TFEB Research Articles

A rare case of TFEB/6p21/VEGFA-amplified renal cell carcinoma diagnosed by whole-exome sequencing: clinicopathological and genetic feature report and literature review

BackgroundTFEB/6p21/VEGFA-amplified renal cell carcinoma (RCC) is rare and difficult to diagnose, with diverse histological patterns and immunohistochemical and poorly defined molecular genetic characteristics.Case presentationWe report a case of a 63-year-old male admitted in 2017 with complex histomorphology, three morphological features of clear cell, eosinophilic and papillary RCC and resembling areas of glomerular and tubular formation. The immunophenotype also showed a mixture of CD10 and P504s. RCC with a high suspicion of collision tumors was indicated according to the 2014 WHO classification system; no precise diagnosis was possible. The patient was diagnosed at a different hospital with poorly differentiated lung squamous cell carcinoma one year after RCC surgery. We exploited molecular technology advances to retrospectively investigate the patient’s molecular genetic alterations by whole-exome sequencing. The results revealed a 6p21 amplification in VEGFA and TFEB gene acquisition absent in other RCC subtypes. Clear cell, papillary, chromophobe, TFE3-translocation, eosinophilic solid and cystic RCC were excluded. Strong TFEB and Melan-A protein positivity prompted rediagnosis as TFEB/6p21/VEGFA-amplified RCC as per 2022 WHO classification. TMB-L (low tumor mutational load), CCND3 gene acquisition and MRE11A and ATM gene deletion mutations indicated sensitivity to PD-1/PD-L1 inhibitor combinations and the FDA-approved targeted agents Niraparib (Grade C), Olaparib (Grade C), Rucaparib (Grade C) and Talazoparib (Class C). GO (Gene Ontology) and KEGG enrichment analyses revealed major mutations and abnormal CNVs in genes involved in biological processes such as the TGF-β, Hippo, E-cadherin, lysosomal biogenesis and autophagy signaling pathways, biofilm synthesis cell adhesion substance metabolism regulation and others. We compared TFEB/6p21/VEGFA-amplified with TFEB-translocated RCC; significant differences in disease onset age, histological patterns, pathological stages, clinical prognoses, and genetic characteristics were revealed.ConclusionWe clarified the patient’s challenging diagnosis and discussed the clinicopathology, immunophenotype, differential diagnosis, and molecular genetic information regarding TFEB/6p21/VEGFA-amplified RCC via exome analysis and a literature review.

Bortezomib modulated the autophagy-lysosomal pathway in a TFEB-dependent manner in multiple myeloma

ObjectiveTo explore the involvement of TFEB-mediated autophagy-lysosomal mechanisms in multiple myeloma (MM) during bortezomib treatment. MethodsMM cells were exposed to bortezomib or subjected to TFEB knockdown. CCK assay was used to assess the cell proliferation. Western blotting and fluorescent staining were conducted to examine autophagy and lysosomes. The TFEB expression pattern was analyzed, and whole transcriptome sequencing was carried out. Additionally, TFEB target genes were predicted using the GTRD(http://gtrd.biouml.org/) website, and pathway analysis was performed. ResultsBortezomib demonstrated a dose-dependent and time dependent inhibition of cell proliferation. In MM cells treated with bortezomib, LC3B, Beclin-1, TFEB, and Lamp1 exhibited upregulation in a time- and concentration-dependent manner. LysoTracker dye labeling showed an increase in lysosomes in the bortezomib-treated group. Moreover, bortezomib elevated the expression of lysosome-associated factor Lamp1. Bortezomib promoted the nuclear translocation of TFEB, leading to decreased cytoplasmic TFEB and increased nuclear TFEB. TFEB gene silencing reversed bortezomib's inhibitory effect on MM cell lines, significantly reducing autophagosome expression and lysosome numbers. Furthermore, bioinformatic analysis identified the MAPK pathway as a potential downstream target of TFEB. ConclusionBortezomib effectively inhibits MM cell proliferation and induces autophagy, partly through TFEB-mediated mechanisms, with potential involvement of the MAPK pathway.

The Mutant Menage- Mit Family Translocation Renal Cell Carcinoma

Microphthalmia associated transcription factor (MiT) family translocation renal cell carcinoma is neoplasm which enunciates genetic fusions within members of MiT family of transcription factors, especially TFE3 gene and TFEB gene. Neoplasm frequently incriminates the adult population ≤ 35 years of age with an intense female predilection. Tumefaction exhibits a papillary, solid or alveolar pattern of tumour configuration and is composed of clear cells or non-cohesive, pseudo stratified, eosinophilic epithelial cells. Tumour cells are pervaded with voluminous cytoplasm and pleomorphic nuclei of advanced grade. Neoplastic cells are intensely immune reactive to TFE3 or TFEB, PAX8, cathepsin K, CD10, alpha methyacyl CoA racemase (AMACR), vimentin or E-cadherin. Tumour cells appear immune non-reactive to cytokeratin, epithelial membrane antigen (EMA), carbonic anhydrase IX(CAIX), CD45, calretinin and smooth muscle actin(SMA). MiT family translocation renal cell carcinoma requires segregation from neoplasms such as clear cell papillary renal cell carcinoma, clear cell renal cell carcinoma, epithelioid angiomyolipoma, chromophobe renal cell carcinoma or papillary renal cell carcinoma. Neoplasm may be appropriately alleviated by cogent manoeuvers as surgical extermination of the neoplasm.

