Methodology for consensus sequence determination.

Supplemental Table S2. Number of PCR cycles used during library preparation and panel capture-enrichment.

Supplemental Figure S15. Distribution of allele frequencies for somatic mutations discovered by True2 in tumor and peritumoral edema of adult diffuse gliomas.

Supplemental Figure S5. Effect of using the dbSNP database to remove potential contamination.

Supplemental Table S4. Truth set of single nucleotide variants in HD701 DNA and associated dilutions.

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.

Supplemental Figure S18. Copy number variation (CNV) detection demonstrates differences in CNVs between samples from the same patient.

Supplemental Figure S8. Alternative experimental and in vitro noise reduction strategies without substantive improvement in error.

Supplemental Figure S3. Effects of proper-pair-plus (PP+), alignment quality (MQ), and base quality (BQ) on read depth and false positives.

Supplemental Figure S10. Comparison of True2 sensitivity using reduced sample complexity and iterative captures.

Supplemental Figure S14. Comparison of tumor burden by histology and True2.

Supplemental Figure S2. Preconsensus metrics of sequencing data.

Supplemental Table S1. Alphabetical listing of 115 genes included on the custom-designed, 114 kb, hotspot panel.

Supplemental Figure S13. Relative to the overall median read depth for each sample, the median read depth for the TERT promoter is less compared to other gene-specific regions (IDH1, IDH2, and NF1).

BED file of custom-designed panel used during analysis.

Supplemental Figure S11. Read depth and total paired reads for each tumor and peritumoral sample.

TXT file of positions on custom-designed panel to exclude during analysis because of high error rate.

AbstractAdult diffuse gliomas commonly recur regardless of therapy. As recurrence typically arises from the peritumoral edema adjacent to the resected bulk tumor, the profiling of somatic mutations from infiltrative malignant cells within this critical, unresected region could provide important insights into residual disease. A key obstacle has been the inability to distinguish between next-generation sequencing (NGS) noise and the true but weak signal from tumor cells hidden among the noncancerous brain tissue of the peritumoral edema. Here, we developed and validated True2 sequencing to reduce NGS-associated errors to <1 false positive/100 kb panel positions while detecting 97.6% of somatic mutations with an allele frequency ≥0.1%. True2 was then used to study the tumor and peritumoral edema of 22 adult diffuse gliomas including glioblastoma, astrocytoma, oligodendroglioma, and NF1-related low-grade neuroglioma. The tumor and peritumoral edema displayed a similar mutation burden, indicating that surgery debulks these cancers physically but not molecularly. Moreover, variants in the peritumoral edema included unique cancer driver mutations absent in the bulk tumor. Finally, analysis of multiple samples from each patient revealed multiple subclones with unique mutations in the same gene in 17 of 22 patients, supporting the occurrence of convergent evolution in response to patient-specific selective pressures in the tumor microenvironment that may form the molecular foundation of recurrent disease. Collectively, True2 enables the detection of ultralow frequency mutations during molecular analyses of adult diffuse gliomas, which is necessary to understand cancer evolution, recurrence, and individual response to therapy.Significance:True2 is a next-generation sequencing workflow that facilitates unbiased discovery of somatic mutations across the full range of variant allele frequencies, which could help identify residual disease vulnerabilities for targeted adjuvant therapies.

Supplemental Figure S7. True2 reduces false positives across all variant allele frequency (VAF) categories.

Supplemental Figure S4. Effects of thresholding on false positives.