A recurrent NTRK1 tyrosine kinase domain mutation pair is characteristic in a subset of dedifferentiated liposarcomas.

INTRODUCTION: Dedifferentiated liposarcoma (DDLPS) is a common form of liposarcoma with challenging treatment modalities. Pan-TRK immunopositivity can be often observed without NTRK gene fusion in soft tissue sarcomas with myogenic differentiation. Expression and the role of NTRK in DDLPS are under-studied. We sought to identify activating mutations of the NTRK genes. MATERIALS AND METHODS: 131 DDLPS patients were selected for pan-TRK immunohistochemistry and positive cases were analyzed by Sanger sequencing for NTRK1, NTRK2 and NTRK3 genes. Functional assays were performed using a lentiviral transduction system to study the effect of NTRK variants in fibroblast, immortalized fibroblast, and dedifferentiated liposarcoma cell lines. RESULTS: Out of the 131 DDLPS cases, 75 immunohistochemical staining positive cases, 46 were successfully Sanger sequenced. A recurrent somatic mutation pair in cis position (NGS) of the NTRK1 c.1810C>T (p.H604Y) and c.1838G>T (p.G613V) was identified in six cases (13%) that have never been reported in DDLPS. NTRK fusions were excluded in all six cases by FISH and NGS. The phospho-AKT immunopositivity among the six mutated cases suggested downstream activation of the NTRK signaling pathway. Functional assays showed no transforming effects, but resistance to first- and second-line TRK inhibitors of the p.G613V and p.H604Y variant. CONCLUSIONS: We detected (de novo/somatic) missense mutation variants in cis position of the NTRK1 gene in a subset of DDLPS indicating modifying mutations that may contribute to tumorigenesis in a subset of DDLPS. These variants beget resistance to TRK inhibitors indicating an interesting biomarker for other studies with TRK inhibitors.

mTOR hyperactivity and RICTOR amplification as targets for personalized treatments in malignancies.

The increasing knowledge of molecular alterations in malignancies, including mutations and regulatory failures in the mTOR (mechanistic target of rapamycin) signaling pathway, highlights the importance of mTOR hyperactivity as a validated target in common and rare malignancies. This review summarises recent findings on the characterization and prognostic role of mTOR kinase complexes (mTORC1 and mTORC2) activity regarding differences in their function, structure, regulatory mechanisms, and inhibitor sensitivity. We have recently identified new tumor types with RICTOR (rapamycin-insensitive companion of mTOR) amplification and associated mTORC2 hyperactivity as useful potential targets for developing targeted therapies in lung cancer and other newly described malignancies. The activity of mTOR complexes is recommended to be assessed and considered in cancers before mTOR inhibitor therapy, as current first-generation mTOR inhibitors (rapamycin and analogs) can be ineffective in the presence of mTORC2 hyperactivity. We have introduced and proposed a marker panel to determine tissue characteristics of mTOR activity in biopsy specimens, patient materials, and cell lines. Ongoing phase trials of new inhibitors and combination therapies are promising in advanced-stage patients selected by genetic alterations, molecular markers, and/or protein expression changes in the mTOR signaling pathway. Hopefully, the summarized results, our findings, and the suggested characterization of mTOR activity will support therapeutic decisions.

Novel RICTOR amplification harbouring entities: FISH validation of RICTOR amplification in tumour tissue after next-generation sequencing.

Alterations in mTOR signalling molecules, including RICTOR amplification, have been previously described in many cancers, particularly associated with poor prognosis. In this study, RICTOR copy number variation (CNV) results of diagnostic next-generation sequencing (NGS) were analysed in 420 various human malignant tissues. RICTOR amplification was tested by Droplet Digital PCR (ddPCR) and validated using the "gold standard" fluorescence in situ hybridisation (FISH). Additionally, the consequences of Rictor protein expression were also studied by immunohistochemistry. RICTOR amplification was presumed in 37 cases with CNV ≥ 3 by NGS, among these, 16 cases (16/420; 3.8%) could be validated by FISH, however, ddPCR confirmed only 11 RICTOR-amplified cases with lower sensitivity. Based on these, neither NGS nor ddPCR could replace traditional FISH in proof of RICTOR amplification. However, NGS could be beneficial to highlight potential RICTOR-amplified cases. The obtained results of the 14 different tumour types with FISH-validated RICTOR amplification demonstrate the importance of RICTOR amplification in a broad spectrum of tumours. The newly described RICTOR-amplified entities could initiate further collaborative studies with larger cohorts to analyse the prevalence of RICTOR amplification in rare diseases. Finally, our and further work could help to improve and expand future therapeutic opportunities for mTOR-targeted therapies.

