Neoadjuvant Chemoradiation Alters Biomarkers of Anticancer Immunotherapy Responses in Locally Advanced Rectal Cancer

Neoadjuvant Chemoradiation Alters Biomarkers of Anticancer Immunotherapy Responses in Locally Advanced Rectal Cancer

Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2020-001610 on 10 March 2021. Downloaded from Neoadjuvant chemoradiation alters biomarkers of anticancer immunotherapy responses in locally advanced rectal cancer 1 2,3 4 2,5 Incheol Seo, Hye Won Lee, Sang Jun Byun , Jee Young Park, 5 6,7 7 5,7,8 Hyeonji Min, Sung Hwan Lee , Ju- Seog Lee, Shin Kim , Sung Uk Bae8,9 To cite: Seo I, Lee HW, ABSTRACT This therapeutic strategy is associated with Byun SJ, et al. Neoadjuvant Background Neoadjuvant chemoradiation therapy (CRT) downstaging of the tumor, more frequent use chemoradiation alters is a widely used preoperative treatment strategy for of sphincter- preserving procedures by preop- biomarkers of anticancer locally advanced rectal cancer (LARC). However, a few immunotherapy responses in eratively shrinking the tumor, decreased local studies have evaluated the molecular changes caused locally advanced rectal cancer. recurrence and reduced toxicity compared by neoadjuvant CRT in these cancer tissues. Here, we Journal for ImmunoTherapy with postoperative adjuvant chemoradi- aimed to investigate changes in immunotherapy-rela ted of Cancer 2021;9:e001610. ation.1–4 CRT delivers ionizing radiation doi:10.1136/jitc-2020-001610 immunogenic effects in response to preoperative CRT in LARC. directly to target cells, with the goal of causing genetic damage, such as radiation- ► Prepublication history and Methods We analyzed 60 pairs of human LARC tissues additional material is published before and after irradiation from three independent induced DNA double- strand breaks, which online only. To view please visit LARC cohorts, including a LARC patient RNA sequencing are repaired through double-strand break the journal online (http:// dx. doi. (RNA-seq) dataset from our cohort and GSE15781 and repair mechanisms.5 In addition to the direct org/ 10. 1136/ jitc- 2020- 001610). GSE94104 datasets. cytotoxic effects of radiotherapy, this treat- Results Gene ontology analysis showed that preoperative ment may increase neoantigens, activate IS and HWL contributed equally. CRT significantly enriched the immune response in LARC the major histocompatibility complex class Accepted 31 January 2021 tissues. Moreover, gene set enrichment analysis revealed six significantly enriched Kyoto Encyclopedia of Genes and I system, activate tumor- infiltrating lympho- Genomes pathways associated with downregulated genes, cytes and induce the abscopal effect, wherein http://jitc.bmj.com/ including mismatch repair (MMR) genes, in LARC tissues localized radiation provokes distant anti- 6–9 after CRT in all three cohorts. Radiation also induced tumor effects. The abscopal effect can be apoptosis and downregulated various MMR system- related enhanced by combining an immune check- genes in three colorectal cancer cells. One patient with point blocker (ICB) with radiotherapy.10 LARC showed a change in microsatellite instability (MSI) Cancer immunotherapy with ICBs, such status after CRT, as demonstrated by the loss of MMR as antiprogrammed death-1/programmed protein and PCR for MSI. Moreover, CRT significantly death- ligand 1 and anticytotoxic T lympho- on September 28, 2021 by guest. Protected copyright. increased tumor mutational burden in LARC tissues. cyte antigen-4 inhibitors, is a novel treatment CIBERSORT analysis revealed that the proportions of M2 11 macrophages and CD8 T cells were significantly increased strategy in the field of immuno- oncology. after CRT in both the RNA- seq dataset and GSE94104. To improve the effectiveness of this treat- Notably, preoperative CRT increased various immune ment strategy, several studies have recently biomarker scores, such as the interferon-γ signature, the evaluate various biomarkers, such as micro- cytolytic activity and the immune signature. satellite instability (MSI),12 13 mismatch 13 © Author(s) (or their Conclusions Taken together, our findings demonstrated repair (MMR) deficiency, tumor mutational employer(s)) 2021. Re- use that neoadjuvant CRT modulated the immune- related burden (TMB)14 15 and immune biomarker permitted under CC BY. characteristics of LARC, suggesting that neoadjuvant scores,16–18 for prediction of responses to Published by BMJ. CRT may enhance the responsiveness of LARC to immunotherapies, such as ICBs, in cancer. For numbered affiliations see immunotherapy. end of article. Identifying molecular signatures using next- generation sequencing (NGS) has Correspondence to BACKGROUND become an emerging focus in the field of Professor Shin Kim; Neoadjuvant chemoradiation therapy (CRT) cancer research, facilitating the diagnosis god98005@ dsmc. or. kr and total