Evaluating TMB Compared to tDNA TMB in Recurrent/Metastatic Head and Neck Squamous Cell Carcinoma

High tumor mutation burden (TMB) is connected to immune checkpoint inhibitor (ICI) response in many solid tumors. A team of researchers evaluated TMB from whole-exome sequencing (WES) of cfDNA as an alternate for tDNA TMB in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) patients treated with Cetuximab & Nivolumab(C+N). Dr. Christine Chung, Chair of the Department of Head and Neck-Endocrine Oncology presented the research findings at the 2023 ASCO Annual Meeting.

The team collected archived formalin fixed parrafin embedded tumors, serially collected plasma from 1.pre-treatment, 2. on-treatment, 3. And at time of disease progression/ end of treatment, peripheral mononuclear blood cells, and clinical data from R/M HNSCC patients treated with C+N from a completed clinical trial (NCT03370276). Nucleic acids were extracted using AllPrep DNA/RNA Mini Kit (Qiagen). Libraries were created using KAPA HyperPrep Kit with Library Amplification (KK8504) and IDT’s duplex UMI adapters. Using the IDT’s XGen hybridization kit, Hybridization and capture were performed. Whole exome sequencing was performed using Illumina NovaSeq S4 flowcells (paired 151bp runs). Point mutation calling, filtering, CNV calling, and purity and ploidy estimation were achieved using the Getz Lab CGA WES Characterization pipeline. TMB for each sample was calculated based on the number of mutations with cancer cell fraction > 0.75 as determined by ABSOLUTE and normalized to the 35Mb targeted for exome capture.

The number of cohorts sequenced included 88 tDNA, 226 cfDNA, and 30 normal DNA (nDNA) from 82. Sequencing data from 13 matched tDNA/cfDNA/nDNA samples and 34 matched cfDNA/nDNA samples with sufficient tumor purity were available for analyses. The median TMB was 2.4 mut/Mb (range 0.46-17.5) in tDNA and 1.8 mut/Mb (range 1.1-12.6) in cfDNA. There was a high correlation between tDNA/cfDNA pairs in TMB (r = 0.826, p = 1.5x10^-16, Pearson correlation). The median TMBs in tDNA and cfDNA obtained at different time points or treatment course did not differ significantly in 22 patients with at least two cfDNA timepoints (p = 0.26 paired t-test of log-transformed TMB). The results presented no difference between responder (R) vs. non-responder (NR) in cfDNA TMBs (two-sample t-test p = 0.7 between 2 R and 12 NR) and tDNA TMBs (two-sample t-test p = 0.25 between 6 R and 21 NR). However, tDNA TMB was higher in R compared NR in a subset of pts who did not have prior treatment with ICI (two-sample t-test p = 0.05 between 5 R and 2 NR).

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