AmiCa: Atlas of miRNA-gene correlations in cancer.

The increasing availability of RNA sequencing data has opened up numerous opportunities to analyze various RNA interactions, including microRNA-target interactions (MTIs). In response to the necessity for a specialized tool to study MTIs in cancer and normal tissues, we developed AmiCa (https://amica.omics.si/), a web server designed for comprehensive analysis of mature microRNA (miRNA) and gene expression in 32 cancer types. Data from 9498 tumor samples and 626 normal samples from The Cancer Genome Atlas were obtained through the Genomic Data Commons and used to calculate differential expression and miRNA-target gene (MTI) correlations. AmiCa provides data on differential expression of miRNAs/genes for cancers for which normal tissue samples were available. In addition, the server calculates and presents correlations separately for tumor and normal samples for cancers for which normal samples are available. Furthermore, it enables the exploration of miRNA/gene expression in all cancer types with different miRNA/gene expression. In addition, AmiCa includes a ranking system for genes and miRNAs that can be used to identify those that are particularly highly expressed in certain cancers compared to other cancers, facilitating targeted and cancer-specific research. Finally, the functionality of AmiCa is illustrated by two case studies.

Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression.

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

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.

Association between Sperm Morphology and Altered Sperm microRNA Expression.

Evaluation of male infertility has been based on semen analysis for years. As this method can be subjective at times, there is a scientific tendency to discover stable and quantifiable biomarkers. This study included 28 couples who underwent an in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle. The couples were assigned into two groups, according to sperm morphology. Couples where the males were normozoospermic were placed in the control group (15 participants), while couples where males had teratozoospermia were placed in the study group (13 participants). Thirteen candidate miRNAs were selected for qPCR analysis, based on our literature search. We determined significant under-expression of nine miRNAs (miR-10a-5p/-15b-5p/-26a-5p/-34b-3p/-122-5p/-125b-5p/-191-5p/-296-5p and let-7a-5p) in spermatozoa from patients with teratozoospermia compared to the controls, whereas expression levels of four miRNAs (miR-92a-3p/-93-3p/-99b-5p/-328-3p) did not significantly differ between the study and control groups. The expression levels of all 13 included miRNAs were significantly positively correlated with each other and significantly positively associated with spermatozoa morphology, excluding miR-99b-5p. There were no other significant associations between miRNA expression and sperm quality parameters. Only expression levels of miR-99b-5p were significantly positively correlated with good-quality day 3 embryo rate (ρ = 0.546; p = 0.003), while other variables of the IVF/ICSI cycle outcome showed no significant associations with miRNA expression profiles. This is one of the rare studies providing an insight directly into miRNA profiles in regard to sperm morphology. We identified nine miRNAs that could serve as biomarkers of spermatozoa quality in regard to teratozoospermia.

Iris Ring Melanoma Presenting as Scleral Pigmentation.

To report a patient with a very rare variant of iris melanoma that grows in the shape of a ring (ring melanoma). A 65-year-old patient was examined because of a pigmented lesion on the sclera. After a complete ophthalmic and ultrasound examination, a ring melanoma was diagnosed. Enucleation of the affected eye was performed, and histology report confirmed iris ring melanoma. This type of malignancy represents an exceedingly rare variant of uveal melanoma, and because of atypical clinical picture, it can be easily overlooked or misdiagnosed, which often delays adequate treatment. Gonioscopy, transillumination, and ultrasound help us to recognize and diagnose ring melanoma. Suspicion should be raised with a clinical picture that shows unilateral pigmentary glaucoma. The objective of this presentation is to describe and outline the challenging diagnosis and management of this rare disease entity.

Intraoperative Intradermal Application of Stromal Vascular Fraction into the Abdominal Suture Line: Histological Analysis of Abdominal Scar Tissue.