SIRT1-NRF2-TFEB axis-mediated hepatic lipophagy alleviates the lipid deposition induced by high glucose in yellow catfish Pelteobagrus fulvidraco.

Metabolic stress induces lipophagy, a crucial process in lipid catabolism, which is under the regulation of autophagy involving transcription factor EB (TFEB). However, the precise mechanisms underlying TFEB's control remain enigmatic. In this study, we focused on yellow catfish (Pelteobagrus fulvidraco) as the model to investigate lipophagy activation under high glucose-induced lipid deposition. We hypothesized that lipophagy mediates high glucose-induced lipid deposition and proposed the involvement of the SIRT1-NRF2-TFEB pathway in the activation of lipophagy. We found that there was a functional antioxidative responsive element (ARE) on the tfeb gene promoter; high glucose (HG) increased the nuclear translocation of nuclear factor E2-related factor 2 (NRF2) recruitment to the tfeb promoter; TFEB, whose expression is regulated by NRF2, mediated the HG-induced activation of lipophagy and lipolysis. Moreover, we found that HG increased the silencing information regulator 2 related enzymes 1 (SIRT1) expression, and that the SIRT1 mediates NRF2 translocation to the nucleus, increased TFEB expression and activated autophagy. In the glucose tolerance test, blood glucose increased rapidly and plateaued at 4-h glucose after injection and then declined until 48-h post-injection. Generally speaking, the transcript level and protein expression of SIRT1, NRF2, TFEB, microtubule-associated proteins 1A/1B light chain 3B (LC3B), and autophagy-related 6 (Beclin1) showed similar trend after glucose injection, and trends to increase and plateau at 4-h injection, then decline until 16-h post-injection, and finally increased until 48-h post-injection. These results indicated that the SIRT1-NRF2-TFEB axis-mediated lipophagy may be an adaptive response to glucose injection. Collectively, for the first time, we found that NRF2 was associated directly with TFEB-mediated transcriptional control of hepatic lipophagy, and that lipophagy helps to alleviate the HG-induced lipid deposition via SIRT1-NRF2-TFEB activation in yellow catfish.

Prognosis and Clinicopathological Characters of Adult TFEB-Altered Renal Cell Carcinoma: A Single Center Experience of 18 Cases

IntroductionTFEB-altered renal cell carcinoma (RCC) is a rare entity characterized by the rearrangement of the TFEB gene or TFEB amplified. The therapeutic implications and long-term survival of TFEB-altered RCC remain unclear, especially for metastatic cases. Materials and MethodsThe current study initially enrolled 7604 consecutive RCC patients at our center and a total of 248 patients were selected for FISH and immunohistochemistry (IHC) analysis. Eventually, eighteen TFEB-altered RCC patients were identified. We then reported the clinical, morphological, IHC, and radiological features of these cases. ResultsThe median age at initial diagnosis was 45 years, ranging from 18 years to 66 years. The majority of the TFEB-altered RCC patients were male (61.1%), with localized disease (T1-2N0M0, 77.8%). The median split TFEB fluorescent signal was 24%, ranging from 15%-80%. The morphological characteristics of TFEB-altered RCC were variable, with acinar, papillary, solid, or nest patterns. IHC and magnetic resonance imaging features of TFEB-altered RCC were nonspecific. Nine patients with localized disease received partial nephrectomy and five patients with localized disease received radical nephrectomy. During the median follow-up of 67 months, no signs of recurrence or metastasis were found in these patients. Two patients had distant metastasis and received axitinib plus PD-1 immunotherapy. One of them died at 40-month follow-up and another still alive at 88-month follow-up. ConclusionTFEB-altered RCC is an extremely rare variant, exhibited mixed morphological characteristics. The radiological feature lack specificity, resembling clear cell RCC or papillary RCC. Genetic analyses including FISH analysis is crucial in the diagnosis of TFEB-altered RCC. For localized TFEB-altered RCC, both radical nephrectomy and partial nephrectomy conferred satisfactory prognosis. For metastatic TFEB-altered RCC, immunotherapy-based drug combinations could be a promising treatment strategy.

