 (AG) is ranged from 0.5 to 2 mg/day.8 That implies that 10 mg is at least a 400% increase from the therapeutic dose without rifampin. Interestingly, most reported cases who reached therapeutic INR during the concomitant warfarin–rifampin use received relatively low warfarin doses around 10 to 15 mg/day.33,35,36 Since the reported cases were not tested for genotype variations; genetic polymorphism might represent an unrevealed explanation of the variable warfarin responses among patients during rifampin use.
Up to our knowledge, this is the first warfarin–rifampin interaction case report to address genetic polymorphism as a contributing factor in the interaction outcome since it was first described in the 1970s. Nevertheless, there are studies of rifampin induction effect on CYP2C9 variants. Vormfelde et al42 used tolbutamide as a probe substrate to evaluate rifampin effect on CYP2C9 activity in 128 healthy volunteers with different genotypes. While the pre-rifampin enzyme activity difference between *1/*1 and *3/*3 was six-folds, the induction effect was around two-fold regardless of the genotype.42 It is important to emphasize that the study duration was only four days,42 which may not represent the subsequent enzyme induction phases. George et al43, using phenytoin as a probe drug, studied the total effect of one month of rifampin therapy on CYP2C9 various genotypes in forty-eight new TB patients. Joined data from twelve mutant patients showed that rifampin’s induction potential was statistically significant regardless of the genotype.43 While these studies suggest that rifampin increases the CYP2C9 concentration with the same ratio,42,43 the catalytic activity would be genotype-dependent.5
Our case may represent an example of phenoconversion, a phenomenon of genotype-phenotype mismatch, in which an individual’s metabolizing enzyme is functionally converted from a poor metabolizer to an intermediate or extensive metabolizer or vice versa due to the use of an enzyme inducer or inhibitor, respectively.44–46 Rifampin shifted the patient from her genotype-based poor metabolizer status to a functional rapid metabolizer status that required warfarin daily dose escalation.46 However, because the patient’s gene-based estimated warfarin dose was 2 mg, escalation to 10 mg, a feasible dose compared with other interaction reports, was sufficient to attain therapeutic INR.
While the patient confirmed complete adherence, the INR dropped to 1.0 on the last day of warfarin therapy and day 97 of rifampin. One explanation may be a late CYP2C9 induction phase by rifampin. CYP2C9 half-life has been reported to be much longer than other CYPs as CYP3A4. Shibata et al35 monitored CYP2C9 and CYP3A activities in two cases who were receiving rifampin and warfarin concomitantly after rifampin discontinuation. The CYP2C9 estimated half-lives were 25.7 and 16.8 days, compared with CYP3A half-lives of 2.4 and 11.5 days, in the first and second case, respectively.35 Indicating that the CYP2C9 turnover can take up to months. Moreover, having a CYP2C9*3*3 genotype might have prolonged the time-course to maximal induction. Since warfarin was stopped at that point, verifying these explanations is not possible.
Conclusion
This case report demonstrated the highly significant effect of rifampin metabolic induction and genetic polymorphism on warfarin dose requirements. Our findings reveal a genetic explanation of the variable patients’ responses to different warfarin doses while on rifampin. While wild-type patients are not likely to respond to extreme warfarin doses due to the drug interaction with rifampin, patients with loss-of-function genetic variants of CYP2C9 and VKORC1 are more likely to respond at a feasible dose level. Future studies are warranted to evaluate the genotype variants’ effect on the interaction, which might benefit in selecting likely warfarin responders when rifampin therapy is needed.
Ethical Approval
Ethical approval for this case report was provided by the Medical Research Committee (MRC) of Hamad Medical Corporation (HMC) (#MRC-04-20-428).
Author Contributions
All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that they have no conflicts of interest in this work. | Oral | DrugAdministrationRoute | CC BY | 33542643 | 18,944,693 | 2021 |
What was the dosage of drug 'HUMAN IMMUNOGLOBULIN G'? | Genetic Polymorphism Effect on Warfarin-Rifampin Interaction: A Case Report and Review of Literature.
Warfarin-rifampin interaction has been reported since the 1970s. Due to rifampin's strong induction of CYP2C9, most cases could not attain the target international normalized ratio (INR) despite warfarin dose escalation. Genetic polymorphisms determine up to 50% of warfarin dose variability. A 38-year-old woman was started on warfarin and rifampin for cerebral venous sinus thrombosis and pulmonary tuberculosis. Over six weeks, the daily warfarin dose was increased from 3 to 10 mg to attain three consecutive in-clinic therapeutic INRs. She completed three complications-free months of warfarin treatment with time in therapeutic range (TTR) of 46%. We performed retrospective genetic testing to determine the patient's CYP2C9, CYP4F2, and VKORC1 genotypes and whether they had affected the interaction outcome. The analysis revealed that the subject carries CYP2C9*3*3 and VKORC1-1639 (GA) mutations, classifying her as a slow metabolizer and, hence, highly warfarin-sensitive. This was reflected on how the case responded to a relatively lower dose than previously reported cases that did not achieve the target on warfarin daily doses up to 35 mg. This is the first report addressing the genotype effect on this interaction. Patients with genetic variants requiring low warfarin doses are more likely to respond at a feasible dose while on rifampin. Future studies to evaluate warfarin-rifampin-gene interaction are warranted.
Introduction
Warfarin, a vitamin K antagonist, remains the preferred oral anticoagulation for atrial fibrillation with prosthetic cardiac valves or rheumatic heart disease with more than mild mitral stenosis, and venous thromboembolism (VTE) at unusual sites.1,2 It is a mixture of two racemic isomers, R and S-warfarin. Both impair the vitamin k-dependent proteins production via inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).3 Cytochrome P450 2C9 (CYP2C9) extensively metabolizes S-warfarin, the stereoisomer of predominant potency, to the inactive 7-hydroxywarfarin.3
VKORC1 and CYP2C9 genetic polymorphisms, with other genetic variants, determine up to 50% of warfarin dose variance.3,4 The most studied and common variant alleles of CYP2C9 are CYP2C9*2 (rs1799853) and *3 (rs1057910), which result from missense mutations associated with diminished catalytic activity, poor warfarin metabolism, and decreased dose requirements.5
VKORC1–1639G>A (rs9923231) genotype variants (GA and AA) contribute majorly to sensitizing warfarin.5 On the other hand, CYP4F2*3, a nonsynonymous variant of the gene coding for the primary liver vitamin K oxidase, CYP4F2, has been associated in some studies with a modest increase of warfarin dose requirements (8–11%).3 These mutations were incorporated, with other clinical factors, into dosing algorithms which were shown to provide better warfarin dose prediction.3 Gage and International Warfarin Pharmacogenetics Consortium (IWPC), are among the most widely studied algorithms and are contained in the website (www.warfarindosing.org), which calculates the initial dose with the ability to adjust for CYP2C9*5, *6, CYP4F2, and GGCX genotypes.3 Genetic-based dosing of warfarin upon initiation was shown in some studies to improve target international normalized ratio (INR) attainment and time in therapeutic range (TTR) during the initial month.6 This was mainly mediated by CYP2C9 and VKORC1 polymorphisms which significantly impact the maintenance dose requirement.3,4,7 The Food and Drug Administration (FDA) has also approved warfarin label modifications with dosing guidance based on the CYP2C9 and VKORC1 genotypes.8 Apart from genetics, numerous warfarin drug interactions have been reported requiring dose adjustments and frequent INR monitoring to avoid bleeding or anticoagulation failure.9
Antituberculous management includes rifampin, isoniazid, ethambutol, and pyrazinamide. While ethambutol and pyrazinamide are neither cytochrome P450s inhibitors nor reported to affect warfarin, isoniazid is a week inhibitor of CYP3A4, which is not FDA classified as a clinical index inhibitor.10 While it has been reported to increase warfarin’s hypoprothrombinemic effect in two cases,11,12 the interaction magnitude is considered minimal, with no action recommended.13
Rifampin, a life-saving antimicrobial for tuberculosis, endocarditis, and meningitis,14 is a clinically significant inducer of CYP2B6, CYP2C19, CYP2C8, CYP2C9, and CYP3A4, as well as P glycoprotein.10 It induces CYP2C9 transcription by binding to its main De-novo synthesis regulatory nuclear receptor, pregnane X receptor (PXR).5,15 The binding increases the CYP2C9 mRNA expression by up to six-folds.15 That leads to a higher amount of the enzyme, and extensive metabolism of the substrate/victim drug.15 While the onset of induction can be few days with rifampin,16 the time required to reach maximal enzyme abundance and new steady state is more than two weeks based on the CYP turnover and degradation half-life.16–19 FDA classifies rifampin as a moderate inducer of CYP2C9, defined as a decrease in the substrates area under the concentration-time curve (AUC) by 50% to less than 80%.10 That was based on two healthy-volunteers studies with probe substrates, S-warfarin and tolbutamide.20,21 However, the first study’s duration was only four days,20 and 12–15 days in the latter,21 which, yet, showed high variability of CYP2C9 activity (1–7.4-fold).22 These short durations may imply that rifampin is rather a strong CYP2C9 inducer as AUC ratio were measured before reaching maximal induction.16–19
Rifampin almost eliminates warfarin’s therapeutic effect, which required extensive dose escalation in all cases and is typically associated with the inability to maintain therapeutic range. The interaction has been described since the 1970s.20,23,24 Till the late 1980s, multiple reports showed a significant increase in warfarin dose requirements with rifampin.25–27 After the INR test was universally adopted,28 several reports demonstrated that most patients who required the anti-infective along with anticoagulation were unable to maintain target INR.14,29–36
This report aims to describe a case who received warfarin and rifampin concomitantly and the interaction outcome, and to perform genetic testing to determine the patient’s CYP2C9, VKORC1, CALU, and CYP4F2 genotypes and whether they could explain the response to warfarin dose escalation.
Case Description
A 38-year-old Ethiopian woman with a weight of 60 kg, a height of 150 cm, and a history of diabetes and immune thrombocytopenic purpura (ITP) on metformin and chronic eltrombopag presented to the emergency of Hamad General Hospital in Qatar on January 13, 2020, with dizziness, severe diffuse headache, photophobia, and multiple vomiting for three days. Intracranial computed tomography (CT) venogram showed cerebral venous sinus thrombosis (CVST). Eltrombopag was stopped. Since the Glasgow Coma Score (GCS) dropped to 11, the patient was admitted to the medical ICU with sedation, analgesia, and close neurologic observation. Because of low platelet count (PC), 32X109/L, she was started on 0.5 gm/kg intravenous immunoglobulin (IVIG) plus steroids for three days to raise the PC above 50X109/L in order to initiate anticoagulation. Two days later, PC reached 75X109/L, and heparin continuous IV infusion was initiated with platelets transfusion as the patient was neurologically deteriorating, and repeated CT showed extension of thrombosis with intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). On January 19, although PC was maintained above 200X109/L, heparin was suspended due to a sharp drop of hemoglobin to 5 gm/dL with no identified source of bleeding. The patient was transfused immediately. The next day hemoglobin increased to 8 then maintained at 9–10 gm/dL. Heparin was resumed on January 23. Hematology planned to start rituximab for ITP, yet the Quantiferone test for tuberculosis (TB) was positive. CT chest on January 26 revealed consolidation patches in the right upper and middle lung lobes suggesting active TB. Since bronchoscopy was not feasible due to the high bleeding risk, the pulmonary, MICU, and infectious diseases teams decided to start empiric anti-TB medications based on radiology and follow response by imaging. The rituximab plan was aborted.
On January 29, the patient was started on daily rifampin 600 mg, isoniazid 300 mg, ethambutol 1100 mg, and pyrazinamide 1600 mg. She was transferred to the medicine ward after sedation withdrawal. On January 31, anticoagulation was shifted to daily oral warfarin 5 mg with twice-daily enoxaparin 60 mg as bridging. Three days later, warfarin was held for one day then resumed with dose reduction to 3 mg due to a sharp INR increase to 3.7. The next day, INR dropped to 1.0, then increased gradually after three days to 1.7, 1.9, and reached 2.0 on February 9. Enoxaparin was stopped, and the patient was discharged on warfarin 3 mg, anti-TB medications, pyridoxine 40 mg, metformin 500 mg twice daily, and metoprolol 50 mg twice daily. Following in anticoagulation clinic, on February 12, after 14 days of rifampin, INR was 2.6; therefore, the same warfarin dose continued. Although decreased to 1.7 on February 16, the warfarin dose remained. A week later, INR dropped to 1.3, so warfarin was escalated to 4 mg/day, and enoxaparin resumed. Over the next three weeks, the dose was gradually escalated up to 10 mg, after four days of which, INR reached 2.3 on March 15. Enoxaparin was stopped, and the patient was maintained on daily warfarin 10 mg. The anti-TB medications were switched to (Rifampin 600 mg/Isoniazid 300 mg) on March 24. INR was maintained in the next two clinic visits at 3 and 2.9 on March 23 and April 6, respectively. On May 4, the INR was 1.0 in the last anticoagulation clinic visit. That could not be explained by non-compliance as per the patient interview. The planned three-month duration of anticoagulation ended, so warfarin was stopped. Rifampin/isoniazid continued till July 14, 2020. The patient was interviewed on each visit and excluded any adverse effects. She had normal follow up laboratory values throughout the treatment. Details of warfarin daily dose and INR are shown in [Figure 1].Figure 1 This graph represents the daily warfarin dose, rifampin days, and INR overtime. The bottom x-axis represents dates. The top x-axis represents rifampin days. The left y-axis represents the daily warfarin dose in milligrams and is shown by the vertical bars. The right Y-axis represents the INR and is shown by the black diamond points. The therapeutic range is indicated between the two dotted lines (2.0–3.0).
Methods
The patient was approached by one of the study investigators and explained the reasons and expectations of the research. The patient confirmed her understanding, and agreement to provide saliva sample for genetic testing as well as to have the case published by signing an Institutional Review Board (IRB) approved informed consent form. She was asked to provide a saliva sample using Oragene•DNA (OG-500) self-collection kit (DNA genotek, USA). Hereafter, the kit was kept in a water bath at 50 C° overnight for DNA extraction. The prepIT®•L2P standard protocol for the purification of DNA was used for DNA extraction.37 The purified DNA’s quality and quantity were evaluated by Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific). Finally, the sample was genotyped for detecting the following genetic variants: CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), CYP4F2*3 (rs2108622),VKORC1 (rs9923231), VKORC1 Asp36Tyr (rs61742245), and CALU (rs339097). This was performed using the QuantStudio™ 5 Real-Time Polymerase Chain Reaction (PCR) system for Human Identification, 96-well, 0.2 mL, desktop manufactured by Applied BiosystemsTM.
Results
The genotyping revealed that the patient is a carrier of CYP2C9*3*3 homozygous, VKORC1-1639 (GA) heterozygous, and CYP4F2 (CC) wild-type homozygous. Based on this genetic profile, the subject is considered a slow metabolizer which indicates high warfarin sensitivity. On the other hand, both VKORC1 Asp36Tyr (CT) and CALU (AG) genotyping were heterozygous indicating partial warfarin resistance.
As it is shown in Figure 1, the sudden rise in INR with a moderate standard warfarin dose of 5 mg is very well explained by the CYP2C9 loss of function genetic variant carried by this patient (CYP2C9*3/*3). A few weeks later and with the interacting effect of rifampin reaching its peak, the daily warfarin dose requirement for the patient increased reaching 10 mg.
Discussion
In this case report, we observed warfarin–rifampin drug interaction in a patient with CVST and pulmonary TB. The interaction management required warfarin dose-escalation, frequent INR monitoring, and low molecular weight heparin (LMWH) bridging over six weeks. On a daily warfarin dose of 10 mg, our patient attained three consecutive therapeutic INR levels in three clinic visits. That is considered a “stable warfarin dose,” as defined in most studies,38 despite the one subtherapeutic INR in the last treatment day. The TTR, calculated by the Rosendaal method,39 was 46.2%. The three-month warfarin anticoagulation treatment, combined with rifampin, was completed with difficulty, yet no complications.
Since the World Health Organization (WHO) adopted INR in the 1980s,28 more than nineteen cases of warfarin–rifampin interaction have been reported.14,29–36 Despite extensive warfarin dose escalation over a prolonged time, the majority could not attain target INR while on the combination.14,29–32,34–36 Cases are summarized in [Table 1]. Excessive warfarin exposure and hemorrhage after rifampin discontinuation have been reported, implying the importance of close monitoring and careful dose de-escalation after rifampin stoppage.33,40 Due to the lack of laboratory monitorable parameters like INR, labels of direct oral anticoagulants (DOACs), that are substrates of CYP3A4 and/or P glycoprotein, recommend avoiding concomitant use with rifampin to avoid unmanageable therapeutic failures.41Table 1 Summary of Studies of Warfarin–Rifampicin Interaction with INR Monitoring
Study Patient’s Data Sequence and Duration During Concomitant Use After Rifampin Stopped
Case No. Age Sex Warfarin Indication Rifampin Start in Relation to Warfarin Start (Days) Concomitant Duration (Days) Average Initial Warfarin Dose (mg/day)a,b Average Last Warfarin Dose (mg/day)a,b Warfarin Dose Increase Target INR Target Attained Time to Stable Warfarin Dosec (Days) TTR Stable Warfarin Dosec (mg/day) Time to Stable Warfarin Dosec (Days)
Casner, 199629 1 36 M PE −2 20 7.8b 20b 169% 2.0–3.0 No NA NA 10 10
Lee & Trasher, 200130 1 58 M LVT −120 113 7.5b 25b 233% 2.0–3.0 No NA NA 7.5 35
Kim et al, 200631 1 79 M DVT −11 30 5a 30b 500% 2.0–3.0 No NA NA 6 60
Krajewski, 201032 1 71 M AF +14 45 5.7a 25b 340% 2.0–3.0 No NA NA 5 120
Martins et al, 201333 1 59 F AF +300 203 6.4a 11.4a 78% 2.0–3.0 Yes 104 50% 5.4 60
Maina et al, 201336 1 17 F DVT −7 UNK 10b 27.7a 177.3% 2.0–3.0 Yes 63 52% UNK UNK
2 24 F RHD/LAT −42 UNK 5b 5.8a 16% 2.0–3.0 Yesd 66 67% UNK UNK
3 36 M DVT −44 UNK 12b 11.4b −4.8% 2.0–3.0 No NA 24% UNK UNK
4 64 F DVT −45 UNK 10b 11.5b 15.3% 2.0–3.0 No NA 47% UNK UNK
5 22 F DVT −88 UNK 10b 4.8a −37% 2.0–3.0 Yese 12 54% UNK UNK
6 9 M DVT 0 UNK 5b 5.3b 5.8% 2.0–3.0 No NA 53% UNK UNK
7 49 M DVT −3 UNK 5b 9.5a 89.3% 2.0–3.0 Yes 49 42% UNK UNK
8 30 F PE −35 UNK 5b 27a 440.9% 2.0–3.0 Yes 67 30% UNK UNK
9 29 F DVT −31 UNK 5b 11.8a 135.8% 2.0–3.0 Yes 7 40% UNK UNK
10 41 M Stroke & DVT −46 UNK 6b 6.5a 8.3% 2.0–3.0 Yes 63 66% UNK UNK
Dawson et al, 201634 1 60 M MV replaced +UNK 42 8a 35b 340% 2.5–3.5 No NA NA 8 28
Fahmi et al, 201614 1 34 F MV replaced +19 years 42 7.5a 30b 300% 2.5–3.5 No NA NA 11.4 35
Shibata et al, 201735 1 70 F Stroke +UNK 365 4a 15a 275% 2.0–3.0 Yes UNK UNK 4 30
2 80 F AF +UNK 330 2.5a 10b 300% 2.0–3.0 No NA NA 3 60
Notes: Maina et al did not report rifampin cessation times or confirmed adherence to medications other than warfarin,36 which may have affected response. aTherapeutic INR attained on the dose. bTherapeutic INR not attained on the dose. cDefined as three consecutive therapeutic INR levels on the same warfarin dose (In Maina et al case series, defined as two consecutive therapeutic INRs). dOccasional warfarin overdoses. eMissed warfarin doses.
Abbreviations: AF, atrial fibrillation; DVT, deep venous thrombosis; INR, international normalized ratio; LAT, left arterial thrombosis; LVT, left ventricular thrombus; MV, mitral valve; NA, not applicable; PE, pulmonary embolism; RHD, rheumatic heart disease; TTR, time in therapeutic range; UNK, unknown.
Our case had one supratherapeutic INR after three warfarin doses of 5 mg and five days of rifampin commencement explained by her CYP2C9 poor metabolizer phenotype. She had two consecutive therapeutic INR readings after 12 and 14 days of rifampin on daily warfarin 3 mg. However, INR dropped below therapeutic after 17 days of rifampin, likely due to the latter’s liver enzyme induction. Comparable patterns have been observed in other cases when the commencement sequence was a few days apart or when rifampin was added to chronic warfarin. In 1996, Casner29 reported a patient who had one therapeutic INR after 13 rifampin days, which declined to subtherapeutic until ten days after the rifampin stoppage. The case reported by Kim et al31 had one in-target INR after warfarin 20 mg, which dropped then increased back to target for a week after escalation to 25 mg on rifampin days 25 and 33. Then, the INR declined steadily even after warfarin dose was raised to 30 mg.31 An INR of 4.4 after three days of rifampin in a mechanical valve replacement case, reported by Fahmi et al14, necessitated the holding of warfarin for five days. Then INR dropped to below the desired 7–14 days from the combination. In the case reported by Dawson et al34, it took 17 days for the interaction to become apparent. These reports, aligned with our report, indicate that the time-course to rifampin CYP2C9 induction is more than 14 days. Studies of this rifampin duration or less are inadequate to evaluate the full impact of such interaction. It is vital to monitor INR frequently during the first two weeks of rifampin and shortly after to avoid exposing the patient to subtherapeutic levels with no bridging.
Our patient’s CYP2C9 genotyping revealed that she is a slow metabolizer with a homozygous CYP2C9*3*3, which indicates high warfarin sensitivity and the lowest dose requirements. Additionally, VKORC1 –1639G>A genotyping showed upstream variant (AG) requiring lower warfarin doses.3 The patient reached three consecutive therapeutic INRs on a daily warfarin dose of 10 mg, which is generally considered a high dose. However, compared with most cases that received rifampin with warfarin doses up to 35 mg and no target attainment, 10 mg represents a relatively reasonable dose. The estimated therapeutic warfarin dose for our case, calculated using clinical factors via www.warfarindosing.org, was 5.8 mg/day. However, when the genotyping results were added to the calculation, the estimated therapeutic dose was 2 mg/day. The FDA warfarin label expected maintenance dosing for CYP2C9*3/*3 combined with VKORC1 (AG) is ranged from 0.5 to 2 mg/day.8 That implies that 10 mg is at least a 400% increase from the therapeutic dose without rifampin. Interestingly, most reported cases who reached therapeutic INR during the concomitant warfarin–rifampin use received relatively low warfarin doses around 10 to 15 mg/day.33,35,36 Since the reported cases were not tested for genotype variations; genetic polymorphism might represent an unrevealed explanation of the variable warfarin responses among patients during rifampin use.
Up to our knowledge, this is the first warfarin–rifampin interaction case report to address genetic polymorphism as a contributing factor in the interaction outcome since it was first described in the 1970s. Nevertheless, there are studies of rifampin induction effect on CYP2C9 variants. Vormfelde et al42 used tolbutamide as a probe substrate to evaluate rifampin effect on CYP2C9 activity in 128 healthy volunteers with different genotypes. While the pre-rifampin enzyme activity difference between *1/*1 and *3/*3 was six-folds, the induction effect was around two-fold regardless of the genotype.42 It is important to emphasize that the study duration was only four days,42 which may not represent the subsequent enzyme induction phases. George et al43, using phenytoin as a probe drug, studied the total effect of one month of rifampin therapy on CYP2C9 various genotypes in forty-eight new TB patients. Joined data from twelve mutant patients showed that rifampin’s induction potential was statistically significant regardless of the genotype.43 While these studies suggest that rifampin increases the CYP2C9 concentration with the same ratio,42,43 the catalytic activity would be genotype-dependent.5
Our case may represent an example of phenoconversion, a phenomenon of genotype-phenotype mismatch, in which an individual’s metabolizing enzyme is functionally converted from a poor metabolizer to an intermediate or extensive metabolizer or vice versa due to the use of an enzyme inducer or inhibitor, respectively.44–46 Rifampin shifted the patient from her genotype-based poor metabolizer status to a functional rapid metabolizer status that required warfarin daily dose escalation.46 However, because the patient’s gene-based estimated warfarin dose was 2 mg, escalation to 10 mg, a feasible dose compared with other interaction reports, was sufficient to attain therapeutic INR.
