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MiT family translocation renal cell carcinoma (MiT-RCC) harbors translocations involving the TFE3or TFEBgenes. RCC with TFEBamplification is also identified and is associated with a more aggressive clinical course. Accurate diagnosis of MiT-RCC is crucial for patient management. In this study, we evaluated the performance of the Archer FusionPlex assay for detection of MiT-RCC with TFE3or TFEBtranslocations and TFEBamplifications. RNA was extracted from 49 RCC FFPE tissue samples with known TFE3/TFEBstatus (26 TFE3FISH positive, 12 TFEBFISH positive, 4 TFEBamplified (1 case both split and amplified), and 8 FISH negative) using the Covaris extraction kit. Target enriched cDNA libraries were prepared using the Archer FusionPlex kit and sequenced on the Illumina NextSeq 550. We demonstrate that the age of the specimen, quality of RNA, and sequencing metrics are important for fusion detection. Fusions were identified in 20 of 21 cases less than 2 years old, and TFE3/TFEBrearrangements were detected in all cases with Fusion QC ≥ 100. The assay identified intrachromosomal inversions in two cases (TFE3-RBM10and NONO-TFE3), usually difficult to identify by FISH assays. TFEBmRNA expression and the TFEB/TFE3mRNA expression ratio were significantly higher in RCCs with TFEBfusion and TFEBgene amplification compared to tumors without TFEBfusion or amplification. A cutoff TFEB/TFE3ratio of 0.5 resulted in 97.3% concordance to FISH results with no false negatives. Our study demonstrates that the FusionPlex assay successfully identifies TFE3and TFEBfusions including intrachromosomal inversions. Age of the specimen and certain sequencing metrics are important for successful fusion detection. Furthermore, mRNA expression levels may be used for predicting cases harboring TFEBamplification, thereby streamlining testing. This assay enables accurate molecular detection of multiple subtypes of MiT-RCCs in a convenient workflow.

Renal cell carcinomas with t(6;11) chromosome translocation has been classically characterized by the rearrangement of the TFEBgene, located on chromosome 6, and MALAT1gene, located on chromosome 11. Recently, a few other genes have been described as fusion partners in TFEBrearranged renal cell carcinomas. Although most of TFEBrearranged 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 TFEBrearranged 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). MALAT1gene 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, ACTBand 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-TFEBrearranged renal cell carcinoma and a novel NEAT1-TFEBrearranged renal cell carcinoma, both without the distinctive biphasic morphology typical of t(6;11) renal cell carcinoma. Finally, PDL1 SP263 was constantly expressed in TFEBrearranged renal cell carcinoma with possible clinical benefit which requires further investigations.