The FUS::DDIT3 fusion oncoprotein inhibits BAF complex targeting and activity in myxoid liposarcoma.

Mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes play critical roles in governing genomic architecture and gene expression and are frequently perturbed in human cancers. Transcription factors (TFs), including fusion oncoproteins, can bind to BAF complex surfaces to direct chromatin targeting and accessibility, often activating oncogenic gene loci. Here, we demonstrate that the FUS::DDIT3 fusion oncoprotein hallmark to myxoid liposarcoma (MLPS) inhibits BAF complex-mediated remodeling of adipogenic enhancer sites via sequestration of the adipogenic TF, CEBPB, from the genome. In mesenchymal stem cells, small-molecule inhibition of BAF complex ATPase activity attenuates adipogenesis via failure of BAF-mediated DNA accessibility and gene activation at CEBPB target sites. BAF chromatin occupancy and gene expression profiles of FUS::DDIT3-expressing cell lines and primary tumors exhibit similarity to SMARCB1-deficient tumor types. These data present a mechanism by which a fusion oncoprotein generates a BAF complex loss-of-function phenotype, independent of deleterious subunit mutations.

Diverse clonal fates emerge upon drug treatment of homogeneous cancer cells.

Even among genetically identical cancer cells, resistance to therapy frequently emerges from a small subset of those cells1-7. Molecular differences in rare individual cells in the initial population enable certain cells to become resistant to therapy7-9; however, comparatively little is known about the variability in the resistance outcomes. Here we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing, to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically and functionally distinct resistant types. These resistant types are largely predetermined by molecular differences between cells before drug addition and not by extrinsic factors. Changes in the dose and type of drug can switch the resistant type of an initial cell, resulting in the generation and elimination of certain resistant types. Samples from patients show evidence for the existence of these resistant types in a clinical context. We observed diversity in resistant types across several single-cell-derived cancer cell lines and cell types treated with a variety of drugs. The diversity of resistant types as a result of the variability in intrinsic cell states may be a generic feature of responses to external cues.

Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Immunohistochemistry Can Be a Useful Ancillary Tool to Identify Perivascular Epithelioid Cell Tumor.

Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal tumors that express smooth muscle and melanocytic makers. Diagnosis of PEComas can be challenging due to focal or lost expression of traditional immunohistochemical markers, limited availability of molecular testing, and morphological overlap with much more common smooth muscle tumors. This study evaluates the use of glycoprotein nonmetastatic melanoma protein B (GPNMB) immunohistochemical staining as a surrogate marker for TSC1/2/MTOR alteration or TFE3 rearrangement to differentiate PEComas from other mesenchymal tumors. Cathepsin K was also assessed for comparison. A total of 399 tumors, including PEComas, alveolar soft part sarcomas, and other histologic PEComa mimics, were analyzed using GPNMB and cathepsin K immunohistochemistry. GPNMB expression was seen in all PEComas and alveolar soft part sarcomas with the majority showing diffuse and moderate-to-strong labeling, whereas other sarcomas were negative or showed focal labeling. When a cutoff of diffuse and at least moderate staining was used, GPNMB demonstrated 95% sensitivity and 97% specificity in distinguishing PEComas from leiomyosarcoma, well-differentiated/dedifferentiated liposarcomas, and undifferentiated pleomorphic sarcomas. Cathepsin K with a cutoff of any labeling had lower sensitivity (78%) and similar specificity (94%) to GPNMB. This study highlights GPNMB as a highly sensitive marker for PEComas and suggests its potential use as an ancillary tool within a panel of markers for accurate classification of these tumors.

Relationships between highly recurrent tumor suppressor alterations in 489 leiomyosarcomas.

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.

Enhancer reprogramming in PRC2-deficient malignant peripheral nerve sheath tumors induces a targetable de-differentiated state.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that frequently harbor genetic alterations in polycomb repressor complex 2 (PRC2) components-SUZ12 and EED. Here, we show that PRC2 loss confers a dedifferentiated early neural-crest phenotype which is exclusive to PRC2-mutant MPNSTs and not a feature of neurofibromas. Neural crest phenotype in PRC2 mutant MPNSTs was validated via cross-species comparative analysis using spontaneous and transgenic MPNST models. Systematic chromatin state profiling of the MPNST cells showed extensive epigenomic reprogramming or chromatin states associated with PRC2 loss and identified gains of active enhancer states/super-enhancers on early neural crest regulators in PRC2-mutant conditions around genomic loci that harbored repressed/poised states in PRC2-WT MPNST cells. Consistently, inverse correlation between H3K27me3 loss and H3K27Ac gain was noted in MPNSTs. Epigenetic editing experiments established functional roles for enhancer gains on DLX5-a key regulator of neural crest phenotype. Consistently, blockade of enhancer activity by bromodomain inhibitors specifically suppressed this neural crest phenotype and tumor burden in PRC2-mutant PDXs. Together, these findings reveal accumulation of dedifferentiated neural crest like state in PRC2-mutant MPNSTs that can be targeted by enhancer blockade.