While the patient confirmed complete adherence, the INR dropped to 1.0 on the last day of warfarin therapy and day 97 of rifampin. One explanation may be a late CYP2C9 induction phase by rifampin. CYP2C9 half-life has been reported to be much longer than other CYPs as CYP3A4. Shibata et al35 monitored CYP2C9 and CYP3A activities in two cases who were receiving rifampin and warfarin concomitantly after rifampin discontinuation. The CYP2C9 estimated half-lives were 25.7 and 16.8 days, compared with CYP3A half-lives of 2.4 and 11.5 days, in the first and second case, respectively.35 Indicating that the CYP2C9 turnover can take up to months. Moreover, having a CYP2C9*3*3 genotype might have prolonged the time-course to maximal induction. Since warfarin was stopped at that point, verifying these explanations is not possible.
Conclusion
This case report demonstrated the highly significant effect of rifampin metabolic induction and genetic polymorphism on warfarin dose requirements. Our findings reveal a genetic explanation of the variable patients’ responses to different warfarin doses while on rifampin. While wild-type patients are not likely to respond to extreme warfarin doses due to the drug interaction with rifampin, patients with loss-of-function genetic variants of CYP2C9 and VKORC1 are more likely to respond at a feasible dose level. Future studies are warranted to evaluate the genotype variants’ effect on the interaction, which might benefit in selecting likely warfarin responders when rifampin therapy is needed.
Ethical Approval
Ethical approval for this case report was provided by the Medical Research Committee (MRC) of Hamad Medical Corporation (HMC) (#MRC-04-20-428).
Author Contributions
All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that they have no conflicts of interest in this work. | 0.5 G/KG | DrugDosageText | CC BY | 33542643 | 18,944,693 | 2021 |
What was the outcome of reaction 'Drug interaction'? | Genetic Polymorphism Effect on Warfarin-Rifampin Interaction: A Case Report and Review of Literature.
Warfarin-rifampin interaction has been reported since the 1970s. Due to rifampin's strong induction of CYP2C9, most cases could not attain the target international normalized ratio (INR) despite warfarin dose escalation. Genetic polymorphisms determine up to 50% of warfarin dose variability. A 38-year-old woman was started on warfarin and rifampin for cerebral venous sinus thrombosis and pulmonary tuberculosis. Over six weeks, the daily warfarin dose was increased from 3 to 10 mg to attain three consecutive in-clinic therapeutic INRs. She completed three complications-free months of warfarin treatment with time in therapeutic range (TTR) of 46%. We performed retrospective genetic testing to determine the patient's CYP2C9, CYP4F2, and VKORC1 genotypes and whether they had affected the interaction outcome. The analysis revealed that the subject carries CYP2C9*3*3 and VKORC1-1639 (GA) mutations, classifying her as a slow metabolizer and, hence, highly warfarin-sensitive. This was reflected on how the case responded to a relatively lower dose than previously reported cases that did not achieve the target on warfarin daily doses up to 35 mg. This is the first report addressing the genotype effect on this interaction. Patients with genetic variants requiring low warfarin doses are more likely to respond at a feasible dose while on rifampin. Future studies to evaluate warfarin-rifampin-gene interaction are warranted.
Introduction
Warfarin, a vitamin K antagonist, remains the preferred oral anticoagulation for atrial fibrillation with prosthetic cardiac valves or rheumatic heart disease with more than mild mitral stenosis, and venous thromboembolism (VTE) at unusual sites.1,2 It is a mixture of two racemic isomers, R and S-warfarin. Both impair the vitamin k-dependent proteins production via inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).3 Cytochrome P450 2C9 (CYP2C9) extensively metabolizes S-warfarin, the stereoisomer of predominant potency, to the inactive 7-hydroxywarfarin.3
VKORC1 and CYP2C9 genetic polymorphisms, with other genetic variants, determine up to 50% of warfarin dose variance.3,4 The most studied and common variant alleles of CYP2C9 are CYP2C9*2 (rs1799853) and *3 (rs1057910), which result from missense mutations associated with diminished catalytic activity, poor warfarin metabolism, and decreased dose requirements.5
VKORC1–1639G>A (rs9923231) genotype variants (GA and AA) contribute majorly to sensitizing warfarin.5 On the other hand, CYP4F2*3, a nonsynonymous variant of the gene coding for the primary liver vitamin K oxidase, CYP4F2, has been associated in some studies with a modest increase of warfarin dose requirements (8–11%).3 These mutations were incorporated, with other clinical factors, into dosing algorithms which were shown to provide better warfarin dose prediction.3 Gage and International Warfarin Pharmacogenetics Consortium (IWPC), are among the most widely studied algorithms and are contained in the website (www.warfarindosing.org), which calculates the initial dose with the ability to adjust for CYP2C9*5, *6, CYP4F2, and GGCX genotypes.3 Genetic-based dosing of warfarin upon initiation was shown in some studies to improve target international normalized ratio (INR) attainment and time in therapeutic range (TTR) during the initial month.6 This was mainly mediated by CYP2C9 and VKORC1 polymorphisms which significantly impact the maintenance dose requirement.3,4,7 The Food and Drug Administration (FDA) has also approved warfarin label modifications with dosing guidance based on the CYP2C9 and VKORC1 genotypes.8 Apart from genetics, numerous warfarin drug interactions have been reported requiring dose adjustments and frequent INR monitoring to avoid bleeding or anticoagulation failure.9
Antituberculous management includes rifampin, isoniazid, ethambutol, and pyrazinamide. While ethambutol and pyrazinamide are neither cytochrome P450s inhibitors nor reported to affect warfarin, isoniazid is a week inhibitor of CYP3A4, which is not FDA classified as a clinical index inhibitor.10 While it has been reported to increase warfarin’s hypoprothrombinemic effect in two cases,11,12 the interaction magnitude is considered minimal, with no action recommended.13
Rifampin, a life-saving antimicrobial for tuberculosis, endocarditis, and meningitis,14 is a clinically significant inducer of CYP2B6, CYP2C19, CYP2C8, CYP2C9, and CYP3A4, as well as P glycoprotein.10 It induces CYP2C9 transcription by binding to its main De-novo synthesis regulatory nuclear receptor, pregnane X receptor (PXR).5,15 The binding increases the CYP2C9 mRNA expression by up to six-folds.15 That leads to a higher amount of the enzyme, and extensive metabolism of the substrate/victim drug.15 While the onset of induction can be few days with rifampin,16 the time required to reach maximal enzyme abundance and new steady state is more than two weeks based on the CYP turnover and degradation half-life.16–19 FDA classifies rifampin as a moderate inducer of CYP2C9, defined as a decrease in the substrates area under the concentration-time curve (AUC) by 50% to less than 80%.10 That was based on two healthy-volunteers studies with probe substrates, S-warfarin and tolbutamide.20,21 However, the first study’s duration was only four days,20 and 12–15 days in the latter,21 which, yet, showed high variability of CYP2C9 activity (1–7.4-fold).22 These short durations may imply that rifampin is rather a strong CYP2C9 inducer as AUC ratio were measured before reaching maximal induction.16–19
Rifampin almost eliminates warfarin’s therapeutic effect, which required extensive dose escalation in all cases and is typically associated with the inability to maintain therapeutic range. The interaction has been described since the 1970s.20,23,24 Till the late 1980s, multiple reports showed a significant increase in warfarin dose requirements with rifampin.25–27 After the INR test was universally adopted,28 several reports demonstrated that most patients who required the anti-infective along with anticoagulation were unable to maintain target INR.14,29–36
This report aims to describe a case who received warfarin and rifampin concomitantly and the interaction outcome, and to perform genetic testing to determine the patient’s CYP2C9, VKORC1, CALU, and CYP4F2 genotypes and whether they could explain the response to warfarin dose escalation.
Case Description
A 38-year-old Ethiopian woman with a weight of 60 kg, a height of 150 cm, and a history of diabetes and immune thrombocytopenic purpura (ITP) on metformin and chronic eltrombopag presented to the emergency of Hamad General Hospital in Qatar on January 13, 2020, with dizziness, severe diffuse headache, photophobia, and multiple vomiting for three days. Intracranial computed tomography (CT) venogram showed cerebral venous sinus thrombosis (CVST). Eltrombopag was stopped. Since the Glasgow Coma Score (GCS) dropped to 11, the patient was admitted to the medical ICU with sedation, analgesia, and close neurologic observation. Because of low platelet count (PC), 32X109/L, she was started on 0.5 gm/kg intravenous immunoglobulin (IVIG) plus steroids for three days to raise the PC above 50X109/L in order to initiate anticoagulation. Two days later, PC reached 75X109/L, and heparin continuous IV infusion was initiated with platelets transfusion as the patient was neurologically deteriorating, and repeated CT showed extension of thrombosis with intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). On January 19, although PC was maintained above 200X109/L, heparin was suspended due to a sharp drop of hemoglobin to 5 gm/dL with no identified source of bleeding. The patient was transfused immediately. The next day hemoglobin increased to 8 then maintained at 9–10 gm/dL. Heparin was resumed on January 23. Hematology planned to start rituximab for ITP, yet the Quantiferone test for tuberculosis (TB) was positive. CT chest on January 26 revealed consolidation patches in the right upper and middle lung lobes suggesting active TB. Since bronchoscopy was not feasible due to the high bleeding risk, the pulmonary, MICU, and infectious diseases teams decided to start empiric anti-TB medications based on radiology and follow response by imaging. The rituximab plan was aborted.
On January 29, the patient was started on daily rifampin 600 mg, isoniazid 300 mg, ethambutol 1100 mg, and pyrazinamide 1600 mg. She was transferred to the medicine ward after sedation withdrawal. On January 31, anticoagulation was shifted to daily oral warfarin 5 mg with twice-daily enoxaparin 60 mg as bridging. Three days later, warfarin was held for one day then resumed with dose reduction to 3 mg due to a sharp INR increase to 3.7. The next day, INR dropped to 1.0, then increased gradually after three days to 1.7, 1.9, and reached 2.0 on February 9. Enoxaparin was stopped, and the patient was discharged on warfarin 3 mg, anti-TB medications, pyridoxine 40 mg, metformin 500 mg twice daily, and metoprolol 50 mg twice daily. Following in anticoagulation clinic, on February 12, after 14 days of rifampin, INR was 2.6; therefore, the same warfarin dose continued. Although decreased to 1.7 on February 16, the warfarin dose remained. A week later, INR dropped to 1.3, so warfarin was escalated to 4 mg/day, and enoxaparin resumed. Over the next three weeks, the dose was gradually escalated up to 10 mg, after four days of which, INR reached 2.3 on March 15. Enoxaparin was stopped, and the patient was maintained on daily warfarin 10 mg. The anti-TB medications were switched to (Rifampin 600 mg/Isoniazid 300 mg) on March 24. INR was maintained in the next two clinic visits at 3 and 2.9 on March 23 and April 6, respectively. On May 4, the INR was 1.0 in the last anticoagulation clinic visit. That could not be explained by non-compliance as per the patient interview. The planned three-month duration of anticoagulation ended, so warfarin was stopped. Rifampin/isoniazid continued till July 14, 2020. The patient was interviewed on each visit and excluded any adverse effects. She had normal follow up laboratory values throughout the treatment. Details of warfarin daily dose and INR are shown in [Figure 1].Figure 1 This graph represents the daily warfarin dose, rifampin days, and INR overtime. The bottom x-axis represents dates. The top x-axis represents rifampin days. The left y-axis represents the daily warfarin dose in milligrams and is shown by the vertical bars. The right Y-axis represents the INR and is shown by the black diamond points. The therapeutic range is indicated between the two dotted lines (2.0–3.0).
Methods
The patient was approached by one of the study investigators and explained the reasons and expectations of the research. The patient confirmed her understanding, and agreement to provide saliva sample for genetic testing as well as to have the case published by signing an Institutional Review Board (IRB) approved informed consent form. She was asked to provide a saliva sample using Oragene•DNA (OG-500) self-collection kit (DNA genotek, USA). Hereafter, the kit was kept in a water bath at 50 C° overnight for DNA extraction. The prepIT®•L2P standard protocol for the purification of DNA was used for DNA extraction.37 The purified DNA’s quality and quantity were evaluated by Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific). Finally, the sample was genotyped for detecting the following genetic variants: CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), CYP4F2*3 (rs2108622),VKORC1 (rs9923231), VKORC1 Asp36Tyr (rs61742245), and CALU (rs339097). This was performed using the QuantStudio™ 5 Real-Time Polymerase Chain Reaction (PCR) system for Human Identification, 96-well, 0.2 mL, desktop manufactured by Applied BiosystemsTM.
Results
The genotyping revealed that the patient is a carrier of CYP2C9*3*3 homozygous, VKORC1-1639 (GA) heterozygous, and CYP4F2 (CC) wild-type homozygous. Based on this genetic profile, the subject is considered a slow metabolizer which indicates high warfarin sensitivity. On the other hand, both VKORC1 Asp36Tyr (CT) and CALU (AG) genotyping were heterozygous indicating partial warfarin resistance.
As it is shown in Figure 1, the sudden rise in INR with a moderate standard warfarin dose of 5 mg is very well explained by the CYP2C9 loss of function genetic variant carried by this patient (CYP2C9*3/*3). A few weeks later and with the interacting effect of rifampin reaching its peak, the daily warfarin dose requirement for the patient increased reaching 10 mg.
Discussion
In this case report, we observed warfarin–rifampin drug interaction in a patient with CVST and pulmonary TB. The interaction management required warfarin dose-escalation, frequent INR monitoring, and low molecular weight heparin (LMWH) bridging over six weeks. On a daily warfarin dose of 10 mg, our patient attained three consecutive therapeutic INR levels in three clinic visits. That is considered a “stable warfarin dose,” as defined in most studies,38 despite the one subtherapeutic INR in the last treatment day. The TTR, calculated by the Rosendaal method,39 was 46.2%. The three-month warfarin anticoagulation treatment, combined with rifampin, was completed with difficulty, yet no complications.
Since the World Health Organization (WHO) adopted INR in the 1980s,28 more than nineteen cases of warfarin–rifampin interaction have been reported.14,29–36 Despite extensive warfarin dose escalation over a prolonged time, the majority could not attain target INR while on the combination.14,29–32,34–36 Cases are summarized in [Table 1]. Excessive warfarin exposure and hemorrhage after rifampin discontinuation have been reported, implying the importance of close monitoring and careful dose de-escalation after rifampin stoppage.33,40 Due to the lack of laboratory monitorable parameters like INR, labels of direct oral anticoagulants (DOACs), that are substrates of CYP3A4 and/or P glycoprotein, recommend avoiding concomitant use with rifampin to avoid unmanageable therapeutic failures.41Table 1 Summary of Studies of Warfarin–Rifampicin Interaction with INR Monitoring
Study Patient’s Data Sequence and Duration During Concomitant Use After Rifampin Stopped
Case No. Age Sex Warfarin Indication Rifampin Start in Relation to Warfarin Start (Days) Concomitant Duration (Days) Average Initial Warfarin Dose (mg/day)a,b Average Last Warfarin Dose (mg/day)a,b Warfarin Dose Increase Target INR Target Attained Time to Stable Warfarin Dosec (Days) TTR Stable Warfarin Dosec (mg/day) Time to Stable Warfarin Dosec (Days)
Casner, 199629 1 36 M PE −2 20 7.8b 20b 169% 2.0–3.0 No NA NA 10 10
Lee & Trasher, 200130 1 58 M LVT −120 113 7.5b 25b 233% 2.0–3.0 No NA NA 7.5 35
Kim et al, 200631 1 79 M DVT −11 30 5a 30b 500% 2.0–3.0 No NA NA 6 60
Krajewski, 201032 1 71 M AF +14 45 5.7a 25b 340% 2.0–3.0 No NA NA 5 120
Martins et al, 201333 1 59 F AF +300 203 6.4a 11.4a 78% 2.0–3.0 Yes 104 50% 5.4 60
Maina et al, 201336 1 17 F DVT −7 UNK 10b 27.7a 177.3% 2.0–3.0 Yes 63 52% UNK UNK
2 24 F RHD/LAT −42 UNK 5b 5.8a 16% 2.0–3.0 Yesd 66 67% UNK UNK
3 36 M DVT −44 UNK 12b 11.4b −4.8% 2.0–3.0 No NA 24% UNK UNK
4 64 F DVT −45 UNK 10b 11.5b 15.3% 2.0–3.0 No NA 47% UNK UNK
5 22 F DVT −88 UNK 10b 4.8a −37% 2.0–3.0 Yese 12 54% UNK UNK
6 9 M DVT 0 UNK 5b 5.3b 5.8% 2.0–3.0 No NA 53% UNK UNK
7 49 M DVT −3 UNK 5b 9.5a 89.3% 2.0–3.0 Yes 49 42% UNK UNK
8 30 F PE −35 UNK 5b 27a 440.9% 2.0–3.0 Yes 67 30% UNK UNK
9 29 F DVT −31 UNK 5b 11.8a 135.8% 2.0–3.0 Yes 7 40% UNK UNK
10 41 M Stroke & DVT −46 UNK 6b 6.5a 8.3% 2.0–3.0 Yes 63 66% UNK UNK
Dawson et al, 201634 1 60 M MV replaced +UNK 42 8a 35b 340% 2.5–3.5 No NA NA 8 28
Fahmi et al, 201614 1 34 F MV replaced +19 years 42 7.5a 30b 300% 2.5–3.5 No NA NA 11.4 35
Shibata et al, 201735 1 70 F Stroke +UNK 365 4a 15a 275% 2.0–3.0 Yes UNK UNK 4 30
2 80 F AF +UNK 330 2.5a 10b 300% 2.0–3.0 No NA NA 3 60
Notes: Maina et al did not report rifampin cessation times or confirmed adherence to medications other than warfarin,36 which may have affected response. aTherapeutic INR attained on the dose. bTherapeutic INR not attained on the dose. cDefined as three consecutive therapeutic INR levels on the same warfarin dose (In Maina et al case series, defined as two consecutive therapeutic INRs). dOccasional warfarin overdoses. eMissed warfarin doses.
Abbreviations: AF, atrial fibrillation; DVT, deep venous thrombosis; INR, international normalized ratio; LAT, left arterial thrombosis; LVT, left ventricular thrombus; MV, mitral valve; NA, not applicable; PE, pulmonary embolism; RHD, rheumatic heart disease; TTR, time in therapeutic range; UNK, unknown.
Our case had one supratherapeutic INR after three warfarin doses of 5 mg and five days of rifampin commencement explained by her CYP2C9 poor metabolizer phenotype. She had two consecutive therapeutic INR readings after 12 and 14 days of rifampin on daily warfarin 3 mg. However, INR dropped below therapeutic after 17 days of rifampin, likely due to the latter’s liver enzyme induction. Comparable patterns have been observed in other cases when the commencement sequence was a few days apart or when rifampin was added to chronic warfarin. In 1996, Casner29 reported a patient who had one therapeutic INR after 13 rifampin days, which declined to subtherapeutic until ten days after the rifampin stoppage. The case reported by Kim et al31 had one in-target INR after warfarin 20 mg, which dropped then increased back to target for a week after escalation to 25 mg on rifampin days 25 and 33. Then, the INR declined steadily even after warfarin dose was raised to 30 mg.31 An INR of 4.4 after three days of rifampin in a mechanical valve replacement case, reported by Fahmi et al14, necessitated the holding of warfarin for five days. Then INR dropped to below the desired 7–14 days from the combination. In the case reported by Dawson et al34, it took 17 days for the interaction to become apparent. These reports, aligned with our report, indicate that the time-course to rifampin CYP2C9 induction is more than 14 days. Studies of this rifampin duration or less are inadequate to evaluate the full impact of such interaction. It is vital to monitor INR frequently during the first two weeks of rifampin and shortly after to avoid exposing the patient to subtherapeutic levels with no bridging.
Our patient’s CYP2C9 genotyping revealed that she is a slow metabolizer with a homozygous CYP2C9*3*3, which indicates high warfarin sensitivity and the lowest dose requirements. Additionally, VKORC1 –1639G>A genotyping showed upstream variant (AG) requiring lower warfarin doses.3 The patient reached three consecutive therapeutic INRs on a daily warfarin dose of 10 mg, which is generally considered a high dose. However, compared with most cases that received rifampin with warfarin doses up to 35 mg and no target attainment, 10 mg represents a relatively reasonable dose. The estimated therapeutic warfarin dose for our case, calculated using clinical factors via www.warfarindosing.org, was 5.8 mg/day. However, when the genotyping results were added to the calculation, the estimated therapeutic dose was 2 mg/day. The FDA warfarin label expected maintenance dosing for CYP2C9*3/*3 combined with VKORC1 (AG) is ranged from 0.5 to 2 mg/day.8 That implies that 10 mg is at least a 400% increase from the therapeutic dose without rifampin. Interestingly, most reported cases who reached therapeutic INR during the concomitant warfarin–rifampin use received relatively low warfarin doses around 10 to 15 mg/day.33,35,36 Since the reported cases were not tested for genotype variations; genetic polymorphism might represent an unrevealed explanation of the variable warfarin responses among patients during rifampin use.
Up to our knowledge, this is the first warfarin–rifampin interaction case report to address genetic polymorphism as a contributing factor in the interaction outcome since it was first described in the 1970s. Nevertheless, there are studies of rifampin induction effect on CYP2C9 variants. Vormfelde et al42 used tolbutamide as a probe substrate to evaluate rifampin effect on CYP2C9 activity in 128 healthy volunteers with different genotypes. While the pre-rifampin enzyme activity difference between *1/*1 and *3/*3 was six-folds, the induction effect was around two-fold regardless of the genotype.42 It is important to emphasize that the study duration was only four days,42 which may not represent the subsequent enzyme induction phases. George et al43, using phenytoin as a probe drug, studied the total effect of one month of rifampin therapy on CYP2C9 various genotypes in forty-eight new TB patients. Joined data from twelve mutant patients showed that rifampin’s induction potential was statistically significant regardless of the genotype.43 While these studies suggest that rifampin increases the CYP2C9 concentration with the same ratio,42,43 the catalytic activity would be genotype-dependent.5
Our case may represent an example of phenoconversion, a phenomenon of genotype-phenotype mismatch, in which an individual’s metabolizing enzyme is functionally converted from a poor metabolizer to an intermediate or extensive metabolizer or vice versa due to the use of an enzyme inducer or inhibitor, respectively.44–46 Rifampin shifted the patient from her genotype-based poor metabolizer status to a functional rapid metabolizer status that required warfarin daily dose escalation.46 However, because the patient’s gene-based estimated warfarin dose was 2 mg, escalation to 10 mg, a feasible dose compared with other interaction reports, was sufficient to attain therapeutic INR.
While the patient confirmed complete adherence, the INR dropped to 1.0 on the last day of warfarin therapy and day 97 of rifampin. One explanation may be a late CYP2C9 induction phase by rifampin. CYP2C9 half-life has been reported to be much longer than other CYPs as CYP3A4. Shibata et al35 monitored CYP2C9 and CYP3A activities in two cases who were receiving rifampin and warfarin concomitantly after rifampin discontinuation. The CYP2C9 estimated half-lives were 25.7 and 16.8 days, compared with CYP3A half-lives of 2.4 and 11.5 days, in the first and second case, respectively.35 Indicating that the CYP2C9 turnover can take up to months. Moreover, having a CYP2C9*3*3 genotype might have prolonged the time-course to maximal induction. Since warfarin was stopped at that point, verifying these explanations is not possible.
Conclusion
This case report demonstrated the highly significant effect of rifampin metabolic induction and genetic polymorphism on warfarin dose requirements. Our findings reveal a genetic explanation of the variable patients’ responses to different warfarin doses while on rifampin. While wild-type patients are not likely to respond to extreme warfarin doses due to the drug interaction with rifampin, patients with loss-of-function genetic variants of CYP2C9 and VKORC1 are more likely to respond at a feasible dose level. Future studies are warranted to evaluate the genotype variants’ effect on the interaction, which might benefit in selecting likely warfarin responders when rifampin therapy is needed.
Ethical Approval
Ethical approval for this case report was provided by the Medical Research Committee (MRC) of Hamad Medical Corporation (HMC) (#MRC-04-20-428).
Author Contributions
All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that they have no conflicts of interest in this work. | Recovering | ReactionOutcome | CC BY | 33542643 | 18,944,693 | 2021 |
What was the outcome of reaction 'International normalised ratio increased'? | Genetic Polymorphism Effect on Warfarin-Rifampin Interaction: A Case Report and Review of Literature.
Warfarin-rifampin interaction has been reported since the 1970s. Due to rifampin's strong induction of CYP2C9, most cases could not attain the target international normalized ratio (INR) despite warfarin dose escalation. Genetic polymorphisms determine up to 50% of warfarin dose variability. A 38-year-old woman was started on warfarin and rifampin for cerebral venous sinus thrombosis and pulmonary tuberculosis. Over six weeks, the daily warfarin dose was increased from 3 to 10 mg to attain three consecutive in-clinic therapeutic INRs. She completed three complications-free months of warfarin treatment with time in therapeutic range (TTR) of 46%. We performed retrospective genetic testing to determine the patient's CYP2C9, CYP4F2, and VKORC1 genotypes and whether they had affected the interaction outcome. The analysis revealed that the subject carries CYP2C9*3*3 and VKORC1-1639 (GA) mutations, classifying her as a slow metabolizer and, hence, highly warfarin-sensitive. This was reflected on how the case responded to a relatively lower dose than previously reported cases that did not achieve the target on warfarin daily doses up to 35 mg. This is the first report addressing the genotype effect on this interaction. Patients with genetic variants requiring low warfarin doses are more likely to respond at a feasible dose while on rifampin. Future studies to evaluate warfarin-rifampin-gene interaction are warranted.
