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Recurrent fusions between OGT and members of the Forkhead box (FOXO) family of genes have been recently described in three cases of hyalinizing epithelioid acral soft tissue tumors in young adults showing co-expression for EMA and CD34. Despite the lack of an established myoepithelial lineage by immunohistochemistry, these lesions have been labeled as myoepithelioma-like due to their epithelioid phenotype and sclerotic background. In this study, we report a novel FOXO4-OGT fusion identified by targeted RNA sequencing in an unclassified shoulder soft tissue mass in a 40-year-old male. The tumor showed nodular foci of increased cellularity in a uniformly hyalinized background. The neoplastic cells were mainly epithelioid and focally spindled, with eosinophilic cytoplasm and indented nuclei with mild atypia. The tumor lacked significant mitotic activity and necrosis. Immunohistochemically, the tumor showed variable positivity for EMA, pan-CK, CD34, ERG and FLI1, while it was negative for CD31, S100, SOX10, desmin, and MUC4. INI1 expression was retained. Due to its unusual histology and conflicting immunoprofile, TruSight RNA fusion panel sequencing was performed which revealed a fusion between FOXO4 exon 2 to OGT exon 2. This is the first example of a soft tissue lesion harboring OGT-related fusions occurring in a non-acral location and associated with FOXO4 gene. Its line of differentiation and biologic potential remain uncertain.

Contains fulltext : 245199.pdf (Publisher’s version ) (Open Access) Undifferentiated mesenchymal tumors arising from the inner lining (intima) of large arteries are classified as intimal sarcomas (ISA) with MDM2 amplification 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 MDM2 amplifications in both neoplasms. However, little is known about ISAs and cardiac UPS without MDM2 amplifications 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/B was 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 MDM4 and CDK6 amplifications in ISAs and UPS of the left atrium, lacking MDM2 amplification. 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.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications., This work was funded by Deutsche Krebshilfe grant 70112499, the NCT Heidelberg and an Illumina Medical Research Grant. Part of this work was funded by the National Institute of Health Research (to S.B. and Z.J.) and to UCLH Biomedical research centre (BRC399/NS/RB/101410). Human tissues were obtained from University College London NHS Foundation Trust as part of the UK Brain Archive Information Network (BRAIN UK, Ref: 18/004) which is funded by the Medical Research Council and Brain Tumour Research UK. The methylation profiling at NYU is supported by a grant from the Friedberg Charitable Foundation (to M.Sn.). M.Mi. would like to thank the Luxembourg National Research Fond (FNR) for the support (FNR PEARL P16/BM/11192868 grant). Open Access funding enabled and organized by Projekt DEAL.

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 BRAF V600E mutations, while PDGFRB mutations 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 NOTCH1-4 and MIR143 gene 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 NOTCH genes are seen in over half of GT, with NOTCH2-MIR143 being 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.

PURPOSE To detect alterations in DNA damage repair (DDR) genes, measure homologous recombination deficiency (HRD), and correlate these findings with clinical outcome in patients with leiomyosarcoma (LMS). PATIENTS AND METHODS Patients with LMS treated at Memorial Sloan Kettering (MSK) Cancer Center who consented to prospective targeted next-generation sequencing with MSK-IMPACT were screened for oncogenic somatic variants in one of 33 DDR genes; where feasible, an experimental HRD score was calculated from IMPACT data. Progression-free survival (PFS) and overall survival (OS) were estimated after stratifying patients by DDR gene alteration status and HRD score. RESULTS Of 211 patients with LMS, 20% had an oncogenic DDR gene alteration. Univariable analysis of PFS in 117 patients who received standard frontline chemotherapy in the metastatic setting found that an altered homologous recombination pathway gene was significantly associated with shorter PFS (hazard ratio [HR], 1.79; 95% CI, 1.04 to 3.07; P = .035). Non- BRCA homologous recombination gene alteration was associated with shorter PFS (HR, 2.61; 95% CI, 1.35 to 5.04; P = .004) compared with BRCA-altered and wild-type homologous recombination genes. Univariable analysis of OS from diagnosis in the entire cohort of 211 patients found that age, tumor size, number of metastatic sites, localized disease, and non- BRCA homologous recombination gene alteration were significantly associated with OS. On multivariable analysis, non- BRCA homologous recombination pathway gene alteration remained significant (HR, 4.91; 95% CI, 2.47 to 9.76; P < .001). High HRD score was not associated with a different PFS or OS. CONCLUSION Patients with LMS with homologous recombination pathway gene alterations have poor clinical outcomes, particularly those with non- BRCA gene alterations. HRD score calculated from a targeted exome panel did not discern disparate clinical outcomes.

SMARCB1-deficient sinonasal carcinoma (SNC) is an aggressive malignancy characterized by INI1 loss mostly owing to homozygous SMARCB1 deletion. With the exception of a few reported cases, these tumors have not been thoroughly studied by massive parallel sequencing (MPS). A retrospective cohort of 22 SMARCB1-deficient SNCs were studied by light microscopy, immunohistochemistry, fluorescence in situ hybridization (n = 9), targeted exome MPS (n = 12), and Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) (n = 10), a bioinformatics pipeline for copy number/zygosity assessment. SMARCB1-deficient SNC was found in 13 (59%) men and 9 (41%) women. Most common growth patterns were the basaloid pattern (59%), occurring mostly in men (77%), and plasmacytoid/eosinophilic/rhabdoid pattern (23%), arising mostly in women (80%). The former group was significantly younger (median age = 46 years, range = 24-54, vs 79 years, range = 66-95, p 0.0001). Clear cell, pseudoglandular, glandular, spindle cell, and sarcomatoid features were variably present. SMARCB1-deficient SNC expressed cytokeratin (100%), p63 (72%), neuroendocrine markers (52%), CDX-2 (44%), S-100 (25%), CEA (4/4 cases), Hepatocyte (2/2 cases), and aberrant nuclear β-catenin (1/1 case). SMARCB1 showed homozygous deletion (68%), hemizygous deletion (16%), or truncating mutations associated with copy neutral loss of heterozygosity (11%). Coexisting genetic alterations were 22q loss including loss of NF2 and CHEK2 (50%), chromosome 7 gain (25%), and TP53 V157F, CDKN2A W110∗, and CTNNB1 S45F mutations. At 2 years and 5 years, the disease-specific survival and disease-free survival were 70% and 35% and 13% and 0%, respectively. SMARCB1-deficient SNCs are phenotypically and genetically diverse, and these distinctions warrant further investigation for their biological and clinical significance.

Nasopharyngeal adenocarcinoma is a rare malignancy that is classified into conventional/surface- and salivary-types. Herein we report the case of a 52-year-old male who presented with a right nasopharyngeal mass and right-sided hearing loss. Diagnostic imaging revealed a circumscribed 1.7 cm mass centred in the right antero-lateral aspect of the nasopharynx. A biopsy showed a gland-forming neoplasm that was in continuity with the surface epithelium. The tumor exhibited a nested to micro-papillary architecture, with mild cytologic atypia. Immunohistochemistry demonstrated diffuse staining for CK7, SOX10, and p16; the abluminal layer was highlighted by CK5 and p63, while the luminal cells expressed CD117. The tumor was not amenable to subclassification and was diagnosed as a low-grade nasopharyngeal adenocarcinoma, not otherwise specified (NOS). Subsequent RNA sequencing was performed which identified a novel GOLGB1-BRAF fusion product. Based on its unique morphology and molecular findings, this is presumed to represent a novel subtype of nasopharyngeal adenocarcinoma. In addition to being of diagnostic relevance, this fusion may ultimately represent a potential therapeutic target.

