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Embryonal rhabdomyosarcoma (ERMS) is the most common subtype of RMS, occurring in soft tissue and visceral sites of young children, and is associated with favorable outcomes. A subset occurs in mucosal-lined luminal structures, displaying a unique grape-like growth termed as “botryoid-type.” To further delineate the differences between conventional (cERMS) and botryoid-type (bERMS) RMS, we performed a comparative histologic review and comprehensive molecular profiling of 48 cases (25 bERMS and 23 cERMS). All tumors were subjected to a hybridization capture-based targeted matched tumor-normal DNA NGS assay. The mean age was 17 and 7 years for bERMS and cERMS, respectively. Most bERMS were female with a predilection for the gynecologic tract (75%), while cERMS had a slight male predominance and were preferentially located in abdominopelvic and paratesticular sites (30%, each). All bERMS exhibited an exophytic, bulbous architecture accompanied by a subepithelial “cambium layer.” Distinctive germline alterations were detected, with DICER1(18%) and FH(6%) mutations only in bERMS, and rare TP53, VHL, and APCmutations in cERMS. Similarly, contrasting somatic genomic landscapes were observed, with frequent DICER1(52%, P**<0.0001) and TP53(36%, P*<0.05) alterations exclusively in bERMS. Cartilaginous differentiation was only observed in DICER1-mutated bERMS. All patients had longitudinal follow-up. bERMS patients with somatic/germline DICER1mutations showed significantly improved recurrence-free survival compared with that of DICER1-wild type patients (P*<0.05). Moreover, bERMS showed improved disease-specific survival compared with that of cERMS, with 8% versus 30% (P*<0.05) dead of disease, respectively. In summary, we compare the molecular underpinnings of the largest cohort of bERMS and cERMS with targeted DNA sequencing and long-term follow-up data. Our findings reveal divergent genomic topographies between the 2 groups, with bERMS showing unique germline and somatic abnormalities, including enrichment in DICER1and TP53alterations, and a trend towards improved survival.

Since their original description as a distinctive neoplastic entity, ~50 TFE3-rearranged perivascular epithelioid cell tumors (PEComas) have been reported. We herein report 25 new TFE3-rearranged PEComas and review the published literature to further investigate their clinicopathologic spectrum. Notably, 5 of the 25 cases were associated with a prior history of chemotherapy treatment for cancer. This is in keeping with prior reports, based mainly on small case series, with overall 11% of TFE3-rearranged PEComas being diagnosed postchemotherapy. The median age of our cohort was 38 years. Most neoplasms demonstrated characteristic features such as nested architecture, epithelioid cytology, HMB45 positive, and muscle marker negative immunophenotype. SFPQwas the most common TFE3fusion partner present in half of the cases, followed by ASPSCR1and NONOgenes. Four of 7 cases in our cohort with meaningful follow-up presented with or developed systemic metastasis, while over half of the reported cases either recurred locally, metastasized, or caused patient death. Follow-up for the remaining cases was limited (median 18.5 months), suggesting that the prognosis may be worse. Size, mitotic activity, and necrosis were correlated with aggressive behavior. There is little evidence that treatment with MTOR inhibitors, which are beneficial against TSC-mutated PEComas, is effective against TFE3-rearranged PEComas: only one of 6 reported cases demonstrated disease stabilization. As co-expression of melanocytic and muscle markers, a hallmark of conventional TSC-mutated PEComa is uncommon in the spectrum of TFE3-rearranged PEComa, an alternative terminology may be more appropriate, such as “TFE3-rearranged PEComa-like neoplasms,” highlighting their distinctive morphologic features and therapeutic implications.

Ossifying fibromyxoid tumors (OFMTs) are rare mesenchymal neoplasms which typically present in the superficial subcutaneous tissues and have not been reported to arise in visceral organs. We now report 4 molecularly confirmed cases of OFMT involving the genitourinary tract. All patients were males, ranging in age from 20 to 66 years (mean: 43 y). One case each arose in the kidney, ureter, perirenal soft tissue, and penis. All neoplasms demonstrated bland epithelioid to spindled cells set in a variably fibrous to fibromyxoid stroma, and only 1 had a peripheral shell of lamellar bone. All cases appeared well-circumscribed on gross/radiologic examination, though the primary renal neoplasm permeated between native renal tubules. By immunohistochemistry, S100 protein was negative in all 4 cases, while desmin was positive in 2 cases. In 2 cases, the Illumina TruSight RNA Fusion Panel demonstrated a PHF1::TFE3and EP400::PHF1fusion, respectively. In the remaining 2 cases, PHF1gene rearrangement was confirmed by fluorescence in situ hybridization analysis. Due to unusual clinical presentation, lack of S100 positivity, and only occasional bone formation, the correct diagnosis was challenging in the absence of molecular testing. In summary, OFMT may rarely present primarily in the genitourinary tract. Given their nonspecific morphology and immunophenotype, molecular analysis is crucial to establish the correct diagnosis.

Despite a wide spectrum of clinical presentations, including primary or secondary, most angiosarcomas are considered high grade. One exception is primary breast angiosarcoma, where historically, histologic grading has shown to predict outcome using the Rosen 3-tier system. However, more recent studies have challenged this concept suggesting that even in this specific clinical context angiosarcomas should be considered high grade. This study aimed to critically reevaluate the impact of histologic grade in a clinically uniform cohort managed at a single institution using a newly proposed grading system. Our study included 49 primary breast angiosarcomas diagnosed during 1994 to 2022 (median follow-up: 33 mo), classified as low grade (29%), intermediate grade (20%), and high grade (51%), based on mitotic count, extent of solid components, and necrosis. At last follow-up, 22% patients developed locoregional recurrences, 63% distant metastases, and 47% patients died of disease. As patients with low and intermediate-grade angiosarcomas had relatively similar outcomes, our cohort was further analyzed using a 2-tier system (low grade and high grade). Targeted-DNA next-generation sequencing (505 cancer gene panel) performed in 11 cases found KDRmutations in 78% and PIK3CAmutations in 44% of high-grade lesions. Histologic grade, by either 3-tier or 2-tier grading systems, had a strong impact on survival, with the 2-tier system being an independent predictor of disease-specific survival and overall survival. Based on 2-tier system, the 5-year overall survival was 38% for high-grade angiosarcoma and 74% for low-grade angiosarcoma. PIK3CAmutations alone or concurrent with KDRalterations were identified in angiosarcomas with worse prognosis.

TRAF7somatic mutations are rare and have been reported in meningiomas, intraneural perineuriomas, and mesotheliomas. Triggered by an index case of an unclassified low-grade mesenchymal tumor with TRAF7mutation as the only genetic alteration, we searched our files and identified 2 additional cases with similar features. The tumors arose in 2 females and 1 male, aged 63 to 75 years old (median: 67 y). They were infiltrative deep soft tissue masses involving the shoulder, chest wall, and thigh, measuring 7.0 to 9.1 cm in greatest dimensions. One tumor was locally aggressive, and 2 were associated with lung and bone metastases. The tumors displayed alternating fibrous and myxoid stroma with mild to moderate cellularity and consisted of uniform spindle cells with open chromatin, inconspicuous nucleoli and scant cytoplasm. Significant mitotic activity or necrosis were not present. However, the metastatic tumor of 1 case showed an epithelioid morphology and brisk mitotic activity. Immunohistochemically, the tumors showed nonspecific and focal smooth muscle actin or CD34 expression. By DNA sequencing, all 3 cases harbored TRAF7missense mutations involving the C-terminal WD40 domains as the only somatic mutations, showed nonrecurrent focal copy number alterations, and were negative for gene fusions by targeted RNA sequencing. On methylation profiling, the tumors clustered with the undifferentiated sarcoma and myxofibrosarcoma methylation classes and were distinct from morphologic mimics. On follow-up (5 to 36 mo), 2 patients died of disease following aggressive chemotherapeutic regimens. We describe a novel TRAF7-mutated mesenchymal tumor characterized by aggressive clinical behavior despite the histologic appearance of a low-grade fibromyxoid spindle cell tumor.

