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The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression. Significance: Mutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1–AXL axis and to stimulate lymphovascular angiogenesis.

Background: Aberrant activation of the pioneer transcription factor (TF) FOXA1 contributes to endocrine resistance and metastasis in ER+ breast cancer (BC) by promoting genome-wide enhancer and transcriptional reprogramming that engages the AP-1 TF complex. Identification of central transcriptional nexuses in this deregulated network is key for developing new therapeutic interventions focusing on transcriptional programs. We previously identified FRA1, among the AP-1 components, as the top super-enhancer target forming a FOXA1/FRA1-centered transcriptional axis to activate genes enriched in luminal B-subtype BC and ER+ metastases. To further dissect the FOXA1/FRA1-centered transcriptional axis and its potential therapeutic role, we employed integrative multi-omics data analysis and functional studies targeting FRA1 using our MCF7-parental (P) and FOXA1-amplifed tamoxifen-resistant (TamR) preclinical models. Methods: RNA-seq data were obtained in MCF7-P and TamR cells with FOXA1 and/or AP-1 (FRA1 and c-Jun) perturbation via overexpression and/or si/shRNA knockdown (KD). Differential gene expression was analyzed using DESeq2 or the limma-voom R package. ChIP-seq-based genome-wide FOXA1 binding sites were further refined by intersection with promoter-tethered regions (PTRs) denoting Hi-C-mapped chromatin looping. Genomic binding of FOXA1/c-Jun and H3K27ac modification at target gene loci were aligned and visualized by IGV. Significance of gene set enrichment was determined using a chi-square test adjusted for multiple comparisons. Clinical relevance was examined using an RNA-seq dataset of ER+ metastatic BC (SABCS19-GS2-02). Clonogenicity, soft agar, and wound-healing assays were performed using MCF7-TamR cell derivatives engineered for doxycycline (Dox)-inducible KD of FRA1 or NS shRNA. Significance of the KD effects in functional assays was determined using a linear mixed-effects model. Results: We found that FRA1 and the two embryonic TFs SOX9 and KLF4 that harbor FOXA1-bound PTRs were commonly down-regulated in TamR cells upon KD of FOXA1, FRA1, or c-Jun. We observed increased c-Jun binding at the FOXA1-bound super-enhancer and the PTR looping to the FRA1 gene locus in TamR vs. P cells, suggesting a feed-forward mechanism by which FRA1 transcription is strengthened by the AP-1-engaged super-enhancer in TamR cells. Among the FOXA1-activated genes in TamR cells, a subset of secretory protein-encoding genes was more enriched in the genes commonly dependent on both FRA1 and c-Jun, vs. the genes depending on either FRA1 or c-Jun alone. This FOXA1/FRA1/c-Jun-activated secretome is enriched for multiple biological processes engaged in tumor metastasis and associated microenvironmental niches, and was upregulated in clinical ER+ metastases vs. primary tumors. A larger proportion of this secretome, including CXCL8 and S100P, also relies on ER preferentially in TamR vs. P cells (40% vs. 16%, respectively). FRA1 KD using two different shRNA sequences in TamR cells reduced SOX9 and KLF4 expression levels, and significantly diminished clonogenicity, soft agar colony formation, and wound healing, compared to the control NS KD. Conclusions: Our integrative bioinformatics analyses reveal a feed-forward mechanism on FRA1 activation in amplifying high-FOXA1-induced transcriptional reprogramming in endocrine resistance. A prometastatic secretome activated by FRA1/c-Jun may represent a main transcriptional output of the FOXA1/FRA1-dependent nexus in promoting ER+ disease progression. These identified key transcriptional nexuses may present network hotspots susceptible to therapeutic interventions in which FRA1 inhibition could be used as a new transcriptional program-oriented therapy to treat advanced ER+ BC. Citation Format: Chia Chia Liu, Lanfang Qin, Carmine De Angelis, Sarmistha Nanda, Resel Pereira, Martin J. Shea, Agostina Nardone, Rinath Jeselsohn, Ofir Cohen, Nikhil Wagle, Zhijie Liu, Mothaffar F. Rimawi, C. Kent Osborne, Rachel Schiff, Xiaoyong Fu. Targeting the FRA1-dependent transcriptional nexus in high FOXA1-driven endocrine-resistant and metastatic 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 PD1-05.

Alterations in DNA methylation are one of the earliest, most common signatures of cancer development, making them ideal biomarkers for early detection. Methylation profiling of plasma cell-free DNA (cfDNA) has significant potential to expand the use of liquid biopsies to cancer screening. The EpiCheck platform combines methylation-sensitive restriction endonuclease (MSRE) digestion with whole genome sequencing to comprehensively map genome-wide DNA methylation changes with high fidelity. In this proof-of-concept study, MSRE-NGS was used to interrogate a liquid biopsy atlas focused on early-stage lung cancers to i) identify informative NGS methylation biomarkers in plasma, and ii) develop a machine learning method to discriminate between cancer and high-risk, non-cancer controls. The EpiCheck lung cancer atlas was constructed using biospecimens from academic (UBC, Vanderbilt, Cleveland Clinic) and commercial biobanks, and from a prospective multi-center study (NCT04968548). Plasma samples were collected from 93 high-risk primary lung cancer patients (62% stage I) and 90 high-risk individuals without cancer. Extracted DNA was digested with methylation-sensitive endonucleases and sequenced at an average depth of 600x. Methylation levels of ~6 million genomic loci were rank ordered using Student's t-test. Gene set enrichment analysis (GSEA) was performed on the top ranking 1000 differentially methylated loci. A logistic regression classifier with Lasso regularization was trained on 100,000 loci, and performance was examined by mean AUC using 5-fold cross-validation. A total of 23,130 loci exhibited significant differential methylation patterns between cancers and controls (p Citation Format: Dvir Netanely, Stephen Lam, Anna McGuire, Stephen Deppen, Eric Grogan, Fabien Maldonado, Michael Gieske, Joseph Seaman, Kimberly Rieger-Christ, Satish Kalanjeri, Luis Herrera, Nichole Tanner, Garrett B. Sherwood, Orna Savin, Shacade Danan, Sarah Zaouch, Nimrod Axelrad, Revital Knirsh, Ofir Shliefer, Keren Manor, Radha Duttagupta, Aharona Shuali, Peter J. Mazzone, Gerard A. Silvestri, Catherine A. Schnabel, Danny Frumkin, Adam Wasserstrom. Machine-learning-based epigenetic detection of early-stage lung cancers using the EpiCheck liquid biopsy platform. [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 5434.

Background: Genomic mechanisms associated with response to ICI in mTNBC are largely unknown. The aim of this work is to assess the genomic and immune profiles of mTNBC samples collected from patients (pts) treated with ICI. Methods: We identified 31 women with mTNBC treated with ICI (pembrolizumab, n=6, NCT02447003; atezolizumab, n=4, NCT01375842; nivolumab + cabozantinib, n = 6, NCT03316586; pembrolizumab + eribulin, n=8, NCT02513472; atezolizumab + nab-paclitaxel, n=7, NCT01633970) who had tumor tissue or blood available for sequencing obtained before and after ICI. Clinical benefit (CB), here defined as any objective response or stable disease (SD) for > 24 weeks, was observed in 20 pts (65%). An extraordinary responder was defined as having CB ≥ 2 yrs; 5 pts were considered extraordinary responders (range 26-60months). Whole exome sequencing (WES) was done on each tumor and on germline DNA from blood (23 pts had successful WES performed on samples collected before ICI; 5 of these had WES on samples taken after disease progression). RNA sequencing (RNAseq) was successfully performed in 18 of the tumors with WES performed on samples before ICI; and 3 of these had RNAseq on samples taken after disease progression. 18 pts had tumors assessed by multiplex immunofluorescence (mIF) panels encompassing CD4, CD8, PD-1, PD-L1, and cytokeratin on samples collected before ICI. WES, deep targeted panel and low coverage whole genome sequencing were performed on serially collected plasma samples from 22 pts to evaluate tumor fraction and specific mutations. The association between biomarkers and clinical benefit to ICI was assessed. Results: 21 of 31 pts (67%) had received ≥1 prior lines of systemic therapy in the metastatic setting before starting ICI. Among the most frequently mutated genes at baseline are: TP53 (57%); PIK3CA (18%); DNAH5, MYH8 (both 13%); KMT2C, AKT1, LAMA2 (all 9%). Pts with CB had a higher tumor mutational burden (TMB) than pts with no CB (p=0.018). Differential expression analysis of RNAseq data revealed an upregulation of several immune-related genes in pts with CB, indicating increased immune infiltration in that group. Gene set enrichment analysis of this expression data using hallmark and canonical pathway gene sets from MSigDB (nominal p-val < 0.05) showed that, compared to samples from pts without CB, extraordinary responders had elevated transcriptional signatures of several cancer-related pathways associated with cell survival, proliferation and metabolism, as well as genes associated with increased immune infiltration and upregulation of inflammatory response programs. The mIF showed that the tumor microenvironment (TME) of pts with CB were enriched in Cytokeratin-negative/PD-L1-positive cells compared to those without CB (p=0.014). Expression of CD4, CD8 and PD-1 was not significantly different between pts with and without CB. Genomic analysis of circulating tumor DNA, and tumor evolutionary analysis for pts with both pre- and post-ICI samples (acquired resistance) will be presented. Conclusions: Clinical benefit to ICI in mTNBC was associated with upregulation of immune-related pathways, enrichment of non-tumoral PD-L1-positive cells in TME, and high TMB. Citation Format: Romualdo Barroso-Sousa, Juliet Forman, Zachary T. Weber, Katherine Collier, Katrina Z. Kao, Edward T. Richardson, III, Tanya Keenan, Ofir Cohen, Michael P. Manos, Ryan C. Brennick, Patrick Ott, F. Steve Hodi, Deborah A. Dillon, Nancy U. Lin, Eliezer E. Van Allen, Scott Rodig, Eric P. Winer, Elizabeth A. Mittendorf, Catherine J. Wu, Daniel Stover, Nikhil Wagle, Sachet Shukla, Sara Tolaney. Comprehensive genomic analysis reveals molecular correlates of response to immune checkpoint inhibitors (ICI) in metastatic triple-negative breast cancer (mTNBC) [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 PS4-25.

