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Effect of probe filtering pre-normalisation | Filtering probes prior to normalisation, compared to filtering after normalisation, led to modest differences in number of DMPs, rankings of probes by logFC and Probe rankings, by logFC and The question of whether probe filtering should be carried out before or after normalisation is one which has received surprisingly little attention in the literature, but our results suggest that it can make a difference to findings in some contexts. In particular there may be a higher risk of spurious findings in pre-filtered data, but there may also be a risk of failing to detect true differences –either any differences, or specifically hypomethylated or hypermethylated loci, depending upon the normalisation method employed. | PMC9901304 | ||
Effect of normalisation method | Normalisation method had a substantial influence on number and identity of DMPs, rankings of probes and There was a fair degree of consistency in rankings of probes by Distributions of Overall, there was little difference between SQN and SWAN methods when adjusting for batch in the model. There is some evidence that SQN would result in fewer significant DMPs than SWAN for known effects (particularly when using supervised ComBat), but (many) more spuriously significant DMPs than either SWAN or BMIQ where effects are absent. The behaviour of BMIQ was more variable depending on other dimensions of the pipeline, but had a wider dispersion of logFC estimates than the other methods, particularly when adjusting for batch in the model. This tended to result in more DMPs in some scenarios, but in general will lead effect estimates derived from BMIQ data to be more extreme (and probably overestimates of the true effect). | PMC9901304 | ||
Effect of batch correction method | There were clear differences in all dimensions between batch correction methods. For all effects (infant sex, BMI, intervention and fake groups), supervised ComBat processing produced a larger number of DMPs compared to either unsupervised ComBat processing or adjustment for batch in the analysis model. The difference between unsupervised ComBat and batch-adjustment was less consistent for infant sex effects, but for BMI, intervention and fake group effects, there were no DMPs in unsupervised ComBat models, whereas there were a few for batch-adjusted models.Rankings of top probes by The distribution of Of particular note is the combination of SQN normalisation and either adjustment for batch in the model, or use of supervised ComBat. These combinations produced an extremely large number of significant DMPs for fake group effects; this was more extreme in the case of supervised ComBat (producing over 6,000 DMPs) than when adjusting for batch (somewhat over 2,000 DMPs). Additional adjustment for cell type proportion ameliorated this effect, as discussed below, but in the case of supervised ComBat data, did not eliminate spurious findings. This suggests that batch adjustment may be particularly ill-advised in the context of SQN normalisation; since SQN involves between-array as well as within-array normalisation, additional adjustment for batch may be over-correcting. | PMC9901304 | ||
Effect of adjustment for estimated cell type proportion | Adjustment for cell type proportion affected results, but the impact was not consistent across the different types of effects studied. Adjustment for batch resulted in a substantially larger number of DMPs (both negative and positive) for infant sex, but reduced the number of DMPs for fake groups (for models where there were DMPs for fake groups effects). For BMI and intervention, the effect of cell type adjustment was mostly but not entirely to produce more DMPs.The effect of cell type adjustment on top probe rankings was fairly modest, although some quite large discrepancies were observed for Overall, adjustment for cell type proportion tended to improve model behaviour regarding spurious results: the number of significantly differentially methylated probes decreased with adjustment for cell type proportion, though they were not always eliminated. The number of differentially methylated probes for infant sex was increased, which may reflect either improvement (greater ability to detect true effects due to removal of noise due to cell type differences) or harm (greater number of spurious effects) depending on whether the extra probes are in fact differentially methylated between males and females; without knowing the true number and identity of DMPs, we cannot be certain. Similarly, adjustment for cell type proportion increased the number of DMPs for BMI effects in BMIQ and SWAN data, in one scenario (BMIQ with Supervised ComBat) by a substantial amount (from 99 to 2017 DMPs) and these are most likely to be false positives. | PMC9901304 | ||
Supplemental Information | PMC9901304 | |||
Supplementary Material | Click here for additional data file. | PMC9901304 | ||
R scripts | Click here for additional data file.We would like to acknowledge the participants in the LIMIT Randomised Controlled Trial, whose samples and data are used in this study, as well as the LIMIT research team who collected data and samples for the study. We would also like to acknowledge Shobana Navaneethabalakrishnan, the Academic Editor at PeerJ and two anonymous reviewers, whose helpful suggestions have substantially improved the manuscript. | PMC9901304 | ||
Additional Information and Declarations | PMC9901304 | |||
Competing Interests | The authors declare there are no competing interests. | PMC9901304 | ||
Human Ethics | The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):The study was reviewed by the ethics committee of each participating institution: the Women’s and Children’s Health Network Human Research Ethics Committee (1839 & 2051), the Central and Northern Adelaide Health Network Human Research Ethics Committee (2008033) and the Southern Adelaide Local Health Network Human Research Ethics Committee (128/08). | PMC9901304 | ||
Data Availability | The following information was supplied regarding data availability:The R scripts contain all code for data processing and analysis are available in the | PMC9901304 | ||
References | PMC9901304 | |||
Subject terms | DCD kidney transplants, death, ischemic injury | END-STAGE RENAL DISEASE, KIDNEY, COLD | Kidney transplantation is the optimal treatment for end-stage renal disease, but it is still severely limited by a lack of suitable organ donors. Kidneys from donation after circulatory death (DCD) donors have been used to increase transplant rates, but these organs are susceptible to cold ischemic injury in the storage period before transplantation, the clinical consequence of which is high rates of delayed graft function (DGF). Normothermic machine perfusion (NMP) is an emerging technique that circulates a warmed, oxygenated red-cell-based perfusate through the kidney to maintain near-physiological conditions. We conducted a randomized controlled trial to compare the outcome of DCD kidney transplants after conventional static cold storage (SCS) alone or SCS plus 1-h NMP. A total of 338 kidneys were randomly allocated to SCS (In an open-label, randomized controlled trial, normothermic machine perfusion of kidneys from donation after circulatory death was found to be feasible and safe but did not reduce the rate of delayed graft function compared to static cold storage. | PMC10287561 |
Main | ischemia, allograft dysfunction, bile duct, acute kidney injury | ISCHEMIA, KIDNEY, COLD, COMPLICATIONS | Kidney transplantation is one of the major successes of modern medicine in the 20th century, transforming and extending the lives of many thousands of patients. However, a longstanding limitation of kidney transplantation is that the supply of transplantable organs does not meet demandFor more than 50 years, the traditional method of pre-transplant kidney preservation has been static cold storage (SCS) in iceDCD takes place after cessation of cardiac activity in the donor, leading to an inevitable period of warm ischemia, which can cause acute kidney injury. In addition, DCD kidneys are more susceptible to cold storage injuryNormothermic machine perfusion (NMP) is an emerging technique that uses cardiopulmonary bypass technology with extracorporeal membrane oxygenation to perfuse kidneys with a warmed and oxygenated red-cell-based plasma-free solutionThe first randomized controlled trial of NMP in liver transplantation demonstrated that, compared to conventional SCS, NMP reduced early allograft dysfunction. There was no effect on bile duct complications, graft survival and patient survivalOur non-randomized pilot study in kidney transplantation suggested that a 1-h period of NMP, delivered at the end of the cold storage period and immediately before transplantation, reduced the rate of DGF in extended criteria donor kidneys | PMC10287561 |
Results | PMC10287561 | |||
Patient population | diabetes | HYPERTENSION, COLD, DIABETES | The characteristics of the donors and recipients are detailed in Table Donor and recipient characteristics for the MITT analysis populationBMI, body mass index; HLA, human leucocyte antigen. The warm ischemic time is defined from the cession of circulation after withdrawal of life-sustaining treatment until the start of the in situ cold flush. The first cold ischemic time is calculated from the time of the in situ cold flush until the start of NMP. The second cold ischemic time is from the end of NMP when the kidney is flushed with cold preservation solution and placed back on ice until removal from ice for transplantation.Summary of missing data: There was a small amount of missing data (fewer than 10 observations) for five characteristics: donor ethnicity, past diabetes, past hypertension, left kidney transplant and cold ischemic time. To note, 63 values were missing for pre- transplant eGFR, and 25 values were missing for duration of NMP.Duplication of donors or recipients: 45 donors appear in the table twice (for left and right kidney).Zero recipients appear in the table twice.Data are | PMC10287561 |
