PRCC-TFE3 Fusion Research Articles

Exploring G-quadruplex structure in PRCC-TFE3 fusion oncogene: Plausible use as anti cancer therapy for translocation Renal cell carcinoma (tRCC)

The TFE3 fusion gene, byproduct of Xp11.2 translocation, is the diagnostic marker for translocation renal cell carcinoma (tRCC). Absence of any clinically recognized therapy for tRCC, pressing a need to create novel and efficient therapeutic approaches. Previous studies shown that stabilization of the G-quadruplex structure in oncogenes suppresses their expression machinery. To combat the oncogenesis caused by fusion genes, our objective is to locate and stabilize the G-quadruplex structure within the PRCC-TFE3 fusion gene. Using the Quadruplex-forming G Rich Sequences (QGRS) mapper and the Non-B DNA motif search tool (nBMST) online server, we found putative G-quadruplex forming sequences (PQS) in the PRCC-TFE3 fusion gene. Circular dichroism demonstrating a parallel G-quadruplex in the targeted sequence. Fluorescence and UV–vis spectroscopy results suggest that pyridostatin binds to this newly discovered G-quadruplex. The PCR stop assay, as well as transcriptional or translational inhibition using real time PCR and Dual luciferase assay, revealed that stable G-quadruplex formation affects biological processes. Confocal microscopy of HEK293T cells transfected with the fusion transcript confirmed G-quadruplexes formation in cell. This investigation may shed light on G-quadruplex’s functions in fusion genes and may help in the development of therapies specifically targeted against fusion oncogenes, which would enhance the capability of current tRCC therapy approach.

Abstract 7580: The antihistamine terfenadine inhibits TFE3 dimerization, has antitumor activity and synergizes with tyrosine kinase inhibitors in translocation renal cell carcinoma

Abstract Translocation renal cell carcinoma is a subtype of kidney cancer characterized by gene fusions involving MiT transcription factor family, TFE3 and TFEB, with various gene partners. In general, these tumors do not respond to conventional therapies, which stresses the importance of developing new therapeutical approaches. To date, several TFE3 fusion gene partners have been identified. However, the mechanism driving TFE3 cooperation with various gene partners remains to be elucidated. We have previously shown the vulnerability of dimerization domain of multiple TFE3-fusion proteins, and we have screened the FDA approved drugs library LOPAC and a small molecule library (Microsource) to identify compounds that inhibit TFE3 dimerization. The antihistamine terfenadine was found to inhibit TFE3 dimerization and had antiproliferative activity in different models of tRCC, both in vitro and in vivo. Interestingly, our more recent data show that terfenadine reduced the amount of TFE3 in the nuclei. We have also observed enhanced antiproliferative activity of terfenadine in HEK293 cells overexpressing PRCC-TFE3, SPFQ-TFE3 or NONO-TFE3 compared to TFE3 alone or vector control, suggesting a targeted effect of terfenadine on cells driven by these TFE3-SF fusions. In addition, knock-down of SFPQ-TFE3 in HEK293 partially reversed this enhanced sensitivity to terfenadine. Cell proliferation and combination index analysis were performed in cell lines endogenously expressing the most common TFE3-fusion proteins cells to determinate the combination with different tyrosine kinase inhibitors. Immunofluorescence and confocal microscopy were performed to assess the impact on TFE3 fusion protein levels. The combination of terfenadine with sunitinib showed a synergistic effect in cells endogenously expressing PRCC-TFE3 (IC50=1.5µM; Fraction Affected > 0.05) and NONO-TFE3 (IC50=1.8µM; Fraction Affected<0.7) fusions. The combination of terfenadine with crizotinib showed a synergistic effect in cells expressing SFPQ-TFE3 fusion (IC50=0.7µM; Fraction Affected>0.7). The combination of terfenadine with dovitinib showed a synergistic effect in cells expressing PRCC-TFE3 fusion (IC50=1µM; Fraction Affected>0.4). These preliminary data suggest a different response to the combination with different TKIs. This observation underlines the importance of identifying the TFE3 partner gene for targeted therapy. Overall, our results suggest that targeting the dimerization domain along with different oncogenic pathways activated in tRCC cells may represent a new therapeutic approach for this disease. Citation Format: Ilaria Delle Fontane, Ricardo Cordova, Christopher Rupert, Sabrina Orsi, Abbas Jawadwala, David E. Heppener, Roberto Pili. The antihistamine terfenadine inhibits TFE3 dimerization, has antitumor activity and synergizes with tyrosine kinase inhibitors in translocation renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7580.