Radiation-associated sarcomas other than malignant peripheral nerve sheath tumours demonstrate loss of histone H3K27 trimethylation†.

BACKGROUND AND AIMS: Complete loss of histone H3 lysine 27 trimethylation (H3K27me3) has recently emerged as a biomarker for malignant peripheral nerve sheath tumours (MPNST). Loss of H3K27me3 staining has also been reported in post-radiation MPNST; however, it has not been evaluated in a large series of radiation-associated sarcomas of different histological subtypes. The aim of this study was to assess H3K27me3 labelling by immunohistochemistry in radiation-associated sarcomas and to determine the prevalence of H3K27me3 loss in these tumours. METHODS AND RESULTS: Radiation-associated sarcomas (n = 119) from two tertiary care referral centres were evaluated for loss of H3K27me3, defined as complete loss of staining within tumour cells in the presence of a positive internal control. Twenty-three cases (19%) showed H3K27me3 loss, including nine of 10 (90%) MPNST, seven of 77 (9%) undifferentiated spindle cell/pleomorphic sarcomas, five of 25 (20%) angiosarcomas, one of five (20%) leiomyosarcomas and one of two (50%) osteosarcomas. CONCLUSIONS: Complete H3K27me3 loss was present in 19% of radiation-associated sarcomas in our series. Our findings demonstrate that loss of H3K27me3 is not specific for radiation-associated MPNST and may also occur in other histological subtypes of RAS, including radiation-associated undifferentiated spindle cell/pleomorphic sarcoma, angiosarcoma, leiomyosarcoma and osteosarcoma.

The degree of sclerosis is associated with prognosis in well-differentiated liposarcoma of the retroperitoneum.

BACKGROUND AND OBJECTIVES: Well-differentiated liposarcomas (WDL) are often partly composed of sclerotic tissue, however, the amount varies widely between tumors, and its prognostic significance is unknown. We hypothesized that tumors with more sclerosis would behave more aggressively. METHODS: Primary retroperitoneal WDL from 29 patients resected at our institution with follow-up were histologically evaluated by soft tissue pathologists blinded to outcome. Tumors with ≥ 10% sclerosis were designated "sclerotic" while tumors with < 10% sclerosis were designated as "minimally sclerotic". Cellular and dedifferentiated tumors were excluded. Clinical parameters and radiologic assessments on computed tomography (CT) were recorded. RESULTS: Histological evaluation identified 13 minimally sclerotic WDL and 16 sclerotic WDL. Median follow-up was 9 years (range, 3-20). Median recurrence-free survival (RFS) and median overall survival (OS) were 6.16 and 13.9 years, respectively. Compared with patients with sclerotic WDL, those with minimally sclerotic WDL had superior RFS (HR = 0.17 [95% CI, 0.06-0.53], P = .002) and OS (log-rank test, P = .002). Sclerotic WDL exhibited higher Houndsfield Units than minimally sclerotic WDL (26 vs 1, P = .040). CONCLUSIONS: Minimally sclerotic WDL were associated with more favorable outcome compared with sclerotic tumors. Assessment of sclerosis in WDL is likely a useful prognostic marker.

Clinicopathological analysis of ATRX, DAXX and NOTCH receptor expression in angiosarcomas.