CONSORT diagram. | COLD, RECRUITMENT | Two eligibility periods are presented due to the COVID-19 pandemic: 4 September 2020 is the date the trial officially closed to recruitment, and 23 March 2020 was the start of the UK national lockdown. No participants were recruited between 23 March 2020 and 4 September 2020. Two participants did not have a cold ischemic time (CIT) reported, and two participants received dual transplants and, hence, do not have a left/right kidney variable populated; these four cases were excluded from all risk-adjusted modeling. **Two additional exclusion criteria (donors who underwent normothermic regional perfusion or one of a pair already randomized as a single kidney in the trial) were introduced on 13 October 2017, and, retrospectively, two participants fulfilled these exclusion criteria (both were randomized to NMP). These patients were included in the MITT analysis and excluded from the per-protocol analysis.In total, 168 kidneys were allocated to SCS and 170 to NMP. In the SCS arm, one kidney received NMP due to the surgeon’s preference. In the NMP arm, 25 kidneys did not receive NMP. In 14 cases, this was due to the inability to secure a cannula into or around the renal artery; in eight cases, this was due to logistics with access to theater or time constraints; in one case, this was due to the surgeon’s preference; in one case, this was due to damage to the renal artery; and in one case, the kidney was transplanted as a dual transplant. A total of 21 kidneys in the SCS arm and 27 kidneys in the NMP arm were not transplanted (details are listed in Fig. There were no withdrawals from the trial. Two participants were randomized in error, as the single kidneys were transplanted as dual kidney transplants. Fifty-five participants experienced a protocol deviation: not receiving the randomized treatment was the most common reason (previously described); 21 kidneys that were one of a pair in the trial that were transplanted in the incorrect order to which they were randomized (due to logistics); and 13 kidneys that were randomized to NMP did not receive 60 min of NMP. All of the participants who experienced a protocol deviation were included in the modified intention-to-treat (MITT) analysis but were excluded from the per-protocol analysis. Four cases had to be excluded from all risk-adjusted modeling, as one of the risk adjustment factors was missing (two participants did not have a cold ischemic time reported, and two participants who received dual transplants did not have a left/right kidney variable populated) (Fig. Two interim analyses were carried out, and the DMC recommended continuation after both ( | PMC10287561 | |
Secondary outcome measures | thrombosis, DGF, necrosis | THROMBOSIS, NECROSIS | Nine participants had PNF (three in the SCS arm and six in the NMP arm) and were excluded from subsequent analyses. In the SCS arm, two patients had a vascular thrombosis, and one had cortical necrosis. In the NMP arm, one patient had a vascular thrombosis; two patients had acute rejection; and, in three cases, the reason was unknown. No significant difference was observed in the number of patients who experienced PNF between the treatment arms for both the MITT analysis (adjusted OR (95% CI) 2.34 (0.56–9.86); Table Patients who experienced DGF were excluded from the outcomes and analyses for functional DGF, creatinine reduction ratio on post-transplant day 2 (CRR2) and creatinine reduction ratio on post-transplant day 5 (CRR5). The median duration of DGF was 6 (2–9) days in the SCS arm and 4 (1–9) days in the NMP arm. No significant difference was observed between the groups in the MITT (adjusted hazard ratio (HR) (95% CI) 0.97 (0.70–1.34); Table The total number of patients who had biopsy-proven acute rejection was 19 in the SCS group and 24 in the NMP group. The unadjusted mean number of biopsy-proven acute rejection episodes per patient was numerically higher in the NMP arm compared to the SCS arm (0.3 per participant versus 0.2, respectively) but was not statistically different (adjusted rate ratio (95% CI) 1.57 (0.83–2.95) for the MITT population; Table Serum creatinine and estimated glomerular filtration rate (eGFR) were not statistically significant at 1, 3, 6 or 12 months after transplant in the MITT or per-protocol analyses ( | PMC10287561 |
Renal function in renal transplant recipients receiving donor kidneys randomized to preservation by SCS or NMP. | Serum creatinine levels (No differences were observed in tacrolimus trough blood levels between the SCS and NMP groups at 1, 3, 6 and 12 months after transplant (Supplementary Table | PMC10287561 | ||
Patient and graft survival | Patient and graft survival were similar between the arms for both the MITT and per-protocol analyses (patient survival: SCS 97.2 versus NMP 96.3 (HR (95% CI) 1.44 (0.33–6.36)); Fig. | PMC10287561 | ||
Patient and allograft survival in renal transplant recipients receiving donor kidneys randomized to preservation by SCS or NMP. | Kaplan–Meier plot for 12-month patient survival ( | PMC10287561 | ||
Exploratory assessment of kidney quality during NMP | COLD | The median renal blood flow was 180 ml/min/100 g (interquartile range (IQR) 120–230), and the median arterial pressure was 76 mmHg (IQR 74–80). The median amount of urine produced was 95 ml (IQR 50–180). The quality assessment score was applied to each of the analyzed kidneys. Forty-six percent had an assessment score of 2 of more. When adjusting for cold ischemic time, donor age, left/right kidney and transplant center, no significant difference in DGF was observed between those kidneys that scored a 1 versus 2 or more in the assessment score (adjusted OR (95% CI) 1.02 (0.47–2.24); Supplementary Table | PMC10287561 | |
Post hoc subgroup analysis of DGF | In a sub-analysis including nine PNF cases in the MITT cohort and six in the per-protocol cohort, no significant difference was observed in rates of DGF between the study arms ( | PMC10287561 | ||
Post hoc analysis of CRR2 | In the MITT and per-protocol analysis, excluding patients who were not on dialysis before transplant, the CRR2 was significantly higher in the NMP arm ( | PMC10287561 | ||
Post hoc analysis of missing eGFR values | A sub-analysis imputing all missing eGFR values with the value 8.5 ml/min/1.73 m | PMC10287561 | ||
Post hoc analysis of the effect of a second period of cold ischemia on DGF rates after NMP | COLD | In the MITT analysis, no statistically significant difference was observed in the duration of the second cold ischemic time in NMP kidneys with initial graft function or DGF (median (range) 113.5 (1–514) minutes in NMP kidneys with initial graft function versus 134.6 (8–696) minutes in NMP kidneys with DGF; | PMC10287561 | |
Discussion | agonal, thrombosis, ischemic injury, DGF.In liver transplantation, atrributable, ischemia, inflammation, infection, hypotension | THROMBOSIS, GRAFT DYSFUNCTION, CIRCULATORY ARREST, ADVERSE EVENTS, ISCHEMIA, INFLAMMATION, CARDIAC ARREST, COMPLICATION, INFECTION, COMPLICATIONS, VENOUS THROMBOSIS, COLD, COMPLICATIONS | We compared a 1-h period of NMP with conventional SCS for kidney transplantation from DCD donors. The NMP protocol had no effect on the primary endpoint, which was the incidence of DGF, defined as the requirement for dialysis in the first 7 d after transplant. No significant statistical differences were observed in the rates of acute rejection, renal function at 12 months, patient survival or graft survival. Our study also demonstrated that NMP is a safe procedure, as no significant differences were observed in complication rates when compared to SCS, and no adverse events were directly atrributable to NMP.After the introduction of ex vivo NMP for donor kidneys into clinical practiceThere are several possible causes of the high rates of DGF described here and in some of the previous literature. Physiological parameters during the agonal period, defined as the time between withdrawal of treatment and circulatory arrest, are likely to be critical. In the UK, withdrawal of treatment comprises disconnection from the ventilator and stopping inotropes but, in many centers, does not include removal of the endotracheal tube. Continuing support of the airway in this way can prolong the agonal period. Even more importantly, severe and prolonged hypotension during the agonal period and the consequent inadequate organ perfusion causes additional warm ischemic injury and has been shown to be associated with higher DGF rates in DCD kidneysThe high rates of DGF reported here are also related to defining DGF as the requirement for dialysis in the first 7 d after transplant. Although this is the simplest and most widely used definition of DGFThe use of normothermic regional perfusion (NRP), which restores oxygenated blood flow to the abdominal organs in situ after cardiac arrest, has yielded even lower DGF rates of 23–30%Conventional SCS was used as the control arm in this study because it is the standard of care in the UK. Although HMP has been shown to reduce the rate of DGF in DCD kidneys when compared to SCSThere are several possible explanations for the lack of effect of NMP on DGF rates. A 1-h period of NMP may not be long enough to reverse the effects of cold ischemia on renal tubular cells. The rationale for our trial design was based on pilot clinical data suggesting that 1 h of NMP could significantly reduce the rate of DGF in DCD kidneysThere is limited evidence to define the optimal duration of NMP. One experimental study suggested that 1 h of NMP is not as beneficial as more prolonged periods of perfusionThe original intention in our protocol was to deliver a short period of NMP immediately before the kidney was transplanted, but this was not always achieved because of the logistics of preparing patients for transplant surgery. After 1 h of NMP, the kidney was re-flushed with cold preservation solution