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

Fibroma of tendon sheath (FTS) is an uncommon benign myofibroblastic neoplasm that arises in association with tenosynovial tissue. Fusions of the USP6 gene have been recently documented in a proportion of so-called "cellular FTS" but not in "classic FTS". It remains unknown whether FTS can be defined by a USP6 fusion, regardless of cellularity, and what are USP6 fusion-negative "cellular FTS". Furthermore, FTS with low cellularity seems to be frequently confused with desmoplastic fibroblastoma. We performed a comprehensive analysis, including targeted RNA sequencing, of 58 consecutive cases originally diagnosed as FTS (n = 49), desmoplastic fibroblastoma (n = 6), or nodular fasciitis (n = 3); the latter two at the predilection sites for FTS. After review of the original slides, 28 lesions were morphologically classified as FTS (13 "classic" and 15 "cellular") and 23 as desmoplastic fibroblastoma. Among originally diagnosed FTS at the more cellular end of the spectrum, we identified seven lesions that shared many morphologic features of FTS but, in addition, showed several distinct morphologic features consistent with myofibroma, such as myoid appearance, branching thin-walled vessels, and perivascular growth. Targeted RNA sequencing showed a USP6 fusion in 17 of 18 analyzed FTS, regardless of cellularity, 0 of 5 desmoplastic fibroblastomas and 0 of 4 myofibromas. MYH9, COL1A1, and ASPN were identified as fusion partners in three cases each, and MIR22HG, CTNNB1, SPARC, CAP1, EMP1, LINC00152, NR1D1, and RAB1A in a single case each. FTS, regardless of cellularity, can be defined by a USP6 fusion with a variety of fusion partners. More cellular lesions exhibiting some morphologic features of FTS but lacking a USP6 fusion tend to be myofibromas.

PRAME expression in melanocytic lesions of the conjunctiva.

AIMS: PRAME (PReferentially expressed Antigen in MElanoma) is a tumour-associated antigen that is preferentially strongly expressed in most cutaneous melanomas but not or only focally in naevi. Our aim was to evaluate PRAME expression in melanocytic lesions of the conjunctiva. METHODS AND RESULTS: Surgical specimens of 114 conjunctival melanocytic naevi of different types (including 67 common, 25 combined deep penetrating and 21 inflamed juvenile naevi), 30 invasive melanomas, 10 in-situ melanomas, 23 primary acquired melanoses (PAM) without atypia and 11 PAM with atypia were analysed for PRAME expression by immunohistochemistry. Nuclear positivity for PRAME in melanocytes was assessed as the percentage of positive nuclei: negative (0%), 1+ (1-25%), 2+ (26-50%), 3+ (51-75%) and 4+ (> 75%). In 113 of 114 conjunctival melanocytic naevi, PRAME was either completely negative or focally 1+ positive. Diffuse 4+ PRAME expression was identified in 17 of 30 (57%) invasive melanomas, seven of 10 (70%) in-situ melanomas, four of five (80%) PAM with severe atypia, none of three PAM with moderate atypia, none of three PAM with mild atypia, one of 23 (4%) PAM without atypia and none of 114 naevi. Diffuse 4+ PRAME expression in invasive melanomas correlated with a higher mitotic count but was not related to age and gender of the patients, Breslow thickness, location or mutational status. CONCLUSION: Diffuse 4+ PRAME positivity is highly specific for malignant conjunctival melanocytic lesions. PRAME is therefore a useful ancillary marker to support the diagnosis of a suspected conjunctival melanoma.

Novel ASAP1-USP6, FAT1-USP6, SAR1A-USP6, and TNC-USP6 fusions in primary aneurysmal bone cyst.

