Lessons from discovery of true ADAR RNA editing sites in a human cell line.

BACKGROUND: Conversion or editing of adenosine (A) into inosine (I) catalyzed by specialized cellular enzymes represents one of the most common post-transcriptional RNA modifications with emerging connection to disease. A-to-I conversions can happen at specific sites and lead to increase in proteome diversity and changes in RNA stability, splicing, and regulation. Such sites can be detected as adenine-to-guanine sequence changes by next-generation RNA sequencing which resulted in millions reported sites from multiple genome-wide surveys. Nonetheless, the lack of extensive independent validation in such endeavors, which is critical considering the relatively high error rate of next-generation sequencing, leads to lingering questions about the validity of the current compendiums of the editing sites and conclusions based on them. RESULTS: Strikingly, we found that the current analytical methods suffer from very high false positive rates and that a significant fraction of sites in the public databases cannot be validated. In this work, we present potential solutions to these problems and provide a comprehensive and extensively validated list of A-to-I editing sites in a human cancer cell line. Our findings demonstrate that most of true A-to-I editing sites in a human cancer cell line are located in the non-coding transcripts, the so-called RNA 'dark matter'. On the other hand, many ADAR editing events occurring in exons of human protein-coding mRNAs, including those that can recode the transcriptome, represent false positives and need to be interpreted with caution. Nonetheless, yet undiscovered authentic ADAR sites that increase the diversity of human proteome exist and warrant further identification. CONCLUSIONS: Accurate identification of human ADAR sites remains a challenging problem, particularly for the sites in exons of protein-coding mRNAs. As a result, genome-wide surveys of ADAR editome must still be accompanied by extensive Sanger validation efforts. However, given the vast number of unknown human ADAR sites, there is a need for further developments of the analytical techniques, potentially those that are based on deep learning solutions, in order to provide a quick and reliable identification of the editome in any sample.

Loss of YAP1 C-terminus expression as an ancillary marker for metaplastic thymoma: a potential pitfall in detecting YAP1::MAML2 gene rearrangement.

AIMS: Metaplastic thymoma is a rare thymic tumour characterized by Yes Associated Protein 1 (YAP1) and Mastermind Like Transcriptional Coactivator 2 (MAML2) gene fusions resulting from an intrachromosomal inversion of chromosome 11. Immunohistochemistry with an antibody directed against the C-terminus of YAP1 has shown loss of expression in YAP1-rearranged vascular neoplasms, poromas, and porocarcinomas. This study aimed to validate an anti-YAP1 C-terminal antibody as an ancillary immunohistochemical marker for the diagnosis of metaplastic thymoma. MATERIALS AND METHODS: Ten metaplastic thymomas were selected for the current study. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed to detect YAP1::MAML2 fusions. We then performed immunohistochemistry to detect YAP1 C-terminus expression in 10 metaplastic thymomas, 50 conventional thymomas (10 each of type A thymoma, type AB thymoma, type B1 thymoma, type B2 thymoma, and type B3 thymoma) and seven thymic carcinomas. RESULTS: All 10 cases showed narrow split signals with a distance of nearly two signal diameters and sometimes had false-negative results in YAP1 and MAML2 break-apart FISH (BA-FISH). Abnormal colocalized signals of the YAP1::MAML2 fusion were observed in all 10 cases using fusion FISH (F-FISH) assays. Eight of 10 cases with adequate nucleic acids were successfully sequenced and all showed YAP1::MAML2 fusions; in two cases the fusions were detected by both DNA and RNA sequencing and in six cases by RNA sequencing only. YAP1::MAML2 fusion transcripts were identified in four cases by RT-PCR. Metaplastic thymoma showed loss of YAP1 C-terminus expression in all 10 (100%) cases. All other thymic neoplasms showed retained YAP1 C-terminus expression. CONCLUSION: YAP1 C-terminus immunohistochemistry is a highly sensitive and specific ancillary marker that distinguishes metaplastic thymoma from its mimics. BA-FISH assays could not effectively detect YAP1::MAML2 fusions due to the proximity of the two genes. Loss of YAP1 C-terminus expression is a reliable surrogate for the detection of YAP1::MAML2 fusions in metaplastic thymoma.