INTRODUCTION: Neoadjuvant imatinib (Neo-IM) therapy may facilitate R0 resection in primary gastrointestinal stromal tumors (GISTs) that are large or in difficult anatomic locations. While response to preoperative tyrosine kinase inhibitors is associated with better outcome in metastatic GIST, little is known about prognostic factors after Neo-IM in primary GIST. STUDY DESIGN: Patients with primary GIST with or without synchronous metastases who underwent Neo-IM were retrospectively analyzed from a prospective maintained institutional database for Response Evaluation Criteria in Solid Tumors (RECIST), tumor viability, and mitotic rate. Overall survival (OS) was estimated by Kaplan-Meier and compared by log-rank test. Cox proportionate hazard models were used for univariate and multivariate analysis. RESULTS: One hundred and fifty patients were treated for a median of 7.1 months (range 0.2–160). By RECIST, partial response, stable disease, and progressive disease were seen in 40%, 51%, and 9%, respectively. By pathologic analysis, ≤50% of the tumor was viable in 72%, and the mitotic rate was ≤5/50HPF in 74%. On multivariate analysis, RECIST response and tumor viability were not associated with OS, while post-treatment high mitotic rate (hazard ratio [HR] for death 5.3, CI 2.3–12.4), R2 margins (HR 6.0, CI 2.3–15.5), and adjuvant imatinib (HR 0.4, CI 0.2–0.9) were (p

The genetic hallmark of epithelioid hemangioendothelioma (EHE) is a recurrent WWTR1-CAMTA1 fusion, which is present in most cases bearing a conventional histology. A subset of cases is characterized by a distinct morphology and harbors instead of YAP1-TFE3 fusion. Nevertheless, isolated cases lack these canonical fusions and remain difficult to classify. Triggered by an index case of a left atrial mass in a 76-year-old female with morphologic features typical of EHE, but which showed a WWTR1-MAML2 fusion by targeted RNA sequencing, we searched our files for similar cases displaying alternative WWTR1 fusions. A total of 6 EHE cases were identified with variant WWTR1 fusions, four of them presenting within the heart. There were three females and three males, with a wide age range at diagnosis (21-76 years, mean 62, median 69). The four cardiac cases occurred in older adults (mean age of 72, equal gender distribution); three involved the left atrium and one the right ventricle. One case presented in the vertebral bone and one in pelvic soft tissue. Microscopically, all tumors had morphologic features within the spectrum of classic EHE; two of the cases appeared overtly malignant. All cases were tested by FISH and four were investigated by targeted RNA sequencing. Two tumors harbored WWTR1-MAML2 fusions, one WWTR1-ACTL6A, and in three cases, no WWTR1 partner was identified. Of the four patients with follow-up, two died of disease, one was alive with lung metastases, and the only patient free of disease was s/p resection of a T11 vertebral mass. Our findings report on additional genetic variants involving WWTR1 rearrangements, with WWTR1-MAML2 being a recurrent event, in a small subset of EHE, which appears to have predilection for the heart.

Adenosarcoma can mimic high-grade endometrial stromal sarcoma with ZC3H7B-BCOR fusion 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 JAZF1 rearrangement. 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 JAZF1 and BCORL1 rearrangements. No BCOR or BCORL1 rearrangement was identified in the remaining tumors. BCOR expression is seen in most adenosarcomas with and without stromal overgrowth. BCORL1 rearrangement is seen in rare tumors with diffuse BCOR expression. Assessment of BCOR or BCORL1 rearrangement status is required in adenosarcomas demonstrating BCOR expression.

Pediatric soft tissue tumors are relatively rare and show significant overlap in morphology and immunoprofile, often posing diagnostic and management challenges. Thus, their classification remains often subjective or lumped under "unclassified categories," as a number of lesions lack objective and reproducible criteria in diagnosis. Although in a subset of cases immunohistochemistry has been proved useful to identify a specific line of differentiation, most tumors lack a readily defined histogenesis, being characterized by a rather non-specific immunoprofile. Furthermore, tumors with an ambiguous diagnosis are difficult to grade and their risk of malignancy or clinical management remains uncertain. Advances in molecular genetics, including the more wide application of next generation sequencing in routine clinical practice, have improved diagnosis and refined classification based on objective molecular markers. Importantly, some soft tissue tumors in children are characterized by recurrent gene fusions involving either growth factors (eg, PDGFB) or protein kinases (eg, ALK, ROS, NTRK, BRAF), which have paved the way for new targeted treatments that block the respective upregulated downstream pathways. However, the majority of gene fusions or mutations detected in soft tissue tumors result in an abnormal function of transcription factors or chromatin remodeling. The present review focuses on the latest genetic discoveries in the spectrum of both benign and malignant pediatric soft tissue neoplasia. These genetic abnormalities promise to provide relevant insight for their proper classification, prognosis, and treatment. The entities discussed herein are grouped either based on their shared genetic mechanism or based on their presumed line of differentiation.

Uterine tumor resembling ovarian sex-cord tumor (UTROSCT) is a rare and distinctive neoplasm of unclear histogenesis, and uncertain malignant potential. These neoplasms morphologically resemble sex-cord stromal tumors of the ovary, and possess a polyphenotypic immunophenotype. Their molecular pathogenesis has yet to be elucidated; notably, however, tumors lack alterations found in other uterine tumors bearing sex-cord-like differentiation, such as endometrial stromal sarcoma. Following identification of an index patient with an ESR1-NCOA3 fusion gene by RNA-sequencing, we undertook a retrospective review for additional cases of UTROSCT. We identified a total of 4 patients, with an average age of 53 years (range, 38 to 68 y). RNA-sequencing was performed in all cases, revealing an ESR1-NCOA3 fusion in 2 cases and one case each with related ESR1-NCOA2 and GREB1-NCOA2 fusions. Each of the tumors showed histologic and an immunophenotype features within the previously reported spectrum of UTROSCT; interestingly, one case contained prominent spindle cell fascicles and another was largely comprised of sheets of small round cells. Our results demonstrate UTROSCT are defined by recurrent fusions involving NCOA2 or NCOA3, a finding that is directly amenable to diagnostic evaluation. This study confirms UTROSCT is molecularly distinct from endometrial stromal sarcoma, and raises intriguing new questions into the pathogenesis of these neoplasms and possible relationship with other NCOA fusion-positive uterine tumors.

Despite significant recent advances in characterizing the molecular pathogenesis of undifferentiated round cell neoplasms, rare cases remain unclassified. Here, we report two distinctive undifferentiated round cell tumors occurring in young adults. One tumor presented intrabdominally and the other arose within the abdominal wall. One patient died of disease following local and distance recurrence, despite aggressive chemotherapy and radiotherapy. Morphologically, both tumors were similarly composed of primitive round to epithelioid cells arranged in nests, sheets, and trabecular patterns. The cytoplasm was scant and amphophilic, while the nuclei were round and uniform with brisk mitotic activity. Focal necrosis was present. Immunohistochemically, both tumors were variably positive for S100 and EMA, and one case focally expressed cytokeratin and TLE1. Targeted RNA sequencing revealed in both an identical SS18-POU5F1 fusion gene. Fluorescence in situ hybridization was performed which confirmed SS18 and POU5F1 gene rearrangements. Expression data, relative to over 200 other mesenchymal neoplasms that had undergone targeted RNA sequencing on the same platform, suggested the SS18-POU5F1 tumors cluster with EWSR1/FUS-POU5F1-positive myoepithelial tumors. In view of our limited sample size, additional studies are needed to characterize the breadth of clinical and pathologic findings in these neoplasms. In addition, further investigation is necessary to determine whether this entity represents a clinically aggressive and phenotypically undifferentiated variant of myoepithelial tumors, or perhaps an altogether novel category of undifferentiated round cell sarcoma.