Aneurysmal bone cyst (ABC) is a benign but locally aggressive neoplasm, with a tendency for local recurrence. In contrast to other bone tumors with secondary cystic change, ABC is characterized by USP6 gene rearrangement. There is a growing list of known USP6 fusion partners, characterization of which has been enabled with the advent of next-generation sequencing (NGS). The list of known fusion partners includes CDH11, CNBP, COL1A1, CTNNB1, EIF1, FOSL2, OMD, PAFAH1B1, RUNX2, SEC31A, SPARC, STAT3, THRAP3, and USP9X. Using NGS, we analyzed a series of 11 consecutive ABCs and identified USP6 fusions in all cases, providing further evidence that USP6 fusions are universally present in primary ABCs. We identified four novel fusion partners in five ABCs and confirmed them by RT-PCR and Sanger sequencing, ASAP1, FAT1, SAR1A, and TNC (in two cases). Because of high sensitivity and specificity, detection of a USP6 fusion by NGS may assist in differentiating between ABC and its mimics, especially in small biopsy samples when a definite diagnosis cannot be achieved on morphological grounds alone. Further studies with a large number of cases and follow-up are needed to determine whether different fusion partners are associated with specific clinical and pathologic features of ABCs.

Triaging Atypical Squamous Cells-Cannot Exclude High-Grade Squamous Intraepithelial Lesion With p16/Ki67 Dual Stain.

OBJECTIVES: The management of women with Pap cytology results categorized as "atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion" (ASC-H) can often be challenging. We assessed the performance of p16/Ki67 dual-stained cytology as a potential triage tool for the detection of biopsy-confirmed high-grade squamous intraepithelial lesions (HSILs) in women with ASC-H. MATERIALS AND METHODS: Women with ASC-H were followed for a period of 36 months by repeat cytology and colposcopy. p16/Ki67 dual staining was performed retrospectively on the initial Pap cytology slide with ASC-H interpretation. Results were compared with the final histological diagnosis and/or cytological follow-up for at least 1 year. All outcomes were defined as clinically significant at the threshold of HSIL (cervical intraepithelial neoplasia grade 2 or worse). RESULTS: One hundred sixty-nine cases fulfilled all conditions to be included in the study group. The rate of histologically confirmed HSIL was 57.4%, low-grade squamous intraepithelial lesion was diagnosed in 17.7% of the patients and normal histology or follow-up in 24.9% of the patients. Overall sensitivity and specificity of p16/Ki67 dual stain were 95% and 72%, respectively. Overall positive likelihood ratio of p16/Ki67 in detection of HSIL was 3.41, considerably increasing pretest probability from 57% to posttest probability of 82%. CONCLUSIONS: A substantial subset of patients with ASC-H interpretations is associated with an appreciable risk of clinically significant cervical disease. p16/Ki67 dual stain can provide additional valuable information that may lead to higher-quality management of women with ASC-H, especially when initial colposcopy or biopsy results do not show HSIL lesions.

Primary cutaneous CD4-positive small or medium T-cell lymphoproliferative disorder: a case report and literature review.

Primary cutaneous CD4-positive small or medium T-cell lymphoproliferative disorder (PCSM-LPD) is characterized by a slow-growing and asymptomatic solitary plaque or tumor, usually involving the head, neck, or upper extremities. The diagnosis is established based on clinical presentation, histopathological features including pleomorphic morphology and CD4-positive immunophenotype of neoplastic T lymphocytes, and molecular analysis showing clonally rearranged T-cell receptor (TCR) genes. Plaques typical of mycosis fungoides are essentially absent. Treatment options include surgical excision, radiotherapy, and topical or intralesional steroids. Because the disease is indolent, aggressive diagnostic tests and systemic treatments are not recommended. We present a case of PCSM-LPD in a previously healthy young man that spontaneously regressed after a biopsy.

Subcutaneous chondromyxoid fibroma with a novel PNISR::GRM1 fusion-report of a primary soft tissue tumour without connection to an underlying bone.