AIMS: Multiple genetic alterations, including alternative lengthening of telomeres (ALT) and NOTCH mutations, have been described in angiosarcoma. Loss of α-thalassaemia/mental retardation syndrome X-linked (ATRX) and death domain-associated protein 6 (DAXX) expression is frequently associated with the ALT phenotype. Additionally, inhibition of NOTCH signalling induces the development of malignant vascular tumours in mice, indicating a tumour suppressive role of the NOTCH pathway in the pathogenesis of angiosarcoma. The aim of this study was to evaluate the immunohistochemical expression of ATRX, DAXX and NOTCH receptors (NOTCH1 and NOTCH2) in a large cohort of angiosarcomas, and study their clinicopathological and prognostic significance. METHODS AND RESULTS: One hundred and forty cases of angiosarcoma were stained for ATRX, DAXX, NOTCH1 and NOTCH2. ATRX loss (<10% labelling) was seen in seven of 118 (6%) cases, and was more frequent in deep soft tissue tumours than in other body sites (P = 0.004). Angiosarcomas with ATRX loss were associated with worse event-free survival than angiosarcomas with retained ATRX expression (P = 0.003). DAXX was retained in all specimens examined. Decreased NOTCH1 expression (≤1+ intensity) was seen in 29 of 123 (24%) cases, and was associated with a cutaneous site of origin (P = 0.013) and advanced disease (P = 0.026). NOTCH2 expression was decreased in 16 of 103 (16%) cases, was associated with visceral tumours (P = 0.001), and correlated with worse disease-specific survival (P = 0.033). CONCLUSIONS: ATRX, NOTCH1 and NOTCH2 expression varies in angiosarcomas and shows significant correlations with site of origin and poor clinical outcome, thus highlighting the biological heterogeneity within this tumour type.

EWSR1 fusion proteins mediate PAX7 expression in Ewing sarcoma.

PAX7 is a paired-box transcription factor that is required for the developmental specification of adult skeletal muscle progenitors in mice. We previously demonstrated PAX7 expression as a marker of skeletal muscle differentiation in rhabdomyosarcoma. Here, using analyses of published whole-genome gene expression microarray data, we identify PAX7 as a gene with significantly increased expression in Ewing sarcoma in comparison to CIC-DUX4 round cell sarcoma. Analysis of PAX7 in a large cohort of 103 Ewing sarcoma cases by immunohistochemistry revealed expression in 99.0% of cases (102/103). PAX7 expression was noted in cases demonstrating three distinct Ewing sarcoma EWSR1 translocations involving FLI1, ERG, and NFATc2. No PAX7 expression was observed in any of 27 cases of CIC-DUX4 sarcoma by immunohistochemistry (0%; 0/27). Exploring the mechanism of PAX7 expression in Ewing sarcoma using curated RNA- and ChIP-sequencing data, we demonstrate that the EWSR1 fusion protein is required for PAX7 expression in Ewing sarcoma and identify a candidate EWSR1-FLI1-bound PAX7 enhancer that coincides with both a consensus GGAA repeat-containing binding site and a peak of regulatory H3K27 acetylation. Taken together, our findings provide mechanistic support for the utility of PAX7 immunohistochemistry in the diagnosis of Ewing sarcoma, while linking this sarcoma of uncertain histogenesis to a key transcriptional regulator of mammalian muscle progenitor cells.

The role of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) in peripheral nerve sheath tumours.

AIMS: STAT3 is a pro-oncogenic transcription factor that requires phosphorylation for transcriptional activation. The aim of this study was to evaluate the role of phosphorylated STAT3 (pSTAT3) expression in neurofibromas, schwannomas, and malignant peripheral nerve sheath tumours (MPNSTs). METHODS AND RESULTS: Twenty-six neurofibromas, 62 schwannomas and 39 MPNSTs from a formalin-fixed paraffin-embedded tissue microarray were examined. Immunohistochemical analysis was performed with an anti-pSTAT3 (Tyr705) antibody. Nuclear expression was reviewed for both intensity and percentage of tumoral labelling. Distributions of disease-specific overall survival (DSOS) and event-free survival (EFS) were estimated with the Kaplan-Meier method, and compared between two pSTAT3 groups by use of the log-rank test. MPNSTs had higher median tumoral labelling than neurofibromas (P = 0.0012) or schwannomas (P = 0.0008). Moderate to strong pSTAT3 expression (defined as at least moderate labelling in ≥50% of cells) was found more frequently in MPNSTs than in neurofibromas (P = 0.026). Among MPNSTs, pSTAT3 expression differed between primary, recurrent and metastatic disease (P = 0.063 with increased expression in recurrent and metastatic cases). pSTAT3 expression (at least moderate labelling in ≥10% of cells) in primary MPNSTs was associated with worse DSOS (P = 0.048) and trended towards being associated with worse EFS (P = 0.063). Paired specimens revealed no increase in pSTAT3 expression in the recurrences or metastases relative to the primary tumour, suggesting that pSTAT3 expression may be an early indicator of aggressive disease at disease onset. CONCLUSIONS: pSTAT3 is expressed in a higher proportion of MPNSTs than neurofibromas and schwannomas. Moderate to strong pSTAT3 expression in ≥10% of cells was found to be a negative prognostic factor for DSOS among primary MPNSTs, suggesting a role of pSTAT3 in the progression of these tumours.

