Genetic alterations of the MYH gene in gastric cancer

Increased oxygen free radicals produced in gastric mucosa by H. pylori induce DNA damage and lead to dyspalsia and gastric cancers. However, only a small percentage of individuals that carry H. pylori develop gastric cancer, indicating that other factors are involved. We have screened a set of 95 sporadic gastric cancers for mutations and allele loss of the DNA glycosylase MYH gene, which excises adenine misincorporated opposite unrepaired 8-oxoG. Two of 95 cancers had bi-allelic mutations of the MYH gene with somatic missense mutation of one allele and loss of the remaining allele. The mutations were missense mutations, P391S and Q400R, in exon 13 encoding NUDIX hydrolase domain of the gene. The patients were H. pylori-positive and the tumors were of advanced intestinal-type gastric cancer with lymph node metastasis. In addition, 4 (17.4%) of 23 informative cases showed allele loss at MYH locus. Therefore, our data suggest that somatic mutations of base excision repair gene MYH contribute to the development of a sub-set of sporadic gastric cancers.     

Genetic and epigenetic analysis of the VHL gene in gastric cancers

The von Hippel-Lindau tumor suppressor gene (VHL), which is located on chromosome 3p25, plays an important role in tumorigenesis, particularly in tumor growth and vascularization. Mutations of the VHL gene have been observed in the hereditary VHL syndrome and a variety of other sporadic cancers. In this study, in order to investigate whether the VHL gene is involved in gastric carcinogenesis, we have examined the genetic alterations, including somatic mutations and allelic loss, with the two microsatellite markers, D3S1038 and D3S1110, as well as promoter hypermethylation of the VHL gene in 88 sporadic gastric adenocarcinomas. No mutation was detected in the coding region of the VHL gene. Allelic loss was found in 20 (33.9%) of 59 informative cancer cases at one or both markers. In addition, promoter hypermethylation was not detected in the gastric cancer samples. This is the first investigation of the genetic and epigenetic alterations of the VHL gene in gastric cancers. Our results suggest that genetic and epigenetic alterations of the VHL gene may be not involved in the development or progression of gastric cancers. The findings also provide evidence for the presence of another gastric cancer specific tumor suppressor gene at the 3p25 region.     

Genetic analysis of the DBC2 gene in gastric cancer

The DBC2 (Deleted in breast cancer, RhoBTB2) has been identified as a tumor suppressor gene that has growth inhibitory function. To investigate whether genetic alterations of the DBC2 gene are involved in the development of gastric cancer, we analyzed mutations and allelic loss in the DBC2 gene in 95 primary gastric cancers by PCR-SSCP, sequencing and LOH analysis. In the mutational analysis, we found one missense somatic mutation (CGG-->TGG, R275W) in the BTB/POZ domain of the gene in a patient with advanced gastric cancer and lymph node metastasis. In addition, we found one known polymorphism and three novel polymorphisms in the coding region of DBC2, which showed an amino acid change, and was detected in both the cancer cells and corresponding normal cells. On LOH analysis, 62 cases were heterozygous for at least one marker and 18 cases (29.0%) showed allelic loss at these markers. In conclusion, the mutations and allelic loss in the DBC2 gene are uncommon in gastric cancers in Korean patients. Further studies to identify the target gene at 8q21 responsible for the development of gastric cancer should be explored.     

Somatic mutations in the BRCA2 gene and high frequency of allelic loss of BRCA2 in sporadic male breast cancer

Breast cancer occurs rarely in men and risk factors for the disease include germline mutations of the BRCA2 gene. High frequency of allelic loss at the BRCA2 locus has been reported in sporadic breast tumors, but somatic mutations of BRCA2 are very rare. Here we report the first case of somatic BRCA2 mutation in male breast cancer with demonstrated loss of heterozygosity. We analyzed a series of 27 archival samples from male breast cancer patients for BRCA2 mutations and loss of heterozygosity at BRCA2 locus. The mutation analysis of BRCA2 gene was performed using SSCA-HA and sequencing methods. PCR was used to detect LOH at 3 highly polymorphic microsatellite markers spanning BRCA2 region on 13q by comparing the allelic pattern in matched tumor and blood DNA samples. In this study LOH at the BRCA2 locus was observed in 82.6% of informative cases, confirming previous observations on high frequency of LOH affecting the BRCA2 region in male breast cancer. We identified 5 somatic BRCA2 mutations in a set of 23 sporadic male breast cancers (21%). Two silent and 1 missense alterations were novel BRCA2 variants. Here we also report first somatic frameshift BRCA2 mutation in male breast cancer 8138del5. In 3 tumors with somatic BRCA2 alterations, 1 missense, 1 silent and frameshift LOH at chromosome 13q12-13 were detected and losses involved a wild-type allele of BRCA2 gene.     

