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Myoepithelial (ME) tumors of soft tissue and bone display a heterogeneous histologic spectrum and in about half of the cases harbor EWSR1 gene rearrangements. Despite rare case reports, the prevalence of fused in sarcoma (FUS) gene abnormalities and its related fusion partners remains undetermined among ME tumors. Therefore, we screened 66 EWSR1-negative ME tumors for FUS abnormalities by fluorescence in situ hybridization (FISH). In an index FUS-rearranged case, 3'-rapid amplification of cDNA ends (RACE) was applied to identify the fusion partner. Results were further confirmed by reverse transcription-PCR, followed by FISH screening the entire cohort of FUS-rearranged and EWSR1-positive ME lesions lacking a known fusion partner. The correlation between genotype and clinicopathological features was also investigated. As a result, six (9%) FUS-rearranged cases were identified, spanning divergent age groups, tumor locations, and morphologic features. A novel FUS-KLF17 fusion was identified by 3'-RACE in an 11-year-old girl with a foot lesion associated with locoregional metastases. Three additional cases with FUS-KLF17 fusions were identified and one KLF17 rearrangement (6.3%) was found among the 16 EWSR1-positive cases tested. The KLF17-related ME tumors affected younger patients and often exhibited trabecular growth in a myxohyaline stroma, but this genotype did not correlate with a malignant phenotype. In conclusion, a small subset of ME tumors harbor FUS rearrangements, two thirds of them being associated with KLF17 fusion. FUS FISH analysis is recommended in EWSR1-negative lesions in which a ME diagnosis is suspected. KLF17 is also a rare gene fusion partner to EWSR1-rearranged ME tumors., (© 2015 Wiley Periodicals, Inc.)

The genetics of myoepithelial tumors (ME) of soft tissue and bone have recently been investigated, with EWSR1-related gene fusions being seen in approximately half of the tumors. The fusion partners of EWSR1 so far described include POU5F1, PBX1, ZNF444 and, in a rare case, ATF1. We investigated by RNA sequencing an index case of EWSR1-rearranged ME of the tibia, lacking a known fusion partner, and identified a novel EWSR1-PBX3 fusion. The fusion was further validated by reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization (FISH). To evaluate if this is a recurrent event, an additional cohort of 22 EWSR1-rearranged ME cases lacking a fusion partner were screened by FISH for abnormalities in PBX3 gene. Thus, two additional cases were identified showing an EWSR1-PBX3 gene fusion. One of them was also intraosseous involving the ankle, while the other occurred in the soft tissue of the index finger. The morphology of the EWSR1-PBX3 fusion positive cases showed similar findings, with nests or sheets of epithelioid to spindle cells in a partially myxoid to collagenous matrix. All three cases showed expression of S100 and EMA by immunohistochemistry. In summary, we report a novel EWSR1-PBX3 gene fusion in a small subset of ME, thereby expanding the spectrum of EWSR1-related gene fusions seen in these tumors. This gene fusion seems to occur preferentially in skeletal ME, with two of the three study cases occurring in intraosseous locations., (© 2014 Wiley Periodicals, Inc.)

