PURPOSE: There are numerous prostate cancer-related genes that involve in carcinogenesis and tumor progression. Among the genes, DNA mismatch repair genes recognize and repair misincorporated nucleotides during DNA replication. In this analysis, we evaluated the association of hMSH2 which is one of the mismatch repair genes, with risk of aggressive prostate cancer and prostate cancer recurrence. MATERIALS AND METHODS: Immunohistochemistry was performed in 46 patients who diagnosed prostate cancer and underwent radical prostatectomy between January 2006 and December 2012 at Kyung Hee University Hospital at Gangdong. We evaluated an association between the degree of hMSH2 immunohistochemical staining and various clinical variables including prostate-specific antigen (PSA), Gleason score, pathological stage, and biochemical recurrence. The intensity of immunostaining for hMSH2 was divided into 2 groups: low expression group (immunostaining score < 2) and high expression group (immunostaining score ≥2). RESULTS: Although seminal vesicle invasion was marginally associated with the degree of hMSH2 immunohistochemical staining, PSA, Gleason score, lymph node metastasis, presence of lymphatic, perineural, vascular invasion, and extracapsular extension were not associated with the degree of hMSH2 immunohistochemical staining. Furthermore, the association of biochemical recurrence free survival with hMSH2 expression was not statistically significant. CONCLUSIONS: The hMSH2 expression was marginally associated with risk of aggressive prostate cancer such as seminal vesicle invasion. Further evaluation with a larger number of cases is needed to verify these results.
Base Pair Mismatch ; Carcinogenesis ; DNA Mismatch Repair ; DNA Repair ; DNA Replication ; Gene Expression ; Humans ; Immunohistochemistry ; Lymph Nodes ; Neoplasm Grading ; Neoplasm Metastasis ; Nucleotides ; Prostate ; Prostate-Specific Antigen ; Prostatectomy ; Prostatic Neoplasms ; Recurrence ; Seminal Vesicles

OBJECTIVETo investigate cyclooxygenase-2 (COX-2) expression and its relationship with mismatch repair (MMR) protein expression and microsatellite instability (MSI) in hereditary nonpolyposis colorectal cancer (HNPCC).

METHODSA total of 28 cases of colorectal adenoma and 14 cases of colorectal carcinoma were collected between July 2003 and July 2007 from 33 HNPCC families. Sporadic colorectal adenoma (n=32) and carcinoma patients (n=24) served as controls. With samples of tumor tissues and normal colonic mucosa collected from the patients, the protein expressions of COX-2 and MMR (hMLH1, hMSH2, and hMSH6) were examined with immunohistochemical assay. Frequency of MSI in five standard MSI loci BAT25, BAT26, D2S123, D5S346, and D17S250 were analyzed by means of polymerase chain reaction.

RESULTSThe rate of COX-2 high-expression was 53.6% (15/28) and 42.9% (6/14) in HNPCC adenoma and carcinoma; 62.5% (20/32) and 91.7% (22/24) in sporadic adenoma and carcinoma, respectively. That rate was lower in HNPCC carcinoma than in sporadic carcinoma (Pü0.05). MMR-deletion rate and percentage of high-frequency MSI (MSI-H) in HNPCC carcinoma were higher than those in sporadic colorectal carcinoma [both 71.4% (10/14) vs. 12.5% (3/24), both Pü0.01]. Among the 10 MMR-deficient HNPCC carcinoma patients, COX-2 low-expression was observed in 8 cases (80.0%), while COX-2 high-expression was observed in all of the 4 MMR-positive HNPCC carcinoma cases (Pü0.05). In comparison to MMR positive HNPCC carcinoma, HNPCC adenoma, and sporadic carcinoma, COX-2 expression was significantly lower in corresponding MMR-deficient cases (all Pü0.05). The rates of COX-2 low-expression in HNPCC adenoma, HNPCC carcinoma, and sporadic carcinoma with MSI-H were significantly higher than those in the cases with microsatellite stability (all Pü0.05).

CONCLUSIONCOX-2 is expressed at a low level in HNPCC carcinoma, different from the high COX-2 expression in sporadic carcinoma.

Adult ; Aged ; Base Pair Mismatch ; Base Sequence ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; Cyclooxygenase 2 ; genetics ; DNA Primers ; DNA Repair ; Female ; Humans ; Immunohistochemistry ; Male ; Microsatellite Repeats ; genetics ; Middle Aged

OBJECTIVETo study the characteristics of germline mutations of hMLH1, hMSH2 and hMSH6 and promoter methylation status of MLH1 in patients with MSI colorectal cancer.

