Lee, Adrian V.

Abstract Patients with metastatic breast cancer (MBC) typically have short survival times and their successful treatment represents one of most challenging aspects of patient care. This poor prognostic behavior is in part due to molecular features including increased tumor cell clonal heterogeneity, multiple drug resistance mechanisms, and alterations of the tumor microenvironment. The AURORA US Metastasis Project was established with the goal to identify molecular features specifically associated with metastasis. We therefore collected and molecularly characterized specimens from 55 metastatic breast cancer (BC) patients representing 51 primary cancers and 102 metastases. The 153 unique tumors were assayed using RNAseq, tumor/germline DNA exomes and low pass whole genome sequencing, and global DNA methylation microarrays. We found intrinsic molecular subtype differences between primary tumors and their matched metastases to be rare in triple negative breast cancer (TNBC)/Basal-like subtype tumors. Conversely, tumor subtype changes were relatively frequent in estrogen receptor positive (ER+) cancers where ~30% of Luminal A cases switched to Luminal B or HER2-enriched (HER2E) subtypes. Clonal evolution studies identified changes in expression subtype coincident with DNA clonality shifts, especially involving HER2 amplification and/or the HER2E expression subtype. We further found evidence for ER-mediated downregulation of genes involved in cell-cell adhesion in metastases. Microenvironment differences varied according to tumor subtype where ER+/Luminal metastases had lower fibroblast and endothelial cell content, while TNBC/Basal-like metastases showed a dramatic decrease in B cells and T cells. In 17% of metastatic tumors, we identified DNA hypermethylation and/or focal DNA deletions near HLA-A that were associated with its’ significantly reduced expression, and with lower immune cell infiltrates. We also identified low immune cell features in brain and liver metastases when compared to other metastatic sites, even within the same patient. These findings have direct implications for the treatment of metastatic breast cancer patients with immune- and HER2-targeting therapies and suggest potential novel therapeutic avenues for the improvement of outcomes for some patients with MBC.

Breast cancer is the most prominent type of cancer among women. Understanding the microenvironment of breast cancer and the interactions between cells and cytokines will lead to better treatment approaches for patients. In this study, we developed a data-driven mathematical model to investigate the dynamics of key cells and cytokines involved in breast cancer development. We used gene expression profiles of tumors to estimate the relative abundance of each immune cell and group patients based on their immune patterns. Dynamical results show the complex interplay between cells and molecules, and sensitivity analysis emphasizes the direct effects of macrophages and adipocytes on cancer cell growth. In addition, we observed the dual effect of IFN-γ on cancer proliferation, either through direct inhibition of cancer cells or by increasing the cytotoxicity of CD8+ T-cells.

Abstract BACKGROUND: Brain metastatic disease occurs in 10-30% of metastatic breast cancer cases. The incidence of brain metastases is increasing, yet overall survival remains < 2 years. Treatment of brain metastases is limited with current clinical practice centered on radiation, chemotherapy and surgery. Although these treatments may prolong survival in the short term, targeting oncogenic alterations in brain metastases may deliver a more sustained clinical benefit. In this multicenter study, we comprehensively characterized DNA and RNA alterations that aberrantly drive specific oncogenic pathway activity pertinent to breast cancer brain metastases (BCBM). METHODS: RNA sequencing was performed on a cohort of patient-matched primary and resected brain metastatic tumours (45 patients; N=90 samples). Whole exome DNA sequencing (WXS) was performed for 18/45 patients (54 trios consisting of primary tumor, brain metastasis and matched normal tissue). An independent brain metastatic WXS cohort (N=21 patients) (PMID: 26410082) was also analysed resulting in a total of 39 patient samples. Recurrent somatic copy number alterations (SCNA), somatic single nucleotide variants (SNVs) and mutational signatures were identified from WXS data. Expressed gene fusions were detected computationally from RNA-Seq (N=45 cases) RESULTS: Of the 45 BCBM patients, median age at diagnosis was 51 years [25, 67], median overall survival 57 months (range 18-255) with median brain metastases free survival 34 months (range 5-216).Clinical molecular subtype of the primary tumour included 13 ER+/HER2- (29%), 16 HER2+ (35.5%) and 16 TNBC (35.5%). Regions of significant recurrent amplifications and deletions in BCBM (N=39 patients) were identified in 4q12, 10q11.21, 8p11.23, 8q23.3 and 17q12 (FDR < 0.10). Recurrent expressed gene fusions identified in known cancer driver genes were associated with chromatin modification, MAPK and HER signaling pathways. Mutational signature analysis of SNVs identified signatures associated with ageing, mismatch repair and homologous recombination deficiency (HRD) mutational processes. The relative contribution of the HRD signature was significantly increased in brain metastases compared to matched primary tumour (p < 0.05). Concordantly increased HRD in the brain metastatic transcriptome was confirmed in these patients by gene set variation analysis (GSVA) of homologous recombination pathway genes from KEGG database in RNA-Seq data. Moreover, in the extended BCBM RNA-Seq (N=45 patients) GSVA pathway scores were elevated in brain metastases relative to primary tumours, validating the functional significance of altered DNA repair defects in brain metastases CONCLUSIONS: Here, we report recurrent genetic drivers, supported by altered functional transcriptome, unique to brain metastasis that may have clinical implications for prognosis and treatment choice. Specifically, targeting defects in the homologous recombination repair mechanism may represent new therapeutic strategies and management opportunities for breast cancer brain metastases patients. Citation Format: Nicola S Cosgrove, Damir Varešlija, Stephen Keelan, Simon Furney, Jodi M Carter, Steven N Hart, Siddhartha Yadav, Arnold DK Hill, Steffi Oesterreich, Adrian V Lee, Fergus J Couch, Leonie Young. Homologous recombination deficiency represents a new therapeutic strategy for breast cancer brain metastases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-01.