Sclerosing epithelioid fibrosarcoma (SEF) is a rare soft tissue tumor exhibiting considerable morphologic overlap with low-grade fibromyxoid sarcoma (LGFMS). Moreover, both SEF and LGFMS show MUC4 expression by immunohistochemistry. While the majority of LGFMS cases are characterized by a FUS-CREB3L1 fusion, both FUS-CREB3L2 and EWSR1-CREB3L1 fusions were recently demonstrated in a small number of LGFMS and SEF/LGFMS hybrid tumors. In contrast, recent studies pointed out that SEF harbor frequent EWSR1 rearrangements, with only a minority of cases showing FUS-CREB3L2 fusions. In an effort to further characterize the molecular characteristics of pure SEF and hybrid SEF/LGFMS lesions, we undertook a clinicopathologic, immunohistochemical and genetic analysis of a series of 10 SEF and 8 hybrid SEF/LGFMS tumors. The mortality rate was similar between the two groups, 44% within the pure SEF group and 37% in the hybrid SEF/LGFMS with a mean overall follow-up of 66 months. All but one pure SEF and all hybrid SEF/LGFMS-tested cases showed MUC4 immunoreactivity. The majority (90%) of pure SEF cases showed EWSR1 gene rearrangements by fluorescence in situ hybridization with only one case exhibiting FUS rearrangement. Of the nine EWSR1 positive cases, six cases harbored CREB3L1 break-apart, two had CREB3L2 rearrangement (a previously unreported finding) and one lacked evidence of CREB3L1/2 abnormalities. In contrast, all hybrid SEF/LGFMS tumors exhibited FUS and CREB3L2 rearrangements. These results further demarcate a relative cytogenetic dichotomy between pure SEF, often characterized by EWSR1 rearrangements, and hybrid SEF/LGFMS, harboring FUS-CREB3L2 fusion; the latter group recapitulating the genotype of LGFMS., (© 2014 Wiley Periodicals, Inc.)

Epithelioid hemangioma (EH) is a benign neoplasm with distinctive vasoformative features, which occasionally shows increased cellularity, cytologic atypia, and/or loco-regional aggressive growth, resulting in challenging differential diagnosis from malignant vascular neoplasms. Based on two intraosseous EH index cases with worrisome histologic features, such as the presence of necrosis, RNA sequencing was applied for possible fusion gene discovery and potential subclassification of a novel atypical EH subset. A ZFP36-FOSB fusion was detected in one case, while a WWTR1-FOSB chimeric transcript in the other, both were further validated by fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR). These abnormalities were then screened by FISH in 44 EH from different locations with seven additional EH revealing FOSB gene rearrangements, all except one being fused to ZFP36. Interestingly, 4/6 penile EH studied showed FOSB abnormalities. Although certain atypical histologic features were observed in the FOSB-rearranged EH, including solid growth, increased cellularity, mild to moderate nuclear pleomorphism, and necrosis in 3/9 cases, no overt sarcomatous areas were discerned to objectively separate the lesions from the fusion-negative EH. No patient has developed recurrence to date, but the follow-up was relatively limited and short to draw definitive conclusions regarding behavior. Although FOSB-rearranged EH do not show significant morphologic overlap with SERPINE1-FOSB fusion-positive pseudomyogenic hemangioendothelioma, FOSB oncogenic activation is emerging as an important event in these benign and intermediate groups of vascular tumors., (© 2014 Wiley Periodicals, Inc.)

PHF1 gene rearrangements have been recently described in around 50% of ossifying fibromyxoid tumors (OFMT) including benign and malignant cases, with a small subset showing EP400-PHF1 fusions. In the remaining cases no alternative gene fusions have been identified. PHF1-negative OFMT, especially if lacking S100 protein staining or peripheral ossification, are difficult to diagnose and distinguish from other soft tissue mimics. In seeking more comprehensive molecular characterization, we investigated a large cohort of 39 OFMT of various anatomic sites, immunoprofiles and grades of malignancy. Tumors were screened for PHF1 and EP400 rearrangements by FISH. RNA sequencing was performed in two index cases (OFMT1, OFMT3), negative for EP400-PHF1 fusions, followed by FusionSeq data analysis, a modular computational tool developed to discover gene fusions from paired-end RNA-seq data. Two novel fusions were identified ZC3H7B-BCOR in OFMT1 and MEAF6-PHF1 in OFMT3. After being validated by FISH and RT-PCR, these abnormalities were screened on the remaining cases. With these additional gene fusions, 33/39 (85%) of OFMTs demonstrated recurrent gene rearrangements, which can be used as molecular markers in challenging cases. The most common abnormality is PHF1 gene rearrangement (80%), being present in benign, atypical and malignant lesions, with fusion to EP400 in 44% of cases. ZC3H7B-BCOR and MEAF6-PHF1 fusions occurred predominantly in S100 protein-negative and malignant OFMT. As similar gene fusions were reported in endometrial stromal sarcomas, we screened for potential gene abnormalities in JAZF1 and EPC1 by FISH and found two additional cases with EPC1-PHF1 fusions., (Copyright © 2013 Wiley Periodicals, Inc.)

