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Supplementary Figure Legends 1-4 from A Feed-Forward Loop Involving Protein Kinase Cα and MicroRNAs Regulates Tumor Cell Cycle

Liquid biopsy has proven valuable in identifying individual genetic alterations; however, the ability of plasma ctDNA to capture complex tumor phenotypes with clinical value is unknown. To address this question, we have performed 0.5X shallow whole-genome sequencing in plasma from 459 patients with metastatic breast cancer, including 245 patients treated with endocrine therapy and a CDK4/6 inhibitor (ET + CDK4/6i) from 2 independent cohorts. We demonstrate that machine learning multi-gene signatures, obtained from ctDNA, identify complex biological features, including measures of tumor proliferation and estrogen receptor signaling, similar to what is accomplished using direct tumor tissue DNA or RNA profiling. More importantly, 4 DNA-based subtypes, and a ctDNA-based genomic signature tracking retinoblastoma loss-of-heterozygosity, are significantly associated with poor response and survival outcome following ET + CDK4/6i, independently of plasma tumor fraction. Our approach opens opportunities for the discovery of additional multi-feature genomic predictors coming from ctDNA in breast cancer and other cancer-types. Plasma ctDNA is a promising method to determine patient outcome in multiple cancer types. Here, the authors use shallow WGS to create machine learning signatures to identify tumor phenotypes and predict therapy response in patients with metastatic breast cancer.

PURPOSE CALGB 40603 ( NCT00861705 ), a 2 × 2 randomized phase II trial, demonstrated that adding carboplatin or bevacizumab to weekly paclitaxel (wP) followed by doxorubicin and cyclophosphamide significantly increased the pathologic complete response (pCR) rate in stage II-III triple-negative breast cancer. We now report long-term outcomes (LTOs) and correlative science end points. PATIENTS AND METHODS The Kaplan-Meier method was used to estimate LTOs in 443 patients who initiated study treatment. Log-rank tests and Cox proportional hazards models evaluated the impact of clinical characteristics, pathologic response, calculated residual cancer burden (RCB) in patients with residual disease (RD), treatment assignment, and dose delivery during wP on LTOs, including event-free survival (EFS). Genomic predictors of treatment response and outcomes were assessed on pretreatment tumor samples by mRNA sequencing. RESULTS Among baseline characteristics, only the clinical stage was associated with LTOs. At a median follow-up of 7.9 years, LTOs were not significantly improved with either carboplatin or bevacizumab, overall or in patients with basal-like subtype cancers by genomic analysis. Patients with pCR (n = 205, 46.3%) had significantly higher 5-year EFS (85.5% v 56.6%, log-rank P < .0001) and overall survival (87.9% v 63.4%, P < .0001) rates compared with patients with RD, even those with RCB class I. Among clinical and genomic features, evidence of immune activation, including tumor-infiltrating lymphocytes and low B-cell receptor evenness, was associated with pCR and improved EFS. CONCLUSION Despite higher pCR rates, neither carboplatin nor bevacizumab appeared to improve LTOs although the study was not powered to assess these secondary end points. pCR was associated with superior LTOs even when compared with minimal RD. Markers of immune activation in pretreatment tumor biopsies were independently associated with higher pCR rates and improved survival.

Model systems are an essential resource in cancer research. They simulate effects that we can infer into humans, but come at a risk of inaccurately representing human biology. This inaccuracy can lead to inconclusive experiments or misleading results, urging the need for an improved process for translating model system findings into human-relevant data. We present a process for applying joint dimension reduction (jDR) to horizontally integrate gene expression data across model systems and human tumor cohorts. We then use this approach to combine human TCGA gene expression data with data from human cancer cell lines and mouse model tumors. By identifying the aspects of genomic variation joint-acting across cohorts, we demonstrate how predictive modeling and clinical biomarkers from model systems can be improved.

Antiretroviral therapy (ART) is not curative due to the existence of cellular reservoirs of latent HIV-1 that persist during therapy. Current research efforts to cure HIV-1 infection include “shock and kill” strategies to disrupt latency using small molecules or latency-reversing agents (LRAs) to induce expression of HIV-1 enabling cytotoxic immune cells to eliminate infected cells. The modest success of current LRAs urges the field to identify novel drugs with increased clinical efficacy. Aminobisphosphonates (N-BPs) that include pamidronate, zoledronate, or alendronate, are the first-line treatment of bone-related diseases including osteoporosis and bone malignancies. Here, we show the use of N-BPs as a novel class of LRA: we found inex vivoassays using primary cells from ART-suppressed people living with HIV-1 that N-BPs induce HIV-1 from latency to levels that are comparable to the T cell activator phytohemagglutinin (PHA). RNA sequencing and mechanistic data suggested that reactivation may occur through activation of the activator protein 1 signaling pathway. Stored samples from a prior clinical trial aimed at analyzing the effect of alendronate on bone mineral density, provided further evidence of alendronate-mediated latency reversal and activation of immune effector cells. Decay of the reservoir measured by IPDA was however not detected. Our results demonstrate the novel use of N-BPs to reverse HIV-1 latency while inducing immune effector functions. This preliminary evidence merits further investigation in a controlled clinical setting possibly in combination with therapeutic vaccination.

Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples.

Summary Few predictors of response to topical corticosteroid (tCS) treatment have been identified in eosinophilic esophagitis (EoE). We aimed to determine whether baseline gene expression predicts histologic response to tCS treatment for EoE. We analyzed prospectively collected samples from incident EoE cases who were treated with tCS for 8 weeks in a development cohort (prospective study) or in an independent validation cohort (clinical trial). Whole transcriptome RNA expression was determined from a baseline (pre-treatment) RNA-later preserved esophageal biopsy. Baseline expression was compared between histologic responders (

Alternative splicing is an RNA processing mechanism involved in skeletal muscle development and pathology. Muscular diseases exhibit splicing alterations and changes in mechanobiology leading us to investigate the interconnection between mechanical forces and RNA processing. We performed deep RNA-sequencing after stretching muscle cells. First, we uncovered transcriptional changes in genes encoding proteins involved in muscle function and transcription. Second, we observed that numerous mechanosensitive genes were part of the MAPK pathway which was activated in response to stretching. Third, we revealed that stretching skeletal muscle cells increased the proportion of alternatively spliced cassette exons and their inclusion. Fourth, we demonstrated that the serine and arginine-rich proteins exhibited stronger transcriptional changes than other RNA-binding proteins and that SRSF4 phosphorylation is mechanosensitive. Identifying SRSF4 as a mechanosensitive RNA-binding protein that might contribute to crosstalk between mechanotransduction, transcription, and splicing could potentially reveal novel insights into muscular diseases, particularly those with unknown etiologies.

The reversibility of the procancer effects of obesity was interrogated in formerly obese C57BL/6 mice that lost weight via a nonrestricted low-fat diet (LFD) or 3 distinct calorie-restricted (CR) regimens (low-fat CR, Mediterranean-style CR, or intermittent CR). These mice, along with continuously obese mice and lean control mice, were orthotopically injected with E0771 cells, a mouse model of triple-negative breast cancer. Tumor weight, systemic cytokines, and incidence of lung metastases were elevated in the continuously obese and nonrestricted LFD mice relative to the 3 CR groups. Gene expression differed between the obese and all CR groups, but not the nonrestricted LFD group, for numerous tumoral genes associated with epithelial-to-mesenchymal transition as well as several genes in the normal mammary tissue associated with hypoxia, reactive oxygen species production, and p53 signaling. A high degree of concordance existed between differentially expressed mammary tissue genes from obese versus all CR mice and a microarray dataset from overweight/obese women randomized to either no intervention or a CR diet. Assessment of differentially methylated regions in mouse mammary tissues revealed that obesity, relative to the 4 weight loss groups, was associated with significant DNA hypermethylation. However, the anticancer effects of the CR interventions were independent of their ability to reverse obesity-associated mammary epigenetic reprogramming. Taken together, these preclinical data showing that the procancer effects of obesity are reversible by various forms of CR diets strongly support translational exploration of restricted dietary patterns for reducing the burden of obesity-associated cancers. Prevention Relevance: Obesity is an established risk and progression factor for triple-negative breast cancer (TNBC). Given rising global rates of obesity and TNBC, strategies to reduce the burden of obesity-driven TNBC are urgently needed. We report the genomic, epigenetic, and procancer effects of obesity are reversible by various calorie restriction regimens.

