Abstract P2-07-01: Integrative analyses of immunophenotypes and multi-omics profiles in breast cancers
Authors: Kan Z , Powell E , Ram S , Ching K , Ding Y , Vizcarra P , Nichols T , Hardwick J , Lee S-H , Cho SY , Choi Y-L , Yu J-H , Park YH 
Published: February 2018

Abstract
The advent of immuno-oncology (IO) therapies has made it an imperative to characterize intratumoral immune microenvironment in addition to oncogenic alterations through molecular profiling of the tumor. To elucidate the baseline profiles of tumor infiltrating leukocytes (TILs) in breast cancer (BC) in the context of molecular subtypes and oncogenic alterations, we performed whole-exome sequencing (WES) and RNA-Seq of an Asian BC cohort (SMC) consisting of 178 treatment naïve primary tumors. A subset of 120 tumors was further analyzed by H&E and IHC using a panel of 8 TIL markers (CD45, CD4, CD8, CD163, PD1, PD-L1, IDO1 and FOXP3). Using expression signatures representing distinct immune cell types, we classified an expression compendium of 2,781 tumor samples, including SMC and multiple cancers from TCGA, into three immune subtypes with high, medium and low levels of TILs. Basal and HER2 subtypes show higher levels of TILs than Luminal subtypes, consistent with observed clinical responses to checkpoint blockade in clinical trials. Moreover, Asian BCs were significantly enriched in TIL-high subtype (35.3%) compared to the primarily Caucasian TCGA BC cohort (20.2%) while 50.6% of the highly immunogenic Lung adenocarcinoma was TIL-high. We then applied machine learning methods to detect and quantify TILs from H&E images of 120 SMC and 349 TCGA BC tumors. The expression signature analysis results were concordant with independently derived histology based TIL data. Taken together, our findings suggest that IO therapies may be more effective in HR negative BC subtypes and Asian BCs.
Leukocyte exclusion (LE), an immunophenotype where TILs concentrate at the tumor periphery, has been linked to worse prognosis and resistance to IO therapies. Visual assessment of whole tumor IHC images identified LE patterns in 25% of SMC cases. We observed differential distribution of LE by molecular subtype and evidence for selective exclusion of immune cell subsets. Covariate analyses with clinical and molecular data while controlling for subtype as a confounder identified significant associations with tumor proliferation index, percent tumor purity and TP53 mutations. LE is also significantly associated with expression signatures of chemokine signaling, macrophages, angiogenesis and hypoxia, indicating that marked distinctions exist in both tumor intrinsic and microenvironment characteristics between TIL excluded and TIL infiltrated tumors. To validate these findings, we independently identified LE for 200 cases of TCGA BCs based on patterns of TILs extracted from H&E images and saw significant concordance of covariate relationships identified between TCGA and SMC. Our study provided a rare comprehensive resource for studying tumor associated immunity in breast cancers by generating the integrated multi-omics and IO profiles for a large cohort of primary tumors. Comparative analyses revealed that TIL activities are highly variable across different intrinsic subtypes and geographic origins of BC, with potential implications for IO therapeutic application. Correlative analyses of immunophenotypes with molecular data further yielded insights into LE's role in immune escape and identified hallmark signatures for LE indicative of causal molecular mechanisms.
Citation Format: Kan Z, Powell E, Ram S, Ching K, Ding Y, Vizcarra P, Nichols T, Hardwick J, Lee S-H, Cho SY, Choi Y-L, Yu J-H, Park YH. Integrative analyses of immunophenotypes and multi-omics profiles in breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-01.