TFEB Research Articles

Contemporary Characterization and Recategorization of Adult Unclassified Renal Cell Carcinoma.

Our recent study of early-onset unclassified eosinophilic renal cell carcinoma (RCC) demonstrated that two third of cases could be reclassified by performing a limited number of immunohistochemistry stains. Following the same approach, we aimed to investigate what proportion of adult unclassified RCC could be reclassified. We identified 79 cases. The mean age at presentation was 58 years (range, 29 to 84 y). Tumors were grouped based on their predominant morphologic features as oncocytic (n=23); papillary (n=22); clear cell (n=22); mucinous tubular and spindle cell (MTSC; n=5); rhabdoid (n=4); or lacking a dominant pattern (n=3). By reviewing the morphologic features and performing ancillary studies, we were able to reclassify 10 cases (13%). Four cases were positive for CK20 and showed morphologic features consistent with eosinophilic solid and cystic RCC. Four cases were reclassified as MTSC based on VSTM2A expression by RNA in situ hybridization. One case was negative for SDHB and reclassified as succinate dehydrogenase-deficient RCC. None of the cases showed loss of expression of fumarate hydratase. One case was diffusely positive for CK7 and negative for CD117 and reclassified as a low-grade oncocytic tumor. Four cases were positive for both cathepsin-K and TFE3 by immunohistochemistry, although fluorescence in situ hybridization failed to identify rearrangement in either TFE3 or TFEB genes. Of the tumors that remained unclassified, those with oncocytic features were less likely to be a high grade (odds ratio [OR]=0.22, P=0.013) or advanced stage (OR=0.19, P=0.039) and were more common in women (OR=3.4, P=0.05) compared with those without oncocytic features. Tumors with rhabdoid morphology were associated with advanced stage (relative risk=3.6, P=0.009), while tumors with clear cell or papillary features had a wide range of grades and stages at presentation. In summary, the most frequent reclassified entity is eosinophilic solid and cystic RCC. Investigation of expression of succinate dehydrogenase or fumarate hydratase in individuals older than 35 years with unclassifiable tumors is low yield in the absence of specific morphologic features. A subset of MTSC without well-developed morphologic features can be reclassified by using RNA-ISH for VSTM2A. Recognition of more-recently described RCC subtypes allows for their distinction from the unclassified subtype and improves the prognostic information provided.

TFEB rearranged renal cell carcinoma. A clinicopathologic and molecular study of 13 cases. Tumors harboring MALAT1-TFEB, ACTB-TFEB, and the novel NEAT1-TFEB translocations constantly express PDL1.

Renal cell carcinomas with t(6;11) chromosome translocation has been classically characterized by the rearrangement of the TFEB gene, located on chromosome 6, and MALAT1 gene, located on chromosome 11. Recently, a few other genes have been described as fusion partners in TFEB rearranged renal cell carcinomas. Although most of TFEB rearranged renal cell carcinomas have an indolent behavior, in the rare cases of advanced metastatic disease targeted therapy and predictive markers remain lacking. In the present study, we collected 13 TFEB rearranged renal cell carcinomas, confirmed by FISH, analyzing their morphology and exploring the novel gene partners. Looking for predictive markers, we have also performed PDL1 immunohistochemical analysis by using four different assays (E1L3N, 22C3, SP142, and SP263). MALAT1 gene rearrangement has been found in ten tumors, five cases showing classical biphasic morphology with "rosettes", five cases without "rosettes" mimicking other renal cell carcinomas or epithelioid angiomyolipoma/pure epithelioid PEComa. We identified two different partner genes, ACTB and NEAT1, the latter previously unreported and occurring in a tumor with an unusual solid and cystic appearance. In both cases, the "rosettes" were absent. In one case no gene partner was identified. Overall, in 12 of 13 TFEB-rearranged renal cell carcinomas staining for PDL1 SP263 was observed, whereas the other antibodies were less reliable or more difficult to interpret. In conclusion, we described the third case of ACTB-TFEB rearranged renal cell carcinoma and a novel NEAT1-TFEB rearranged renal cell carcinoma, both without the distinctive biphasic morphology typical of t(6;11) renal cell carcinoma. Finally, PDL1 SP263 was constantly expressed in TFEB rearranged renal cell carcinoma with possible clinical benefit which requires further investigations.

