TFE3 Fusion Research Articles

CNSC-51. PRIMARY INTRACRANIAL ALVEOLAR SOFT PART SARCOMA (PIASPS): A SYSTEMATIC REVIEW OF CASE REPORTS AND CASE SERIES FROM 2000 – 2023

Abstract BACKGROUND AND OBJECTIVE Alveolar soft part sarcoma is a rare malignancy of the lower extremities considered to be of unknown cellular origin. Common in young female patients, the ASPS usually has a strong metastatic potential, mostly involving the lungs at the time of presentation. Cases involving the CNS primarily, without any evidence of systemic disease have emerged i.e. the primary intracranial alveolar soft part sarcoma (PIASPS). We aim to determine the clinical manifestations, natural progression and management of primary intracranial alveolar soft part sarcoma. MATERIALS AND METHODS After an extensive literature search using PubMed Central and Google Scholar, seven case reports and two case series of 14 patients with PIASPS were retrieved and included using the Preferred Reporting Items for Systemic review and Meta-analyses guidelines, published between 2000 and 2023. RESULTS Female preponderance of the neoplasm (57.14%) with an average age of 24.9 years was noted. Headache (36.36%) and papilledema (25%) were the most common presenting complaint and sign, respectively. Brain parenchymal involvement was noted in 71.42% cases, mostly the frontal lobe and left hemisphere. The lesion appeared isointense to hypointense on T1-weighted images and hyperintense on T2-weighted images (66.67%) on MRI. Gross Total Resection was possible in 57.14% lesions. On immunohistochemistry, the tumor cells manifested TFE3 and PAS positivity in 25% and 20% of the cases respectively. An average survival after follow up duration was found to be 31.79 months. CONCLUSION Surgery and radiotherapy are the mainstay of treatment of PIASPS, while meningioma an important differential diagnosis. TFE3 fusion product is readily been helpful in diagnosis. Evidence of a primary lesion in the CNS cannot rule out the presence of an occult primary elsewhere in the body. This, along with the slow growing nature of the neoplasm and delayed localized recurrences, require a long-term follow-up.

TFE3-Rearranged Tumors of the Kidney: An Emerging Conundrum

Background: Identical translocations involving the TFE3 gene and various partners have been found in both renal and soft tissue tumors, like alveolar soft part sarcoma (ASPSCR1), ossifying fibromyxoid tumor (PHF1), epithelioid hemangioendothelioma, and the clear cell stromal tumor of the lung (YAP1). Methods: Herein, we review in detail the clinicopathologic and molecular data of TFE3-rearranged renal tumors and propose our perspective, which may shed light on this emerging conundrum. Results: Among the kidney tumors carrying TFE3 translocations, most are morphologically heterogeneous carcinomas labeling for the tubular marker PAX8. The others are mesenchymal neoplasms known as PEComas, characterized by epithelioid cells co-expressing smooth muscle actin, cathepsin-K, melanogenesis markers, and sometimes melanin pigment deposition. Over the past 30 years, numerous TFE3 fusion partners have been identified, with ASPL/ASPSCR1, PRCC, SFPQ/PSF, and NONO being the most frequent. Conclusions: It is not well understood why similar gene fusions can give rise to renal tumors with different morpho-immunophenotypes, which may contribute to the recent disagreement regarding their classification. However, as these two entities, respectively, epithelial and mesenchymal in nature, are widely recognized by the pathology community and their clinicopathologic features well established, we overall believe it is still better to retain the names TFE3-rearranged renal cell carcinoma and TFE3-rearranged PEComa.

