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Soft tissue sarcomas comprise a heterogeneous group of neoplasms. Although soft tissue malignancies make up only 2% of adult cancers, classification based on histomorphology presents a diagnostic challenge. Characterisation of soft tissue sarcomas by molecular analysis is rapidly evolving to improve diagnostic accuracy and develop targeted therapies. This review highlights the advances in molecular techniques, including current next-generation sequencing-based assays (fusion detection by RNA sequencing, targeted/whole exome sequencing, microRNA profiling), as well as emerging methods (liquid biopsies, DNA methylation profiling, single-cell molecular profiling and next-generation immunohistochemistry) for future clinical applications.

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

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

Pleomorphic liposarcoma (PLPS) is a highly aggressive sarcoma comprising variable numbers of pleomorphic lipoblasts mixed with undifferentiated pleomorphic sarcoma (UPS)-like areas. Morphologic variants, such as myxofibrosarcoma-like or epithelioid, may cause diagnostic confusion, especially on a core biopsy, but there are few data on the prognostic significance of these features. A total of 120 PLPS biopsies and resection specimens were reviewed and catalogued based on the presence of myxofibrosarcoma-like, UPS-like, and epithelioid foci, in 10% increments. The clinical parameters were collected. Cases occurred in 75 males and 45 females, ranging from 8 to 98 years (median, 62.5 y). Cases arose in the extremities (n=72), trunk (n=32), head/neck (n=10), bone (n=4), mediastinum (n=1), or viscera (colon polyp, n=1). Of those with known depth (n=81), 40 were intramuscular, 34 were subcutaneous, and 7 arose in the dermis. Their sizes ranged from 1 to 24.5 cm (median, 7 cm). Of the patients with ≥1 month of follow-up (n=70), 5 had recurrence and 15 had metastasis. The 5-year overall survival and event-free survival rates were 66.2% and 63.1%, respectively. Tumors ≥5 cm had inferior overall survival compared with tumors <5 cm. The presence of epithelioid areas was also statistically significant in terms of poorer overall survival and event-free survival, while tumors with ≥50% undifferentiated pleomorphic-like areas had better overall survival. There was a trend towards poorer outcomes in tumors with necrosis (≥1%). PLPS is an aggressive adipocytic malignancy that is most commonly present in the extremities of older adults. The morphologic features of these tumors are diverse, and they may be mistaken for UPS or myxofibrosarcoma, carcinoma, and melanomas, particularly on biopsies. Tumor size, necrosis, and epithelioid morphology are associated with adverse prognosis.

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

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

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

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

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

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::MRTFBgene fusions in all cases. We suggest that myxoid mesenchymal tumors with myogenic differentiation harboring a CRTC1::MRTFBfusion 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.

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

YAP1-TFE3-fused hemangioendothelioma is an extremely rare malignant vascular tumor. We present the largest multi-institutional clinicopathologic study of YAP1-TFE3-fused hemangioendothelioma to date. The 24 cases of YAP1-TFE3-fused hemangioendothelioma showed a female predominance (17 female, 7 male) across a wide age range (20–78 years old, median 44). Tumors were most commonly located in soft tissue (50%), followed by bone (29%), lung (13%), and liver (8%), ranging from 3 to 115 mm in size (median 40 mm). About two-thirds presented with multifocal disease, including 7 cases with distant organ metastasis. Histopathologically, we describe three dominant architectural patterns: solid sheets of coalescing nests, pseudoalveolar and (pseudo)vasoformative pattern, and discohesive strands and clusters of cells set in a myxoid to myxohyaline stroma. These patterns were present in variable proportions across different tumors and often coexisted within the same tumor. The dominant cytomorphology (88%) was large epithelioid cells with abundant, glassy eosinophilic to vacuolated cytoplasm, prominent nucleoli and well-demarcated cell borders. Multinucleated or binucleated cells, prominent admixed erythrocytic and lymphocytic infiltrates, and intratumoral fat were frequently present. Immunohistochemically, ERG, CD31, and TFE3 were consistently expressed, while expression of CD34 (83%) and cytokeratin AE1/AE3 (20%) was variable. CAMTA1 was negative in all but one case. All cases were confirmed by molecular testing to harbor YAP1-TFE3gene fusions: majority with YAP1exon 1 fused to TFE3exon 4 (88%), or less commonly, TFE3exon 6 (12%). Most patients (88%) were treated with primary surgical resection. Over a follow-up period of 4–360 months (median 36 months) in 17 cases, 35% of patients remained alive without disease, and 47% survived many years with stable, albeit multifocal and/or metastatic disease. Five-year progression-free survival probability was 88%. We propose categorizing YAP1-TFE3-fused hemangioendothelioma as a distinct disease entity given its unique clinical and histopathologic characteristics in comparison to conventional epithelioid hemangioendothelioma.

