Hepatocyte growth factor (HGF/SF) is a potent renal proximal tubular cell (PTEC) mitogen involved in renal development. HGF/SF is the functional ligand for the c-met proto-oncogene, and germline c-met mutations are associated with familial papillary renal cell carcinoma. Somatic von Hippel-Lindau disease tumour-suppressor gene (VHL) mutations are frequently detected in sporadic clear cell renal cell carcinomas (RCC), and germline VHL mutations are the commonest cause of familial clear cell RCC. pVHL binds to the positive regulatory components of the trimeric elongin (SIII) complex (elongins B and C) and has been observed to deregulate expression of the vascular endothelial growth factor (VEGF) gene. HGF/SF has similarly been reported to up-regulate expression of the VEGF gene in non-renal experimental systems. To investigate the mechanism of HGF/SF action in PTECs and, specifically, to examine potential interactions between the HGF/c-met and the VHL-mediated pathways for renal tubular growth control, we have isolated untransformed PTECs from normal kidneys, developed conditions for their culture in vitro and used these cells to investigate changes in mRNA levels of the VHL, elongin A, B and C, VEGF, c-myc, c-fos and c-met genes after HGF/SF exposure. Significant elevations in the mRNA levels of VEGF, c-myc, c-fos, c-met and elongins A, B and C, but not VHL, were detected after HGF/SF stimulation of human PTECs (P < 0.02), with a consistent order of peak levels observed over successive replicates (c-fos at 1 h, VEGF at 2-4 h, c-myc, at 4 h, followed by c-met and all three elongin subunits at 8 h). This study highlights the spectrum of changes in gene expression observed in PTECs after HGF/SF stimulation and has identified possible candidate mediators of the HGF/SF-induced mitogenic response. Our evidence would suggest that the changes in PTEC VEGF expression induced by HGF/SF are mediated by a VHL-independent pathway.
Claudin-7 has recently been suggested to be a distal nephron marker. We tested the possibility that expression of claudin-7 could be used as a marker of renal tumors originating from the distal nephron. We examined the immunohistochemical expression of claudin-7 and parvalbumin in 239 renal tumors, including 179 clear cell renal cell carcinoma (RCC)s, 29 papillary RCCs, 20 chromophobe RCCs, and 11 renal oncocytomas. In addition, the methylation specific-PCR (MSP) of claudin-7 was performed. Claudin-7 and parvalbumin immunostains were positive in 3.4%, 7.8% of clear cell RCCs, 34.5%, 31.0% of papillary RCCs, 95.0%, 80.0% of chromophobe RCCs, and 72.7%, 81.8% of renal oncocytomas, respectively. The sensitivity and specificity of claudin-7 in diagnosing chromophobe RCC among subtypes of RCC were 95.0% and 92.3%. Those of parvalbumin were 80.0% and 88.9%. The expression pattern of claudin-7 was mostly diffuse in chromophobe RCC and was either focal or diffuse in oncocytoma. All of the cases examined in the MSP revealed the presence of unmethylated promoter of claudin-7 without regard to claudin-7 immunoreactivity. Hypermethylation of the promoter might not be the underlying mechanism for loss of its expression in RCC. Claudin-7 can be used as a useful diagnostic marker in diagnosing chromophobe RCC and oncocytoma.
Chromophobe Renal Cell Carcinoma; Oncocytoma; Claudin-7
Parafibromin, encoded by HRPT2 gene, is a recently identified tumor suppressor. Complete and partial loss of its expression have been observed in hyperparathyroidism-jaw tumor (HPT-JT), parathyroid carcinoma, breast carcinoma, lung carcinoma, gastric and colorectal carcinoma. However, little has been known about its expression in renal tumors. In order to study the expression of parafibromin in a series of the 4 major renal cell tumors - clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC), chromophobe renal cell carcinoma (chRCC) and oncocytoma, one hundred thirty nine renal tumors including 61 ccRCCs, 37 pRCCs, 22 chRCCs and 19 oncocytomas were retrieved and used for the construction of renal tissue microarrays (TMAs). The expression of parafibromin was detected by immunohistochemical method on the constructed TMAs. Positive parafibromin stains are seen in 4 out of 61 ccRCCs (7%), 7 out of 37 pRCCs (19%), 12 out of 23 chRCCs (52%) and all 19 oncocytomas (100%). Parafibromin expression varies significantly (P<8.8×10−16) among the four major renal cell tumors and were correlated closely with tumor types. No correlation of parafibromin expression with tumor staging in ccRCCs, pRCCs and chRCCs, and Fuhrman nuclear grading in ccRCCs and pRCCs was seen. In summary, parafibromin expression was strongly correlated with tumor types, which may suggest that it plays a role in the tumorigenesis in renal cell tumors.
parafibromin; renal epithelial tumors.
