Modeling Extraordinary Response Through Targeting Secondary Alterations in Fusion-Associated Sarcoma.

PURPOSE: Targeted therapy in translocation-associated sarcomas has been limited to oncogenic activation of tyrosine kinases or ligands while gene fusions resulting in aberrant expression of transcription factors have been notoriously difficult to target. Moreover, secondary genetic alterations in sarcomas driven by translocations are uncommon, comprising mostly alterations in tumor suppressor genes (TP53, CDKN2A/B). Our study was triggered by an index patient showing a dramatic clinical response by targeting the secondary BRAF V600E mutation in a metastatic angiomatoid fibrous histiocytoma (AFH) harboring the typical EWSR1::CREB1 fusion. MATERIALS AND METHODS: The patient, a 28-year-old female, was diagnosed with an AFH of the thigh and followed a highly aggressive clinical course, with rapid multifocal local recurrence within a year and widespread distant metastases (adrenal, bone, liver, lung). The tumor showed characteristic morphologic features, with histiocytoid cells intermixed with hemorrhagic cystic spaces and lymphoid aggregates. In addition to the pathognomonic EWSR1::CREB1 fusion, targeted DNA sequencing revealed in both primary and adrenal metastatic sites a hot spot BRAF V600E mutation and a CDKN2A/B deletion. Accordingly, the patient was treated with a BRAF-MEK inhibitor combination (encorafenib/binimetinib) showing an excellent but short-lived response. RESULTS: Using a CRISPR-Cas9 approach, we introduced the BRAF c.1799 T>A point mutation in human embryonic stem (hES) cells harboring a conditional EWSR1 (exon7)::CREB1 (exon7) translocation and further differentiated to mesenchymal progenitors (hES-MP) before fusion expression. The cells maintained the fusion transcript expression and the AFH core gene signature while responding to treatment with encorafenib and binimetinib. CONCLUSION: These results highlight that additional targeted DNA NGS in chemotherapy-resistant translocation-associated sarcomas may reveal actionable oncogenic drivers occurring as secondary genetic events during disease progression.

[Detection of EWSR1 gene rearrangement by fluorescence in situ hybridization in bone and soft tissue tumors: clinical application evaluation and atypical signal analysis].

Objective: To investigate the clinical application of EWSR1 gene rearrangement by fluorescence in situ hybridization (FISH) in bone and soft tissue tumors and to analyze the cases with atypical signal pattern. Methods: The cases detected for EWSR1 gene rearrangement by FISH in Beijing Jishuitan Hospital, Capital Medical University from 2014 to 2021 were collected, and the value of detecting EWSR1 gene rearrangement for diagnosing bone and soft tissue tumors was analyzed. The cases with atypical positive signals were further analyzed by next generation sequencing (NGS). Results: FISH using EWSR1 break-apart probe kit was successfully performed in 97% (205/211) of cases, 6 cases failed. Four of the 6 failures were due to improper decalcification, 1 case due to signal overlap caused by thick slices, and 1 case due to signal amplification and disorder. EWSR1 gene rearrangements were positive in 122 cases (122/205, 59%), atypical positive signal in 8 cases (8/205, 4%), and negative in 75 cases (75/205, 37%). In cases testing positive, the percentage of positive cells ranged from 34% to 98%, with 120 cases (120/122, 98%) showing a positive cell percentage greater than 50%. Among the 205 successfully tested cases, 156 cases were histologically diagnosed as Ewing's sarcoma, of which 110 were positive (110/156, 71%), 7 were atypical positive (7/156, 4%), and 39 were negative (39/156, 25%). Nine cases were histologically diagnosed as clear cell sarcoma of soft tissue, of which 6 were positive (6/9), 1 was atypical positive (1/9), and 2 were negative (2/9). Five cases were histologically diagnosed as extraskeletal myxoid chondrosarcoma, of which 2 were positive (2/5) and 3 were negative (3/5). Three cases were histologically diagnosed as angiomatoid fibrous histiocytoma, of which 2 were positive (2/3) and 1 was negative (1/3). Two cases were histologically diagnosed as myoepithelioma of soft tissue, of which 1 was positive (1/2) and 1 was negative (1/2). One case was histologically diagnosed as olfactory neuroblastoma with a positive result. The 29 other tumor cases including osteosarcoma, synovial sarcoma, and malignant melanoma and others were all negative. Basing on histology as the standard for diagnosis and considering atypical positive cases as negative, comparing with the 29 cases of other tumors as control group, the sensitivity for diagnosing Ewing's sarcoma through the detection of EWSR1 gene rearrangement was 71%, and the specificity was 100%; the sensitivity for diagnosing clear cell sarcoma of soft tissue was 67%, and the specificity was 100%; the sensitivity for diagnosing extraskeletal myxoid chondrosarcoma was 40%, and the specificity was 100%; the sensitivity for diagnosing angiomatoid fibrous histiocytoma was 67%, and the specificity was 100%; the sensitivity for diagnosing myoepithelioma of soft tissue was 50%, and the specificity was 100%; the sensitivity for diagnosing olfactory neuroblastoma was 100%, and the specificity was 100%. Four of 8 cases with atypical positive signals analyzed by NGS showed EWSR1 rearrangement, including EWSR1::FLI1 in one case of Ewing sarcoma, EWSR1::NFATC2 in one case of EWSR1::NFATC2-rearranged sarcoma, EWSR1::ATF1 in one case of clear cell sarcoma of soft tissue and EWSR1::NR4A3 in one case of extraskeletal myxoid chondrosarcoma. Conclusions: Detection of EWSR1 rearrangement by FISH is of utmost significance in the diagnosis of bone and soft tissue tumors. Cases with atypical positive signals should be further scrutinized, correlating with their histomorphology and verifying by NGS if necessary.