BACKGROUND: Stem cell therapy is a promising new approach to wound healing. Stromal vascular fraction is a heterogeneous collection of cells, including adipose-derived stem cells, which are traditionally isolated using a manual collagenase-based technique. To our knowledge, this is the first human study that histologically assesses the potential of intraoperative intradermal injection of stromal vascular fraction on skin regeneration. METHODS: In this controlled study, 20 patients undergoing deep inferior epigastric perforator flap breast reconstruction and bilateral flank liposuction were included. Stromal vascular fraction was injected intradermally into one side of the abdominal suture line, while the other side served as a control. Outcome measures included analysis of stromal vascular fraction by flow cytometry, histological analysis of scar tissue, and scar photography. RESULTS: Cell yield for application and cell viability were 55.9 ± 28.5 × 106 and 75.1% ± 14.5%, respectively. Age and body mass index were positively correlated with the number of cells for application and adipose-derived stem cells. Mean vascular density, elastic fiber content, collagen maturity (scar index), epidermal thickness, and number of rete ridges all showed higher values on the treated side. Furthermore, the injected number of adipose-derived stem cells and pericytes positively correlated with vascular density. CONCLUSIONS: It is safe to speculate that intradermal stromal vascular fraction injection at the beginning of the healing process increases vascular density, collagen maturity and organization, elastic fiber content, epidermal thickness, epidermal-dermal anchoring of the scarring skin and is therefore responsible for improved skin regeneration. It is a viable and safe method that can be used as an adjunctive treatment in plastic surgery procedures where suboptimal wound healing is anticipated. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Giant Cell Tumor of Bone Versus Tenosynovial Giant Cell Tumor - Similarities and Differences.

Giant cell tumor of bone (GCTB) and tenosynovial giant cell tumor (TGCT) share misleadingly similar names, soft texture and brown color macroscopically, osteoclast-like multinucleated giant cells microscopically and localisation in the musculoskeletal system. However, these two tumor types are biologically and clinically two distinct entities with different natural courses of progression and considerably different modes of surgical and medical treatment. In this article, we provide a detailed update on the similarities and the differences between both tumor types.GCTB is a locally aggressive osteolytic bone tumor, commonly seen in patients in their third decade of life. It usually occurs as a solitary lesion in the meta-epiphyseal region of long bones. It can be diagnosed using plain radiographic imaging, CT radiography or MRI to estimate the tumor extent, soft tissue and joint involvement. GCTB is usually treated with intralesional excision by curettage. Systemically, it can be treated with bisphosphonates and denosumab or radiotherapy.TGCT is a rare, slowly progressing tumor of synovial tissue, affecting the joint, tendon sheath or bursa, mostly seen in middle-aged patients. TGCT is usually not visible on radiographs and MRI is mostly used to enable assessment of potential bone involvement and distinguishing between two TGCT types. Localised TGCT is mostly treated with marginal surgical resection, while diffuse TGCT is optimally treated with total synovectomy and is more difficult to remove. Additionally, radiotherapy, intraarticular injection of radioactive isotopes, anti-TNF-α antibodies and targeted medications may be used.

Desmoid-type fibromatosis of paranasal sinuses with intracranial extension in a child-acase-based review.

PURPOSE: Desmoid-type fibromatosis (DF) is clonal fibroblastic proliferation that arises in the deep soft tissues, tends to reoccur, and is locally invasive. Desmoid-type fibromatosis of paranasal sinuses with intracranial extension is a rare condition that is even rarer in a small child. We aim to share with the reader our literature review, decision-making, and endoscopic endonasal operation procedure that combined gained us favorable results against this benign tumor with unpredictable natural history and disease course. CASE REPORT: We describe the decision-making process in the management of a 3-year-old boy with a history of sudden vision loss and vomiting. MR showed an expansive well-delineated homogeneous tumor in the sphenoid sinus with intracranial extension and optic nerves compression. The diagnosis of a sporadic form of desmoid-type fibromatosis was made using genetic testing of tumor tissue. A total gross removal was carried out with endoscopic endonasal microsurgical approach. At a 3-month follow-up, the patient is without any signs of recurrance. CONCLUSION: The treatment of children with desmoid-type fibromatosis requires a multidisciplinary approach by clinicians experienced with the management of pediatric cancer. While the desmoid-type fibromatosis is a benign, locally invasive tumor, observation should be the first step in the management. In case of life-threatening or symptomatic cases, operations that preserve function and structure should be the first choice for this benign tumor with unpredictable natural history and disease course.

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.

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.

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.

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.

Clinical and Histopathological Features of Gelsolin Amyloidosis Associated with a Novel GSN Variant p.Glu580Lys.

