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The interaction between neoplastic and stromal cells within a tumor mass plays an important role in cancer biology. However, it is challenging to distinguish between tumor and stromal cells in mesenchymal tumors because lineage-specific cell surface markers typically used in other cancers do not distinguish between the different cell subpopulations. Desmoid tumors consist of mesenchymal fibroblast-like cells driven by mutations stabilizing beta-catenin. Here we aimed to identify surface markers that can distinguish mutant cells from stromal cells to study tumor–stroma interactions. We analyzed colonies derived from single cells from human desmoid tumors using a high-throughput surface antigen screen, to characterize the mutant and nonmutant cells. We found that CD142 is highly expressed by the mutant cell populations and correlates with beta-catenin activity. CD142-based cell sorting isolated the mutant population from heterogeneous samples, including one where no mutation was previously detected by traditional Sanger sequencing. We then studied the secretome of mutant and nonmutant fibroblastic cells. PTX3 is one stroma-derived secreted factor that increases mutant cell proliferation via STAT6 activation. These data demonstrate a sensitive method to quantify and distinguish neoplastic from stromal cells in mesenchymal tumors. It identifies proteins secreted by nonmutant cells that regulate mutant cell proliferation that could be therapeutically. Significance: Distinguishing between neoplastic (tumor) and non-neoplastic (stromal) cells within mesenchymal tumors is particularly challenging, because lineage-specific cell surface markers typically used in other cancers do not differentiate between the different cell subpopulations. Here, we developed a strategy combining clonal expansion with surface proteome profiling to identify markers for quantifying and isolating mutant and nonmutant cell subpopulations in desmoid tumors, and to study their interactions via soluble factors.

Densitometry of selected secreted proteome from mutant and non-mutant conditioned media

Cell surface profiling data of mutant and non-mutant desmoid tumor-derived colonies. Name of the sample indicates the mutation status (mutant (MUT) or wildtype (WT)) of the pooled colonies and the genotype (T41A or S45F) of the original primary sample.

The interaction between neoplastic and stromal cells within a tumor mass plays an important role in cancer biology. However, it is challenging to distinguish between tumor and stromal cells in mesenchymal tumors because lineage-specific cell surface markers typically used in other cancers do not distinguish between the different cell subpopulations. Desmoid tumors consist of mesenchymal fibroblast-like cells driven by mutations stabilizing beta-catenin. Here we aimed to identify surface markers that can distinguish mutant cells from stromal cells to study tumor–stroma interactions. We analyzed colonies derived from single cells from human desmoid tumors using a high-throughput surface antigen screen, to characterize the mutant and nonmutant cells. We found that CD142 is highly expressed by the mutant cell populations and correlates with beta-catenin activity. CD142-based cell sorting isolated the mutant population from heterogeneous samples, including one where no mutation was previously detected by traditional Sanger sequencing. We then studied the secretome of mutant and nonmutant fibroblastic cells. PTX3 is one stroma-derived secreted factor that increases mutant cell proliferation via STAT6 activation. These data demonstrate a sensitive method to quantify and distinguish neoplastic from stromal cells in mesenchymal tumors. It identifies proteins secreted by nonmutant cells that regulate mutant cell proliferation that could be therapeutically.Significance:Distinguishing between neoplastic (tumor) and non-neoplastic (stromal) cells within mesenchymal tumors is particularly challenging, because lineage-specific cell surface markers typically used in other cancers do not differentiate between the different cell subpopulations. Here, we developed a strategy combining clonal expansion with surface proteome profiling to identify markers for quantifying and isolating mutant and nonmutant cell subpopulations in desmoid tumors, and to study their interactions via soluble factors.

Type B (mutant) cells exhibit higher alpha-smooth muscle actin levels at the protein and RNA level.

Summary of double-color cell sorting experiments.

Supplementary Figure S1. ALK copy number data from TCGA-FX-A48G and TCGA-IW-A3M6 LMS visualized using IGV (Broad, MIT) Supplementary Table S1. Identification of ALK rearrangements using ChimeraScan algorithm. Supplementary Figure S2. Results from the fusion analysis pipeline run on RNA-seq data for TCGA-FX-A48G identifying KANK2-ALK Supplementary Figure S3. Results from the fusion analysis pipeline run on RNA-seq data for TCGA-IW-A3M6 identifying ACTG2-ALK Supplementary Figure S4. Confirmation of KANK2-ALK transgene expression from transformed Ba/F3 cells. Supplementary Figure S5. Dose-response inhibitor assay with ALK inhibitors with Ba/F3 parental cells in presence of IL-3 Supplementary Figure S6. Body weight of mice treated with vehicle, crizotinib or lorlatinib

BARX1-/HAND1- GIST

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.

Purpose:Gastrointestinal stromal tumor (GIST) arises from interstitial cells of Cajal (ICC) or their precursors, which are present throughout the gastrointestinal tract. Although gastric GIST is commonly indolent and small intestine GIST more aggressive, a molecular understanding of disease behavior would inform therapy decisions in GIST. Although a core transcription factor (TF) network is conserved across GIST, accessory TFs HAND1 and BARX1 are expressed in a disease state-specific pattern. Here, we characterize two divergent transcriptional programs maintained by HAND1 and BARX1, and evaluate their association with clinical outcomes.Experimental Design:We evaluated RNA sequencing and TF chromatin immunoprecipitation with sequencing in GIST samples and cultured cells for transcriptional programs associated with HAND1 and BARX1. Multiplexed tissue-based cyclic immunofluorescence and IHC evaluated tissue- and cell-level expression of TFs and their association with clinical factors.Results:We show that HAND1 is expressed in aggressive GIST, modulating KIT and core TF expression and supporting proliferative cellular programs. In contrast, BARX1 is expressed in indolent and micro-GISTs. HAND1 and BARX1 expression were superior predictors of relapse-free survival, as compared with standard risk stratification, and they predict progression-free survival on imatinib. Reflecting the developmental origins of accessory TF programs, HAND1 was expressed solely in small intestine ICCs, whereas BARX1 expression was restricted to gastric ICCs.Conclusions:Our results define anatomic and transcriptional determinants of GIST and molecular origins of clinical phenotypes. Assessment of HAND1 and BARX1 expression in GIST may provide prognostic information and improve clinical decisions on the administration of adjuvant therapy.