Introduction
Warfarin, a vitamin K antagonist, remains the preferred oral anticoagulation for atrial fibrillation with prosthetic cardiac valves or rheumatic heart disease with more than mild mitral stenosis, and venous thromboembolism (VTE) at unusual sites.1,2 It is a mixture of two racemic isomers, R and S-warfarin. Both impair the vitamin k-dependent proteins production via inhibition of vitamin K epoxide reductase complex subunit 1 (VKORC1).3 Cytochrome P450 2C9 (CYP2C9) extensively metabolizes S-warfarin, the stereoisomer of predominant potency, to the inactive 7-hydroxywarfarin.3
VKORC1 and CYP2C9 genetic polymorphisms, with other genetic variants, determine up to 50% of warfarin dose variance.3,4 The most studied and common variant alleles of CYP2C9 are CYP2C9*2 (rs1799853) and *3 (rs1057910), which result from missense mutations associated with diminished catalytic activity, poor warfarin metabolism, and decreased dose requirements.5
VKORC1–1639G>A (rs9923231) genotype variants (GA and AA) contribute majorly to sensitizing warfarin.5 On the other hand, CYP4F2*3, a nonsynonymous variant of the gene coding for the primary liver vitamin K oxidase, CYP4F2, has been associated in some studies with a modest increase of warfarin dose requirements (8–11%).3 These mutations were incorporated, with other clinical factors, into dosing algorithms which were shown to provide better warfarin dose prediction.3 Gage and International Warfarin Pharmacogenetics Consortium (IWPC), are among the most widely studied algorithms and are contained in the website (www.warfarindosing.org), which calculates the initial dose with the ability to adjust for CYP2C9*5, *6, CYP4F2, and GGCX genotypes.3 Genetic-based dosing of warfarin upon initiation was shown in some studies to improve target international normalized ratio (INR) attainment and time in therapeutic range (TTR) during the initial month.6 This was mainly mediated by CYP2C9 and VKORC1 polymorphisms which significantly impact the maintenance dose requirement.3,4,7 The Food and Drug Administration (FDA) has also approved warfarin label modifications with dosing guidance based on the CYP2C9 and VKORC1 genotypes.8 Apart from genetics, numerous warfarin drug interactions have been reported requiring dose adjustments and frequent INR monitoring to avoid bleeding or anticoagulation failure.9
Antituberculous management includes rifampin, isoniazid, ethambutol, and pyrazinamide. While ethambutol and pyrazinamide are neither cytochrome P450s inhibitors nor reported to affect warfarin, isoniazid is a week inhibitor of CYP3A4, which is not FDA classified as a clinical index inhibitor.10 While it has been reported to increase warfarin’s hypoprothrombinemic effect in two cases,11,12 the interaction magnitude is considered minimal, with no action recommended.13
Rifampin, a life-saving antimicrobial for tuberculosis, endocarditis, and meningitis,14 is a clinically significant inducer of CYP2B6, CYP2C19, CYP2C8, CYP2C9, and CYP3A4, as well as P glycoprotein.10 It induces CYP2C9 transcription by binding to its main De-novo synthesis regulatory nuclear receptor, pregnane X receptor (PXR).5,15 The binding increases the CYP2C9 mRNA expression by up to six-folds.15 That leads to a higher amount of the enzyme, and extensive metabolism of the substrate/victim drug.15 While the onset of induction can be few days with rifampin,16 the time required to reach maximal enzyme abundance and new steady state is more than two weeks based on the CYP turnover and degradation half-life.16–19 FDA classifies rifampin as a moderate inducer of CYP2C9, defined as a decrease in the substrates area under the concentration-time curve (AUC) by 50% to less than 80%.10 That was based on two healthy-volunteers studies with probe substrates, S-warfarin and tolbutamide.20,21 However, the first study’s duration was only four days,20 and 12–15 days in the latter,21 which, yet, showed high variability of CYP2C9 activity (1–7.4-fold).22 These short durations may imply that rifampin is rather a strong CYP2C9 inducer as AUC ratio were measured before reaching maximal induction.16–19
Rifampin almost eliminates warfarin’s therapeutic effect, which required extensive dose escalation in all cases and is typically associated with the inability to maintain therapeutic range. The interaction has been described since the 1970s.20,23,24 Till the late 1980s, multiple reports showed a significant increase in warfarin dose requirements with rifampin.25–27 After the INR test was universally adopted,28 several reports demonstrated that most patients who required the anti-infective along with anticoagulation were unable to maintain target INR.14,29–36
This report aims to describe a case who received warfarin and rifampin concomitantly and the interaction outcome, and to perform genetic testing to determine the patient’s CYP2C9, VKORC1, CALU, and CYP4F2 genotypes and whether they could explain the response to warfarin dose escalation.
Case Description
A 38-year-old Ethiopian woman with a weight of 60 kg, a height of 150 cm, and a history of diabetes and immune thrombocytopenic purpura (ITP) on metformin and chronic eltrombopag presented to the emergency of Hamad General Hospital in Qatar on January 13, 2020, with dizziness, severe diffuse headache, photophobia, and multiple vomiting for three days. Intracranial computed tomography (CT) venogram showed cerebral venous sinus thrombosis (CVST). Eltrombopag was stopped. Since the Glasgow Coma Score (GCS) dropped to 11, the patient was admitted to the medical ICU with sedation, analgesia, and close neurologic observation. Because of low platelet count (PC), 32X109/L, she was started on 0.5 gm/kg intravenous immunoglobulin (IVIG) plus steroids for three days to raise the PC above 50X109/L in order to initiate anticoagulation. Two days later, PC reached 75X109/L, and heparin continuous IV infusion was initiated with platelets transfusion as the patient was neurologically deteriorating, and repeated CT showed extension of thrombosis with intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH). On January 19, although PC was maintained above 200X109/L, heparin was suspended due to a sharp drop of hemoglobin to 5 gm/dL with no identified source of bleeding. The patient was transfused immediately. The next day hemoglobin increased to 8 then maintained at 9–10 gm/dL. Heparin was resumed on January 23. Hematology planned to start rituximab for ITP, yet the Quantiferone test for tuberculosis (TB) was positive. CT chest on January 26 revealed consolidation patches in the right upper and middle lung lobes suggesting active TB. Since bronchoscopy was not feasible due to the high bleeding risk, the pulmonary, MICU, and infectious diseases teams decided to start empiric anti-TB medications based on radiology and follow response by imaging. The rituximab plan was aborted.
On January 29, the patient was started on daily rifampin 600 mg, isoniazid 300 mg, ethambutol 1100 mg, and pyrazinamide 1600 mg. She was transferred to the medicine ward after sedation withdrawal. On January 31, anticoagulation was shifted to daily oral warfarin 5 mg with twice-daily enoxaparin 60 mg as bridging. Three days later, warfarin was held for one day then resumed with dose reduction to 3 mg due to a sharp INR increase to 3.7. The next day, INR dropped to 1.0, then increased gradually after three days to 1.7, 1.9, and reached 2.0 on February 9. Enoxaparin was stopped, and the patient was discharged on warfarin 3 mg, anti-TB medications, pyridoxine 40 mg, metformin 500 mg twice daily, and metoprolol 50 mg twice daily. Following in anticoagulation clinic, on February 12, after 14 days of rifampin, INR was 2.6; therefore, the same warfarin dose continued. Although decreased to 1.7 on February 16, the warfarin dose remained. A week later, INR dropped to 1.3, so warfarin was escalated to 4 mg/day, and enoxaparin resumed. Over the next three weeks, the dose was gradually escalated up to 10 mg, after four days of which, INR reached 2.3 on March 15. Enoxaparin was stopped, and the patient was maintained on daily warfarin 10 mg. The anti-TB medications were switched to (Rifampin 600 mg/Isoniazid 300 mg) on March 24. INR was maintained in the next two clinic visits at 3 and 2.9 on March 23 and April 6, respectively. On May 4, the INR was 1.0 in the last anticoagulation clinic visit. That could not be explained by non-compliance as per the patient interview. The planned three-month duration of anticoagulation ended, so warfarin was stopped. Rifampin/isoniazid continued till July 14, 2020. The patient was interviewed on each visit and excluded any adverse effects. She had normal follow up laboratory values throughout the treatment. Details of warfarin daily dose and INR are shown in [Figure 1].Figure 1 This graph represents the daily warfarin dose, rifampin days, and INR overtime. The bottom x-axis represents dates. The top x-axis represents rifampin days. The left y-axis represents the daily warfarin dose in milligrams and is shown by the vertical bars. The right Y-axis represents the INR and is shown by the black diamond points. The therapeutic range is indicated between the two dotted lines (2.0–3.0).
Methods
The patient was approached by one of the study investigators and explained the reasons and expectations of the research. The patient confirmed her understanding, and agreement to provide saliva sample for genetic testing as well as to have the case published by signing an Institutional Review Board (IRB) approved informed consent form. She was asked to provide a saliva sample using Oragene•DNA (OG-500) self-collection kit (DNA genotek, USA). Hereafter, the kit was kept in a water bath at 50 C° overnight for DNA extraction. The prepIT®•L2P standard protocol for the purification of DNA was used for DNA extraction.37 The purified DNA’s quality and quantity were evaluated by Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific). Finally, the sample was genotyped for detecting the following genetic variants: CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), CYP4F2*3 (rs2108622),VKORC1 (rs9923231), VKORC1 Asp36Tyr (rs61742245), and CALU (rs339097). This was performed using the QuantStudio™ 5 Real-Time Polymerase Chain Reaction (PCR) system for Human Identification, 96-well, 0.2 mL, desktop manufactured by Applied BiosystemsTM.
Results
The genotyping revealed that the patient is a carrier of CYP2C9*3*3 homozygous, VKORC1-1639 (GA) heterozygous, and CYP4F2 (CC) wild-type homozygous. Based on this genetic profile, the subject is considered a slow metabolizer which indicates high warfarin sensitivity. On the other hand, both VKORC1 Asp36Tyr (CT) and CALU (AG) genotyping were heterozygous indicating partial warfarin resistance.
As it is shown in Figure 1, the sudden rise in INR with a moderate standard warfarin dose of 5 mg is very well explained by the CYP2C9 loss of function genetic variant carried by this patient (CYP2C9*3/*3). A few weeks later and with the interacting effect of rifampin reaching its peak, the daily warfarin dose requirement for the patient increased reaching 10 mg.
Discussion
In this case report, we observed warfarin–rifampin drug interaction in a patient with CVST and pulmonary TB. The interaction management required warfarin dose-escalation, frequent INR monitoring, and low molecular weight heparin (LMWH) bridging over six weeks. On a daily warfarin dose of 10 mg, our patient attained three consecutive therapeutic INR levels in three clinic visits. That is considered a “stable warfarin dose,” as defined in most studies,38 despite the one subtherapeutic INR in the last treatment day. The TTR, calculated by the Rosendaal method,39 was 46.2%. The three-month warfarin anticoagulation treatment, combined with rifampin, was completed with difficulty, yet no complications.
Since the World Health Organization (WHO) adopted INR in the 1980s,28 more than nineteen cases of warfarin–rifampin interaction have been reported.14,29–36 Despite extensive warfarin dose escalation over a prolonged time, the majority could not attain target INR while on the combination.14,29–32,34–36 Cases are summarized in [Table 1]. Excessive warfarin exposure and hemorrhage after rifampin discontinuation have been reported, implying the importance of close monitoring and careful dose de-escalation after rifampin stoppage.33,40 Due to the lack of laboratory monitorable parameters like INR, labels of direct oral anticoagulants (DOACs), that are substrates of CYP3A4 and/or P glycoprotein, recommend avoiding concomitant use with rifampin to avoid unmanageable therapeutic failures.41Table 1 Summary of Studies of Warfarin–Rifampicin Interaction with INR Monitoring
Study Patient’s Data Sequence and Duration During Concomitant Use After Rifampin Stopped
Case No. Age Sex Warfarin Indication Rifampin Start in Relation to Warfarin Start (Days) Concomitant Duration (Days) Average Initial Warfarin Dose (mg/day)a,b Average Last Warfarin Dose (mg/day)a,b Warfarin Dose Increase Target INR Target Attained Time to Stable Warfarin Dosec (Days) TTR Stable Warfarin Dosec (mg/day) Time to Stable Warfarin Dosec (Days)
Casner, 199629 1 36 M PE −2 20 7.8b 20b 169% 2.0–3.0 No NA NA 10 10
Lee & Trasher, 200130 1 58 M LVT −120 113 7.5b 25b 233% 2.0–3.0 No NA NA 7.5 35
Kim et al, 200631 1 79 M DVT −11 30 5a 30b 500% 2.0–3.0 No NA NA 6 60
Krajewski, 201032 1 71 M AF +14 45 5.7a 25b 340% 2.0–3.0 No NA NA 5 120
Martins et al, 201333 1 59 F AF +300 203 6.4a 11.4a 78% 2.0–3.0 Yes 104 50% 5.4 60
Maina et al, 201336 1 17 F DVT −7 UNK 10b 27.7a 177.3% 2.0–3.0 Yes 63 52% UNK UNK
2 24 F RHD/LAT −42 UNK 5b 5.8a 16% 2.0–3.0 Yesd 66 67% UNK UNK
3 36 M DVT −44 UNK 12b 11.4b −4.8% 2.0–3.0 No NA 24% UNK UNK
4 64 F DVT −45 UNK 10b 11.5b 15.3% 2.0–3.0 No NA 47% UNK UNK
5 22 F DVT −88 UNK 10b 4.8a −37% 2.0–3.0 Yese 12 54% UNK UNK
6 9 M DVT 0 UNK 5b 5.3b 5.8% 2.0–3.0 No NA 53% UNK UNK
7 49 M DVT −3 UNK 5b 9.5a 89.3% 2.0–3.0 Yes 49 42% UNK UNK
8 30 F PE −35 UNK 5b 27a 440.9% 2.0–3.0 Yes 67 30% UNK UNK
9 29 F DVT −31 UNK 5b 11.8a 135.8% 2.0–3.0 Yes 7 40% UNK UNK
10 41 M Stroke & DVT −46 UNK 6b 6.5a 8.3% 2.0–3.0 Yes 63 66% UNK UNK
Dawson et al, 201634 1 60 M MV replaced +UNK 42 8a 35b 340% 2.5–3.5 No NA NA 8 28
Fahmi et al, 201614 1 34 F MV replaced +19 years 42 7.5a 30b 300% 2.5–3.5 No NA NA 11.4 35
Shibata et al, 201735 1 70 F Stroke +UNK 365 4a 15a 275% 2.0–3.0 Yes UNK UNK 4 30
2 80 F AF +UNK 330 2.5a 10b 300% 2.0–3.0 No NA NA 3 60
Notes: Maina et al did not report rifampin cessation times or confirmed adherence to medications other than warfarin,36 which may have affected response. aTherapeutic INR attained on the dose. bTherapeutic INR not attained on the dose. cDefined as three consecutive therapeutic INR levels on the same warfarin dose (In Maina et al case series, defined as two consecutive therapeutic INRs). dOccasional warfarin overdoses. eMissed warfarin doses.
Abbreviations: AF, atrial fibrillation; DVT, deep venous thrombosis; INR, international normalized ratio; LAT, left arterial thrombosis; LVT, left ventricular thrombus; MV, mitral valve; NA, not applicable; PE, pulmonary embolism; RHD, rheumatic heart disease; TTR, time in therapeutic range; UNK, unknown.
Our case had one supratherapeutic INR after three warfarin doses of 5 mg and five days of rifampin commencement explained by her CYP2C9 poor metabolizer phenotype. She had two consecutive therapeutic INR readings after 12 and 14 days of rifampin on daily warfarin 3 mg. However, INR dropped below therapeutic after 17 days of rifampin, likely due to the latter’s liver enzyme induction. Comparable patterns have been observed in other cases when the commencement sequence was a few days apart or when rifampin was added to chronic warfarin. In 1996, Casner29 reported a patient who had one therapeutic INR after 13 rifampin days, which declined to subtherapeutic until ten days after the rifampin stoppage. The case reported by Kim et al31 had one in-target INR after warfarin 20 mg, which dropped then increased back to target for a week after escalation to 25 mg on rifampin days 25 and 33. Then, the INR declined steadily even after warfarin dose was raised to 30 mg.31 An INR of 4.4 after three days of rifampin in a mechanical valve replacement case, reported by Fahmi et al14, necessitated the holding of warfarin for five days. Then INR dropped to below the desired 7–14 days from the combination. In the case reported by Dawson et al34, it took 17 days for the interaction to become apparent. These reports, aligned with our report, indicate that the time-course to rifampin CYP2C9 induction is more than 14 days. Studies of this rifampin duration or less are inadequate to evaluate the full impact of such interaction. It is vital to monitor INR frequently during the first two weeks of rifampin and shortly after to avoid exposing the patient to subtherapeutic levels with no bridging.
Our patient’s CYP2C9 genotyping revealed that she is a slow metabolizer with a homozygous CYP2C9*3*3, which indicates high warfarin sensitivity and the lowest dose requirements. Additionally, VKORC1 –1639G>A genotyping showed upstream variant (AG) requiring lower warfarin doses.3 The patient reached three consecutive therapeutic INRs on a daily warfarin dose of 10 mg, which is generally considered a high dose. However, compared with most cases that received rifampin with warfarin doses up to 35 mg and no target attainment, 10 mg represents a relatively reasonable dose. The estimated therapeutic warfarin dose for our case, calculated using clinical factors via www.warfarindosing.org, was 5.8 mg/day. However, when the genotyping results were added to the calculation, the estimated therapeutic dose was 2 mg/day. The FDA warfarin label expected maintenance dosing for CYP2C9*3/*3 combined with VKORC1 (AG) is ranged from 0.5 to 2 mg/day.8 That implies that 10 mg is at least a 400% increase from the therapeutic dose without rifampin. Interestingly, most reported cases who reached therapeutic INR during the concomitant warfarin–rifampin use received relatively low warfarin doses around 10 to 15 mg/day.33,35,36 Since the reported cases were not tested for genotype variations; genetic polymorphism might represent an unrevealed explanation of the variable warfarin responses among patients during rifampin use.
Up to our knowledge, this is the first warfarin–rifampin interaction case report to address genetic polymorphism as a contributing factor in the interaction outcome since it was first described in the 1970s. Nevertheless, there are studies of rifampin induction effect on CYP2C9 variants. Vormfelde et al42 used tolbutamide as a probe substrate to evaluate rifampin effect on CYP2C9 activity in 128 healthy volunteers with different genotypes. While the pre-rifampin enzyme activity difference between *1/*1 and *3/*3 was six-folds, the induction effect was around two-fold regardless of the genotype.42 It is important to emphasize that the study duration was only four days,42 which may not represent the subsequent enzyme induction phases. George et al43, using phenytoin as a probe drug, studied the total effect of one month of rifampin therapy on CYP2C9 various genotypes in forty-eight new TB patients. Joined data from twelve mutant patients showed that rifampin’s induction potential was statistically significant regardless of the genotype.43 While these studies suggest that rifampin increases the CYP2C9 concentration with the same ratio,42,43 the catalytic activity would be genotype-dependent.5
Our case may represent an example of phenoconversion, a phenomenon of genotype-phenotype mismatch, in which an individual’s metabolizing enzyme is functionally converted from a poor metabolizer to an intermediate or extensive metabolizer or vice versa due to the use of an enzyme inducer or inhibitor, respectively.44–46 Rifampin shifted the patient from her genotype-based poor metabolizer status to a functional rapid metabolizer status that required warfarin daily dose escalation.46 However, because the patient’s gene-based estimated warfarin dose was 2 mg, escalation to 10 mg, a feasible dose compared with other interaction reports, was sufficient to attain therapeutic INR.
While the patient confirmed complete adherence, the INR dropped to 1.0 on the last day of warfarin therapy and day 97 of rifampin. One explanation may be a late CYP2C9 induction phase by rifampin. CYP2C9 half-life has been reported to be much longer than other CYPs as CYP3A4. Shibata et al35 monitored CYP2C9 and CYP3A activities in two cases who were receiving rifampin and warfarin concomitantly after rifampin discontinuation. The CYP2C9 estimated half-lives were 25.7 and 16.8 days, compared with CYP3A half-lives of 2.4 and 11.5 days, in the first and second case, respectively.35 Indicating that the CYP2C9 turnover can take up to months. Moreover, having a CYP2C9*3*3 genotype might have prolonged the time-course to maximal induction. Since warfarin was stopped at that point, verifying these explanations is not possible.
Conclusion
This case report demonstrated the highly significant effect of rifampin metabolic induction and genetic polymorphism on warfarin dose requirements. Our findings reveal a genetic explanation of the variable patients’ responses to different warfarin doses while on rifampin. While wild-type patients are not likely to respond to extreme warfarin doses due to the drug interaction with rifampin, patients with loss-of-function genetic variants of CYP2C9 and VKORC1 are more likely to respond at a feasible dose level. Future studies are warranted to evaluate the genotype variants’ effect on the interaction, which might benefit in selecting likely warfarin responders when rifampin therapy is needed.
Ethical Approval
Ethical approval for this case report was provided by the Medical Research Committee (MRC) of Hamad Medical Corporation (HMC) (#MRC-04-20-428).
Author Contributions
All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.
Disclosure
The authors declare that they have no conflicts of interest in this work. | Recovering | ReactionOutcome | CC BY | 33542643 | 18,944,693 | 2021 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Haemoglobin decreased'. | Giant hepatic haemangioma rupture in a patient on direct oral anticoagulant therapy.
Rupture of a liver haemangioma is extremely rare, with less than 100 cases reported in the literature. This is the first case known to date reporting a rupture occurring with direct oral anticoagulant therapy. A 76-year-old woman presented with acute abdominal pain and syncope in the context of commencing apixaban 4 weeks prior. Abdominal computed tomography and angiography demonstrated a large heterogenous mass in the left liver lobe with contrast blush suggestive of a bleeding tumour. Urgent transcatheter arterial embolization successfully ceased haemorrhage. Due to persistent compressive symptoms, a left lateral hepatectomy was performed. CD34 immunostaining of the tissue revealed variably sized vessels, which was consistent with a ruptured giant hepatic haemangioma. Our case substantiates the limited available literature regarding ruptured haemangiomas. Combined with previous case reports, this report will significantly contribute to our understanding of the risk factors, diagnosis and surgical indications for ruptured hepatic haemangiomas.
INTRODUCTION
Hepatic haemangiomas are the most common benign tumour of the liver [1]. Mesenchymal in origin, haemangiomas are composed of masses of blood vessels that are atypical in arrangement and size [1]. Most cases are asymptomatic, while few may present with symptoms related to tumour growth or compression of adjacent structures [2]. Rupture is a rare complication either occurring spontaneously or as a consequence of trauma [1–3]. Donati et al. [3] reviewed the 97 cases of ruptured liver haemangiomas published between 1898 and 2010, including two cases occurring with antithrombotic therapy. We present the case of a giant hepatic haemangioma rupture in the context of recent apixaban treatment that was subsequently treated with transcatheter arterial embolization (TAE) followed by left lateral hepatectomy. This is the first case report of a liver haemangioma rupture as a potential complication of direct oral anticoagulant (DOAC) use.
CASE REPORT
A 76-year-old woman presented with three syncopal episodes and a 2-day history of worsening abdominal pain. She had no nausea, vomiting, bowel changes or history of recent trauma. Apixaban (2.5 mg twice a day) was commenced 4 weeks prior for atrial fibrillation. Other past medical history included Parkinson’s disease. She had no history of malignancy or chronic liver disease. On presentation, she was pale, hypotensive (114/44 mmHg) and tachycardic (106 beats/minute). Physical examination demonstrated diffuse abdominal tenderness and distension without any signs of peritoneal irritation. Laboratory investigations revealed low haemoglobin (60 g/L), red blood cell count (2.35 × 1012/L) and haematocrit (0.215 L/L). Mean cell volume (92 fL) and platelets (190 × 109/L) were within range. Coagulation and liver function tests were unremarkable. Contrast-enhanced computerized tomography (CECT) reported a 62 × 83 × 90 mm heterogenous mass in the left lobe of the liver demonstrating contrast blush with large volume haemoperitoneum concerning for a bleeding hepatic neoplasm (Fig. 1). She was stabilized with five units of packed red blood cells before being transferred to our institute.
Figure 1 Axial and coronal reformats in non-contrast, arterial and delayed phases demonstrating contrast blush, with pooling within the left hepatic lobe haemangioma consistent with active bleeding.
