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Background: Metastasis is the leading cause of cancer related deaths in breast cancer patients. Lymphovascular invasion represents one of the earliest stages of metastasis wherein the cells are introduced to a very different and distinct microenvironment. Methods: We leveraged spatial techniques developed for limited specimens in archival tissue to study patient matched cross-sectional tumor samples from different stages of breast neoplasia including normal breast, ductal carcinoma in situ (DCIS), primary invasive carcinoma (IBC), lymphovascular invasion (LVI) and regional lymph node metastasis. We selected a set of 21 patients with ER+ breast cancer to generate cross-sectional samples of each of these stages, for a total of 331 samples. The areas of LVI were identified by a combination of H&E review and immunohistochemistry for podoplanin. We performed smart-3SEQ for gene expression profiling and light pass whole genome sequencing for DNA copy number alterations. Results: We profiled the spectrum of neoplasia for transcriptome-wide gene expression. Principal component analysis of all 252 DCIS, LVI, IBC, or metastasis samples using the top 500 genes with the highest variance demonstrated that clustering was roughly based on the diagnostic stage (i.e. DCIS, LVI, IBC, or metastasis). Differential gene expression profiling identified thousands of genes increased or decreased in expression across the transitional stages with the largest change in gene expression being the transition from normal breast to DCIS, dominated by gene expression down regulation. We next performed NMF clustering on 62 samples of LVI from 18 cases and identified two patterns of gene expression which define two subgroups. Gene ontology analysis revealed that one cluster was associated with increased proliferation and metabolism, whereas the second cluster was dominated by an immune response. When we analyzed the immune and proliferative LVI subgroups separately, we found that the immune profiles in the patient matched IBC and LVI samples from the LVI Immune cluster were similar, whereas the immune profiles in the patient matched IBC and LVI samples from the Proliferative cluster were significantly different. At the LVI stage, all immune cell populations estimated by CibersortX were decreased in the Proliferative LVI cluster. These changes were validated using immunofluorescence for proliferation (Ki67), T cells (CD3) and macrophages (CD68) on the same samples. Using the LVI centroids, we built a model that could predict the same clusters in the METABRIC IBC. Kaplan-Meier analysis showed a significant difference between groups, with the Proliferative-like IBC group having a worse prognosis than the Immune-like IBC group. Conclusions: We observed a dichotomy at the LVI stage with a more proliferative cluster that may escape the immune response and an immune cluster which has a microenvironment with a similar pattern to its primary IBC. The recognition of two groups of LVI, differing in immune association and proliferation, raises the possibility that the risk of metastasis could be different in these two groups, leading to different biological pathways of progression. Citation Format: Belén Rivero-Gutiérrez, Diego Mallo, Almudena Espín-Pérez, Sujay Vennam, Chunfang Zhu, Sushama Varma, Greg Scott, Joseph Foley, E Shelley Hwang, Carlo Maley, Robert West. Characterization of the lymphovascular invasion microenvironment reveals immune response dichotomy [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 PD2-09.

Purpose: A major component of cells in tenosynovial giant cell tumor (TGCT) consists of bystander macrophages responding to CSF1 that is overproduced by a small number of neoplastic cells with a chromosomal translocation involving the CSF1 gene. An autocrine loop was postulated where the neoplastic cells would be stimulated through CSF1R expressed on their surface. Here, we use single-cell RNA sequencing (scRNA-seq) to investigate cellular interactions in TGCT. Experimental Design: A total of 18,788 single cells from three TGCT and two giant cell tumor of bone (GCTB) samples underwent scRNA-seq. The three TGCTs were additionally analyzed using long-read RNA sequencing. Immunofluorescence and IHC for a range of markers were used to validate and extend the scRNA-seq findings. Results: Two recurrent neoplastic cell populations were identified in TGCT that are highly similar to nonneoplastic synoviocytes. We identified GFPT2 as a marker that highlights the neoplastic cells in TCGT. We show that the neoplastic cells themselves do not express CSF1R. We identified overlapping MAB features between the giant cells in TGCT and GCTB. Conclusions: The neoplastic cells in TGCT are highly similar to nonneoplastic synoviocytes. The lack of CSF1R on the neoplastic cells indicates they may be unaffected by current therapies. High expression of GFPT2 in the neoplastic cells is associated with activation of the YAP1/TAZ pathway. In addition, we identified expression of the platelet-derived growth factor receptor in the neoplastic cells. These findings suggest two additional pathways to target in this tumor.

Background. DCIS consists of a molecularly heterogeneous group of premalignant lesions, with variable risk of invasive progression. Understanding biomarkers for invasive progression could help individualize treatment recommendations based upon tumor biology. As part of the NCI Human Tumor Atlas Network (HTAN), we conducted comprehensive genomic analyses on two large DCIS case-control cohorts. Methods. We performed smart3-seq and low-pass whole genome sequencing on two independent, retrospective, longitudinally sampled DCIS case-control cohorts. TBCRC 038 was a multicenter cohort diagnosed with DCIS between 1998 and 2016 at one of the Translational Breast Cancer Research sites; the RAHBT (Resource of Archival Human Breast Tissue) cohort included women identified through the St. Louis Breast Tissue Repository, and the Women’s Health Repository diagnosed between 1997 and 2001. We studied the spectrum of molecular changes present and sought genomic predictors of subsequent ipsilateral breast events (iBEs: DCIS recurrence or invasive progression) in both DCIS epithelium and stroma in formalin fixed paraffin embedded tissue. We generated de novo tumor and stroma-centric subtypes for DCIS that represents fundamental transcriptomic organization. Copy number analysis was performed using low-pass DNA sequencing. Non-negative matrix factorization (NMF) was applied to the RNA expression of all coding genes to identify clusters. A negative-binomial regression model was used to identify differentially expressed genes. Results. We analyzed 677 DCIS samples from 481 patients with 7.1 years median follow-up. In TBCRC samples, we identified three clusters via NMF in TBCRC referred to as ER low, quiescent, and ER high. The ER-low cluster had significantly higher levels of ERBB2 and lower levels of ESR1 compared to quiescent and ER-high clusters. Quiescent cluster lesions were less proliferative and less metabolically active than ER high and ER low subtypes. These findings were replicated in the RAHBT cohort. Focusing on the stromal component of DCIS from laser capture microdissection in RAHBT samples, we identified four distinct DCIS-associated stromal clusters. A “normal-like” stromal cluster with ECM organization and PI3K-AKT signaling; a “collagen-rich” stromal cluster; a “desmoplastic” stromal cluster with high fibroblast and total myeloid abundance, mostly associated with macrophages and myeloid dendritic cells (mDC); and an “immune-dense” stromal cluster. Further, we compared differentially expressed genes in patients with or without subsequent iBEs within 5 years of diagnosis. Hypothesizing that the resulting 812 DE genes (DESeq2) represent multiple routes to subsequent iBEs, we leveraged NMF to identify paths to progression. In both TBCRC and RAHBT cohorts, poor outcome groups exhibited increased ER, MYC signaling, and oxidative phosphorylation, supporting that these pathways are important for DCIS recurrence and progression. Conclusion. Comprehensive genomic profiling in two independent DCIS cohorts with longitudinal outcomes shows distinct DCIS stromal expression patterns and immune cell composition. RNA expression profiles reveal underlying tumor biology that is associated with later iBEs in both cohorts. These studies provide new insight into DCIS biology and will guide the design of diagnostic strategies to prevent invasive progression. Citation Format: Siri H Strand, Belén Rivero-Gutiérrez, Kathleen E Houlahan, Jose A Seoane, Lorraine M King, Tyler Risom, Lunden Simpson, Sujay Vennam, Aziz Khan, Timothy Hardman, Bryan E Harmon, Fergus J Couch, Kristalyn Gallagher, Mark Kilgore, Shi Wei, Angela DeMichele, Tari King, Priscilla F McAuliffe, Julie Nangia, Joanna Lee, Jennifer Tseng, Anna Maria Storniolo, Alastair Thompson, Gaorav Gupta, Robyn Burns, Deborah J Veis, Katherine DeSchryver, Chunfang Zhu, Magdalena Matusiak, Jason Wang, Shirley X Zhu, Jen Tappenden, Daisy Yi Ding, Dadong Zhang, Jingqin Luo, Shu Jiang, Sushama Varma, Cody Straub, Sucheta Srivastava, Christina Curtis, Rob Tibshirani, Robert Michael Angelo, Allison Hall, Kouros Owzar, Kornelia Polyak, Carlo Maley, Jeffrey R Marks, Graham A Colditz, E Shelley Hwang, Robert B West. The Breast PreCancer Atlas DCIS genomic signatures define biology and correlate with clinical outcomes: An analysis of TBCRC 038 and RAHBT cohorts [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 GS4-07.