Novel, clinically relevant genomic patterns identified by comprehensive genomic profiling in ATRX-deficient IDH-wildtype adult high-grade gliomas.

Glioblastomas are the most common IDH-wildtype adult high-grade gliomas, frequently harboring mutations in the TERT gene promoter (pTERT) and utilizing the subsequent telomerase overexpression for telomere length maintenance. However, some rare cases show loss of ATRX and use alternative mechanisms of telomere lengthening. In this study, we performed the first complex genomic analysis specifically concentrating on the latter subgroup. Comprehensive genomic profiling of 12 ATRX-deficient and 13 ATRX-intact IDH-wildtype adult high-grade gliomas revealed that ATRX and pTERT mutations are mutually exclusive. DNMT3A alterations were confined to ATRX-deficient, while PTEN mutations to ATRX-intact cases. RAS-MAPK pathway alterations, including NF1 mutations, were more characteristic in the ATRX-deficient group. Variants of genes related to homologous recombination repair showed different patterns of affected genes. Two ATRX-deficient tumors with high tumor mutational burden and mismatch repair deficiency were found. One of these contained a novel fusion involving the NTRK2 and LRRFIP2 genes, while the other showed loss of MSH2 and MSH6 without genetic alterations in the encoding genes suggesting an epigenetic background. Genetic characteristics of ATRX-deficient IDH-wildtype adult high-grade gliomas suggest that these tumors are particularly intriguing targets of potential future therapeutic interventions including immunotherapies combined with MAPK pathway inhibition and DNA repair inhibitors.

[he importance of mTOR hyperactivity and RICTOR amplification, and the associated targeted therapy possibilities in malignant tumours]. / Az mTOR-hiperaktivitás és a RICTORamplifikáció jelentosége, és az ezzel összefüggo célzott terápiás lehetoségek rosszindulatú daganatokban.

Failures of anti-tumour therapies and drug resistance initiate difficulties in cancer treatments often caused by alterations in signalling network activity, including PI3K/Akt/mTOR hyperactivity due to oncogenic mutations. In this review, we summarise the relevance of mTOR (mechanistic target of rapamycin) dysregulation identified decades ago, which is now known to be characteristic of many tumours. In this context, we present differences in activity, function and testability of mTOR kinase complexes (mTORC1 and mTORC2) differing in structure, regulatory mechanisms and inhibitor sensitivity. We highlight that genetic alterations, including RICTOR amplification and associated mTOR hyperactivity, are relevant in targeted therapy development. It is recommended to investigate mTOR profile activity in patients for whom mTOR inhibitor therapies are considered since the current first-generation mTOR inhibitors (rapamycin and analogues) may be ineffective in case of mTORC2 hyperactivity. Ongoing phase trials of new inhibitors and combination therapies are promising in advanced stage patients selected by molecular markers.

Potential molecular mechanism in self-renewal is associated with miRNA dysregulation in sacral chordoma - A next-generation RNA sequencing study.

Background: Chordoma, the most frequent malignant primary spinal neoplasm, characterized by a high rate of recurrence, is an orphan disease where the clarification of the molecular oncogenesis would be crucial to developing new, effective therapies. Dysregulated expression of non-coding RNAs, especially microRNAs (miRNA) has a significant role in cancer development. Methods: Next-generation RNA sequencing (NGS) was used for the combinatorial analysis of mRNA-miRNA gene expression profiles in sacral chordoma and nucleus pulposus samples. Advanced bioinformatics workflow was applied to the data to predict miRNA-mRNA regulatory networks with altered activity in chordoma. Results: A large set of significantly dysregulated miRNAs in chordoma and their differentially expressed target genes have been identified. Several molecular pathways related to tumorigenesis and the modulation of the immune system are predicted to be dysregulated due to aberrant miRNA expression in chordoma. We identified a gene set including key regulators of the Hippo pathway, which is targeted by differently expressed miRNAs, and validated their altered expression by RT-qPCR. These newly identified miRNA/RNA interactions are predicted to have a role in the self-renewal process of chordoma stem cells, which might sustain the high rate of recurrence for this tumor. Conclusions: Our results can significantly contribute to the designation of possible targets for the development of anti-chordoma therapies.