Hydrochlorothiazide and increased risk of atypical fibroxanthoma and pleomorphic dermal sarcoma.

BACKGROUND AND OBJECTIVES: Previous work has demonstrated that hydrochlorothiazide (HCTZ) is a risk factor for squamous cell carcinomas (SCC) and basal cell carcinomas (BCC) due to pro-photocarcinogenic effects. Atypical fibroxanthoma (AFX) and pleomorphic sarcoma (PDS), both ultraviolet-induced cancers, display a rare but rising cutaneous tumor entity. This study aimed to evaluate if the use of HCTZ is higher in patients with AFX/PDS than in patients with SCC/BCC and subsequently may be a risk factor for AFX/PDS-development. PATIENTS AND METHODS: In a retrospective study of four German skin cancer centers, AFX/PDS cases and SCC/BCC controls were sex and age matched (1:3) over a time-period of 7 years (2013-2019) to evaluate the use of HCTZ, immunosuppressive medication, second malignancies, and presence of diabetes mellitus. RESULTS: Overall, 146 AFX/PDS and 438 controls (SCC/BCC) were included in the study. The use of HCTZ was significantly higher in patients with AFX/PDS (44.5%) compared to patients with SCC/BCC (25.3%). Additionally, the presence of diabetes mellitus was significantly higher in AFX/PDS patients. CONCLUSIONS: This study demonstrates a significantly higher use of HCTZ in patients with AFX/PDS compared to SCC/BCC. This result suggests that HCTZ may be a risk factor for AFX/PDS. Additionally, diabetes mellitus or its comorbidities may be associated with an increased risk for AFX/PDS.

Analysis of clinical factors impacting recurrence in myxofibrosarcoma.