Establishment and Characterization of a TFE3-rearranged Renal Cell Carcinoma Cell Line (FU-UR-2) With the PRCC-TFE3 Fusion Transcript.

Xp11.2-RCC was classified into molecularly defined renal carcinomas and named TFE3-rearranged renal cell carcinoma (TFE3-rRCC) in the 2022 World Health Organization classification of renal tumors. In this study, we established and characterized a TFE3-rRCC cell line from a right-sided renal tumor of a 35-year-old female patient and named it FU-UR-2. FU-UR-2 had been initially diagnosed as a papillary RCC because the patient was 35 years old, a routine immunohistochemical staining for TFE3 was negative, and its morphology was papillary. The G-band analysis revealed an X-chromosome aberration, thus we performed immunohistochemical re-staining for TFE3 and examined the aberration in the TFE3 gene by reverse-transcriptase polymerase chain reaction and fluorescence in situ hybridization. FU-UR-2 was confirmed as a TFE3-rRCC with a PRCC-TFE3 fusion transcript. Cultured FU-UR-2 cells continuously propagated over 90 passages and may provide a new permanent culture model to study pathogenetic mechanisms, investigate biological behavior, and develop new treatments such as molecular-targeting antitumor agents or immunological drugs for TFE3-rRCCs.

Phase I results of the INFORM2 combination study of nivolumab and entinostat in children and adolescents: INFORM2 NivEnt.

10034 Background: Pediatric patients with relapsed or refractory high-risk solid and CNS tumors have dismal survival. To date treatment with immune checkpoint inhibitors in this population has been disappointing. This study exploits the immune enhancing effects of entinostat on nivolumab in biomarker enriched subpopulations. The study aims to determine the pediatric recommended phase II dose (pRP2D) and to evaluate activity and safety. Methods: This is an exploratory non-randomized, open-label, multinational seamless phase I/II trial in children and adolescents with relapsed / refractory or progressive high-risk solid and CNS tumors. The phase I is divided in 2 age cohorts: 12–21 years (y) and 6–11y and follows a 3 + 3 design with two dose levels for entinostat (dose level 1: 2 mg/m2 and dose level 2: 4 mg/m2 once per week) and fixed dose nivolumab (3 mg/kg every 2 weeks). Patients entering the trial on pRP2D can seamlessly enter phase II which consists of a biomarker defined four group basket trial: high mutational load (group A), high PD-L1 mRNA expression (group B), focal MYC(N) amplification (group C), low mutational load and low PD-L1 mRNA expression and no MYC(N) amplification (group D). Results: The first patient was enrolled in May 2020 and at the time of the data cut (21-JAN-2022), 19 patients were treated. The median age at enrollment was 14 y. In the 12 – 21y cohort 15 patients were enrolled and four patients in the 6 – 11y cohort. The most frequent treatment-related AEs to date were thrombocytopenia in six (32%), nausea and vomiting both in four (21%), and neutropenia in three patients (16%). Five patients (26%) experienced grade 3/4 mostly reversible treatment-related AEs, e.g. neutropenia/leukopenia. No treatment related deaths were reported. In the 6 – 11y cohort dose escalation is ongoing. In the 12 – 21y cohort, one DLT (CTCAE grade 3 thrombocytopenia) was observed in six patients on dose level two, which was determined as the pRP2D of the combination. At the time of the data cut, 10 patients (six in arm D and four patients in which the biomarker group was not yet determined) had received at least one RECIST/RANO response evaluation by central review in phase II. One patient (17%) in arm D with metastatic relapsed renal cell carcinoma (RCC) harboring a typical PRCC-TFE3 fusion showed a PR after two cycles and finally achieved an ongoing CR. Extensive explorative analyses of immune signatures derived from INFORM RNA-Seq and WES data revealed that both the primary diagnosis and the current relapse samples harbored a remarkable high immune cell infiltration, especially CD8+ T-cells. Conclusions: The first and ongoing global INFORM2 trial has identified the pRP2D for the nivolumab and entinostat combination in the older age cohort with good tolerability. A patient with metastasized relapsed RCC experienced a CR. The role of immune infiltration as a potential predictive biomarker is currently being explored. Clinical trial information: NCT03838042.