Ductal carcinoma in situ; Tumor microenvironment; Whole genome sequencing Carcinoma ductal in situ; Microambiente tumoral; Secuenciación del genoma completo Carcinoma ductal in situ; Microambient tumoral; Seqüenciació del genoma complet Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We perform multiscale, integrated molecular profiling of DCIS with clinical outcomes by analyzing 774 DCIS samples from 542 patients with 7.3 years median follow-up from the Translational Breast Cancer Research Consortium 038 study and the Resource of Archival Breast Tissue cohorts. We identify 812 genes associated with ipsilateral recurrence within 5 years from treatment and develop a classifier that predicts DCIS or IBC recurrence in both cohorts. Pathways associated with recurrence include proliferation, immune response, and metabolism. Distinct stromal expression patterns and immune cell compositions are identified. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome. This publication is part of the HTAN (Human Tumor Atlas Network) Consortium paper package. A list of HTAN members is available at humantumoratlas.org/htan-authors/. R01 CA185138-01 (E.S.H.); U2C CA-17-035 Pre-Cancer Atlas (PCA) Research Centers (E.S.H., R.B.W., C.M., K.P., G.A.C., K.O.); UO1 CA214183 (J.R.M.); DOD BC132057 (E.S.H., C.M.); BCRF 19-074 (E.S.H.); BCRF 19-028 (G.A.C.); PRECISION CRUK Grand Challenge (E.S.H.); R01CA193694 (R.B.W., G.A.C.), BCRF PPI-18-006 (R.B.W.). AEI RYC2019- 026576-I, "LaCaixa" Foundation LCF/PR/PR17/51120011 (J.A.S.). S.H.S. was supported by the Lundbeck Foundation (R288-2018-35) and the Danish Cancer Society (R229-A13616). K.E.H. was supported by a CIHR Banting Postdoctoral Fellowship. TBCRC 038 was conducted by the TBCRC, which receives major funding support from The Breast Cancer Research Foundation and Susan G. Komen. Some results in this paper are based upon data generated by the TCGA Research Network.

Metabolic reprogramming of tumor cells and the increase of glucose uptake is one of the hallmarks of cancer. In order to identify metabolic pathways activated in leiomyosarcoma (LMS), we analyzed transcriptomic profiles of distinct subtypes of LMS in several datasets. Primary, recurrent and metastatic tumors in the subtype 2 of LMS showed consistent enrichment of genes involved in hexosamine biosynthesis pathway (HBP). We demonstrated that glutamine-fructose-6-phosphate transaminase 2 (GFPT2), the rate-limiting enzyme in HBP, is expressed on protein level in a subset of LMS and the expression of this enzyme is frequently retained in patient-matched primary and metastatic tumors. In a new independent cohort of 327 patients, we showed that GFPT2 is associated with poor outcome of uterine LMS but not extra-uterine LMS. Based on the analysis of a small group of patients studied by 18F-FDG-PET imaging, we propose that strong expression of GFPT2 in primary LMS may be associated with high metabolic activity. Our data suggest that HBP is a potential new therapeutic target in one of the subtypes of LMS.

Background The acquisition of oncogenic drivers is a critical feature of cancer progression. For some carcinomas, it is clear that certain genetic drivers occur early in neoplasia and others late. Why these drivers are selected and how these changes alter the neoplasia’s fitness is less understood. Methods Here we use spatially oriented genomic approaches to identify transcriptomic and genetic changes at the single-duct level within precursor neoplasia associated with invasive breast cancer. We study HER2 amplification in ductal carcinoma in situ (DCIS) as an event that can be both quantified and spatially located via fluorescence in situ hybridization (FISH) and immunohistochemistry on fixed paraffin-embedded tissue. Results By combining the HER2-FISH with the laser capture microdissection (LCM) Smart-3SEQ method, we found that HER2 amplification in DCIS alters the transcriptomic profiles and increases diversity of copy number variations (CNVs). Particularly, interferon signaling pathway is activated by HER2 amplification in DCIS, which may provide a prolonged interferon signaling activation in HER2-positive breast cancer. Multiple subclones of HER2-amplified DCIS with distinct CNV profiles are observed, suggesting that multiple events occurred for the acquisition of HER2 amplification. Notably, DCIS acquires key transcriptomic changes and CNV events prior to HER2 amplification, suggesting that pre-amplified DCIS may create a cellular state primed to gain HER2 amplification for growth advantage. Conclusion By using genomic methods that are spatially oriented, this study identifies several features that appear to generate insights into neoplastic progression in precancer lesions at a single-duct level.