Mesenchymal tumors harboring various kinase fusions were recently recognized as emerging entities mainly in the soft tissues. We herein investigate the clinicopathologic and molecular characteristics of head and neck mesenchymal tumors harboring kinase fusions. The study cohort included 15 patients with a median age of 13 years (ranging from congenital to 63 y). The kinase genes involved in descending order were NTRK1(n=6), NTRK3(n=5), BRAF(n=2), and 1 each with MET, and RET. The anatomic locations were broad involving all tissue planes, including skin (n=4), intraosseous (n=4), major salivary glands (n=2), sinonasal tract (n=2), soft tissue of face or neck (n=2), and oral cavity (n=1). The histologic spectrum ranged from benign to high grade, in descending order including tumors resembling malignant peripheral nerve sheath tumor (MPNST)-like, fibrosarcoma (infantile or adult-type), lipofibromatosis-like neural tumor (LPFNT), inflammatory myofibroblastic tumor-like, and a novel phenotype resembling myxoma. Perivascular hyalinization/stromal keloid-like collagen bands and staghorn vasculature were common features in MPNST-like and LPFNT-like tumors. Two tumors (1 each with NTRK1 or BRAFrearrangement) were classified as high grade. By immunohistochemistry, S100 and CD34 positivity was noted in 71% and 60%, frequently in MPNST-like and LPFNT-like phenotypes. Pan-TRK was a sensitive marker for NTRK-translocated tumors but was negative in tumor with other kinase fusions. One patient with a high-grade tumor developed distant metastasis. Molecular testing for various kinase fusions should be considered for S100+/CD34+spindle cell neoplasms with perivascular hyalinization and staghorn vessels, as pan-TRK positivity is seen only in NTRKfusions.

SMARCB1biallelic inactivation resulting in SMARCB1/INI1 deficiency drives a wide range of malignancies, including many mesenchymal tumors. However, the specific types of SMARCB1alterations and spectrum of cooperating mutations among various types of sarcomas has not been well investigated. We profiled SMARCB1genetic alterations by targeted DNA sequencing and fluorescence in situ hybridization (FISH) in a large cohort of 118 soft tissue and bone tumors, including SMARCB1-deficient sarcomas (78, 66%): epithelioid sarcomas, epithelioid peripheral nerve sheath tumors, poorly differentiated chordomas, malignant rhabdoid tumors, and soft tissue myoepithelial tumors, as well as non-SMARCB1-deficient sarcomas (40, 34%) with various SMARCB1genetic alterations (mutations, copy number alterations). SMARCB1 loss by immunohistochemistry was present in 94% SMARCB1pathogenic cases. By combined sequencing and FISH assays, 80% of SMARCB1-deficient tumors harbored homozygous (biallelic) SMARCB1loss, while 14% demonstrated heterozygous SMARCB1loss-of-function (LOF) alterations, and 6% showed no demonstrable SMARCB1alterations. FISH and sequencing were concordant in the ability to detect SMARCB1loss in 48% of cases. Epithelioid sarcomas most commonly (75%) harbored homozygous deletions, while a subset showed focal intragenic deletions or LOF mutations (nonsense, frameshift). In contrast, most soft tissue myoepithelial tumors (83%) harbored SMARCB1nonsense point mutations without copy number losses. Additionally, clinically significant, recurrent co-occurring genetic events were rare regardless of histotype. By sequencing, extended 22q copy number loss in genes flanking the SMARCB1locus (22q11.23) occurred in one-third of epithelioid sarcomas and the majority of poorly differentiated chordomas. Poorly differentiated chordomas and soft tissue myoepithelial tumors showed significantly worse overall and disease-free survival compared to epithelioid sarcomas. Overall, SMARCB1LOF alterations predominate and account for SMARCB1 protein loss in most cases: majority being biallelic but a subset were heterozygous. In contrast, SMARCB1alterations of uncertain significance can be seen in diverse sarcomas types and does not indicate a SMARCB1-deficient entity.

Myxoid pleomorphic liposarcoma (MPLPS) is a recently described and extremely rare subtype of liposarcoma with a predilection for the mediastinum. However, the genomic features of MPLPS remain poorly understood. We performed comprehensive genomic profiling of MPLPS in comparison with pleomorphic liposarcoma (PLPS) and myxoid/round cell liposarcoma (MRLPS). Of the 8 patients with MPLPS, 5 were female and 3 were male, with a median age of 32 years old (range 10–68). All except one were located in the mediastinum, with invasion of surrounding anatomic structures, including chest wall, pleura, spine, and large vessels. All cases showed an admixture of morphologies reminiscent of PLPS and MRLPS, including myxoid areas with plexiform vasculature admixed with uni- and/or multivacuolated pleomorphic lipoblasts. Less common features included well-differentiated liposarcoma-like areas, and in one case fascicular spindle cell sarcoma reminiscent of dedifferentiated LPS. Clinically, 4 experienced local recurrence, 4 had distant metastases and 5 died of disease. Compared to PLPS and MRLPS, patients with MPLPS had worse overall and progression-free survival. Recurrent TP53mutations were present in all 8 MPLPS cases. In contrast, in PLPS, which also showed recurrent TP53mutations (83%), RB1and ATRXlosses were more common. MRLPS was highly enriched in TERTpromoter mutations (88%) and PI3K/AKT pathway mutations. Copy number profiling in MPLPS revealed multiple chromosomal gains with recurrent amplifications of chromosomes 1, 19 and 21. Importantly, allele-specific copy number analysis revealed widespread loss of heterozygosity (80% of the genome on average) in MPLPS, but not in PLPS or MRLPS. Our findings revealed genome-wide loss of heterozygosity co-existing with TP53mutations as a characteristic genomic signature distinct from other liposarcoma subtypes, which supports the current classification of MPLPS as a stand-alone pathologic entity. These results further expand the clinicopathologic features of MPLPS, including older age, extra-mediastinal sites, and a highly aggressive outcome.

POU2F3 is a recent marker of a small cell lung carcinoma (SCLC) subtype related to chemosensory tuft cells (SCLC-P). The characteristics of SCLC-P have not been fully defined, and the data on POU2F3 expression in other lung tumors are scarce.

With the widespread application of next-generation sequencing, the genetic landscape of uterine mesenchymal neoplasms has been evolving rapidly to include several recently identified fusion genes. Although chromosomal rearrangements involving the 10q22 and 17q21.31 loci have been reported in occasional uterine leiomyomas decades ago, the corresponding KAT6B::KANSL1fusion has been only recently identified in 2 uterine tumors diagnosed as leiomyoma and leiomyosarcoma. We herein describe 13 uterine stromal neoplasms carrying a KAT6B::KANSL1(n=11) and KAT6A::KANSL1(n=2) fusion. Patient ages ranged from 33 to 81 years (median, 49 y). Tumor size was 2.6 to 23.5 cm (median, 8.2 cm). Nine tumors were myometrium-centered, and 3 had an intracavitary component. Original diagnoses were mostly low-grade endometrial stromal sarcoma (LG-ESS; 10 cases) with atypical features (limited CD10 expression, sex cord-like features, pericytic vasculature, and frequent myxoid changes). Treatment was hysterectomy±bilateral salpingo-oophorectomy (10), myomectomy (1), and curettage (2). Five patients were disease-free at 6 to 34 months, 3 (27%) died of disease at 2 to 47 months, and 3 were alive with disease at 2, 17, and 17 years. Histologically, most tumors showed variable overlap with LG-ESS, but they were generally well-circumscribed lacking the extensive permeative and angioinvasive growth typical of LG-ESS. They were composed of monotonous medium-sized oval and spindle cells arranged into diffuse sheets with prominent spiral-type arterioles and frequent pericytoma-like vascular pattern. Variable myxoid stromal changes were frequent. Mitotic activity ranged from 1 to >20 in 10 HPFs. Immunohistochemistry showed variable expression of CD10 (12/13), estrogen receptor (8/11), progesterone receptor (8/11), smooth muscle actin (9/11), desmin (4/12), h-caldesmon (2/10), calretinin (3/8), inhibin (1/7), WT1 (4/7), cyclin D1 (5/11; diffuse in only 1 case), and pankeratin (5/10). This series characterizes a KAT6B/A::KANSL1fusion-positive uterine stromal neoplasm within the morphologic spectrum of LG-ESS but with atypical features. The relationship of these neoplasms to genuine LG-ESS remains unclear. This molecular subtype of uterine endometrial stromal sarcoma has the potential for an unfavorable clinical course despite the absence of widely invasive growth; nevertheless, analysis of more cases is necessary to delineate the phenotypic spectrum and biological potential of this tumor.