BackgroundThe clinical approvals of KRAS G12C inhibitors has been a revolutionary advance in precision oncology, but response rates are often modest. To improve patient selection, we developed an integrated model to predict KRAS dependency.Methods and FindingsBy integrating molecular profiles of a large panel of cell lines from the DEMETER2 dataset, we built a binary classifier to predict a tumor’s KRAS dependency. Monte Carlo cross validation via ElasticNet within the training set was used to compare model performance and to tune parameters. The final model was then applied to the validation set. We validated the model with genetic depletion assays and an external dataset of lung cancer cells treated with a G12C inhibitor. We then applied the model to several Cancer Genome Atlas (TCGA) datasets. The final “K20” model contains 20 features, including expression of 19 genes and KRAS mutation status. In the validation cohort, K20 had an AUC of 0.94 and accurately predicted KRAS dependency in both mutant and KRAS wild-type cell lines following genetic depletion. It was also highly predictive across an external dataset of lung cancer lines treated with KRAS G12C inhibition. When applied to TCGA datasets, specific subpopulations such as the invasive subtype in colorectal cancer and copy number high pancreatic adenocarcinoma were predicted to have higher KRAS dependency.ConclusionThe K20 model has simple yet robust predictive capabilities that may provide a useful tool to select patients with KRAS mutant tumors that are most likely to respond to direct KRAS inhibitors.

The AURORA US Metastasis Project was established with the goal to identify molecular features associated with metastasis. We assayed 55 females with metastatic breast cancer (51 primary cancers and 102 metastases) by RNA sequencing, tumor/germline DNA exome and low-pass whole-genome sequencing and global DNA methylation microarrays. Expression subtype changes were observed in ~30% of samples and were coincident with DNA clonality shifts, especially involving HER2. Downregulation of estrogen receptor (ER)-mediated cell-cell adhesion genes through DNA methylation mechanisms was observed in metastases. Microenvironment differences varied according to tumor subtype; the ER

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.

Background Human papillomavirus-positive (HPV+) squamous cell carcinoma of the oropharynx (OPSCC) is the most prevalent HPV-associated malignancy in the United States. Favorable treatment outcomes have led to increased interest in treatment de-escalation to reduce treatment morbidity as well as the development of prognostic markers to identify appropriately low-risk patients. Intratumoral genomic heterogeneity and copy number alteration burden have been demonstrated to be predictive of poor outcomes in many other cancers; therefore, we sought to determine whether intratumor heterogeneity and genomic instability are associated with poor outcomes in HPV+ OPSCC. Methods Tumor heterogeneity estimates were made based on targeted exome sequencing of 45 patients with HPV+ OPSCC tumors. Analysis of an additional cohort of HPV+ OPSCC tumors lacking matched normal sequencing allowed copy number analysis of 99 patient tumors. Results High intratumorally genomic heterogeneity and high numbers of copy number alterations were strongly associated with worse recurrence-free survival. Tumors with higher heterogeneity and frequent copy number alterations were associated with loss of distal 11q, which encodes key genes related to double-strand break repair, including ATM and MRE11A. Conclusions Both intratumor genomic heterogeneity and high-burden copy number alterations are strongly associated with poor recurrence-free survival in patients with HPV+ OPSCC. The drivers of genomic instability and heterogeneity in these tumors remains to be elucidated. However, 11q loss and defective DNA double-strand break repair have been associated with genomic instability in other solid tumors. Copy number alteration burden and intratumoral heterogeneity represent promising avenues for risk stratification of patients with HPV+OPSCC.

Cancer cells rewire regulatory elements scattered throughout the genome (such as enhancers) to drive aberrant gene expression. Thus, deconvoluting the regulatory mechanisms that contribute to oncogenic gene expression in cancer cells is key to understanding tumor biology. To this end, we have charted the transcriptional and epigenetic landscape of breast cancer at single-cell resolution to quantitatively link variation in chromatin accessibility to gene expression across malignant and non-malignant cell types. Our comprehensive dataset profiles the chromatin landscape (scATAC-seq) in concert with the transcriptional profiles (scRNA-seq) of 4 breast cancer cell lines, 12 primary breast tumors, and 4 normal mammary reduction tissue specimens collected and processed immediately after surgical resection. This dataset, encompassing over 250,000 individual cells, allowed us to define the regulatory logic of cancer cells by 1) revealing how the epigenome underlies cellular heterogeneity of these tumors in comparison to normal mammary tissue, 2) defining how malignant cells hijack enhancer elements to drive key transcriptional programs in a subtype-specific manner, and 3) annotating which cancer-specific enhancer-to-gene connections portend a worse outcome in patients. Notably, we discovered that cancer cells acquire de novo non-coding enhancer elements to modulate hallmark cancer pathways that were previously hidden using bulk genomics approaches. This highlights the potential for cancer-specific enhancers to serve as markers with diagnostic and prognostic potential, or even serve as tractable targets for therapeutic intervention. Together these data enable the annotation of the cellular composition, transcriptional, and epigenetic landscape of breast tumors to help pinpoint clinically relevant mechanisms of tumorigenesis. Citation Format: Matthew J. Regner, Aatish Thennavan, Susana Garcia-Recio, Kamila Wisniewska, Philip M. Spanheimer, Joel S. Parker, Charles M. Perou, Hector L. Franco. Determining the regulatory logic of breast cancer cells using single-cell multi-omics. [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 5754.

Background The TNT trial (NCT00532727) showed no evidence of carboplatin (C) superiority over docetaxel (D) overall in metastatic triple negative breast cancers (TNBC), but a C benefit was observed in the pre-specified sub-group analysis in patients with a gBRCA1/2 mutation (Tutt et al, Nat Med 2018). Given only ~30% of patients have a gBRCA1/2 mutation, broader predictive biomarkers of response are needed. In this cohort we previously found that DNA Damage Response (DDR) signatures were associated with improved C response in chemotherapy (CT) naïve patients only (Tovey et al, ASCO 2020). Since DDR activities influence tumour immune-microenvironment, we explored the predictive ability of immune cell markers and performed integrative analyses on multi-omics features to identify novel TNBC subgroups. Patients and Methods Tumour infiltrating lymphocytes (TILs) were evaluated on haematoxylin and eosin stained primary tumour (PT) slides for 222/376 TNT patients. Formalin-fixed paraffin-embedded PT tissues from 186/376 TNT patients were successfully profiled using total RNA-sequencing. Matched recurrence (REC) was also sequenced for 13 patients. Twenty-five immune signatures were assessed. Logistic regression and restricted mean progression free survival (PFS) were applied to delineate the relationship of these features with treatment outcomes. Random forest clustering of multi-omics DDR and immune biology markers, including gene expression signatures and mutation/methylation status, was applied to identify subgroups. We further molecularly characterised these clusters through supervised clustering of 693 gene expression “modules” (sets of co-expressed genes), immune cell deconvolution and genomic scars. Results Immune gene expression signatures and TILs were highly correlated. Average immune infiltration based on ConsensusTME was lower in mutated/methylated tumours compared with BRCA1 wildtype tumours (p=0.04). Immune signature score markers decreased from PT to REC, demonstrating a dynamic immune microenvironment. In the overall population and when restricting to prior CT treated patients, high immune infiltration (gene expression based & TILs) was associated with response to D while C response rates were not associated with immune scores (interaction p-values< 0.05). This did not translate to a PFS benefit. Multi-omics clustering identified 6 biological subgroups including immune enriched, immune depleted, DDR deficient and proficient clusters as well as 2 small clusters with no obvious distinguishing features. Immune enriched TNBC were predominantly basal-like immune activated with high B-cell/T-cell diversity. Immune depleted TNBC showed higher activity of proliferation and DDR pathway modules. DDR proficient tumours had low expression of immune markers and enrichment for ESR1/PGR expression, markers of extra cellular formation, cell structure, lipid metabolism and proliferation. The DDR deficient cluster was enriched for proliferation and demonstrated high number of TILs despite no apparent enrichment for gene expression-based immune modules. In the prior CT treated cohort, the immune enriched cluster had preferential response to D (62.5% (D) vs. 29.4% (C); p=0.02). The immune depleted cluster had preferential response to C (8.0% (D) vs. 40.0% (C); p=0.01). Numbers were too small to assess differential response within the other clusters or in the CT naïve cohort. Conclusions Tumours with high immune features have high response to D while those with low immune features have preferential response to C in advanced TNBC. Combining multi-omics markers of DDR deficiency and immune biology can identify clusters of patients with distinct biological profiles and differential treatment specific response rates. Citation Format: Holly Tovey, Orsolya Sipos, Katherine A Hoadley, Joel S Parker, Jelmar Quist, Sarah Kernaghan, Lucy Kilburn, Roberto Salgado, Sherene Loi, Richard D Kennedy, Ioannis Roxanis, Patrycja Gazinska, Sarah E. Pinder, Judith Bliss, Charles M. Perou, Syed Haider, Andrew Tutt, Anita Grigoriadis, Maggie Chon U Cheang. Histopathological and molecular immune landscape and DNA damage response signatures to predict response to carboplatin and docetaxel in TNT trial TNBC cohort [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-06.

