TAPS chemistry: A novel, ultrasensitive pan-CNS cancer liquid biopsy assay.

e14034 Background: The gold standard of diagnosis for central nervous system (CNS) malignancy is biopsy of the tumor, an invasive procedure that, at times, comes with considerable risk or is not feasible. Treatment of CNS malignancies is now heavily based on the molecular profiling of tumor tissue. Without molecular profiling of the tumor, effective treatment decisions cannot be made. We report our development of a pan-CNS cancer liquid biopsy assay, which interrogates cell-free, tumor-derived DNA (cfDNA) found in cerebrospinal fluid (CSF). Our assay uses a novel chemistry, known as TAPS, which enables ultrasensitive and concurrent base sequencing and DNA methylation profiling. This enables detection of the tumor molecular profile from CSF based on sparse tumor-derived cfDNA with minimally invasive collection methods for CSF. Methods: DNA extraction and library preparation from a CSF sample begins with manual isolation of cfDNA from 1-5mL of CSF/sample, processed no more than 48 hours after sample collection. CfDNA is bound to magnetic nanoparticles, washed, and eluted, and cfDNA content is then quantified. Library preparation is performed using TET-assisted pyridine borane sequencing (TAPS chemistry), effectively converting all methylated cytosine (Cs) to thymine (Ts). Samples are then deep sequenced on the NOVASeq 2000.Our bioinformatics workflow leverages a multimodal approach to process and interpret sequencing data derived from TAPS. We align paired-end reads to the hg38 reference genome using BWA-MEM, followed by quality control, to achieve ~10x coverage. Post-processing steps include fragCounter and Dryclean, which implement GC and mappability bias correction and denoising through rPCA, respectively. Methylation signals are extracted using Rastair, a TAPS-specific module enabling genome-wide DNA methylation profiling. Results: TAPS demonstrates robust performance with high sensitivity for detecting CNS tumor biomarkers, enabling concurrent identification of mutations (IDH1/2, TP53, TERT), copy number aberrations (1p19q loss, EGFR amplification, CDKN2A loss), gene fusions (EGFRvIII, MN1:CXXC5), and targeted methylation analysis (including that of the MGMT promoter) within a single assay. Further, principal component analysis of 32 CSF samples showed clear methylation-based classification of CNS tumors, including glioblastomas, meningiomas, and metastatic carcinomas. Conclusions: Using our novel, ultrasensitive pan-CNS cancer CSF liquid biopsy assay, we are able to identify major drivers of CNS disease, both genomic and epigenetic, enabling minimally invasive diagnosis and longitudinal monitoring of CNS cancers. The approach's low DNA input requirements and minimal destructive effects highlight its potential for clinical implementation, particularly for pediatric and metastatic CNS tumors.