To elucidate the mechanisms underlying the divergent clinicopathologic spectrum of EWSR1/FUS::CREBtranslocation-associated tumors, we performed a comprehensive genomic analysis of fusion transcript variants, recurrent genetic alterations (mutations, copy number alterations), gene expression, and methylation profiles across a large cohort of tumor types. The distribution of the EWSR1/FUSfusion partners—ATF1, CREB1, and CREM—and exon involvement was significantly different across different tumor types. Our targeted sequencing showed that secondary genetic events are associated with tumor type rather than fusion type. Of the 39 cases that underwent targeted NGS testing, 18 (46%) had secondary OncoKB mutations or copy number alterations (29 secondary genetic events in total), of which 15 (52%) were recurrent. Secondary recurrent, but mutually exclusive, TERTpromoter and CDKN2Amutations were identified only in clear cell sarcoma (CCS) and associated with worse overall survival. CDKN2A/Bhomozygous deletions were recurrent in angiomatoid fibrous histiocytoma (AFH) and restricted to metastatic cases. mRNA upregulation of MITF, CDH19, PARVB, and PFKPwas found in CCS, compared to AFH, and correlated with a hypomethylated profile. In contrast, S100A4and XAF1were differentially upregulated and hypomethylated in AFH but not CCS. Unsupervised clustering of methylation profiles revealed that CREB family translocation-associated tumors form neighboring but tight, distinct clusters. A sarcoma methylation classifier was able to accurately match 100% of CCS cases to the correct methylation class; however, it was suboptimal when applied to other histologies. In conclusion, our comprehensive genomic profiling of EWSR1/FUS::CREBtranslocation-associated tumors uncovered mostly histotype, rather than fusion-type associated correlations in transcript variants, prognostically significant secondary genetic alterations, and gene expression and methylation patterns.

Identical TFE3-related gene fusions may be found in renal cell carcinoma and mesenchymal neoplasms such as alveolar soft part sarcoma and TFE3-rearranged perivascular epithelioid cell tumor (PEComa). Among mesenchymal neoplasms, the ASPSCR1-TFE3gene fusion has previously been described only in alveolar soft part sarcoma. We report 3 unusual mesenchymal neoplasms harboring the ASPSCR1-TFE3gene fusion, the morphologic phenotype of which more closely matches PEComa rather than alveolar soft part sarcoma. All 3 neoplasms occurred in females ranging in age from 18 to 34 years and were located in the viscera (kidney, bladder, and uterus). All 3 contained nests of epithelioid cells bounded by fibrovascular septa. However, all were associated with hyalinized stroma, tight nested architecture, mixed spindle cell and epithelioid pattern, clear cytoplasm, and lacked significant discohesion. Overall, morphologic features closely resembled PEComa, being distinct from the typical alveolar soft part sarcoma phenotype. While none of the neoplasms labeled for HMB45, cytokeratin, or PAX8 all showed positivity for TFE3 and cathepsin K, and all except 1 were positive for smooth muscle actin. One patient developed a liver metastasis 7 years after nephrectomy. These cases bridge the gap between 2 TFE3-rearranged neoplasms, specifically alveolar soft part sarcoma and Xp11 translocation PEComa, highlighting the relatedness and overlap among Xp11 translocation neoplasms. While most TFE3-rearranged neoplasms can be confidently placed into a specific diagnostic category such as alveolar soft part sarcoma, PEComa, or Xp11 translocation renal cell carcinoma, occasional cases have overlapping features, highlighting the potential role that the cell of origin and the specific gene fusion play in the phenotype of these neoplasms.

We report 4 neoplasms of the kidney (2 cases) and uterus (2 cases) harboring rearrangements or amplifications of the GLI1gene, which because of their unusual clinical presentation, morphology, and immunoprofile mimicked other neoplasms, causing significant diagnostic challenge. The neoplasms occurred in 4 female patients ages 33 to 88 years. Histologically they all demonstrated nodular growth, solid architecture, bland epithelioid to ovoid-spindle cells with pale cytoplasm set in a variably myxoid or hyalinized stroma. One uterine tumor also demonstrated a focal round cell pattern, while another demonstrated focal pleomorphism. Unlike most previously reported neoplasms with these genetic abnormalities, the neoplasms in the current series were negative for S100 protein and minimally reactive for actin. All labeled for CD10 and cyclin D1, while 2 labeled for estrogen receptor and BCOR and 1 labeled for desmin, raising consideration of endometrial stromal sarcoma, myxoid leiomyosarcoma, metastatic breast carcinoma, and glomus tumor. One renal neoplasm demonstrated a GLI1-FOXO4gene fusion and the other harbored a GLI1gene rearrangement (unknown partner). The 2 uterine neoplasms exhibited GLI1gene amplifications. GLI1-altered neoplasms (particularly those with GLI1amplification) show variable morphology and lack a consistent immunophenotype, and thus may trigger diagnostic challenges which can be resolved by molecular testing.

Uterine PEComas often present a diagnostic challenge as they share morphological and immunohistochemical features with smooth muscle tumors. Herein we evaluated a series of 19 uterine PEComas to compare the degree of melanocytic marker expression with their molecular profile. Patients ranged from 32–77 (median 48) years, with six tumors classified as malignant based on the modified gynecologic-specific prognostic algorithm. All patients with malignant PEComas were alive with disease or dead  of disease at last follow-up, while all those of uncertain malignant potential were alive and well (median follow-up, 47 months).

Undifferentiated mesenchymal tumors arising from the inner lining (intima) of large arteries are classified as intimal sarcomas (ISA) with MDM2amplification as their molecular hallmark. Interestingly, undifferentiated pleomorphic sarcomas (UPS) of the heart have recently been suggested to represent the cardiac analog of ISA due to morphological overlap and high prevalence of MDM2amplifications in both neoplasms. However, little is known about ISAs and cardiac UPS without MDM2amplifications and molecular data supporting their common classification is sparse. Here, we report a series of 35 cases comprising 25 ISAs of the pulmonary artery, one ISA of the renal artery and 9 UPS of the left atrium. Tumors were analyzed utilizing the Illumina Infinium MethylationEPIC BeadChip array, enabling copy number profile generation and unsupervised DNA methylation analysis. DNA methylation patterns were investigated using t-distributed stochastic neighbor embedding (t-SNE) analysis. Histologically, all ISAs and UPS of the left atrium resembled extra-cardiac UPS. All cases exhibited highly complex karyotypes with overlapping patterns between ISA and UPS. 29/35 cases showed mutually exclusive amplifications in the cell-cycle associated oncogenes MDM2(25/35), MDM4(2/35), and CDK6(2/35). We further observed recurrent co-amplifications in PDGFRA(21/35), CDK4(15/35), TERT(11/35), HDAC9(9/35), and CCND1(4/35). Sporadic co-amplifications occurred in MYC, MYCN,and MET(each 1/35). The tumor suppressor CDKN2A/Bwas frequently deleted (10/35). Interestingly, DNA methylation profiling (t-SNE) revealed an overlap of ISA and cardiac UPS. This “ISA” methylation signature was distinct from potential histologic and molecular mimics. In conclusion, our data reveal MDM4and CDK6amplifications in ISAs and UPS of the left atrium, lacking MDM2amplification. We further report novel co-amplifications of various oncogenes, which may have therapeutic implications. Finally, the genetic and epigenetic concordance of ISAs and UPS of the left atrium further supports a shared pathogenesis and common classification.