CD34-negative solitary fibrous tumors (SFTs) are rare and have not been comprehensively studied. We retrospectively reviewed all cases of SFT confirmed with STAT6 immunohistochemistry and/or STAT6gene fusion between 2013 and 2020 and collected pertinent clinicopathologic parameters. Of a total of 244 cases, 25 (10%) lacked CD34 expression by immunohistochemistry. Compared with CD34-positive SFT, CD34-negative SFT are more likely to arise in the head and neck area (32% vs. 24%, P=0.02) and present as metastatic disease (28% vs. 1%, P<0.0001). A significantly higher percentage of CD34-negative SFT exhibit high-grade cytologic atypia (hypercellularity, round cell or anaplastic morphology, nuclear pleomorphism, etc.) (48% vs. 22%, P=0.0073). There are no significant differences in the distributions of age, sex, tumor size, mitotic count, tumor necrosis, or risk stratification between CD34-negative and CD34-positive SFT. In addition, only 56% of CD34-negative SFT display a typical hemangiopericytoma-like vascular pattern. Special histologic features among CD34-negative SFT include prominent alternating hypercellular or fibrous and hypocellular myxoid areas with curvilinear vessels mimicking low-grade fibromyxoid sarcoma, pulmonary edema-like microcystic changes, and prominent amianthoid collagen fibers. In conclusion, compared with their CD34-positive counterparts, CD34-negative SFT is more likely to present as metastatic disease, show high-grade nuclear atypia, and lack the characteristic hemangiopericytoma-like vasculature, posing a unique diagnostic challenge. The use of STAT6 immunohistochemistry and/or molecular studies may be prudent in soft tissue tumors that appear CD34 negative and lack conventional SFT histopathologic characteristics.

YAP1-TFE3-fused hemangioendothelioma is an extremely rare malignant vascular tumor. We present the largest multi-institutional clinicopathologic study of YAP1-TFE3-fused hemangioendothelioma to date. The 24 cases of YAP1-TFE3-fused hemangioendothelioma showed a female predominance (17 female, 7 male) across a wide age range (20–78 years old, median 44). Tumors were most commonly located in soft tissue (50%), followed by bone (29%), lung (13%), and liver (8%), ranging from 3 to 115 mm in size (median 40 mm). About two-thirds presented with multifocal disease, including 7 cases with distant organ metastasis. Histopathologically, we describe three dominant architectural patterns: solid sheets of coalescing nests, pseudoalveolar and (pseudo)vasoformative pattern, and discohesive strands and clusters of cells set in a myxoid to myxohyaline stroma. These patterns were present in variable proportions across different tumors and often coexisted within the same tumor. The dominant cytomorphology (88%) was large epithelioid cells with abundant, glassy eosinophilic to vacuolated cytoplasm, prominent nucleoli and well-demarcated cell borders. Multinucleated or binucleated cells, prominent admixed erythrocytic and lymphocytic infiltrates, and intratumoral fat were frequently present. Immunohistochemically, ERG, CD31, and TFE3 were consistently expressed, while expression of CD34 (83%) and cytokeratin AE1/AE3 (20%) was variable. CAMTA1 was negative in all but one case. All cases were confirmed by molecular testing to harbor YAP1-TFE3gene fusions: majority with YAP1exon 1 fused to TFE3exon 4 (88%), or less commonly, TFE3exon 6 (12%). Most patients (88%) were treated with primary surgical resection. Over a follow-up period of 4–360 months (median 36 months) in 17 cases, 35% of patients remained alive without disease, and 47% survived many years with stable, albeit multifocal and/or metastatic disease. Five-year progression-free survival probability was 88%. We propose categorizing YAP1-TFE3-fused hemangioendothelioma as a distinct disease entity given its unique clinical and histopathologic characteristics in comparison to conventional epithelioid hemangioendothelioma.