Background: The CDK4/6 inhibitors have emerged as standard first- or second-line regimens in combination with an antiestrogen for patients with HR+/HER2- MBC. While these agents convey significant clinical benefit in many patients, intrinsic resistance can occur and, in patients who respond, acquired resistance is unfortunately inevitable. Despite their widespread use, we have limited insight into the molecular mechanisms governing response and resistance to these agents. Methods: Whole exome sequencing (WES) was performed on metastatic tumor biopsies from 58 patients (pts) with HR+/HER2- MBC who received a CDK4/6 inhibitor with or without an antiestrogen at the Dana-Farber Cancer Institute, including 7 pts with pre/post-exposure biopsy pairs. Among these biopsies, 69.5% were characterized as resistant (intrinsic or acquired) and 30.5% were characterized as sensitive. To validate putative resistance mediators identified in patient samples, HR+/HER2- breast cancer cells were modified via CRISPR knockout or lentiviral overexpression. Sensitivity of these cells to antiestrogens and CDK4/6i was interrogated via cell-titer-glo assay. In parallel, HR+/HER2- breast cancer cells were cultured to resistance in the presence of an escalating dose of CDK4/6i. Derivative cell lines were subjected to western blotting in an effort to interrogate the putative resistance mediators identified in pts. Novel dependencies were identified in these derivative cell lines via treatment with targeted therapeutic agents in vitro. Results: WES of tumors with CDK4/6i exposure revealed candidate mechanisms of resistance including biallelic RB1 disruption (n=4, 10%) and activating events in AKT1 (n=5, 12.5%), RAS (n=4, 10%), aurora kinase A (AURKA, n=11, 27.5%), and cyclin E2 (CCNE2, n=6, 15%). Convergent evolution toward biallelic RB1 disruption was identified in a single patient with one pre- and two post-exposure biopsies, while acquisition of AKT1 mutation and amplification was identified in two separate instances. Knockout of RB1 and overexpression of AKT1, KRAS G12D, AURKA, and CCNE2 provoked CDK4/6i and antiestrogen resistance in vitro. Breast cancer cells cultured to resistance in CDK4/6i demonstrated concordant acquisition of RB1 downregulation, RAS/ERK activation, AURKA overexpression, and CCNE2 overexpression. Derivative resistant cell lines with RB1 loss or AURKA gain demonstrated enhanced sensitivity to a novel AURKA inhibitor (LY3295668), while cells with RAS activation were highly sensitive to ERK inhibition (via LY3214996). CCNE2-overexpressing cells were highly sensitive to prexasertib, a CHEK1 inhibitor. Conclusions: The genomic landscape of resistance to CDK4/6i is heterogeneous with multiple potential mediators that play well-established roles in cell division and oncogenic signal transduction. We present novel mechanisms of clinical resistance including activation of AKT1 and RAS family oncogenes as well as amplification of AURKA and CCNE2. These drivers were able to provoke resistance to CDK4/6i in vitro. Finally, in each case, a novel dependency was identified which is readily translatable into the clinic. These results underscore the potential of next-generation sequencing as a critical tool to enable identification of resistance mediators, while also suggesting that the presence of specific genomic alterations may define new therapeutic opportunities in CDK4/6i-resistant HR+ MBC. Citation Format: Seth A. Wander, Ofir Cohen, Xueqian Gong, Gabriela N. Johnson, Jorge Buendia-Buendia, Maxwell Lloyd, Dewey Kim, Flora Luo, Pingping Mao, Karla Helvie, Kailey Kowalski, Utthara Nayar, Stephen Parsons, Ricardo Martinez, Lacey Litchfield, Xiang Ye, Chun Ping Yu, Valerie Jansen, Levi A. Garraway, Eric P. Winer, Sara M. Tolaney, Nancy U. Lin, Sean Buchanan, Nikhil Wagle. The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in patients with hormone receptor-positive (HR+)/HER2- metastatic breast cancer (MBC) [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 PD2-09.

Combinatorial inhibition of MEK1/2 and CDK4/6 is currently undergoing clinical investigation in NRAS-mutant melanoma. To prospectively map the landscape of resistance to this investigational regimen, we utilized a series of gain- and loss-of-function forward genetic screens to identify modulators of resistance to clinical inhibitors of MEK1/2 and CDK4/6 alone and in combination. First, we identified NRAS-mutant melanoma cell lines that were dependent on NRAS for proliferation and sensitive to MEK1/2 and CDK4/6 combination treatment. We then used a genome-scale ORF overexpression screen and a CRISPR knockout screen to identify modulators of resistance to each inhibitor alone or in combination. These orthogonal screening approaches revealed concordant means of achieving resistance to this therapeutic modality, including tyrosine kinases, RAF, RAS, AKT, and PI3K signaling. Activated KRAS was sufficient to cause resistance to combined MEK/CDK inhibition and to replace genetic depletion of oncogenic NRAS. In summary, our comprehensive functional genetic screening approach revealed modulation of resistance to the inhibition of MEK1/2, CDK4/6, or their combination in NRAS-mutant melanoma. Significance: These findings reveal that NRAS-mutant melanomas can acquire resistance to genetic ablation of NRAS or combination MEK1/2 and CDK4/6 inhibition by upregulating activity of the RTK–RAS–RAF and RTK–PI3K–AKT signaling cascade.

This abstract was withdrawn by the authors. Citation Format: Bjelic-Radisic V, Bottomley A, Cardoso F, Cameron D, Brain E, Kuljanic K, de Costa RA, Conroy T, Deville V, Inwald E, Serpentini S, Pinto M, Bleiker E, Arrares J, Duhoux F, Weiss J, Morag O, Lindviksmoen Astrup G, Tomaszweksi K, Velikova G, Pogoda K, Nagele E, Bliem B, Sinai P, Sprangers M, Aaronson N, Greimel E. Withdrawn [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-12-01.

Metastatic breast cancer (MBC) remains incurable due to inevitable development of therapeutic resistance. Although tumor cell intrinsic mechanisms of resistance in MBC are beginning to be elucidated by bulk sequencing studies, the roles of the tumor microenvironment and intratumor heterogeneity in therapeutic resistance remain underexplored due to both technological barriers and limited availability of samples. To comprehensively capture these characteristics we have adapted a research biopsy protocol to collect tissue for an array of single-cell and spatio-molecular assays whose performance we have optimized for MBC, including single-cell and single-nucleus RNA sequencing, Slide-Seq, Multiplexed Error-Robust FISH (MERFISH), Expansion Sequencing (ExSEQ), Co-detection by Indexing (CODEX) and Multiplexed Ion Beam Imaging (MIBI). To date, we have successfully performed single-cell or single-nucleus RNAseq in 67 MBC biopsies and generated detailed accompanying clinical annotations for each. These samples provide a representation of the clinicopathological diversity of MBC including different breast cancer subtypes (44 HR+/HER2-, 3 HR-/HER2+, 3 HR+/HER2+, 16 TNBC, 1 unknown), common anatomic sites of metastasis (37 liver, 9 axilla, 7 breast, 5 bone, 3 chest wall, 3 neck, 1 brain, 1 lung, 1 skin), metastatic presentations (53 recurrent, 14 de novo) and histologic subtypes in the breast (45 IDC, 7 ILC, 6 mixed, 3 DCIS, 1 mucinous, 5 unknown/NA). Following optimization, both single-cell and single-nucleus RNA seq perform well in these MBC biopsies recovering all expected cell types including the malignant, stromal (e.g. fibroblasts, endothelial cells), myeloid (e.g. monocytes, macrophages) and lymphoid compartments (e.g. T cells, B cells, NK cells) as well as relevant oncogenic programs (e.g. cell cycle programs in all compartments; EMT-like and ER signaling programs in the malignant compartment, immune checkpoint programs in the lymphoid compartment; and fibroblast activation and vascular homeostasis programs in the stromal compartment). In addition to differences between the two techniques, these data demonstrate substantial intratumor heterogeneity in cell type composition. For example in liver biopsies the average number of cells per sample compartment by single nucleus RNA-seq was 6745 malignant (56%, SD 4216), 4637 stromal (41%, SD 3727), 1196 lymphoid (8%, SD 1617) and 874 myeloid (6%, SD 852); in breast biopsies the average number of cells per compartment by single nucleus RNA-seq was 6421 malignant (70%, SD 3497), 1628 stromal (24%, SD 117), 333 lymphoid (4%, SD 170) and 213 myeloid (3%, SD 117). Additionally, we find both inter- and intra-tumor heterogeneity in expression patterns and programs including, for example, expression of ER, PR and HER2 within clinical receptor subtypes (log normalized counts for ER expression in tumor cells by single cell RNA-seq: HR+/HER2- 0.921 (SD 0.714); HR+/HER2+ 0.768 (SD 0.624); HR-/HER2+ 0.018 (SD 0.122); and HR-/HER2- 0.005 (SD 0.066). For a subset of 13 biopsies we are also completing the spatiomolecular characterization methods on serial sections of a single adjacent biopsy. This unique experimental setup was designed to enable efficient comparison and integration of these assays. In spite of differences between experimental techniques and readouts, cell typing can be approached by annotation transfer from matching single cell or single nucleus RNAseq data, enabling exploratory analyses including evaluation of spatial phenotypes and cell type colocalization. Overall, these single cell and spatial data afford a comprehensive atlas including cell types, cell states/programs, cell interactions and spatial organization in MBC lesions. Future analyses will include serial biopsies over time and integration of clinicopathologic data including therapeutic response and resistance. Citation Format: Daniel L Abravanel, Johanna Klughammer, Timothy Blosser, Yury Goltsev, Sizun Jiang, Yunjao Bai, Evan Murray, Shahar Alon, Yi Cui, Daniel R Goodwin, Anubhav Sinha, Ofir Cohen, Michal Slyper, Orr Ashenberg, Danielle Dionne, Judit Jané-Valbuena, Caroline BM Porter, Asa Segerstolpe, Julia Waldman, Sébastien Vigneau, Karla Helvie, Allison Frangieh, Laura DelloStritto, Miraj Patel, Jingyi We, Kathleen Pfaff, Nicole Cullen, Ana Lako, Madison Turner, Isaac Wakiro, Sara Napolitano, Abhay Kanodia, Rebecca Ortiz, Colin MacKichan, Stephanie Inga, Judy Chen, Aaron R Thorner, Asaf Rotem, Scott Rodig, Fei Chen, Edward S Boyden, Garry P Nolan, Xiaowei Zhuang, Orit Rozenblatt-Rosen, Bruce E Johnson, Aviv Regev, Nikhil Wagle. Spatio-molecular dissection of the breast cancer metastatic microenvironment [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 PD6-03.

BACKGROUND - Interleukin(IL)-12 activates T cells pivots the switch that turns lingering inflammation into acute inflammation and cancer rejection. However, its clinical utilization is limited by severe systemic toxicity. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-2-12) that induces minimally effective doses of IL-12 for autocrine activation of CAR-T cells. METHODS - RB-2-12 is a CAR T cell engineered to express the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. The circuit includes a lentiviral constructs encoding an anti-HER2 (4D5) single chain variable fragment, with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the promoter region for IL-12A orL-12B. A second constructs encodes linker for activation of T cells, complexed to nuclease-deactivated/dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) via a TEV-cleavable linker (LdCV). Activation of CAR brings CAR-TEV in proximity to LdCV releasing dCas9 for nuclear localization to the regulatory regions and conditionally and reversibly induce nanoscale expression of the p70 heterodimer. RB-2-12 was compared in vitro to control (cRB-2-12, lacking the IL-12 sgRNAs). Results - RB-2-12 induced low concentrations of IL-12 upon exposure to HER2+ FaDu cancer cells resulting in significantly enhanced production of interferon (IFN)-γ, cytotoxic activity and proliferation (Figure 1a). These effects were comparable to co-culturing conventional HER2-specific CAR-T cells with a modified FaDu cell line expressing high doses of IL-12. In vivo administration of RB-2-12 significantly enhanced survival of mice carrying FaDu xenografts compared to mice treated with the respective conventional CAR-t cells or RB-2-12 lacking the guide RNAs targeting the IL-12 gene subunits CONCLUSIONS - We have previously shown that tandem suppression of PD-1 expression upon HER-2 CAR activation using CRISPR interference enhances anti-cancer properties of CAR-T cells in vivo against HER2-FaDu xenografts by promoting their persistence and long-term tumor colonization (companion abstract submitted to SITC annual meeting). We hypothesize that addition of a Th1 polarizing component such as IL-12 will exponentially increase the efficacy of reprogrammed CAR-T cells by combining enhancement of effector functions to cellular fitness. At the same time, the autocrine effects of nanoscale IL-12 production limit the risk of off-tumor leakage and systemic toxicity. Such cumulative synthetic biology approaches are currently investigated in vitro and in vivo model systems. Current work is testing the effectiveness of RB-2-12 in vivo against FaDu xenografts. Citation Format: Zhifen Yang, Maggie Bobbin, Hana Choi, Ofir Stefanson, Khristina Magallanes, Jin Yang, Bing Wang, Alessandra Cesano, Lei Stanley Qi, Francesco M. Marincola. Nanoscale, antigen-dependent, reversible IL-12 secretion by CAR T cells for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB026.