Clear Cell Stromal Tumor of the Lung: Clinicopathologic, Immunohistochemical, and Molecular Characterization of Eight Cases

Clear cell stromal tumor is a recently described mesenchymal neoplasm of the lung, characterized by spindle cells with variably clear-to-pale eosinophilic cytoplasm and prominent vascularity, as well as a recurrent YAP1::TFE3 gene fusion in most cases. Diagnosis can be challenging given its rarity and the lack of supportive immunohistochemical (IHC) markers aside from TFE3. To date, less than 20 cases have been reported, and data on clinical behavior are also limited. Although most appear to be benign, aggressive behavior has been reported rarely. In this study, we present the largest multiinstitutional series of clear cell stromal tumor to date, comprising a total of 8 cases and including 6 previously unpublished cases. We investigate its clinicopathologic and genomic features, while also assessing the diagnostic use of IHC for YAP1 C-terminus. Five patients were men and 3 were women. The median age was 59 years (range: 35-84 years). In all cases, a TFE3 rearrangement was demonstrated by either fluorescence in situ hybridization or DNA/RNA sequencing. In 7 tumors, the YAP1::TFE3 fusion was identified by sequencing. We demonstrate that the combination of YAP1 C-terminus loss and TFE3 overexpression using IHC reliably predicts an underlying YAP1::TFE3 fusion in these neoplasms and may be more sensitive than TFE3 fluorescence in situ hybridization. Although the median follow-up time for our study was short (18 months, available in 7 cases), all cases pursued a benign clinical course, with no recurrences or metastases. Our study provides further characterization of this novel entity, supporting its wider recognition.

ASPSCR1::TFE3-mediated upregulation of insulin receptor substrate 2 (IRS-2) activates PI3K/AKT signaling and promotes malignant phenotype

The ASPSCR1::TFE3 fusion gene, resulting from chromosomal translocation, is detected in alveolar soft part sarcoma (ASPS) and a subset of renal cell carcinomas (RCC). The ASPSCR1::TFE3 oncoprotein, functioning as an aberrant transcription factor, contributes to tumor development and progression by inappropriately upregulating target genes. Here, we identified insulin receptor substrate 2 (IRS-2), a cytoplasmic adaptor protein, as a novel transcriptional target of ASPSCR1::TFE3. Ectopic expression of ASPSCR1::TFE3 led to increased IRS-2 mRNA and protein levels. Chromatin immunoprecipitation and luciferase assays demonstrated that ASPSCR1::TFE3 bound to the IRS-2 promoter region and enhanced its transcription. Moreover, IRS-2 was highly expressed in the ASPSCR1::TFE3-positive RCC cell line FU-UR1, while small interfering RNA-mediated depletion of ASPSCR1::TFE3 markedly decreased IRS-2 mRNA and protein levels. Functionally, IRS-2 knockdown attenuated activation of the PI3K/AKT pathway and reduced proliferation, migration, invasion, adhesion, and clonogenicity in FU-UR1 cells. Pharmacological inhibition of IRS-2 also reduced AKT activation as well as cell viability, migration, invasion, and adhesion. These findings suggest that IRS-2, regulated by ASPSCR1::TFE3, promotes tumor progression by activating PI3K/AKT signaling and enhancing the malignant phenotype.

Inflammatory spindle cell PEComa of the lung with YAP1::TFE3 fusion: a report of two cases and a potential relationship with clear cell stromal tumour.

The PEComa family of tumours is defined by spindle/epithelioid cells with myomelanocytic differentiation. A small subset harbours TFE3 fusion; however, YAP1::TEE3 has not been reported. Clear cell stromal tumour of the lung (CCST-L) is an emerging entity characterized by spindle to epithelioid cells with focal cytoplasmic clearing, inflammatory infiltrates, no myomelanocytic differentiation, and YAP1::TFE3 fusion. Herein, we report two cases of lung tumours with myomelanocytic differentiation that showed inflammatory spindle cell histology, focal epithelioid clear cells, as well as YAP1::TFE3 fusion. The patients were both men, aged 61 and 68 years. The tumours in both cases presented as well-circumscribed solid masses involving the lung hilum. After lobectomy, no recurrence was observed at 7 and 32 months. Both tumours shared storiform to short fascicular growth of long spindle cells, with a minor component of epithelioid cells showing clear cytoplasm in the background of substantial intratumoral chronic inflammation and dilated blood vessels. One tumour showed focal melanin deposition. Both tumours were immunohistochemically positive for HMB45, Melan A, and h-caldesmon. Fluorescence in situ hybridization assays indicated the presence of YAP1::TFE3 fusions, which was confirmed by RNA sequencing in one case tested, and by immunohistochemical TFE3 expression and loss of YAP1 C-terminus staining. We present two cases of inflammatory spindle to epithelioid cell tumours of the lungs with myomelanocytic differentiation and YAP1::TFE3 fusion. This unique morphology and gene fusion suggest that these tumours may constitute a distinct subset of lung PEComa. Furthermore, morphological and molecular overlap with CCST-L gives rise to a hypothesis of a potential inherent relationship between PEComa and CCST-L.