Background: The HER2DX assay is a genomic test in early-stage HER2-positive (HER2+) breast cancer that provides prognostic and predictive information. HER2DX is a supervised learning algorithm incorporating tumor size, nodal staging, and 4 gene expression signature scores (immune/IGG, tumor cell proliferation, luminal differentiation and the expression of the HER2 amplicon). Among them, the IGG signature is associated with both overall survival and probability of achieving a pathologic complete response (pCR). Here, we studied the association of percentage (%) of tumor infiltrating lymphocytes (TILs) with HER2DX scores, immune genes and other breast cancer-related genes. Methods: HER2DX and %TILs were evaluated in 670 formalin-fixed paraffin-embedded (FFPE) samples of HER2+ breast cancer, including in 3 clinical studies SHORTHER (n=437), PAMELA (n=86), a cohort of patients treated with anti-HER2 therapy plus chemotherapy at Hospital Clínic of Barcelona (n=147). The %TILs were quantified by histological evaluation with hematoxylin eosin staining according to International TILs Working Group guidelines. The nCounter platform determined the expression of 192 genes and HER2DX scores. Pearson correlations (Cor) and Significance Analysis of Microarrays (SAM) with a false-discovery rate (FDR) < 5% assessed the association between %TILs and the expression of individual genes or HER2DX signature scores. Results: A moderate correlation was observed between %TILs and the immune IGG signature (Cor=0.56, p< 0.001). Of note, 171 (25.52%) cases had low %TILs (< 30%) and high IGG score, while 1 (0.15%) case had high %TILs (≥30%) and low IGG score. The %TILs were significantly associated with the expression of immune genes, ERBB2, IGG signature and HER2 amplicon score, and negatively associated with the expression of luminal genes (i.e., ESR1, PRG and BCL2). An unclear relationship between TILs and proliferation genes was observed. Finally, moderate correlations were observed between %TILs and HER2DX pCR score (Cor=0.48, p< 0.001) and between %TILs and HER2DX risk score (Cor=0.33, p< 0.001). Conclusions: Important differences exist between the %TILs and the HER2DX IGG signature in early-stage HER2+ breast cancer. The %TILs should not be used to predict the HER2DX scores. Biologically, a higher %TILs indirectly capture a higher ERBB2 expression and a lower expression of luminal genes, both associated with response to anti-HER2 treatment. Citation Format: Esther Sanfeliu, Fara Brasó-Maristany, Maria Vittoria Dieci, Mercedes Marín-Aguilera, Blanca González-Farré, Gaia Griguolo, Tomas Pascual, Patricia Galván, Laura Angelats, Oleguer Castillo, Paula Blasco, Valeria Sirenko, Pedro Jares, Joan Antón Puig-Butillé, Laia Paré, Antonio Martínez, Antonio Llombart-Cussac, Javier Cortés, Ana Vivancos, Patricia Villagrasa, Joel S Parker, Charles M Perou, Aleix Prat, PierFranco Conte, Valentina Guarneri. Association between tumor infiltrating lymphocytes (TILs) and the HER2DX assay in early-stage of HER2-positive (HER2+) breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-30.

Background: HER2DX (Prat et al. EBiomedicine 2022) is a 27-gene prognostic (risk-score) and predictive (pathological complete response [pCR]-score) assay in early-stage HER2+ breast cancer based on clinical data and the expression of 4 gene signatures (immune, proliferation, luminal differentiation and HER2 amplicon). Here, we aim to evaluate, for the first time, the ability of HER2DX to predict pCR following neoadjuvant TCH or TCHP in HER2+ disease. Methods: Standardized HER2DX was performed in a central lab on baseline pre-treatment FFPE tumor biopsies from the GOM-HGUGM-2018-05 study in Spain, a consecutive retrospective series of patients (pts) with newly diagnosed stage I-III HER2+ breast cancer eligible for neoadjuvant therapy. Pts received standard 6 cycles of docetaxel, carboplatin and trastuzumab (TCH) or TCH with pertuzumab (TCHP) regimens. Primary aim was to test the ability of HER2DX pCR score to predict pCR (ypT0/is ypN0). Secondary objectives were to test the ability of HER2DX pCR score to predict pCR independently of clinical-pathological variables and the PAM50 subtype (HER2-enriched versus not), and to evaluate the association of HER2DX pCR score with the HER2DX risk-score. Logistic regression and receiver-operator curve (ROC) analysis were assessed. Statistical analyses were performed in R code 4.0.5. Results: HER2DX was evaluated in 155 pts (97%) enrolled in the study with available RNA (as of June 2022). Mean age of pts was 50 (range 22-74) and 55.2% of pts (n=85) were pre-menopausal. Clinical T2-4 disease represented 77.4% of cases (n=120), clinical node-positive disease (cN1-3) represented 63.9% of cases (n=99), and 68.0% of tumors (n=105) were hormone receptor-positive. The overall pCR rate was 57.4% (95% confidence interval [CI] 50-65): 52.2% (95% CI 40-64) with TCH (n=67) and 61.4% (95% CI 50-72) with TCHP (n=88). The proportion of HER2DX low-, medium- and high-pCR groups was 34.2%, 34.8% and 31.0%, respectively. HER2DX pCR score (as a continuous variable from 0 to 100) was significantly associated with pCR (odd ratio [OR]=1.03, p=5.91e-07). The pCR rates in HER2DX pCR-high and pCR-low groups were 75.0% and 28.0% (OR=7.6, 95% CI 3.2-19.1, p=7.14e-06), respectively. In pts treated with TCHP, the pCR rates in HER2DX pCR-high and pCR-low groups were 85.7% and 27.3% (OR=16.0, 95% CI 4.3-59.01, p=3.2e-05), respectively. The AUC ROC of HER2DX pCR score (as a continuous variable) and pCR status was 0.746 (in all pts) and 0.812 (in pts treated with TCHP). HER2DX pCR score was significantly associated with pCR independently of hormone receptor status, Ki67, age, menopausal status, pertuzumab use, clinical stage and PAM50 HER2-enriched subtype. The proportion of HER2DX low- and high-risk of relapse disease was 32.0% and 68.0%, respectively. The correlation of HER2DX pCR score and HER2DX risk-score was weak (coefficient=-0.17), as previously described. Proportion of cases according to both HER2DX scores and absolute difference of pCR rates between TCHP and TCH in each combined group is shown in Table. Conclusion: The HER2DX genomic test predicts pCR following neoadjuvant TCH or TCHP regimens independently of clinical-pathological variables and intrinsic subtype. The combination of both HER2DX scores might help better tailor systemic therapy in patients with newly diagnosed stage I-III HER2+ breast cancer. Citation Format: Coralia Bueno-Muiño, Isabel Echavarria, Sara López-Tarruella, Roche-Molina Marta, María del Monte-Millán, Tatiana Massarrah, Yolanda Jerez Gilarranz, Blanca Herrero, Salvador Gámez, Iván Márquez-Rodas, María Cebollero-Presmanes, Nevado Santos Manuel, Pilar de la Morena Barrio, Francisco Ayala de la Peña, José Ángel García-Sáenz, Fernando Moreno Antón, Álvaro Rodríguez Lescure, Teresa Quintanar, Diego Malón-Giménez, Laura Rodriguez-Lajusticia, Ana Isabel Ballesteros García, Dulce Bañón Torres, Lucía Villarejo, Nerea Lobato, Ainhoa Arias, Inmaculada Ocaña, Enrique Álvarez, Laia Paré, Mercedes Marín-Aguilera, Patricia Galván, Fara Brasó-Maristany, Ana Vivancos, Patricia Villagrasa, Joel S Parker, Charles M. Perou, Aleix Prat, Miguel Martín. Independent validation of the HER2DX genomic test in HER2-positive breast cancer treated with neoadjuvant docetaxel, carboplatin, trastuzumab +/- pertuzumab (TCH/TCHP): a correlative analysis from a multicenter academic study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-46.

