Abstract 5593: Clonal mutation burden and evolutionary dynamics analysis in metastatic gastro-esophageal adenocarcinoma (GEA) by error corrected whole-exome circulating tumor DNA sequencing
Authors: McCafferty Neil , Fong Caroline , Barber Louise J. , Woolston Andrew , Kleftogiannis Dimitrios , Rana Taqia , Cromarty Susan , Kidd Shannon , Begum Ruwaida , Chau Ian , Starling Naureen , Cunningham David , Gerlinger Marco 
Published: June 2023

Abstract
Introduction: The evolution of metastatic cancers over time can be assessed in circulating tumor DNA (ctDNA) but sequencing and bioinformatics tools for ultra-deep ctDNA whole-exome sequencing (WES) analysis are lacking.
Methods: We developed ctDNA WES that only requires 15ng DNA to achieve sequencing depths of 1000-2000x. Error correction with molecular barcodes and duplex DNA detection allowed calling of mutations ≥0.5% variant frequency (VF). This pilot study applied ctDNA WES to plasma from 20 EGA patients (pts) and standard WES to matched biopsies in order to assess ctDNA WES performance and whether clonal mutation burden (cMB), a critical immunotherapy biomarker and important for neoantigen vaccine designs, differed between ctDNA and biopsies. We furthermore established a mutation and copy number data analysis pipeline using Bayesian clustering to define subclones and track their evolution during therapy in 3pts.
Results: The median age of pts was 71y, 95% had distant metastases and 5% locally advanced EGAs. At a median sequencing depth of ????x after de-duplication, VFs of mutations in pre-treatment ctDNA was low (&lt;2% VF) in 8 pts, and intermediate (2-10%) to high (&gt;10%) in 12. Whether cMB differed in biopsies vs ctDNA was assessed in 7 pts with high VFs in ctDNA and good cancer purity in matching biopsy WES. The median cMB was 82 in biopsies and 111 in ctDNA. The increase was driven by 3 cases with 30%, 35% and 59% higher cMB in ctDNA vs biopsies. In two pts, most mutations that only appeared clonal in ctDNA were subclonal rather than absent in the primary tumor, indicating the subclonal presence of the metastasis progenitor clone. Subclonal intermixing in primary tumors hence limits the accurate identification of mutations that are clonal in metastatic disease. Evolutionary dynamics analyses in 3 pts who had good responses to chemotherapy before progression showed a major clonal sweep in one, supporting monoclonal resistance, and evolution of small subclones in two, indicating polyclonal resistance. A MEK1 K57T mutation evolved at resistance in a HER2 amplified EGA treated with trastuzumab+chemotherapy, demonstrating the utility to identify mechanisms of acquired resistance.
Conclusions: ctDNA WES can assess the genetics of entire metastatic cancer cell populations over time and deconvolute their evolutionary trajectories. 43% pts had higher cMB in ctDNA compared to biopsies. Liquid biopsy analyses by WES may be superior to tissue-based WES for mutation burden analysis and neoantigen vaccine designs. ctDNA WES identified distinct evolutionary modes of resistance. A larger cohort is being analyzed to define how these differ clinically and whether they can be predicted from pre-treatment ctDNA WES. Efforts to increase the ctDNA WES sensitivity are ongoing as 8 pts had low ctDNA VFs which increased false negative rates.
Citation Format: Neil McCafferty, Caroline Fong, Louise J. Barber, Andrew Woolston, Dimitrios Kleftogiannis, Taqia Rana, Susan Cromarty, Shannon Kidd, Ruwaida Begum, Ian Chau, Naureen Starling, David Cunningham, Marco Gerlinger. Clonal mutation burden and evolutionary dynamics analysis in metastatic gastro-esophageal adenocarcinoma (GEA) by error corrected whole-exome circulating tumor DNA sequencing. [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 5593.