Cancer of unknown primary (CUP) refers to metastatic tumors for which the primary tumor of origin cannot be determined at the time of diagnosis, despite extensive clinicopathologic investigations. Molecular profiling is increasingly able to predict a probable primary tumor type for CUP when clinicopathologic workup is inconclusive. Numerous studies have explored the use of various molecular profiling techniques for identification of site/tissue of origin of CUP. These techniques include gene expression profiling utilizing microarray, reverse transcriptase polymerase chain reaction, RNA-sequencing, somatic gene mutation profiling with next-generation DNA sequencing, and epigenomics including DNA methylation profiling. Despite the generally poor prognosis of CUP, a minority of patients can expect to benefit from targeted therapy despite being agnostic to the tissue of origin. Studies have explored the use of various molecular profiling techniques to predict prognostic and therapeutic biomarkers, with the goal of improving outcome for patients with CUP. However, discordant results between non-randomized and randomized clinical trials in evaluating tumor-type specific therapies raise uncertainties of the benefits of molecularly-predicted tissue of origin-based treatment in routine clinical use. Nevertheless, the current overall trend is in favor of using molecular tools to refine the diagnosis and clinical management of patients with CUP. More large-cohort, randomized prospective studies are needed to assess and validate the utility and feasibility of molecular profiling to uncover potentially targetable genetic alterations. These efforts will also yield further biological insights into the biology and pathogenesis of CUP (Graphical Abstract).

Gene rearrangements involving the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase gene have been identified in various neoplasms, including inflammatory myofibroblastic tumor and epithelioid fibrous histiocytoma. We present an ALK-rearranged cutaneous soft tissue tumor with unique morphologic and immunophenotypic features that are not shared by other entities with ALKrearrangements. The six cases involved two females and four males, aged 18–84 (mean 51) years old. Three tumors were on the back and three on the lower extremities (thigh, knee, shin); ranging from 0.5 to 5.6 (mean 2.1) cm. Four were confined to the dermis; two involved the subcutis. All six cases were characterized by the presence of spindled to ovoid cells arranged in concentric whorls and cords against a myxoid to myxohyaline stroma and relatively cellular aggregates of plump ovoid to epithelioid cells. Four cases showed distinct hyalinized blood vessels. Both cases that involved the subcutis showed peripheral lipofibromatosis-like areas. Tumor-infiltrating lymphocytes were absent to moderate. Severe cytologic atypia or conspicuous mitotic activity was not identified. Immunohistochemically, all tumors diffusely expressed ALK (D5F3) and CD34. All but one tumor was diffusely positive for S100 protein. All tumors were negative for EMA, AE1/AE3, SMA, and SOX10. Next-generation sequencing revealed ALKfusions with FLNA(3 cases), MYH10(2 cases), and HMBOX1(1 case) as the partner genes. In all six cases, the breakpoints involved exon 20 of ALK, which preserves the receptor tyrosine kinase domains of ALK in the fusion product. Of the four cases with limited follow-up information (2–18 months), none recurred. In conclusion, we report an ALK-rearranged cutaneous soft tissue tumor characterized by the presence of myxoid spindle cell whorls and cords, and co-expression of ALK, CD34, and frequently S100 protein, we term “superficial ALK-rearranged myxoid spindle cell neoplasm”.

