Neoadjuvant chemoradiation alters biomarkers of anticancer immunotherapy responses in locally advanced rectal cancer

Neoadjuvant chemoradiation therapy (CRT) is a widely used preoperative treatment strategy for locally advanced rectal cancer (LARC). However, a few studies have evaluated the molecular changes caused by neoadjuvant CRT in these cancer tissues. Here, we aimed to investigate changes in immunotherapy-related immunogenic effects in response to preoperative CRT in LARC. We analyzed 60 pairs of human LARC tissues before and after irradiation from three independent LARC cohorts, including a LARC patient RNA sequencing (RNA-seq) dataset from our cohort and GSE15781 and GSE94104 datasets. Gene ontology analysis showed that preoperative CRT significantly enriched the immune response in LARC tissues. Moreover, gene set enrichment analysis revealed six significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways associated with downregulated genes, including mismatch repair (MMR) genes, in LARC tissues after CRT in all three cohorts. Radiation also induced apoptosis and downregulated various MMR system-related genes in three colorectal cancer cells. One patient with LARC showed a change in microsatellite instability (MSI) status after CRT, as demonstrated by the loss of MMR protein and PCR for MSI. Moreover, CRT significantly increased tumor mutational burden in LARC tissues. CIBERSORT analysis revealed that the proportions of M2 macrophages and CD8 T cells were significantly increased after CRT in both the RNA-seq dataset and GSE94104. Notably, preoperative CRT increased various immune biomarker scores, such as the interferon-γ signature, the cytolytic activity and the immune signature. Taken together, our findings demonstrated that neoadjuvant CRT modulated the immune-related characteristics of LARC, suggesting that neoadjuvant CRT may enhance the responsiveness of LARC to immunotherapy. Formalin-fixed paraffin-embedded (FFPE) block specimens from preoperative biopsy via sigmoidoscopy and surgical resection of the primary tumor were obtained from the 11 pairs of human locally advanced rectal cancer. Total RNA was extracted using TRIzol RNA Isolation Reagent (Life Technologies) from the samples. The quantity and quality of the total RNA were evaluated using an Agilent 2100 bioanalyzer RNA kit. The isolated total RNA was processed for preparation of an RNA-seq library using an Illumina TruSeq Stranded mRNA Sample Preparation kit according to the manufacturer’s protocol. The quality and size of libraries were assessed using an Agilent 2100 bioanalyzer DNA kit. All libraries were quantified by quantitative real-time PCR using a CFX96 Real Time System and sequenced on NextSeq500 sequencers with a paired-end 76 bp plus single 6 bp index read run. For quality control, low-quality bases and adapter sequences were trimmed from the raw sequencing reads using Trimmomatic software with default parameters, except that the minimum length of reads to be dropped was 38 bp. Trimmed sequencing reads were mapped to the hg38 human reference genome using STAR aligner with default parameters. Reads per gene was counted simultaneously by STAR using the ‘--quantMode GeneCounts’ parameter. Count normalization were performed DESeq2.