—  SPECIALTY CONFERENCE  —

Genitourinary Pathology

Case 1 - t(6;11)(p21;q12) Renal Carcinoma Characterized by Alpha-TFEB Gene Fusion

Pedram Argani
Johns Hopkins University
Baltimore, MD


Click on each slide thumbnail image for an enlarged view
Clinical History
An 18-year-old male presents with hematuria, and is found to have a 7cm left renal mass.


Case 1 - Figure 1 - Note entrapped native elongated renal tubule within the neoplasm.
Case 1 - Figure 2 - Low and intermediate power views of the tumor showing sheet like architecture and biphasic histology. The majority of the cells are epithelioid with either clear or eosinophilic cytoplasm. A second population of smaller cells is clustered around nodules of hyaline basement membrane material.
Case 1 - Figure 3 - Low and intermediate power views of the tumor showing sheet like architecture and biphasic histology. The majority of the cells are epithelioid with either clear or eosinophilic cytoplasm. A second population of smaller cells is clustered around nodules of hyaline basement membrane material.



Case 1 - Figure 4 - Cytological touch preparation highlights the smaller cell population, clustered around basement membrane material.
Case 1 - Figure 5 - PAS-diastase stain highlights the nested pattern and hyaline nodules
Case 1 - Figure 6 - Immunostain for type 4 collagen labels the hyaline nodules, supporting ultrastructural observations that these nodules represent duplicated basement membrane



Case 1 - Figure 7 - Tumor cells are non-immunoreactive for cytokeratin
Case 1 - Figure 8 - Tumor cells are focally immunoreactive for HMB45.

Diagnosis: t(6;11)(p21;q12) Renal Carcinoma Characterized by Alpha-TFEB Gene Fusion

Discussion:
The 2004 WHO Classification of Renal Carcinomas [1] includes several newly recognized entities. These include:

Mucinous tubular and spindle cell carcinoma
This low-grade lesion has been described in the literature as "low grade myxoid renal epithelial neoplasm of distal nephron differentiation" [2], "low-grade tubular-mucinous renal neoplasms" [3, 4] and was often referred to as "Henle loopoma" by Dr. J. Bruce Beckwith. These tumors are usually circumscribed and feature branching, elongated tubules in a bubbly, basophilic, myxoid stroma. Tumor cells are predominantly low cuboidal, but may be spindle. The combination of collapsed, parallel tubules with spindle cell change creates a mimic of a sarcomatoid carcinoma. Only rare cases have metastasized to lymph nodes: most are cured by excision and no tumor-related deaths are on record [2, 3, 4]

Renal cell carcinomas after neuroblastoma
These rare tumors typically affect children (median 13.5 years) who are long term survivors of neuroblastoma. They feature oncocytoid cytoplasm that is often voluminous, and have solid and papillary architecture. Several cases have arisen in children who never received chemotherapy for their neuroblastoma (stage 4S cases), so it is possible that a genetic susceptibility is at play here [5].

Xp11-translocation carcinomas
These carcinomas are characterized by chromosome translocations involving Xp11.2, creating gene fusions involving the TFE3 transcription factor. The most common Xp11.2 translocations are the t(X;1)(p11.2;q21), which fuses the PRCC and TFE3 genes [6, 7] , and the t(X;17)(p11.2;q25), which fuses the ASPL and TFE3 genes [8, 9] . Of note, an unbalanced translocation with identical breakpoints, der(17)t(X;17)(p11.2;q25), is characteristic of Alveolar Soft Part Sarcoma (ASPS), and results in the same ASPL-TFE3 gene fusion [10] . While there are intriguing similarities, ASPL-TFE3 positive renal tumors differ from ASPS at the clinical, histopathologic, and cytogenetic level. Other reported translocations, all three very rare, have included the t(X;1)(p11.2;p34), fusing the PSF and TFE3 genes, the inv (X)(p11;q12), fusing the NonO (p54nrb) and TFE3 genes [11], and the t(X;17)(p11.2;q23), fusing the CLTC and TFE3 genes [12].