MITF and TFEB cross-regulation in melanoma cells.

The MITF, TFEB, TFE3 and TFEC (MiT-TFE) proteins belong to the basic helix-loop-helix family of leucine zipper transcription factors. MITF is crucial for melanocyte development and differentiation, and has been termed a lineage-specific oncogene in melanoma. The three related proteins MITF, TFEB and TFE3 have been shown to be involved in the biogenesis and function of lysosomes and autophagosomes, regulating cellular clearance pathways. Here we investigated the cross-regulatory relationship of MITF and TFEB in melanoma cells. Like MITF, the TFEB and TFE3 genes are expressed in melanoma cells as well as in melanoma tumors, albeit at lower levels. We show that the MITF and TFEB proteins, but not TFE3, directly affect each other's mRNA and protein expression. In addition, the subcellular localization of MITF and TFEB is subject to regulation by the mTOR signaling pathway, which impacts their cross-regulatory relationship at the transcriptional level. Our work shows that the relationship between MITF and TFEB is multifaceted and that the cross-regulatory interactions of these factors need to be taken into account when considering pathways regulated by these proteins.

Abstract 3525: Epidemiology and clinicopathology prognostic factors of TFEB translocation renal cell carcinoma (TBRCC): Analysis of updated pooled database

Abstract Renal cell carcinomas harboring the t(6;11) (p21;q12) translocation with a specific Alpha(MALAT1)-TFEB gene fusion is a recognized rare subtype of the Microphthalmia Transcription factor (MiT) family of translocation RCC. TBRCC is an indolent disease that occurs at a younger age. Though it may take on a wide spectrum of morphology, TBRCC has been classically characterized by distinctive biphasic morphology with larger epithelioid cells and smaller cells clustered around eosinophilic spheres formed by basement membrane material. This analysis was conducted to update and expand our existing knowledge of this rare disease. In order to study the demographic characteristics, cytogenetic and molecular signatures, survival, and prognostic factors, we compiled a pooled database of 140 cases. Kaplan-Meier survival curves were constructed. Cox proportional-hazards model and Log-rank tests were used to assess the influence of demographic and clinicopathologic factors on survival. The median age was 32 (3-78) years with a peak incidence between ages 17 and 30 years. Males and females were afflicted equally. The median overall survival (OS) of the whole group was not reached with more than 78% alive at 10 years. The group's median disease-free survival (DFS) was 96 months. Six percent of this cohort recurred. Among patients who recurred, the median time to recurrence was 24(6-75) months. Sex did not impact OS or DFS. Similarly, the molecular partner of the TFEB gene fusion, Alpha (MALAT1) versus others, did not seem to impact OS or DFS. The presence of necrosis seemed to adversely impact OS (HR:14.3, p<0.0001) and DFS (HR:14.4, p<0.0001). Nuclear grade negatively impacted DFS, the higher the grade the worse the DFS. The presence of eosinophilia, psammoma bodies, and pigmentation did not affect OS or DFS. Similarly, the histologic morphology, biphasic vs others, did not affect OS or DFS. Multivariate analysis revealed older age (HR:1.08, p=0.01) and larger tumor size (HR:10.12, p=0.002) adversely impacted OS. This study presents an updated epidemiologic and clinicopathologic data from a pooled cohort of patients with TBRCC. Age, size, and necrosis were found to adversely impact the OS. However, higher nuclear grade only adversely impacted the cohort's DFS. Citation Format: Philip A. Haddad, Dalia Hammoud, Kevin Gallagher. Epidemiology and clinicopathology prognostic factors of TFEB translocation renal cell carcinoma (TBRCC): Analysis of updated pooled database [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3525.