The Metastatic Breast Cancer Project (MBCproject) is an ongoing research study that directly engages patients (pts) through social media and advocacy groups, and empowers them to share their samples, clinical information, and experiences. The goal is to create a publicly available dataset of linked genomic, clinical, and pt-reported data to enable research. In collaboration with pts, advocates, and advocacy groups, a website (MBCproject.org) was developed that allows pts with metastatic breast cancer (MBC) anywhere in the US or Canada to register. From 10/20/15-3/31/20, 5708 women and men with MBC registered for the MBCproject. Registered pts are sent an online consent form that asks for permission to obtain and analyze their medical records and samples. Consented pts are sent a saliva and/or blood kit and asked to mail back a saliva sample, which is used to extract germline DNA, and/or a blood sample, which is used to extract germline DNA and cell free DNA (cfDNA). We contact participants’ medical providers to obtain medical records and a portion of their stored tumor biopsies. 3245 pts receiving care at over 1700 different institutions have consented to share medical records and tumor/saliva/blood samples and to have genomic analysis performed. Whole exome sequencing (WES) is performed on tumor DNA, germline DNA, and cfDNA; transcriptome sequencing (RNA-seq) is performed on tumor RNA. Medical records and pt-reported data are abstracted to create a detailed clinical record for each pt. Table 1 highlights clinical data collection, biospecimen acquisition, and genomic data generation to date. Examples of clinicogenomic analyses are shown in Table 2. De-identified linked genomic, clinical, and pt-reported data is shared regularly via public and semi-public databases (mbcproject.org, cBioPortal, dbGaP, NCI Genomic Data Commons). To date, this data has been cited in over 20 published journal articles. Study updates are shared with participants regularly. The MBCproject continues to enroll new patients, generate additional data, and perform integrated clinical and genomic analyses with the goal of building a dataset that is representative of patients with MBC. We have partnered with over 30 non-profit breast cancer advocacy groups. We also have several community engagement efforts underway to more directly reach patients in underrepresented communities, including partnerships with faith-based organizations and colleges/universities, as well as targeted engagement with the African American community. In addition, in partnership with Latinx patients, advocates, and researchers, the project has been translated into Spanish and is expected to launch in late 2020. Partnering directly with pts rapidly enables thousands of pts to remotely share tumors, blood, saliva, and medical records to accelerate research. The resulting publicly shared clinically annotated database is a resource that allows researchers to identify patients with specific phenotypes, who have often been challenging to identify with traditional approaches. Clinical data collection, biospecimen acquisition, and genomic data generation:NumberConsent signed (US & CA)3245 ptsSurvey #1 submitted(demographics, diagnosis details, receptor status, clinical experiences)3245 ptsSurvey #2 submitted(pathology details, sites of metastasis, treatments with start and stop dates)1638 ptsMedical record received1352 ptsSaliva sample received2004 ptsBlood sample received1121 ptsTumor samples received585 tumor samples from 424 ptsDigital image of tumor slide H&E generated585 tumor samplesWES from germline complete458 germline samplesWES from tumor (primary and metastatic) samples complete343 tumor samplesRNA-seq from tumor (primary and metastatic) samples complete228 tumor samplesULP-WGS from cfDNA (taken in metastatic setting) complete993 blood samplesWES from circulating tumor DNA (taken in metastatic setting) complete143 blood samples CohortConsented (US & CA)Tumor WES completeTumor RNA-seq completePts diagnosed < 40 yrs of age107312071De novo MBC112712183Late recurrence (>5 years after dx)8307752Long term survivors (MBC > 10yrs)158115Resistance to CDK4/6 inhibitors70914839NED at time of f/u survey4238939Triple Negative Breast Cancer3107531Patients with 2 or more tumor biopsies / cfDNA samples collected by the MBCproject2876138 Citation Format: Nikhil Wagle, Corrie Painter, Elana Anastasio, Michael Dunphy, Mary McGillicuddy, Esha Jain, Brett Tomson, Tania G. Hernandez, Beena Thomas, Dewey Kim, Alyssa L. Damon, Shahrayz Shah, Rafael Ramos, Colleen Nguyen, Lee O'Neil, Sarah Winnicki, Sara Balch, Rachel Stoddard, Taylor Cusher, Parker Chastain, Jorge Gomez Tejeda Zanudo, Jorge Buendia-Buendia, Ofir Cohen, Netsanet Tsegai, Lauren Sterlin, Ulcha F. Ulysse, Imani Boykin, Kate Sine, Oyin Alao, Jacqueline Lucia, Eric S. Lander, Todd R. Golub. The metastatic breast cancer project: Generating the clinical and genomic landscape of metastatic breast cancer through patient-partnered research [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 OT-18-01.

Background: Forkhead box A1 (FOXA1) is an essential pioneer transcription factor (TF) evoking other key TFs-mediated lineage-specific transcriptional programs in several endoderm-derived organs. Aberrant FOXA1 augmentation, via genetic alterations, occurs in 10-15% of ER+ primary and metastatic breast cancer (BC). We have recently shown that high levels of FOXA1 (H-FOXA1) induces enhancer and transcriptional reprogramming to promote endocrine-resistant (EndoR) and pro-metastatic phenotypes. Using the core transcriptional regulatory circuitry (CRC) mapping method, we identified the AP-1 TF JUNB as a key CRC component in BC cells expressing H-FOXA1. In this study, we aimed to further characterize key AP-1 components that play a role in mediating H-FOXA1-induced transcriptional reprogramming in EndoR and metastatic BC. Methods: The ROSE and HOMER tools were used to identify super-enhancers (SEs) and the predicted SE-harboring TFs in MCF7-parental (P) cells with ectopic FOXA1 overexpression (OE), and in MCF7 tamoxifen-resistant (TamR) cells with endogenous FOXA1 amplification and OE. Cell growth, migration, and co-immunoprecipitation assays, were performed using ER+ BC P cells with ectopic FRA1 OE. A FOXA1 core gene signature (GS) was deduced using the BETA.plus algorithm to analyze our previously reported RNA-seq and ChIP-seq data derived from MCF7-P cells with ectopic FOXA1 OE. Additional RNA-seq analyses include MCF7-P cells with ectopic FRA1 OE, FOXA1 OE and simultaneous FRA1 siRNA knockdown (KD), and MCF7-TamR cells with FRA1 KD. We identified a FOXA1/FRA1-centered GS and its clinical relevance was examined using expression profiles of TCGA, METABRIC, and a metastatic biopsy study from cohort of patients with ER+ metastatic BC from Dana-Farber Cancer Institute.Results: We identified FRA1 as one of the top TFs selectively harboring SEs at their gene loci in MCF7-TamR vs. P cells. Both FRA1 and JUNB expression was elevated in TamR vs. P cells and altered concordantly with FOXA1 in P and TamR cells upon FOXA1 OE or KD, respectively. As we identified JUNB as a CRC component with binding sites enriched at the SEs in BC cells expressing H-FOXA1, we hypothesized that FRA1 and JUNB form a feed-forward transcriptional axis amplifying H-FOXA1-induced enhancer reprogramming. We found that JUNB co-immunoprecipitated with FRA1 in MCF7-TamR and MCF7-P cells with ectopic FRA1 OE, suggesting that FRA1 forms a heterodimer with JUNB to exert AP-1 activity. Ectopic FRA1 OE reduced P cell endocrine sensitivity, increased cell migration, and elicited a transcriptome enriched for the FOXA1-induced core GS. In P cells with ectopic FOXA1 OE, we identified a FRA1-dependent GS (n = 27) that is highly enriched for interferon signaling. This FOXA1/FRA1 GS was highly expressed in luminal B vs. A subtype of primary tumors, further elevated in ER+ metastases, where its expression was positively correlated with FRA1 mRNA levels. Notably, this FOXA1/FRA1 GS was not dependent on FRA1 in P cells without FOXA1 OE, suggesting its relevance in the context of H-FOXA1.Conclusions: Here we show that a FOXA1/FRA1-centered transcriptional axis induces an interferon signaling-enriched GS associated with poor outcome of ER+ BC and metastasis. A FRA1/JUNB AP-1 complex may form a feed-forward transcriptional axis to amplify H-FOXA1 signaling. The FOXA1/FRA1-centered GS could be used to stratify patients with ER+ BC who may need additional targeted therapies. Further studies are warranted to elucidate the interplay between FOXA1 and FRA1/JUNB in regulating interferon signaling, which may guide approaches to improve patient outcomes, possibly with immunotherapy using immune checkpoint inhibitors. Citation Format: Xiaoyong Fu, Resel Pereira, Lanfang Qin, Carmine De Angelis, Sarmistha Nanda, Martin Shea, Agostina Nardone, Rinath Jeselsohn, Ofir Cohen, Nikhil Wagle, Mothaffar Rimawi, C Kent Osborne, Rachel Schiff. A FOXA1/FRA1-centered transcriptional axis regulates interferon signaling in high FOXA1-associated endocrine-resistant and metastatic breast cancer [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 PD8-03.

Background: Breast cancer (BC) may recur years to decades after initial treatment, leading to incurable, metastatic disease. Prior studies have shown blood biopsy can detect minimal residual disease (MRD), but not in all patients and often with very short lead time. Most efforts thus far have focused on tracking one or a few mutations via ctDNA, which may limit sensitivity. Here we sought to maximize the detection sensitivity of blood biopsies by tracking up to hundreds of individualized tumor mutations in cell-free DNA (cfDNA). Doing so enables us to break the detection ceiling imposed by the limited copies of each gene in the cell-free DNA in a blood draw. Methods: cfDNA was extracted from archival plasma samples (n=271) from 142 patients with stage 0-III BC enrolled prospectively onto two IRB-approved studies. We applied whole-exome sequencing (WES) to define up to several hundred mutations from each patient’s tumor. To limit potential errors, we employed strict criteria to select somatic SNVs to track using duplex sequencing in cfDNA. We required detection of ≥ 2 mutations for a cfDNA sample to be MRD+ and excluded any mutations also found in a patient’s own germline DNA. Results: We identified 142 patients treated for stage 0-III BC, who had postoperative blood and plasma samples available, and were monitored for distant recurrences for up to thirteen years. All patients had biopsy-proven BC, with 86 (61%) having HR+/HER2- BC, 31 (22%) having HR-/HER2-, or “triple negative” BC (TNBC), and 25 (18%) having HER2-positive disease. Three (2%) patients had stage 0 disease, 32 (23%) had stage I, 68 (42%) had stage II, and 39 (27%) had stage III BC at diagnosis. Archived plasma samples were collected post-operatively, at year 1, and at year 4. We created individualized assays targeting a median of 57 mutations (range 2 - 346) per patient. Reasoning that MRD status after completion of all local therapy and chemotherapy might best predict for distant recurrence, we conducted a landmark analysis at one year following surgery, and found all patients (n=6/6) with detectable MRD experienced recurrence (HR=21.2 (7.43-60.35)) in a median of 6.7 (range 3.4 - 15.8) months. Median lead time including all timepoints from first positive blood sample to recurrence was 18.9 (range: 3.4 - 39.2) months. Finally, in a multivariate model, MRD remained highly statistically significant independent of stage, subtype, and age at diagnosis. Conclusions: We present a novel approach to MRD tracking based on the premise that tracking many - rather than one or a handful of - mutations inherently increases sensitivity and that assay personalization maximizes sensitivity in a disease without multiple, recurrent mutations. To our knowledge, the lead time we show here is significantly longer than that seen in prior investigations, and if confirmed, could offer an opportunity to treat MRD long before the development of clinically apparent metastatic disease. Prospective studies are needed to determine whether earlier detection of MRD is clinically meaningful for patients. Citation Format: Heather A Parsons, Justin Rhoades, Sarah C. Reed, Greg Gydush, Priyanka Ram, Pedro Exman, Kan Xiong, Christopher Lo, Tianyu Li, Mark Fleharty, Greg Kirkner, Denisse Rotem, Ofir Cohen, Melissa Hughes, Shoshanna Rosenberg, Laura Collins, Kathy Miller, Brendan Blumenstiel, Carrie Cibulskis, Donna Neuberg, Samuel S. Freeman, Niall Lennon, Nikhil Wagle, Gavin Ha, Daniel G. Stover, Atish D. Choudhury, Gad Getz, Matthew Meyerson, Nancy U. Lin, Ian E. Krop, J. Christopher Love, G. Mike Makrigiorgos, Ann Partridge, Erika Mayer, Todd R. Golub, Viktor A. Adalsteinsson. Ultrasensitive detection of minimal residual disease in patients treated for breast cancer [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 P5-01-03.

