Complementary value of molecular analysis to expert review in refining classification of uncommon soft tissue tumors.

The classification of many soft tissue tumors remains subjective due their rarity, significant overlap in microscopic features and often a non-specific immunohistochemical (IHC) profile. The application of molecular genetic tools, which leverage the underlying molecular pathogenesis of these neoplasms, have considerably improved the diagnostic abilities of pathologists and refined classification based on objective molecular markers. In this study, we describe the results of an international collaboration conducted over a 3-year period, assessing the added diagnostic value of applying molecular genetics to sarcoma expert pathologic review in a selected series of 84 uncommon, mostly unclassifiable mesenchymal tumors, 74 of which originated in soft tissues and 10 in bone. The case mix (71% historical, 29% contemporary) included mostly unusual and challenging soft tissue tumors, which remained unclassified even with the benefit of expert review and routine ancillary methods, including broad IHC panels and a limited number of commercially available fluorescence in situ hybridization (FISH) probes. All cases were further tested by FISH using a wide range of custom bacterial artificial chromosome probes covering most of known fusions in sarcomas, whereas targeted RNA sequencing was performed in 13 cases negative by FISH, for potential discovery of novel fusion genes. Tumor-defining molecular alterations were found in 48/84 tumors (57%). In 27 (32%) cases the tumor diagnosis was refined or revised by the additional molecular work-up, including five cases (6%), in which the updated diagnosis had clinical implications. Sarcoma classification is rapidly evolving due to an increased molecular characterization of these neoplasms, so unsurprisingly 17% of the tumors in this series harbored abnormalities only very recently described as defining novel molecularly defined soft tissue tumor subsets.

Kinase fusion positive intra-osseous spindle cell tumors: A series of eight cases with review of the literature.

Mesenchymal spindle cell tumors with kinase fusions, often presenting in superficial or deep soft tissue locations, may rarely occur in bone. Herein, we describe the clinicopathologic and molecular data of eight bone tumors characterized by various kinase fusions from our files and incorporate the findings with the previously reported seven cases, mainly as single case reports. In the current series all but one of the patients were young children or teenagers, with an age range from newborn to 59 years (mean 19 years). Most tumors (n = 5) presented in the head and neck area (skull base, mastoid, maxilla, and mandible), and remaining three in the tibia, pelvic bone, and chest wall. The fusions included NTRK1 (n = 3), RET (n = 2), NTRK3 (n = 2), and BRAF (n = 1). In the combined series (n = 15), most tumors (73%) occurred in children and young adults (<30 years) and showed a predilection for jaw and skull bones (40%), followed by long and small tubular bones (33%). The fusions spanned a large spectrum of kinase genes, including in descending order NTRK3 (n = 6), NTRK1 (n = 4), RET (n = 2), BRAF (n = 2), and RAF1 (n = 1). All fusions confirmed by targeted RNA sequencing were in-frame and retained the kinase domain within the fusion oncoprotein. Similar to the soft tissue counterparts, most NTRK3-positive bone tumors in this series showed high-grade morphology (5/6), whereas the majority of NTRK1 tumors were low-grade (3/4). Notably, all four tumors presenting in the elderly were high-grade spindle cell sarcomas, with adult fibrosarcoma (FS)-like, malignant peripheral nerve sheath tumor (MPNST)-like and MPNST phenotypes. Overall, 10 tumors had high-grade morphology, ranging from infantile and adult-types FS, MPNST-like, and MPNST, whereas five showed benign/low-grade histology (MPNST-like and myxoma-like). Immunohistochemically (IHC), S100 and CD34 positivity was noted in 57% and 50%, respectively, while co-expression of S100 and CD34 in 43% of cases. One-third of tumors (4 high grade and the myxoma-like) were negative for both S100 and CD34. IHC for Pan-TRK was positive in all eight NTRK-fusion positive tumors tested and negative in two tumors with other kinase fusions. Clinical follow-up was too limited to allow general conclusions.

Malignant undifferentiated epithelioid neoplasms with MAML2 rearrangements: A clinicopathologic study of seven cases demonstrating a heterogenous entity.

