Exploring the immune microenvironment in small bowel adenocarcinoma using digital image analysis

PLOS ONE RESEARCH ARTICLE Exploring the immune microenvironment in small bowel adenocarcinoma using digital image analysis Fatima Abdullahi Sidi ID1‡, Victoria Bingham1‡, Stephen McQuaid1,2,3, Stephanie G. Craig1, Richard C. Turkington4, Jacqueline A. James1,2,3, Matthew P. Humphries ID1,5,6‡*, Manuel Salto-Tellez1,2,7‡* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Precision Medicine Centre of Excellence, The Patrick G. Johnston Centre for Cancer Research, Queen’s University Belfast, Antrim, United Kingdom, 2 Cellular Pathology, Belfast Health and Social Care Trust, Belfast City Hospital, Lisburn Road, Belfast, Antrim, United Kingdom, 3 Northern Ireland Biobank, The Patrick G. Johnston Centre for Cancer Research, Queen’s University Belfast, Antrim, United Kingdom, 4 The Patrick G. Johnston Centre for Cancer Research, Queen’s University Belfast, Antrim, United Kingdom, 5 Leeds Teaching Hospitals NHS Trust, Leeds, West Yorkshire, United Kingdom, 6 University of Leeds, St James’ University Hospital, Leeds, West Yorkshire, United Kingdom, 7 Division of Molecular Pathology, The Institute for Cancer Research, London, Greater London, United Kingdom ‡ FAS and VB are joint first authors. MPH and MST are joint senior authors. * (MPH); (MST) OPEN ACCESS Citation: Sidi FA, Bingham V, McQuaid S, Craig SG, Turkington RC, James JA, et al. (2023) Exploring the immune microenvironment in small bowel adenocarcinoma using digital image analysis. PLoS ONE 18(8): e0289355. https://doi.org/10.1371/ journal.pone.0289355 Editor: Vincenzo L’Imperio, Universita degli Studi di Milano-Bicocca, ITALY Received: November 15, 2022 Accepted: July 18, 2023 Abstract Background Small bowel adenocarcinoma (SBA) is a rare malignancy of the small intestine associated with late stage diagnosis and poor survival outcome. High expression of immune cells and immune checkpoint biomarkers especially programmed cell death ligand-1 (PD-L1) have been shown to significantly impact disease progression. We have analysed the expression of a subset of immune cell and immune checkpoint biomarkers in a cohort of SBA patients and assessed their impact on progression-free survival (PFS) and overall survival (OS). Published: August 1, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0289355 Copyright: © 2023 Sidi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The data underlying the results presented in the study are available from The Precision Medicine Centre of Excellence (PMC) at Queen’s University Belfast: Queen’s Methods 25 patient samples in the form of formalin fixed, paraffin embedded (FFPE) tissue were obtained in tissue microarray (TMAs) format. Automated immunohistochemistry (IHC) staining was performed using validated antibodies for CD3, CD4, CD8, CD68, PD-L1, ICOS, IDO1 and LAG3. Slides were scanned digitally and assessed in QuPath, an open source image analysis software, for biomarker density and percentage positivity. Survival analyses were carried out using the Kaplan Meier method. Results Varying expressions of biomarkers were recorded. High expressions of CD3, CD4 and IDO1 were significant for PFS (p = 0.043, 0.020 and 0.018 respectively). High expression of ICOS was significant for both PFS (p = 0.040) and OS (p = 0.041), while high PD-L1 expression in tumour cells was significant for OS (p = 0.033). High correlation was observed between PD-L1 and IDO1 expressions (Pearson correlation co-efficient = 1) and PLOS ONE | https://doi.org/10.1371/journal.pone.0289355 August 1, 2023 1 / 14 PLOS ONE The immune microenvironment of small bowel adenocarcinoma University Belfast. Health Sciences Building. 97 Lisburn Road. Belfast. BT9 7AE. Tel: +44(0)28 9097 2293. . subsequently high IDO1 expression in tumour cells was found to be significant for PFS (p = 0.006) and OS (p = 0.034). Funding: This study was funded by a Cancer Research UK (CRUK) Accelerator Grant (A20256) to JJ and MST. CRUK (https://www. cancerresearchuk.org/) had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Northern Ireland Biobank has received funds from HSC Research and Development Division of the Public Health Agency in Northern Ireland. Conclusions Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Manuel Salto-Tellez is a scientific advisor to Mindpeak and Sonrai Analytics, and has received honoraria recently from BMS, MSD and Incyte. None of these disclosures are related to this work. The remaining authors declare no potential conflicts of interest. Abbreviations: FFPE, Formalin Fixed Paraffin Embedded; ICI, Immune Checkpoint Inhibitor; ICOS, Inducible T-cell COStimulator; IDO1, Indoleamine 2, 3-DiOxygenase 1; OS, Overall Survival; PD-L1, Programmed cell Death Ligand 1; PFS, Progression-Free Survival; SBA, Small Bowel Adenocarcinoma; TMA, Tissue Micro-Array. High levels of immune cells and immune checkpoint proteins have a significant impact on patient survival in SBA. These data could provide an insight into the immunotherapeutic management of patients with SBA. Introduction Aetiology and incidence Small bowel cancers (SBCs) are malignant lesions found in the small intestine, which makes up about 80% of the intestinal tract’s length. Although an uncommon neoplasm, SBCs result in approximately 1700 new cancer cases every year in the UK, accounting for less than 1% of total cancer cases. In the last two decades, the incidence rates for SBCs in the UK have risen by about 163%. Though occurring more commonly in men, women have seen larger increased incidence over that time period [1–6]. Potential influences on the rising incidence of SBC include increased use of imaging tests, inflammatory bowel diseases, changes in the microbiome, environmental factors, and genetic predisposition [7–11]. Small bowel adenocarcinoma (SBA) is the most common histological type of small bowel tumour, being more common in the duodenum, and is associated with late stage diagnosis, poor prognosis and high mortality rates [1, 2, 12, 13]. Five year overall survival for early stage disease has been reported to be between 35%– 80%, with 8%– 47% for locally advanced disease and 5% for disseminated disease [14]. The management of SBA clinically is similar to that of colorectal cancer (CRC) adenocarcinomas. Differences have been observed in incidence and disease progression within the two cancer groups, w (...truncated)