Genomic Evolution and Acquired Resistance to Pre-Operative Chemoradiation Therapy in Locally Advanced Rectal Cancer

Abstract

Pre-operative CRT is the standard for locally advanced rectal cancers. However, at the time of surgery, patients have mixed responses with approximately 20% achieving a pathologic complete response (CR) and 20-40% having little to no response (NR). Pathologic downstaging after CRT is associated with improved outcomes but molecular predictors of response are incompletely understood. The long-term goal of this project is to characterize somatic mutations and derive mutational signatures distinguishing CR vs NR through whole exome sequencing analysis in pre- and post-CRT samples. In addition, we seek to describe the evolution of mutational load in tumors undergoing CRT, which may have implications for the role of immune checkpoint inhibitor therapies. Locally advanced rectal cancer patients treated with neoadjuvant CRT were identified. DNA extraction from clinical FFPE samples and whole exome sequencing was performed on pre- and post-treatment tumor and matched germline DNA followed by standard mutation analysis pipelines to identify somatic mutations, insertion/deletions, and copy number variations (CNV). We identified 40 pre- and post-CRT matched tumor samples from 20 patients. All were uniformly treated with pre-operative fluoropyrimidine-based CRT (50.4 Gy) followed by surgery within 8-10 weeks. Ten patients were classified as NR while 10 patients were partial or complete responders (PR/CR) at the time of surgery. In our initial cohort, all FFPE samples (n = 12) were successfully sequenced and passed quality control metrics. Mean somatic mutational burden pre-CRT was 7.26 mutations/Mb (range, 5.37-9.85), which is consistent with prior reports for colorectal tumors. Unexpectedly, mean somatic mutational burden in the post-CRT samples was not increased (mean, 6.40 mutations/Mb; range, 3.22-10.2) even though 83% of samples were from potentially aggressive tumors harboring mutations in the KRAS oncogene. Among the NR cases, 75% had concurrent KRAS/TP53 mutations. This observation is consistent with our recent findings on the radioresistance of this tumor genotype in lung and colon cancer patients, which confirms the usefulness of the current study cohort. However, our sample size is currently too small for meaningful statistical comparisons. Preliminary CNV analysis showed no major chromosomal changes between pre- and post-CRT samples. We have established feasibility and a pipeline for performing whole exome sequencing on FFPE pre- and post-CRT rectal samples. Our initial, KRAS mutation-enriched cohort demonstrates no significant change in mutational burden between pre- and post-CRT samples, suggesting that the evolution of treatment resistant subclones is not through hypermutation.