Among mesenchymal tumors, MAML2 gene rearrangements have been described in a subset of composite hemangioendothelioma and myxoinflammatory fibroblastic sarcoma (MIFS). However, we have recently encountered MAML2-related fusions in a group of seven undifferentiated malignant epithelioid neoplasms that do not fit well to any established pathologic entities. The patients included five males and two female, aged 41-71 years old (median 65 years). The tumors involved the deep soft tissue of extremities (hip, knee, arm, hand), abdominal wall, and the retroperitoneum. Microscopically, the tumors consisted of solid sheets of atypical epithelioid to histiocytoid cells with abundant cytoplasm. Prominent mitotic activity and necrosis were present in 4 cases. In 3 cases, the cells displayed hyperchromatic nuclei or conspicuous macronucleoli, and were admixed with background histiocytoid cells and a lymphoplasmacytic infiltrate. By immunohistochemistry (IHC), the neoplastic cells had a nonspecific phenotype. On targeted RNA sequencing, MAML2 was the 3' partner and fused to YAP1 (4 cases), ARHGAP42 (2 cases), and ENDOD1 (1 case). Two cases with YAP1::MAML2 harbored concurrent RAF kinase fusions (RBMS3::RAF1 and AGK::BRAF, respectively). In 2 cases with targeted DNA sequencing, mutations in TP53, RB1 and PTEN were detected in 1 case, and PDGFRB mutations, CCNE1 amplifications and CDKN2A/2B deletion were detected in another case, which showed strong and diffuse PDGFRB expression by IHC. Of the 4 cases with detailed clinical history (median follow-up period 8 months), three developed distant metastatic disease (one of which died of disease); one case remained free of disease 3 years following surgical excision. In conclusion, we describe a heterogeneous series of MAML2-rearranged undifferentiated malignant epithelioid neoplasms, a subset of which may overlap with a recently described MIFS variant with YAP1::MAML2 fusions, further expanding the clinicopathologic spectrum of mesenchymal neoplasms with recurrent MAML2 gene rearrangements.

Psammomatoid Ossifying Fibroma Is Defined by SATB2 Rearrangement.

Psammomatoid ossifying fibroma (PsOF), also known as juvenile PsOF, is a benign fibro-osseous neoplasm predominantly affecting the extragnathic bones, particularly the frontal and ethmoid bones, with a preference for adolescents and young adults. The clinical and morphologic features of PsOF may overlap with those of other fibro-osseous lesions, and additional molecular markers would help increase diagnostic accuracy. Because identical chromosomal breakpoints at bands Xq26 and 2q33 have been described in 3 cases of PsOF located in the orbita, we aimed to identify the exact genes involved in these chromosomal breakpoints and determine their frequency in PsOF using transcriptome sequencing and fluorescence in situ hybridization (FISH). We performed whole RNA transcriptome sequencing on frozen tissue in 2 PsOF index cases and identified a fusion transcript involving SATB2, located on chromosome 2q33.1, and AL513487.1, located on chromosome Xq26, in one of the cases. The fusion was validated using reverse transcription (RT)-PCR and SATB2 FISH. The fusion lead to a truncated protein product losing most of the functional domains. Subsequently, we analyzed an additional 24 juvenile PsOFs, 8 juvenile trabecular ossifying fibromas (JTOFs), and 11 cemento-ossifying fibromas (COFs) for SATB2 using FISH and found evidence of SATB2 gene rearrangements in 58% (7 of 12) of the evaluable PsOF cases but not in any of the evaluable JTOF (n = 7) and COF (n = 7) cases. A combination of SATB2 immunofluorescence and a 2-color SATB2 FISH in our index case revealed that most tumor cells harboring the rearrangement lacked SATB2 expression. Using immunohistochemistry, 65% of PsOF, 100% of JTOF, and 100% of COF cases showed moderate or strong staining for SATB2. In these cases, we observed a mosaic pattern of expression with >25% of the spindle cells in between the bone matrix, with osteoblasts and osteocytes being positive for SATB2. Interestingly, 35% (8 of 23) of PsOFs, in contrast to JTOFs and COFs, showed SATB2 expression in <5% of cells. To our knowledge, this is the first report that shows the involvement of SATB2 in the development of a neoplastic lesion. In this study, we have showed that SATB2 rearrangement is a recurrent molecular alteration that appears to be highly specific for PsOF. Our findings support that PsOF is not only morphologically and clinically but also genetically distinct from JTOF and COF.

Novel EWSR1::GFI1B gene fusion in angiofibroma of soft tissue.

