Stefano Gobbo

Clear Cell Papillary Renal Cell Carcinoma : A Distinct Histopathologic and Molecular Genetic Entity

A group of renal tumors composed mainly of cells with clear cytoplasm arranged in papillary patterns and arising in end-stage kidneys has recently been identified. The aim of our study is to investigate the cytogenetic and immunohistochemical phenotypes of these unusual renal tumors, and of morphologically similar tumors arising in kidneys unaffected by end-stage renal disease. Seven tumors from 5 patients (age range: 53 to 64 y, mean: 60 y; 3 men and 2 women) were identified. Sections were obtained from paraffin blocks, including the tumors and adjacent non-neoplastic renal parenchyma. Interphase fluorescence in situ hybridization was performed with centromeric probes for chromosomes 3, 7, 17, Y, and with a subtelomeric probe for 3p25. Immunohistochemistry was performed with antibodies against cytokeratin 7, carbonic anhydrase IX, α-methylacyl-CoA racemase, CD10, and transcription factor E3. Four of the tumors were from patients who did not have end-stage renal disease. One patient had end-stage renal disease and presented with 3 morphologically identical tumors, composed of clear cells arranged in a mixture of cystic and papillary structures. Follow-up data were available from all patients and none showed recurrence or metastasis (mean follow-up: 24 mo). All 7 tumors (ranging from 4 to 50 mm in diameter) were stage pT1. All tumors lacked the gains of chromosome 7 and losses of chromosome Y that are typical of papillary renal cell carcinoma. Only 1 tumor showed gain of chromosome 17. Deletion of 3p, usually seen in clear cell renal cell carcinoma, was not detected. All tumors showed strongly positive immunohistochemical staining for cytokeratin 7 and carbonic anhydrase IX and negative immunostaining with antibodies against α-methylacyl-CoA racemase, CD10, and transcription factor E3. In conclusion, clear cell papillary renal cell carcinoma can arise in otherwise normal kidneys and in kidneys with end-stage renal disease. This tumor has immunophenotypic and genetic profiles distinct from those of either classic papillary or clear cell renal cell carcinoma, and should be considered a distinct entity in the spectrum of renal cell neoplasia.

Cathepsin-K immunoreactivity distinguishes MiTF/TFE family renal translocation carcinomas from other renal carcinomas

The microphthalmia transcription factor/transcription factor E (TFE)-family translocation renal cell carcinomas bear specific translocations that result in overexpression of TFE3 or TFEB. TFE3 fusion gene product overexpression occurs as consequence of different translocations involving chromosome Xp11.2, whereas TFEB overexpression is the result of the specific translocation t(6;11)(p21;q12), which fuses the Alpha gene to TFEB. Both TFE3 and TFEB are closely related members of the microphthalmia transcription factor/TFE-family, which also includes TFEC and microphthalmia transcription factor. These transcription factors have overlapping transcriptional targets. Overexpression of microphthalmia transcription factor has been shown to mediate the expression of cathepsin-K in osteoclasts. We hypothesize that the overexpression of the related TFE3 fusion proteins and TFEB in translocation renal cell carcinomas may have the same effect. We studied cathepsin-K in 17 cytogenetically confirmed microphthalmia transcription factor/TFE-family translocation renal cell carcinomas. Seven cases showed a t(6;11)(p21;q12), ten cases showed translocations involving Xp11.2; five cases t(X;1)(p11;q21) resulting in a PRCC–TFE3 gene fusion; three cases t(X;1)(p11;p34) resulting in a PSF–TFE3 gene fusion, one t(X;17)(p11;q25) resulting in an ASPL–TFE3 gene fusion, and one t(X;3)(p11;q23) with an unknown TFE3 gene fusion. As control we analyzed cathepsin-K in 210 clear cell, 40 papillary, 25 chromophobe renal cell carcinomas and 30 oncocytomas. All seven TFEB translocation renal cell carcinomas were labeled for cathepsin-K. Among the cytogenetically confirmed TFE3 translocation renal cell carcinomas, 6 out of 10 were positive. None of the other renal neoplasms expressed cathepsin-K. We conclude the following: (1) cathepsin-K is consistently and strongly expressed in TFEB translocation renal cell carcinomas and in 6 of 10 TFE3 translocation renal cell carcinomas. (2) Cathepsin-K immunolabeling in both TFE3 and TFEB translocation renal cell carcinomas distinguishes these neoplasms from the more common adult renal cell carcinomas, and may be a specific marker of these neoplasms. (3) These results further support the concept that the overexpression of TFE3 or TFEB in these neoplasms activates the expression of genes normally regulated by microphthalmia transcription factor in other cell types.

