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Original research
Epigenetic promoter alterations in GI tumour immune-editing and resistance to immune checkpoint inhibition
  1. Raghav Sundar1,2,3,4,5,
  2. Kie-Kyon Huang3,
  3. Vikrant Kumar3,
  4. Kalpana Ramnarayanan3,
  5. Deniz Demircioglu6,
  6. Zhisheng Her7,
  7. Xuewen Ong3,
  8. Zul Fazreen Bin Adam Isa3,6,8,
  9. Manjie Xing3,6,8,
  10. Angie Lay-Keng Tan3,
  11. David Wai Meng Tai9,
  12. Su Pin Choo9,10,
  13. Weiwei Zhai6,
  14. Jia Qi Lim6,
  15. Meghna Das Thakur11,
  16. Luciana Molinero11,
  17. Edward Cha11,
  18. Marcella Fasso11,
  19. Monica Niger12,
  20. Filippo Pietrantonio12,
  21. Jeeyun Lee13,
  22. Anand D Jeyasekharan1,14,
  23. Aditi Qamra15,16,
  24. Radhika Patnala17,
  25. Arne Fabritius17,
  26. Mark De Simone18,
  27. Joe Yeong7,14,
  28. Cedric Chuan Young Ng19,
  29. Sun Young Rha20,21,
  30. Yukiya Narita22,
  31. Kei Muro22,
  32. Yu Amanda Guo6,
  33. Anders Jacobsen Skanderup6,
  34. Jimmy Bok Yan So5,23,24,
  35. Wei Peng Yong1,5,
  36. Qingfeng Chen7,25,
  37. Jonathan Göke6,
  38. Patrick Tan3,5,6,14,26,27
  1. 1 Department of Haematology-Oncology, National University Cancer Institute, Singapore, National University Hospital, Singapore
  2. 2 Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  3. 3 Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore
  4. 4 The N.1 Institute for Health, National University of Singapore, Singapore
  5. 5 Singapore Gastric Cancer Consortium, Singapore
  6. 6 Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore
  7. 7 Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore
  8. 8 Diagnostic Development Hub (DxD), Agency for Science, Technology and Research, Singapore
  9. 9 Division of Medical Oncology, National Cancer Centre, Singapore
  10. 10 Curie Oncology, Singapore
  11. 11 Department of Development Sciences, Genentech, San Francisco, California, USA
  12. 12 Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
  13. 13 Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea
  14. 14 Cancer Science Institute of Singapore, National University of Singapore, Singapore
  15. 15 Statistical Programming and Analytics, Roche Canada, Mississauga, Ontario, Canada
  16. 16 University Health Network, Toronto, Ontario, Canada
  17. 17 Sci-illustrate, Munich, Germany
  18. 18 InSilico Genomics, Phoenix, Arizona, USA
  19. 19 Laboratory of Cancer Epigenome, Department of Medical Sciences, National Cancer Centre, Singapore
  20. 20 Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, South Korea
  21. 21 Songdang Institute for Cancer Research, Yonsei University College of Medicine, Seoul, South Korea
  22. 22 Department of Clinical Oncology, Aichi Cancer Center Hospital, Nagoya, Japan
  23. 23 Department of Surgery, National University Hospital, Singapore
  24. 24 Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  25. 25 Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  26. 26 SingHealth/Duke-NUS Institute of Precision Medicine, National Heart Centre, Singapore
  27. 27 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  1. Correspondence to Dr Patrick Tan, Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore, Singapore; gmstanp{at}duke-nus.edu.sg; Dr Raghav Sundar, Department of Haematology-Oncology, National University Cancer Institute, Singapore; National University Hospital, Singapore, Singapore; raghav_sundar{at}nuhs.edu.sg

Abstract

Objectives Epigenomic alterations in cancer interact with the immune microenvironment to dictate tumour evolution and therapeutic response. We aimed to study the regulation of the tumour immune microenvironment through epigenetic alternate promoter use in gastric cancer and to expand our findings to other gastrointestinal tumours.

Design Alternate promoter burden (APB) was quantified using a novel bioinformatic algorithm (proActiv) to infer promoter activity from short-read RNA sequencing and samples categorised into APBhigh, APBint and APBlow. Single-cell RNA sequencing was performed to analyse the intratumour immune microenvironment. A humanised mouse cancer in vivo model was used to explore dynamic temporal interactions between tumour kinetics, alternate promoter usage and the human immune system. Multiple cohorts of gastrointestinal tumours treated with immunotherapy were assessed for correlation between APB and treatment outcomes.

Results APBhigh gastric cancer tumours expressed decreased levels of T-cell cytolytic activity and exhibited signatures of immune depletion. Single-cell RNAsequencing analysis confirmed distinct immunological populations and lower T-cell proportions in APBhigh tumours. Functional in vivo studies using ‘humanised mice’ harbouring an active human immune system revealed distinct temporal relationships between APB and tumour growth, with APBhigh tumours having almost no human T-cell infiltration. Analysis of immunotherapy-treated patients with GI cancer confirmed resistance of APBhigh tumours to immune checkpoint inhibition. APBhigh gastric cancer exhibited significantly poorer progression-free survival compared with APBlow (median 55 days vs 121 days, HR 0.40, 95% CI 0.18 to 0.93, p=0.032).