Gene rearrangements involving the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase gene have been identified in various neoplasms, including inflammatory myofibroblastic tumor and epithelioid fibrous histiocytoma. We present an ALK-rearranged cutaneous soft tissue tumor with unique morphologic and immunophenotypic features that are not shared by other entities with ALKrearrangements. The six cases involved two females and four males, aged 18–84 (mean 51) years old. Three tumors were on the back and three on the lower extremities (thigh, knee, shin); ranging from 0.5 to 5.6 (mean 2.1) cm. Four were confined to the dermis; two involved the subcutis. All six cases were characterized by the presence of spindled to ovoid cells arranged in concentric whorls and cords against a myxoid to myxohyaline stroma and relatively cellular aggregates of plump ovoid to epithelioid cells. Four cases showed distinct hyalinized blood vessels. Both cases that involved the subcutis showed peripheral lipofibromatosis-like areas. Tumor-infiltrating lymphocytes were absent to moderate. Severe cytologic atypia or conspicuous mitotic activity was not identified. Immunohistochemically, all tumors diffusely expressed ALK (D5F3) and CD34. All but one tumor was diffusely positive for S100 protein. All tumors were negative for EMA, AE1/AE3, SMA, and SOX10. Next-generation sequencing revealed ALKfusions with FLNA(3 cases), MYH10(2 cases), and HMBOX1(1 case) as the partner genes. In all six cases, the breakpoints involved exon 20 of ALK, which preserves the receptor tyrosine kinase domains of ALK in the fusion product. Of the four cases with limited follow-up information (2–18 months), none recurred. In conclusion, we report an ALK-rearranged cutaneous soft tissue tumor characterized by the presence of myxoid spindle cell whorls and cords, and co-expression of ALK, CD34, and frequently S100 protein, we term “superficial ALK-rearranged myxoid spindle cell neoplasm”.

Intravascular lobular capillary hemangioma (ILCH), or intravascular pyogenic granuloma, is relatively rare and likely underrecognized. We reviewed all ILCH cases from our institution confirmed pathologically from 2006 to 2019. Immunostains for smooth muscle actin and Wilms tumor 1 were performed on all cases and prior immunohistochemical stains were reviewed. Forty cases were identified (22 females; 18 males) with a median age of 53 years (range: 13 to 85 y). Clinically, all were well-circumscribed, stable to slow-growing raised/cystic skin lesions ranging from 1 to 40 mm. Most were located on the upper extremities (n=18), followed by head and neck (n=16). Vascular lesions were suspected clinically in one third. Of the consultation cases, provided differential diagnoses included Masson tumor, other hemangiomas, and vascular tumors of intermediate malignancy. The common histologic features were a well-circumscribed, lobular proliferation of closely packed capillaries with central ectatic vessels. The surrounding vascular wall was well-visualized in only half of the cases. About 30% of the cases were mitotically active (mean: 8 to 9 mitotic figures/10 HPFs), and 34% showed mild to moderate cytologic atypia. Hobnail features were present in 40% of cases. All cases were diffusely and strongly positive for Wilms tumor 1. Smooth muscle actin stains highlighted pericytes in all cases. Of the 20 cases with clinical follow-up (median: 40 mo), none recurred. ILCH commonly involves the upper extremities and a vascular tumor is suspected clinically in the minority. Mitotic activity and cytologic atypia, when present, can cause confusion with more aggressive vascular tumors. Recognition of this entity is essential as it is a benign lesion with no risk of recurrence following limited local excision.