Clear cell (hemangioblastoma-like) stromal tumor of the lung (CCST-L) is a recently described distinctive rare pulmonary neoplasm of unknown histogenesis and molecular pathogenesis. Only 7 cases have been reported in 2 recent studies, although additional cases might have been reported under the heading of extraneural pulmonary hemangioblastoma. We herein describe 4 CCST-L cases, 3 of them harboring a YAP1-TFE3fusion. The fusion-positive tumors occurred in 3 women, aged 29, 56, and 69 years. All presented with solitary lung nodules measuring 2.3 to 9.5 cm. Histologically, all tumors showed similar features being composed of relatively uniform medium-sized epithelioid to ovoid cells with clear cytoplasm and small round monomorphic nuclei. Scattered larger cells with enlarged hyperchromatic nuclei and marked pleomorphism were noted in 2 cases. The tumors were associated with a hypervascularized stroma with variable but essentially subtle resemblance to capillary hemangioblastoma and perivascular epithelioid cell tumor (PEComa). Immunohistochemistry was negative for all lineage-specific markers. Targeted RNA sequencing showed a YAP1-TFE3fusion in 3 of 4 cases. All 3 tumors showed homogeneous nuclear TFE3 immunoreactivity. Two patients were disease free at 36 and 12 months. The third patient had biopsy-proven synchronous renal and hepatic metastases, but extended follow-up is not available (recent case). The fourth case lacking the fusion affected a 66-year-old woman and showed subtle histologic differences from the fusion-positive cases, but had comparable TFE3 immunoreactivity. CCST-L represents a distinctive entity unrelated to hemangioblastoma and likely driven by recurrent YAP1-TFE3fusions in most cases. The relationship of our cases to the recently reported “hemangioblastoma-like” CCST-L remains to be determined. Analysis of larger series is paramount to delineate the morphologic spectrum and biological behavior of this poorly characterized entity.

Supplemental Digital Content is available in the text.We report a novel NIPBL-NACC1gene fusion in a rare primary hepatic neoplasm previously described as the “cholangioblastic variant of intrahepatic cholangiocarcinoma.” The 2 index cases were identified within our consultation files as morphologically distinctive primary hepatic neoplasms in a 24-year-old female and a 54-year-old male. The neoplasms each demonstrated varied architecture, including trabecular, organoid, microcystic/follicular, and infiltrative glandular patterns, and biphasic cytology with large, polygonal eosinophilic cells and smaller basophilic cells. The neoplasms had a distinctive immunoprofile characterized by diffuse labeling for inhibin, and patchy labeling for neuroendocrine markers (chromogranin and synaptophysin) and biliary marker cytokeratin 19. RNA sequencing of both cases demonstrated an identical fusion of NIBPLexon 8 to NACC1exon 2, which was further confirmed by break-apart fluorescence in situ hybridization assay for each gene. Review of a tissue microarray including 123 cases originally diagnosed as well-differentiated neuroendocrine neoplasm at one of our hospitals resulted in identification of a third case with similar morphology and immunophenotype in a 52-year-old male, and break-apart fluorescence in situ hybridization probes confirmed rearrangement of both NIPBLand NACC1. Review of The Cancer Genome Atlas (TCGA) sequencing data and digital images from 36 intrahepatic cholangiocarcinomas (www.cbioportal.org) revealed one additional case with the same gene fusion and the same characteristic solid, trabecular, and follicular/microcystic architectures and biphasic cytology as seen in our genetically confirmed cases. The NIPBL-NACC1fusion represents the third type of gene fusion identified in intrahepatic cholangiocarcinoma, and correlates with a distinctive morphology described herein.

Gene fusions have been discussed in the scientific literature since they were first detected in cancer cells in the early 1980s. There is currently no standardized way to denote the genes involved in fusions, but in the majority of publications the gene symbols in question are listed either separated by a hyphen (-) or by a forward slash (/). Both types of designation suffer from important shortcomings. HGNC has worked with the scientific community to determine a new, instantly recognizable and unique separator—a double colon (::)—to be used in the description of fusion genes, and advocates its usage in all databases and articles describing gene fusions.

Chromosomal translocations constitute driver mutations in solid tumors and leukemias. The mechanisms of how related or even identical gene fusions drive the pathogenesis of various tumor types remain elusive. One remarkable example is the presence of EWSR1fusions with CREB1and ATF1, members of the CREB family of transcription factors, in a variety of sarcomas, carcinomas and mesotheliomas. To address this, we have developed in vitro models of oncogenic fusions, in particular, EWSR1-CREB1and EWSR1-ATF1, in human embryonic stem (hES) cells, which are capable of multipotent differentiation, using CRISPR-Cas9 technology and HDR together with conditional fusion gene expression that allows investigation into the early steps of cellular transformation. We show that expression of EWSR1-CREB1/ATF1fusion in hES cells recapitulates the core gene signatures, respectively, of angiomatoid fibrous histiocytoma (AFH) and gastrointestinal clear cell sarcoma (GI-CCS), although both fusions lead to cell lethality. Conversely, expression of the fusions in hES cells differentiated to mesenchymal progenitors is compatible with prolonged viability while maintaining the core gene signatures. Moreover, in the context of a mesenchymal lineage, the proliferation of cells expressing the EWSR1-CREB1fusion is further extended by deletion of the tumor suppressor TP53. We expect the generation of isogenic lines carrying oncogenic fusions in various cell lineages to expand our general understanding of how those single genetic events drive tumorigenesis while providing valuable resources for drug discovery.

Despite extraordinary advances in the molecular characterization of soft tissue tumors as a result of the widespread application of next generation sequencing in clinical practice, a subset of lesions remain difficult to diagnose. In this study we describe 3 unclassified spindle cell sarcomas with a monomorphic cytomorphology and distinctive storiform growth, characterized by novel fusions between EWSR1or FUS1, and NACC1genes. The tumors occurred in 3 young adult females (age range: 29–31) involving deep soft tissues, two located in the lower extremity and one in the abdominal wall. All three tumors showed patchy positivity for S100 protein, while being negative for SOX10 and retained H3K27me3 expression. All cases were negative for epithelial or muscle markers. As the findings were non-specific, molecular studies using targeted panels of RNA sequencing were performed, including one case tested by TruSight RNA Fusion Panel and 2 cases by Archer FusionPlex. The results showed 2 cases were positive for FUS-NACC1and one for EWSR1-NACC1fusions. These findings were further confirmed by FISH using custom BAC probes for a dual-color fusion assay. These results suggest the possibility of a previously undescribed soft tissue neoplasm characterized by a uniform spindle cell phenotype arranged in a storiform and fascicular pattern, expressing S100 protein and harboring NACC1-related fusions. The biologic behavior of this tumor remains to be determined.

Benign peripheral nerve tumors include schwannoma, neurofibroma, and perineurioma, as well as a recently recognized group of tumors with dual patterns of differentiation. The molecular pathogenesis of these so-called “hybrid” tumors remains poorly understood. Following identification of a novel CHD7-VGLL3fusion gene in a hybrid schwannoma–perineurioma, we evaluated an expanded cohort of this tumor-type—as well as tumors with VGLL3rearrangement identified from a curated molecular database—to characterize the prevalence of fusion genes among these tumors. Eighteen tumors met the inclusion criteria for this study. RNA sequencing identified VGLL3rearrangement in 14 of these cases; the partner genes included CHD7(ten cases), CHD9(two cases), and MAMLD1(two cases). Two cases possessed altogether unrelated fusions, including: DST-BRAFand SQSTM1-CDX1fusion genes. Finally, two cases lacked identifiable fusion products. These findings highlight the molecular diversity of these neoplasms, with frequent rearrangement of VGLL3. More importantly, despite their dual pattern of differentiation, our results reveal the pathogenesis of hybrid schwannoma–perineurioma is unrelated to conventional schwannoma and perineurioma, thereby implying this tumor represents an altogether pathologically distinct entity.