Germline mutations in E-cadherin do not explain association of hereditary prostate cancer, gastric cancer and breast cancer

Somatic mutations in the E-cadherin (CDH1) gene have frequently been reported in cases with diffuse gastric and lobular breast cancers. Recently, germline mutations have been identified in families with diffuse gastric cancers. In families with hereditary prostate cancer (HPC), a significant association of prostate cancer, gastric and/or breast cancer has been observed in epidemiological studies. The aim of this study was to investigate if germline mutations in CDH1 could explain the risk for cancer in HPC families with an excess of gastric and breast cancer. In total, 17 members from 13 HPC families and 3 members from 3 families with hereditary gastric cancer (HGC) were screened for germline CDH1 sequence alterations using PCR/Denaturing HPLC for initial screening of nucleotide variants followed by confirmatory direct sequencing analysis. The frequency of identified novel germline mutations were tested for in 136 cases with hereditary prostate cancer and 215 cases of sporadic prostate cancer with 422 age matched controls in an allelic discrimination assay. In total, 8 sequence variants were detected in 20 samples tested. In the HPC families, we found 2 missense mutations, A592T in exon 12 and a novel D777N in exon 15 and a mutation in intron 5, 687+92T>A. A previously known polymorphism in exon 13 and 3 sequence variations in introns and untranslated regions were also found, of which the significance is unknown. In HGC-023 with early onset diffuse gastric cancer a truncating mutation, R335X, was identified in exon 7. None of the missense mutations or 687+92T>A were found in the extended HPC material or in the sporadic prostate cancer cases with age-matched controls in the allelic discrimination assay. We found several germline mutations of unknown clinical significance in the CDH1 gene that probably do not explain the association of prostate, gastric and/or breast cancers in the HPC-families. Two missense mutations and a mutation in intron 5 were identified that do not influence the risk of hereditary or sporadic prostate cancer in general and are considered to be pedigree specific. In a family with hereditary gastric cancer of the diffuse type, we identified the first truncating germline mutation in a Scandinavian family.     

鼻咽癌中KLF6基因突变的检测

背景与目的：鼻咽癌（nasopharyngeal carcinoma,NPC)高发于中国南方和东南亚地区,其发病是一个多因素,多步骤的过程,与遗传因素,EB病毒感染和环境因子有关,但分子机理仍不清楚,KLF6基因编码一种广泛表达的核转录因子,在某些前列腺癌肿瘤中发现有高频的等位基因的丢失和基因突变,本研究通过对KLF6基因在NPC肿瘤中的突变检测,探讨KLF6基因与NPC发病的关系。方法：采用PCR-直接测序分析方法。在19例散发性NPC组织细胞和3个NPC细胞系（CNE1,CNE2,SUNE1）中对KLF6编码区和拼接位点进行检测。结果：在3你散发性NPC肿瘤组织DNA中检测到KLF6基因的3个不同位置的错义改变,分别为GAG→TG（Glu75Val),AGG→AGG（Ser136Arg)和AGA→AAA（Arg243Lys),在50个随机正常个体（100条染色体）和3个NPC细胞系中没有发现这3个位点的碱基变异,结论：这3个变异可能是KLF6基因的新的突变位点,在某些NPC发病中可能有作用。     

Allele loss occurs frequently at hMLH1, but rarely at hMSH2, in sporadic colorectal cancers with microsatellite instability.

Mutations at the hMSH2 and hMLH1 mismatch repair loci have been implicated in the pathogenesis of colorectal cancer. Tumours with two allelic mutations at a mismatch repair locus develop replication errors (RERs). In the hereditary non-polyposis colorectal cancer (HNPCC) syndrome, one mutation is inherited and the other acquired somatically: in RER+ sporadic colorectal cancers, both mutations are somatic. RER+ tumours tend to have a low frequency of allele loss, presumably because they acquire most mutations through RERs. However, before a second mismatch repair mutation has occurred somatically, there is no reason to suppose that allele loss occurs less frequently in tumours that are to become RER+. Indeed, this second mutation might itself occur by allele loss. We have searched for allele loss at the hMSH2 and hMLH1 loci in RER+ and RER- sporadic colorectal cancers. Loss occurred at the hMLH1 locus in 7/17 (41%) RER+ tumours, compared with 6/40 (15%) RER- cancers (chi2=3.82, P approximately 0.05). At hMSH2, 2/22 RER+ sporadic cancers (9%) had lost an allele, compared with 2/40 (5%) RER- cancers (chi2=0.03, P>0.5). Taken together with previous studies which focused on colorectal cancers from HNPCC families, the data suggest that allele loss at hMLH1, but not at hMSH2, contributes to defective mismatch repair in inherited and sporadic colorectal cancer.     