Index report of cutaneous angiosarcomas with strong positivity for tyrosinase mimicking melanoma with further evaluation of melanocytic markers in a large angiosarcoma series.

Cutaneous angiosarcoma can be challenging to diagnose particularly when poorly vasoformative and studied on biopsies. We report a case of a cutaneous angiosarcoma with strong positivity for tyrosinase, the first to our knowledge, initially misdiagnosed as melanoma. We subsequently evaluated the reactivity of panmelanocytic cocktail (tyrosinase, HMB-45 and Melan-A), SOX10, tyrosinase and MITF in a large tissue microarray (TMA) of angiosarcoma. The TMA included 142 cases of angiosarcomas (29 cutaneous, 22 primary breast, 41 post-radiation breast, 15 visceral, 26 deep soft tissue and bone, 5 chronic lymphedema-associated and 4 angiosarcomas arising in other sarcomas). Immunohistochemical studies were performed with anti-panmelanocytic cocktail, anti-SOX10, anti-MITF and anti-tyrosinase antibodies. TMA staining results were scored on intensity and percentage of tumoral labeling. Aside from the index case, no cases (0 of 133) showed positivity for tyrosinase including 28 cutaneous angiosarcomas. One breast angiosarcoma (1 of 131) was positive for MITF. All cases were negative for SOX10 and panmelanocytic cocktail (0 of 132). Angiosarcomas can rarely be positive for tyrosinase and MITF. Pathologists should be cognizant of these rare exceptions to prevent confusion with melanoma. Additional immunohistochemical markers for vascular and melanocytic differentiation, thorough histological examination for vasoformative and in situ areas as well as clinical impression are helpful in these exceptionally problematic cases.

Establishment and characterization of a new human myxoid liposarcoma cell line (DL-221) with the FUS-DDIT3 translocation.

Myxoid liposarcoma has the pathognomonic fusion oncogene FUS-DDIT3 encoding a chimeric transcription factor. Metastatic risk is higher with an increased round cell component and has been linked to aberrations involving the IGFR/PI3K/AKT pathway. These molecular insights have yet to translate to targeted therapies, and the lack of experimental models is a major hindrance. We describe the initial in-depth characterization of a new cell line (DL-221) and establishment of a mouse xenograft model. The cell line DL-221 was derived from a metastatic pleural lesion showing myxoid and round cell histology. This newly established cell line was characterized for phenotypic properties and molecular cytogenetic profile, using PCR, COBRA-FISH, and western blot. Next-generation whole-exome sequencing was performed to further characterize the cell line and the parent tumor. NOD-SCID-IL2R gamma knockout mice were xenograft hosts. DL-221 cells grew an adhering monolayer and COBRA-FISH showed an aneuploid karyotype with t(12;16)(q13;p11) and several other rearrangements; RT-PCR demonstrated a FUS-DDIT3 fusion transcript type 1. Both the cell line and the original tumor harbored a TP53 compound heterozygous mutation in exon 4 and 7, and were wild-type for PIK3CA. Moreover, among the 1254 variants called by whole-exome sequencing, there was 77% concordance between the cell line and parent tumor. The recently described hotspot mutation in the TERT promoter region in myxoid liposarcomas was also found at C228T in DL-221. Xenografts suitable for additional preclinical studies were successfully established in mice after subcutaneous injection. The established DL-221 cell line is the only published available myxoid liposarcoma cell line that underwent spontaneous immortalization, without requiring SV40 transformation. The cell line and its xenograft model are unique and helpful tools to study the biology and novel potential-targeted treatment approaches for myxoid liposarcoma.