Figure S1 Summary of CAPP-Seq and GRP findings in plasma specimens of 7 LMS patients. Figure S2 Specificity and sensitivity of ctDNA detection by CAPP-Seq in LMS patients. Figure S3 Individual DNA copy number alteration profiles of 22 tumor specimens from 7 LMS patients. CN - copy number Figure S4 UCSC Genome Browser view of genomic region 11q24.3-q25 that was consistently lost across all tumor specimens in patients LMS2, LMS3, LMS4, LMS5 and LMS7. Figure S5 Correlation between ctDNA levels detected by CAPP-Seq (represented as VAF) and GRP (represented as percent of genome altered by copy number alterations). Table S1 Summary of clinical features of 9 LMS patients Table S2 LMS-specific CAPP-Seq selector. Table S3 Tumor specimens used for CAPP-Seq and SNP array analysis. Table S4 Depth of sequencing and % of on-target reads by CAPP-Seq. Table S5 Summary statistics for GRP. Table S6 Input of cfDNA used for CAPP-Seq and GRP. Table S7 Summary of quality control metrics from OncoScan FFPE Assay and the extent of CNAs in 22 LMS tumor specimens.

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.

Cancer precision medicine implies identification of tumor-specific vulnerabilities associated with defined oncogenic pathways. Desmoid tumors are soft-tissue neoplasms strictly driven by Wnt signaling network hyperactivation. Despite this clearly defined genetic etiology and the strict and unique implication of the Wnt/β-catenin pathway, no specific molecular targets for these tumors have been identified. To address this caveat, we developed fast and semi-high throughput genetic Xenopus tropicalis desmoid tumor models to identify and characterize novel drug targets. We used multiplexed CRISPR/Cas9 genome editing in these models to simultaneously target a tumor suppressor gene (apc) and candidate dependency genes. Our methodology CRISPR/Cas9 Selection mediated Identification of Dependencies (CRISPR-SID) uses calculated deviations between experimentally observed gene editing outcomes and deep-learning-predicted double strand break repair patterns, to identify genes under negative selection during tumorigenesis. This revealed EZH2 and SUZ12, both encoding polycomb repressive complex 2 components, and the transcription factor CREB3L1, as genetic dependencies for desmoid tumors. In vivo EZH2 inhibition by Tazemetostat induced partial regression of established autochthonous tumors. In vitro models of patient desmoid tumor cells revealed a direct effect of Tazemetostat on Wnt pathway activity. CRISPR-SID represents a potent novel approach for in vivo mapping of tumor vulnerabilities and drug target identification.Significance StatementCRISPR-SID was established in the diploid frog Xenopus tropicalis for in vivo elucidation of cancer cell vulnerabilities. CRISPR-SID uses deep learning predictions and binomial theory to identify genes under positive or negative selection during autochthonous tumor development. Using CRISPR-SID in a genetic model for desmoid tumors, treatment-recalcitrant mesenchymal tumors driven by hyper-activation of the Wnt signaling pathway, we identified EZH2 and SUZ12, both encoding critical components of the polycomb repressive complex 2, as dependency genes for desmoid. Finally, we demonstrate the promise of EZH2 inhibition as a novel therapeutic strategy for desmoid tumors. With the simplicity of CRISPR sgRNA multiplexing in Xenopus embryos the CRISPR-SID method may be applicable to reveal vulnerabilities in other tumor types.

High-level amplification of MDM2 and other genes in the 12q13–15 locus is a hallmark genetic feature of well-differentiated and dedifferentiated liposarcomas (WDLPS and DDLPS, respectively). Detection of this genomic aberration in plasma cell-free DNA may be a clinically useful assay for non-invasive distinction between these liposarcomas and other retroperitoneal tumors in differential diagnosis, and might be useful for the early detection of disease recurrence. In this study, we performed shallow whole genome sequencing of cell-free DNA extracted from 10 plasma samples from 3 patients with DDLPS and 1 patient with WDLPS. In addition, we studied 31 plasma samples from 11 patients with other types of soft tissue tumors. We detected MDM2 amplification in cell-free DNA of 2 of 3 patients with DDLPS. By applying a genome-wide approach to the analysis of cell-free DNA, we also detected amplification of other genes that are known to be recurrently affected in DDLPS. Based on the analysis of one patient with DDLPS with longitudinal plasma samples available, we show that tracking MDM2 amplification in cell-free DNA may be potentially useful for evaluation of response to treatment. The patient with WDLPS and patients with other soft tissue tumors in differential diagnosis were negative for the MDM2 amplification in cell-free DNA. In summary, we demonstrate the feasibility of detecting amplification of MDM2 and other DDLPS-associated genes in plasma cell-free DNA using technology that is already routinely applied for other clinical indications. Our results may have clinical implications for improved diagnosis and surveillance of patients with retroperitoneal tumors.

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.