Eosinophilic solid and cystic renal cell carcinoma with TSC2 gene mutations in children

Objective: To study clinical pathological characteristics, immunohistochemical, molecular genetical changes and prognosis in pediatric eosinophilic solid and cystic renal cell carcinoma (ESC RCC) with TSC2 gene mutations. Methods: The tissue samples were collected from two pediatric ESC RCC patients between 2017 and 2018. The tissues were subjected to histological examination and immunohistochemistry using EnVision system. The TFE3, TFEB gene rearrangements were tested using FISH and molecular genetic study. The paraffin sections were used for DNA extraction, PCR amplification and NGS sequencing. Results: The two patients with ESC RCC were both male, aged at 9 years and 8 months, and 13 years, respectively. The tumors were from the right kidney, 5 cm and 7 cm in size, respectively, with solid and cystic changes in cross section, and grey-reddish or grey-whitish fish meat appearance. Microscopic observation revealed the tumors had fibrous capsules, which were infiltrated by the tumor cells. The tumor cells were diffusely distributed, round-shaped, or polygon-shaped, and had voluminous cytoplasm, eosinophilic cytoplasm, various sizes of vacuoles and clear cell-like appearance. There were papillary structures in some areas, with visible fiber septa. The nuclei were round and vesicular, with multi-nucleated cells and megakaryocytes. The mitoses were not seen. A few cystic structures were visible in different sizes, and capsule walls were covered with a single layer of spike-like tumor cells. Thick-walled blood vessels were seen in the stroma, with focal lymphocytic infiltration, eosinophilic necrosis, calcifications and cholesterol crystals. Immunohistochemistry of the tumor cells was positive for PAX8 (diffuse), CK20 (focal), CKpan (focal), CK10 (1 focal, 1 diffuse), INI1, vimentin, CD68, and Ki-67 (5%~10%); the tumor cells were negative for HMB45, S-100, Melan A, p53, desmin, TFE3, CK7, CK19, EMA, CD56, CgA, Syn, CD30, CD117, WT1 and SMA. Molecular genetic study showed that TFE3 and TFEB gene rearrangements were not detected by FISH. NGS sequencing showed TSC2 p.Lys574Ter (0.198) was found in patient one and TSC2 p.Arg406Ter (0.355) in patient two. Conclusions: ESC RCC in children is a rare disease, and can be misdiagnosed easily. It has unique pathological characteristics, and immunohistochemical, molecular and genetic changes. The prognosis is relatively good.

Biogenesis of the Spacious Coxiella-Containing Vacuole Depends on Host Transcription Factors TFEB and TFE3.

Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates inside the lysosome-derived Coxiella-containing vacuole (CCV). To establish this unique niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS) to translocate a cohort of effector proteins into the host cell, which modulate multiple cellular processes. To characterize the host-pathogen interactions that occur during C. burnetii infection, stable-isotope labeling by amino acids in cell culture (SILAC)-based proteomics was used to identify changes in the host proteome during infection of a human-derived macrophage cell line. These data revealed that the abundances of many proteins involved in host cell autophagy and lysosome biogenesis were increased in infected cells. Thus, the role of the host transcription factors TFEB and TFE3, which regulate the expression of a network of genes involved in autophagy and lysosomal biogenesis, were examined in the context of C. burnetii infection. During infection with C. burnetii, both TFEB and TFE3 were activated, as demonstrated by the transport of these proteins from the cytoplasm into the nucleus. The nuclear translocation of these transcription factors was shown to be dependent on the T4SS, as a Dot/Icm mutant showed reduced nuclear translocation of TFEB and TFE3. This was supported by the observation that blocking bacterial translation with chloramphenicol resulted in the movement of TFEB and TFE3 back into the cytoplasm. Silencing of the TFEB and TFE3 genes, alone or in combination, significantly reduced the size of the CCV, which indicates that these host transcription factors facilitate the expansion and maintenance of the organelle that supports C. burnetii intracellular replication.

Clinicopathologic and Molecular Analysis of the TFEB Fusion Variant Reveals New Members of TFEB Translocation Renal Cell Carcinomas (RCCs): Expanding the Genomic Spectrum.