PURPOSE The preoperative distinction between uterine leiomyoma (LM) and leiomyosarcoma (LMS) is difficult, which may result in dissemination of an unexpected malignancy during surgery for a presumed benign lesion. An assay based on circulating tumor DNA (ctDNA) could help in the preoperative distinction between LM and LMS. This study addresses the feasibility of applying the two most frequently used approaches for detection of ctDNA: profiling of copy number alterations (CNAs) and point mutations in the plasma of patients with LM. PATIENTS AND METHODS By shallow whole-genome sequencing, we prospectively examined whether LM-derived ctDNA could be detected in plasma specimens of 12 patients. Plasma levels of lactate dehydrogenase, a marker suggested for the distinction between LM and LMS by prior studies, were also determined. We also profiled 36 LM tumor specimens by exome sequencing to develop a panel for targeted detection of point mutations in ctDNA of patients with LM. RESULTS We identified tumor-derived CNAs in the plasma DNA of 50% (six of 12) of patients with LM. The lactate dehydrogenase levels did not allow for an accurate distinction between patients with LM and patients with LMS. We identified only two recurrently mutated genes in LM tumors ( MED12 and ACLY). CONCLUSION Our results show that LMs do shed DNA into the circulation, which provides an opportunity for the development of ctDNA-based testing to distinguish LM from LMS. Although we could not design an LM-specific panel for ctDNA profiling, we propose that the detection of CNAs or point mutations in selected tumor suppressor genes in ctDNA may favor a diagnosis of LMS, since these genes are not affected in LM.

Cancer cells rewire metabolic pathways and energy production to support the enhanced proliferation, invasion and resistance to treatment. The three main glucose metabolism pathways that support growth of cancer cells are: a) the glycolysis pathway for energy production; b) the pentose phosphate pathway for biomass production; and c) the hexosamine biosynthesis pathway (HBP) for protein glycosylation. It is known that the activation of HBP leads to altered glycosylation of oncogenes, transcription factors and kinases in many types of cancer. These aberrations may lead to increased proliferation and survival of tumor cells, and may be associated with resistance to therapy. A better understanding of the role of HBP in malignancies has the potential for clinical implications. Several studies demonstrated that pharmacological inhibition of GFPT2 (glutamine-fructose-6-phosphate transaminase 2, the first and rate-limiting enzyme in HBP) and the enzymes that act downstream of HBP may exhibit anti-tumorigenic effect both in vitro and in vivo, and may modulate sensitivity to chemo-, radio- and immunotherapy. Most of these studies focused on carcinomas and the role of HPB in sarcoma has not been studied. We recently reported a remarkable enrichment of genes involved in HBP in a subset of leiomyosarcoma (LMS) and demonstrated that expression of GFPT2 in LMS is associated with poor clinical outcome. We identified the c-Myc oncoprotein as a potential target of HPB that may be stabilized by aberrant glycosylation in LMS. Here we show the results of a large-scale screening of 260 primary specimens of 33 types of soft tissue lesions. In addition to expression in a subset of LMS, we observed near universal expression of GFPT2 in 34 of 35 desmoid type fibromatosis (DTF), independent of the mutation type of the CTNNB1 gene. Gene Set Enrichment Analysis of a previously published 3SEQ transcriptomic dataset composed of DTF and 9 other types of fibrotic lesions identified significant enrichment of other genes implicated in HBP and multiple glycosylation-associated pathways in DTF compared to the other types of fibrotic lesions. Our analysis identified ATF6 (activating transcription factor 6) as a possible target regulated by aberrant glycosylation as a consequence of HBP activation in DTF. ATF6 is a glycoprotein that has been demonstrated to underlie the resistance to chemotherapy in osteosarcoma, to have a pro-oncogenic role in primary liver cancers and has been proposed as a therapeutic target in cystic fibrosis. Others have shown that targeting HBP can provide therapeutic benefit in a number of preclinical models of carcinoma. Our studies offer new insights into the mechanisms of DTF tumorigenesis and, when confirmed by in vitro studies, will provide a rationale to explore the potential of therapeutic targeting of HBP in DTF. Citation Format: Joanna Przybyl, Angela Tolwani, Sushama Varma, Matt van de Rijn. Targeting hexosamine biosynthesis pathway for the treatment of desmoid tumors [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B015.

Objective There are no effective systemic therapies for adenoid cystic cancer (ACC) and lack of tumor lines and mouse models have hindered drug development. We aim to develop MYB-activated models for testing new therapeutic agents. Materials and methods We studied new ACC patient-derived xenograft (PDX) models and generated a matched cell line from one patient. In addition, we generated a genetically-engineered MYB-NFIB mouse model (GEMM) that was crossed with Ink4a+/−/Arf+/− mice to study tumor spectrum and obtain tumor lines. Using human and murine ACC-like tumor lines, we analyzed MYB expression by RNA-Seq and immunoblot and tested efficacy of new MYB inhibitors. Results We detected MYB-NFIB transcripts in both UFH1 and UFH2 PDX and observed tumor inhibition by MYB depletion using shRNA in vivo. We observed rapid loss of MYB expression when we cultured UFH1 in vitro, but were able to generate a UFH2 tumor cell line that retained MYB expression for 6 months. RNA-Seq expression detected an ACC-like mRNA signature in PDX samples and we confirmed an identical KMT2A/MLL variant in UFH2 PDX, matched cell line, and primary biopsy. Although the predominant phenotype of the MYB-NFIB GEMM was B-cell leukemia, we also generated a MYB-activated ACC-like mammary tumor cell line. We observed tumor inhibition using a novel MYB peptidomimetic in both human and murine tumor models. Conclusions We generated and studied new murine and human MYB-activated tumor samples and detected growth inhibition with MYB peptidomimetics. These data provide tools to define treatment strategies for patients with advanced MYB-activated ACC.

The invasive leading edge represents a potential gateway for tumor metastasis. The role of fibroblasts from the tumor edge in promoting cancer invasion and metastasis has not been comprehensively elucidated. We hypothesize that cross-talk between tumor and stromal cells within the tumor microenvironment results in activation of key biological pathways depending on their position in the tumor (edge vs. core). Here we highlight phenotypic differences between tumor-adjacent-fibroblasts (TAF) from the invasive edge and tumor core fibroblasts from the tumor core, established from human lung adenocarcinomas. A multiomics approach that includes genomics, proteomics, and O-glycoproteomics was used to characterize cross-talk between TAFs and cancer cells. These analyses showed that O-glycosylation, an essential posttranslational modification resulting from sugar metabolism, alters key biological pathways including the cyclin-dependent kinase 4 (CDK4) and phosphorylated retinoblastoma protein axis in the stroma and indirectly modulates proinvasive features of cancer cells. In summary, the O-glycoproteome represents a new consideration for important biological processes involved in tumor–stroma cross-talk and a potential avenue to improve the anticancer efficacy of CDK4 inhibitors. Significance: A multiomics analysis of spatially distinct fibroblasts establishes the importance of the stromal O-glycoproteome in tumor–stroma interactions at the leading edge and provides potential strategies to improve cancer treatment. See related commentary by De Wever, p. 537

Ductal carcinomain situ(DCIS) is a pre-invasive lesion that is thought to be a precursor to invasive breast cancer (IBC). To understand how the tumor microenvironment (TME) changes with transition to IBC, we used Multiplexed Ion Beam Imaging by time of flight (MIBI-TOF) and a 37-plex antibody staining panel to analyze 140 clinically annotated surgical resections covering the full spectrum of breast cancer progression. We compared normal, DCIS, and IBC tissues using machine learning tools for multiplexed cell segmentation, pixel-based clustering, and object morphometrics. Transition from DCIS to IBC was found to occur along a trajectory marked by coordinated shifts in location and function of myoepithelium, fibroblasts, and infiltrating immune cells in the surrounding stroma. Taken together, this comprehensive study within the HTAN Breast PreCancer Atlas offers insight into the etiologies of DCIS, its transition to IBC, and emphasizes the importance of the TME stroma in promoting these processes.

