Brian Hutchinson

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.

PRCC-TFE3 renal carcinomas: morphologic, immunohistochemical, ultrastructural, and molecular analysis of an entity associated with the t(X;1)(p11.2;q21).

The reappraisal of genetically defined subsets of renal tumors can help to highlight the key pathologic features of specific neoplastic entities. We report the morphologic, immunophenotypic, ultrastructural, and molecular features of 11 renal carcinomas bearing a t(X;1)(p11.2;q21) and/or the resulting PRCC-TFE3 gene fusion. The male/female ratio was 4:7. Ten patients were in the age range of 9–29 years and one was 64 years old (mean 21.3 years, median 15 years). The predominant histologic pattern was nested, with islands of tumor cells compartmentalized by thin-walled capillary vasculature. Minor variations on this pattern yielded solid, acinar, alveolar, and tubular architecture. Papillary architecture was seen in nine cases, usually as a minor component. Neoplastic cells were typically characterized by irregularly shaped nuclei with vesicular chromatin and small nucleoli not visible with a 10× objective, and cytoplasm that ranged from clear to densely granular and eosinophilic. Mitoses were extremely rare; 5 were found in 900 high power fields examined from the 11 neoplasms. The most distinctive immunohistochemical feature of these neoplasms was moderate to intense nuclear labeling for TFE3 protein. These tumors were also consistently immunoreactive for the RCC antigen (10 of 11) and CD10 (9 of 9), whereas cytokeratin and epithelial membrane antigen were negative in four cases and were positive focally in the others. Ultrastructurally, all of the six neoplasms examined showed features consistent with conventional-type (clear cell) renal carcinoma, although two demonstrated distinctive intracisternal microtubules. Both tumors tested contained PRCC-TFE3 fusion transcripts. The differential diagnosis includes conventional-type papillary renal cell carcinoma, conventional-type (clear cell) renal carcinoma, and the ASPL-TFE3 renal carcinomas associated with the t(X;17)(p11.2;q25), with the latter two being morphologically the most similar to the t(X;1) renal carcinomas. Aside from their distinctive clinicopathologic features described here, there is experimental evidence suggesting that these tumors may show differential sensitivity to certain chemotherapeutic agents.

Renal carcinomas with the t(6;11)(p21;q12): Clinicopathologic features and demonstration of the specific alpha-TFEB gene fusion by immunohistochemistry, RT-PCR, and DNA PCR

A highly distinctive subset of renal neoplasms of children and young adults contains a t(6;11)(p21;q12), a translocation recently been shown to result in fusion of Alpha, a gene on 11q12, with the transcription factor gene TFEB on 6p21. To define the clinicopathologic spectrum of this nascent entity and to establish immunohistochemical (IHC) and molecular methods for the detection of the specific Alpha-TFEB fusion, we studied 7 renal neoplasms that showed the t(6;11) by cytogenetic or molecular analysis (patient age: range, 9-33 years; mean, 17 years). While all tumors were confined to the kidney, 3 tumors demonstrated vascular invasion. In limited follow-up, none has metastasized. We postulated that the Alpha-TFEB gene fusion may result in deregulated expression of TFEB protein that would be detectable by IHC. Using a polyclonal antibody to TFEB on formalin-fixed, paraffin-embedded tissue sections, we found that all 7 renal neoplasms with the t(6;11) demonstrated moderate (2 cases) or strong (5 cases) nuclear TFEB immunoreactivity. In contrast, none of 1089 other tumors (of 74 histologic types from 16 sites) labeled significantly for TFEB. Nuclear immunoreactivity for TFEB in normal tissues was extremely rare, limited to weak labeling of scattered benign lymphocytes. We also show that the Alpha-TFEB fusion RNAs are highly variable in size and structure, making detection by reverse-transcriptase polymerase chain reaction (RT-PCR) less reliable than for other gene fusions. Because Alpha is an intronless gene and therefore lacks splice signals, we hypothesized that DNA PCR and RT-PCR products would be identical, allowing for the use of more robust molecular assays based on genomic DNA. Indeed, in 2 cases with available frozen tissue, we showed the genomic Alpha-TFEB junction detected by DNA PCR to be identical to the Alpha-TFEB fusion mRNA detected by RT-PCR. In summary, renal neoplasms with the t(6;11) are a distinctive neoplastic entity with many similarities to the Xp11 translocation carcinomas, and together with the latter form a growing “MiTF/TFE family” of translocation carcinomas. Nuclear immunoreactivity for TFEB protein is a highly sensitive and specific diagnostic marker for these renal neoplasms. Finally, the special molecular features of the Alpha-TFEB gene fusion allow its molecular detection by DNA PCR as a robust alternative to RT-PCR in clinical tumor samples.