Xp11 renal cell carcinoma (RCC) with different gene fusions may have different clinicopathologic features. We sought to identify variant fusions in TFEB translocation RCC. A total of 31 cases of TFEB RCCs were selected for the current study; MALAT1-TFEB fusion was identified in 25 cases (81%, 25/31) using fusion probes. The remaining 6 cases (19%, 6/31) were further analyzed by RNA sequencing and 5 of them were detected with TFEB-associated gene fusions, including 2 ACTB-TFEB, 1 EWSR1-TFEB, 1 CLTC-TFEB, and 1 potential PPP1R10-TFEB (a paracentric inversion of the TFEB gene, consistent with "negative" TFEB split FISH result, and advising a potential diagnostic pitfall in detecting TFEB gene rearrangement). Four of the 5 fusion transcripts were successfully validated by reverse transcription-polymerase chain reaction and Sanger sequencing. Morphologically, approximately one third (29%, 9/31) of TFEB RCCs showed typical biphasic morphology. The remaining two thirds of the cases (71%, 22/31) exhibited nonspecific morphology, with nested, sheet-like, or papillary architecture, resembling other types of renal neoplasms, such as clear cell RCC, Xp11 RCC, perivascular epithelioid cell tumor (PEComa), or papillary RCC. Although cases bearing a MALAT1-TFEB fusion demonstrated variable morphologies, all 9 cases featuring typical biphasic morphology were associated with MALAT1-TFEB genotype. Accordingly, typical biphasic morphology suggests MALAT1-TFEB fusion, whereas atypical morphology did not suggest the specific type of fusion. Isolated or clustered eosinophilic cells were a common feature in TFEB RCCs, which may be a useful morphology diagnostic clue for TFEB RCCs. Clinicopathologic variables assessment showed that necrosis was the only morphologic feature that correlated with the aggressive behavior of TFEB RCC (P=0.004). In summary, our study expands the genomic spectrum and the clinicopathologic features of TFEB RCCs, and highlights the challenges of diagnosis and the importance of subtyping of this tumor by combining morphology and multiple molecular techniques.

TFEB Expression Profiling in Renal Cell Carcinomas: Clinicopathologic Correlations.

TFEB is overexpressed in TFEB-rearranged renal cell carcinomas as well as in renal tumors with amplifications of TFEB at 6p21.1. As recent literature suggests that renal tumors with 6p21.1 amplification behave more aggressively than those with rearrangements of TFEB, we compared relative TFEB gene expression in these tumors. This study included 37 TFEB-altered tumors: 15 6p21.1-amplified and 22 TFEB-rearranged (including 5 cases from The Cancer Genome Atlas data set). TFEB status was verified using a combination of fluorescent in situ hybridization (n=27) or comprehensive molecular profiling (n=13) and digital droplet polymerase chain reaction was used to quantify TFEB mRNA expression in 6p21.1-amplified (n=9) and TFEB-rearranged renal tumors (n=19). These results were correlated with TFEB immunohistochemistry. TFEB-altered tumors had higher TFEB expression when normalized to B2M (mean: 168.9%, n=28), compared with non-TFEB-altered controls (mean: 7%, n=18, P=0.005). Interestingly, TFEB expression in tumors with rearrangements (mean: 224.7%, n=19) was higher compared with 6p21.1-amplified tumors (mean: 51.2%, n=9; P=0.06). Of note, classic biphasic morphology was only seen in TFEB-rearranged tumors and when present correlated with 6.8-fold higher TFEB expression (P=0.00004). Our results suggest that 6p21.1 amplified renal tumors show increased TFEB gene expression but not as much as t(6;11) renal tumors. These findings correlate with the less consistent/diffuse expression of downstream markers of TFEB activation (cathepsin K, melan A, HMB45) seen in the amplified neoplasms. This suggests that the aggressive biological behavior of 6p21.1 amplified renal tumors might be secondary to other genes at the 6p21.1 locus that are co-amplified, such as VEGFA and CCND3, or other genetic alterations.

MiT family translocation renal cell carcinomas: A 15th anniversary update.

Microphthalmia (MiT) family translocation renal cell carcinomas (RCCs) are a heterogeneous category of renal tumors which all express MiT transcription factors, typically from chromosomal translocation and rarely from gene amplification. This tumor family has two major subtypes [i.e., Xp11 translocation RCC and t(6;11) RCC] and several related neoplasms (i.e., TFEB amplification RCC and melanotic Xp11 translocation renal cancers). Increased understanding of the clinical, pathological, molecular and prognostic heterogeneity of these tumors, since their official recognition in 2004, provides the opportunity to identify prognostic biomarkers and to understand the reasons for tumor aggression. We will review the literature from the past 15 years and highlight the need for a greater understanding of the molecular mechanisms underpinning heterogeneous tumor behavior.

