Journal of Molecular Pathology · Published 2026-05-12 · DOI 10.3390/jmp7020018
Viriya Keo, Sakshi Khurana, Vindhya Udhane, Alexandra Larson, Jennifer N. Adams, Daniel Sanchez, Tarin Peltier, Anthony Acevedo, Kathleen Mitchell, Kala F. Schilter, Qian Nie, Honey V. Reddi
Background: Evaluation of gene-level copy number variants (CNVs) for diagnosis and therapeutic decision making has become standard of care with next-generation sequencing (NGS), immunohistochemistry (IHC), and/or fluorescence in situ hybridization (FISH) being used to detect gene amplifications/deletions in tumor tissue. In contrast to most solid tumors, CNS cancers are challenging to evaluate by resection and/or biopsy due to the associated risks with invasive brain surgery that can also result in death or associated morbidity and therefore alternate methods are required.Methods: This study presents the analytical validation of using quantitative PCR (qPCR) to detect gene CNVs directly from cerebrospinal fluid (CSF)-derived DNA and from the Ascent<sup>TM</sup> low-pass whole genome sequencing (LP-WGS) libraries, demonstrating concordance with the gold standard of NGS/IHC/FISH used in tumor tissue. Results: The analytical sensitivity of qPCR to detect gene amplification calls for <i>ERBB2</i> (erb-b2 receptor tyrosine kinase 2) was demonstrated to be 100% and that of <i>EGFR</i> (epidermal growth factor receptor) was 83%, with specificities of 96% and 100%, respectively. The analytical sensitivity of qPCR to detect gene deletions for <i>CDKN2A/2B</i> (cyclin-dependent kinase inhibitor 2A/2B) was 60% and that for <i>MTAP</i> (methylthioadenosine phosphorylase) was 100% with a specificity of 100% for all three genes. Ascent<sup>TM</sup> was demonstrated to have a higher sensitivity (100%) when compared to qPCR for the same genes evaluated and demonstrated 100% positive agreement and 100% negative agreement with known CNV status. Conclusions: The results demonstrate that given the paucity of cells in CSF limiting the use of IHC and FISH, qPCR and Ascent<sup>TM</sup> provide highly sensitive, novel, minimally invasive methods for the evaluation of gene copy number (CN) status to inform the diagnosis and management of CNS cancers.
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Keo, V., Khurana, S., Udhane, V., et al. (2026). Analytical Validation of Quantitative Polymerase Chain Reaction and AscentTM Low-Pass Whole Genome Sequencing to Report on Gene Copy Number Variants in Cerebrospinal Fluid Tumor-Derived DNA. Journal of Molecular Pathology. https://doi.org/10.3390/jmp7020018