Regulatory Relationship between the Keap1/Nrf2/ARE Signaling System and Transcriptional Regulators of Lysosomal Biogenesis

Despite the key role of the Keap1/Nrf2/ARE redox-sensitive signaling system in cellular metabolism, little is known about its relationship to lysosome biogenesis. In this paper, a theoretical and experimental analysis of the possibility of such a link has been carried out. By forming a position frequency matrix in the transcription factor genes TFEB and TFE3, the presence of a large number of ARE-like sequences was found in the non-coding regions. In vitro exposure to J774 cells by Keap1/Nrf2/ARE activators (original synthetic monophenol TS-13 and tert-butylhydroquinone as comparison compound) results in dose-dependent induction of Tfe3 and Tfeb genes, accompanied by a gradual increase in the lysosome number and autosomal-lysosomal fusion intensity. Thus, it can be assumed that the proteins controlling the ARE-dependent genes are able to influence lysosome biogenesis.

Activation of PPARα Exhibits Therapeutic Efficacy in a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal inherited neurodegenerative disease of children that occurs because of defective function of the lysosomal membrane glycoprotein CLN3. JNCL features glial activation and accumulation of autofluorescent storage material containing subunit c of mitochondrial ATP synthase (SCMAS), ultimately resulting into neuronal loss. Until now, no effective therapy is available for JNCL. This study underlines the possible therapeutic importance of gemfibrozil, an activator of peroxisome proliferator-activated receptor α (PPARα) and a lipid-lowering drug approved by the Food and Drug Administration in an animal model of JNCL. Oral gemfibrozil treatment reduced microglial and astroglial activation, attenuated neuroinflammation, restored the level of transcription factor EB (TFEB; the master regulator of lysosomal biogenesis), and decreased the accumulation of storage material SCMAS in somatosensory barrel field (SBF) cortex of Cln3Δex7/8 (Cln3ΔJNCL) mice of both sexes. Accordingly, gemfibrozil treatment also improved locomotor activities of Cln3ΔJNCL mice. While investigating the mechanism, we found marked loss of PPARα in the SBF cortex of Cln3ΔJNCL mice, which increased after gemfibrozil treatment. Oral gemfibrozil also stimulated the recruitment of PPARα to the Tfeb gene promoter in vivo in the SBF cortex of Cln3ΔJNCL mice, indicating increased transcription of Tfeb in the CNS by gemfibrozil treatment via PPARα. Moreover, disease pathologies aggravated in Cln3ΔJNCL mice lacking PPARα (Cln3ΔJNCLΔPPARα) and gemfibrozil remained unable to decrease SCMAS accumulation, reduce glial activation, and improve locomotor performance of Cln3ΔJNCLΔPPARα mice. These results suggest that activation of PPARα may be beneficial for JNCL and that gemfibrozil may be repurposed for the treatment of this incurable disease.SIGNIFICANCE STATEMENT Despite intense investigations, no effective therapy is available for JNCL, an incurable inherited lysosomal storage disorder. Here, we delineate that oral administration of gemfibrozil, a lipid-lowering drug, decreases glial inflammation, normalizes and/or upregulates TFEB, and reduces accumulation of autofluorescent storage material in SBF cortex to improve locomotor activities in Cln3Δex7/8 (Cln3ΔJNCL) mice. Aggravation of disease pathology in Cln3ΔJNCL mice lacking PPARα (Cln3ΔJNCLΔPPARα) and inability of gemfibrozil to decrease SCMAS accumulation, reduce glial activation, and improve locomotor performance of Cln3ΔJNCLΔPPARα mice delineates an important role of PPARα in this process. These studies highlight a new property of gemfibrozil and indicate its possible therapeutic use in JNCL patients.

TFEB Rearranged Renal Cell Carcinoma: Pathological and Molecular Characterization of 10 Cases, with Novel Clinical Implications: A Single Center 10-Year Experience.