Diagnosis of uncommon renal epithelial neoplasms: performances of fluorescence in situ hybridization

Renal cell carcinomas (RCC) are divided in several subtypes, characterized by morphological and histological features, protein expression patterns and genetics criteria. The main subtypes include Clear cell renal cell carcinoma (CCRCC), Papillary RCC (PRCC), Chromophobe RCC (ChRCC), oncocytoma, TFE3 and TFEB Translocation renal cell carcinoma (TRCC). In most cases, RCC can be easily classified according to histological criteria and immunohistochemistry. Nevertheless, the subtyping process can be more complex in some cases: differential diagnosis (CCRCC or TFE3 TRCC, PRCC or TFE3 TRCC, oncocytic tumors corresponding to ChRCC or oncocytoma), molecular confirmation (TFEB TRCC) and unclassified RCC. Complementary analyses are required such as fluorescence in situ hybridization (FISH) for the detection of chromosomal abnormalities associated to each subtype. In this aim, this study assessed the performance of FISH analysis in the histological classification of 359 RCC exhibiting unusual histological characteristics and/or occurring in young people. FISH probes were selected according to the histological features of each tumor. FISH analysis contributed to the histological classification in 73% of the RCC (261/359). Conversely, FISH did not contribute to the diagnosis in 19% of the cases (69/359) and a hybridization failure was observed for the remaining tumors (8%; 29/359). Considering the different RCC subtypes, FISH analysis was highly efficient to confirm the histological diagnosis of CCRCC, PRCC, and TFE3 TRCC and to identify abnormalities of the TFEB gene. However, this strategy showed some limitations for the diagnosis of oncocytic tumors and unclassified RCC, suggesting that additional molecular assays should be evaluated in these cases.

MiT Family Translocation Renal Cell Carcinoma: from the Early Descriptions to the Current Knowledge.

The new category of MiT family translocation renal cell carcinoma has been included into the World Health Organization (WHO) classification in 2016. The MiT family translocation renal cell carcinoma comprises Xp11 translocation renal cell carcinoma harboring TFE3 gene fusions and t(6;11) renal cell carcinoma harboring TFEB gene fusion. At the beginning, they were recognized in childhood; nevertheless, it has been demonstrated that these neoplasms can occur in adults as well. In the nineties, among Xp11 renal cell carcinoma, ASPL, PRCC, and SFPQ (PSF) were the first genes recognized as partners in TFE3 rearrangement. Recently, many other genes have been identified, and a wide spectrum of morphologies has been described. For this reason, the diagnosis may be challenging based on the histology, and the differential diagnosis includes the most common renal cell neoplasms and pure epithelioid PEComa/epithelioid angiomyolipoma of the kidney. During the last decades, many efforts have been made to identify immunohistochemical markers to reach the right diagnosis. To date, staining for PAX8, cathepsin K, and melanogenesis markers are the most useful identifiers. However, the diagnosis requires the demonstration of the chromosomal rearrangement, and fluorescent in situ hybridization (FISH) is considered the gold standard. The outcome of Xp11 translocation renal cell carcinoma is highly variable, with some patients surviving decades with indolent disease and others dying rapidly of progressive disease. Despite most instances of t(6;11) renal cell carcinoma having an indolent clinical course, a few published cases demonstrate aggressive behavior. Recently, renal cell carcinomas with TFEB amplification have been described in connection with t(6;11) renal cell carcinoma. Those tumors appear to be associated with a more aggressive clinical course. For the aggressive cases of MiT family translocation carcinoma, the optimal therapy remains to be determined; however, new target therapies seem to be promising, and the search for predictive markers is mandatory.