AIMS: Angiofibroma of soft tissue is a benign soft tissue tumour characterised by bland spindle cells and a distinct branching vascular network. The majority of soft tissue angiofibromas harbour AHRR::NCOA2 gene fusions. Here we present three cases of EWSR1::GFI1B-fused soft tissue tumours that are morphologically most reminiscent of soft tissue angiofibroma. METHODS AND RESULTS: All three cases presented in male patients with an age range of 35-78 years (median = 54 years). Two cases presented as subcutaneous nodules on the trunk (posterior neck and chest wall); one was an intramuscular foot mass. The tumours were unencapsulated nodules with infiltrative margins ranging from 2.2 to 3.4 cm in greatest dimension. Histologically, the tumours contained uniformly bland fibroblastic spindle cells with ovoid to fusiform nuclei and delicate cytoplasmic processes embedded in a myxoid to myxocollagenous stroma. All three cases were characterised by a thin-walled, branching vascular network evenly distributed throughout the tumour. Overt cytological atypia or conspicuous mitotic activity was absent. The spindle cells had an essentially null immunophenotype. By targeted RNA sequencing, an in-frame gene fusion between EWSR1 exons 1-7 and GFI1B exons 6-11 or 7-11 was detected in all three cases. The tumours were marginally excised. For all three cases, there were no documented local recurrence or distant metastases during a limited follow-up period of 6-10 months. CONCLUSIONS: We propose that EWSR1::GFI1B may represent a novel fusion variant of soft tissue angiofibroma.

Mesenchymal chondrosarcoma of the head and neck with HEY1::NCOA2 fusion: A clinicopathologic and molecular study of 13 cases with emphasis on diagnostic pitfalls.

BACKGROUND: Mesenchymal chondrosarcoma (MCS) is a rare translocation-associated sarcoma, driven by a canonical HEY1::NCOA2 fusion. The tumors typically have a biphasic phenotype of primitive small blue round cells intermixed with hyaline cartilage. The head and neck (HN) region is a common site for MCS, accounting for 12-45% of all cases reported. AIMS: We assembled a relatively large cohort of 13 molecularly confirmed HN MCS for a detailed clinicopathologic analysis. The underlying fusion events were determined using fluorescence in situ hybridization and/or targeted RNA sequencing. RESULTS: The median age of presentation was 19 years. Five MCSs (39%) had an intraosseous presentation (skull, maxilla, palate, and mandible), while the remaining eight cases occurred in the brain/meninges, orbit, and nasal cavity. Microscopically, HN MCSs were characterized by primitive round cells arranged in a distinctive nested architecture and a rich staghorn vasculature. A cartilaginous component of hyaline cartilage islands and/or single chondrocytes were present in 69% cases. A combined immunoprofile of CD99(+)/SATB2(+)/CD34(-)/STAT6(-) was typically noted. As this immunoprofile is non-specific, the referral diagnoses in cases lacking a cartilaginous component included Ewing sarcoma family and osteosarcoma. Among the seven patients with follow-up data, three developed distant metastasis and one died of disease. CONCLUSION: HN MCS may arise at intra- or extra-osseous sites. The HN MCS appears to have a more prolonged survival compared other MCS sites. Testing for HEY1::NCOA2 fusion is recommended in HN tumors with nested round cell morphology and staghorn vasculature that lack a distinctive cartilaginous component.

Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy.

BACKGROUND: Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy with a high prevalence of ventricular arrhythmias. How the R14 deletion causes dilated cardiomyopathy is poorly understood, and there are no disease-specific therapies. METHODS: We used single-cell RNA sequencing to uncover PLN R14del disease mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We used both 2-dimensional and 3-dimensional functional contractility assays to evaluate the impact of modulating disease-relevant pathways in PLN R14del hiPSC-CMs. RESULTS: Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro. Single-cell RNA sequencing revealed the induction of the unfolded protein response (UPR) pathway in PLN R14del compared with isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the 3 main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP (binding immunoglobulin protein) inducer X. PLN R14del hiPSC-CMs treated with BiP protein inducer X showed a dose-dependent amelioration of the contractility deficit in both 2-dimensional cultures and 3-dimensional engineered heart tissues without affecting calcium homeostasis. CONCLUSIONS: Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically.

Epithelioid hemangioma of bone harboring FOS and FOSB gene rearrangements: A clinicopathologic and molecular study.