Renal Cell Carcinomas With Papillary Architecture and Clear Cell Components: The Utility of Immunohistochemical and Cytogenetical Analyses in Differential Diagnosis

Although histologic features enable an accurate diagnosis in most renal carcinomas, overlapping morphologic findings between some renal neoplasms make subclassification difficult. Some renal carcinomas show papillary architecture but are composed extensively of cells with clear cytoplasm, and it is unclear whether they should be classified as clear cell renal cell carcinomas or papillary renal cell carcinomas. We analyzed the immunohistochemical profiles and the cytogenetic patterns of 14 renal carcinomas showing papillary architecture in which there were variable amounts of cells with clear cytoplasm. The patients were 8 women and 6 men (mean age: 54 y). Immunohistochemistry and fluorescence in situ hybridization analysis distinguished 2 different groups. The first consisted of 10 renal cell carcinomas with strong immunoreactivity for α-methyl coenzyme A racemase, of which 9 also expressed cytokeratin 7. All of these neoplasms showed gains of chromosome 7 or 17 and chromosome Y was lost in all the male patients whereas 3p deletion was detected only in one case. In the other 4 renal cell carcinomas, cytokeratin 7 was not detected and α-methylacyl-CoA racemase was positive in only 1. In these neoplasms, no gain of chromosome 7 or 17 and no loss of chromosome Y were observed, whereas 3p deletion was detected in 3 of them. None of the 14 neoplasms showed immunoreactivity for TFE3. The combined use of immunohistochemistry and cytogenetics enabled us to provide a definitive diagnosis for 12 of 14 renal cell carcinomas with papillary architecture and clear cell components: 9 cases were confirmed to be papillary renal cell carcinomas and 3 cases were confirmed to be clear cell renal cell carcinomas. Despite these ancillary techniques, 2 cases remained unclassified. Our study establishes the utility of these procedures in accurately classifying the great majority of renal cell carcinomas with these findings.

Loss of chromosome 9p is an independent prognostic factor in patients with clear cell renal cell carcinoma

Loss of chromosome 9p has been implicated in the progression of renal cell carcinoma. We evaluated the clinical utility of fluorescence in situ hybridization analysis of loss of chromosome 9p in 73 patients with clear cell renal cell carcinomas with varied stage, size, grade, necrosis (SSIGN) scores. Loss of chromosome 9p was observed in 13 tumors (18%). The 5-year cancer-specific survival of patients without loss of chromosome 9p was 88% and was 43% in those with loss of chromosome 9p (P<0.001). Local extension of the primary tumor according to the 2002 TNM staging system, lymph node involvement, the presence of distant metastases, and the SSIGN score were the other variables that predicted cancer-specific survival in univariate analysis. Loss of chromosome 9p was an independent prognostic factor in multivariate analysis. Our data indicate that the detection of chromosome 9p loss by fluorescence in situ hybridization analysis of clear cell renal cell carcinoma adds prognostic information beyond the pathological factors included in the current predictive models for renal cell carcinoma, such as SSIGN score.

Chromosomal gains in the sarcomatoid transformation of chromophobe renal cell carcinoma.

The hallmark of chromophobe renal cell carcinoma is multiple chromosomal losses from among chromosomes 1, 2, 6, 10 and 17. Chromophobe renal cell carcinoma with distant metastases or sarcomatoid transformation are uncommon and little is known about their chromosomal abnormalities. We collected six sarcomatoid chromophobe renal cell carcinomas and three primary chromophobe renal cell carcinomas with distant metastases. A cytogenetic analysis by fluorescent in situ hybridization on paraffin-embedded tissue was performed using centromeric probes for chromosomes 1, 2, 6, 10 and 17. We found more than one signal in four of six (66%) sarcomatoid chromophobe renal cell carcinomas, in both sarcomatoid and adjacent epithelial components. Both primary chromophobe renal cell carcinomas and matched metastases showed single signals for all chromosomes studied in two cases and no abnormalities in the remaining case. We concluded that: (1) both epithelial and sarcomatoid components of sarcomatoid chromophobe renal cell carcinoma show different genetic abnormalities from those characteristic of chromophobe renal cell carcinoma; (2) sarcomatoid chromophobe renal cell carcinomas frequently have multiple gains (polysomy) of chromosomes 1, 2, 6, 10 and 17; (3) distant metastases show the same genetic patterns, usually chromosomal losses (monosomy), found in the primary tumors.