NEAT1-TFE3 and KAT6A-TFE3 renal cell carcinomas, new members of MiT family translocation renal cell carcinoma

Microphthalmia-associated transcription factor (MiT) family translocation renal cell carcinoma harbors variable gene fusions involving either TFE3 or TFEB genes. Multiple 5′ fusion partners for TFE3 have been reported, including ASPSCR1, CLTC, DVL2, LUC7L3, KHSRP, PRCC, PARP14, NONO, SFPQ1, MED15, and RBM10. Each of these fusion genes activates TFE3 transcription which can be detected by immunostaining. Using targeted RNA-sequencing, TFE3 fusion gene partners were identified in 5 cases of TFE3 immunohistochemistry positive translocation renal cell carcinoma. Three cases demonstrated known fusions: ASPSCR1-TFE3, MED15-TFE3 and RBM10-TFE3. However, two cases showed unreported NEAT1-TFE3 and KAT6A-TFE3 fusion transcripts. The NEAT1-TFE3 RCC arose in a 59-year-old male; which demonstrated overlapping morphological features seen in NEAT2(MALAT1)-TFEB t(6;11) renal cell carcinoma, including biphasic alveolar/nested tumor cells with eosinophilic cytoplasm. The KAT6A-TFE3 renal cell carcinoma demonstrated typical morphological features of TFE3/Xp11 renal cell carcinoma including papillae, eosinophilic cytoplasm with focal clearing and abundant psammoma bodies. KAT6A gene fusion was reported in some cases of acute myeloid leukemia, which has not been previously reported in solid tumors. This report highlights the genetic complexity of TFE3 translocation renal cell carcinoma; and RNA-sequencing is a powerful approach for elucidating the underlying genetic alterations.

Renal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†.

Renal cell carcinoma (RCC) accounts for 2%–3% of all adultmalignancies, representing the seventh most common cancer inmen and the ninth most common cancer in women [1].Worldwide, there are ∼209000 new cases and 102000 deathsper year. The incidence of all stages of RCC has increased overthe past several years, contributing to a steadily increasing mor-tality rate per unit population. Active and passive cigarettesmoking is an established risk factor for RCC as well as hyper-tension. However, anti-hypertensive medications such as diure-tics are not independently associated with RCC development.RCC also appears to be more common in patients with obesity,end-stage renal failure, acquired renal cystic disease and tuber-ous sclerosis.Approximately 2%–3% of RCC are hereditary and severalautosomal dominant syndromes are described, each with a dis-tinct genetic basis and phenotype, the most common one beingVon Hippel Lindau disease.In recent years, many new genes associated with RCC havebeen reported (such as PBRM1, SETD2, BAP1). Their roles inpathogenesis and as prognostic biomarkers are currently underinvestigation.

VEGFA amplification/increased gene copy number and VEGFA mRNA expression in renal cell carcinoma with TFEB gene alterations

Amplification of vascular endothelial growth factor A (VEGFA) has been recently reported in TFEB-amplified renal cell carcinomas regardless the level of TFEB amplification. We sought to determine VEGFA amplification by fluorescent in situ hybridization (FISH) and VEGFA mRNA expression by in situ hybridization (RNAscope 2.5) in a series of 10 renal cell carcinomas with TFEB gene alterations, either amplification and/or rearrangement (t(6;11) renal cell carcinoma). TFEB gene rearrangement was demonstrated in eight cases, whereas the remaining two cases showed a high level of TFEB (> 10 copies of fluorescent signals) gene amplification without evidence of rearrangement. Among the eight t(6;11) renal cell carcinomas (TFEB-rearranged cases), one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3–4 copies of fluorescent signals). Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, 3 cases showed high-level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, whereas four showed TFEB gene rearrangement without increased copy number. In summary, VEGFA amplification/increased gene copy number and VEGFA mRNA expression occur in TFEB-amplified renal cell carcinoma, but also in a subset of t(6;11) renal cell carcinoma demonstrating aggressive behavior, and in unamplified conventional t(6;11) renal cell carcinoma suggesting VEGFA as potential therapeutic target in these neoplasms even in the absence of TFEB amplification. We finally propose that all the renal tumors showing morphological characteristics suggesting t(6;11) renal cell carcinoma and all unclassified renal cell carcinomas, either high grade or low grade, should immunohistochemically be evaluated for cathepsin K and/or Melan-A and if one of them is positive, tested for TFEB gene alteration and VEGFA gene amplification.

Therapeutic Targeting of TFE3/IRS-1/PI3K/mTOR Axis in Translocation Renal Cell Carcinoma.

Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB, or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease. We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models. The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3-tRCC models was confirmed by significantly higher expression of phospho-S6 (P < 0.0001) and phospho-4EBP1 (P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro (P < 0.0001) and in vivo (P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation. These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3-tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3-tRCC.