Nodular fasciitis: a comprehensive, time-correlated investigation of 17 cases.

The self-limited nature of nodular fasciitis (NF) is well-known but its precise mechanism has not yet been clarified. We observed that "young" NF (preoperative duration <1 month) consistently contains a higher percentage (~80%) of USP6 break-apart FISH signals than "old" NF (preoperative duration >3 months) (~20%). Thus, we hypothesized that our original observation may reflect a connection with the self-limited nature of NF. Seventeen cases with reliable data concerning the onset were selected, thus approximating the lifetime of each tumor. Besides the USP6 interphase FISH examination, we also checked the most common MYH9-USP6 fusion using RT-PCR. Because of the known pathways of the tumorigenesis of NF, the mRNA level of USP6, TRAIL, IFN-beta, JAK1, STAT1, STAT3, JUN, and CDKN2A was measured using qRT-PCR. Regarding proteins, USP6, p16, p27, TRAIL, and IFN-beta were examined using immunohistochemistry. Targeted gene panel next-generation sequencing (NGS) of three cases was additionally performed. We found a strong negative correlation (p = 0.000) between the lifetime and percentage of USP6 break-apart signals and a strong positive relationship (p = 0.000) between USP6 break-apart signals and mitotic counts. Results of immunostainings, along with qRT-PCR results, favored the previously-suggested USP6-induced negative feedback mechanism through activation of TRAIL and IFN-beta, likely resulting in apoptosis and senescence of tumor cells harboring USP6 fusions. Targeted-NGS resulted in the detection of several variants, but no additional recurrent changes in the pathogenesis of these tumors. We revealed on a cellular level the USP6-induced negative feedback mechanism. In conclusion, we emphasize that in "old" NF, the percentage of USP6 break-apart FISH signals can be as low as 14-27% which can be very important from a differential diagnostic point of view. We emphasize that a careful examination and interpretation of the NGS data is needed before clinical decision-making on treatment.

Efficacy of carboplatin chemotherapy in a metastatic, castration-resistant BRCA2 mutation positive prostate cancer patient / A karboplatin-kemoterápia hatékonysága egy áttétes, kasztrációrezisztens, BRCA2-mutáció-pozitív prosztatarákos betegben.

Összefoglaló. A sérült BRCA1/2 gént hordozó prosztatadaganatok klinikai szempontból elkülönülo, agresszív altípust képviselnek. Ugyanakkor a BRCA1/2 gén sérülése a DNS-támadáspontú kemoterápiákkal szemben érzékennyé teszi a daganatot, ami terápiás szempontból kihasználható. A platinaalapú kemoterápia hatékonysága prosztatarákban klinikai vizsgálatokkal nincs alátámasztva, ezért annak alkalmazására igen ritkán kerül sor. Közleményünkben egy elorehaladott stádiumú, agresszív prosztata adenocarcinomával diagnosztizált beteg esetét mutatjuk be, akinél a BRCA2-gén patogén mutációját találtuk, és akinél az elozoleg alkalmazott androgénmegvonásos, valamint docetaxelkezelések sikertelensége miatt karboplatinkezelést alkalmaztunk - ez a beteg állapotának, valamint radiológiai és biokémiai paramétereinek látványos javulásához vezetett. Ez az eset rámutat a DNS-hiba-javító mechanizmusban szerepet játszó gének terápiás szempontból történo felhasználásának potenciális elonyeire prosztatarákban. Orv Hetil. 2021; 162(25): 1004-1008. Summary. BRCA1/2 deficient prostate cancers represent a clinically distinct aggressive subtype. However, the presence of BRCA1/2 alterations enhance the sensitivity to platinum-based chemotherapies. The efficacy of platinum-based chemotherapies in prostate cancer has not been proven in prospective clinical studies and therefore these treatments are rarely used in prostate adenocarcinomas. Here we present a case of BRCA2 mutant prostate cancer, which was diagnosed at a metastatic stage and showed no or only little response to androgen deprivation and docetaxel therapies. Therefore, we started carboplatin chemotherapy which resulted in an exceptional response regarding biochemical, radiographic parameters accompanied by significant improvement of patients' physical condition. This case underlines the potential therapeutic benefits of testing for genes involved in the DNA repair mechanism. Orv Hetil. 2021; 162(25): 1004-1008.

