A morphomolecular study of 175 'USP6-associated neoplasms': The USP6 fusion partner strongly depends on morphology and location.

USP6 gene rearrangement has been identified in the most of Nodular Fasciitis (NF) cases. Though the detection of several USP6 fusion partners has been identified, most of nodular fasciitis cases have exhibited the presence of MYH9- USP6 gene fusion. Recently, a literature review of rare intradermal nodular fasciitis focusing on the genetic analysis has been provided, suggesting that the detection of USP6 gene rearrangement may be the very useful and important genetic analysis for accurate diagnosis in even intradermal nodular fasciitis. The result provided MYH9, EIF5A and TPM4 as a fusion partner with USP6 in rare intradermal nodular fasciitis. In this review, the author summarized the rare nodular fasciitis accompanied by atypical clinicopathological findings including recurrence and/or metastasis, malignant morphology, atypical mitosis and different fusion partners with USP6. The result provided that USP6 rearrangement is a potential tool for accurate diagnosis in atypical nodular fasciitis. Several fusion partners including PPP6R3, MYH9, EIF5A, CALD1, COL6A2, PAFAH1B1 and SREBF1 with USP6 have been identified by Fluorescence In Situ Hybridization (FISH) and/or Next Generation Sequencing (NGS), suggesting that these fusion genes may have different biological spectrum reflecting unusual clinicopathological features. Further study is needed to clarify the biological spectrum of USP6 fusion partner to avoid aggressive and invasive treatment for nodular fasciitis associated with atypical clinicopathological features. Meanwhile, a few cases of nodular fasciitis with atypical manifestations and typical MYH9- USP6 fusion have been revealed, therefore it is significant to identify USP6 rearrangement for diagnostic conformation of nodular fasciitis by molecular analysis. Keywords Nodular fasciitis; USP6-associated neoplasia; Biological spectrum; USP6 fusion gene partner; Atypical clinicopathological features

USP6 gene rearrangement has been identified in the most of Nodular Fasciitis (NF) cases. Though the detection of several USP6 fusion partners has been identified, most of nodular fasciitis cases have exhibited the presence of MYH9- USP6 gene fusion. Recently, a literature review of rare intradermal nodular fasciitis focusing on the genetic analysis has been provided, suggesting that the detection of USP6 gene rearrangement may be the very useful and important genetic analysis for accurate diagnosis in even intradermal nodular fasciitis. The result provided MYH9, EIF5A and TPM4 as a fusion partner with USP6 in rare intradermal nodular fasciitis. In this review, the author summarized the rare nodular fasciitis accompanied by atypical clinicopathological findings including recurrence and/or metastasis, malignant morphology, atypical mitosis and different fusion partners with USP6. The result provided that USP6 rearrangement is a potential tool for accurate diagnosis in atypical nodular fasciitis. Several fusion partners including PPP6R3, MYH9, EIF5A, CALD1, COL6A2, PAFAH1B1 and SREBF1 with USP6 have been identified by Fluorescence In Situ Hybridization (FISH) and/or Next Generation Sequencing (NGS), suggesting that these fusion genes may have different biological spectrum reflecting unusual clinicopathological features. Further study is needed to clarify the biological spectrum of USP6 fusion partner to avoid aggressive and invasive treatment for nodular fasciitis associated with atypical clinicopathological features. Meanwhile, a few cases of nodular fasciitis with atypical manifestations and typical MYH9- USP6 fusion have been revealed, therefore it is significant to identify USP6 rearrangement for diagnostic conformation of nodular fasciitis by molecular analysis. Keywords Nodular fasciitis; USP6-associated neoplasia; Biological spectrum; USP6 fusion gene partner; Atypical clinicopathological features

