We report the aberrantly strong nuclear immunoreactivity for the C-terminal portion of TFE3 protein in tumors characterized by chromosome translocations involving the TFE3 gene at Xp11.2. This group of tumors includes alveolar soft part sarcoma and a specific subset of renal carcinomas that tend to affect young patients. They contain fusion genes that encode chimeric proteins consisting of the N-terminal portion of different translocation partners fused to the C-terminal portion of TFE3. We postulated that expression of these fusion proteins may be dysregulated in these specific tumors and detectable by immunohistochemistry. We performed immunohistochemistry using a polyclonal antibody to the C-terminal portion of TFE3 in 40 formalin-fixed, paraffin-embedded tumors characterized by TFE3 gene fusions, including 19 alveolar soft part sarcoma (of which nine were molecularly confirmed) and 21 renal carcinomas with cytogenetically confirmed characteristic Xp11.2 translocations and/or fusion transcripts involving TFE3 (11 PRCC-TFE3, 7 ASPL-TFE3, 3 PSF-TFE3). We also screened 1476 other tumors of 64 histologic types from 16 sites for TFE3 immunoreactivity using tissue microarrays and evaluated a broad range of normal tissues. Thirty-nine of 40 neoplasms characterized by TFE3 gene fusions (19 of 19 alveolar soft part sarcoma, 20 of 21 renal carcinomas) demonstrated moderate or strong nuclear TFE3 immunoreactivity. In contrast, only 6 of 1476 other neoplasms labeled for TFE3 (sensitivity 97.5%, specificity 99.6%). Nuclear immunoreactivity in normal tissues was extremely rare. We then applied this assay to a set of 11 pediatric renal carcinomas for which only paraffin-embedded tissue was available, to assess if morphologic features could predict TFE3 immunoreactivity. Of the eight cases in which we suspected that a TFE3 gene rearrangement might be present based on morphology, seven scored positive for nuclear TFE3 labeling. Of the three tumors whose morphology did not suggest the presence of a TFE3 gene fusion, none showed nuclear TFE3 labeling. In summary, we find that nuclear immunoreactivity for TFE3 protein by routine immunohistochemistry is a highly sensitive and specific assay for neoplasms bearing TFE3 gene fusions. Furthermore, the finding in our set of test cases (i.e., that morphologic features can be used to predict TFE3 immunoreactivity) further supports the notion that renal carcinomas with TFE3 gene fusions have a distinctive morphology that corresponds to their genetic distinctiveness. Carcinomas associated with TFE3 gene fusions may account for a significant proportion of pediatric renal carcinomas, and this immunohistochemistry assay may help to clarify their true prevalence.
Alveolar soft part sarcoma (ASPS) is an unusual tumor with highly characteristic histopathology and ultrastructure, controversial histogenesis, and enigmatic clinical behavior. Recent cytogenetic studies have identi®ed a recurrent der(17) due to a non-reciprocal t(X;17)(p11.2;q25) in this sarcoma. To de®ne the interval containing the Xp11.2 break, we ®rst performed FISH on ASPS cases using YAC probes for OATL1 (Xp11.23) and OATL2 (Xp11.21), and cosmid probes from the intervening genomic region. This localized the breakpoint to a 160 kb interval. The prime candidate within this previously fully sequenced region was TFE3, a transcription factor gene known to be fused to translocation partners on 1 and X in some papillary renal cell carcinomas. Southern blotting using a TFE3 genomic probe identi®ed non-germline bands in several ASPS cases, consistent with rearrangement and possible fusion of TFE3 with a gene on 17q25. Ampli®cation of the 5' portion of cDNAs containing the 3' portion of TFE3 in two di erent ASPS cases identi®ed a novel sequence, designated ASPL, fused in-frame to TFE3 exon 4 (type 1 fusion) or exon 3 (type 2 fusion). Reverse transcriptase PCR using a forward primer from ASPL and a TFE3 exon 4 reverse primer detected an ASPL-TFE3 fusion transcript in all ASPS cases (12/12: 9 type 1, 3 type 2), establishing the utility of this assay in the diagnosis of ASPS. Using appropriate primers, the reciprocal fusion transcript, TFE3-ASPL, was detected in only one of 12 cases, consistent with the non-reciprocal nature of the translocation in most cases, and supporting ASPL-TFE3 as its oncogenically signi®cant fusion product. ASPL maps to chromosome 17, is ubiquitously expressed, and matches numerous ESTs (Unigene cluster Hs.84128) but no named genes. The ASPL cDNA open reading frame encodes a predicted protein of 476 amino acids that contains within its carboxy-terminal portion of a UBXlike domain that shows signi®cant similarity to predicted proteins of unknown function in several model organisms. The ASPL-TFE3 fusion replaces the N-terminal portion of TFE3 by the fused ASPL sequences, while retaining the TFE3 DNA-binding domain, implicating transcriptional deregulation in the pathogenesis of this tumor, consistent with the biology of several other translocationassociated sarcomas. Oncogene (2001) 20, 48 ± 57.