and placed back on ice for a variable second period of cold storage until removal from ice for implantation. The mean duration of this second cold time was just over 2 h, and, although this may not influence outcome, longer additional cold storage would eventually counteract any beneficial effects of NMP. Post hoc analysis demonstrated that there was no difference in the duration of the second cold ischemic period in NMP kidneys with or without DGF.In liver transplantation, donor livers have been maintained by NMP for the full duration of the preservation periodThe induction of inflammation during NMP is another potential reason for the lack of its effect on the rate of DGF. The red-cell-based perfusate used in the NMP system was designed to create an anti-inflammatory environment without platelets, white cells or complementOur trial has several limitations. It was designed as an open-label study because of the logistics of the NMP technique. Blinding of the surgical team was not possible because NMP was performed in the operating room while the transplant recipient was being prepared for surgery. However, the perfusion teams were not involved in data analysis. We were unable to perform NMP in 14 kidneys (8.2%) randomized to this treatment arm because of concerns over the technical aspects of cannulating the renal arterial system. The alternatives for arterial perfusion are the use of an aortic patch clamp or direct cannulation of the renal artery. The former is more favorable as it allows perfusion of multiple renal arteries without loss of the aortic patch for subsequent anastomosis in the recipientAs NMP requires cannulation of the renal blood vessels, there is potential to cause endothelial damage. There is also a risk of transmitting infection during the period in which the kidney is perfused in an ex vivo organ chamber. In our trial, NMP was not associated with any increase in transplant thrombosis, infectious complications or any other adverse events. No significant differences were observed between the groups in terms of recipient safety outcomes. The incidence of renal arterial or venous thrombosis was very low, and there were few complications related to the renal transplant biopsies. Complications associated with kidney transplantation that require hospitalization are common and were categorized into graft dysfunction, infection, related to surgery or immunosuppression based.In conclusion, a 1-h period of NMP after SCS does not reduce the risk of DGF in DCD kidney transplants. Nonetheless, we have demonstrated that this new technology for kidney preservation is feasible, safe and suitable for clinical application. This trial delivers the first, essential step in exploring the broader potential of NMP in kidney transplantation. | PMC10287561 |
Methods | PMC10287561 | |||
Trial design | This investigator-led, randomized controlled, open-label trial was approved by the UK National Research Ethics Service and local institutional review boards (REC 15/EE/0356), with trial registration number | PMC10287561 | ||
Trial patients | multi-organ, end-stage renal failure | END-STAGE RENAL FAILURE | Eligible patients enrolled on the transplant waiting list and allocated a suitably matched kidney were enrolled at four UK transplant centers. Inclusion criteria included recipients 18 years of age or older with end-stage renal failure requiring their first or second kidney transplant who received a kidney from Maastricht category III or IV DCD donors 18 years of age or older. Exclusion criteria included recipients receiving a third or subsequent kidney transplant, multi-organ transplants, dual kidney transplants, pediatric en bloc kidney transplants and kidneys preserved by HMP. Two additional exclusion criteria were introduced on 13 October 2017: donors who underwent normothermic regional perfusion or one of a pair already randomized as a single kidney in the trial. This was approved by the Research Ethics Service, local institutional review boards and the TSC. All patients provided written informed consent.Patients were randomly assigned in a 1:1 ratio to 1-h NMP or SCS. The randomization list was created by the trial statistician in SAS Enterprise Guide (version 5.1) with SAS 9.4, stratified by transplant center and using randomly permuted blocks of fixed size 2 and 4 for single and pairs of kidneys, respectively. In cases where paired kidneys from the same donor were transplanted in the same transplant center, the randomization was stratified by kidney (right or left) so that one kidney was randomly allocated to each treatment and in which order they should be transplanted. The randomization process was facilitated using an Interactive Web Response System. After the assignment of treatment arms, no one in the trial was blinded to the treatment allocation. | PMC10287561 |
SCS | BLOOD | Kidneys were retrieved by National Health Service Blood and Transplant (NHSBT) National Organ Retrieval Service teams, and, after flushing with cold preservation solution, they were stored on ice until transplanted. | PMC10287561 | |
NMP | NMP was performed at the transplanting center for 1 h using a customized pediatric cardiopulmonary bypass system. Kidneys were perfused with an oxygenated red-cell-based solution supplemented with a crystalloid solution and amino acids. Details are documented in the | PMC10287561 | ||
Transplantation | Kidneys were transplanted into either iliac fossa with anastomosis of the artery to the common, external or internal iliac arteries. The vein was anastomosed to either the common or the external iliac vein. The ureteric anastomosis was performed as an extravesical onlay over a double J stent. | PMC10287561 | ||
Immunosuppression | A standard immunosuppressive protocol was used in all four trial centers. All patients received induction therapy with basiliximab 20 mg IV given on the day of transplantation and on the fourth postoperative day. All patients received methylprednisolone 500 mg IV at induction of anaesthesia. Maintenance immunosuppression was given as triple therapy with tacrolimus, mycophenolic acid and prednisolone. Tacrolimus was administered at a dose of 0.1 mg/kg/day orally either in two divided doses (adoport) or as a single daily dose (advagraf). Tacrolimus trough blood levels were measured at least twice weekly, and the therapeutic target range in the first 3 months after transplant was 5–10 ng ml | PMC10287561 | ||
Clinical outcomes | infection, renal graft dysfunction | COMPLICATION, INFECTION, VENOUS THROMBOSIS, RENAL DISEASE, COMPLICATIONS | The primary outcome measure was DGF, defined as the requirement for dialysis in the first week after transplantation. Secondary outcome measures included incidence of PNF; duration of DGF; functional DGF defined as less than 10% fall in serum creatinine for three consecutive days in the first week after transplantation; CRR2 (creatinine day 1 − creatinine day 2 / creatinine day 1 × 100), CRR5 (creatinine day 1 − creatinine day 5 / creatinine day 1 × 100); duration of hospital stay; serum creatinine and eGFR at 1, 3, 6 and 12 months using the Modification of Diet in Renal Disease (MDRD) 4 variable equation; and patient and allograft survival up to 12 months after transplant. For safety outcomes, the total number of incidences of biopsy-proven acute rejection, renal artery or venous thrombosis, complications of the renal transplant biopsy and the number of hospital admissions for any recognized complication of the renal transplant, including renal graft dysfunction, infection, surgery related or due to the immunosuppression, were recorded.Data were collected by each of the participating transplant centers using an online secure database hosted by the NHSBT Clinical Trials Unit. | PMC10287561 |
Statistical analysis | PMC10287561 | |||
Study design | RECRUITMENT | The NHSBT Clinical Trials Unit supported the design, data management and analysis of the trial. Historical data spanning a 5-year period for three participating centers showed that the overall rate of DGF in DCD kidney transplants was 50%. This was used as the baseline rate. In a pilot series of kidney transplants from extended criteria donors (ECDs), 18 kidneys undergoing SCS followed by 1 h of NMP were compared to a historical control group of 47 ECD transplants after SCS alone. The DGF rates were 1/18 (6%) in the NMP group compared to 17/47 (36%) in the SCS group. Using a fixed sample size study, with interim analyses after 124 and 248 participants had been enrolled and reached 7 d after transplant, a total of 370 patients receiving a DCD kidney were required to detect a 30% relative reduction in DGF (from 50% to 35%) with a power of 80%, a statistical significance of α = 0.05 and 1:1 allocation. To allow for a study withdrawal rate of 7.5%, a maximum of 400 patients were needed for recruitment. There would be no sample size re-estimation during the trial. | PMC10287561 | |
Interim analysis | A group sequential design, with O’Brien–Fleming stopping rules (which preserved the 5% significance level in the final analysis), was used to allow the DMC to review the primary outcome for evidence of harm, benefit or futility. Two unadjusted interim analyses were performed—the first after 124 patients were randomized and reached 7 d after randomization and the second after 248 patients were randomized and reached 7 d after randomization. The stopping rules were used as a guideline, alongside the other safety data available to the DMC, as an overall assessment of the trial. The interim analyses were performed by the trial statistician who was unblinded to the treatment arm, and these results were presented to the DMC only. The DMC reported its recommendations, without disclosing any trial results, to the TSC, which made the final decision regarding continuation of the trial. | PMC10287561 | ||