Alveolar rhabdomyosarcoma (ARMS) with FOXO1gene rearrangements is an aggressive pediatric rhabdomyosarcoma subtype that is prognostically distinct from embryonal rhabdomyosarcoma and fusion-negative ARMS. Herein, we report two cases of ARMS with PAX3::MAML3fusions. The tumors arose in an infant and an adolescent as stage IV metastatic disease (by Children’s Oncology Group staging system). Histologically, both cases were small round blue cell tumors arranged in vague nests and solid sheets that were diffusely positive for desmin and myogenin. By methylation profiling and unsupervised clustering analysis, the tumors clustered with ARMS with classic FOXO1rearrangements and ARMS with variant PAX3::NCOA1/INO80Dfusions, but not with biphenotypic sinonasal sarcoma (BSNS) with PAX3::MAML3/NCOA2/FOXO1/YAP1fusions, nor with other small round blue cell tumors, including embryonal rhabdomyosarcoma. The differentially methylated genes between ARMS and BSNS were highly enriched in genes involved in myogenesis, and 21% of these genes overlap with target genes of the PAX3::FOXO1 fusion transcription factor. On follow-up after initiation of vincristine/actinomycin/cyclophosphamide chemotherapy, the tumors showed partial and complete clinical response, consistent with typical upfront chemotherapy responsiveness of ARMS with the classic FOXO1rearrangement. We conclude that PAX3::MAML3is a novel variant fusion of ARMS, which displays a methylation signature distinct from BSNS despite sharing similar PAX3fusions. These findings highlight the utility of methylation profiling in classifying ARMS with non-canonical fusions.

Adamantinoma-like Ewing sarcoma (ALES) has traditionally been considered a variant of Ewing sarcoma because it generally harbors EWSR1::FLI1fusions despite showing diffuse positivity for keratins and p40. However, it has become increasingly recognized that different tumors can have identical translocations, including shared fusions between carcinomas and sarcomas, raising questions as to whether ALES might represent a separate entity. Using methylation profiling, we further explored the relationship between Ewing sarcoma and ALES. The archives of multiple institutions were searched for candidate cases of ALES. DNA methylation profiling was performed and results were compared to corresponding data from conventional Ewing sarcoma. Twelve cases of ALES (5 previously reported) were identified in 10 men and 2 women (aged 20-72 years; median age, 41.5 years). Cases included tumors arising in the parotid gland (3), sinonasal cavity (2), submandibular gland (2), thyroid gland (1), neck (1), gingiva (1), hypopharynx (1), and mandible (1). Histologic review consistently showed sheets and nests of basaloid cells within a fibromyxoid or hyalinized stroma. All tumors were positive for at least 1 keratin and CD99 expression, whereas all 10 cases tested were positive for p63 or p40; S100 protein expression was noted in 2 cases. Cases harbored either EWSR1::FLI1fusions (n = 6), FUS::FLI1fusions (n = 1), and/or EWSR1rearrangements (n = 6). Methylation profiling was successful in 11/12 cases evaluated. Unsupervised clustering and dimensionality reduction (Uniform Manifold Approximation and Projection) of DNA methylation data revealed a distinct methylation cluster for all 11 cases, including the tumor with the FUS::FLI1fusion, which clearly segregated them from the conventional Ewing sarcoma. Follow-up (n = 11, 1-154 months) revealed that 4 patients experienced recurrence and 6 developed metastatic disease. ALES demonstrates a distinct methylation signature from conventional Ewing sarcoma. This finding adds to the distinctive immunoprofile of ALES, suggesting that these 2 tumors should be considered distinct entities rather than histologic extremes of the same disease.

Pulmonary carcinoid tumors are an uncommon tumor in the lung, representing <1% to 2% of all primary lung cancers, and have a relatively indolent clinical course. Because of their low incidence, these tumors do not have a specific staging system. However, since the 7th edition, the TNM Classification for Lung Cancer has been used in these tumors, though the ability of this staging classification to predict prognosis in carcinoid tumors is not well-studied. We report the largest single institution study of typical and atypical carcinoid tumors with recurrence as a measure of outcome and compared the ability of the 7th and 8th TNM Classification of Lung Cancer to predict recurrence in typical and atypical carcinoid tumors of the lung. All surgical lung resection cases from 1995 to 2016 with a diagnosis of primary lung carcinoid tumor were reviewed and clinicopathologic parameters, including tumor size, nodal status, histology (mitotic counts), and recurrence were recorded. The final cohort consists of 205 carcinoid tumors: 188 (92%) typical carcinoids and 17 (8%) atypical carcinoids. Pulmonary carcinoid tumors have an excellent prognosis with a low recurrence rate of 8%. Atypical carcinoids were significantly more likely to recur (median time to recurrence: 3 y) compared with typical carcinoids. By placing more emphasis on tumor size and nodal status in the staging classification, the TNM 8th edition was superior in predicting outcome compared with the TNM 7th edition.