EGFR variant allele frequency predicts EGFR-TKI efficacy in lung adenocarcinoma: a multicenter study.

BACKGROUND: Although lung adenocarcinoma (LADC) with sensitizing mutations of the epidermal growth factor receptor (EGFR) is highly sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs), in most cases disease progression inevitably occurs. Our aim was to investigate the predictive and prognostic significance of adjusted tumoral EGFR variant allele frequency (EGFR-aVAF) in the above setting. METHODS: Eighty-nine Caucasian advanced-stage LADC patients with known exon-specific EGFR mutations undergoing EGFR-TKI treatment were included. The correlations of EGFR-aVAF with clinicopathological variables including progression-free and overall survival (PFS and OS, respectively) were retrospectively analyzed. RESULTS: Of 89 EGFR-mutant LADC patients, 46 (51.7%) had exon 19 deletion, while 41 (46.1%) and 2 (2.2%) patients had exon 21- and exon 18-point mutations, respectively. Tumoral EGFR-aVAF was significantly higher in patients harboring EGFR exon 19 mutations than in those with exon 21-mutant tumors (P<0.001). Notably, patients with EGFR exon 19 mutant tumors demonstrated significantly improved PFS (P=0.003) and OS (P=0.02) compared to patients with exon 21 mutations. Irrespective of specific exon mutations, a statistically significant positive linear correlation was found between EGFR-aVAF of tumoral tissue and PFS (r=0.319; P=0.002). High (≥70%) EGFR-aVAF was an independent predictor of longer PFS [vs. low (<70%) EGFR-aVAF; median PFSs were 52 vs. 26 weeks, respectively; P<0.001]. Additionally, patients with high EGFR-aVAF also had significantly improved OS than those with low EGFR-aVAF (P=0.011). CONCLUSIONS: Our study suggests that high (≥70%) EGFR-aVAF of tumoral tissue predicts benefit from EGFR-TKI treatment in advanced LADC and, moreover, that exon 19 EGFR mutation is associated with high EGFR-aVAF and improved survival outcomes.

Cross-testing of major molecular markers indicates distinct pathways of tumorigenesis in gastric adenocarcinomas and synchronous gastrointestinal stromal tumors.

Small subtype of the gastrointestinal stromal tumor (micro-GIST, MG) is usually asymptomatic and is frequently found incidentally in association with gastric adenocarcinoma (GAC). The background of this coincidence is still an open question. This study comprehensively characterized nine MGs and GACs present in the same surgical specimen by cross-testing the markers of the major pathogenetic pathways of both tumor types. All of the MGs were immunohistochemically positive for CD117/KIT, CD34, and DOG1. DOG1 was also detected in four GACs. Four MGs carried mutations in c-KIT (exons 9, 11, and 13) and two cases in PDGFRα (exon 18). None of the GACs carried activating mutations in c-KIT or PDGFRα. MMR immunopanel identified one GAC as microsatellite unstable tumor. No EBV-positive tumor was found. According to the TCGA molecular classification, one GAC was categorized in the MSI subgroup, three GACs in the genomically stable subgroup, and the rest into the chromosomal instability subgroup. Although a common carcinogenic effect cannot be ruled out, our data suggest a distinct molecular background in the evolvement of the synchronous MGs and GACs. The presence of a MG in gastric resection specimens may be indicative of the development of synchronous malignant tumors in or outside the stomach.

The Presence of ALK Alterations and Clinical Relevance of Crizotinib Treatment in Pediatric Solid Tumors.