It has been documented that some tissues, such as salivary gland, liver, cardiac and skeletal muscles and kidney, have high level endogenous biotin or endogenous avidin binding activity (EABA). Limited data is available on EABA in renal cell neoplasms. A tissue microarray (TMA) was constructed that included oncocytoma (n=30), chromophobe renal cell carcinoma (RCC) (n=18), clear cell RCC (n=45), clear cell RCC with granular/eosinophilic (G/E) features (n=19), papillary RCC (n=21), papillary RCC with G/E features (n=29) and benign renal tissues (n=31). The TMA slides were stained with or without biotin blocker and analyzed using the automated cellular imaging system (ACIS®). Without biotin blocker, a high positive rate of EABA was found in oncocytoma (56/60, 93%) and normal renal tubules (46/60, 77%). A moderate positive rate of EABA was found in clear cell and papillary RCCs with G/E features (13/39, 33% and 19/55, 35%, respectively). Chromophobe RCC and RCC without G/E features had essentially no EABA. With biotin blocker, benign renal tissue and clear cell RCC were negative for EABA; but a significant number of renal oncocytoma (29/60, 48%) and a few papillary RCC with G/E features (5/52, 10%) remained positive for EABA. In conclusion, high EABA may be used to differentiate oncocytoma from chromophobe RCC, and the staining results must be interpreted with caution when avidin-biotin detection system is used in diagnosing renal neoplasms.
Oncocytoma; endogenous avidin binding activity (EABA); chromophobe renal cell carcinoma
Kidney cancer is one of the 10 most common forms of cancer in both men and women. Ninety percent or more of these cancers are believed to be of epithelial cell origin, and are referred to as renal cell carcinoma (RCC). RCCs can be further subdivided, based on their histologic appearance, into clear-cell renal carcinomas (~75%), papillary renal carcinomas (15%), chromophobe tumors (5%), and oncocytomas (5%).1,2 Studies of hereditary kidney cancer families led to the identification of genes that, when mutated in the germline, confer an increased risk of these various histologic RCC subtypes and hence a glimpse at the molecular circuits that are deregulated in these different forms of RCC.2 In practice, there is some overlap among the histologic subtypes (eg, a tumor with predominantly clear-cell features might contain areas more typical of papillary RCC). Similarly, there are some shared molecular features among these tumor types (see later discussion). This review focuses primarily on the most common form of RCC, clear-cell renal carcinoma, while making note of some recent advances in the other histologic subtypes.
Renal cell carcinoma; Clear-cell renal carcinoma; Hypoxia-inducible factor; HIF-responsive gene products
The presence of cytoplasmic sialyl glycoproteins is a conspicuous feature in chromophobe renal cell carcinoma (RCC). We compared the immunohistochemical expression of sialyl glycoproteins in chromophobe RCC with that in other types of renal tumors. Formalin-fixed, paraffin-embedded tissues of surgically resected renal tumors (chromophobe RCC, 14 cases [10 cases of classic type and 4 cases of eosinophilic variant]; oncocytoma, 7 cases; and clear cell RCC, 9 cases) and kidneys from immature infants (4 cases) were immunostained with antibodies against sialyl glycoproteins (anti-KL-6 and anti-sialyl MUC1 antibodies). Cytoplasmic expression of KL-6 and sialyl MUC1 was distinctive in the chromophobe RCC and renal oncocytoma cells, and in the intercalated cells in collecting duct epithelia. Apical-surface staining of these sialyl glycoproteins was predominantly observed in clear RCC, in the epithelia of the distal tubule and collecting duct, and in the neonatal renal proximal tubule, but not in those of the adult renal proximal tubule. The above-mentioned observations provide additional evidence for similar phenotypic profiles of chromophobe RCC and renal oncocytoma, and the intercalated cells in collecting ducts and the oncofetal expression of sialyl glycoproteins in clear cell RCC. KL-6 is a potential tumor marker for renal tumors.
chromophobe renal cell carcinoma; immunohistochemistry; KL-6; MUC1; renal cell tumor
Renal tumors consist of heterogeneous groups that frequently show complex and overlapping morphology, thus making it difficult to make a correct diagnosis. One of the most problematic differential diagnoses is to distinguish chromophobe renal cell carcinoma (RCC) from oncocytoma. These should be distinguished by differences in their behavior and clinical outcome. Our study was performed to identify whether caveolin-1 and MOC-31 are useful immunohistochemical markers for differentiating chromophobe RCC from oncocytoma.