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 KIT V559Δ 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 KIT V654A mutation. To determine the biologic impact of this second-site mutation in vivo, we created a mouse model with the corresponding V558Δ;V653A Kit double 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 V558Δ;V653A Kit double mutation were resistant to imatinib, while cabozantinib was more effective in overcoming resistance than sunitinib. Compared to control mice with a single V558Δ Kit mutation, mice with a double V558Δ; V653A Kit mutation 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.

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 TFEB gene 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 NF2 mutations 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 genetic hallmark of classic Ewing sarcoma is a recurrent fusion between EWSR1 and FUS gene with a member of the ETS transcription factor family. In contrast, tumors with non-ETS gene partners have been designated until recently "Ewing-like sarcoma," as a provisional molecular entity, as their clinical and pathologic features were still evolving. However, this group was reclassified as "round cell sarcoma with EWSR1-non-ETS fusions" in the latest 2020 WHO classification. Moreover, round cell sarcomas with either CIC or BCOR gene abnormalities, initially classified under Ewing family of tumors, are now regarded as stand-alone pathologic entities based on their distinct features. In this study we investigated the clinical characteristics of 226 confirmed Ewing sarcoma patients (EWSR1-FLI1 [n = 176], EWSR1/FUS-ERG [n = 35], EWSR1/FUS-FEV [n = 12], and EWSR1-ETV1/4 [n = 3]) and 14 round cell sarcoma patients with EWSR1-non-ETS fusion (EWSR1/FUS-NFATC2 [n = 10], EWSR1-PATZ1 [n = 3], and EWSR1-VEZF1 [n = 1]). The impact on overall survival (OS) was assessed in 90 patients with available follow-up, treated between 2011 and 2018. Patients with fusions involving FEV and NFATC2 genes showed an older median age at diagnosis, compared to those with EWSR1-FLI1 (P = .005), while extraskeletal location was more common in tumors with noncanonical EWSR1-FLI1 fusions (P = .001). Axial and pelvic primary sites were more common in patients with EWSR1-FLI1 (72%), while tumors with NFATC2 fusions were more frequent in the limb (78%, P = .006). The 3-year OS in patients with EWSR1-FLI1 was 91%, compared to only 60% in patients with alternative fusions (P = .037); the latter group showing a higher rate of metastases at presentation. However, this OS difference was not significant in patients with localized tumor (P = .585). Our study demonstrates significant correlations between fusion subtype and age at presentation, primary tumor sites, and OS, in both conventional Ewing sarcoma and round cell sarcoma with EWSR1-non ETS fusions patients. Larger studies are needed to determine survival differences in localized tumors.

A recent breakthrough in the classification of soft tissue tumors (STT) has been a significant expansion in the number of neoplasms associated with NTRK and other kinase related fusions. This is important not only for diagnostic purposes, but also because it opens new avenues for targeted therapy. Indeed, recent clincal trials have shown significant benefit across multiple tumor-types, prompting approval of NTRK inhibitors for clinical use in the setting of advanced/metastatic NTRK-rearranged neoplasms. Despite these therapeutic oportunities, diagnostic challenges have transpired in recognizing these emerging new histologic subtypes of kinase fusions positive-STT prospectively. This, in part, is attributable to their wide morphologic spectrum, variable risk of malignancy, and non-specific immunoprofile. As such, recommendations for pathologic criteria and immunohistochemical testing are needed to improve classification and streamline the small subset of potential candidates for further molecular validation. This overview summarizes the key histologic features of various STT associated with NTRK and other kinase fusions, which appear to share a similar morphologic spectrum. Immunohistochemically, many of these tumors, regardless of the kinase fusion type, notably show co-expression of S100 and CD34; issues related to the utility of pan-NTRK and NTRK1 immunostaining are therefore summarized. Finally, I discuss the role of confirmatory molecular testing and how, in some instances, this may also be of prognostic value. This review is intended as a critical summary of the current literature to emphasize pathologic criteria for improving recognition of this emerging and complex group of kinase fusion associated STT.

Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare primary bone sarcoma that lacks a specific line of differentiation. There is very little information about the genetic alterations leading to tumourigenesis or malignant transformation. Distinguishing between UPSb and other malignant bone sarcomas, including dedifferentiated chondrosarcoma and osteosarcoma, can be challenging due to overlapping features. To explore the genomic and transcriptomic landscape of UPSb tumours, whole-exome sequencing (WES) and RNA sequencing (RNA-Seq) were performed on UPSb tumours. All tumours lacked hotspot mutations in IDH1/2 132 or 172 codons, thereby excluding the diagnosis of dedifferentiated chondrosarcoma. Recurrent somatic mutations in TP53 were identified in four of 14 samples (29%). Moreover, recurrent mutations in histone chromatin remodelling genes, including H3F3A, ATRX and DOT1L, were identified in five of 14 samples (36%), highlighting the potential role of deregulated chromatin remodelling pathways in UPSb tumourigenesis. The majority of recurrent mutations in chromatin remodelling genes identified here are reported in COSMIC, including the H3F3A G34 and K36 hotspot residues. Copy number alteration analysis identified gains and losses in genes that have been previously altered in UPSb or UPS of soft tissue. Eight somatic gene fusions were identified by RNA-Seq, two of which, CLTC-VMP1 and FARP1-STK24, were reported previously in multiple cancers. Five gene fusions were genomically characterised. Hierarchical clustering analysis, using RNA-Seq data, distinctly clustered UPSb tumours from osteosarcoma and other sarcomas, thus molecularly distinguishing UPSb from other sarcomas. RNA-Seq expression profiling analysis and quantitative reverse transcription-polymerase chain reaction showed an elevated expression in FGF23, which can be a potential molecular biomarker for UPSb. To our knowledge, this study represents the first comprehensive WES and RNA-Seq analysis of UPSb tumours revealing novel protein-coding recurrent gene mutations, gene fusions and identifying a potential UPSb molecular biomarker, thereby broadening the understanding of the pathogenic mechanisms and highlighting the possibility of developing novel targeted therapeutics. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pseudomyogenic hemangioendothelioma (PHE) is an uncommon, rarely metastasizing vascular neoplasm with predilection to affect young adults. The tumors often present as multiple nodules involving various tissue planes, including superficial and deep soft tissues as well as bone. Recurrent SERPINE1-FOSB gene fusions have been reported as the hallmark genetic abnormality in PHE, however, in our experience, a number of cases with typical histology lack this genetic abnormality. In this study, we identify a novel ACTB-FOSB gene fusion, which is as prevalent as the initial translocation reported. We selected 15 consecutive cases of PHE with typical morphologic features which had material for molecular testing. The cohort included 10 males and 5 females, ranging in age from 17 to 58 years (median age: 33 y; mean age: 35.3 y). Eight (53%) cases were located in the lower extremities (foot, calf, tibia, thigh), 5 (33%) were located in the trunk, abdomen or pelvis (abdominal wall-2, shoulder, back, ischium) and 2 (13%) were located in the upper extremity (humerus and hand). Ten (67%) cases had multifocal presentation and 5 (33%) presented as solitary lesions. Three (20%) cases were located only in the superficial dermis and subcutaneous tissues, 4 (27%) involved the superficial and deep soft tissue and 8 (53%) cases involved only the deep soft tissue and bone. Using fluorescence in situ hybridization and ARCHER fusionplex analysis we identified a novel ACTB-FOSB gene fusion in 7 cases, while the remaining 8 had the previously described SERPINE1-FOSB fusion. The clinicopathologic features and behavior of PHE associated with the ACTB-FOSB gene fusion were similar to those harboring the SERPINE1-FOSB; except that tumors with the ACTB variant were more often associated with solitary presentation. In conclusion, our results expand the spectrum of genetic alterations in PHE with a novel gene fusion identified in half of the cases. We speculate that some of the novel targeted therapies that have shown promise in SERPINE1-FOSB-positive PHE might also be beneficial in this molecular subset.