Myxofibrosarcoma (MFS) is a malignant fibroblastic/myofibroblastic neoplasm with a prominent myxoid area. It has the clinical features of frequent local recurrence (LR) and occasional distant metastasis. Robust epidemiological data on MFS in China are lacking. The aim of this retrospective analysis was to determine the natural history of MFS, identify prognostic factors for recurrence and describe the real-life outcomes of MFS. We reviewed 52 patients with primary MFS from the First Affiliated Hospital of Nanjing Medical University diagnosed between 2016 and 2020. All tumors were subjected to retrospective univariate analysis for prognostic factors of the disease, including tumor size, grade, location and sex; patient age; planned operation; surgical margin; and laboratory results. The significant factors identified by univariate analysis were subsequently analyzed via multivariate analysis. Overall survival (OS), post-treatment LR and metastatic-free survival were assessed as outcomes. The median age was 61 years (range, 13-93). Fourteen (26.92%) patients exhibited low grade disease, and 38 (73.08%) exhibited high grade disease. Among the 29 males, and 23 females, 15 (28.85%) had tumors in the trunk, 37 (71.15%) had tumors in the extremities, 26 had undergone planned surgery, and 26 had unexpected unplanned operation. The margin was negative in 39 (75%) patients and positive in 13 patients (25%). The serum creatine kinase (CK) concentration was high level in 33 (63.46%) patients and low level in 19 (36.54%) patients. The serum lactate dehydrogenase (LDH) levels were low in 23 (44.23%) patients and high in 29 (55.77%) patients. LR was observed in 25 patients (48.08%), and 4 patients developed metastasis. A worse LR rate was found for patients with a low CK level (84.21%) than for those with a high CK level (27.27%) at 5 years (p < 0.05). The LR rate of patients who underwent planned surgery was lower than that of patients who underwent unplanned surgery (p < 0.05). There were significantly more patients with positive margins than patients with negative margins (92.30%, and 33.33%, respectively; p < 0.05). Moreover, superficial tumors were also associated with greater recurrence rate (2/20 [10%]) than deep tumors, (23/32 [71.86%]) [p < 0.05]. The probability of LR in patients with MFS was significantly greater in association with unplanned operations, positive margins, low serum CK levels or superficial tumor depth. These data could help identify high-risk patients; thus, more careful follow-up should be performed for higher-risk patients. Diagnosis and treatment at qualified regular medical centers can reduce the local recurrence rate of MFS.

DDIT3-amplified or low-polysomic pleomorphic sarcomas without MDM2 amplification: Clinicopathological review and immunohistochemical profile of nine cases.

Several high-grade pleomorphic sarcoma cases that cannot be classified into any existing established categories have been reported. These cases were provisionally classified into undifferentiated pleomorphic sarcoma (UPS). Some dedifferentiated liposarcoma (DDLS) cases may also have been classified into the UPS category due to the absence of MDM2 amplification or an atypical lipomatous tumor/well-differentiated liposarcoma component. We retrieved and reviewed 77 high-grade pleomorphic sarcoma cases, initially diagnosed as UPS in 66 cases and DDLS in 11 cases. Fluorescence in situ hybridization (FISH) analyses of DDIT3 and MDM2 were performed for available cases. Of the cases successfully subjected to DDIT3 FISH (n = 56), nine (7 UPS and 2 DDLS) showed DDIT3 amplification but no MDM2 amplification. Two UPS cases showed both telomeric (5') and centromeric (3') amplification of DDIT3 or low polysomy of chromosome 12, whereas 5 UPS and 2 DDLS cases showed 5'-predominant DDIT3 amplification. Histopathologically, all cases showed UPS-like proliferation of atypical pleomorphic tumor cells. Immunohistochemically, only one case showed focal nuclear positivity for DDIT3, supporting the previous finding that DDIT3 expression was not correlated with DDIT3 amplification. All three cases with focal MDM2 expression involved 5'-predominant amplification, two of which showed DDLS-like histological features. The majority of cases (7/9) showed decreased expression in p53 staining, suggesting that DDIT3 amplification regulates the expression of TP53 like MDM2. From a clinicopathological perspective, we hypothesize that DDIT3-amplified sarcoma, especially with 5'-predominant amplification, can be reclassified out of the UPS category.

Primary Pulmonary Myxoid Sarcoma and Thoracic Angiomatoid Fibrous Histiocytoma: Two Sides of the Same Coin?

