Comprehensive clinicopathological, molecular, and methylation analysis of mesenchymal tumors with NTRK and other kinase gene aberrations.

Alterations in kinase genes such as NTRK1/2/3, RET, and BRAF underlie infantile fibrosarcoma (IFS), the emerging entity 'NTRK-rearranged spindle cell neoplasms' included in the latest WHO classification, and a growing set of tumors with overlapping clinical and pathological features. In this study, we conducted a comprehensive clinicopathological and molecular analysis of 22 cases of IFS and other kinase gene-altered spindle cell neoplasms affecting both pediatric and adult patients. Follow-up periods for 16 patients ranged in length from 10 to 130 months (mean 38 months). Six patients were treated with targeted therapy, achieving a partial or complete response in five cases. Overall, three cases recurred and one metastasized. Eight patients were free of disease, five were alive with disease, and two patients died. All cases showed previously reported morphological patterns. Based on the cellularity and level of atypia, cases were divided into three morphological grade groups. S100 protein and CD34 were at least focally positive in 12/22 and 14/22 cases, respectively. Novel PWWP2A::RET, NUMA1::RET, ITSN1::RAF1, and CAPZA2::MET fusions, which we report herein in mesenchymal tumors for the first time, were detected by RNA sequencing. Additionally, the first uterine case with BRAF and EGFR mutations and CD34 and S100 co-expression is described. DNA sequencing performed in 13 cases uncovered very rare additional genetic aberrations. The CNV profiles showed that high-grade tumors demonstrate a significantly higher percentage of copy number gains and losses across the genome compared with low- and intermediate-grade tumors. Unsupervised clustering of the tumors' methylation profiles revealed that in 8/9 cases, the methylation profiles clustered with the IFS methylation class, irrespective of their clinicopathological or molecular features. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Calcifying Spindle Cell Soft Tissue Tumor With SOX10::PLAG1 Fusion: A Case Report of a Morphologically Distinctive and Potentially Novel Soft Tissue Tumor.

The widespread use of advanced molecular techniques has led to the identification of several tumor types with PLAG1 gene fusions some of which also affect the skin and soft tissues. Herein, we present a 38-year-old female with a subcutaneous tumor affecting her forearm, which does not seem to fit into any currently recognized entity. It was a well-circumscribed tumor measuring 6 × 4,5 × 4 cm. It had a thick capsule composed of bland spindle cells forming palisades and Verocay body-like structures within a myxocollagenous background. Scattered calcifications were dispersed throughout the lesion. No cytological atypia, mitotic activity, or necrosis were present. Targeted NGS revealed a SOX10::PLAG1 fusion and fluorescent in situ hybridization confirmed the presence of PLAG1 gene rearrangement. The neoplastic cells showed a diffuse immunohistochemical expression of S100, SOX10, and PLAG1, as well as patchy desmin and CD34 positivity. The methylation profile of this tumor did not match any other entity covered by the DKFZ sarcoma classifier and apart from the gain of chromosome 12, the copy number profile was normal. The tumor was completely excised, and the patient has been free of disease for 4 years since the excision. While more cases are needed to confirm this tumor as a distinct entity, we propose a provisional name "SOX10::PLAG1-rearranged calcifying spindle cell tumor."

Sarcomas Harboring EWSR1::PATZ1 Fusions: A Clinicopathologic Study of 17 Cases.

Soft tissue sarcomas harboring EWSR1::PATZ1 are a recently recognized entity with variable morphology and a heterogeneous immunohistochemical profile. We studied 17 such tumors. The tumors occurred in 12 men and 5 women (median age, 50 years; range, 15-71 years), involved the thoracoabdominal soft tissues (14 cases; 82%), lower extremities (2 cases; 12%), and tongue (1 case; 6%), and ranged from 0.7 to 11.3 cm (median, 4.7 cm). All but 1 patient received complete surgical resection; 7 were also treated with neoadjuvant chemo/radiotherapy. All cases showed typical features of EWSR1::PATZ1 sarcoma, including uniform round to spindled cells, fibromyxoid matrix, fibrous bands, hyalinized vessels, and pseudoalveolar/microcystic spaces. Unusual features, seen in a subset of cases, included degenerative-appearing nuclear atypia, epithelioid cytomorphology, mature fat, abundant rhabdomyoblasts, high mitotic activity, and foci with increased cellularity and nuclear atypia. Positive immunohistochemical results were desmin (16/17, 94%), MyoD1 (13/14, 93%), myogenin (6/14, 43%), GFAP (10/10, 100%), S100 protein (15/17, 88%), SOX10 (7/13, 54%), keratin (10/17, 59%), CD99 (4/11, 36%), H3K27me3 (retained expression 9/9, 100%), p16 (absent expression 1/4, 25%), and p53 (wild type 3/3, 100%). Fusion events included EWSR1 exon 8::PATZ1 exon 1 (14/17, 82%), EWSR1 exon 9::PATZ1 exon 1 (2/17, 12%), and EWSR1 exon 7::PATZ1 exon 1 (1/17, 6%). No evaluated tumor had alterations of CDKN2A/B and/or TP53, or MDM2 amplification. Clinical follow-up (16 patients: median, 13.5 months; range, 1-77 months) showed distant metastases in 3 patients (1/3 at time of presentation) and no local recurrences. At the time of last follow-up, 14 patients were disease free, 1 was alive with disease, 1 was dead of disease (at 13 months), and 1 had an indeterminant pulmonary nodule. We conclude that the morphologic spectrum of EWSR1::PATZ1 is broader than has been previously appreciated. Although more long-term follow-up is needed, the prognosis of these very rare sarcomas may be more favorable than previously reported.