We describe two cases of a distinct sarcoma characterized by a novel MEIS1-NCOA2 gene fusion. This gene fusion was identified in the renal neoplasms of two adults (21 year-old male, 72 year-old female). Histologically, the resected renal neoplasms had a distinctively nodular appearance, and while one renal neoplasm was predominantly cystic, the other demonstrated solid architecture, invasion of perirenal fat, and renal sinus vasculature invasion. The neoplasms were characterized predominantly by monomorphic plump spindle cells arranged in vague fascicles with a whorling pattern; however, a more primitive small round cell component was also noted. Both neoplasms were mitotically active and one case showed necrosis. The neoplasms did not have a distinctive immunohistochemical profile, though both labeled for TLE1. The morphologic features are distinct from other sarcomas associated with NCOA2 gene fusions, including mesenchymal chondrosarcoma, congenital/infantile spindle cell rhabdomyosarcoma, and soft tissue angiofibroma. While we have minimal clinical follow-up, the aggressive histologic features of these neoplasms indicate malignant potential, thus warranting classification as a novel subtype of sarcoma.

OBJECTIVE. To describe the imaging features of plexiform fibrohistiocytic tumor and its associated clinical findings. MATERIALS AND METHODS. An institutional database was searched to identify all patients with a pathological diagnosis of plexiform fibrohistiocytic tumor. The electronic medical record was reviewed for relevant clinical data. Radiologic images of the primary tumor site were reviewed by two radiologists to assess primary, residual, or recurrent tumor with respect to tumor location, size, morphology, MR signal characteristics and enhancement, and involvement of adjacent structures. RESULTS. Thirteen patients with imaging of the primary tumor site were identified (8 female, 5 male; mean age, 15.9 years (range, 3–41 years)). Plexiform fibrohistiocytic tumor typically manifested as a solitary, painless, firm, slow-growing lesion centered in the subcutaneous tissues, with a predilection for the upper extremity or head and neck region. Most tumors had a purely plaque-like or infiltrative morphology at MRI; some demonstrated no round or oval mass. Tumors were predominantly isointense to muscle on T1-weighted imaging and hyperintense on fluid-sensitive imaging, and enhanced after gadolinium contrast administration. Five patients (38%) had residual tumor after initial surgery, resembling postoperative changes. No patient had recurrent tumor. One patient (8%) developed metastases, to local lymph nodes and to lung. No patient died from plexiform fibrohistiocytic tumor. CONCLUSION. Plexiform fibrohistiocytic tumor often manifests as a plaque-like or infiltrative process, sometimes without a round or oval mass, most commonly in the subcutaneous tissues of the upper extremity or head and neck region. Residual tumor is often present after initial surgery, indistinguishable from postoperative changes.

Background Preclinical studies suggest that imatinib resistance in gastrointestinal stromal tumor (GIST) can be mediated by MAP-kinase activation via fibroblast growth factor (FGF) signaling. In FGF stimulated GIST cell lines, BGJ398, a pan-FGFR kinase inhibitor in combination with imatinib, was cytotoxic and superior to imatinib therapy alone. In FGF-dependent GIST, the combination of BGJ398 and imatinib may provide a mechanism to overcome imatinib resistance. Methods This phase Ib study of BGJ398 and imatinib was performed in patients with imatinib refractory advanced GIST. A standard 3 + 3 dosing schema was utilized to determine the recommended phase II dose (RP2D). Two treatment schedules were evaluated incorporating imatinib 400 mg daily in combination with (A) BGJ398 daily 3 weeks on, 1 week off or (B) BGJ398 daily 1 week on, 3 weeks off. Results 16 patients enrolled. The median age was 54 years (range: 44-77), 81% were male, and the median number of lines of prior therapy was 4 [range: 2-6, 13 patients had ≥3 prior therapies]. 12 patients received treatment on schedule A [BGJ398 dose range: 25 - 75 mg]: 2 patients experienced dose limiting toxicities (DLT) (n = 1, myocardial infarction & grade (G)4 CPK elevation; n = 1, G3 ALT elevation) on schedule A (BGJ398 75 mg), significant hyperphosphatemia, an on-target effect, was not observed, implying the maximum tolerated dose was below the therapeutic dose. Following protocol amendment, 4 patients enrolled on schedule B [BGJ398 dose range: 75 - 100 mg]: no DLTs were observed. The most common treatment related adverse events occurring in >15% of patients included CPK elevation (50%), lipase elevation (44%), hyperphosphatemia (24%), anemia (19%), and peripheral edema (19%). Among the 12 evaluable patients, stable disease (SD) was the best response observed in 7 patients by RECIST v1.1 and 9 patients by CHOI. Stable disease ≥ 32 weeks was observed in 3 patients (25%). Median progression free survival was 12.1 weeks (95% CI 4.7-19.5 weeks). Conclusions Toxicity was encountered with the combination therapy of BGJ398 and imatinib. Due to withdrawal of sponsor support the study closed before the RP2D or dosing schedule of the combination therapy was identified. In heavily pre-treated patients, stable disease ≥ 32 weeks was observed in 3 of 12 evaluable patients. Trial Registration: NCT02257541 .

Undifferentiated solid tumors with small blue round cell histology and expression of CD99 mostly resemble Ewing sarcoma. However, they also may include other tumors such as mesenchymal chondrosarcoma, synovial sarcoma, or small cell osteosarcoma. Definitive classification usually requires detection of entity-specific mutations. While this approach identifies the majority of Ewing sarcomas, a subset of lesions remains unclassified and, therefore, has been termed "Ewing-like sarcomas" or small blue round cell tumors not otherwise specified. We developed an approach for further characterization of small blue round cell tumors not otherwise specified using an array-based DNA-methylation profiling approach. Data were analyzed by unsupervised clustering and t-distributed stochastic neighbor embedding analysis and compared with a reference methylation data set of 460 well-characterized prototypical sarcomas encompassing 18 subtypes. Verification was performed by additional FISH analyses, RNA sequencing from formalin-fixed paraffin-embedded material or immunohistochemical marker analyses. In a cohort of more than 1,000 tumors assumed to represent Ewing sarcomas, 30 failed to exhibit the typical EWS translocation. These tumors were subjected to methylation profiling and could be assigned to Ewing sarcoma in 14 (47%), to small blue round cell tumors with CIC alteration in 6 (20%), to small blue round cell tumors with BCOR alteration in 4 (13%), to synovial sarcoma and to malignant rhabdoid tumor in 2 cases each. One single case each was allotted to mesenchymal chondrosarcoma and adamantinoma. 12/14 tumors classified as Ewing sarcoma could be verified by demonstrating either a canonical EWS translocation evading initial testing, by identifying rare breakpoints or fusion partners. The methylation-based assignment of the remaining small blue round cell tumors not otherwise specified also could be verified by entity-specific molecular alterations in 13/16 cases. In conclusion, array-based DNA-methylation analysis of undifferentiated tumors with small blue round cell histology is a powerful tool for precisely classifying this diagnostically challenging tumor group.