Resistance to therapy in metastatic breast cancer patients results from acquired mutations and selection of resistant clones. Understanding the mechanisms of resistance in the clinical setting is complicated by the lack of genomic characterization of tumor specimens. This case report describes a patient with ER+ and dynamic HER2+ status with exposure to a range of different therapies, suggesting that inference of tumor subclonal composition and evolution can be leveraged to study treatment resistance. A female patient was diagnosed with estrogen receptor positive (ER+) and human epidermal growth factor receptor positive (HER2+) stage I (T1N0) breast cancer. After receiving standard of care adjuvant chemotherapy (AC-TH) and Tamoxifen for two years, the patient developed metastatic disease to the liver. Upon metastatic disease, she received first line treatment with the trastuzumab emtansine antibody-drug conjugate (T-DM1). After fifteen months, enlargement of supraclavicular and retroperitoneal lymph nodes was observed, Letrozole was added to the treatment regimen until the patient progressed after eight months. Subsequent therapy with Fulvestrant and the CDK4/6 inhibitor Palbocicilb was administered for three months until the patient developed brain metastases. We analyzed biopsies taken at different time points of metastatic disease and, using whole exome sequencing (WES), reconstructed the subclonal architecture of each of the tumor specimens and inferred the evolutionary history of these clones. These results suggest potential mechanisms of resistance to the different therapies administered during disease progression. Biopsies obtained originated from a retroperitoneal lymph node at 3 years after diagnosis, a liver metastasis at 4 years after diagnosis and a brain metastasis after 4 years and 3 months after diagnosis. WES was performed on these tumor specimens and characterization of mutational events and copy number alterations was made using standard analytical pipelines. Inference of subclonal populations in the three tumor specimens analyzed revealed presence of a founding clone with mutations common to the three metastatic biopsies with alterations in TP53 p.K305* and PIK3CA p.E545K. A clone unique to the ER+/HER2- retroperitoneal lymph node metastasis shows no known driver mutations. In the liver metastasis (ER+/HER2-), we observed the presence of a dominant clone (cellular prevalence 93.5%) with mutation in ESR1 p.G442R and another subclone detected at low cellular prevalence (~5%) with an amplification in the ERBB2 gene. In the brain metastasis (ER+/HER2-), the latest tumor specimen obtained, we observed clonal selection of the ERBB2-amplified subpopulation (clonal prevalence 60%). The subclonal population dynamics and the different alterations in the clones present at different time points of tumor evolution provide a rationale to understand the therapeutic sensitivity and resistance observed in this patient. The ER+/HER2- retroperitoneal lymph node metastasis which appeared under T-DM1 treatment, suggests that this subclone is resistant to T-DM1, explained by the HER2 negative status, and was selected while the patient was on this regime. At the time of appearance of this clone, Letrozole was added to the treatment and a clone with an ESR1 p.G442R mutation appeared and dominated the tumor population, suggesting that this mutation provides resistance to the aromatase inhibitor. When treatment was switched to Fulvestrant and Palbociclib, a potentially dormant clone with ER+ and HER2+ features re-appeared in the tumor. Evolutionary analysis of this patient's tumor specimens underscores the importance of multiple therapies directed at all tumor vulnerabilities present, since targeting a single driver feature of the tumor will result in selection of other resistant subclones. Citation Format: Jorge E. Buendia-Buendia, Sonia Pernas, Ofir Cohen, Ping Ping Mao, Kailey Kowalski, Nancy Lin, Eric Winer, Nikhil Wagle. Clonal heterogeneity and tumor evolution inform treatment resistance in a patient with HER2+ breast cancer [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 P3-01-09.

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.

Background: The Metastatic Breast Cancer Project (MBCproject) is a research study that directly engages patients (pts) through social media and advocacy groups, and empowers them to share their samples, clinical information, and experiences. The goal is to create a publicly available dataset of linked genomic, clinical, and pt-reported data to enable research. Methods: In collaboration with pts, advocates, and advocacy groups, a website (MBCproject.org) was developed that allows pts with metastatic breast cancer (MBC) anywhere in the U.S. or Canada to register. Registered pts are sent an online consent form that asks for permission to obtain and analyze their medical records and samples. Once enrolled, pts are sent a saliva kit and a blood kit and asked to mail back a saliva sample, which is used to extract germline DNA, and/or a blood sample, which is used to extract germline DNA and cell free DNA (cfDNA). We contact participants’ medical providers and obtain medical records and a portion of their stored tumor biopsies. Whole exome sequencing (WES) is performed on tumor DNA, germline DNA, and cfDNA; transcriptome sequencing (RNA-seq) is performed on tumor RNA. Medical records and pt-reported data are abstracted to create a detailed clinical record for each pt. All de-identified data are shared regularly via public databases (cbioportal.org, mbcproject.org, dbGaP, NCI Genomic Data Commons) without restrictions. Study updates are shared with participants regularly. Results: From 10/20/15-7/8/19, 5357 women and men with MBC registered. 3290 pts receiving care at over 1700 different institutions consented to share medical records and tumor/saliva/blood samples, and to have genomic analysis performed. Details of clinical data collection, biospecimen acquisition, and genomic data generation to date are outlined in the Table. WES from 463 tumors obtained from 326 pts have been generated (with matched germline WES), including 61 pts with 2 timepoints, 19 pts w 3 timepoints, and 11 pts w 4+ timepoints. 278 tumor exomes were from the breast/regional lymph nodes, 63 from distant metastatic sites and 122 from cfDNA. 110 tumor exomes were from samples obtained before the diagnosis of MBC, 258 from after the diagnosis of MBC, and 95 to be determined (TBD). 161 tumor exomes were obtained prior to any therapy, 204 following some therapy, and 98 TBD. Clinically annotated genomic data are used to study specific pt cohorts (including rare subsets and outliers) and to identify mechanisms of response and resistance to therapies. Examples of the clinical and genomic analyses that will be presented include: - Pts diagnosed 5 yrs after diagnosis (830 pts enrolled; 77 with tumor WES) - Long-term survivors, >10 yrs with MBC (159 pts enrolled; 11 with tumor WES) - Resistance to CDK4/6 inhibitors (709 pts enrolled; 148 with tumor WES) Conclusions: Partnering directly with pts enables rapid identification of thousands of pts willing to share tumors, blood, saliva, and medical records to accelerate research. This approach allows for identification of patients with specific phenotypes, who have been challenging to identify with traditional approaches. Remote acquisition of medical records and saliva/blood/tumor samples is feasible. This clinically annotated dataset is a shared resource for the research community. Table 1Clinical data collection, biospecimen acquisition, and genomic data generation:NumberConsent signed3290 ptsSurvey #1 submitted3290 pts(demographics, diagnosis details, receptor status, clinical experiences)Survey #2 submitted1435 pts(pathology details, sites of metastasis, treatments with start and stop dates)Medical record received1307 ptsSaliva sample received1976 ptsBlood sample received1121 ptsTumor samples received482 tumor samples from 346 ptsDigital image of tumor slide H&E generated482 tumor samplesWES from germline complete310 germline samplesWES from tumor sample complete341 tumor samplesRNA-seq from tumor sample complete229 tumor samplesULP-WGS from cfDNA complete947 blood samplesWES from circulating tumor DNA complete122 blood samples Citation Format: Nikhil Wagle, Corrie Painter, Elana Anastasio, Michael Dunphy, Mary McGillicuddy, Esha Jain, Tania G Hernandez, Sara Balch, Beena Thomas, Dewey Kim, Alyssa L. Damon, Shahrayz Shah, Brett N. Tomson, Rachel Stoddard, Colleen Nguyen, Jorge Buendia-Buendia, Ofir Cohen, Jorge Gomez Tejeda Zanudo, Netsanet Tsegai, Lauren Sterlin, Ulcha Fergie Ulysse, Kathryn Sine, Oyin Alao, Jacqueline Lucia, Eric S. Lander, Todd R. Golub. The metastatic breast cancer project: Generating the clinical and genomic landscape of metastatic breast cancer through patient-partnered research [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 PD8-01.