Background Tumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway. Result To develop a deeper understanding of the interactions between cells within human lung tumors, we perform RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We map the cell-specific differential expression of prognostically associated secreted factors and cell surface genes, and computationally reconstruct cross-talk between these cell types to generate a novel resource called the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identify and validate a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also find a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior. Conclusion These results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance.

Purpose: The clinical utility of circulating tumor DNA (ctDNA) monitoring has been shown in tumors that harbor highly recurrent mutations. Leiomyosarcoma represents a type of tumor with a wide spectrum of heterogeneous genomic abnormalities; thus, targeting hotspot mutations or a narrow genomic region for ctDNA detection may not be practical. Here, we demonstrate a combinatorial approach that integrates different sequencing protocols for the orthogonal detection of single-nucleotide variants (SNV), small indels, and copy-number alterations (CNA) in ctDNA. Experimental Design: We employed Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for the analysis of SNVs and indels, together with a genome-wide interrogation of CNAs by Genome Representation Profiling (GRP). We profiled 28 longitudinal plasma samples and 25 tumor specimens from 7 patients with leiomyosarcoma. Results: We detected ctDNA in 6 of 7 of these patients with >98% specificity for mutant allele fractions down to a level of 0.01%. We show that results from CAPP-Seq and GRP are highly concordant, and the combination of these methods allows for more comprehensive monitoring of ctDNA by profiling a wide spectrum of tumor-specific markers. By analyzing multiple tumor specimens in individual patients obtained from different sites and at different times during treatment, we observed clonal evolution of these tumors that was reflected by ctDNA profiles. Conclusions: Our strategy allows for the comprehensive monitoring of a broad spectrum of tumor-specific markers in plasma. Our approach may be clinically useful not only in leiomyosarcoma but also in other tumor types that lack recurrent genomic alterations. Clin Cancer Res; 24(11); 2688–99. ©2018 AACR.

Tumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway. To develop a deeper understanding of the interactions between cells within human lung tumors we performed RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We mapped the cell-specific differential expression of prognostically-associated secreted factors and cell surface genes, and computationally reconstructed cross-talk between these cell types to generate a novel resource we call the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identified and validated a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also found a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior. These results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance.SummaryRNA-seq profiling of sorted populations from primary lung cancer samples identifies prognostically relevant cross-talk between cell types in the tumor microenvironment.

PURPOSE: The detection rate of breast ductal carcinoma in situ (DCIS) has increased significantly, raising the concern that DCIS is over-diagnosed and over-treated. Therefore, there is an unmet clinical need to better predict the risk of progression among DCIS patients. Our hypothesis is that by combining molecular signatures with clinicopathologic features, we can elucidate the biology of breast cancer progression, and risk-stratify patients with DCIS. METHODS: Targeted exon-sequencing with a custom panel of 223 genes/regions was performed for 125 DCIS cases. Among them, 60 were from cases having concurrent or subsequent invasive breast cancer (IBC) (DCIS+IBC group), and 65 from cases with no IBC development over a median follow-up of 13 years (DCIS-only group). Copy number alterations in chromosome 1q32, 8q24, 11q13 were analyzed using fluorescence in situ hybridization (FISH). Multivariable logistic regression models were fit to the outcome of DCIS progression to IBC as a function of demographic and clinical features. RESULTS: We observed recurrent variants of known IBC-related mutations, and the most commonly mutated genes in DCIS were PIK3CA (34.4%) and TP53 (18.4%). There was an inverse association between PIK3CA kinase domain mutations and progression (Odds Ratio [OR]=10.2, p

Increased tissue stiffness is a driver of breast cancer progression. The transcriptional regulator YAP is considered a universal mechanotransducer, based largely on 2D culture studies. However, the role of YAP during in vivo breast cancer remains unclear. Here, we find that mechanotransduction occurs independently of YAP in breast cancer patient samples and mechanically tunable 3D cultures. Mechanistically, the lack of YAP activity in 3D culture and in vivo is associated with the absence of stress fibers and an order of magnitude decrease in nuclear cross-sectional area relative to 2D culture. This work highlights the context-dependent role of YAP in mechanotransduction, and establishes that YAP does not mediate mechanotransduction in breast cancer., The transcriptional regulator YAP is regarded as the universal mechanotransducer, largely from 2D culture studies. Here the authors show that in breast cancer patient tissues and cells in 3D culture, mechanical signals are transduced independently of YAP, questioning YAP as a therapeutic target.

Ductal Carcinoma in Situ (DCIS) is a pre-invasive lesion that accounts for nearly 20% of new breast cancer diagnoses. Of these cases, about half will progress to invasive breast cancer (IBC) within ten years. However, because diagnostic criteria for delineating low risk lesions from those likely to progress to IBC have not been identified, many patients are receiving unnecessary chemotherapy and surgery that can result in therapy-related morbidity and death. With this in mind, we used Multiplexed Ion Beam Imaging by time of flight (MIBI-TOF) and RNA-seq laser-capture microdissection (SMART-3SEQ) to construct a comprehensive spatial atlas describing the structure, function, and cellular composition of DCIS. MIBI-TOF and SMART-3SEQ were used to compare lesions from patients that later developed IBC with those from age- and history matched DCIS controls without recurrence. Using a 37-marker staining panel to interrogate 137 lesions, we identified 30 distinct cell populations of the epithelial, stromal, and immune lineages that were arranged in recurrent cellular microenvironments specific for DCIS or invasive disease. We observe a coordinated shift in the immune and stromal compartments as invasive disease arises, including an expansion of immune cell diversity and transition to reactive stromal phenotypes in synchronous DCIS + IBC, which was distinct from the macrophage-dominant microenvironment of recurrent IBC. Single-cell segmentation using a deep learning model was combined with pixel-level coexpression analysis to determine how thickness, continuity, and phenotype of ductal myoepithelium changes as tumors progress from a pre-invasive state. These data were incorporated into a comprehensive model which was subsequently used to identify a subset of features that correlate with disease-free survival following DCIS tumor resection. Taken together, these features represent important prognostic metrics that can be used to separate pre-invasive from indolent DCIS tumors, and allow for tailored therapy that improves patient outcomes and quality of life in this disease. Citation Format: Tyler Risom, Belen Rivero, Candace Liu, Alex Baranski, Siri Strand, Noah Greenwald, Erin McCaffrey, Sushama Varma, Leeat Keren, Sucheta Srivastava, Chunfang Zhu, Sujay Vennam, Shelley Hwang, Graham Colditz, Sean Bendall, Robert West, Michael Angelo. Mapping the tumor and microenvironmental evolution underlying DCIS progression through multiplexed ion beam imaging [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PR05.

Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial cancer common in the Southern Chinese population. Patients with recurrent or metastatic NPC treated with PD-1 blockade showed a response rate of 20.5%, with no correlation observed between the expression of PD-L1 and treatment response. In an effort to identify biomarkers for anti-PD-1 treatment response, we evaluated a cohort of 10 NPC patients with recurrent or metastatic NPC treated with nivolumab or pembrolizumab. Laser capture microdissection was performed on pretreatment biopsies, with tumor and microenvironment compartments separately microdissected. Nasopharyngeal epithelium from normal nasopharyngeal biopsies were also microdissected as controls. Smart-3SEQ, a novel 3´ end RNA-Seq technique that allows for the accurate quantification of transcript abundance in FFPE samples, was performed to obtain gene expression libraries. ERAP2, a zinc metalloaminopeptidase that performs N-terminal trimming of antigenic epitopes for presentation by MHC class I, was among the most highly upregulated genes in the tumor compartment of nonresponders (adjusted p = 0.033). ERAP2 overexpression was also observed in NPC tumors compared to normal controls. Immunohistochemistry validated the overexpression of ERAP2 in nonresponders, while responders had similar expression to normal controls. In conclusion, ERAP2 is overexpressed in NPC tumors and is a promising biomarker for reduced anti-PD-1 response in NPC. Citation Format: Joshua K. Tay, Brigette B.Y. Ma, Sushama Varma, Yaw Chyn Lim, Chee Seng Tan, Kwok Wai Lo, John B. Sunwoo, Kevin J. Cullen, Boon Cher Goh, Robert B. West. ERAP2 overexpression is a marker for reduced anti-PD-1 response in nasopharyngeal carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B28.

Soft-tissue sarcomas such as leiomyosarcoma pose a clinical challenge because systemic treatment options show only modest therapeutic benefit. Discovery and validation of targetable vulnerabilities is essential. To discover putative kinase fusions, we analyzed existing transcriptomic data from leiomyosarcoma clinical samples. Potentially oncogenic ALK rearrangements were confirmed by application of multiple RNA-sequencing fusion detection algorithms and FISH. We functionally validated the oncogenic potential and targetability of discovered kinase fusions through biochemical, cell-based (Ba/F3, NIH3T3, and murine smooth muscle cell) and in vivo tumor modeling approaches. We identified ALK rearrangements in 9 of 377 (2.4%) patients with leiomyosarcoma, including a novel KANK2-ALK fusion and a recurrent ACTG2-ALK fusion. Functional characterization of the novel ALK fusion, KANK2-ALK, demonstrates it is a dominant oncogene in Ba/F3 or NIH3T3 model systems, and has tumorigenic potential when introduced into smooth muscle cells. Oral monotherapy with targeted ALK kinase inhibitor lorlatinib significantly inhibits tumor growth and prolongs survival in a murine model of KANK2-ALK leiomyosarcoma. These results provide the first functional validation of a targetable oncogenic kinase fusion as a driver in a subset of leiomyosarcomas. Overall, these findings suggest that some soft-tissue sarcomas may harbor previously unknown kinase gene translocations, and their discovery may propel new therapeutic strategies in this treatment-refractory cancer. Implications: A subset of leiomyosarcomas harbor previously unrecognized oncogenic ALK fusions that are highly responsive to ALK inhibitors and thus these data emphasize the importance of detailed genomic investigations of leiomyosarcoma tumors.

The immune system is critical in modulating cancer progression, but knowledge of immune composition, phenotype, and interactions with tumor is limited. We used multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to simultaneously quantify in situ expression of 36 proteins covering identity, function, and immune regulation at sub-cellular resolution in 41 triple-negative breast cancer patients. Multi-step processing, including deep-learning-based segmentation, revealed variability in the composition of tumor-immune populations across individuals, reconciled by overall immune infiltration and enriched co-occurrence of immune subpopulations and checkpoint expression. Spatial enrichment analysis showed immune mixed and compartmentalized tumors, coinciding with expression of PD1, PD-L1, and IDO in a cell-type- and location-specific manner. Ordered immune structures along the tumor-immune border were associated with compartmentalization and linked to survival. These data demonstrate organization in the tumor-immune microenvironment that is structured in cellular composition, spatial arrangement, and regulatory-protein expression and provide a framework to apply multiplexed imaging to immune oncology.

Purpose: Leiomyosarcoma is a malignant neoplasm with smooth muscle differentiation. Little is known about its molecular heterogeneity and no targeted therapy currently exists for leiomyosarcoma. Recognition of different molecular subtypes is necessary to evaluate novel therapeutic options. In a previous study on 51 leiomyosarcomas, we identified three molecular subtypes in leiomyosarcoma. The current study was performed to determine whether the existence of these subtypes could be confirmed in independent cohorts. Experimental Design: Ninety-nine cases of leiomyosarcoma were expression profiled with 3′end RNA-Sequencing (3SEQ). Consensus clustering was conducted to determine the optimal number of subtypes. Results: We identified 3 leiomyosarcoma molecular subtypes and confirmed this finding by analyzing publically available data on 82 leiomyosarcoma from The Cancer Genome Atlas (TCGA). We identified two new formalin-fixed, paraffin-embedded tissue-compatible diagnostic immunohistochemical markers; LMOD1 for subtype I leiomyosarcoma and ARL4C for subtype II leiomyosarcoma. A leiomyosarcoma tissue microarray with known clinical outcome was used to show that subtype I leiomyosarcoma is associated with good outcome in extrauterine leiomyosarcoma while subtype II leiomyosarcoma is associated with poor prognosis in both uterine and extrauterine leiomyosarcoma. The leiomyosarcoma subtypes showed significant differences in expression levels for genes for which novel targeted therapies are being developed, suggesting that leiomyosarcoma subtypes may respond differentially to these targeted therapies. Conclusions: We confirm the existence of 3 molecular subtypes in leiomyosarcoma using two independent datasets and show that the different molecular subtypes are associated with distinct clinical outcomes. The findings offer an opportunity for treating leiomyosarcoma in a subtype-specific targeted approach. Clin Cancer Res; 21(15); 3501–11. ©2015 AACR.

