Purpose: Fusion-positive myoepithelial tumors (MET) are clinicopathologically heterogeneous and variably termed mixed tumors and myoepithelial carcinomas. As FET-rearranged METs lack ductal/epithelial differentiation, we test whether FET-rearranged METs are epigenetically distinct from adnexal PLAG1-rearranged METs, which we hypothesize to be analogues of salivary gland METs. Experimental Design: DNA methylation profiling from a multi-institutional cohort of 52 fusion-positive skin, soft-tissue, and bone MET cases was performed and compared with diverse tumor types, including salivary METs. The MET subgroups harbored EWSR1::KLF15, EWSR1/FUS::KLF17, EWSR1::PBX1, EWSR1::PBX3, EWSR1/FUS::POU5F1, SS18::POU5F1, EWSR1::ZNF444, and PLAG1 rearrangements. Pooled clinicopathologic and outcome analysis with new and published cases (total 185) was performed. Results: The MET subgroups showed significant heterogeneity in age, site, and histology. Specifically, EWSR1::KLF15 METs affected predominantly young children (<5 years); EWSR1::PBX1/PBX3 METs were enriched in skin/bone; and EWSR1/FUS::POU5F1, SS18::POU5F1, and EWSR1::KLF15 METs tended to display malignant histology. Conversely, PLAG1-rearranged tumors were predominantly benign, arising in older adults and located in the skin. DNA methylation profiling revealed that FET-rearranged METs were epigenetically related to SS18::POU5F1 METs and FET::NFATC2 sarcomas but entirely distinct from PLAG1-rearranged adnexal and salivary METs. Histologic features were correlated with the degree of genome-wide copy-number variation. The median disease-specific survival was shortest in SS18::POU5F1 (31 months), EWSR1::PBX3 (38 months), and EWSR1::KLF15 (45 months) METs. On multivariate analysis, age <25 years was a significant predictor of worse progression-free survival. Conclusions: FET-rearranged METs are epigenetically unrelated to cutaneous and salivary gland METs, and their malignant counterparts are best classified as sarcomas rather than carcinomas.

Background Sarcomas are malignancies of mesodermal origin that have a propensity for aggressive invasion of local structures and hematogenous spread. Sarcomas of the posterior trunk are problematic because of their insidious growth, lack of patient realization, and proximity to the vertebral column and spinal cord. Reconstruction often requires well-vascularized tissue that can withstand the demands of radiotherapy, without significant complications. Methods We assessed the outcomes of 47 patients over a 7-year period with soft-tissue sarcomas of the posterior trunk extending from the neck to the sacrum and their reconstructions. Nine were direct closures, and 2 required split-thickness skin grafts. Thirty-seven patients underwent 39 flap reconstructions (26 myocutaneous, 12 fasciocutaneous, 1 muscle-only). Thirty-five patients required a single flap reconstruction, and 2 required multiple flaps. Results There were no recorded flap failures. Thirty-three patients (70%) healed without complications. Twenty-one patients required radiotherapy (preoperative 3, postoperative 18) and wound healing complications occurred in one (33%) and 5 (27%%) patients, respectively. Complications: seroma (10%), dehiscence (12.8%), return to theater (12.8%), infection (12.8%), and partial flap necrosis (10%). No statistical significance was found when comparing sex, smoking status, diabetes, defect location, or radiotherapy exposure with wound complication outcomes. There was no statistical significance when comparing wound complications, return to theater or healing time between those who had received preoperative and postoperative radiotherapy. Conclusions Patients with posterior trunk sarcomas often require well-vascularized robust reconstruction that can withstand the potential complications of radiotherapy and pressure. Reconstruction can include fasciocutaneous or musculocutaneous flaps, or a combination of both, to help reduce morbidity.