Loss of H3K27 tri-methylation is a diagnostic marker for malignant peripheral nerve sheath tumors and an indicator for an inferior survival.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that can show overlapping features with benign neurofibromas as well as high-grade sarcomas. Additional diagnostic markers are needed to aid in this often challenging differential diagnosis. Recently mutations in two critical components of the polycomb repressor 2 (PRC2) complex, SUZ12 and EED, were reported to occur specifically in MPNSTs while such mutations are absent in neurofibromas, both in the setting of neurofibromatosis (NF) and sporadic cases. Furthermore, both SUZ12 and EED mutations in MPNSTs were associated with loss of H3K27 tri-methylation, a downstream target of PRC2. Therefore, we tested whether H3K27me3 immunohistochemistry is useful as a diagnostic and prognostic marker for MPNSTs. We performed H3K27me3 immunohistochemistry in 162 primary MPNSTs, 97 neurofibromas and 341 other tumors using tissue microarray. We observed loss of H3K27me3 in 34% (55/162) of all MPNSTs while expression was retained in all neurofibromas including atypical (n=8) and plexiform subtypes (n=24). Within other tumors we detected loss of H3K27me3 in only 7% (24/341). Surprisingly, 60% (9/15) of synovial sarcomas and 38% (3/8) of fibrosarcomatous dermatofibrosarcoma protuberans (DFSP) showed loss of H3K27 trimethylation. Only 1 out of 44 schwannomas showed loss of H3K27me3 and all 4 perineuriomas showed intact H3K27me3. Furthermore, MPNSTs with loss of H3K27 tri-methylation showed inferior survival compared with MPNSTs with intact H3K27 tri-methylation, which was validated in two independent cohorts. Our results indicate that H3K27me3 immunohistochemistry is useful as a diagnostic marker, in which loss of H3K27me3 favors MPNST above neurofibroma. However, H3K27me3 immunohistochemistry is not suitable to distinguish MPNST from its morphological mimicker synovial sarcoma or fibrosarcomatous DFSP. Since loss of H3K27 tri-methylation was related to poorer survival in MPNST, chromatin modification mediated by this specific histone seems to orchestrate more aggressive tumour biology.

Analysis of Clinical and Molecular Factors Impacting Oncologic Outcomes in Undifferentiated Pleomorphic Sarcoma.

BACKGROUND: Undifferentiated pleomorphic sarcomas (UPS) present a diagnostic and therapeutic challenge. Identification of prognostic molecular markers is required for the discovery of novel treatment approaches. The purpose of this study was to correlate clinicopathologic variables, expression of tyrosine kinase receptors, and markers of cell cycle progression and survival with oncologic outcomes. METHODS: A tissue microarray containing 208 primary UPS samples was analyzed by immunohistochemistry for protein markers and in situ hybridization for microRNA. Staining results were correlated with clinicopathologic features and oncologic outcomes. Univariate and multivariate analyses were conducted to assess associations between expression of protein markers, mi-RNA, and outcome. RESULTS: At a median follow-up of 3.9 years (9 years for survivors), 5-year disease-specific survival (DSS) was 63 %. Clinical variables associated with improved DSS included age <61 years, tumor size <10 cm, margin-negative resection, and sporadic-tumor status. At the protein level, loss of cyclin D1 (p = 0.06), pEGFR (p = 0.023), pIGF-1R (p = 0.022), and PTEN (p < 0.001) and overexpression of AXL (p = 0.015) were associated with reduced DSS on univariate analysis. Ki67, PCNA, and pEGFR were more highly expressed in sporadic UPS than radiation-associated (RA-UPS), whereas RA-UPS samples expressed higher levels of both phosphorylated and total IGF-1R. DISCUSSION: Loss of cyclin D1, overexpression of AXL, and loss of PTEN are associated with poor cancer-specific outcomes and warrant further investigation in UPS. The differences in protein expression in sporadic versus RA-UPS may indicate that the activated molecular signaling nodes may be different for each specific histology and also could explain the aggressive phenotype seen in RA-UPS compared with the sporadic lesions.