To report our experience with the cases of TFEB rearranged RCC, with particular attention to the clinicopathological, immunohistochemical and molecular features of these tumors and to their predictive markers of response to therapy. We have retrieved the archives of 9749 renal cell carcinomas in the Institute of Urology, Peking University and found 96 rearranged RCCs between 2013 and 2022. Among these renal tumors, ten cases meet the morphologic, immunohistochemical and FISH characterization for TFEB rearranged RCC. The 10 patients' mean and median age is 34.9 and 34 years, respectively (range 23-55 years old), and the male to female ratio is 1:1.5. Macroscopically, these tumors generally have a round shape and clear boundary. They present with variegated, grayish yellow and grayish brown cut surface. The average maximum diameter of the tumor is 8.5 cm and the median 7.7 (ranged from 3.4 to 16) cm. Microscopically, the tumor is surrounded by a thick local discontinuous pseudocapsule. All tumors exhibit two types of cells: voluminous, clear and eosinophilic cytoplasm cells arranged in solid sheet, tubular growth pattern with local cystic changes, and papillary, pseudopapillary and compact nested structures are also seen in a few cases. Non-neoplastic renal tubules are entrapped in the tumor. A biphasic "rosette-like" pattern, psammomatous calcifications, cytoplasmic vacuolization, multinucleated giant cells and rhabdomyoid phenotype can be observed in some tumors. A few tumors may be accompanied by significant pigmentation or hemorrhage and necrosis. The nucleoli are equivalent to the WHO/ISUP grades 2-4. All tumors are moderately to strongly positive for Melan-A, TFEB, Vimentin and SDHB, and negative for CK7, CAIX, CD117, EMA, SMA, Desmin and Actin. CK20 and CK8/18 are weakly positive. In addition, AE1/AE3, P504s, HMB45 and CD10 are weakly moderately positive. TFE3 is moderately expressed in half of the cases. PAX8 can be negative, weakly positive or moderately-strongly positive. The therapy predictive marker for PD-L1 (SP263) is moderately to strongly positive membranous staining in all cases. All ten tumors demonstrate a medium frequency of split TFEB fluorescent signals ranging from 30 to 50% (mean 38%). In two tumors, the coincidence of the TFEB gene copy number gains are observed (3-5 fluorescent signals per neoplastic nuclei). Follow-up is available for all patients, ranging from 4 to 108 months (mean 44.8 and median 43.4 months). All patients are alive, without tumor recurrences or metastases. We described a group of TFEB rearranged RCC identified retrospectively in a large comprehensive Grade III hospital in China. The incidence rate was about 10.4% of rearranged RCCs and 0.1% of all the RCCs that were received in our lab during the ten-year period. The gross morphology, histological features, and immunohistochemistry of TFEB rearranged RCC overlapped with other types of RCC such as TFE3 rearranged RCC, eosinophilic cystic solid RCC, or epithelioid angiomyolipoma, making the differential diagnosis challenging. The diagnosis was based on TFEB fluorescence in situ hybridization. At present, most of the cases reported in the literature have an indolent clinical behavior, and only a small number of reported cases are aggressive. For this small subset of aggressive cases, it is not clear how to plan treatment strategies, or which predictive markers could be used to assess upfront responses to therapies. Between the possible options, immunotherapy currently seems a promising strategy, worthy of further exploration. In conclusion, we described a group of TFEB rearranged RCC identified in a large, comprehensive Grade III hospital in China, in the last 10 years.

TFEB coordinates autophagy and pyroptosis as hepatotoxicity responses to ZnO nanoparticles

Zinc oxide nanoparticles (ZnO NPs) have drawn serious concerns about their biotoxicity due to their extensive applications in biological medicine, clinical therapeutic, daily chemical production, food and agricultural additives. In our present study, we clarified hepatotoxic mechanism of ZnO NPs through investigating the crosstalk between autophagy and pyroptosis, a remaining enigma in hepatocyte stimulated by ZnO NPs. Based on the effects of autophagy intervention by Rapamycin (Rap) and 3-Methyladenine (3-MA), and the observation of pyroptosis morphology and related indexes, the autophagy and pyroptosis simultaneously initiated by ZnO NPs were interrelated and the autophagy characterized by autophagosome production and increased expression of autophagy proteins was identified as a protective response of ZnO NPs against pyroptosis. According to the analysis of protein expression and fluorescence localization, the NLRP3 inflammasome assemble and the classical Caspase-1/GSDMD-dependent pyroptosis induced by ZnO NPs was modulated by autophagy. In this process, the adjustment of TFEB expression and nuclear translocation by gene knockout and gene overexpression, further altered the tendency of ZnO NPs-induced pyroptosis via the regulation of autophagy and lysosomal biogenesis. The knockout of TFEB gene exacerbated the pyroptosis via autophagy elimination and lysosome inhibition. While the alleviation of NLRP3 generation and pyroptosis activation was observed after treatment of TFEB gene overexpression. Additionally, the siRNA interference confirmed that TRAF-6 was involved in the TFEB-mediated global regulation of autophagy-lysosome-pyroptosis in response to ZnO NPs. Accordingly, pyroptosis induced by ZnO NPs in hepatocyte could be significantly avoided by TFEB-regulated autophagy and lysosome, further providing new insights for the risk assessment and therapeutic strategy.

TFE3 and TFEB-rearranged renal cell carcinomas: an immunohistochemical panel to differentiate from common renal cell neoplasms.