PHF1::TFE3-positive fibromyxoid sarcoma? Report of 2 cases and review of 13 cases of PHF1::TFE3-positive ossifying fibromyxoid tumor in the literature.

Ossifying fibromyxoid tumor (OFMT) is a rare soft tissue neoplasm of uncertain histogenesis. Most OFMTs have benign behavior, andmany harbor gene fusions involving the PHD finger protein 1 (PHF1), such as EP400::PHF1, MEAF6::PHF1, EPC1::PHF1, and PHF1::TFE3. The PHF1::TFE3 fusion is unique because PHF1 is at 5' instead of residing at 3' in the other fusions. In this study, we describe 2 cases of OFMT harboring PHF1::TFE3 fusions and review 13 published cases. Two cases of PHF1::TFE3-positive OFMT were investigated using RNA Next-Generation Sequencing and immunohistochemistry. Most (12/15) of the PHF1::TFE3 OFMTs we studied were located at proximal and distal extremities, with a multinodular growth pattern. Only 1 case (1/10) had a shell of bone at the periphery. Areas morphologically similar to sclerosing epithelioid fibrosarcoma or low-grade fibromyxoid sarcoma were found in 8 of 12 (66.7%) cases. Eleven cases (11/15 [73.3%]) were regarded as malignant based on more than 2/50 high-power field mitotic figures, increased cellularity, or the presence of necrosis. Among the 9 cases with follow-up data, 2 patients died of disease (with metastases), 1 patient is alive with metastases, and 1 patient had multiple local recurrences. Because PHF1 is located at 3' in all the PHF1 fusions in OFMTs except PHF1::TFE3, the different driver molecular alterations suggest that OFMTs with 3'-PHF1 fusions and OFMTs with PHF1::TFE3 are different tumors. Immunohistochemistry confirmed TFE3 expression in all PHF1::TFE3 OFMTs. Because PHF1::TFE3-positive OFMTs have increased mitotic figures and tumor cellularity, with a high rate of metastasis, using the name PHF1::TFE3 positive fibromyxoid sarcoma may be appropriate.

TFE3-rearranged nonmelanotic renal PEComa: a case series expanding their phenotypic and fusion landscape.

