Abstract A20: Analysis of tumor infiltrating lymphocytes and expression of PD1 and PD-L1 in breast tumors prior to and after neoadjuvant chemotherapy
Authors: Wesolowski Robert , Li Zaibo , McQuinn Christopher , Lustberg Maryam , Ramaswamy Bhuvaneswari , Noonan Anne , Reinbolt Raquel , Sardesai Sagar , VanDeusen Jeffrey B. , Williams Nicole , Carson William E. 
Published: August 2018

Abstract
Introduction: The effect of chemotherapy on the presence of tumor-infiltrating lymphocytes (TILs) and expression of PD1 and PD-L1 is unclear. We sought to describe the differences in the percentage (%) of TILs, cytotoxic T lymphocytes (CTL), and expression of PD1/PD-L1 in tumors of patients (pts) with operable breast cancer (BC) treated with neoadjuvant chemotherapy (NAC) who were enrolled in a biomarker study at our institution (IRB protocol# 2010C0036).
Methods: A multicolor immune-histochemical multiplex assay simultaneously detecting PD1, PD-L1, and CD8 expressing cells was performed on formalin-fixed, paraffin-embedded diagnostic pretreatment biopsy or resected tumor specimen following NAC in 18 of 26 pts participating in the study. A cut-off of ≥1% was considered positive for PD1 and PD-L1 expression. We evaluated stromal and intratumoral CTLs by estimating % of stroma and tumor that contained CD8+ cells. In addition, stromal TILs (sTILs) were identified on full-face hematoxylin and eosin stained sections and defined as the % of tumor stroma containing infiltrating lymphocytes. Pathologic complete or near-complete response (pCR) was analyzed based on residual cancer burden (RCB) score and defined as RCB class 0 or I. Analysis of all slides was performed by an expert breast pathologist. Since the number of pts was limited, we only provide descriptive statistics (mean, range). In addition, because most pts had only a biopsy or only residual tumor available for the analysis, we divided pts into 2 separate cohorts based on what tissue was available.
Results: Of 18 pts, biopsy was analyzed in 7 (Bx cohort) and residual tumor was analyzed in 11 pts (RT cohort). The median age of study pts was 48 (range 32-70); 11 (61%), 6 (33%), and 1 (6%) of pts were Caucasian, African American, and Hispanic, respectively. Ten pts (5 in Bx and RT cohorts each) had triple-negative BC (TNBC), 4 had HER2+ BC (1 and 3 in Bx and RT cohorts, respectively), and 4 had hormone receptor-positive, HER2- BC (1 and 3 in Bx and RT cohorts, respectively). Eight pts (44%) had pCR. In the bx cohort, 85% of pts had pCR while 18% of pts in the RT cohort had minimal residual disease (RCB class I). In the Bx cohort, average % of sTILs was 30% (range 2-70%), including 1 pt (14%) with lymphocyte predominant tumor (≥50% of sTILs). The % of sTILs was similar in the 11 residual tumors (mean 22, range 2-60) with 2 pts having lymphocyte predominant tumors. Average % of CTLs was 19 (range 1-50) in the Bx cohort and 14 (range 1-50) in the RT cohort. An average % of intratumoral CTLs was 8 (range 1-30) and 7.5 (range 0-40) while the average % of stromal CTLs was 25 (range 1-60) and 19 (range 1-60) in the Bx and RT cohorts, respectively. Similar % and trends were seen in 10 TNBC pts. PD-L1 expression was seen in 86% and 36% of tumors in the Bx and RT cohorts, respectively, with majority of expression present in the stroma. All cases of intratumoral PD-L1 expression were also positive for stromal PD-L1 expression. This difference was also seen in the TNBC pts (80% vs. 40% of tumors were PD-L1+ in Bx and RT cohorts, respectively). An average PD-L1 intensity was approximately 3% in both cohorts (range 1-20%). Expression of PD1 was very low (1% intensity in 3 pts in Bx cohort and in 1 pt in RT cohort) and it was seen on CD8+ CTLs.
Conclusion: Our study preliminarily shows that percent of sTILs and stromal and intratumoral CTLs does not differ between pretreatment biopsy and residual tumors following NAC. Lower proportion of residual tumors were PD-L1+ compared to pretreatment biopsy specimen. The study limitations include small number of subjects and lack of comparison in the same pts. Future studies are needed to confirm these findings.
Citation Format: Robert Wesolowski, Zaibo Li, Christopher McQuinn, Maryam Lustberg, Bhuvaneswari Ramaswamy, Anne Noonan, Raquel Reinbolt, Sagar Sardesai, Jeffrey B. VanDeusen, Nicole Williams, William E. Carson, III. Analysis of tumor infiltrating lymphocytes and expression of PD1 and PD-L1 in breast tumors prior to and after neoadjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A20.