Abstract P5-02-11: Immune cell profile of tumors from patients with metastatic (met) HER2+ breast cancer (BC) with &lt; 30 months overall survival (OS)
Authors: Collins Denis M. , McCormack Janet , Ivers Laura P. , Pina Jose Javier Berenguer , Ballot Jo , Quinn Cecily , Skrobo Darko , Eustace Alex J. , Walsh Naomi , Fabre Aurelie , Crown John 
Published: March 2023

Abstract
Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin has been used to identify HER2+ BC patients with durable complete response (never relapsed) to trastuzumab-based therapy. ~10% of met HER2+ BC patients achieve a durable complete response to trastuzumab, meaning the majority of patients progress on treatment. Higher stromal tumour immune infiltrate has been associated with longer OS in met HER2+ BC. There is limited tumor immune profile and PD-1 expression data available for patients with met HER2+ BC with short OS. Using the SVUH database, we have identified a preliminary cohort of 21 met HER2+ BC patients that received trastuzumab and had an OS &lt; 30 months. This study examines the levels of pan T cell marker CD3, cytotoxic T cell marker CD8, Natural Killer (NK) cell marker CD56 and immune checkpoint PD-1 by immunohistochemistry (IHC) in this preliminary cohort. Methods: Formalin-fixed, paraffin-embedded (FFPE) biopsy specimens (n=21 primary, n=7 matched metastatic biopsies) and associated clinico-pathological data were curated. Tumor biopsies were processed for IHC staining of CD8 (Agilent IR62361-2), CD3 (Agilent IR50361-2), CD56 (Agilent IR62861-2) and PD-1 (Roche 07099029001). PD-1 staining was available for 20/21 samples. Staining was performed using the DAKO Link 48 Autostainer as per the manufacturer’s instructions using positive (tonsil tissue) and negative controls (isotype controls). Slides were processed using the Aperio AT2 Digital Slide Scanner (Leica Biosystems), reviewed using Aperio ImageScope 12.4 software (Leica Biosystems) and analyzed in QuPath (University of Edinburgh). Images were annotated to outline tumor areas and an algorithm was trained to identify cells and classify them as either tumor or stromal. Data was expressed as number of positively stained cells/mm2 breast tumor or stromal tissue. Survival studies utilized the Kaplan Meier method. The paired Student’s T test was utilized for primary vs metastatic site comparisons. Results: Designating samples with &gt; 1 stained cell/mm2 breast tumor as positive (pos) and zero stained cells as negative (neg), 19/21 (90.5%) primary samples were pos for CD3, 15/21 (71.4%) for CD56, 14/21 (66.6%) for CD8, and 10/20 (50%) for PD-1. Within the stromal compartment, 20/21 (95.2%) primary samples were pos for CD8, 18/21 (85.7%) for CD56, 16/21 (76.2%) for CD3 and 8/20 (40%) for PD-1. PD-1 expression in the primary tumor (median OS PD-1pos 7.85 mo vs PD-1neg 5.39 mo, hazard ratio (HR) 0.642 (95% CI 0.256-1.613), p=0.346) or the stroma (median OS PD-1pos 8.84 mo vs PD-1neg 5.39 mo, HR 0.495 (95% CI 0.197-1.244), p=0.135) was not significantly associated with OS. When comparing matched primary and metastatic samples (n=7), increased stromal levels of CD3 (4/7), CD8 (4/7), CD56 (5/7) and PD-1 (4/7) were observed. Increased levels of CD3 and CD8 were observed for 2/7 samples, and increased levels of CD56 and PD-1 for 4/7 samples. With the exception of tumor CD8 levels which decreased, mean values for tumor and stromal CD3, CD56, PD-1 and stromal CD8 levels were higher in metastatic sites but all differences were not found to be significant (p&gt;0.05). Conclusions: Our results suggest that met HER2+ BC patients with &lt; 30 months OS have significant T cell and NK cell presence in the tumor and stromal compartments in both primary and metastatic sites. Further expansion of this limited dataset is planned to gain greater insight in to the immune cell profiles and PD-1 status of met HER2+ BC patients with short OS.
Citation Format: Denis M. Collins, Janet McCormack, Laura P. Ivers, Jose Javier Berenguer Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex J. Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Immune cell profile of tumors from patients with metastatic (met) HER2+ breast cancer (BC) with &lt; 30 months overall survival (OS). [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-11.