Immunophenotype of High-Grade Prostatic Adenocarcinoma and Urothelial Carcinoma

Morphologic features alone can usually be used to distinguish prostatic adenocarcinoma and urothelial carcinoma of the urinary bladder. Poorly differentiated tumors, however, can occasionally have features of both neoplasms, making determination of site of origin difficult. No study has provided a panel of antibodies to assist in the distinction of these two tumors. For this study, 73 examples of moderately and poorly differentiated prostatic adenocarcinoma and 46 examples of high-grade urothelial carcinoma were obtained from radical resection specimens. Immunohistochemical studies were performed using the following panel of antibodies: cytokeratin (CK) 7, CK 20, 34βE12, Leu M1, carcinoembryonic antigen (CEA)m, CEAp, p53, Leu 7, prostate-specific acid phosphatase (PSAP), prostate-specific antigen (PSA), and B72.3. Mucicarmine was also performed. Intermediate and high-grade prostatic carcinoma were compared and then high-grade prostatic carcinoma was compared with high-grade urothelial carcinoma. PSA and PSAP each stained 94% of prostatic adenocarcinomas, but no urothelial carcinomas. Leu 7 stained 94% of prostate and 17% of urothelial carcinomas. Over half of the urothelial carcinomas showed positivity for 34βE12 (65%), as did two cases of prostatic carcinoma (6%). Eighty-three percent of urothelial carcinomas and 12% of prostatic adenocarcinomas stained with CK 7. Forty-one percent of urothelial carcinomas and 12% of prostatic carcinomas were reactive for CEAm, and p53 stained 33% and 3% of urothelial and prostatic adenocarcinomas, respectively. No significant difference was seen in the expression of CEAp, CK 20, B72.3, Leu M1, or mucicarmine between prostate and urothelial carcinoma. We propose a panel of six antibodies to assist in the distinction of high-grade prostatic adenocarcinoma from high grade urothelial carcinoma: PSA, PSAP, 34βE12, Leu 7, CK 7, and p53. The first three antibodies should be used initially; if results are negative, the remaining antibodies may be employed.

Alpha-methylacyl-CoA racemase as a marker in the differential diagnosis of metanephric adenoma

Metanephric adenoma (MA), a well-described renal neoplasm, usually behaves in a benign fashion. It may have areas that are morphologically similar to papillary renal cell carcinoma (RCC) type, or epithelial (tubular predominant) type Wilms’ tumor. Prior immunohistochemical studies of MA have reported variable staining patterns. Alpha-methylacyl-CoA racemase (AMACR), a molecular marker for prostate carcinoma, has subsequently been found to be overexpressed in breast, colorectal and ovarian cancers, among others. Recent microarray analysis of renal tumors has shown an increase of AMACR mRNA levels in papillary RCC but not in other subtypes. We investigated the utility of immunohistochemical staining for AMACR, cytokeratin 7(CK7), CD57 and WT1 to differentiate between the above-mentioned three neoplasms. Immunohistochemical stains were performed on paraffin-embedded tissue sections from 25 papillary RCC, 10 MAs and eight Wilms’ tumors. AMACR was positive in one (10%) of 10 MAs and 24 (96%) of 25 papillary RCC, while it was negative in all Wilms’ tumors. CK7 was positive in 20 of 25 papillary RCCs, focally positive in one Wilms’ tumor and was negative in all MAs. CD57 was positive in all six MAs that were stained, focally positive in one of 25 papillary RCC and one of eight Wilms’ tumors. WT1 was positive in seven of 10 MAs, three of 25 papillary RCCs and all eight Wilms’ tumors. In conclusion, diffuse and strong immunoreactivity for AMACR may be useful in differentiating papillary RCC from MA but a panel which includes AMACR, CK7 and CD57 is better in this differential diagnosis. AMACR is not helpful in differentiating MA from Wilms’ tumor, but CD57 is helpful in this differential diagnosis. WT1 may be useful in separating Wilms’ tumor from MA and papillary RCC but is not helpful in differentiating MA from papillary RCC.

Conventional (clear cell) renal carcinoma metastases have greater bcl-2 expression than high-risk primary tumors

Bcl-2 antagonizes p53-induced apoptosis and may contribute to chemoresistance. In renal cell carcinoma (RCC), the role of bcl-2 is not well-defined, though its expression is reportedly low in primary tumors and lacks prognostic value. This study evaluates patterns of bcl-2 expression in high-risk (pT(3)) primary tumors and in matched patient metastases. Immunohistochemical analysis of bcl-2 was performed on 149 cases of conventional (clear cell) RCC (112 pT(3) primaries, 37 metastases). Paraffin-embedded tissues were obtained from nephrectomies and metastatic resections. Median follow up was 48 months in the entire cohort and 69 months in living patients. We evaluated associations between bcl-2 expression and tumor recurrence or patient survival with the Cox regression test, and used the t-test and Pearson correlation methods to evaluate bcl-2 expression in primary and metastatic cases. Bcl-2 expression was observed at a higher frequency in metastases (21/37 cases; 57%) compared to primary tumors (24/112 cases; 21%; P < 0.001). The percentage of cells stained was greater in metastases than primary tumors (P = 0.003). This finding was also noted when expression in metastatic cases was compared with matched primaries (P = 0.05). Bcl-2 expression did not predict disease-free (P = 0.30), disease-specific (P = 0.90), or overall (P = 0.51) survival. Most RCC primary tumors have low-to-absent levels of bcl-2 protein, whereas most RCC metastases display greater protein levels. Bcl-2 expression in primary tumors does not predict clinical outcome. However, expression of bcl-2 protein occurs at a high frequency in RCC metastases when compared to primary tumors. It may be reasonable to target RCC patients displaying altered bcl-2 levels for molecular therapies, such as anti-bcl2, should metastatic disease develop.