The diagnosis of epithelioid hemangioma (EH) remains challenging due to its rarity, worrisome histologic features, and locally aggressive clinical and radiographic presentation. Especially in the bone, EH can be misdiagnosed as a malignant vascular neoplasm due its lytic, often destructive or multifocal growth, as well as atypical morphology. The discovery of recurrent FOS and FOSB gene fusions in the pathogenesis of most EH has strengthened its stand-alone classification, distinct from other malignant epithelioid vascular lesions, such as epithelioid hemangioendothelioma or angiosarcoma. In this study we investigate a group of molecularly confirmed skeletal EH by the presence of FOS or FOSB gene rearrangements to better define its clinical and pathologic characteristics within a homogenous molecular subset. The cohort included 38 patients (25 males, 13 females), with a mean age at diagnosis of 38 years (range, 4-75). Regional, multifocal presentation was noted in 10 cases. Only six cases were correctly recognized as EH by the referring institutions, while most were misdiagnosed as other vascular tumors. Of the 17 patients with follow-up data available, five patients (29%) developed local recurrence after marginal en bloc excision (n = 3) or curettage (n = 2). Local recurrence-free survival rates were 84% at 3 years and 38% at 5 years. No metastasis or disease-related death was identified. Imaging studies exhibited no specific features, showing cortical bone destruction and soft-tissue extension in 14 (38%) cases. FOS gene rearrangements were detected in 28 (74%) of cases, while FOSB rearrangements in 10 (26%) cases. Our results highlight the significant challenges encountered in establishing a correct diagnosis exclusive of the molecular testing, mainly due to its overlap to other malignant epithelioid vascular tumors. Skeletal EH emerges as a genetically defined locally aggressive vascular neoplasm, with a high rate of local recurrence, but lacking the propensity for distant spread.

P62-positive aggregates are homogenously distributed in the myocardium and associated with the type of mutation in genetic cardiomyopathy.

Genetic cardiomyopathy is caused by mutations in various genes. The accumulation of potentially proteotoxic mutant protein aggregates due to insufficient autophagy is a possible mechanism of disease development. The objective of this study was to investigate the distribution in the myocardium of such aggregates in relation to specific pathogenic genetic mutations in cardiomyopathy hearts. Hearts from 32 genetic cardiomyopathy patients, 4 non-genetic cardiomyopathy patients and 5 controls were studied. Microscopic slices from an entire midventricular heart slice were stained for p62 (sequestosome-1, marker for aggregated proteins destined for autophagy). The percentage of cardiomyocytes with p62 accumulation was higher in cardiomyopathy hearts (median 3.3%) than in healthy controls (0.3%; P < .0001). p62 accumulation was highest in the desmin (15.6%) and phospholamban (7.2%) groups. P62 accumulation was homogeneously distributed in the myocardium. Fibrosis was not associated with p62 accumulation in subgroup analysis of phospholamban hearts. In conclusion, accumulation of p62-positive protein aggregates is homogeneously distributed in the myocardium independently of fibrosis distribution and associated with desmin and phospholamban cardiomyopathy. Proteotoxic protein accumulation is a diffuse process in the myocardium while a more localized second hit, such as local strain during exercise, might determine whether this leads to regional myocyte decay.

Recurrent MEIS1-NCOA2/1 fusions in a subset of low-grade spindle cell sarcomas frequently involving the genitourinary and gynecologic tracts.

Sarcomas with MEIS1-NCOA2 fusions 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-NCOA2 or NCOA1 fusions 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-NCOA2 fusions were recurrently identified in 5 cases, and a novel MEIS1-NCOA1 fusion 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-NCOA2/1 fusions, providing evidence of an undifferentiated spindle cell phenotype with nonspecific immunoprofile and low-grade clinical behavior.

Head and neck rhabdomyosarcoma with TFCP2 fusions and ALK overexpression: a clinicopathological and molecular analysis of 11 cases.