Relationship between compression time and long-term hypoesthesia in primary trigeminal neuralgia treated with percutaneous balloon compression.

Percutaneous balloon compression is a surgical method for the treatment of trigeminal neuralgia, but one of the surgical parameters, compression time, is inconclusive. To investigate the effect of compression time during balloon compression on long-term postoperative hypoesthesia in patients with primary trigeminal neuralgia and to provide guidance on relevant parameters for balloon compression in the treatment of primary trigeminal neuralgia, we conducted a nested case-control study. Patients with primary trigeminal neuralgia treated by balloon compression from March 2013 to September 2013 were divided into case group and control group according to whether there were still symptoms of hypoesthesia at present. The relationship between the compression time of balloon compression and long-term hypoesthesia was analyzed. A total of 289 trigeminal neuralgia patients treated with percutaneous balloon compression were included in this study. Multivariate logistic regression showed that compression time was significantly correlated with long-term hypoesthesia (OR = 1.91, 95% CI = 1.13-3.23, P = 0.02), and compression time was greater than one. The risk of hypoesthesia in the long-term when the compression time is longer than 1 min is 1.93 times that of 1 min. PBC is a safe and effective surgical method, and the long-term hypoesthesia is related to the compression time during operation. The longer the compression time during operation, the greater the risk of long-term hypoesthesia.

Malignant melanotic Xp11 neoplasms exhibit a clinicopathologic spectrum and gene expression profiling akin to alveolar soft part sarcoma: a proposal for reclassification.

The classification of the distinct group of mesenchymal neoplasms, first described as 'Xp11 translocation perivascular epithelioid cell tumor (PEComa)' and for which the term 'melanotic Xp11 neoplasm' or 'Xp11 neoplasm with melanocytic differentiation' has recently been proposed, remains challenging and controversial. We collected 27 melanotic Xp11 neoplasms, the largest series to date, for a comprehensive evaluation. Fourteen of the cases, together with eight alveolar soft part sarcomas (ASPS), nine conventional PEComas and a control group of seven normal tissues were submitted to RNA sequencing. Follow-up available in 22 patients showed 5-year overall survival and 5-year disease-free survival of 47.6 and 35.7%, respectively, which were similar to ASPS and significantly worse than conventional PEComa. Univariate analysis of location (occurring in the kidney versus not kidney), infiltrative growth pattern, nuclear pleomorphism, mitotic activity ≥2/50 high-power fields (HPF), necrosis and lymphovascular invasion were found to be associated with overall survival and/or disease-free survival. Multivariate analysis identified that location was the only factor found to independently correlate with disease-free survival. More importantly, RNA sequencing-based clustering analysis segregated melanotic Xp11 neoplasm and ASPS from other tumors, including conventional PEComa and Xp11 translocation renal cell carcinoma, and formed a compact cluster representative of the largely similar expression signature. Here we clearly define the true biologic nature of melanotic Xp11 neoplasms which are distinctive malignant mesenchymal tumors, rather than simply PEComa variants with occasionally unpredictable behavior. Meanwhile, melanotic Xp11 neoplasm and ASPS more likely represent phenotypic variants of the same entity, which is distinct from conventional PEComa and Xp11 translocation renal cell carcinoma. Based on these important findings, melanotic Xp11 neoplasm might be reclassified into a distinctive entity together with ASPS, independent from PEComa, in future revisions of the current WHO categories of tumors of soft tissue and bone for the improved reclassification. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genetic and epigenetic alterations of SDH genes in patients with sporadic succinate dehydrogenase-deficient gastrointestinal stromal tumors.

This study aimed to investigate the association of SDH gene mutations and promoter methylation with succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GISTs) and to further discuss the potential molecular mechanisms underlying SDHB expression loss in these tumors. First, a total of 26 patients with SDH-deficient GISTs were selected by identifying the loss of SDHB protein expression and wild-type for KIT and PDGFRa mutations. Then SDH gene mutations and promoter methylation were detected by DNA sequencing and methylation-specific polymerase chain reaction, respectively, and the clinical and pathological data of SDH-deficient GISTs patients were collected and analyzed accordingly. The results of genetic testing demonstrated that 38.46% (10/26) of these patients harbored mutations in SDHB, SDHC, and SDHD genes (3 cases with double mutations). Besides, aberrant promoter methylation of SDH genes was detected in 10 out of 26 cases (38.46%), including 8 cases in SDHA gene, 3 cases in SDHB gene, 1 case in both SDHA and SDHB genes. It is suggested that SDH gene mutations and promoter methylation may contribute to the loss of SDH protein expression in sporadic SDH-deficient GISTs. This study indicated that the genetic and epigenetic alterations of SDH genes may occur during tumor formation.