Background Sarcomas are a rare, heterogeneous group of tumors with variable tendencies for aggressive behavior. Molecular markers for prognosis are needed to risk stratify patients and identify those who might benefit from more intensive therapeutic strategies. Patients and methods We analyzed somatic tumor genomic profiles and clinical outcomes of 152 soft tissue (STS) and bone sarcoma (BS) patients sequenced at Stanford Cancer Institute as well as 206 STS patients from The Cancer Genome Atlas. Genomic profiles of 7733 STS from the Foundation Medicine database were used to assess the frequency of CDKN2A alterations in histological subtypes of sarcoma. Results Compared to all other tumor types, sarcomas were found to carry the highest relative percentage of gene amplifications/deletions/fusions and the lowest average mutation count. The most commonly altered genes in STS were TP53 (47%), CDKN2A (22%), RB1 (22%), NF1 (11%), and ATRX (11%). When all genomic alterations were tested for prognostic significance in the specific Stanford cohort of localized STS, only CDKN2A alterations correlated significantly with prognosis, with a hazard ratio (HR) of 2.83 for overall survival (p = 0.017). These findings were validated in the TCGA dataset where CDKN2A altered patients had significantly worse overall survival with a HR of 2.7 (p = 0.002). Analysis of 7733 STS patients from Foundation One showed high prevalence of CDKN2A alterations in malignant peripheral nerve sheath tumors, myxofibrosarcomas, and undifferentiated pleomorphic sarcomas. Conclusion Our clinico-genomic profiling of STS shows that CDKN2A deletion was the most prevalent DNA copy number aberration and was associated with poor prognosis.

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.

Purpose: Gastrointestinal stromal tumor (GIST) arises from interstitial cells of Cajal (ICC) or their precursors, which are present throughout the gastrointestinal tract. Although gastric GIST is commonly indolent and small intestine GIST more aggressive, a molecular understanding of disease behavior would inform therapy decisions in GIST. Although a core transcription factor (TF) network is conserved across GIST, accessory TFs HAND1 and BARX1 are expressed in a disease state-specific pattern. Here, we characterize two divergent transcriptional programs maintained by HAND1 and BARX1, and evaluate their association with clinical outcomes. Experimental Design: We evaluated RNA sequencing and TF chromatin immunoprecipitation with sequencing in GIST samples and cultured cells for transcriptional programs associated with HAND1 and BARX1. Multiplexed tissue-based cyclic immunofluorescence and IHC evaluated tissue- and cell-level expression of TFs and their association with clinical factors. Results: We show that HAND1 is expressed in aggressive GIST, modulating KIT and core TF expression and supporting proliferative cellular programs. In contrast, BARX1 is expressed in indolent and micro-GISTs. HAND1 and BARX1 expression were superior predictors of relapse-free survival, as compared with standard risk stratification, and they predict progression-free survival on imatinib. Reflecting the developmental origins of accessory TF programs, HAND1 was expressed solely in small intestine ICCs, whereas BARX1 expression was restricted to gastric ICCs. Conclusions: Our results define anatomic and transcriptional determinants of GIST and molecular origins of clinical phenotypes. Assessment of HAND1 and BARX1 expression in GIST may provide prognostic information and improve clinical decisions on the administration of adjuvant therapy.

BACKGROUND: Leiomyosarcoma (LMS) is the most common soft tissue and uterine sarcoma, but no standard therapy is available for recurrent or metastatic LMS. TP53, p16/RB1, and PI3K/mTOR pathway dysregulations are recurrent events, and some LMS express estrogen receptor (ER) and/or progesterone receptor (PR). To characterize relationships between these pathway perturbations, the authors evaluated protein expression in soft tissue and uterine nonprimary leiomyosarcoma (np-LMS), including local recurrences and distant metastases. METHODS: TP53, RB1, p16, and PTEN expression aberrations were determined by immunohistochemistry (IHC) in tissue microarrays (TMAs) from 227 np-LMS and a comparison group of 262 primary leiomyosarcomas (p-LMS). Thirty-five of the np-LMS had a matched p-LMS specimen in the TMAs. Correlative studies included differentiation scoring, ER and PR IHC, and CDKN2A/p16 fluorescence in situ hybridization. RESULTS: Dysregulation of TP53, p16/RB1, and PTEN was demonstrated in 90%, 95%, and 41% of np-LMS, respectively. PTEN inactivation was more common in soft tissue np-LMS than uterine np-LMS (55% vs 31%; P = .0005). Moderate-strong ER expression was more common in uterine np-LMS than soft tissue np-LMS (50% vs 7%; P < .0001). Co-inactivation of TP53 and RB1 was found in 81% of np-LMS and was common in both soft tissue and uterine np-LMS (90% and 74%, respectively). RB1, p16, and PTEN aberrations were nearly always conserved in p-LMS and np-LMS from the same patients. CONCLUSIONS: These studies show that nearly all np-LMS have TP53 and/or RB1 aberrations. Therefore, therapies targeting cell cycle and DNA damage checkpoint vulnerabilities should be prioritized for evaluations in LMS.

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.

Objective Low-grade endometrial stromal sarcomas (LGESS) harbor chromosomal translocations that affect proteins associated with chromatin remodeling Polycomb Repressive Complex 2 (PRC2), including SUZ12, PHF1 and EPC1. Roughly half of LGESS also demonstrate nuclear accumulation of β-catenin, which is a hallmark of Wnt signaling activation. However, the targets affected by the fusion proteins and the role of Wnt signaling in the pathogenesis of these tumors remain largely unknown. Methods Here we report the results of a meta-analysis of three independent gene expression profiling studies on LGESS and immunohistochemical evaluation of nuclear expression of β-catenin and Lef1 in 112 uterine sarcoma specimens obtained from 20 LGESS and 89 LMS patients. Results Our results demonstrate that 143 out of 310 genes overexpressed in LGESS are known to be directly regulated by SUZ12. In addition, our gene expression meta-analysis shows activation of multiple genes implicated in Wnt signaling. We further emphasize the role of the Wnt signaling pathway by demonstrating concordant nuclear expression of β-catenin and Lef1 in 7/16 LGESS. Conclusions Based on our findings, we suggest that LGESS-specific fusion proteins disrupt the repressive function of the PRC2 complex similar to the mechanism seen in synovial sarcoma, where the SS18-SSX fusion proteins disrupt the mSWI/SNF (BAF) chromatin remodeling complex. We propose that these fusion proteins in LGESS contribute to overexpression of Wnt ligands with subsequent activation of Wnt signaling pathway and formation of an active β-catenin/Lef1 transcriptional complex. These observations could lead to novel therapeutic approaches that focus on the Wnt pathway in LGESS.