PLAG1-Rearranged Uterine Sarcomas: A Study of 11 Cases Showing a Wide Phenotypical Spectrum Not Limited to Myxoid Leiomyosarcoma-like Morphology.

PLAG1 gene fusions were recently identified in a subset of uterine myxoid leiomyosarcomas (M-LMS). However, we have encountered cases of PLAG1-rearranged uterine sarcomas lacking M-LMS-like morphology and/or any expression of smooth muscle markers. To better characterize their clinicopathologic features, we performed a multiinstitutional search that yielded 11 cases. The patients ranged in age from 34 to 72 years (mean, 57 years). All tumors arose in the uterine corpus, ranging in size from 6.5 to 32 cm (mean, 15 cm). The most common stage at presentation was pT1b (n = 6), and 3 cases had stage pT1 (unspecified), and 1 case each presented in stages pT2a and pT3b. Most were treated only with hysterectomy and adnexectomy. The follow-up (range, 7-71 months; median, 39 months) was available for 7 patients. Three cases (7-21 months of follow-up) had no evidence of disease. Three of the 4 remaining patients died of disease within 55 to 71 months, while peritoneal spread developed in the last patient, and the patient was transferred for palliative care at 39 months. Morphologically, the tumors showed a high intertumoral and intratumoral heterogeneity. M-LMS-like and epithelioid leiomyosarcoma-like morphology were present in 3 and 5 primary tumors, respectively, the remaining mostly presented as nondescript ovoid or spindle cell sarcomas. Unusual morphologic findings included prominently hyalinized stroma (n = 3), adipocytic differentiation with areas mimicking myxoid liposarcoma (n = 2), osteosarcomatous differentiation (n = 1), and undifferentiated pleomorphic sarcoma-like areas (n = 1). The mitotic activity ranged from 3 to 24 mitoses per 10 high-power fields (mean, 9); 3 of 10 cases showed necrosis. In 3 of 11 cases, no expression of smooth muscle actin, h-caldesmon, or desmin was noted, whereas 5 of 5 cases expressed PLAG1. By RNA sequencing, the following fusion partners were identified: PUM1, CHCHD7 (each n = 2), C15orf29, CD44, MYOCD, FRMD6, PTK2, and TRPS1 (each n = 1). One case only showed PLAG1 gene break by fluorescence in situ hybridization. Our study documents a much broader morphologic spectrum of PLAG1-rearranged uterine sarcomas than previously reported, encompassing but not limited to M-LMS-like morphology with occasional heterologous (particularly adipocytic) differentiation. As it is currently difficult to precisely define their line of differentiation, for the time being, we suggest using a descriptive name "PLAG1-rearranged uterine sarcoma."

Novel CRTC1::MRTFB(MKL2) Gene Fusion Detected in Myxoid Mesenchymal Neoplasms With Myogenic Differentiation Involving Bone and Soft Tissues.

Appropriate classification of fusion-driven bone and soft tissue neoplasms continues to evolve, often relying on the careful integration of morphologic findings with immunohistochemical, molecular, and clinical data. Herein, we present 3 cases of a morphologically distinct myxoid mesenchymal neoplasm with myogenic differentiation and novel CRTC1::MRTFB (formerly MKL2) gene fusion. Three tumors occurred in 1 male and 2 female patients with a median age of 72 years (range: 28-78). Tumors involved the left iliac bone, the right thigh, and the left perianal region with a median size of 4.0 cm (4.0-7.6 cm). Although 1 tumor presented as an incidental finding, the other 2 tumors were noted, given their persistent growth. At the time of the last follow-up, 1 patient was alive with unresected disease at 6 months, 1 patient was alive without evidence of disease at 12 months after surgery, and 1 patient died of disease 24 months after diagnosis. On histologic sections, the tumors showed multinodular growth and were composed of variably cellular spindle to round-shaped cells with distinct brightly eosinophilic cytoplasm embedded within a myxoid stroma. One tumor showed overt smooth muscle differentiation. Cytologic atypia and mitotic activity ranged from minimal (2 cases) to high (1 case). By immunohistochemistry, the neoplastic cells expressed focal smooth muscle actin, h-caldesmon, and desmin in all tested cases. Skeletal muscle markers were negative. Next-generation sequencing detected nearly identical CRTC1::MRTFB gene fusions in all cases. We suggest that myxoid mesenchymal tumors with myogenic differentiation harboring a CRTC1::MRTFB fusion may represent a previously unrecognized, distinctive entity that involves soft tissue and bone. Continued identification of these novel myxoid neoplasms with myogenic differentiation will be important in determining appropriate classification, understanding biologic potential, and creating treatment paradigms.

