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PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
CoQ10 is synthesised within mitochondria and must then be relocated to other subcellular and cell membranes.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
The mechanism(s) by which CoQ10 is transported within the cell is not completely understood but is thought to involve the lipid transfer protein STARD7 (steroidogenic acute regulatory protein related lipid transfer domain 7).
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
The interaction between CoQ10 and STARD7 has been investigated in model systems using several types of human cell lines, including SH-SY5Y neuroblastoma and HEK293 cells.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Using the latter model system, two key roles for STARD7 were identified: firstly, in the transport of phosphatidylcholine to mitochondria to support the biosynthesis of CoQ10, and secondly, to transport newly synthesised CoQ10 from mitochondria to subcellular and cellular membranes.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
The function of STARD7 was, in turn, shown to be regulated by the mitochondrial protease PARL, which releases the STARD7-CoQ10 complex from the mitochondria for transport within the cell.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
CRISPR/Cas9 technology to create STARD7 or PARL knockout HEK 293 cell lines was used to establish how the absence of these proteins affects CoQ10 production, transport, and mitochondrial health .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
A model system based on the human liver cancer cell line HepG2 has been used to investigate the intracellular transport of CoQ10 by another type of lipid carrier, Saposin B, and its precursor prosaposin .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Saposin B transports newly synthesised CoQ10 from the mitochondria to other sub-cellular organelles for subsequent membrane incorporation, including lysosomes.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
The binding of CoQ10 to Saposin B is pH-dependent, such that the acidic internal pH of the lysosomes causes Saposin B to release the transported CoQ10 .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
It is of note that Saposin B is also an intracellular carrier of vitamin E, suggesting the possibility of competition in the transport of CoQ10, as noted previously in this article .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Human neuronal model systems based on the neuroblastoma cell line SH-SY5Y have similarly been used to investigate the mechanism of intracellular CoQ10 transport by STARD7 and Saposin B, as well as by the Cqd1 protein, which promotes the export of CoQ10 from mitochondria .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
It is also of note that human-cell-based model systems have been used to investigate vesicle-mediated intracellular transport of CoQ10, which operates in tandem with the lipid transporters above, particularly regarding transport to the endoplasmic reticulum and Golgi apparatus .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Intranasal drug delivery provides a mechanism by which drugs can be delivered directly into the brain via transport along the olfactory and trigeminal nerves, bypassing the BBB.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
While this route has been used for the administration of several prescription-type medicinal products relevant to the treatment of CNS disorders, to date, there have been no clinical studies relating to intranasal administration of CoQ10 .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Although animal (primarily rodent)-based models are available for studying intranasal drug administration, the applicability of such systems for studying the delivery of CoQ10 in humans has been questioned because of differences in nasal anatomy between rodents and humans ; there is, therefore, a rationale for the use of human-cell-based model systems for studying the intranasal delivery of CoQ10.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Cell-based model systems for studying intranasal drug delivery include cell lines or primary cell cultures derived from human nasal epithelium.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Cell lines represent a simpler system in use, particularly for preliminary screening, but may be lacking in some functional characteristics.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Primary cultures represent a more complex system to use but contain all the cell types found in the human nasal mucosa, including ciliated cells and mucus-producing goblet cells, and are used to study drug transport mechanisms .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
No studies were identified to date in which this type of model system had been used to study the intranasal administration of CoQ10.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
One area of importance to CoQ10 metabolism, although sometimes overlooked, is the interaction between CoQ10 and selenium.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
As noted earlier in this article, CoQ10 exists in both oxidised (ubiquinone) and reduced (ubiquinol) forms.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
The normal functioning of CoQ10 requires the continual interconversion of ubiquinone and ubiquinol CoQ10 forms (approximately 1.4 cycles per second ).
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Contrary to the marketing literature provided by some supplement companies supplying CoQ10, the ubiquinol form of CoQ10 is, therefore, not more important than the ubiquinone form in cellular metabolism.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
The importance of selenium in the CoQ10 recycling process is in its role as an essential cofactor of the flavoenzyme thioredoxin reductase, which converts ubiquinone to ubiquinol to help facilitate the continual recycling of CoQ10.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
It is of note that thioredoxin reductase is not the only enzyme converting ubiquinone to ubiquinol, but is one of the most important .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Thioredoxin reductase uses NADPH as an electron donor, transferring electrons to CoQ10; the amino acid derivative, selenocysteine, forms part of the active site and is vital for the activity of this enzyme.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
This interaction at the cellular level explains the synergistic action of CoQ10 and selenium reported in clinical studies .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
In general terms, both 2D and 3D cell cultures have been used as model systems to study selenium metabolism in human cells, including embryonic kidney cells (HEK 293), hippocampal progenitor cells (HPCs), and colon cancer cells (Caco 2, HT 29).