METHODSSequence analysis of germline mutation and promoter methylation of MLH1 in 34 prospective collected patients with MSI colorectal cancer were performed.

RESULTSNineteen out of 34 patients with MSI colorectal cancer were detected with hypermethylation of MLH1,which accounted for 55.9%. 73.7% MSI-H colorectal cancer cases and 33.3% MSI-L colorectal cancer cases were detected with hypermethylation of MLH1 and the difference was significant. Eight germline mutations were found, including 3 MSH6 mutations and 5 MSH2 mutations.

CONCLUSIONThere are some different characteristics of the germline mutations of hMLH1, hMSH2 and hMSH6 and promoter methylation of MLH1 in Chinese MSI colorectal patients.

Adaptor Proteins, Signal Transducing ; genetics ; Aged ; Base Pair Mismatch ; Colorectal Neoplasms ; genetics ; DNA Methylation ; DNA, Neoplasm ; DNA-Binding Proteins ; genetics ; Female ; Germ-Line Mutation ; Humans ; Male ; Microsatellite Instability ; Middle Aged ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; genetics ; Nuclear Proteins ; genetics ; Sequence Analysis, DNA

OBJECTIVETo investigate the effect of the protocol recommended by NCCN-2007 on the diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) in China.

METHODSNCCN protocol consists of identifying HNPCC characteristics according to the revised Bethesda Guidelines,genetic counseling with immunohistochemistry and finally genetic testing. Four hundred and nineteen patients diagnosed as colorectal cancer from January 2002 to February 2006 were selected. The hMLH1 and hMSH2 immunostaining were implemented for 90 patients who fulfilled the revised Bethesda Guidelines, in whom 8 patients fulfilling the Amsterdam II (Criteria were classified as group A and the other 82 patients as group B. The frozen tissues were collected from patients who showed loss of hMLH1 or hMSH2 protein expression, then RNA was extracted, and RT-PCR and cDNA sequencing were adopted to detect the germline mutations of hMLH1 and hMSH2.

RESULTSTumor tissues from 18 patients showed loss of hMLH1 or hMSH2 protein expression (5 patients in group A and 13 in group B). Finally, 21 patients(8 in group A and 13 in group B showed loss expression of MMR protein) were diagnosed as HNPCC, including 2 cases of hMLH1 and 1 case of hMSH2 mutations. These 3 cases with cDNA mutations did not fulfill the Amsterdam II( Criteria, and were finally diagnosed as HNPCC.

CONCLUSIONThe protocol recommended by NCCN-2007 offers a useful approach to identify HNPCC patients,and reduces the possibility of missed diagnosis of HNPCC.

Adult ; Aged ; Aged, 80 and over ; Base Pair Mismatch ; China ; Colorectal Neoplasms, Hereditary Nonpolyposis ; diagnosis ; genetics ; Female ; Gene Deletion ; Genetic Testing ; methods ; Guidelines as Topic ; Humans ; Male ; Middle Aged ; Young Adult

OBJECTIVETo detect the germline mutation of mismatch repair gene (MSH6) in hereditary nonpolyposis colorectal cancer (HNPCC) kindreds fulfilling different clinical criteria.

METHODSThe germline mutations of MSH6 gene were detected by PCR based DNA sequencing in 39 unrelated HNPCC probands fulfilling different clinical criteria in which MSH2 and MLH1 mutations were excluded. The exons with missense mutations were analyzed using PCR sequencing in the germline genomic DNA of 137 healthy persons. The expression of MSH6 protein was detected by Envision immunohistochemistry staining in the tumor tissues of the mutational probands.

RESULTSSix germline mutations of MSH6 gene were detected in 39 probands of Chinese HNPCC kindreds, and the mutations distributed in the exon 4, 6, 9 and 10. Four out of six mutations were missense mutation, one was nonsense mutation and the remaining one was insertion mutation in splice site. The results of sequecing for the exons with above four missense mutations in 137 healthy persons' genomic DNA showed that 5 of 137 persons had the missense mutation of c.3488 A to T at codon 1163 of the 6th exon. The mutational rate was approximately 3.65% (5/137), so the mutation could be a single nucleotide polymorphism (SNP). The remaining missense mutations were not found in any germline genomic DNA of 137 healthy persons. Positive expression of MSH6 protein had been identified in the tumor of the SNP proband while the tumors had negative MSH6 protein expression in the rest probands of germline mutation MSH6 gene. The types of mutations and their potential significance were determined by comparing the following databases: http://www.ncbi.nlm.nih.gov/, http://www.ensembl.org/homo-sapies, and http://www.insight-group.org. Five out of the six mutations had not been reported previously and they were new pathological mutations, the rest one was a new SNP.