Soft tissue sarcomas (STS) and neuroblastomas (NBL), are childhood malignancies still associated with poor prognoses despite the overall improvement in childhood tumor survival of the past decades. Anaplastic lymphoma kinase (ALK) inhibition is promising new strategy to improve the outcome of these pediatric tumors. Eighteen histologic samples of pediatric STS and 19 NBL patients were analyzed for ALK abnormalities using fluorescent in situ hybridization (FISH) with break-apart probes and immunohistochemistry (IHC). ALK alterations were presented in 20 of the 37 sections. The presence of ALK alteration in NBL samples were detected using IHC in 84,2% of all cases compared to 21,1% FISH positivity. In STS cases the results were less different (IHC 16,7% vs FISH 22,2%). The difference can be explained by the different type of molecular alterations. FISH method detected translocation and amplification, but not the point mutation of ALK gene. IHC confirmed the diagnosis by detecting the expression of ALK protein.After ALK positivity was proven, the effectiveness and safety of the crizotinib therapy was examined in 4 patients (1 alveolar rhabdomyosarcoma (RMA), 1 embryonal rhabdomyosarcoma (RME), 1 inflammatory myofibroblastic tumor (IMT), 1 NBL). We observed continuous remission of the IMT patient, all other cases the inhibitor treatment was not curative.Our findings underline the importance of screening the ALK status parallel with both IHC and FISH. Crizotinib treatment had a long-term effect in ALK positive IMT patients, however itwas only temporary efficient in relapsed, progressive STS and NBL.

Release of circulating tumor cells and cell-free nucleic acids is an infrequent event in synovial sarcoma: liquid biopsy analysis of 15 patients diagnosed with synovial sarcoma.

BACKGROUND: Synovial sarcoma is a rare soft tissue tumor which contains the unique SS18-SSX1, SS18-SSX2 - or, rarely, SS18-SSX4 - fusion transcripts. It is well known that some soft tissue tumors, like Ewing sarcomas and myxoid liposarcomas, can spread via the blood with free circulating tumor cells (CTC); this can be detected by several sensitive molecular biology methods. Here we report a study of fifteen synovial sarcoma patients with varied clinical backgrounds. METHOD: After blood withdrawal and nucleic acid isolation, we attempted to detect the SS18-SSX fusion genes from circulating tumor cells or cell-free nucleic acids with nested PCR and droplet digital PCR. RESULTS: SS18-SSX2 fusion transcript was identified in a small copy number with droplet digital PCR in one case. Nested PCR could not detect any of the fusion transcripts in the examined 15 synovial sarcoma cases. CONCLUSIONS: Heretofore two case reports could detect CTCs in synovial sarcoma - in the first paper, the patient was diagnosed with poorly differentiated type while the other had a rare primary gastric synovial sarcoma. However, until now, no other studies have detected CTCs in the peripheral blood of synovial sarcoma patients. Based on our findings, we can conclude that detection of the chimeric SS18-SSX fusion gene after surgical excision and/or chemotherapy/radiotherapy is a rare circumstance and hence in itself is not sufficient for monitoring the tumor recurrence. Therefore, monitoring of other possible biomarkers - for example synovial sarcoma specific miRNAs - is recommended.

The oncomir face of microRNA-206: A permanent miR-206 transfection study.