Materials and Methods
We selected 23 chromophobe RCCs, 8 oncocytomas, and 25 clear cell RCCs and performed immunohistochemical staining for caveolin-1 and MOC-31.
Caveolin-1 was positive in 20 (87%) of 23 chromophobe RCCs, 0 of 8 oncocytomas, and 21 (84%) of 25 clear cell RCCs. MOC-31 was positive in 22 (96%) of 23 chromophobe RCCs, 2 (25%) of 8 oncocytomas, and 14 (56%) of 25 clear cell RCCs. There was a statistically significant difference in the expression of caveolin-1 and MOC-31 between chromophobe RCC and oncocytoma (p<0.001). In addition, clear cell RCC was also significantly different from oncocytoma in the expression of caveolin-1 (p<0.001) and was significantly different from chromophobe RCC in the expression of MOC-31 (p<0.001).
Caveolin-1 and MOC-31 can be useful markers in the differential diagnosis of chromophobe RCC, oncocytoma, and clear cell RCC.
Adenoma, oxyphilic; Caveolin 1; MOC-31 monoclonal antibody, human; Renal cell carcinoma
Renal cell carcinoma (RCC) is not a single entity, but comprises a group of tumors including clear cell RCC, papillary RCC and chromophobe RCC, which arise from the epithelium of renal tubules. The majority of clear cell RCCs, the major histological subtype, have genetic or epigenetic inactivation of the von Hippel-Lindau (VHL) gene. Germline mutations in the MET and fumarate hydratase (FH) genes lead to the development of type 1 and type 2 papillary RCCs, respectively, and such mutations of either the TSC1 or TSC2 gene increase the risk of RCC. Genome-wide copy number alteration analysis has suggested that loss of chromosome 3p and gain of chromosomes 5q and 7 may be copy number aberrations indispensable for the development of clear cell RCC. When chromosome 1p, 4, 9, 13q or 14q is also lost, more clinicopathologically aggressive clear cell RCC may develop. Since renal carcinogenesis is associated with neither chronic inflammation nor persistent viral infection, and hardly any histological change is evident in corresponding non-tumorous renal tissue from patients with renal tumors, precancerous conditions in the kidney have been rarely described. However, regional DNA hypermethylation on C-type CpG islands has already accumulated in such non-cancerous renal tissues, suggesting that, from the viewpoint of altered DNA methylation, the presence of precancerous conditions can be recognized even in the kidney. Genome-wide DNA methylation profiles in precancerous conditions are basically inherited by the corresponding clear cell RCCs developing in individual patients: DNA methylation alterations at the precancerous stage may further predispose renal tissue to epigenetic and genetic alterations, generate more malignant cancers, and even determine patient outcome. The list of tumor-related genes silenced by DNA hypermethylation has recently been increasing. Genetic and epigenetic profiling provides an optimal means of prognostication for patients with RCCs. Recently developed high-throughput technologies for genetic and epigenetic analyses will further accelerate the identification of key molecules for use in the prevention, diagnosis and therapy of RCCs.
Renal cell carcinoma; copy number alteration; DNA methylation; precancerous condition; prognostication
Adult renal epithelial neoplasms are a heterogeneous group with varying prognosis and outcome requiring sub-classification.
Cases of renal cell carcinoma (RCC) in a 10 years period were analyzed with regard to the clinical features and histology. Sections were reviewed by four pathologists and the discordant cases were resolved with the help of Hale's colloidal iron stain, vimentin, CK 7, and vinculin immunostains and electron microscopy.
Amongst the total of 278 cases, clear cell renal cell carcinoma was the commonest tumor with 74.8% cases, followed by papillary RCC 12.2%, chromophobe RCC 7.9%, oncocytoma 1.8%, and one case of collecting duct RCC. Eight cases were of sarcomatoid renal cell carcinoma. In 28/278 cases, diagnoses varied amongst the four pathologists and the discordance was resolved by Hale's colloidal iron stain, CK7 immunostain and electron microscopy. Vimentin and vinculin did not contribute much in differentiating subtypes of renal cell carcinomas. Relative incidence of sub-types of RCCs was compared with other series
To accurately subclassify renal cell carcinomas, simple ancillary techniques would possibly resolve all difficult cases. The relative incidence of sub-types of renal cell carcinoma is relatively consistent the world over. However, in India, RCCs afflict the patients two decades earlier.