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Specifically, regarding the brain, model systems using human cells to study how selenium crosses the human BBB have used brain microvascular endothelial cells in static transwell systems, as well as BBB-on-a-chip models.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Using such model systems, it has been shown that selenium (transported in the form selenoprotein P) enters cells via a receptor-mediated endocytosis pathway involving the low-density lipoprotein receptor ApoER2; this process also allows selenium (in selenoprotein P form) to access the BBB .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Several types of model systems have been used to study the interaction between CoQ10 and selenium, including selenium-deficient cell cultures.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Cell lines grown in selenium-deficient media mimic the effect of dietary selenium deficiency on mitochondrial function and oxidative stress.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Cell types with high energy or antioxidant demands, such as liver cells, are particularly sensitive to selenium deficiency; human hepatocytes cultured in selenium-deficient media have reduced levels of thioredoxin reductase, as well as the antioxidant selenoenzyme glutathione peroxidase .
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
For obvious reasons, the ability to study CoQ10 metabolism in the human brain in vivo is extremely limited.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Non-invasive imaging techniques such as phosphorus-31 magnetic resonance spectroscopy can be used to assess brain energy metabolism in vivo.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
However this technique evaluates the effect of CoQ10, for example, on mitochondrial function and ATP generation (by measuring phosphorylated compounds such as phosphocreatine), rather than measuring CoQ10 levels directly.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
At present there are no suitable techniques available for monitoring CoQ10 directly in the human brain in vivo, particularly whether it can access the human BBB.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
This, in turn, raises the question of the most suitable model systems relevant to CoQ10 metabolism in the human brain; as noted earlier in this article, perhaps surprisingly, there is an argument that in vitro model systems based on human cell lines are more appropriate than studies in animal models, particularly regarding BBB accessibility, although data obtained from such studies needs to be interpreted in context.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
The advantages and disadvantages of the various cell-line-based BBB model systems described earlier in this article are summarised in Table 1.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
The model systems described in this review have been used to further investigate the access of CoQ10 across the BBB and its metabolism within neurons, including mechanisms of intracellular transport.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
A 2D BBB model system based on endothelial cells has demonstrated the transport of CoQ10 in the apical-to-basal direction (i.e., blood-to-brain side) via lipoprotein-associated transcytosis, interacting with the SR-B1 and RAGE receptors.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
However, CoQ10 is simultaneously effluxed back to the blood side via the LDLR transporter, leading to no net accumulation of CoQ10 in the brain.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Similarly a 2D neuronal model system based on CoQ10-deficient human SH-SY5Y cells has demonstrated the beneficial effects (in terms of improved mitochondrial function, reduced oxidative stress, and improved lysosomal function) of CoQ10 supplementation.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Model systems based on the human SH-SY5Y neuroblastoma cell line have also been used to elucidate the mechanism(s) of intracellular CoQ10 transport; however, there are still many aspects of CoQ10 intracellular transport (and other transport situations) that require further elucidation, but the known CoQ10 transporters identified to date are summarised in Table 2.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
Finally, based on the model systems described in this article, a potential strategy to enhance CoQ10 transport across the human BBB and subsequently improve neuronal metabolism, based on activation of SRB1 and inhibition of LRP-1, respectively, is shown in Figure 2.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
With regard to SRB1 and LRP-1, broad-spectrum activation or inhibition, respectively, is not considered advisable because of their multiple functions.
PMC12837543
Blood–Brain Barrier and Neuronal Model Systems for Studying CoQ10 Metabolism
However, SRB1 and LRP-1 still constitute potential targets for improving CoQ10 BBB access using more specific activators or inhibitors of their interaction with CoQ10, and this remains an area for future research.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Bone marrow dissemination of tumor cells, common in various cancers, including neuroblastoma, is associated with poor outcome, necessitating sensitive detection methods for bone marrow minimal residual disease (MRD) and offer detection of biomarkers for therapy stratification.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Current standard-of-care diagnostics, involving cytomorphological and histological assessment of bone marrow aspirates and trephine biopsies, lack sensitivity, leading to undetected MRD in many patients, and do not allow molecular biomarker assessment.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
This study evaluates advanced multi-modal high-sensitivity MRD detection techniques in 509 bone marrow specimens from 108 high-risk neuroblastoma patients across two centers.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
We employed automatic immunofluorescence plus interphase fluorescence in situ hybridization (AIPF) and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) panels to quantify disseminated tumor cells (DTCs), disialoganglioside 2 (GD2) and CD56/Neural cell adhesion molecule (NCAM) levels, and adrenergic (ADRN) and mesenchymal (MES)-phenotype mRNA markers.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
This multi-modal analysis significantly improved MRD detection compared to standard-of-care methods; 395 samples yielded results for RT-qPCR-ADRN, AIPF and CM/histology and 223 showed concordant results (64 positive, 159 negative).