Most renal carcinomas with Xp11 translocations have occurred in children and young adults, though occasional adult cases have also been reported.While renal cell carcinomas comprise less than 5% of pediatric renal neoplasms, Xp11.2-associated carcinomas likely comprise approximately one-third of pediatric renal cell carcinomas. [13, 14, 15] . Their clinical behavior remains to be determined. Several Xp11-translocation carcinomas have arisen in patients previously exposed to chemotherapy, including a unique case in which a child with a prior ASPL-TFE3 renal carcinoma developed a PRCC-TFE3 renal carcinoma in the opposite kidney [16].

A papillary carcinoma comprised of clear cells is the most distinctive histopathologic appearance of an Xp11 translocation carcinoma, since this combination is uncommon in other defined types of renal carcinomas. The histology of Xp11-translocation carcinomas associated with specific chromosome translocation breakpoints differ. The ASPL-TFE3 renal carcinomas feature cells with voluminous, clear to eosinophilic cytoplasm, discrete cell borders, vesicular nuclear chromatin and prominent nucleoli. Tumor cells are often discohesive, leading to alveolar and pseudopapillary architecture. Psammoma bodies are almost universal and sometimes extensive, usually forming upon characteristic hyaline nodules [8]. In contrast, the PRCC-TFE3 renal carcinomas typically have less abundant cytoplasm, fewer psammoma bodies, fewer hyaline nodules, and a more nested, compact architecture [17]. A calcified fibrous pseudocapsule, which may be grossly apparent, usually surrounds these tumors, but entrapped renal tubules may be present within. The morphologic features of other Xp11.2 translocation carcinomas (PSF-TFE3, NonO-TFE3, CLTC-TFE3) have not been clearly defined, since so few have been reported.

Renal carcinomas with Xp11.2-associated translocations characteristically underexpress epithelial immunohistochemical markers such as cytokeratin and epithelial membrane antigen. Only approximately one half of cases will be positive with these markers, and the labeling is often focal. Rare Xp11.2-translocation carcinomas, specifically ones with variant gene fusions such as PSF-TFE3 and CLTC-TFE3, have labeled for melanocytic markers HMB45 and Melan A [12]. The most distinctive immunohistochemical feature of these neoplasms is nuclear labeling for TFE3 protein using an antibody to the C-terminal portion of TFE3, which is retained in the gene fusions [18]. Nuclear labeling for TFE3 is a common feature in all Xp11.2-associated carcinomas and ASPS but does not occur in conventional or papillary renal carcinomas. Since native TFE3 is known to be ubiquitously expressed but is not detectable in normal tissues by immunohistochemistry, we suspect that the genes fused 5' to TFE3 contribute strong promoters that cause overexpression of the chimeric protein relative to native TFE3.

t(6:11)(p21;q12) renal neoplasms-a new member of the translocation carcinoma family?
An abstract in the cytogenetics literature first reported this translocation in renal neoplasms in 1996 [19], but the distinctive clinicopathologic features of these neoplasms were not described until 2001 [20].Only 9 cases have been reported in the literature [19, 20, 21, 22, 23, 24] , and I know of 2 additional unreported cases. Of the 11 cases, there have been 4 males and 7 females and the mean age has been 21.8 years (median=18 years, range: 6-53 years). No patient has presented with or has developed metastases in the limited follow-up. However, these neoplasms have demonstrated vascular invasion, suggesting that they have metastatic potential.

On microscopic examination, these neoplasms feature nests and tubules of polygonal epithelioid cells, separated by thin capillaries. Papillary architecture is usually not well-developed, but we have seen it in several recent cases. The majority of the tumor cells have abundant clear to granular eosinophilic cytoplasm, well-defined cell borders and round nuclei with small nucleoli. However, a second population of smaller epithelioid cells is also characteristic, typically (but not always) clustered around nodules of hyaline basement membrane material within larger acini. Mitoses are rare and necrosis is absent. The cases examined have generally been negative for cytokeratins by immunohistochemistry, but all have labeled at least focally for HMB45 and Melan A. We have found these tumors to be negative for Microphthalmia Transcription Factor (MiTF) (0 of 3 cases) [24].