TFE3/TFEB-rearranged renal cell carcinomas are characterized by translocations involving TFE3 and TFEB genes. Despite the initial description of typical morphology, their histological spectrum is wide, mimicking common subtypes of renal cell tumors. Thus, the diagnosis is challenging requiring the demonstration of the gene rearrangement, usually by FISH. However, this technique is limited in most laboratories and immunohistochemical TFE3/TFEB analysis is inconsistent. We sought to identify a useful immunohistochemical panel using the most common available markers to recognize those tumors. We performed an immunohistochemical panel comparing 27 TFE3-rearranged and 10 TFEB-rearranged renal cell carcinomas to the most common renal cell tumors (150 clear cell, 100 papillary, 50 chromophobe renal cell carcinomas, 18 clear cell papillary renal cell tumors, and 50 oncocytomas). When dealing with neoplasms characterized by cells with clear cytoplasm, CA9 is a helpful marker to exclude clear cell renal cell carcinoma. GATA3, AMACR, and CK7 are useful to rule out clear cell papillary renal cell tumor. CK7 is negative in TFE3/TFEB-rearranged renal cell carcinoma and positive in papillary renal cell carcinoma, being therefore useful in this setting. Parvalbumin and CK7/S100A1 respectively are of paramount importance when TFE3/TFEB-rearranged renal cell carcinoma resembles oncocytoma and chromophobe renal cell carcinoma. Moreover, in TFEB-rearranged renal cell carcinoma, cathepsin K and melanogenesis markers are constantly positive, whereas TFE3-rearranged renal cell carcinoma stains for cathepsin K in roughly half of the cases, HMB45 in 8% and Melan-A in 22%. In conclusion, since TFE3/TFEB-rearranged renal cell carcinoma may mimic several histotypes, an immunohistochemical panel to differentiate them from common renal cell tumors should include cathepsin K, CA9, CK7, and parvalbumin.

Aberrant SPOP-CHAF1A ubiquitination axis triggers tumor autophagy that endows a therapeutical vulnerability in diffuse large B cell lymphoma

PurposeAberrant epigenetic changes, like DNA methylation, histone modifications, or ubiquitination, could trigger metabolic disorders in human cancer cells. This study planed to uncover the biological roles of epigenetic SPOP/CHAF1A axis in modulating tumor autophagy during Diffuse large B-cell lymphoma (DLBCL) tumorigenesis.Materials and methodsThe Immunohistochemistry (IHC) was performed to assess the CHAF1A expressions. The expression data of CHAF1A was derived from The Cancer Genome Atlas (TCGA), GSE32918 and GSE83632 datasets. Bioinformatic assays contain differential analysis, functional enrichment analysis and Kaplan–Meier survival curve analysis. The colony generation assay, Transwell assay and CCK-8 assays were conducted for the in vitro assays. The in vivo ubiquitination assays were used to assess regulations of SPOP on CHAF1A. The Chromatin immunoprecipitation (ChIP) assays were used to uncover epigenetic regulations of CHAF1A on TFEB. The relevant DLBCL cells were subcutaneously injected to SCID beige mice to establish the xenograft models.ResultsBioinformatic results revealed that CHAF1A expressed highly in DLBCL that were validated in patients samples. Patients with high CHAF1A suffered from inferior prognosis with shorter survival months relative to those with low CHAF1A. High CHAF1A enhanced DLBCL aggressiveness, including cell proliferation, migration and in vivo growth. Mechanistically, E3 ubiquitin ligase SPOP binds to and induces the degradative ubiquitination of CHAF1A via recognizing a consensus SPOP-binding motif in CHAF1A. SPOP is down-regulated in DLBCL and habours two DLBCL-associated mutations. Deficient SPOP leads to accumulated CHAF1A proteins that promote malignant features of DLBCL. Subsequently, ChIP-qPCR assay revealed that CHAF1A directly binds to TFEB promoters to activate the expressions. High CHAF1A could enhance the transcriptional activity of TFEB and downstream genes. The SPOP/CHAF1A axis modulates TFEB-dependent transactivation to regulate the lysosomal biogenesis and autophagy. The in vivo models suggested that TFEB inhibition is effective to suppress growth of SPOP-deficient DLBCLs.ConclusionsCHAF1A is aberrantly elevated in SPOP-deficient DLBCL. The in‐depth mechanism understanding of SPOP/CHAF1A/TFEB axis endows novel targets for DLBCL treatment.

Immunohistochemistry for the diagnosis of renal epithelial neoplasms.

Despite the increasing number of newly identified renal neoplasms, the diagnosis of renal cell carcinoma (RCC) can usually be reached with careful histologic examination and a limited immunohistochemical (IHC) panel. Clear cell, papillary, chromophobe RCC and oncocytoma account for more than 90% of renal neoplasia in adults, and sophisticated ancillary tools are usually unnecessary. Renal tumors with entity-defining genetic alterations may ultimately require molecular confirmation via cytogenetics or sequencing technologies, such as RCC with TFE3, TFEB, or ALK gene rearrangements, or TFEB amplified RCC. In fumarate hydratase-deficient and succinate dehydrogenase-deficient RCC, highly specific IHC markers can strongly suggest the diagnosis. In the metastatic setting, PAX8 and carbonic anhydrase 9 are among the most helpful markers for confirming RCC and clear cell type, respectively; however, caution should be exercised in the absence of a current or historical renal mass. In diagnostically challenging cases, such as renal eosinophilic tumors with low-grade nuclear features, or infiltrative high-grade tumors, careful examination coupled with a judicious panel of IHC markers usually resolves the diagnosis. This review offers concise algorithms for diagnosis of kidney neoplasia with the latest recognized, provisional, and emerging entities to daily pathology practice.

