Kum H. Koo

Genotyping possible polymorphic variants of human mismatch repair genes in healthy Korean individuals and sporadic colorectal cancer patients.

The genotypic consequences of numerous single-nucleotide variants in human mismatch repair genes are mostly undetermined. We examined 27 reported single-nucleotide variants, rarely or ambiguously verified in a population-based study, to identify single-nucleotide polymorphisms (SNPs), haplotypes, and the genotype–phenotype association in Korean populations of 330 healthy individuals, 107 sporadic colorectal cancer patients, and 107 of their first-degree relatives. Real-time PCR 5′-nuclease assays (TaqMan® MGB assay) were used to determine 24 single-nucleotide variants, and restriction fragment length polymorphism (RFLP) assays were used to determine 3 variants. Of these 27 variants, 4 (hMSH2 gIVS12-6, hMLH1 655, hMLH1 1151, and hMSH2 1168, in descending order) were identified as SNPs occurring in 4.5 to 53.1% of healthy individuals, with polymorphism levels of 0.023–0.3 (mean, 0.092). East Asian populations had an ethnic predilection for the hMLH1 1151 SNP. The genotype distribution for all four SNPs showed no association with sporadic colorectal cancer. Twenty-three variants were not identified in the Korean population, suggesting that fifteen of these variants are colorectal cancer-related mutations and eight are SNPs. Two haplotype patterns existed exclusively, but with rare frequency, in sporadic colorectal cancer patients. The hMLH1 655 allele was closely correlated with hMLH1 protein expression (P= 0.02), but none of the four SNPs was associated with clinicopathologic variables. Among the 27 single nucleotide variants of mismatch repair genes, 12 were suggestive of nonfunctional SNPs and 15 may be colorectal cancer-related mutations. Further verification in other ethnic groups may provide the genotypic and phenotypic significance of single nucleotide variants found in mismatch repair genes.

MYH, OGG1, MTH1, and APC alterations involved in the colorectal tumorigenesis of Korean patients with multiple adenomas

This study was done to characterize base excision repair (BER) genes and adenomatous polyposis coli (APC) alterations in the tumorigenesis of multiple colorectal adenomas in Korean patients. In total, 217 adenomas (mean number = 10) and 117 cancers were available from 143 patients. The heterozygous genotype of OGG1 c.1-18G>T was closely associated with multiple adenoma families (P < 0.001), while MYH A359V mutation exhibited a tendency (P = 0.053). MYH R170G mutation was exclusively identified in one patient. The G:C>T:A transversion or attenuated familial adenomatous polyposis (AFAP) mutations of APC was identified in the specific genotypes of BER variants. Tubular adenomas or adenomas with none-to-mild dysplasia were significantly associated with polymorphic genotypes of OGG1 IVS4-15 and S326C. In addition, large and pedunculated adenomas were more frequent in patients with G:C>T:A transversion and AFAP mutations of APC, respectively. However, BER variants were not associated with mismatch repair or altered p53 protein expression. Conclusively, two novel mutations of MYH and a novel OGG1 polymorphism seemed to be associated with multiple colorectal adenomas in Korean families, differing from those in other ethnic groups. Some BER variants involved in specific APC mutations are associated with characteristics of histogenesis other than altered mismatch repair or p53 pathway.

Enhancement of colorectal tumor targeting using a novel biparatopic monoclonal antibody against carcinoembryonic antigen in experimental radioimmunoguided surgery

Biparatopic CEA, carcinoembryonic antigen (MAb) was newly designed and tested as to whether it enhanced the accuracy of tumor detection by reducing non‐specific binding in experimental radioimmunoguided surgery. Biparatopic MAb was prepared by using cross‐linking of reduced Fab′ fragments from PR1A3 and T84.66. Fifty‐nine tumors from 2 human colorectal carcinoma cell lines with high (KM‐12c) and low (Clone A) carcinoembryonic antigen (CEA) expression were successfully implanted subcutaneously on the backs of 42 nude mice. Tumors were localized using 125I‐labeled MAbs: IgG, F(ab′)2 and Fab′ of PR1A3, and biparatopic MAb of PR1A3 and T84.66. Radioactivity counted on a portable radioisotope detector correlated well with that counted on a gamma counter (p < 0.001). Accumulations of radioactivity in control mice without tumorigenesis were the greatest in PR1A3 IgG‐pretreated mice and the least in biparatopic MAb‐pretreated mice. Tumors of 2 cell lines did not differ in the distribution of radiolabeled MAbs. Localization indices of the tumor in various organs revealed 1.3 to 4.1 in PR1A3 IgG‐pretreated mice, 2.4 to 6.6 in fragment MAbs of PR1A3‐pretreated mice and 2 to 4.6 in biparatopic MAb‐pretreated mice. Silver grains and immune staining were predominantly distributed in tumor cells of all types of MAb‐pretreated mice. Sensitivity and specificity of tumor localization by radioimmunoguided surgery (RIGS) were the highest in the biparatopic MAb‐pretreated mice (90.9% and 94.5%, respectively) and the least in the PR1A3 IgG‐pretreated mice (50% and 72%). The biparatopic MAb using 2 anti‐CEA MAbs against different epitopes achieved a great affinity and avidity with accurate localization of colorectal carcinoma in experimental radioimmunoguided surgery.