A subset of exceptionally rare primary renal perivascular epithelioid cell tumours (PEComas) that harbour Xp11.2 translocation have been reported, but no larger series devoted to this topic have been published. We describe the clinicopathological and molecular features of 10 renal PEComas, collected from our routine and consultation files. There were five female and five male patients aged 14-65 (median: 32 years). One patient had a history of childhood neuroblastoma, but no patients were known to have a tuberous sclerosis complex or other hereditary disorder. Complete surgical excision was the treatment for all patients. The available follow-up in five patients indicated a favourable outcome in 4/5 cases. Tumour size ranged from 2.8 to 15.2 cm (median, 5.2 cm). Immunohistochemistry revealed consistently strong TFE3 expression in all tumours, whereas PAX8 and keratin cocktails were uniformly negative. Other positive markers included HMB45 (7/9 tumours), CathepsinK (7/9 tumours), and CD117 (KIT) (3/5 tumours). TFE3 rearrangements were detected in 8/9 tumours (by targeted RNA sequencing in seven and by FISH in one). The identified fusion partners included SFPQ (n = 2) and one tumour each with ASPSCR1, ZC3H4, MED15, RBMX, and PRCC. One tumour that lacked TFE3 rearrangement by next-generation sequencing (NGS) and fluorescence in situ hybridization (FISH) revealed a large intrachromosomal deletion involving PKD1 and TSC2 by DNA-based NGS. This study highlights the morphologic and genetic diversity of TFE3-rearranged primary renal PEComas and underlines the value of surrogate TFE3 immunohistochemistry in identifying them. The lack of PAX8 and keratin expression represents the mainstay for distinguishing these tumours from MiTF-associated renal cell carcinomas. In addition, we report rare (ZC3H4, RBMX) and novel (MED15) TFE3 fusion partners in PEComa.

TFE3-rearranged perivascular epithelioid cell tumors: a clinicopathological analysis of eight cases

Objective: To investigate the clinicopathological, immunohistochemical and molecular genetic characteristics of TFE3-rearranged perivascular epithelioid cell tumor (PEComa). Methods: Eight cases of PEComa with TFE3 rearrangement diagnosed in the First Affiliated Hospital of Air Force Medical University from January 2014 to July 2022 were collected. Three were consultation cases and 5 were collected from our hospital; 7 cases were resection specimens and 1 case was a needle biopsy specimen. Routine histolopathological analysis, immunohistochemical staining, fluorescence in situ hybridization (FISH) and the next-generation sequencing were performed. Clinical data were collected and the prognosis was assessed. Results: The 8 patients consisted of 5 females and 3 males with a median age of 45 years (ranged from 25 to 65 years). The tumor location included 1 uterus, 1 liver, 1 urachus, 2 kidneys, 1 abdominal cavity, 1 colon, and 1 retroperitoneum (3 subsequent recurrences in the abdominal cavity, pelvis and ovary, and abdominal cavity, respectively). Morphologically, the tumor cells were uniform and epithelioid with translucent or eosinophilic cytoplasm. They were arranged in nests or sheets, most of which were separated by thin-walled blood vessels. There were no papillary structures, and no overt smooth muscle or fat components. Atypical features were seen in 3 cases, with bizarre nuclei and tumor giant cells. Large areas of necrosis were visible, and mitosis was common (up to 28/50 HPF). Melanin deposition was present in 3 cases. Immunohistochemical staining showed diffuse and strong positivity for TFE3 in 8/8 cases and for HMB45 in 6/8 cases; focal positivity for Cathepsin K and Melan-A in 6/8 cases and for SMA in 2/8 of cases. All cases were negative for CKpan, PAX8 and Desmin. TFE3 gene break-apart was detected by FISH in all 8 cases, 4 of which underwent next-generation sequencing, and it revealed that 2 cases presented with SFPQ::TFE3 fusion, 1 case with ASPSCR1::TFE3 fusion, and 1 case with no chimeric fusion. Seven cases were followed up for 4-94 months. All cases were alive; 4 cases were disease-free, 2 cases showed recurrence, and 1 case had metastasis at initial diagnosis. Conclusions: TFE3-rearranged PEComa has unique histomorphological, immunohistochemical and molecular characteristics. The biological behavior is aggressive, which could lead to recurrence and metastasis, and warrants close clinical follow-up.

NMRK2 is an efficient diagnostic indicator for Xp11.2 translocation renal cell carcinoma.