To refine treatment recommendations for patients with metastatic gastrointestinal stromal tumors (GISTs) treated with tyrosine kinase inhibitors (TKIs) and surgery.Early reports suggested that patients with metastatic GIST responding to TKIs treated with surgery may have favorable outcomes. However, identification of prognostic factors was limited by small cohorts.Progression-free survival (PFS) and overall survival (OS) from time of surgery and from start of initial TKI was determined. Multivariate analysis was conducted on all patients undergoing GIST metastasectomy between 2001 and 2014 at 2 institutions.We performed 400 operations on 323 patients with metastatic GIST on TKIs. Radiographic response at time of surgery was classified as responsive disease (RD, n = 64, 16%), stable disease (SD, n = 100, 25%), unifocal progressive disease (UPD, n = 132, 33%), and multifocal progressive disease (MPD, n = 104, 26%). For patients on imatinib before surgery, radiographic response was predictive of PFS from time of surgery (RD 36 months, SD 30 months, UPD 11 months, MPD 6 months; P0.001) and from imatinib initiation (RD 71 months, SD 51 months, UPD 47 months, MPD 33 months; P0.001). Radiographic response was predictive of OS from time of surgery (RD not reached, SD 110 months, UPD 59 months, MPD 24 months; P0.001), and from imatinib initiation (RD not reached, SD 144 months, UPD 105 months, MPD 66 months; P = 0.005). Radiographic response was not predictive of PFS/OS for patients on sunitinib. Metastatic mitotic index ≥5/50 HPF, MPD, and R2 resection were prognostic of worse PFS/OS; primary mutation was not.Surgery in metastatic GIST patients in the absence of MPD on imatinib is associated with outcomes at least comparable with second-line sunitinib and may be considered in select patients.

NUT midline carcinoma is an aggressive tumor that occurs mainly in the head and neck and, less frequently, the mediastinum and lung. Following identification of an index case of a NUTM1 fusion positive undifferentiated soft tissue tumor, we interrogated additional cases of primary undifferentiated soft tissue and visceral tumors for NUTM1 abnormalities. Targeted next-generation sequencing was performed on RNA extracted from formalin-fixed paraffin-embedded tissue, and results validated by fluorescence in situ hybridization using custom bacterial artificial chromosome probes. Six patients were identified: mean age of 42 years (range, 3 to 71 y); equal sex distribution; and, tumors involved the extremity soft tissues (N=2), kidney (N=2), stomach, and brain. On systemic work-up at presentation all patients lacked a distant primary tumor. Morphologically, the tumors were heterogenous, with undifferentiated round-epithelioid-rhabdoid cells arranged in solid sheets, nests, and cords. Mitotic activity was generally brisk. Four cases expressed pancytokeratin, but in only 2 cases was this diffuse. Next-generation sequencing demonstrated the following fusions: BRD4-NUTM1 (3 cases), BRD3-NUTM1, MXD1-NUTM1, and BCORL1-NUTM1. Independent testing by fluorescence in situ hybridization confirmed the presence of NUTM1 and partner gene rearrangement. This study establishes that NUT-associated tumors transgress the midline and account for a subset of primitive neoplasms occurring in soft tissue and viscera. Tumors harboring NUTM1 gene fusions are presumably underrecognized, and the extent to which they account for undifferentiated mesenchymal, neuroendocrine, and/or epithelial neoplasms is unclear. Moreover, the relationship, if any, between NUT-associated tumors in soft tissue and/or viscera, and conventional NUT carcinoma, remains to be elucidated.

In recent years, undifferentiated small round cell sarcomas (USRCSs) have been divided into a variety of new, rare, sarcoma subtypes, including the group of Ewing-like sarcomas, which have the morphological appearance of Ewing sarcomas, but carry CIC-DUX4, BCOR-CCNB3 and other gene fusions different from the classic EWSR1-ETS gene fusion. Using high-throughput RNA-sequencing (RNA-seq) analyses, we identified a novel recurrent gene fusion, CRTC1-SS18, in two cases of USRCS that lacked any known translocation. RNA-seq results were confirmed by reverse transcription polymerase chain reaction, long-range polymerase chain reaction, and fluorescence in situ hybridization. In vitro, we showed that the cells expressing the gene fusion were morphologically distinct and had enhanced oncogenic potential as compared with control cells. Expression profile comparisons with tumours of other sarcoma subtypes demonstrated that both cases clustered close to EWSR1-CREB1-positive tumours. Moreover, these analyses indicated enhanced NTRK1 expression in CRTC1-SS18-positive tumours. We conclude that the novel gene fusion identified in this study adds a new subtype to the USRCSs with unique gene signatures, and may be of therapeutic relevance. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Item does not contain fulltext Benign/low-grade fibroblastic tumors encompass a broad spectrum of tumors with different morphologies and molecular genetic abnormalities. However, despite significant progress in recent genomic characterization, there are still tumors in this histologic spectrum that are difficult to classify, lacking known molecular characteristics. Triggered by a challenging congenital spindle cell neoplasm arising in the heel of a 1-year-old boy, we applied RNA sequencing for genetic discovery and identified a novel EWSR1-SMAD3 gene fusion. On the basis of the index case superficial acral location and fibroblastic appearance with a nonspecific immunophenotype, we searched our files for similar cases and screened them by fluorescence in situ hybridization for these abnormalities. Thus an identical EWSR1-SMAD3 fusion was identified in 2 additional spindle cell tumors with similar clinicopathologic features. Both cases occurred in the feet of adult women (58 and 61 y old) and were characterized by distinctive nodular growth with zonation pattern of peripheral hypercellular areas arranged in short fascicles, transitioning to hypocellular central areas of hyalinization and infarction. Focal stippled calcification in the collagenous area was present in 1 case. All 3 tumors had similar immunoprofiles, being negative for SMA, CD34, CD31, and S100, but showing consistent ERG positivity of uncertain significance. Follow-up information was available in 2 patients who developed local recurrences after incomplete initial excisions, at 5 and 14 months, respectively. None developed metastatic disease. In summary, we report a group of locally recurrent superficial acral tumors, characterized by bland spindle cell fascicular growth, occasional zonation pattern, ERG positivity, and recurrent EWSR1-SMAD3 gene fusions.

Tyrosine kinase inhibition of gastrointestinal stromal tumors (GIST) is effective but typically culminates in resistance and is rarely curative. Immunotherapy has potential application to GIST, as we previously showed that T-cell checkpoint blockade increases the antitumor effects of imatinib. Here, we showed that ligation of CD40 using an agonistic antibody (anti-CD40) activated tumor-associated macrophages (TAMs) in vivo in a knock-in mouse model of GIST harboring a germline mutation in Kit exon 11. Activated TAMs had greater TNFα production and NFκB signaling and directly inhibited tumor cells in vitro. Anti-CD40 required concomitant therapy with imatinib for efficacy and depended on TAMs, and to a lesser extent CD8+ T cells, but not on CD4+ T cells or B cells. In an analysis of 50 human GIST specimens by flow cytometry, we found that CD40 was expressed on human TAMs and tumor cells yet was downregulated after response to imatinib. CD40 ligation did not have a direct inhibitory effect on human GIST cells. Our findings provide the rationale for combining anti-CD40 and tyrosine kinase inhibition to treat human GIST. Cancer Immunol Res; 6(4); 434–47. ©2018 AACR.