Determining how cells vary with their local signaling environment and organize into distinct cellular communities is critical for understanding processes as diverse as development, aging, and cancer. Here we introduce EcoTyper, a machine learning framework for large-scale identification and validation of cell states and multicellular communities from bulk, single-cell, and spatially resolved gene expression data. When applied to 12 major cell lineages across 16 types of human carcinoma, EcoTyper identified 69 transcriptionally defined cell states. Most states were specific to neoplastic tissue, ubiquitous across tumor types, and significantly prognostic. By analyzing cell-state co-occurrence patterns, we discovered ten clinically distinct multicellular communities with unexpectedly strong conservation, including three with myeloid and stromal elements linked to adverse survival, one enriched in normal tissue, and two associated with early cancer development. This study elucidates fundamental units of cellular organization in human carcinoma and provides a framework for large-scale profiling of cellular ecosystems in any tissue.

Tumors are complex ecosystems consisting of malignant, immune, and stromal elements whose dynamic interactions drive patient survival and response to therapy. A comprehensive understanding of the diversity of cellular states within the tumor microenvironment (TME), and their patterns of co-occurrence, could provide new diagnostic tools for improved disease management and novel targets for therapeutic intervention. To address this challenge, we developed EcoTyper, a novel machine learning framework for large-scale identification of TME cell states and their co-association patterns from bulk, single-cell, and spatially resolved tumor expression data. EcoTyper starts by “purifying” cell type-specific gene expression profiles of epithelial cells, immune, and stromal cell types from bulk tissue transcriptomes using CIBERSORTx (Newman et al., Nat Biotechnol 2019). It then identifies transcriptional states for each cell type, validates them in scRNA-seq data, and uncovers co-occurrence patterns between cell states in order to define tumor cellular ecosystems. Applied to 6,475 tumor and adjacent normal samples from solid tumor types profiled by The Cancer Genome Atlas (TCGA), EcoTyper identified robust transcriptional states across 12 major cell types, including epithelial, fibroblast, endothelial, and 9 immune subsets. These states included both known and novel cellular phenotypes, nearly all of which could be validated in a compendium of scRNA-seq tumor atlases spanning ~140,000 cells. Most cell states were specific to neoplastic tissue, ubiquitous across tumor types, and significantly associated with overall survival, both in TCGA and in 9,062 held-out tumor specimens (Gentles/Newman et al., Nat Medicine 2015). We found that specific cell states co-occur in distinct cellular communities with characteristic patterns of ligand-receptor interactions, genomic features, clinical outcomes, and spatial organization. One such ecosystem defined a normal-like state that was strongly enriched in non-malignant samples. Others delineated novel pro- and anti-tumor inflammatory environments involving specific fibroblast, endothelial, and immune cell transcriptional programs. In summary, large-scale deconvolution of cell type-specific transcriptomes across thousands of solid tumors revealed a comprehensive atlas of TME cell states and cellular ecosystems. Our results provide a high-resolution portrait of cellular heterogeneity in the TME across multiple solid tumor types, with implications for novel diagnostics and immunotherapeutic targets. References: 1. Newman, A.M., et al., Determining cell type abundance and expression from bulk tissues with digital cytometry. Nat Biotechnol, 2019. 37(7): p. 773-782. 2. Gentles, A.J., et al., The prognostic landscape of genes and infiltrating immune cells across human cancers. Nature medicine, 2015. 21(8): p. 938. Citation Format: Bogdan A. Luca, Chloé B. Steen, Armon Azizi, Magdalena Matusiak, Joanna Przybyl, Nastaran Neishaboori, Almudena Espín Pérez, Maximilian Diehn, Ash A. Alizadeh, Matt van de Rijn, Andrew J. Gentles, Aaron M. Newman. Atlas of clinically-distinct cell states and cellular ecosystems across human solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3443.

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.

Endometrial stromal sarcomas (ESSs) are mesenchymal uterine tumors characterized by recurrent genetic events, most commonly chromosomal rearrangements, that create oncogenic gene fusions. High-grade endometrial stromal sarcomas (HG-ESSs), as defined in the 2014 World Health Organization Classification, typically contain oncogenic YWHAE-NUTM2 fusions; however, although not well characterized, there are tumors morphologically overlapping with HG-ESS that do not contain the YWHAE-NUTM2 fusions. These fusions are also found in certain pediatric primitive sarcomas, including clear cell sarcoma of the kidney and soft tissue undifferentiated round cell sarcoma of infancy. A subset of these same pediatric sarcomas lack YWHAE-NUTM2 fusions and instead have internal tandem duplications (ITDs) involving exon 15 of BCOR (BCOR ITD). We investigated the presence of BCOR ITD by targeted sequencing in a series of 31 uterine sarcomas, comprising 5 low-grade ESS, 13 uterine sarcomas diagnosed as HG-ESS, and 13 undifferentiated uterine sarcomas. BCOR ITD were present in 1 uterine sarcoma diagnosed as HG-ESS and 2 undifferentiated sarcomas with uniform nuclear features, all of which lacked any of the recurrent chromosome translocations known to occur in ESS. These 3 high-grade sarcomas with BCOR ITD affected young patients (average age, 24) and morphologically were composed of nonpleomorphic spindle cells admixed with epithelioid and round cell areas. Focal myxoid stroma was present in 2 cases. Mitotic activity was brisk, necrosis was present, and there was lymphovascular involvement in all cases. The 3 uterine sarcomas with BCOR ITD exhibited diffuse cyclin D1 immunohistochemical expression and there was diffuse BCOR expression in the 2 cases tested. Long-term follow-up in 2 patients revealed 1 to be tumor-free after 22 years and the other to die of disease after 8 years. In conclusion, BCOR ITD is an oncogenic alternative to YWHAE-NUTM2 fusion in high-grade uterine sarcomas with uniform nuclear features. We propose that neoplasms with the morphology described and BCOR ITD be regarded as a unique subtype of high-grade uterine sarcoma, possibly within the family of endometrial stromal neoplasia. ispartof: The American Journal of Surgical Pathology vol:42 issue:3 pages:335-341 ispartof: location:United States status: published