Spindle cell rhabdomyosarcomas: With TFCP2 rearrangements, and novel EWSR1::ZBTB41 and PLOD2::RBM6 gene fusions. A study of five cases and review of the literature.

AIMS: Spindle-cell/sclerosing rhabdomyosarcomas (SS-RMS) are clinically and genetically heterogeneous. They include three well-defined molecular subtypes, of which those with EWSR1/FUS::TFCP2 rearrangements were described only recently. This study aimed to evaluate five new cases of SS-RMS and to perform a clinicopathological and statistical analysis of all TFCP2-rearranged SS-RMS described in the English literature to more comprehensively characterize this rare tumour type. METHODS AND RESULTS: Cases were retrospectively selected and studied by immunohistochemistry, fluorescence in situ hybridization with EWSR1/FUS and TFCP2 break-apart probes, next-generation sequencing (Archer FusionPlex Sarcoma kit and TruSight RNA Pan-Cancer Panel). The PubMed database was searched for relevant peer-reviewed English reports. Five cases of SS-RMS were found. Three cases were TFCP2 rearranged SS-RMS, having FUSex6::TFCP2ex2 gene fusion in two cases and triple gene fusion EWSR1ex5::TFCP2ex2, VAX2ex2::ALKex2 and VAX2intron2::ALKex2 in one case. Two cases showed rhabdomyoblastic differentiation and spindle-round cell/sclerosing morphology, but were characterized by novel genetic fusions including EWSR1ex8::ZBTB41ex7 and PLOD2ex8::RBM6ex7, respectively. In the statistical analysis of all published cases, CDKN2A or ALK alterations, the use of standard chemotherapy and age at presentation in the range of 18-24 years were negatively correlated to overall survival. CONCLUSION: EWSR1/FUS::TFCP2-rearranged SS-RMS is a rare rhabdomyosarcoma subtype, affecting predominantly young adults with average age at presentation 34 years (median 29.5 years; age range 7-86 years), with a predilection for craniofacial bones, rapid clinical course with frequent bone and lung metastases, and poor prognosis (3-year overall survival rate 28%).

TFG::MET-rearranged soft tissue tumor: A rare infantile neoplasm with a distinct low-grade triphasic morphology.

This article presents 2 cases of TFG::MET-rearranged mesenchymal tumor, an extremely rare molecular subset among an emerging group of mesenchymal neoplasms with kinase gene (NTRK, BRAF, RET and others) alterations. Both tumors were congenital, occurred in female patients and presented as huge masses on the trunk and thigh, measuring 18 and 20 cm in the largest dimension. Both cases showed identical areas with a distinctive triphasic morphology resembling fibrous hamartoma of infancy (FHI), consisting of haphazardly arranged ovoid to spindled cells traversed by variably cellular and hyalinized fascicles admixed with (most likely non-neoplastic) adipose tissue. In other areas, a high-grade infantile fibrosarcoma/malignant peripheral nerve sheath tumor-like (IFS/MPNST-like) morphology was present in both cases. While the first case co-expressed CD34 and S100 protein, the other case did not. When combined with the three previously reported MET-rearranged cases (of which two harbored TFG::MET fusion), 3/5 and 3/4 of MET-rearranged and TFG::MET fusion-associated tumors, respectively exhibited similar triphasic FHI-like low-grade morphology. This points toward the existence of a relatively distinct morphological subset among kinase-fusion-associated tumors which seems to be strongly associated with MET fusions. It seems some of these low-grade cases may transform into a high-grade variant with IFS/MPNST-like morphology as has been observed in other tumors with kinase gene fusions. While most cases seem to follow an indolent clinical course, the recognition of these tumors is clinically relevant as MET tyrosine kinase inhibitors might represent an effective treatment option for clinically aggressive or unresectable cases.

Clear cell mesotheliomas with inactivating VHL mutations and near-haploid genomic features.

