Abstract 4049: Assessment of immune biomarkers by digital pathological analysis across a large colorectal cancer patient cohort predicts patient outcome and may provide a clinically relevant therapeutic index for immunotherapeutic treatment stratification
Authors: Humphries Matthew P. , Craig Stephanie , Bingham Victoria , Loughrey Maurice , Coleman Helen , Murray Liam , Tim Maughan , McQuaid Stephen , James Jacqueline , Salto-Tellez Manuel 
Published: August 2018

Abstract
Introduction: Colorectal cancer (CRC) is a heterogeneous malignancy, treatment for which has principally been cytotoxic chemotherapy. Herein we describe the assessment of immune-biomarkers in a large patient cohort, demonstrating the therapeutic potential of immunotherapy in CRC. Methods: By immunohistochemistry, employing REMARK criteria, using well-validated antibodies, 4 biomarkers (CD3, CD4, CD8, FOXP3) in over 1000 cases, represented across 33 TMAs, were assessed for their protein expression. Digital pathological assessment of immune biomarker densities were quantified via the application of QuPath, an open source digital pathology platform, developed at Queens University, Belfast (https://qupath.github.io.). Biomarker densities were dichotomised by R.O.C analysis. Kaplan-Meier curves (Log-rank p values) were used to assess the impact of immune cell densities on overall survival (OS) and progression-free survival (PFS). Hazard ratios were assessed using the Cox proportional hazards regression model for univariate analysis. Spearman's rank was used to assess correlations of the biomarkers. Results: Expression of two biomarkers were found to vary by stage and three significantly affected OS. Across the patient cohort CD3, CD4 and CD8 were individually shown to be positively prognostic for OS alone (log-rank p = 0.0007, p = 8.65e-06, p = 0.006, respectively). CD4 demonstrated a more significant impact on OS in later stage disease (Log-rank stage II p = 0.0017, stage III p = 0.0002), while conversely CD8 only showed significance in earlier stage disease (Log-rank stage II p = 0.009, stage III p = 0.546). Although significantly associated with OS and within all stages, CD3 showed no differential expression in a particular stage. Correlative analysis demonstrated a statistically significant association between biomarkers. Upon univariate analysis, all biomarkers remained significantly prognostic for OS. All analyses were performed using R. Conclusions: These data demonstrate the prognostic significance of adaptive immune markers in a large CRC cohort on OS. Moreover, stage specific expression maybe clinically relevant and assessment within stages II and III may provide important stratifications for specific immunotherapy treatment regimes. We additionally add weight to the beneficial utility of digital pathology and its potential for integration into routine tissue biomarker assessment. Additional analysis of immune checkpoint and adaptive immune biomarkers in CRC is warranted.
Citation Format: Matthew P. Humphries, Stephanie Craig, Victoria Bingham, Maurice Loughrey, Helen Coleman, Liam Murray, Maughan Tim, Stephen McQuaid, Jacqueline James, Manuel Salto-Tellez. Assessment of immune biomarkers by digital pathological analysis across a large colorectal cancer patient cohort predicts patient outcome and may provide a clinically relevant therapeutic index for immunotherapeutic treatment stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4049.