Among those tumors with consistent USP6 rearrangement, some arise from soft tissue and show bone metaplasia, including myositis ossificans (MO), fibro-osseous pseudotumor of digits (FOPD), soft tissue aneurysmal bone cyst (ST-ABC) and fasciitis ossificans (FO). These lesions are easily confused with malignancies because they show a rapid growth rate and brisk mitoses. Here, we aim to clarify the clinicopathologic and genetic characteristics of this entity and analyze the correlations among the different subtypes in one of the largest cohorts. The clinicopathologic features of 73 cases of MO, FOPD, ST-ABC and FO diagnosed at West China Hospital, Sichuan University from January 2010 to December 2021 were retrospectively analyzed. Forty-three undecalcified samples were analyzed by systematic genetic studies, including fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), Sanger sequencing and next-generation-based sequencing were performed. This series included 40 males and 33 females aged 2 to 80 years old (median: 31 years). FOPD occurred in extremal soft tissue, while lower extremities (38/58, 65.5%) were the most commonly involved lesions in the other three subgroups. Histologically, proliferative myofibroblasts/fibroblasts with varying degrees of osteoid tissue were present. Fluorescence in situ hybridization (FISH) results indicated that 22 cases (22/27, 81.5%) were positive for USP6 rearrangement, and 5 cases were negative. Among those cases with positive FISH results, 18 underwent reverse transcription-polymerase chain reaction (RT-PCR) detection that successfully detected common USP6 fusion types. Thirteen cases showed COL1A1::USP6 fusion, one showed MYH9::USP6 fusion, and 4 were negative for common fusion types. Next-generation-based sequencing technology was performed on two lesions with negative RT-PCR results and novel fusion partners SNHG3 and UBE2G1 were discovered. Our findings revealed that COL1A1 is the most common fusion partner in this entity, unlike primary aneurysmal bone cysts and nodular fasciitis. Notably, we believed that FO may demonstrate more similar clinicopathologic and genetic manifestations with MO/FOPD and ST-ABC instead of nodular fasciitis for involving lower limbs most frequently and showing recurrent COL1A1::USP6 fusion. Additionally, this study also found two novel USP6 fusion partners, which further expanded our knowledge of this neoplastic spectrum.

USP6 rearrangement underpins self-limiting fibroblastic/myofibroblastic neoplasms, including nodular fasciitis (NF), myositis ossificans (MO), aneurysmal bone cyst (ABC), and related variants. The aim of this study was to characterise UPS6 and fusion partners in order to delineate the clinicopathological, genetic and bone-forming features in such lesions of soft tissue (ST). Break-apart fluorescence in-situ hybridisation (FISH) validated USP6 rearrangement in 31 of 35 NF [comprising three of three fasciitis ossificans (FO) cases, seven of eight cellular variant of fibroma of tendon sheath (C-FTS), four of six MO, three of three ST-ABC, and two of two fibro-osseous pseudotumours of digits (FOPD)]. As determined with FISH and reverse transcription polymerase chain reaction, MYH9-USP6 was the commonest fusion in four C-FTS and 20 NF, including one intravascular case and two infantile (one retroperitoneal) cases. The presence of MYH9-USP6 confirmed the diagnosis of two NFs>50mm with prominent ischaemic necrosis. COL1A1-USP6 was predominant in ossifying lesions, including all FO, MO, ST-ABC and FOPD with identified partner genes, and was also present in non-ossifying head and neck NF (HN-NF) and C-FTS in two cases each. A cervical NF of a 14-month-old girl harboured the novel COL1A2-USP6. Ossifying lesions showed considerable genetic and morphological overlaps. Sharing COL1A1-USP6, FO and FOPD showed similar central or haphazard bone matrix deposition. Besides zonation of outward bone maturation, four COL1A1-USP6-positive MO had incipient to sieve-like pseudocysts reminiscent of ST-ABC. MYH9-USP6 is present in some C-FTS and most NF, including rare variants, but is unrelated to bone formation. All bone-forming USP6-rearranged lesions adopt COL1A1 as the 5' partner, indicating close genetic kinships. However, COL1A1/COL1A2 also contributes to the pathogenesis of minor subsets of non-ossifying USP6-rearranged HN-NF and C-FTS.