PD43-09 PHENOTYPICAL SCREENING ONMETASTATIC PRCC-TFE3 FUSIONTRANSLOCATION RENAL CELL CARCINOMA ORGANOIDS REVEALSPOTENTIAL THERAPEUTIC AGENTS

You have accessJournal of UrologyCME1 May 2022PD43-09 PHENOTYPICAL SCREENING ONMETASTATIC PRCC-TFE3 FUSIONTRANSLOCATION RENAL CELL CARCINOMA ORGANOIDS REVEALSPOTENTIAL THERAPEUTIC AGENTS Chuanzhen Cao, Xiaomei Lan, Weixing Jiang, Lei Guo, Xingang Bi, Shan Zheng, Aiping Zhou, Zhijian Sun, and Jianzhong Shou Chuanzhen CaoChuanzhen Cao More articles by this author , Xiaomei LanXiaomei Lan More articles by this author , Weixing JiangWeixing Jiang More articles by this author , Lei GuoLei Guo More articles by this author , Xingang BiXingang Bi More articles by this author , Shan ZhengShan Zheng More articles by this author , Aiping ZhouAiping Zhou More articles by this author , Zhijian SunZhijian Sun More articles by this author , and Jianzhong ShouJianzhong Shou More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002604.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Translocation renal cell carcinoma (tRCC) is a subtypethat occurred predominantly in children and young. Metastatic tRCC occurred in young is more aggressive and there are still no effective therapies. Organoidscan mimic original tissues then adopt to high-throughput screening (HTS). We aim to utilize patient-derived organoid and HTS to screen repurposing drugs for metastatic PRCC-TFE3 fusion tRCC. METHODS: Tumor tissues were obtained from treatment-naïve metastatic tRCC patients who underwent surgeries.Pathology, fluorescence in situ hybridization (FISH) confirmed the PRCC-TFE3 fusion tRCC. Organoids were cultured and verified byFISH and RNA-seq.HTS was performed to seek promising drugs and potential mechanisms were analyzed by RNA-seq. RESULTS: We successfully established a metastatic PRCC-TFE3 fusion tRCC organoid of a common fusion subtypeand characterization was verified by histopathology, FISH, and RNA-seq. HTS assay was developed and the robustness was confirmed. A compound library of 1816 drugs was screened. Eventually, axitinib, crizotinib, and JQ-1were selected for further validation, which could induce cell cycle arrest and apoptosis. RNA-seq analyses of post-treatment organoids indicated crizotinib induced significant autophagy-related gene alternations which were consistent with tRCC potential pathogenesis. CONCLUSIONS: We established and validated the organoid cultures from tissues starting from a metastatic PRCC-TFE3 fusion tRCC patient and achieved the HTS process for the first time. Crizotinib might be the targeted therapy worthy of exploring in clinic for metastatic PRCC-TFE3 fusion tRCC.Such organoid and HTS assay may represent a promising model system in translational research assisting clinical strategies. Source of Funding: This work was supported by theNational Science and Technology Major Project of China (ID Number: 2016ZX09101094) and the National Natural Science Foundation of China (ID Number: 82072837) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e705 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chuanzhen Cao More articles by this author Xiaomei Lan More articles by this author Weixing Jiang More articles by this author Lei Guo More articles by this author Xingang Bi More articles by this author Shan Zheng More articles by this author Aiping Zhou More articles by this author Zhijian Sun More articles by this author Jianzhong Shou More articles by this author Expand All Advertisement PDF DownloadLoading ...