Chondromyxoid fibroma (CMF) is a rare benign bone tumour. While CMF located entirely on the surface of a bone (i.e. juxtacortical CMF) has been well characterised, CMF has not so far been convincingly documented to arise in soft tissues without connection to an underlying bone.We report a subcutaneous CMF in a 34-year-old male, located on the distal medial aspect of the right thigh without any connection with the femur. The tumour measured 15 mm, it was well-circumscribed and displayed typical morphological features of a CMF. At the periphery, there was a small area of metaplastic bone. Immunohistochemically, the tumour cells were diffusely positive for smooth muscle actin and GRM1, and negative for S100 protein, desmin and cytokeratin AE1AE3. Whole transcriptome sequencing revealed a novel PNISR::GRM1 gene fusion.Our case indicates that CMF should be included in the differential diagnosis of soft tissue (including subcutaneous) tumours composed of spindle/ovoid cells, with a lobular architecture and chondromyxoid matrix. The diagnosis of CMF arising in soft tissues can be confirmed by identifying a GRM1 gene fusion or GRM1 expression by immunohistochemistry.

A Unique Case of Multicentric Infantile Myofibromatosis with Radiologic-Pathologic Correlation.

Infantile myofibromatosis (IM) is a rare condition with a variable clinical presentation that characteristically affects young children. Most frequently it presents as one or more benign nodules of the skin, bones, soft tissues, or, rarely, visceral organs. According to the location and number of lesions, there are three different forms: solitary, multicentric without visceral involvement, and multicentric with visceral involvement (generalised), with the latter having the least favourable prognosis. We present a unique case of severe congenital generalised IM in a new-born male who required intubation and mechanical ventilation immediately after the birth due to respiratory distress. A chest radiograph showed numerous tumours involving the entire lung, resembling a metastatic lung disease. Additionally, the neonate had multiple, bluish, papular skin nodules and a biopsy of a skin nodule ultimately led to the diagnosis of IM. Diffuse lung involvement prevented adequate ventilation which resulted in multiorgan failure and death before targeted treatment could have been initiated. The presented case is unique, as such atypical extensive involvement of the lung and leptomeninges in IM has not been reported before. In this brief report, we present the findings of radiographic and ultrasonographic examinations in correlation with autopsy and histopathology.

A traumatized melanocytic nevus with atypical clinical and dermoscopic features: a case report and review of the literature.

We report the case of an adolescent girl that presented with an atypical melanocytic lesion on the left gluteal region, suspicious for melanoma. She was healthy with no associated diseases, and there was no history of skin cancer in the family. The nevus had been present for several years, but she had noted a change and growth of it in the last few months. She reported that the nevus was injured about 2 years earlier and it had appeared different ever since. Although dermoscopic examination showed the lesion to be highly suspicious for melanoma and it was therefore surgically excised on the same day, pathohistological examination showed a compound melanocytic nevus with extensive dermal fibrosis/regression and overlying atypical junctional hyperplasia of melanocytes consistent with pseudomelanoma, also known as recurrent melanocytic nevus.

An Update on Molecular Genetic Aberrations in Spitz Melanocytic Proliferations: Correlation with Morphological Features and Biological Behavior.

The aim of the paper is to give an update on molecular genetic aberrations in Spitz melanocytic proliferations with special emphasis on their correlation with morphological features and biological behavior. The Spitz group of melanocytic proliferations is defined by a combination of distinctive morphological features and driver molecular genetic events. Morphologically, these neoplasms are characterized by large, oval, polygonal, or spindled melanocytes with abundant eosinophilic cytoplasm, vesicular nuclei with prominent nucleoli, often in association with epidermal hyperplasia. Molecular aberrations in Spitz melanocytic proliferations can be divided into two main groups, according to the driver genetic change: 1) 11p amplification/HRAS mutation, present in about 20% of cases, and 2) kinase fusions, present in about 50%, further subdivided into tyrosine kinase fusions (ALK, ROS1, NTRK1, NTRK3, MET, RET) or serine-threonine kinase fusions (MAP3K8, BRAF). Driver genetic aberrations can be detected along the whole biological spectrum of Spitz melanocytic proliferations, and are mutually exclusive. Although driver genetic aberrations enable proliferation of melanocytes, additional genetic events (often biallelic inactivation of CDKN2A and TERT promoter mutations) are necessary for the development of overt Spitz malignancy. CONCLUSIONS: Recent studies have demonstrated that certain driver genetic aberrations are more often associated with the benign spectrum of Spitz melanocytic proliferations and indolent biological behavior (11p amplification/HRAS mutation, tyrosine kinase fusions). In contrast, some driver aberrations are more frequent in the atypical/malignant spectrum of Spitz melanocytic proliferations with a potential for aggressive biological behavior (serine-threonine kinase fusions). In addition, certain driver aberrations are often associated with distinctive morphological features. However, none of the morphological features is entirely specific for any of these driver genetic aberrations. Immunohistochemistry for ALK, ROS1, and pan-TRK can be used for screening purposes to detect corresponding fusion proteins.