Clear cell mesothelioma is uncommon and shows predominance of clear cells with resemblance to clear cell carcinomas. Clinicopathologic and molecular descriptions of clear cell mesothelioma remained limited. In this study, we identified an index patient with clear cell mesothelioma, confirmed by immunohistochemical and ultrastructural studies. Targeted next-generation sequencing revealed the presence of an inactivating VHL mutation. We then systematically searched for VHL-mutant mesotheliomas in a comprehensive genomic profiling database of 1532 mesotheliomas. Collectively, we identified a cohort of four VHL-mutant clear cell mesotheliomas, including three peritoneal and one pleural tumors from three females and one male, with age range of 47-68 (median 63) years. Histologically, each tumor showed a microcystic to tubulopapillary architecture with prominent clear cells. By next-generation DNA sequencing, each of the four clear cell mesotheliomas harbored inactivating VHL mutations, while lacking other alterations typical of mesotheliomas such as BAP1, NF2, SETD2, CDKN2A, CDKN2B, TP53, and PTEN. By using low-pass whole genome sequencing on the index case and targeted next-generation sequencing on the remaining three cases, we identified extensive loss of heterozygosity throughout the genome but consistently sparing chromosomes 5, 7, and 20, characteristic of genomic near-haploidization. In summary, clear cell mesotheliomas were characterized by inactivating VHL mutations and genomic near-haploidization and appeared to represent a distinct clinicopathologic and molecular category of mesotheliomas. Our findings implicate VHL in the pathogenesis of a subset of mesotheliomas, particularly those with clear cell morphology.

NR4A3 fusions characterize a distinctive peritoneal mesothelial neoplasm of uncertain biological potential with pure adenomatoid/microcystic morphology.

A focal adenomatoid-microcystic pattern is not uncommon in peritoneal mesothelioma, but tumors composed almost exclusively of this pattern are distinctly rare and have not been well characterized. A small subset of mesotheliomas (mostly in children and young adults) are characterized by gene fusions including EWSR1/FUS::ATF1, EWSR1::YY1, and NTRK and ALK rearrangements, and often have epithelioid morphology. Herein, we describe five peritoneal mesothelial neoplasms (identified via molecular screening of seven histologically similar tumors) that are pure adenomatoid/microcystic in morphology and unified by the presence of an NR4A3 fusion. Patients were three males and two females aged 31-70 years (median, 40 years). Three presented with multifocal/diffuse and two with a localized disease. The size of the individual lesions ranged from 1.5 to 8 cm (median, 4.7). The unifocal lesions originated in the small bowel mesentery and the mesosigmoid. Treatment included surgery, either alone (three) or combined with hyperthermic intraperitoneal chemotherapy (two), and neoadjuvant or adjuvant chemotherapy (one case each). At the last follow-up (6-13 months), all five patients were alive and disease-free. All tumors were morphologically similar, characterized by extensive sieve-like microcystic growth with bland-looking flattened cells lining variably sized microcystic spaces and lacked a conventional epithelioid or sarcomatoid component. Immunohistochemistry confirmed mesothelial differentiation, but most cases showed limited expression of D2-40 and calretinin. Targeted RNA sequencing revealed an NR4A3 fusion (fusion partners were EWSR1 in three cases and CITED2 and NIPBL in one case each). The nosology and behavior of this morphomolecularly defined novel peritoneal mesothelial neoplasm of uncertain biological potential and its distinction from adenomatoid variants of conventional mesothelioma merit further delineation as more cases become recognized.

Mesenchymal skin tumors - Novel entities in the 5th edition of WHO classification of skin tumors.

The section on mesenchymal tumors in 5th edition of WHO classification of skin tumors has undergone several changes, the most important of which, as usual, is the inclusion of newly identified tumor entities, which will be the main focus of this review article. These specifically include three novel cutaneous mesenchymal tumors with melanocytic differentiation, and rearrangements of the CRTC1::TRIM11, ACTIN::MITF, and MITF::CREM genes. In addition, EWSR1::SMAD3-rearranged fibroblastic tumors, superficial CD34-positive fibroblastic tumors, and NTRK-rearranged spindle cell neoplasms were newly included. Of the other changes, only the most important ones will be briefly mentioned.

Lipoblastoma-Like Tumor and Fibrosarcoma-Like Lipomatous Neoplasm Represent the Same Entity: A Clinicopathologic and Molecular Genetic Study of 23 Cases Occurring in Both Men and Women at Diverse Locations.