Both SUMOylation and ubiquitination of TFE3 fusion protein regulated by androgen receptor are the potential target in the therapy of Xp11.2 translocation renal cell carcinoma.

ABSTRACTBackgroundThe aggressiveness of renal cell carcinoma (RCC) associated with Xp11.2 translocation/TFE3 gene fusion (Xp11.2 translocation RCC [Xp11.2 tRCC]) is age‐dependent, which is similar to the overall trend of reproductive endocrine hormones. Therefore, this study focused on the effect and potential mechanism of androgen and androgen receptor (AR) on the progression of Xp11.2 tRCC.MethodsThe effects of androgen and AR on the proliferation and migration of Xp11.2 tRCC cells were first evaluated utilising Xp11.2 tRCC cell lines and tissues. Because Transcription factor enhancer 3 (TFE3) fusion proteins play a key role in Xp11.2 tRCC, we focused on the regulatory role of AR and TFE3 expression and transcriptional activity.ResultsWhen Xp11.2 tRCC cells were treated with dihydrotestosterone, increased cell proliferation, invasion and migration were observed. Compared with clear cell RCC, the positive rate of AR in Xp11.2 tRCC tissues was higher, and its expression was negatively associated with the progression‐free survival of Xp11.2 tRCC. Further studies revealed that AR could positively regulate the transcriptional activity of TFE3 fusion proteins by small ubiquitin‐related modifier (SUMO)‐specific protease 1, inducing the deSUMOylation of TFE3 fusion. On the other hand, UCHL1 negatively regulated by AR plays a role in the deubiquitination degradation of the PRCC‐TFE3 fusion protein. Therefore, the combination of the AR inhibitor MDV3100 and the UCHL1 inhibitor 6RK73 was effective in delaying the progression of Xp11.2 tRCC, especially PRCC‐TFE3 tRCC.ConclusionsAndrogen and AR function as facilitators in Xp11.2 tRCC progression and may be a novel therapeutic target for Xp11.2 tRCC.The combined use of AR antagonist MDV3100 and UCHL1 inhibitor 6RK73 increased both the SUMOylation and ubiquitination of the PRCC‐TFE3 fusion protein

Phenotypical screening on metastatic PRCC-TFE3 fusion translocation renal cell carcinoma organoids reveals potential therapeutic agents

PurposeTranslocation renal cell carcinoma (tRCC) is a subtype that occurs predominantly in children and young individuals. Metastatic tRCC occurring in young patients is more aggressive than that occurring in older patients, and there are still no effective therapies. Organoids can mimic original tissues and be assessed by high-throughput screening (HTS). We aimed to utilize patient-derived organoids and HTS to screen drugs that can be repurposed for metastatic tRCC with PRCC-TFE3 fusion.MethodsTumor tissues were obtained from treatment-naïve metastatic tRCC patients who underwent surgery. Histopathology and fluorescence in situ hybridization (FISH) confirmed the tRCC. Organoids derived from the dissected tissues were cultured and verified by FISH and RNA-seq. HTS was performed to seek promising drugs, and potential mechanisms were explored by RNA-seq and cell-based studies.ResultsWe successfully established a metastatic tRCC organoid with PRCC-TFE3 fusion, a common fusion subtype, and its characteristics were verified by histopathology, FISH, and RNA-seq. An HTS assay was developed, and the robustness was confirmed. A compound library of 1816 drugs was screened. Eventually, axitinib, crizotinib, and JQ-1 were selected for further validation and were found to induce cell cycle arrest and apoptosis. RNA-seq analyses of posttreatment organoids indicated that crizotinib induced significant changes in autophagy-related genes, consistent with the potential pathogenesis of tRCC.ConclusionsWe established and validated organoids derived from tissues dissected from a patient with metastatic tRCC with PRCC-TFE3 fusion and achieved the HTS process for the first time. Crizotinib might be a targeted therapy worthy of exploration in the clinic for metastatic tRCC with PRCC-TFE3 fusion. Such organoid and HTS assays may represent a promising model system in translational research assisting in the development of clinical strategies.