Primary pulmonary myxoid sarcoma (PPMS) and thoracic angiomatoid fibrous histiocytoma (AFH) are rare neoplasms with EWSR1 fusions and overlapping morphology. Both tumor types often show epithelial membrane antigen expression, but AFH characteristically co-expresses desmin. We encountered a case of PPMS with the unexpected finding of patchy, strong anaplastic lymphoma kinase (ALK) (previously reported in AFH) and synaptophysin expression. We evaluated a cohort of PPMS and thoracic AFH with systematic morphologic comparison and surveyed for aberrant expression of ALK and synaptophysin. Medical records and slides were reviewed for 16 molecularly confirmed cases of PPMS (n=5) and thoracic AFH (n=11). Each case was scored for morphologic characteristics typical of PPMS and/or AFH. ALK, synaptophysin, chromogranin, desmin, and epithelial membrane antigen immunostains were performed on cases with available tissue. AFH and PPMS cases showed similar age at presentation and long-term tumor behavior. Almost all cases of PPMS and AFH had a fibrous pseudocapsule and lymphoid rim. All PPMS had myxoid stroma and reticular growth pattern, but these features were also present in a subset of AFH. Synaptophysin expression was present in 6 of 11 AFH and 1 of 5 PPMS; all tested cases were negative for chromogranin (n=15). One case of AFH and 1 case of PPMS showed focally strong coexpression of synaptophysin and ALK. AFH and PPMS show considerable clinicopathologic overlap. When supportive, the immunohistochemical findings described may aid in diagnosis before molecular confirmation. PPMS and AFH may be morphologic variants of the same clinicopathologic entity, which can show more immunophenotypic variability than previously reported.

Potential prognostic determinants for FET::CREB fusion-positive intracranial mesenchymal tumor.

Intracranial mesenchymal tumor (IMT), FET::CREB fusion-positive is a provisional tumor type in the 2021 WHO classification of central nervous system tumors with limited information available. Herein, we describe five new IMT cases from four females and one male with three harboring an EWSR1::CREM fusion and two featuring an EWSR1::ATF1 fusion. Uniform manifold approximation and projection of DNA methylation array data placed two cases to the methylation class "IMT, subclass B", one to "meningioma-benign" and one to "meningioma-intermediate". A literature review identified 74 cases of IMTs (current five cases included) with a median age of 23 years (range 4-79 years) and a slight female predominance (female/male ratio = 1.55). Among the confirmed fusions, 25 (33.8%) featured an EWSR1::ATF1 fusion, 24 (32.4%) EWSR1::CREB1, 23 (31.1%) EWSR1::CREM, one (1.4%) FUS::CREM, and one (1.4%) EWSR1::CREB3L3. Among 66 patients with follow-up information available (median: 17 months; range: 1-158 months), 26 (39.4%) experienced progression/recurrences (median 10.5 months; range 0-120 months). Ultimately, three patients died of disease, all of whom underwent a subtotal resection for an EWSR1::ATF1 fusion-positive tumor. Outcome analysis revealed subtotal resection as an independent factor associated with a significantly shorter progression free survival (PFS; median: 12 months) compared with gross total resection (median: 60 months; p < 0.001). A younger age (< 14 years) was associated with a shorter PFS (median: 9 months) compared with an older age (median: 49 months; p < 0.05). Infratentorial location was associated with a shorter overall survival compared with supratentorial (p < 0.05). In addition, the EWSR1::ATF1 fusion appeared to be associated with a shorter overall survival compared with the other fusions (p < 0.05). In conclusion, IMT is a locally aggressive tumor with a high recurrence rate. Potential risk factors include subtotal resection, younger age, infratentorial location, and possibly EWSR1::ATF1 fusion. Larger case series are needed to better define prognostic determinants in these tumors.

Does Wound VAC Temporization Offer Patient-Reported Outcomes Similar to Single-Stage Excision Reconstruction After Myxofibrosarcoma Resection?

