Abstract P1-04-05: Multiplexed immunofluorescence staining of intra-tumoral immune cell populations and associations with immunohistochemical, clinical, and pathologic variables in breast cancer
Authors: O'Meara Tess A. , Keenan Tanya E. , Waks Adrienne G. , Felt Kristen D. , Sharma Bijaya , Rodig Scott , Hughes Melissa , Lin Nancy U. , Agudo Judith , Guerriero Jennifer L. , McAllister Sandra S. , Mittendorf Elizabeth , Tolaney Sara 
Published: March 2022

Abstract
Background: Although immune checkpoint inhibitors (ICI) are most widely used in PD-L1 positive, metastatic triple-negative breast cancer (TNBC), the clinical utility of ICI in other tumor subtypes and patient populations is not well defined. In this study, we measured infiltration of multiple immune cell populations in primary and metastatic HR+ tumors to infer potential benefit from ICI. We investigated how risk stratifying features such as pathologic grade and Oncotype Dx scores affect immune cell infiltration across subtypes. Lastly, we compared immune cell infiltration in breast tumors from younger versus older women, as our pre-clinical studies demonstrated differential enrichment of immune response pathways by age and association with improved response to ICI. Methods: ImmunoProfile, a multiplexed immunofluorescence (IF) assay, was performed on FFPE tissue from 167 archived breast cancer tumors from the Dana-Farber Cancer Institute (HR+/HER2- n=124, HER2+ n=21, TNBC n=22). Unmatched samples were obtained from either primary breast (HR+/HER2- n=104, HER2+ n=13, TNBC n=15) or metastatic core biopsies. IF staining signal was measured for T cell markers CD8 and co-localized CD8/PD-1, T regulatory cell marker FOXP3, PD-L1 and PD1 expression on tumor cells as well as PD-L1 expression on inflammatory cells. Inflammatory, tumoral (TPS) and combined inflammatory and tumor (CPS) pathologic PD-L1 scores were calculated. PD-L1 staining was also assessed by IHC (E1L3N) staining and compared to multiplexed IF stains. IF staining signal (Vectra analyzed, log2+1 transformed) was analyzed against clinical and pathologic variables, including tumor subtype, primary versus metastatic status, pathologic grade, and Oncotype Dx score, using non-parametric statistical comparisons. Given the decline in adaptive immunity that occurs with normal physiological aging and our own group’s previous reports, we also explored whether immune cell staining signal associated with age. Results: TPS, CPS, and inflammatory PD-L1 scores strongly correlated between IHC and IF staining methods (Spearman r = 0.660, 0.683, 0.638 respectively, p &lt; 2.2e-16). When HR+/HER2- (n=124), HER2+ (n=21) and TNBC (n=22) cases were analyzed separately, there was no statistical difference between unmatched primary and metastatic cases in any of the analyzed immune cell population stains for any subtype. When all primary cases were analyzed, TNBC showed higher CD8, CD8/PD1, PD1 (all p &lt; 0.05), and FOXP3 staining as well as IF CPS, TPS and inflammatory PD-L1 scores (all p &lt; 0.005) than HR+ cases. HR+/HER2- cases from women &lt; age 65 (n=56) showed higher intra-tumoral CD8 (p=0.039), PD1 (p=0.043), and CD8/PD1 (p=0.022) than cases from women &gt; age 65 (n=38). There was a statistically significant step-wise increase in IF staining of all immune cell populations with increasing pathologic grade (I, II, III) as well as Oncotype Dx scores (&lt; 11, 11-25, &gt;25) among primary HR+/HER2- cases (n=74). Conclusions: Strong correlations between PD-L1 scores based on IF and IHC methods suggest clinical utility for multiplexed IF immune cell staining in therapeutic planning. ImmunoProfile demonstrated the previously known pattern that primary TNBC contains higher immune cell infiltration than HR+ or HER2+ tumors. Women &lt; age 65 with HR+ cancer showed higher levels of CD8 and PD1 stained populations, suggesting there may be greater utility of ICI in younger patients. Lastly, the association of higher pathologic grade and Oncotype Dx score with increasing immune cell infiltration supports exploration of ICI in biologically unfavorable primary HR+ cancers.
Citation Format: Tess A. O'Meara, Tanya E. Keenan, Adrienne G. Waks, Kristen D. Felt, Bijaya Sharma, Scott Rodig, Melissa Hughes, Nancy U. Lin, Judith Agudo, Jennifer L. Guerriero, Sandra S. McAllister, Elizabeth Mittendorf, Sara Tolaney. Multiplexed immunofluorescence staining of intra-tumoral immune cell populations and associations with immunohistochemical, clinical, and pathologic variables in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-05.