Creighton, Chad J.

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.

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.

Abstract RNA sequencing detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor positive (ER+) breast cancer but their role in disease pathogenesis remains unclear. Herein we examined multiple in-frame and out-of-frame ESR1 fusions and found only two, both identified in advanced endocrine treatment resistant disease, encoded stable and functional in-frame fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, the N-terminal, DNA binding and dimerization motifs encoded by exons 2-6 were fused to C-terminal sequences from the partner gene. Functional properties included estrogen-independent growth, constitutive expression of ER target genes, anti-estrogen resistance, induction of cellular motility in vitro and the development of lung metastasis in vivo. Chromatin immunoprecipitation and RNA sequencing experiments showed both fusions uniquely activated a metastasis-associated transcriptional program. ESR1-e6>YAP1 and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, palbociclib, and a patient-derived xenograft (PDX) expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective. Citation Format: Jonathan T. Lei, Jieya Shao, Jin Zhang, Michael Iglesia, Doug W. Chan, Jin Cao, Meenakshi Anurag, Purba Singh, Xiaping He, Yoshimasa Kosaka, Ryoichi Matsunuma, Robert Crowder, Jeremy Hoog, Chanpheng Phommaly, Rodrigo Goncalves, Susana Romalho, Raquel M. Peres, Nindo Punturi, Cheryl Schmidt, Alex Bartram, Eric Jou, W V. Lai, Oliver Hampton, Anna Rogers, Ethan Tobias, Poojan Parikh, Sherri R. Davies, Shunqiang Li, Cynthia X. Ma, Vera Suman, Kelly K. Hunt, Mark A. Watson, Katherine A. Hoadley, E A. Thompson, Xi Chen, Shyam M. Kavuri, Chad J. Creighton, Christopher A. Maher, Charles M. Perou, Svasti Haricharan, Matthew J. Ellis. Functional and therapeutic significance of ESR1 gene fusions in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5240.

Abstract Dysregulation of estrogen receptor gene (ESR1) is an established mechanism of inducing endocrine therapy resistance. We previously discovered a chromosomal translocation event generating an estrogen receptor gene fused in-frame to C-terminal sequences of YAP1 (ESR1-YAP1) that contributed to endocrine therapy resistance in estrogen receptor positive (ER+) breast cancer models. This current study compares functional and pharmacological properties of additional ESR1 gene fusion events of both early stage (ESR1-NOP2) and advanced endocrine therapy resistant (ESR1-YAP1 and ESR1-PCDH11x) breast cancers. The YAP1 and PCDH11x fusions conferred estrogen-independent and fulvestrant-resistant growth in T47D, an ER+ breast cancer cell line in vitro and in vivo, in contrast to the NOP2 fusion which was sensitive to hormone deprivation. Immunohistochemical (IHC) staining of mouse lungs revealed significantly higher numbers of micrometastatic ER+ cells from the T47D tumors expressing the YAP1 and PCDH11x fusions than YFP control and NOP2 fusion. Estrogen response element (ERE) reporter and pull down assays revealed that although all ESR1 fusions studied bound EREs, only the YAP1 and PCDH11x caused ERE activation. Cell lines containing these “canonical” ESR1 fusions upregulated expression of ER responsive genes such as TFF1 and GREB1 in hormone deprived conditions. In contrast, the NOP2 fusion neither induced ERE activity nor upregulated TFF1 and GREB1 gene expression. The proliferative ability of canonical fusion-containing T47D cells was inhibited by palbociclib, a CDK4/6 inhibitor, in a dose-dependent manner. In vivo growth of patient-derived xenograft tumors naturally harboring the ESR1-YAP1 fusion (WHIM18) was significantly reduced in mice fed palbociclib-containing chow. Mice transplanted with WHIM18 also formed lung micrometastases, with an ER IHC staining pattern similar to lungs from YAP1 and PCDH11x fusion expressing T47D xenografts. In conclusion, in-frame ERE activating canonical fusions occur in end-stage, drug resistant, advanced breast cancer and can be added to ESR1 point mutations as a class of somatic mutation that may cause acquired resistance. Endocrine therapy resistant growth induced by these fusions can be treated with CDK4/6 inhibition, using an FDA approved drug, palbociclib, which could potentially improve outcomes in patients with ESR1 translocated tumors. Citation Format: Jonathan T. Lei, Jieya Shao, Jin Zhang, Michael Iglesia, Doug W. Chan, Ryoichi Matsunuma, Xiaping He, Purba Singh, Yoshimasa Kosaka, Robert Crowder, Svasti Haricharan, Shyam Kavuri, Jeremy Hoog, Chanpheng Phommaly, Rodrigo Goncalves, Susana Romalho, Wei-Chu Lai, Oliver Hampton, Anna Rogers, Ethan Tobias, Poojan Parikh, Sherri Davies, Cynthia Ma, Vera Suman, Kelly Hunt, Mark Watson, Katherine A. Hoadley, Aubrey Thompson, Charles Perou, Chad J. Creighton, Chris Maher, Matthew J. Ellis. Estrogen receptor gene fusions drive endocrine therapy resistance in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1033. doi:10.1158/1538-7445.AM2017-1033