Study population | SECONDARY, COLD | The population used for efficacy analyses was a MITT population including all randomized patients who received a transplant. This was a change from the original protocol because it was deemed illogical to include those participants who did not receive a transplant, as no outcome data were available. Primary and secondary outcomes were also analyzed per protocol, which excluded any participant who did not receive a transplant, was randomized in error, experienced a protocol deviation or was withdrawn from the trial (details are provided in the statistical analysis plan). For both analysis populations, results were presented by randomized treatment, and all ratios and mean differences were presented as NMP versus SCS.All analyses were adjusted for cold ischemic time, donor age, left/right kidney and transplant center (all as fixed effects). All tests were two-sided, and | PMC10287561 | |
Primary and secondary outcome measures | REGRESSION, SECONDARY | The primary outcome was analyzed using an adjusted logistic regression model and excluded participants who experienced PNF. The data for this outcome were complete, and, therefore, it was not necessary to undertake any of the methods proposed in the statistical analysis plan for assessing the impact of these missing data.Secondary and safety outcome measures were analyzed using logistic regression model (PNF and functional DGF), Cox proportional hazards model (duration of DGF, length of hospital stay, allograft and patient survival), normal linear regression model (CRR at day 2 and day 5, serum creatinine and eGFR at 1, 3, 6 and 12 months) and negative binomial model (biopsy-proven acute rejection and safety outcomes). Missing secondary outcome measures were not imputed and were excluded from the relevant analyses, except for eGFR. Full details can be found in the statistical analysis plan. To ensure model assumptions were met, residual plots were examined. | PMC10287561 | |
NMP assessment score | mean renal blood flow | After NMP, kidneys were allocated a score of 1–5 based on the macroscopic appearance, mean renal blood flow and urine production. A lower value indicated a better score (details in the | PMC10287561 | |
Post hoc subgroup analyses | hyperkalemia | COLD, FLUID OVERLOAD |
PNF was included in the DGF groups to determine the impact of the pre-transplant preservation interventions on rates of non-function.To determine the effect of pre-transplant recipient dialysis status (receiving dialysis versus pre-dialysis) on DGF in the MITT analysis, we used the same model as that used for the primary outcome but with the inclusion of the pre-transplant dialysis term, and we also assessed the interaction between treatment group and pre-dialysis status.Some patients received a single post-transplant dialysis as a safety measure in response to hyperkalemia or fluid overload, irrespective of renal function. To take account of this, we analyzed the effect of excluding patients who received a single post-transplant dialysis on DGF rates.We also analysed the effect of excluding pre-dialysis patients from CRR2 calculations in both the MITT and per-protocol analyses.The duration of the second cold ischemic period after NMP was variable, and this might have influenced the rate of DGF. We, therefore, compared the duration of second cold ischemic time in kidneys with initial function and DGF after NMP.To take account of missing eGFR data in the MITT analysis, we imputed a value of 8.5 ml/min/1.73 m | PMC10287561 |
Reporting Summary | Further information on research design is available in the | PMC10287561 | ||
Online content | Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41591-023-02376-7. | PMC10287561 | ||
Supplementary information | The online version contains supplementary material available at 10.1038/s41591-023-02376-7. | PMC10287561 | ||
Acknowledgements | KIDNEY | The authors give sincere thanks to the donor families. Without their generous gifts, this study would not have been possible. We would like to thank all the patients and the multidisciplinary teams involved in their care. We would like to acknowledge all the participating investigators from the Kidney Research UK Normothermic Machine Perfusion Network who provided and cared for study patients and collected data (full details in the | PMC10287561 | |
Author contributions | S.A.H. | S.A.H. and M.L.N. designed the study, acquired funding, carried out the perfusion techniques and project administration, collected the data and wrote and reviewed the manuscript. L.S. assisted in the design of the study, provided the statistical plan, analyzed and validated the data and co-wrote, reviewed and edited the manuscript. J. Mullings and J. Mehew analyzed and validated the data. C.J.C. and C.H.W. assisted in the design of the study, carried out the perfusion techniques, collected data and reviewed and edited the manuscript. G.C.O., B.L.P. and L.B. carried out the perfusion techniques, collected data and reviewed and edited the manuscript. L.S. and J. Mullings directly accessed and verified the underlying data. | PMC10287561 | |
Peer review | PMC10287561 | |||
Data availability | Data from the trial are stored in an online secure database hosted by the NHSBT Clinical Trials Unit. The protocol, consent form, statistical analysis plan, definition and derivation of clinical characteristics and outcomes, training materials, regulatory documents and other relevant study materials are available online and were published elsewhere. The datasets generated during analysis will be available upon reasonable request from the NHSBT Clinical Trials Unit after de-identification (text, tables, figures and appendices) 9 months after publication and ending 5 years after article publication. Data will be shared with investigators whose use of the data has been assessed and approved by an NHSBT review committee as a methodologically sound proposal. The NHSBT Clinical Trials Unit can be contacted at CTU@nhsbt.nhs.uk. The Clinical Trials Unit will be able to provide a copy of our data-sharing policy and arrange a data use agreement, which will need to be signed. All data use agreements will be in line with the consent given by participants upon agreeing to take part in the trial. | PMC10287561 | ||
Competing interests | The authors declare no competing interests. | PMC10287561 | ||
References | PMC10287561 | |||
Subject terms | HER2-negative, adenocarcinoma | ADENOCARCINOMA, GASTROESOPHAGEAL JUNCTION ADENOCARCINOMA | There is an urgent need for first-line treatment options for patients with human epidermal growth factor receptor 2 (HER2)-negative, locally advanced unresectable or metastatic gastric or gastroesophageal junction (mG/GEJ) adenocarcinoma. Claudin-18 isoform 2 (CLDN18.2) is expressed in normal gastric cells and maintained in malignant G/GEJ adenocarcinoma cells. GLOW (closed enrollment), a global, double-blind, phase 3 study, examined zolbetuximab, a monoclonal antibody that targets CLDN18.2, plus capecitabine and oxaliplatin (CAPOX) as first-line treatment for CLDN18.2-positive, HER2-negative, locally advanced unresectable or mG/GEJ adenocarcinoma. Patients (In the randomized, double-blind, phase 3 GLOW trial, capecitabine and oxaliplatin combined with zolbetuximab, a monoclonal antibody that targets CLDN18.2, significantly improved progression-free survival and overall survival in patients with CLDN18.2-positive, HER2-negative, untreated, locally advanced unresectable or metastatic gastric or gastroesophageal junction adenocarcinoma. | PMC10427418 |
Main | Gastric adenocarcinoma, cancer, HER2-negative, adenocarcinoma, gastroesophageal junction adenocarcinomas | GASTRIC ADENOCARCINOMA, CANCER, ADENOCARCINOMA, GASTROESOPHAGEAL JUNCTION ADENOCARCINOMA, CYTOTOXICITY | Gastric adenocarcinoma is the fifth most commonly diagnosed cancer worldwide, and incidence of gastroesophageal junction adenocarcinomas has markedly increased in the last few decadesThe combination of targeted therapies or immunotherapies with chemotherapy can improve overall survival (OS) in some patients with metastatic diseaseCLDN18.2 is a tight junction protein exclusively expressed in normal gastric mucosa cells and is retained in most G/GEJ adenocarcinomasZolbetuximab is a first-in-class immunoglobulin G1 monoclonal antibody that targets CLDN18.2 and mediates antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in CLDN18.2-positive G/GEJ adenocarcinoma cellsHere we report the primary analysis of GLOW, a global, randomized, double-blind, phase 3 study, which evaluated the efficacy and safety of first-line treatment with zolbetuximab plus CAPOX compared to placebo plus CAPOX in patients with CLDN18.2-positive, HER2-negative, locally advanced unresectable or mG/GEJ adenocarcinoma. | PMC10427418 |
Results | PMC10427418 | |||
Patients and treatment | HER2-negative, tumor, tumors, adenocarcinoma | TUMOR, TUMORS, ADENOCARCINOMA | Between 28 November 2018 and 18 February 2022, a total of 2,333 patients with previously untreated, locally advanced unresectable or mG/GEJ adenocarcinoma were screened at 166 sites in 18 countries. CLDN18.2 tumor status was assessed in 2,104 patients, of whom 808 (38.4%) had tumors that met the cutoff for CLDN18.2 positivity (≥75% of tumor cells with moderate-to-strong CLDN18 membranous staining as determined by central immunohistochemistry using the investigational VENTANA CLDN18 (43-14A) RxDx assay). Among patients whose tumors were assessed for CLDN18.2 status, 1,701 had tumors that were HER2-negative; 729 of 1,701 (42.9%) patients had tumors that met the cutoff for CLDN18.2 positivity. Of screened patients with tumors that were HER2-negative and met the cutoff for CLDN18.2 positivity, 222 were not randomized owing to failure to meet other inclusion criteria or the patient’s decision to withdraw from the study. Ultimately, 507 patients with CLDN18.2-positive tumors were randomly assigned to receive either zolbetuximab plus CAPOX ( | PMC10427418 |
PFS | PFS as the primary endpoint was statistically significantly prolonged in patients randomized to receive zolbetuximab versus placebo (median, 8.21 months versus 6.80 months, respectively; hazard ratio (HR) = 0.687; 95% confidence interval (CI), 0.544–0.866; | PMC10427418 | ||