Very recently, the gene fusion that results from the characteristic t(6;11) translocation has been characterized [22, 23] . The translocation fuses the Alpha gene, an intronless gene of unknown function at 11q12, with the first intron of the TFEB transcription factor gene at 6p21. The breakpoint on TFEB is just upstream of the TFEB initiation ATG codon, which results in retention of the entire TFEB coding region in the fusion. Although the Alpha promoter drives expression of the fusion gene, the Alpha gene does not contribute to the open reading frame. Therefore, the consequence of the Alpha-TFEB fusion is dysregulated expression of the normal full-length TFEB protein. Along these lines, we have recently found that the t(6;11) renal neoplasms demonstrate specific nuclear labeling for TFEB protein by IHC while other neoplasms and normal tissues do not [24]. Hence, nuclear labeling for TFEB is a sensitive and specific diagnostic marker for this renal neoplasm with a TFEB gene fusion, just as nuclear labeling for TFE3 is a sensitive and specific marker for tumors bearing TFE3 gene fusions. Based upon these results, strong nuclear labeling provides presumptive evidence of the t(6;11) translocation. Such analysis of archival cases may help to further define the clinicopathologic spectrum of this nascent entity.

TFEB, TFE3, TFEC and Mitf comprise the members of the microphthalmia transcription factor subfamily, which have homologous DNA binding domains and in fact bind to a common DNA sequence. These four transcription factors may homo- or heterodimerize to bind DNA, and they may have functional overlap. MiTF is a transcription factor that regulates expression of genes encoding enzymes necessary for melanogenesis, such as tyrosinase, tyrosinase related protein (TRP) 1 and 2. Nuclear labeling for MiTF has been shown to be a sensitive and specific diagnostic immunohistochemical marker of melanocytic lesions and lesions in the perivascular epithelioid cell family (PEComas) such as angiomyolipoma and lymphangioleiomyomatosis [25]. Homozygous loss of Mitf in mouse models results in failure of melanocytic development [26].

While we considered possible a relationship of the t(6;11) renal neoplasm to epithelioid angiomyolipoma in the initial description of this entity, we now believe that the t(6;11) renal neoplasms are related to the Xp11-translocation carcinomas based upon:

  1. Their similar tendency to affect children and young adults , unlike most renal cell carcinomas.

  2. Their overlapping morphology. Both neoplasms are composed of nests of predominantly clear to eosinophilic epithelioid cells, typical of a conventional renal cell carcinoma on routine H & E sections. With more experience, we have seen more overtly epithelial features (papillae, tubule formation) within the t(6;11) neoplasms, similar to those seen in the Xp11-translocation carcinomas. Along these lines, an additional t(6;11) neoplasm analyzed ultrastructurally has shown poorly formed lumens with junctional complexes and rudimentary microvilli, consistent with an epithelial neoplasm [22]. Furthermore, occasional Xp11-translocation carcinomas have had a second population of smaller cells similar to those seen in the t(6;11) neoplasms-one such case has been illustrated [27].

  3. Their similar immunohistohemical profiles. Given their epithelioid morphology, both tumors underexpress epithelial immunohistochemical IHC markers (cytokeratins, EMA). Several Xp11-translocation carcinomas (specifically, those with PSF-TFE3 and CLTC-TFE3 fusions) have expressed melanocytic markers by immunohistochemistry, just as the t(6;11) renal neoplasms characteristically do. Both tumors overexpress proteins in the MiTF/TFE transcription factor family (TFE3 fusion proteins or native TFEB), and in each case a routine IHC assay detects this overexpression in a highly sensitive and specific fashion.