Background ONC201 is a dopamine receptor D2 (DRD2) antagonist that inhibits tumor growth in preclinical models through ClpP activation to induce integrated stress response pathway and mitochondrial events related to inhibition of cell growth, which is being explored in clinical trials for solid tumors and hematological malignancies. In this study, we investigated the anti-tumorigenic effect of ONC201 in endometrial cancer cell lines and a genetically engineered mouse model of endometrial cancer. Methods Cell proliferation was assessed by MTT and colony formation assays. Cell cycle and apoptosis were evaluated by Cellometer. Invasion capacity was tested using adhesion, transwell and wound healing assays. LKB1fl/flp53fl/fl mouse model of endometrial cancer were fed a control low fat diet versus a high fat diet to mimic diet-induced obesity. Following tumor onset, mice were treated with placebo or ONC201. Metabolomics and lipidomics were used to identify the obesity-dependent effects of ONC201 in the mouse endometrial tumors. DRD2 expression was analyzed by immunohistochemistry in human endometrioid and serous carcinoma specimens. DRD2 mRNA expression from the Cancer Genome Atlas (TCGA) database was compared between the four molecular subtypes of endometrial cancer. Results Increasing DRD2 expression in endometrial cancer was significantly associated with grade, serous histology and stage, as well as worse progression free survival and overall survival. Higher expression of DRD2 mRNA was found for the Copy Number High (CNH) subtype when compared to the other subtypes. ONC201 inhibited cell proliferation, induced cell cycle G1 arrest, caused cellular stress and apoptosis and reduced invasion in endometrial cancer cells. Diet-induced obesity promoted endometrial tumor growth while ONC201 exhibited anti-tumorigenic efficacy in the obese and lean LKB1fl/fl/p53fl/fl mice. Metabolomic analysis demonstrated that ONC201 reversed the obesity-driven upregulation of lipid biosynthesis and reduced protein biosynthesis in obese and lean mice. Conclusion ONC201 has anti-tumorigenic effects in endometrial cancer cells and a transgenic mouse model of endometrial cancer, and DRD2 expression was documented in both human serous and endometrioid endometrial cancer. These studies support DRD2 antagonism via ONC201 as a promising therapeutic strategy for endometrial cancer that has already demonstrated pharmacodynamic activity and clinical benefit in both serous and endometrioid endometrial cancer patients.

PURPOSE CALGB 40601 assessed whether dual versus single human epidermal growth factor receptor 2 (HER2) –targeting drugs added to neoadjuvant chemotherapy increased pathologic complete response (pCR). Here, we report relapse-free survival (RFS), overall survival (OS), and gene expression signatures that predict pCR and survival. PATIENTS AND METHODS Three hundred five women with untreated stage II and III HER2-positive breast cancer were randomly assigned to receive weekly paclitaxel combined with trastuzumab plus lapatinib (THL), trastuzumab (TH), or lapatinib (TL). The primary end point was pCR, and secondary end points included RFS, OS, and gene expression analyses. mRNA sequencing was performed on 264 pretreatment samples. RESULTS One hundred eighteen patients were randomly allocated to THL, 120 to TH, and 67 to TL. At more than 7 years of follow-up, THL had significantly better RFS and OS than did TH (RFS hazard ratio, 0.32; 95% CI, 0.14 to 0.71; P = .005; OS hazard ratio, 0.34; 95% CI, 0.12 to 0.94; P = .037), with no difference between TH and TL. Of 688 previously described gene expression signatures, significant associations were found in 215 with pCR, 45 with RFS, and only 22 with both pCR and RFS (3.2%). Specifically, eight immune signatures were significantly correlated with a higher pCR rate and better RFS. Among patients with residual disease, the immunoglobulin G signature was an independent, good prognostic factor, whereas the HER2-enriched signature, which was associated with a higher pCR rate, showed a significantly shorter RFS. CONCLUSION In CALGB 40601, dual HER2-targeting resulted in significant RFS and OS benefits. Integration of intrinsic subtype and immune signatures allowed for the prediction of pCR and RFS, both overall and within the residual disease group. These approaches may provide means for rational escalation and de-escalation treatment strategies in HER2-positive breast cancer.

Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2(–)). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/ER(+) tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification.

We hypothesized that human genes differ by their sensitivity to ultraviolet (UV) exposure. We used somatic mutations detected by genome-wide screens in melanoma and reported in the Catalog Of Somatic Mutations In Cancer. As a measure of UV sensitivity, we used the number of silent mutations generated by C>T transitions in pyrimidine dimers of a given transcript divided by the number of potential sites for this type of mutations in the transcript. We found that human genes varied by UV sensitivity by two orders of magnitude. We noted that the melanoma-associated tumor suppressor gene CDKN2A was among the top five most UV-sensitive genes in the human genome. Melanoma driver genes have a higher UV-sensitivity compared with other genes in the human genome. The difference was more prominent for tumor suppressors compared with oncogene. The results of this study suggest that differential sensitivity of human transcripts to UV light may explain melanoma specificity of some driver genes. Practical significance of the study relates to the fact that differences in UV sensitivity among human genes need to be taken into consideration whereas predicting melanoma-associated genes by the number of somatic mutations detected in a given gene.

BackgroundPan-cancer genomic analyses based on the magnitude of pathway activity are currently lacking. Focusing on the cell cycle, we examined the DNA mutations and chromosome arm-level aneuploidy within tumours with low, intermediate and high cell cycle activity.Patients and methodsMatching mRNA, gene mutational status, chromosomal arm-level aberrations and clinico-pathological data was assembled from pan-cancer studies of 9,515 patients with 32 different cancers. Cell cycle activity was estimated from mRNA data using the cell cycle score (CCS) signature. Barplots were used to visualise mutation and chromosomal aberration frequency within CCS subgroups. Kaplan-Meier and multivariable Cox-regression analyses were used to determine survival differences between CCS subgroups.ResultsCell cycle activity varied broadly across and within all cancers. TP53 and PIK3CA mutations were common in all CCS subgroups but with increasing frequency as cell cycle activity levels increased (P < 0.001). Mutations in BRAF and gains in 16p were less frequent CCS high tumours (P < 0.001). In Kaplan-Meier analysis, patients whose tumours were CCS Low had a longer PFI relative to intermediate or high (P < 0.001) and this significance remained in multivariable analysis (CCS intermediate: HR = 1.37; 95% CI 1.17 – 1.60, CCS high: 1.54; 1.29 – 1.84, CCS Low = Ref).ConclusionsCell cycle activity varies across and within cancers and whilst similar DNA alterations can be found at all activity levels, some notable exceptions exist. These data also demonstrate that independent prognostic information can be derived on a pan-cancer level from a simple measure of cell cycle activity.

Background: It has become increasingly clear that effective treatment of metastatic breast cancer (MBC) requires an in-depth understanding of the molecular differences between primary tumors and metastases. The AURORA US Network was established to collect primary breast cancer-metastasis pairs for multi-platform genomic profiling in order to identify the molecular drivers of metastatic disease. AURORA US has both a retrospective and prospective phase. This is the first report of the retrospective phase. Methods: Archived tissue samples from the primary tumor and at least one distant metastasis were retrospectively collected from 83 MBC patients. Following internal quality assessment, samples from 55 pts, including 105 distinct metastatic lesions, were subject to DNA low pass whole genome and exome sequencing, DNA methylation arrays, and RNA sequencing. Early analyses of these multi-platform data include: DNA methylation, tumor gene expression and microenvironmental signatures, somatic and germline variants, DNA copy number changes, and structural variants between breast primaries and matched metastases. Results: Median age at diagnosis was 49 years (25-76); 32 (58%) were Stage I or II at presentation, 27 (49%) had a family history of breast cancer, and 20 (36%) had a second breast primary. Median disease-free interval before developing MBC was 2 years (range 0-36, 5 patients presented with Stage IV). Median overall survival from initial presentation was 4 years (range 0-37). Median survival after developing MBC was 1 year (range 0-13), with a median of three treatments. Primary tissue samples were banked from 1977-2017 and metastases were banked from 1999-2017. Clinical phenotypes of the primaries included 27 HR+ (49%), 15 triple negative (TNBC, 27%), and 11 HER2+ (20%, 12 missing HER2 status). Intrinsic subtype distribution of the primaries included 17 Basal-like (31%), 17 Luminal A (31%), 7 Normal-like (13%), 5 HER2-enriched (9%), and 1 Luminal B, with 8 pending. All metastases from the Basal-like cases remained Basal-like, while metastases from luminal primaries tended to gain HER2-Enriched subtype features (5/18, p = 0.01). Overall, we identified significant metastasis-enriched alterations in metabolism pathways, an increase in proliferation, and the loss of differentiation signatures and immune infiltrates with progression; the latter being the most pronounced in brain metastases. The most frequent somatic mutations in this cohort were in TP53, NCOR1, and RUNX1. Interestingly, ERBB2, EGFR, and ATM were also mutated in ≥10% of the tumors sequenced. In almost all cases, CpG island hypermethylation was clonally present in the primary tumor and persisted stably in the majority of metastatic lesions. Promoter CpG island hypermethylation was also identified in some metastatic lesions at JAM3, an important cellular adhesion molecule,and this was accompanied by reduced mRNA expression. CONCLUSIONS: Collection of banked primary and metastatic tissue pairs identified a young MBC cohort with a high frequency of breast cancer family history and second breast primaries. Molecular characterization of luminal tumor pairs highlighted acquisition of aggressive traits including increased proliferation and loss of differentiation in the metastases. In contrast, basal-like pairs remained relatively unchanged, except for the loss of immune activation. Ongoing analyses to be presented include clonal heterogeneity and phylogeny, novel metastasis signature discovery, gene fusion, and endogenous retrovirus detection. Citation Format: Tari A King, Minetta C Liu, Marni B McClure, Toshinori Hinoue, Benjamin J Kelly, Chad J Creighton, Jay Bowen, Kristen Leraas, Robyn T Burns, Sara Coppens, Salma Rezk, Amy L Garrett, Justin M Balko, Joel S Parker, Ben H Park, Ian Krop, Carey Anders, Katherine A Hoadley, Julie Gastier-Foster, Mothaffar F Rimawi, Rita Nanda, Nancy U Lin, Claudine Isaacs, P. Kelly Marcom, Anna Maria Storniolo, Fergus J Couch, Elaine R Mardis, Adrian V Lee, Uma Chandran, Peter W Laird, Susan G Hilsenbeck, Larry Norton, Andrea L Richardson, W. Fraser Symmans, Lisa A Carey, Antonio C Wolff, Nancy E Davidson, Charles M Perou, the AURORA US Network. Multiplatform analysis of matched primary and metastatic breast tumors from the AURORA US Network [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS3-08.