Malignant chondroblastoma is a recently described variant of chondroblastoma showing a distinct age/site distribution and morphology along with the typical H3-3B p.K36M mutation. We sought to further compare conventional and malignant chondroblastoma. Malignant chondroblastomas were collected. H3-3 K36M immunohistochemistry, as well as DNA methylation and copy number profiling, were performed and compared with conventional chondroblastoma. Forty-one samples from 26 patients were identified (20 males, 6 females; age: 19-62 years; median: 39 years). Anatomical sites included rib (7), pelvis (4), acromion (4), scapula (4), spine (2), long bone (3), calcaneus (1), and talus (1). Imaging showed an expansile mass with variable cortical erosion and/or breakthrough. Most patients (n = 17) showed sheets of atypical ovoid cells deposited in a myxoid stroma. Osteoclast-like giant cells/matrix formation was scarce. Six patients had tumors with features of conventional chondroblastoma but significant cytologic atypia. Three tumors demonstrated a morphology indistinguishable from conventional chondroblastoma except for extensive permeation, and one of these cases showed a transition to high-grade sarcoma. The final case was composed only of high-grade pleomorphic sarcoma that harbored an H3F3A p.K36M mutation. When an adjacent host bone was present, permeative growth through the cortex and native trabeculae was noted. All samples tested positive for the H3-3 K36M-specific antibody (26 of 26). Methylome profiling of 28 specimens from 24 patients revealed that 26 of these specimens formed a distinct cluster on a uniform manifold approximation and projection dimension reduction plot separate from conventional chondroblastoma and high-grade osteosarcoma. Of 26 methylomes from which interpretable copy number profiles can be derived, 12 had no variations, whereas 14 had copy number changes. Of 22 patients, 11 experienced local recurrence, 8 patients developed metastasis, and 3 patients died of disease. Malignant chondroblastoma is a rare, clinically and epigentically distinct variant of chondroblastoma with a predilection for the flat bones of skeletally mature individuals and high rates of local recurrence and distant metastases. A subset transforms into high-grade pleomorphic sarcoma, either de novo or in subsequent recurrences. Malignant chondroblastoma with high-grade features and/or copy number variations appears to have a higher propensity for adverse events, including death from disease.

Malignant melanoma is a common and often aggressive neoplasm of the skin arising from melanocytes. Metastatic melanoma is known for its diverse clinical manifestations, and can present with atypical features prior to diagnosis of the primary lesion, which can pose a diagnostic challenge. We report a rare case of metastatic melanoma in a 67 year-old male who presented with a painless, enlarging mass in the right axilla over a 4 week period. Ultrasound and magnetic resonance imaging (MRI) scans revealed a well-defined solitary, cystic appearing lesion in the right axilla with a distinct fluid–fluid level. An ultrasound guided biopsy of the lesion diagnosed a metastatic melanoma. While haemorrhagic distant metastases are a well-recognised complication of malignant melanoma, particularly in the brain and lung, soft tissue metastases presenting with fluid–fluid levels is not well described in the literature. The case highlights the importance of considering the differential of melanoma metastasis when encountered with such a lesion and importance of ultrasound guided biopsy for histopathological confirmation, as the imaging features can mimic that of a haemorrhagic soft tissue sarcoma, the management of which differs substantially from that of melanoma.

Ewing sarcoma (ES) is the second most common primary malignant bone tumour in children and adolescents. About 14.5

TP53 is the most frequently mutated gene in human cancer. This gene shows not only loss-of-function mutations but also recurrent missense mutations with gain-of-function activity. We have studied the primary bone malignancy osteosarcoma, which harbours one of the most rearranged genomes of all cancers. This is odd since it primarily affects children and adolescents who have not lived the long life thought necessary to accumulate massive numbers of mutations. In osteosarcoma, TP53 is often disrupted by structural variants. Here, we show through combined whole-genome and transcriptome analyses of 148 osteosarcomas that TP53 structural variants commonly result in loss of coding parts of the gene while simultaneously preserving and relocating the promoter region. The transferred TP53 promoter region is fused to genes previously implicated in cancer development. Paradoxically, these erroneously upregulated genes are significantly associated with the TP53 signalling pathway itself. This suggests that while the classical tumour suppressor activities of TP53 are lost, certain parts of the TP53 signalling pathway that are necessary for cancer cell survival and proliferation are retained. In line with this, our data suggest that transposition of the TP53 promoter is an early event that allows for a new normal state of genome-wide rearrangements in osteosarcoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Chondrosarcoma (CS) is the second most frequent primary malignant bone tumour, characterized by production of non‐osteoid cartilage matrix. Up to more than 30

