L. Frydrych1, L. H. Maguire2, P. Ulintz3, A. Bankhead4,8, J. K. Greenson5, C. J. Sifuentes3, E. Fearon6,7, K. M. Hardiman2 1University Of Michigan,Department of Surgery,Ann Arbor, MI, USA 2University Of Michigan,Colorectal Surgery,Ann Arbor, MI, USA 3University Of Michigan,Bioinformatics Core,Ann Arbor, MI, USA 4University Of Michigan,Biostatistics,Ann Arbor, MI, USA 5University Of Michigan,Pathology,Ann Arbor, MI, USA 6University Of Michigan,Internal Medicine,Ann Arbor, MI, USA 7University Of Michigan,Human Genetics,Ann Arbor, MI, USA 8University Of Michigan,Computational Medicine And Bioinformatics,Ann Arbor, MI, USA
Introduction: Recommendations for treatment of locally advanced rectal cancer include chemotherapy, radiation and surgery. Response to neo-adjuvant therapy is variable. We have previously shown rectal cancers are made up of multiple genetically distinct sub-clones. Unique sub-clones within primary tumors may harbor mutations which contribute to treatment resistance, relapse, and inter-patient variations in response to standard-of-care neo-adjuvant therapy. Analysis of the influence of neoadjuvant therapy on the molecular evolution of rectal cancer may provide insight about mechanisms of resistance and highlight new therapeutic approaches for patients.
Methods: Rectal cancer patients with an indication for neo-adjuvant chemoradiotherapy were identified at a tertiary referral center. Primary tumor biopsies from multiple discrete geographic locations were obtained prior to treatment. At the time of surgical intervention after standard-of-care chemoradiotherapy, tissue from the treated tumor was obtained and analyzed. Pre- and post- treatment specimens were subjected to whole exome sequencing followed by confirmatory deep sequencing to define somatic mutations. Copy number variation was assessed in all samples using Oncoscan SNP arrays. Genomic data were analyzed using Pyclone to identify sub-clonal tumor populations that differed in frequency after neo-adjuvant chemoradiotherapy. Recurrent drivers of resistance were identified in the sub-clones across tumors. Using Hotnet2, pathway analysis was performed on integrated resistant gene mutation and copy number alteration data.
Results: 32 samples were obtained from 10 patients. Pyclone identified 2-9 individual subclonal populations per tumor. Neoadjuvant therapy resulted in substantial change in the relative proportions of individual sub-clones within the primary tumor. Resistant sub-clones recurrently (>30%) contained mutations in TP53, APC, ABCA13, ITIH5, MUC16, PTEN, THSD4, and TNS1. Recurrent copy number variations for selected chromosome regions were seen in cancers after therapy. Pathway analysis revealed significantly altered pathways in resistant tumor specimens associated with RNA splicing and processing, regulation of transcription, APC-mediated pathways, mTOR signaling, fatty acid biosynthesis, and histone modification.
Conclusion: Intra-tumoral heterogeneity is evident in pre-treatment rectal cancer and sub-clonal populations are selectively modified by treatment. Resistant sub-clones demonstrate high frequencies of somatic mutation in multiple known tumorigenesis driver genes, and intra-tumoral heterogeneity persists post-treatment. Our analyses reveal complex and dynamic genomic architectures in pre- and post-treatment rectal cancers. These data indicate that novel treatment strategies must take into account a changing and heterogeneous tumor mutational profile when attempting to target rectal cancers.