Pathogenic gene fusions have been identified in several histologic types of salivary gland neoplasia, but not previously in acinic cell carcinoma (AcCC). To discover novel gene fusions, we performed whole-transcriptome sequencing surveys of three AcCC archival cases. In one specimen we identified a novel HTN3-MSANTD3 gene fusion, and in another a novel PRB3-ZNF217 gene fusion. The structure of both fusions was consistent with the promoter of the 5' partner (HTN3 or PRB3), both highly expressed salivary gland genes, driving overexpression of full-length MSANTD3 or ZNF217. By fluorescence in situ hybridization of an expanded AcCC case series, we observed MSANTD3 rearrangements altogether in 3 of 20 evaluable cases (15%), but found no additional ZNF217 rearrangements. MSANTD3 encodes a previously uncharacterized Myb/SANT domain-containing protein. Immunohistochemical staining demonstrated diffuse nuclear MSANTD3 expression in 8 of 27 AcCC cases (30%), including the three cases with MSANTD3 rearrangement. MSANTD3 displayed heterogeneous expression in normal salivary ductal epithelium, as well as among other histologic types of salivary gland cancer though without evidence of translocation. In a broader survey, MSANTD3 showed variable expression across a wide range of normal and neoplastic human tissue specimens. In preliminary functional studies, engineered MSANTD3 overexpression in rodent salivary gland epithelial cells did not enhance cell proliferation, but led to significant upregulation of gene sets involved in protein synthesis. Our findings newly identify MSANTD3 rearrangement as a recurrent event in salivary gland AcCC, providing new insight into disease pathogenesis, and identifying a putative novel human oncogene.

Currently, only patients with HER2-positive tumors are candidates for HER2-targeted therapies. However, recent clinical observations suggest that the survival of patients with HER2-low breast cancers, who lack HER2 amplification, may benefit from adjuvant therapy that targets HER2. In this study, we explored a mechanism through which these benefits may be obtained. Prompted by the hypothesis that HER2/HER3 signaling in breast tumor-initiating cells (TIC) promotes self-renewal and survival, we obtained evidence that neuregulin 1 (NRG1) produced by TICs promotes their proliferation and self-renewal in HER2-low tumors, including in triple-negative breast tumors. Pharmacologic inhibition of EGFR, HER2, or both receptors reduced breast TIC survival and self-renewal in vitro and in vivo and increased TIC sensitivity to ionizing radiation. Through a tissue microarray analysis, we found that NRG1 expression and associated HER2 activation occurred in a subset of HER2-low breast cancers. Our results offer an explanation for why HER2 inhibition blocks the growth of HER2-low breast tumors. Moreover, they argue that dual inhibition of EGFR and HER2 may offer a useful therapeutic strategy to target TICs in these tumors. In generating a mechanistic rationale to apply HER2-targeting therapies in patients with HER2-low tumors, this work shows why these therapies could benefit a considerably larger number of patients with breast cancer than they currently reach. Cancer Res; 74(1); 341–52. ©2013 AACR.

Cancer evolution involves cycles of genomic damage, epigenetic deregulation, and increased cellular proliferation that eventually culminate in the carcinoma phenotype. Early breast neoplasias include usual ductal hyperplasia, columnar cell lesions, and flat epithelial atypia and some are thought to represent precursor stages. To elucidate their role in cancer evolution we performed comparative whole genome sequencing of early neoplasias, matched normal tissue, and carcinomas from six patients. The identified somatic mutations served as lineage markers to build trees that relate the tissue samples. On the basis of the lineage trees we inferred the order, timing, and rates of mutational events. We find that in a subset of cases, the last common ancestor of an early neoplasm and a carcinoma was hypermutated and had several aneuploidies, and that evolution further accelerated in the carcinoma lineage. In contrast to highly advanced tumors that are the focus of much of current cancer genome sequencing, the early neoplasia genomes, and the carcinomas as well, harbor a striking paucity of potentially functional somatic point mutations. The earliest significant events we could detect, those changes that occurred in common ancestors of neoplastic and tumor cells, are aneuploidies. Several aneuploidies are recurrent, suggesting that they are among the earliest genomic events that predispose breast tissue to eventual development of invasive carcinoma. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD05-09.

Adenoid cystic carcinoma is a locally aggressive salivary gland neoplasm, which has a poor long-term prognosis. A chromosomal translocation involving the genes encoding the transcription factors, MYB and NFIB, has been recently discovered in these tumors.MYB translocation and protein expression were studied in 37 adenoid cystic carcinomas, 112 other salivary gland neoplasms, and 409 nonsalivary gland neoplasms by fluorescence in situ hybridization and immunohistochemistry. MYB translocation and expression status in adenoid cystic carcinoma was correlated with clinicopathologic features including outcome, with a median follow-up of 77.1 months (range, 23.2 to 217.5 mo) for living patients.A balanced translocation between MYB and NFIB is present in 49% of adenoid cystic carcinomas but is not identified in other salivary gland tumors or nonsalivary gland neoplasms. There is no apparent translocation of MYB in 35% of the cases. Strong Myb immunostaining is very specific for adenoid cystic carcinomas but is only present in 65% of all cases. It is interesting to note that Myb immunostaining is confined to the basal cell component although the translocation is present in all the cells. Neoplasms with MYB translocation show a trend toward higher local relapse rates, but the results are not statistically significant with the current number of cases.MYB translocation and expression are useful diagnostic markers for a subset of adenoid cystic carcinomas. The presence of the translocation may be indicative of local aggressive behavior, but a larger cohort may be required to show statistical significance.

