Abstract 4528: Evaluation of plasma circulating tumor DNA (ctDNA)-based whole genome sequencing (pWGS) and whole exome sequencing (pWES) and concordance with tumor tissue whole exome sequencing (tWES): a pilot study in patients with recurrent or metastatic head and neck squamous cell carcinoma or metastatic urothelial carcinoma
Authors: Cristescu Razvan , Albright Andrew , Townson Steven , Chen Cai , Moreno Blanca Homet , Lerman Nati , Cao Z. Alexander , Peña Carol 
Published: April 2023

Abstract
Background: The genetic heterogeneity and clonal evolution of tumors is hypothesized to influence disease response and acquisition of resistance during treatment. However, comprehensive analysis of the changing tumor molecular landscape to support evaluation of such a hypothesis is often difficult to quantify due to the lack of accessibility to longitudinal biopsy samples in a large number of patients (pts). Longitudinal analyses using pWGS or pWES may allow the delineation of the genetic evolution of tumors and its impact on treatment outcomes. We conducted a pilot study to evaluate the feasibility of obtaining pWGS and pWES data from ctDNA in pretreatment samples from pts enrolled in 2 phase 3 trials that evaluated second-line pembrolizumab versus chemotherapy/standard-of-care therapy: KEYNOTE-040 (recurrent or metastatic head and neck squamous cell carcinoma; NCT02252042) and KEYNOTE-045 (metastatic urothelial carcinoma, NCT02256436). Concordance between pWES and tWES was also evaluated.
Methods: Cell-free DNA was isolated from 1-3 mL of plasma; pWGS (via low-pass WGS) and pWES were performed on samples from pts with available tWES data generated as part of planned tissue biomarker analyses for the respective studies. Tumor burden in ctDNA was estimated from pWES data using single nucleotide variant (SNV)-based maximum somatic allele frequency (MSAF) and from pWGS data using a copy number variation-based measure of mutational burden in the tumor fraction. Concordance between the pWES and tWES mutational spectrum was determined using the Jaccard index. Concordance of key driver somatic events (FGFR3, TP53, and HPV status) was evaluated.
Results: Plasma samples from 47 pts (KEYNOTE-040, n = 24; KEYNOTE-045, n = 23) were analyzed. Tumor burden estimates from pWGS (median coverage, 5.84; range, 4.28-8.18) and pWES (median coverage, 1806; range, 1038-2413) data were highly correlated (Spearman ρ = 0.94). A range of 0 to 956 nonsynonymous SNVs (median, 50) per pt was identified in ctDNA. When the ctDNA tumor burden was high (MSAF ≥5%, observed in two-thirds of pts), a relatively high concordance was observed for individual alterations in TP53, FGFR3, and HPV status (overall agreement: 0.87, 0.97, and 0.96, respectively); between tissue- and plasma-based tumor mutational burden (R = 0.49); and in overall mutational landscape overlap (Jaccard index: median, 0.30; range, 0.002-0.53).
Conclusions: These data show that pWES exhibits reasonably high concordance with tWES when the tumor burden in ctDNA is relatively high and may be informative regarding tumor genomic characteristics. Extensive longitudinal studies are still needed to understand tumor genetic evolution using plasma ctDNA.
Citation Format: Razvan Cristescu, Andrew Albright, Steven Townson, Cai Chen, Blanca Homet Moreno, Nati Lerman, Z. Alexander Cao, Carol Peña. Evaluation of plasma circulating tumor DNA (ctDNA)-based whole genome sequencing (pWGS) and whole exome sequencing (pWES) and concordance with tumor tissue whole exome sequencing (tWES): a pilot study in patients with recurrent or metastatic head and neck squamous cell carcinoma or metastatic urothelial carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4528.