RNA sequencing of Xp11 translocation-associated cancers reveals novel gene fusions and distinctive clinicopathologic correlations.

Both Xp11 translocation renal cell carcinomas and the corresponding mesenchymal neoplasms are characterized by a variety of gene fusions involving TFE3. It has been known that tumors with different gene fusions may have different clinicopathologic features; however, further in-depth investigations of subtyping Xp11 translocation-associated cancers are needed in order to explore more meaningful clinicopathologic correlations. A total of 22 unusual cases of Xp11 translocation-associated cancers were selected for the current study; 20 cases were further analyzed by RNA sequencing to explore their TFE3 gene fusion partners. RNA sequencing identified 17 of 20 cases (85%) with TFE3-associated gene fusions, including 4 ASPSCR1/ASPL-TFE3, 3 PRCC-TFE3, 3 SFPQ/PSF-TFE3, 1 NONO-TFE3, 4 MED15-TFE3, 1 MATR3-TFE3, and 1 FUBP1-TFE3. The results have been verified by fusion fluorescence in situ hybridization (FISH) assays or reverse transcriptase polymerase chain reaction (RT-PCR). The remaining 2 cases with specific pathologic features highly suggestive of MED15-TFE3 renal cell carcinoma were identified by fusion FISH assay. We provide the detailed morphologic and immunophenotypic description of the MED15-TFE3 renal cell carcinomas, which frequently demonstrate extensively cystic architecture, similar to multilocular cystic renal neoplasm of low malignant potential, and expressed cathepsin K and melanotic biomarker Melan A. This is the first time to correlate the MED15-TFE3 renal cell carcinoma with specific clinicopathologic features. We also report the first case of the corresponding mesenchymal neoplasm with MED15-TFE3 gene fusion. Additional novel TFE3 gene fusion partners, MATR3 and FUBP1, were identified. Cases with ASPSCR1-TFE3, SFPQ-TFE3, PRCC-TFE3, and NONO-TFE3 gene fusion showed a wide variability in morphologic features, including invasive tubulopapillary pattern simulating collecting duct carcinoma, extensive calcification and ossification, and overlapping and high columnar cells with nuclear grooves mimicking tall cell variant of papillary thyroid carcinoma. Furthermore, we respectively evaluated the ability of TFE3 immunohistochemistry, TFE3 FISH, RT-PCR, and RNA sequencing to subclassify Xp11 translocation-associated cancers. In summary, our study expands the list of TFE3 gene fusion partners and the clinicopathologic features of Xp11 translocation-associated cancers, and highlights the importance of subtyping Xp11 translocation-associated cancers combining morphology, immunohistochemistry, and multiple molecular techniques.

Novel gene fusion of PRCC-MITF defines a new member of MiT family translocation renal cell carcinoma: clinicopathological analysis and detection of the gene fusion by RNA sequencing and FISH.

AIMS: MITF, TFE3, TFEB and TFEC belong to the same microphthalmia-associated transcription factor family (MiT). Two transcription factors in this family have been identified in two unusual types of renal cell carcinoma (RCC): Xp11 translocation RCC harbouring TFE3 gene fusions and t(6;11) RCC harbouring a MALAT1-TFEB gene fusion. The 2016 World Health Organisation classification of renal neoplasia grouped these two neoplasms together under the category of MiT family translocation RCC. RCCs associated with the other two MiT family members, MITF and TFEC, have rarely been reported. Herein, we identify a case of MITF translocation RCC with the novel PRCC-MITF gene fusion by RNA sequencing. METHODS AND RESULTS: Histological examination of the present tumour showed typical features of MiT family translocation RCCs, overlapping with Xp11 translocation RCC and t(6;11) RCC. However, this tumour showed negative results in TFE3 and TFEB immunochemistry and split fluorescence in-situ hybridisation (FISH) assays. The other MiT family members, MITF and TFEC, were tested further immunochemically and also showed negative results. RNA sequencing and reverse transcription-polymerase chain reaction confirmed the presence of a PRCC-MITF gene fusion: a fusion of PRCC exon 5 to MITF exon 4. We then developed FISH assays covering MITF break-apart probes and PRCC-MITF fusion probes to detect the MITF gene rearrangement. CONCLUSIONS: This study both proves the recurring existence of MITF translocation RCC and expands the genotype spectrum of MiT family translocation RCCs.