Objective: Nasopharyngeal carcinoma (NPC) is a common Epstein-barr virus-associated epithelial malignancy in several parts of the world including Southeast Asia. While the majority of patients are treated uniformly with a combination of chemo and radiation therapy, 20% of patients experience recurrence, most commonly in the form of distant metastasis. NPC subtypes based on underlying differences in biology are unexplored and are likely responsible for the heterogeneous clinical response. As NPC biopsies are small and have significant stromal infiltrate, obtaining pure epithelial cells for genomic profiling is a challenge. We aim to overcome these limitations to identify biological subtypes and dysregulated molecular pathways in NPC. Methods: We first evaluated 217 whole exomes sequences, including 102 microdissected tumors, from three previous studies for single nucleotide variants and copy number changes, following GATK standards and using next generation sequencing copy number callers. We then applied a novel method for gene expression profiling developed in our lab to a new cohort of EBV-positive primary NPC cases from our institution. We performed laser capture microdissection, separately dissecting tumor, normal and microenvironment for each case. We applied Smart-3SEQ, a novel 3' end RNA-Seq technique which allows for the accurate quantification of transcript abundance in dissected FFPE samples comprising only a few hundred cells. Results: We achieved a per-base concordance of 80.4% between copy number profiles by SNP array and whole exome sequencing. Unsupervised clustering identified three distinct copy-number groups of NPC tumors, with a low copy-number group demonstrating an 18.7% better 5-year disease-specific survival, not attributable to stage. Apart from broad cytogenetic changes, narrow regions of amplifications (e.g. 1q21, 11q13) and deletions (e.g. 9p21, 11q22) were important for defining copy-number subtypes. Preliminary differential gene expression analysis showed that genes involved in cell cycle and cellular differentiation were significantly dysregulated in tumor cells (p < 0.001 and p = 0.03 respectively), while genes involved in cilia assembly and flagella transport were upregulated in normal cells (p < 0.001 for both). Our further analysis includes defining tumor subtypes based on gene expression, identifying key driver pathways, and correlating with EBV-latent gene expression, the immune environment, as well as clinical outcome. Conclusion: NPC tumors are biologically heterogeneous and can be classified based on their mutational and gene expression profiles. This provides an important basis for the consideration of escalation and de-escalation of therapy in selected patient groups. Citation Format: Kai Xun Joshua Tay, Chunfang Zhu, Sujay Vennam, Sushama Varma, Quynh-Thu Le, John Sunwoo, Robert West. Biological subtypes of nasopharyngeal carcinoma by genomic profiling [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 3411.

11563Background: Leiomyosarcoma (LMS), a type of soft tissue tumor that originates from smooth muscle, is the second most common type of sarcoma. Despite multimodal treatment including surgery, che...

Circulating tumor DNA (ctDNA) has significant potential for several clinical applications, including assessment of treatment response and monitoring of recurrent/residual disease. We performed a pilot study to explore the feasibility of ctDNA monitoring in patients with leiomyosarcoma (LMS). We profiled matching plasma and FFPE tumor specimens from 9 LMS patients. We analyzed between 2 to 6 longitudinal plasma samples (median of 5) and between 1 to 7 tumor specimens (median of 2) per patient. ctDNA analysis was performed on plasma samples collected pre-/post-surgery, throughout chemo-/radiotherapy and during follow-up. We used two separate approaches in our study: 1) targeted deep sequencing of ctDNA, tumor DNA and germline DNA to detect single nucleotide variants and indels using Cancer Personalized Profiling by deep Sequencing with integrated digital error suppression (CAPP-Seq; with a median deduplicated depth of sequencing of 2,136x); 2) copy number variant analysis in ctDNA by genome representation profiling (GRP; median coverage across the whole genome 0.23x) and in the matched tumors by SNP arrays. One patient was excluded from the analysis due to inadequate sequencing coverage in tumor specimen. For CAPP-Seq analysis, we designed a custom 184kb capture panel targeting 89 genes that are recurrently mutated in LMS. Using strict variant calling criteria (requiring that variants be present on each strand of the original DNA “duplex” molecule) our panel identified a median of one nonsynonymous coding/splicing variant per tumor. We detected the same variants in TP53, RB1 and ATRX genes in ctDNA of 6/8 patients (with a baseline sensitivity of 87.5% and overall specificity of 98.96% calculated using plasma from 24 healthy donors). These six patients presented with advanced disease at the time of the first blood collection and were progressing throughout multiple lines of therapy. Two patients who did not have any variants detectable by CAPP-Seq in plasma had localized disease at the time of the first blood collection and/or responded well to the therapy. We found that changes in ctDNA levels appear to correspond with the extent of disease and response to treatment. Specifically, ctDNA levels decreased in a subset of patients after surgery or at the time of temporary response to chemo- and/or radiotherapy. Congruently, increases in ctDNA levels correlated with progression in most of the patients. There was a high correlation between ctDNA levels detected by CAPP-Seq (quantified as mutant molecules/mL plasma) and GRP (quantified as percent of genome showing copy number aberrations) across all plasma samples (Pearson's r= 0.88, p < 0.0001), but in a few samples ctDNA was detected by only one of the two assays. Our results suggest that serial analysis of ctDNA is a promising approach for evaluation of treatment response in LMS patients. Validation of these findings in a prospective study on a larger group of patients will be required to determine the use of this approach in a clinical setting. References: CAPP-Seq: PMIDs 24705333, 27018799 GRP: PMIDs 25585704, 26687610 Citation Format: Joanna Przybyl, Jacob J. Chabon, Lien Spans, Kristen Ganjoo, Sujay Vennam, Aaron M. Newman, Erna Forgó, Sushama Varma, Shirley Zhu, Maria Debiec-Rychter, Ash Alizadeh, Maximilian Diehn, Matt van de Rijn. Circulating tumor DNA levels correlate with response to treatment in LMS patients [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A05.

Purpose: Macrophages play an important role in breast carcinogenesis. The pathways that mediate the macrophage contribution to breast cancer and the heterogeneity that exists within macrophages are incompletely understood. Macrophage colony-stimulating factor 1 (CSF1) is the primary regulator of tissue macrophages. The purpose of this study was to define a novel CSF1 response signature and to evaluate its clinical and biological significance in breast cancer. Experimental Design: We defined the CSF1 response signature by identifying genes overexpressed in tenosynovial giant cell tumor and pigmented villonodular synovitis (tumors composed predominantly of macrophages recruited in response to the overexpression of CSF1) compared with desmoid-type fibromatosis and solitary fibrous tumor. To characterize the CSF1 response signature in breast cancer, we analyzed the expression of CSF1 response signature genes in eight published breast cancer gene expression data sets (n = 982) and did immunohistochemistry and in situ hybridization for CSF1 response genes on a breast cancer tissue microarray (n = 283). Results: In both the gene microarray and tissue microarray analyses, a consistent subset (17-25%) of breast cancers shows the CSF1 response signature. The signature is associated with higher tumor grade, decreased expression of estrogen receptor, decreased expression of progesterone receptor, and increased TP53 mutations (P < 0.001). Conclusions: Our data show that the CSF1 response signature is consistently seen in a subset of breast carcinomas and correlates with biological features of the tumor. Our findings provide insight into macrophage biology and may facilitate the development of personalized therapy for patients most likely to benefit from CSF1-targeted treatments.

Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation and that its effect on Hedgehog-pathway activity can be inhibited by arsenic trioxide (ATO), an anti-leukemia drug approved by the US Food and Drug Administration (FDA) that is currently in clinical trials for its Hedgehog-inhibitory activity. In a similar manner, ameloblastoma cells harboring an activating BRAF mutation encoding p.Val600Glu are sensitive to the BRAF inhibitor vemurafenib. Our findings establish a new paradigm for the diagnostic classification and treatment of ameloblastomas.