Recent evidence suggests a role for the nuclear marker INSM1 in the diagnosis of neuroendocrine lung neoplasms. The aim of this study was to determine the utility of INSM1 as a marker of neuroendocrine differentiation using a large series of whole-tissue sections of primary lung neoplasms. We stained 345 primary lung neoplasms with INSM1, including 292 whole-tissue sections. Most cases were also stained with synaptophysin, chromogranin, and CD56. The tumors included 64 small cell lung carcinomas, 24 large cell neuroendocrine carcinomas, 64 carcinoid tumors (48 typical, 16 atypical), 130 adenocarcinomas, and 33 squamous cell carcinomas. For small cell lung carcinoma, the sensitivity of INSM1 (98%) was similar to synaptophysin (100%) and CD56 (95%) but considerably higher than chromogranin (83%). For large cell neuroendocrine carcinoma, CD56 (92%) and synaptophysin (88%) were more sensitive than INSM1 (75%), while chromogranin was less sensitive (46%). All markers stained 100% of carcinoid tumors, except one atypical carcinoid tumor, which was negative for INSM1. The sensitivity of INSM1 for neuroendocrine lung neoplasms as a group (95%) was similar to synaptophysin (98%) and CD56 (97%), but higher than chromogranin (84%). The specificity of INSM1 for neuroendocrine lung neoplasms (97%) was similar to chromogranin (98%) but higher than synaptophysin (90%) and CD56 (87%). INSM1 staining was concordant in primary tumors and matched metastases. In conclusion, INSM1 is a reliable marker of neuroendocrine differentiation in primary lung neoplasms, with sensitivity similar to synaptophysin and CD56, and specificity similar to chromogranin.

Capicua transcriptional repressor (CIC)-rearranged sarcoma represents a distinct pathologic entity and constitutes the second most prevalent category of undifferentiated round cell sarcomas (URCSs) after Ewing sarcoma. The 2 most common translocations are t(4;19) and t(10;19), resulting in CICfusions with either DUX4and DUX4L paralog, respectively; however, other rare variant fusions have also been reported. In this study, we expand the molecular spectrum of CIC-gene partners, reporting on 5 cases of URCSs showing CICfusions with AXL, CITED1, SYK, and LEUTXby targeted RNA or DNA sequencing. There were 4 female patients and 1 male patient with a wide age range (12-70 years; median, 36 years). Four cases occurred in the deep soft tissues (lower extremity, 3; neck, 1) and 1 case in the central nervous system (midbrain/thalamus). All cases showed similar histologic findings within the spectrum of URCSs. Immunohistochemistry, showed variable positivity for ETV4 in 4 of the 4 cases and positive results for ERG in 3 of the 4 cases and for WT1 in 1 of the 4 cases. CD31 showed positivity in 2 of the 3 cases, including one coexpressing ERG. Unsupervised clustering of methylation profiles by T-distributed stochastic neighborhood embedding performed in 4 cases showed that all clustered tightly together and along the CICsarcoma methylation class. RNA-sequencing data showed consistent upregulation of ETV1and ETV4mRNA in all cases examined, at similar levels to CIC::DUX4URCSs. Our study expands the molecular diversity of CIC-rearranged URCSs to include novel and rare partners, providing morphologic, immunohistochemical, gene expression, and methylation evidence supporting their classification within the family of tumors harboring the more common DUX4/DUX4Lpartner genes.