OS | SECONDARY | At the interim analysis, 318 out of 507 (62.7%) patients had died: 144 of the 254 (56.7%) patients randomized to receive zolbetuximab, and 174 of the 253 (68.8%) patients randomized to receive placebo. OS as a key secondary endpoint was statistically significantly prolonged in patients in the zolbetuximab arm versus the placebo arm (median, 14.39 months versus 12.16 months, respectively; HR = 0.771; 95% CI, 0.615–0.965; | PMC10427418 | |
Discussion | HER2-negative, nausea and vomiting, tumors | EVENTS, DISEASE, TUMORS, ADENOCARCINOMA | In GLOW, the addition of first-line zolbetuximab to CAPOX significantly improved PFS and OS in patients with CLDN18.2-positive, HER2-negative, locally advanced unresectable or mG/GEJ adenocarcinoma. GLOW confirmed the increased survival benefit of adding zolbetuximab to chemotherapy observed in previous phase 2 and 3 studiesAlthough the control arm in SPOTLIGHT performed better than expected, in GLOW, the control arm performed as expected, which is in line with other studies that suggest that CLDN18.2 is not a prognostic biomarkerGLOW demonstrated that CLDN18.2 is a prevalent biomarker in HER2-negative, locally advanced unresectable or mG/GEJ adenocarcinoma. In GLOW, 38.4% of screened patients with tumors assessable for CLDN18.2 expression had tumors that met the cutoff for CLDN18.2 positivity. In SPOTLIGHT, 38.4% of screened patients with tumors assessable for CLDN18.2 expression also had CLDN18.2-positive tumorsIn GLOW, the most common TEAEs observed in patients who received zolbetuximab plus CAPOX were nausea and vomiting; these events occurred at a more than 10% difference compared with patients who received placebo plus CAPOX. These results are consistent with the safety profile of zolbetuximab monotherapy and zolbetuximab plus chemotherapy observed in previous phase 1, 2 and 3 studiesThis study had some limitations. First, this study was underpowered to statistically determine the effectiveness of zolbetuximab plus CAPOX in the pre-specified subgroups. In all cases, these subgroups were relatively small, and so interpretations should be made with caution. Next, this study did not evaluate the combination of zolbetuximab with nivolumab. Chemotherapy was selected as a relevant comparator arm in GLOW because nivolumab was not approved in this patient population at the time of study initiation. Furthermore, there is a substantial number of patients in this population whose disease does not respond to nivolumab. The ILUSTRO study (Overall, treatment with zolbetuximab plus CAPOX led to statistically significantly prolonged PFS and OS compared to placebo plus CAPOX in patients with CLDN18.2-positive, HER2-negative, locally advanced unresectable or mG/GEJ adenocarcinoma. These results further confirm the survival benefits observed in the phase 3 SPOTLIGHT study | PMC10427418 |
Methods | PMC10427418 | |||
Trial oversight | Astellas (the study sponsor) collaborated with the academic authors on the design of the study and on the collection and interpretation of the data after analysis. The protocol and all amendments were approved by the appropriate independent ethics committee (IEC) or institutional review board (IRB) at each participating institution (Supplementary Table | PMC10427418 | ||
Patients | HER2-negative, tumor, tumors | DISEASE, TUMOR, TUMORS | Eligible patients were adults according to local regulations and had CLDN18.2-positive (defined as ≥75% of tumor cells with moderate-to-strong membranous CLDN18 staining as determined by central immunohistochemistry using the investigational VENTANA CLDN18 (43-14A) RxDx Assay), HER2-negative (per local or central testing), previously untreated, locally advanced unresectable or mG/GEJ tumors with radiologically evaluable disease according to RECIST version 1.1. Patients had an ECOG performance status score of 0 or 1 and adequate organ function. Full inclusion and exclusion criteria are as follows: | PMC10427418 |
Inclusion criteria | WOCBP, tumor, ≤28 | TUMOR, LIVER METASTASES, DISEASE, METASTATIC DISEASE, ADENOCARCINOMA, APPENDIX | General criteria:IRB/IEC-approved written informed consent and privacy language as per national regulations (for example, Health Insurance Portability and Accountability Act (HIPAA) authorization for US sites) must be obtained from the patient or legally authorized representative (if applicable) before any study-related procedures.Patient is considered an adult (for example, ≥18 years of age in the USA) according to local regulation at the time of signing the informed consent.A female patient is eligible to participate if she is not pregnant (negative serum pregnancy test at screening; female patients with elevated serum beta human chorionic gonadotropin (βhCG) and a demonstrated non-pregnant status through additional testing are eligible) and at least one of the following conditions applies.Not a woman of childbearing potential (WOCBP) as defined in Protocol Appendix 12.3 Contraception RequirementsORWOCBP who agrees to follow the contraceptive guidance as defined in Protocol Appendix 12.3 Contraception Requirements throughout the treatment period and for 9 months after the final administration of oxaliplatin and 6 months after the final administration of all other study drugsFemale patient must agree not to breastfeed starting at screening and throughout the study period and for 6 months after the final study treatment administration.Female patient must not donate ova starting at screening and throughout the study period and for 9 months after the final administration of oxaliplatin and for 6 months after the final administration of all other study drugs.Male patient with female partner(s) of childbearing potential must agree to use contraception as detailed in Protocol Appendix 12.3 Contraception Requirements during the treatment period and for 6 months after the final study treatment administration.Male patient must not donate sperm during the treatment period and for 6 months after the final study treatment administration.Male patient with a pregnant or breastfeeding partner(s) must agree to remain abstinent or use a condom for the duration of the pregnancy or time partner is breastfeeding throughout the study period and for 6 months after the final study treatment administration.Patient agrees not to participate in another interventional study while receiving study drug in the present study.Disease-specific criteria:Patient has histologically confirmed diagnosis of G/GEJ adenocarcinoma.Patient has radiologically confirmed locally advanced unresectable or metastatic disease within 28 days before randomization.Patient has radiologically evaluable disease (measurable and/or non-measurable) according to RECIST version 1.1, per local assessment, ≤28 days before randomization. For patients with only one evaluable lesion and prior radiotherapy ≤3 months before randomization, the lesion must either be outside the field of prior radiotherapy or have documented progression after radiation therapy.Patient’s tumor expresses CLDN18.2 in ≥75% of tumor cells, demonstrating moderate-to-strong CLDN18 membranous staining as determined by central immunohistochemistry testing.Patient has a HER2-negative tumor as determined by local or central testing on a G/GEJ tumor specimen.Physical or laboratory findings:Patient has a ECOG performance status score 0 or 1.Patient has predicted life expectancy ≥12 weeks, in the opinion of the investigator.Patient must meet all of the following criteria based on the centrally or locally analyzed laboratory tests collected within 14 days before randomization. In the case of multiple sample collections within this period, the most recent sample collection with available results should be used to determine eligibility.Hemoglobin ≥9 g dlAbsolute neutrophil count (ANC) ≥1.5 × 10Platelets ≥100 × 10Albumin ≥2.5 g dlTotal bilirubin ≤1.5× upper limit of normal (ULN) without liver metastases (or <3.0× ULN if liver metastases are present)Aspartate aminotransferase and alanine aminotransferase ≤2.5× ULN without liver metastases (or ≤5× ULN if liver metastases are present)Estimated creatinine clearance ≥30 ml minProthrombin time/international normalized ratio and partial thromboplastin time ≤1.5× ULN (except for patients receiving anti-coagulation therapy) | PMC10427418 |
Exclusion criteria | Torsades de Pointes)QTc interval, malignancy, ≤28, autoimmune disease, coronary stenting, allergic reaction, toxicity, ventricular arrhythmias, cancer, infections, gastric outlet syndrome, persistent/recurrent vomiting, infection, adenocarcinoma, long QT syndromeCardiac arrhythmias, peripheral sensory neuropathy grade, psychiatric illness, human immunodeficiency virus (HIV) infection, coronary angioplasty | DIHYDROPYRIMIDINE DEHYDROGENASE DEFICIENCY, CARCINOMATOUS MENINGITIS, CARDIOVASCULAR DISEASE, UNSTABLE ANGINA, HEART, AUTOIMMUNE DISEASE, ALLERGIC REACTION, GASTRIC BLEEDING, VENTRICULAR ARRHYTHMIA, CANCER, INFECTIONS, HEPATITIS C INFECTION, HEPATITIS B, GASTRIC ULCERS, INFECTION, HEPATITIS C, ADENOCARCINOMA, METASTASES, CONGESTIVE HEART FAILURE, HYPERTENSIVE CRISIS, FIBRILLATION, VIRUS, MYOCARDIAL INFARCTION, DISEASE, CEREBROVASCULAR ACCIDENT, DELAYED HYPERSENSITIVITY, CONTROLLED ATRIAL FIBRILLATION | Prohibited treatment or therapies:Patient has received prior systemic chemotherapy for locally advanced unresectable or mG/GEJ adenocarcinoma. However, patient may have received either neoadjuvant or adjuvant chemotherapy, immunotherapy or other systemic anti-cancer therapies as long as it was completed at least 6 months before randomization.Patient has received radiotherapy for locally advanced unresectable or mG/GEJ adenocarcinoma ≤14 days before randomization and has not recovered from any related toxicity.Patient has received treatment with herbal medications or other treatments that have known anti-tumor activity within 28 days before randomization.Patient has received systemic immunosuppressive