Gene fusions, like BCR/ABL1 in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a “breakpoint analysis” pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving ROS1 tyrosine kinase in angiosarcoma (CEP85L/ROS1), SLC1A2 glutamate transporter in colon cancer (APIP/SLC1A2), RAF1 kinase in pancreatic cancer (ATG7/RAF1) and anaplastic astrocytoma (BCL6/RAF1), EWSR1 in melanoma (EWSR1/CREM), CDK6 kinase in T-cell acute lymphoblastic leukemia (FAM133B/CDK6), and CLTC in breast cancer (CLTC/VMP1). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including EGFRvIII and FIP1L1/PDGFRA. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis., Author Summary Gene fusions represent an important class of cancer genes, created by rearrangements of the genome that bring together two different genes. Because they are unique to cancer cells, gene fusions are ideal diagnostic markers and therapeutic targets. While gene fusions were once thought restricted mainly to blood cancers, recent discoveries suggest they are more widespread. Here, we have developed an approach for mining DNA microarray data to detect the tell-tale signatures of gene fusions, as “breakpoints” occurring within the encoding DNA or expressed transcripts. We apply this approach to a large collection of nearly 1,000 human cancer specimens. From this analysis, we discover and verify twelve new gene fusions occurring in diverse cancer types. We verify that some of these rearrangements recur in other samples of the same cancer type (supporting a causal role) and that the cancers show dependency on the fusion for cancer cell growth. Notably, some of these fusions (e.g. CEP85L/ROS1 in angiosarcoma) represent the first for that cancer type and thus provide important new biological insight. Some are also good drug targets (including rearrangements of ROS1, RAF1, and CDK6 kinases), with clear implications for therapy.

Sarcomas of the uterus are derived from uterine smooth muscle (leiomyosarcomas) or from endometrial stromal cells. The latter group includes three subtypes: the low-grade (LG) and high-grade (HG) endometrial stromal sarcomas (ESS), each defined by characteristic chromosomal translocations, and undifferentiated uterine sarcoma (UUS), the most aggressive form. The majority of UUS patients present with high-stage disease and even patients with stage I tumors often die within 2 years from diagnosis. Adjuvant radio- and chemotherapy do not improve the clinical outcome. No prior comprehensive molecular analysis of all three types of endometrial stromal tumors has been reported. We performed genomic analysis (whole exome sequencing and aCGH), gene expression profiling (RNA-seq and microarrays), immunohistochemistry (IHC) and FISH on 31 endometrial stromal tumors, including 17 UUS and 14 LG and HG ESS. All ESS cases carried characteristic JAZF1, PHF1, MBTD1 or YWHAE rearrangements. Selected findings were validated by IHC, qRT-PCR and Sanger sequencing. Genomic analysis revealed that UUS are characterized by complex chromosomal aberrations. The most frequent somatic mutations (in TP53, KRAS, FBXW7, PIK3CA and ERBB3) and copy number changes (e.g. gains of 19q, 8q11.1-q24.3 and 3q26.2-q29) in UUS resemble those seen in uterine carcinosarcomas and carcinomas. UUS over-expressed numerous genes encoding M2 macrophage-specific markers, including CD163, CCL18, mannose receptor, stabilin-1, and macrophage galactose-type C-type lectin 2. Immunohistochemistry for CD163 and CD68 confirmed high tumor-associated macrophages (TAM) counts in UUS tissues compared to the ESS. In UUS, we also observed significantly higher mRNA expression for genes implicated in angiogenesis (CD34, MMP9), immunosuppression and tumor invasion (e.g. CCR2, IL10RA, IRAK3, CCL13, CXCL9, CXCL10). TAMs are associated with progression, resistance to cytotoxic therapy and metastatic spread in most human tumor types. In animal models, TAMs were shown to induce increased angiogenesis. CSF1 is a major regulator of macrophage function and is implicated in these tumor-promoting effects. Clinical trials have shown an effect of inhibitors of the CSF1 pathway in tenosynovial giant cell tumors and our data indicate that these inhibitors may be considered for the treatment of UUS. Our study also showed significantly elevated expression in UUS of two additional therapeutic targets involving macrophage pathways: CCR2 (C-C chemokine receptor type 2) that can be targeted by monoclonal antibodies, and BTK (Bruton's tyrosine kinase) that can be targeted by ibrutinib. In conclusion, our findings demonstrate abundant presence of TAMs and over-expression of TAM-associated markers in UUS compared to the other tumors derived from endometrial stroma. Moreover, our results indicate novel potential therapeutic targets for UUS patients management. Citation Format: Joanna Przybyl, Magdalena Kowalewska, Anna Quattrone, Barbara Dewaele, Vanessa Vanspauwen, Sushama Varma, Robert T. Sweeney, Michal Swierniak, Elwira Bakula-Zalewska, Janusz A. Siedlecki, Mariusz Bidzinski, Jan Cools, Matt van de Rijn, Maria Debiec-Rychter. Tumor associated macrophages in undifferentiated uterine sarcoma: association with angiogenesis and therapeutic implications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3182.

Rare cancer types are not only understudied, but are typically represented by formalin-fixed paraffin-embedded (FFPE) (rather than freshly-frozen) specimens that are suboptimal for genomic analysis. Ameloblastoma is one such rare tumor type, thought to arise from ameloblasts, the cells that deposit enamel during tooth development. Though typically benign, ameloblastomas are locally destructive to the jaw and face, and new non-surgical interventions are needed. To discover novel driver mutations and therapeutic targets, we optimized methods and performed whole-transcriptome sequencing and/or targeted exon sequencing (TruSeq Cancer Panel) of 8 FFPE cases. Identified mutations were verified, and then evaluated on a larger, independent set of 21 FFPE cases by PCR and Sanger sequencing. From the analysis, we identified recurrent somatic mutations in three key developmental or signaling pathways, including Hedgehog, fibroblast growth factor, and MAP kinase pathways. Functional interrogation of a novel Hedgehog pathway mutation confirmed increased basal pathway activity, and defined the response profile to various pharmacologic Hedgehog inhibitors. Together, our results define new ameloblastoma drivers and nominate new molecularly-directed therapies for this rare but disfiguring disease. More generally, our findings validate a robust approach for discovering driver mutations in rare cancers. Citation Format: Andrew C. McClary, Robert T. Sweeney, Jewison Biscocho, Benjamin R. Myers, Lila Neahring, Kevin A. Kwei, Kunbin Qu, Xue Gong, Tony Ng, Carol D. Jones, Sushama Varma, Justin I. Odegaard, Brian Rubin, Megan L. Troxell, Robert J. Pelham, James L. Zehnder, Philip A. Beachy, Jonathan R. Pollack, Robert B. West. Ameloblastoma driver mutations revealed by next-generation sequencing of formalin-fixed paraffin-embedded specimens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3436. doi:10.1158/1538-7445.AM2014-3436

Hypoxic Induction of Vascular Endothelial Growth Factor (VEGF) and HIF-1 Protein in Retinal Pigment Epithelial Cells