Purpose Claudin-low molecular subtypes have been identified in breast and bladder cancers and are characterized by low expression of claudins, enrichment for epithelial-to-mesenchymal transition (EMT), and tumor-initiating cell (TIC) features. We evaluated whether the claudin-low subtype also exists in gastric cancer. Materials and Methods Four hundred fifteen tumors from The Cancer Genome Atlas (TCGA) gastric cancer mRNA data set were clustered on the claudin, EMT, and TIC gene sets to identify claudin-low tumors. We derived a 24-gene predictor that classifies gastric cancer into claudin-low and non–claudin-low subtypes. This predictor was validated with the Asian Cancer Research Group (ACRG) data set. We characterized molecular and clinical features of claudin-low tumors. Results We identified 46 tumors that had consensus enrichment for claudin-low features in TCGA data set. Claudin-low tumors were most commonly diffuse histologic type (82%) and originally classified as TCGA genomically stable (GS) subtype (78%). Compared with GS subtype, claudin-low subtype had significant activation in Rho family of GTPases signaling, which appears to play a key role in its EMT and TIC properties. In the ACRG data set, 28 of 300 samples were classified as claudin-low tumors by the 24-gene predictor and were phenotypically similar to the initially derived claudin-low tumors. Clinically, claudin-low subtype had the worst overall survival. Of note, the hazard ratios that compared claudin-low versus GS subtype were 2.10 (95% CI, 1.07 to 4.11) in TCGA and 2.32 (95% CI, 1.18 to 4.55) in the ACRG cohorts, with adjustment for age and pathologic stage. Conclusion We identified a gastric claudin-low subtype that carries a poor prognosis likely related to therapeutic resistance as a result of its EMT and TIC phenotypes.

BACKGROUNDThe KRAS proto-oncogene is among the most frequently mutated genes in cancer, yet for 40 years it remained an elusive therapeutic target. Recently, allosteric inhibitors that covalently bind to KRAS G12C mutations have been approved for use in lung adenocarcinomas. Although responses are observed, they are often short-lived, thus making in-depth characterization of the mechanisms of resistance of paramount importance.METHODSHere, we present a rapid-autopsy case of a patient who had a KRASG12C-mutant lung adenocarcinoma who initially responded to a KRAS G12C inhibitor but then rapidly developed resistance. Using deep-RNA and whole-exome sequencing comparing pretreatment, posttreatment, and matched normal tissues, we uncover numerous mechanisms of resistance to direct KRAS inhibition.RESULTSIn addition to decreased KRAS G12C-mutant allele frequency in refractory tumors, we also found reactivation of the MAPK pathway despite no new mutations in KRAS or its downstream mediators. Tumor cell-intrinsic and non-cell autonomous mechanisms included increased complement activation, coagulation, and tumor angiogenesis, and several lines of evidence of immunologic evasion.CONCLUSIONTogether, our findings reveal numerous mechanisms of resistance to current KRAS G12C inhibitors through enrichment of clonal populations, KRAS-independent downstream signaling, and diverse remodeling of the tumor microenvironment.FUNDINGRichard and Fran Duley, Jimmy and Kay Mann, the NIH, and the North Carolina Biotechnology Center.

Gene expression; Breast cancer; Prognosis Expressió gènica; Càncer de mama; Pronòstic Expresión génica; Cáncer de mama; Pronóstico Background: Both clinical and genomic data independently predict survival and treatment response in early-stage HER2-positive breast cancer. Here we present the development and validation of a new HER2DX risk score, and a new HER2DX pathological complete response (pCR) score, both based on a 27-gene expression plus clinical feature-based classifier. Methods: HER2DX is a supervised learning algorithm incorporating tumour size, nodal staging, and 4 gene expression signatures tracking immune infiltration, tumour cell proliferation, luminal differentiation, and the expression of the HER2 amplicon, into a single score. 434 HER2-positive tumours from the Short-HER trial were used to train a prognostic risk model; 268 cases from an independent cohort were used to verify the accuracy of the HER2DX risk score. In addition, 116 cases treated with neoadjuvant anti-HER2-based chemotherapy were used to train a predictive model of pathological complete response (pCR); two independent cohorts of 91 and 67 cases were used to verify the accuracy of the HER2DX pCR likelihood score. Five publicly available independent datasets with >1,000 patients with early-stage HER2-positive disease were also analysed. Findings: In Short-HER, HER2DX variables were associated with good risk outcomes (i.e., immune, and luminal) and poor risk outcomes (i.e., proliferation, and tumour and nodal staging). In an independent cohort, continuous HER2DX risk score was significantly associated with disease-free survival (DFS) (p=0·002); the 5-year DFS in the low-risk group was 97·4% (94·4-100·0%). For the neoadjuvant pCR predictor training cohort, HER2DX variables were associated with pCR (i.e., immune, proliferation and HER2 amplicon) and non-pCR (i.e., luminal, and tumour and nodal staging). In both independent test set cohorts, continuous HER2DX pCR likelihood score was significantly associated with pCR (p

BackgroundTriple negative breast cancer (TNBC) is an aggressive variant of breast cancer that lacks the expression of estrogen and progesterone receptors (ER and PR) and HER2. Nearly 50% of patients with advanced TNBC will develop brain metastases (BrM), commonly with progressive extracranial disease. Immunotherapy has shown promise in the treatment of advanced TNBC; however, the immune contexture of BrM remains largely unknown. We conducted a comprehensive analysis of TNBC BrM and matched primary tumors to characterize the genomic and immune landscape of TNBC BrM to inform the development of immunotherapy strategies in this aggressive disease.MethodsWhole-exome sequencing (WES) and RNA sequencing were conducted on formalin-fixed, paraffin-embedded samples of BrM and primary tumors of patients with clinical TNBC (n = 25, n = 9 matched pairs) from the LCCC1419 biobank at UNC—Chapel Hill. Matched blood was analyzed by DNA sequencing as a comparison for tumor WES for the identification of somatic variants. A comprehensive genomics assessment, including mutational and copy number alteration analyses, neoantigen prediction, and transcriptomic analysis of the tumor immune microenvironment were performed.ResultsPrimary and BrM tissues were confirmed as TNBC (23/25 primaries, 16/17 BrM) by immunohistochemistry and of the basal intrinsic subtype (13/15 primaries and 16/19 BrM) by PAM50. Compared to primary tumors, BrM demonstrated a higher tumor mutational burden. TP53 was the most frequently mutated gene and was altered in 50% of the samples. Neoantigen prediction showed elevated cancer testis antigen- and endogenous retrovirus-derived MHC class I-binding peptides in both primary tumors and BrM and predicted that single-nucleotide variant (SNV)-derived peptides were significantly higher in BrM. BrM demonstrated a reduced immune gene signature expression, although a signature associated with fibroblast-associated wound healing was elevated in BrM. Metrics of T and B cell receptor diversity were also reduced in BrM.ConclusionsBrM harbored higher mutational burden and SNV-derived neoantigen expression along with reduced immune gene signature expression relative to primary TNBC. Immune signatures correlated with improved survival, including T cell signatures. Further research will expand these findings to other breast cancer subtypes in the same biobank. Exploration of immunomodulatory approaches including vaccine applications and immune checkpoint inhibition to enhance anti-tumor immunity in TNBC BrM is warranted.