  4. Their related molecular pathology. Members of the MiTF/TFE transcription factor family homodimerize and heterodimerize in all combinations to bind the same E-box consensus similar or identical DNA sequences CA[C/T]GTG. Therefore, it seems likely that the transcription factors that are overexpressed in these two tumors (TFE3 fusion proteins and native TFEB) have similar downstream targets. Most of these targets remain to be determined; however, genes normally expressed in melanocytic differentiation may be one such example. In cell line transfection assays, overexpression of TFE3 activates the promoter of the tyrosinase gene, while both TFEB and TFE3 activate the promoter of the tyrosinase related protein-1 gene [28]. The genes encoding tyrosinase and tyrosinase related-protein-1 are normally expressed in melanocyte differentiation, where they are regulated by another MiTF/TFE family member, MiTF [29]. These results suggest that aberrant overexpression of TFEB may be responsible for the expression of melanocytic markers, a characteristic and distinctive feature of the t(6;11) renal carcinomas, and that some specific TFE3 fusion proteins may do so as well. Mouse knockout studies provide further evidence that MiTF family members may functionally overlap in certain cell types. In these studies, severe osteopetrosis occurs in mice with combined TFE3 and MiTF inactivation, but there is no effect of TFE3 or MiTF loss individually on osteoclasts [30].
Therefore, based upon the above, we now refer to these lesions as t(6;11) renal carcinomas or Alpha-TFEB renal carcinomas, and propose to classify these neoplasms and the Xp11 translocation carcinomas as members of the "MiTF/TFE translocation carcinoma family."

Differential Diagnosis:
The differential diagnosis for the t(6;11) renal carcinomas includes adult type renal carcinomas (specifically clear cell carcinoma), Xp11 translocation carcinomas, and epithelioid angiomyolipoma. Conventional clear cell carcinomas typically affect older patients, lack the biphasic morphology, do not express melanocytic markers, and do not show nuclear labeling for TFEB. Xp11 translocation carcinomas may overlap morphologically with the t(6;11) renal carcinomas, which speaks to their relatedness. Papillary architecture is more typical of the Xp11 translocation carcinomas, while the biphasic morphology is more typical of the t(6;11) carcinomas. Immunohistochemistry for TFE3 and TFEB can resolve this differential. Epithelioid angiomyolipomas typically affect older patients, lack clear cells, and do not show nuclear labeling for TFEB.

Table 1: Diagnostic immunohistochemical markers from the MiTF/TFE transcription factor family
Gene Translocation? IHC Assay Neoplasm
MiTF No Yes Malignant Melanoma, Angiomyolipoma
TFE3 Yes Yes Alveolar Soft Part Sarcoma, Xp11 translocation renal carcinomas
TFEB Yes Yes t(6;11)(p21;q12) renal carcinomas
TFEC ? ? ?

Table 2: MiTF/TFE translocation renal carcinomas
Tumor Translocation Reference
ASPL-TFE3 renal carcinomas t(X;17)(p11.2;q25) Argani et al. (8);Heimann et al. (9)
PRCC-TFE3 renal carcinomas t(X;1)(p11.2;q21) Sidhar et al. (6); Weterman et al. (7); Argani et al. (17)
PSF-TFE3 renal carcinomas t(X;1)(p11.2;p34) Clark et al. (11)
NoNo-TFE3 renal carcinomas inv(X)(p11;q12) Clark et al. (11)
CLTC-TFE3 renal carcinomas t(X;17)(p11.2;q23) Argani et al. (12)
Alpha-TFEB renal carcinomas t(6;11)(p21;q12) Argani et al. (20); Davis et al. (22); Kuiper et al. (23)

Table 3: WHO renal tumor classification 2004: renal cell carcinomas

Familial renal cell carcinoma
Clear cell renal cell carcinoma
Multilocular cystic renal cell carcinoma
Papillary renal cell carcinoma
Chromophobe renal cell carcinoma
Collecting duct carcinoma
Renal medullary carcinoma
Renal carcinomas associated with Xp11.2 translocations/TFE3 gene fusions
Renal cell carcinoma associated with neuroblastoma
Mucinous tubular spindle cell carcinomas
Renal cell carcinoma unclassified

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