Chromophobe renal cell carcinoma (chRCC) typically shows ~7 chromosome losses (1, 2, 6, 10, 13, 17, and 21) and ~31 exonic somatic mutations, yet carries ~5%–10% metastatic incidence. Since extensive chromosomal losses can generate proteotoxic stress and compromise cellular proliferation, it is intriguing how chRCC, a tumor with extensive chromosome losses and a low number of somatic mutations, can develop lethal metastases. Genomic features distinguishing metastatic from nonmetastatic chRCC are unknown. An integrated approach, including whole-genome sequencing (WGS), targeted ultradeep cancer gene sequencing, and chromosome analyses (FACETS, OncoScan, and FISH), was performed on 79 chRCC patients including 38 metastatic (M-chRCC) cases. We demonstrate that TP53 mutations (58%), PTEN mutations (24%), and imbalanced chromosome duplication (ICD, duplication of ≥ 3 chromosomes) (25%) were enriched in M-chRCC. Reconstruction of the subclonal composition of paired primary-metastatic chRCC tumors supports the role of TP53 , PTEN , and ICD in metastatic evolution. Finally, the presence of these 3 genomic features in primary tumors of both The Cancer Genome Atlas kidney chromophobe (KICH) ( n = 64) and M-chRCC ( n = 35) cohorts was associated with worse survival. In summary, our study provides genomic insights into the metastatic progression of chRCC and identifies TP53 mutations, PTEN mutations, and ICD as high-risk features.

Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) –positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was > 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was > 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of > 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred > 2) versus PEPI > 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI > 0 (recurrence hazard ratio [PEPI = 0 v PEPI > 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A Phase III Study; clinical trial information: NCT01953588).

Abstract Purpose: DNA methylation is a heritable covalent modification that is developmentally regulated and is critical in tissue-type definition. Although genotype–phenotype correlations have been described for different subtypes of renal cell carcinoma (RCC), it is unknown if DNA methylation profiles correlate with morphological or ontology based phenotypes. Here, we test the hypothesis that DNA methylation signatures can discriminate between putative precursor cells in the nephron. Experimental Designs: We performed deep profiling of DNA methylation and transcriptome in diverse histopathological RCC subtypes and validated DNA methylation in an independent dataset as well as in The Cancer Genome Atlas Clear Cell and Chromophobe Renal Cell Carcinoma Datasets. Results: Our data provide the first mapping of methylome epi-signature and indicate that RCC subtypes can be grouped into two major epi-clusters: C1, which encompasses clear-cell RCC, papillary RCC, mucinous and spindle cell carcinomas and translocation RCC; C2, which comprises oncocytoma and chromophobe RCC. Interestingly, C1 epi-cluster displayed 3-fold more hypermethylation as compared with C2 epi-cluster. Of note, differentially methylated regions between C1 and C2 epi-clusters occur in gene bodies and intergenic regions, instead of gene promoters. Transcriptome analysis of C1 epi-cluster suggests a functional convergence on Polycomb targets, whereas C2 epi-cluster displays DNA methylation defects. Furthermore, we find that our epigenetic ontogeny signature is associated with worse outcomes of patients with clear-cell RCC. Conclusions: Our data define the epi-clusters that can discriminate between distinct RCC subtypes and for the first time define the epigenetic basis for proximal versus distal tubule derived kidney tumors. Clin Cancer Res; 22(24); 6236–46. ©2016 AACR.