Quantitative Proteomics Analysis of Ischemia/Reperfusion Injury-Modulated Proteins in Cardiac Microvascular Endothelial Cells and the Protective Role of Tongxinluo.

BACKGROUND: The protection of endothelial cells (ECs) against reperfusion injury has received little attention. In this study, we used Tandem Mass Tag (TMT) labeling proteomics to investigate the modulated proteins in an in vitro model of cardiac microvascular endothelial cells (CMECs) subjected to ischemia/reperfusion (I/R) injury and their alteration by traditional Chinese medicine Tongxinluo (TXL). METHODS: Human CMECs were subjected to 2 h of hypoxia followed by 2 h of reoxygenation with different concentrations of TXL Protein expression profiles of CMECs were determined using tandem mass spectrometry. We evaluated several proteins with altered expression in I/R injury and summarized some reported proteins related to I/R injury. RESULTS: TXL dose-dependently decreased CMEC apoptosis, and the optimal concentration was 800 µg/mL. I/R significantly altered proteins in CMECs, and 30 different proteins were detected between a normal group and a hypoxia and serum deprivation group. In I/R injury, TXL treatment up-regulated 6 types of proteins including acyl-coenzyme A synthetase ACSM2B mitochondrial (ACSM2B), cyclin-dependent kinase inhibitor 1B (CDKN1B), heme oxygenase 1 (HMOX1), transcription factor SOX-17 (SOX17), sequestosome-1 isoform 1 (SQSTM1), and TBC1 domain family member 10B (TBC1D10B). Also, TXL down-regulated 5 proteins including angiopoietin-2 isoform c precursor (ANGPT2), cytochrome c oxidase assembly factor 5 (COA5), connective tissue growth factor precursor (CTGF), cathepsin L1 isoform 2 (CTSL), and eukaryotic elongation factor 2 kinase (LOC101930123). These types of proteins mainly had vital functions, including cell proliferation, stress response, and regulation of metabolic process. CONCLUSIONS: The study presented differential proteins upon I/R injury through a proteomic analysis. TXL modulated the expression of proteins in CMECs and has a protective role in response to I/R.

Xp11.2 translocation renal cell carcinoma with NONO-TFE3 gene fusion: morphology, prognosis, and potential pitfall in detecting TFE3 gene rearrangement.

Xp11 translocation renal cell carcinomas are characterized by several different translocations involving the TFE3 gene. Tumors with different specific gene fusions may have different clinicopathological manifestations. Fewer than 10 renal cell carcinoma cases with NONO-TFE3 have been described. Here we examined eight additional cases of this rare tumor using clinicopathological, immunohistochemical, and molecular analyses. The male-to-female ratio of our study cohort was 1:1, and the median age was 30 years. The most distinctive feature of the tumors was that they exhibited glandular/tubular or papillary architecture that was lined with small-to-medium cuboidal to high columnar cells with indistinct cell borders and an abundantly clear or flocculent eosinophilic cytoplasm. The nuclei were oriented toward the luminal surface and were round and uniform in shape, which resulted in the appearance of secretory endometrioid subnuclear vacuolization. The distinct glandular/tubular or papillary architecture was often accompanied by sheets of epithelial cells that presented a biphasic pattern. Immunohistochemically, all eight cases demonstrated moderate (2+) or strong (3+) positive staining for TFE3, CD10, RCC marker, and PAX-8. None of the tumors were immunoreactive for CK7, Cathepsin K, Melan-A, HMB45, Ksp-cadherin, Vimentin, CA9, 34ßE12 or CD117. NONO-TFE3 fusion transcripts were identified in six cases by RT-PCR. All eight cases showed equivocal split signals with a distance of nearly 2 signal diameters and sometimes had false-negative results. Furthermore, we developed a fluorescence in situ hybridization (FISH) assay to serve as an adjunct diagnostic tool for the detection of the NONO-TFE3 fusion gene and used this method to detect the fusion gene in all eight cases. Long-term follow-up (range, 10-102 months) was available for 7 patients. All 7 patients were alive with no evidence of recurrent disease or disease progression after their initial resection. This report adds to the known data regarding NONO-TFE3 renal cell carcinoma.