While recent studies have begun characterizing the metastatic breast cancer (MBC) genomics, our understanding of mechanisms of acquired endocrine-resistance, their induced cell-state, and their altered drug-response profile, remains lacking. We collected biopsies from patients with MBC with detailed clinicopathologic features. To date we profiled 520 exomes, and 291 transcriptomes, with 126 patients having multiple biopsy exomes. Curated endocrine-relapse set include 60 patients with pre-treatment and post-relapse exomes. Characterization of candidate mechanisms of resistance (MOR) included 909 RNA-seq profiles of T47D cells with introduced MOR under various drugs. In the acquired endocrine resistance cohort, as expected, we found frequent ESR1 acquired mutations (13 pt, 22%). Additionally, we identified acquired activating SNVs and amplifications in oncogenic receptor tyrosine kinases (RTKs) in 18/60 (30%), including EGF family - HER2 (n=7), ERRB3 (R525Q), and EGFR (S116F), and FGF family - FGFR1 (n=5), FGFR2 (n=4), and FGF3 amplicon (n=4). RNA-seq of T47D cells overexpressing HER2 activating mutations revealed a distinct cell-state (HER2-ACT). Similarly, FGFR activation revealed a district FGFR-ACT state. The transcriptional signatures of these HER2 and FGFR states were remarkably similar (odd-ratio= 91, p= 2.64E-94). To characterize the cell-state common to HER and FGFR, we defined RTK-ACT with 358 overlapping marker genes (see table). Canonical (estradiol) ER signaling is slightly elevated in RTK-ACT, however this state is strongly associated with growth-factor driven ER signaling, suggesting reprogramming of ER from AF2, to AF1 signaling. Consistent with this, RTK-ACT had significantly higher MAPK activation. Additionally, RTK-ACT Induced stronger similarity to Basal-state, enrichment in motility/migration, mesenchymal, and stem-like features, with top genes including CDH3, MBP7, and S100, ETV, DUSP, SPRY families (see table). To study RTK-driven state in-vivo, we analyzed RNA-seq from our MBC biopsies, and compared tumors with activating RTK mutations (n=38) with WT (n=118), and inferred activated RTK in-vivo (RTK.ACT.iv). Our in-vitro and in-vivo states show significant overlap (OR=4.71, p=9.42E-20). Furthermore, characterization of RTK.ACT.iv, recapitulated all the cell-state features observed in RTK.ACT - including higher growth-factors ER signaling, MAPK, and basal-like state (see table). We further studied the viability and transcription of these RTKs under various drugs (12 treatments), including fulvestrant, palbociclib, and specific tyrosine kinase inhibitors (TKIs) - Neratinib (pan-HER inhibitor) and FIIN-3 (FGFR-i). We found that HER2-ACT and FGFR-ACT signatures remain robust when treated with fulvestrant, palbociclib, and their combinations, as compared to TKIs suppression (see table). in-line with our viability results - suggesting intrinsic resistance to CDK4/6i and sensitivity to TKIs. This study demonstrated that activating RTKs constitute of prevalent modality of acquired resistance to endocrine therapies, inducing a distinct state with clinical implications - suggesting the potential benefit of combination therapies with specific TKIs over CDK4/6i. The common MAPK activity and our preliminary results - suggests the potential of convergence-node targeting strategy with added MEK or SHP2 inhibition. TableGene set AGene set BOdds-ratioP-value (two-sided)Fisher''s Exact test matHER2-ACT -FGFR-ACT -912.64E-9469, 131, 131, 22704HER2 S653C, L755S, V777L, L869R vs. GFP, under DMSO, rank genes based on the logFC (top 200)FGFR1, FGFR2 (WT, N550K, M538I, K660N) vs. GFP, Parental, Under DMSO, rank genes based on the logFC (top 200)HER2-ACT_1000 -FGFR-ACT_1000 -18.98.28E-248358, 642, 642, 21758HER2 S653C, L755S, V777L, L869R vs. GFP, under DMSO, rank genes based on the logFC- top 1000FGFR1, FGFR2 (WT, N550K, M538I, K660N) vs. GFP, Parental, Under DMSO, rank genes based on the logFC - top 1000RTK-ACTHALLMARK_ESTROGEN_RESPONSE_EARLY, MSigDB3.030.004229, 191, 349, 22474RTK-ACTER driven by Growth Factors, PMID: 208897186.771.74E-059, 86, 349, 22585RTK-ACTMEK_UP.V1_UP (MAPK), MSigDB10.95.72E-1827, 169, 331, 22496RTK-ACTRAS ONCOGENE (MAPK), PMID: 162730928.445.77E-1220, 158, 338, 22553RTK-ACTWU_CELL_MIGRATION, PMID 187243907.648.96E-1119, 165, 339, 22502RTK-ACTHUPER_BREAST_BASAL_VS_LUMINAL_UP, PMID 17409405131.38E-079, 45, 349, 22621RTK-ACTHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, MSigDB3.770.00031411, 189, 347, 22477RTK-ACTLIM_MAMMARY_STEM_CELL_UP, PMID 203461513.119.56E-0622, 467, 336, 22205RTK-ACTRTK-ACT.iv -4.719.42E-2060, 940, 298, 21992In MBC biopsies, compare RTK mutations with WT, rank genes based on the logFC - top 1000RTK-ACT.iv (in-vivo)HALLMARK_ESTROGEN_RESPONSE_EARLY, MSigDB1.950.020316, 184, 984, 22088RTK-ACT.ivER driven by Growth Factors, PMID: 208897182.640.0076310, 85, 990, 22193RTK-ACT.ivMEK_UP.V1_UP (MAPK), MSigDB2.863.91E-0522, 174, 978, 22098RTK-ACT.ivRAS ONCOGENE (MAPK), PMID: 162730921.620.13212, 166, 988, 22152RTK-ACT.ivWU_CELL_MIGRATION, PMID 187243903.573.60E-0725, 159, 975, 22115RTK-ACT.ivHUPER_BREAST_BASAL_VS_LUMINAL_UP, PMID 174094059.515.43E-1016, 38, 984, 22235RTK-ACT.ivHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, MSigDB2.090.0073817, 183, 983, 22090RTK-ACT.ivLIM_MAMMARY_STEM_CELL_UP, PMID 203461511.420.089229, 460, 971, 21819HER2-ACTHER2-ACT - Fulv3006.53E-183109, 91, 91, 22750HER2-ACTHER2-ACT - Palbo2331.46E-163101, 99, 99, 22749HER2-ACTHER2.ACT - Fulv + Palbo2197.64E-15999, 101, 101, 22742HER2-ACTHER2.ACT - Neratinib63.19.78E-7257, 143, 143, 22707HER2-ACTHER2.ACT - Fulv + Neratinib59.33.53E-6855, 145, 145, 22724FGFR-ACTFGFR.ACT - Palbo19003.2e-319157, 43, 43, 22778FGFR-ACTFGFR.ACT - Fulv + Palbo12501.38E-290148, 52, 52, 22767FGFR-ACTFGFR.ACT - Fulv8661.72E-266140, 60, 60, 22764FGFR-ACTFGFR.ACT - Palbo + FIIN.31062.88E-10474, 126, 126, 22710FGFR-ACTFGFR.ACT - Fulv + Palbo + FIIN.378.41.14E-8464, 136, 136, 22705FGFR-ACTFGFR.ACT - FIIN.367.42.16E-7559, 141, 141, 22730FGFR-ACTFGFR.ACT - Fulv + FIIN.336.89.47E-4541, 159, 159, 22733 Citation Format: Ofir Cohen, Pingping Mao, Utthara Nayar, Jorge E Buendia-Bue, Dewey Kim, Esha Jain, Karla Helvie, Daniel Abravanel, Kailey J Kowalski, Christian Kapstad, Samuel Freeman, Victor Adalsteinsson, Seth A Wander, Adrienne G Waks, Gad Getz, Aviv Regev, Eric Winer P Winer, Nancy U Nancy U. Lin, Nikhil Wagle. Acquired activating mutations in RTKs confer endocrine resistance in ER+ metastatic breast cancer through ER-reprogramming, MAPK signaling, and an induced stem-like cell state [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 GS2-02.

Introduction: Tumor Treating Fields (TTFields) is an antimitotic cancer treatment approved for the treatment of Glioblastoma Multiforme (GBM). TTFields are delivered using 2 pairs of transducer arrays positioned on the shaved scalp of the GBM patient to optimize the TTFields dose delivered to the tumor. This results in an increased dose of TTFields to the affected tumor region, whilst reducing the intensity of the fields to other regions. Brain metastases, or secondary brain tumors, occur in 10 to 30 percent of adults with cancer. Cancer types most likely to cause brain metastases are lung, breast, colon, kidney and melanoma. When using TTFields to treat brain metastases, it may be desirable to deliver TTFields at therapeutic intensities to the entire brain, in order to treat multiple rather than a single lesion. We investigated novel transducer array layouts designed to deliver a uniform distribution of TTFields to the entire brain. Methods Computer simulations were used to calculate the field distributions generated by different array layouts. The simulations utilized a realistic computerized head model of a 40+ years old human male prepared in-house from a T1 MRI series. Using Sim4Life v3.0 (ZMT Zürich), we simulated the delivery of TTFields using pairs of array layouts placed at different locations on the head and neck. To analyze the field distributions, the brain was divided into five regions: (1) the cerebellum, brain stem and other infra-tentorial anatomical regions; and (2-5) the four quadrants of the cerebrum. The mean and median field intensities in the five regions generated by each layout were calculated and compared. Results Median intensities between 1.5 V/cm to 1.7 V/cm within all regions were achieved using a layout in which one pair of arrays was placed on the right temple and left scapula, while the second pair was placed on the left temple and right scapula. This layout yielded a uniform intensity distribution within the brain. Conclusion We have identified a novel TTFields array layout that could potentially be used to treat the entire brain with sufficiently high intensity to treat for brain metastases. The ongoing Phase 3 METIS study [NCT02831959 is investigating radiosurgery with TTFields for 1-10 brain metastases from non-small cell lung cancer. Citation Format: Ofir Yesharim, Ariel Naveh, Ze'ev Bomzon. Novel transducer array layouts to optimize the treatment of multiple brain metastases with tumor treating fields [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4450.

In estrogen-receptor positive (ER+) breast cancer, therapies that target the ER have led to reductions in recurrence and mortality. However, in the metastatic setting, resistance to ER-targeted therapies is near universal. Various resistance mechanisms have been proposed, including ESR1 activating mutations and upregulation of alternative signal transduction pathways, yet clinically relevant resistance mechanisms have not been clearly defined. We conducted a genome-scale gain-of-function screen in ER+ breast cancer cell lines, spanning 17,255 overexpressed open reading frames (ORFs), to investigate genes whose overexpression is sufficient to confer resistance to selective estrogen receptor degraders (SERDs). Among several validated resistance genes, the fibroblast growth factor receptor (FGF/FGFR) pathway was among the top pathways, with several FGF ligands scoring as top resistance genes in this screen. In parallel, we performed whole exome sequencing (WES) in paired pre-treatment and post-resistance biopsies from 60 patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-targeted therapy. The FGFR pathway was altered in 40% (24 of 60) of the post-resistance biopsies; alterations included FGFR1 amplifications (9 patients), FGFR2 amplifications and activating mutations (4 patients), and FGF3 amplifications (17 patients). In 12 out of the 24 cases (5 FGFR1, 4 FGFR2, 3 FGF3), the FGF/FGFR pathway alterations were present in the resistant tumors and not detected in the pre-treatment tumors, suggesting that these alterations were acquired under the selective pressure of ER-directed therapy. Overexpression of wildtype FGFR1, FGFR2, FGF3 in ER+ breast cancer cells (T47D and MCF7) led to resistance to fulvestrant as well as the combination of fulvestrant and the CDK4/6 inhibitor palbociclib. This resistance phenotype was reversed by the FGFR inhibitor PD173074, and, to a lesser extent, the MEK inhibitor trametinib and mTOR inhibitor everolimus, suggesting several potential treatment strategies. The three FGFR2 mutations (M538I, N550K and K660N) identified in these patients induced hyperactive FGFR signaling and conferred resistance to fulvestrant, which was abrogated by the irreversible FGFR inhibitors FIIN-2 and FIIN-3. Together, these results offer new insights into endocrine resistance and suggest new clinical approaches to overcome or preempt therapeutic resistance. Citation Format: Pingping Mao, Ofir Cohen, Kailey Kowalski, Justin Kusiel, Jorge Buendia, Pedro Exman, Seth A. Wander, Adrienne G. Waks, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired FGF and FGFR alterations confer resistance to estrogen receptor (ER) targeted therapy in metastatic ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 314.