Frequent somatic mutations of the beta-catenin gene in intestinal-type gastric cancer.

The increased level of cytoplasmic beta-catenin through the mutations to either beta-catenin or adenomatous polyposis coli (APC) has been proposed as an important oncogenic step in various tumors. Gastric cancer showed frequent genetic alterations of the APC gene, and the risk for gastric cancer in familial adenomatosus polyposis patients is 10 times higher than that in the general population. These findings raise the possibility that mutations of beta-catenin may also be associated with the development of gastric cancer. We detected seven somatic mutations in a portion of exon 3 encoding for the glycogen synthase kinase 3beta phosphorylation consensus region of the beta-catenin gene in 43 gastric cancers. All of these mutations were missense mutations, of which five are in the highly conserved aspartic acid 32 and two are in serine 29; all of these seven mutations were detected exclusively in intestinal-type gastric cancers (7 of 26; 26.9%), but not in the diffuse-type (0 of 17). We concluded that disruption of the APC/beta-catenin/T cell factor-lymphoid enhancer binding factor pathway might play an important role especially in the development of intestinal-type gastric cancer.     

Alterations of the base excision repair gene MUTYH in sporadic colorectal cancer

The base excision repair gene MUTYH encodes glycosylase which removes adenine residues mispaired with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHG). Biallelic germline mutations of the MUTYH gene are known to cause multiple colorectal adenomas including polyposis and cancer, mostly due to G:C?T:A transversions in proto-oncogenes or tumor suppressor genes. The risk of colorectal cancer (CRC) in monoallelic mutation carriers of MUTYH is estimated to be higher in comparison with non-carriers. To investigate the possible role in sporadic CRC, we examined alterations of the MUTYH gene including somatic mutations and allelic loss in 101 cases of sporadic CRC, together with the KRAS mutation in some cases. MUTYH mutations in cancer DNA were detected in 3 cases, while mutations were also found in DNA samples from normal tissues, indicating that all were germline mutations. Allelic loss at the MUTYH locus was found in 10 of 51 (20.0%) CRC cases and KRAS mutations were found in 33 of the 101 (32.7%) samples. There was no significant difference in the rate of G:C?T:A transversion in KRAS between cases with allelic loss (1 of 10, 10.0%) and without allelic loss (9 of 41, 22.0%). Investigation of quantitative allelic imbalance at SNP rs3219489 of MUTYH showed that CRC cases with C allele dominance (minor type corresponding to His) were more frequently detected with G:C?T:A transversions than in those with G allele dominance (major type corresponding to Gln). In conclusion, somatic alterations of MUTYH in sporadic CRC were rare, similar to other DNA repair genes. However, it is possible that unknown mutations of regions not analyzed in this study and epigenetic changes of the promoter region of MUTYH may contribute to the disease.     

Somatic von Hippel-Lindau gene mutations detected in sporadic endolymphatic sac tumors

Endolymphatic sac tumors (ELSTs) occur sporadically or in association with an autosomal dominantly inherited tumor syndrome, von Hippel-Lindau (VHL) disease. In VHL disease, a germline mutation of the VHL tumor suppressor gene is inherited, and loss of function of the wild-type allele occurs through genetic deletion with subsequent development of neoplastic growth. Genetic alterations associated with sporadic ELSTs are less well understood. In this study, we used tissue microdissection to selectively analyze neoplastic cells from four sporadic ELSTs. In two cases, we detected somatic mutations involving VHL gene exons 1 and 2, respectively. Additionally, one of these cases revealed deletion of the VHL gene locus. Two cases did not reveal VHL gene mutation; one of these two cases showed VHL gene deletion. These results suggest that mutations and allelic deletions of the VHL tumor suppressor gene play a role in the tumorigenesis of sporadic ELSTs.     