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
114 samples did not produce results as either no cytospins were prepared (n = 96) or results were inconclusive (all techniques n = 18).
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
AIPF and RT-qPCR complemented each other in detecting MRD and characterizing ADRN- and MES-phenotypes and GD2 immunotherapy target.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
RT-qPCR-ADRN alone frequently detected low tumor cell burden.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
High DTC infiltration at diagnosis showed bilateral bone marrow disease, whereas MRD settings often involved only one side.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
RT-qPCR-MES, despite lower sensitivity, identified 37 additional cases and showed delayed clearance of MES markers post-chemotherapy, with increases prior to relapse.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Our findings demonstrate the feasibility of integrating high-sensitivity techniques with standard-of-care assessments in an international multicenter setting.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Advanced multi-modal MRD detection, monitoring phenotype switches and assessing immunotherapy targets are crucial for improving patient outcomes in neuroblastoma and other cancers.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
The findings from this study have significant implications for the future practice of neuroblastoma management.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
By showing the feasibility and effectiveness of using multi-modal, high-sensitivity minimal residual disease (MRD) detection techniques, i.e. automatic immunofluorescence plus interphase fluorescence in situ hybridization (AIPF) and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), in an international multicenter setting, this research paves the way for more standardized and accurate MRD assessments.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
These methods allow for early detection of disease progression and relapse, enabling timely intervention and personalized treatment adjustments.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Additionally, the ability to monitor the plasticity of tumor cell states and the expression of disialoganglioside 2 (GD2) immunotherapy targets provides critical insights into therapy resistance and potential relapse.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
This approach not only enhances the precision of current MRD detection but also supports the optimization of immunotherapy by identifying appropriate targets on rare, therapy-resistant cells (in 5/108 patients).
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Overall, these advances in MRD detection and monitoring could significantly improve patient outcomes by facilitating tailored treatment strategies and earlier interventions.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
It will be important to assess the impact on patient survival prospectively in future studies.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Liquid biopsies are minimally invasive and allow for frequent and less burdensome disease diagnosis, prognosis and monitoring of cancer .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
A variety of liquids and analytes have demonstrated benefits in preclinical and clinical studies, e.g. tumor-specific mRNA, circulating or disseminated tumor cells, therapeutic targets or circulating cell-free DNA in bone marrow, blood, urine and other body fluids [3–5].
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Bone marrow dissemination is frequently found in adult and pediatric cancers, such as breast cancer, prostate cancer, sarcomas and neuroblastoma, and is associated with poor prognosis in patients [6–8].
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Therefore, monitoring disease in bone marrow liquid biopsies is critical for assessing therapeutic targets, therapy response and imminent disease recurrence.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Neuroblastoma is the most common extra-cranial solid tumor in children and patient outcomes largely depend on risk-group allocation according to international guidelines .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
High-risk patients are characterized by widely metastatic disease, including bone marrow involvement, genetic alterations (such as MYCN oncogene amplification), older age at initial diagnosis and other clinical parameters .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Despite intensive multi-modal treatment in international study groups, less than 50% of patients survive [10–12].
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
At diagnosis, more than 90% of high-risk patients present with bone marrow infiltration and the majority of recurrent disease originates in the bone marrow, often deriving from a clone that was already present at time of diagnosis [15–17].
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
The current standard-of-care for assessing bone marrow disease advises to obtain two bone marrow aspirates and two trephine biopsies, one each from the right and left iliac crest .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Bone trephine biopsies are histologically analyzed for tumor cells and bone marrow aspirates are assessed by cytomorphological staining of smears followed by visual inspection, which yields limited sensitivity and specificity.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Yet, around 60% of patients assumed to be in complete remission by standard-of-care diagnostics experience recurrent disease, indicating that minimal residual disease (MRD) remains undetected .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Thus, there is a need for more sensitive and specific techniques to detect MRD in bone marrow that are also less labor-intensive and easy to standardize.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
The effective quantification of tumor cells and therapeutic targets in the bone marrow is further challenged by the plasticity of neuroblastoma cells, which can assume adrenergic (ADRN) and mesenchymal (MES) cell states [20–22].