Xp11.2 translocation renal cell carcinomas (tRCC) are a rare and highly malignant type of renal cancer, lacking efficient diagnostic indicators and therapeutic targets. Through the analysis of public databases and our cohort, we identified NMRK2 as a potential diagnostic marker for distinguishing Xp11.2 tRCC from kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP) due to its specific upregulation in Xp11.2 tRCC tissues. Mechanistically, we discovered that TFE3 fusion protein binds to the promoter of the NMRK2 gene, leading to its upregulation. Importantly, we established RNA- and protein-based diagnostic methods for identifying Xp11.2 tRCC based on NMRK2 expression levels, and the diagnostic performance of our methods was comparable to a dual-color break-apart fluorescence in situ hybridization assay. Moreover, we successfully identified fresh Xp11.2 tRCC tissues after surgical excision using our diagnostic methods and established an immortalized Xp11.2 tRCC cell line for further research purposes. Functional studies revealed that NMRK2 promotes the progression of Xp11.2 tRCC by upregulating the NAD+/NADH ratio, and supplementation with β-nicotinamide mononucleotide (NMN) or nicotinamide riboside chloride (NR), effectively rescued the phenotypes induced by the knockdown of NMRK2 in Xp11.2 tRCC. Taken together, these data introduce a new diagnostic indicator capable of accurately distinguishing Xp11.2 tRCC and highlight the possibility of developing novel targeted therapeutics. © 2024 The Pathological Society of Great Britain and Ireland.

Unusual PEComa With PRCC :: TFE3 Fusion Mimicking Sinonasal Tract Melanoma.

We report a nasal cavity unusual perivascular epithelioid cell tumor (PEComa) mimicking mucosal melanoma. Immunohistochemistry was performed using BenchMark Ultra and panel of antibodies. The Ion Torrent platform and Ion AmpliSeq cancer hotspot panel were utilized for DNA genotyping. Target-specific RNA libraries for the detection of fusion transcripts were constructed using Archer Universal RNA Reagent Kit v2 and Archer FusionPlex Solid Tumor panel and sequenced on the MiSeqDx instrument. The tumor, diagnosed in 46-year-old female, was composed of spindle cells, and lacked pigmentation. Immunohistochemically, it showed a patchy HMB-45 positivity. Other melanocytic markers (S100 protein, Melan-A, SOX10) were negative. The tumor cells were weakly positive for KIT (CD117) while negative for smooth muscle actin, pancytokeratin cocktail (AE1/AE3), and synaptophysin. Diagnosis of primary sinonasal tract mucosal melanoma was favored. Additional molecular studies detected PRCC :: TFE3 fusion as the sole genetic change, and suggested the diagnosis of unusual PEComa. Previously, TFE3 fusions were reported in a subset of PEComas but not in melanomas, while PRCC involvement has only been documented once in an ocular PEComa. Immunohistochemistry revealed strong nuclear TFE3 expression concordant with the molecular findings. This report emphasis the importance of molecular testing in the differential diagnosis between PEComa and melanoma, especially when the tumor arises in a site typical of melanoma but showing an unusual morphology and immunophenotype. The detection of TFE3 fusion transcripts suggested the diagnosis of SNT PEComa, although it cannot be excluded that this and similar tumors represent a distinct diagnostic category.

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.

ASPSCR1::TFE3 Drives Alveolar Soft Part Sarcoma by Inducing Targetable Transcriptional Programs.

Alveolar soft part sarcoma (ASPS) is a rare mesenchymal malignancy driven by the ASPSCR1::TFE3 fusion. A better understanding of the mechanisms by which this oncogenic transcriptional regulator drives cancer growth is needed to help identify potential therapeutic targets. In this study, we characterized the transcriptional and chromatin landscapes of ASPS tumors and preclinical models, identifying the essential role of ASPSCR1::TFE3 in tumor cell viability by regulating core transcriptional programs involved in cell proliferation, angiogenesis, and mitochondrial biology. ASPSCR1::TFE3 directly interacted with key epigenetic regulators at enhancers and promoters to support ASPS-associated transcription. Among the effector programs driven by ASPSCR1::TFE3, cell proliferation was driven by high levels of cyclin D1 expression. Disruption of cyclin D1/CDK4 signaling led to a loss of ASPS proliferative capacity, and combined inhibition of CDK4/6 and angiogenesis halted tumor growth in xenografts. These results define the ASPS oncogenic program, reveal mechanisms by which ASPSCR1::TFE3 controls tumor biology, and identify a strategy for therapeutically targeting tumor cell-intrinsic vulnerabilities. Significance: The ASPSCR1::TFE3 fusion propels the growth of alveolar soft part sarcoma by activating transcriptional programs that regulate proliferation, angiogenesis, mitochondrial biogenesis, and differentiation and can be therapeutically targeted to improve treatment.