Cathepsin-k expression in pulmonary lymphangioleiomyomatosis

Lymphangioleiomyomatosis is a rare and progressive lung cystic disease, caused by the infiltration of lung parenchyma by mesenchymal cells characterized by co-expression of contractile proteins and melanocytic markers. The pathogenesis of lymphangioleiomyomatosis is determined by mutations affecting tuberous sclerosis complex (TSC) genes, with eventual deregulation of the Rheb/mTOR/p70S6K pathway, and the potential therapeutic activity of mTOR inhibitors is currently under investigation. To better understand the molecular mechanisms involved in the pathogenesis of lymphangioleiomyomatosis, we investigated the expression of cathepsin-k (a papain-like cysteine protease with high matrix-degrading activity). The rationale of this choice was based on the recent demonstration that mTOR inhibitors can regulate major functional activities of osteoclasts, including the expression of cathepsin-k. The immunohistochemical study included 12 cases of lymphangioleiomyomatosis. Twelve angiomyolipomas and several lung diseases (sarcoidosis, organizing pneumonia, usual interstitial pneumonia, emphysema) were investigated as controls. In all lymphangioleiomyomatosis cases, strong cathepsin-k immunoreactivity was demonstrated, restricted to lymphangioleiomyomatosis cells. Similar expression levels were observed in renal angiomyolipomas. These observations extend the knowledge regarding the immunophenotypic profile of lymphangioleiomyomatosis cells, and provide a useful new marker for diagnosis in difficult cases (eg, in small transbronchial biopsies). The strong expression of such a potent papain-like cysteine protease in lymphangioleiomyomatosis cells can significantly contribute to the progressive remodelling of lung parenchyma observed in this deadly disease, with eventual formation of lung cysts. It is possible to speculate that mTOR inhibitors may exert part of their action by limiting the destructive remodelling of lung structure.

Diagnostic utility of S100A1 expression in renal cell neoplasms: an immunohistochemical and quantitative RT-PCR study.

S100A1 is a calcium-binding protein, which has been recently found in renal cell neoplasms. We evaluated the diagnostic utility of immunohistochemical detection of S100A1 in 164 renal cell neoplasms. Forty-one clear cell, 32 papillary, and 51 chromophobe renal cell carcinomas, and 40 oncocytomas, 164 samples of normal renal parenchyma adjacent to the tumors and 13 fetal kidneys were analyzed. The levels of S100A1 mRNA detected by quantitative RT-PCR analysis of frozen tissues from seven clear cell, five papillary, and six chromophobe renal cell carcinomas, four oncocytomas, and nine samples of normal renal tissues adjacent to neoplasms were compared with the immunohistochemical detection of protein expression. Clear cell and papillary renal cell carcinomas showed positive reactions for S100A1 in 30 out of 41 tumors (73%) and in 30 out of 32 (94%) tumors, respectively. Thirty-seven renal oncocytomas out of 40 (93%) were positive for S100A1, whereas 48 of 51 (94%) chromophobe renal cell carcinomas were negative. S100A1 protein was detected in all samples of unaffected and fetal kidneys. S100A1 mRNA was detected by RT-PCR in all normal kidneys and renal cell neoplasms, although at very different levels. Statistical analyses comparing the different expression of S100A1 in clear cell and chromophobe renal cell carcinomas observed by immunohistochemical and RT-PCR methods showed significant values (P<0.001), such as when comparing by both techniques the different levels of S100A1 expression in chromophobe renal cell carcinomas and oncocytomas (P<0.001). Our study shows that S100A1 protein is expressed in oncocytomas, clear cell and papillary renal cell carcinomas but not in chromophobe renal cell carcinomas. Its immunodetection is potentially useful for the differential diagnosis between chromophobe renal cell carcinoma and oncocytoma. Further, S100A1 protein expression is constantly detected in the normal parenchyma of the adult and fetal kidney.

Cathepsin K expression in the spectrum of perivascular epithelioid cell (PEC) lesions of the kidney

The perivascular epithelioid cell (PEC) is a unique cell type coexpressing contractile proteins (mainly α-smooth muscle actin), melanocytic markers, including microphthalmia-associated transcription factor (MITF), and estrogen and progesterone receptors. It is constantly present in a group of tumors called PEComas. Renal PEComas include the common angiomyolipoma as well as less common lesions such as microscopic angiomyolipoma, intraglomerular lesions, angiomyolipoma with epithelial cysts, epithelioid angiomyolipoma, oncocytoma-like angiomyolipoma and lymphangioleiomyomatosis of the renal sinus. It has been demonstrated that most of these lesions are determined by mutations affecting genes of the tuberous sclerosis complex, tuberous sclerosis 1 (TSC1) and tuberous sclerosis 2 (TSC2), with eventual deregulation of the RHEB/MTOR/RPS6KB2 pathway, and it has been observed that some PEComas regressed during sirolimus therapy, an MTOR inhibitor. Recently, overexpression of MITF has been related to the expression of the papain-like cysteine protease cathepsin K in osteoclasts where it has inhibited MTOR. The aim of this study is to evaluate cathepsin K immunohistochemically in the entire spectrum of PEComa lesions in the kidney. The study population consisted of 84 renal PEComa lesions, including 5 composed predominantly of fat (lipoma-like angiomyolipoma), 15 almost exclusively composed of spindle-shaped smooth muscle cells (leiomyoma-like angiomyolipoma) and 31 common angiomyolipomas composed of a mixture of fat, spindle and epithelioid smooth muscle cells, and abnormal thick-walled blood vessels, 15 microscopic angiomyolipomas, 5 intraglomerular lesions, 2 oncocytoma-like angiomyolipomas, 8 epithelioid angiomyolipomas, 2 angiomyolipomas with epithelial cysts and 1 example of lymphangioleiomyomatosis of the renal sinus. In all of the renal PEComas, cathepsin K was found to be constantly and strongly expressed and seems to be a more powerful marker than other commonly used markers for their identification, especially to confirm the diagnosis on needle biopsies.