Analysis of genetic alterations associated with DNA diploidy, aneuploidy and multiploidy in gastric cancers

OBJECTIVE: Recent studies have shown a close association between DNA ploidy status (diploidy, aneuploidy and multiploidy) identified by the crypt isolation technique and specific genetic alterations in colorectal carcinomas. However, such an association has not been elucidated for gastric tumors, even though they share common genetic features with colorectal carcinomas. In the present study, we established an association between DNA ploidy status and genetic alterations in gastric cancer. METHOD: The DNA ploidy status of gastric tumors was classified as diploid, aneuploid or multiploid using the crypt isolation technique, which allows isolation of pure tumor crypt from tumor tissue. Crypt isolation combined with DNA cytometric sorting, polymerase chain reaction assay using 26 microsatellite markers and direct sequencing of the p53 gene were used to detect allelic imbalances [loss of heterozygosity (LOH) or allelic loss], microsatellite imbalance (MSI) and mutation of p53 in 54 gastric cancers (13 diploid, 12 aneuploid, 29 multiploid). RESULT: Diploid tumors showed few genetic alterations, including allelic imbalances and p53 mutations. In contrast, aneuploid tumors and multiploid tumors (in particular, aneuploid populations of multiploid tumors) exhibited multiple genetic alterations, including allelic imbalances and p53 mutations. In addition, the frequencies of genetic alterations observed in the corresponding diploid fractions of multiploid tumors were relatively higher than in diploid tumors. MSI was commonly observed in diploid, aneuploid and multiploid carcinomas. CONCLUSIONS: The present results indicate that in gastric carcinomas, diploid tumors are generally non-LOH and MSI, whereas aneuploid and multiploid tumors are associated with LOH and MSI, suggesting that the genetic profile of these carcinomas is dependent on the tumor's ploidy status.     

Alterations of p73 preferentially occur in gastric adenocarcinomas with foveolar epithelial phenotype.

To establish the possible involvement of p73, a newly discovered p53-related candidate as a tumor-suppressor gene in human stomach carcinogenesis, the allelic status, allele-specific expression and mutations of the gene were investigated using PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, RT-PCR SSCP analysis and direct DNA sequencing in 95 gastric adenocarcinomas. Of these, 32 exhibited the heterozygous p73 allele for the StyI restriction site in exon 2. Among these, the cancer DNA of 12 revealed loss of heterozygosity (LOH) of p73. All of the cancers with p73 LOH exhibited phenotypes of foveolar epithelium of the stomach. RT-PCR SSCP analysis of p73 heterozygous cases demonstrated not only bi-allelic expression of the gene but also relatively reduced expression of the affected allele in 6 of 8 tumors with p73 LOH. No gene mutation was detected in the remaining allele of LOH-positive cancers. Our results suggest that alterations of p73, including LOH and abnormal expression, may play roles in the genesis of foveolar-type gastric adenocarcinomas, though this is not in line with a classical Knudson's "2-hit" model.     

PTEN mutations are common in sporadic microsatellite stable colorectal cancer

The tumour suppressor gene PTEN, located at chromosome sub-band 10q23.3, encodes a dual-specificity phosphatase that negatively regulates the phosphatidylinositol 3'-kinase (PI3 K)/Akt-dependent cellular survival pathway. PTEN is frequently inactivated in many tumour types including glioblastoma, prostate and endometrial cancers. While initial studies reported that PTEN gene mutations were rare in colorectal cancer, more recent reports have shown an approximate 18% incidence of somatic PTEN mutations in colorectal tumours exhibiting microsatellite instability (MSI+). To verify the role of this gene in colorectal tumorigenesis, we analysed paired normal and tumour DNA from 41 unselected primary sporadic colorectal cancers for PTEN inactivation by mutation and/or allelic loss. We now report PTEN gene mutations in 19.5% (8/41) of tumours and allele loss, including all or part of the PTEN gene, in a further 17% (7/41) of the cases. Both PTEN alleles were affected in over half (9/15) of these cases showing PTEN genetic abnormalities. Using immunohistochemistry, we have further shown that all tumours harbouring PTEN alterations have either reduced or absent PTEN expression and this correlated strongly with later clinical stage of tumour at presentation (P=0.02). In contrast to previous reports, all but one of the tumours with PTEN gene mutations were microsatellite stable (MSI-), suggesting that PTEN is involved in a distinct pathway of colorectal tumorigenesis that is separate from the pathway of mismatch repair deficiency. This work therefore establishes the importance of PTEN in primary sporadic colorectal cancer.     