TFE3 -Rearranged PEComa/PEComa-like Neoplasms : Report of 25 New Cases Expanding the Clinicopathologic Spectrum and Highlighting its Association With Prior Exposure to Chemotherapy.

Since their original description as a distinctive neoplastic entity, ~50 TFE3 -rearranged perivascular epithelioid cell tumors (PEComas) have been reported. We herein report 25 new TFE3 -rearranged PEComas and review the published literature to further investigate their clinicopathologic spectrum. Notably, 5 of the 25 cases were associated with a prior history of chemotherapy treatment for cancer. This is in keeping with prior reports, based mainly on small case series, with overall 11% of TFE3 -rearranged PEComas being diagnosed postchemotherapy. The median age of our cohort was 38 years. Most neoplasms demonstrated characteristic features such as nested architecture, epithelioid cytology, HMB45 positive, and muscle marker negative immunophenotype. SFPQ was the most common TFE3 fusion partner present in half of the cases, followed by ASPSCR1 and NONO genes. Four of 7 cases in our cohort with meaningful follow-up presented with or developed systemic metastasis, while over half of the reported cases either recurred locally, metastasized, or caused patient death. Follow-up for the remaining cases was limited (median 18.5 months), suggesting that the prognosis may be worse. Size, mitotic activity, and necrosis were correlated with aggressive behavior. There is little evidence that treatment with MTOR inhibitors, which are beneficial against TSC -mutated PEComas, is effective against TFE3 -rearranged PEComas: only one of 6 reported cases demonstrated disease stabilization. As co-expression of melanocytic and muscle markers, a hallmark of conventional TSC -mutated PEComa is uncommon in the spectrum of TFE3 -rearranged PEComa, an alternative terminology may be more appropriate, such as " TFE3 -rearranged PEComa-like neoplasms," highlighting their distinctive morphologic features and therapeutic implications.

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.

Abstract 4430: Metabolic reprogramming by ASPSCR1::TFE3 and VCP

Abstract Alveolar soft part sarcoma (ASPS) is a lethal soft tissue malignancy typically incident among adolescents and young adults, distinguished by the t(X;17) ASPSCR1::TFE3 fusion gene (AT3). Conventional chemotherapy has shown limited efficacy against ASPS, necessitating exploration of alternative strategies. Previous investigations have shown the pivotal role of the fusion gene in sarcomagenesis in mice, particularly in lactate-rich environments. This study aims to elucidate the metabolic alterations actuated by the AT3 fusion gene, both in vitro and in vivo. To model in vitro metabolic profiles, ASPS (ASPS-1) and renal cell carcinoma (FU-UR1) cell lines, each expressing AT3, were employed. Utilizing siRNA-mediated gene silencing, AT3 and VCP, a cofactor, were targeted in vitro. Metabolomic profiling through GC-MS analyzed the impact of depletion of AT3 or its transcriptional co-factor, VCP. C13-labeled metabolites were introduced and traced in vitro and in vivo to enhance comprehension of carbon utilization in aerobic respiration. While metabolic activity and O2 consumption differed between cell lines, inhibition of AT3 and VCP led to similar metabolite alterations. Significant changes in amino acid metabolism, notably glutamate, aspartate, and alanine were observed in both cell lines. There was notable diversion of glucose carbon utilization away from typical entry into the tricarboxylic acid (TCA) cycle, with minimal carbon incorporation beyond alpha-ketoglutarate. Despite prior data indicating heightened mitophagy activity in AT3 driven cells, inhibition of lysosomal activity did not yield significant changes in cellular respiration or O2 consumption. Citation Format: Rahi Patel, Amir Pozner, Kyle Dunlap, Lara Carroll, Jeff Brown, Greg Ducker, Kevin Jones. Metabolic reprogramming by ASPSCR1::TFE3 and VCP [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 4430.