AIMS: Primary intraosseous rhabdomyosarcoma (RMS) is a rare entity defined by EWSR1/FUS-TFCP2 or, less commonly, MEIS1-NCOA2 fusions. The lesions often show a hybrid spindle and epithelioid phenotype, frequently coexpress myogenic markers, ALK, and cytokeratin, and show a striking propensity for the pelvic and craniofacial bones. The aim of this study was to investigate the clinicopathological and molecular features of 11 head and neck RMSs (HNRMSs) characterised by the genetic alterations described in intraosseous RMS. METHODS AND RESULTS: The molecular abnormalities were analysed with fluorescence in-situ hybridisation and/or targeted RNA/DNA sequencing. Seven cases had FUS-TFCP2 fusions, four had EWSR1-TFCP2 fusions, and none had MEIS1-NCOA2 fusions. All except one case were intraosseous, affecting the mandible (n = 4), maxilla (n = 3), and skull (n = 3). One case occurred in the superficial soft tissue of the neck. The median age was 29 years (range, 16-74 years), with an equal sex distribution. All tumours showed mixed epithelioid and spindle morphology. Immunohistochemical coexpression of desmin, myogenin, MyoD1, ALK, and cytokeratin was seen in most cases. An intragenic ALK deletion was seen in 43% of cases. Regional and distant spread were seen in three and four patients, respectively. Two patients died of their disease. CONCLUSIONS: We herein present the largest series of HNRMSs with TFCP2 fusions to date. The findings show a strong predilection for the skeleton in young adults, although we also report an extraosseous case. The tumours are characterised by a distinctive spindle and epithelioid phenotype and a peculiar immunoprofile, with coexpression of myogenic markers, epithelial markers, and ALK. They are associated with a poor prognosis, including regional or distant spread and disease-related death.

MRI after Whoops procedure: diagnostic value for residual sarcoma and predictive value for an incomplete second resection.

OBJECTIVE: To determine the value of MRI for the detection and assessment of the anatomic extent of residual sarcoma after a Whoops procedure (unplanned sarcoma resection) and its utility for the prediction of an incomplete second resection. MATERIALS AND METHODS: This study included consecutive patients who underwent a Whoops procedure, successively followed by gadolinium chelate-enhanced MRI and second surgery at a tertiary care sarcoma center. RESULTS: Twenty-six patients were included, of whom 19 with residual tumor at the second surgery and 8 with an incomplete second resection (R1: n = 6 and R2: n = 2). Interobserver agreement for residual tumor at MRI after a Whoops procedure was perfect (κ value: 1.000). MRI achieved a sensitivity of 47.4% (9/19), a specificity of 100% (7/7), a positive predictive value of 100% (9/9), and a negative predictive value of 70.0% (7/17) for the detection of residual tumor. MRI correctly classified 2 of 19 residual sarcomas as deep-seated (i.e., extending beyond the superficial muscle fascia) but failed to correctly classify 3 of 19 residual sarcomas as deep-seated. There were no significant associations between MRI findings (presence of residual tumor, maximum tumor diameter, anatomic tumor extent, tumor margins, tumor spiculae, and tumor tail on the superficial fascia) with an incomplete (R1 or R2) second resection. CONCLUSION: Gadolinium chelate-enhanced MRI is a reproducible method to rule in residual sarcoma, but it is insufficiently accurate to rule out and assess the anatomic extent or residual sarcoma after a Whoops procedure. Furthermore, MRI has no utility in predicting an incomplete second resection.

Diagnostic yield of NanoString nCounter FusionPlex profiling in soft tissue tumors.

Diagnostic histopathology of soft tissue tumors can be troublesome as many entities are quite rare and have overlapping morphologic features. Many soft tissue tumors harbor tumor-defining gene translocations, which may provide an important ancillary tool for tumor diagnosis. The NanoString nCounter platform enables multiplex detection of pre-defined gene fusion transcripts in formalin-fixed and paraffin-embedded tissue. A cohort of 104 soft tissue tumors representing 20 different histological types was analyzed for the expression of 174 unique gene fusion transcripts. A tumor-defining gene fusion transcript was detected in 60 cases (58%). Sensitivity and specificity of the NanoString assay calculated against the result of an alternative molecular method were 85% and 100%, respectively. Highest diagnostic coverage was obtained for Ewing sarcoma, synovial sarcoma, myxoid liposarcoma, alveolar rhabdomyosarcoma, and desmoplastic small round cell tumor. For these tumor types, the NanoString assay is a rapid, cost-effective, sensitive, and specific ancillary screening tool for molecular diagnosis. For other sarcomas, additional molecular testing may be required when a translocation transcript is not identified with the current 174 gene fusion panel.

Variant WWTR1 gene fusions in epithelioid hemangioendothelioma-A genetic subset associated with cardiac involvement.

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.

A morphologic and molecular reappraisal of myoepithelial tumors of soft tissue, bone, and viscera with EWSR1 and FUS gene rearrangements.

