Molecular Profiling of Serially Collected Cerebrospinal Fluid Uncovers Therapy-Resistant Subclones in Recurrent Leptomeningeal Metastatic Disease

Abstract Leptomeningeal metastatic disease (LMD) is the spread of cancer cells to the cerebrospinal fluid (CSF)-filled spaces surrounding the central nervous system. After symptom onset, LMD patients survive for only weeks to months while enduring devastating neurologic symptoms. Current clinical tools to diagnose LMD, such as CSF cytology and magnetic resonance imaging, do not detect mutations susceptible to treatment, quantitatively monitor response to therapy, or identify mutation-based resistance mechanisms. Here, we sought to profile LMD using next-generation sequencing by studying CSF-derived cell-free DNA (CSF-cfDNA) from 9 breast cancer and 5 lung cancer patients with LMD and 4 LMD-negative controls. To conduct an unbiased search for somatic mutations, we used a custom-designed gene panel and our validated True2 sequencing workflow with a specificity of <1 false positive per 100kb panel positions and a 97.6% sensitivity for detecting single nucleotide variants (SNVs) with a variant allele frequency (VAF) ≥0.1%. Although CSF-cfDNA quantity ranged widely (<1 to 91 ng), genomic alterations were observed in 100% of LMD patients vs. none in the controls. In 13 patients with a SNV, an average of 4.5±3.3 variants were detected with a VAF ranging from 0.051% (HER2 p.S1151L associated with a 12-fold amplification and read depth of 25,542X) to 98.7% (PTEN p.Q733X associated with a loss of heterozygosity). Importantly, subclones in cancer driver genes were detected in 10 of 13 (76.9%) samples consistent with a high prevalence in LMD. Somatic mutations in genes common to both cancers were discovered (e.g., TP53, KMT2D, and EGFR), while other affected genes segregated based on cancer type (e.g., PIK3CA, ATM, and CDH1 in breast cancer; PTEN, MTOR and PDGFRA in lung cancer). An amplification or deletion was detected in 10 of 14 patients (71.4%), a subset of which segregated by cancer type (e.g., HER2 and ESR1 amplifications in breast cancer; CDKN2A and TP53 codeletions in lung cancer). Using serially collected CSF samples, we observed: (1) loss of subclones in response to therapy followed by resurgence at clinical recurrence showing suppression of disease but not eradication; (2) multiple subclones in the same gene supporting evidence of convergent evolution; and (3) emergence of new subclones at clinical recurrence consistent with mutation evolution in response to therapy. For example, in a breast cancer patient with a known HER2 amplification receiving intrathecal Trastuzumab, a previously absent HER2 p.V777L mutation was present at the end of therapy with a VAF of 18.6% which increased at clinical recurrence to 94.8% in association with a 2.4-fold HER2 amplification indicating the mutated HER2 subclone was already therapy-resistant. Our findings support a panel-based approach to detect and monitor LMD-derived somatic mutations using CSF-cfDNA. Moreover, the untargeted search for somatic mutations via True2 has the potential to guide personalized, adaptive therapeutics for LMD patients which may prove essential in improving patient survival. Citation Format: Yingqi Zhang, Rachna Malani, Charlie S Dean, Laura A Boatz, Brion E Harrison, Mei Wei, Wallace Akerley, Mary P Bronner, Hunter R Underhill. Molecular profiling of serially collected cerebrospinal fluid uncovers therapy-resistant subclones in recurrent leptomeningeal metastatic disease [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B043.