Spectrum of biphasic renal cell carcinomas with hyalinized stroma and psammoma bodies associated and not associated with NF2 alteration

A unique subtype of biphasic renal cell carcinoma (RCC) was recently described and termed biphasic hyalinizing psammomatous RCC (BHPRCC). This tumor shows a dual population of larger cells and small cells surrounding basement membrane-like materials and invariably has papillary features, hyalinized stroma, and psammoma calcifications. The biphasic pattern in BHPRCC may resemble that of RCC associated with TFEB gene fusion or t (6;11) RCC. However, all reported BHPRCCs had no TFEB alterations and all were associated with neurofibromin 2 (NF2) mutations. Herein, we present three biphasic RCCs encompassing the reported BHPRCC morphologies. One RCC showed solid, nested, papillary, and tubular growths, with biphasic pattern of larger cells surrounding clusters of smaller cells arranged around basement membrane-like materials, and harbored NF2 mutation consistent with BHPRCC. This patient developed bone metastasis 59 months after surgery. The two other biphasic RCCs showed morphologic overlap to BHPRCC, but in addition had other features not seen in BHPRCC, such as lack of papillary pattern, having large tubules containing mucinous to collagenous spherules (mucicarmine and collagen IV positive) bordered by a single layer of small cells with occasional central targetoidpsammoma bodies, and with widespread nuclear grooves. Interestingly, these two tumors also did not show alterations in NF2 or TFEB including translocation or amplification. In conclusion, we report another example of the novel BHPRCC that had metastasized and two biphasic RCCs not associated with NF2 or TFEB alterations; the latter two shared additional distinct morphological features and may represent a unique biphasic RCC distinct from the novel BHPRCC.

Renal cell tumor with sex-cord/gonadoblastoma-like features: analysis of 6 cases.

Renal tumors are one of the most diverse groups of tumors in pathology. Many emerging and important entities have been described recently. Here, we describe a series of renal tumors occurring in adult patients, with distinct histologic features, and with a striking resemblance to gonadal sex cord-stromal tumors. Patients were three males and three females aged 39-82years; tumor size ranged from 0.9 to 3.6cm. Five tumors were organ-confined, while one case had a focal perinephric invasion. No aggressive behavior was noted. Microscopically, all the tumors were composed of loose or compact tubular structures with elongated or angulated shapes. The tumor cells were cylindrical or cuboidal, with pale eosinophilic cytoplasm, irregular nuclear membranes, and ISUP/WHO grade 2-3 nuclei. The stroma showed focal or prominent collagen deposition with prominent basement membrane-like material. In all cases, the tumor cells were positive for PAX8, CD10, and vimentin and retained positivity for FH and SDHB. Cathepsin K and AMACR were variably positive. Tumors were negative for HMB45, Melan A, TFE3, SF1, inhibin, calretinin, ER, PR, CD117, OCT3/4, SALL4, ALK, and WT1. Molecular studies showed no abnormalities in TFEB, TFE3, or FH genes. In 3/4 tested cases, mutation of the NF2 gene was present. In all the tested male cases, loss of the Y chromosome was found. In the relatively short follow-up, these tumors appear to have indolent behavior. This study expands the clinicopathologic diversity of renal cell tumors by describing a series of potentially novel tumors morphologically resembling gonadal sex-stromal tumors, with negativity for sex cord-stromal markers. Potential relationship to recently described biphasic hyalinizing psammomatous renal cell carcinoma is discussed.

Diagnostic utility of one-stop fusion gene panel to detect TFE3/TFEB gene rearrangement and amplification in renal cell carcinomas