MiR-206 is a remarkable miRNA because it functions as a suppressor miRNA in rhabdomyosarcoma while at the same time, as previously showed, it can act as an oncomiRNA in SMARCB1 immunonegative soft tissue sarcomas. The aim of this study was to investigate the effect of miR-206 on its several target genes in various human tumorous and normal cell lines. In the current work, we created miR-206-overexpressing cell lines (HT-1080, Caco2, iASC, and SS-iASC) using permanent transfection. mRNA expression of the target genes of miR-206 (SMARCB1, ACTL6A, CCND1, POLA1, NOTCH3, MET, and G6PD) and SMARCB1 protein expression were examined with quantitative real-time polymerase chain reaction, immunoblotting, immunocytochemistry, and flow cytometry. MiRNA inhibition was used to validate our results. We found a diverse silencing effect of miR-206 on its target genes. While an overall tendency of downregulation was noted, expression profiles of individual cell lines showed large variability. Only CCND1 and MET were consistently downregulated. MiR-206 had an antiproliferative effect on a normal human fibroblast cell line. A strong silencing effect of SMARCB1 in miR-206 transfected SS-iASC was most likely caused by the synergic influence of the SS18-SSX1 fusion protein and miR-206. In the same cell line, a moderate decrease of SMARCB1 protein expression could be observed with immunocytochemistry and flow cytometry. In the most comprehensive analysis of miR-206 effects so far, a modest but significant downregulation of miR-206 targets on the mRNA level was confirmed across all cell lines. However, the variability of the effect shows that the action of this miRNA is largely cell context-dependent. Our results also support the conception that the oncomiR effect of miR-206 on SMARCB1 plays an important but not exclusive role in SMARCB1 immunonegative soft tissue sarcomas so it can be considered important in planning the targeted therapy of these tumors in the future. Impact statement Mir-206 is a very unique microRNA because it can act as a suppressor miRNA or as an oncomiRNA depending on the tumor tissue. In SMARCB1 negative soft tissue sarcomas miR-206 is overexpressed, so thus in epithelioid and synovial sarcomas it functions as an oncomiRNA. MiR-206 has diverse silencing effects on its target genes. We found that the action of miR-206 is largely cell context dependent. The oncomiR role of miR-206 is crucial but not exclusive in SMARCB1 negative soft tissue sarcomas and miR-206 has an antiproliferative effect on a normal human fibroblast cell line. Expressions of miR-206 targets observed in tumors can only be reproduced in the corresponding tumorous cell lines. This is the first study which examined the permanent effect of miR-206 on its target genes in normal, tumor, and genetically engineered cell lines.

Concomitant 1p36 deletion and TNFRSF14 mutations in primary cutaneous follicle center lymphoma frequently expressing high levels of EZH2 protein.

Primary cutaneous follicle center lymphoma (PCFCL) is an indolent variant of follicular lymphoma (FL) with limited information available on the genetic background of the disease. The genetic hallmark of nodal FL, the t(14;18) translocation, affecting the BCL2 gene, is rare in PCFCL. Loss of 1p36, the most common secondary chromosomal abnormality in nodal FL, has been recently reported in 16.7% of PCFCL cases. In order to further characterize PCFCL, 21 cases were analyzed using interphase fluorescence in situ hybridization with BCL2 break apart and 1p36/1q25 dual color probes. Sanger sequencing was used to investigate TNFRSF14 and EZH2 mutations and immunohistochemistry to assess BCL2, EZH2 protein expressions.1p36 deletion occurred in 22% (5/21), BCL2 gene break in 10% (2/20) of the PCFCL cases. Mutations of the candidate tumor suppressor gene of the 1p36 region, TNFRSF14 mutations were detected in 4/17 (23.5%) cases with 2 cases presenting with concurrent 1p36 deletion. EZH2 hotspot mutations at Y641, A682, and A692 were not found. High EZH2 protein expression associated with a BCL2 negative phenotype was observed in 43% (9/21) of the cases. BCL2 gene break or 1p36 deletion did not impact the prognosis; however, they showed association with advanced stages at diagnosis (p = 0.016) and a tendency with shorter event free survival (p = 0.052).In conclusion, 1p36 deletion co-occurs with acquired TNFRSF14 mutations, suggesting a role of this tumor suppressor gene in the development of a subgroup of PCFCL. High EZH2 protein expression associated with BCL2 negative phenotype is common and might represent an ideal therapeutic target.

First cloned human immortalized adipose derived mesenchymal stem-cell line with chimeric SS18-SSX1 gene (SS-iASC).

The SS18-SSX chimeric gene is unique to synovial sarcoma. Multiple model systems including mouse cell lines expressing SS18-SSX, and genetically engineered mouse models of synovial sarcoma have been developed to elucidate the role of the chimeric gene in synovial sarcomagenesis. Although several cell lines stably expressing human SS18-SSX exist, there is an ongoing need for cell culture models enabling researchers to investigate the molecular mechanism of SS18-SSX action in a relevant cellular context. Here we report the establishment of a novel SS18-SSX1-expressing cell line created from immortalized human adipose tissue-derived mesenchymal stem cells via lentiviral transduction of the chimeric gene. Our cell line, termed SS-iASC, has been characterized by karyotyping and cell line identification, and stable expression of SS18-SSX1 has been verified using real-time PCR (RT-PCR), nested PCR, immunofluorescence, and immunoblotting. Focal cytokeratin positivity characteristic of synovial sarcoma but no ß-Catenin, Bcl-2 or cyclin D1 expression was observed in SS-iASC. The novel cell line expressing SS18-SSX1 on a human adipose-derived stromal cell background is expected to be helpful in addressing the question whether the chimeric gene alone is sufficient to trigger the formation of synovial sarcoma.