Figure 2 DSA demonstrating hepatic vessels during TAE.
The patient was transferred to the interventional operating room for embolization of the left hepatic artery. Digital subtraction angiography (DSA) showed active contrast agent extravasation (Fig. 2). The tumour was embolized with 500–700 μm beads followed by gel foam. The patient remained hemodynamically stable throughout the procedure.
Figure 3 Pathological examination of hepatic mass revealing infarcted tissue and haemorrhage. Preserved areas of variably sized vessels shown.
Given the large size of mass and the patient’s continued symptoms of suppressed satiety and anorexia, a decision was made to perform an open left lateral hepatectomy Day 3 post-embolization. Laparotomy through rooftop incision revealed moderate hemoperitoneum and confinement of a necrotic liver lesion to Segment III. The lesion was not actively bleeding. A stapled left lateral sectionectomy was performed.
Histology showed variably sized dilated spaces within the infarcted and haemorrhagic tissue, mostly showing necrotic nuclei with focal viable bland nuclei lining the spaces. Immunohistochemistry with CD34 staining was positive in these cells (Fig. 3): (a) H&E, ×40, (b) H&E, ×400, (c) CD34 immunohistochemistry ×40 and (d) CD34 immunohistochemistry ×400. Adjacent liver parenchyma demonstrated preserved architecture. The overall features were consistent with an infarcted haemangioma.
Postoperatively, the patient successfully recovered and was transferred to rehabilitation.
DISCUSSION
Hepatic haemangiomas have an estimated prevalence of 0.4% to 7.3–20% in the general population [1, 2]. They are often asymptomatic and detected incidentally on imaging as small (<4 cm) solitary nodules. Diameters exceeding 4 or 5 cm are considered ‘giant haemangiomas’, with few reaching 20 cm [2, 3]. Spontaneous rupture is an extremely rare complication which can present as sudden abdominal pain and hypovolemic shock [1, 4]. Current literatures regarding risk factors for rupture are limited and conflicting. A 2011 literature review reported no correlation between haemangioma sizes with the risk of rupture [3]. Conversely, a more recent retrospective cohort study found that haemangiomas greater than 4 cm, when peripherally located and exophytic, were more likely to rupture [1].
This is the first case report concerning the rupture of a hepatic haemangioma occurring with recent DOAC treatment. As literature in this area is limited, a causative link may be speculated. A PubMed search yielded two relevant cases regarding antithrombotic therapy and hepatic haemangioma rupture. This may be partially due to the insufficient reporting of concurrent medications in some published cases, which is compounded by the fact that rupture is extremely rare. The small number of cases limits any meaningful statistical measure. In one case, bleeding from a cavernous liver haemangioma occurred with streptokinase therapy [5]. Another case documented a giant haemangioma rupture in a woman after acenocoumarol (vitamin K antagonist) treatment [6]. Unlike vitamin K antagonists, the plasma concentration of DOACs is not detectable by a simple blood test nor is there a reversal agent available. Hence, the risk of bleeding prior to rupture is not quantifiable.
As histology was limited post-embolization, clinical and radiological correlation was required. CD34 staining highlighted poorly preserved vessels of varying calibres, which is often seen in haemangiomas [7]. In the setting of haemorrhage, appropriate radiological features and no evidence of cirrhosis or liver disease, a haemangioma was favoured over hepatocellular carcinoma.
The early use of TAE in managing intraperitoneal haemorrhage was shown in our case to be successful in stopping haemorrhage. However, the tumour continued to elicit compressive symptoms and hence surgical intervention was pursued. Surgical resection and enucleation are traditionally the treatments of choice for symptomatic or ruptured haemangiomas [2, 8]. Surgery also provides useful tissue confirmation as needle liver biopsy carries the risk of haemorrhage [2]. Embolization performed as a bridging technique prior to surgical resection has been shown to stabilize patients and reduce intraoperative blood loss [3, 9, 10]. The long-term success rate of embolization alone, however, has not been well studied [1, 3]. Other less common therapies include surgical ligation, radiofrequency ablation and liver transplant [4, 8].
In conclusion, rupture of a liver haemangioma is rare, with less than 100 reported cases in literature. This is the first report known to date concerning a rupture occurring with recent DOAC therapy. Our case substantiates the limited available literature regarding validity of preoperative TAE and its utility in improving surgical outcomes. Previous case reports combined with this report will significantly contribute to our understanding of the risk factors and surgical indications for ruptured hepatic haemangiomas.
CONSENT
Full informed consent was provided by the patient for the writing of this case and the use of associated images.
ACKNOWLEDGEMENTS
Histology images provided by Pathology Queensland.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None. | APIXABAN | DrugsGivenReaction | CC BY-NC | 33542805 | 19,381,085 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Hepatic haemangioma rupture'. | Giant hepatic haemangioma rupture in a patient on direct oral anticoagulant therapy.
Rupture of a liver haemangioma is extremely rare, with less than 100 cases reported in the literature. This is the first case known to date reporting a rupture occurring with direct oral anticoagulant therapy. A 76-year-old woman presented with acute abdominal pain and syncope in the context of commencing apixaban 4 weeks prior. Abdominal computed tomography and angiography demonstrated a large heterogenous mass in the left liver lobe with contrast blush suggestive of a bleeding tumour. Urgent transcatheter arterial embolization successfully ceased haemorrhage. Due to persistent compressive symptoms, a left lateral hepatectomy was performed. CD34 immunostaining of the tissue revealed variably sized vessels, which was consistent with a ruptured giant hepatic haemangioma. Our case substantiates the limited available literature regarding ruptured haemangiomas. Combined with previous case reports, this report will significantly contribute to our understanding of the risk factors, diagnosis and surgical indications for ruptured hepatic haemangiomas.
INTRODUCTION
Hepatic haemangiomas are the most common benign tumour of the liver [1]. Mesenchymal in origin, haemangiomas are composed of masses of blood vessels that are atypical in arrangement and size [1]. Most cases are asymptomatic, while few may present with symptoms related to tumour growth or compression of adjacent structures [2]. Rupture is a rare complication either occurring spontaneously or as a consequence of trauma [1–3]. Donati et al. [3] reviewed the 97 cases of ruptured liver haemangiomas published between 1898 and 2010, including two cases occurring with antithrombotic therapy. We present the case of a giant hepatic haemangioma rupture in the context of recent apixaban treatment that was subsequently treated with transcatheter arterial embolization (TAE) followed by left lateral hepatectomy. This is the first case report of a liver haemangioma rupture as a potential complication of direct oral anticoagulant (DOAC) use.
CASE REPORT
A 76-year-old woman presented with three syncopal episodes and a 2-day history of worsening abdominal pain. She had no nausea, vomiting, bowel changes or history of recent trauma. Apixaban (2.5 mg twice a day) was commenced 4 weeks prior for atrial fibrillation. Other past medical history included Parkinson’s disease. She had no history of malignancy or chronic liver disease. On presentation, she was pale, hypotensive (114/44 mmHg) and tachycardic (106 beats/minute). Physical examination demonstrated diffuse abdominal tenderness and distension without any signs of peritoneal irritation. Laboratory investigations revealed low haemoglobin (60 g/L), red blood cell count (2.35 × 1012/L) and haematocrit (0.215 L/L). Mean cell volume (92 fL) and platelets (190 × 109/L) were within range. Coagulation and liver function tests were unremarkable. Contrast-enhanced computerized tomography (CECT) reported a 62 × 83 × 90 mm heterogenous mass in the left lobe of the liver demonstrating contrast blush with large volume haemoperitoneum concerning for a bleeding hepatic neoplasm (Fig. 1). She was stabilized with five units of packed red blood cells before being transferred to our institute.
Figure 1 Axial and coronal reformats in non-contrast, arterial and delayed phases demonstrating contrast blush, with pooling within the left hepatic lobe haemangioma consistent with active bleeding.
Figure 2 DSA demonstrating hepatic vessels during TAE.
The patient was transferred to the interventional operating room for embolization of the left hepatic artery. Digital subtraction angiography (DSA) showed active contrast agent extravasation (Fig. 2). The tumour was embolized with 500–700 μm beads followed by gel foam. The patient remained hemodynamically stable throughout the procedure.
Figure 3 Pathological examination of hepatic mass revealing infarcted tissue and haemorrhage. Preserved areas of variably sized vessels shown.
Given the large size of mass and the patient’s continued symptoms of suppressed satiety and anorexia, a decision was made to perform an open left lateral hepatectomy Day 3 post-embolization. Laparotomy through rooftop incision revealed moderate hemoperitoneum and confinement of a necrotic liver lesion to Segment III. The lesion was not actively bleeding. A stapled left lateral sectionectomy was performed.
Histology showed variably sized dilated spaces within the infarcted and haemorrhagic tissue, mostly showing necrotic nuclei with focal viable bland nuclei lining the spaces. Immunohistochemistry with CD34 staining was positive in these cells (Fig. 3): (a) H&E, ×40, (b) H&E, ×400, (c) CD34 immunohistochemistry ×40 and (d) CD34 immunohistochemistry ×400. Adjacent liver parenchyma demonstrated preserved architecture. The overall features were consistent with an infarcted haemangioma.
Postoperatively, the patient successfully recovered and was transferred to rehabilitation.
DISCUSSION
Hepatic haemangiomas have an estimated prevalence of 0.4% to 7.3–20% in the general population [1, 2]. They are often asymptomatic and detected incidentally on imaging as small (<4 cm) solitary nodules. Diameters exceeding 4 or 5 cm are considered ‘giant haemangiomas’, with few reaching 20 cm [2, 3]. Spontaneous rupture is an extremely rare complication which can present as sudden abdominal pain and hypovolemic shock [1, 4]. Current literatures regarding risk factors for rupture are limited and conflicting. A 2011 literature review reported no correlation between haemangioma sizes with the risk of rupture [3]. Conversely, a more recent retrospective cohort study found that haemangiomas greater than 4 cm, when peripherally located and exophytic, were more likely to rupture [1].
This is the first case report concerning the rupture of a hepatic haemangioma occurring with recent DOAC treatment. As literature in this area is limited, a causative link may be speculated. A PubMed search yielded two relevant cases regarding antithrombotic therapy and hepatic haemangioma rupture. This may be partially due to the insufficient reporting of concurrent medications in some published cases, which is compounded by the fact that rupture is extremely rare. The small number of cases limits any meaningful statistical measure. In one case, bleeding from a cavernous liver haemangioma occurred with streptokinase therapy [5]. Another case documented a giant haemangioma rupture in a woman after acenocoumarol (vitamin K antagonist) treatment [6]. Unlike vitamin K antagonists, the plasma concentration of DOACs is not detectable by a simple blood test nor is there a reversal agent available. Hence, the risk of bleeding prior to rupture is not quantifiable.
As histology was limited post-embolization, clinical and radiological correlation was required. CD34 staining highlighted poorly preserved vessels of varying calibres, which is often seen in haemangiomas [7]. In the setting of haemorrhage, appropriate radiological features and no evidence of cirrhosis or liver disease, a haemangioma was favoured over hepatocellular carcinoma.
The early use of TAE in managing intraperitoneal haemorrhage was shown in our case to be successful in stopping haemorrhage. However, the tumour continued to elicit compressive symptoms and hence surgical intervention was pursued. Surgical resection and enucleation are traditionally the treatments of choice for symptomatic or ruptured haemangiomas [2, 8]. Surgery also provides useful tissue confirmation as needle liver biopsy carries the risk of haemorrhage [2]. Embolization performed as a bridging technique prior to surgical resection has been shown to stabilize patients and reduce intraoperative blood loss [3, 9, 10]. The long-term success rate of embolization alone, however, has not been well studied [1, 3]. Other less common therapies include surgical ligation, radiofrequency ablation and liver transplant [4, 8].
In conclusion, rupture of a liver haemangioma is rare, with less than 100 reported cases in literature. This is the first report known to date concerning a rupture occurring with recent DOAC therapy. Our case substantiates the limited available literature regarding validity of preoperative TAE and its utility in improving surgical outcomes. Previous case reports combined with this report will significantly contribute to our understanding of the risk factors and surgical indications for ruptured hepatic haemangiomas.
CONSENT
Full informed consent was provided by the patient for the writing of this case and the use of associated images.
ACKNOWLEDGEMENTS
Histology images provided by Pathology Queensland.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None. | APIXABAN | DrugsGivenReaction | CC BY-NC | 33542805 | 18,901,738 | 2021-01 |
What was the administration route of drug 'APIXABAN'? | Giant hepatic haemangioma rupture in a patient on direct oral anticoagulant therapy.
Rupture of a liver haemangioma is extremely rare, with less than 100 cases reported in the literature. This is the first case known to date reporting a rupture occurring with direct oral anticoagulant therapy. A 76-year-old woman presented with acute abdominal pain and syncope in the context of commencing apixaban 4 weeks prior. Abdominal computed tomography and angiography demonstrated a large heterogenous mass in the left liver lobe with contrast blush suggestive of a bleeding tumour. Urgent transcatheter arterial embolization successfully ceased haemorrhage. Due to persistent compressive symptoms, a left lateral hepatectomy was performed. CD34 immunostaining of the tissue revealed variably sized vessels, which was consistent with a ruptured giant hepatic haemangioma. Our case substantiates the limited available literature regarding ruptured haemangiomas. Combined with previous case reports, this report will significantly contribute to our understanding of the risk factors, diagnosis and surgical indications for ruptured hepatic haemangiomas.
INTRODUCTION
Hepatic haemangiomas are the most common benign tumour of the liver [1]. Mesenchymal in origin, haemangiomas are composed of masses of blood vessels that are atypical in arrangement and size [1]. Most cases are asymptomatic, while few may present with symptoms related to tumour growth or compression of adjacent structures [2]. Rupture is a rare complication either occurring spontaneously or as a consequence of trauma [1–3]. Donati et al. [3] reviewed the 97 cases of ruptured liver haemangiomas published between 1898 and 2010, including two cases occurring with antithrombotic therapy. We present the case of a giant hepatic haemangioma rupture in the context of recent apixaban treatment that was subsequently treated with transcatheter arterial embolization (TAE) followed by left lateral hepatectomy. This is the first case report of a liver haemangioma rupture as a potential complication of direct oral anticoagulant (DOAC) use.
CASE REPORT
A 76-year-old woman presented with three syncopal episodes and a 2-day history of worsening abdominal pain. She had no nausea, vomiting, bowel changes or history of recent trauma. Apixaban (2.5 mg twice a day) was commenced 4 weeks prior for atrial fibrillation. Other past medical history included Parkinson’s disease. She had no history of malignancy or chronic liver disease. On presentation, she was pale, hypotensive (114/44 mmHg) and tachycardic (106 beats/minute). Physical examination demonstrated diffuse abdominal tenderness and distension without any signs of peritoneal irritation. Laboratory investigations revealed low haemoglobin (60 g/L), red blood cell count (2.35 × 1012/L) and haematocrit (0.215 L/L). Mean cell volume (92 fL) and platelets (190 × 109/L) were within range. Coagulation and liver function tests were unremarkable. Contrast-enhanced computerized tomography (CECT) reported a 62 × 83 × 90 mm heterogenous mass in the left lobe of the liver demonstrating contrast blush with large volume haemoperitoneum concerning for a bleeding hepatic neoplasm (Fig. 1). She was stabilized with five units of packed red blood cells before being transferred to our institute.
Figure 1 Axial and coronal reformats in non-contrast, arterial and delayed phases demonstrating contrast blush, with pooling within the left hepatic lobe haemangioma consistent with active bleeding.
Figure 2 DSA demonstrating hepatic vessels during TAE.
The patient was transferred to the interventional operating room for embolization of the left hepatic artery. Digital subtraction angiography (DSA) showed active contrast agent extravasation (Fig. 2). The tumour was embolized with 500–700 μm beads followed by gel foam. The patient remained hemodynamically stable throughout the procedure.
Figure 3 Pathological examination of hepatic mass revealing infarcted tissue and haemorrhage. Preserved areas of variably sized vessels shown.
Given the large size of mass and the patient’s continued symptoms of suppressed satiety and anorexia, a decision was made to perform an open left lateral hepatectomy Day 3 post-embolization. Laparotomy through rooftop incision revealed moderate hemoperitoneum and confinement of a necrotic liver lesion to Segment III. The lesion was not actively bleeding. A stapled left lateral sectionectomy was performed.
Histology showed variably sized dilated spaces within the infarcted and haemorrhagic tissue, mostly showing necrotic nuclei with focal viable bland nuclei lining the spaces. Immunohistochemistry with CD34 staining was positive in these cells (Fig. 3): (a) H&E, ×40, (b) H&E, ×400, (c) CD34 immunohistochemistry ×40 and (d) CD34 immunohistochemistry ×400. Adjacent liver parenchyma demonstrated preserved architecture. The overall features were consistent with an infarcted haemangioma.
Postoperatively, the patient successfully recovered and was transferred to rehabilitation.
DISCUSSION
Hepatic haemangiomas have an estimated prevalence of 0.4% to 7.3–20% in the general population [1, 2]. They are often asymptomatic and detected incidentally on imaging as small (<4 cm) solitary nodules. Diameters exceeding 4 or 5 cm are considered ‘giant haemangiomas’, with few reaching 20 cm [2, 3]. Spontaneous rupture is an extremely rare complication which can present as sudden abdominal pain and hypovolemic shock [1, 4]. Current literatures regarding risk factors for rupture are limited and conflicting. A 2011 literature review reported no correlation between haemangioma sizes with the risk of rupture [3]. Conversely, a more recent retrospective cohort study found that haemangiomas greater than 4 cm, when peripherally located and exophytic, were more likely to rupture [1].
This is the first case report concerning the rupture of a hepatic haemangioma occurring with recent DOAC treatment. As literature in this area is limited, a causative link may be speculated. A PubMed search yielded two relevant cases regarding antithrombotic therapy and hepatic haemangioma rupture. This may be partially due to the insufficient reporting of concurrent medications in some published cases, which is compounded by the fact that rupture is extremely rare. The small number of cases limits any meaningful statistical measure. In one case, bleeding from a cavernous liver haemangioma occurred with streptokinase therapy [5]. Another case documented a giant haemangioma rupture in a woman after acenocoumarol (vitamin K antagonist) treatment [6]. Unlike vitamin K antagonists, the plasma concentration of DOACs is not detectable by a simple blood test nor is there a reversal agent available. Hence, the risk of bleeding prior to rupture is not quantifiable.
As histology was limited post-embolization, clinical and radiological correlation was required. CD34 staining highlighted poorly preserved vessels of varying calibres, which is often seen in haemangiomas [7]. In the setting of haemorrhage, appropriate radiological features and no evidence of cirrhosis or liver disease, a haemangioma was favoured over hepatocellular carcinoma.
The early use of TAE in managing intraperitoneal haemorrhage was shown in our case to be successful in stopping haemorrhage. However, the tumour continued to elicit compressive symptoms and hence surgical intervention was pursued. Surgical resection and enucleation are traditionally the treatments of choice for symptomatic or ruptured haemangiomas [2, 8]. Surgery also provides useful tissue confirmation as needle liver biopsy carries the risk of haemorrhage [2]. Embolization performed as a bridging technique prior to surgical resection has been shown to stabilize patients and reduce intraoperative blood loss [3, 9, 10]. The long-term success rate of embolization alone, however, has not been well studied [1, 3]. Other less common therapies include surgical ligation, radiofrequency ablation and liver transplant [4, 8].
In conclusion, rupture of a liver haemangioma is rare, with less than 100 reported cases in literature. This is the first report known to date concerning a rupture occurring with recent DOAC therapy. Our case substantiates the limited available literature regarding validity of preoperative TAE and its utility in improving surgical outcomes. Previous case reports combined with this report will significantly contribute to our understanding of the risk factors and surgical indications for ruptured hepatic haemangiomas.
CONSENT
Full informed consent was provided by the patient for the writing of this case and the use of associated images.
ACKNOWLEDGEMENTS
Histology images provided by Pathology Queensland.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None. | Oral | DrugAdministrationRoute | CC BY-NC | 33542805 | 18,901,713 | 2021-01 |
What was the outcome of reaction 'Haemoglobin decreased'? | Giant hepatic haemangioma rupture in a patient on direct oral anticoagulant therapy.
Rupture of a liver haemangioma is extremely rare, with less than 100 cases reported in the literature. This is the first case known to date reporting a rupture occurring with direct oral anticoagulant therapy. A 76-year-old woman presented with acute abdominal pain and syncope in the context of commencing apixaban 4 weeks prior. Abdominal computed tomography and angiography demonstrated a large heterogenous mass in the left liver lobe with contrast blush suggestive of a bleeding tumour. Urgent transcatheter arterial embolization successfully ceased haemorrhage. Due to persistent compressive symptoms, a left lateral hepatectomy was performed. CD34 immunostaining of the tissue revealed variably sized vessels, which was consistent with a ruptured giant hepatic haemangioma. Our case substantiates the limited available literature regarding ruptured haemangiomas. Combined with previous case reports, this report will significantly contribute to our understanding of the risk factors, diagnosis and surgical indications for ruptured hepatic haemangiomas.
INTRODUCTION
Hepatic haemangiomas are the most common benign tumour of the liver [1]. Mesenchymal in origin, haemangiomas are composed of masses of blood vessels that are atypical in arrangement and size [1]. Most cases are asymptomatic, while few may present with symptoms related to tumour growth or compression of adjacent structures [2]. Rupture is a rare complication either occurring spontaneously or as a consequence of trauma [1–3]. Donati et al. [3] reviewed the 97 cases of ruptured liver haemangiomas published between 1898 and 2010, including two cases occurring with antithrombotic therapy. We present the case of a giant hepatic haemangioma rupture in the context of recent apixaban treatment that was subsequently treated with transcatheter arterial embolization (TAE) followed by left lateral hepatectomy. This is the first case report of a liver haemangioma rupture as a potential complication of direct oral anticoagulant (DOAC) use.
CASE REPORT
A 76-year-old woman presented with three syncopal episodes and a 2-day history of worsening abdominal pain. She had no nausea, vomiting, bowel changes or history of recent trauma. Apixaban (2.5 mg twice a day) was commenced 4 weeks prior for atrial fibrillation. Other past medical history included Parkinson’s disease. She had no history of malignancy or chronic liver disease. On presentation, she was pale, hypotensive (114/44 mmHg) and tachycardic (106 beats/minute). Physical examination demonstrated diffuse abdominal tenderness and distension without any signs of peritoneal irritation. Laboratory investigations revealed low haemoglobin (60 g/L), red blood cell count (2.35 × 1012/L) and haematocrit (0.215 L/L). Mean cell volume (92 fL) and platelets (190 × 109/L) were within range. Coagulation and liver function tests were unremarkable. Contrast-enhanced computerized tomography (CECT) reported a 62 × 83 × 90 mm heterogenous mass in the left lobe of the liver demonstrating contrast blush with large volume haemoperitoneum concerning for a bleeding hepatic neoplasm (Fig. 1). She was stabilized with five units of packed red blood cells before being transferred to our institute.
Figure 1 Axial and coronal reformats in non-contrast, arterial and delayed phases demonstrating contrast blush, with pooling within the left hepatic lobe haemangioma consistent with active bleeding.
Figure 2 DSA demonstrating hepatic vessels during TAE.
The patient was transferred to the interventional operating room for embolization of the left hepatic artery. Digital subtraction angiography (DSA) showed active contrast agent extravasation (Fig. 2). The tumour was embolized with 500–700 μm beads followed by gel foam. The patient remained hemodynamically stable throughout the procedure.
Figure 3 Pathological examination of hepatic mass revealing infarcted tissue and haemorrhage. Preserved areas of variably sized vessels shown.
Given the large size of mass and the patient’s continued symptoms of suppressed satiety and anorexia, a decision was made to perform an open left lateral hepatectomy Day 3 post-embolization. Laparotomy through rooftop incision revealed moderate hemoperitoneum and confinement of a necrotic liver lesion to Segment III. The lesion was not actively bleeding. A stapled left lateral sectionectomy was performed.
Histology showed variably sized dilated spaces within the infarcted and haemorrhagic tissue, mostly showing necrotic nuclei with focal viable bland nuclei lining the spaces. Immunohistochemistry with CD34 staining was positive in these cells (Fig. 3): (a) H&E, ×40, (b) H&E, ×400, (c) CD34 immunohistochemistry ×40 and (d) CD34 immunohistochemistry ×400. Adjacent liver parenchyma demonstrated preserved architecture. The overall features were consistent with an infarcted haemangioma.
Postoperatively, the patient successfully recovered and was transferred to rehabilitation.
DISCUSSION
Hepatic haemangiomas have an estimated prevalence of 0.4% to 7.3–20% in the general population [1, 2]. They are often asymptomatic and detected incidentally on imaging as small (<4 cm) solitary nodules. Diameters exceeding 4 or 5 cm are considered ‘giant haemangiomas’, with few reaching 20 cm [2, 3]. Spontaneous rupture is an extremely rare complication which can present as sudden abdominal pain and hypovolemic shock [1, 4]. Current literatures regarding risk factors for rupture are limited and conflicting. A 2011 literature review reported no correlation between haemangioma sizes with the risk of rupture [3]. Conversely, a more recent retrospective cohort study found that haemangiomas greater than 4 cm, when peripherally located and exophytic, were more likely to rupture [1].