CONCLUSIONGermline mutations of MSH6 gene may play an important role in Chinese HNPCC kindreds fulfilling different clinical criteria. It is necessary to analyze the germline mutations of MSH6 gene using sequencing to identify HNPCC families in the probands in which MSH2 and MLH1 mutation were excluded.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Pair Mismatch ; genetics ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; pathology ; DNA Mutational Analysis ; DNA Repair Enzymes ; genetics ; Female ; Germ-Line Mutation ; genetics ; Humans ; Male ; Middle Aged ; MutS DNA Mismatch-Binding Protein ; genetics ; MutS Homolog 2 Protein ; genetics ; Pedigree ; Polymerase Chain Reaction

OBJECTIVETo confirm that PCR products with heterozygous mutations contain not only wide-type and mutant homoduplexes, but also two types of heteroduplexes.

METHODSAn insertion-deletion mutation in the exon 1 of KRT9 gene (497delAinsGGCT), which caused Chinese epidermolytic palmoplantar keratoderma (EPPK) was investigated by polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and denaturing high-performance liquid chromatography(DHPLC).

RESULTSTwo heteroduplexes and two homoduplexes in the PCR product from the heterozygous mutation of the exon 1 of KRT9 (497delAinsGGCT) were detected.

CONCLUSIONPCR products from KRT9 gene with heterozygous mutations contain two types of heteroduplexes. It is without the need to perform heating and cooling PCR products obtained from heterozygous mutations in advance before the mutation screening steps such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), conformation-sensitive gel electrophoresis (CSGE), DHPLC and heteroduplex analysis (HA), etc.

Base Pair Mismatch ; Chromatography, High Pressure Liquid ; DNA Mutational Analysis ; Electrophoresis, Polyacrylamide Gel ; Heteroduplex Analysis ; Heterozygote ; Humans ; Keratin-9 ; Keratins ; genetics ; Mutation ; Nucleic Acid Heteroduplexes ; Polymerase Chain Reaction

OBJECTIVETo detect methylation in promoter region of hMSH2 gene in esophageal cancer.

METHODSSpecimens of cancer and normal tissues freshly removed from 32 cases of esophageal cancer patients without previous radiotherapy, chemotherapy or other treatment were preserved at -80 degrees C within 30 min. Methylation specific PCR (MSP) was used to detect methylation of mismatch repair gene (MMR) hMSH2 in promoter region in esophageal cancer and normal esophageal tissues.

RESULTSThe frequencies of methylation of hMSH2 gene in promoter region of cancer and normal esophageal tissues were 32.4% (11/32) and 0/30 (0%), respectively, and significant difference was found between the two groups (P < 0.01). The frequency of methylation in elder patients (> or = 70 years old) was significantly higher than that in younger patients (< 70 years old) (P < 0.05). Methylation was less frequently found in grade I-II (18.2%) than in grade III-IV (70.0%) (P < 0.05).

CONCLUSIONMethylation of hMSH2 gene in promoter region is related to patients' age and histopathological grade of the esophageal cancer.

Aged ; Base Pair Mismatch ; Carcinoma, Squamous Cell ; genetics ; pathology ; DNA Methylation ; Esophageal Neoplasms ; genetics ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; MutS Homolog 2 Protein ; genetics ; Promoter Regions, Genetic ; Transfection

BACKGROUND: Although there have been some reports on microsatellite alterations in gastric cancer, findings are inconsistent regarding the associations between histological classification and microsatellite instability (MSI). In the present study, we attempted to determine whether Lauren's histological subtypes are related with MSI status. METHODS: Paraffin-embedded tissue samples from 14 diffuse-type and 14 intestinal-type gastric adenocarcinomas were matched up according to patient gender and age. Mononucleotide markers (BAT25 and BAT26) and dinucleotide markers (D2S123, D5S346, and D17S250) were used for MSI analyses. Microsatellite genotypes were categorized in terms of high MSI incidence (MSI-H, > 30% positive marker) or low MSI incidence (MSI-L, < 30% positive marker). Losses of hMLH1 and hMSH2 protein expression were immunohistochemically studied. RESULTS: MSI-H was observed in 11 cases (78%) of the 14 intestinal-type cases as compared to 3 (21%) of the 14 diffuse-type cases (p=0.007). In MSI-H tumors, 10 cases (71%) showed losses of hMLH1 protein expression, while 2 cases (14%) in MSI-L tumors showed losses of hMLH1 protein expression (p=0.006). CONCLUSION: MSI-H tumors are more frequently found in intestinal-type gastric cancer, which suggests the possibility that there are different pathogenic pathways in gastric carcinogenesis according to histologic type.
Adenocarcinoma/epidemiology/*genetics/pathology ; Aged ; Base Pair Mismatch/*genetics ; Comparative Study ; Female ; Gene Expression Regulation, Neoplastic ; Genotype ; Humans ; Incidence ; Korea/epidemiology ; Male ; Microsatellite Repeats/*genetics ; Neoplasm Proteins/genetics ; Nuclear Proteins/genetics ; Polymerase Chain Reaction ; RNA, Messenger/genetics ; Retrospective Studies ; Stomach Neoplasms/epidemiology/*genetics/pathology