EGFR gene mutation in gastrointestinal stromal tumours.

AIMS: The aim of this study was to evaluate the mutation status of epidermal growth factor receptor (EGFR) in gastrointestinal stromal tumours (GISTs) and its association with various clinicopathological variables, as well as to discuss further the effects of EGFR mutations on tumour formation and progression. METHODS AND RESULTS: A well-characterized cohort of 323 GISTs, obtained between 2010 and 2015 from the surgical pathology files of at the Department of Pathology of the Nanjing Jinling Hospital, was screened for mutations in exons 19 and 21 of the EGFR gene. Patient clinical data and clinicopathological features were collected if available in the medical records. Among the 323 primary GISTs, we identified three cases (0.93%) of EGFR mutations; these mutations never occurred together with KIT, PDGFRα, KRAS or BRAF mutations. In two cases, tumour cells exhibited spindle cell morphology and, in one case, epithelioid cell morphology. Additionally, the morphology and immunophenotype of these three cases did not show significant differences compared to common GISTs. The clinical results in summary were that two cases of EGFR-mutated GISTs occurred in females and in the stomach. The mean age of EGFR-mutated cases was 54.33 years, and the follow-up data indicated that these tumours were low risk and exhibited low recurrence. CONCLUSIONS: We first established that GISTs carrying EGFR mutation are relatively benign tumours. Although EGFR mutations were rarely present in GIST, EGFR seems to play a significant role in the development and progression of GIST.

BRM/SMARCA2-negative clear cell renal cell carcinoma is associated with a high percentage of BRM somatic mutations, deletions and promoter methylation.

AIMS: The aim of this study was to investigate potential molecular mechanisms associated with loss of BRM expression in poorly differentiated clear cell renal cell carcinoma (ccRCC). METHODS AND RESULTS: Nineteen previously selected BRM-negative RCC tissues were examined by DNA sequencing, fluorescence in-situ hybridization (FISH) and methylation-specific polymerase chain reaction (PCR) of the BRM gene. BRM mutation was identified in 78.9% (15 of 19) cases, chromosome 9 monosomy or BRM deletion in 43.8% (seven of 16) and BRM promoter region cytosine-phosphate-guanine (CpG) methylation in 42.8% (six of 14). These results indicated that 89.5% (17 of 19) of the cases harboured at least one type of BRM genetic alteration, with two or more types of alteration in 47.4% (nine of 19). Such alterations were found rarely in adjacent non-neoplastic tissues and low-grade areas of composite tumours. CONCLUSIONS: BRM gene mutation, chromosome 9 monosomy or BRM deletion and CpG methylation contribute collectively to the loss of BRM expression in ccRCC. This work focusing on composite tumours indicated that BRM abnormality occurred during tumour progression.

Tongxinluo exerts protective effects via anti-apoptotic and pro-autophagic mechanisms by activating AMPK pathway in infarcted rat hearts.