High-grade endometrial stromal sarcoma likely encompasses underrecognized tumors harboring genetic abnormalities besides YWHAE-NUTM2 fusion. Triggered by three initial endometrial stromal sarcomas with ZC3H7B-BCOR fusion characterized by high-grade morphology and aggressive clinical behavior, we herein investigate the clinicopathologic features of this genetic subset by expanding the analysis to 17 such tumors. All of them occurred in adult women with a median age of 54 (range, 28-71) years. They were predominantly based in the endomyometrium and demonstrated tongue-like and/or pushing myometrial invasion. Most were uniformly cellular and displayed haphazard fascicles of spindle cells with mild to moderate nuclear atypia. Myxoid matrix was seen in 14 of 17 (82%) tumors, and collagen plaques were seen in 8 (47%). The mitotic index was ≥10 mitotic figures/10 high-power fields (HPFs) in 14 of 17 (82%) tumors with a median of 14.5 mitotic figures/10 HPFs. No foci of conventional or variant low-grade endometrial stromal sarcoma were seen. All tumors expressed CD10 with only limited or absent desmin, SMA and/or h-caldesmon staining. ER and PR expression in >5% of cells was seen in 4 of 12 (33%) tumors. Diffuse cyclin D1 and BCOR immunoreactivity was present in 7 of 8 (88%) and 7 of 14 (50%) tumors, respectively. Fluorescence in situ hybridization or targeted RNA sequencing confirmed ZC3H7B-BCOR fusion in all tumors, including four and two previously diagnosed as myxoid leiomyosarcoma and undifferentiated uterine sarcoma, respectively. Limited clinical data suggest that patients present at higher stage and have worse prognosis compared with published outcomes in low-grade endometrial stromal sarcoma. Tumors with ZC3H7B-BCOR fusion constitute a distinct group of endometrial stromal sarcomas with high-grade morphology that should be distinguished from other uterine mesenchymal neoplasms that may demonstrate myxoid morphology.

ACTB-GLI1 fusions have been reported as the pathognomonic genetic abnormality defining an unusual subset of actin-positive, perivascular myoid tumors, known as "pericytoma with the t(7;12) translocation." In addition, GLI1 oncogenic activation through a related MALAT1-GLI1 gene fusion has been recently reported in 2 unrelated gastric tumors, namely plexiform fibromyxoma and gastroblastoma. Triggered by unexpected targeted RNA-sequencing results detecting GLI1-related fusions in a group of malignant neoplasms with round to epithelioid morphology, and frequently strong S100 protein immunoreactivity, we investigated their clinicopathologic features in relation to other known pathologic entities sharing similar genetics. On the basis of a combined approach of targeted RNA sequencing and fluorescence in situ hybridization screening, we identified 6 cases with GLI1 gene fusions, including 4 fused to ACTB, 1 with MALAT1 and 1 with PTCH1 gene. Patients had a mean age of 36 years at diagnosis (range, 16 to 79 y) and slight female predilection all except 1 tumor originated in the soft tissue. Microscopically, the tumors had a monomorphic epithelioid phenotype arranged in a distinctive nested or cord-like architecture, separated by thin septae and delicate capillary network. All except 2 cases were strongly positive for S100 protein, whereas being negative for SOX10, SMA, and EMA. Only 1 tumor showed focal cytokeratin positivity in rare cells. Although the tumors showed some resemblance to pericytic/glomus tumors or myoepithelial tumors, the immunoprofile was not supportive of either lineage. Moreover, in contrast to the benign course of so-called pericytoma with t(7;12), 3 patients in this series developed metastatic disease to either lymph nodes or lung. In fact the only patient with lung metastases showed a novel PTCH1-GLI1 gene fusion. It remains to be determined whether these tumors represent a clinically and immunohistologically distinct subset of pericytoma, or an altogether novel soft tissue sarcoma. Our findings open new opportunities for targeted therapy, as tumors with GLI1 oncogenic activation, and subsequent PTCH1 overexpression, might be sensitive to sonic hedgehog pathway inhibitors.

A 14-month-old boy presented with a slow-growing, asymptomatic back plaque, which was biopsied and found to have S100 positivity, sparse CD34 staining, and no significant mitotic activity, nuclear pleomorphism, or necrosis; genetic workup found LMNA-NTRK1 gene fusion, overall consistent with lipofibromatosis-like neural tumor (LPF-NT). LPF-NT is rare, with 14 cases previously reported, and our patient is the first report of this diagnosis in infancy. This case report and literature review includes comparison of similar diagnoses including lipofibromatosis, low-grade malignant peripheral nerve sheath tumor, infantile fibrosarcoma, and dermatofibrosarcoma protuberans and serves to aid detection of LPF-NT presenting in pediatric patients by highlighting similarities and differences that should prompt consideration. LPF-NT shows locally aggressive behavior only and should not be confused with conditions that have potential for distant spread. However, case reports of metastasizing LMNA-NTRK1 tumors draw into question whether growths with this gene fusion exist on a spectrum of disease severity. Our patient was treated with wide local excision and has developed no complications or evidence of recurrence with 6 months of follow-up time.

Alveolar soft part sarcoma (ASPS) represents

We report 2 primary renal sarcomas demonstrating BCOR-CCNB3 gene fusions that have recently been identified in undifferentiated round cell sarcomas of bone and soft tissue. These neoplasms occurred in male children aged 11 and 12 years, and both were cystic as a result of entrapment and dilatation of native renal tubules. Both cases were composed of variably cellular bland spindle cells with fine chromatin set in myxoid stroma and separated by a branching capillary vasculature. Both neoplasms demonstrated immunoreactivity for BCOR, cyclin D1, TLE1, and SATB2 in the spindle neoplastic cells and negativity in the prominent capillary vasculature. One case was extensively cystic and had hypocellular areas that simulated cystic nephroma; this neoplasm recurred 3 years later as a solid, highly cellular spindle cell sarcoma in the abdominal cavity. The morphology and immunoprofile of these renal neoplasms was compared with a control group of other sarcomas with BCOR genetic abnormalities, including clear cell sarcoma of the kidney (CCSK), infantile undifferentiated round cell sarcomas of soft tissue/primitive myxoid mesenchymal tumor of infancy, and bone/soft tissue sarcomas with BCOR-CCNB3 gene fusion; along with primary renal synovial sarcoma. Our findings show that the renal sarcomas with BCOR-CCNB3 gene fusion overlap with CCSK. These results are in keeping with a "BCOR-alteration family" of renal and extrarenal neoplasms which includes CCSK and undifferentiated round cell sarcomas of soft tissue/primitive myxoid mesenchymal tumor of infancy (which typically harbor BCOR internal tandem duplication), and BCOR-CCNB3 sarcomas, all of which are primarily driven by BCOR overexpression and have overlapping (but not identical) clinicopathologic features.

Poorly differentiated chordomas (PDCs) represent a rare subset of notochordal neoplasms, affecting primarily children and associated with an aggressive outcome. In contrast to conventional chordomas, PDC show solid growth and increased cellularity, cytologic atypia, and mitotic activity. Recent studies have shown that PDCs are characterized by recurrent deletions encompassing the SMARCB1 locus, resulting in consistent loss of nuclear SMARCB1 expression. Thus PDC joined the expanding family of SMARCB1-deficient tumors characterized by various SMARCB1 structural abnormalities, ranging from large homozygous deletions to small intragenic mutations. In the present study, we investigate the SMARCB1 abnormalities in a group of nine well-characterized PDCs and to establish the sensitivity of the FISH method in detecting these changes in the clinical setting. We further assessed the pathologic features and clinical behavior of this cohort managed at our referral center over a 20-year period. The mean age at diagnosis was 10 years-of-age. All except one case occurred in the cranial region. All demonstrated strong nuclear expression of brachyury and loss of SMARCB1 expression. FISH identified homozygous SMARCB1 deletions in all except one case; additionally two cases revealed a heterozygous EWSR1 locus co-deletion. Clinical follow-up information was available in five patients. Two patients presented with distant metastases at initial diagnosis. Two of the three remaining patients with primary disease failed both locally and distantly after multimodality therapy. We conclude that PDCs are highly aggressive tumors and the dominant mechanism of loss of SMARCB1 expression is through large, homozygous SMARCB1 deletions that can be readily detected by FISH.