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
These cell states are characterized by distinct gene expression profiles and the activity of gene regulatory networks, such as (high) expression of paired-like homeobox 2b (PHOX2B), tyrosine hydroxylase (TH), cholinergic receptor nicotinic alpha 3 (CHRNA3) and growth-associated protein 43 (GAP43) in ADRN cells, and periostin (POSTN) and paired related homeobox 1 (PRRX1) in MES tumor cells .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
MES neuroblastoma cells have been implicated in therapy resistance, at least in vitro and MES-related gene expression signatures are enriched in tumors during induction chemotherapy and in patients who experience relapse .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
In vitro, the MES cell state also presents with low or absent levels of disialoganglioside 2 (GD2) and CD56 (neural cell adhesion molecule (NCAM)), both targets for immunotherapies , likely leading to therapy escape.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Anti-GD2 antibody therapy has been the standard-of-care maintenance therapy for patients with high-risk neuroblastoma for the past decade and has led to a significant increase in event-free and overall survival .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
However, anti-GD2 antibody therapy has been administered without stratifying for GD2 levels, which might explain the limited response in some patients .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Therefore, the assessment of MES cell states and the abundance of immunotherapy targets might be essential for accurately evaluating immunotherapy efficacy.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
While multiparameter flow cytometry is a sensitive method for assessing CD56 and GD2 expression and quantification for treatment purpose, techniques that enable more precise evaluation of tumor cell states and therapeutic targets are needed [27–29].
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
We have previously developed highly sensitive and specific assays to quantify (1) disseminated tumor cells (DTCs) in bone marrow aspirates, (2) GD2 and CD56 (NCAM) cellular levels and (3) ADRN- and MES-type tumor cells based on expression of specific mRNAs .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Automatic immunofluorescence plus interphase fluorescence in situ hybridization (iFISH) (AIPF), developed by Méhes et al .,
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
allows the exact quantification of DTCs in the bone marrow using an automated microscopy imaging platform and unambiguous genetic verification.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
This technique sensitively detects up to one DTC in one million healthy bone marrow cells of neuroblastoma patients .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Another technique that can very sensitively detect tumor-derived mRNA in the bone marrow of patients with neuroblastoma is reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) [19, 20, 31–40].
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
This technique reliably detects tumor cells with a sensitivity of one in one million normal nucleated bone marrow cells .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
We have previously designed two panels of neuroblastoma-mRNA markers for highly sensitive MRD monitoring in bone marrow: an ADRN- and a MES mRNA-panel .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
In this prospective study across two centers, the main aim was to evaluate quantitative MRD in patients with high-risk neuroblastoma using AIPF and RT-qPCR against the standard-of-care, i.e. cytomorphological and histological assessment.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Second, in the same sequentially collected bone marrow samples, we determined ADRN and MES cell states as well as GD2 and CD56 (NCAM) cellular expression.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Furthermore, we evaluated and reported quality assessment and key performance parameters of these diagnostic tests, which will facilitate their rapid implementation as new standard-of-care.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Bone marrow samples from high-risk neuroblastoma patients enrolled in the SIOPEN/HR-NBL1 (NCT01704716) or Dutch DCOG NB2009 (NCT01704716) trials were prospectively collected at diagnosis and during treatment between 2018 and 2022 (Supplemental Fig. 1).
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
The study was approved by the Medical Research Ethics Committees of the Academic Medical Center (Amsterdam, the Netherlands; MEC07/219#08.17.0836) and the Medical University of Vienna (Vienna, Austria; EK#1853/2016, EK#1216/2018), following the Declaration of Helsinki, with written informed consent obtained from parents/guardians .
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Patients were diagnosed and staged per the International Neuroblastoma Staging System (INSS) , with high-risk patient cases defined as stage 4 patients over one year old or any stage with MYCN amplification.
PMC12317575
Sensitive detection of minimal residual disease and immunotherapy targets by multi-modal bone marrow analysis in high-risk neuroblastoma – a multi-center study
Bilateral bone marrow aspirates were obtained from left and right iliac crests and collected in ethylenediaminetetraacetic acid (EDTA) tubes.