This is the first case report concerning the rupture of a hepatic haemangioma occurring with recent DOAC treatment. As literature in this area is limited, a causative link may be speculated. A PubMed search yielded two relevant cases regarding antithrombotic therapy and hepatic haemangioma rupture. This may be partially due to the insufficient reporting of concurrent medications in some published cases, which is compounded by the fact that rupture is extremely rare. The small number of cases limits any meaningful statistical measure. In one case, bleeding from a cavernous liver haemangioma occurred with streptokinase therapy [5]. Another case documented a giant haemangioma rupture in a woman after acenocoumarol (vitamin K antagonist) treatment [6]. Unlike vitamin K antagonists, the plasma concentration of DOACs is not detectable by a simple blood test nor is there a reversal agent available. Hence, the risk of bleeding prior to rupture is not quantifiable.
As histology was limited post-embolization, clinical and radiological correlation was required. CD34 staining highlighted poorly preserved vessels of varying calibres, which is often seen in haemangiomas [7]. In the setting of haemorrhage, appropriate radiological features and no evidence of cirrhosis or liver disease, a haemangioma was favoured over hepatocellular carcinoma.
The early use of TAE in managing intraperitoneal haemorrhage was shown in our case to be successful in stopping haemorrhage. However, the tumour continued to elicit compressive symptoms and hence surgical intervention was pursued. Surgical resection and enucleation are traditionally the treatments of choice for symptomatic or ruptured haemangiomas [2, 8]. Surgery also provides useful tissue confirmation as needle liver biopsy carries the risk of haemorrhage [2]. Embolization performed as a bridging technique prior to surgical resection has been shown to stabilize patients and reduce intraoperative blood loss [3, 9, 10]. The long-term success rate of embolization alone, however, has not been well studied [1, 3]. Other less common therapies include surgical ligation, radiofrequency ablation and liver transplant [4, 8].
In conclusion, rupture of a liver haemangioma is rare, with less than 100 reported cases in literature. This is the first report known to date concerning a rupture occurring with recent DOAC therapy. Our case substantiates the limited available literature regarding validity of preoperative TAE and its utility in improving surgical outcomes. Previous case reports combined with this report will significantly contribute to our understanding of the risk factors and surgical indications for ruptured hepatic haemangiomas.
CONSENT
Full informed consent was provided by the patient for the writing of this case and the use of associated images.
ACKNOWLEDGEMENTS
Histology images provided by Pathology Queensland.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None. | Recovered | ReactionOutcome | CC BY-NC | 33542805 | 19,381,085 | 2021-01 |
What was the outcome of reaction 'Hepatic haemangioma rupture'? | Giant hepatic haemangioma rupture in a patient on direct oral anticoagulant therapy.
Rupture of a liver haemangioma is extremely rare, with less than 100 cases reported in the literature. This is the first case known to date reporting a rupture occurring with direct oral anticoagulant therapy. A 76-year-old woman presented with acute abdominal pain and syncope in the context of commencing apixaban 4 weeks prior. Abdominal computed tomography and angiography demonstrated a large heterogenous mass in the left liver lobe with contrast blush suggestive of a bleeding tumour. Urgent transcatheter arterial embolization successfully ceased haemorrhage. Due to persistent compressive symptoms, a left lateral hepatectomy was performed. CD34 immunostaining of the tissue revealed variably sized vessels, which was consistent with a ruptured giant hepatic haemangioma. Our case substantiates the limited available literature regarding ruptured haemangiomas. Combined with previous case reports, this report will significantly contribute to our understanding of the risk factors, diagnosis and surgical indications for ruptured hepatic haemangiomas.
INTRODUCTION
Hepatic haemangiomas are the most common benign tumour of the liver [1]. Mesenchymal in origin, haemangiomas are composed of masses of blood vessels that are atypical in arrangement and size [1]. Most cases are asymptomatic, while few may present with symptoms related to tumour growth or compression of adjacent structures [2]. Rupture is a rare complication either occurring spontaneously or as a consequence of trauma [1–3]. Donati et al. [3] reviewed the 97 cases of ruptured liver haemangiomas published between 1898 and 2010, including two cases occurring with antithrombotic therapy. We present the case of a giant hepatic haemangioma rupture in the context of recent apixaban treatment that was subsequently treated with transcatheter arterial embolization (TAE) followed by left lateral hepatectomy. This is the first case report of a liver haemangioma rupture as a potential complication of direct oral anticoagulant (DOAC) use.
CASE REPORT
A 76-year-old woman presented with three syncopal episodes and a 2-day history of worsening abdominal pain. She had no nausea, vomiting, bowel changes or history of recent trauma. Apixaban (2.5 mg twice a day) was commenced 4 weeks prior for atrial fibrillation. Other past medical history included Parkinson’s disease. She had no history of malignancy or chronic liver disease. On presentation, she was pale, hypotensive (114/44 mmHg) and tachycardic (106 beats/minute). Physical examination demonstrated diffuse abdominal tenderness and distension without any signs of peritoneal irritation. Laboratory investigations revealed low haemoglobin (60 g/L), red blood cell count (2.35 × 1012/L) and haematocrit (0.215 L/L). Mean cell volume (92 fL) and platelets (190 × 109/L) were within range. Coagulation and liver function tests were unremarkable. Contrast-enhanced computerized tomography (CECT) reported a 62 × 83 × 90 mm heterogenous mass in the left lobe of the liver demonstrating contrast blush with large volume haemoperitoneum concerning for a bleeding hepatic neoplasm (Fig. 1). She was stabilized with five units of packed red blood cells before being transferred to our institute.
Figure 1 Axial and coronal reformats in non-contrast, arterial and delayed phases demonstrating contrast blush, with pooling within the left hepatic lobe haemangioma consistent with active bleeding.
Figure 2 DSA demonstrating hepatic vessels during TAE.
The patient was transferred to the interventional operating room for embolization of the left hepatic artery. Digital subtraction angiography (DSA) showed active contrast agent extravasation (Fig. 2). The tumour was embolized with 500–700 μm beads followed by gel foam. The patient remained hemodynamically stable throughout the procedure.
Figure 3 Pathological examination of hepatic mass revealing infarcted tissue and haemorrhage. Preserved areas of variably sized vessels shown.
Given the large size of mass and the patient’s continued symptoms of suppressed satiety and anorexia, a decision was made to perform an open left lateral hepatectomy Day 3 post-embolization. Laparotomy through rooftop incision revealed moderate hemoperitoneum and confinement of a necrotic liver lesion to Segment III. The lesion was not actively bleeding. A stapled left lateral sectionectomy was performed.
Histology showed variably sized dilated spaces within the infarcted and haemorrhagic tissue, mostly showing necrotic nuclei with focal viable bland nuclei lining the spaces. Immunohistochemistry with CD34 staining was positive in these cells (Fig. 3): (a) H&E, ×40, (b) H&E, ×400, (c) CD34 immunohistochemistry ×40 and (d) CD34 immunohistochemistry ×400. Adjacent liver parenchyma demonstrated preserved architecture. The overall features were consistent with an infarcted haemangioma.
Postoperatively, the patient successfully recovered and was transferred to rehabilitation.
DISCUSSION
Hepatic haemangiomas have an estimated prevalence of 0.4% to 7.3–20% in the general population [1, 2]. They are often asymptomatic and detected incidentally on imaging as small (<4 cm) solitary nodules. Diameters exceeding 4 or 5 cm are considered ‘giant haemangiomas’, with few reaching 20 cm [2, 3]. Spontaneous rupture is an extremely rare complication which can present as sudden abdominal pain and hypovolemic shock [1, 4]. Current literatures regarding risk factors for rupture are limited and conflicting. A 2011 literature review reported no correlation between haemangioma sizes with the risk of rupture [3]. Conversely, a more recent retrospective cohort study found that haemangiomas greater than 4 cm, when peripherally located and exophytic, were more likely to rupture [1].
This is the first case report concerning the rupture of a hepatic haemangioma occurring with recent DOAC treatment. As literature in this area is limited, a causative link may be speculated. A PubMed search yielded two relevant cases regarding antithrombotic therapy and hepatic haemangioma rupture. This may be partially due to the insufficient reporting of concurrent medications in some published cases, which is compounded by the fact that rupture is extremely rare. The small number of cases limits any meaningful statistical measure. In one case, bleeding from a cavernous liver haemangioma occurred with streptokinase therapy [5]. Another case documented a giant haemangioma rupture in a woman after acenocoumarol (vitamin K antagonist) treatment [6]. Unlike vitamin K antagonists, the plasma concentration of DOACs is not detectable by a simple blood test nor is there a reversal agent available. Hence, the risk of bleeding prior to rupture is not quantifiable.
As histology was limited post-embolization, clinical and radiological correlation was required. CD34 staining highlighted poorly preserved vessels of varying calibres, which is often seen in haemangiomas [7]. In the setting of haemorrhage, appropriate radiological features and no evidence of cirrhosis or liver disease, a haemangioma was favoured over hepatocellular carcinoma.
The early use of TAE in managing intraperitoneal haemorrhage was shown in our case to be successful in stopping haemorrhage. However, the tumour continued to elicit compressive symptoms and hence surgical intervention was pursued. Surgical resection and enucleation are traditionally the treatments of choice for symptomatic or ruptured haemangiomas [2, 8]. Surgery also provides useful tissue confirmation as needle liver biopsy carries the risk of haemorrhage [2]. Embolization performed as a bridging technique prior to surgical resection has been shown to stabilize patients and reduce intraoperative blood loss [3, 9, 10]. The long-term success rate of embolization alone, however, has not been well studied [1, 3]. Other less common therapies include surgical ligation, radiofrequency ablation and liver transplant [4, 8].
In conclusion, rupture of a liver haemangioma is rare, with less than 100 reported cases in literature. This is the first report known to date concerning a rupture occurring with recent DOAC therapy. Our case substantiates the limited available literature regarding validity of preoperative TAE and its utility in improving surgical outcomes. Previous case reports combined with this report will significantly contribute to our understanding of the risk factors and surgical indications for ruptured hepatic haemangiomas.
CONSENT
Full informed consent was provided by the patient for the writing of this case and the use of associated images.
ACKNOWLEDGEMENTS
Histology images provided by Pathology Queensland.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None. | Recovered | ReactionOutcome | CC BY-NC | 33542805 | 18,901,738 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Disease progression'. | Rare presentation of growing teratoma syndrome in patient with remote history of testicular cancer resection.
Growing teratoma syndrome (GTS) is documented in literature to be a rare complication of non-seminomatous germ cell tumors that arises following chemotherapeutic treatment. Though represented through multiple case reports, the condition is rare that it evades observation and diagnosis, leading to complications secondary to metastasis and unchecked growth. GTS is identified via incidental finding on imaging (e.g. CT) or due to complications involving mass obstruction. Due to the potential severity of undiagnosed malignancy, it is important to effectively diagnose GTS in those presenting with non-specific symptoms and a history of testicular/ovarian cancer. It is also necessary to develop a method on how to monitor those considered to be at increased risk for developing such a condition. Here, we present a case of a middle-aged male who presented with complaints of a left lower quadrant abdominal mass and incidental finding of right retroperitoneal lesion, consistent with GTS.
INTRODUCTION
Growing teratoma syndrome (GTS) is a rare complication of non-seminomatous germ cell tumors (NSGCTs) that arises following chemotherapy, comprised entirely of mature teratomatous cells. GTS typically presents within 2 years of initial treatment after recurrence of mass growth and/or metastasis. Here, we present a case of a GTS presenting as a left lower quadrant mass with ulceration, 19 years after treatment of a primary testicular cancer.
CASE REPORT
A 56-year-old male with a history of left testicular cancer treated with chemotherapy (Bleomycin, Etoposide, Cisplatin; BEP), radiotherapy, retroperitoneal lymph node dissection, and partial left nephrectomy in 1992 presented to hospital with complaints of left lower abdominal mass for 3–5 years, associated with ulceration and active drainage. He was asymptomatic with β-hCG and alpha-fetoprotein (AFP) levels within reference ranges (<2.39 and 1.4, respectively). On examination, inspection of abdomen revealed well-healed midline scar with bilateral incisional hernias and a 10 × 8 cm mass in the left lower quadrant (Fig. 1) firm and fixed to the abdominal wall. An open lesion characterized by yellow, non-purulent drainage was noted in the left lower quadrant. CT imaging of abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm (Fig. 2) along with a similar soft tissue nodule in the right inguinal area. A right retroperitoneal soft tissue mass in the mid abdomen was also identified, measuring 3.3 × 4.2 × 5.7 cm (Fig. 3). Changes of prior left nephrectomy and orchiectomy consistent with treatment of previous left-sided testicular cancer were also noted.
Figure 1 Left lower quadrant mass on physical examination.
Figure 2 CT imaging abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm.
Figure 3 CT scan axial cut evidencing a right retroperitoneal mass measuring 3.3 × 4.2 × 5.7 cm.
Figure 4 Final operative specimen.
Biopsy of right retroperitoneal mass revealed squamous cell-lined cystic lesion followed by left lower quadrantabdominal mass excision and complex wound closure. An elliptical incision was made in the left groin incorporating the lesion, and subcutaneous flaps were created. Dissection was carried down to fascia where the mass extended into inguinal ring and the lower edge of the external oblique aponeurosis, extending into the abdomen. The mass was excised and noted to be multi-loculated and heterogeneous in consistency. The fascia and peritoneum that was adhered to the mass were then resected. A superficial inguinal lymph node was excised and sent to pathology as a separate specimen, and no other inguinal or femoral enlarged lymph nodes were found. Final pathology results revealed a mucinous cystic neoplasm with a negative lymph node for malignancy, consistent with potential spermatic cord mucinous cystadenoma (Fig. 4). The presence of retroperitoneal and contralateral inguinal masses, however, with history of teratoma raised suspicion of bilateral mucinous cystadenoma vs. residual/recurrent teratomatous component of testicular tumor. With this patient’s history and laboratory findings, diagnosis of recurrent teratoma was favored. The patient was asked to follow up with surgery two weeks after the procedure. At follow-up, patient denied any new onset of symptoms and was deemed to be experiencing appropriate postoperative recovery.
DISCUSSION
As originally defined by Logothetis et al., GTS is a rare condition characterized by an increasing mass, caused by teratomatous cells, following administration of chemotherapy. Diagnostic criteria include normalization of β-hCG and AFP, persistent enlargement of a mass following chemotherapy, and mature teratoma on pathological assessment, void of additional germ cell precursors, often originating from the testicles or ovaries [1]. The patient described in this report is a 56-year-old male who presented approximately 27 years after chemotherapeutic treatment for testicular cancer in 1992 with development of ulceration and drainage from abdominal mass that had been present for approximately 3–5 years. This is significant considering that development of GTS has been previously documented to typically arise within two-year period following completion of a chemotherapeutic regimen, specifically between 15 and 27 months [2]. Though there is reference in literature of cases being discovered as far as 19 years post-therapy, it is noted that this is a particularly rare occurrence in the setting of a pathology that, itself, is very underrepresented, with an approximate incidence of GTS ranging between 1.9 and 7.6 percent of confirmed NSGCTs [3]. Given our patient’s rare case, we seek to provide another uncommon presentation of GTS. Additionally, we perform a brief literature review for further insight into the characterization of GTS.
The exact pathophysiology/etiology of GTS is unclear, of which two of the more popular theories suggest the following: chemotherapy may induce malignant NSGCT cells to progress into mature teratomas [4] or it may lead to the death of all tumor cells excluding those associated with mature teratoma [5]. The primordial germ cell provides the origin for germ cell tumors (GCT), where successful genetic divergence (either via abnormal division, retention of embryonic features, or genomic instability) leads to the formation of malignant precursors defined as germ cell neoplasia in situ (GCNIS). Continuous alterations occur (e.g. chromosome 12p amplification and/or CCND2/KRAS/MDM2 mutations) to yield GCT, as described by Michelksi et al. [6]. GCNIS-derived teratomas are remarkable for the fact that they arise from these germ cell precursors, as proven by Jones et al., who discovered a correlation between metastatic mature teratomas and non-teratomatous GCT components by identifying several chromosomal locations (1p36, 9p21, 9q21, 13q22-q31, 18q21 and 18q22) that revealed identical genetic alterations between the mature teratoma and non-teratomatous samples alike [7]. This discovery provides a possible explanation for the theory suggesting that growing teratomas arise from teratomatous remnants of chemotherapy. Following the pattern of cell differentiation, it is reasonable to consider that mature teratomas may simultaneously reside with additional germ cell components in an individual diagnosed with NSGCT. Following eradication of germ cell components with chemotherapy, teratomatous components continue to differentiate, leading to recurrence of symptoms secondary to mass effect and/or metastasis.
CONCLUSION
GTS is a rare, evasive diagnosis that is difficult to qualify and develop standardized guidelines for. Though the consensus remains that complete excision of the mass is necessary, there is lack of appropriate guidelines for surveillance after treatment. This necessitates the development of standardized guidelines for GTS, regarding both post-treatment observation and pre-diagnostic screening for individuals with a history of NSGCTs.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None.
INFORMED CONSENT
Obtained from patient. | BLEOMYCIN SULFATE, CISPLATIN, ETOPOSIDE | DrugsGivenReaction | CC BY-NC | 33542815 | 19,419,231 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Second primary malignancy'. | Rare presentation of growing teratoma syndrome in patient with remote history of testicular cancer resection.
Growing teratoma syndrome (GTS) is documented in literature to be a rare complication of non-seminomatous germ cell tumors that arises following chemotherapeutic treatment. Though represented through multiple case reports, the condition is rare that it evades observation and diagnosis, leading to complications secondary to metastasis and unchecked growth. GTS is identified via incidental finding on imaging (e.g. CT) or due to complications involving mass obstruction. Due to the potential severity of undiagnosed malignancy, it is important to effectively diagnose GTS in those presenting with non-specific symptoms and a history of testicular/ovarian cancer. It is also necessary to develop a method on how to monitor those considered to be at increased risk for developing such a condition. Here, we present a case of a middle-aged male who presented with complaints of a left lower quadrant abdominal mass and incidental finding of right retroperitoneal lesion, consistent with GTS.
INTRODUCTION
Growing teratoma syndrome (GTS) is a rare complication of non-seminomatous germ cell tumors (NSGCTs) that arises following chemotherapy, comprised entirely of mature teratomatous cells. GTS typically presents within 2 years of initial treatment after recurrence of mass growth and/or metastasis. Here, we present a case of a GTS presenting as a left lower quadrant mass with ulceration, 19 years after treatment of a primary testicular cancer.
CASE REPORT
A 56-year-old male with a history of left testicular cancer treated with chemotherapy (Bleomycin, Etoposide, Cisplatin; BEP), radiotherapy, retroperitoneal lymph node dissection, and partial left nephrectomy in 1992 presented to hospital with complaints of left lower abdominal mass for 3–5 years, associated with ulceration and active drainage. He was asymptomatic with β-hCG and alpha-fetoprotein (AFP) levels within reference ranges (<2.39 and 1.4, respectively). On examination, inspection of abdomen revealed well-healed midline scar with bilateral incisional hernias and a 10 × 8 cm mass in the left lower quadrant (Fig. 1) firm and fixed to the abdominal wall. An open lesion characterized by yellow, non-purulent drainage was noted in the left lower quadrant. CT imaging of abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm (Fig. 2) along with a similar soft tissue nodule in the right inguinal area. A right retroperitoneal soft tissue mass in the mid abdomen was also identified, measuring 3.3 × 4.2 × 5.7 cm (Fig. 3). Changes of prior left nephrectomy and orchiectomy consistent with treatment of previous left-sided testicular cancer were also noted.
Figure 1 Left lower quadrant mass on physical examination.
Figure 2 CT imaging abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm.
Figure 3 CT scan axial cut evidencing a right retroperitoneal mass measuring 3.3 × 4.2 × 5.7 cm.
Figure 4 Final operative specimen.
Biopsy of right retroperitoneal mass revealed squamous cell-lined cystic lesion followed by left lower quadrantabdominal mass excision and complex wound closure. An elliptical incision was made in the left groin incorporating the lesion, and subcutaneous flaps were created. Dissection was carried down to fascia where the mass extended into inguinal ring and the lower edge of the external oblique aponeurosis, extending into the abdomen. The mass was excised and noted to be multi-loculated and heterogeneous in consistency. The fascia and peritoneum that was adhered to the mass were then resected. A superficial inguinal lymph node was excised and sent to pathology as a separate specimen, and no other inguinal or femoral enlarged lymph nodes were found. Final pathology results revealed a mucinous cystic neoplasm with a negative lymph node for malignancy, consistent with potential spermatic cord mucinous cystadenoma (Fig. 4). The presence of retroperitoneal and contralateral inguinal masses, however, with history of teratoma raised suspicion of bilateral mucinous cystadenoma vs. residual/recurrent teratomatous component of testicular tumor. With this patient’s history and laboratory findings, diagnosis of recurrent teratoma was favored. The patient was asked to follow up with surgery two weeks after the procedure. At follow-up, patient denied any new onset of symptoms and was deemed to be experiencing appropriate postoperative recovery.
DISCUSSION
As originally defined by Logothetis et al., GTS is a rare condition characterized by an increasing mass, caused by teratomatous cells, following administration of chemotherapy. Diagnostic criteria include normalization of β-hCG and AFP, persistent enlargement of a mass following chemotherapy, and mature teratoma on pathological assessment, void of additional germ cell precursors, often originating from the testicles or ovaries [1]. The patient described in this report is a 56-year-old male who presented approximately 27 years after chemotherapeutic treatment for testicular cancer in 1992 with development of ulceration and drainage from abdominal mass that had been present for approximately 3–5 years. This is significant considering that development of GTS has been previously documented to typically arise within two-year period following completion of a chemotherapeutic regimen, specifically between 15 and 27 months [2]. Though there is reference in literature of cases being discovered as far as 19 years post-therapy, it is noted that this is a particularly rare occurrence in the setting of a pathology that, itself, is very underrepresented, with an approximate incidence of GTS ranging between 1.9 and 7.6 percent of confirmed NSGCTs [3]. Given our patient’s rare case, we seek to provide another uncommon presentation of GTS. Additionally, we perform a brief literature review for further insight into the characterization of GTS.
The exact pathophysiology/etiology of GTS is unclear, of which two of the more popular theories suggest the following: chemotherapy may induce malignant NSGCT cells to progress into mature teratomas [4] or it may lead to the death of all tumor cells excluding those associated with mature teratoma [5]. The primordial germ cell provides the origin for germ cell tumors (GCT), where successful genetic divergence (either via abnormal division, retention of embryonic features, or genomic instability) leads to the formation of malignant precursors defined as germ cell neoplasia in situ (GCNIS). Continuous alterations occur (e.g. chromosome 12p amplification and/or CCND2/KRAS/MDM2 mutations) to yield GCT, as described by Michelksi et al. [6]. GCNIS-derived teratomas are remarkable for the fact that they arise from these germ cell precursors, as proven by Jones et al., who discovered a correlation between metastatic mature teratomas and non-teratomatous GCT components by identifying several chromosomal locations (1p36, 9p21, 9q21, 13q22-q31, 18q21 and 18q22) that revealed identical genetic alterations between the mature teratoma and non-teratomatous samples alike [7]. This discovery provides a possible explanation for the theory suggesting that growing teratomas arise from teratomatous remnants of chemotherapy. Following the pattern of cell differentiation, it is reasonable to consider that mature teratomas may simultaneously reside with additional germ cell components in an individual diagnosed with NSGCT. Following eradication of germ cell components with chemotherapy, teratomatous components continue to differentiate, leading to recurrence of symptoms secondary to mass effect and/or metastasis.
CONCLUSION
GTS is a rare, evasive diagnosis that is difficult to qualify and develop standardized guidelines for. Though the consensus remains that complete excision of the mass is necessary, there is lack of appropriate guidelines for surveillance after treatment. This necessitates the development of standardized guidelines for GTS, regarding both post-treatment observation and pre-diagnostic screening for individuals with a history of NSGCTs.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None.
INFORMED CONSENT
Obtained from patient. | BLEOMYCIN SULFATE, CISPLATIN, ETOPOSIDE | DrugsGivenReaction | CC BY-NC | 33542815 | 19,436,311 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Teratoma'. | Rare presentation of growing teratoma syndrome in patient with remote history of testicular cancer resection.