Endometrial stromal tumors include translocation-associated low- and high-grade endometrial stromal sarcomas (ESS) and highly malignant undifferentiated uterine sarcomas (UUS). UUS is considered a poorly defined group of aggressive tumors and is often seen as a diagnosis of exclusion after ESS and leiomyosarcoma (LMS) have been ruled out. We performed a comprehensive analysis of gene expression, copy number variation, point mutations, and immune cell infiltrates in the largest series to date of all major types of uterine sarcomas to shed light on the biology of UUS and to identify potential novel therapeutic targets. We show that UUS tumors have a distinct molecular profile from LMS and ESS. Gene expression and immunohistochemical analyses revealed the presence of high numbers of tumor-associated macrophages (TAMs) in UUS, which makes UUS patients suitable candidates for therapies targeting TAMs. Our results show a high genomic instability of UUS and downregulation of several TP53-mediated tumor suppressor genes, such as NDN, CDH11, and NDRG4. Moreover, we demonstrate that UUS carry somatic mutations in several oncogenes and tumor suppressor genes implicated in RAS/PI3K/AKT/mTOR, ERBB3, and Hedgehog signaling.

Introduction Synovial sarcoma (SynSa) is an aggressive mesenchymal tumor, comprising approximately 10% of all soft tissue sarcomas. Over half of SynSa patients develop metastasis or local recurrence, but the underlying molecular mechanisms of the aggressive clinical behavior remain poorly characterized. Materials and methods Sixty-four frozen tumor specimens from 54 SynSa patients were subjected to array comparative genomic hybridization (aCGH) and gene expression profiling. The examined set of tumor specimens included 16 primary tumors from untreated patients who did not develop metastasis/local recurrence (SynSa1 group), 26 primary tumors from untreated patients who developed metastases or local recurrence during follow-up (SynSa2 group), and 22 metachronous metastatic/recurrent SynSa tumors (SynSa3 group). Results AURKA and KIF18A, which play important roles in various mitotic events, were the two most up-regulated genes in SynSa2 and SynSa3 groups compared to the SynSa1 group. Expression profiles of SynSa2 and SynSa3 tumors did not show any significant differences. Analysis of genomic index (GI) based on aCGH profiles demonstrated that the SynSa1 group consisted of tumors with significantly less complex genomes compared to SynSa2 and SynSa3 groups. There was no significant difference in genome complexity between SynSa2 and SynSa3 tumors. Conclusions Primary SynSa tumors from patients who develop metastases or local recurrence share common molecular features with metastatic/recurrent tumors. Presented data suggest that the aggressive clinical SynSa behavior is determined early in tumorigenesis and might be related to impaired regulation of mitotic mechanisms. This article is part of a Directed Issue entitled: Rare Cancers.

Castleman disease is a rare lymphoproliferative disorder known to represent at least four distinct clinico-pathologic sub-types. Large advancements in our clinical and histopathologic description of these diverse diseases have been made, resulting in subtyping based on degree of nodal involvement (unicentric versus multicentric) and according to viral infection (human herpes virus 8 - HHV8 and Human Immunodeficiency Virus - HIV). However, we continue to lack a foundational understanding of the biologic mechanisms driving at least a subset of patients with these diseases. In recent years, significant molecular and genetic abnormalities that may be associated with this disease have been described; yet we are still unclear as to a unified vision of what loci and genes are involved in at least a subset of cases of Castleman disease. In our study we performed a meta-analysis of all cases (unicentric and multicentric) of Castleman disease described in literature to date, and correlate cytogenetic, molecular and genetic abnormalities with disease subtypes, phenotypes and clinical outcomes. We further performed targeted DNA deep sequencing and RNA-sequencing on a cohort of Castleman Disease cases. Our results demonstrate that genetic abnormalities in the MAPK-pathways, PI3K-pathway and Interleukin signaling are frequent in unicentric Castleman disease, whereas abnormalities in DNA methylation are seen more commonly in idiopathic multicentric Castleman disease, along with abnormalities in MAPK-pathways. We conclude that UCD and iMCD may share similar pathways of MAPK pathogenesis but can also be distinguished by cytokine and other DNA modification pathways that differ. Disclosures Ohgami: Agilent technologies: Other: received support/funding.

On the basis of the recently published results of a clinical trial comparing 12 and 36 months of imatinib in adjuvant therapy for gastrointestinal stromal tumors (GISTs), which demonstrated clinical benefit of longer imatinib treatment in terms of delaying recurrences and improving overall survival, both the US Food and Drug Administration and the European Medicines Agency have updated their recommendations and approved 36 months of imatinib treatment in patients with v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT)-positive GISTs (also known as CD117-positive GISTs) at high risk of recurrence after surgical resection of a primary tumor. This article discusses patient selection criteria for extended adjuvant therapy with imatinib, different classifications of risk of recurrence, and assessment of the response to therapy.

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...