Sarcomas with MEIS1-NCOA2fusions have been so far reported in 2 cases each of primitive renal sarcomas and intraosseous pelvic rhabdomyosarcomas. Their histologic spectrum, anatomic distribution, and clinical behavior remain poorly defined. In this study, we report 6 additional spindle cell sarcomas with MEIS1-NCOA2or NCOA1fusions that fall into the same disease spectrum with the previously reported renal sarcomas. The patients’ age range was wide (20–76 years, mean 46) and all except one were female. The tumors arose in the kidney (n= 2), and one each in the uterine corpus, vagina, scrotum, and para-rectal region. The consistent morphology was that of monomorphic spindle to ovoid cells in a storiform, whorling, or solid pattern. Alternating cellularity, myxoid stroma, and microcystic changes were seen in some cases. Mitotic activity varied greatly (<1–33/10 high power fields). The immunophenotype was nonspecific, with most cases expressing variable degrees of TLE1, WT1, cyclin D1, CD56, and CD10. Using various platforms of RNA-based targeted sequencing, MEIS1-NCOA2fusions were recurrently identified in 5 cases, and a novel MEIS1-NCOA1fusion was found in one renal tumor. The gene fusions were validated by fluorescence in situ hybridization using custom BAC probes. Of the 5 patients with available follow-up (5 months to 8 years), all experienced local recurrences, but no distant spread or death from disease. Our results expand the clinicopathologic spectrum of sarcomas with MEIS1-NCOA2/1fusions, providing evidence of an undifferentiated spindle cell phenotype with nonspecific immunoprofile and low-grade clinical behavior.

Recurrent gene fusions have been observed in epithelioid and myxoid variants of uterine leiomyosarcoma. PGR::NR4A3fusions were recently described in a subset of epithelioid leiomyosarcomas exhibiting rhabdoid morphology. In this study, we sought to expand the clinical, morphologic, immunohistochemical, and genetic features of gynecologic leiomyosarcomas harboring NR4A3rearrangements with PGRand novel fusion partners. We identified 9 gynecologic leiomyosarcomas harboring PGR::NR4A3, CARMN::NR4A3, ACTB::NR4A3, and possible SLCO5A1::NR4A3fusions by targeted RNA sequencing. Tumors frequently affected premenopausal women, involving the uterine corpus, uterine cervix, or pelvis. All were similarly characterized by lobules of monomorphic epithelioid and/or spindled cells arranged in sheets, cords, trabeculae, and micro- and macrocysts associated with abundant myxoid matrix and hemorrhage, creating labyrinth-like or pulmonary edema–like architecture. Myogenic differentiation with frequent estrogen receptor and progesterone receptor staining and no CD10 expression characterized all tumors. All cases showed high NR4A3RNA expression levels and NOR1 (NR4A3) nuclear staining similar to salivary gland acinic cell carcinomas and a subset of extraskeletal myxoid chondrosarcomas harboring NR4A3rearrangements. NOR1 (NR4A3) immunohistochemistry may serve as a useful diagnostic marker of NR4A3fusion–positive gynecologic leiomyosarcomas.

High-grade endometrial stromal sarcoma (HGESS) may harbor YWHAE-NUTM2A/Bfusion, ZC3H7B-BCORfusion, and BCORinternal tandem duplication (ITD). NTRK3upregulation and pan-Trk expression were reported in soft tissue lesions that share similar morphology and genetic abnormalities. To confirm these findings in HGESS, differential expression analysis was performed at gene level comparing 11 HGESS with 48 other uterine sarcomas, including 9 low-grade endometrial stromal sarcomas, 23 undifferentiated uterine sarcomas, and 16 leiomyosarcomas, using targeted RNA sequencing data. Pan-Trk immunohistochemistry was performed on 35 HGESS, including 10 tumors with RNA expression data, with genotypes previously confirmed by targeted RNA sequencing, fluorescence in situ hybridization, and/or genomic PCR. Unsupervised hierarchical clustering of the top 25% of differentially expressed probes identified three molecular groups: (1) high NTRK3, FGFR3, RET, BCOR, GLI1, and PTCH1and low ESR1expression; (2) low NTRK3, FGFR3, RET, BCOR, GLI1, and PTCH1and high ESR1expression; and (3) low NTRK3, FGFR3, RET, BCOR, GLI1, PTCH1, and ESR1expression. Among HGESS, 64% of tumors clustered in group 1, while 27% clustered in group 2. Cytoplasmic and/or nuclear pan-Trk staining of variable extent and intensity was seen in 91% of HGESS regardless of cyclin D1 and/or BCOR positivity. ER and PR expression was seen in 44% of HGESS despite ESR1downregulation. Two patients with ER and PR positive but ESR1downregulated stage I HGESS were treated with endocrine therapy, and both recurred at 12 and 36 months after primary resection. By RNA expression, HGESS appear homogenous and distinct from other uterine sarcomas by activation of kinases, including NTRK3, and sonic hedgehog pathway genes along with downregulation of ESR1. Most HGESS demonstrate pan-Trk staining which may serve as a diagnostic biomarker. ESR1downregulation is seen in some HGESS that express ER and PR which raises implications in the utility of endocrine therapy in these patients.

The genetic hallmark of epithelioid hemangioma (EH) is the presence of recurrent gene fusions involving FOS and FOSB transcription factors, which occur in one-third of the cases. Certain clinical, pathologic, and genotypic correlations have been described, with FOS-related fusions being more often detected in skeletal and cellular variants of EH, while FOSBgene rearrangements are more commonly associated with atypical histologic features and penile location. These fusions are infrequently detected in the cutaneous or head and neck EH. Overall, two-thirds of EH lack these canonical fusions and remain difficult to classify, especially when associated with atypical features and/or clinical presentations. Triggered by an index case of an intravascular soft tissue EH with a novel GATA6-FOXO1gene fusion by targeted RNA sequencing (Archer® FusionPlex® Sarcoma Panel), we have investigated 27 additional EH cases negative for FOSand FOSBgene rearrangements for this novel abnormality to determine its recurrent potential, and its association with clinical and pathologic features. Four additional EH cases were found to display GATA6-FOXO1fusions (18%). There were three females and two males, with a mean age of 32 years old. Three lesions occurred in the head and neck (dura, nasopharyngeal, and cheek), one in the back and one in the leg. Two of these lesions were cutaneous and one was intravascular in the subcutis of the leg. Microscopically, the tumors showed a variegated morphology, with alternating vasoformative and solid components, extravasated red blood cells and mild to moderate cytologic atypia. None showed brisk mitotic activity or necrosis. Tumors were negative for FOS and FOSB by immunohistochemistry. In conclusion, we report a new GATA6-FOXO1fusion in a subset of EH, with a predilection for skin, and head and neck location. The relationship of this novel molecular subset with the more common FOS/FOSBfusion-positive EH remains to be determined.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and arises in the gastrointestinal tract. Most GISTs are caused by activating mutations in the KIT receptor tyrosine kinase, such as the exon 11 KITV559? mutation. The small molecule imatinib inhibits KIT and has been a mainstay of therapy in GIST. Unfortunately, imatinib-treated patients typically relapse, most often due to clonal emergence of the resistance-associated KITV654A mutation. To determine the biologic impact of this second-site mutation in vivo, we created a mouse model with the corresponding V558?;V653A Kitdouble mutation restricted (a) spatially to ETV1+cells, which include the interstitial cells of Cajal (ICCs) from which GISTs presumably originate, and (b) temporally through tamoxifen treatment after birth. This resulted in the first in vivo model of the most common second-site mutation associated with imatinib resistance in GIST and the first in vivo demonstration that cell-autonomous expression of mutant KIT in the ICC lineage leads to GIST. GISTs driven by the V558?;V653A Kitdouble mutation were resistant to imatinib, while cabozantinib was more effective in overcoming resistance than sunitinib. Compared to control mice with a single V558? Kitmutation, mice with a double V558?; V653A Kitmutation had increased tumor oncogenesis and associated KIT-dependent STAT activation. Our findings demonstrate that the biologic consequences of a second-site mutation in an oncogenic driver may include not only a mechanism for drug resistance, but changes in tumor oncogenic potential and differential activation of signaling pathways.