Unusual Signal Patterns of Break-apart FISH Probes Used in the Diagnosis of Soft Tissue Sarcomas.

Break-apart FISH probes are the most popular and reliable type of FISH probes used to confirm certain pathological diagnoses. The interpretation is usually easy, however, in some instances it is not so unequivocal. Our aim was to reveal and elucidate the problems occurring in the process of evaluation of the break-apart probe results. Altogether 301 soft tissue sarcomas with confirmed molecular tests using break-apart probes were assessed to reveal the frequency and type of unusual signal pattern. Among 89 synovial sarcoma (SS18) 11%, 12 alveolar rhabdomyosarcoma (FOXO1) 50%, 53 myxoid liposarcoma (DDIT3) 7.5%, 6 low grade fibromyxoid sarcoma (FUS) 67%, 93 Ewing sarcoma (EWSR1) 3%, 12 clear cell sarcoma (EWSR1) 8%, 5 desmoplastic small round cell tumor (EWSR1) 0%, 9 extraskeletal myxoid chondrosarcoma (EWSR1) 0%, 2 myoepithelial carcinoma (EWSR1) 50%, 14 dermatofibrosarcoma protuberans (COL1A1) 86% and 6 nodular fasciitis (USP6) 17% atypical break-apart signals were detected. Despite the unusual signal pattern type, the fusion genes were detected using either metaphase FISH, interphase FISH with translocation/TriCheck probe or RT-PCR methods. Although the interpretation problems in the process to evaluate the break-apart probe results is well known from sporadic case reports, a systemic overview to detect their frequency has not been performed so far. In our work we highlighted the relative frequency of this problem and pinpointed those signal-patterns which, despite their unusual appearance, can still confirm the diagnosis.

Epigenetic regulation of SMARCB1 By miR-206, -381 and -671-5p is evident in a variety of SMARCB1 immunonegative soft tissue sarcomas, while miR-765 appears specific for epithelioid sarcoma. A miRNA study of 223 soft tissue sarcomas.

Complete/partial loss of SMARCB1 nuclear-immunopositivity is characteristic of a certain subset of soft tissue sarcomas (STSs). Our previous work showed that oncomiRs-206,-381, and 671-5p could silence the SMARCB1 mRNA and protein expression and that they display significant overexpression in epithelioid sarcomas (ESs). MiR-765 was overexpressed too, but functionally was inactive in the silencing. In the current work, using quantitative PCR, we conducted a miRNA study of 51 ESs, 20 rhabdoid tumors (RTs), 20 synovial sarcomas (SSs), 15 malignant peripheral nerve sheath tumors (MPNSTs), 11 myoepithelial carcinomas (MECs), and 10 extraskeletal myxoid chondrosarcomas (EMCSs) with complete/partial loss of SMARCB1 nuclear immunostain, in contrast to controls (SMARCB1-immunopositive) of 96 STSs, 13 melanomas and 10 sarcomatoid carcinomas. The SMARCB1 genetic status of ESs was determined by MLPA and FISH. A subset of ESs (5/51) showed biallelic deletion of SMARCB1 with no overexpression of any miRNA, suggesting these tumors could be the counterpart of pediatric RT, at least genetically. Another subset (5/51) was genetically either intact or monoallelic deleted with at least threefold overexpression of one of miR-206,-381,-671-5p, suggesting epigenetic regulation only. 39/51 ESs had a biallelic deletion (>20% by FISH and/or by MLPA) but with overexpressed miR-206,-381, and 671-5p, suggesting intratumoral heterogeneity, i.e., both genetic and epigenetic regulation. At least threefold overexpression of one of miR-206,-381, and 671-5p was detected in all MPNSTs, EMCSs, SSs and 7 MCs. Except for ESs, four SSs and one MPNST, there was no event above threefold overexpression of miR-765 among all 195 tested tumors. Our results suggest a general role of miR-206,-381, and 671-5p in SMARCB1 gene silencing of ES, MC, EMCS, MPNST and SS. In the future, miR-765 could possibly be a diagnostic tool for ES because of its 97% specificity and 80% sensitivity. © 2016 Wiley Periodicals, Inc.

