Abstract P5-07-09: Heterogeneity of tumor infiltrating lymphocytes in breast cancer and its impact for use as a biomarker
Authors: Mani NL , Schalper K , Hatzis C , Chagpar A , Pusztai L , Rimm DL 
Published: May 2016

Abstract
Background: In breast cancer, elevated tumor infiltrating lymphocytes (TILs) is associated with PD-L1 expression, hormone receptors negativity, and better outcome. The presence of numerous CD8+ cytotoxic T cells in pre-treatment specimens is associated with clinical benefit from PD-1 axis blockade in melanoma and lung cancer, suggesting its predictive value. Despite recent efforts to standardize the pathologist evaluation of TILs in breast cancer, objective determination of lymphocyte subpopulations and their distribution/uniformity within tumor tissues remains largely unexplored. Here, we simultaneously measured diverse TIL subpopulations using quantitative immunofluorescence (QIF) in different areas of breast tumors to determine the heterogeneity of TILs and its possible impact for use as biomarker.
Methods: Using a multiplexed QIF-based assay for simultaneous detection of DAPI (all cells), Cytokeratin (epithelial cells, M3515-DAKO), CD3 (T lymphocytes, E272--Novus), CD8 (cytotoxic T cells, C8/144B--DAKO), and CD20 (B cells, clone L26-DAKO), we measured the levels of TIL subpopulations in whole tissue section slides of 3 tumor cores obtained from different areas of 31 breast carcinomas. The levels of the markers were measured using the AQUA method of QIF and the heterogeneity was studied using numerical correlations of log2 transformed scores and variance component analysis with linear mixed effects (LME). The concordance (kappa index [κ]) between binarized scores obtained measuring 1 vs 3 cores of the same tumor was also evaluated.
Results: As expected, we found a positive correlation between CD3 and CD8 levels across all patients (Pearson correlation coefficient [CC]=0.827). The levels of CD3 and CD8 showed weaker association with CD20 signal (CC=0.446 and 0.363, respectively). For all the TIL markers, the intra-tumor variation was higher than the inter-tumor differences with intraclass correlation coefficients (ICC) of 0.411 for CD3, 0.324 for CD8, and 0.252 for CD20. In the variance component analysis, 66-69% of the variance was attributable to signal differences between areas of the same tumor core and 30-33% was due to differences between cores from different areas. Consistent with this and using the median score as cutpoint to stratify cases in high/low marker levels, the concordance of measuring TILs in 1 vs 3 cores of the same tumor was κ=0.705 for CD3, κ=0.655 for CD8, and κ=0.603 for CD20.
Conclusion: Objective measurement of TIL markers indicates that T and B lymphocytes show heterogeneity in breast cancer. The tumor variation of the markers is driven predominantly by differences within the same tumor core. The data from our study suggests that although a single core biopsy of tumors provides considerable information regarding the degree of lymphocyte infiltration in breast cancer patients, caution should be taken when using this as a clinical biomarker.
Citation Format: Mani NL, Schalper K, Hatzis C, Chagpar A, Pusztai L, Rimm DL. Heterogeneity of tumor infiltrating lymphocytes in breast cancer and its impact for use as a biomarker. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-09.