Small cell osteosarcoma (SCOS), a variant of conventional high-grade osteosarcoma (COS), may mimic fusion-driven round cell sarcomas (FDRCS) by overlapping clinico-radiological and histomorphological/immunohistochemical characteristics, hampering accurate diagnosis and consequently proper therapy. We retrospectively analyzed decalcified formalin-fixed paraffin-embedded (FFPE) samples of 18 bone tumors primarily diagnosed as SCOS by methylation profiling, fusion gene analysis, and immunohistochemistry. In eight cases, the diagnosis of SCOS was maintained, and in 10 cases it was changed into FDRCS, including three Ewing sarcomas ( EWSR1::FLI1 in two cases and no identified fusion gene in the third case), two sarcomas with BCOR alterations ( KMT2D::BCOR, CCNB3::BCOR , respectively), three mesenchymal chondrosarcomas (HEY1::NCOA2 in two cases and one case with insufficient RNA quality), and two sclerosing epithelioid fibrosarcomas ( FUS::CREBL3 and EWSR1 rearrangement, respectively). Histologically, SCOS usually possessed more pleomorphic cells in contrast to the FDRCS showing mainly monomorphic cellular features. However, osteoid was seen in the latter tumors as well, often associated with slight pleomorphism. Also, the immunohistochemical profile (CD99, SATB2, and BCOR) overlapped. Clinically and radiologically, similarities between SCOS and FDRCS were observed, with by imaging only minimal presence or lack of (mineralized) osteoid in most of the SCOSs. In conclusion, discrimination of SCOS, epigenetically related to COS, versus FDRCS of bone can be challenging but is important due to different biology and therefore therapeutic strategies. Methylation profiling is a reliable and robust diagnostic test especially on decalcified FFPE material. Subsequent fusion gene analysis and/or use of specific immunohistochemical surrogate markers can be used to substantiate the diagnosis.

How massive genome rearrangements confer a competitive advantage to a cancer cell has remained an enigma. The malignant bone tumour osteosarcoma harbours an extreme number of structural variations and thereby holds the key to understand complex cancer genomes. Genome integrity in osteosarcoma is generally lost together with disruption of normal TP53 gene function, the latter commonly through either missense mutations or structural alterations that separate the promoter region from the coding parts of the gene. To unravel the consequences of a TP53 promoter relocated in this manner, we performed in-depth genetic analyses of osteosarcoma biopsies ( n =148) and cell models. We show that TP53 structural variations are early events that not only facilitate further chromosomal alterations, but also allow the TP53 promoter to upregulate genes erroneously placed under its control. Paradoxically, many of the induced genes are part of the TP53 -associated transcriptome, suggesting a need to counterbalance loss of TP53 function through ‘separation-of-function’ mutations via promoter swapping. Our findings demonstrate how massive genome errors can functionally turn the promoter region of a tumour suppressor gene into a constitutively active oncogenic driver.