Intraarticular nodular fasciitis-detection of USP6 gene fusions in three cases by targeted RNA sequencing.

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 cases located 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.

The role of molecular diagnostics in aneurysmal and simple bone cysts - a prospective analysis of 19 lesions.

Aneurysmal (ABC) and simple bone cysts (SBC) have been traditionally distinguished by radiological and histopathological features. However, there is some radiological and histopathological overlap between ABC and SBC. ABC is characterised by USP6 fusions while, recently, NFATC2 fusions have been found in a large proportion of SBC. Identifying these fusions may assist in confirming the diagnosis of either ABC or SBC. To elaborate the potential benefit of molecular testing, we report a prospective series of 19 consecutive bone cysts with comprehensive radiological, histopathological and molecular diagnostics. Integrating radiological, histopathological and molecular findings, 11 cysts were diagnosed as SBC and 8 as ABC. Radiologically, 6 of 11 SBC and 6 of 8 ABC were diagnosed as ABC. Fibrin-like collagen deposits were identified in 8 of 11 (73%) SBC and 3 of 8 (38%) ABC. Nodular fasciitis-like areas were identified in 6 of 8 (75%) ABC and in 7 of 11 (64%) SBC. A USP6 fusion was identified in all 8 ABC, including a novel RBM5-USP6 fusion. An NFATC2 fusion was found in 7 of 11 SBC (FUS-NFATC2 fusion in 5 and EWSR1-NFATC2 in 2 cases). There is radiological and histopathological overlap between SBC and ABC in a significant proportion of cases. A diagnosis of ABC is frequently suggested radiologically in SBC, and fibrin-like deposits, thought to be specific for SBC, may be found in some ABC. Molecular testing may significantly improve diagnostic accuracy in bone cysts.

Combined deep penetrating nevi of the conjunctiva are relatively common lesions characterised by BRAFV600E mutation and activation of the beta catenin pathway: a clinicopathological analysis of 34 lesions.

BACKGROUND: Deep penetrating nevus (DPN) is not a widely recognised lesion on the conjunctiva and only a few cases consistent with combined DPN have been reported. METHODS: A review of all excised and histopathologically diagnosed conjunctival melanocytic lesions between 2003 and 2018 was performed in order to identify melanocytic nevi morphologically consistent with DPN. RESULTS: Thirty-four DPN were identified among 361 histopathologically examined conjunctival nevi (9.4%), including 33 (97%) combined with a common nevus and 1 (3%) pure DPN. The patients' age ranged from 7 to 51 years (median, 22 years). Clinically, 21 of 29 (72%) lesions with available data were darkly pigmented, and an increase in size and/or pigmentation was noted in 13 of 18 (72%) lesions with known history. All 24 lesions in which an immunohistochemical analysis was possible were diffusely positive for BRAFV600E (in DPN and common nevus components) and showed a diffuse nuclear positivity for beta catenin and cyclin D1 in the DPN component. None of the 21 lesions with available follow-up data recurred during a follow-up period from 0.3 to 16.3 years (median, 7.5 years). CONCLUSIONS: DPN of the conjunctiva is a relatively common lesion and usually presents as a combined nevus. Genetically, DPN of the conjunctiva are characterised by a combination of BRAFV600E mutation and activation of the beta catenin pathway. Recognition of DPN of the conjunctiva is important in order not to overdiagnose it as a melanoma, and to explain its potential atypical clinical features. DPN of the conjunctiva seems to be a benign lesion.