OBJECTIVESTo establish DHPLC method in detecting mutations of mismatch repair genes, hMLH1 and hMSH2, and to identify germline mutations of hMLH1 and hMSH2 in HNPCC kindreds fulfilling Chinese HNPCC criteria.

METHODSFourteen peripheral blood DNA samples from 14 unrelated HNPCC probands fulfilling Chinese HNPCC criteria were obtained respectively. PCR amplified 35 exons of two main mismatch repair genes, hMLH1 and hMSH2. DHPLC followed by DNA sequencing was used to detect and confirm mutations.

RESULTSa total of 41 colorectal cancers and 19 extracolonic tumors were developed in 14 HNPCC kindreds, and gastric cancer was the most common extracolonic tumor type. Twelve single nucleotide changes were identified by DHPLC in 14 probands. Among them, three were missense mutations, one was a nonsense mutation. Other single nucleotide changes included five single nucleotide polymorphisms, two intron single nucleotide changes, one synonymous mutation. hMLH1 EXON19 CODON749 TAC-->TAG (Tyr-->X), hMSH2 EXON12 CODON629 CAA-->CGA (Gln-->Arg) and hMSH2 EXON15 CODON839 CAT-->CGT (His-->Arg) were new discovered mutations.

CONCLUSIONS(1) DHPLC was considered to be highly effective, convenient technique with consistent results for the mutation detection of hMLH1 and hMSH2 genes. (2) Valid mutations of hMLH1 and hMSH2 genes were identified in about one-third HNPCC kindreds fulfilling Chinese HNPCC criteria and missense mutation was the most common mutational types in this cohort of families.

Adaptor Proteins, Signal Transducing ; Asian Continental Ancestry Group ; genetics ; Base Pair Mismatch ; Carrier Proteins ; genetics ; Chromatography, High Pressure Liquid ; Codon, Nonsense ; Colorectal Neoplasms, Hereditary Nonpolyposis ; genetics ; DNA Mutational Analysis ; Female ; Genetic Testing ; Germ-Line Mutation ; Humans ; Male ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; genetics ; Mutation, Missense ; Nuclear Proteins ; genetics ; Pedigree

OBJECTIVETo investigate the frequency of large fragment aberrations of MSH2 and MLH1 genes from Chinese colorectal cancer (CRC) patients with family history.

METHODSSixteen exons of MSH2, nineteen exons of MLH1 and seven DNA sequences from the other genes of the samples were screened and checked by multiplex ligation dependent probe amplification (MLPA). First, the methodology was confirmed by testing the positive and negative control samples. Then, 32 CRC or hereditary nonpolyposis colorectal cancer (HNPCC) patients with family history and 20 cases of sporadic CRC were applied to investigate for the large fragment aberrations of MSH2 and MLH1 genes.

RESULTSThe genomic DNA fragment deletions of all positive controls were identified and verified by MLPA. Three cases of 32 familial (hereditary) CRC/HNPCC were detected and identified to be the germline heterozygous deletions of MSH2 gene, of which exons 1-7 were deleted from patient No.3, exon 11 from No.25 and exons 2-6 from No.11. However, no genomic DNA fragment aberration of either MSH2 or MLH1 gene was uncovered from 20 sporadic CRC.

CONCLUSIONLarge DNA fragment aberrations of MSH2 gene was a frequent cause of Chinese HNPCC and CRC patients with family history, and the identification of those aberrations should be included in the regular genetic analysis for CRC/HNPCC patients.

Adaptor Proteins, Signal Transducing ; genetics ; Asian Continental Ancestry Group ; genetics ; Base Pair Mismatch ; China ; Colorectal Neoplasms, Hereditary Nonpolyposis ; ethnology ; genetics ; DNA Mutational Analysis ; Humans ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; genetics ; Mutation ; Nuclear Proteins ; genetics