Differential expression of cathepsin K in neoplasms harboring TFE3 gene fusions

Cathepsin K is a protease whose expression is driven by microphthalmia transcription factor (MITF) in osteoclasts. TFE3 and TFEB are members of the same transcription factor subfamily as MITF and all three have overlapping transcriptional targets. We have shown that all t(6;11) renal cell carcinomas, which harbor an Alpha-TFEB gene fusion, as well as a subset of the Xp11 translocation renal carcinomas, which harbor various TFE3 gene fusions, express cathepsin K, while no other common renal carcinoma does. We have hypothesized that overexpression of TFEB or certain TFE3 fusion proteins function like MITF in these neoplasms, and thus activate cathepsin K expression. However, the expression of cathepsin K in specific genetic subtypes of Xp11 translocation carcinomas, as well as alveolar soft part sarcoma, which harbors the same ASPSCR1-TFE3 gene fusion as some Xp11 translocation carcinomas, has not been addressed. We performed immunohistochemistry for cathepsin K on 14 genetically confirmed t(X;1)(p11;q21) carcinomas, harboring the PRCC-TFE3 gene fusion; eight genetically confirmed t(X;17)(p11;q25) carcinomas, harboring the ASPSCR1-TFE3 gene fusion; and 18 alveolar soft part sarcomas (12 genetically confirmed), harboring the identical ASPSCR1-TFE3 gene fusion. All 18 alveolar soft part sarcomas expressed cathepsin K. In contrast, all eight ASPSCR1-TFE3 carcinomas were completely negative for cathepsin K. However, 12 of 14 PRCC-TFE3 carcinomas expressed cathepsin K. Expression of cathepsin K distinguishes alveolar soft part sarcoma from the ASPSCR1-TFE3 carcinoma, harboring the same gene fusion. The latter can be useful diagnostically, especially when alveolar soft part sarcoma presents in an unusual site (such as bone) or with clear cell morphology, which raises the differential diagnosis of metastatic ASPSCR1-TFE3 renal cell carcinoma. The difference in expression of cathepsin K between the PRCC-TFE3 and ASPSCR1-TFE3 carcinomas, together with the observed clinical differences between these subtypes of Xp11 translocation carcinomas, suggests the possibility of functional differences between these two related fusion proteins.

Molecular Pathology of Lymphangioleiomyomatosis and Other Perivascular Epithelioid Cell Tumors

CONTEXT Lymphangioleiomyomatosis (LAM) is a cystic lung disease that can be included in the wide group of proliferative lesions named PEComas (perivascular epithelioid cell tumors). These proliferative tumors are characterized by the coexpression of myogenic and melanogenesis-related markers. In all these lesions, genetic alterations related to the tuberous sclerosis complex (TSC) have been demonstrated. Striking improvements in the understanding of the genetic basis of this autosomal dominant genetic disease are coupled to the understanding of the mechanisms that link the loss of TSC1 (9q34) or TSC2 (16p13.3) genes with the regulation of the Rheb/m-TOR/p70S6K pathway. These data have opened a new era in the comprehension of the pathogenesis of LAM and have also suggested new therapeutic strategies for this potentially lethal disease. OBJECTIVE To present and discuss the pathologic and molecular features of LAM within the spectrum of PEComas, providing a rational approach to their diagnosis. DATA SOURCES The published literature and personal experience. CONCLUSIONS The inclusion of LAM within the PEComa category is supported by a variety of biologic data and can significantly help in providing a comprehensive view of this interesting and clinically relevant group of lesions. The demonstration of molecular alterations of the mTOR pathway in LAM and other PEComas represents a rational basis for innovative therapeutic approaches with inhibitors of mTOR signaling.