Pigmented (melanotic) myoepithelial tumor of soft tissue with EWSR1-KLF17 fusion.

Myoepithelial tumors of soft tissue are rare, morphologically and biologically heterogeneous tumors. EWSR1 fusion is found in about half of the cases, followed by PLAG1 and FUS fusions. EWSR1-KLF17 fusion has so far been reported in one benign myoepithelial tumor. Using next generation sequencing we identified another myoepithelial tumor of soft tissue with EWSR1-KLF17 fusion, located on the foot in a 55-year-old male. It was composed predominantly of spindle cells with multiple small areas of epithelioid and multinucleated cells in myxohyaline stroma and areas of melanin pigment in the cytoplasm of tumor cells. The pigmented tumor cells were positive for HMB45 and, ultrastructurally, melanosomes were identified in their cytoplasm. Melanin production has not been previously documented in myoepithelial tumors of soft tissue. Our case extends the spectrum of myoepithelial tumors of soft tissue and emphasizes the importance of molecular characterization of fusions, including determination of fusion partners in myoepithelial tumors and their mimics.

FUS-NFATC2 or EWSR1-NFATC2 Fusions Are Present in a Large Proportion of Simple Bone Cysts.

A simple bone cyst (SBC) is a benign bone lesion of unknown etiology. It can be differentiated from an aneurysmal bone cyst (ABC) by radiologic and histopathologic features, as well as by the absence of fusions of the USP6 gene characteristic of an ABC. In an attempt to differentiate between ABC and SBC in a recurrent bone cyst, we performed targeted RNA sequencing and found an EWSR1-NFATC2 fusion and no fusion of the USP6 gene. We subsequently analyzed additional 10 cysts, consistent with SBCs after radiologic-pathologic correlation, for the presence of an NFATC2 gene fusion, by targeted RNA sequencing, reverse-transcription polymerase chain reaction (RT-PCR) and Sanger sequencing, and fluorescent in situ hybridization. Targeted RNA sequencing showed a FUS-NFATC2 fusion in 4 of 11 SBCs and an EWSR1-NFATC2 fusion in 2 of 11 SBCs. No fusion was identified in 3 SBCs and the analysis was not successful in 2 SBCs because of the low quantity or poor quality of isolated RNA. All the 6 fusions detected by targeted RNA sequencing were confirmed by RT-PCR and Sanger sequencing, and 5 of the 6 fusions by fluorescent in situ hybridization. An additional FUS-NFATC2 fusion was identified by RT-PCR, Sanger sequencing, and fluorescent in situ hybridization in 1 of the 3 cases negative for fusions by targeted RNA sequencing. At least a large subset of SBCs represents cystic neoplasms characterized by FUS-NFATC2 or EWSR1-NFATC2 fusions, which also define a group of distinct, rare "Ewing-like" sarcomas that predominantly arise in long bones. Our results provide additional evidence of the existence of benign lesions with FUS-NFATC2 or EWSR1-NFATC2 fusions. Although they can recur locally in a nondestructive manner, their clinical course and possible relation to sarcoma with EWSR1-NFATC2 or FUS-NFATC2 fusion remains to be elucidated.

Primary aneurysmal bone cyst with a novel SPARC-USP6 translocation identified by next-generation sequencing.

Aneurysmal bone cyst (ABC) is a benign but locally aggressive, mostly pediatric neoplasm, with characteristic USP6 gene rearrangement that distinguishes it from a secondary ABC and other primary bone tumors. With the advent of next-generation sequencing (NGS) technology, several hitherto unknown USP6 fusion partners have been identified in ABC. Accordingly, we present a case of an 18-year-old male with a solid sub-periosteal primary ABC in the diaphysis of the left femur. Using an NGS-based assay, we identified SPARC-USP6 fusion, which has not previously been described in ABC. Including our case, the list of currently known USP6 fusion partners in primary ABC include: CDH11, CNBP, COL1A1, CTNNB1, EIF1, FOSL2, OMD, PAFAH1B1, RUNX2, SEC31A, SPARC, STAT3 and THRAP3.