Retiform and composite hemangioendotheliomas (CHEs) are both locally aggressive, rarely metastasizing vascular neoplasms characterized by arborizing vascular channels lined by endothelial cells with a hobnail morphology. CHE displays additional cytologic and architectural components, including often vacuolated epithelioid cells, solid areas, or features reminiscent of well-differentiated angiosarcoma. Triggered by an index case of a soft tissue retiform hemangioendothelioma (RHE) which revealed a YAP1-MAML2gene fusion by targeted RNA sequencing, we sought to investigate additional cases in this morphologic spectrum for this genetic abnormality. A total of 24 cases, 13 RHE and 11 CHE involving skin and soft tissue were tested by fluorescence in situ hybridization using custom BAC probes for rearrangements involving these genes. An additional visceral CHE with neuroendocrine differentiation was tested by targeted RNA sequencing. Among the soft tissue cohort, 5/13 (38%) RHE and 3/11 (27%) CHE showed YAP1gene rearrangements, with 5 cases showing a YAP1-MAML2fusion, including all 3 CHE. The single neuroendocrine CHE showed the presence of a PTBP1-MAML2fusion. All YAP1-positive CHE lesions occurred in female children at acral sites, compared with fusion-negative cases which occurred in adults, with a wide anatomic distribution. YAP1-positive RHE occurred preferentially in males and lower limb, compared with negative cases. These results suggest that RHE and CHE represent a morphologic continuum, sharing abnormalities in YAP1and MAML2genes. In contrast, the neuroendocrine CHE occurring in a 37-year-old male harbored a distinct PTBP1-MAML2fusion and showed aggressive clinical behavior (pancreatic mass with multiple liver and lung metastases). These preliminary findings raise the possibility that neuroendocrine CHE may be genetically distinct from the conventional RHE/CHE spectrum. Further studies are needed to investigate the pathogenetic relationship of fusion-negative cases with this subset and, less likely, with other members of the HE family of tumors.

Gene fusions constitute pivotal driver mutations often encoding aberrant chimeric transcription factors. However, an increasing number of gene fusion events have been shown not to be histotype specific and shared among different tumor types, otherwise completely unrelated clinically or phenotypically. One such remarkable example of chromosomal translocation promiscuity is represented by fusions between EWSR1or FUSwith genes encoding for CREB-transcription factors family (ATF1, CREB1, and CREM), driving the pathogenesis of various tumor types spanning mesenchymal, neuroectodermal, and epithelial lineages. In this study, we investigate a group of 13 previously unclassified malignant epithelioid neoplasms, frequently showing an epithelial immunophenotype and marked predilection for the peritoneal cavity, defined by EWSR1/FUS–CREBfusions. There were seven females and six males, with a mean age of 36 (range 9–63). All except three cases occurred intra-abdominally, including one each involving the pleural cavity, upper, and lower limb soft tissue. All tumors showed a predominantly epithelioid morphology associated with cystic or microcystic changes and variable lymphoid cuffing either intermixed or at the periphery. All except one case expressed EMA and/or CK, five were positive for WT1, while being negative for melanocytic and other mesothelioma markers. Nine cases were confirmed by various RNA-sequencing platforms, while in the remaining four cases the gene rearrangements were detected by FISH. Eleven cases showed the presence of CREM-related fusions (EWSR1–CREM, 7; FUS–CREM, 4), while the remaining two harbored EWSR1–ATF1fusion. Clinically, seven patients presented with and/or developed metastases, confirming a malignant biologic potential. Our findings expand the spectrum of tumors associated with CREB-related fusions, defining a novel malignant epithelioid neoplasm with an immunophenotype suggesting epithelial differentiation. This entity appears to display hybrid features between angiomatoid fibrous histiocytoma (cystic growth and lymphoid cuffing) and mesothelioma (peritoneal/pleural involvement, epithelioid phenotype, and cytokeratin and WT1 co-expression).

Supplemental Digital Content is available in the text.Glomus tumors (GTs), together with myofibroma (MF), myopericytoma (MP), and angioleiomyoma (AL) are classified as members of the perivascular myoid family of tumors. The reported genetic abnormalities across these neoplasms is dissimilar, arguing against a pathogenetically unified family; half of the GT showing NOTCH-gene fusions and a smaller subset BRAFV600Emutations, while PDGFRBmutations are noted in a subset of MF and MP. This study aimed to investigate the prevalence and specificity of NOTCH-gene fusions in a large group of GT and correlate with clinical features. BRAF-VE1 and PDGFRB immunoexpression was also investigated in this cohort. A total of 93 GT and 43 other pericytic lesions (11 MP, 13 MF, and 19 AL) were selected. All cases were tested by fluorescence in situ hybridization for NOTCH1-4and MIR143gene abnormalities and 6 cases were investigated by targeted RNA-sequencing. Fluorescence in situ hybridization revealed NOTCH-gene rearrangements in 50 (54%) GT, 2 MP (18%), and 2 AL (11%). NOTCH-rearrangements were present in 34 (68%) benign and 16 (32%) malignant GT. Fusion-positive benign GT were overwhelmingly seen in males with a predilection for extremities, while the malignant GT occurred mostly in viscera. Among the fusion-negative GT, 88% were benign, 9% uncertain malignant potential, and 2% malignant. Half of the fusion-negative GTs occurred in the finger/subungual region. In summary, rearrangements of NOTCHgenes are seen in over half of GT, with NOTCH2-MIR143being the most common fusion (73%), while only a small subset of AL and MP share these abnormalities. The common subungual GT subset lack NOTCH-gene fusions suggesting an alternative pathogenesis. BRAF-VE1 was negative in all 37 cases studied, while strong PDGFRB staining was seen in 14 (21%) cases. Additional studies are needed to investigate the genetic alterations in the fusion-negative cases.

High-grade histologic transformation of low-grade endometrial stromal sarcoma (LGESS) is rare. Here, we describe the clinicopathologic features and gene fusion status of 12 cases (11 primary uterine corpus and 1 primary vaginal), 11 diagnosed prospectively from 2016, and 1 retrospectively collected. Targeted RNA sequencing and/or fluorescence in situ hybridization was employed in all cases. High-grade transformation was seen at the time of initial diagnosis in eight patients and at the time of recurrence in four patients, 4–11 years after initial diagnosis of LGESS. High-grade morphology consisted of generally uniform population of round to epithelioid cells with enlarged nuclei one to two times larger than a lymphocyte, visible nucleoli, and increased mitotic index (range, 6–30; median, 16 per 10 high-power fields); there was often an associated sclerotic and/or myxoid stroma. Estrogen receptor, progesterone receptor, and CD10 expression was absent or significantly decreased (compared with the low-grade component) in the high-grade foci of five tumors. One tumor demonstrated positive (diffuse and strong) cyclin D1 and BCOR staining. p53 staining was wild type in both components of all eight tumors tested. JAZF1-SUZ12(n= 6), JAZF1-PHF1(n= 3), EPC1-PHF1, (n= 1), or BRD8-PHF1(n= 1) fusions were detected in 11 tumors; no fusions were found in one by targeted RNA sequencing. Patients presented with FIGO stages I (n= 4), II (n= 4), III (n= 1), and IV disease (n= 2). Median overall survival calculated from the time of histologic transformation was 22 months (range, 8 months to 8 years) with five patients who died of disease 8–18 months after transformation. High-grade transformation may occur in LGESS with JAZF1and PHF1rearrangements at the time of or years after initial diagnosis. Such high-grade transformation is characterized by nuclear enlargement, prominent nucleoli, and increased mitotic index compared with typical LGESS. Histologic high-grade transformation may herald aggressive behavior.

Until recently, undifferentiated round cell sarcomas (URCS) in infants have been considered a wastebasket diagnosis, composed of various pathologic entities and lacking consistent genetic alterations. The recent identification of recurrent BCORinternal tandem duplications (ITD) and less common alternative YWHAE–NUTM2B/Efusions in half of infantile URCS and the majority of so-called primitive myxoid mesenchymal tumors of infancy (PMMTI) suggests a common pathogenesis with clear cell sarcoma of the kidney which also harbors the same genetic alterations. These tumors also share a similar morphology and immunoprofile, including positivity for BCOR, cyclin D1, and SATB2. In this study, we investigate the largest cohort to date of genetically confirmed URCS and PMMTI with BCORITD or YWHAEfusions to better define their morphologic spectrum and clinical behavior. Twenty-eight cases harbored BCORITD and five YWHAEfusions, occurring in 29 infants and 4 children, 19 males and 14 females. Microscopically, 20 were classified as URCS and 13 as PMMTI. Follow-up was available in 25 patients, with 14 (56%) succumbing to their diseases at a mean duration of 18-months follow-up (range: 2–62). Six patients remained with no evidence of disease at a mean follow-up of 63 months (range: 4–192), four patients were still alive with disease (mean follow-up: 46 months, range: 4–120), and one died of other causes. Local recurrence and distant metastasis were each observed in 11/25 (44%) of the patients. The overall survival was 42% at 3 years and 34% at 5 years (median survival: 26 months). There was no statistically significant survival difference between cases diagnosed as URCS and PMMTI and between those with BCORITD and YWHAEfusions.