Genetic and epigenetic changes in the APC gene in sporadic colorectal carcinoma with synchronous adenoma.

Abstract Background and aims. Somatic APC mutation, frequently associated with colorectal tumors, is implicated in the early stage of tumorigenesis. This study was performed to identify APC-related colorectal tumorigenesis in sporadic colorectal carcinomas with synchronous adenoma. Materials and methods. We screened the entire coding region of APC and also assessed 5q LOH, 5q MSI, and promoter hypermethylation in fresh colorectal tissue and the lymphocytes of 31 patients with synchronous colorectal adenoma and carcinoma. Results. The APC mutation prevalence was greater in carcinomas (70%) than in adenomas (45%). The 5q LOH and MSI were identified in 7 and in 5 of 31 carcinomas and in 6 each of 43 adenomas, respectively. The APC promoter methylation was identified in 3 cases each of both carcinomas and adenomas. Mutations in cases with 5q LOH were identified exclusively from codons 959 to the 3′ end of exon 15. Otherwise mutations identified between exons 1 and 14 showed additional mutation on exon 15 and no additional mutation in two cases. All carcinomas with 5q LOH, 5q MSI, or methylation included at least one APC mutation, whereas 5 carcinomas and 6 adenomas showed solely an APC mutation. Both alleles were disrupted in 1 of 31 normal mucosa (3.2%), 12 of 40 adenomas (30%), and 18 of 33 carcinomas (54.5%). Conclusion. Genetic and epigenetic events encompassing APC occur variously among patients and tissues in sporadic colorectal cancer patients with synchronous colorectal adenoma. Moreover, these changes sometimes appear to be accumulated in all of the stages of colorectal tumorigenesis.

Mutations at the APC exon 15 in the colorectal neoplastic tissues of serial array

Although the APC protein is known to participate in cellular proliferation and apoptosis, APC mutations have been thought to play a major role in the early stage of colorectal tumorigenesis. The somatic APC mutation of exon 15 was assessed to determine its impact on various stages of colorectal tumorigenesis. The colorectal neoplastic tissues of serial array studied included sporadic adenomas (group 1, n=36), adenomas (group 2, n=33), and carcinomas (group 3, n=32) in the synchronous adenoma and carcinoma as well as sporadic carcinomas (group 4, n=36). Aberrant DNA was detected by protein truncation test and confirmed by direct sequencing. The mutation prevalence was 36.1% in group 1, 45.5% in group 2, 59.4% in group 3, and 41.7% in group 4 with no differences among the groups. Among the 18 patients with synchronous adenoma and carcinoma, 9 had mutation in their adenomas and 12 in their carcinomas. The mutation loci and patterns did not differ in adenomas and carcinomas. Mutations in the mutation cluster region (MCR) were much more frequent than in the preceding region of MCR, i.e., 85.7% vs. 14.3%. The mutation prevalence of villous adenomas appeared greater than that of tubular adenoma (3/21 vs. 3/4). Predominant pathogenic mutations at MCR suggest that the APC mutation is implicated in all stages of colorectal tumorigenesis.

Characterization of mutator phenotype in Familial colorectal cancer patients not fulfilling Amsterdam criteria