Prognostic impact of reduced connexin43 expression and gap junction coupling of neoplastic stromal cells in giant cell tumor of bone.

Missense mutations of the GJA1 gene encoding the gap junction channel protein connexin43 (Cx43) cause bone malformations resulting in oculodentodigital dysplasia (ODDD), while GJA1 null and ODDD mutant mice develop osteopenia. In this study we investigated Cx43 expression and channel functions in giant cell tumor of bone (GCTB), a locally aggressive osteolytic lesion with uncertain progression. Cx43 protein levels assessed by immunohistochemistry were correlated with GCTB cell types, clinico-radiological stages and progression free survival in tissue microarrays of 89 primary and 34 recurrent GCTB cases. Cx43 expression, phosphorylation, subcellular distribution and gap junction coupling was also investigated and compared between cultured neoplastic GCTB stromal cells and bone marow stromal cells or HDFa fibroblasts as a control. In GCTB tissues, most Cx43 was produced by CD163 negative neoplastic stromal cells and less by CD163 positive reactive monocytes/macrophages or by giant cells. Significantly less Cx43 was detected in α-smooth muscle actin positive than α-smooth muscle actin negative stromal cells and in osteoclast-rich tumor nests than in the adjacent reactive stroma. Progressively reduced Cx43 production in GCTB was significantly linked to advanced clinico-radiological stages and worse progression free survival. In neoplastic GCTB stromal cell cultures most Cx43 protein was localized in the paranuclear-Golgi region, while it was concentrated in the cell membranes both in bone marrow stromal cells and HDFa fibroblasts. In Western blots, alkaline phosphatase sensitive bands, linked to serine residues (Ser369, Ser372 or Ser373) detected in control cells, were missing in GCTB stromal cells. Defective cell membrane localization of Cx43 channels was in line with the significantly reduced transfer of the 622 Da fluorescing calcein dye between GCTB stromal cells. Our results show that significant downregulation of Cx43 expression and gap junction coupling in neoplastic stromal cells are associated with the clinical progression and worse prognosis in GCTB.

SMARCB1 expression in epithelioid sarcoma is regulated by miR-206, miR-381, and miR-671-5p on Both mRNA and protein levels.

Proximal type epithelioid sarcoma shares similarities with malignant rhabdoid tumor, including the lack of nuclear immunoreactivity of SMARCB1. Biallelic mutation of SMARCB1 has been convincingly established as the cause of loss of protein expression in rhabdoid tumor, but the cause in epithelioid sarcoma remains unknown. In our previous work, we demonstrated that DNA hypermethylation and post-translational modification mechanisms were not involved. In this current work, we explored the hypothesis that miRNAs regulate SMARCB1 gene expression in epithelioid sarcomas. In silico target prediction analysis revealed eight candidate miRNAs, and quantitative PCR-in 32 formalin-fixed, paraffin-embedded tumor samples comprising 30 epithelioid sarcomas and two malignant rhabdoid tumors-demonstrated significant (P < 0.001) overexpression of four miRNAs in epithelioid sarcomas: miR-206, miR-381, miR-671-5p, and miR-765. Two human tumors (fibrosarcoma and colon adenocarcinoma) and a normal cell line (human dermal fibroblast) with retained SMARCB1 expression were cultured for miRNA transient transfection (electroporation) experiments. SMARCB1 mRNA expression was analyzed by quantitative real-time PCR and immunostaining of SMARCB1 was performed to examine the effect of miRNAs transfections on both RNA and protein levels. Only three of the overexpressed miRNAs (miR-206, miR-381, and miR-671-5p) could silence the SMARCB1 mRNA expression in cell cultures; most effectively miR-206. Transfection of miR-206, miR-381, miR-671-5p, and some combination of them also eliminated SMARCB1 nuclear staining, demonstrating a strong effect on not only mRNA but also protein levels. Our results suggest loss of SMARCB1 protein expression in epithelioid sarcoma is due to the epigenetic mechanism of gene silencing by oncomiRs.