Gelsolin amyloidosis typically presents with corneal lattice dystrophy and is most frequently associated with pathogenic GSN variant p.Asp214Asn. Here we report clinical and histopathological features of gelsolin amyloidosis associated with a novel GSN variant p.Glu580Lys. We studied DNA samples of seven members of a two-generation family. Exome sequencing was performed in the proband, and targeted Sanger sequencing in the others. The heterozygous GSN variant p.Glu580Lys was identified in six patients. The patients exhibited corneal dystrophy (5/6), loose skin (5/6) and/or heart arrhythmia (3/6) and one presented with bilateral optic neuropathy. The impact of the mutation on the protein structure was evaluated in silico. The substitution is located in the fifth domain of gelsolin protein, homologous to the second domain harboring the most common pathogenic variant p.Asp214Asn. Structural investigation revealed that the mutation might affect protein folding. Histopathological analysis showed amyloid deposits in the skin. The p.Glu580Lys is associated with corneal dystrophy, strengthening the association of the fifth domain of gelsolin protein with the typical amyloidosis phenotype. Furthermore, optic neuropathy may be related to the disease and is essential to identify before discussing corneal transplantation.

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.

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.

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.

Identification of microRNAs and their target gene networks implicated in arterial wall remodelling in giant cell arteritis.

OBJECTIVES: To identify dysregulated microRNAs (miRNAs) and their gene targets in temporal arteries from GCA patients, and determine their association with GCA pathogenesis and related arterial wall remodelling. METHODS: We included 93 formalin-fixed, paraffin-embedded temporal artery biopsies (TABs) from treatment-naïve patients: 54 positive and 17 negative TABs from clinically proven GCA patients, and 22 negative TABs from non-GCA patients. miRNA expression analysis was performed with miRCURY LNA miRNome Human PCR Panels and quantitative real-time PCR. miRNA target gene prediction and pathway enrichment analysis was performed using the miRDB and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) databases, respectively. RESULTS: Dysregulation of 356 miRNAs was determined in TAB-positive GCA arteries, among which 78 were significantly under-expressed and 22 significantly overexpressed above 2-fold, when compared with non-GCA controls. Specifically, TAB-positive GCA arteries were characterized by a significant overexpression of 'pro-synthetic' (miR-21-3p/-21-5p/-146a-5p/-146b-5p/-424-5p) and under-expression of 'pro-contractile' (miR-23b-3p/-125a-5p/-143-3p/-143-5p/-145-3p/-145-5p/-195-5p/-365a-3p) vascular smooth muscle cell phenotype-associated regulatory miRNAs. These miRNAs targeted gene pathways involved in the arterial remodelling and regulation of the immune system, and their expression correlated with the extent of intimal hyperplasia in TABs from GCA patients. Notably, the expression of miR-21-3p/-21-5p/-146a-5p/-146b-5p/-365a-3p differentiated between TAB-negative GCA arteries and non-GCA temporal arteries, revealing these miRNAs as potential biomarkers of GCA. CONCLUSION: Identification of dysregulated miRNAs involved in the regulation of the vascular smooth muscle cell phenotype and intimal hyperplasia in GCA arterial lesions, and detection of their expression profiles, enables a novel insight into the complexity of GCA pathogenesis and implies their potential utilization as diagnostic and prognostic biomarkers of GCA.

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.

Frequent alterations in p16/CDKN2A identified by immunohistochemistry and FISH in chordoma.

The expression of p16/CDKN2A, the second most commonly inactivated tumour suppressor gene in cancer, is lost in the majority of chordomas. However, the mechanism(s) leading to its inactivation and contribution to disease progression have only been partially addressed using small patient cohorts. We studied 384 chordoma samples from 320 patients by immunohistochemistry and found that p16 protein was lost in 53% of chordomas and was heterogeneously expressed in these tumours. To determine if CDKN2A copy number loss could explain the absence of p16 protein expression we performed fluorescence in situ hybridisation (FISH) for CDKN2A on consecutive tissue sections. CDKN2A copy number status was altered in 168 of 274 (61%) of samples and copy number loss was the most frequent alteration acquired during clinical disease progression. CDKN2A homozygous deletion was always associated with p16 protein loss but only accounted for 33% of the p16-negative cases. The remaining immunonegative cases were associated with disomy (27%), monosomy (12%), heterozygous loss (20%) and copy number gain (7%) of CDKN2A, supporting the hypothesis that loss of protein expression might be achieved via epigenetic or post-transcriptional regulatory mechanisms. We identified that mRNA levels were comparable in tumours with and without p16 protein expression, but other events including DNA promoter hypermethylation, copy number neutral loss of heterozygosity and expression of candidate microRNAs previously implicated in the regulation of CDKN2A expression were not identified to explain the protein loss. The data argue that p16 loss in chordoma is commonly caused by a post-transcriptional regulatory mechanism that is yet to be defined.

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.