Purpose: Although the mutator phenotype, including genetic and epigenetic alterations of the mismatch repair (MMR) system, seems to be pronounced in familial colorectal cancer, there have been few integrative studies comprising the entire mutator pathway. This study was done to identify the entire mutator pathway determining risk factors in patients with familial colorectal cancer not fulfilling the Amsterdam criteria. Experimental Design: We consecutively recruited 134 colorectal cancer patients with a family history of accompanying cancers. Patients with hereditary nonpolyposis colorectal cancer meeting the Amsterdam criteria, familial adenomatous polyposis, or those receiving preoperative radiotherapy were excluded. Mutator phenotype was assessed by assaying microsatellite instability (MSI) at 24 markers, hMLH1-promoter methylation, mutations at MMR genes (hMLH1, hMSH2, hMSH6, and hPMS2), and immune staining of MMR proteins (hMLH1, hMSH2, hMSH6, hPMS1, and hPMS2). Results: Of the 208 cancers in first-degree and/or second-degree relatives of patients, colorectal and gastric cancers (81%) were most common. Of the 134 proband colorectal cancers, 23 (17%) were MSI in high level, and 32 (24%) were MSI in low level. MMR alterations, including known polymorphism and splicing substitution, were identified in eight patients (6%). Twenty-eight tumors with mutator phenotype were further identified by hMLH1-promoter methylation and/or loss of MMR protein expression. In 51 tumors (38%), mutator phenotype was associated with right-sided colon cancer (P < 0.001) and younger age at onset (P = 0.032), but the number of patients with a mutator phenotype did not differ with respect to inheritance patterns of accompanying cancers, either successive or horizontal transmission (P = 0.815). Familial impact value, which differentially associated the degree of relatives with all accompanying cancers, effectively discriminated MSI in high level from microsatellite stable/MSI in low level tumors. Conclusion: Familial colorectal cancer may be associated with multiple occurrences of colorectal or accompanying cancers inherited by dominant or recessive transmission. MMR gene mutations, however, are less associated with mutator phenotype in familial colorectal cancer.

Carcino-embryonic antigen may function as a chemo-attractant in colorectal-carcinoma cell lines

Locomotion of colorectal‐carcinoma cells was tested in order to establish whether it might be affected by carcino‐embryonic antigen (CEA). CEA production, cell growth and DNA ploidy were measured in 22 colorectal‐carcinoma cell lines. A cell‐invasion assay was adapted using a transfilter chamber, the lower surface of which was coated with various substrates in the amount of 5 μg/filter (CEA, type‐IV collagen, laminin). Cells infiltrated into the lower surface of the filter were counted over 9‐microscope fields (×400). All cell lines produced CEA, 9 producing more than 100 ng/ml medium. Of the total, 8 cell lines were diploid and 14 were aneuploid. Invasiveness, measured by the number of infiltrated cells, was highest in CEA‐coated filters, and next highest in type‐IV‐collagen‐ and laminin‐coated filters, in descending order (p < 0.001–0.05). Invasiveness of each cell line was closely correlated with 2 substrates. Poorly differentiated or advanced‐stage tumors were more invasive than well‐differentiated or early‐stage tumors (p < 0.001–0.05). However, invasiveness was not associated with DNA ploidy or CEA production. CEA may function as a chemo‐attractant as well as an adhesion molecule in colorectal‐carcinoma cell lines. In addition, adhesion to CEA appears to be related to type‐IV collagen and laminin. Int. J. Cancer 82:880–885, 1999.

Geno- and pheno-typic characterization in ten patients with double-primary gastric and colorectal adenocarcinomas

AbstractBackground and aimsAlthough a number of double-primary gastric and colorectal cancers have been known to correlate with the mutator pathway, a possible association with known hereditary cancers regarding geno-pathogenesis has rarely been investigated. This study was intended to identify a possible association of hereditary cancers and the implications of the mutator or tumor-suppressor pathway in double-primary gastric and colorectal cancers.Materials and methodsFresh colorectal tissues and lymphocytes from ten patients with double-primary gastric and colorectal cancers were obtained consecutively. The mutator pathway was evaluated by detecting hMSH2 and hMLH1 mutations, microsatellite instability (MSI) using 12 microsatellite markers, and hMLH1 promoter region methylation. Protein expressions of hMSH2, hMLH1, APC, E-cadherin, and β-catenin were identified by immune staining.ResultsThere was no pathogenic mutation in any introns or exons of hMSH2, hMLH1 or CDH1, and in exons 3, 5, 6, and 15 of CTNNB1. Either MSI or methylator phenotype was found in five gastric cancers and in four colorectal cancers. No patients met the Amsterdam criteria of hereditary nonpolyposis colorectal cancer (HNPCC) or its equivalent of hereditary gastric cancer. Two patients with gastric cancers among their first-degree relatives showed no E-cadherin expression. The two of the three patients with rectal cancers among their first-degree relatives showed mutator phenotype either in the gastric or in the colorectal cancer. A subset of double-primary gastric and colorectal cancers may thereby be categorized as variant forms of hereditary gastric or colorectal cancer. Both cytoplasmic and nuclear β-catenins were expressed in all gastric and colorectal cancers. Among the gastric and colorectal cancers with either MSI or methylator phenotype, four of five gastric cancers showed both APC and E-cadherin expression, whereas one of four colorectal cancers showed them.ConclusionThis may suggest that the mutator pathway and the aberrant tumor suppressor pathway of the APC-E-cadherin may be cooperative or separately activated in the geno-pathogenesis of double-primary gastric and colorectal cancers.