Background: Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare primary bone sarcoma that lacks a specific line of differentiation. Distinguishing between UPSb and other malignant bone sarcomas, including dedifferentiated chondrosarcoma and osteosarcoma, is challenging due to their overlapping features. We have previously identified that UPSb tumours have elevated mRNA levels of Fibroblast Growth Factor 23 (FGF23) transcripts compared to other sarcomas including osteosarcoma. In the present study, we evaluated the specificity and practicality of FGF23 immunoreactivity as a specific diagnostic tool to differentiate UPSb tumours from osteosarcomas and dedifferentiated chondrosarcomas. Methods: A total of 10 UPSb, 10 osteosarcoma, and 10 dedifferentiated chondrosarcoma cases (all high-grade), were retrieved and immunohistochemistry for FGF23 was performed. Results: FGF23 protein was expressed at high levels in 80–90% of undifferentiated pleomorphic sarcoma of the bone cases, whereas it was expressed at significantly lower levels in dedifferentiated chondrosarcoma and osteosarcoma cases. A semiquantitative analysis, considering the intensity of immunoreactivity, confirmed significantly elevated FGF23 expression levels in UPSb tissues compared to those observed in osteosarcoma and dedifferentiated chondrosarcoma tissues. Conclusions: The results we present here suggest that FGF23 immunohistochemistry may be a useful tool to aid in differentiating UPSb from morphologically similar malignant bone sarcomas, especially in situations where sampling is restricted and there is limited clinical information available.

Background: Chondrosarcoma (CS) is the second most common surgically treated primary malignancy of the bone. The current study explored the effect of the margin and extraosseous tumor component in CS in the femur on local recurrence (LR), LR-free survival (LRFS), and disease-specific survival (DSS). Methods: Among 202 patients, 115 were in the proximal extremity of the femur, 4 in the corpus of the femur, and 83 in the distal extremity of femur; 105 patients had an extraosseous tumor component. Results: In the Kaplan–Meier analysis, factors significant for decreased LRFS were the extraosseous tumor component (p < 0.001), extraosseous tumor component arising from the superior aspect (p < 0.001), histological grade (p = 0.031), and narrow surgical margin < 3 mm (p < 0.001). Factors significantly affecting DSS were the histological grade (p < 0.001), extraosseous component (p < 0.001), LR (p < 0.001), metastases (p < 0.001), and surgical margin (p < 0.001). Conclusions: In CS of the femur, the presence of an extraosseous tumor component has a predictive role in LRFS, and extraosseous tumor component arising from the superior aspect was significant for decreased LRFS. Wide margins were more commonly achieved when the tumor had only an intraosseous component, and the rate of LR was significantly higher in cases with an extraosseous tumor component. When the extraosseous component arose from the superior aspect of the femur, LR occurred more frequently despite achieving adequate margins.

Osseous metastatic disease is commonly encountered in breast carcinoma, which typically presents as either osteolytic, osteoblastic, or mixed lesions on imaging. Osseous metastasis presenting as a multiloculated cystic lesion with fluid-fluid levels resembling that of an aneurysmal bone cyst (ABC) is sparsely described in the literature, and even less so in the case of breast carcinoma. We report an unusual case of fluid-fluid levels in a bone metastasis to the spine in a 66-year-old female with a prior history of breast carcinoma. Magnetic resonance imaging (MRI) demonstrated a cystic lesion with fluid levels resembling that of an ABC. Computed tomography (CT)-guided biopsy revealed the lesion to be a metastasis from breast carcinoma. The management of ABCs and osseous metastases differ drastically. Accurate diagnosis and distinction between these lesions is paramount as the management of metastatic disease can have a significant impact on the quality and length of life. The presentation, differential diagnoses and imaging features of this atypical case are discussed.