11543Background: Sarcomas are a heterogeneous group of tumors with variable tendencies for aggressive behavior. Molecular markers for prognosis are needed to help risk stratify patients and potenti...

BACKGROUND: Despite conventional neuromonitoring, the recurrent laryngeal nerve (RLN) is still at risk for damage during thyroid surgery. The feasibility of continuous RLN monitoring by vagal nerve (VN) stimulation with a new anchor electrode should be shown, and electromyographic signal alterations of stressed RLN were analyzed to be alerted to imminent nerve failure whereby the nerve damage becomes reversible. METHODS: VN stimulation was achieved in 23 pigs. Sensed signals were analyzed and stored as real-time audio/video feedback EMG system. RLN was stressed by mechanical and thermal injury; signal alterations were evaluated. RESULTS: VNs were successfully real-time stimulated by using the anchor electrode. No complications or side effects during stimulation were detected. RLN injury led to an alteration of signal amplitude and latency period but signal restitution after injury. CONCLUSIONS: Real-time monitoring of the RLN is technically feasible to perceive imminent nerve failure. The anchor electrode was safely and easy to handle. Its implementation is being tested in an ongoing clinical trial.

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.

Advances in the understanding of the molecular mechanisms of gastrointestinal stromal tumors (GISTs) pathogenesis have resulted in the development of a treatment approach which has become a model of targeted therapy in oncology. The introduction of imatinib mesylate [inhibiting KIT/PDGFRA (platelet-derived growth factor receptor-α) and their downstream signaling cascade] has dramatically improved the therapy of advanced (inoperable and/or metastatic) GIST. Imatinib has now become the standard of care in the treatment of patients with advanced GIST and its efficacy has been proven also in adjuvant setting after resection of primary high-risk tumors. However, a majority of patients eventually develop resistance to imatinib. Over the last few years, significant progress has been made in elucidating the mechanism of disease progression (as secondary mutations in KIT and/or PDGFRA kinase domains) and resistance to imatinib. Currently, the second-line approved drug is sunitinib—a multikinase inhibitor of KIT and PDGFRA/B and of the vascular endothelial growth factor receptors (VEGFRs)-1, -2, and 3, FMS-like tyrosine kinase-3 (FLT3), colony stimulating factor 1 receptor (CSF-1R), and glial cell-line derived neurotrophic factor receptor [Rearranged during Transfection (RET)], and in the third line, regorafenib was approved recently. However, a number of new generation tyrosine kinase inhibitors (as ponatinib) are being evaluated at present alongside treatment options alternative to inhibiting the KIT signaling pathway (as heat shock protein 90 or insulin-like growth factor 1 receptor).

Soft tissue and bone sarcomas comprise a heterogeneous group of mesenchymal tumors that include roughly 130 distinct diagnostic entities. Many of them are exceptionally rare, with only few cases diagnosed worldwide each year. Development of novel targeted treatment in this group of tumors is of special importance since many sarcoma subtypes are resistant to conventional chemotherapy and the effective therapeutic options are limited. In this review we aim to discuss the molecular implications for targeted therapy in selected rare soft tissue and bone sarcoma subtypes, including dermatofibrosarcoma protuberans (DFSP), alveolar soft part sarcoma (ASPS), clear cell sarcoma (CCS), giant cell tumor of bone (GCTB) and perivascular epithelioid cell neoplasms (PEComas). This article is part of a Directed Issue entitled: Rare cancers.

Endometrial stromal sarcomas (ESSs) are a genetically heterogeneous group of rare uterine neoplasms that are commonly driven by recurrent gene rearrangements. In conventional low-grade ESS, JAZF1-SUZ12, PHF1-JAZF1, EPC1-PHF1 and MEAF6-PHF1, and recently described ZC3H7-BCOR chimeric fusions have been reported in > 50% of cases. Conversely, oncogenic t(10;17)(q22;p13) translocation yields YWHAE-FAM22A/B chimeric proteins that are associated with histologically high-grade and clinically more aggressive ESS. Integrating whole-transcriptome paired-end RNA sequencing with fluorescence in situ hybridization (FISH) and banding cytogenetics, we identified MBTD1 (malignant brain tumor domain-containing 1) and CXorf67 (chromosome X open reading frame 67) as the genes involved in the novel reciprocal t(X;17)(p11.2;q21.33) translocation in two independent low-grade ESS of classical histology. The presence of the MBTD1-CXorf67 fusion transcript was validated in both cases using reverse-transcription polymerase chain reaction followed by Sanger sequencing. A specific FISH assay was developed to detect the novel t(X;17) translocation in formalin-fixed paraffin-embedded material, and resulted in identification of an additional low-grade ESS case positive for the MBTD1-CXorf67 fusion among 25 uterine stromal tumors [14 ESS and 11 undifferentiated endometrial sarcomas (UESs)] that were negative for JAZF1 and YWHAE rearrangements. Gene expression profiles of seven ESS (including three with YWHAE and two with JAZF1 rearrangements) and four UES without specific chromosomal aberrations indicated clustering of tumors with MBTD1-CXorf67 fusion together with low-grade JAZF1-associated ESS. The chimeric MBTD1-CXorf67 fusion identifies yet another cytogenetically distinct subgroup of low-grade ESS and offers the opportunity to shed light on the functions of two poorly characterized genes.