The most common renal tumours are clear cell, papillary, chromophobe and collecting duct renal cell carcinomas (RCCs), and benign oncocytomas and angiomyolipomas. Tumours with hybrid features between some of these entities have been recognised; in particular, tumours with features of both chromophobe RCC and oncocytoma. Case reports describing one distinct type of primary renal tumour actually within another are very rare. The incidental finding of a papillary RCC located in an oncocytoma in a nephrectomy specimen from a 75‐year‐old man is described. Morphological criteria for each tumour type were completely satisfied and fluorescence in situ hybridisation detected the expected number of copies of chromosome 7 in the cells of each tumour type. The cells in the papillary tumour contained three copies, whereas the oncocytoma cells contained only two per nucleus. To our knowledge, this is the first report of a papillary RCC being identified within an oncocytoma.
Signal transducer and activator of transcription 3 (Stat3) plays a vital role in signal transduction pathways that mediate transformation and inhibit apoptosis. Oncogenic Stat3 is persistently activated in several human cancers and transformed cell lines. Previous studies indicate activation of Stat3 in renal cell carcinoma (RCC). However, the detailed characterization of the Stat3 expression pattern in different histologic types of RCC is lacking. We have analyzed the immunoprofile of activated or phosphorylated Stat3 (pStat3) in a tissue microarray of renal tumors of different histologic types, including 42 cases of conventional clear cell type, 24 chromophobe, and 7 papillary, 15 oncocytoma, 7 urothelial carcinoma and 21 normal kidney tissues using an anti-pStat3 antibody (recognizes only activated STAT3). pStat3 nuclear staining was observed in 25 of 42 conventional clear cell RCC (59.5 %), 8 of 24 chromophobe RCC (33.3%), 4 of 7 papillary RCC (57.1%). In the other tumor groups, 4 of 15 oncocytomas (26.7%) and 6 of 7 urothelial carcinomas (85.7%) showed positive nuclear staining. Weak nuclear immunoreactivity for pStat3 was seen in 4 of 21 cases of non-neoplastic kidney tissue (19.0%). The extent of Stat3 activation as determined by nuclear expression of its phosphorylated form is increased in histologic types of renal tumors with greater malignant potential, specifically conventional clear cell RCC, papillary RCC and urothelial carcinoma, only slightly increased in chromophobe RCC, and not increased in oncocytoma. These results suggest a role of Stat3 activation in different types of renal neoplasia, possibly serving as a prognostic marker or therapeutic target.
Signal Transducer and activator of transcription 3 (Stat3); signal transduction; phosphorylation; renal tumors; kidney cancers
Familial renal cell carcinoma (RCC) is genetically heterogeneous. Genetic predisposition to clear cell RCC (CCRCC) is a major feature of von Hippel-Lindau (VHL) disease (MIM 193300) and has rarely been associated with chromosome 3 translocations. In addition, familial papillary (non-clear cell) RCC may result from germline mutations in the MET proto-oncogene (MIM 164860). However, rare kindreds with familial CCRCC (FCRC) not linked to the VHL tumour suppressor gene have been described suggesting that further familial RCC susceptibility genes exist. To investigate the genetic epidemiology of FCRC, we undertook a clinical and molecular study of FCRC in nine kindreds with two or more cases of CCRCC in first degree relatives. FCRC was characterised by an earlier age at onset (mean 47.1 years, 52% of cases <50 years of age) than sporadic cases. These findings differ from the only previous report of two FCRC kindreds and have important implications for renal surveillance in FCRC. The molecular basis of CCRCC susceptibility was investigated in nine FCRC kindreds and seven isolated cases with features of possible genetic susceptibility to CCRCC (four bilateral CCRCC aged <50 years and three with unilateral CCRCC aged <30 years). No germline mutations were detected in the VHL or MET genes, suggesting that FCRC is not allelic with VHL disease or HPRC. As binding of the VHL gene product to the CUL2 protein is important for pVHL function, we then searched for germline CUL2 mutations. Although CUL2 polymorphisms were identified, no pathogenic mutations were detected. These findings further define the clinical features of FCRC and exclude a major role for mutations in VHL, MET, or CUL2 in this disorder.
Keywords: familial clear cell renal carcinoma; VHL; MET; CUL2
Urothelial carcinoma (UC) of the kidney is a relatively rare but aggressive form of kidney cancer. Differential diagnosis of renal UC from renal cell carcinoma (RCC) can be difficult, but is critical for correct patient management. We aimed to use global gene expression profiling to identify genes specifically expressed in urothelial carcinoma (UC) of the kidney, with purpose of finding new biomarkers for differential diagnosis of UC of both upper and lower tract from normal tissues.
Materials and methods:
Microarray gene expression profiling was performed on a variety of human kidney tumor samples, including clear cell, papillary, chromophobe, oncocytoma, renal UC and normal kidney controls. Differentially expressed mRNAs in various kidney tumor subtypes were thus identified. Protein expression in human UC tumor samples from both upper and lower urinary tract was evaluated by immunohistochemistry.
FXYD3 (MAT-8) mRNA was specifically expressed in UC of the kidney and not in normal kidney tissue or in any RCC tumor subtypes. FXYD3 mRNA levels displayed equal or better prediction rate for the detection of renal UC than the mRNA levels of selected known UC markers as p63, vimentin, S100P, KRT20 and KRT7. Finally, immunohistochemical staining of clinical UC samples showed that FXYD3 protein is overexpressed in majority of UC of the upper genitourinary tract (encompassing the kidney, ∼90%) and in majority of high grade bladder UC (∼84%, it’s <40% in low grade tumors, P < 0.001) compared to normal kidney and bladder tissues.
FXYD3 may be a promising novel biomarker for the differential diagnosis of renal UC and a promising prognosis marker of UC from bladder. Because it was identified genome-widely, FXYD3 may have important biological ramifications for the genetic study of UC.
FXYD3; FXYD; urothelial carcinoma; kidney; marker; microarray
In this study, we aimed to evaluate the protein expression profile of a spectrum of renal cell carcinomas (RCC) to find potential biomarkers for disease onset and progression and therefore, prospective therapeutic targets. A 2D-gel based proteomic analysis was used to outline differences in protein levels among different subtypes of renal cell carcinomas, including clear cell carcinomas, papillary lesions, chromophobe tumors and renal oncocytomas. Spot pattern was compared to the corresponding normal kidney from the same patients and distinctive, differentially expressed proteins were characterized by mass spectrometry. Twenty-one protein spots were found differentially expressed between clear cell RCC and normal tissue and 38 spots were found expressed in chromophobe tumors. Eleven proteins were identified, with most differentially expressed -by fold change- between clear cell tumors and the corresponding normal tissue. Two of the identified proteins, Triosephosphate isomerase 1 (TPI-1) and Heat Shock protein 27 (Hsp27), were further validated in a separate set of tumors by immunohistochemistry and expression levels were correlated with clinicopathologic features of the patients. Hsp27 was highly expressed in 82% of the tumors used for validation, and all cases showed strong immunoreactivity for TPI-1. In both Hsp27 and TPI-1, protein expression positively correlated with histologic features of the disease. Our results suggest that the subjacent cytogenetic abnormalities seen in different histological types of RCC are followed by specific changes in protein expression. From these changes, Hsp27 and TPI-1 emerged as potential candidates for the differentiation and prognosis in RCC.
Renal cell carcinoma; proteomics; protein profiling; biomarker; Hsp27; TPI-1
Previous studies have revealed altered expression of epidermal growth factor receptor (EGFR)-family members and their endogenous inhibitor leucine-rich and immunoglobulin-like domains 1 (LRIG1) in renal cell carcinoma (RCC). In this study, we analyzed the gene expression levels of EGFR-family members and LRIG1, and their possible associations with clinical parameters in various types of RCC.
Gene expression levels of EGFR–family members and LRIG1 were analyzed in 104 RCC samples, including 81 clear cell RCC (ccRCC), 15 papillary RCC (pRCC), and 7 chromophobe RCC (chRCC) by quantitative real-time RT-PCR. Associations between gene expression levels and clinical data, including tumor grade, stage, and patient survival were statistically assessed.
Compared to kidney cortex, EGFR was up-regulated in ccRCC and pRCC, LRIG1 and ERBB2 were down-regulated in ccRCC, and ERBB4 was strongly down-regulated in all RCC types. ERBB3 expression did not differ between RCC types or between RCC and the kidney cortex. The expression of the analyzed genes did not correlate with patient outcome.
This study revealed that the previously described up-regulation of EGFR and down-regulation of ERBB4 occurred in all analyzed RCC types, whereas down-regulation of ERBB2 and LRIG1 was only present in ccRCC. These observations illustrate the need to evaluate the different RCC types individually when analyzing molecules of interest and potential biological markers.
Renal cell carcinoma; EGFR; ERBB2; ERBB3; ERRB4; LRIG1; Survival
Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are two distinct but closely related entities with strong morphologic and genetic similarities. While chRCC is a malignant tumor, oncocytoma is usually regarded as a benign entity. The overlapping characteristics are best explained by a common cellular origin, and the biologic differences between chRCC and oncocytoma are therefore of considerable interest in terms of carcinogenesis, diagnosis and clinical management. Previous studies have been relatively limited in terms of examining the differences between oncocytoma and chromophobe RCC.
Gene expression profiling using the Affymetrix HGU133Plus2 platform was applied on chRCC (n = 15) and oncocytoma specimens (n = 15). Supervised analysis was applied to identify a discriminatory gene signature, as well as differentially expressed genes. High throughput single-nucleotide polymorphism (SNP) genotyping was performed on independent samples (n = 14) using Affymetrix GeneChip Mapping 100 K arrays to assess correlation between expression and gene copy number. Immunohistochemical validation was performed in an independent set of tumors.
A novel 14 probe-set signature was developed to classify the tumors internally with 93% accuracy, and this was successfully validated on an external data-set with 94% accuracy. Pathway analysis highlighted clinically relevant dysregulated pathways of c-erbB2 and mammalian target of rapamycin (mTOR) signaling in chRCC, but no significant differences in p-AKT or extracellular HER2 expression was identified on immunohistochemistry. Loss of chromosome 1p, reflected in both cytogenetic and expression analysis, is common to both entities, implying this may be an early event in histogenesis. Multiple regional areas of cytogenetic alterations and corresponding expression biases differentiating the two entities were identified. Parafibromin, aquaporin 6, and synaptogyrin 3 were novel immunohistochemical markers effectively discriminating the two pathologic entities.
Gene expression profiles, high-throughput SNP genotyping, and pathway analysis effectively distinguish chRCC from oncocytoma. We have generated a novel transcript predictor that is able to discriminate between the two entities accurately, and which has been validated both in an internal and an independent data-set, implying generalizability. A cytogenetic alteration, loss of chromosome 1p, common to renal oncocytoma and chRCC has been identified, providing the opportunities for identifying novel tumor suppressor genes and we have identified a series of immunohistochemical markers that are clinically useful in discriminating chRCC and oncocytoma.
Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G0) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.
Renal cell carcinoma (RCC) is histopathologically heterogeneous with clear cell and papillary the most common subtypes. The most frequent molecular abnormality in clear cell RCC is VHL inactivation but promoter methylation of tumour suppressor genes is common in both subtypes of RCC. To investigate whether RCC CpG methylation status was influenced by histopathology and VHL status we performed high-throughput epigenetic profiling using the Illumina Goldengate Methylation Array in 62 RCC (29 RCC from von Hippel-Lindau (VHL) disease patients, 20 sporadic clear cell RCC with wild type VHL and 13 sporadic papillary RCC).
43 genes were methylated in >20% of primary RCC (range 20–45%) and most (37/43) of these had not been reported previously to be methylated in RCC. The distribution of the number of methylated CpGs in individual tumours differed from the expected Poisson distribution (p < 0.00001; log-likelihood G test) suggesting that a subset of RCC displayed a CpG Island Methylator Phenotype. Comparison of RCC subtypes revealed that, on average, tumour specific CpG methylation was most prevalent in papillary RCC and least in VHL RCC. Many of the genes preferentially methylated in pRCC were linked to TGFβ or ERK/Akt signalling.
These findings demonstrate differing patterns of tumour-specific CpG methylation in VHL and non VHL clear cell RCC and papillary RCC, and identify multiple novel potential CpG methylation biomarkers for RCC.
We demonstrate the constitutive expression of interleukin 6 (IL-6), IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in normal kidney cells, and in the majority of renal oncocytomas, papillary and non-papillary renal cell carcinomas (RCCs) by reverse transcriptase polymerase chain reaction (RT-PCR) technique. No expression of IL-6 and TGF-alpha and variable expression of GM-CSF, IL-8, EGF and EGFR was seen in chromophobe RCCs. The lack of expression of IL-6 and TGF-alpha might be correlated with the growth pattern, poor vascularity and low malignancy of chromophobe RCCs.
This study investigated the expression of hypoxia-inducible protein 2 (HIG2), a novel renal cell carcinoma (RCC)-associated molecule and an essential growth factor for RCC, in kidneys to elucidate its clinical significance in RCC. An immunohistochemical study of HIG2 was conducted in 93 surgical samples of RCC tissues and 10 samples of normal kidney tissues obtained after nephrectomies for localized RCC. HIG2 expression was also correlated with clinicopathological characteristics and survival. Only faint or weak immunostaining for HIG2 was observed in normal kidney samples. HIG2 expression was found in 86% of RCC tissues (80/93). When analyzed by histological type, positive staining for HIG2 was detected in all papillary (7/7), chromophobe (1/1) and cyst-associated (3/3) RCC. In contrast, the HIG2 expression was observed in 85% of clear cell (68/80) and 50% of spindle cell (1/2) RCC. Labeling indices were 74.1, 45.4, 39, 24.8 and 12.1% in papillary, spindle, clear cell, cyst-associated and chromophobe RCC, respectively. A significant increase in HIG2 expression was noted in RCC tissues obtained from patients with high stage RCC, lymph node metastasis and high nuclear grade (p<0.001, p<0.02 and p<0.006, respectively). RCC patients with a negative HIG2 staining had prolonged 5-year cancer-specific survival. In conclusion, HIG2 expression was extensively observed in RCC tissues and was higher in advanced RCC, suggesting that HIG2 is a candidate for the development of molecular targeting therapy.
renal cell carcinoma; hypoxia-inducible protein 2; tumor stage; nuclear grade; cancer-specific survival
Germline mutations in the folliculin (FLCN) gene are associated with the development of Birt-Hogg-Dubé syndrome (BHDS), a disease characterized by papular skin lesions, a high occurrence of spontaneous pneumothorax, and the development of renal neoplasias. The majority of renal tumors that arise in BHDS-affected individuals are histologically similar to sporadic chromophobe renal cell carcinoma (RCC) and sporadic renal oncocytoma. However, most sporadic tumors lack FLCN mutations and the extent to which the BHDS-derived renal tumors share genetic defects associated with the sporadic tumors has not been well studied.
BHDS individuals were identified symptomatically and FLCN mutations were confirmed by DNA sequencing. Comparative gene expression profiling analyses were carried out on renal tumors isolated from individuals afflicted with BHDS and a panel of sporadic renal tumors of different subtypes using discriminate and clustering approaches. qRT-PCR was used to confirm selected results of the gene expression analyses. We further analyzed differentially expressed genes using gene set enrichment analysis and pathway analysis approaches. Pathway analysis results were confirmed by generation of independent pathway signatures and application to additional datasets.
Renal tumors isolated from individuals with BHDS showed distinct gene expression and cytogenetic characteristics from sporadic renal oncocytoma and chromophobe RCC. The most prominent molecular feature of BHDS-derived kidney tumors was high expression of mitochondria-and oxidative phosphorylation (OXPHOS)-associated genes. This mitochondria expression phenotype was associated with deregulation of the PGC-1α-TFAM signaling axis. Loss of FLCN expression across various tumor types is also associated with increased nuclear mitochondrial gene expression.
Our results support a genetic distinction between BHDS-associated tumors and other renal neoplasias. In addition, deregulation of the PGC-1α-TFAM signaling axis is most pronounced in renal tumors that harbor FLCN mutations and in tumors from other organs that have relatively low expression of FLCN. These results are consistent with the recently discovered interaction between FLCN and AMPK and support a model in which FLCN is a regulator of mitochondrial function.
Whether an oncofetal protein, IMP3, can serve as a prognostic biomarker to predict metastasis for patients with localized papillary and chromophobe subtypes of renal cell carcinomas (RCCs) was investigated.
The expression of IMP3 in 334 patients with primary papillary and chromophobe RCC from multiple medical centers was evaluated by immunohistochemistry. The 317 patients with localized papillary and chromophobe RCCs were further evaluated for outcome analyses.
IMP3 was significantly increased in a subset of localized papillary and chromophobe RCCs that subsequently metastasized. Patients with localized IMP3-positive tumors (n = 33; 10%) were over 10 times more likely to metastasize (risk ratio [RR], 11.38; 95% confidence interval [CI], 5.40–23.96; P <.001) and were nearly twice as likely to die (RR, 1.91; 95% CI, 1.13–3.22; P =.016) compared with patients with localized IMP3 negative tumors. The 5-year metastasis-free and overall survival rates were 64% and 58% for patients with IMP3-positive localized papillary and chromophobe RCCs compared with 98% and 85% for patients with IMP3 negative tumors, respectively. In multivariable analysis adjusting for the TNM stage and nuclear grade, patients with IMP3-positive tumors were still over 10 times more likely to progress to distant metastasis (RR, 13.45; 95% CI, 6.00–30.14; P <.001) and were still nearly twice as likely die (RR, 1.95; 95% CI, 1.15–3.31; P =.013) compared with patients with IMP3-negative tumors.
IMP3 is an independent prognostic biomarker that can be used to identify a subgroup of patients with localized papillary and chromophobe RCC who are at high risk for developing distant metastasis.
IMP3; oncofetal protein; mRNA binding protein; prognostic biomarker; papillary renal cell carcinoma; chromophobe renal cell carcinoma
To elucidate the novel and common genetic changes in histopathologic subtype-specific profiles of renal cell carcinomas (RCCs), a newly developed high-resolution whole-genome array analysis was applied. Human CNV370-Duo DNA Analysis BeadChip (genotype 370K) was used in a panel of 22 primary clear cell RCCs (ccRCCs), seven papillary RCCs (PRCCs) (six type II and one type I) and eight chromophobe RCCs (ChRCCs). In ccRCC, a chromosome 3p loss was identified in 95% of the carcinomas, suggesting that 3p loss is the first stage in ccRCC carcinogenesis. Other frequent changes included losses of 1p (23%), 3q (46%) and 8p (32%), and gains of 5q (32%), 7p (27%), 7q (27%) and 1q (23%). The most frequent chromosomal losses in PRCC (43%) were noted in 3p and 3q, followed by 29% of losses of 1p, 1q, 11q, 18q, 22p and 22q, and gains of 20q (57%), 20p (43%), 8q (43%) and 12q (43%). Loss of the entire chromosomes 1, 2, 6, 8, 10, 13 or 17 was noted in patients with ChRCC. A high-density single nucleotide polymorphism array analysis confirmed that partial chromosomal changes rarely occur in ChRCC. Additionally, 32 microdeletions and 10 microamplifications of less than 1 Mb were detected, which may represent potential candidate tumor suppressor genes and oncogenes, respectively.
single nucleotide polymorphism array; comparative genomic hybridization; renal cell carcinoma
Renal Cell Carcinoma (RCC) has the highest mortality rate of the genitourinary cancers and the incidence of RCC has risen steadily. If detected early, RCC is curable by surgery although a minority are at risk of recurrence. Increasing incidental detection and an ageing population has led to active surveillance as an option for patients with small renal masses. RCC is heterogeneous and comprises several histological cell types with different genetics, biology and behavior. The identification of the genes predisposing to inherited syndromes with RCC has provided much of our knowledge of the molecular basis of early sporadic RCC. Many of the oncogenes and tumor suppressor genes that are mutated leading to pathway dysregulation in RCC remain to be elucidated. Global studies of copy number, gene sequencing, gene expression, miRNA expression and gene methylation in primary RCC will lead towards this goal. The natural history of RCC indicated by candidate precursor lesions, multifocal or bilateral disease, growth rate of small renal masses under surveillance, and high risk populations provide insight into the behavior of this disease. The use of molecular markers for early detection and prognosis merits more attention with ongoing advances in omics technologies. This review focuses on early RCC, that is disease confined within the renal capsule.
RCC; Genetics; Epigenetics; Natural history
Clear cell renal cell carcinoma (ccRCC) and chromophobe renal cell carcinoma (chRCC) can usually be distinguished by histologic characteristics. Occasionally, diagnosis proves challenging and diagnostic difficulty will likely increase as needle biopsies of renal lesions become more common.
To identify markers that aid in differentiating ccRCC from chRCC, we used gene expression profiles to identify candidate markers that correlate with histology. 39 antisera and antibodies, including 35 for transcripts identified from gene expression profiling, were evaluated. Promising markers were tested on a tissue microarray (TMA) containing 428 renal neoplasms. Strength of staining of each core on the TMA was formally scored and the distribution of staining across different types of renal neoplasms was analyzed.
Based on results from initial immunohistochemical staining of multitissue titer arrays, 23 of the antisera and antibodies were selected for staining of the TMA. For 7 of these markers, strength of staining of each core on the TMA was formally scored. Vimentin (positive in ccRCC) and CD9 (positive in chRCC) best distinguished ccRCC from chRCC. The combination of vimentin negativity and CD9 positivity was found to distinguish chRCC from ccRCC with a sensitivity of 100.0% and a specificity of 95.2%.
Based on gene expression analysis, we identify CD9 and vimentin as candidate markers for distinguishing between ccRCC and chRCC. In difficult cases and particularly when the amount of diagnostic tissue is limited, vimentin and CD9 staining could serve as a useful adjunct in the differential diagnosis of ccRCC and chRCC.