Inflammatory myofibroblastic tumor (IMT) is a rare, but distinctive mesenchymal neoplasm composed of fascicles of bland myofibroblasts admixed with a prominent inflammatory component. Genetic studies of IMTs have demonstrated chromosomal abnormalities of 2p23 and rearrangement of the anaplastic lymphoma kinase (ALK) gene locus. In a subset of IMTs, the ALK C-terminal kinase domain is fused with a tropomyosin N-terminal coiled-coil domain. In the current study, fusion of ALK with the clathrin heavy chain (CTLC) gene localized to 17q23 was detected in two cases of IMT. One of these cases exhibited a 2;17 translocation in addition to other karyotypic anomalies [46,XX,t(2;17)(p23;q23),add(16)(q24)].
A distinctive subset of renal carcinomas is associated with Xp11.2 translocations and resulting TFE3 gene fusions (PRCC-TFE3, PSF-TFE3, NONO-TFE3, ASPL-TFE3), encoding related aberrant transcription factors. We report the cloning of a novel clathrin heavy-chain gene (CLTC)-TFE3 gene fusion resulting from a t(X;17)(p11.2;q23) in a renal carcinoma arising in a 14-year-old boy. The fusion transcript joined the 5 0 exons of CLTC on chromosome band 17q23 to the 3 0 exons of TFE3. CLTC encodes a major subunit of clathrin, a multimeric protein on cytoplasmic organelles, and is a known recurrent fusion partner of the ALK tyrosine kinase gene in anaplastic large-cell lymphoma and inflammatory myofibroblastic tumors. The predicted CLTC-TFE3 product retains the nuclear localization and DNA-binding domains of TFE3, but lacks the multimerization domain of CLTC. The present renal tumor demonstrated morphologic and immunohistochemical features of both PRCC-TFE3 and ASPL-TFE3 carcinomas, including strong nuclear immunoreactivity for the TFE3 C-terminal and only minimal expression of epithelial proteins. However, unlike most renal carcinomas, it also focally expressed melanocytic proteins. The present report highlights the promiscuity of certain genes involved in chromosomal translocations. Further analysis of the shared features of CLTC and other TFE3 fusion partners may shed light on the essential biology of TFE3 fusion proteins.
The unbalanced translocation, der(17)t(X;17)(p11.2;q25), is characteristic of alveolar soft part sarcoma (ASPS). We have recently shown that this translocation fuses the TFE3 transcription factor gene at Xp11.2 to ASPL, a novel gene at 17q25. We describe herein eight morphologically distinctive renal tumors occurring in young people that bear the identical ASPL-TFE3 fusion transcript as ASPS, with the distinction that the t(X;17) translocation is cytogenetically balanced in these renal tumors. A relationship between these renal tumors and ASPS was initially suggested by the cytogenetic finding of a balanced t(X;17)(p11.2;q25) in two of the cases, and the ASPL-TFE3 fusion transcripts were then confirmed by reverse transcriptase-polymerase chain reaction. The morphology of these eight ASPL-TFE3 fusion-positive renal tumors, although overlapping in some aspects that of classic ASPS, more closely resembles renal cell carcinoma (RCC), which was the a priori diagnosis in all cases. These tumors demonstrate nested and pseudopapillary patterns of growth, psammomatous calcifications, and epithelioid cells with abundant clear cytoplasm and well-defined cell borders. By immunohistochemistry, four tumors were negative for all epithelial markers tested, whereas four were focally positive for cytokeratin and two were reactive for epithelial membrane antigen (EMA) (one diffusely, one focally). Electron microscopy of six tumors demonstrated a combination of ASPS-like features (dense granules in four cases, rhomboid crystals in two cases) and epithelial features (cell junctions in six cases, microvilli and true glandular lumens in three cases). Overall, although seven of eight tumors demonstrated at least focal epithelial features by electron microscopy or immunohistochemistry, the degree and extent of epithelial differentiation was notably less than expected for typical RCC. We confirmed the balanced nature of the t(X;17) translocation by fluorescence in situ hybridization in all seven renal tumors thus analyzed, which contrasts sharply with the unbalanced nature of the translocation in ASPS. In summary, a subset of tumors previously considered to be RCC in young people are in fact genetically related to ASPS, although their distinctive morphological and genetic features justify their classification as a distinctive neoplastic entity. Finally, the finding of distinctive tumors being associated with balanced and unbalanced forms of the same translocation is to our knowledge, unprecedented.
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