RNA sequencing of Xp11 translocation-associated cancers reveals novel gene fusions and distinctive clinicopathologic correlations

Abstract

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.

RBM10–TFE3 renal cell carcinoma characterised by paracentric inversion with consistent closely split signals in break‐apart fluorescence in‐situ hybridisation: study of 10 cases and a literature review

“…Cumulative studies have identified several gene fusion partners of TFE3 , including UBX domain containing tether for SLC2A4 ( ASPSCR1 ), proline‐rich mitotic checkpoint control factor ( PRCC ), and splicing factor proline and glutamine rich ( SFPQ ) . Less frequent fusion partners such as mediator complex subunit 15 ( MED15 ) and dishevelled segment polarity protein 2 ( DVL2 ) have also been identified . The histological diagnosis of Xp11 translocation RCCs is based on a characteristic morphology, such as papillary architecture composed of clear epithelioid cells with psammoma bodies, and strong nuclear immunostaining of TFE3.…”

“…1,7,8 Less frequent fusion partners such as mediator complex subunit 15 (MED15) and dishevelled segment polarity protein 2 (DVL2) have also been identified. [8][9][10] The histological diagnosis of Xp11 translocation RCCs is based on a characteristic morphology, such as papillary architecture composed of clear epithelioid cells with psammoma bodies, and strong nuclear immunostaining of TFE3. Nuclear TFE3 immunoreactivity is highly sensitive and specific for diagnosing Xp11 translocation RCC 11 ; however, suboptimal fixation or high endogenous TFE3 expression may result in equivocal immunostaining.…”

Gene fusion analysis in renal cell carcinoma by FusionPlex RNA‐sequencing and correlations of molecular findings with clinicopathological features

“…Immunohistochemical stains for TFE3, TFEB, cathepsin‐K, and MITF are often positive in these tumors, although the specificity and sensitivity of these analytes for tRCC's are far from perfect. Prolonged fixation time, presence of necrosis, antibody specificity, variation in technical methods, and scoring systems are sources of error—leading to both false positive and false negative results . Morphologic and immunohistochemical findings can prompt reflex fluorescence in situ hybridization (FISH) with break‐apart probes that are currently considered a gold standard in diagnosing tRCC .…”

“…However, break‐apart FISH analysis has significant limitations, such as fixation and cutting artifacts, borderline results due to low‐frequency split signals—especially for inversions/intrachromosomal fusions, interpretation subjectivity, and an inability to detect precise breakpoints, cryptic fusions or partner genes in rearrangements . Likewise, RT‐PCR and cytogenetic studies have numerous drawbacks and challenges in routine diagnostic application often requiring higher quality fresh‐frozen specimens for adequate results, A study by Wang et al showed no failed samples by RNA‐seq method vs a 41% failure rate of RT‐PCR followed by Sanger sequencing in the same set of cases. Moreover, RT‐PCR can only identify known fusion with known breakpoints, which is a major limitation as the spectrum of new fusion partners and diverse breakpoints constantly expands causing false negative results …”

“…Recent studies have shown that partnering genes in tRCC could be associated with specific pathologic features and also predict clinical behavior . Therefore, it is especially desirable to validate sensitive molecular technique for formalin‐fixed paraffin‐embedded tissues (FFPE) allowing reliable identification of both known and novel fusion partners with precise knowledge of exons/domains in fusion at single‐nucleotide resolution.…”