A1129 - Phenotypical screening on metastatic PRCC-TFE3 fusion translocation renal cell carcinoma organoids reveals potential therapeutic agents

A1129 - Phenotypical screening on metastatic PRCC-TFE3 fusion translocation renal cell carcinoma organoids reveals potential therapeutic agents

The positive regulation loop between NRF1 and NONO-TFE3 fusion promotes phase separation and aggregation of NONO-TFE3 in NONO-TFE3 tRCC

TFE3 gene fusions often place TFE3 under the control of a more active promoter and cause overexpression of the TFE3 proteins in renal cell carcinoma associated with Xp11.2 translocations (Xp11.2 tRCC). The purpose of this study was to investigate the transcriptional regulation and aggregation mechanism of NONO-TFE3 in NONO-TFE3 tRCC. In this study, we found that the nuclear aggregation of NONO-TFE3 fusion was significantly more than that of intact TFE3 or PRCC-TFE3 fusion. We observed that NONO fragment mediated-phase separation promoted stabilization and aggregation of NONO-TFE3 fusion. Meantime, we revealed that the positive regulation loop between NONO-TFE3 and NRF1 increased mitochondrial biosynthesis and metabolism in NONO-TFE3 tRCC. Therefore, the present study raises the possibility that mitochondrial metabolism is potentially a fruitful arena for NONO-TFE3 tRCC therapy.

PRCC-TFE3 fusion-mediated PRKN/parkin-dependent mitophagy promotes cell survival and proliferation in PRCC-TFE3 translocation renal cell carcinoma

ABSTRACT TFE3 (transcription factor binding to IGHM enhancer 3) nuclear translocation and transcriptional activity has been implicated in PINK1-PRKN/parkin-dependent mitophagy. However, the transcriptional control governing the mitophagy in TFE3/Xp11.2 translocation renal cell carcinoma (TFE3 tRCC) is largely unknown. Here, we investigated the role and mechanisms of PRCC-TFE3 fusion protein, one of TFE3 fusion types in TFE3 tRCC, in governing mitophagy to promote development of PRCC-TFE3 tRCC. We observed and analyzed mitophagy, transcriptional control of PRCC-TFE3 on PINK1-PRKN-dependent mitophagy, PRCC-TFE3 fusions nuclear translocation, cancer cell survival and proliferation under mitochondrial oxidative damage in PRCC-TFE3 tRCC cell line. We found that nuclear-aggregated PRCC-TFE3 fusions constitutively activated expression of the target gene E3 ubiquitin ligase PRKN, leading to rapid PINK1-PRKN-dependent mitophagy that promoted cell survival under mitochondrial oxidative damage as well as cell proliferation through decreasing mitochondrial ROS formation. However, nuclear translocation of TFE3 fusions escaped from PINK1-PRKN-dependent mitophagy. Furthermore, we confirmed that PRCC-TFE3 fusion accelerated mitochondrial turnover by activating PPARGC1A/PGC1α-NRF1. In conclusion, our findings indicated a major role of PRCC-TFE3 fusion-mediated mitophagy and mitochondrial biogenesis in promoting proliferation of PRCC-TFE3 tRCC.

Genetic diversity in alveolar soft part sarcoma: A subset contain variant fusion genes, highlighting broader molecular kinship with other MiT family tumors.