Lipoblastoma-like tumor (LLT) is a benign soft tissue tumor demonstrating mixed morphologic features of lipoblastoma, myxoid liposarcoma, and spindle cell lipoma but lacking genetic alterations associated with those tumors. LLT was originally thought to be specific to the vulva but has since been reported in the paratesticular region. The morphologic features of LLT overlap with those of "fibrosarcoma-like lipomatous neoplasm" (FLLN), a rare, indolent adipocytic neoplasm considered by some to form part of the spectrum of atypical spindle cell and pleomorphic lipomatous tumor. We compared the morphologic, immunohistochemical, and genetic features of 23 tumors previously classified as LLT (n = 17) and FLLN (n = 6). The 23 tumors occurred in 13 women and 10 men (mean age, 42 years; range, 17 to 80 years). Eighteen (78%) cases arose in the inguinogenital region, whereas 5 tumors (22%) involved noninguinogenital soft tissue, including the flank (n = 1), shoulder (n = 1), foot (n = 1), forearm (n = 1), and chest wall (n = 1). Microscopically, the tumors were lobulated and septated, with variably collagenized fibromyxoid stroma, prominent thin-walled vessels, scattered univacuolated or bivacuolated lipoblasts, and a minor component of mature adipose tissue. Using immunohistochemistry, 5 tumors (42%) showed complete RB1 loss, with partial loss in 7 cases (58%). RNA sequencing, chromosomal microarray, and DNA next-generation sequencing study results were negative for significant alterations. There were no clinical, morphologic, immunohistochemical, or molecular genetic differences between cases previously classified as LLT or FLLN. Clinical follow-up (11 patients [48%]; range, 2-276 months; mean, 48.2 months) showed all patients were alive without disease, and only one patient had experienced a single local recurrence. We conclude that LLT and FLLN represent the same entity, for which "LLT" seems most appropriate. LLT may occur in either sex and any superficial soft tissue location. Careful morphologic study and appropriate ancillary testing should allow for the distinction of LLT from its potential mimics.

EWSR1::POU2AF3(COLCA2) Sarcoma: An Aggressive, Polyphenotypic Sarcoma With a Head and Neck Predilection.

EWSR1::POU2AF3 (COLCA2) sarcomas are a recently identified group of undifferentiated round/spindle cell neoplasms with a predilection for the head and neck region. Herein, we report our experience with 8 cases, occurring in 5 men and 3 women (age range, 37-74 years; median, 60 years). Tumors involved the head/neck (4 cases), and one each the thigh, thoracic wall, fibula, and lung. Seven patients received multimodal therapy; 1 patient was treated only with surgery. Clinical follow-up (8 patients; range, 4-122 months; median, 32 months) showed 5 patients with metastases (often multifocal, with a latency ranging from 7 to 119 months), and 3 of them also with local recurrence. The median local recurrence-free and metastasis-free survival rates were 24 months and 29 months, respectively. Of the 8 patients, 1 died of an unknown cause, 4 were alive with metastatic disease, 1 was alive with unresectable local disease, and 2 were without disease. The tumors were composed of 2 morphologic subgroups: (1) relatively bland tumors consisting of spindled to stellate cells with varying cellularity and fibromyxoid stroma (2 cases) and (2) overtly malignant tumors composed of nests of "neuroendocrine-appearing" round cells surrounded by spindled cells (6 cases). Individual cases in the second group showed glandular, osteogenic, or rhabdomyoblastic differentiation. Immunohistochemical results included CD56 (4/4 cases), GFAP (5/8), SATB2 (4/6), keratin (AE1/AE3) (5/8), and S100 protein (4/7). RNA sequencing identified EWSR1::POU2AF3 gene fusion in all cases. EWSR1 gene rearrangement was confirmed by fluorescence in situ hybridization in 5 cases. Our findings confirm the head/neck predilection and aggressive clinical behavior of EWSR1::POU2AF3 sarcomas and widen the morphologic spectrum of these rare lesions to include relatively bland spindle cell tumors and tumors with divergent differentiation.

Alterations in key signaling pathways in sinonasal tract melanoma. A molecular genetics and immunohistochemical study of 90 cases and comprehensive review of the literature.

Sinonasal mucosal melanoma is a rare tumor arising within the nasal cavity, paranasal sinuses, or nasopharynx (sinonasal tract). This study evaluated 90 cases diagnosed in 29 males and 61 females with median age 68 years. Most tumors involved the nasal cavity and had an epithelioid morphology. Spectrum of research techniques used in this analysis includes targeted-DNA and -RNA next-generation sequencing, Sanger sequencing, fluorescence in situ hybridization and immunohistochemistry. Sinonasal melanomas were commonly driven by RAS (38/90, 42%), especially NRAS (n = 36) mutations and rarely (4/90, 4%) displayed BRAF pathogenic variants. BRAF/RAS mutants were more frequent among paranasal sinuses (10/14, 71%) than nasal (26/64, 41%) tumors. BRAF/RAS-wild type tumors occasionally harbored alterations of the key components and regulators of Ras-MAPK signaling pathway: NF1 mutations (1/17, 6%) or NF1 locus deletions (1/25, 4%), SPRED1 (3/25, 12%), PIK3CA (3/50, 6%), PTEN (4/50, 8%) and mTOR (1/50, 2%) mutations. These mutations often occurred in a mutually exclusive manner. In several tumors some of which were NRAS mutants, TP53 was deleted (6/48, 13%) and/or mutated (5/90, 6%). Variable nuclear accumulation of TP53, mirrored by elevated nuclear MDM2 expression was seen in >50% of cases. Furthermore, sinonasal melanomas (n = 7) including RAS/BRAF-wild type tumors (n = 5) harbored alterations of the key components and regulators of canonical WNT-pathway: APC (4/90, 4%), CTNNB1 (3/90, 3%) and AMER1 (1/90, 1%). Both, TERT promoter mutations (5/53, 9%) and fusions (2/40, 5%) were identified. The latter occurred in BRAF/RAS-wild type tumors. No oncogenic fusion gene transcripts previously reported in cutaneous melanomas were detected. Eight tumors including 7 BRAF/RAS-wild type cases expressed ADCK4::NUMBL cis-fusion transcripts. In summary, this study documented mutational activation of NRAS and other key components and regulators of Ras-MAPK signaling pathway such as SPRED1 in a majority of sinonasal melanomas.