Pure intravascular growth of epithelioid hemangioma (EH) is exceptional. Herein, we report a series of 21 intravascular EHs, representing a potential serious diagnostic pitfall by mimicking malignant vascular neoplsms with epithelioid morphology. The tumors developed in 12 males and 4 females, aged from 11 to 71 years (mean age 40.2 years) with a predilection for the extremities (13 of 21, 61.9%), followed by the head and neck (8 of 21, 38.1%). Lesions ranged in size from 2 to 30 mm (mean size 13 mm). The most common presenting feature was a slowly growing nodule. Most neoplasms were solitary (13 of 16 patients, 81.2%) but three patients developed more than one intravascular EH (3 of 16, 18.8%). Treatment consisted of complete surgical excision and was generally curative. Follow-up was available for 13 lesions that had developed in ten patients (range 4–72 months, mean 27.3 months). No recurrences or development of additional tumors were observed. All 21 lesions developed in subcutaneous veins. Two morphological patterns of intravascular epithelioid endothelial cell proliferation were observed: (1) a lobular capillary hemangioma-like proliferation with variable formation of open vascular lumina and (2) a solid proliferation generally lacking open vascular spaces. A lobular capillary hemangioma-like pattern was the sole pattern in nine lesions, a mixed lobular hemangioma-like pattern, and solid pattern in eight and a pure solid pattern in four intravascular EHs. Mitotic activity in epithelioid endothelial cells ranged from 0 to 7 mitoses per 10 high-power field (mean 2.1 mitoses per 10 HPFs). Six lesions displayed brisk mitotic activity of five or more mitoses per 10 HPF (6 of 21, 28.5%). The number of mitoses was usually more prominent in areas with solid growth. Atypical mitoses were not observed. No intratumoral necroses were seen. Cytological atypia was mild (20 out of 21 cases). By immunohistochemistry, all tumors were positive for CD31 (14 out of 14) and ERG (5 out of 5). While all tested cases were FOS negative by immunohistochemistry (6 out of 6), one out of six cases (case 6) displayed FOSB nuclear positivity in about 30% of the lesional endothelial cells. Eight cases were analysed by FISH for the presence of FOSand FOSBgene rearrangements. While all cases were negative for FOSBrearrangements, a single case proved positive for FOSgene break-apart. In conclusion, intravascular growth of EH is not associated with adverse biological behavior. Solid intravascular proliferations of endothelial cells can mimic a malignant vascular tumor with epithelioid morphology. Nevertheless, intravascular EHs display mild cytological atypia coupled with low mitotic activity, and a lack of atypical mitoses, pronounced nuclear atypia, multilayering or tumor necrosis. Finally, the FOSgene is infrequently rearranged, and there are no FOSBgene abnormalities in this subset of EHs, suggesting a potential distinct pathogenesis than most classic EHs.

Supplemental Digital Content is available in the text.We report 8 cases of a distinctive, previously undescribed renal cell carcinoma associated with somatic mutations in the neurofibromin 2(NF2) gene. All patients were adults, ranging from 51 to 78 years of age and of cases of known sex 6 of 7 were males. The carcinomas were predominantly unencapsulated, and all had a rounded, nodular interface with the native kidney. The neoplasms were all solid with papillary architecture evident in most cases (7/8), while 1 was only tubular. All cases were biphasic, characterized by larger and smaller carcinoma cells. The smaller cells clustered around basement membrane material similar to the characteristic pattern of the t(6;11) renal cell carcinoma associated with TFEBgene fusions. In 6 of 8 carcinomas, branching nodules of small cells clustered around basement membrane material within larger acini yielding a distinctive glomeruloid pattern. In 6 of 8 carcinomas, the small cells were focally spindle-shaped and unassociated with the basement membrane material. The stroma was sclerotic in all 8 carcinomas, and all 8 contained psammoma bodies that were abundant in 2. In some carcinomas, focal or predominant areas had a less distinctive appearance; 2 had areas that resembled clear cell renal cell carcinoma, 2 had high-grade eosinophilic areas, while 1 had branching tubular architecture that resembled mucinous tubular and spindle cell carcinoma. Two carcinomas demonstrated cellular necrosis. Although we have minimal clinical follow-up, 1 case presented with distant metastasis, progressed and resulted in patient death. While NF2mutations may be found in other established renal cell carcinoma subtypes (often as secondary genetic alterations), they are potentially the genetic driver of this distinctive entity.

The BCOR family of tumors includes a number of undifferentiated sarcomas, occurring in various age groups and anatomic sites, characterized by a spindle and round cell phenotype and diffuse immunoreactivity for BCOR. Prior RNA sequencing data revealed that NTRK3was a top-upregulated gene in BCOR-CCNB3sarcomas. In this study, we investigate a large cohort of tumors harboring BCOR/YWHAEgenetic alterations for NTRK3 upregulation at both the mRNA and protein levels, compared with other sarcoma types. Pan-Trk immunohistochemistry was assessed for intensity and extent. A correlation between NTRK3 expression and the type of BCORalteration and BCOR immunoreactivity was also performed. Most soft tissue undifferentiated round cell sarcomas with YWHAEor BCORrearrangements or BCORinternal tandem duplications (ITD) showed NTRK3, but not NTRK1or NTRK2, upregulation by RNA sequencing data analysis. Cytoplasmic pan-Trk immunoreactivity was also observed in most soft tissue round cell sarcomas with YWHAErearrangements (100%), BCORITD (80%), and BCOR-CCNB3fusions (67%), as well as clear cell sarcomas of kidney (75%), another BCOR family tumor, and ossifying fibromyxoid tumors with ZC3H7B-BCORfusion (100%), with variable staining intensity and extent. Pan-Trk staining was also seen in solitary fibrous tumors (100%) and less frequently in synovial sarcoma and Ewing sarcoma, but rarely in other sarcomas tested. Tumors harboring rare fusion variants of BCOR, such as BCOR-CHD9, a novel fusion identified by targeted RNA sequencing, and KMT2D-BCOR, were also positive for pan-Trk staining and NTRK3overexpression. In conclusion, NTRK3upregulation resulting in pan-Trk overexpression is common in the BCOR family of tumors as well as in subsets of BCOR-expressing sarcomas through alternative mechanisms. The therapeutic implication of this finding awaits further investigation.

Adenosarcoma can mimic high-grade endometrial stromal sarcoma with ZC3H7B-BCORfusion that may show entrapped glands and often exhibits diffuse BCOR expression. We encountered diffuse BCOR expression in rare adenosarcomas and sought to define its frequency among a larger cohort of these tumors. BCOR immunohistochemistry was performed on archival formalin-fixed paraffin-embedded tumor tissue in 13 of 14 adenosarcomas with and without stromal overgrowth arising in the uterus or ovary. The staining intensity and percentage of positive tumor nuclei in the mesenchymal component were evaluated. Eleven cases with sufficient tumoral tissue were subjected to fluorescence in situ hybridization for the detection of BCOR, BCORL1, NUTM1, ZC3H7B, and JAZF1rearrangement. Three cases were subjected to targeted RNA sequencing. BCOR was expressed in 9 of 13 (70%) tumors, including 6 with and 3 without stromal overgrowth. Moderate to strong staining in >70% of cells was seen throughout in 1 low-grade and 6 high-grade tumors, 5 of which had stromal overgrowth. No staining was seen in 3 low-grade and 1 high-grade tumors with stromal overgrowth. One tumor demonstrating extensive sex cord-like differentiation and diffuse BCOR expression harbored JAZF1and BCORL1rearrangements. No BCORor BCORL1rearrangement was identified in the remaining tumors. BCOR expression is seen in most adenosarcomas with and without stromal overgrowth. BCORL1rearrangement is seen in rare tumors with diffuse BCOR expression. Assessment of BCORor BCORL1rearrangement status is required in adenosarcomas demonstrating BCOR expression.