therapy, including systemic corticosteroids, within 14 days before randomization. Patient using a physiologic replacement dose of hydrocortisone or its equivalent (defined as up to 30 mg per day of hydrocortisone or up to 10 mg per day of prednisone), receiving a single dose of systemic corticosteroids or receiving systemic corticosteroids as premedication for radiologic imaging contrast use is eligible.Patient has received other investigational agents or devices within 28 days before randomization.Medical history or concurrent disease:Patient has prior severe allergic reaction or intolerance to known ingredients of zolbetuximab or other monoclonal antibodies, including humanized or chimeric antibodies.Patient has known immediate or delayed hypersensitivity, intolerance or contraindication to any component of study treatment.Patient has prior severe allergic reaction or intolerance to any component of CAPOX.Patient has known dihydropyrimidine dehydrogenase deficiency. (Note that screening for dihydropyrimidine dehydrogenase deficiency should be conducted per local requirements.)Patient has a complete gastric outlet syndrome or a partial gastric outlet syndrome with persistent/recurrent vomiting.Per investigator judgment, patient has significant gastric bleeding and/or untreated gastric ulcers that exclude the patient from participation.Patient has a known history of a positive test for human immunodeficiency virus (HIV) infection or known active hepatitis B (HB; positive HB surface antigen (HBs Ag)) or hepatitis C infection. (Note: Screening for these infections should be conducted per local requirements.)For patients who are negative for HBs Ag but HB core antibody (HBc Ab) positive, an HB DNA test will be performed, and, if positive, the patient will be excluded.Patients with positive hepatitis C virus (HCV) serology but negative HCV RNA test are eligible.Patients treated for HCV with undetectable viral load results are eligible.Patient has an active autoimmune disease that has required systemic treatment within the past 3 months before randomization.Patient has an active infection requiring systemic therapy that has not completely resolved within 7 days before randomization.Patient has significant cardiovascular disease, including any of the following.Congestive heart failure (defined as New York Heart Association class III or IV), myocardial infarction, unstable angina, coronary angioplasty, coronary stenting, coronary artery bypass graft, cerebrovascular accident or hypertensive crisis within 6 months before randomizationHistory of clinically significant ventricular arrhythmias (that is, sustained ventricular tachycardia, ventricular fibrillation or Torsades de Pointes)QTc interval >450 ms for male patients; QTc interval >470 ms for female patientsHistory or family history of congenital long QT syndromeCardiac arrhythmias requiring anti-arrhythmic medications (patients with rate controlled atrial fibrillation for >1 month before randomization are eligible)Patient has history of central nervous system (CNS) metastases and/or carcinomatous meningitis from G/GEJ cancer.Patient has known peripheral sensory neuropathy grade >1 unless the absence of deep tendon reflexes is the sole neurological abnormality.Patient has had a major surgical procedure ≤28 days before randomization.Patient without complete recovery from a major surgical procedure ≤14 days before randomization.Patient has psychiatric illness or social situations that would preclude study compliance, per investigator judgment.Patient has another malignancy for which treatment is required, per investigator judgment.Patient has any concurrent disease, infection or comorbid condition that interferes with the ability of the patient to participate in the study, which places the patient at undue risk or complicates the interpretation of data, in the opinion of the investigator. | PMC10427418 |
Study design and treatment | non-Asia | METASTASES | GLOW is a global, randomized, double-blind, phase 3 trial. Patients were randomly assigned 1:1 to receive intravenous infusion of zolbetuximab 800 mg/mRandomization was performed by blinded site staff using interactive response technology by block randomization with block sizes of two and was stratified according to region (Asia versus non-Asia), number of organs with metastases (0–2 versus ≥3) and prior gastrectomy (yes versus no). Countries in the Asia subgroup analysis were mainland China, Japan, South Korea, Malaysia, Taiwan (province of China) and Thailand, and countries in the non-Asia subgroup analysis were Argentina, Canada, Croatia, Greece, Ireland, The Netherlands, Portugal, Romania, Spain, Turkey, UK and USA. The randomization list and study drug blinding were maintained by the interactive response technology system. The sponsor, investigators, clinical staff and patients remained blinded to treatment throughout the study. To maintain blinding, zolbetuximab and placebo, which were identical in appearance and form, were provided to investigators or designees by an unblinded pharmacist and administered in identical volumes, routes and schedules. | PMC10427418 |
Endpoints | abdominal pain, Cancer | SECONDARY, CANCER | The primary endpoint was PFS per RECIST version 1.1 as determined by an IRC. Key secondary endpoints were OS and time to confirmed deterioration in scores for European Organization for Research and Treatment of Cancer global health status and quality of life, physical functioning and abdominal pain and discomfort assessments, which were determined as clinically meaningful to patients; time to confirmed deterioration data are pending the clinically meaningful threshold obtained from the ongoing exit survey study per protocol and will be reported in a future publication. Additional secondary endpoints were ORR and DOR per RECIST version 1.1 as determined by an IRC, safety and tolerability of zolbetuximab, additional patient-reported outcomes and pharmacokinetics and immunogenicity of zolbetuximab; patient-reported outcomes will also be reported in a future publication.PFS, OS, ORR and DOR were assessed in the ITT population, which consisted of all randomized patients. Safety was assessed in all patients who received at least one dose of any study drug. | PMC10427418 |
Assessments | Tumor, Pain, Cancer | DISEASE PROGRESSION, ADVERSE EVENT, TUMOR, ADVERSE EVENT, CANCER | Tumor response was assessed by imaging at screening, every 9 weeks for the first 54 weeks of treatment and every 12 weeks thereafter until disease progression or start of another anti-cancer treatment. Survival was assessed at least every 12 weeks during follow-up. Patients completed health-related quality of life assessments, including the EuroQOL EQ-5D-5L and the European Organization for Research and Treatment of Cancer QLQ-C30, QLQ-OG25 plus STO22 and Global Pain at screening, every 3 weeks during study treatment, at study treatment discontinuation and 30 and 90 days after study treatment discontinuation. Adverse events, graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03, were evaluated throughout the trial and for 90 days after study treatment discontinuation. Data were collected at study sites where study treatment was administered. | PMC10427418 |
Statistical analysis | deaths, death | DISEASE PROGRESSION | The Kaplan–Meier method was used to estimate the median and distribution of PFS, OS and DOR; stratified log-rank tests were used to assess between-group differences, and the stratified Cox proportional hazard model was used to calculate HRs and associated 95% CIs. The Cochran–Mantel–Haenszel test was used to assess between-group differences in ORR. Pre-specified multiplicity adjustment methods were used to control the overall one-sided type I error rate at 0.025. Efficacy boundaries were calculated based on the information fraction at the time of analysis. The reported 95% CIs describe the precision of the point estimates and may not correspond to the significance of the test. The study aimed to enroll 500 patients. The final analysis of PFS was planned when 300 patients experienced disease progression or death to provide 93.4% power to detect a between-group difference with the assumption of median PFS of 9 months with zolbetuximab plus CAPOX versus 6 months with placebo plus CAPOX (HR = 0.67) at an overall one-sided alpha level of 0.025. An interim analysis of OS was planned at the final PFS analysis, and a final analysis of OS was planned after 386 deaths to provide 80% power to detect a between-group difference with the assumption of median OS of 14.7 months with zolbetuximab plus CAPOX versus 11 months with placebo plus CAPOX (HR = 0.75) at an overall one-sided alpha level of 0.025. An efficacy boundary was calculated for the interim OS based on the information fraction at the time of the interim analysis; a one-sided level of significance of 0.0135 was used with an 82.4% information fraction. To strictly control the type I error rate at an alpha level of 0.025, OS was tested only if the null hypothesis of the final PFS analysis was rejected. Full details of the statistical analysis plan are provided in the protocol (Collected data were entered using the RAVE electronic data collection system. Sample size calculations were performed with East version 6.4 software. Statistical data analyses were performed with SAS version 9.3 or higher software. | PMC10427418 |
Reporting summary | Further information on research design is available in the | PMC10427418 | ||
Online content | Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41591-023-02465-7. | PMC10427418 | ||
Supplementary information |
Supplementary Tables 1–6, Study Protocol, Statistical Analyis Plan (SAP) and SAP amendmentsReporting Summary | PMC10427418 | ||
Extended data | PMC10427418 | |||
PFS in the ITT population by investigator. | Kaplan-Meier curve of PFS in all patients. CAPOX, capecitabine plus oxaliplatin regimen; CI, confidence interval; HR, hazard ratio; ITT, intent-to-treat; mo., months; No., number; PFS, progression-free survival.