Growing teratoma syndrome (GTS) is documented in literature to be a rare complication of non-seminomatous germ cell tumors that arises following chemotherapeutic treatment. Though represented through multiple case reports, the condition is rare that it evades observation and diagnosis, leading to complications secondary to metastasis and unchecked growth. GTS is identified via incidental finding on imaging (e.g. CT) or due to complications involving mass obstruction. Due to the potential severity of undiagnosed malignancy, it is important to effectively diagnose GTS in those presenting with non-specific symptoms and a history of testicular/ovarian cancer. It is also necessary to develop a method on how to monitor those considered to be at increased risk for developing such a condition. Here, we present a case of a middle-aged male who presented with complaints of a left lower quadrant abdominal mass and incidental finding of right retroperitoneal lesion, consistent with GTS.
INTRODUCTION
Growing teratoma syndrome (GTS) is a rare complication of non-seminomatous germ cell tumors (NSGCTs) that arises following chemotherapy, comprised entirely of mature teratomatous cells. GTS typically presents within 2 years of initial treatment after recurrence of mass growth and/or metastasis. Here, we present a case of a GTS presenting as a left lower quadrant mass with ulceration, 19 years after treatment of a primary testicular cancer.
CASE REPORT
A 56-year-old male with a history of left testicular cancer treated with chemotherapy (Bleomycin, Etoposide, Cisplatin; BEP), radiotherapy, retroperitoneal lymph node dissection, and partial left nephrectomy in 1992 presented to hospital with complaints of left lower abdominal mass for 3–5 years, associated with ulceration and active drainage. He was asymptomatic with β-hCG and alpha-fetoprotein (AFP) levels within reference ranges (<2.39 and 1.4, respectively). On examination, inspection of abdomen revealed well-healed midline scar with bilateral incisional hernias and a 10 × 8 cm mass in the left lower quadrant (Fig. 1) firm and fixed to the abdominal wall. An open lesion characterized by yellow, non-purulent drainage was noted in the left lower quadrant. CT imaging of abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm (Fig. 2) along with a similar soft tissue nodule in the right inguinal area. A right retroperitoneal soft tissue mass in the mid abdomen was also identified, measuring 3.3 × 4.2 × 5.7 cm (Fig. 3). Changes of prior left nephrectomy and orchiectomy consistent with treatment of previous left-sided testicular cancer were also noted.
Figure 1 Left lower quadrant mass on physical examination.
Figure 2 CT imaging abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm.
Figure 3 CT scan axial cut evidencing a right retroperitoneal mass measuring 3.3 × 4.2 × 5.7 cm.
Figure 4 Final operative specimen.
Biopsy of right retroperitoneal mass revealed squamous cell-lined cystic lesion followed by left lower quadrantabdominal mass excision and complex wound closure. An elliptical incision was made in the left groin incorporating the lesion, and subcutaneous flaps were created. Dissection was carried down to fascia where the mass extended into inguinal ring and the lower edge of the external oblique aponeurosis, extending into the abdomen. The mass was excised and noted to be multi-loculated and heterogeneous in consistency. The fascia and peritoneum that was adhered to the mass were then resected. A superficial inguinal lymph node was excised and sent to pathology as a separate specimen, and no other inguinal or femoral enlarged lymph nodes were found. Final pathology results revealed a mucinous cystic neoplasm with a negative lymph node for malignancy, consistent with potential spermatic cord mucinous cystadenoma (Fig. 4). The presence of retroperitoneal and contralateral inguinal masses, however, with history of teratoma raised suspicion of bilateral mucinous cystadenoma vs. residual/recurrent teratomatous component of testicular tumor. With this patient’s history and laboratory findings, diagnosis of recurrent teratoma was favored. The patient was asked to follow up with surgery two weeks after the procedure. At follow-up, patient denied any new onset of symptoms and was deemed to be experiencing appropriate postoperative recovery.
DISCUSSION
As originally defined by Logothetis et al., GTS is a rare condition characterized by an increasing mass, caused by teratomatous cells, following administration of chemotherapy. Diagnostic criteria include normalization of β-hCG and AFP, persistent enlargement of a mass following chemotherapy, and mature teratoma on pathological assessment, void of additional germ cell precursors, often originating from the testicles or ovaries [1]. The patient described in this report is a 56-year-old male who presented approximately 27 years after chemotherapeutic treatment for testicular cancer in 1992 with development of ulceration and drainage from abdominal mass that had been present for approximately 3–5 years. This is significant considering that development of GTS has been previously documented to typically arise within two-year period following completion of a chemotherapeutic regimen, specifically between 15 and 27 months [2]. Though there is reference in literature of cases being discovered as far as 19 years post-therapy, it is noted that this is a particularly rare occurrence in the setting of a pathology that, itself, is very underrepresented, with an approximate incidence of GTS ranging between 1.9 and 7.6 percent of confirmed NSGCTs [3]. Given our patient’s rare case, we seek to provide another uncommon presentation of GTS. Additionally, we perform a brief literature review for further insight into the characterization of GTS.
The exact pathophysiology/etiology of GTS is unclear, of which two of the more popular theories suggest the following: chemotherapy may induce malignant NSGCT cells to progress into mature teratomas [4] or it may lead to the death of all tumor cells excluding those associated with mature teratoma [5]. The primordial germ cell provides the origin for germ cell tumors (GCT), where successful genetic divergence (either via abnormal division, retention of embryonic features, or genomic instability) leads to the formation of malignant precursors defined as germ cell neoplasia in situ (GCNIS). Continuous alterations occur (e.g. chromosome 12p amplification and/or CCND2/KRAS/MDM2 mutations) to yield GCT, as described by Michelksi et al. [6]. GCNIS-derived teratomas are remarkable for the fact that they arise from these germ cell precursors, as proven by Jones et al., who discovered a correlation between metastatic mature teratomas and non-teratomatous GCT components by identifying several chromosomal locations (1p36, 9p21, 9q21, 13q22-q31, 18q21 and 18q22) that revealed identical genetic alterations between the mature teratoma and non-teratomatous samples alike [7]. This discovery provides a possible explanation for the theory suggesting that growing teratomas arise from teratomatous remnants of chemotherapy. Following the pattern of cell differentiation, it is reasonable to consider that mature teratomas may simultaneously reside with additional germ cell components in an individual diagnosed with NSGCT. Following eradication of germ cell components with chemotherapy, teratomatous components continue to differentiate, leading to recurrence of symptoms secondary to mass effect and/or metastasis.
CONCLUSION
GTS is a rare, evasive diagnosis that is difficult to qualify and develop standardized guidelines for. Though the consensus remains that complete excision of the mass is necessary, there is lack of appropriate guidelines for surveillance after treatment. This necessitates the development of standardized guidelines for GTS, regarding both post-treatment observation and pre-diagnostic screening for individuals with a history of NSGCTs.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None.
INFORMED CONSENT
Obtained from patient. | BLEOMYCIN SULFATE, CISPLATIN, ETOPOSIDE | DrugsGivenReaction | CC BY-NC | 33542815 | 19,457,499 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Testicular germ cell tumour'. | Rare presentation of growing teratoma syndrome in patient with remote history of testicular cancer resection.
Growing teratoma syndrome (GTS) is documented in literature to be a rare complication of non-seminomatous germ cell tumors that arises following chemotherapeutic treatment. Though represented through multiple case reports, the condition is rare that it evades observation and diagnosis, leading to complications secondary to metastasis and unchecked growth. GTS is identified via incidental finding on imaging (e.g. CT) or due to complications involving mass obstruction. Due to the potential severity of undiagnosed malignancy, it is important to effectively diagnose GTS in those presenting with non-specific symptoms and a history of testicular/ovarian cancer. It is also necessary to develop a method on how to monitor those considered to be at increased risk for developing such a condition. Here, we present a case of a middle-aged male who presented with complaints of a left lower quadrant abdominal mass and incidental finding of right retroperitoneal lesion, consistent with GTS.
INTRODUCTION
Growing teratoma syndrome (GTS) is a rare complication of non-seminomatous germ cell tumors (NSGCTs) that arises following chemotherapy, comprised entirely of mature teratomatous cells. GTS typically presents within 2 years of initial treatment after recurrence of mass growth and/or metastasis. Here, we present a case of a GTS presenting as a left lower quadrant mass with ulceration, 19 years after treatment of a primary testicular cancer.
CASE REPORT
A 56-year-old male with a history of left testicular cancer treated with chemotherapy (Bleomycin, Etoposide, Cisplatin; BEP), radiotherapy, retroperitoneal lymph node dissection, and partial left nephrectomy in 1992 presented to hospital with complaints of left lower abdominal mass for 3–5 years, associated with ulceration and active drainage. He was asymptomatic with β-hCG and alpha-fetoprotein (AFP) levels within reference ranges (<2.39 and 1.4, respectively). On examination, inspection of abdomen revealed well-healed midline scar with bilateral incisional hernias and a 10 × 8 cm mass in the left lower quadrant (Fig. 1) firm and fixed to the abdominal wall. An open lesion characterized by yellow, non-purulent drainage was noted in the left lower quadrant. CT imaging of abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm (Fig. 2) along with a similar soft tissue nodule in the right inguinal area. A right retroperitoneal soft tissue mass in the mid abdomen was also identified, measuring 3.3 × 4.2 × 5.7 cm (Fig. 3). Changes of prior left nephrectomy and orchiectomy consistent with treatment of previous left-sided testicular cancer were also noted.
Figure 1 Left lower quadrant mass on physical examination.
Figure 2 CT imaging abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm.
Figure 3 CT scan axial cut evidencing a right retroperitoneal mass measuring 3.3 × 4.2 × 5.7 cm.
Figure 4 Final operative specimen.
Biopsy of right retroperitoneal mass revealed squamous cell-lined cystic lesion followed by left lower quadrantabdominal mass excision and complex wound closure. An elliptical incision was made in the left groin incorporating the lesion, and subcutaneous flaps were created. Dissection was carried down to fascia where the mass extended into inguinal ring and the lower edge of the external oblique aponeurosis, extending into the abdomen. The mass was excised and noted to be multi-loculated and heterogeneous in consistency. The fascia and peritoneum that was adhered to the mass were then resected. A superficial inguinal lymph node was excised and sent to pathology as a separate specimen, and no other inguinal or femoral enlarged lymph nodes were found. Final pathology results revealed a mucinous cystic neoplasm with a negative lymph node for malignancy, consistent with potential spermatic cord mucinous cystadenoma (Fig. 4). The presence of retroperitoneal and contralateral inguinal masses, however, with history of teratoma raised suspicion of bilateral mucinous cystadenoma vs. residual/recurrent teratomatous component of testicular tumor. With this patient’s history and laboratory findings, diagnosis of recurrent teratoma was favored. The patient was asked to follow up with surgery two weeks after the procedure. At follow-up, patient denied any new onset of symptoms and was deemed to be experiencing appropriate postoperative recovery.
DISCUSSION
As originally defined by Logothetis et al., GTS is a rare condition characterized by an increasing mass, caused by teratomatous cells, following administration of chemotherapy. Diagnostic criteria include normalization of β-hCG and AFP, persistent enlargement of a mass following chemotherapy, and mature teratoma on pathological assessment, void of additional germ cell precursors, often originating from the testicles or ovaries [1]. The patient described in this report is a 56-year-old male who presented approximately 27 years after chemotherapeutic treatment for testicular cancer in 1992 with development of ulceration and drainage from abdominal mass that had been present for approximately 3–5 years. This is significant considering that development of GTS has been previously documented to typically arise within two-year period following completion of a chemotherapeutic regimen, specifically between 15 and 27 months [2]. Though there is reference in literature of cases being discovered as far as 19 years post-therapy, it is noted that this is a particularly rare occurrence in the setting of a pathology that, itself, is very underrepresented, with an approximate incidence of GTS ranging between 1.9 and 7.6 percent of confirmed NSGCTs [3]. Given our patient’s rare case, we seek to provide another uncommon presentation of GTS. Additionally, we perform a brief literature review for further insight into the characterization of GTS.
The exact pathophysiology/etiology of GTS is unclear, of which two of the more popular theories suggest the following: chemotherapy may induce malignant NSGCT cells to progress into mature teratomas [4] or it may lead to the death of all tumor cells excluding those associated with mature teratoma [5]. The primordial germ cell provides the origin for germ cell tumors (GCT), where successful genetic divergence (either via abnormal division, retention of embryonic features, or genomic instability) leads to the formation of malignant precursors defined as germ cell neoplasia in situ (GCNIS). Continuous alterations occur (e.g. chromosome 12p amplification and/or CCND2/KRAS/MDM2 mutations) to yield GCT, as described by Michelksi et al. [6]. GCNIS-derived teratomas are remarkable for the fact that they arise from these germ cell precursors, as proven by Jones et al., who discovered a correlation between metastatic mature teratomas and non-teratomatous GCT components by identifying several chromosomal locations (1p36, 9p21, 9q21, 13q22-q31, 18q21 and 18q22) that revealed identical genetic alterations between the mature teratoma and non-teratomatous samples alike [7]. This discovery provides a possible explanation for the theory suggesting that growing teratomas arise from teratomatous remnants of chemotherapy. Following the pattern of cell differentiation, it is reasonable to consider that mature teratomas may simultaneously reside with additional germ cell components in an individual diagnosed with NSGCT. Following eradication of germ cell components with chemotherapy, teratomatous components continue to differentiate, leading to recurrence of symptoms secondary to mass effect and/or metastasis.
CONCLUSION
GTS is a rare, evasive diagnosis that is difficult to qualify and develop standardized guidelines for. Though the consensus remains that complete excision of the mass is necessary, there is lack of appropriate guidelines for surveillance after treatment. This necessitates the development of standardized guidelines for GTS, regarding both post-treatment observation and pre-diagnostic screening for individuals with a history of NSGCTs.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None.
INFORMED CONSENT
Obtained from patient. | BLEOMYCIN SULFATE, CISPLATIN, ETOPOSIDE | DrugsGivenReaction | CC BY-NC | 33542815 | 19,457,499 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Testicular malignant teratoma'. | Rare presentation of growing teratoma syndrome in patient with remote history of testicular cancer resection.
Growing teratoma syndrome (GTS) is documented in literature to be a rare complication of non-seminomatous germ cell tumors that arises following chemotherapeutic treatment. Though represented through multiple case reports, the condition is rare that it evades observation and diagnosis, leading to complications secondary to metastasis and unchecked growth. GTS is identified via incidental finding on imaging (e.g. CT) or due to complications involving mass obstruction. Due to the potential severity of undiagnosed malignancy, it is important to effectively diagnose GTS in those presenting with non-specific symptoms and a history of testicular/ovarian cancer. It is also necessary to develop a method on how to monitor those considered to be at increased risk for developing such a condition. Here, we present a case of a middle-aged male who presented with complaints of a left lower quadrant abdominal mass and incidental finding of right retroperitoneal lesion, consistent with GTS.
INTRODUCTION
Growing teratoma syndrome (GTS) is a rare complication of non-seminomatous germ cell tumors (NSGCTs) that arises following chemotherapy, comprised entirely of mature teratomatous cells. GTS typically presents within 2 years of initial treatment after recurrence of mass growth and/or metastasis. Here, we present a case of a GTS presenting as a left lower quadrant mass with ulceration, 19 years after treatment of a primary testicular cancer.
CASE REPORT
A 56-year-old male with a history of left testicular cancer treated with chemotherapy (Bleomycin, Etoposide, Cisplatin; BEP), radiotherapy, retroperitoneal lymph node dissection, and partial left nephrectomy in 1992 presented to hospital with complaints of left lower abdominal mass for 3–5 years, associated with ulceration and active drainage. He was asymptomatic with β-hCG and alpha-fetoprotein (AFP) levels within reference ranges (<2.39 and 1.4, respectively). On examination, inspection of abdomen revealed well-healed midline scar with bilateral incisional hernias and a 10 × 8 cm mass in the left lower quadrant (Fig. 1) firm and fixed to the abdominal wall. An open lesion characterized by yellow, non-purulent drainage was noted in the left lower quadrant. CT imaging of abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm (Fig. 2) along with a similar soft tissue nodule in the right inguinal area. A right retroperitoneal soft tissue mass in the mid abdomen was also identified, measuring 3.3 × 4.2 × 5.7 cm (Fig. 3). Changes of prior left nephrectomy and orchiectomy consistent with treatment of previous left-sided testicular cancer were also noted.
Figure 1 Left lower quadrant mass on physical examination.
Figure 2 CT imaging abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm.
Figure 3 CT scan axial cut evidencing a right retroperitoneal mass measuring 3.3 × 4.2 × 5.7 cm.
Figure 4 Final operative specimen.
Biopsy of right retroperitoneal mass revealed squamous cell-lined cystic lesion followed by left lower quadrantabdominal mass excision and complex wound closure. An elliptical incision was made in the left groin incorporating the lesion, and subcutaneous flaps were created. Dissection was carried down to fascia where the mass extended into inguinal ring and the lower edge of the external oblique aponeurosis, extending into the abdomen. The mass was excised and noted to be multi-loculated and heterogeneous in consistency. The fascia and peritoneum that was adhered to the mass were then resected. A superficial inguinal lymph node was excised and sent to pathology as a separate specimen, and no other inguinal or femoral enlarged lymph nodes were found. Final pathology results revealed a mucinous cystic neoplasm with a negative lymph node for malignancy, consistent with potential spermatic cord mucinous cystadenoma (Fig. 4). The presence of retroperitoneal and contralateral inguinal masses, however, with history of teratoma raised suspicion of bilateral mucinous cystadenoma vs. residual/recurrent teratomatous component of testicular tumor. With this patient’s history and laboratory findings, diagnosis of recurrent teratoma was favored. The patient was asked to follow up with surgery two weeks after the procedure. At follow-up, patient denied any new onset of symptoms and was deemed to be experiencing appropriate postoperative recovery.
DISCUSSION
As originally defined by Logothetis et al., GTS is a rare condition characterized by an increasing mass, caused by teratomatous cells, following administration of chemotherapy. Diagnostic criteria include normalization of β-hCG and AFP, persistent enlargement of a mass following chemotherapy, and mature teratoma on pathological assessment, void of additional germ cell precursors, often originating from the testicles or ovaries [1]. The patient described in this report is a 56-year-old male who presented approximately 27 years after chemotherapeutic treatment for testicular cancer in 1992 with development of ulceration and drainage from abdominal mass that had been present for approximately 3–5 years. This is significant considering that development of GTS has been previously documented to typically arise within two-year period following completion of a chemotherapeutic regimen, specifically between 15 and 27 months [2]. Though there is reference in literature of cases being discovered as far as 19 years post-therapy, it is noted that this is a particularly rare occurrence in the setting of a pathology that, itself, is very underrepresented, with an approximate incidence of GTS ranging between 1.9 and 7.6 percent of confirmed NSGCTs [3]. Given our patient’s rare case, we seek to provide another uncommon presentation of GTS. Additionally, we perform a brief literature review for further insight into the characterization of GTS.
The exact pathophysiology/etiology of GTS is unclear, of which two of the more popular theories suggest the following: chemotherapy may induce malignant NSGCT cells to progress into mature teratomas [4] or it may lead to the death of all tumor cells excluding those associated with mature teratoma [5]. The primordial germ cell provides the origin for germ cell tumors (GCT), where successful genetic divergence (either via abnormal division, retention of embryonic features, or genomic instability) leads to the formation of malignant precursors defined as germ cell neoplasia in situ (GCNIS). Continuous alterations occur (e.g. chromosome 12p amplification and/or CCND2/KRAS/MDM2 mutations) to yield GCT, as described by Michelksi et al. [6]. GCNIS-derived teratomas are remarkable for the fact that they arise from these germ cell precursors, as proven by Jones et al., who discovered a correlation between metastatic mature teratomas and non-teratomatous GCT components by identifying several chromosomal locations (1p36, 9p21, 9q21, 13q22-q31, 18q21 and 18q22) that revealed identical genetic alterations between the mature teratoma and non-teratomatous samples alike [7]. This discovery provides a possible explanation for the theory suggesting that growing teratomas arise from teratomatous remnants of chemotherapy. Following the pattern of cell differentiation, it is reasonable to consider that mature teratomas may simultaneously reside with additional germ cell components in an individual diagnosed with NSGCT. Following eradication of germ cell components with chemotherapy, teratomatous components continue to differentiate, leading to recurrence of symptoms secondary to mass effect and/or metastasis.
CONCLUSION
GTS is a rare, evasive diagnosis that is difficult to qualify and develop standardized guidelines for. Though the consensus remains that complete excision of the mass is necessary, there is lack of appropriate guidelines for surveillance after treatment. This necessitates the development of standardized guidelines for GTS, regarding both post-treatment observation and pre-diagnostic screening for individuals with a history of NSGCTs.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None.
INFORMED CONSENT
Obtained from patient. | BLEOMYCIN SULFATE, CISPLATIN, ETOPOSIDE | DrugsGivenReaction | CC BY-NC | 33542815 | 19,415,437 | 2021-01 |
What was the outcome of reaction 'Disease progression'? | Rare presentation of growing teratoma syndrome in patient with remote history of testicular cancer resection.
Growing teratoma syndrome (GTS) is documented in literature to be a rare complication of non-seminomatous germ cell tumors that arises following chemotherapeutic treatment. Though represented through multiple case reports, the condition is rare that it evades observation and diagnosis, leading to complications secondary to metastasis and unchecked growth. GTS is identified via incidental finding on imaging (e.g. CT) or due to complications involving mass obstruction. Due to the potential severity of undiagnosed malignancy, it is important to effectively diagnose GTS in those presenting with non-specific symptoms and a history of testicular/ovarian cancer. It is also necessary to develop a method on how to monitor those considered to be at increased risk for developing such a condition. Here, we present a case of a middle-aged male who presented with complaints of a left lower quadrant abdominal mass and incidental finding of right retroperitoneal lesion, consistent with GTS.
INTRODUCTION
Growing teratoma syndrome (GTS) is a rare complication of non-seminomatous germ cell tumors (NSGCTs) that arises following chemotherapy, comprised entirely of mature teratomatous cells. GTS typically presents within 2 years of initial treatment after recurrence of mass growth and/or metastasis. Here, we present a case of a GTS presenting as a left lower quadrant mass with ulceration, 19 years after treatment of a primary testicular cancer.
CASE REPORT
A 56-year-old male with a history of left testicular cancer treated with chemotherapy (Bleomycin, Etoposide, Cisplatin; BEP), radiotherapy, retroperitoneal lymph node dissection, and partial left nephrectomy in 1992 presented to hospital with complaints of left lower abdominal mass for 3–5 years, associated with ulceration and active drainage. He was asymptomatic with β-hCG and alpha-fetoprotein (AFP) levels within reference ranges (<2.39 and 1.4, respectively). On examination, inspection of abdomen revealed well-healed midline scar with bilateral incisional hernias and a 10 × 8 cm mass in the left lower quadrant (Fig. 1) firm and fixed to the abdominal wall. An open lesion characterized by yellow, non-purulent drainage was noted in the left lower quadrant. CT imaging of abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm (Fig. 2) along with a similar soft tissue nodule in the right inguinal area. A right retroperitoneal soft tissue mass in the mid abdomen was also identified, measuring 3.3 × 4.2 × 5.7 cm (Fig. 3). Changes of prior left nephrectomy and orchiectomy consistent with treatment of previous left-sided testicular cancer were also noted.
Figure 1 Left lower quadrant mass on physical examination.
Figure 2 CT imaging abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm.
Figure 3 CT scan axial cut evidencing a right retroperitoneal mass measuring 3.3 × 4.2 × 5.7 cm.
Figure 4 Final operative specimen.
Biopsy of right retroperitoneal mass revealed squamous cell-lined cystic lesion followed by left lower quadrantabdominal mass excision and complex wound closure. An elliptical incision was made in the left groin incorporating the lesion, and subcutaneous flaps were created. Dissection was carried down to fascia where the mass extended into inguinal ring and the lower edge of the external oblique aponeurosis, extending into the abdomen. The mass was excised and noted to be multi-loculated and heterogeneous in consistency. The fascia and peritoneum that was adhered to the mass were then resected. A superficial inguinal lymph node was excised and sent to pathology as a separate specimen, and no other inguinal or femoral enlarged lymph nodes were found. Final pathology results revealed a mucinous cystic neoplasm with a negative lymph node for malignancy, consistent with potential spermatic cord mucinous cystadenoma (Fig. 4). The presence of retroperitoneal and contralateral inguinal masses, however, with history of teratoma raised suspicion of bilateral mucinous cystadenoma vs. residual/recurrent teratomatous component of testicular tumor. With this patient’s history and laboratory findings, diagnosis of recurrent teratoma was favored. The patient was asked to follow up with surgery two weeks after the procedure. At follow-up, patient denied any new onset of symptoms and was deemed to be experiencing appropriate postoperative recovery.