Purpose Synovial sarcoma (SS) occurs in both children and adults, although metastatic events are much more common in adults. Whereas the importance of the t(X;18) translocation in SS oncogenesis is well established, the genetic basis of SS metastasis is still poorly understood. We recently reported expression (CINSARC; Complexity Index in Sarcoma) and Genomic Index prognostic signatures related to chromosome integrity in sarcomas and GI stromal tumors. Here we investigate whether these signatures can also predict outcomes in SS. Patients and Methods One hundred patients who had primary untreated SS tumors were selected for expression and genomic profiling in a training/validation approach. Results CINSARC and Genomic Index have strong independent and validated prognostic values (P < .001). By comparing expression profiles of tumors with or without metastasis, 14 genes that are common to the CINSARC signature were identified, and the two top-ranked genes, KIF14 and CDCA2, were validated as prognostic markers in an independent cohort. Comparing genomic profiles of adult versus pediatric SS, we show that metastasis is associated with genome complexity in both situations and that the adult genome is more frequently rearranged. Accordingly, pediatric patients with an even genomic profile do not develop metastasis. Conclusion Metastasis development in SS is strongly associated with chromosome complexity, and CINSARC and Genomic Index are validated independent prognostic factors. The differences in metastasis frequency between adults and children are associated with genome instability, which is much more frequent in adults. Genomic Index is potentially the best overall biomarker and clearly the most clinically relevant, considering that genome profiling from formalin-fixed samples is already used in pathology.

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.

Synovial sarcoma (SS), an aggressive type of soft tissue tumor, occurs mostly in adolescents and young adults. The origin and molecular mechanism of the development of SS remain only partially known. Over 90% of SS cases are characterized by the t(X;18)(p11.2;q11.2) translocation, which results mainly in the formation ofSS18-SSX1orSS18-SSX2fusion genes. In recent years, several reports describing direct and indirect interactions ofSS18-SSX1/SSX2oncoproteins have been published. These reports suggest that the fusion proteins particularly affect the cell growth, cell proliferation, TP53 pathway, and chromatin remodeling mechanisms, contributing to SS oncogenesis. Additional research efforts are required to fully explore the protein-protein interactions of SS18-SSX oncoproteins and the pathways that are regulated by these partnerships for the development of effective targeted therapy.

Gastrointestinal stromal tumors (GISTs) constitute a rare heterogeneous group of the most common mesenchymal neoplasm of gastrointestinal tract (GI). GISTs have emerged during the recent years as a distinct sarcoma entity due to advances in the understanding of molecular mechanism of their pathogenesis. They are believed to originate from precursors shared with interstitial cells of Cajal (ICC) – the pacemaker cells of the gut (for which CD117 antigen is the immunohistochemical marker), and they may arise along all GI (most commonly in the stomach or the small bowel) or rarely elsewhere. Their biological behavior is difficult to predict, ranging from clinically benign to malignant. The treatment of choice in primary resectable GISTs is radical surgery, but majority of GISTs are associated with a risk of recurrences. The main criteria of aggressive behavior of GIST are based on the presence of invasion of surrounding structures and/or metastases (overtly malignant cases), as well as on primary tumor site, size, and mitotic index.

Intraoperative neuromonitoring has the limitation that the recurrent laryngeal nerve (RLN) is still at risk for damage between two stimulations with a handheld bipolar stimulation electrode. The purpose of this study was to establish the vagal anchor electrode for real-time monitoring of the RLN in surgical routine and to be alerted to imminent nerve failure by electromyography (EMG) signal analysis whereby the nerve damage becomes reversible.This fully implantable electrode has been used in addition to a conventional handheld bipolar stimulation electrode during thyroid surgery on 45 consecutive patients (78 nerves at risk) stratified to low- and high-risk groups. The signal analysis was performed as real-time audio/video feedback by the use of a new multichannel EMG system.No complications were attributable to the use of the anchor electrode. The mean delay to place the anchor electrode was 1.45 min, whereas the mean stimulation time of the vagus nerve was 38 min. Stable and repeatable signals were evocable in all cases with one exception. No permanent RLN paralyses occurred in this study.The vagal anchor electrode is safe and easy to use. It allows continuous neuromonitoring without any threats. The new technique will provide more security, especially during preparation steps on the RLN that are difficult for the surgeon.

Introduction Endometrial stromal tumors (EST) are the second most prevalent uterine sarcomas. EST are divided into three histosubtypes: benign endometrial stromal nodules (ESN), low-grade endometrial stromal sarcomas (ESS) and the most malignant undifferentiated stromal sarcomas (UES). Moreover, due to clinicopathological features intermediate between classical low-grade ESS and UES, some tumors have been described in the literature as “high-grade ESS”. The histological distinction between these three subtypes has important prognostic implications. The molecular biology of these tumors remains poorly understood because of their low incidence. Patients and Methods In the present study, we performed gene expression profiling of 22 EST using microarray, RNA-Seq and qRT-PCR techniques. Our cohort included 10 classical low-grade ESS (8 with ESS-associated gene fusions), 8 UES and 4 tumors which were classified as high-grade ESS based on the presence of YWHAE-FAM22A/B fusion and histological features. Results Using gene expression microrarrays, we have identified 81 entities differentially expressed in analyzed samples (fold change > 2, p < 0.005). Hierarchical clustering analysis revealed that high-grade ESS formed a separate sub-cluster with a distinct molecular profile as compared with the low-grade ESS and UES cases. Low-grade ESS cases formed 2 distinct sub-clusters, each containing 5 cases, one of which presented gene expression profile partially corresponding to high-grade ESS cases. GO analysis of genes strongly up-regulated in UES specimens showed over-representation of immune response (GO:0006955) and immune system process (GO:0002376) pathways (MARCO, MYO1F, FCN1, GPR183, HLA-DPB1, FCGR2B, NCF1, CCL18, CD28, PTAFR, CCL13, STK4, CD14, NCF4 and CTLA4 genes) (p < 0.05). UES cases were also characterized by significant down-regulation of WT1 tumor suppressor gene as compared to the low- and high-grade ESS. Moreover, both high-grade ESS and UES cases were characterized by significantly lower expression level of PEG3 gene, which has been reported as a tumor suppressor gene. All low-grade ESS cases presented significant over-expression of estrogen receptor ESR1 gene. Microarray results were validated by RNA-Seq and qRT-PCR experiments. Conclusions This study provides gene expression profiles that distinguish between EST subtypes and sheds light on the biology of these tumors. Our results point to immune system involvement in the pathogenesis of UES and support the notion that the current EST classification should discriminate high-grade ESS as a distinct subtype. Citation Format: Joanna Przybyl, Magdalena Kowalewska, Anna Quattrone, Barbara Dewaele, Vanessa Vanspauwen, Julio Finalet-Ferreiro, Michal Swierniak, Elwira Bakula-Zalewska, Janusz A. Siedlecki, Mariusz Bidzinski, Jan Cools, Maria Debiec-Rychter. Gene expression profiling distinguishes between endometrial stromal tumors subtypes. [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 4698. doi:10.1158/1538-7445.AM2014-4698