MED15::TFE3 fusion renal cell carcinoma with extensive cystic change: A clinicopathologic and molecular genetic study of 2 cases, with an emphasis on differential diagnosis.

TFE3-rearranged renal cell carcinomas (RCCs) harbor gene fusions between TFE3 and 1 of many partner genes. MED15::TFE3 fusion RCC is rare, often cystic, and easily misdiagnosed. This study aimed to characterize 2 cases of MED15::TFE3 fusion RCC with extensive cystic change using fluorescence in situ hybridization and targeted RNA sequencing. Both patients were young adult women aged 29 and 35 years. Radiologically, both presented with a cystic Bosniak category II renal lesion. The cysts measured 9.3 cm and 4.8 cm in greatest dimension. Both patients underwent cyst enucleation, and neither had tumor recurrence or metastasis at 26 and 6 months of follow-up, respectively. Microscopically, both tumors were entirely cystic, with thick, fibrous cystic walls lined by small clusters of cells with clear to eosinophilic cytoplasm and uniform, round nuclei with inconspicuous nucleoli. There were also small aggregations of similar clear cells within the cystic walls. Foci of basement membrane-like material depositions were noted in 1 case; calcifications were observed in both cases. Both cases demonstrated nuclear positivity for PAX8 and TFE3 and cytoplasmic staining for Melan-A; HMB45, CAIX, and CK7 were negative. Fluorescence in situ hybridization revealed that both tumors were positive for TFE3 rearrangements. RNA sequencing identified MED15::TFE3 gene fusions in both cases. The main differential diagnosis of MED15::TFE3 fusion RCC includes multilocular cystic renal neoplasm of low malignant potential and atypical renal cysts. Molecular confirmation of TFE3 fusion is essential for establishing the correct diagnosis.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP’s potential as a novel therapeutic target.

Epithelioid hemangioendothelioma (EHE) with WWTR1::TFE3 gene fusion, a novel fusion variant.

Epithelioid hemangioendothelioma (EHE) is a rare endothelial sarcoma associated with a high incidence of metastases and for which there are no standard treatment options. Based on disease-defining mutations, most EHEs are classified into two subtypes: WWTR1::CAMTA1-fused EHE or YAP1::TFE3-fused EHE. However, rare non-canonical fusions have been identified in clinical samples of EHE cases and are challenging to classify. In this study, we report the identification of a novel WWTR1::TFE3 fusion variant in an EHE patient using targeted RNA sequencing. Histologically, the tumor exhibited hybrid morphological characteristics between WWTR1::CAMTA1-fused EHE and YAP1::TFE3-fused EHE. In addition to the driver fusion, there were six additional secondary mutations identified, including a loss-of-function FANCA mutation. Furthermore, in vitro studies were conducted to investigate the tumorigenic function of the WWTR1::TFE3 fusion protein in NIH3T3 cells and demonstrated that WWTR1::TFE3 promotes colony formation in soft agar. Finally, as the wild-type WWTR1 protein relies on binding the TEAD family of transcription factors to affect gene transcription, mutation of the WWTR1 domain of the fusion protein to inhibit such binding abrogates the transformative effect of WWTR1::TFE3. Overall, we describe a novel gene fusion in EHE with a hybrid histological appearance between the two major genetic subtypes of EHE. Further cases of this very rare subtype of EHE will need to be identified to fully elucidate the clinical and pathological characteristics of this unusual subtype of EHE.

PEComa with ASPSCR1::TFE3 fusion: expanding the molecular genetic spectrum of TFE3-rearranged PEComa with an emphasis on overlap with alveolar soft part sarcoma.