NEW FINDINGS: What is the central question of this study? In a rat model of acute myocardial infarction (AMI), we investigated the effect of Tongxinluo (TXL) treatment. Does TXL activate autophagy and attenuate apoptosis of cardiomyocytes through the AMPK pathway to facilitate survival of cardiomyocytes and improve cardiac function? What is the main finding and its importance? Major findings are as follows: (i) TXL treatment preserved cardiac function and reduced ventricular remodelling, infarct size and inflammation in rat hearts after AMI; (ii) TXL treatment dramatically increased autophagy and inhibited apoptosis in myocardium; and (iii) the AMPK signalling pathway played a crucial role in mediating the beneficial effects of TXL. Tongxinluo (TXL) has been demonstrated to have a protective role during ischaemia-reperfusion after acute myocardial infarction, but the long-term effects and underlying mechanisms are still unknown. The aim of this study was to investigate whether TXL could have an effect on apoptosis or autophagy of cardiomyocytes through the AMP-activated protein kinase (AMPK) pathway. Male Sprague-Dawley rats (n = 75) were randomly divided to sham, control, TXL (4 mg kg-1  day-1 orally), compound C (i.p. injection of 10 mg kg-1  day-1 ) and TXL + compound C groups. The extent of fibrosis, infarct size and angiogenesis were determined by pathological and histological studies. Four weeks after acute myocardial infarction, TXL treatment significantly increased ejection fraction, promoted angiogenesis in the peri-infarct region and substantially decreased fibrosis and the size of the infarcted area (P < 0.05). Treatment with TXL also increased AMPK/mTOR phosphorylation, upregulated expression of the autophagic protein LC3 and downregulated expression of the apoptotic protein Bax in the infarcted myocardium (P < 0.05). Addition of the AMPK inhibitor, compound C, counteracted these beneficial effects significantly (P < 0.05). The cardioprotective benefits of TXL against myocardial infarction are related to the inhibition of apoptosis and promotion of autophagy in rat hearts after acute myocardial infarction. This effect may occur through the AMPK signalling pathway.

Cyclophosphamide enhances anti-tumor effects of a fibroblast activation protein α-based DNA vaccine in tumor-bearing mice with murine breast carcinoma.

Cyclophosphamide (CY) is a DNA alkylating agent, which is widely used with other chemotherapy drugs in the treatment of various types of cancer. It can be used not only as a chemotherapeutic but also as an immunomodulatory agent to inhibit IL-10 expression and T regulatory cells (Tregs). Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts in the tumor microenvironment. Immunotherapy based on FAPα, as a tumor stromal antigen, typically induces specific immune response targeting the tumor microenvironment. This study evaluated the efficacy of a previously unreported CY combination strategy to enhance the limited anti-tumor effect of a DNA vaccine targeting FAPα. The results suggested CY administration could promote the percentage of splenic CD8+ T cells and decrease the proportion of CD4 + CD25 + Foxp3+ Tregs in spleen. In tumor tissues, levels of immunosuppressive cytokines including IL-10 and CXCL-12 were also reduced. Meanwhile, the CY combination did not impair the FAPα-specific immunity induced by the DNA vaccine and further reduced tumor stromal factors. Most importantly, FAP-vaccinated mice also treated with CY chemotherapy showed a marked suppression of tumor growth (inhibition ratio =80%) and a prolongation of survival time. Thus, the combination of FAPα immunotherapy and chemotherapy with CY offers new insights into improving cancer therapies.

EWSR1 rearrangement is present in a subset of myoepithelial tumors of salivary glands with variable morphology and does not correlate with clinical behavior.

To investigate that whether myoepithelial tumors of salivary glands (MTs) with EWSR1 rearrangement display distinctive morphological characteristics and whether EWSR1 detection aids to distinguish malignant myoepithelial tumors (MMTs) from benign myoepithelial tumors (BMTs) of salivary glands. We examined 37 cases of MTs, including 24 BMTs, 13 MMTs, by histological, immunohistochemical, and molecular analysis. All of 37 cases were immunoreactive for CKpan, and at least one myoepithelial marker. 26 of 37 cases of MTs were available to be analyzed for EWSR1 rearrangement, with the result that EWSR1 gene break was detected in 4 cases of 15 BMTs, and 4 cases of 11 MMTs. In addition, the 8 EWSR1-rearranged cases displayed not exactly similar morphological features, covering 4 clear-cell cases, 1 plasmacytoid-cell case, 1 spindle-cell case, 1 epithelioid-cell case, and 1 chordoid-cell case. Our study proposed that EWSR1 rearrangement was present in a subset of MTs, with variable morphological features. Moreover, the presence of EWSR1 rearrangement could not be a forceful evidence to distinguish MMTs from BBTs.