| PMC10427418 | ||
First occurrence of nausea and vomiting in the safety analysis set. | EVENTS | Events were counted if they did not occur in any previous onset interval. Individual data points are reported in Supplementary Tables
| PMC10427418 | |
Extended data | is available for this paper at 10.1038/s41591-023-02465-7. | PMC10427418 | ||
Supplementary information | The online version contains supplementary material available at 10.1038/s41591-023-02465-7. | PMC10427418 | ||
Acknowledgements | J. Donnelly | The study was funded by Astellas Pharma Inc. The authors thank the patients, caregivers, investigators and site staff who participated in this study. We thank J. Raizer; study managers J. Donnelly, S. Koshy Hunt, A. Shrivastava and C. Muench; and steering committee members S. Kim and S. Klempner. We thank S. Moran, C. Beckman, J. McDonald, S. Arora, J. Pugh, M. Kouzova, L. Pang and the CLDN18 project team at Roche Tissue Diagnostics for their collaboration in assay development, clinical trial implementation and technical review of the manuscript. We thank A. Ferguson from Oxford PharmaGenesis, Inc., who provided medical writing support that was funded by Astellas Pharma Inc. | PMC10427418 | |
Author contributions | M.A.S., J.A., Y.-J.B., P.E., D.I., F.L., K.S., E.V.C., M.O., J.W.P., D.M., P.B., A.A. and R.-H.X. made substantial contributions to the study design. M.A.S., J.A., Y.-J.B., P.E., D.I., F.L., K.S., E.V.C., J.G.P., J.H., L.S., S.C.O., P.S., H.F.S.H., H.M.T. and R.-H.X. acquired the study data. M.O., J.W.P., D.M., P.B. and A.A. analyzed and interpreted the study data. All authors had access to the data, participated in reviewing and editing the manuscript and gave final approval to submit the manuscript for publication. | PMC10427418 | ||
Peer review | PMC10427418 | |||
Data availability | Upon request, and subject to certain criteria, conditions and exceptions, Astellas will provide access to anonymized patient-level data from completed Astellas-sponsored phase 1 to phase 4 interventional clinical studies conducted for products and indications that have been approved in any country and also for studies conducted for terminated compounds. Approval must have been granted by the agencies of the main regions: the USA the European Union and Japan. If approval is sought in only one or two regions, approval must have been granted by those agencies. Where available, the following anonymized patient-level data and information are provided for each clinical study: raw dataset, analysis-ready dataset, protocols with any amendments or addenda, annotated case report form, statistical analysis plan, dataset specifications and clinical study report. Additionally, data may be available upon reasonable request. Researchers may request access to anonymized participant-level data, trial-level data and protocols from Astellas-sponsored clinical trials at | PMC10427418 | ||
Competing interests | SE, Pierre | ONCOLOGY | M.A.S. reports receiving research funding from Astellas Pharma Inc., Merck, Bristol Myers Squibb and Oncolys BioPharma and serving a leadership or judiciary role in board, society, committee or advocacy groups for the American Society of Clinical Oncology Leadership Council. K.S. reports receiving research funding from Astellas Pharma Inc., Ono Pharmaceutical, Daiichi Sankyo, Taiho Pharmaceutical, Chugai Pharmaceutical Co., Merck Sharp & Dohme, Amgen, Eisai and Medi Science; receiving consulting fees from Eli Lilly and Company, Bristol Myers Squibb, Takeda Pharmaceutical Company, Pfizer, Ono Pharmaceutical, Novartis, AbbVie, Daiichi Sankyo, Taiho Pharmaceutical, GlaxoSmithKline, Amgen, Boehringer Ingelheim, Merck Sharp & Dohme, Astellas Pharma Inc., Guardant Health Japan and Janssen Pharmaceuticals; and receiving payment or honoraria from Bristol Myers Squibb, Takeda Pharmaceutical Company and Janssen Pharmaceuticals. J.A.A. reports receiving study funding from Astellas Pharma Inc., Turning Point Therapeutics, Inc., Bristol Myers Squibb, Merck, Taiho Pharmaceutical, Delta-Fly Pharma, Inc., Roche, ProLynx, Inc., Zymeworks, Daiichi Sankyo, Leap Therapeutics, Inc., Gilead Sciences, Inc. and Lanova Pharma; receiving consulting fees from Bristol Myers Squibb, Merck, Astellas Pharma Inc., Amgen, Taiho Pharmaceutical, Zymeworks, BeiGene, AstraZeneca, Daiichi Sankyo, Bayer, GRAIL, Novartis, Geneos, Servier Laboratories and Gilead Sciences, Inc.; receiving support for travel and/or meeting attendance from Daiichi Sankyo, Bristol Myers Squibb and Merck; and participating on data safety monitoring board or advisory board for BeiGene. Y.-J.B. reports receiving research funding from Astellas Pharma Inc., Genentech, Roche, Merck Serono, Daiichi Sankyo, Merck Sharp & Dohme, Amgen and BeiGene and receiving consulting fees from Merck Sharp & Dohme, Daiichi Sankyo, ALX Oncology, Hanmi Pharmaceutical, Merck Serono, Astellas Pharma Inc., Samyang Biopharm Corporation and Daewoong Pharmaceutical. P.E. reports receiving research funding from Astellas Pharma Inc. and receiving consulting fees from ALX Oncology, Arcus Biosciences, Astellas Pharma Inc., AstraZeneca, Blueprint Medicines, Bristol Myers Squibb, Chimeric Therapeutics, Celgene, Coherus BioSciences, Daiichi Sankyo, Five Prime Therapeutics, Inc., IDEAYA Biosciences, Istari Oncology, Legend Biotech, Eli Lilly and Company, Loxo Oncology, Merck, Novartis, Ono Pharmaceutical, Servier Laboratories, Taiho Pharmaceutical, Takeda Pharmaceutical Company, Turning Point Therapeutics, Inc., Xencor and Zymeworks. D.I. reports receiving research funding from Astellas Pharma Inc.; receiving consulting fees from Amgen, Bayer, Astellas Pharma Inc., Merck, Daiichi Sankyo, Taiho Pharmaceutical, Natera, Inc., Bristol Myers Squibb, Eli Lilly and Company, Roche and AstraZeneca; and participating on data safety monitoring boards or advisory boards for MacroGenics and Merck. F.L. reports receiving research funding from Astellas Pharma Inc.; receiving consulting fees from Amgen, Astellas Pharma Inc., Bristol Myers Squibb, Daiichi Sankyo, Eli Lilly and Company, Merck Sharp & Dohme, Novartis and Roche; receiving payment or honoraria from Amgen, Astellas Pharma Inc., AstraZeneca, Bayer, Bristol Myers Squibb, Daiichi Sankyo, Eli Lilly and Company, Elsevier, the Falk Foundation, Incyte Corporation, Medscape, MedUpdate GmbH, Merck, Merck Sharp & Dohme, Novartis, Roche, Servier Laboratories, Springer Nature and Streamed Up; receiving support for travel and/or meeting attendance from Bristol Myers Squibb; and participating on data safety monitoring boards or advisory boards for BioNTech SE. E.V.C. reports receiving research funding from Astellas Pharma Inc., Amgen, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Ipsen, Eli Lilly and Company, Merck Sharp & Dohme, Merck KGaA, Novartis, Roche and Servier Laboratories and receiving consulting fees from AbbVie, Array BioPharma, Astellas Pharma Inc., AstraZeneca, Bayer, BeiGene, Biocartis, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Daiichi Sankyo, Halozyme, GlaxoSmithKline, Helsinn Healthcare SA, Incyte Corporation, Ipsen, Janssen Pharmaceuticals, Eli Lilly and Company, Merck Sharp & Dohme, Merck KGaA, Mirati Therapeutics, Inc., Novartis, Laboratoires Pierre Fabre, Roche, Seagen, Servier Laboratories, Sirtex Medical, Terumo Corporation, Taiho Pharmaceutical, TRIGR and Zymeworks. J.G.P. reports receiving research funding from Astellas Pharma Inc.; receiving consulting fees from Amgen, Bristol Myers Squibb and Eisai; receiving payment or honoraria from Amgen, Bayer, Bristol Myers Squibb, Merck and Servier Laboratories; and receiving support for travel and/or meeting attendance from Amgen and Novartis. J.H. reports receiving study funding from Astella Pharma Inc. L.S. reports receiving study funding from Astella Pharma Inc., Beijing Xiantong Biomedical Technology, Qilu Pharmaceutical Co., Ltd., Zai Lab Pharmaceutical (Shanghai), Beihai Kangcheng (Beijing) Medical Technology, Yaojie Ankang (Nanjing) Technology Co., Ltd., Baiji Shenzhou (Beijing) Biotechnology Co., Ltd. and Jacobio Pharmaceuticals; consulting fees from Mingji Biopharmaceuticals, Haichuang Pharmaceutical and Herbour Biomed; receiving payment or honoraria from Hutchison Whampoa, Hengrui, Zai Lab Pharmaceutical and CStone Pharmaceutical; and participating on data safety monitoring board or advisory board for Merck Sharp & Dohme, Merck, Bristol Myers Squibb, Boehringer Ingelheim, Sanofi, Roche, Servier Laboratories and AstraZeneca. S.C.O. reports receiving study funding from Astellas Pharma Inc. P.S. reports receiving study funding from Astellas Pharma Inc. H.F.S.H. reports receiving study funding from Astellas Pharma Inc. H.M.T. reports receiving study funding from Astellas Pharma Inc. M.O., J.W.P., D.M. and P.B. are full-time employees of Astellas Pharma Inc. A.A. is a full-time employee and stock holder of Astellas Pharma Inc. R.-H.X. reports receiving research funding from Astellas Pharma Inc. | PMC10427418 |
References | PMC10427418 | |||
Background | HIV infection, HIV in Kilimanjaro and Arusha Region, Tanzania., ®, HIV and retention | HIV INFECTION | Adherence to antiretroviral (ARV) treatment for HIV infection is challenging because of many factors. The World Health Organization (WHO) has recommended using digital adherence technologies (DATs). However, there is limited evidence on how DATs improve adherence. Wisepill® is an internet-enabled medication dispenser found feasible and acceptable in several studies. However, limited evidence is available on its effectiveness in improving ART adherence, specifically among children and adolescents. Furthermore, DATs are often developed without involving the target groups. We propose a two-stage project consisting of a formative study to customize an existing Wisepill DAT intervention and a randomized clinical trial to investigate the effectiveness of DAT combined with reminder cues and tailored feedback on adherence to ARV treatment among children and adolescents living with HIV and retention in care among breastfeeding women living with HIV in Kilimanjaro and Arusha Region, Tanzania. | PMC10120095 |
Methods | We will conduct a formative mixed-methods study and three sub-trials in Kilimanjaro and Arusha Regions among (1) children aged 0–14 years and their caregivers, (2) adolescents aged 15–19 years and (3) breastfeeding women and their HIV-negative infants. In the formative study, we will collect and analyse data on needs and contents for DATs, including the contents of short message service (SMS) texts and tailored feedback. The results will inform the customization of the DAT to be tested in the sub-trials. In the trials, participants will be randomized in the intervention arm, where the DAT will be implemented or the control arm, where standard care will be followed. Participants in the intervention arm will take their medication from the Wisepill box and receive daily reminder texts and tailored feedback during clinic visits. | PMC10120095 | ||
Discussion | If the intervention improves adherence to ART and the devices are acceptable, accurate and sustainable, the intervention can be scaled up within the National Aids Control Programmes. | PMC10120095 | ||