Aims Surgical excision of all benign vascular lesions of the breast identified by core needle biopsy has been recommended in the past to rule out a more serious lesion. In this study we investigated the clinical, radiologic, and pathologic findings in patients diagnosed with a benign vascular lesion at our institution to assess whether excision may be spared for lesions without atypia. Methods and results We searched the electronic medical record for patients with a vascular lesion of the breast diagnosed between 2000 and 2015. The study population consisted of 84 patients, 83 females and 1 male. The index diagnoses included 76 benign vascular lesions, 5 vascular lesions with cytologic atypia, and 3 angiosarcomas. A radiologist reviewed all pre and post-biopsy imaging studies; all cases had concordant radiologic and pathologic findings. Based on radiologic and histologic correlation, the vascular lesion accounted for the radiologic target in 40 (48%) cases and was deemed an incidental finding in 44 (52%). 7 of 32 (22%) targeted and 10 of 44 (23%) incidental benign vascular lesions underwent surgical excision; there were no upgrades at excision. No recurrences or clinical events were observed in patients with a targeted or incidental benign vascular lesion with a median followup of 39 months and 40.6 months, respectively. Conclusion Our data suggest that benign vascular lesions diagnosed on core biopsy with concordant radiologic and pathologic findings do not warrant surgical excision. This article is protected by copyright. All rights reserved.

Recognition of high-grade endometrial stromal sarcoma is important because of its aggressive clinical behavior. Morphologic features of YWHAE-NUTM2 high-grade endometrial stromal sarcoma may overlap with other uterine sarcoma types. BCOR immunoexpression was studied in these tumors and their morphologic mimics to assess its diagnostic utility. BCOR immunohistochemical staining was performed on archival tissue from 28 high-grade endometrial stromal sarcomas with classic morphology (20 YWHAE-NUTM2, 5 ZC3H7B-BCOR, 3 BCOR-ZC3H7B), 3 high-grade endometrial stromal sarcomas with unusual morphology and unknown gene rearrangement status, 66 low-grade endometrial stromal sarcomas, 21 endometrial stromal nodules, 38 uterine leiomyosarcomas, and 19 uterine leiomyomas. Intensity of nuclear staining and percentage of positive tumor cells were recorded. Strong diffuse nuclear BCOR staining (defined as >95% of tumor cells) was seen in the round cell component of all 20 (100%) classic YWHAE-NUTM2 high-grade endometrial stromal sarcomas and the 3 unusual high-grade endometrial stromal sarcomas which prompted FISH studies confirming YWHAE rearrangement in 2 tumors. Genomic PCR confirmed the presence of BCOR exon 16 internal tandem duplication in the third case. Diffuse BCOR staining was strong in three and weak in one BCOR-rearranged high-grade endometrial stromal sarcoma while absent in the remaining four BCOR-rearranged tumors. BCOR staining was weakly positive in

Patients with neurofibromatosis 1 (NF1) develop multiple neurofibromas, with 8% to 15% of patients experiencing malignant peripheral nerve sheath tumor (MPNST) during their lifetime. Prediction of transformation, typically from plexiform neurofibroma, is clinically and histologically challenging. In this overview, after a consensus meeting in October 2016, we outline the histopathologic features and molecular mechanisms involved in the malignant transformation of neurofibromas. Nuclear atypia alone is generally insignificant. However, with atypia, loss of neurofibroma architecture, high cellularity, and/or mitotic activity >1/50 but

Gastrointestinal stromal tumor (GIST) is the most common subtype of sarcoma. Despite clinical advances in the treatment of KIT/PDGFRA–mutant GIST, similar progress against KIT/PDGFRA wild-type GIST, including mutant BRAF-driven tumors, has been limited by a lack of model systems. ETV1 is a master regulator in the intestinal cells of Cajal (ICC), thought to be the cells of origin of GIST. Here, we present a model in which the ETV1 promoter is used to specifically and inducibly drive Cre recombinase in ICC as a strategy to study GIST pathogenesis. Using a conditional allele for BrafV600E, a mutation observed in clinical cases of GIST, we observed that BrafV600E activation was sufficient to drive ICC hyperplasia but not GIST tumorigenesis. In contrast, combining BrafV600E activation with Trp53 loss was sufficient to drive both ICC hyperplasia and formation of multifocal GIST-like tumors in the mouse gastrointestinal tract with 100% penetrance. This mouse model of sporadic GIST model was amenable to therapeutic intervention, and it recapitulated clinical responses to RAF inhibition seen in human GIST. Our work offers a useful in vivo model of human sporadic forms of BRAF-mutant GIST to help unravel its pathogenesis and therapeutic response to novel experimental agents. Cancer Res; 77(14); 3758–65. ©2017 AACR.

CIC-DUX4 gene fusion, resulting from either a t(4;19) or t(10;19) translocation, is the most common genetic abnormality detected in EWSR1-negative small blue round cell tumors. Following their discovery it was debated if these tumors should be classified as variants of Ewing sarcoma (ie, atypical Ewing sarcoma) or as a stand-alone pathologic entity. As such the WHO classification temporarily grouped the CIC-rearranged tumors under undifferentiated sarcomas with round cell phenotype, until further clinical evidence was available. However, most studies reported so far include small series with limited follow-up information, which preclude a more definitive assessment. The present work investigates the clinicopathologic features of a large cohort of sarcomas with CIC gene rearrangement, to define their clinical presentation, morphologic spectrum, and outcome. Our study further examines the overall survival of the CIC-positive cohort compared with a control group of EWSR1-rearranged Ewing sarcoma matched for age and stage. The study cohort included 115 patients, with a mean age of 32 years and a slight male predominance. Most tumors occurred in the soft tissue (86%), predominantly deep-seated and equally divided among trunk and extremity, followed by visceral locations (12%) and rarely in the bone (3%). Microscopically, most tumors showed round to ovoid cytomorphology but half of the cases showed also focal areas of spindling and epithelioid/rhabdoid phenotype, with frequent myxoid stromal changes. Variable CD99 reactivity was seen in 84% cases, with a diffuse pattern only in 23% of cases, whereas nuclear WT1 was seen in 92%. A CIC-DUX4 fusion was detected in 57% of cases, with either DUX4 on 4q35 (35%) or on 10q26 in 25 (22%) cases. No FOXO4 gene rearrangements were present in 39 cases tested. Clinical follow-up was available in 57 patients, with a 5-year survival of 43%, which was significantly lower than the 77% 5-year survival in the control Ewing sarcoma group (P=0.002). Our findings show that CIC-DUX4 sarcomas occur most commonly in young adults within the somatic soft tissues, having a wide spectrum of morphology including round, epithelioid and spindle cells, and associated with an aggressive clinical course, with an inferior overall survival compared with Ewing sarcoma. The results support the classification of CIC-rearranged tumors as an independent molecular and clinical subset of small blue round cell tumors distinct from Ewing sarcoma.

An increasing number of congenital and infantile sarcomas displaying a primitive, monomorphic spindle cell phenotype have been characterized to harbor recurrent gene fusions, including infantile fibrosarcoma and congenital spindle cell rhabdomyosarcoma. Here, we report an unusual spindle cell sarcoma presenting as a large and infiltrative pelvic soft tissue mass in a 4-month-old girl, which revealed a novel TFG-MET gene fusion by whole transcriptome RNA sequencing. The tumor resembled the morphology of an infantile fibrosarcoma with both fascicular and patternless growth, however, it expressed strong S100 protein immunoreactivity, while lacking SOX10 staining and retaining H3K27me3 expression. Although this immunoprofile suggested partial neural/neuroectodermal differentiation, overall features were unusual and did not fit into any known tumor types (cellular schwannoma, MPNST), raising the possibility of a novel pathologic entity. The TFG-MET gene fusion expands the genetic spectrum implicated in the pathogenesis of congenital spindle cell sarcomas, with yet another example of kinase oncogenic activation through chromosomal translocation. The discovery of this new fusion is significant since the resulting MET activation can potentially be inhibited by targeted therapy, as MET inhibitors are presently available in clinical trials.