Myoepithelial tumors (MET) represent a clinicopathologically heterogeneous group of tumors, ranging from benign to highly aggressive lesions. Although MET arising in soft tissue, bone, or viscera share morphologic and immunophenotypic overlap with their salivary gland and cutaneous counterparts, there is still controversy regarding their genetic relationship. Half of MET of soft tissue and bone harbor EWSR1 or FUS related fusions, while MET arising in the salivary gland and skin often show PLAG1 and HMGA2 gene rearrangements. Regardless of the site of origin, the gold standard in diagnosing a MET relies on demonstrating its "myoepithelial immunophenotype" of positivity for EMA/CK and S100 protein or GFAP. However, the morphologic spectrum of MET in soft tissue and bone is quite broad and the above immunoprofile is nonspecific, being shared by other pathogenetically unrelated neoplasms. Moreover, rare MET lack a diagnostic immunoprofile but shows instead the characteristic gene fusions. In this study, we analyzed a large cohort of 66 MET with EWSR1 and FUS gene rearrangements spanning various clinical presentations, to better define their morphologic spectrum and establish relevant pathologic-molecular correlations. Genetic analysis was carried out by FISH for EWSR1/FUS rearrangements and potential partners, and/or by targeted RNA sequencing. Then, 82% showed EWSR1 rearrangement, while 18% had FUS abnormalities. EWSR1-POU5F1 occurred with predilection in malignant MET in children and young adults and these tumors had nested epithelioid morphology and clear cytoplasm. In contrast, EWSR1/FUS-PBX1/3 fusions were associated with benign and sclerotic spindle cell morphology. Tumors with EWSR1-KLF17 showed chordoma-like morphology. Our results demonstrate striking morphologic-molecular correlations in MET of bone, soft tissue and viscera, which might have implications in their clinical behavior.

Soft tissue tumors characterized by a wide spectrum of kinase fusions share a lipofibromatosis-like neural tumor pattern.

Gene fusions resulting in oncogenic activation of various receptor tyrosine kinases, including NTRK1-3, ALK, and RET, have been increasingly recognized in soft tissue tumors (STTs), displaying a wide morphologic spectrum and therefore diagnostically challenging. A subset of STT with NTRK1 rearrangements were recently defined as lipofibromatosis-like neural tumors (LPFNTs), being characterized by mildly atypical spindle cells with a highly infiltrative growth in the subcutis and expression of S100 and CD34 immunostains. Other emerging morphologic phenotypes associated with kinase fusions include infantile/adult fibrosarcoma and malignant peripheral nerve sheath tumor-like patterns. In this study, a large cohort of 73 STT positive for various kinase fusions, including 44 previously published cases, was investigated for the presence of an LPFNT phenotype, to better define the incidence of this distinctive morphologic pattern and its relationship with various gene fusions. Surprisingly, half (36/73) of STT with kinase fusions showed at least a focal LPFNT component defined as >10%. Most of the tumors occurred in the subcutaneous tissues of the extremities (n = 25) and trunk (n = 9) of children or young adults (<30 years old) of both genders. Two-thirds (24/36) of these cases showed hybrid morphologies with alternating LPFNT and solid areas of monomorphic spindle to ovoid tumor cells with fascicular or haphazard arrangement, while one-third (12/36) had pure LPFNT morphology. Other common histologic findings included lymphocytic infiltrates, staghorn-like vessels, and perivascular or stromal hyalinization, especially in hybrid cases. Mitotic activity was generally low (<4/10 high power fields in 81% cases), being increased only in a minority of cases. Immunoreactivity for CD34 (92% in hybrid cases, 89% in pure cases) and S100 (89% in hybrid cases, 64% in pure cases) were commonly present. The gene rearrangements most commonly involved NTRK1 (75%), followed by RET (8%) and less commonly NTRK2, NTRK3, ROS1, ALK, and MET.

Clinical and molecular characterization of primary sclerosing epithelioid fibrosarcoma of bone and review of the literature.

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.

EWSR1/FUS-CREB fusions define a distinctive malignant epithelioid neoplasm with predilection for mesothelial-lined cavities.

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 EWSR1 or FUS with 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-CREB fusions. 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-ATF1 fusion. 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).

Undifferentiated round cell sarcoma with BCOR internal tandem duplications (ITD) or YWHAE fusions: a clinicopathologic and molecular study.

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.

New advances in the molecular classification of pediatric mesenchymal tumors.

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.