MiT family translocation renal cell carcinoma (MiT-RCC) harbors translocations involving the TFE3 or TFEB genes. RCC with TFEB amplification is also identified and is associated with a more aggressive clinical course. Accurate diagnosis of MiT-RCC is crucial for patient management. In this study, we evaluated the performance of the Archer FusionPlex assay for detection of MiT-RCC with TFE3 or TFEB translocations and TFEB amplifications. RNA was extracted from 49 RCC FFPE tissue samples with known TFE3/TFEB status (26 TFE3 FISH positive, 12 TFEB FISH positive, 4 TFEB amplified (1 case both split and amplified), and 8 FISH negative) using the Covaris extraction kit. Target enriched cDNA libraries were prepared using the Archer FusionPlex kit and sequenced on the Illumina NextSeq 550. We demonstrate that the age of the specimen, quality of RNA, and sequencing metrics are important for fusion detection. Fusions were identified in 20 of 21 cases less than 2 years old, and TFE3/TFEB rearrangements were detected in all cases with Fusion QC ≥ 100. The assay identified intrachromosomal inversions in two cases (TFE3-RBM10 and NONO-TFE3), usually difficult to identify by FISH assays. TFEB mRNA expression and the TFEB/TFE3 mRNA expression ratio were significantly higher in RCCs with TFEB fusion and TFEB gene amplification compared to tumors without TFEB fusion or amplification. A cutoff TFEB/TFE3 ratio of 0.5 resulted in 97.3% concordance to FISH results with no false negatives. Our study demonstrates that the FusionPlex assay successfully identifies TFE3 and TFEB fusions including intrachromosomal inversions. Age of the specimen and certain sequencing metrics are important for successful fusion detection. Furthermore, mRNA expression levels may be used for predicting cases harboring TFEB amplification, thereby streamlining testing. This assay enables accurate molecular detection of multiple subtypes of MiT-RCCs in a convenient workflow.

Dysregulation of miR-342-3p in plasma exosomes derived from convalescent AMI patients and its consequences on cardiac repair

Plasma exosomes derived from healthy people have been shown to be beneficial in terms of protecting against ischemia-reperfusion injury or acute myocardial infarction (AMI). However, a pathological condition may severely affect the constitution and biological activity of exosomes. In our study, we isolated plasma exosomes from healthy volunteers and convalescent AMI patients (3–7 d after onset). Compared to exosomes from healthy controls (Nor-Exo), exosomes from convalescent AMI patients (AMI-Exo) exhibited an impaired ability to repair damaged cardiomyocytes both in vitro and in vivo. miRNA sequencing and PCR analysis indicated that miR-342-3p was significantly downregulated in AMI-Exo. Moreover, miR-342-3p alleviated H2O2-induced injury and reduced apoptosis and autophagy in H9c2 cardiomyocytes, while in vivo restoration of miR-342-3p expression enhanced the reparative function of AMI-Exo. Further mechanistic studies revealed that the SOX6 and TFEB genes were two direct and functional targets of miR-342-3p. Taken together, during the early convalescent phase after AMI, dysregulated miR-342-3p in plasma exosomes might be responsible for their impaired cardioprotective potential. miR-342-3p contributed to exosome-mediated heart repair by inhibiting cardiomyocyte apoptosis and autophagy through targeting SOX6 and TFEB, respectively. Our work provided novel insights on the role of plasma exosomes in the natural process of cardiac repair after AMI and suggestions for therapy development.

TRIM63 is a sensitive and specific biomarker for MiT family aberration-associated renal cell carcinoma.

Microphthalmia-associated transcription factor (MiT) family aberration-associated renal cell carcinoma (MiTF-RCC) is a subtype of renal cell carcinoma harboring recurrent chromosomal rearrangements involving TFE3 or TFEB genes. MiTF-RCC is morphologically diverse, can histologically resemble common RCC subtypes like clear cell RCC and papillary RCC, and often poses a diagnostic challenge in genitourinary clinical and pathology practice. To characterize the MiTF-RCC at the molecular level and identify biomarker signatures associated with MiTF-RCC, we analyzed RNAseq data from MiTF-RCC, other RCC subtypes and benign kidney. Upon identifying TRIM63 as a cancer-specific biomarker in MiTF-RCC, we evaluated its expression independently by RNA in situ hybridization (RNA-ISH) in whole tissue sections from 177 RCC cases. We specifically included 31 cytogenetically confirmed MiTF-RCC cases and 70 RCC cases suspicious for MiTF-RCC in terms of clinical and morphological features, to evaluate and compare TRIM63 RNA-ISH results with theresults from TFE3/TFEB fluorescence in situ hybridization (FISH), which is the current clinical standard. We confirmed that TRIM63 mRNA was highly expressed in all classes of MiTF-RCC compared to other renal tumor categories, where it was mostly absent to low. While the TRIM63 RNA-ISH and TFE3/TFEB FISH results were largely concordant, importantly, TRIM63 RNA-ISH was strongly positive in TFE3 FISH false-negative cases with RBM10-TFE3 inversion. In conclusion, TRIM63 can serve as a diagnostic marker to distinguish MiTF-RCC from other renal tumor subtypes with overlapping morphology. We suggest a combination of TFE3/TFEB FISH and TRIM63 RNA-ISH assays to improve the accuracy and efficiency of MiTF-RCC diagnosis. Accurate diagnosis of MiTF-RCC and other RCC subtypes would enable effective targeted therapy and avoid poor therapeutic response due to tumor misclassification.