"High-grade oncocytic renal tumor": morphologic, immunohistochemical, and molecular genetic study of 14 cases.

The spectrum of the renal oncocytic tumors has been expanded in recent years to include several novel and emerging entities. We describe a cohort of novel, hitherto unrecognized and morphologically distinct high-grade oncocytic tumors (HOT), currently diagnosed as "unclassified" in the WHO classification. We identified 14 HOT by searching multiple institutional archives. Morphologic, immunohistochemical (IHC), molecular genetic, and molecular karyotyping studies were performed to investigate these tumors. The patients included 3 men and 11 women, with age range from 25 to 73years (median 50, mean 49years). Tumor size ranged from 1.5 to 7.0cm in the greatest dimension (median 3, mean 3.4cm). The tumors were all pT1 stage. Microscopically, they showed nested to solid growth, and focal tubulocystic architecture. The neoplastic cells were uniform with voluminous oncocytic cytoplasm. Prominent intracytoplasmic vacuoles were frequently seen, but no irregular (raisinoid) nuclei or perinuclear halos were present. All tumors demonstrated prominent nucleoli (WHO/ISUP grade 3 equivalent). Nine of 14 cases were positive for CD117 and cytokeratin (CK) 7 was either negative or only focally positive in of 6/14 cases. All tumors were positive for AE1-AE3, CK18, PAX 8, antimitochondrial antigen, and SDHB. Cathepsin K was positive in 13/14 cases and CD10 was positive in 12/13 cases. All cases were negative for TFE3, HMB45, Melan-A. No TFEB and TFE3 genes rearrangement was found in analyzable cases. By array CGH, complete chromosomal losses or gains were not found in any of the cases, and 3/9 cases showed absence of any abnormalities. Chromosomal losses were detected on chromosome 19 (4/9), 3 with losses of the short arm (p) and 1 with losses of both arms (p and q). Loss of chromosome 1 was found in 3/9 cases; gain of 5q was found in 1/9 cases. On molecular karyotyping, 3/3 evaluated cases showed loss of heterozygosity (LOH) on 16p11.2-11.1 and 2/3 cases showed LOH at 7q31.31. Copy number (CN) losses were found at 7q11.21 (3/3), Xp11.21 (3/3), Xp11.22-11.21 (3/3), and Xq24-25 (2/3). CN gains were found at 13q34 (2/3). Ten patients with available follow up information were alive and without disease progression, after a mean follow-up of 28months (1 to 112months). HOT is a tumor with unique morphology and its IHC profile appears mostly consistent. HOT should be considered as an emerging renal entity because it does not meet the diagnostic criteria for other recognized eosinophilic renal tumors, such as oncocytoma, chromophobe renal cell carcinoma (RCC), TFE3 and TFEB RCC, SDH-deficient RCC, and eosinophilic solid and cystic RCC.

Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.

Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux and a massive enlargement of lysosomes. Collectively, this leads to various physiological defects, including embryonic lethality, neurodegeneration and overt inflammation. The reasons for such drastic lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF, enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition, and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.

T(6;11) renal cell carcinoma: a study of seven cases including two with aggressive behavior, and utility of CD68 (PG-M1) in the differential diagnosis with pure epithelioid PEComa/epithelioid angiomyolipoma

