Abstract 7019: Identifying cell free DNA methylation signatures in cerebrospinal fluids for the early detection of brain metastasis in non-small cell lung cancer

Abstract Brain metastasis (BM) is major complication for adult cancer patients. Up to 2% of all cancer patients develop BM. Non-small cell lung cancer (NSCLC) contributes up to 55% of all BM. Approximately 15-20% of NSCLC patients show metastases at diagnosis, and approximately half will develop BM during their clinical course. Even after treatments like surgery, radiation and immunotherapy, the prognosis is dismal with median overall survival less than 13 months. No standard screening or early diagnostic testing exists for BM now. There is an urgent need to identify genomic features that predispose towards brain metastasis - these biomarkers could be used for early detection and disease monitoring. The cfDNA methylation and fragmentation patterns provide potential epigenetic biomarkers for detecting cancer. These patterns are specific to cell/tissue origin and cancer types. DNA methylation refers to a chemical modification of a specific carbon with an added methyl group in the cytosine. Hypomethylation in the gene body and hypermethylation in the promoter region lead to cancer development. We hypothesize that there are specific cfDNA methylation and fragmentation signatures in CSF that reflect the epigenetic states of BMs, and cancer predisposition for brain metastasis. We applied a novel single molecule sequencing approach to study CSF cfDNA methylation of BM from NSCLC. Our cohort consisted of ten patients with NSCLC-BM (samples N=14) and eleven healthy controls (N=11). Briefly, extracted cfDNA was obtained from CSF samples. Multiplexed sequencing libraries were prepared following an optimized protocol, and sequenced on PromethION flow cell for 72hr by Oxford nanopore technologies. Data were processed with the onboard base and methylation calling, followed by our bioinformatic pipelines for cfDNA size and methylation analysis. Distinct methylation patterns were found on significant CpG sites (fdr-corrected p0. 01) between cancer patients and controls, in both gene-body and promoter region. Biological pathway analysis showed enrichment in essential functions involving PI3K, ERBB2, TNF-α signaling (p0. 03), all of which play a role in NSCLC. Enriched mono-nucleosome levels (p = 1. 07e-03) and differentiated mono-/tri- nucleosome ratios (p = 1. 34e-03) were found in cancer vs. controls. Overall, our results suggest that a combination of cfDNA methylation and fragmentation can detect NSCLC-BM. This study will benefit BM patients with early detection and better prognosis, and extend our understanding in brain metastases in NSCLC. Citation Format: Tianqi (Kiki) Chen, Billy T. Lau, Xiangqi Bai, Melanie Hayden Gephart, Hanlee P. Ji. Identifying cell free DNA methylation signatures in cerebrospinal fluids for the early detection of brain metastasis in non-small cell lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 7019.