Trial registration | PACTR202301844164954, date 27 January 2023. | PMC10120095 | ||
Keywords | PMC10120095 | |||
Introduction | HIV in SSA [ | DISEASE | Children and adolescents form a significant part of the world population of people living with human immunodeficiency virus (HIV), with the World Health Organization (WHO) reporting 1.8 million children (age < 15) and 1.8 million adolescents (ages 10–19) in Sub-Saharan Africa in 2019 [Despite high access for pregnant women to HIV treatment of 92%, the percentage of infected infants has not decreased much since 2010 [It is estimated that only 59% of children (0–14 years) living with HIV are on anti-retroviral (ARV) treatment in Eastern and Southern Africa [Adolescents (aged 15–19) are more independent than children below 14 and depend less on their parents. Reports from nurse counsellors in our setting imply that older children, including adolescents, visit clinics independently and often do not have their parents/caregivers as treatment supporters. Virological suppression is more difficult in this group, and levels differ significantly between studies: 27 to 89% [Digital adherence tools (DATs) have been proposed for monitoring adherence and have been investigated in several studies [Using the common way of communication through SMS has a high potential for disease management. The number of mobile phone subscriptions per 100 inhabitants in Tanzania was 86% in 2020. Additionally, over 25 billion local SMS were sent in the second quarter of 2019 [Only limited evidence exists for interventions improving adherence to treatment and retention in care for children and adolescents with HIV in SSA [ | PMC10120095 |
Methods/design | PMC10120095 | |||
Study area | We will conduct our project in care and treatment clinics (CTC) and postnatal clinics serving clients on antiretroviral treatment in the Kilimanjaro region in Tanzania, which has an area of 13,250 km | PMC10120095 | ||
Design | This project will evaluate a DAT consisting of real-time medication monitoring with the Wisepill® device and SMS messages and tailored feedback to participants on their adherence data. The project will consist of two stages (see Fig. Schematic display of the study | PMC10120095 | ||
Overall aim and objectives | HIV in Kilimanjaro, TanzaniaTo | SECONDARY | We aim to investigate whether our customized digital adherence tool will improve retention in care among breastfeeding women and adherence to treatment among children and adolescents.The specific objectives for the first stage are as follows:
To identify the enablers and barriers to using DAT among children and their caregivers/parents, adolescents and breastfeeding women living with HIV in Kilimanjaro, TanzaniaTo identify the needed content of SMS schemes based on the participants’ preferencesTo identify the preferred timing and frequency for SMS, including reminder cues to take medication and to visit the clinicTo understand the need for additional information for different groups to be included in the SMS, i.e. education on sexual and reproductive health importance of breastfeeding, amongst othersTo design the blueprint for the DAT to be tested in the second phase of the projectIn the second stage of clinical trials, we will investigate the effect of our developed DAT according to the blueprint. This will be established through the following specific objectives:
Breastfeeding women: to assess the percentage retention in care at 18 months post-partumChildren: to assess the percentage of children being 95% adherent to treatment based on pharmacy refill counts and self-reported adherenceAdolescents: to assess the percentage of adolescents being 95% adherent to treatment based on pharmacy refill counts and self-reported adherenceOur secondary objectives for the second stage are as follows:
To assess the percentage of breastfeeding women, children and adolescents living with HIV reporting 95% adherence at 18, 12 and 12 months, respectivelyTo assess the percentage of breastfeeding women, children and adolescents living with HIV being virologically suppressed (<20 copies/ml) at 18, 12 and 12 months, respectivelyTo determine the trend in adherence over time based on self-report and pharmacy refill counts for breastfeeding women, children and adolescents living with HIVTo establish different cut-off points of adherence levels and their association with virological suppressionTo determine the HIV status of the infants of breastfeeding women living with HIV at 6 weeks, 9 months and 18 monthsTo investigate the feasibility and acceptability of the DATTo determine the implementation costs of the DAT | PMC10120095 |
Formative stage | The objective of this stage is to evoke feedback from the key intervention stakeholders to guide on designing and developing the newly proposed DAT. We will first survey 142 participants from each group to investigate enablers and barriers to using DAT. This figure is based on selecting an arbitrary percentage of 50% experiencing barriers for using DAT with Next, we will purposively select 20 participants from each group, considered enough to reach ‘saturation’ of information, to use the DAT for 1 month. After signing informed consent, we will provide mothers, parents/caregivers of children and adolescents with a Wisepill® box which they will use for a month. After a month, we will interview each of them, including the children whose status has been disclosed on their experience with the box and the need for SMS content to be developed for the customized digital tool. We will also hold two focus group discussions with each group of breastfeeding mothers, parents/caregivers and adolescents. The outcomes of the formative research will be as follows:
An overview of needs for SMS contents and timing for reminder cues to take medicationAn overview of needs for SMS contents and timing for reminder cues for clinic visitsAn overview of further needs for other SMSs, such as educational and informational texts, including timing and frequencyAn overview of enablers and barriers to the success of the interventionQuantitative data from the survey will be analysed descriptively using frequency tables, chi-square tests and | PMC10120095 | ||
Clinical trials | We will conduct three clinical trials in each target groups to investigate whether the developed DATs improve adherence to treatment and virological outcomes. The study procedures can be found in the | PMC10120095 | ||
Ethical approval | The formative study and the trial have been separately approved by the College Research and Ethical Review Committee (CRERC) of Kilimanjaro Christian Medical University College (KCMUCo) and the National Health Research Ethics Sub-Committee (NatHREC) of the National Medical Research Institute (NIMR) of Tanzania. Furthermore, the regional representatives of the Ministry of Health and the regional medical officer have been permitted to conduct the study. Approval for the trials was requested and obtained after we had designed the customized DAT. | PMC10120095 | ||
Participants | HIV 0–14 | We will have three different study populations, which are the following:
Breastfeeding mothers living with HIV with their uninfected infants (for the survey of the formative phase, we will also include pregnant women to understand their views)Children aged 0–14 and their caregivers/parentsAdolescents aged 15–19The inclusion criteria are as follows:
Mother (of HIV-exposed infant)/child/adolescent being HIV positive, with the infant of the mother not being positiveAttending Care and Treatment Centres (CTC) or postnatal clinics in the Kilimanjaro region at the start of the studyAge of mother between 18–50 years, age of children living with HIV 0–14 years and adolescents living with HIV between 15–19 yearsMother, caregiver/parents and adolescents willing to use RTMM and/or receive SMSMother, caregiver/parent and adolescents able to read and understand SMSMother, caregiver/parent and adolescents aged 18–19 able to understand and willing to sign the informed consentAdolescents aged 15–17 able and willing to sign informed assentThe exclusion criteria are as follows:
Admission to a hospital at study entryParticipation in other trials related to adherence and retention in carePrevious participation in digital health research | PMC10120095 | |
Intervention | The basis for the intervention is the Wisepill® device and our previously developed SMS scheme [ | PMC10120095 | ||
Attending the nurse; feedback on adherence data | Velicer | Nurse counsellors will recruit participants at their respective care and treatment clinics, follow standard care according to their normal schedule of care. After signing the informed consent, we will provide parents/caregivers of children and adolescents with a Wisepill® box which they will use for 12 months, while mothers will use it for 18 months.Study nurses will attend extensive training on all study procedures. Participants will receive feedback on their adherence to ART during their clinic visit. Study nurses will share and discuss adherence reports generated by the device with the participant during the clinic visit. We will use the ‘stages of change’ model by Prochaska and Velicer [ | PMC10120095 | |
Blinding | BLIND | Due to the nature of the intervention, which is behavioural based, and the fact that assessing the outcome, which is adherence, is part of the behavioural intervention, it is impossible to blind any person in the study. During the handling of missing data, the transformation of variables, doing subgroup analyses and covariate selection, our data analysts will be blinded. | PMC10120095 | |
Sample size calculation and randomization | We calculated the sample size for each group separately as the outcomes differed. In a previous study, the proportion of breastfeeding women that stayed in care was 41% in Kilimanjaro [The proportion of children reaching 95% adherence was 72% in a study among orphans in Dar es Salaam [The proportion of adolescents reaching 95% was 67% in a study among adolescents in the Kilimanjaro region [Randomization will be done using stratified block randomization on the strata of inclusion site and sex. We will use computer-generated random numbers, which were then programmed in the randomization module of the redcap. Those who enrol participants or assign the interventions only press a button in the database after entering the inclusion site and sex of the screened participant to give them the randomisation outcome and, therefore, cannot predict the allocation. | PMC10120095 | ||
Study outcomes | ADVERSE EVENT, EVENTS, ADVERSE EVENT | The primary study outcome for breastfeeding women is the percentage retained in care at 18-month post-partum. For children and adolescents, the primary outcome is mean adherence to ARV treatment.Adverse events will be monitored through an adverse event form as part of the case report form. Adverse events will be recorded by the data collectors, which are mainly the nurse counsellors in the field sites. Due to the nature of the intervention, we do not expect serious events. An incidental findings policy is in place. | PMC10120095 |
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