Glomus tumors (GT) have been classified among tumors of perivascular smooth muscle differentiation, together with myopericytoma, myofibroma/tosis, and angioleiomyoma, based on their morphologic overlap. However, no molecular studies have been carried out to date to investigate their genetic phenotype and to confirm their shared pathogenesis. RNA sequencing was performed in three index cases (GT1, malignant GT; GT2, benign GT and M1, multifocal myopericytoma), followed by FusionSeq data analysis, a modular computational tool developed to discover gene fusions from paired-end RNA-seq data. A gene fusion involving MIR143 in band 5q32 was identified in both GTs with either NOTCH2 in 1p13 in GT1 or NOTCH1 in 9q34 in GT2, but none in M1. After being validated by FISH and RT-PCR, these abnormalities were screened on 33 GTs, 6 myopericytomas, 9 myofibroma/toses, 18 angioleiomyomas and in a control group of 5 sino-nasal hemangiopericytomas. Overall NOTCH2 gene rearrangements were identified in 52% of GT, including all malignant cases and one NF1-related GT. No additional cases showed NOTCH1 rearrangement. As NOTCH3 shares similar functions with NOTCH2 in regulating vascular smooth muscle development, the study group was also investigated for abnormalities in this gene by FISH. Indeed, NOTCH3 rearrangements were identified in 9% of GTs, all present in benign soft tissue GT, one case being fused to MIR143. Only 1/18 angioleiomyomas showed NOTCH2 gene rearrangement, while all the myopericytomas and myofibroma/toses were negative. In summary, we describe novel NOTCH1-3 rearrangements in benign and malignant, visceral, and soft tissue GTs., (Copyright © 2013 Wiley Periodicals, Inc.)

Conventional epithelioid hemangioendotheliomas (EHE) have a distinctive morphologic appearance and are characterized by a recurrent t(1;3) translocation, resulting in a WWTR1-CAMTA1 fusion gene. We have recently encountered a fusion-negative subset characterized by a somewhat different morphology, including focally well-formed vasoformative features, which was further investigated for recurrent genetic abnormalities. Based on a case showing strong transcription factor E3 (TFE3) immunoreactivity, fluorescence in situ hybridization (FISH) analysis for TFE3 gene rearrangement was applied to the index case as well as to nine additional cases, selected through negative WWTR1-CAMTA1 screening. A control group, including 18 epithelioid hemangiomas, nine pseudomyogenic HE, and three epithelioid angiosarcomas, was also tested. TFE3 gene rearrangement was identified in 10 patients, with equal gender distribution and a mean age of 30 years old. The lesions were located in somatic soft tissue in six cases, lung in three and one in bone. One case with available frozen tissue was tested by RNA sequencing and FusionSeq data analysis to detect novel fusions. A YAP1-TFE3 fusion was thus detected, which was further validated by FISH and reverse transcription polymerase chain reaction (RT-PCR). YAP1 gene rearrangements were then confirmed in seven of the remaining nine TFE3-rearranged EHEs by FISH. No TFE3 structural abnormalities were detected in any of the controls. The TFE3-rearranged EHEs showed similar morphologic features with at least focally, well-formed vascular channels, in addition to a variably solid architecture. All tumors expressed endothelial markers, as well as strong nuclear TFE3. In summary, we are reporting a novel subset of EHE occurring in young adults, showing a distinct phenotype and YAP1-TFE3 fusions., (Copyright © 2013 Wiley Periodicals, Inc.)