CIC-DUX4 sarcoma (CDS) or CIC-rearranged sarcoma is a subcategory of small round cell sarcoma resembling the morphological phenotypes of Ewing sarcoma (ES). However, recent clinicopathologic and molecular genetic analyses indicate that CDS is an independent disease entity from ES. Few ancillary markers have been used in the differential diagnosis of CDS, and additional CDS-specific biomarkers are needed for more definitive classification. Here, we report the generation of an ex vivo mouse model for CDS by transducing embryonic mesenchymal cells (eMC) with human CIC-DUX4 cDNA. Recipient mice transplanted with eMC-expressing CIC-DUX4 rapidly developed an aggressive, undifferentiated sarcoma composed of small round to short spindle cells. Gene-expression profiles of CDS and eMC revealed upregulation of CIC-DUX4 downstream genes such as PEA3 family genes, Ccnd2, Crh, and Zic1. IHC analyses for both mouse and human tumors showed that CCND2 and MUC5AC are reliable biomarkers to distinguish CDS from ES. Gene silencing of CIC-DUX4 as well as Ccnd2, Ret, and Bcl2 effectively inhibited CDS tumor growth in vitro. The CDK4/6 inhibitor palbociclib and the soft tissue sarcoma drug trabectedin also blocked the growth of mouse CDS. In summary, our mouse model provides important biological information about CDS and provides a useful platform to explore biomarkers and therapeutic agents for CDS. Cancer Res; 77(11); 2927–37. ©2017 AACR.

Primary pulmonary myxoid sarcoma (PPMS) is a recently described, exceedingly rare low-grade lung sarcoma that tends to present in young females as an endobronchial mass and shows evidence of an EWSR1- CREB1 fusion. Herein, we present a case of PPMS with fluorescence in situ hybridization (FISH) analysis for EWSR1 and CREB1 rearrangements. An 80-year-old woman presented with an endobronchial, multinodular tumor exhibiting spindle, ovoid and epithelioid cells arranged in reticular/lattice-like and alveolar-like patterns in a myxoid background. The tumor showed focal epithelial membrane antigen immunoreactivity as well as an Alcian blue–positive stroma that was sensitive to digestion with hyaluronidase. EWSR1 and CREB1 rearrangements were detected by break-apart FISH probes. The patient showed persistence of disease 36 months after diagnosis and was discharged to hospice care. We contribute with a report of an additional case of this very unusual entity and perform a brief review of the literature published so far on the subject.

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 JAZF1 and PHF1 rearrangements 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.

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 × 106plaque-forming units [PFU]/mL; second and subsequent doses, ≤4 mL × 108PFU/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

Uterine tumors resembling ovarian sex cord tumor (UTROSCT) are rare mesenchymal neoplasms, of uncertain biologic potential, that were recently reported to exhibit recurrent gene fusions involving NCOA2–3. The purpose of this study was to, using a larger sample size, better characterize the histopathologic and molecular diversity of UTROSCT. Twenty-six cases of UTROSCT from five institutions were selected for further study. Fluorescence in situ hybridization for NCOA1, NCOA2, NCOA3, ESR1 and GREB1, and targeted RNA sequencing was performed on seventeen and eight UTROSCTs, respectively. Eight cases underwent massively parallel sequencing to detect single nucleotide variants (SNV), copy number variations (CNV), and structural variants using a targeted hybrid-capture based assay. NCOA1–3 rearrangement was identified in 81.8% (18/22) of cases. The most common fusion was ESR1-NCOA3, occurring in 40.9% (9/22). GREB1-NCOA1 (n=4), ESR1-NCOA2 (n=3), and GREB1-NCOA2 (n=1) rearrangements were also identified. No recurrent SNVs were identified and no tumor had SNVs in FOXL2, DICER1, STK11, or AKT1, which can be seen in ovarian sex cord-stromal tumors. CNVs were infrequent. Clinical follow-up was available for eleven cases with a mean follow-up interval of 94.4 (range 1 – 319) months. Only one case had a recurrence 66 months after the initial diagnosis and this was the single case with a with a GREB1-NCOA2 fusion. This study reports the morphologic spectrum of UTROSCT and confirms the recently reported recurrent NCOA2/3 gene fusions, in addition to identifying novel rearrangements involving NCOA1 in these tumors.

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 BCOR internal tandem duplications (ITD) and less common alternative YWHAE-NUTM2B/E fusions 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 BCOR ITD or YWHAE fusions to better define their morphologic spectrum and clinical behavior. Twenty-eight cases harbored BCOR ITD and five YWHAE fusions, 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 BCOR ITD and YWHAE fusions.

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 NTRK3 was a top-upregulated gene in BCOR-CCNB3 sarcomas. In this study, we investigate a large cohort of tumors harboring BCOR/YWHAE genetic 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 BCOR alteration and BCOR immunoreactivity was also performed. Most soft tissue undifferentiated round cell sarcomas with YWHAE or BCOR rearrangements or BCOR internal tandem duplications (ITD) showed NTRK3, but not NTRK1 or NTRK2, upregulation by RNA sequencing data analysis. Cytoplasmic pan-Trk immunoreactivity was also observed in most soft tissue round cell sarcomas with YWHAE rearrangements (100%), BCOR ITD (80%), and BCOR-CCNB3 fusions (67%), as well as clear cell sarcomas of kidney (75%), another BCOR family tumor, and ossifying fibromyxoid tumors with ZC3H7B-BCOR fusion (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 NTRK3 overexpression. In conclusion, NTRK3 upregulation 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.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare sarcoma subtype characterized by monomorphic epithelioid cells embedded in a densely sclerotic collagenous matrix. The overwhelming majority of tumors arise in soft tissues; however, rare cases have been documented to occur primarily in bone. The hallmarks of soft tissue SEF include MUC4 immunoreactivity and the presence of an EWSR1-CREB3L1 fusion. Rare cases with alternative fusions have also been reported such as EWSR1-CREB3L2 and FUS-CREB3L2 transcripts. The molecular alterations of skeletal SEF have not been well-defined, with only rare cases analyzed to date. In this study we investigated the clinicopathologic and molecular features of seven patients presenting with primary osseous SEF. There were 3 males and 4 females, with a mean age at diagnosis of 38 years. All cases had microscopic features within the histologic spectrum of SEF and showed strong and diffuse MUC4 positivity, while lacking SATB2 expression. However, due to its unusual presentation within bone, four cases were initially misinterpreted as either osteosarcoma, Ewing sarcoma or chondroblastoma. Half of the patients with follow-up data developed metastasis. The cases were tested by targeted RNA sequencing, MSK-IMPACT, and/or fluorescence in situ hybridization, showing EWSR1-CREB3L1 in six cases and EWSR1-CREB3L2 in one case. The fusion transcripts were composed of EWSR1 exon 11 to either exon 6 of CREB3L1 or CREB3L2. In summary, due to their rarity in the bone, skeletal SEF are often misdiagnosed, resulting in inadequate treatment modalities. Similar to their soft tissue counterpart, bone SEF follow an aggressive clinical behavior and show similar EWSR1-CREB3L1/CREB3L2 fusions.