Background Clinical trials have shown minimal activity of immune checkpoint blockade (ICB) in HR+ breast cancer. While this is, in part, attributable to low tumor-infiltrating lymphocyte (TIL) levels in HR+ breast cancer, a thorough investigation of the immunologic aspects of the tumor microenvironment (TME) is required to fully understand the limited efficacy of ICB in HR+ disease. We sought to characterize the TME of HR+ breast cancer at single cell resolution using single-cell RNA-seq (scRNA-seq), paired with tissue-based cyclic immunofluorescence (t-CyCIF), a multiplexed imaging technology for formalin-fixed paraffin-embedded (FFPE) tissue sections across up to 30 distinct antigen channels. We hypothesized that integrative analysis of clinical samples using these technologies would elucidate inter-patient heterogeneity of the TME of HR+ breast cancer. Methods Surgically excised primary tumor samples were obtained from five, treatment-naïve, HR+ breast cancer patients. Each tumor was divided with half preserved as a FFPE block for t-CyCIF and bulk RNA-seq, and half frozen and later dissociated into single cells for scRNA-seq. For each t-CyCIF cycle, slides were stained with 3 antibodies and a DNA dye. The resultant image was captured with a CyteFinder (RareCyte) slide scanning fluorescence microscope. Each slide was stained for 3-7 cycles (9-21 antibodies), depending on tissue stability. Acquired images were processed (illumination correction, alignment, stitching, and segmentation) using mcmicro (https://mcmicro.org/) and the fluorescence intensity per cell per antibody was determined. After identifying regions where cells were appropriately stained throughout the cycles, the tumor, stromal, and immune cell types were determined based on expressed antigens. Samples were further characterized based on the spatial distribution of T cells within the tumor and categorized into three classes: immunologically cold (lacking T cells), immune stromal-restricted (T cells located predominantly in the stromal) and highly inflamed (T cells located throughout entire tumor). CyCIF data was compared to bulk RNA-seq data for each sample. For scRNA-seq, two samples with high cellularity were identified. The single-cell barcoded library was constructed with 10X Chromium and sequenced with NextSeq (Illumina). The cell types were compared as were gene expressions of the corresponding T cell populations between the two samples. For all experiments, downstream analysis was performed in R. Results CyCIF analysis revealed diverse compositions of immune cell types. In particular, the spatial distributions of T cells revealed that the five samples were classified into the three TME classes: 2 immune cold, 1 immune stromal-restricted, and 2 highly inflamed. This trend was consistent in the corresponding bulk RNA-seq data, in which we found higher expression of an interferon-gamma signaling signature in the two highly-inflamed tumors. For the two scRNA-seq samples, one corresponds to the immunologically cold class in CyCIF and the other to the immune stromal-restricted class. Consistent with CyCIF analysis, scRNA-seq revealed that the immune stromal-restricted tumor had higher T cell infiltration than the immune cold tumor, particularly with a higher abundance of naïve T cells, Th17 cells, and helper T cells. Conclusions While HR+ breast cancer is generally considered immunologically cold, our pilot analysis integrating CyCIF, scRNA-seq, and bulk RNA-seq revealed considerable diversity between the five HR+ samples, including the distribution of T cells relative to tumor cells. Additional samples are being collected to allow for more detailed exploration of the TME heterogeneity of HR+ breast cancer. Citation Format: Kenichi Shimada, Yvonne X Cui, Jonathan S Goldberg, Ricardo Pastorello, Janae Davis, Tuulia Vallius, Lukas Kania, Ashka Patel, Mckenna Moore, Esther R Ogayo, Deborah Dillon, Peter K Sorger, Jennifer L Guerriero, Elizabeth A Mittendorf. Integrative analysis of single-cell transcriptomic and spatial profiles characterized distinct tumor microenvironment phenotypes in hormone receptor positive (HR+) 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 P4-04-06.

