Primary renal neoplasms with the ASPL-TFE3 gene fusion of alveolar soft part sarcoma: a distinctive tumor entity previously included among renal cell carcinomas of children and adolescents

Abstract

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.

Figure 1.

Low-power view of case 1 showing circumscription, nested, and pseudopapillary growth patterns (H&E; original magnification, ×100).

Figure 2.

Low-power view of case 2 showing solid, sheet-like growth pattern where the alveolar septa do not connect (H&E; original magnification, ×100).

Figure 3.

Intermediate-power view of case 1 showing pseudopapillary growth pattern and psammomatous calcification that occurs in hyaline cores of small pseudopapillae (H&E; original magnification, ×160).

Figure 4.

A: Trabecular growth pattern in case 6 (H&E; original magnification, ×160X). B: Diffuse EMA immunoreactivity in case 6 (DAKO Envision labeling; original magnification, ×160).

Figure 5.

High-power view of case 1, showing fine and coarse cytoplasmic granularity, vesicular nuclei with prominent nucleoli (H&E; original magnification, ×630).

Figure 6.

A: Unencapsulated tumor in case 1 abuts native renal tubules (H&E; original magnification, ×160). B: Cytokeratin AE1/AE3 labels native tubules, but not the tumor (avidin-biotin peroxidase; original magnification, × 160).

Figure 7.

Case 1. A: Ultrastructural appearance of tumor cells showing an intercellular lumen with projecting slender microvilli. There is an intracytoplasmic membrane-bound crystal in the lower-right corner (original magnification, ×57,460). B: Detail of a membrane-bound rhomboid crystal from same case, composed of parallel rigid fibrils of 5-nm diameter and a periodicity of 10 nm, typical of ASPS crystals (original magnification, ×98,600).

Figure 8.

Case 6. Dense, membrane-bound cytoplasmic secretory granules (original magnification, ×36,400), similar to those seen in typical ASPS; inset shows detail of early crystallization of the secretory material in one of the granules, as in ASPS (see text) (original magnification, ×71,120).

Figure 9.

Partial karyotype of case 1 showing the t(X;17)(p11.2;q25).

Figure 10.

Detection of ASPL-TFE3 fusion transcripts by RT-PCR. RT-PCR was performed as described in the text. The positive result (195-bp product) corresponding to the type 1 ASPL-TFE3 fusion is seen in all four cases illustrated, and positive results were also obtained in the remaining four cases (not illustrated). Negative controls lacking RNA or containing sample RNA but lacking reverse transcriptase (no RT) were appropriately negative. M is the marker lane (HaeIII digest of PhiX174 marker).

Figure 11.

Detection of TFE3-ASPL fusion transcripts by RT-PCR. RT-PCR was performed as described in the text. Different primer combinations were used according to the type of ASPL-TFE3 fusion detected in each tumor (see text). Positive results in four cases are illustrated (positive result in case 8 not shown). The product band in case 2 was faint (arrow). M is the marker lane (HaeIII digest of PhiX174 marker). RT-PCR products were confirmed by sequencing. Partial sequences of the products are shown in Figure 12 ▶ .

Figure 12.

Partial sequences of fusion point of three types of reciprocal TFE3-ASPL fusion transcripts detected in individual renal tumors with the ASPL-TFE3 fusion transcript. All eight tumors contained in-frame ASPL-TFE3 fusion transcripts (see Results and Figure 10 ▶ ). The junction sequences of types 1 and 2 ASPL-TFE3 fusion transcripts have been published elsewhere. Five tumors also contained the reciprocal TFE3-ASPL fusion product (Figure 11) ▶ , consistent with a simple reciprocal t(X;17)(p11;q25). The fusion transcripts in cases 1, 2, 5, and 8 were in frame and are predicted to encode a functional fusion protein. The transcript in case 6 contained an inserted ALU repeat sequence (see text), altering the reading frame of the ASPL portion of the transcript, which is otherwise identical to that in cases 1, 2, 5, and 8. This altered reading frame leads to premature termination within the portion encoded by ASPL (termination codon not shown).

Figure 13.

Bicolor interphase FISH analysis of copy number of 17qter distal to ASPL, to evaluate the balanced versus unbalanced nature of the t(X;17). Images shown are from a representative ASPL-TFE3 renal tumor (case 1) and a representative ASPS case (case ASPS-2 from our previous study 1 ). In these images, the 17qter probe [BAC 525L23 from 17q25.3, known to map telomeric to ASPL (see Materials and Methods)], appears red, whereas the centromere 17-specific probe, CEP 17, appears blue-green. The renal tumor cells show two green and two red signals throughout, as expected for a balanced t(X;17). In contrast, the ASPS cells show generalized loss of one red signal, as expected for an unbalanced t(X;17).

Figure 14.

Schematic representation of the differences between the unbalanced der(17)t(X;17)(p11.2;q25) of soft tissue ASPS and the balanced translocation identified in the t(X;17)(p11.2;q25) renal tumors. Both translocations are shown occurring in a 46, XX female in this illustration. Chromosome X material is colored green, whereas chromosome 17 material is colored purple. Whereas soft tissue ASPS has an extra copy of the majority of the short arm of the X chromosome (distal to Xp11.2) and is missing one copy of the small amount of chromosome 17 that is telomeric to 17q25, the ASPL-TFE3 renal tumors show neither loss nor gain of genetic material at these loci. The locations of the ASPL-TFE3 and TFE3-ASPL fusion genes are shown and their orientation is indicated by the arrows (artwork by Jennifer L. Parsons, M. A.).