Document resource
Background Tumour budding (TB), a single cell or small cluster of up to 4 cells found at the invasive front of tumours, 1 has been well studied as an independent prognostic marker in CRC and has been consistently reported as a prognostic factor associated with tumour recurrence and metastases across solid tumours.2–4 However the underlying mechanism of TB has not yet been fully understood.Methods Multi-omic approaches from bulk RNA as well as regional GeoMx bulk RNA were used to identify the underlying mechanism of TB and its possible correlation with tumour microenvironment in CRC tissue. The results were validated using immunohistochemistry (IHC) and high plex sequential immunofluorescence (20-plex) staining using SPYRE Immuno-oncology from COMET TMLunaphore platform together with the SPYRETM Signal Amplification kit.Results Patients with high TB were associated with worse outcome in two individual CRC cohorts and with a strong correlation with late stage of disease (p=0.003), venous invasion (p=0.006), distant recurrence (p=0.028), peritoneal involvement (p=0.002) and local inflammation (p=0.015) ( figure 1). Bulk RNA expression revealed that tumours with high TB are significantly enriched for gene signatures related to epithelial-mesenchymal transition (EMT), TNF-α and TGF-β, in both independent CRC cohorts. Regional bulk RNA expression revealed that cell cycle related protein cyclinD1 was highly expressed within cytokeratin positive regions at the tumour invasive margin, and that high cyclinD1, within budding cells, associated with poorer outcomes compared to those with low cyclinD1 in CRC patients. Additionally, regional bulk RNA expression from non-tumour (PanCK-) invasive areas showed that tumours with high TB were significantly associated with a decreased immune activity. Using SPYRETM Antibody Immuno-Oncology panels plus EMT and TNF-α related antibodies on the Lunaphore COMET platform, we were able to not only validate the associations observed with EMT, cyclinD1 and TNF-α at the protein level, but also understand the immune microenvironment of the tumour buds, with close proximity of regulatory T cells and pan-macrophages to individual buds being associated with prognosis, demonstrating how the immune landscape can determine the fate of the tumour bud and metastatic potential (figure 2).Conclusions This study confirms the prognostic role of TB in CRC and gives an insight into its underlying mechanism and potential interactions with the tumour microenvironment in CRC, with key insights being provided using high plex sequential immunofluorescenceAcknowledgements The authors wish to acknowledge the Glasgow Tissue Research Facility and NHS Greater Glasgow and Clyde biorepository for helping in preparing the tissue for this study. The authors gratefully acknowledge for Mahidol University and National Research Council of Thailand (2022) for funding and financial support (PH) (N41B650195). The authors would also like to acknowledge the Academic unit of Surgery at the Glasgow Royal Infirmary. The authors also wish to acknowledge CRUK funding which enable this research (Scottish Cancer Centre CTRQQR-2021/100006) (JE) as well as Chief Scientific Office (CSO) Scotland funding (EPD/22/13) (KP, JE).References Lugli A, Kirsch R, Ajioka Y, Bosman F, Cathomas G, Dawson H, et al. Recommendations for reporting tumor budding in colorectal cancer based on the international tumor budding consensus conference (ITBCC) 2016. Mod Pathol. 2017;30(9):1299–311.Zlobec I, Hädrich M, Dawson H, Koelzer VH, Borner M, Mallaev M, et al. Intratumoural budding (ITB) in preoperative biopsies predicts the presence of lymph node and distant metastases in colon and rectal cancer patients. British Journal of Cancer. 2013;110(4):1008–13.Koelzer VH, Zlobec I, Lugli A. Tumor budding in colorectal cancer--ready for diagnostic practice? Hum Pathol. 2016;47(1):4–19.Lugli A, Zlobec I, Berger MD, Kirsch R, Nagtegaal ID. Tumour budding in solid cancers. Nat Rev Clin Oncol. 2021;18(2):101–15.Ethics Approval The study was approved by the West of Scotland Research Ethics Committee (REC 4: 22/ws/0207) and data are stored in Greater Glasgow and Clyde Safehaven (SH21ON012).Abstract 1253 Figure 1(A) Representative image of tumours with low (top) and high (bottom) budding phenotype in CRC H&E stained sections. (B) Kaplan-Meier survival analysis based on tumour budding phenotype for cancer specific survival (CSS) in discovery cohort. (C) The correlation plot for the Pearson’s chi square test showed the residuals for TB phenotypes against clinical factors; positive residuals are in blue, suggesting a positive association and negative residuals are red, suggesting a negative association. The bigger size of the circle, the more significant association was found, red arrow indicates the statistically significant associationAbstract 1253 Figure 2(A) 20-plex immunofluorescence Lunaphore COMET™ in CRC tissue with high budding phenotype. (B) Representative image of the distance measured from individual budding cluster to immune cells. (C-D) Boxplots shows the distance from budding clusters to each immune phenotype (um)