DISCUSSION
As originally defined by Logothetis et al., GTS is a rare condition characterized by an increasing mass, caused by teratomatous cells, following administration of chemotherapy. Diagnostic criteria include normalization of β-hCG and AFP, persistent enlargement of a mass following chemotherapy, and mature teratoma on pathological assessment, void of additional germ cell precursors, often originating from the testicles or ovaries [1]. The patient described in this report is a 56-year-old male who presented approximately 27 years after chemotherapeutic treatment for testicular cancer in 1992 with development of ulceration and drainage from abdominal mass that had been present for approximately 3–5 years. This is significant considering that development of GTS has been previously documented to typically arise within two-year period following completion of a chemotherapeutic regimen, specifically between 15 and 27 months [2]. Though there is reference in literature of cases being discovered as far as 19 years post-therapy, it is noted that this is a particularly rare occurrence in the setting of a pathology that, itself, is very underrepresented, with an approximate incidence of GTS ranging between 1.9 and 7.6 percent of confirmed NSGCTs [3]. Given our patient’s rare case, we seek to provide another uncommon presentation of GTS. Additionally, we perform a brief literature review for further insight into the characterization of GTS.
The exact pathophysiology/etiology of GTS is unclear, of which two of the more popular theories suggest the following: chemotherapy may induce malignant NSGCT cells to progress into mature teratomas [4] or it may lead to the death of all tumor cells excluding those associated with mature teratoma [5]. The primordial germ cell provides the origin for germ cell tumors (GCT), where successful genetic divergence (either via abnormal division, retention of embryonic features, or genomic instability) leads to the formation of malignant precursors defined as germ cell neoplasia in situ (GCNIS). Continuous alterations occur (e.g. chromosome 12p amplification and/or CCND2/KRAS/MDM2 mutations) to yield GCT, as described by Michelksi et al. [6]. GCNIS-derived teratomas are remarkable for the fact that they arise from these germ cell precursors, as proven by Jones et al., who discovered a correlation between metastatic mature teratomas and non-teratomatous GCT components by identifying several chromosomal locations (1p36, 9p21, 9q21, 13q22-q31, 18q21 and 18q22) that revealed identical genetic alterations between the mature teratoma and non-teratomatous samples alike [7]. This discovery provides a possible explanation for the theory suggesting that growing teratomas arise from teratomatous remnants of chemotherapy. Following the pattern of cell differentiation, it is reasonable to consider that mature teratomas may simultaneously reside with additional germ cell components in an individual diagnosed with NSGCT. Following eradication of germ cell components with chemotherapy, teratomatous components continue to differentiate, leading to recurrence of symptoms secondary to mass effect and/or metastasis.
CONCLUSION
GTS is a rare, evasive diagnosis that is difficult to qualify and develop standardized guidelines for. Though the consensus remains that complete excision of the mass is necessary, there is lack of appropriate guidelines for surveillance after treatment. This necessitates the development of standardized guidelines for GTS, regarding both post-treatment observation and pre-diagnostic screening for individuals with a history of NSGCTs.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None.
INFORMED CONSENT
Obtained from patient. | Recovered | ReactionOutcome | CC BY-NC | 33542815 | 19,419,231 | 2021-01 |
What was the outcome of reaction 'Second primary malignancy'? | Rare presentation of growing teratoma syndrome in patient with remote history of testicular cancer resection.
Growing teratoma syndrome (GTS) is documented in literature to be a rare complication of non-seminomatous germ cell tumors that arises following chemotherapeutic treatment. Though represented through multiple case reports, the condition is rare that it evades observation and diagnosis, leading to complications secondary to metastasis and unchecked growth. GTS is identified via incidental finding on imaging (e.g. CT) or due to complications involving mass obstruction. Due to the potential severity of undiagnosed malignancy, it is important to effectively diagnose GTS in those presenting with non-specific symptoms and a history of testicular/ovarian cancer. It is also necessary to develop a method on how to monitor those considered to be at increased risk for developing such a condition. Here, we present a case of a middle-aged male who presented with complaints of a left lower quadrant abdominal mass and incidental finding of right retroperitoneal lesion, consistent with GTS.
INTRODUCTION
Growing teratoma syndrome (GTS) is a rare complication of non-seminomatous germ cell tumors (NSGCTs) that arises following chemotherapy, comprised entirely of mature teratomatous cells. GTS typically presents within 2 years of initial treatment after recurrence of mass growth and/or metastasis. Here, we present a case of a GTS presenting as a left lower quadrant mass with ulceration, 19 years after treatment of a primary testicular cancer.
CASE REPORT
A 56-year-old male with a history of left testicular cancer treated with chemotherapy (Bleomycin, Etoposide, Cisplatin; BEP), radiotherapy, retroperitoneal lymph node dissection, and partial left nephrectomy in 1992 presented to hospital with complaints of left lower abdominal mass for 3–5 years, associated with ulceration and active drainage. He was asymptomatic with β-hCG and alpha-fetoprotein (AFP) levels within reference ranges (<2.39 and 1.4, respectively). On examination, inspection of abdomen revealed well-healed midline scar with bilateral incisional hernias and a 10 × 8 cm mass in the left lower quadrant (Fig. 1) firm and fixed to the abdominal wall. An open lesion characterized by yellow, non-purulent drainage was noted in the left lower quadrant. CT imaging of abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm (Fig. 2) along with a similar soft tissue nodule in the right inguinal area. A right retroperitoneal soft tissue mass in the mid abdomen was also identified, measuring 3.3 × 4.2 × 5.7 cm (Fig. 3). Changes of prior left nephrectomy and orchiectomy consistent with treatment of previous left-sided testicular cancer were also noted.
Figure 1 Left lower quadrant mass on physical examination.
Figure 2 CT imaging abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm.
Figure 3 CT scan axial cut evidencing a right retroperitoneal mass measuring 3.3 × 4.2 × 5.7 cm.
Figure 4 Final operative specimen.
Biopsy of right retroperitoneal mass revealed squamous cell-lined cystic lesion followed by left lower quadrantabdominal mass excision and complex wound closure. An elliptical incision was made in the left groin incorporating the lesion, and subcutaneous flaps were created. Dissection was carried down to fascia where the mass extended into inguinal ring and the lower edge of the external oblique aponeurosis, extending into the abdomen. The mass was excised and noted to be multi-loculated and heterogeneous in consistency. The fascia and peritoneum that was adhered to the mass were then resected. A superficial inguinal lymph node was excised and sent to pathology as a separate specimen, and no other inguinal or femoral enlarged lymph nodes were found. Final pathology results revealed a mucinous cystic neoplasm with a negative lymph node for malignancy, consistent with potential spermatic cord mucinous cystadenoma (Fig. 4). The presence of retroperitoneal and contralateral inguinal masses, however, with history of teratoma raised suspicion of bilateral mucinous cystadenoma vs. residual/recurrent teratomatous component of testicular tumor. With this patient’s history and laboratory findings, diagnosis of recurrent teratoma was favored. The patient was asked to follow up with surgery two weeks after the procedure. At follow-up, patient denied any new onset of symptoms and was deemed to be experiencing appropriate postoperative recovery.
DISCUSSION
As originally defined by Logothetis et al., GTS is a rare condition characterized by an increasing mass, caused by teratomatous cells, following administration of chemotherapy. Diagnostic criteria include normalization of β-hCG and AFP, persistent enlargement of a mass following chemotherapy, and mature teratoma on pathological assessment, void of additional germ cell precursors, often originating from the testicles or ovaries [1]. The patient described in this report is a 56-year-old male who presented approximately 27 years after chemotherapeutic treatment for testicular cancer in 1992 with development of ulceration and drainage from abdominal mass that had been present for approximately 3–5 years. This is significant considering that development of GTS has been previously documented to typically arise within two-year period following completion of a chemotherapeutic regimen, specifically between 15 and 27 months [2]. Though there is reference in literature of cases being discovered as far as 19 years post-therapy, it is noted that this is a particularly rare occurrence in the setting of a pathology that, itself, is very underrepresented, with an approximate incidence of GTS ranging between 1.9 and 7.6 percent of confirmed NSGCTs [3]. Given our patient’s rare case, we seek to provide another uncommon presentation of GTS. Additionally, we perform a brief literature review for further insight into the characterization of GTS.
The exact pathophysiology/etiology of GTS is unclear, of which two of the more popular theories suggest the following: chemotherapy may induce malignant NSGCT cells to progress into mature teratomas [4] or it may lead to the death of all tumor cells excluding those associated with mature teratoma [5]. The primordial germ cell provides the origin for germ cell tumors (GCT), where successful genetic divergence (either via abnormal division, retention of embryonic features, or genomic instability) leads to the formation of malignant precursors defined as germ cell neoplasia in situ (GCNIS). Continuous alterations occur (e.g. chromosome 12p amplification and/or CCND2/KRAS/MDM2 mutations) to yield GCT, as described by Michelksi et al. [6]. GCNIS-derived teratomas are remarkable for the fact that they arise from these germ cell precursors, as proven by Jones et al., who discovered a correlation between metastatic mature teratomas and non-teratomatous GCT components by identifying several chromosomal locations (1p36, 9p21, 9q21, 13q22-q31, 18q21 and 18q22) that revealed identical genetic alterations between the mature teratoma and non-teratomatous samples alike [7]. This discovery provides a possible explanation for the theory suggesting that growing teratomas arise from teratomatous remnants of chemotherapy. Following the pattern of cell differentiation, it is reasonable to consider that mature teratomas may simultaneously reside with additional germ cell components in an individual diagnosed with NSGCT. Following eradication of germ cell components with chemotherapy, teratomatous components continue to differentiate, leading to recurrence of symptoms secondary to mass effect and/or metastasis.
CONCLUSION
GTS is a rare, evasive diagnosis that is difficult to qualify and develop standardized guidelines for. Though the consensus remains that complete excision of the mass is necessary, there is lack of appropriate guidelines for surveillance after treatment. This necessitates the development of standardized guidelines for GTS, regarding both post-treatment observation and pre-diagnostic screening for individuals with a history of NSGCTs.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None.
INFORMED CONSENT
Obtained from patient. | Recovering | ReactionOutcome | CC BY-NC | 33542815 | 19,436,311 | 2021-01 |
What was the outcome of reaction 'Testicular germ cell tumour'? | Rare presentation of growing teratoma syndrome in patient with remote history of testicular cancer resection.
Growing teratoma syndrome (GTS) is documented in literature to be a rare complication of non-seminomatous germ cell tumors that arises following chemotherapeutic treatment. Though represented through multiple case reports, the condition is rare that it evades observation and diagnosis, leading to complications secondary to metastasis and unchecked growth. GTS is identified via incidental finding on imaging (e.g. CT) or due to complications involving mass obstruction. Due to the potential severity of undiagnosed malignancy, it is important to effectively diagnose GTS in those presenting with non-specific symptoms and a history of testicular/ovarian cancer. It is also necessary to develop a method on how to monitor those considered to be at increased risk for developing such a condition. Here, we present a case of a middle-aged male who presented with complaints of a left lower quadrant abdominal mass and incidental finding of right retroperitoneal lesion, consistent with GTS.
INTRODUCTION
Growing teratoma syndrome (GTS) is a rare complication of non-seminomatous germ cell tumors (NSGCTs) that arises following chemotherapy, comprised entirely of mature teratomatous cells. GTS typically presents within 2 years of initial treatment after recurrence of mass growth and/or metastasis. Here, we present a case of a GTS presenting as a left lower quadrant mass with ulceration, 19 years after treatment of a primary testicular cancer.
CASE REPORT
A 56-year-old male with a history of left testicular cancer treated with chemotherapy (Bleomycin, Etoposide, Cisplatin; BEP), radiotherapy, retroperitoneal lymph node dissection, and partial left nephrectomy in 1992 presented to hospital with complaints of left lower abdominal mass for 3–5 years, associated with ulceration and active drainage. He was asymptomatic with β-hCG and alpha-fetoprotein (AFP) levels within reference ranges (<2.39 and 1.4, respectively). On examination, inspection of abdomen revealed well-healed midline scar with bilateral incisional hernias and a 10 × 8 cm mass in the left lower quadrant (Fig. 1) firm and fixed to the abdominal wall. An open lesion characterized by yellow, non-purulent drainage was noted in the left lower quadrant. CT imaging of abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm (Fig. 2) along with a similar soft tissue nodule in the right inguinal area. A right retroperitoneal soft tissue mass in the mid abdomen was also identified, measuring 3.3 × 4.2 × 5.7 cm (Fig. 3). Changes of prior left nephrectomy and orchiectomy consistent with treatment of previous left-sided testicular cancer were also noted.
Figure 1 Left lower quadrant mass on physical examination.
Figure 2 CT imaging abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm.
Figure 3 CT scan axial cut evidencing a right retroperitoneal mass measuring 3.3 × 4.2 × 5.7 cm.
Figure 4 Final operative specimen.
Biopsy of right retroperitoneal mass revealed squamous cell-lined cystic lesion followed by left lower quadrantabdominal mass excision and complex wound closure. An elliptical incision was made in the left groin incorporating the lesion, and subcutaneous flaps were created. Dissection was carried down to fascia where the mass extended into inguinal ring and the lower edge of the external oblique aponeurosis, extending into the abdomen. The mass was excised and noted to be multi-loculated and heterogeneous in consistency. The fascia and peritoneum that was adhered to the mass were then resected. A superficial inguinal lymph node was excised and sent to pathology as a separate specimen, and no other inguinal or femoral enlarged lymph nodes were found. Final pathology results revealed a mucinous cystic neoplasm with a negative lymph node for malignancy, consistent with potential spermatic cord mucinous cystadenoma (Fig. 4). The presence of retroperitoneal and contralateral inguinal masses, however, with history of teratoma raised suspicion of bilateral mucinous cystadenoma vs. residual/recurrent teratomatous component of testicular tumor. With this patient’s history and laboratory findings, diagnosis of recurrent teratoma was favored. The patient was asked to follow up with surgery two weeks after the procedure. At follow-up, patient denied any new onset of symptoms and was deemed to be experiencing appropriate postoperative recovery.
DISCUSSION
As originally defined by Logothetis et al., GTS is a rare condition characterized by an increasing mass, caused by teratomatous cells, following administration of chemotherapy. Diagnostic criteria include normalization of β-hCG and AFP, persistent enlargement of a mass following chemotherapy, and mature teratoma on pathological assessment, void of additional germ cell precursors, often originating from the testicles or ovaries [1]. The patient described in this report is a 56-year-old male who presented approximately 27 years after chemotherapeutic treatment for testicular cancer in 1992 with development of ulceration and drainage from abdominal mass that had been present for approximately 3–5 years. This is significant considering that development of GTS has been previously documented to typically arise within two-year period following completion of a chemotherapeutic regimen, specifically between 15 and 27 months [2]. Though there is reference in literature of cases being discovered as far as 19 years post-therapy, it is noted that this is a particularly rare occurrence in the setting of a pathology that, itself, is very underrepresented, with an approximate incidence of GTS ranging between 1.9 and 7.6 percent of confirmed NSGCTs [3]. Given our patient’s rare case, we seek to provide another uncommon presentation of GTS. Additionally, we perform a brief literature review for further insight into the characterization of GTS.
The exact pathophysiology/etiology of GTS is unclear, of which two of the more popular theories suggest the following: chemotherapy may induce malignant NSGCT cells to progress into mature teratomas [4] or it may lead to the death of all tumor cells excluding those associated with mature teratoma [5]. The primordial germ cell provides the origin for germ cell tumors (GCT), where successful genetic divergence (either via abnormal division, retention of embryonic features, or genomic instability) leads to the formation of malignant precursors defined as germ cell neoplasia in situ (GCNIS). Continuous alterations occur (e.g. chromosome 12p amplification and/or CCND2/KRAS/MDM2 mutations) to yield GCT, as described by Michelksi et al. [6]. GCNIS-derived teratomas are remarkable for the fact that they arise from these germ cell precursors, as proven by Jones et al., who discovered a correlation between metastatic mature teratomas and non-teratomatous GCT components by identifying several chromosomal locations (1p36, 9p21, 9q21, 13q22-q31, 18q21 and 18q22) that revealed identical genetic alterations between the mature teratoma and non-teratomatous samples alike [7]. This discovery provides a possible explanation for the theory suggesting that growing teratomas arise from teratomatous remnants of chemotherapy. Following the pattern of cell differentiation, it is reasonable to consider that mature teratomas may simultaneously reside with additional germ cell components in an individual diagnosed with NSGCT. Following eradication of germ cell components with chemotherapy, teratomatous components continue to differentiate, leading to recurrence of symptoms secondary to mass effect and/or metastasis.
CONCLUSION
GTS is a rare, evasive diagnosis that is difficult to qualify and develop standardized guidelines for. Though the consensus remains that complete excision of the mass is necessary, there is lack of appropriate guidelines for surveillance after treatment. This necessitates the development of standardized guidelines for GTS, regarding both post-treatment observation and pre-diagnostic screening for individuals with a history of NSGCTs.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None.
INFORMED CONSENT
Obtained from patient. | Recovered | ReactionOutcome | CC BY-NC | 33542815 | 19,457,499 | 2021-01 |
What was the outcome of reaction 'Testicular malignant teratoma'? | Rare presentation of growing teratoma syndrome in patient with remote history of testicular cancer resection.
Growing teratoma syndrome (GTS) is documented in literature to be a rare complication of non-seminomatous germ cell tumors that arises following chemotherapeutic treatment. Though represented through multiple case reports, the condition is rare that it evades observation and diagnosis, leading to complications secondary to metastasis and unchecked growth. GTS is identified via incidental finding on imaging (e.g. CT) or due to complications involving mass obstruction. Due to the potential severity of undiagnosed malignancy, it is important to effectively diagnose GTS in those presenting with non-specific symptoms and a history of testicular/ovarian cancer. It is also necessary to develop a method on how to monitor those considered to be at increased risk for developing such a condition. Here, we present a case of a middle-aged male who presented with complaints of a left lower quadrant abdominal mass and incidental finding of right retroperitoneal lesion, consistent with GTS.
INTRODUCTION
Growing teratoma syndrome (GTS) is a rare complication of non-seminomatous germ cell tumors (NSGCTs) that arises following chemotherapy, comprised entirely of mature teratomatous cells. GTS typically presents within 2 years of initial treatment after recurrence of mass growth and/or metastasis. Here, we present a case of a GTS presenting as a left lower quadrant mass with ulceration, 19 years after treatment of a primary testicular cancer.
CASE REPORT
A 56-year-old male with a history of left testicular cancer treated with chemotherapy (Bleomycin, Etoposide, Cisplatin; BEP), radiotherapy, retroperitoneal lymph node dissection, and partial left nephrectomy in 1992 presented to hospital with complaints of left lower abdominal mass for 3–5 years, associated with ulceration and active drainage. He was asymptomatic with β-hCG and alpha-fetoprotein (AFP) levels within reference ranges (<2.39 and 1.4, respectively). On examination, inspection of abdomen revealed well-healed midline scar with bilateral incisional hernias and a 10 × 8 cm mass in the left lower quadrant (Fig. 1) firm and fixed to the abdominal wall. An open lesion characterized by yellow, non-purulent drainage was noted in the left lower quadrant. CT imaging of abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm (Fig. 2) along with a similar soft tissue nodule in the right inguinal area. A right retroperitoneal soft tissue mass in the mid abdomen was also identified, measuring 3.3 × 4.2 × 5.7 cm (Fig. 3). Changes of prior left nephrectomy and orchiectomy consistent with treatment of previous left-sided testicular cancer were also noted.
Figure 1 Left lower quadrant mass on physical examination.
Figure 2 CT imaging abdomen and pelvis with contrast revealed a multiloculated large cystic mass in the left inguinal canal measuring 11 × 7 × 7.7 cm.
Figure 3 CT scan axial cut evidencing a right retroperitoneal mass measuring 3.3 × 4.2 × 5.7 cm.
Figure 4 Final operative specimen.
Biopsy of right retroperitoneal mass revealed squamous cell-lined cystic lesion followed by left lower quadrantabdominal mass excision and complex wound closure. An elliptical incision was made in the left groin incorporating the lesion, and subcutaneous flaps were created. Dissection was carried down to fascia where the mass extended into inguinal ring and the lower edge of the external oblique aponeurosis, extending into the abdomen. The mass was excised and noted to be multi-loculated and heterogeneous in consistency. The fascia and peritoneum that was adhered to the mass were then resected. A superficial inguinal lymph node was excised and sent to pathology as a separate specimen, and no other inguinal or femoral enlarged lymph nodes were found. Final pathology results revealed a mucinous cystic neoplasm with a negative lymph node for malignancy, consistent with potential spermatic cord mucinous cystadenoma (Fig. 4). The presence of retroperitoneal and contralateral inguinal masses, however, with history of teratoma raised suspicion of bilateral mucinous cystadenoma vs. residual/recurrent teratomatous component of testicular tumor. With this patient’s history and laboratory findings, diagnosis of recurrent teratoma was favored. The patient was asked to follow up with surgery two weeks after the procedure. At follow-up, patient denied any new onset of symptoms and was deemed to be experiencing appropriate postoperative recovery.
DISCUSSION
As originally defined by Logothetis et al., GTS is a rare condition characterized by an increasing mass, caused by teratomatous cells, following administration of chemotherapy. Diagnostic criteria include normalization of β-hCG and AFP, persistent enlargement of a mass following chemotherapy, and mature teratoma on pathological assessment, void of additional germ cell precursors, often originating from the testicles or ovaries [1]. The patient described in this report is a 56-year-old male who presented approximately 27 years after chemotherapeutic treatment for testicular cancer in 1992 with development of ulceration and drainage from abdominal mass that had been present for approximately 3–5 years. This is significant considering that development of GTS has been previously documented to typically arise within two-year period following completion of a chemotherapeutic regimen, specifically between 15 and 27 months [2]. Though there is reference in literature of cases being discovered as far as 19 years post-therapy, it is noted that this is a particularly rare occurrence in the setting of a pathology that, itself, is very underrepresented, with an approximate incidence of GTS ranging between 1.9 and 7.6 percent of confirmed NSGCTs [3]. Given our patient’s rare case, we seek to provide another uncommon presentation of GTS. Additionally, we perform a brief literature review for further insight into the characterization of GTS.
The exact pathophysiology/etiology of GTS is unclear, of which two of the more popular theories suggest the following: chemotherapy may induce malignant NSGCT cells to progress into mature teratomas [4] or it may lead to the death of all tumor cells excluding those associated with mature teratoma [5]. The primordial germ cell provides the origin for germ cell tumors (GCT), where successful genetic divergence (either via abnormal division, retention of embryonic features, or genomic instability) leads to the formation of malignant precursors defined as germ cell neoplasia in situ (GCNIS). Continuous alterations occur (e.g. chromosome 12p amplification and/or CCND2/KRAS/MDM2 mutations) to yield GCT, as described by Michelksi et al. [6]. GCNIS-derived teratomas are remarkable for the fact that they arise from these germ cell precursors, as proven by Jones et al., who discovered a correlation between metastatic mature teratomas and non-teratomatous GCT components by identifying several chromosomal locations (1p36, 9p21, 9q21, 13q22-q31, 18q21 and 18q22) that revealed identical genetic alterations between the mature teratoma and non-teratomatous samples alike [7]. This discovery provides a possible explanation for the theory suggesting that growing teratomas arise from teratomatous remnants of chemotherapy. Following the pattern of cell differentiation, it is reasonable to consider that mature teratomas may simultaneously reside with additional germ cell components in an individual diagnosed with NSGCT. Following eradication of germ cell components with chemotherapy, teratomatous components continue to differentiate, leading to recurrence of symptoms secondary to mass effect and/or metastasis.
CONCLUSION
GTS is a rare, evasive diagnosis that is difficult to qualify and develop standardized guidelines for. Though the consensus remains that complete excision of the mass is necessary, there is lack of appropriate guidelines for surveillance after treatment. This necessitates the development of standardized guidelines for GTS, regarding both post-treatment observation and pre-diagnostic screening for individuals with a history of NSGCTs.
CONFLICT OF INTEREST STATEMENT
None declared.
FUNDING
None.
INFORMED CONSENT
Obtained from patient. | Recovering | ReactionOutcome | CC BY-NC | 33542815 | 19,415,437 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Aneurysm ruptured'. | An atypical presentation of Salmonella enterica ser Dublin in an immunocompromised patient.
Mycotic aneurysms of the carotid artery are a rare entity that can be fatal if not diagnosed promptly. We present a 60-year-old man with a tender left-sided neck mass due to a ruptured aneurysm of the left internal carotid artery. Cultures taken intraoperatively grew Salmonella enterica ser Dublin.
INTRODUCTION
Infection with Salmonella enterica usually results in a self-limited gastroenteritis characterized by fever, diarrhea, and less often nausea, vomiting and headache. However, according to CDC data, ~8% of laboratory confirmed cases are complicated by bacteremia, arteritis, meningitis and osteomyelitis [1]. Occasionally, extraintestinal infections result in infected or mycotic aneurysms. In the USA, Staphylococcus is the most common organism associated with mycotic aneurysms, whereas Salmonella is much more common in East Asian countries such as China [2]. Mycotic aneurysms associated with Salmonella infection rupture over 50% of the time and if untreated have a mortality ranging from 16 to 44% [2]. Thus, prompt recognition and treatment are key to improved patient outcomes. We report the first published case of a middle-aged man who developed a carotid mycotic aneurysm due S. enterica ser Dublin, a serovar that is usually associated with cattle (ground beef and unpasteurized milk).