OBJECTIVES:We aimed to analyze survival, predictors of outcome, and the long-term functional status of patients with a diagnosis of primary chest wall sarcoma who undergo chest wall resection and reconstruction. METHODS:We analyzed a prospectively maintained database, including all patients operated on between 2008 and 2021. The primary outcome measures were overall and disease-free survival and analyses were employed to determine the risk factors for poor survival and recurrence. RESULTS:One hundred thirty-nine patients were included, 55% were men. The majority (96%) had an R0 resection and 75.1% had no postoperative complications up to 30 days postprocedure; median length of hospital stay was 7 days (range, 6-10 days). Median overall and disease-free survival was 58.8 and 53.6 months, respectively. For those alive, at long-term follow-up, 80% had a Medical Research Council dyspnea score of 0 and Karnofsky index >80%. Survival and mortality rates were better in chondrosarcomas compared with nonchondromatous sarcomas (P < .05). Previous history of radiotherapy, previous history of cancer, the type of sarcoma (Ewing's or soft tissue), the need for adjuvant treatment and tumor grade were significant predictors of mortality and recurrence on univariate testing. Extended resection, a higher number of ribs removed, and the incidence of postoperative complications were significantly associated with a worse postoperative Medical Research Council dyspnea score. CONCLUSIONS:Careful patient selection and multidisciplinary decision making is crucial. This leads to clear resection margins, good overall, and disease-free survival and good functional outcomes.

Nodular fasciitis is a benign soft tissue pseudotumour typically occurring in the upper extremities, head and neck, thigh and trunk. It is most commonly seen in subcutaneous locations but also can be present in intramuscular and intermuscular (fascial) locations. Its occurrence in the hand is rare, and while it can occur in close proximity to tendons, its presentation as an intra-tendinous mass has not been previously described. We present a unique and rare case of nodular fasciitis arising within the flexor digitorum profundus (FDP) tendon of the hand in a 16-year-old female. The patient presented with a painful swelling in the volar aspect of the base of her left middle finger, with progressive flexion deformity of the finger. Ultrasound and magnetic resonance imaging revealed a mass within the FDP tendon of the middle finger. An ultrasound-guided biopsy revealed a diagnosis of nodular fasciitis. Given the self-limiting nature of the condition, she was managed conservatively with close clinical and imaging follow-up. This case highlights the importance of considering nodular fasciitis in the differential diagnosis of an intra-tendinous lesion in the hand, even though it is a rare occurrence in this location. The clinical presentation, diagnostic workup, and management of this unique case are discussed, emphasising the potential for its misdiagnosis as a malignancy which can have important implications in management.

Expression of the SEF vs controls 100-top up-regulated genes in Tet-On 3G cells expressing the EWSR1-CREB3L1 fusion

Introduction:Chondrosarcoma of the synovium is a rare and malignant form of cartilaginous tumor that originates in synovial tissue. There have only been a limited number of reported cases of malignant transformation of synovial chondromatosis (SC) into secondary chondrosarcoma (SCH), primarily in the hip and knee, in patients with resistant illness. The occurrence of chondrosarcoma in SC of the wrist is highly uncommon, as evidenced by only a single previous case study that has been documented in the literature.Case Report:This study presents a case series of two patients with primary SC who developed SCH at the wrist joint.Conclusion:Clinicians treating localized swellings of the hand and wrist should be alert to the likelihood of a sarcoma diagnosis to minimize delays to definitive therapy.

Primary bone cancer (PBC) comprises several subtypes each underpinned by distinctive genetic drivers. This driver diversity produces novel morphological features and clinical behaviour that serendipitously makes PBC an excellent metastasis model. Here, we report that some transfer RNA-derived small RNAs termed tRNA fragments (tRFs) perform as a constitutive tumour suppressor mechanism by blunting a potential pro-metastatic protein-RNA interaction. This mechanism is reduced in PBC progression with a gradual loss of tRNAGlyTCC cleavage into 5' end tRF-GlyTCC when comparing low-grade, intermediate-grade and high-grade patient tumours. We detected recurrent activation of miR-140 leading to upregulated RUNX2 expression in high-grade patient tumours. Both tRF-GlyTCC and RUNX2 share a sequence motif in their 3' ends that matches the YBX1 recognition site known to stabilise pro-metastatic mRNAs. Investigating some aspects of this interaction network, gain- and loss-of-function experiments using small RNA mimics and antisense LNAs, respectively, showed that ectopic tRF-GlyTCC reduced RUNX2 expression and dispersed 3D micromass architecture in vitro. iCLIP sequencing revealed YBX1 physical binding to the 3' UTR of RUNX2. The interaction between YBX1, tRF-GlyTCC and RUNX2 led to the development of the RUNX2 inhibitor CADD522 as a PBC treatment. CADD522 assessment in vitro revealed significant effects on PBC cell behaviour. In xenograft mouse models, CADD522 as a single agent without surgery significantly reduced tumour volume, increased overall and metastasis-free survival and reduced cancer-induced bone disease. Our results provide insight into PBC molecular abnormalities that have led to the identification of new targets and a new therapeutic.