Aberrant activation of the androgen receptor (AR) may play a critical role in castration resistant prostate cancer. After ligand binding, AR is recruited to the androgen responsive element (ARE) sequences on the DNA where AR interaction with coactivators and corepressors modulates transcription. We demonstrated that phosphorylation of AR at Tyr-267 by Ack1/TNK2 tyrosine kinase results in nuclear translocation, DNA binding, and androgen-dependent gene transcription in a low androgen environment. In order to dissect downstream mechanisms, we searched for proteins whose interaction with AR was regulated by Ack1. SLIRP (SRA stem-loop interacting RNA binding protein) was identified as a candidate protein. Interaction between AR and SLIRP was disrupted by Ack1 kinase activity as well as androgen or heregulin treatment. The noncoding RNA, SRA, was required for AR-SLIRP interaction. SLIRP was bound to ARE’s of AR target genes in the absence of androgen. Treatment with androgen or heregulin led to dissociation of SLIRP from the ARE. Whole transcriptome analysis of SLIRP knockdown in androgen responsive LNCaP cells showed that SLIRP affects a significant subset of androgen-regulated genes. Our data suggest that Ack1 kinase and androgen regulate interaction between AR and SLIRP and that SLIRP functions as a coregulator of AR with properties of a corepressor in a context-dependent manner.

Cutaneous melanoma can be lethal even if detected at an early stage. Epigenetic profiling may facilitate the identification of aggressive primary melanomas with unfavorable outcomes. We performed clustering of whole-genome methylation data to identify subclasses that were then assessed for survival, clinical features, methylation patterns, and biological pathways. Among 89 cutaneous primary invasive melanomas, we identified three methylation subclasses exhibiting low methylation, intermediate methylation, or hypermethylation of CpG islands, known as the CpG island methylator phenotype (CIMP). CIMP melanomas occurred as early as tumor stage 1b and, compared with low-methylation melanomas, were associated with age at diagnosis ≥65 years, lentigo maligna melanoma histologic subtype, presence of ulceration, higher American Joint Committee on Cancer stage and tumor stage, and lower tumor-infiltrating lymphocyte grade (all P0.05). Patients with CIMP melanomas had worse melanoma-specific survival (hazard ratio = 11.84; confidence interval = 4.65‒30.20) than those with low-methylation melanomas, adjusted for age, sex, American Joint Committee on Cancer stage, and tumor-infiltrating lymphocyte grade. Genes hypermethylated in CIMP compared with those in low-methylation melanomas included PTEN, VDR, PD-L1, TET2, and gene sets related to development/differentiation, the extracellular matrix, and immunity. CIMP melanomas exhibited hypermethylation of genes important in melanoma progression and tumor immunity, and although present in some early melanomas, CIMP was associated with worse survival independent of known prognostic factors.

Despite years of progress, mutation detection in cancer samples continues to require significant manual review as a final step. Expert review is particularly challenging in cases where tumors are sequenced without matched normal control DNA. Attempts have been made to call somatic point mutations without a matched normal sample by removing well-known germline variants, utilizing unmatched normal controls, and constructing decision rules to classify sequencing errors and private germline variants. With budgetary constraints related to computational and sequencing costs, finding the appropriate number of controls is a crucial step to identifying somatic variants. Our approach utilizes public databases for canonical somatic variants as well as germline variants and leverages information gathered about nearby positions in the normal controls. Drawing from our cohort of targeted capture panel sequencing of tumor and normal samples with varying tumortypes and demographics, these served as a benchmark for our tumor-only variant calling pipeline to observe the relationship between our ability to correctly classify variants against a number of unmatched normals. With our benchmarked samples, approximately ten normal controls were needed to maintain 94% sensitivity, 99% specificity and 76% positive predictive value, far outperforming comparable methods. Our approach, called UNMASC, also serves as a supplement to traditional tumor with matched normal variant calling workflows and can potentially extend to other concerns arising from analyzing next generation sequencing data.

Interactions between WD40 repeat domain protein 5 (WDR5) and its various partners such as mixed lineage leukemia (MLL) and c-MYC are essential for sustaining oncogenesis in human cancers. However, inhibitors that block protein-protein interactions (PPIs) between WDR5 and its binding partners exhibit modest cancer cell killing effects and lack in vivo efficacy. Here, we present pharmacological degradation of WDR5 as a promising therapeutic strategy for treating WDR5-dependent tumors and report two high-resolution crystal structures of WDR5-degrader-E3 ligase ternary complexes. We identified an effective WDR5 degrader via structure-based design and demonstrated its in vitro and in vivo antitumor activities. On the basis of the crystal structure of an initial WDR5 degrader in complex with WDR5 and the E3 ligase von Hippel–Lindau (VHL), we designed a WDR5 degrader, MS67, and demonstrated the high cooperativity of MS67 binding to WDR5 and VHL by another ternary complex structure and biophysical characterization. MS67 potently and selectively depleted WDR5 and was more effective than WDR5 PPI inhibitors in suppressing transcription of WDR5-regulated genes, decreasing the chromatin-bound fraction of MLL complex components and c-MYC, and inhibiting the proliferation of cancer cells. In addition, MS67 suppressed malignant growth of MLL-rearranged acute myeloid leukemia patient cells in vitro and in vivo and was well tolerated in vivo. Collectively, our results demonstrate that structure-based design can be an effective strategy to identify highly active degraders and suggest that pharmacological degradation of WDR5 might be a promising treatment for WDR5-dependent cancers.

Chromosome 11q13-14 amplification is a defining feature of high-risk hormone receptor-positive (HR+) breast cancer; however, the mechanism(s) by which this amplicon contributes to breast tumorigenesis remains unclear. In the current study, proteogenomic analyses of >3000 breast tumors from the TCGA, METABRIC and CPTAC studies demonstrated that carnitine palmitoyltransferase 1A (CPT1A), which is localized to this amplicon, is overexpressed at the mRNA and protein level in aggressive luminal tumors, strongly associated with indicators of tumor proliferation and a predictor of poor prognosis. In vitro genetic studies demonstrated that CPT1A is required for and can promote luminal breast cancer proliferation, survival, as well as colony and mammosphere formation. Since CPT1A is the rate-limiting enzyme during fatty acid oxidation (FAO), our data indicate that FAO may be essential for these tumors. Pharmacologic inhibition of FAO prevented in vitro and in vivo tumor growth and cell proliferation as well as promoted apoptosis in luminal breast cancer cells and orthotopic xenograft tumor models. Collectively, our data establish an oncogenic role for CPT1A and FAO in HR+ luminal tumors and provide preclinical evidence and rationale supporting further investigation of FAO as a potential therapeutic opportunity for the treatment of HR+ breast cancer.

Current tumor neoantigen calling algorithms primarily rely on epitope/major histocompatibility complex (MHC) binding affinity predictions to rank and select for potential epitope targets. These algorithms do not predict for epitope immunogenicity using approaches modeled from tumor-specific antigen data. Here, we describe peptide-intrinsic biochemical features associated with neoantigen and minor histocompatibility mismatch antigen immunogenicity and present a gradient boosting algorithm for predicting tumor antigen immunogenicity. This algorithm was validated in two murine tumor models and demonstrated the capacity to select for therapeutically active antigens. Immune correlates of neoantigen immunogenicity were studied in a pan-cancer data set from The Cancer Genome Atlas and demonstrated an association between expression of immunogenic neoantigens and immunity in colon and lung adenocarcinomas. Lastly, we present evidence for expression of an out-of-frame neoantigen that was capable of driving antitumor cytotoxic T-cell responses. With the growing clinical importance of tumor vaccine therapies, our approach may allow for better selection of therapeutically relevant tumor-specific antigens, including nonclassic out-of-frame antigens capable of driving antitumor immunity.

Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy., Polymerase theta is a widely conserved DNA polymerase that mediates Theta Mediated End Joining. Here authors present a synthetic lethal CRISPR screen to identify DDR gene mutations that induce cellular addiction to Pol theta.

Lymph node (LN) metastases correspond with a worse prognosis in nearly all cancers, yet the occurrence of cancer spreading from LNs remains controversial. Additionally, the mechanisms explaining how cancers survive and exit LNs are largely unknown. Here, we show that breast cancer patients frequently have LN metastases that closely resemble distant metastases. In addition, using a microsurgical model, we show how LN metastasis development and dissemination is regulated by the expression of a chromatin modifier, histone deacetylase 11 (HDAC11). Genetic and pharmacologic blockade of HDAC11 decreases LN tumor growth, yet substantially increases migration and distant metastasis formation. Collectively, we reveal a mechanism explaining how HDAC11 plasticity promotes breast cancer growth as well as dissemination from LNs and suggest caution with the use of HDAC inhibitors., The prognosis of cancer patients with lymph node (LN) metastasis is worse than those without. Here, the authors report that while histone deacetylase 11 (HDAC11) inhibition suppresses tumor growth within the LN, it also promotes cancer cell migration out of the LN to form distant metastasis, and therefore suggest caution with HDAC inhibitors.