Renal cell carcinomas with t(6;11) chromosome translocation involving the TFEB gene are indolent neoplasms which often occur in young patients. In this study, we report seven cases of renal cell carcinoma with TFEB rearrangement, two of whom had histologically proven metastasis. Patients (4F, 3M) ranged in age from 19 to 55 years (mean 37). One patient developed paratracheal and pleural metastases 24 months after surgery and died of disease after 46 months; another one recurred with neoplastic nodules in the perinephric fat and pelvic soft tissue. Histologically, either cytological or architectural appearance was peculiar in each case whereas one tumor displayed the typical biphasic morphology. By immunohistochemistry, all tumors labelled for cathepsin K, Melan-A and CD68 (KP1 clone). HMB45 and PAX8 staining were detected in six of seven tumors. All tumors were negative for CD68 (PG-M1 clone), CKAE1-AE3, CK7, CAIX, and AMACR. Seven pure epithelioid PEComa/epithelioid angiomyolipomas, used as control, were positive for cathepsin K, melanocytic markers, and CD68 (PG-M1 and KP1) and negative for PAX8. Fluorescence in situ hybridization results showed the presence of TFEB gene translocation in all t(6;11) renal cell carcinomas with a high frequency of split TFEB fluorescent signals (mean 74%). In the primary and metastatic samples of the two aggressive tumors, increased gene copy number was observed (3–5 fluorescent signals per neoplastic nuclei) with a concomitant increased number of CEP6. Review of the literature revealed older age and larger tumor size as correlating with aggressive behavior in these neoplasms. In conclusion, we present the clinical, morphological and molecular features of seven t(6;11) renal cell carcinomas, two with histologically demonstrated metastasis. We report the high frequency of split signals by FISH in tumors with t(6;11) chromosomal rearrangement and the occurrence of TFEB gene copy number gains in the aggressive cases, analyzing either the primary or metastatic tumor. Finally, we demonstrate the usefulness of CD68 (PG-M1) immunohistochemical staining in distinguishing t(6;11) renal cell carcinoma from pure epithelioid PEComa/epithelioid angiomyolipoma.

Comprehensive genetic characterization of TFE3-positive renal cell carcinoma.

635 Background: MiT family translocation renal cell carcinoma (RCC) is very rare variant which has translocation of Xp11.2/TFE3 gene or t(6;11)/TFEB gene. Xp11.2 translocation RCC is predominantly reported in younger age and has aggressive features. However, its pathophysiology or genetic characteristics are rarely understood. The aim of the study is to describe the comprehensive genetic characteristics of TFE3-positive RCC diagnosed by immunohistochemistry. Methods: We identified patients who were clinically diagnosed as Xp11.2 translocation RCC by immunostaining positive of TFE3 from two tertiary referral hospitals. Among them, a total of 19 patients whose fresh frozen tissue and normal DNA were achieved were included in the analysis. Whole exome sequencing (WES) was performed to evaluate somatic mutations and copy-number variation (CNV). We also performed RNAseq on the TFE3-positive RCC, 7 clear cell RCC, and 4 normal kidney tissue for comparison. Fusion partners were identified and clustering analysis was done by RNAseq. Results: Mutational landscape of TFE3-positive RCC is unique by low somatic mutation rate and very rare COSMIC variants. High prevalence of CNV with loss of 3p, 6q, 9, gain of 5, 7, and 12 were most frequent. We identified NONO, RBM10, SFPQ, ASPSCR1 and PRCC as fusion partner as previously described. We could not find TFE3 fusion in 6 out of 19 patients by RNAseq, and they were older and had more mutations than the others. They were also genetically clustered with clear cell RCC. Oxidative reducatase pathway was upregulated and immune response and cell adhesion were downregulated by gene ontology results from heatmap clusters. These may promote metastasis, resistant to immunity and cell survival in poorer condition which could be potential mechanism of Xp11.2 translocation RCC aggressiveness. Conclusions: TFE3-positive RCC showed distinctive mutation pattern with low somatic mutation and rare common driver gene. One-third clinically diagnosed Xp11.2 translocation RCC (6/19) did not have real gene fusion even in TFE3 overexpression. They were older and had more somatic mutations than the others. Furthermore, they were clustered with clear cell RCC.

Carcinoma de células renales asociado a amplificación del gen TFEB. Presentación de un caso y revisión de la literatura

Renal carcinomas associated with translocation of transcription factors of the MiT/TFE family include, according to the latest World Health Organization classification, carcinomas with Xp11 translocation that involve the TFE3 gene and those with translocation t(6;11)(p21;q12) that affect the TFEB gene. Each one of these sub-types have well-defined clinicopathological and molecular characteristics. Currently, progress in molecular techniques has led to the description of neoplasms with molecular changes in these same genes but with alterations different to translocation. Thus, recently, cases have been published of TFEB-amplified renal carcinomas with prognoses that vary from cases associated with translocation and could therefore represent a new entity. We present a case of TFEB-amplified renal carcinoma with a full description of the clinicopathological characteristics and an updated revision of these neoplasms.