The primary aim of this study was to compare the clinical outcomes of osteoid osteoma (OO) between the group of patients with the presence of nidus on biopsy samples from radiofrequency ablation (RFA) with those without nidus. Secondly, we aimed to examine other factors that may affect the outcomes of OO reflecting our experience as a tertiary orthopaedic oncology centre.We retrospectively reviewed 88 consecutive patients diagnosed with OO treated with RFA between November 2005 and March 2015, consisting of 63 males (72%) and 25 females (28%). Sixty-six patients (75%) had nidus present in their biopsy samples. Patients’ mean age was 17.6 years (4-53). Median duration of follow-up was 12.5 months (6-20.8). Lesions were located in the appendicular skeleton in seventy-nine patients (90%) while nine patients (10%) had an OO in the axial skeleton. Outcomes assessed were based on patients’ pain alleviation (partial, complete, or no pain improvement) and the need for further interventions.Pain improvement in the patient group with nidus in histology sample was significantly better than the group without nidus (OR 7.4, CI 1.35-41.4, p=0.021). The patient group with nidus on biopsy demonstrated less likelihood of having a repeat procedure compared to the group without nidus (OR 0.092, CI 0.016-0.542, p=0.008). Our study showed significantly better outcomes in pain improvement in appendicular lesions compared to the axially located lesions (p = 0.005). Patients with spinal lesions tend to have relatively poor pain relief than those with appendicular or pelvic lesions (p=0.007).Patients with nidus on histology had better pain alleviation compared to patients without nidus. The histological presence of nidus significantly reduces the chance of repeat interventions. The pain alleviation of OO following RFA is better in patients with appendicular lesions than spinal or axially located lesions.

Epithelioid sarcoma is a rare and aggressive mesenchymal tumour, the genetic hallmark of which is the loss of expression of SMARCB1, a key member of the SWItch/Sucrose Non‐Fermentable (SWI/SNF) chromatin remodelling complex. Hampered by its rarity, epithelioid sarcoma has received little research attention and therapeutic options for this disease remain limited. SMARCB1‐deficient tumours also include malignant rhabdoid tumour, atypical teratoid and rhabdoid tumour, epithelioid malignant peripheral nerve sheath tumour, and poorly differentiated chordoma. Histologically, it can be challenging to distinguish epithelioid sarcoma from malignant rhabdoid tumour and other SMARCB1‐deficient tumours, whereas methylation profiling shows that they represent distinct entities and facilitates their classification. Methylation studies on SMARCB1‐deficient tumours, although not including epithelioid sarcomas, reported methylation subgroups which resulted in new clinical stratification and therapeutic approaches. In addition, emerging evidence indicates that immunotherapy, including immune checkpoint inhibitors, represents a promising therapeutic strategy for SMARCB1‐deficient tumours. Here, we show that some epithelioid sarcomas share methylation patterns of malignant rhabdoid tumours indicating that this could help to distinguish these entities and guide treatment. Using gene expression data, we also showed that the immune environment of epithelioid sarcoma is characterised by a predominance of CD8+ lymphocytes and M2 macrophages. These findings have potential implications for the management of patients with epithelioid sarcoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.