PDGFRB-activating mutations have been reported in pediatric myofibroma and myofibromatosis. However, recurrent gain-of-function PDGFRBmutations have not been documented in sarcomas with myogenic differentiation. Driven by occasional sarcomas harboring PDGFRBmutations, we investigated their prevalence and clinicopathologic and genomic features in a large cohort of sarcomas. An institutional targeted DNA next-generation sequencing database was searched for sarcomas with myogenic differentiation harboring hotspot PDGFRBgene alterations. Among 3300 patients with sarcomas, 21 (0.6%) patients were identified (17 women, 4 men) with an age range of 35 to 88 years. The site distribution included 13 gynecologic tract (12 uteri, 1 vagina), 4 bone and soft tissue, and 4 viscera. All except 1 were high grade. Most patients were diagnosed as sarcomas with myogenic differentiation based on partial staining for 1 or more muscle markers, whereas 6 were labeled as leiomyosarcoma (LMS). Most tumors showed monomorphic spindle morphology, with either heterogeneous features of myofibroblastic and smooth muscle differentiation or an undifferentiated phenotype. Hormone receptors were negative in all uterine cases. PDGFRB immunostaining in all cases tested was strong and diffuse, whereas PDGFRA was negative/focal. The most frequent PDGFRBmutations were exon 12 (43%), exon 14 (N666K/S/T) (38%), and exon 18 (D850Y/H/V or insertion/deletion) (19%). The most frequent co-existing genetic alterations (26% to 37%) occurred in CDKN2A/B, TP53, TERT, and MED12. Moreover, PDGFRB-mutant sarcomas had an overall distinct genomic landscape compared with both uterine and soft tissue LMS control groups. These tumors were associated with a highly aggressive clinical course, with frequent distant metastases (81%) and death (76%), regardless of anatomic location, and worse overall survival compared with the 2 LMS control groups. This is the first study documenting recurrent hotspot PDGFRBalterations in high-grade sarcomas, which show a predilection for uterine location and myogenic differentiation that fall short of the diagnostic criteria for LMS. Further studies are needed to investigate the therapeutic potential of kinase inhibitors in this group of tumors.

Myxoid pleomorphic liposarcoma (MPLPS) shows a strong predilection for the mediastinum and can affect a wide age range. Clinically, MPLPS exhibits aggressive behavior and demonstrates a worse overall and progression-free survival than myxoid/round cell liposarcoma (MRLPS) and pleomorphic liposarcoma (PLPS). Histologically, MPLPS is characterized by hybrid morphologic features of MRLPS and PLPS, including myxoid stroma, chicken wire-like vasculature, univacuolated and multivacuolated lipoblasts, and high-grade pleomorphic sarcomatous components. In terms of molecular features, MPLPS is distinct from other lipomatous tumors as it harbors genome-wide loss of heterozygosity.

This article focuses on various recently described or emerging cutaneous soft tissue neoplasms. These entities encompass a wide range of clinical and histologic characteristics. Emphasis is placed on their distinguishing morphologic and immunophenotypic features compared with entities that enter into their differential diagnosis, as well as novel immunophenotypic and molecular tests that are often necessary for accurate diagnosis of these entities. Entities discussed include EWSR1-SMAD3-rearranged fibroblastic tumor, superficial CD34-positive fibroblastic tumor, epithelioid fibrous histiocytoma, CIC-rearranged sarcomas, and NTRK-rearranged spindle cell tumors.

Myxofibrosarcoma is a malignant pleomorphic fibroblastic sarcoma with variably myxoid stroma, and is characterised by a distinctive curvilinear vascular pattern. In the head and neck area, myxofibrosarcoma is extremely rare, with only a handful of case reports in the literature to date. We report the first case series of primary sinonasal myxofibrosarcoma across two institutions. Among the five cases (2 female, 3 males, aged 52–82 years old), four arose from the maxillary sinus and one from the sphenoid sinus. Four patients received surgical resection and three with adjuvant radiotherapy. The tumours ranged from 2.9 to 5.6 cm in greatest dimensions. All tumours demonstrated extensive myxoid stroma (>50% myxoid component) with a characteristic curvilinear, elongated, thin-walled vasculature with perivascular condensation of tumour cells. All but one were classified as intermediate to high grade myxofibrosarcoma. Among the four patients with follow-up information, three reported no local recurrence or distal metastasis, and one had local recurrence. Myxofibrosarcoma should be included in the differential diagnosis of sinonasal tumours with a pleomorphic spindle cell morphology and a ‘null’ immunophenotype.