Introduction: Tumor Treating Fields (TTFields) is an anti-mitotic technology utilizing low-intensity (1-3 V/cm) electric fields within the medium frequency range (100-300 kHz) to treat solid tumors. TTFields are currently approved for the treatment of Glioblastoma (GBM). Some cancer types, such as breast cancer, lung cancer, and melanoma, frequently result in metastases to the spinal cord. Transducer array positioning used to treat abdominopelvic diseases (e.g. pancreatic cancer), in which one pair of arrays are placed on the anterior and posterior of the patient, and another pair of arrays placed on the lateral aspects of the patient have been shown to be inefficient for treating the spinal cord because the clinical threshold for field intensity of 1V/cm is not reached in the spine when these layouts. The structure of the spine, in which a relatively resistive bone structure encapsulates a highly conductive layer of fluid (cerebrospinal fluid), shunts the current delivered across the body by the arrays away from the spine, reducing the field within the spine to below the therapeutic threshold. To overcome this limitation, a new approach of array placement on the body is needed. Here we present a simulation-based study designed to identify array layouts that effectively deliver TTFields to the spine. Methods: To simulate delivery of TTFields to the spine, we used a human male 34 years old realistic computerized model (DUKE v3.1 by ITI’S, Zurich). In order to generate TTFields, an alternating current with current density of 200mA/disk with a frequency of 150 kHz was imposed on the outer surfaces of the disks of each pair of arrays. The simulations were performed using ZMT's Sim4Life v4.0 electro-quasi-static solver. Results: One of the array layouts tested was placement of a pair of arrays on the back of the patient. One array is placed above the target region in the spine to which treatment would be delivered, and the other array is placed below the region. With this configuration, a high electric field is induced within the spinal cord and surrounding CSF. Mean field intensity within the spine and nerves in between vertebrae T8-T9 at the top, and L3-L4 at the bottom were calculated at a value of 1.77 V/cm. It is important to note that the electric field is oriented along the spine in this instance (a vertical field). In general, the whole spine could be treated by placing one array on the neck, and one at the bottom of the spine. Conclusions: Placing two transducer arrays on the patient’s back allows single-direction treatment of the spinal cord and nerves. Further work should be conducted to find an effective treatment in the perpendicular direction. Citation Format: Ofir Yesharim, Ariel Naveh, Ze'ev Bomzon. Treating spinal cord metastases with tumor treating fields [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 692.

Background: Many patients with upper-abdominal malignancies suffer from severe low back / epigastric pain caused by infiltration of the celiac plexus. The celiac plexus is a network of nociceptive nerves, located along the aorta. Contemporary approaches (opioids, celiac plexus chemical neurolysis, systemic chemotherapy) are often inadequate. The celiac plexus has not previously been targeted using radiation. We hypothesized that ablative radiation targeted to the celiac plexus would alleviate pain and improve quality of life (QOL). Methods: We conducted a single arm prospective clinical trial. Eligible patients had celiac-pain > 4/10 on Numerical Rating Scale (NRS) and completed treatment per protocol with at least one post-treatment visit. The celiac plexus was irradiated from D12 to L2. Radiation was given as either five fractions of 9 Gy or a single-fraction 25 Gy. The primary endpoint was NRS pain 3 weeks post-treatment. Secondary endpoints were toxicity, pain at 6w, analgesic use, and pain interference with daily activities as evaluated by ‘Brief pain assessment short-form’. Analgesic use was not restricted. Here we report results from those who received single fraction treatment. Results: 19 patients were evaluable. The median age of the study population was 67 yr with a median ECOG of 2, 89% had pancreatic cancer. Patients were a median 8 months from diagnosis, and had received a median of one systemic treatment. Toxicity was limited to grade 1-2. Average pain decreased from 5.9/10 at baseline, to 3.1/10 at 3w, and to 1.8/10 (both p < 0.0001) at 6w post-treatment. Number of rescue analgesic doses decreased from 3.6 (baseline) to 1.9 at 3w and 1.7 at 6w (both p Conclusions: Single fraction celiac plexus radiosurgery alleviates pain, and improves quality of life among patients with advanced upper-GI cancer. An international phase II trial is accruing. Pain interference measuresdomainweek 0 (baseline)week 3p (w0 vs v3)week 6p (w0 vs v6)General Activity7.93.4 Citation Format: Yaacov R. Lawrence, Liat Hammer, Ofir Morag, Maoz Ben-Ailan, Dror Alezra, Ofer Margalit, Naama Halpern, Ben Boursi, Einat Shacham Shmueli, Galia Jacobson, Uri Amit, Tamar Katzman, Kinneret Shefer, Ilana Weiss, Inessa Yanovsky, Adam P. Dicker, Talia Golan, Zvi Symon, David Hausner. Celiac plexus radiosurgery a new palliative modality for upper gastrointestinal malignancies - final quality of life results from a proof-of-concept clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT147.

Background: While great strides have been made in the treatment of ER+ MBC, therapeutic resistance is nearly universal. The genomic evolution of ER+ breast cancer in the metastatic setting under the selective pressure of multiple lines of therapies is not well understood. To address this, we analyzed the clonal dynamics of serial metastatic samples (mets) to evaluate how tumors evolve and to identify acquired resistance mechanisms. Methods: We performed WES on 462 clinically annotated samples from 208 patients (pts) with ER+ MBC, including 67 primary tumor biopsies, 229 metastatic biopsies and 160 blood samples (cfDNA). Pts with multiple mets included cases with temporally concordant metastatic tumor and blood samples (48 pts) and cases with serial mets obtained over the course of treatment in the metastatic setting (69 pts). Treatments given between the serial mets included CDK4/6 inhibitors (23 pts), and selective estrogen receptor degraders (19 pts), among others. Results: In the temporally-concordant mets, we found that cfDNA mutations (muts) largely overlap with muts found in tumor biopsies, capturing >85% of clonal tumor muts. However, we observed a higher level of heterogeneity in cfDNA compared to biopsies (p.value< 1.05e-19, Welch test) and a subset of high-confidence muts that were only detected in cfDNA, including in clinically important genes such as ESR1, PIK3CA, KRAS, and ERBB2. Analysis of serial mets was used to elucidate the evolutionary dynamics within the metastatic setting under the selective pressure of treatment. The median duration between mets was 112 days and the median number of inter-biopsy unique treatments was two. Most tumors continued to evolve within the metastatic setting, with 50 out of 69 pts (72%) acquiring a meaningful sub-clone (50% increase in relative cancer cell fraction) and 31 out of 69 (45%) acquiring muts in known cancer genes, including a subset acquiring a plausible resistance alteration such as alterations that dysregulate ER (5 out of 69 pts, 7%; ESR1 mut, FOXA1 amplification (amp), NCOR1 bi-allelic deletion (del)), ERBB (4%; ERBB2 amp, ERBB3 mut), RAS (4%; KRAS mut, NRAS amp, NF1 del), FGF/FGFR (12%; FGFR2 mut, FGFR1/2 amp, FGF3 amp), and cell cycle (13%; RB1 del, CDK4 amp, AURKA amp, CDKN2A del). Finally, in pts who had multiple mets, we observed several cases of evolutionary convergence toward equivalent resistance mechanisms including convergent RB1 loss as a mechanism of resistance to a CDK4/6 inhibitor and convergent BRCA2 reversion following resistance to a PARP inhibitor. Conclusions: This study demonstrates that ER+ MBC continues to evolve under the selective pressure of treatments in the metastatic setting. These findings elucidate the challenge of studying high complexity and heavily treated tumors, while also highlighting some commonalities in the evolutionary trajectories selected by these treatments. The multiplicity of clinically relevant genomic alterations acquired in these advanced stages highlights the need for serial biopsies and the potential to inform post-progression therapeutic choices through targeting the acquired dependencies in post-progression tumors. Citation Format: Cohen O, Buendia-Buendia J, Wander S, Nayar U, Mao P, Waks A, Kim D, Freeman S, Adalsteinsson V, Helvie K, Livitz D, Rosebrock D, Leshchiner I, Dellostritto L, Garrido-Castro A, Jain E, Periyasamy S, Mackichan C, Lloyd M, Marini L, Krop I, Garraway L, Getz G, Winer E, Lin N, Wagle N. Evolutionary analysis of 462 serial metastatic biopsies from 208 patients with estrogen receptor-positive (ER+) metastatic breast cancer (MBC) using whole exome sequencing (WES) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-02.

Background: Approximately 5-10% of newly diagnosed breast cancers (BC) are de novo MBC, which means that metastatic disease was identified at the time of initial diagnosis. Patients with de novo MBC are underrepresented in currently available genomic studies. In The Cancer Genome Atlas (TCGA) dataset, only 15 out of ˜980 BC patients can be classified as having de novo MBC. The objective of this study is to analyze the genomic landscape of de novo MBC and to study the genomic differences of this cohort with early stage BC. To enhance our ability to study de novo MBC, we utilized data from the Metastatic Breast Cancer Project (MBCproject), a patient-partnered research project that aims to generate a large public database of clinical, genomic, and patient reported data (PRD) from patients with MBC. Methods: We defined de novo MBC as patients diagnosed with metastatic disease less than 4 months after their initial diagnosis with BC.Out of 127 patients in the MBCproject with publicly released whole exome sequencing (WES) data, we identified 33 patients with de novo MBC. We combined this data with 15 de novo MBC patients in TCGA. For patients with de novo MBC with multiple tumor biopsies available, we used WES from breast biopsies to enable appropriate comparison to the early stage biopsies. Somatic mutations were evaluated and significantly recurring genes were identified using MutSig2CV. We compared the mutations found in the de novo cohort with early stage tumors. 10 patients in the de novo MBC cohort had paired simultaneous primary and metastatic biopsies; genomic alterations in these samples were compared. Finally, we used RNA sequencing data to compare cytolytic signatures among the de novo and early stage biopsies. Results: Among the 48 patients analyzed the receptor subtype distribution was: HR+/HER2-(23), HR+/HER2+(13), HR-/HER2+(2), HR-/HER2-(3), HR+/HER2 unknown(5), and HR-/HER2 unknown(2). Histology subtype distribution was as follows: IDC(39), MDLC(6), ILC(2) and Other (1). Significantly recurrent genes in the de novo MBC cohort (q Gene% Mutation Rate in De Novo MBC (N=48)% Mutation rate in Early Stage BC (N= 997)p-valuePTEN10.403.510.0324EGFR6.250.500.00435MDM44.170.300.0189 Conclusions: Our initial results highlight genomic differences between de novo MBC and early stage BC, including increased frequency of PTEN, EGFR, and MDM4 mutations. Enrichment of PTEN mutations (implicated in tumor immune surveillance), and downregulation of cytolytic signature potentially suggests that de novo MBC may have immunosuppressive tumor microenvironment. To date, ˜1200 patients with self-reported de novo MBC have registered for the MBCproject. We anticipate that additional study of genomic and clinical data from these patients will greatly improve our understanding of de novo MBC. Citation Format: Jain E, Kim D, Buendia JB, Cohen O, Sousa RB, Anastasio E, Dunphy M, McGillicuddy M, Stoddard R, Balch S, Thomas B, Di Lascio S, Tomson BN, Nguyen C, Painter C, Wagle N. The genomic landscape of de novo metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-03.

Resistance to endocrine therapies in estrogen receptor positive (ER+) metastatic breast cancer is widespread, and understanding the mechanisms whereby these tumors acquire resistance is a critical need. Through whole-exome sequencing of metastatic tumor biopsies from patients with endocrine resistant ER+ metastatic breast cancer, we identified 13 different HER2 mutations, including five in the kinase domain, four in the signaling domain, three in the extracellular domain, and one in the transmembrane region of the protein. Two of the kinase domain mutations (L755S and V777L) have been previously described and shown to be activating and resistant to reversible anti-HER2 targeted therapies; the remaining mutations have not been reported. In several of these patients, whole exome sequencing of a pre-treatment primary tumor did not identify the HER2 mutations seen in the corresponding metastatic tumor, suggesting that they were acquired during therapy. To examine the role of HER2 mutations in endocrine resistance, we generated ER+ breast cancer cell lines (MCF7 and T47D) stably expressing the HER2 mutants observed in our clinical data. Several mutants promoted enhanced growth in charcoal dextran-stripped media, which lacks estradiol and mimics treatment with aromatase inhibitor. In addition, several mutants conferred varying degrees of resistance to fulvestrant and tamoxifen. Taken together, these results suggest that HER2 mutations are associated with acquired resistance to endocrine therapies in patients with ER+ breast cancer. The ability of irreversible anti-HER2 agents as well as other agents that target the HER2 pathway to overcome this resistance is being tested for individual HER2 mutations in vitro. The results from these studies may provide a clinical rationale for therapeutic combination strategies in patients with refractory tumors that have acquired endocrine resistance through HER2 mutations. Citation Format: Nayar U, Cohen O, Oh C, Wagle N. The role of HER2 mutations in resistance to endocrine therapy in ER+ breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-08.

