Differential and longitudinal immune gene patterns associated with reprogrammed microenvironment and viral mimicry in response to neoadjuvant radiotherapy in rectal cancer.

Background: Rectal cancers show a highly varied response to neoadjuvant radiotherapy/chemoradiation (RT/CRT) and the impact of the tumor immune microenvironment on this response is poorly understood. Current clinical tumor regression grading systems attempt to measure radiotherapy response but are subject to interobserver variation. An unbiased and unique histopathological quantification method (change in tumor cell density (ΔTCD)) may improve classification of RT/CRT response. Furthermore, immune gene expression profiling (GEP) may identify differences in expression levels of genes relevant to different radiotherapy responses: (1) at baseline between poor and good responders, and (2) longitudinally from preradiotherapy to postradiotherapy samples. Overall, this may inform novel therapeutic RT/CRT combination strategies in rectal cancer.

Methods: We generated GEPs for 53 patients from biopsies taken prior to preoperative radiotherapy. TCD was used to assess rectal tumor response to neoadjuvant RT/CRT and ΔTCD was subjected to k-means clustering to classify patients into different response categories. Differential gene expression analysis was performed using statistical analysis of microarrays, pathway enrichment analysis and immune cell type analysis using single sample gene set enrichment analysis. Immunohistochemistry was performed to validate specific results. The results were validated using 220 pretreatment samples from publicly available datasets at metalevel of pathway and survival analyses.

Results: ΔTCD scores ranged from 12.4% to -47.7% and stratified patients into three response categories. At baseline, 40 genes were significantly upregulated in poor (n=12) versus good responders (n=21), including myeloid and stromal cell genes. Of several pathways showing significant enrichment at baseline in poor responders, epithelial to mesenchymal transition, coagulation, complement activation and apical junction pathways were validated in external cohorts. Unlike poor responders, good responders showed longitudinal (preradiotherapy vs postradiotherapy samples) upregulation of 198 immune genes, reflecting an increased T-cell-inflamed GEP, type-I interferon and macrophage populations. Longitudinal pathway analysis suggested viral-like pathogen responses occurred in post-treatment resected samples compared with pretreatment biopsies in good responders.

Conclusion: This study suggests potentially druggable immune targets in poor responders at baseline and indicates that tumors with a good RT/CRT response reprogrammed from immune "cold" towards an immunologically "hot" phenotype on treatment with radiotherapy.