Background Measures of the adaptive immune response have prognostic and predictive associations in melanoma and other cancer types. Specifically, intratumoral T cell density and function have considerable prognostic and predictive value in skin cutaneous melanoma (SKCM). Less is known about the significance of tumor-infiltrating B cells in SKCM. Our goal was to understand the prognostic and predictive value of B cell phenotypic subsets in SKCM using RNA sequencing. Methods We used our previously published algorithm, V’DJer, to assemble B cell receptor (BCR) repertoires and estimate diversity from short-read RNA sequencing (RNA-seq). We applied machine learning-based cellular phenotype classifiers to measure relative similarity of bulk tumor sample gene expression profiles and different B cell phenotypes. We assessed these aspects of B cell biology in 473 SKCM from the Cancer Genome Atlas Project (TCGA) as well as in RNA-seq data corresponding to tumor samples procured from patients who received CTLA-4 and PD-1 inhibitors for metastatic SKCM. Results We found that the BCR repertoire was associated with different clinical factors, such as tumor tissue site and sex. However, increased clonality of the BCR repertoire was favorably prognostic in SKCM and was prognostic even after first conditioning on various clinical factors. Mutation burden was not correlated with any BCR measurement, and no specific mutation had an altered BCR repertoire. Lack of an assembled BCR in pre-treatment tumor tissues was associated with a lack of anti-tumor response to a CTLA-4 inhibitor in metastatic SKCM. Conclusions These findings suggest an important prognostic and predictive role for B cell characteristics in SKCM. This has implications for melanoma immunobiology and potential development of immunogenomics features to predict survival and response to immunotherapy. Electronic supplementary material The online version of this article (10.1186/s13073-019-0647-5) contains supplementary material, which is available to authorized users.

In tumors, extravascular fibrin forms provisional scaffolds for endothelial cell (EC) growth and motility during angiogenesis. We report that fibrin-mediated angiogenesis was inhibited and tumor growth delayed following postnatal deletion of Tgfbr2 in the endothelium of Cdh5-Cre(ERT2) Tgfbr2(fl/fl) mice (Tgfbr2(iECKO) mice). ECs from Tgfbr2(iECKO) mice failed to upregulate the fibrinolysis inhibitor plasminogen activator inhibitor 1 (Serpine1, also known as PAI-1), due in part to uncoupled TGF-β–mediated suppression of miR-30c. Bypassing TGF-β signaling with vascular tropic nanoparticles that deliver miR-30c antagomiRs promoted PAI-1–dependent tumor growth and increased fibrin abundance, whereas miR-30c mimics inhibited tumor growth and promoted vascular-directed fibrinolysis in vivo. Using single-cell RNA-Seq and a NanoString miRNA array, we also found that subtypes of ECs in tumors showed spectrums of Serpine1 and miR-30c expression levels, suggesting functional diversity in ECs at the level of individual cells; indeed, fresh EC isolates from lung and mammary tumor models had differential abilities to degrade fibrin and launch new vessel sprouts, a finding that was linked to their inverse expression patterns of miR-30c and Serpine1 (i.e., miR-30c(hi) Serpine1(lo) ECs were poorly angiogenic and miR-30c(lo) Serpine1(hi) ECs were highly angiogenic). Thus, by balancing Serpine1 expression in ECs downstream of TGF-β, miR-30c functions as a tumor suppressor in the tumor microenvironment through its ability to promote fibrin degradation and inhibit blood vessel formation.

Immune checkpoint inhibitor (ICI) therapies have led to remarkable clinical responses in cancers such as melanoma and non-small cell lung cancer. In breast cancer, current immunotherapy trials have placed an emphasis on triple negative breast cancers (TNBC), where early results suggest response rates of 10-20%. Thus, it is critical to identify predictive biomarkers to enhance patient selection for immunotherapy. With this goal in mind, we simulated a clinical trial employing anti-PD1 and anti-CTLA therapies in immune-intact genetically engineered mouse models (GEMMs) of TNBC. Testing of ICI therapies on 8 different GEMMs demonstrated that each model was resistant. Whole exome sequencing showed that each model also harbored a low mutation burden. Given that mutation load is predictive of immunotherapy response in other cancer types, and that Apobec3B activity is associated with higher tumor mutation burden (TMB) in breast cancer, we created two different tumor lines with overexpression of murine Apobec3. In contrast to the parental lines, the Apobec3 overexpressing lines showed an elevated tumor mutation burden and new mutations were consistent with the Apobec mutation signature. These TNBC lines with new mutations resulting from Apobec3 activity were exquisitely sensitive to anti-PD1/anti-CTLA4 combination therapy; as assessed by reduction in tumor volume and extended overall survival. To identify features that predict response, we examined resistant and sensitive tumors at pretreatment, at 1 week of treatment, and at end stage by flow cytometry and mRNA-seq. Gene expression profiling identified multiple immune signatures as predictive of response to ICI therapy; specifically CD8+ T-effector memory cells, CD4+ T-cells, and activated B-Cells. Similarly, gene expression analysis showed that these cell types increased at 1 week of therapy in sensitive models but not in resistant models. Flow cytometry confirmed these predictions. Next, we used an antibody based approach to separately deplete CD4+ T-Cells, CD8+ T-cells, or B-cells in Apobec3 mutagenized murine tumors receiving aPD1/aCTLA4 combination therapy. In each case, depletion of these populations significantly reduced the therapeutic response. However, mice receiving combination immunotherapy and depleted for CD8+ T-cells still exhibited a significant extension in overall survival compared to non-treated controls. In contrast, the CD4+ T-cell depleted mice and B-cell depleted mice exhibited no ICI therapeutic benefit. Together, these data point to key immune biomarkers of response to anti-PD1/anti-CTLA4 therapy; we have further developed a genomic predictor of ICI response using our murine models and will test this on a human TNBC data set. Lastly, this GEMM system provides a rich RNA-seq resource, and new immune-activated models for TNBC, which uncovered a key role for B-cells and CD4+ T-cells in response to ICI therapies. Citation Format: Hollern DP, Xu N, Mott KR, He X, Carey-Ewend K, Marron DS, Ford J, Parker JS, Vincent BG, Serody JS, Perou CM. Apobec3 induced mutagenesis sensitizes murine models of triple negative breast cancer to immunotherapy by activating B-cells and CD4+ T-cells [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 GS1-05.

Background: The expression of ER, PR and HER2 in breast cancer is determined by routine pathology assessment to indicate systemic treatment. Here, we investigated the ability of a standardized mRNA-based assay to predict ER, PR and HER2 status and treatment response. Methods: ESR1, PGR and ERBB2 expression was analyzed from the standardized nCounter-based PAM50 assay in 1,544 FFPE breast tumors obtained from 13 independent studies (NeoEribulin, GEICAM 2012-09, TBCRC023/006, LPT109096, EGF117165, PAMELA, PerELISA, GEICAM 2003-11, VENTANA, IBIMA and HCB). All immunohistochemical and in situ hybridization analyses followed ASCO/CAP criteria and were performed in central labs except for Neoeribulin and VENTANA studies. To explore the best cutoff for each gene, we used Monte Carlo cross validation (repeated 1000 times, 2/3 training, 1/3 testing) to achieve the highest kappa values when gene expression was compared to pathology assessment. Receiver operating characteristic analysis and area under the ROC curve (AUC) was used to evaluate the performance of each gene to predict ER, PR and HER2 status. Finally, the association of each gene (using the pre-established cutoffs) with pathological complete response (pCR) was evaluated using univariate logistic regression analyses in 2 neoadjuvant cohorts: 1) A combined HER2+ cohort using 191 tumor samples from PAMELA/PerELISA phase II trials, where patients received neoadjuvant dual HER2 blockade without chemotherapy for 15-18 weeks and 2) a combined cohort of hormone receptor-positive/HER2-negative using 205 tumor samples from IBIMA/HCB consecutive series, where patients received anthracycline/taxane-based chemotherapy. Results: Concordance between ESR1 and ER was 95.3% (95% confidence interval [95CI] 94.0-96.4%; AUC=0.98). ER+ and ER- cases were classified as ESR1- and ESR1+ in 5.5% and 4.8% of the cases, respectively. Concordance between PR and PGR was 85.2% (95CI 83.1-87.1%; AUC=0.95), and between ERBB2 and HER2 was 92.8% (95CI 91.4-94.1%; AUC=0.95). HER2+ and HER2- cases were classified ERBB2- and ERBB2+ in 16.7% and 2.9% of the cases, respectively. In the neoadjuvant HER2+ cohort, the pCR rates were 44.6% in ESR1- and 18.8% in ESR1+ (odds ratio [OR] 3.9; 95CI 1.9-8.4; p Conclusion: ESR1, PGR and ERBB2 standardized mRNA levels show high concordance with ER, PR and HER2, and might provide a more objective and quantitative prediction of response to chemotherapy and anti-HER2 therapy. The level of concordance between GE and pathology-based assessments is similar to 2 FDA 510(k) cleared pathology-based ER assays (e.g. SP1 versus 6F11 antibody clones), local versus central HER2 testing, and ER or PR status when determined by the ligand-binding assay versus immunohistochemistry. Citation Format: Pascual T, Tsai YS, Martín M, Lluch A, Cortes J, Llombart A, Conte P, Guarneri V, Rimawi MF, Alba E, Ruiz-Borrego M, Rojo F, de la Haba J, Schiff R, Adamo B, Vidal M, Paré L, Chic N, Muñoz M, Galvan P, Gonzalez-Farre B, Brauer HA, Sullivan A, Nuciforo P, Parker JS, Prat A. Standardized nCounter-based determination of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor status in breast cancer [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 P2-08-06.