Myositis ossificans – Another condition with USP6 rearrangement, providing evidence of a relationship with nodular fasciitis and aneurysmal bone cyst

USP6 and CDH11 Oncogenes Identify the Neoplastic Cell in Primary Aneurysmal Bone Cysts and Are Absent in So-Called Secondary Aneurysmal Bone Cysts

Intraarticular nodular fasciitis arising in the joint synovium is an uncommon lesion. Most cases have been reported in the knee and rarely in other joints. A USP6 gene fusion has so far been documented in only four cases of intraarticular nodular fasciitis, three were located in the knee and one in the proximal interphalangeal joint. In all three caseslocated in the knee, MYH9 was detected as a USP6 fusion partner. We analysed three cases of intraarticular nodular fasciitis for the presence of USP6 fusion by targeted RNA sequencing. Two cases were located in the hip (a 25-year-old female and 48-year-old male) and one in the shoulder (a 38-year-old male). We detected a MYH9-USP6 fusion in the two hip cases and a COL1A1-USP6 fusion in the shoulder case. Our findings provide additional evidence that intraarticular nodular fasciitis is a form of nodular fasciitis arising in the joint synovium, harbouring a USP6 fusion. Although a MYH9-USP6 fusion seems to predominate in intraarticular nodular fasciitis, other fusion partners of the USP6 gene may also be involved. Detection of a USP6 fusion by targeted RNA sequencing may assist in confirming the diagnosis in selected cases.

MYOFIBROBLASTIC LESIONS IN THE ORAL CAVITY: IMMUNOHISTOCHEMICAL ANALYSIS

Recurrent and novel USP6 fusions in cranial fasciitis identified by targeted RNA sequencing.

Fibroma of tendon sheath is defined by a USP6 gene fusion—morphologic and molecular reappraisal of the entity

Nodular fasciitis is a benign myofibroblastic proliferation with a predilection for the subcutaneous tissues of the upper extremities, trunk, and head and neck of young adults. Nodular fasciitis is not generally recognized to arise within joints. In this study, the clinicopathologic and immunohistochemical features of 10 cases of intraarticular nodular fasciitis are described. Six patients were female and 4 were male, with a median age of 33 years (range, 9-50 years). Lesional size ranged from 2 to 4 cm (median, 2.6 cm). Seven tumors arose in the knee, 2 in the hand, and 1 in the ankle. Most patients complained of joint pain; 4 presented with a palpable mass. Only 1 patient reported antecedent trauma. The duration of symptoms prior to surgery ranged from 2 months to 1 year (median, 6 months). The clinical differential diagnoses included giant cell tumor of tendon sheath, pigmented villonodular synovitis, synovial chondromatosis, inflammatory arthritis, and lymphoma. Grossly, the lesions were solid, nodular, rubbery, or firm masses. Histologically, all tumors were circumscribed but unencapsulated and showed typical features of nodular fasciitis, being composed of cytologically bland plump spindle cells arranged in short, intersecting bundles within a variably loose myxoid to collagenous stroma, containing extravasated red blood cells and scattered lymphocytes. Five lesions showed prominent stromal hyalinization, in 2 cases keloidal in appearance. In 4 cases, the tissue at the periphery of the lesion showed hemosiderin deposition. By immunohistochemistry, all tumors examined were positive for SMA, 1 was positive for desmin, and all were negative for caldesmon and S-100 protein; none showed nuclear staining for beta-catenin. Clinical follow-up information was available for 5 patients, ranging from 2 to 86 months. No lesion recurred. In summary, intraarticular nodular fasciitis occurs most commonly in the knees of young adults, and often appears to have a somewhat longer preoperative duration than typical subcutaneous or intramuscular nodular fasciitis. Intraarticular lesions show morphologic features similar to other cases of nodular fasciitis, with the exception that stromal hyalinization and adjacent hemosiderin deposition are common, likely attributable to frictional trauma in this location.

Fibro-osseous pseudotumor of digits - Expanding the spectrum of clonal transient neoplasms harboring USP6 rearrangement.