Alveolar soft part sarcoma (ASPS) is a rare malignancy that, since its initial description, remains a neoplasm of uncertain histogenesis. The disease-defining molecular event characterizing the diagnosis of ASPS is the ASPSCR1-TFE3 fusion gene. Following identification of an index case of ASPS with a novel TFE3 fusion partner, we performed a retrospective review to determine whether this represents an isolated event. We identified two additional cases, for a total of three cases lacking ASPSCR1 partners. The average patient age was 46 years (range, 17-65); two patients were female. The sites of origin included the transverse colon, foot, and dura. Each case exhibited a histomorphology typical of ASPS, and immunohistochemistry was positive for TFE3 in all cases. Routine molecular testing of the index patient demonstrated a HNRNPH3-TFE3 gene fusion; the remaining cases were found to have DVL2-TFE3 or PRCC-TFE3 fusion products. The latter two fusions have previously been identified in renal cell carcinoma; to our knowledge, this is the first report of a HNRNPH3-TFE3 gene fusion. These findings highlight a heretofore underrecognized genetic diversity in ASPS, which appears to more broadly molecularly overlap with that of translocation-associated renal cell carcinoma and PEComa. These results have immediate implications in the diagnosis of ASPS since assays reliant upon ASPSCR1 may yield a false negative result. While these findings further understanding of the molecular pathogenesis of ASPS, issues related to the histogenesis of this unusual neoplasm remain unresolved.

MicroRNA-204-5p: A novel candidate urinary biomarker of Xp11.2 translocation renal cell carcinoma.

Xp11.2 translocation renal cell carcinoma (Xp11 tRCC) is a rare sporadic pediatric kidney cancer caused by constitutively active TFE3 fusion proteins. Tumors in patients with Xp11 tRCC tend to recur and undergo frequent metastasis, in part due to lack of methods available to detect early‐stage disease. Here we generated transgenic (Tg) mice overexpressing the human PRCC‐TFE3 fusion gene in renal tubular epithelial cells, as an Xp11 tRCC mouse model. At 20 weeks of age, mice showed no histological abnormalities in kidney but by 40 weeks showed Xp11 tRCC development and related morphological and histological changes. MicroRNA (miR)‐204‐5p levels in urinary exosomes of 40‐week‐old Tg mice showing tRCC were significantly elevated compared with levels in control mice. MicroRNA‐204‐5p expression also significantly increased in primary renal cell carcinoma cell lines established both from Tg mouse tumors and from tumor tissue from 2 Xp11 tRCC patients. All of these lines secreted miR‐204‐5p‐containing exosomes. Notably, we also observed increased miR‐204‐5p levels in urinary exosomes in 20‐week‐old renal PRCC‐TFE3 Tg mice prior to tRCC development, and those levels were equivalent to those in 40‐week‐old Tg mice, suggesting that miR‐204‐5p increases follow expression of constitutively active TFE3 fusion proteins in renal tubular epithelial cells prior to overt tRCC development. Finally, we confirmed that miR‐204‐5p expression significantly increases in noncancerous human kidney cells after overexpression of a PRCC‐TFE3 fusion gene. These findings suggest that miR‐204‐5p in urinary exosomes could be a useful biomarker for early diagnosis of patients with Xp11 tRCC.

Monensin induces cell death by autophagy and inhibits matrix metalloproteinase 7 (MMP7) in UOK146 renal cell carcinoma cell line.

Monensin is a metal ionophore used as anticancer agent in many types of cancer cells. In this study, therapeutic potential of monensin was evaluated in TFE3 translocated renal cell carcinoma (RCC) cell line UOK146. UOK146 cells were treated with different concentrations of monensin, and cell death was induced as shown by MTT assay. Autophagy was studied by LC3 western, FACS and LC3 puncta formation after monensin treatment. Mitochondrial potential was studied by staining with TMRM and FACS. Antimetastatic potential of monensin was checked by inhibition of wound closure and MMP7 expression at RNA level. Dead and floating cells after the 10μM monensin treatment were observed under phase contrast microscope. FACS analysis following TMRM staining showed that mitochondrial membrane gets depolarized after monensin treatment. FACS analysis after acridine orange staining showed increased double positive (green and red) cells, and LC3 upregulation and increased LC3 punta displayed autophagy activation in UOK146 cell line after monensin treatment. These findings showed that monensin acts as antiproliferative agent, activating autophagy and downregulates PRCC-TFE3 fusion transcript in Xp11.2 translocated tumor cell line.

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.