Introduction: TTFields is an antimitotic cancer treatment that utilizes low intensity (1-3 V/cm) alternating electric fields in the intermediate frequency (100-300 kHz) that are delivered in two orthogonal directions using 2 pairs of transducer arrays. A phase II clinical trial (PANOVA, NCT01971281) showed that TTFields in combination with chemotherapy was safe in patients with locally advanced pancreatic cancer. Preclinical studies show that the effect of TTFields is intensity-dependent with a therapeutic threshold of 1 V/cm. The field distribution within the body is known to changes with array placement. Therefore, there is a need to develop principles for personalizing array placement to optimize TTFields delivery when treating pancreatic cancer. The aim of such guidelines should be to ensure delivery of maximal field intensities to the region of disease, whilst minimizing the size of the arrays placed on the body. Minimizing array size is important to enable periodic shifting of the arrays for potentially reducing skin irritation associated with TTFields therapy, as well as potentially improving overall patient comfort. Here we present a systematic study investigating how the location and size of the arrays on the abdomen influences the field distribution. Methods: To simulate delivery of TTFields to the abdomen, we used 3 realistic computerized models (from ZMT_Zurich) of: a male (DUKE 3.0); b) , a female (ELLA 3.0); and an obese male (FATS 3.0). For each model, 6-8 different layouts utilizing combinations of arrays with either 13 or 20 disks per-array were tested. The arrays were placed over the upper 6 standard abdominopelvic, and field intensity distributions within these regions were evaluated. In order to generate TTFields, an alternating voltage at a frequency of 150 KHz was imposed on the outer surfaces of the disks of each pair of arrays. The voltage was set to deliver a current of 200 mA peak to peak per-disk (total current 2.6 A for 13-disk arrays, 4.0 A for 20-disk arrays). The simulations were performed using ZMT's Sim4Life V3.0 electro-quasi-static solver. Results: In all simulations, the large arrays generated higher field intensities than the small arrays. However, On ELLA and DUKE, the large arrays covered most of the skin around the abdomen, leaving little to no room for shifting the arrays. FATs body surface is large enough so that even when large arrays are used, there is still ample space for shifting the arrays. On DUKE and ELLA, when using small arrays, the average field intensity in the abdominopelvic region over which the arrays were placed was above the therapeutic threshold of 1 V/cm. Conclusion: This work shows that TTFields array placement can be optimized to deliver therapeutic field intensities to specific abdominopelvic regions. Tailoring the size and position of the arrays based on disease site and patient size may help to improve overall treatment outcome. Citation Format: Ariel Naveh, Ze'ev Bomzon, Ori Farber, Noa Urman, Ofir Yesharim, Eilon Kirson, Uri Weinberg. Transducer array configuration optimization for treatment of pancreatic cancer using Tumor Treating Fields (TTFields) [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 3204.

Despite the reduction in cancer recurrence and mortality provided by therapies that target the estrogen receptor (ER), resistant ER+ breast cancer remains the most common cause of breast cancer death. Beyond mutations in ER in 25-30% of patients treated with aromatase inhibitors, which deplete circulating estrogen, our understanding of clinical mechanisms of resistance to agents that target the ER remains incomplete. We used whole exome sequencing (WES) of metastatic biopsies to identify mechanisms of resistance in patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-directed agents, including aromatase inhibitors, tamoxifen, and fulvestrant. We noted mutations in human epidermal growth factor receptor 2 (HER2) in metastatic biopsies from 12 patients out of 168. Of these, eight mutations had previously been described as activating. Examination of the treatment-naïve primary tumors in the five patients with activating mutants where WES from the matched treatment-naïve primary tumor was available revealed no evidence of pre-existing mutations in four of the five patients, suggesting that these four mutations were acquired under the selective pressure of ER-directed therapy. These acquired HER2 mutations were mutually exclusive with ER mutations, suggesting a distinct mechanism of resistance to ER-directed therapies. In vitro analysis through expression in ER+/HER2- cell lines confirmed that these mutations conferred estrogen independence. In addition, and in contrast to ER mutations, these HER2 mutations resulted in resistance to tamoxifen, fulvestrant, and the CDK4/6 inhibitor palbociclib. The mutants result in increased AKT and ERK phosphorylation in ER+ cell lines, while simultaneously repressing ER levels and ER-dependent transcriptional targets. One mutation observed in two patients, S653C, occurring in the transmembrane domain and not previously observed in breast cancer, was shown to likely function through constitutive dimerization, a mechanism of activation not previously described for this mutation. Resistance caused by all four mutations was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib, suggesting a novel effective treatment strategy in these patients. Thus, we have shown that acquired activating HER2 mutations can confer endocrine resistance in ER+/HER2- breast cancer, and that such resistance can be effectively reversed by combination therapies that include an anti-HER2 inhibitor. Citation Format: Utthara Nayar, Ofir Cohen, Christian Kapstad, Adrienne Waks, Seth A. Wander, Corrie Painter, Samuel Freeman, Priyanka Ram, Nicole Persky, Lori Marini, Karla Helvie, Nelly Oliver, Cynthia X. Ma, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired HER2 mutations in ER+ metastatic breast cancer confer resistance to ER-directed therapies [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 4952.

The Metastatic Breast Cancer Project (MBCproject) is a research study that directly engages patients (pts) through social media and advocacy groups, and empowers them to share samples, clinical data, and experiences. The goal is to create a publicly available database of genomic, molecular, clinical, and patient-reported data to enable research. Working with pts and advocates, a website (MBCproject.org) was developed that allows pts with metastatic breast cancer (MBC) to register. Registered pts are sent an online consent form that asks for permission to obtain and analyze their medical records and samples. Once enrolled, pts are sent a saliva kit and asked to mail back a saliva sample, which is used to extract germline DNA. We contact participants' medical providers and obtain medical records and a portion of their stored tumor biopsies. Pts may be asked to mail in a blood sample, which is used to extract cell free DNA (cfDNA). Whole-exome sequencing (WES) is performed on tumor DNA, germline DNA, and cfNDA; transcriptome sequencing is performed on tumor RNA. Clinically annotated genomic data are used to study specific pt cohorts (including outliers) and to identify mechanisms of response and resistance to therapies. All de-identified data are shared via public databases. Study updates are shared with participants regularly. From 10/2015-11/2017, 4237 MBC pts registered, representing over 1,000 institutions. 95% answered the 16-question survey about their cancer, treatments, and demographic information. 2471 (58%) completed the consent form. 2,136 saliva kits were mailed to pts and 1,523 saliva samples were sent in (71%). 408 blood kits were mailed to pts and 175 blood samples have been received for cfDNA analysis. To date, we have obtained medical records from 311 pts and 190 tumors from 127 pts. In 10/2017, all data generated so far were publicly released on cbioportal.org, including WES for 103 tumors from 78 pts linked to clinical data including pathology (22 elements), medical record abstraction including all treatments and timelines/durations (67 elements), and patient-reported data (11 elements). 81% of biopsies included in this release were from the breast and 19% from metastatic sites. 75% were obtained prior to any therapy, 24% following therapy. New data will be released 4/2018 and every six months thereafter, as they are generated. Additional patient-reported data, including treatments, side effects, quality of life, family history, pregnancies, and sites of metastasis, will also be collected and shared. In summary, a patient-driven approach enabled rapid identification of thousands of MBC pts willing to share samples and clinical data. Remote acquisition of medical records, saliva, blood, and tumor tissue for pts across the U.S. is feasible. This shared clinico-genomic database should enable research in MBC and may serve as a model for patient-driven research in other cancers. Citation Format: Nikhil Wagle, Corrie Painter, Elana Anastasio, Michael Dunphy, Mary McGillicuddy, Rachel Stoddard, Esha Jain, Dewey Kim, Simona Di Lascio, Brett N. Tompson, Sara Balch, Beena Thomas, Priti Kumari, Shawn Johnson, Jamie Holloway, Ofir Cohen, Erik H. Knelson, Katie Larkin, Sam Pollock, Alicia Wong, Samira Bahl, Simone Maiwald, Andrew Zimmer, Esme O. Baker, Jen Hendry Lapan, Scott Sutherland, Scott Sassone, Viktor Adalsteinsson, Eric S. Lander, Todd R. Golub. The Metastatic Breast Cancer Project: Partnering with patients to accelerate progress in cancer research [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 5371.

Approximately 70% of breast cancers express the estrogen receptor (ER), and estrogen signaling drives breast cancer cell growth and progression. ER-directed therapies are commonly used to treat ER+ breast cancer and have improved survival for patients, yet resistance to those therapies inevitably occurs. Mutations in the estrogen receptor itself occur in ∼25-30% of patients with ER+ metastatic breast cancer that has developed resistance to aromatase inhibitors. Beyond these ER mutations, other resistance mechanisms are not well described. Moreover, clinical mechanisms of resistance to another class of ER-targeted agents, selective estrogen receptor degraders (SERDs), such as fulvestrant have not been clearly identified. Here we report two FGFR2 mutations identified in patients with resistant ER+ metastatic breast cancer, N550K and M538I. N550K is a well-known activating FGFR2 mutation; M538I stabilizes the active kinase conformation and it has not previously been described in breast cancer. When expressed in ER+ T47D cells, FGFR2 M538I and N550K led to resistance to fulvestrant, and the CDK4/6 inhibitor palbociclib and the combination of the two agents. FGFR2 M538I induced hyperactivity of p-FRS2, p-ERK and p-AKT, which is higher than wildtype FGFR2 and comparable to other known activating mutations N550K and K660N. In addition, overexpression of M538I mutant reduced sensitivity to FGFR inhibitors PD173074 and dovitinib in T47D cells, suggesting M538I is also functionally activating. Due to the hyperactive downstream signaling elicited by the mutation, cells overexpressing FGFR2 M538I achieved optimal growth in the presence of low dose of FGFR inhibitor. Under such conditions, FGFR2 M538I conferred more potent resistance to fulvestrant as compared to wildtype FGFR2. However, drug resistance resulting from M538I mutant can be fully resensitized to fulvestrant and/or palbociclib with high dose of FGFR inhibitors. In summary, we have identified activating FGFR2 mutations (M538I and N550K) in ER+ breast cancer patients, which may contribute to the development of resistance to SERDs and CDK4/6 inhibitors. Additional FGFR2 mutations have been recently identified in other cohorts of patients with resistant ER+ metastatic breast cancer, suggesting that this may be a clinical mechanism of resistance in some patients. Patients with activating FGFR2 mutations may benefit from the treatment with an FGFR inhibitor in combination with SERDs and CDK4/6 inhibitors. Citation Format: Mao P, Kusiel J, Cohen O, Wagle N. The role of FGF/FGFR axis in resistance to SERDs and CDK4/6 inhibitors in ER+ breast cancer [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 PD4-01.