Several morphologically overlapping (myo)fibroblastic neoplasms harbour USP6 fusions, including aneurysmal bone cysts, nodular fasciitis, myositis ossificans, cranial fasciitis, fibro-osseous pseudotumour of the digits, and cellular fibroma of the tendon sheath. USP6-induced neoplasms are almost universally benign and cured by local excision. We aim to highlight the diagnostic value of USP6 fusion detection in a series of aggressive-appearing paediatric myofibroblastic tumours. Three deep-seated, radiographically aggressive, and rapidly growing childhood myofibroblastic neoplasms were morphologically and molecularly characterised by USP6 break-apart fluorescence in-situ hybridisation (FISH), transcriptome sequencing, and targeted capture analysis. Each tumour occurred in the lower-extremity deep soft tissue of a child presenting with pain, limping, or a mass. In all three patients, imaging studies showed a solid mass that infiltrated into surrounding skeletal muscle or involved/eroded underlying bone. The biopsied tumours consisted of variably cellular myofibroblastic proliferations with variable mitotic activity that lacked overt malignant cytological features. FISH showed that all tumours had USP6 rearrangements. On the basis of these results, all three patients were treated with conservative excision with positive margins. The excised tumours had foci resembling nodular fasciitis, fibromatosis, and pseudosarcomatous proliferation. Next-generation sequencing revealed COL1A1-USP6 fusions in two tumours and a COL3A1-USP6 fusion in the third tumour. One tumour had a subclonal somatic APC in-frame deletion. No recurrence was observed during follow-up (8-40months). We present a series of benign, but aggressive-appearing, USP6-rearranged myofibroblastic tumours. These deep-seated tumours had concerning clinical and radiographic presentations and did not fit into one distinct histological category. These cases highlight the diagnostic value of USP6 fusion detection to identify benign nondescript tumours of this group, especially those with aggressive features, to avoid overtreatment.

Nodular fasciitis (NF) is a self-limiting benign disease that is characterized by rapid proliferation of fibroblastic and myofibroblastic cells. The characteristic gene fusion containing the USP6 gene is a genetic hallmark of NF and MYH9-USP6 is the most frequent fusion, suggesting that NF is not a reactive condition but a neoplastic disease. Malignant transformation of NF has been reported rarely as a single case associated with the PPP6R3-USP6 fusion. Here we report a case of soft part tumor of which the histological feature was a typical NF but showed aggressive and non-regressing growth with local invasion. Targeted RNA sequencing and fluorescence in situ hybridization analysis identified PPP6R3-USP6 with gene amplification. These findings indicate that the present case is the second case of malignant NF, and we suggest potential malignant transformation in certain NF cases.

Background: Nodular fasciitis (NF) is currently considered a self-limited clonal neoplastic process. It shares the rearrangement of USP6-gene with aneurysmal bone cysts and myositis ossificans. The presented case is of interest as this is a rare site of presentation of NF; so far only few single cases of intraarticular NF have been reported with documented USP6-gene rearrangement. Intraarticular neoplasias of the knee joint are rare; the most frequent being tenosynovial giant cell tumor (TSGCT). Given a nationwide annual incidence rate of 14 for the lower extremity and about 75% affecting the knee joint about 10 new cases involving the knee joint can be expected per 1 million persons/year. All other types of benign neoplasms are comparably rare while malignant intraarticular processes are extremely rare with most of them reported as single case studies. Aim: We report our case to emphasize the importance of preoperative diagnostics including the option of biopsy. Intraarticular malignant processes are extremely rare and frequently are operated on accidently with negative consequences for the patient. Tactics and techniques to treat benign processes depend on the correct pathologic diagnosis. Case presentation: The 38 year old man noticed slowly increasing swelling of his left knee joint after wakeboarding. Because of continuing discomfort 2 months later MRI diagnostic revealed, apart from retropatellar cartilage lesions, a popliteal mass compatible with a Baker cyst. The lesion of interest (later diagnosed as NF) was neither recognized by the radiologist nor the treating clinician. During the following 8 months the patient felt increasing swelling of the knee joint. The repeat MRI documented the crescent intraarticular solid synovial mass in the medial patellofemoral recess without signs of hemosiderin impregnation. A percutaneous sonographically guided 16G needle biopsy was performed. Histologically, bland myofibroblastic proliferation suggestive of nodular fasciitis (NF) was found. The next generation sequencing (NGS) demonstrated the presence of MYH9-USP6 gene fusion, confirming the diagnosis of NF. The lesion was excised under arthroscopic control. At 1 year follow-up the patient is asymptomatic. Conclusion: The case is of interest because of its rare pathology. The decision how to treat was based on pathologic biopsy diagnostics including the USP6-gene rearrangement. In view of similar presentation of the rare malignancies we also want to stress the importance of definitive diagnostics which generally are possible only through biopsy.