Antitumor effects of pharmacological EZH2 inhibition on malignant peripheral nerve sheath tumor through the miR-30a and KPNB1 pathway.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2) is a key epigenetic regulator in cancer cell survival, epithelial-mesenchymal transition, and tumorigenesis. Inhibition of EZH2 has become a promising therapeutic option for various human malignancies. Previously, we demonstrated that the EZH2/miR-30d/karyopherin (importin) beta 1 (KPNB1) signaling pathway is critical for malignant peripheral nerve sheath tumor (MPNST) cell survival in vitro and for tumorigenesis in vivo. Here, we sought to determine the antitumor effects of pharmacological inhibition of EZH2 on MPNST in vitro and in vivo. METHODS: We investigated the effects of an EZH2 inhibitor, 3-deazaneplanocin A (DZNep), on MPNST cell cycle, survival and apoptosis in vitro and on MPNST xenograft tumor growth in vivo. RESULTS: We found that DZNep treatment impaired MPNST cell viability and proliferation by inducing apoptosis and cell cycle arrest in vitro. Consistently, DZNep treatment also reduced EZH2 and KPNB1 protein levels and upregulated miR-30d expression in MPNST cells. Intraperitoneal administration of DZNep significantly suppressed MPNST tumor initiation and growth rates in a MPNST xenograft mouse model. Immunoblot and immunohistochemical analyses showed that DZNep downregulated EZH2/KPNB1 signaling in vivo, thereby inhibiting MPNST tumor cell proliferation, and induced cell death. We also found that EZH2 inhibited expression of another miR-30 family member, miR-30a, in MPNST cells. Similar to miR-30d, miR-30a inhibited KPNB1 by targeting the KPNB1 3' untranslated region in MPNST cells. Our data also showed that EZH2 suppressed miR-200b expression and induced epithelial-mesenchymal transition in MPNST cells. CONCLUSION: These findings demonstrated that DZNep, an inhibitor of S-adenosyl-methionine-dependent methyltransferase, suppressed EZH2/miR-30a,d/KPNB1 signaling and blocked MPNST tumor cell growth and survival in vitro and in vivo. More importantly, our study indicated that pharmacological interference of EZH2 is a potential therapeutic approach for MPNST.

NY-ESO-1 (CTAG1B) expression in mesenchymal tumors.

New York esophageal squamous cell carcinoma 1 (NY-ESO-1, CTAG1B) is a cancer-testis antigen and currently a focus of several targeted immunotherapeutic strategies. We performed a large-scale immunohistochemical expression study of NY-ESO-1 using tissue microarrays of mesenchymal tumors from three institutions in an international collaboration. A total of 1132 intermediate and malignant and 175 benign mesenchymal lesions were enrolled in this study. Immunohistochemical staining was performed on tissue microarrays using a monoclonal antibody for NY-ESO-1. Among mesenchymal tumors, myxoid liposarcomas showed the highest positivity for NY-ESO-1 (88%), followed by synovial sarcomas (49%), myxofibrosarcomas (35%), and conventional chondrosarcomas (28%). Positivity of NY-ESO-1 in the remaining mesenchymal tumors was consistently low, and no immunoreactivity was observed in benign mesenchymal lesions. On the basis of these findings, nearly 90% of myxoid liposarcomas, as well as a significant proportion of synovial sarcomas, myxofibrosarcomas, and conventional chondrosarcomas are good candidates for immunotherapy targeting NY-ESO-1.

AXL is a potential therapeutic target in dedifferentiated and pleomorphic liposarcomas.

BACKGROUND: AXL is a well-characterized, protumorigenic receptor tyrosine kinase that is highly expressed and activated in numerous human carcinomas and sarcomas, including aggressive subtypes of liposarcoma. However, the role of AXL in the pathogenesis of well-differentiated (WDLPS), dedifferentiated (DDLPS), and pleomorphic liposarcoma (PLS) has not yet been determined. METHODS: Immunohistochemical analysis of AXL expression was conducted on two tissue microarrays containing patient WDLPS, DDLPS, and PLS samples. A panel of DDLPS and PLS cell lines were interrogated via western blot for AXL expression and activity and by ELISA for growth arrest-specific 6 (GAS6) production. AXL knockdown was achieved by siRNA or shRNA. The effects of AXL knockdown on cell proliferation, migration, and invasion were measured in vitro. In addition, AXL shRNA-containing DDLPS cells were assessed for their tumor-forming capacity in vivo. RESULTS: In this study, we determined that AXL is expressed in a subset of WDLPS, DDLPS, and PLS patient tumor samples. In addition, AXL and its ligand GAS6 are expressed in a panel of DDLPS and PLS cell lines. We show that the in vitro activation of AXL via stimulation with exogenous GAS6 resulted in a significant increase in cell proliferation, migration, and invasion in DDLPS and PLS cell lines. Transient knockdown of AXL resulted in attenuation of these protumorigenic phenotypes in vitro. Stable AXL knockdown not only decreased migratory and invasive characteristics of DDLPS and PLS cells in vitro but also significantly diminished tumorigenicity of two dedifferentiated liposarcoma xenograft models in vivo. CONCLUSIONS: Our results suggest that AXL signaling contributes to the aggressiveness of DDLPS and PLS, and that AXL is therefore a potential therapeutic target for treatment of these rare, yet devastating tumors.