Supplemental Digital Content is available in the text.Soft tissue tumors with GLI1gene fusions or amplifications have been recently described as a unique pathologic entity with an established risk of malignancy. We herein expand these findings by investigating a cohort of 11 head and neck lesions with GLI1alterations, including 8 from the tongue, for their clinicopathologic and molecular features. The tumors commonly affected males in their 30s (male:female ratio 2.7:1; range: 1 to 65). Tumors showed a multinodular growth pattern, nested architecture separated by a delicate, arborizing vascular network, monotonous round to ovoid nuclei, and clear cytoplasm. Tumor protrusion into vascular spaces was common. Genetic alterations were investigated by fluorescence in situ hybridization and/or targeted RNA sequencing. Seven tumors harbored GLI1fusions with the following partners: ACTB(n=4), PTCH1(n=2), or MALAT1(n=1). The remaining 4 cases showed coamplifications of GLI1with CDK4and MDM2genes. Tumors were commonly positive for S100 protein and CD56. CDK4, MDM2, and STAT6 were positive in GLI1-amplified tumors. Two of 6 patients with available follow-up (1 each with GLI1amplification and PTCH1-GLI1fusion) developed distant metastases. Both tumors showed a high mitotic index and tumor necrosis. The head and neck region, particularly tongue, is a common location for GLI1-related mesenchymal tumors. Although a morphologic overlap was noted with the previously reported “pericytoma with t(7,12) translocation,” often occurring in the tongue, our findings expand the original findings, to include a more variable immunophenotype, propensity for late distant metastases, and alternative mechanisms of GLI1 oncogenic activation, such as various GLI1fusion partners or GLI1coamplifications with MDM2and CDK4genes.

NK3 homeobox 1 (NKX3-1) is widely accepted as a highly sensitive and specific marker for prostatic adenocarcinoma. Prompted by published transcriptome data showing upregulation of NKX3-1mRNA expression in EWSR1-NFATC2sarcoma, we explored the utility of NKX3-1 immunohistochemistry in sarcoma diagnosis. We applied NKX3-1 immunohistochemistry to 11 EWSR1-NFATC2sarcomas and 168 mimics using whole tissue sections. All EWSR1-NFATC2sarcomas consisted of uniform small round or ovoid cells, all except 1 showing at least focally the typical growth pattern of nests, cords, or trabeculae within a fibrous/myxoid background. A variable eosinophilic infiltrate was common. NKX3-1 was expressed in 9 of 11 (82%) EWSR1-NFATC2sarcomas, often diffuse and of moderate or strong intensity. All 12 mesenchymal chondrosarcomas tested were also positive for NKX3-1, with over half showing diffuse staining and moderate or strong intensity. The positive staining was seen only in the primitive small round cell component, whereas the cartilaginous component was mostly negative. Although 1 of 30 osteosarcomas showed focal NKX3-1 positivity, all the remaining 155 cases tested, including 20 Ewing sarcomas, 20 myoepithelial tumors, 11 ossifying fibromyxoid tumors, and 1 FUS-NFATC2sarcoma were negative for NKX3-1. Our study provides the first evidence that EWSR1-NFATC2sarcoma and Ewing sarcoma could be distinguished immunohistochemically, adding to the accumulating data that these tumors are phenotypically distinct. We suggest that NKX3-1 may have a diagnostic utility in the evaluation of sarcoma and we also call attention to potential pitfalls in the use of this well-known marker of prostatic adenocarcinoma.

Epithelioid hemangioendothelioma is a low-grade malignant vascular tumor with an intermediate clinical behavior between benign hemangiomas and high-grade angiosarcomas. Pathologic or molecular factors to predict this clinical heterogeneity are not well defined. A WWTR1-CAMTA1fusion is present in most classic epithelioid hemangioendothelioma, regardless of their clinical behavior, suggesting that additional genetic abnormalities might be responsible in driving a more aggressive biology. A small subset of cases show distinct morphology and are characterized genetically by a YAP1-TFE3fusion. Two histologic grades have been described in classic epithelioid hemangioendothelioma of the soft tissue. However, proposed criteria do not apply to other clinical presentations and have not been assessed in the YAP1-TFE3positive tumors. Furthermore, no previous studies have compared the survival of these two molecular subsets. In this study we investigate the clinicopathologic and molecular findings of a large cohort of 93 translocation-positive epithelioid hemangioendothelioma managed at our institution. Patient characteristics, histologic features, treatment outcomes, and genetic abnormalities were investigated and these factors were correlated with overall survival. In 18 patients (15 with WWTR1-CAMTA1and 3 with YAP1-TFE3) Memorial Sloan Kettering-IMPACT targeted DNA sequencing was performed to identify secondary genetic alterations showing more than half of tumors had a genetic alteration beyond the disease-defining gene fusion. Patients with conventional epithelioid hemangioendothelioma with WWTR1-CAMTA1fusion had a less favorable outcome compared with the YAP1-TFE3subset, the 5-year overall survival being 59% versus 86%, respectively. Soft tissue epithelioid hemangioendothelioma were frequently solitary, followed an uneventful clinical course being often managed with curative surgery. Multifocality, pleural involvement, lymph node or distant metastases had a significantly worse outcome. Patients with pleural disease or lymph node metastases had an aggressive clinical course akin to high-grade sarcomas, with 22% and 30%, respectively, alive at 5 years, compared with >70% survival rate in patients lacking these two adverse factors.

IMPORTANCE: Patients with advanced sarcoma have limited treatment options. Talimogene laherparepvec (T-VEC) has been shown to increase tumor-specific immune activation via augmenting antigen presentation and T-cell priming. OBJECTIVE: To examine whether T-VEC in combination with pembrolizumab is associated with increased tumor-infiltrating lymphocyte infiltration and programmed death-ligand 1 expression and thus with increased antitumor activity in patients with locally advanced or metastatic sarcoma. DESIGN, SETTING, AND PARTICIPANTS: This open-label, single-institution phase 2 interventional trial of T-VEC plus pembrolizumab enrolled 20 patients with locally advanced or metastatic sarcoma between March 16 and December 4, 2017, for whom at least 1 standard systemic therapy had failed. The median duration of therapy was 16 weeks (range, 7-67 weeks). Reported analyses include data through December 14, 2018. INTERVENTION: Patients received pembrolizumab (200-mg flat dose) intravenously and T-VEC (first dose, ≤4 mL × 106 plaque-forming units [PFU]/mL; second and subsequent doses, ≤4 mL × 108 PFU/mL) injected into palpable tumor site(s) on day 1 of each 21-day cycle. MAIN OUTCOMES AND MEASURES: The primary end point was objective response rate (ORR; complete response and partial response) at 24 weeks determined by Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1, criteria. Secondary end points included best ORR by immune-related RECIST criteria, progression-free survival rate at 24 weeks, overall survival, and safety. RESULTS: All 20 patients (12 women [60%]; median age, 63.5 years [range, 24-90 years]) were evaluable for response. The study met its primary end point of evaluating the best ORR at 24 weeks determined by RECIST, version 1.1, criteria; the best ORR was 30% (95% CI, 12%-54%; n = 6). The ORR overall was 35% (95% CI, 15%-59%; n = 7). The incidence of grade 3 treatment-related adverse events was low (4 patients [20%]). There were no grade 4 treatment-related adverse events or treatment-related deaths. CONCLUSIONS AND RELEVANCE: In this phase 2 clinical trial, treatment with T-VEC plus pembrolizumab was associated with antitumor activity in advanced sarcoma across a range of sarcoma histologic subtypes, with a manageable safety profile. This combination therapy met its predefined primary study end point; further evaluation of T-VEC in combination with pembrolizumab for patients with select sarcoma subtypes is planned. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03069378