Abstract 5787: Modelling the spatial heterogeneity of CD45-positive tumor infiltrating lymphocytes in early-stage, estrogen receptor-positive breast cancer
Authors: Kinsella Zak , Blümel Anna , Lucas Mairi , Lindner Andreas , Gonzalez Claudia A. , Rahman Arman , Fay Joanna , O'Grady Tony , Murphy Verena , Crown John , Kelly Cathy , Gallagher William , O'Connor Darran 
Published: April 2023

Abstract
The frequency of lymphocytes infiltrating tumors is a known prognostic in estrogen receptor (ER) negative cancers. ER+ disease is putatively believed to be immune cold, however, there exists a subset of ER+ tumors with high immune infiltrate and with a significant spatial heterogeneity. The clinic impact of such infiltrate - especially between the Oncotype Dx Recurrence Score risk categories - remains unclear. Moreover, the distribution of tumor and stromal tissues, while noted as significantly heterogenous, is still ill-defined and not yet clinically used prognostically, despite evidence to support its utility. Using a cohort (n=450) of serial sections taken from early-stage, ER+/HER2- breast tumors of the Irish arm of the TAILORx clinical trial, we aimed to investigate the tumor architecture and spatial distribution of tumor immune infiltrate and proliferating tumor cells using digital image analysis. Antibodies against Ki67 (proliferation marker) and CD45 (leukocyte common antigen), and a routine Haematoxylin and Eosin stain were applied to serial sections of 450 full-face tumors via chromogenic immunohistochemistry, as outlined previously [1]. Digital image analysis was performed using open-source software, QuPath [2]. Pixel classifiers were trained and validated against an expert pathologist in order to define observed lymphocytes as tumor or stromal-infiltrating, and to establish a classifier to quantify the tumor-stroma ratio (TSR) and infiltrating tumor area. Distances of CD45-positive cells from tumor were computed, along with autocorrelation statistics [3,4] of CD45 and Ki67 hotspots; firstly in order to quantify spatial heterogeneity, and secondly to examine whether Ki67 as a component gene in the Oncotype Dx assay has a foundation in tumor biology or is being confounded by potentially Ki67-positive lymphocytes. Subdividing by Oncotype Dx risk categories, no significant difference in TSR was observed (p=0.09799), neither for intermediary risk patients receiving hormone therapy alone or in combination with chemotherapy (p=0.3873). While there was an observed trend overall (p=0.092), no significance was found for recurrence between intermediary risk subcategories (HT alone: p=0.393, HT+CT: p=0.288). However, in the cohort as a whole, median TSR was 0.3215 (range 0 - 5.023), with statistically significant differences in recurrence risk observed (cohort high v low by median TSR. HR: 6.356, 95CI: 2.263-17.84, p&lt;0.0001).
Citation Format: Zak Kinsella, Anna Blümel, Mairi Lucas, Andreas Lindner, Claudia A. Gonzalez, Arman Rahman, Joanna Fay, Tony O'Grady, Verena Murphy, John Crown, Cathy Kelly, William Gallagher, Darran O'Connor. Modelling the spatial heterogeneity of CD45-positive tumor infiltrating lymphocytes in early-stage, estrogen receptor-positive breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5787.