Globular Adiponectin Inhibits the Apoptosis of Mesenchymal Stem Cells Induced by Hypoxia and Serum Deprivation via the AdipoR1-Mediated Pathway.

BACKGROUND/AIMS: Poor viability of transplanted mesenchymal stem cells (MSCs) within the ischemic heart limits their therapeutic potential for cardiac repair. Globular adiponectin (gAPN) exerts anti-apoptotic effects on several types of stem cells. Herein, we investigated the effect of gAPN on the MSCs against apoptosis induced by hypoxia and serum deprivation (H/SD). METHODS: MSCs exposed to H/SD conditions were treated with different concentrations of gAPN. To identify the main type of receptor, MSCs were transfected with siRNA targeting adiponectin receptor 1 or 2 (AdipoR1 or AdipoR2). To elucidate the downstream pathway, MSCs were pre-incubated with AMPK inhibitor Compound C. Apoptosis, caspase-3 activity and mitochondrial membrane potential were evaluated. RESULTS: H/SD-induced MSCs apoptosis and caspase-3 activation were attenuated by gAPN in a concentration-dependent manner. gAPN increased Bcl-2 and decreased Bax expressions. The loss of mitochondrial membrane potential induced by H/SD was also abolished by gAPN. The protective effect of gAPN was significantly attenuated after the knockdown of AdipoR1 rather than AdipoR2. Moreover, Compound C partly suppressed the anti-apoptotic effect of gAPN. CONCLUSIONS: gAPN inhibits H/SD-induced apoptosis in MSCs via AdipoR1-mediated pathway, possibly linked to the activation of AMPK. gAPN may be a novel survival factor for MSCs in the ischemic engraftment environment.

Anti-tumor effects of DNA vaccine targeting human fibroblast activation protein α by producing specific immune responses and altering tumor microenvironment in the 4T1 murine breast cancer model.

Fibroblast activation protein α (FAPα) is a tumor stromal antigen overexpressed by cancer-associated fibroblasts (CAFs). CAFs are genetically more stable compared with the tumor cells and immunosuppressive components of the tumor microenvironment, rendering them excellent targets for cancer immunotherapy. DNA vaccines are widely applied due to their safety. To specifically destroy CAFs, we constructed and examined the immunogenicity and anti-tumor immune mechanism of a DNA vaccine expressing human FAPα. This vaccine successfully reduced 4T1 tumor growth through producing FAPα-specific cytotoxic T lymphocyte responses which could kill CAFs, and the decrease in FAPα-expressing CAFs resulted in markedly attenuated expression of collagen I and other stromal factors that benefit the tumor progression. Based on these results, a DNA vaccine targeting human FAPα may be an attractive and effective cancer immunotherapy strategy.

Improvement of anti-tumor immunity of fibroblast activation protein α based vaccines by combination with cyclophosphamide in a murine model of breast cancer.

Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts (CAFs), which are the main type of cells in the tumor microenvironment. CAFs exert immunosuppressive activity, which can weaken the effects of cancer immunotherapy and mainly account for poor outcomes with therapeutic vaccines. To better target and destroy CAFs, a FAPα vaccine using a modified vaccinia ankara (MVA) vector was constructed and used with a DNA vaccine reported in our previous work for heterologous prime-boost immunizations in mice. This strategy to generate anti-tumor immunity partly reduced 4T1 tumor growth through producing FAPα-specific cytotoxic T lymphocyte responses in a preventive model, but the effect required improvement. Combining the FAPα-based cancer vaccines (CpVR-FAP/MVA-FAP) with cyclophosphamide (CY), which can be used not only as a chemotherapeutic but also an immunomodulatory agent to promote a shift from immunosuppression to immunopotentiation, resulted in markedly enhanced tumor growth inhibition compared with the CpVR-FAP/MVA-FAP group. This strategy achieved synergistic effects in a therapeutic model by improving the tumor inhibition rate by 2.5-fold (90.2%), significantly enhancing cellular immunity and prolonging the survival of 4T1 tumor-bearing mice by 35% compared with the PBS group. Furthermore, CAFs, stromal factors and immunosuppressive factors such as IL-10 and Tregs were also markedly decreased by the CY combination. These results indicated that FAPα-targeted MVA boosting in combination with CY is an effective approach to improving specific anti-tumor immune responses through overcoming immunosuppression. This study may offer important advances in research on clinical cancer immunotherapies by modulating immunosuppressive factors.