Background: PARP inhibitors (PARPi) significantly extend progression-free survival (PFS) compared to chemotherapy in pts with BRCA1/2 mutated (BRCA1/2m) ABC, but responses are not durable. PARPi activate the cGAS-STING pathway leading to increased PD-L1 expression and cytotoxic T-cell recruitment, creating a tumor microenvironment (TME) that may be more vulnerable to immunotherapy. This study was conducted to evaluate the safety, efficacy and effects on the TME of the PARPi tala combined with the PD-L1 inhibitor avelumab in ABC, and to assess the impact of BRCA1/2 status on clinical outcomes. Methods: TALAVE was an open-label, multi-institutional trial (NCT03964532) for pts with HER2-negative ABC. Pts were enrolled in two cohorts: cohort 1 - BRCA1/2m and HER2-negative ABC; cohort 2 - BRCA1/2 wildtype TNBC. Pts received a 4-week induction of tala (1mg po daily D1-D28), followed by a combination of daily tala and avelumab (800mg IV D1, D15). The primary objective was the safety and tolerability of the combination. Secondary objectives included ORR, OS and PFS. Pts underwent serial biopsies to investigate molecular signatures associated with BRCA status or clinical benefit using multiple omics techniques: RNA profiling by NanoString PanCancer IO 360™ Panel, GeoMx® Digital Spatial Profiler (DSP) Whole Transcriptome Atlas (WTA) and protein spatial analysis by multiplex immunofluorescence (mIF) and Cyclic Immunofluorescence (CyCIF). Results: 12 pts were enrolled in each cohort. In cohort 1, 5 pts had gBRCA1, 6 had gBRCA2 and 1 had sBRCA2 mutation. The median age was 50 [IQR:43-59.5]; all pts were female, with median of 1 prior therapy for ABC [IQR: 0-2.5]. 42% pts had prior platinum. ORR was 42% (83% in cohort 1; 0% in cohort 2). There were 10 PRs, all in cohort 1. mPFS was 5.1 months (mo) (95% CI: 3.7-7.3 mo); 9.3mo in cohort 1 and 2.9mo in cohort 2. 5 out of 24 pts remain on treatment, all in cohort 1. Treatment related adverse events (TRAEs) included anemia 33%, neutropenia 25% (gr3+ 13%), thrombocytopenia 21% (gr3+ 13%), fatigue 33% and nausea 29%. Other TRAEs gr3+ included dyspnea (4%) and AST elevation (4%). There were no gr5 events. RNA analysis showed that in cohort 1, tala monotherapy disrupted MMEJ, induced antiproliferative effects and expression of genes in the cGAS-STING pathway, including TBK1-mediated IRF3 activation, with downstream induction of T-cell, dendritic cell and cytokine gene expression. These effects were not seen in biopsies post tala monotherapy in cohort 2. mIF analyses demonstrated T-cell and macrophage infiltration in BRCA1/2m tumors. Analyses of post-combination biopsies is ongoing. Conclusions: There were no new safety signals of PARPi combined with immunotherapy. Responses were limited to pts with BRCA1/2m. RNA and protein analyses indicate cGAS-STING activation and immune cell infiltration in BRCA1/2m tumors, validating murine preclinical findings. Citation Format: Filipa Lynce, Kenichi Shimada, Xue Geng, Edward T. Richardson, Candace Mainor, Mei Wei, Julie M. Collins, Paula P. Pohlmann, Arielle L. Heeke, Kelly F. Zheng, Madeline Townsend, Jane Staunton, Stuart J. Schnitt, Joan S. Brugge, Hongkun Wang, Claudine Isaacs, Geoffrey I. Shapiro, Jennifer L. Guerriero. TALAVE: Induction talazoparib (tala) followed by combined tala and avelumab in patients (pts) with advanced breast cancer (ABC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT142.

Many chemotherapeutic agents target cellular components or processes that are present in all cancers, yet clinical responses to these agents vary greatly between cancer types and even patient age - the basis for these broad-scale differences are unclear. The vast majority of targeted and cytotoxic cancer therapies including ionizing radiation produce pro-apoptotic signaling in exposed cells, suggesting that the mitochondrial apoptosis sensitivity of cancer cells could act as a central signaling “node” to broadly impact therapy outcomes. To test this, we used BH3 profiling and complementary chemosensitivity assays to analyze hundreds of primary cancer specimens across twelve major cancer types. We find that cancers with typically favorable outcomes including certain hematologic malignancies, testicular cancer, and some pediatric cancers contain mitochondria that are highly primed for apoptosis, which renders them hypersensitive to cytotoxic as well as targeted agents and radiation therapy. Priming levels in many epithelial cancers including ovarian cancer and non-small cell lung cancer are highly heterogeneous, mirroring their variability in clinical outcomes. Finally, many tumor types that are typically chemoresistant including adult soft tissue sarcomas, hepatocellular carcinoma and pancreatic cancer are almost completely resistant to pro-apoptotic signaling. By analyzing in vitro and in vivo pancreatic, ovarian, hepatocellular and sarcoma tumorigenesis models, we find that apoptotic priming generally increases during neoplastic transformation, in part due to consistent upregulation of pro-apoptotic proteins BAX and BAK. However, the level of apoptotic priming in cancer cells is constrained by the baseline apoptosis sensitivity of normal cells prior to transformation. Remarkably, we find that apoptotic priming is dynamically regulated by cell lineage and differentiation state but can also be modulated by oncogenes. For instance, Myc activation typically increases apoptotic priming while activation of mutant Ras signaling decreases it - these changes in priming alter the chemosensitivity of cancer cells. Finally, we use inducible mouse tumor models to demonstrate that neoplastic transformation of cells from developmentally immature tissues yields pediatric tumors that are more primed for apoptosis than equivalent tumors arising in adults. This difference in priming causes pediatric tumors to be more sensitive to front-line therapies and BH3 mimetics targeting pro-survival BCL-2 family proteins in vitro and in vivo. Thus, lineage-determined regulation of apoptosis prior to and during neoplastic transformation leads to broad-scale differences in cancer cell chemosensitivity and can be exploited therapeutically by targeting BCL-2 family proteins. Citation Format: Cameron Fraser, Xingping Qin, Kenichi Shimada, Johan Spetz, Mary Heather Florido, Rumani Singh, Stacey Yu, Adam Presser, Zintis Inde, Gaurav Joshi, Jennifer Guerriero, Francisco Sanchez-Rivera, Alison Karst, Omar Lopez, Chendi Li, Peter Winter, Ying Yue, Peter Sorger, Jingwei Cheng, Izidore Lossos, Aaron Hata, Ronny Drapkin, Adam Palmer, James Decaprio, Manisha Thakuria, Charles Yoon, Ursula Matulonis, Matthew Meyerson, Elizabeth Stover, Diana Cardona, Kris Wood, Shayna Sarosiek, David Kirsch, Joseph Mancias, Andrew Cherniack, Anthony Letai, Kristopher Sarosiek. Cancer sensitivity to therapy is constrained by apoptosis regulation in cells of origin. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6130.