Abstract GS6-05: A joint atlas of single-cell and bulk RNA-seq in metastatic breast cancer allows inference of oncogenic and drug-resistant transcriptional programs in malignant cells and the tumor microenvironment
Authors: Cohen Ofir , Abravanel Daniel , Slyper Michal , Klughammer Johanna , Jane-Valbuena Judit , Helvie Karla , Dellostritto Laura , Frangieh Allison , Vigneau Sebastien , Wu Jingyi , Mayorga Angie , Waldman Julia , Nguyen Lan , Dionne Danielle , Ashenberg Orr , Hofree Matan , Cuoco Mike , Rodman Christopher , Winer Eric P , Lin Nancy , Johnson Bruce , Rotem Asaf , Rozenblatt-Rosen Orit , Regev Aviv , Wagle Nikhil 
Published: March 2020

Abstract
While recent studies have begun to elucidate the genomics of metastatic breast cancer (MBC), the transcriptional programs (progs) that drive the drug-resistant phenotype remain poorly understood.
We prospectively collected biopsies from patients with MBC with detailed clinicopathologic features, including treatment and response characteristics. We profiled 29 biopsies by single-cell RNA seq, as well as bulk RNA-seq and whole exome sequencing on an additional 207 biopsies (analyzed snapshot, ongoing study). We analyzed these data to generate an atlas, delineating the cell-types, cell-states, and transcriptional progs.
We profiled 100,470 single cell transcriptomes and generated a comprehensive MBC atlas of the tumor and tumor microenvironment (TME). We next inferred cell types and progs associated with clinicopathologic characteristics. For example, we found significant differences in the TME of liver metastases compared to other sites, consistent with immunosuppression in the hepatic space. In particular, liver metastases were depleted in activated B-cells (lower CD69, TCL1A, VPREB3, BCNP1, and several activation-related chemokines - CXCR4/5/6/7), and their infiltrating T-cells expressed lower levels of effector and cytotoxicity markers (table=T) including CD8, CD3, beta chemokines CCL4/CCL5, IFI16, PRF1, granzymes, and GNLY, and exceptionally low expression of antigen processing genes (T).
To increase our power to make clinically relevant associations, we performed a joint analysis of the single-cell and bulk RNA-Seq data, to identify malignant cell progs related to specific oncogenic mutations, with implications for metastatic and drug-resistance phenotypes. For example, we characterized the oncogenic prog associated with activating estrogen receptor mutation (ESR1-mut). As expected, ESR1-mut prog overlaps with many known ER and luminal B markers (T). ESR1-mut prog also included specific Interferon-stimulated genes (ISGs) - IFI6, ISG15, IFIT1, STAT1, which are associated with tamoxifen resistance (T) and ECM-mediated regulation of apoptosis (T). These ISGs are predictive of poor prognosis among endocrine-treated patients (HR=1.69. p=1e-04, n=929, kmplotter). The ESR1-mut prog also included genes associated with cell-migration (SOX9, AGR2, TXNIP and several S100 genes). This suggests a role for ESR1 mutation in pathogenicity, beyond ligand-independent activation of ER signaling. A second malignant cell prog was associated with RB-null. While most of these genes were highly correlated with cell-cycle (R=0.4), some do not (HINT1, RHOC, SNRPB, ANAPC11, TMEM208, POLR2J). In spite of decoupling from proliferation, these still predict of poor outcome across breast cancer (HR=1.41, p= 6.0e-10, n=3951, kmplotter). We characterized the prog of high-grade tumors in our cohort, and found a strong association with RB-null prog (T). Similarly, we recovered additional mutation-specific oncogenic progs, including for TP53, GATA3, FOXA1, HER2, and FGFR mutants, forming a compendium of in-vivo oncogenic signatures.
To the best of our knowledge these data represent the first integration of single cell and bulk RNA-seq data in MBC, resulting in a comprehensive single-cell-resolution transcriptional atlas, and a catalog of drug-resistance oncogenic progs with implications for immunotherapy and precision-oncology.
Single-cell In-vivo programComparison Gene setAssociationP-value(top genes)(MSigDB name, PubMed ID, when applicable)Odds-ratio(Fisher''''s Exact test, two-sided)Depleted in liver T-cellsEffectors-cells markers4.077.29E-07(compared to other metastatic site - Bone, Skin, Chest-wall, Lymph node, Breast-met)(PMID: 28052254)""Antigen processing markers32.17.26E-07(PMID: 28052254)ESR1-mutVANTVEER_BREAST_CANCER_ESR1_UP5.82.30E-11(Enriched in ESR1-mutatated)(PMID: 11823860)“SMID_BREAST_CANCER_LUMINAL_B_UP5.898.10E-12“”DOANE_BREAST_CANCER_ESR1_UP6.30812.10E-09""BOWIE_RESPONSE_TO_TAMOXIFEN10.360.00036(PMID 17016442)""BECKER_TAMOXIFEN_RESISTANCE_UP7.81.20E-06(PMID 15657362)""BOWIE_RESPONSE_TO_EXTRACELLULAR_MATRIX8.240.0028(PMID 17016442)Enriched in RB-nullEnriched in High-grade (from our study)23.97.50E-05
Citation Format: Ofir Cohen, Daniel Abravanel, Michal Slyper, Johanna Klughammer, Judit Jane-Valbuena, Karla Helvie, Laura Dellostritto, Allison Frangieh, Sebastien Vigneau, Jingyi Wu, Angie Mayorga, Julia Waldman, Lan Nguyen, Danielle Dionne, Orr Ashenberg, Matan Hofree, Mike Cuoco, Christopher Rodman, Eric P Winer, Nancy Lin, Bruce Johnson, Asaf Rotem, Orit Rozenblatt-Rosen, Aviv Regev, Nikhil Wagle. A joint atlas of single-cell and bulk RNA-seq in metastatic breast cancer allows inference of oncogenic and drug-resistant transcriptional programs in malignant cells and the tumor microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS6-05.