Cytotoxic T cells (CTL) play a major role in tumor rejection. Expansion of CTLs, either by immunization or adoptive transfer, is a prominent goal in current immunotherapy. The antigen-specific nature of these expansion processes inevitably initiates a clonotypic attack on the tumor. By injecting an Ovalbumin-expressing melanoma into OT-I mice, in which >90% of CTLs recognize an Ovalbumin peptide, we show that an increased number of tumor-specific CTLs causes emergence of escape variants. We show that these escape variants are a result of antigen silencing via a yet undetermined epigenetic mechanism, which occurs frequently and is spontaneously reversible. We further show that an increase in the time of tumor onset in OT-I compared with C57BL/6J is a result of immune selection. [Cancer Res 2008;68(9):3450–7]

Introduction TTFields is an antimitotic cancer treatment that utilizes low intensity (1-3 V/cm) alternating electric fields in the intermediate frequency (100-300 kHz) that are delivered in two orthogonal directions using 2 pairs of transducer arrays. TTFields are currently approved for Glioblastoma Multiforme (GBM). A phase II clinical trial (EF-20) showed that TTFields in combination with gemcitabine was safe in patients with locally advanced pancreatic cancer. Preclinical studies show that the effect of TTFields is intensity-dependent with a therapeutic threshold of 1 V/cm. Simulation-based studies show that the field distribution changes with array placement. Treatment planning with arrays positioned on the scalp to maximize field intensity to the tumor is standard of practice when treating GBM. Studies to examine array layout on the abdomen to maximize the field distribution in this region have not been done. In the EF-20 study, a generic layout was used. We used computer simulations to test how altering the transducer array layout on the mid-body alters the field distribution within the abdomen and pancreas. Methods To simulate delivery of TTFields to the abdomen, we used a realistic computerized model of a human male (DUKE 3.0 from ZMT-Zurich). Eight different layouts utilizing combinations of arrays with either 13 or 20 disks placed at different locations on the abdomen were simulated. In order to generate TTFields, an alternating voltage difference with a peak to peak magnitude of 100V and a frequency of 150 KHz was imposed on the outer surfaces of the disks of each pair of arrays. The simulations were performed using ZMT's Sim4Life V3.0 electro-quasi-static solver. For each pair of transducer arrays, the mean field intensity in the abdomen and chest, and in the pancreas and liver, was calculated. Results All eight layouts delivered fields with mean intensities of about 2 V/cm to the abdomen. The average field intensity delivered to the pancreas and the liver exceeded the therapeutic threshold of 1 V/cm for all layouts. The highest intensities were delivered to the pancreas by layouts in which a 20-disk array was placed at the middle of the back, and either a 20 disk array placed at the middle of the abdomen or a 13 disk array placed on the front-left side of the abdomen over the pancreas. Generally, field intensities in the pancreas are lower than in the rest of the abdomen because of the high electric conductivity of this organ. Conclusion This work shows that TTFields can be delivered to the pancreas with intensities well above the therapeutic threshold of 1 V/cm, and that altering the location of the arrays on the abdomen can influence the field intensity and distribution within the body. This work forms the basis for developing optimal strategies for delivering TTFields to the pancreas. Citation Format: Ariel Naveh, Ze'ev Bomzon, Noa Urman, Ofir Yesharim, Eilon D. Kirson, Uri Weinberg. Optimizing transducer array configuration for treatment of pancreatic cancer using Tumor Treating Fields (TTFields) [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 1569. doi:10.1158/1538-7445.AM2017-1569

Therapies that target the estrogen receptor provide clinical benefit and improved survival for patients with estrogen receptor-positive (ER+) breast cancer, yet drug resistance remains a challenging problem, leading to disease relapse and mortality. In recent years, the selective estrogen receptor degrader (SERD) fulvestrant has become an important therapeutic option for patients with resistant ER+ metastatic breast cancer, and newer oral SERDs such as GDC-810 are currently being tested in clinical trials. The mechanisms of intrinsic and acquired resistance to SERDs remain to be fully elucidated. We conducted a large-scale lentiviral open reading frame (ORF) screen to identify genes whose overexpression confers drug resistance to either fulvestrant or GDC-810 in the ER+ breast cancer cell line T47D. The lentivral ORF expression library used in this study consists of 16,544 barcoded ORFs, including 2,767 ORFs with mutations. The initial screen yielded 72 genes resulting in resistance to fulvestrant and 85 genes resulting in resistance to GDC-0810, with 44 genes overlapping. The top ranked-genes included multiple genes belonging to the PI3K/Akt, ERbB/HER, and FGF/FGFR pathways as well as genes involved in cell cycle progression. Fibroblast growth factor receptor 1 (FGFR1) amplifications are frequently observed in patients with ER+ breast cancer, and have previously been implicated in resistance to endocrine therapies. Several FGFs (FGF3, FGF6, FGF10, and FGF22) were among the top-ranked resistance genes for both fulvestrant and GDC-0810, suggesting that activation of the FGFR signaling pathway may render cells resistant to fulvestrant and GDC-810. In the presence of FGF2, overexpression of FGFR1 in ER+ breast cancer cells resulted in resistance to both fulvestrant and GDC-0810. The ability of an FGFR inhibitor to overcome FGFR-mediated resistance to SERDs is being tested. Additional potential resistance genes identified in the ORF screen are also being validated. In summary, a whole-genome functional resistance screen has identified several candidate genes and pathways that may cause resistance to fulvestrant and GDC-810. Several of these candidates, such as FGFR1, are also found in patients who develop resistance to SERDs, suggesting rational combination therapies to overcome or preempt SERD resistance. Citation Format: Mao P, Quartey Q, Cohen O, Piccioni F, Wagle N. A large-scale functional screen to identify resistance mechanisms to selective estrogen receptor degraders fulvestrant and GDC-810 in ER+ breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-08.

Background: While great strides have been made in the treatment of estrogen receptor-positive (ER+) metastatic breast cancer (MBC), therapeutic resistance invariably occurs. A better understanding of the underlying resistance mechanisms is critical to enable durable control of this disease. Methods: We performed whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) on metastatic tumor biopsies from 88 patients with ER+ MBC who had developed resistance to one or more ER-directed therapies. For 27 of these patients, we sequenced the treatment-naïve primary tumors for comparison to the resistant specimens. Tumors were analyzed for point mutations, insertions/deletions, copy number alterations, translocations, and gene expression. Detailed clinicopathologic data was collected for each patient and linked to the genomic information. Results: WES of all metastatic samples demonstrated several recurrently altered genes whose incidence differed significantly from primary, treatment-naïve ER+ breast cancers sequenced in the TCGA study (TCGA). These include ESR1 mutations (n=17, 19.3%; 32.86 fold enrichment, q.value Comparing to matched primary samples from the same patient, many alterations were found to be acquired in several cases, including for ESR1, ERBB2, PIK3CA, PTEN, RB1, AKT1, and others. Initial analysis of RNA-seq data from metastatic samples (n=59) allowed classification of individual resistance mechanisms into broader resistance modes based on the observed transcriptional state. Conclusions: We present a genomic landscape of resistant ER+ MBC using WES and RNA-seq. Multiple genes were recurrently altered in these tumors at significantly higher rates than in ER+ primary breast cancer. When compared with matched primary tumors from the same patient, alterations in these and other genes were often found to be acquired after treatment, suggesting a role in resistance to ER-directed therapies and/or metastasis. Potential resistance mechanisms appear to fall into several categories; integrating RNA-seq data may enhance the ability to identify these categories even when genomic alterations are not identified. Multiple clinically relevant genomic and molecular alterations are identified in metastatic biopsies– with implications for choice of next therapy, clinical trial eligibility, and novel drug targets. Citation Format: Cohen O, Kim D, Oh C, Waks A, Oliver N, Helvie K, Marini L, Rotem A, Lloyd M, Stover D, Adalsteinsson V, Freeman S, Ha G, Cibulskis C, Anderka K, Tamayo P, Johannessen C, Krop I, Garraway L, Winer E, Lin N, Wagle N. Whole exome and transcriptome sequencing of resistant ER+ metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-01.

Ubiquitin proteasome pathways are emerging as a growing source for novel anti-cancer therapeutics. In this respect, by depleting a number of deubiquitylating enzymes (DUBs) through a combination of synthetic lethality screens and isogenic cell line models, MISSION has identified several novel DUBs essential for a range of different tumor types, including platinum-resistant ovarian cancers, DNA damage response (DDR) pathway deficient tumors (e.g. ATM, ATR or BRCA2 defective) and haematological tumors such as multiple myeloma. Illustrating the diversity of DUBs as oncology targets we identified USP11 and UCHL1 using our target validation platform. USP11 was identified as essential for proliferation of platinum-resistant cells but not platinum-sensitive tumor cells in an isogenic model derived from patient tumors before and after development of resistance. In addition, USP11 depletion displayed synthetic lethality in isogenic models of BRCA2 or ATR loss, supporting USP11's previously identified association with homologous recombination proteins as well as the importance such processes in platinum resistance mechanisms. UCHL1 on the other hand is a prototypical oncogene target that selectively drives proliferation in many tumor types, such as multiple myeloma and lung cancer. UCHL1 depletion leads to selective killing of a number of tumors whose proliferation is driven by UCHL1. Validation of targets like USP11 or UCHL1 for the selective treatment of cancer bearing genetic deficiencies or resistant to standard-of-care supports the rationale of developing DUB inhibitors for cancers with unmet medical need. A broad drug discovery platform combining unique biochemical, cellular, biophysical and structural assays was designed by MISSION to identify and optimise potent and selective DUB inhibitors. Early selective DUB hits, developed for USP11 and UCHL1, are successfully recapitulating target validation biology, including synthetic lethality in matched isogenic backgrounds. Lead optimisation of chemical series has recently been translated into compounds with adequate properties for in vivo proof-of-concept studies. The challenges and advances in demonstrating in vivo DUB target engagement will be discussed. MISSION's integrated drug discovery methodologies exemplify the tractability of DUBs to pharmacologic intervention and the potential scope for DUB inhibitors in a number of cancer types, including those characterised by DDR deficiencies. Citation Format: Xavier Jacq, Niall MB Martin, Lisa Smith, Jeanine Harrigan, Charlotte Knights, Helen Robinson, Yaara Ofir-Rosenfeld, Aaron Cranston, Mark I. Kemp, Stephen P. Jackson. Discovery of highly selective DUB inhibitors with in vivo pre-clinical anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2015-1728

Specific immunotherapy of prostate cancer may be an alternative or be complementary to other approaches for treatment of recurrent or metastasized disease. This study aims at identifying and characterizing prostate cancer–associated peptides capable of eliciting specific CTL responses in vivo. Evaluation of peptide-induced CTL activity in vitro was done following immunization of HLA-A2 transgenic (HHD) mice. An in vivo tumor rejection was tested by adoptive transfer of HHD immune lymphocytes to nude mice bearing human tumors. To confirm the existence of peptide-specific CTL precursors in human, lymphocytes from healthy and prostate cancer individuals were stimulated in vitro in the presence of these peptides and CTL activities were assayed. Two novel immunogenic peptides derived from overexpressed prostate antigens, prostatic acid phosphatase (PAP) and six-transmembrane epithelial antigen of prostate (STEAP), were identified; these peptides were designated PAP-3 and STEAP-3. Peptide-specific CTLs lysed HLA-A2.1+ LNCaP cells and inhibited tumor growth on adoptive immunotherapy. Furthermore, peptide-primed human lymphocytes derived from healthy and prostate cancer individuals lysed peptide-pulsed T2 cells and HLA-A2.1+ LNCaP cells. Based on the results presented herein, PAP-3 and STEAP-3 are naturally processed CTL epitopes possessing anti–prostate cancer reactivity in vivo and therefore may constitute vaccine candidates to be investigated in clinical trials.