Lipomatous Solitary Fibrous Tumors Harbor Rare NAB2-STAT6 Fusion Variants and Show Up-Regulation of the Gene PPARG, Encoding for a Regulator of Adipocyte Differentiation.

Solitary fibrous tumors (SFTs) harbor activating NAB2-STAT6 gene fusions. Different variants of the NAB2-STAT6 gene fusion have been associated with distinct clinicopathologic features. Lipomatous SFTs are a morphologic variant of SFTs, characterized by a fat-forming tumor component. Our aim was to evaluate NAB2-STAT6 fusion variants and to further study the molecular genetic features in a cohort of lipomatous SFTs. A hybrid-capture-based next-generation sequencing panel was employed to detect NAB2-STAT6 gene fusions at the RNA level. In addition, the RNA expression levels of 507 genes were evaluated using this panel, and were compared with a control cohort of nonlipomatous SFTs. Notably, 5 of 11 (45%) of lipomatous SFTs in the current series harbored the uncommon NAB2 exon 4-STAT6 exon 4 gene fusion variant, which is observed in only 0.9% to 1.4% of nonlipomatous SFTs. Furthermore, lipomatous SFTs displayed significant differences in gene expression compared with their nonlipomatous counterparts, including up-regulation of the gene peroxisome proliferator activated receptor-γ (PPARG). Peroxisome proliferator activated receptor-γ is a nuclear receptor regulating adipocyte differentiation, providing a possible explanation for the fat-forming component in lipomatous SFTs. In summary, the current study provides a possible molecular genetic basis for the distinct morphologic features of lipomatous SFTs.

Recurrent novel HMGA2-NCOR2 fusions characterize a subset of keratin-positive giant cell-rich soft tissue tumors.

Giant cell tumors of soft tissue (GCT-ST) are rare low-grade neoplasms that were at one time thought to represent the soft tissue counterparts of GCT of bone (GCT-B) but are now known to lack the H3F3 mutations characteristic of osseous GCT. We present six distinctive giant cell-rich soft tissue neoplasms that expressed keratins and carried a recurrent HMGA2-NCOR2 gene fusion. Patients were five females and one male aged 14-60 years (median, 29). All presented with superficial (subcutaneous) masses that were removed by conservative marginal (3) or wide (2) local excision. The tumors originated in the upper extremity (2), lower extremity (2), head/neck (1), and trunk (1). Five patients with follow-up (median, 21 months; range, 14-168) remained disease-free. Grossly, all tumors were well-demarcated but not encapsulated with variable lobulation. Histologically, they were composed of bland plump epithelioid or ovoid to spindled mononuclear cells admixed with evenly distributed multinucleated osteoclast-type giant cells. Foci of stromal hemorrhage and hemosiderin were seen in all cases. The mitotic activity ranged from 2 to 14/10 high power fields (median: 10). Foci of necrosis and vascular invasion were seen in one case each. The mononuclear cells were immunoreactive with the AE1/AE3 keratin cocktail and less frequently/less diffusely for K7 and K19 but lacked expression of other lineage-associated markers. RNA-based next-generation sequencing revealed an HMGA2-NCOR2 fusion in all tumors. None of the keratin-negative conventional GCT-ST showed the HMGA2-NCOR2 fusion (0/7). Metaplastic bone (4/9) and SATB2 expression (3/4) were frequent in keratin-negative conventional GCT-ST but were lacking in keratin-positive HMGA2-NCOR2 fusion-positive tumors. The distinctive immunophenotype and genotype of these tumors strongly suggest that they represent a discrete entity, differing from conventional GCT-ST and other osteoclast-rich morphologic mimics. Their natural history appears favorable, although a study of additional cases and longer follow-up are warranted.

Novel BRAF gene fusions and activating point mutations in spindle cell sarcomas with histologic overlap with infantile fibrosarcoma.