Asparagus Racemosus Leaf Extract Inhibits Growth of UOK 146 Renal Cell Carcinoma Cell Line: Simultaneous Oncogenic PRCCTFE3 Fusion Transcript Inhibition and Apoptosis Independent Cell Death

To evaluate anti-cancer activity of Asparagus racemosus (AR) leaf extract on UOK146, a renal cell carcinoma cell line, and explore its mechanism of action. Dried AR leaves were extracted with chloroform and dissolved in DMSO. This extract was applied to UOK146 and cell death was estimated by MTT assay. In addition PRCC-TFE3 fusion transcripts were detected by real time PCR. Extract was found to be cytotoxic with an IC50 of 0.9 mg/ml as estimated by dose response curve. Antitumor activity of the permissible doses of the extract was assessed by the down regulation of PRCC-TFE3 fusion transcript (38%) responsible for oncogenicity of the UOK146 cell line. No increment in the BAX, a proapoptotic marker level was observed. Evidence of antiproliferative effect, PRCC-TFE3 fusion transcript inhibition and static BAX level clearly indicate that AR extract provides or elicits an apoptosis independent anticancer effect on RCC cells by some specific mechanism of regulation.

Renal Cell Carcinoma with t(X;17)(p11.2;q25) in a 5-year-old Taiwanese Boy. A Case Report and Review of the Literature

The classification of adult renal cell carcinomas (RCCs) is well established and is mainly based on histopathologic features and genetic abnormalities [1]. In comparison, pediatric RCCs are more difficult to classify. In the past few years, it has become evident that a great majority of pediatric RCCs belong to the newly-recognized family of Xp11 translocation RCCs [2, 3]. These tumors are genetically characterized by different chromosome translocations, resulting in gene fusions between Xp11.2 and TFE3 transcription factor genes [2]. At least 5 genetic variants have been identified, including t(X;1)(p11.2;q21) translocation with PRCC-TFE3 fusion [4], t(X;1)(p11.2; p34) translocation with PSF-TFE3 fusion [5], inv(X)(p11; q12) translocation with NonO (p54)-TFE3 fusion [5], t (X;17)(p11.2;q25) translocation with ASPL-TFE3 fusion [6],and t(X;17)(p11.2;q23) with CLTC-TFE3 fusion [7]. Additionally, a subset of epithelioid RCCs that harbor t (6:11)(p11.2;q12), which fuses the Alpha gene and TFEB transcription factor gene, has been described [8]. A novel translocation of t(X,3)(p11;q23) has recently been reported in a 32-year-old female patient [9]. As TFEB and TFE3 are closely-related members of the microphthamia transcription factor (MiTF) family (a subfamily of basic helix-loop-helix leucine zipper transcription factors), it has been proposed that these tumors are classified as “MiTF/TFE translocation carcinomas” [2]. In the English literature, most case reports and series studies originate from Western countries [3, 6, 9–14], and case reports and series studies of pediatric RCCs in Asia are rare [15, 16]. Recently, Chen et al described a molecularlydocumented Xp11.2 translocated RCC in a 6-year-old Taiwanese boy [17]. Herein, we report a case of an Xp11.2 translocated RCC in a 5-year-old Taiwanese boy. The diagnosis was established based on the histopathologic features, nuclear expression of TFE3 protein, and the presence of the type 1 TFE3-ASPL fusion gene as detected by reverse transcriptase-polymerase chain reaction. A literature review of translocation RCCs is also included.

The renal cell carcinoma-associated oncogenic fusion protein PRCCTFE3 provokes p21 WAF1/CIP1-mediated cell cycle delay

Previously, we found that in t(X;1)(p11;q21)-positive renal cell carcinomas the bHLH-LZ transcription factor TFE3 is fused to a novel protein designated PRCC. In addition, we found that the PRCCTFE3 fusion protein, which has retained all known functional domains of TFE3, acts as a more potent transcriptional activator than wild type TFE3. We also found that PRCCTFE3 expression confers in vitro and in vivo transformation onto various cell types, including those of the kidney. Here we show that de novo expression of the PRCCTFE3 fusion protein provokes cell cycle delay. This delay, which is mediated by induction of the cyclin-dependent kinase inhibitor p21 WAF1/CIP1 , affects both the G1/S and the G2/M phases of the cell cycle and prevents the cells from undergoing polyploidization. We also show that the PRCCTFE3 fusion protein binds directly to the p21 WAF1/CIP1 promoter and that the PRCCTFE3-induced up-regulation of p21 WAF1/CIP1 leads to activation of the pRB pathway. Finally, we show that in t(X;1)(p11;q21)-positive renal tumor cells several processes that link PRCCTFE3 expression to p21 WAF1/CIP1 -mediated cell cycle delay are abrogated. Our data suggest a scenario in which, during the course of renal cell carcinoma development, an initial PRCCTFE3-induced cell cycle delay must be numbed, thus permitting continued proliferation and progression towards full-blown malignancy.