Mesenchymal neoplasms involving TFE3 gene fusions are diverse, mainly include alveolar soft part sarcoma (ASPS) that is characterised by ASPSCR1::TFE3 fusion, and a small subset of perivascular epithelioid cell tumours (PEComas) referred to as TFE3-rearranged PEComa, that most frequently harbours SFPQ::TFE3 fusion. Historically, ASPS and TFE3-rearranged PEComa are considered two distinctive entities despite their known morphological overlap. However, recent studies have suggested a potential histogenetic relationship between them, and several neoplasms that showed morphological features more closely fit PEComa rather than ASPS but harboured ASPSCR1::TFE3 fusion have been documented. In this study, we report three cases of PEComa with ASPSCR1::TFE3 fusion. Clinicopathological features were assessed and partner agnostic targeted next-generation sequencing on clinically validated platforms were performed. The patients are two females and one male with age at presentation ranging from21 to 51 years. All three tumours were located in the viscera (rectum, kidney and cervix). On a relatively limited follow-up period (range = 9-15 months), all patients are alive without evidence of recurrent or metastatic disease. The neoplasms were composed of tight nested architecture of epithelioid clear cells separated by a delicate vascular network, two of which were associated with sheets of plump spindle cells, and none showed significant discohesive tumour morphology. Immunohistochemically, in addition to TFE3 protein, all three neoplasms demonstrated co-expression of melan-A and smooth muscle actin. RNA-sequencing identified ASPSCR1::TFE3 fusion in all three cases that were confirmed by subsequent fluorescence in-situ hybridisation analyses. Our study expands the molecular genetic spectrum of TFE3-rearranged PEComa and further indicates its close relationship to ASPS.

Melanotic PEComa: A Rare But Distinctive Subtype Analyzed in a Series of 7 Cases.

Perivascular epithelioid cell neoplasms (PEComas) are tumors of uncertain cell lineage that show a strong female predominance. Their hallmark is the presence of combined smooth muscle and melanocytic differentiation. In most cases, melanocytic differentiation is detectable only by immunohistochemistry, but there are rare reports of PEComa with extensive melanin accumulation (so-called "melanotic PEComa"). Here we report a clinicopathologic series of 7 melanotic PEComas that occurred across a wide patient age range of 21 to 82 years (median: 41y) and with a wide anatomic distribution, including 2 cases in the pelvis and 1 case each in the gallbladder, cervix, eyelid, epidural space, and femur. All tumors were heavily pigmented and, like conventional PEComas, were composed of variably sized neoplastic cells with voluminous granular, or less commonly clear, cytoplasm with prominent nucleoli. All tumors expressed HMB45 by immunohistochemistry, and 6 of 7 showed nuclear TFE3 expression. Where tested, tumors were uniformly negative for Mart-1/Melan-A, S100, desmin, and smooth muscle actin. Molecular analysis identified TFE3 gene rearrangement in 5 of 7 cases, 4 of which were demonstrated by fluorescence in situ hybridization and one by whole-exome RNA sequencing which revealed a SFPQ::TFE3 fusion. The one tumor negative for TFE3 by immunohistochemistry was found instead to harbor a SFPQ::TFEB fusion, the first reported example to our knowledge of TFEB fusion in a PEComa. Clinical follow-up was available for 6 of 7 patients (median: 2.5y: range: 0.75 to 7y). The patient whose tumor harbored SFPQ::TFEB died of metastatic disease 9 months after diagnosis. The other tumors behaved in an indolent fashion: 4 patients were alive without evidence of disease at the most recent follow-up and 1 patient died of an unrelated cancer 4 years after diagnosis of the melanotic PEComa. Our results expand the morphologic and molecular spectrum of melanotic PEComa, and awareness of this rare but distinctive subtype is important to ensure accurate diagnosis within the broader family of heavily pigmented neoplasms.