Abstract Background: It has become increasingly clear that effective treatment of metastatic breast cancer (MBC) requires an in-depth understanding of the molecular differences between primary tumors and metastases. The AURORA US Network was established to collect primary breast cancer-metastasis pairs for multi-platform genomic profiling in order to identify the molecular drivers of metastatic disease. AURORA US has both a retrospective and prospective phase. This is the first report of the retrospective phase. Methods: Archived tissue samples from the primary tumor and at least one distant metastasis were retrospectively collected from 83 MBC patients. Following internal quality assessment, samples from 55 pts, including 105 distinct metastatic lesions, were subject to DNA low pass whole genome and exome sequencing, DNA methylation arrays, and RNA sequencing. Early analyses of these multi-platform data include: DNA methylation, tumor gene expression and microenvironmental signatures, somatic and germline variants, DNA copy number changes, and structural variants between breast primaries and matched metastases. Results: Median age at diagnosis was 49 years (25-76); 32 (58%) were Stage I or II at presentation, 27 (49%) had a family history of breast cancer, and 20 (36%) had a second breast primary. Median disease-free interval before developing MBC was 2 years (range 0-36, 5 patients presented with Stage IV). Median overall survival from initial presentation was 4 years (range 0-37). Median survival after developing MBC was 1 year (range 0-13), with a median of three treatments. Primary tissue samples were banked from 1977-2017 and metastases were banked from 1999-2017. Clinical phenotypes of the primaries included 27 HR+ (49%), 15 triple negative (TNBC, 27%), and 11 HER2+ (20%, 12 missing HER2 status). Intrinsic subtype distribution of the primaries included 17 Basal-like (31%), 17 Luminal A (31%), 7 Normal-like (13%), 5 HER2-enriched (9%), and 1 Luminal B, with 8 pending. All metastases from the Basal-like cases remained Basal-like, while metastases from luminal primaries tended to gain HER2-Enriched subtype features (5/18, p = 0.01). Overall, we identified significant metastasis-enriched alterations in metabolism pathways, an increase in proliferation, and the loss of differentiation signatures and immune infiltrates with progression; the latter being the most pronounced in brain metastases. The most frequent somatic mutations in this cohort were in TP53, NCOR1, and RUNX1. Interestingly, ERBB2, EGFR, and ATM were also mutated in ≥10% of the tumors sequenced. In almost all cases, CpG island hypermethylation was clonally present in the primary tumor and persisted stably in the majority of metastatic lesions. Promoter CpG island hypermethylation was also identified in some metastatic lesions at JAM3, an important cellular adhesion molecule,and this was accompanied by reduced mRNA expression. CONCLUSIONS: Collection of banked primary and metastatic tissue pairs identified a young MBC cohort with a high frequency of breast cancer family history and second breast primaries. Molecular characterization of luminal tumor pairs highlighted acquisition of aggressive traits including increased proliferation and loss of differentiation in the metastases. In contrast, basal-like pairs remained relatively unchanged, except for the loss of immune activation. Ongoing analyses to be presented include clonal heterogeneity and phylogeny, novel metastasis signature discovery, gene fusion, and endogenous retrovirus detection. Citation Format: Tari A King, Minetta C Liu, Marni B McClure, Toshinori Hinoue, Benjamin J Kelly, Chad J Creighton, Jay Bowen, Kristen Leraas, Robyn T Burns, Sara Coppens, Salma Rezk, Amy L Garrett, Justin M Balko, Joel S Parker, Ben H Park, Ian Krop, Carey Anders, Katherine A Hoadley, Julie Gastier-Foster, Mothaffar F Rimawi, Rita Nanda, Nancy U Lin, Claudine Isaacs, P. Kelly Marcom, Anna Maria Storniolo, Fergus J Couch, Elaine R Mardis, Adrian V Lee, Uma Chandran, Peter W Laird, Susan G Hilsenbeck, Larry Norton, Andrea L Richardson, W. Fraser Symmans, Lisa A Carey, Antonio C Wolff, Nancy E Davidson, Charles M Perou, the AURORA US Network. Multiplatform analysis of matched primary and metastatic breast tumors from the AURORA US Network [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 GS3-08.