Renal cell carcinoma in a pediatric patient with an inherited mitochondrial mutation

Renal cell carcinoma (RCC) is a rare pediatric renal cancer. Recent molecular genetic studies discovered a tumor-specific mutation involving translocation of a transcription factor TFE3 in a subset of pediatric RCC with distinct histopathology. We reported a case of a 2-year-old boy with RCC associated with TFE3 translocation resulting in a PRCC-TFE3 fusion gene. Interestingly, the case carried a maternally inherited mitochondrial DNA (mtDNA) alteration at the position which is usually found in MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like Episodes) syndrome (A3243G). Although evidence of somatic alterations in mtDNA existed in various cancers, association between inherited mtDNA mutation and pediatric renal cancer has not been reported. Our case provided the first evidence of a co-occurrence between a germ line mutation in mtDNA and the somatic mutation of pediatric RCC. With this information, we speculated a role of mitochondria mutation in the pathogenesis of this cancer.

P-121 Concomitant mitochondrial DNA alteration and PRCC-TFE3 fusion in pediatric renal cell carcinoma, A case report

P-121 Concomitant mitochondrial DNA alteration and PRCC-TFE3 fusion in pediatric renal cell carcinoma, A case report

PRCC-TFE3 renal carcinomas: morphologic, immunohistochemical, ultrastructural, and molecular analysis of an entity associated with the t(X;1)(p11.2;q21).

The reappraisal of genetically defined subsets of renal tumors can help to highlight the key pathologic features of specific neoplastic entities. We report the morphologic, immunophenotypic, ultrastructural, and molecular features of 11 renal carcinomas bearing a t(X;1)(p11.2;q21) and/or the resulting PRCC-TFE3 gene fusion. The male/female ratio was 4:7. Ten patients were in the age range of 9-29 years and one was 64 years old (mean 21.3 years, median 15 years). The predominant histologic pattern was nested, with islands of tumor cells compartmentalized by thin-walled capillary vasculature. Minor variations on this pattern yielded solid, acinar, alveolar, and tubular architecture. Papillary architecture was seen in nine cases, usually as a minor component. Neoplastic cells were typically characterized by irregularly shaped nuclei with vesicular chromatin and small nucleoli not visible with a 10x objective, and cytoplasm that ranged from clear to densely granular and eosinophilic. Mitoses were extremely rare; 5 were found in 900 high power fields examined from the 11 neoplasms. The most distinctive immunohistochemical feature of these neoplasms was moderate to intense nuclear labeling for TFE3 protein. These tumors were also consistently immunoreactive for the RCC antigen (10 of 11) and CD10 (9 of 9), whereas cytokeratin and epithelial membrane antigen were negative in four cases and were positive focally in the others. Ultrastructurally, all of the six neoplasms examined showed features consistent with conventional-type (clear cell) renal carcinoma, although two demonstrated distinctive intracisternal microtubules. Both tumors tested contained PRCC-TFE3 fusion transcripts. The differential diagnosis includes conventional-type papillary renal cell carcinoma, conventional-type (clear cell) renal carcinoma, and the ASPL-TFE3 renal carcinomas associated with the t(X;17)(p11.2;q25), with the latter two being morphologically the most similar to the t(X;1) renal carcinomas. Aside from their distinctive clinicopathologic features described here, there is experimental evidence suggesting that these tumors may show differential sensitivity to certain chemotherapeutic agents.