Optimizing pre-analytical variables for reliable mass spectrometry-based quantification of immunomodulatory proteins in cerebrospinal fluid in pediatric neuro-oncology trials

PROJECT SUMMARY/ABSTRACT The core aim of this study is to systematically investigate and mitigate the impact of preanalytical variables on the accuracy and reliability of targeted, multiple reaction monitoring mass spectrometry (MRM-MS)-based immune protein measurements in cerebrospinal fluid (CSF) from pediatric brain tumor patients participating in clinical trials. Recently, immune therapies such as immune checkpoint inhibitors or intrathecal (i.e., introduced directly into the cerebrospinal fluid) delivery of chimeric antigen receptor T-cell (CAR-T) therapies have shown promise for treating poor prognosis pediatric brain tumors, yielding improved outcomes for some. However, more research is needed to optimize safety, to identify patients most likely to respond, to monitor response in real time, & to understand immune response timing and mechanisms (pharmacodynamics) to develop strategies to increase efficacy. This study will: (i) identify and characterize sources of CSF biospecimen preanalytical variation affecting MRM-based assay performance for quantifying immunomodulatory protein biomarkers in CSF, (ii) develop, validate, and disseminate a preanalytical quality control standard operating protocol (SOP) to counteract preanalytical variation impact on the performance of MRM assays quantifying immunomodulatory proteins in CSF, and (iii) validate the biospecimen SOP through real-world testing by analyzing prospectively collected clinical trial samples at two CLIA (Clinical Laboratory Improvement Amendments) labs to demonstrate harmonization. Tools established through this work will enhance the interpretation of clinical trial outcomes through data reliability & provide valuable insights into the immunological mechanisms underlying the effects of immune-based treatments in pediatric brain tumor patients enrolled in clinical trials. The work also has broader significance for immune analyses of CSF in primary and metastatic adult brain tumors, as well as in neuroinflammatory disorders where immune activation either causes or contributes to the pathogenesis (e.g., multiple sclerosis, Alzheimer disease, Parkinson disease, Huntington's disease, amyotrophic lateral sclerosis, stroke and traumatic brain injuries myelitis).