e22046 Background: EFTs are characterized by oncogenic EWSR1-FLI1 fusion gene, which can be detected in 85-90% of tumors and in 20-70% of peripheral blood specimens. It has been previously reported that downregulation of CDH2 (encoding N-cadherin) and overexpression of CDT2 [also known as DTL, encoding denticleless homolog (Drosophila)]in EFTs tumors are associated with poor prognosis. The aim of the study was to evaluate the expression levels of these markers in the circulating tumor cells and assess their utility as prognostic markers in EFTs. Methods: 10mL of PB was collected from 24 untreated adult EFTs patients at the diagnosis (period: 2009-2011, median age 30 years). 9 blood specimens from healthy individuals, and 5 untreated frozen EFTs tumor samples were used as controls. 8 EFTs patients presented metastatic disease (M1) at the diagnosis and 5 patients died during the follow-up period. Median follow-up was 22 months. Quantitative reverse transcription PCR (qRT-PCR) for CDH2 and CDT2 expression were performed in triplicate using the TaqMan Gene Expression Assays (Applied Biosystems). Mean expression level of 2 reference genes PSMC4 and EIF2B1 served as endogenous control. The fold change was calculated using ddCt method with pooled healthy individuals as calibrator. Results: At least 2-fold decrease (range 2.11-16.35) of CDH2 expression level in PB has been observed in 79% (n=19) of EFTs patients (p=0.04) as compared to healthy control. In general, CDH2 expression level in PB of EFTs patients was 3.08-fold lower compared with healthy individuals (p=0.07). All of the patients who had M1 at diagnosis (n=8) and who died during the follow-up (n=5) had CDH2 gene underexpression. CDT2 overexpression (FC range 3.28-6.97) has been observed in 17% (n=4) of EFTs patients (p=0.01). Three of these patients had M1 at diagnosis and two of these patients died during the follow-up. PB specimens presenting CDT2 overexpression clustered together with the tumor specimens. Conclusions: Abnormalities in the expression of CDH2 and CDT2 genes in PB specimens are associated with aggressive clinical course of EFTs. Diagnostic and prognostic significance of these biomarkers warrants further studies.

Introduction. Synovial sarcoma (SynSa) is an aggressive type of tumor, comprising approximately 10% of soft tissue sarcomas. Over half of SynSa patients develop metastases or local recurrence, but the molecular mechanism of aggressive SynSa outcome remains poorly characterized. Methods.Sixty five frozen tumor specimens from 54 SynSa patients [34 males; median age 37 years (range 5-86 years)] were subjected to aCGH and gene expression profiling. Examined set of tumor specimens included 16 primary tumors from untreated patients who did not develop metastasis/local recurrence (“SynSa1 group;" median follow-up 76 months), 26 primary tumors from untreated patients who developed metastases or local recurrence during follow-up (“SynSa2 group;" median follow-up 29 months), and 23 metastatic/recurrent SynSa tumors (“SynSa3 group;" median follow-up 46 months). For 8 patients primary tumor specimen was paired with metastatic/recurrent tumor specimens. Median follow-up for all patients was 40 months (range 3-218 months). Results. AURKA and KIF18A, which play important roles in various mitotic events, were the two most up-regulated genes in SynSa2 and SynSa3 groups compared to the SynSa1 group (corr p = 4.48e-6 and corr p = 8.65e-6, respectively). Moreover, we identified 125 genes differentially expressed between SynSa1 and SynSa2 groups (t test unpaired, corr p value cut-off = 0.05, fold change ≥ 2), 38 of which were common with recently published CINSARC (Complexity Index in Sarcomas) prognostic signature (Ref. 1, 2). Comparison of expression profiles between SynSa2 and SynSa3 groups did not show any significant differences. Analysis of genomic index (GI) based on aCGH profiles demonstrated that the SynSa1 group consisted of tumors with significantly less complex genomes compared to SynSa2 and SynSa3 groups (p = 0.021 and p = 0.020, respectively). The median GI in SynSa1, SynSa2 and SynSa3 groups was 9 (range 0-49), 16 (range 0-123) and 28 (range 0-130), respectively. Of note, there was no statistically significant difference in genome complexity between SynSa2 and SynSa3 groups, which suggests that progressive disease is not associated with accumulation of secondary genomic rearrangements/copy number changes. Conclusions. Primary SynSa tumors from patients who develop metastases or local recurrence share common molecular features with metastatic/recurrent SynSa. Presented data confirm that aggressive SynSa behavior is determined early in SynSa development and may be related to impaired mitotic regulatory mechanism. Citation Format: Joanna Przybyl, Raf Sciot, Agnieszka Wozniak, Patrick Schöffski, Vanessa Vanspauwen, Ignace Samson, Janusz A. Siedlecki, Piotr Rutkowski, Maria Debiec-Rychter. Metastatic potential is determined early in synovial sarcoma development and reflected by tumor molecular features. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 19. doi:10.1158/1538-7445.AM2013-19