Estrogen receptor alpha and androgen receptor are commonly expressed in well-differentiated liposarcoma.

BACKGROUND: Liposarcoma (LS) is the second-most common type of soft-tissue sarcoma. Despite advances in knowledge and treatment of this disease, there remains a need for more effective LS therapy. Steroid hormone receptors regulate metabolism in adipocytes. Estrogen receptor alpha (ER), progesterone receptor (PR), and androgen receptor (AR) have been implicated in the pathophysiology of other cancer types. We sought to comprehensively determine temporal expression patterns of these receptors in LS. METHODS: We analyzed 561 histologically subtyped LS specimens from 354 patients for expression of ER, PR, and AR by immunohistochemistry (IHC) using diagnostic-grade reagents and protocols. The fractions of positively stained tumor cells were scored within each specimen. IHC scores were compared across LS subtypes using the Kruskal-Wallis test, and subtypes were compared using Dunn's post-hoc test. Ages of patients with receptor-positive vs. -negative LS were compared by t-test. Genders and races were compared for hormone receptor positivity using Fisher's exact test and Chi-square analysis, respectively. Recurrence-free survival was compared between receptor-positive and negative patients by log-rank test. p< 0.05 was considered significant. RESULTS: ER and AR were frequently expressed in LS, while few tumors expressed PR. Most of the ER + and AR + samples were of the well-differentiated LS subtype. A smaller fraction of de-differentiated LS expressed ER or AR, but expression was common within well-differentiated regions of tumors histologically classified as de-differentiated LS. In LS specimens from patients who underwent multiple surgeries over time, receptor expression frequently changed over time, which may be attributable in part to intratumor heterogeneity, varying degrees of de-differentiation, and biopsy bias. ER and AR were frequently co-expressed. Receptor status was not significantly associated with gender or race, but AR and PR expression were associated with earlier age at diagnosis. Receptor expression was not associated with altered recurrence-free survival. CONCLUSIONS: ER and AR are commonly expressed in LS, particularly in well-differentiated tumors. These data warrant further functional study to determine receptor function in LS, and the potential efficacy of anti-hormone therapies for the treatment of patients with LS.

Loss of the DNA Repair Gene RNase H2 Identifies a Unique Subset of DDR-Deficient Leiomyosarcomas.

Targeting the DNA damage response (DDR) pathway is an emerging therapeutic approach for leiomyosarcoma (LMS), and loss of RNase H2, a DDR pathway member, is a potentially actionable alteration for DDR-targeted treatments. Therefore, we designed a protein- and genomic-based RNase H2 screening assay to determine its prevalence and prognostic significance. Using a selective RNase H2 antibody on a pan-tumor microarray (TMA), RNase H2 loss was more common in LMS (11.5%, 9/78) than across all tumors (3.8%, 32/843). In a separate LMS cohort, RNase H2 deficiency was confirmed in uterine LMS (U-LMS, 21%, 23/108) and soft-tissue LMS (ST-LMS; 30%, 39/102). In the TCGA database, RNASEH2B homozygous deletions (HomDels) were found in 6% (5/80) of LMS cases, with a higher proportion in U-LMS (15%; 4/27) compared with ST-LMS (2%; 1/53). Using the SNiPDx targeted-NGS sequencing assay to detect biallelic loss of function in select DDR-related genes, we found RNASEH2B HomDels in 54% (19/35) of U-LMS cases with RNase H2 loss by IHC, and 7% (3/43) HomDels in RNase H2 intact cases. No RNASEH2B HomDels were detected in ST-LMS. In U-LMS patient cohort (n = 109), no significant overall survival difference was seen in patients with RNase H2 loss versus intact, or RNASEH2B HomDel (n = 12) versus Non-HomDel (n = 37). The overall diagnostic accuracy, sensitivity, and specificity of RNase H2 IHC for detecting RNA-SEH2B HomDels in U-LMS was 76%, 93%, and 71%, respectively, and it is being developed for future predictive biomarker driven clinical trials targeting DDR in U-LMS.