CASE REPORT
A 60-year-old man with a history of metastatic prostate cancer (treated with docetaxel ~2 weeks prior to presentation), hypertension, hyperlipidemia, paroxysmal atrial fibrillation (on rivaroxaban) and right knee osteoarthritis (status post total knee replacement) presented to the emergency room with left sided neck pain and swelling. He first noted left sided neck pain 1 to 2 weeks prior to presentation. Initially, he noted some discomfort and a small nodule on his left neck. However, the nodule grew over time and the pain progressed to the point where he noted discomfort with swallowing and breathing. Accordingly, he presented to our medical center for care. On the day of admission, he was febrile (38.7°C), tachycardic (122), hypertensive (152/94). Fortunately, despite the tender neck swelling, there was no airway compromise.
The physical exam revealed a tender non-pulsatile mass on the left neck extending to the angle of the mandible. A computed tomography (CT) scan of the neck with contrast revealed a focal outpouching of the left carotid artery (Fig. 1). There also appeared to be a mass encircling the left carotid artery (4.3 cm × 2.5 cm × 3 cm) with lateral displacement and compression of the left jugular vein.
Figure 1 CT scan (coronal) of neck with contrast showing thrombosed aneurysm of the left internal carotid artery (arrow)
A vascular surgery consultation was obtained and given concern for a contained ruptured aneurysm, the patient was taken to the operating room (OR). During the surgery, an aneurysm with a posterior perforation and contained rupture was noted (Fig. 2). Pathological specimens from the surgical site were notable for significant atherosclerotic disease. Also, purulence was noted along with extravasated blood and cultures taken from intraoperative specimens revealed pan sensitive S. enterica ser Dublin. The affected and ulcerated portion of the carotid artery was excised and replaced via end-to-end anastomoses with a bovine mesenteric vein graft. Interestingly, all peripheral blood cultures were negative for bacterial growth. He was treated with ceftriaxone 2 grams IV daily for at least 6 weeks.
Figure 2 Perforated posterior wall of the left internal carotid artery seen intraoperatively (arrow)
DISCUSSION
We report the case of a middle-aged man who presented with a subacute painful neck swelling that was due to a ruptured mycotic aneurysm of the left carotid artery. Given his history of cancer, a malignant metastasis was among the differential diagnoses. However, CT imaging helped us to quickly narrow the list of possibilities.
The concern for a ruptured aneurysm prompted a vascular surgery consultation and ultimately surgical exploration. The aneurysm was found to be infected with Salmonella. Mycotic aneurysms of the carotid artery are relatively rare and those due to Salmonella spp. are even rarer. Our review of the literature revealed ~22 published cases of a carotid mycotic aneurysm due to Salmonella spp. Mycotic aneurysms tend to involve the aorta causing aortitis with or without aneurysm formation [3]. Only ~5% of mycotic aneurysms involve the carotid artery. In general, male gender is a risk factor for development of mycotic aneurysm [4]. Other risk factors include age >50, diabetes, hypertension and atherosclerosis [2, 4]. Our patient had many of the traditional risk factors. He was also fully anticoagulated while the aneurysm was developing and had recently been treated with chemotherapy. We suspect that these factors likely contributed to his presentation as well.
We did not identify a discrete episode of gastroenteritis or bacteremia. He had been taking chemotherapy ~2 weeks prior to presentation so typical symptoms such as diarrhea, nausea and vomiting might have been misattributed to medication side effects. There are a few published case reports of mycotic aneurysms due to Salmonella with negative blood cultures [5–7]. This may reflect the fact that gram negative bacteremia is frequently transient.
The standard management of mycotic aneurysms is open surgical repair. There are multiple case reports describing an endovascular approach, but studies comparing endovascular versus open repairs are lacking [4]. A minimum of 6 weeks of antibiotic therapy is often recommended although there are no data to support a specific duration of treatment [6].
The Maryland State Health Department performed the microbiological testing in this case and confirmed the Dublin serotype, which usually causes gastroenteritis in both cattle and humans [8]. There are very few case reports of mycotic abdominal aneurysms due to the Dublin serotype [2]. To our knowledge, this is the first published case of carotid mycotic aneurysm associated with the Dublin serotype. Although most Salmonella spp outbreaks have been linked to eggs, poultry and occasionally pet reptiles, outbreaks due to S. enterica ser Dublin have been linked to the consumption of contaminated ground beef and raw or unpasteurized milk. The Centers for Disease Control reported an outbreak of the Dublin serotype in 2019 that involved eight states. The outbreak was linked to ground beef [9].
This case report brings into focus several important clinical and epidemiological points. First, an infected aneurysm should be considered in the differential diagnosis for a patient who is febrile and presents with a tender neck mass. Second, since mycotic aneurysms frequently rupture, prompt referral to a vascular surgeon is paramount. Finally, since our patient likely contracted S. enterica ser Dublin from ground beef or milk, it is imperative that we continue surveillance for this organism in our environment.
ACKNOWLEDGMENTS
None.
FUNDING STATEMENT
The authors received no specific funding for this work.
ETHICAL APPROVAL
This Case Report does not require IRB approval as it is not considered research and does not contain any of the 18 HIPAA identifiers.
CONSENT
A signed written consent was obtained from the patient for this case report.
GUARANTOR
Adrien L. Janvier. | DOCETAXEL, RIVAROXABAN | DrugsGivenReaction | CC BY-NC | 33542826 | 19,062,457 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Aortitis salmonella'. | An atypical presentation of Salmonella enterica ser Dublin in an immunocompromised patient.
Mycotic aneurysms of the carotid artery are a rare entity that can be fatal if not diagnosed promptly. We present a 60-year-old man with a tender left-sided neck mass due to a ruptured aneurysm of the left internal carotid artery. Cultures taken intraoperatively grew Salmonella enterica ser Dublin.
INTRODUCTION
Infection with Salmonella enterica usually results in a self-limited gastroenteritis characterized by fever, diarrhea, and less often nausea, vomiting and headache. However, according to CDC data, ~8% of laboratory confirmed cases are complicated by bacteremia, arteritis, meningitis and osteomyelitis [1]. Occasionally, extraintestinal infections result in infected or mycotic aneurysms. In the USA, Staphylococcus is the most common organism associated with mycotic aneurysms, whereas Salmonella is much more common in East Asian countries such as China [2]. Mycotic aneurysms associated with Salmonella infection rupture over 50% of the time and if untreated have a mortality ranging from 16 to 44% [2]. Thus, prompt recognition and treatment are key to improved patient outcomes. We report the first published case of a middle-aged man who developed a carotid mycotic aneurysm due S. enterica ser Dublin, a serovar that is usually associated with cattle (ground beef and unpasteurized milk).
CASE REPORT
A 60-year-old man with a history of metastatic prostate cancer (treated with docetaxel ~2 weeks prior to presentation), hypertension, hyperlipidemia, paroxysmal atrial fibrillation (on rivaroxaban) and right knee osteoarthritis (status post total knee replacement) presented to the emergency room with left sided neck pain and swelling. He first noted left sided neck pain 1 to 2 weeks prior to presentation. Initially, he noted some discomfort and a small nodule on his left neck. However, the nodule grew over time and the pain progressed to the point where he noted discomfort with swallowing and breathing. Accordingly, he presented to our medical center for care. On the day of admission, he was febrile (38.7°C), tachycardic (122), hypertensive (152/94). Fortunately, despite the tender neck swelling, there was no airway compromise.
The physical exam revealed a tender non-pulsatile mass on the left neck extending to the angle of the mandible. A computed tomography (CT) scan of the neck with contrast revealed a focal outpouching of the left carotid artery (Fig. 1). There also appeared to be a mass encircling the left carotid artery (4.3 cm × 2.5 cm × 3 cm) with lateral displacement and compression of the left jugular vein.
Figure 1 CT scan (coronal) of neck with contrast showing thrombosed aneurysm of the left internal carotid artery (arrow)
A vascular surgery consultation was obtained and given concern for a contained ruptured aneurysm, the patient was taken to the operating room (OR). During the surgery, an aneurysm with a posterior perforation and contained rupture was noted (Fig. 2). Pathological specimens from the surgical site were notable for significant atherosclerotic disease. Also, purulence was noted along with extravasated blood and cultures taken from intraoperative specimens revealed pan sensitive S. enterica ser Dublin. The affected and ulcerated portion of the carotid artery was excised and replaced via end-to-end anastomoses with a bovine mesenteric vein graft. Interestingly, all peripheral blood cultures were negative for bacterial growth. He was treated with ceftriaxone 2 grams IV daily for at least 6 weeks.
Figure 2 Perforated posterior wall of the left internal carotid artery seen intraoperatively (arrow)
DISCUSSION
We report the case of a middle-aged man who presented with a subacute painful neck swelling that was due to a ruptured mycotic aneurysm of the left carotid artery. Given his history of cancer, a malignant metastasis was among the differential diagnoses. However, CT imaging helped us to quickly narrow the list of possibilities.
The concern for a ruptured aneurysm prompted a vascular surgery consultation and ultimately surgical exploration. The aneurysm was found to be infected with Salmonella. Mycotic aneurysms of the carotid artery are relatively rare and those due to Salmonella spp. are even rarer. Our review of the literature revealed ~22 published cases of a carotid mycotic aneurysm due to Salmonella spp. Mycotic aneurysms tend to involve the aorta causing aortitis with or without aneurysm formation [3]. Only ~5% of mycotic aneurysms involve the carotid artery. In general, male gender is a risk factor for development of mycotic aneurysm [4]. Other risk factors include age >50, diabetes, hypertension and atherosclerosis [2, 4]. Our patient had many of the traditional risk factors. He was also fully anticoagulated while the aneurysm was developing and had recently been treated with chemotherapy. We suspect that these factors likely contributed to his presentation as well.
We did not identify a discrete episode of gastroenteritis or bacteremia. He had been taking chemotherapy ~2 weeks prior to presentation so typical symptoms such as diarrhea, nausea and vomiting might have been misattributed to medication side effects. There are a few published case reports of mycotic aneurysms due to Salmonella with negative blood cultures [5–7]. This may reflect the fact that gram negative bacteremia is frequently transient.
The standard management of mycotic aneurysms is open surgical repair. There are multiple case reports describing an endovascular approach, but studies comparing endovascular versus open repairs are lacking [4]. A minimum of 6 weeks of antibiotic therapy is often recommended although there are no data to support a specific duration of treatment [6].
The Maryland State Health Department performed the microbiological testing in this case and confirmed the Dublin serotype, which usually causes gastroenteritis in both cattle and humans [8]. There are very few case reports of mycotic abdominal aneurysms due to the Dublin serotype [2]. To our knowledge, this is the first published case of carotid mycotic aneurysm associated with the Dublin serotype. Although most Salmonella spp outbreaks have been linked to eggs, poultry and occasionally pet reptiles, outbreaks due to S. enterica ser Dublin have been linked to the consumption of contaminated ground beef and raw or unpasteurized milk. The Centers for Disease Control reported an outbreak of the Dublin serotype in 2019 that involved eight states. The outbreak was linked to ground beef [9].
This case report brings into focus several important clinical and epidemiological points. First, an infected aneurysm should be considered in the differential diagnosis for a patient who is febrile and presents with a tender neck mass. Second, since mycotic aneurysms frequently rupture, prompt referral to a vascular surgeon is paramount. Finally, since our patient likely contracted S. enterica ser Dublin from ground beef or milk, it is imperative that we continue surveillance for this organism in our environment.
ACKNOWLEDGMENTS
None.
FUNDING STATEMENT
The authors received no specific funding for this work.
ETHICAL APPROVAL
This Case Report does not require IRB approval as it is not considered research and does not contain any of the 18 HIPAA identifiers.
CONSENT
A signed written consent was obtained from the patient for this case report.
GUARANTOR
Adrien L. Janvier. | DOCETAXEL, RIVAROXABAN | DrugsGivenReaction | CC BY-NC | 33542826 | 19,062,457 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Arteriosclerosis'. | An atypical presentation of Salmonella enterica ser Dublin in an immunocompromised patient.
Mycotic aneurysms of the carotid artery are a rare entity that can be fatal if not diagnosed promptly. We present a 60-year-old man with a tender left-sided neck mass due to a ruptured aneurysm of the left internal carotid artery. Cultures taken intraoperatively grew Salmonella enterica ser Dublin.
INTRODUCTION
Infection with Salmonella enterica usually results in a self-limited gastroenteritis characterized by fever, diarrhea, and less often nausea, vomiting and headache. However, according to CDC data, ~8% of laboratory confirmed cases are complicated by bacteremia, arteritis, meningitis and osteomyelitis [1]. Occasionally, extraintestinal infections result in infected or mycotic aneurysms. In the USA, Staphylococcus is the most common organism associated with mycotic aneurysms, whereas Salmonella is much more common in East Asian countries such as China [2]. Mycotic aneurysms associated with Salmonella infection rupture over 50% of the time and if untreated have a mortality ranging from 16 to 44% [2]. Thus, prompt recognition and treatment are key to improved patient outcomes. We report the first published case of a middle-aged man who developed a carotid mycotic aneurysm due S. enterica ser Dublin, a serovar that is usually associated with cattle (ground beef and unpasteurized milk).
CASE REPORT
A 60-year-old man with a history of metastatic prostate cancer (treated with docetaxel ~2 weeks prior to presentation), hypertension, hyperlipidemia, paroxysmal atrial fibrillation (on rivaroxaban) and right knee osteoarthritis (status post total knee replacement) presented to the emergency room with left sided neck pain and swelling. He first noted left sided neck pain 1 to 2 weeks prior to presentation. Initially, he noted some discomfort and a small nodule on his left neck. However, the nodule grew over time and the pain progressed to the point where he noted discomfort with swallowing and breathing. Accordingly, he presented to our medical center for care. On the day of admission, he was febrile (38.7°C), tachycardic (122), hypertensive (152/94). Fortunately, despite the tender neck swelling, there was no airway compromise.
The physical exam revealed a tender non-pulsatile mass on the left neck extending to the angle of the mandible. A computed tomography (CT) scan of the neck with contrast revealed a focal outpouching of the left carotid artery (Fig. 1). There also appeared to be a mass encircling the left carotid artery (4.3 cm × 2.5 cm × 3 cm) with lateral displacement and compression of the left jugular vein.
Figure 1 CT scan (coronal) of neck with contrast showing thrombosed aneurysm of the left internal carotid artery (arrow)
A vascular surgery consultation was obtained and given concern for a contained ruptured aneurysm, the patient was taken to the operating room (OR). During the surgery, an aneurysm with a posterior perforation and contained rupture was noted (Fig. 2). Pathological specimens from the surgical site were notable for significant atherosclerotic disease. Also, purulence was noted along with extravasated blood and cultures taken from intraoperative specimens revealed pan sensitive S. enterica ser Dublin. The affected and ulcerated portion of the carotid artery was excised and replaced via end-to-end anastomoses with a bovine mesenteric vein graft. Interestingly, all peripheral blood cultures were negative for bacterial growth. He was treated with ceftriaxone 2 grams IV daily for at least 6 weeks.
Figure 2 Perforated posterior wall of the left internal carotid artery seen intraoperatively (arrow)
DISCUSSION
We report the case of a middle-aged man who presented with a subacute painful neck swelling that was due to a ruptured mycotic aneurysm of the left carotid artery. Given his history of cancer, a malignant metastasis was among the differential diagnoses. However, CT imaging helped us to quickly narrow the list of possibilities.
The concern for a ruptured aneurysm prompted a vascular surgery consultation and ultimately surgical exploration. The aneurysm was found to be infected with Salmonella. Mycotic aneurysms of the carotid artery are relatively rare and those due to Salmonella spp. are even rarer. Our review of the literature revealed ~22 published cases of a carotid mycotic aneurysm due to Salmonella spp. Mycotic aneurysms tend to involve the aorta causing aortitis with or without aneurysm formation [3]. Only ~5% of mycotic aneurysms involve the carotid artery. In general, male gender is a risk factor for development of mycotic aneurysm [4]. Other risk factors include age >50, diabetes, hypertension and atherosclerosis [2, 4]. Our patient had many of the traditional risk factors. He was also fully anticoagulated while the aneurysm was developing and had recently been treated with chemotherapy. We suspect that these factors likely contributed to his presentation as well.
We did not identify a discrete episode of gastroenteritis or bacteremia. He had been taking chemotherapy ~2 weeks prior to presentation so typical symptoms such as diarrhea, nausea and vomiting might have been misattributed to medication side effects. There are a few published case reports of mycotic aneurysms due to Salmonella with negative blood cultures [5–7]. This may reflect the fact that gram negative bacteremia is frequently transient.
The standard management of mycotic aneurysms is open surgical repair. There are multiple case reports describing an endovascular approach, but studies comparing endovascular versus open repairs are lacking [4]. A minimum of 6 weeks of antibiotic therapy is often recommended although there are no data to support a specific duration of treatment [6].
The Maryland State Health Department performed the microbiological testing in this case and confirmed the Dublin serotype, which usually causes gastroenteritis in both cattle and humans [8]. There are very few case reports of mycotic abdominal aneurysms due to the Dublin serotype [2]. To our knowledge, this is the first published case of carotid mycotic aneurysm associated with the Dublin serotype. Although most Salmonella spp outbreaks have been linked to eggs, poultry and occasionally pet reptiles, outbreaks due to S. enterica ser Dublin have been linked to the consumption of contaminated ground beef and raw or unpasteurized milk. The Centers for Disease Control reported an outbreak of the Dublin serotype in 2019 that involved eight states. The outbreak was linked to ground beef [9].
This case report brings into focus several important clinical and epidemiological points. First, an infected aneurysm should be considered in the differential diagnosis for a patient who is febrile and presents with a tender neck mass. Second, since mycotic aneurysms frequently rupture, prompt referral to a vascular surgeon is paramount. Finally, since our patient likely contracted S. enterica ser Dublin from ground beef or milk, it is imperative that we continue surveillance for this organism in our environment.
ACKNOWLEDGMENTS
None.
FUNDING STATEMENT
The authors received no specific funding for this work.
ETHICAL APPROVAL
This Case Report does not require IRB approval as it is not considered research and does not contain any of the 18 HIPAA identifiers.
CONSENT
A signed written consent was obtained from the patient for this case report.
GUARANTOR
Adrien L. Janvier. | DOCETAXEL, RIVAROXABAN | DrugsGivenReaction | CC BY-NC | 33542826 | 19,058,832 | 2021-01 |
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Carotid artery aneurysm'. | An atypical presentation of Salmonella enterica ser Dublin in an immunocompromised patient.
Mycotic aneurysms of the carotid artery are a rare entity that can be fatal if not diagnosed promptly. We present a 60-year-old man with a tender left-sided neck mass due to a ruptured aneurysm of the left internal carotid artery. Cultures taken intraoperatively grew Salmonella enterica ser Dublin.
INTRODUCTION
Infection with Salmonella enterica usually results in a self-limited gastroenteritis characterized by fever, diarrhea, and less often nausea, vomiting and headache. However, according to CDC data, ~8% of laboratory confirmed cases are complicated by bacteremia, arteritis, meningitis and osteomyelitis [1]. Occasionally, extraintestinal infections result in infected or mycotic aneurysms. In the USA, Staphylococcus is the most common organism associated with mycotic aneurysms, whereas Salmonella is much more common in East Asian countries such as China [2]. Mycotic aneurysms associated with Salmonella infection rupture over 50% of the time and if untreated have a mortality ranging from 16 to 44% [2]. Thus, prompt recognition and treatment are key to improved patient outcomes. We report the first published case of a middle-aged man who developed a carotid mycotic aneurysm due S. enterica ser Dublin, a serovar that is usually associated with cattle (ground beef and unpasteurized milk).
CASE REPORT
A 60-year-old man with a history of metastatic prostate cancer (treated with docetaxel ~2 weeks prior to presentation), hypertension, hyperlipidemia, paroxysmal atrial fibrillation (on rivaroxaban) and right knee osteoarthritis (status post total knee replacement) presented to the emergency room with left sided neck pain and swelling. He first noted left sided neck pain 1 to 2 weeks prior to presentation. Initially, he noted some discomfort and a small nodule on his left neck. However, the nodule grew over time and the pain progressed to the point where he noted discomfort with swallowing and breathing. Accordingly, he presented to our medical center for care. On the day of admission, he was febrile (38.7°C), tachycardic (122), hypertensive (152/94). Fortunately, despite the tender neck swelling, there was no airway compromise.
The physical exam revealed a tender non-pulsatile mass on the left neck extending to the angle of the mandible. A computed tomography (CT) scan of the neck with contrast revealed a focal outpouching of the left carotid artery (Fig. 1). There also appeared to be a mass encircling the left carotid artery (4.3 cm × 2.5 cm × 3 cm) with lateral displacement and compression of the left jugular vein.
Figure 1 CT scan (coronal) of neck with contrast showing thrombosed aneurysm of the left internal carotid artery (arrow)
A vascular surgery consultation was obtained and given concern for a contained ruptured aneurysm, the patient was taken to the operating room (OR). During the surgery, an aneurysm with a posterior perforation and contained rupture was noted (Fig. 2). Pathological specimens from the surgical site were notable for significant atherosclerotic disease. Also, purulence was noted along with extravasated blood and cultures taken from intraoperative specimens revealed pan sensitive S. enterica ser Dublin. The affected and ulcerated portion of the carotid artery was excised and replaced via end-to-end anastomoses with a bovine mesenteric vein graft. Interestingly, all peripheral blood cultures were negative for bacterial growth. He was treated with ceftriaxone 2 grams IV daily for at least 6 weeks.
Figure 2 Perforated posterior wall of the left internal carotid artery seen intraoperatively (arrow)
DISCUSSION
We report the case of a middle-aged man who presented with a subacute painful neck swelling that was due to a ruptured mycotic aneurysm of the left carotid artery. Given his history of cancer, a malignant metastasis was among the differential diagnoses. However, CT imaging helped us to quickly narrow the list of possibilities.
The concern for a ruptured aneurysm prompted a vascular surgery consultation and ultimately surgical exploration. The aneurysm was found to be infected with Salmonella. Mycotic aneurysms of the carotid artery are relatively rare and those due to Salmonella spp. are even rarer. Our review of the literature revealed ~22 published cases of a carotid mycotic aneurysm due to Salmonella spp. Mycotic aneurysms tend to involve the aorta causing aortitis with or without aneurysm formation [3]. Only ~5% of mycotic aneurysms involve the carotid artery. In general, male gender is a risk factor for development of mycotic aneurysm [4]. Other risk factors include age >50, diabetes, hypertension and atherosclerosis [2, 4]. Our patient had many of the traditional risk factors. He was also fully anticoagulated while the aneurysm was developing and had recently been treated with chemotherapy. We suspect that these factors likely contributed to his presentation as well.
We did not identify a discrete episode of gastroenteritis or bacteremia. He had been taking chemotherapy ~2 weeks prior to presentation so typical symptoms such as diarrhea, nausea and vomiting might have been misattributed to medication side effects. There are a few published case reports of mycotic aneurysms due to Salmonella with negative blood cultures [5–7]. This may reflect the fact that gram negative bacteremia is frequently transient.
The standard management of mycotic aneurysms is open surgical repair. There are multiple case reports describing an endovascular approach, but studies comparing endovascular versus open repairs are lacking [4]. A minimum of 6 weeks of antibiotic therapy is often recommended although there are no data to support a specific duration of treatment [6].
The Maryland State Health Department performed the microbiological testing in this case and confirmed the Dublin serotype, which usually causes gastroenteritis in both cattle and humans [8]. There are very few case reports of mycotic abdominal aneurysms due to the Dublin serotype [2]. To our knowledge, this is the first published case of carotid mycotic aneurysm associated with the Dublin serotype. Although most Salmonella spp outbreaks have been linked to eggs, poultry and occasionally pet reptiles, outbreaks due to S. enterica ser Dublin have been linked to the consumption of contaminated ground beef and raw or unpasteurized milk. The Centers for Disease Control reported an outbreak of the Dublin serotype in 2019 that involved eight states. The outbreak was linked to ground beef [9].
This case report brings into focus several important clinical and epidemiological points. First, an infected aneurysm should be considered in the differential diagnosis for a patient who is febrile and presents with a tender neck mass. Second, since mycotic aneurysms frequently rupture, prompt referral to a vascular surgeon is paramount. Finally, since our patient likely contracted S. enterica ser Dublin from ground beef or milk, it is imperative that we continue surveillance for this organism in our environment.
ACKNOWLEDGMENTS
None.
FUNDING STATEMENT
The authors received no specific funding for this work.
ETHICAL APPROVAL
This Case Report does not require IRB approval as it is not considered research and does not contain any of the 18 HIPAA identifiers.
CONSENT
A signed written consent was obtained from the patient for this case report.
GUARANTOR
Adrien L. Janvier. | DOCETAXEL, RIVAROXABAN | DrugsGivenReaction | CC BY-NC | 33542826 | 19,062,457 | 2021-01 |
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