Conclusion These findings demonstrate an association between alternate promoter use and the tumour microenvironment, leading to immune evasion and immunotherapy resistance.

  • gastric cancer
  • hepatocellular carcinoma
  • immunotherapy

Data availability statement

Data used in this manuscript includes previously published studies with genomic data files from public repositories: European Nucleotide Archive: PRJEB25780 and PRJEB34724 The Cancer Genome Atlas Research Network: dbGaP: phs000178.v10.p8 Data used in this manuscript includes previously published studies with genomic data files from public repositories:European Nucleotide Archive: PRJEB25780 and PRJEB34724The Cancer Genome Atlas Research Network: dbGaP: phs000178.v10.p8NanoString data file is provided as a Supplementary Table (attached) https://www.ebi.ac.uk/ena/data/view/PRJEB25780; https://www.ebi.ac.uk/ena/data/view/PRJEB34724https://www-ncbi-nlm-nih-gov.libproxy1.nus.edu.sg/projects/gap/cgi-bin/study.cgi?study_id=phs000178.v10.p8.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/gutjnl-2021-324420

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    Data availability statement

    Data used in this manuscript includes previously published studies with genomic data files from public repositories: European Nucleotide Archive: PRJEB25780 and PRJEB34724 The Cancer Genome Atlas Research Network: dbGaP: phs000178.v10.p8 Data used in this manuscript includes previously published studies with genomic data files from public repositories:European Nucleotide Archive: PRJEB25780 and PRJEB34724The Cancer Genome Atlas Research Network: dbGaP: phs000178.v10.p8NanoString data file is provided as a Supplementary Table (attached) https://www.ebi.ac.uk/ena/data/view/PRJEB25780; https://www.ebi.ac.uk/ena/data/view/PRJEB34724https://www-ncbi-nlm-nih-gov.libproxy1.nus.edu.sg/projects/gap/cgi-bin/study.cgi?study_id=phs000178.v10.p8.

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    Supplementary materials

    Footnotes

    • RS and PT are joint senior authors.

    • K-KH, VK, KR, DD and ZH contributed equally.

    • Contributors Conceptualisation: RS, AQ, JG and PT; data curation: RS, KKH, DD, VK, KR, ZH, XO, ZFBAI, MX, AL-KT, DWMT, WZ, SPC, JQL, MDT, LM, EC, MF, MN, FP, JL, JY, CCYN, SYR, JS, KM, YN, WPY, QC and JG; formal analysis: RS, KKH, AQ, VK, ADJ, MDS, YAG, AJS, JG and PT; funding acquisition: JG and PT; methodology: RS, VK, MDS, QC, DD, AQ, JG and PT; project administration: RS and PT; resources and supervision: QC, JG and PT; visualisation: RS, AQ, KKH, RP and AF; writing of the original draft: RS; writing (review and editing): ZH, QC, JG and PT; approval of final version of manuscript: all authors.

    • Funding RS is supported by a National Medical Research Council (NMRC) Fellowship (NMRC/Fellowship/0059/2018 and MOH-000627), Singapore. FP is supported by a research grant from Associazione Italiana per la Ricerca sul Cancro (AIRC IG 2019 Id.23624). PT is supported by Duke-NUS Medical School and the Biomedical Research Council, Agency for Science, Technology and Research. Humanised mouse work was supported by National Research Foundation Singapore Fellowship (NRF-NRFF2017-03) to QC. This work was also supported by National Medical Research Council grants OF-LCG18May-0023, NR13NMR111OM, and NMRC/STaR/0026/2015.

    • Competing interests The subject matter in this manuscript was submitted as a technology disclosure to the institutional Technology Transfer Office for potential intellectual property protection. PT had stock and other ownership interests in HealthSeq, research funding from Kyowa Hakko Kirin and Thermo Fisher Scientific, and patents/other intellectual property through the Agency for Science and Technology Research, Singapore (all outside the submitted work). RS received honoraria from Bristol-Myers Squibb, Lilly, Roche, Taiho, Astra Zeneca, DKSH and MSD; had advisory activity with Bristol-Myers Squibb, Eisai, Merck, Bayer, Taiho, Novartis, MSD and AstraZeneca; received research funding from Paxman Coolers and MSD; received travel grants from AstraZeneca, Roche, Eisai and Taiho Pharmaceutical (all outside the submitted work). FP received honoraria for speakers’ bureau/advisory activity from Amgen, Merck-Serono, Roche, Lilly, Sanofi, Bayer and Servier, and received research funding from BMS (all outside the submitted work).

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.