Nonrandom cell-cycle timing of a somatic chromosomal translocation: The t(X;17) of alveolar soft-part sarcoma occurs in G2.

The cell-cycle timing of somatic chromosomal translocations in cancer remains poorly understood but may be relevant to their etiology and the mechanism of their formation. Alveolar soft-part sarcoma (ASPS) is a rare malignant soft-tissue tumor of uncertain lineage that provides an opportunity to address this question. The great majority of ASPSs have relatively simple near-diploid karyotypes characterized by an unbalanced der(17)t(X;17)(p11.2;q25), resulting in nonreciprocal fusion of TFE3 with ASPSCR1 (a.k.a. ASPL), with consequent net gain of Xp11.2-->pter and loss of 17q25-->qter. The presence of a normal X along with the der(17)t(X;17) in ASPSs that occur in men has been well described in previous cytogenetic reports and is most readily explained by a translocation in the G2 phase of the cell cycle. To establish whether formation in G2 is a general feature of the t(X;17), we examined polymorphic loci in Xp11.2-->qter in ASPS from 9 women, including 7 with an unbalanced t(X;17). Our analysis showed that all 7 displayed retention of heterozygosity at all informative markers on Xp11.2-->qter, supporting preferential formation of the t(X;17) in the G2 phase of the cell cycle. Given that the two derivative chromosomes of a translocation in G2 would be expected to segregate together half the time, the predominance of an unbalanced der(17)t(X;17) also raises the possibility of a selective advantage in ASPS cells for gain of Xp11.2-->pter or loss of 17q25.3-->qter or retention of an active copy of TFE3.

A novel CLTC-TFE3 gene fusion in pediatric renal adenocarcinoma with t(X;17)(p11.2;q23).

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' exons of CLTC on chromosome band 17q23 to the 3' 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.

Aberrant nuclear immunoreactivity for TFE3 in neoplasms with TFE3 gene fusions: a sensitive and specific immunohistochemical assay.

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.

The precrystalline cytoplasmic granules of alveolar soft part sarcoma contain monocarboxylate transporter 1 and CD147.

Alveolar soft part sarcoma (ASPS) is an unusual tumor of young adults with the characteristic presence on ultrastructural analysis of rhomboid or rectangular cytoplasmic crystals. These membrane-bound crystals are known to form within specific PAS-diastase-resistant electron-dense cytoplasmic granules. The composition of these crystals and the dense granules from which they are derived has remained elusive. After the detection of strong discrete granular cytoplasmic immunoreactivity in ASPS for monocarboxylate transporter 1 (MCT1) in the course of a broad immunohistochemical characterization of an MCT1 antibody, we studied the expression of MCT1 and its interacting partner, CD147, in a panel of 10 ASPS cases using appropriate antibodies. MCT1 is one of a family of widely expressed proton-linked transporters for monocarboxylates such as lactate and pyruvate. In all normal and neoplastic tissues studied to date, MCT1 immunoreactivity is limited to the cell surface. We find that the periodic acid-Schiff-diastase-resistant cytoplasmic granules of ASPS are strongly immunoreactive for MCT1 and CD147. Specifically, intense cytoplasmic granular positivity for MCT1 and CD147 was found in 7 of 10 and 8 of 10 ASPSs, respectively. Ultrastructural immunohistochemistry with immunogold labeling confirmed that the MCT1 immunoreactivity localized to the cytoplasmic electron-dense granules in ASPS. Western blot analysis of several ASPS cases confirmed that the protein reactive with the MCT1 antibody and that reactive with the CD147 antibody both migrated at the size expected for MCT1 and CD147, respectively. Thus, ASPS cells seem to accumulate MCT1-CD147 complexes in the specific cytoplasmic granules known to undergo crystallization. The possible basis for the overproduction or impaired surface localization of these proteins in ASPS remains unclear.