BACKGROUND: Vacuum-assisted closure (VAC) temporization is a promising technique to achieve local control in aggressive soft tissue sarcomas. Despite its previously reported efficacy, adoption of VAC temporization remains limited, primarily due to the scarce literature on patient-reported outcomes (PROs) supporting its efficacy. This study compared the postoperative PROs after VAC temporization or single-stage (SS) excision and reconstruction for patients undergoing surgical resection for myxofibrosarcoma management. METHODS: A retrospective analysis of myxofibrosarcoma patients who underwent surgical resections at our institution from 2016 to 2022 was performed. Postoperative PROs collected prospectively for those treated with VAC temporization or SS excision/reconstruction were compared using a visual analog scale (VAS) for pain and three Patient-Reported Outcomes Measurement Information System (PROMIS) questionnaires: Global Health Short-Form Mental (SF Mental), Global Health Short-Form Physical (SF Physical), and Physical Function Short-Form 10a (SF 10a). Absolute and differential (postoperative minus preoperative) scores at the 1-month, 3-month, 6-month, 1-year, and 2-year time points were compared. RESULTS: The analysis included 79 patients (47 treated with VAC temporization and 32 treated with SS excision/reconstruction). All outcomes were similar between the groups except for physical function 1 year after surgery, in which the differential PROMIS SF 10a scores were higher in the SS group (p = 0.001). All the remaining absolute and differential PROMIS and VAS pain scores were similar between the groups at all time points. Postoperative complications did not differ between the groups. CONCLUSION: The PROs for physical and mental health, physical function, and pain were similar between the myxofibrosarcoma patients who had VAC temporization and those who had SS excision/reconstruction after surgical resection.

Pleomorphic Dermal Sarcoma.

Pleomorphic dermal sarcoma (PDS) is a rare cutaneous/subcutaneous neoplasm of purported mesenchymal differentiation that exists along a clinicopathologic spectrum with atypical fibroxanthoma (AFX). While PDS and AFX share histopathologic and immunohistochemical features, PDS exhibits deeper tissue invasion and has a higher rate of metastasis and local recurrence than AFX. Given its aggressive clinical course, early recognition and clinical management of PDS are essential for optimizing patient outcomes. This review aims to provide a brief overview of the clinicopathologic and molecular features, prognosis, and treatment of PDS.

Kinase expression in angiomatoid fibrous histiocytoma: panTRK is commonly expressed in the absence of NTRK rearrangement.

Angiomatoid fibrous histiocytoma (AFH) is a soft tissue tumour of intermediate (rarely metastasising) malignant potential, which harbours EWSR1/FUS gene fusions. These tumours can express anaplastic lymphoma kinase (ALK) in the absence of gene rearrangement or copy number alteration and can also coexpresses Pan-TRK immunohistochemistry (IHC). All EWSR1/FUS-rearranged AFH were retrieved from the files of three institutions and Pan-TRK (EPR17341), ALK and BRAF V600E IHC were performed. Fourteen AFH cases were identified, which included three cases of intracranial mesenchymal tumours with FET-CREB fusions. PanTRK and ALK positive immunostaining was identified in 9 (64.2%) and 12 (85.7%) cases, respectively. No NTRK or ALK translocations or increased copy number/amplification were identified in all eight cases which had fluorescence in situ hybridisation and/or next generation sequencing for NTRK1-3 and ALK available for assessment. None of the cases expressed BRAF-V600E.Although our study is limited, our report is the first to document PanTRK expression in AFH in the absence of identifiable NTRK1-3 gene alterations.

Expanding the Spectrum of EWSR1::CREM Fusion Tumors: An Unusual Pediatric Intranasal Myxoid Tumor.

EWSR1::CREM gene fusions are increasingly being recognized in a diverse number of soft tissue tumors, including well-defined entities such as angiomatoid fibrous histiocytoma or clear cell sarcoma, and other unclassifiable tumors. As a group, EWSR1::CREM fused tumors often demonstrate primitive spindle or epithelioid cells, myxoid stroma, and a broad immunophenotype. Herein we present an unusual case of a child diagnosed with an intranasal malignant myxoid tumor harboring an EWSR1::CREM gene fusion. To the best of our knowledge, this is the first case of intranasal myxoid tumor with this particular fusion. Diagnosis and management of the case is discussed.

Pleomorphic dermal sarcoma presenting in a child with Li-Fraumeni syndrome: A case report and review of the literature.

Pleomorphic dermal sarcoma (PDS) is an uncommon malignant soft-tissue tumor that occurs mostly in elderly patients, with only 5% of cases occurring in children. However, pediatric patients with Li-Fraumeni syndrome (LFS) can develop several types of cancer, particularly sarcomas. Here, we describe a young LFS patient who presented with early-onset PDS and review the literature.