mesorectal excision are commonly and prediction of prognosis for patients. Professor Sung Uk Bae; incorporated into the multimodal treatment However, a few studies have performed sabiston0000@ hanmail. net of locally advanced rectal cancer (LARC). transcriptomic profiling of biomarkers for Seo I, et al. J Immunother Cancer 2021;9:e001610. doi:10.1136/jitc-2020-001610 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-001610 on 10 March 2021. Downloaded from predicting responses to immunotherapy in rectal cancer in the work flow shown in figure 1. All patients received before and after preoperative CRT. Accordingly, in this conventional long- course neoadjuvant CRT (five cycles of study, we investigated changes in the molecular profiles 5- fluorouracil (5- FU)- based chemotherapy and 50.4 Gy of LARC after CRT with regard to improved responses to radiation) online supplemental method 1. Additionally, immunotherapy through bioinformatics analysis of NGS all patients underwent staging abdominopelvic and chest transcriptome and Gene Expression Omnibus (GEO) CT scan, rectal MRI, colonoscopy, biopsy and positron datasets. Furthermore, we validated the results from these emission tomography scans. Total mesorectal excision analyses using colorectal cancer (CRC) cell lines. was performed within 6–8 weeks of the final CRT. Cell cultures and materials METHODS DLD-1 and SW480 human colorectal adenocarcinoma Patients and sample collection cells and HCT-116 human CRC cells were obtained from Formalin- fixed paraffin- embedded (FFPE) block speci- the American Type Culture Collection (Rockville, Mary- mens from preoperative biopsy via sigmoidoscopy and land, USA) and grown in GIBCO RPMI1640 medium surgical resection of the primary tumor were obtained (Thermo Fisher Scientific, Wilmington, Delaware, USA) from 54 patients with rectal adenocarcinoma who under- supplemented with 10% heat-inactivated fetal bovine went neoadjuvant concurrent CRT between August serum (Thermo Fisher Scientific), 2 mM L- glutamine, 2016 and December 2017. Preoperative clinical stage 100 µg/mL streptomycin and 100 µg/mL penicillin. 5- FU was determined by abdominal and chest CT scans and was purchased from Sigma-Aldrich (St. Louis, Missouri, pelvic MRI and the inclusion criterion was clinical stages USA). 2–3 (T3 or T4 and/or node positive) rectal adenocar- cinoma. The exclusion criteria were stage IV tumors, Gene expression databases cancer related to familial adenomatous polyposis or The rectal cancer gene expression profiles used in this hereditary nonpolyposis CRC, synchronous or previous study were downloaded from the publicly available malignancies, distant metastasis during neoadjuvant CRT GEO database (National Institutes of Health, Bethesda, and patients who refused the definite surgery or were Maryland, USA; http://www. ncbi. nlm. nih. gov/ geo/), lost to follow-up. Based on these criteria, we included with accession numbers GSE1578119 and GSE94104.20 11 pairs of human CRC tissues obtained before and The gene expression profiles (ID, GSE15781) were after CRT (online supplemental table 1) and performed previously produced using the ABI Human Genome RNA sequencing (RNA- seq) analysis. The detailed study Survey Microarray V.2 (Thermo Fisher Scientific). design and enrolled samples at each stage are illustrated Briefly, specimens from GSE15781 were obtained from http://jitc.bmj.com/ on September 28, 2021 by guest. Protected copyright. Figure 1 Diagram of patient selection and study workflow. Samples before neoadjuvant CRT were obtained at the time of diagnosis using endoscopy. Samples after neoadjuvant CRT were obtained after surgical resection. Samples were obtained from 11 patients after enrollment and were prepared and used for RNA sequencing analysis. The 11 patients underwent curative surgery. CRC, colorectal cancer; CRT, chemoradiation therapy; IHC, immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes; LARC, locally advanced rectal cancer; MMR, mismatch repair; MSI, microsatellite instability. 2 Seo I, et al. J Immunother Cancer 2021;9:e001610. doi:10.1136/jitc-2020-001610 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-001610 on 10 March 2021. Downloaded from 13 patients with LARC who received 50 Gy (delivered Analysis of differentially expressed genes as 25 fractions of 2 Gy) over a 5- week period. In addi- Count normalization and differentially expressed gene tion, patients received Capecitabine (Xeloda; Roche) (DEG) identification in pairwise comparisons between 2500 mg/mL daily throughout the treatment period. before and after CRT were performed using R software Resection of the rectum was performed 4–6 weeks after V.3.5.0 (The R Foundation for Statistical Computing, preoperative radiation therapy. Among the specimens 2018) using DESeq2 package.24 For GSE15781 and in the GSE15781

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