Clinicopathological analysis of clear cell renal cell carcinoma with hemangioblastoma component

Objective: To investigate the clinicopathological features, differential diagnosis and molecular characteristics of clear cell renal cell carcinoma (ccRCC) with hemangioblastoma component (ccRCC-HBc). Methods: Two ccRCC-HBc cases diagnosed at Fujian Provincial Hospital in September 2015 and March 2016, respectively, were included. Their morphological, immunohistochemical and molecular features were analyzed, including fluorescence in situ hybridization (FISH) detection of TFE3, TFEB and VHL genes. Related literature was reviewed to reveal the characteristics of this tumor. Results: The two cases occurred in 2 women, aged 33 and 66 years, respectively. The maximum diameters of the tumors were 4.0 cm and 8.5 cm, respectively. Histologically, the ccRCC component, representing approximate 10%-20% of the neoplasm, while the tumor cells arranged in flaky, nested, and solid distribution. The tumor cells had conspicuous nucleoli, with rich thin-wall capillary network in the stroma. The hemangioblastoma-like component, representing approximate 60%-70% of the neoplasm, showed a rich capillary network of single-layered flat endothelial cells enclosing stromal cells. The latter cell type showed a pale or eosinophilic cytoplasm exhibiting occasional lipid droplets. Rare cell nuclei appeared enlarged, pleomorphic, or bizarre. The two components were intermingled with each other. Immunohistochemically, the tumor cells were positive for PAX8, CKpan, EMA, vimentin, CD10, RCC, CAⅨ, and P504s in ccRCC area; in another area, the tumor cells were positive for α-inhibin, CD34 and vimentin, while CD10 were weakly positive. Neither TFE3 or TFEB gene split signal was detected in the 2 cases (0/2), nor was VHL gene mutation in case 2 (0/1). Conclusion: ccRCC-HBc is an extremely rare entity of ccRCC. The diagnosis is mainly based on clinical and pathological characteristics, as well as immunohistochemistry. Molecular pathology is helpful for its differential diagnosis. The primary approach of treating ccRCC-HBc is complete surgical excision and chemotherapy. The targeted treatment is helpful if possible.

Review of TFEB-amplified renal cell carcinoma with focus on clinical and pathobiological aspects.

The disease entity of TFEB-amplified renal cell carcinoma (RCC) has been recently established. In this article, we review such cases. Clinically, the age of patients ranged from 28 to 83 years with a mean age of 62.8 years. The size of the tumor ranged from 1.9 to 19.5 cm with a mean size of 8.7 cm. The tumor demonstrated a variety of architectural patterns such as solid, alveolar, papillary, pseudopapillary, nested or tubular. The International Society of Urological Pathology (ISUP) grade usually corresponds to grade 3 or 4. Cytomorphology shows eosinophilic, clear, amphophilic or even oncocytic cytoplasm. Necrosis can be frequently observed. Neoplastic cells with TFEB-amplified RCC show diffuse or patchy positivity for TFEB. Fluorescence in situ hybridization frequently show the amplification of more than 10 or 20 copies of the TFEB gene. Most TFEB-amplified RCCs behave in an aggressive fashion. Metastasis frequently occurs. In conclusion, this tumor seems to be characterized by occurrence in older patients, frequent necrosis, papillary/pseudopapillary growth pattern, high-grade nuclear grade, TFEB gene amplification, and aggressive clinical behavior. In order to clarify whether this tumor is a distinct entity from previously described renal tumors or not, a further examination in a large scale study will be required in the future.

Quercetin alleviates Cadmium-induced autophagy inhibition via TFEB-dependent lysosomal restoration in primary proximal tubular cells

Autophagy dysregulation plays a pivotal role in cadmium (Cd)-induced nephrotoxicity. Quercetin (Qu), a flavonoid antioxidant with autophagy-enhancing effect, has protective effect on Cd-induced toxicity, but whether it can prevent Cd-induced nephrotoxicity via restoration of autophagy remains unknown. Here, primary rat proximal tubular (rPT) cells were exposed to Cd and/or Qu in vitro to clarify this issue. Data first showed that Cd-impaired autophagic flux was markedly alleviated by Qu, including decreased levels of autophagy marker proteins and recovery of autophagosome-lysosome fusion targeted for lysosomes. Meanwhile, Cd-induced lysosomal alkalization due to v-ATPases inhibition was prominently recovered by Qu. Accordingly, Qu enhanced Cd-diminished lysosomal degradation capacity and lysosome-related gene transcription levels. Notably, Qu improved Cd-inhibited TFEB nuclear translocation and its gene transcription level. Furthermore, data showed that the restoration of Cd-impaired autophagy-lysosome pathway and resultant alleviation of cytotoxicity by Qu are TFEB-dependent using TFEB gene silencing and overexpression technologies. In summary, these data provide novel evidences that the protective action of Qu against Cd-induced autophagy inhibition is attributed to its restoration of lysosomal dysfunction, which is dependent on TFEB.