This abstract was not presented at the conference. Citation Format: Hardeman A, Grushko T, Clayton W, Hurley I, Khramtsova G, Parker J, Perou C, Olopade O. Not presented [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 P3-07-05.

The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This theory has been limited, however, by an inability to identify and isolate individual senescent cells within an intact organism. Toward that end, we generated a murine reporter strain by “knocking-in” a fluorochrome, tandem-dimer Tomato (tdTom), into exon 1α of the p16INK4a locus. We used this allele (p16tdTom) for the enumeration, isolation, and characterization of individual p16INK4a-expressing cells (tdTom+). The half-life of the knocked-in transcript was shorter than that of the endogenous p16INK4a mRNA, and therefore reporter expression better correlated with p16INK4a promoter activation than p16INK4a transcript abundance. The frequency of tdTom+ cells increased with serial passage in cultured murine embryo fibroblasts from p16tdTom/+ mice. In adult mice, tdTom+ cells could be readily detected at low frequency in many tissues, and the frequency of these cells increased with aging. Using an in vivo model of peritoneal inflammation, we compared the phenotype of cells with or without activation of p16INK4a and found that tdTom+ macrophages exhibited some features of senescence, including reduced proliferation, senescence-associated β-galactosidase (SA-β-gal) activation, and increased mRNA expression of a subset of transcripts encoding factors involved in SA-secretory phenotype (SASP). These results indicate that cells harboring activation of the p16INK4a promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence.

Motivation Genomic variant detection from next-generation sequencing has become established as an extremely important component of research and clinical diagnoses in both cancer and Mendelian disorders. Insertions and deletions (indels) are a common source of variation and can frequently impact functionality, thus making their detection vitally important. While substantial effort has gone into detecting indels from DNA, there is still opportunity for improvement. Further, detection of indels from RNA-Seq data has largely been an afterthought and offers another critical area for variant detection. Results We present here ABRA2, a redesign of the original ABRA implementation that offers support for realignment of both RNA and DNA short reads. The process results in improved accuracy and scalability including support for human whole genomes. Results demonstrate substantial improvement in indel detection for a variety of data types, including those that were not previously supported by ABRA. Further, ABRA2 results in broad improvements to variant calling accuracy across a wide range of post-processing workflows including whole genomes, targeted exomes and transcriptome sequencing. Availability and implementation ABRA2 is implemented in a combination of Java and C/C++ and is freely available to all from: https://github.com/mozack/abra2. Supplementary information Supplementary data are available at Bioinformatics online.

2636 Background: The HER2DX prognostic assay in early-stage HER2-positive breast cancer integrates clinical variables and 4 gene expression signatures (GES) tracking IGG, tumor proliferation, luminal cell differentiation, and the expression of the HER2 amplicon. Here, we assessed the prognostic value across cancer-types of each of these four individual GES and a research-based version of the HER2DX risk-score. Methods: RSEM batch normalized RNA-sequencing gene expression data from The Cancer Genome Atlas (TCGA) project were downloaded from cBioPortal. The association between a research-based version of the HER2DX risk-score, and each GES, with overall survival (OS) was assessed as continuous variables using univariate Cox regression model. The research-based version of the HER2DX risk-score tested did not include clinical variables. The Cox model was applied to estimate hazard ratios (HR) with 95% confidence intervals. The threshold for statistical significance was set at p< 0.05. Results: Gene expression data from 9,852 patients representing 30 different cancer-types were evaluated. The proliferation, IGG, luminal and HER2 amplicon GES were significantly associated with OS in 40.0%, 33.3%, 23.0% and 6.7% of the cancer-types tested, respectively. The IGG GES was found significantly associated with a favorable OS in breast cancer (HR: 0.73, p < 0.001), cervical and head & neck squamous cell carcinomas (HR: 0.75, p = 0.021 and HR: 0.78, p < 0.001), lung adenocarcinoma (HR: 0.84, p = 0.020), skin melanoma (HR: 0.73, p < 0.001) and sarcomas (HR: 0.78, p = 0.029). Conversely, association of IGG GES with unfavorable OS was observed in uveal melanoma (HR: 1.74, p = 0.008), kidney clear cell and papillary cell carcinomas (HR: 1.29, p < 0.001 and HR: 1.44, p = 0.017, respectively), as well as brain low-grade gliomas (LGG) (HR: 1.56, p < 0.001). Finally, the research-based HER2DX risk-score was found significantly associated with OS in 11 of 30 (36.7%) cancer-types, including breast (HR: 1.42, p < 0.001) cervical and head & neck squamous cells cancers (HR: 1.36, p = 0.018 and HR: 1.30, p < 0.001), lung adenocarcinoma (HR: 1.34, p < 0.001), skin melanoma (HR: 1.34, p < 0.001), sarcomas (HR: 1.41, p = 0.003), adrenocortical carcinoma (HR: 2.31, p < 0.001), endometrial carcinoma (HR: 1.36, p = 0.005), hepatocarcinoma (HR: 1.26, p = 0.012), LGG (HR: 1.37, p = 0.001) and mesothelioma (HR: 1.48, p = 0.005). Conclusions: The 14-gene IGG and proliferation signatures are strong prognostic biomarkers across cancer-types. The opposite association of IGG with OS depending on the cancer-type warrants further investigation.

Purpose: In this era of precision-based medicine, for optimal patient care, results reported from commercial next-generation sequencing (NGS) assays should adequately reflect the burden of somatic mutations in the tumor being sequenced. Here, we sought to determine the prevalence of clonal hematopoiesis leading to possible misattribution of tumor mutation calls on unpaired Foundation Medicine NGS assays. Experimental Design: This was a retrospective cohort study of individuals undergoing NGS of solid tumors from two large cancer centers. We identified and quantified mutations in genes known to be frequently altered in clonal hematopoiesis (DNMT3A, TET2, ASXL1, TP53, ATM, CHEK2, SF3B1, CBL, JAK2) that were returned to physicians on clinical Foundation Medicine reports. For a subset of patients, we explored the frequency of true clonal hematopoiesis by comparing mutations on Foundation Medicine reports with matched blood sequencing. Results: Mutations in genes that are frequently altered in clonal hematopoiesis were identified in 65% (1,139/1,757) of patients undergoing NGS. When excluding TP53, which is often mutated in solid tumors, these events were still seen in 35% (619/1,757) of patients. Utilizing paired blood specimens, we were able to confirm that 8% (18/226) of mutations reported in these genes were true clonal hematopoiesis events. The majority of DNMT3A mutations (64%, 7/11) and minority of TP53 mutations (4%, 2/50) were clonal hematopoiesis. Conclusions: Clonal hematopoiesis mutations are commonly reported on unpaired NGS testing. It is important to recognize clonal hematopoiesis as a possible cause of misattribution of mutation origin when applying NGS findings to a patient's care. See related commentary by Pollyea, p. 5790

The human genome contains regulatory elements, such as enhancers, that are often rewired by cancer cells for the activation of genes that promote tumorigenesis and resistance to therapy. This is especially true for cancers that have little or no known driver mutations within protein coding genes, such as ovarian cancer. Herein, we have utilized an integrated set of genomic and epigenomic datasets to identify clinically relevant super-enhancers that are preferentially amplified in ovarian cancer patients. We have systematically probed the top 86 super-enhancers, using CRISPR-interference and CRISPR-deletion assays coupled to RNA-sequencing, to nominate two salient super-enhancers that drive proliferation and migration of cancer cells. Utilizing Hi-C, we constructed chromatin interaction maps that enabled the annotation of direct target genes for these super-enhancers and later confirmed their activity specifically within the cancer cell compartment of human tumors using single-cell genomics data. Together, our multi-omic approach has examined a number of fundamental questions about how regulatory information encoded into super-enhancers drives gene expression networks that underlie the biology of ovarian cancer.

Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5–10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2–46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0–7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation.