Genetic changes of the E-cadherin and APC tumour suppressor genes in clear cell renal cell carcinoma

AIMS: The roles of tumour suppressor genes: adenomatous polyposis coli (APC) and E-cadherin (CDH1) were investigated in clear cell renal cell carcinoma. METHODS: Forty-five human clear cell renal cell carcinomas were tested for APC gene instability by polymerase chain reaction/loss of heterozygosity using the restriction fragment length polymorphism method. E-cadherin gene was analysed by PCR amplification of tetranucleotide marker (D16S752) and the alleles were visualised by PAGE/silver staining. RESULTS: The overall proportion of loss of heterozygosity of the APC gene was 37.5% (9/24). D16S752 marker linked to E-cadherin gene (informativeness 91%) revealed three samples with loss of heterozygosity (7.5%). Interestingly, replication error phenotype was detected in 9.1% of clear cell renal cell carcinoma samples. Multivariate statistical analysis of samples informative for both APC and E-cadherin genes showed that, in this data set, loss of heterozygosity of the APC gene is correlated with advanced age and more severe TNM stages. Genetic changes of the E-cadherin gene, on the other hand, appear to be correlated with younger age groups and less severe TNM stages. CONCLUSIONS: Our results suggest that alterations, both in APC and E-cadherin genes, are involved in the evolution and progression of clear cell renal cell carcinoma. Microsatellite genetic instability of the E-cadherin gene indicates that another cellular mechanism, mismatch repair, may also be targeted in this malignancy.     

The tumour suppressor gene CEACAM1 is completely but reversibly downregulated in renal cell carcinoma

CEACAM1 acts as a tumour suppressor in various epithelial tumours. On the other hand, de novo expression of CEACAM1 is strongly associated with reduced disease-free survival of melanoma and non-small cell lung carcinoma patients. Since effector functions of natural killer and T cells are inhibited by homophilic CEACAM1 interaction, immune escape could be responsible for the poor prognosis of these patients. Here, we describe CEACAM1 expression in normal kidney, renal adenomas and renal cell carcinomas (RCC) using a novel antibody generated by genetic immunization. In normal kidney, CEACAM1 was found in epithelial cells of proximal tubules and in endothelial cells. In contrast, tumour cells of 30 clear cell, three chromophobic, and two chromophilic RCCs were completely devoid of CEACAM1. Renal adenomas also lacked CEACAM1 expression. Similarly, RCC cell lines CaKi1, CaKi2, A498, and RCC26 exhibited no or low-level CEACAM1 expression. However, CEACAM1 expression was transiently induced in A498 cells upon contact with allogeneic CD8+ T cells, mediated at least in part by interferon-gamma. Furthermore, the majority of tumour-infiltrating T and NK cells expressed CEACAM1 upon stimulation. Thus, transient expression of the tumour suppressor CEACAM1 by tumour cells and subsequent homophilic interaction with CEACAM1 on tumour-infiltrating lymphocytes could represent a novel immune escape mechanism in RCC.     

Role of chromosome 3p12-p21 tumour suppressor genes in clear cell renal cell carcinoma: analysis of VHL dependent and VHL independent pathways of tumorigenesis.

AIMS: Chromosome 3p deletions and loss of heterozygosity (LOH) for 3p markers are features of clear cell renal cell carcinoma but are rare in non-clear cell renal cell carcinoma. The VHL tumour suppressor gene, which maps to 3p25, is a major gatekeeper gene for clear cell renal cell carcinoma and is inactivated in most sporadic cases of this disease. However, it has been suggested that inactivation of other 3p tumour suppressor genes might be crucial for clear cell renal cell carcinoma tumorigenesis, with inactivation (VHL negative) and without inactivation (VHL positive) of the VHL tumour suppressor gene. This study set out to investigate the role of non-VHL tumour suppressor genes in VHL negative and VHL positive clear cell renal cell carcinoma. METHODS: Eighty two clear cell renal cell carcinomas of known VHL inactivation status were analysed for LOH at polymorphic loci within the candidate crucial regions for chromosome 3p tumour suppressor genes (3p25, LCTSGR1 at 3p21.3, LCTSGR2 at 3p12 and at 3p14.2). RESULTS: Chromosome 3p12-p21 LOH was frequent both in VHL negative and VHL positive clear cell renal cell carcinoma. However, although the frequency of 3p25 LOH in VHL negative clear cell renal cell carcinoma was similar to that at 3p12-p21, VHL positive tumours demonstrated significantly less LOH at 3p25 than at 3p12-p21. Although there was evidence of LOH for clear cell renal cell carcinoma tumour suppressor genes at 3p21, 3p14.2, and 3p12, both in VHL negative and VHL positive tumours, the major clear cell renal cell carcinoma LOH region mapped to 3p21.3, close to the lung cancer tumour suppressor gene region 1 (LCTSGR1). There was no association between tumour VHL status and tumour grade and stage. CONCLUSIONS: These findings further indicate that VHL inactivation is not sufficient to initiate clear cell renal cell carcinoma and that loss of a gatekeeper 3p21 tumour suppressor gene is a crucial event for renal cell carcinoma development in both VHL negative and VHL positive clear cell renal cell carcinoma.     

Tid1 functions as a tumour suppressor in head and neck squamous cell carcinoma

Human tumourous imaginal disc (Tid1), a human homologue of the Drosophila tumour suppressor protein Tid56, is involved in multiple intracellular signalling pathways such as apoptosis, cell proliferation, and cell survival. Here, we investigated the anti‐tumourigenic activity of Tid1 in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Firstly, the clinical association between Tid1 expression and progression of HNSCC was explored. It was found that expression of Tid1 was negatively associated with tumour status, recurrence, and survival prognosis using immunohistochemical analysis of primary HNSCC patient tumour tissue. Secondly, ectopic expression of Tid1 in HNSCC cells was shown to significantly inhibit cell proliferation, migration, invasion, anchorage‐independent growth, and xenotransplantation tumourigenicity. Thirdly, we showed that overexpression of Tid1 attenuated EGFR activity and blocked the activation of AKT in HNSCC cells, which are known to be involved in the regulation of survival in HNSCC cells. On the other hand, ectopic expression of constitutively active AKT greatly reduced apoptosis induced by Tid1 overexpression. Together, these findings suggest that Tid1 functions as a tumour suppressor in HNSCC tumourigenesis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Familial clear cell renal cell carcinoma (FCRC): clinical features and mutation analysis of the VHL, MET, and CUL2 candidate genes

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     

Immunoexpression of SDHB,FH, and CK20 among eosinophilic renal tumors: A tissue microarray study

BackgroundDifferential diagnosis can be a challenge for eosinophilic subtypes of renal cell tumors due to their overlapping histomorphological and immunohistochemical features. We aimed to investigate the frequency of rare variants of renal cell carcinomas (RCCs) such as succinate dehydrogenase-deficient RCC (SDDRCC), hereditary leiomyomatosis and RCC (HLRCC)-associated RCC, and eosinophilic, solid, and cystic RCC (ESCRCC) in our population.Materials and methodsRenal tumors which could be considered in the eosinophilic tumor category were included: 91 conventional clear cell RCCs with eosinophilic cytoplasm, 72 papillary RCCs, 74 chromophobe RCCs, 88 oncocytomas, and 37 other rare subtypes. Using the tissue microarray method, succinate dehydrogenase B (SDHB), fumarate hydratase (FH), and cytokeratin 20 (CK20) antibodies were performed by immunohistochemistry. Immunohistochemistry was repeated on whole block sections for selected cases. The utility of these antibodies in the differential diagnosis was also investigated.ResultsLoss of SDHB expression was detected in three tumors, two of which showed typical morphology for SDDRCC. In additional two tumors, SDHB showed weak cytoplasmic expression without a mitochondrial pattern (possible-SDHB deficient). None of the tumors showed loss of FH expression. Heterogeneous reactions were observed with SDHB and FH antibodies. Only one ESCRCC was detected with diffuse CK20 positivity.ConclusionSDDRCCs, HLRCC-associated RCCs, and ESCRCCs are very rare tumors depending on the population. Possible weak staining and focal loss of SDHB and FH expression should be kept in mind and genetic testing must be included for equivocal results.     

Molecular genetic analysis of chromosome 9 candidate tumor-suppressor loci in bladder cancer cell lines

Underrepresentation of chromosome 9 is a common finding in bladder cancer. Frequent loss of the whole chromosome suggests the presence of at least one relevant tumor suppressor gene on each arm. Candidate regions identified by loss of heterozygosity (LOH) analysis include a region at 9p21 containing CDKN2A, which encodes p16 and p14(ARF), a large region at 9q12-31 including PTCH and many other genes, a small region at 9q32-33, which includes the DBCCR1 gene, and a region at 9q34 including the TSC1 gene. Experimental replacement of genes or chromosomes into tumor cells with appropriate deletions or mutations represents an important approach to test the functional significance of candidate tumor suppressor genes. Loss of an entire copy of chromosome 9 in many bladder tumor cell lines provides no indication of which gene or genes are affected, and selection of appropriate recipient cells for gene replacement is difficult. We have investigated three candidate tumor suppressor genes on chromosome 9 (CDKN2A, DBCCR1, and TSC1), at the DNA level and by expression analysis in a panel of bladder tumor cell lines, many of which have probable LOH along the length of the chromosome, as indicated by homozygosity for multiple polymorphic markers. Cytogenetically, we found no reduction in the numbers of chromosomes 9 relative to total chromosome count. Homozygous deletion of the CDKN2A locus was frequent but homozygous deletion of TSC1 was not found. A new cell line, DSH1, derived from a pT1G2 transitional cell carcinoma with known homozygous deletion of DBCCR1, is described. This study identifies suitable cell lines for future functional analysis of both CDKN2A and DBCCR1.     

肿瘤抑制基因VHL、低氧诱导因子与肾细胞癌

肿瘤抑制基因von Hippel—Lindau（VHL）的突变是VHL综合征相关的透明细胞肾细胞癌（clear cell renal cell carcinoma,CCRCC）的重要分子病理变化。34％-56％散发性CCRCC也存在VHL基因突变。低氧诱导因子（hypoxia inducible factor,HIF）是受VHL调控的转录因子,具有调节细胞增殖、肿瘤血管生成、代谢等重要功能。HIF的调控紊乱与CCRCC的发生发展密切相关,是一个应予重视的研究领域。本文简要介绍VHL和HIF的结构和功能,以及VHL和HIF异常与CCRCC的关系的研究进展。     

Somatic mutations of the von Hippel -- Lindau disease tumour suppressor gene in non-familial clear cell renal carcinoma

Loss of heterozygosity (LOH) studies have suggested that somaticmutations of a tumour suppressor gene or genes on chromosome3p are a critical event In the pathogenesls of non-familialrenal cell carcinoma (RCC). Germllne mutations of the von Hippel— Lindau (VHL) disease gene predispose to early onsetand multifocal clear cell renal cell carcinoma, and the mechanismof tumorigenesls In VHL disease is consistent with a one-hitmutation model. To Investigate the role of somatic VHL genemutations in non-familial RCC, we analysed 99 primary RCC forVHL gene mutations by SSCP and heteroduplex analysis. SomaticVHL gene mutations were Identified In 30 of 65 (46%) sporadicRCC with chromosome 3p allele loss and one of 34 (3%) tumourswith no LOH for chromosome 3p. The VHL gene mutations were heterogeneous(17 frameshift deletions, eight missense mutations, four frameshiftinsertions, one nonsense and one splice site mutation), butno mutations were detected in the first 120 codons of clonedcoding sequence. Most RCCs with somatic VHL mutations (23 of27 (85%) informative cases) had chromosome 3p25 allele lossin the region of the VHL gene so that both alleles of the VHLgene had been inactivated as expected from a two–hit modelof tumorigenesis. Detailed histopathology was available for59 of the tumours investigated: 18 of 43 (42%) RCC with a clearcell appearance had a somatic VHL gene mutation but none of16 non–clear cell RCC (eight chromophilic, three chromophobeand five oncocytoma) (x²= 7.77, P<0.025). These results suggestthat somatic mutations of the VHL gene are a critical eventin the pathogenesis of non-familial clear cell renal carcinoma,but do not exclude a role for other chromosome 3p tumour suppressorgenes.     

多房性囊性肾细胞癌的临床病理学特征和分子遗传学观察

目的 探讨多房性囊性肾细胞癌(MCRCC)的临床病理及分子遗传学改变特征.方法 对1990年1月至2006年12月间第二军医大学附属长海医院病理科病理诊断为肾细胞癌(706例)中的21例MCRCC进行了光镜、免疫表型观察和微卫星不稳定性(MSI)及杂合性缺失(LOH)检测.结果 21例MCRCC患者年龄34～72岁(平均年龄50岁),男19例,女2例.肉眼或镜下血尿3例,18例因体检而偶然发现.10例肿瘤位于左肾,11例位于右肾.T1期18例,T2期2例,T3期1例.其中1例侵犯肾包膜,均无淋巴结转移.随访20例,无一例死于肾癌,未发现肾癌转移或复发病例.组织学观察,肿块最大直径0.3～10.0 cm(平均3.6 cm).切面呈多房性囊性,囊腔大小不等,囊腔间隔厚薄不均(0.1～0.5 cm.平均0.2 cm),内含淡黄色液体或胶冻样或血性液体.囊腔内衬单层或多层上皮细胞,胞质透明或淡嗜酸性.囊腔间隔内也可见呈巢状或腺泡状排列的透明细胞.Fuhrman 1级16例,Fuhrman 2级5例.免疫组织化学观察,瘤细胞波形蛋白、ABC、CAM5.2、上皮细胞膜抗原(EMA)均呈阳性表达,CD10阳性表达6例.神经元特异性烯醇化酶(NSE)阳性表达16例.分子遗传学观察,21例标本中,PCR扩增成功的有11例,5例发现微卫星变异,3例有LOH,其中D3S1560基因座2例,D14S617基因座1例;2例有MSI,分别为D9S168和D14S617各1例.结论 MCRCC是一种少见的肾囊性肿瘤,具有独特的临床和病理学特征,肿瘤预后良好,可通过手术切除而治愈.     

Papillary pattern in clear cell renal cell carcinoma: Clinicopathologic,morphologic, immunohistochemical and molecular genetic analysis of 23 cases

Clear cell renal cell carcinoma (ccRCC), the most common histologic subtype of RCCs, demonstrates a wide spectrum of morphologic features (i.e., low-grade spindle cell, syncytial giant cells, and mucin-producing cells). However, papillary growth pattern in ccRCCs is rather a rare finding, which can present challenges in differential diagnostic work up. The aim of this study was to investigate ccRCCs with predominant papillary features from morphologic, immunohistochemical and molecular genetic perspectives.23 clear cell renal cell carcinomas with papillary architecture were selected. Tumors were evaluated morphologically, immunohistochemically, and molecularly by next-generation sequencing (NGS). The diagnosis of MiT family translocation RCC was excluded by TFE3 immunohistochemistry.Mean age of patients was 65.2 years (range 42–81 years), and 19/23 were male. Tumor size ranged from 1.6 to 12.8 cm (median 6.5 cm). At a median follow-up of 2.5 years (range 1.5–9 years), 2 patients (8.7%) died of disease, 2 developed metastasis. Areas of papillary pattern accounted for approximately 40–100% of the tumor. CK7 was negative in non-papillary areas in majority of cases (20/23, 87%), and was only focally positive in 3/23 cases (13%). In papillary areas, AMACR was positive/focally positive in 17/23 (73.9%) cases and in the non-papillary areas it was positive/focally positive in 22/23 (95.6%) cases. CAIX was mainly negative in both non-papillary and papillary areas (15/23 [65%] and 16/23 [69.5%], respectively). Molecular analysis of 15 analyzable cases revealed the most frequently mutated gene to be VHL (in 9 cases), followed by PRBM1 (in 2 cases) and 29 other different mutations in various genes.Papillary growth pattern in ccRCC is not an uncommon situation. Papillary RCC with clear cells and MiT family (TFE3) translocation RCCs are the major differential diagnostic considerations in such scenarios. Our NGS molecular analysis supported classifying such tumors as a morphologic variant of ccRCC.     

Association analysis of chromosome 1 migraine candidate genes

Background  

Migraine with aura (MA) is a subtype of typical migraine. Migraine with aura (MA) also encompasses a rare severe subtype Familial Hemiplegic Migraine (FHM) with several known genetic loci. The type 2 FHM (FHM-2) susceptibility locus maps to chromosome 1q23 and mutations in the ATP1A2 gene at this site have recently been implicated. We have previously provided evidence of linkage of typical migraine (predominantly MA) to microsatellite markers on chromosome 1, in the 1q31 and 1q23 regions. In this study, we have undertaken a large genomic investigation involving candidate genes that lie within the chromosome 1q23 and 1q31 regions using an association analysis approach.     

DRAM1 plays a tumor suppressor role in clear cell renal cell carcinoma through modulating Akt signaling

BackgroundClear cell renal carcinoma (ccRCC) is one of the most common malignant tumors worldwide. DNA damage-regulated autophagy modulator1 (DRAM1) plays an important roles in apoptosis and tumor progression. However, the role of DRAM1 in ccRCC is still unknown. In our study, we aimed to investigate the effect of DRAM1 in the progression of ccRCC.MethodsThe expression and prognostic information of DRAM1 in ccRCC were obtained by immunohistochemistry staining and bioinformatics database. Cell proliferation, migration, invasion were detected by CCK-8 assay, wound-healing and transwell assays, and the cell apoptosis was examined by tunel assay and flow cytometry analysis. Western blot was used to detect the expression of DRAM1, Bax, Bcl2, Akt, p53,E-cadherin, N-cadherin of ccRCC cells.ResultsDecreased expression of DRAM1 was found in ccRCC tissues, which predicted a shorter survival rate in ccRCC patient. We confirmed that DRAM1 inhibited the proliferation, migration, invasion and epithelial mesenchymal transformation (EMT), while enhanced the apoptosis of ccRCC cells. In addition, the results of inhibition of Akt signaling were consistent with the above. We further proved that DRAM1 over-expression decreased the phosphorylation of Akt signaling, and overexpression of DRAM1 could reverse oncogenic function induced by the over-activating of Akt in ccRCC cells.Conclusionoverexpression of DRAM1 plays a tumor suppressive role in ccRCC through inactivation of Akt and highlights the potential role of DRAM1 as a prognostic biomarker in ccRCC.     

Analysis of alterations of WFDC1, a new putative tumour suppressor gene,in hepatocellular carcinoma

WFDC1 is a recently isolated human gene identified as a tumour suppressor gene candidate. It is not known whether alterations in this gene are associated with human cancers. The WFDC1 gene maps in human chromosome 16q24, an area of frequent loss of heterozygosity (LOH) in several tumour types, in particular in hepatocellular carcinoma (HCC). We investigated its role in 46 European HCC by means of the detection of LOH at the WFDC1 locus. We describe here an assay for the detection of loss of heterozygosity at this locus using two dinucleotide repeat polymorphisms identified in WFDC1 introns, with a combined informativity of 86%. LOH was observed in 4/40 informative HCC samples. We further investigated the role of WFDC1 as a tumour suppressor gene candidate in five hepatocellular cell lines and in tumours exhibiting LOH by means of mutation, promoter methylation and gene expression analysis. In HCC samples showing LOH, no mutation of the remaining allele was observed. No significant up or down gene expression was observed in tumour samples comparatively to normal liver and gene expression did not seem related to promoter methylation. These results suggest a minor role, if any, of WFDC1 in hepatocarcinogenesis. However, this approach might be useful for investigating the role of this candidate tumour suppressor gene in other tumour types.     

Mutation of the p53 tumour suppressor gene occurs preferentially in the chromophobe type of renal cell tumour

Genomic DNA from 30 non-papillary and 20 chromophobe renal cell carcinomas (RCCs), 30 papillary renal cell tumours, and 20 renal oncocytomas was screened for the presence of mutations in exons 5–8 of the p53 tumour suppressor gene by polymerase chain reaction–single strand conformation polymorphism analysis and direct DNA sequencing. Mutations leading to an amino acid change were found only in 6 out of 20 chromophobe RCCs. Microsatellite analysis of chromophobe RCCs revealed the loss of one allele at chromosome 17p in 14 out of 18 informative cases. No mutation of the p53 gene was found in five sarcomatous RCCs or in seven tumours of stage IV. This study shows that mutation of the p53 tumour suppressor gene does not correlate with the specific loss of DNA sequences at chromosome 17 in chromophobe RCCs, nor can it be used as a prognostic parameter for RCCs in general. © 1997 John Wiley & Sons, Ltd.