Infantile fibrosarcoma (IFS)/cellular congenital mesoblastic nephroma (cCMN) commonly harbors the classic ETV6-NTRK3 translocation. However, there are recent reports of mesenchymal tumors with IFS-like morphology harboring fusions of other receptor tyrosine kinases or downstream effectors, including NTRK1/2/3, MET, RET, and RAF1 fusions as well as one prior series with BRAF fusions. Discovery of these additional molecular drivers contributes to a more integrated diagnostic approach and presents important targets for therapy. Here we report the clinicopathologic and molecular features of 14 BRAF-altered tumors, of which 5 had BRAF point mutations and 10 harbored one or more BRAF fusions. Of the BRAF fusion-positive tumors, one harbored two BRAF fusions (FOXN3-BRAF, TRIP11-BRAF) and another harbored three unique alternative splice variants of EPB41L2-BRAF. Tumors occurred in ten males and four females, aged from birth to 32 years (median 6 months). Twelve were soft tissue based; two were visceral including one located in the kidney (cCMN). All neoplasms demonstrated ovoid to short spindle cells most frequently arranged haphazardly or in intersecting fascicles, often with collagenized stroma and a chronic inflammatory infiltrate. No specific immunophenotype was observed; expression of CD34, S100, and SMA was variable. To date, this is the largest cohort of BRAF-altered spindle cell neoplasms with IFS-like morphology, including not only seven novel BRAF fusion partners but also the first description of oncogenic BRAF point mutations in these tumors.

EWSR1-PATZ1-rearranged sarcoma: a report of nine cases of spindle and round cell neoplasms with predilection for thoracoabdominal soft tissues and frequent expression of neural and skeletal muscle markers.

The knowledge of clinical features and, particularly, histopathological spectrum of EWSR1-PATZ1-rearranged spindle and round cell sarcomas (EPS) remains limited. For this reason, we report the largest clinicopathological study of EPS to date. Nine cases were collected, consisting of four males and five females ranging in age from 10 to 81 years (average: 49 years). Five tumors occurred in abdominal wall soft tissues, three in the thorax, and one in the back of the neck. Tumor sizes ranged from 2.5 to 18 cm (average 6.6 cm). Five patients had follow-up with an average of 38 months (range: 18-60 months). Two patients had no recurrence or metastasis 19 months after diagnosis. Four patients developed multifocal pleural or pulmonary metastasis and were treated variably by surgery, radiotherapy, and chemotherapy. The latter seemed to have little to no clinical benefit. One of the four patients was free of disease 60 months after diagnosis, two patients were alive with disease at 18 and 60 months, respectively. Morphologically, low, intermediate, and high-grade sarcomas composed of a variable mixture of spindled, ovoid, epithelioid, and round cells were seen. The architectural and stromal features also varied, resulting in a broad morphologic spectrum. Immunohistochemically, the following markers were most consistently expressed: S100-protein (7/9 cases), GFAP (7/8), MyoD1 (8/9), Pax-7 (4/5), desmin (7/9), and AE1/3 (4/9). By next-generation sequencing, all cases revealed EWSR1-PATZ1 gene fusion. In addition, 3/6 cases tested harbored CDKN2A deletion, while CDKN2B deletion and TP53 mutation were detected in one case each. Our findings confirm that EPS is a clinicopathologic entity, albeit with a broad morphologic spectrum. The uneventful outcome in some of our cases indicates that a subset of EPS might follow a more indolent clinical course than previously appreciated. Additional studies are needed to validate whether any morphological and/or molecular attributes have a prognostic impact.

Superficial CD34-positive fibroblastic tumor and PRDM10-rearranged soft tissue tumor are overlapping entities: a comprehensive study of 20 cases.

AIMS: Superficial CD34-positive fibroblastic tumor (SCD34FT) and PRDM10-rearranged soft tissue tumor (PRDM10-STT) are rare mesenchymal tumors. These lesions have clinicopathological similarities, but their relationship remains controversial. This study aimed to characterise a series of cases of SCD34FT and PRDM10-STT. METHODS AND RESULTS: Ten lesions each of SCD34FT and PRDM10-STT were studied using immunohistochemistry, array-comparative genomic hybridisation (aCGH), RNA sequencing and exome sequencing. Tumors mainly occurred in young adults, were generally small (< 5 cm) and arose predominantly in the superficial soft tissues of the lower extremities. Follow-up data were available in 15 cases (SCD34FT, n = 7, median 16 months; PRDM10-STT, n = 8, median 14 months), local recurrences occurred in four cases (SCD34FT, two of 10; PRDM10-STT, two of 10), while no distant spread was documented. Morphologically, tumors were relatively well-circumscribed and composed of sheets and fascicles of spindle and pleomorphic cells showing low mitotic activity (< 1/mm²) without necrosis. Other findings included: granular cell change, lipoblast-like cells, ectatic blood vessels with fibrinous material, myxoid stromal changes, metaplastic bone and increased mitotic activity (> 1/mm²). All tumors diffusely expressed CD34, while pan-keratin and desmin were commonly seen focally. SynCAM3 was diffusely expressed in 12 cases (SCD34FT, n = 5; PRDM10-STT, n = 7), independently of fusion status. aCGH profiles were 'flat' (PRDM10-STT, n = 4; SCD34FT, n = 2) and exome sequencing showed no recurrent pathogenic mutations (PRDM10-STT, n = 2; SCD34FT, n = 4). Overall, the only morphological features seen exclusively in PRDM10-STT were myxoid stromal changes (three of 10) and metaplastic bone (two of 10). CONCLUSION: We expand the current knowledge on PRDM10-STT and SCD34FT and provide additional evidence for considering them as overlapping entities.

