Abstract 2332: Tumor infiltrating lymphocytes, immunoSeq, and CMS classification in the molecular epidemiology of colorectal cancer study
Authors: Melas Marilena , Lazaris Charalampos , Schmit Stephanie L. , Maoz Asaf , Pamplona Rebeca Sanz , Qu Chenxu , Greenson Joel K. , Kuick Rork , Lejbkowicz Flavio , Rennert Hedy S. , Walker Christopher P. , Bowen Chase M. , Silva Diane M. Da , Kast W. Martin , Idos Gregory E. , McDonnell Kevin J. , Moreno Victor , Rennert Gad , Gruber Stephen B. 
Published: August 2019

Abstract
Background: Tumor infiltrating lymphocytes (TILs) are prognostic and predictive biomarkers in colorectal cancer and are associated with improved prognosis and response to immunotherapy. While TILs are routinely assessed by pathologists, a standardized technique (immunoSEQ, Adaptive Biotechnologies) that leverages targeted next-generation sequencing can also be used to quantify and characterize the T-cell receptor (TCR) repertoire of individual colorectal cancers. In a large, population-based study of incident colorectal cancer, the host immune responses were measured by an expert pathologist and ImmunoSEQ to understand the relationships between TILs, TCRs/cell and specific subgroups of colorectal cancer. Methods: Incident cases of adenocarcinoma of the colon or rectum from the Molecular Epidemiology of Colorectal Cancer (MECC) study included 1,000 cancers that were uniformly evaluated for TILs and other histopathologic features by one pathologist. FFPE-derived DNA from microdissected tumor tissue was extracted and sequenced using ImmunoSEQ analysis for the same 1,000 individuals. A resulting quantitative metric from this assay includes TCRs/cell, a measure of rearranged T cell quantity relative to all nucleated cells in a tumor sample. Gene expression in snap-frozen tissue available from 342/1,000 MECC colorectal cancers was measured with Affymetrix Human Genome U133 Arrays (U133A and U133 Plus2.0) as previously described. CMS classification was performed using the R package 3.5.1, CMS classifier, randomForest 4.6-14. Multivariate analysis assessed CMS by age, gender, TILs/HPF, TCRs/cell, MSI status, BRAF and KRAS mutational status. Results: TILs/HPF and TCRs/cell were significantly correlated among all 1000 cases (r=0.5, p&lt;0.001). Among the 342 cases with available expression profiles, CMS1 constituted 12.0% of all CRC, with CMS2 (41.8%), CMS3 (8.5%), and CMS4 (13.7%) and unclassified (24%) representing the remaining distribution. There were statistically significant differences in the molecular and histopathologic features of colorectal cancers by CMS subgroups. MSI-H tumors were most frequently observed within CMS1 cancers (56.6% of CMS1 were MSI-H), with lower representation among CMS2 (1.5%), CMS3 (10%), CMS4 (3.5%), and unclassified CRC (9.5%) (p&lt;0.0001). In addition, BRAF positive tumors were more frequently observed within the CMS1 group (12.2%, p =0.0065) and KRAS positive tumors within the CMS3 group (31%, p&lt;0.0001). Consistent with prior reports, TILs/HPF were significantly higher in the CMS1 group (mean=7.7, p&lt;0.0001). Similar statistically significant trends were observed across classes for TCRs/cell (mean=0.16, p=0.04). Conclusions: Subtypes of CRC have distinct histopathologic and molecular features that can be distinguished by expression profiles and immunoSEQ.
Citation Format: Marilena Melas, Charalampos Lazaris, Stephanie L. Schmit, Asaf Maoz, Rebeca Sanz Pamplona, Chenxu Qu, Joel K. Greenson, Rork Kuick, Flavio Lejbkowicz, Hedy S. Rennert, Christopher P. Walker, Chase M. Bowen, Diane M. Da Silva, W. Martin Kast, Gregory E. Idos, Kevin J. McDonnell, Victor Moreno, Gad Rennert, Stephen B. Gruber. Tumor infiltrating lymphocytes, immunoSeq, and CMS classification in the molecular epidemiology of colorectal cancer study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2332.