P05.12.B ULTRASENSITIVE INTEGRATED GENOMIC-EPIGENETIC CTDNA CNS TUMOR ASSAY USING TET-ASSISTED PYRIDINE BORANE SEQUENCING (TAPS)

Abstract BACKGROUND Liquid biopsy analyzing cell free tumor DNA (ctDNA) from Cerebrospinal Fluid (CSF) is a feasible and relatively low-risk alternative to a tissue biopsy for diagnosis of Central Nervous System (CNS) tumors. CNS liquid biopsy is challenging due to molecular and clinical diversity of primary CNS tumors, the multitude of type of metastatic tumors and molecular drivers, and the lack of assays that distinguish tumors from non-neoplastic diseases. An integrated epigenetic and genomic analysis could provide simultaneous DNA methylation classification with mutational, copy number and gene fusion analysis. Current methylation techniques lead to DNA loss due to bisulfite conversion destruction. Enzymatic 5-base chemistries enable simultaneous high throughput genomic analysis and DNA methylation classification without DNA loss. MATERIAL AND METHODS We analyzed ctDNA from 60 CSF samples from patients with primary and metastatic CNS tumors. Library preparation was performed using TET-assisted pyridine borane sequencing (TAPS) chemistry (Watchmakers Genomics). Libraries were sequenced on the NOVASeq 2000 with 500 mil reads/sample. Fastq files were processed using NF-CaseReports pipeline, and coverage bias corrected using fragCounter and rPCA denoising. Methylation signals were extracted using Rastair, followed by integration with the Infinium MethylationEPIC v2.0 array probe set, facilitating downstream clustering analyses and clinical DNA methylation Classifier. Copy number analysis was performed with CBS, and structural variant, using GRIDSS. RESULTS TAPS provides comprehensive genomic landscape analysis including mutation, genome-wide and focal copy number analysis, and gene fusions and DNA methylation classifier in the same assay. For example, we detected 1p19q losses in oligodendroglioma (N=3), trisomy of chromosome 7 (N=7) and loss of chromosome 10 (N=8), as well as high level amplifications of EGFR, MET in GBM (N=11) and ERBB2 (Her2) in metastatic breast cancer. Mutational analysis clearly classified IDH mutant (N=15) and wild-type (N=25) gliomas, based on detection of IDH1/2, ATRX, TP53 and TERT promoter mutations, amplifications, and CDKN2A/B, PTEN loss. Structural variant analysis detected EGFRvIII (N=3) and MET fusions (N=1) in GBM. Furthermore, TAPS enabled targeted analysis of the MGMT promoter concordant with methylation array. In pediatric tumors we detected MN1: : CXXC5 fusion in a case of astroblastoma and isochromosome 17 in Group 4 medulloblastoma, notably a tumor lacking somatic mutations. In 13 cases (21%), we did not detect any informative mutations or epigenetic changes, likely due to low level of CSF ctDNA. CONCLUSION Integrated genomic-epigenetic ctDNA analysis using TAPS chemistry is highly sensitive liquid biopsy approach that provides simultaneous genetic and epigenetic analysis from CSF ctDNA and requires minimum ctDNA input.