Analytical Validation of Quantitative Polymerase Chain Reaction and Belay AscentTM Low-Pass Whole Genome Sequencing to Report on Gene Copy Number Variants in Cerebrospinal Fluid Tumor-Derived DNA
Authors:
Viriya Keo, Sakshi Khurana, Vindhya Udhane, Alexandra Larson, Jennifer N. Adams, Daniel Sanchez, Tarin Peltier, Anthony Acevedo, Kathleen Mitchell, Kala F. Schilter, Qian Nie and Honey V. Reddi
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 Belay Ascent 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 ERBB2 (erb-b2 receptor tyrosine kinase 2) was demonstrated to be 100% and that of EGFR (epidermal growth factor receptor) was 83%, with specificities of 96% and 100%, respectively. The analytical sensitivity of qPCR to detect gene deletions for CDKN2A/2B (cyclin-dependent kinase inhibitor 2A/2B) was 60% and that for MTAP (methylthioadenosine phosphorylase) was 100% with a specificity of 100% for all three genes. Belay Ascent 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 Belay Ascent 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.
Figure 1: Analytical validation of using quantitative PCR to report on gene-level copy number variants (CNVs) in cerebrospinal fluid (CSF) tumor-derived DNA (tDNA) and establishing concordance/equivalence of Belay Ascent low-pass whole genome sequencing (LP-WGS) data to detect focal gene CNVs—schema and specimen cohort.
Figure 2: Representative ichor plots of gene-level amplifications and deletions for 5 genes validated by qPCR for amplifications (EGFR—top panels, ERBB2—middle panels) and deletions (CDKN2A/2B, MTAP—bottom panels). Left panels (all plots)—normal gene copy (no amplification or deletion). Right panels—amplifications (seg.mean > 0.1) and deletions (seg.mean ≤ −0.2). Dotted lines on bottom panels outline locations of MTAP and CDKN2A genes.