Molecular biology of colorectal cancer

Colorectal cancer is a significant cause of morbidity and mortality in Western populations. This cancer develops as a result of the pathologic transformation of normal colonic epithelium to an adenomatous polyp and ultimately an invasive cancer. The multistep progression requires years and possibly decades and is accompanied by a number of recently characterized genetic alterations. Mutations in two classes of genes, tumor-suppressor genes and proto-oncogenes, are thought to impart a proliferative advantage to cells and contribute to development of the malignant phenotype. Inactivating mutations of both copies (alleles) of the adenomatous polyposis coli (APC) gene—a tumor-suppressor gene on chromosome 5q—mark one of the earliest events in colorectal carcinogenesis. Germline mutation of the APC gene and subsequent somatic mutation of the second APC allele cause the inherited familial adenomatous polyposis syndrome. This syndrome is characterized by the presence of hundreds to thousands of colonic adenomatous polyps. If these polyps are left untreated, colorectal cancer develops.Mutation leading to dysregulation of the K-ras protooncogene is also thought to be an early event in colon cancer formation. Conversely, loss of heterozygosity on the long arm of chromosome 18 (18q) occurs later in the sequence of development from adenoma to carcinoma, and this mutation may predict poor prognosis. Loss of the 18q region is thought to contribute to inactivation of the DCC tumor-suppressor gene. More recent evidence suggests that other tumor-suppressor genes—DPC4 and MADR2 of the transforming growth factor β (TGF-β) pathway—also may be inactivated by allelic loss on chromosome 18q. In addition, mutation of the tumor-suppressor gene p53 on chromosome 17p appears to be a late phenomenon in colorectal carcinogenesis. This mutation may allow the growing tumor with multiple genetic alterations to evade cell cycle arrest and apoptosis. Neoplastic progression is probably accompanied by additional, undiscovered genetic events, which are indicated by allelic loss on chromosomes 1q, 4p, 6p, 8p, 9q, and 22q in 25% to 50% of colorectal cancers.Recently, a third class of genes, DNA repair genes, has been implicated in tumorigenesis of colorectal cancer. Study findings suggest that DNA mismatch repair deficiency, due to germline mutation of the hMSH2, hMLH1, hPMS1, or hPMS2 genes, contributes to development of hereditary nonpolyposis colorectal cancer. The majority of tumors in patients with this disease and 10% to 15% of sporadic colon cancers display microsatellite instability, also know as the replication error positive (RER+) phenotype. This molecular marker of DNA mismatch repair deficiency may predict improved patient survival. Mismatch repair deficiency is thought to lead to mutation and inactivation of the genes for type II TGF-β receptor and insulin-like growth-factor II receptor: Individuals from families at high risk for colorectal cancer (hereditary nonpolyposis colorectal cancer or familial adenomatous polyposis) should be offered genetic counseling, predictive molecular testing, and when indicated, endoscopic surveillance at appropriate intervals.Recent studies have examined colorectal carcinogenesis in the light of other genetic processes. Telomerase activity is present in almost all cancers, including colorectal cancer, but rarely in benign lesions such as adenomatous polyps or normal tissues. Furthermore, genetic alterations that allow transformed colorectal epithelial cells to escape cell cycle arrest or apoptosis also have been recognized. In addition, hypomethylation or hypermethylation of DNA sequences may alter gene expression without nucleic acid mutation.     

Inactivating mutations of the Siah-1 gene in gastric cancer

SIAH-1: is the mammalian homolog of Drosophila seven in absentia (sina) and has been identified as a p53-inducible gene. Siah-1 can induce cell cycle arrests, tumor suppression, and apoptosis through a novel beta-catenin degradation pathway. To determine whether genetic alterations of Siah-1 gene are involved in the development and/or progression of gastric cancer, we searched for mutation of the Siah-1 gene in 95 gastric cancers by single-strand conformational polymorphism and sequencing. The effect of Siah-1 on beta-catenin degradation was further examined in wild- and mutant-type Siah-1-transfected HEK 293T cells. We found two missense mutations of the Siah-1 gene. The cases with Siah-1 mutation showed nuclear translocation and cytoplasmic staining of beta-catenin. Interestingly, two mutants of Siah-1 stabilized cytoplasmic levels of beta-catenin, even after treatment of adriamycin. Furthermore, both mutants failed to suppress cyclin D1 expression and to induce apoptosis. These data suggest that inactivating mutations of the Siah-1 may contribute to the development of gastric cancer through beta-catenin stabilization and apoptosis block.