Xp11 neoplasm with melanocytic differentiation of the prostate harbouring the novel NONO-TFE3 gene fusion: report of a unique case expanding the gene fusion spectrum.

Recently, an increasing number of TFE3 rearrangement-associated tumours have been reported, such as TFE3 rearrangement-associated perivascular epithelioid cell tumours (PEComas), melanotic Xp11 translocation renal cancers and melanotic Xp11 neoplasms. We have suggested that these tumours belong to a single clinicopathological spectrum. 'Xp11 neoplasm with melanocytic differentiation' or 'melanotic Xp11 neoplasm' have been proposed to designate this unique neoplasm. Herein, we describe the first case of an Xp11 neoplasm with melanocytic differentiation to be described in the prostate, bearing the novel NONO-TFE3 gene fusion. This study both adds to the spectrum regarding melanotic Xp11 neoplasms and expands its gene fusion spectrum. Moreover, we discuss the relationship of these rare tumours to neoplasms such as conventional PEComas, alveolar soft part sarcomas, malignant melanomas, clear cell sarcomas and Xp11 translocation renal cancers.

[Diagnostic value of immunohistochemistry and FISH for chromosome 12p in type â ¡ testicular germ cell tumors].

OBJECTIVE: To study the pathological morphology, immunohistochemical characteristics, and molecular changes of type Ⅱ testicular germ cell tumors (TGCT) and investigate the possible value of immunohistochemistry and fluorescence in situ hybridization (FISH) in the diagnosis of TGCT. METHODS: We collected for this study 97 cases of TGCT, including 75 cases of seminoma, 17 cases of embryonal carcinoma, 11 cases of yolk sac tumor, 16 cases of mature teratoma, 3 cases of immature teratoma, and 1 case of epidermoid cyst, in which normal testicular tissue was found in 20 and non-TGCT in 6. We detected the expressions of different antibodies in various subtypes of TGCT by immunohistochemistry and determined the rate of chromosome 12p abnormality using FISH. RESULTS: The immunophenotypes varied with different subtypes of TGCT. SALL4 and PLAP exhibited high sensitivity in all histological subtypes. CD117 and OCT4 showed strongly positive expressions in invasive seminoma and germ cell neoplasia in situ (GCNIS) but not in normal seminiferous tubules. GPC3 was significantly expressed in the yolk sac tumor, superior to GATA3 and AFP in both range and intensity. CKpan, OCT4, and CD30 were extensively expressed in embryonal carcinoma, while HCG expressed in choriocarcinoma. The positivity rate of isochromosome 12p and 12p amplification in TGCT was 96.7% (29/30). CONCLUSIONS: The majority of TGCT can be diagnosed by histological observation, but immunohistochemical staining is crucial for more accurate subtypes and valuable for selection of individualized treatment options and evaluation of prognosis. Chromosome 12p abnormality is a specific molecular alteration in type Ⅱ TGCT, which is useful for ruling out other lesions.

Molecular genetics and immunohistochemistry characterization of uncommon and recently described renal cell carcinomas.

Renal cell carcinoma (RCC) compromises multiple types and has been emerging dramatically over the recent several decades. Advances and consensus have been achieved targeting common RCCs, such as clear cell carcinoma, papillary RCC and chromophobe RCC. Nevertheless, little is known on the characteristics of several newly-identified RCCs, including clear cell (tubulo) papillary RCC, Xp11 translocation RCC, t(6;11) RCC, succinate dehydrogenase (SDH)-deficient RCC, acquired cystic disease-associated RCC, hereditary leiomyomatosis RCC syndrome-associated RCC, ALK translocation RCC, thyroid-like follicular RCC, tubulocystic RCC and hybrid oncocytic/chromophobe tumors (HOCT). In current review, we will collect available literature of these newly-described RCCs, analyze their clinical pathologic characteristics, discuss their morphologic and immunohistologic features, and finally summarize their molecular and genetic evidences. We expect this review would be beneficial for the understanding of RCCs, and eventually promote clinical management strategies.