Poorly differentiated chordoma showing loss of SMARCB1/INI1: Clinicopathological and radiological spectrum of nine cases, including uncommon features of a relatively under-recognized entity.

Poorly differentiated chordoma is a newly recognized entity in the recent World Health Organization (WHO) classification of tumors of soft tissue and bone. Slightly over 60 such cases have been documented. Herein, we present a clinicopathological profile, including radiological features, of nine cases, which occurred in five males and four females, with age varying from 1 to 29 years (median = 43), in the cervical spine (n = 2), skull base (n = 2), clivus (n = 2), thoracic spine (n = 1) lumbar spine (n = 1) and coccyx (n = 1) Average tumor size was 4.8 cm. None of the 6-referral cases was diagnosed as a poorly differentiated chordoma at the referring laboratory. Histopathologically, all cases displayed a cellular tumor comprising polygonal cells (n = 9) displaying moderate to marked nuclear pleomorphism with prominent nucleoli (n = 7), eosinophilic (n = 9) to vacuolated cytoplasm (n = 7), rhabdoid morphology (n = 4), interspersed mitotic figures (n = 5), focal necrosis (n = 6) and inflammatory cells (n = 9). A single tumor displayed areas resembling classic chordoma, transitioning into poorly differentiated areas. There were multinucleate giant cells and physaliphorous cells in two tumors, each, respectively. Immunohistochemically, tumor cells were positive for AE1/AE3 (7/7), EMA (7/7), cytokeratin (CK) MNF116 (1/1), OSCAR (1/1), brachyury (9/9, diffusely), S100P (4/7, mostly focally), and glypican 3(2/4). SMARCB1/INI1 was completely lost in all nine tumors. A single case tested by FISH showed homozygous deletion of the SMARCB1 gene. Therapeutically (n = 7), all patients were treated with surgical resection (invariably incomplete) (n = 5), followed by adjuvant radiation therapy (n = 4) and chemotherapy (n = 4). While a single patient partially responded to treatment and another patient is alive with no evidence of disease after 23 years, three patients died of disease, six, eight, and 11 months post-diagnosis, despite adjuvant treatments. A single patient presented with a metastatic lung nodule, while another developed widespread metastasis. Poorly differentiated chordomas display a spectrum of features, are associated with a lower index of suspicion for a diagnosis, and display aggressive outcomes. Critical analysis of radiological and histopathological features, including necessary immunostains (brachyury and SMARCB1/INI1), is necessary for their timely diagnosis. These tumors show loss of SMARCB1/INI1 immunostaining and homozygous deletion of INI1/SMARCB1 gene.

Targeted next generation sequencing of MLH1-deficient, MLH1 promoter hypermethylated, and BRAF/RAS-wild-type colorectal adenocarcinomas is effective in detecting tumors with actionable oncogenic gene fusions.

Oncogenic gene fusions represent attractive targets for therapy of cancer. However, the frequency of actionable genomic rearrangements in colorectal cancer (CRC) is very low, and universal screening for these alterations seems to be impractical and costly. To address this problem, several large scale studies retrospectivelly showed that CRC with gene fusions are highly enriched in groups of tumors defined by MLH1 DNA mismatch repair protein deficiency (MLH1d), and hypermethylation of MLH1 promoter (MLH1ph), and/or the presence of microsatellite instability, and BRAF/KRAS wild-type status (BRAFwt/KRASwt). In this study, we used targeted next generation sequencing (NGS) to explore the occurence of potentially therapeutically targetable gene fusions in an unselected series of BRAFwt/KRASwt CRC cases that displayed MLH1d/MLH1ph. From the initially identified group of 173 MLH1d CRC cases, 141 cases (81.5%) displayed MLH1ph. BRAFwt/RASwt genotype was confirmed in 23 of 141 (~16%) of MLH1d/MLH1ph cases. Targeted NGS of these 23 cases identified oncogenic gene fusions in nine patients (39.1%; CI95: 20.5%-61.2%). Detected fusions involved NTRK (four cases), ALK (two cases), and BRAF genes (three cases). As a secondary outcome of NGS testing, we identified PIK3K-AKT-mTOR pathway alterations in two CRC cases, which displayed PIK3CA mutation. Altogether, 11 of 23 (~48%) MLH1d/MLH1ph/BRAFwt/RASwt tumors showed genetic alterations that could induce resistance to anti-EGFR therapy. Our study confirms that targeted NGS of MLH1d/MLH1ph and BRAFwt/RASwt CRCs could be a cost-effective strategy in detecting patients with potentially druggable oncogenic kinase fusions.