Frequent Microsatellite Instabilities and Analyses of the Related Genes in Familial Gastric Cancers

Microsatellite instability or replication error seems to be related to defective DNA mismatch repair genes, such as hMSH2 , hMLH1 , hPMS1 and hPMS2 , which have been identified as causative genes of hereditary nonpolyposis colorectal cancers (HNPCC). Recently, it was reported that mutations at the simple repeated sequences in the transforming growth factor-β type II receptor ( TGF-β RII ) gene occurred in replication error-positive colorectal cancers. To determine genetic alterations in familial gastric cancers (FGC), we examined replication error using eight microsatellite DNA markers, and screened mutations in the hMSH2 , hMLH1 and TGF-β RII genes in six cases from four FGC kindreds. Moreover, hMTH1 , a human homolog of the bacterial mutT gene, was also screened. Four of six (67%) cancers showed the replication error-positive phenotype, indicating that microsatellite instability is highly associated with not only HNPCC, but also FGC. No germline mutation was found in the whole coding sequences of hMSH2 and hMTH1 , or in the conservative regions of hMLH1 in any patient, while one cancer DNA showed a somatic mutation at codon 682 (threonine to alanine) in hMSH2 . No alteration was found at the small repeated sequences in TGF-β RII in FGC tumor DNA. These results indicate that the carcinogenetic process of FGC may be different from that of HNPCC.     

Clonal Heterogeneity in Human Esophageal Squamous Cell Carcinomas on DNA Analysis

Cancers are thought to arise through multistep accumulation of somatic mutations in the progeny of a single cell. Multiple mutations may induce molecular intratumor heterogeneity. Therefore, we examined molecular clonal heterogeneity in esophageal squamous cell carcinomas. Twenty-four esophageal squamous cell carcinomas and associated lymph node metastases were examined for microsatellite alterations, and abnormalities of the p53 and transforming growth factor-β type II receptor ( TGF-β RII ) genes. There were eight cases (33%) showing different patterns of loss of heterozygosity in primary tumors and metastatic lymph nodes with microsatellite markers. On the other hand, the abnormalities of p53 were identical in all these cases. No mutation was detected in the simple repeated sequences of the TGF-β RII gene. These results indicate that molecular clonal heterogeneity exists in esophageal squamous cell carcinomas. Therefore, care is necessary in preoperative genetic diagnosis using biopsy samples.     

Microsatellite instability is associated with the macroscopic configuration of neoplasms in patients with multiple colorectal adenomas

BACKGROUND: The management of patients with multiple colorectal adenomas, 10-100 in number, is often troublesome clinically. To establish the reasonable management of such cases, genetic backgrounds should be made clear. METHODS: For a total of 19 adenomas and four carcinomas from four patients with multiple colorectal adenomas, we analyzed genetic instability at four selected microsatellite loci and screened exon 1-4 of the APC gene with special reference to macroscopic configurations of adenomas and carcinomas. RESULTS: RER-positive phenotypes were detected in none of 13 protruded type adenomas, two (33%) out of six superficial elevated type adenomas and two (50%) out of four carcinomas. We detected no mutation of exon 1-4 of the APC gene in any sample. In patients with multiple colorectral adenomas, the proportion of superficial elevated type adenomas exhibiting genetic instability was significantly higher than that of protruded type adenomas. CONCLUSIONS: The results suggested that RER-positive phenotype was an early event in tumorigenesis through superficial elevated type adenomas.      

Mutational analysis of the transforming growth factor beta receptor type II gene in hereditary nonpolyposis colorectal cancer and early-onset colorectal cancer patients.

Somatic mutations in the transforming growth factor beta receptor type II (TGF-beta RII) gene have been observed in various human cancers showing microsatellite instability. Most of the mutations observed were additions or deletions of the mononucleotide repeat sequence present in TGF-beta RII coding region, suggesting that the TGF-beta RII may be a target gene of genomic instability in tumorigenesis. Recently, we reported germ-line frameshift mutations in the mononucleotide repeat sequence of the hMSH6 gene, which is believed to be one of the target genes of genomic instability in tumorigenesis, suggesting the possibility of germ-line mutation in mononucleotide repeat sequences. Moreover, one case of germ-line mutation in the TGF-beta RII gene was identified in a hereditary nonpolyposis colorectal cancer (HNPCC) kindred, indicating the involvement of TGF-beta RII inactivation in tumorigenesis of HNPCC. However, germ-line mutation analysis of all of the coding sequences and the mononucleotide repeat sequence of the TGF-beta RII in HNPCC patients has not yet been fully elucidated. Therefore, to further investigate the presence of germ-line mutations, we screened all of the coding region sequences and mononucleotide repeat sequence of TGF-beta RII from 35 HNPCC, 44 suspected HNPCC, and 45 sporadic early-onset colorectal cancer patients. However, no pathogenic mutations other than silent mutations, introgenic mutation, and polymorphisms were identified. Two silent mutations at codons 309 (ACG to ACA) and 340 (CAT to CAC) in the kinase domain located in exon 4 were detected. A 1-bp cytidine deletion was observed 6 bases from the 3' end of intron. Two polymorphisms were identified at codon 389 (AAC to AAT) and at the fourth-to-last base in intron 3. The polymorphism at codon 389 was more frequent in HNPCC (20%; 7 of 35) and suspected HNPCC patients (18%; 8 of 44) than in nonmalignant control group (10%; 5 of 50). Moreover, the frequency was significantly higher in early-onset colorectal cancer patients (31%; 14 of 45). This is the first report of a different frequency of polymorphism in HNPCC, suspected HNPCC, early-onset colorectal cancer patients, and healthy normal individuals. This result suggests that: (a) germ-line mutation of the TGF-beta RII gene may be a rare event during tumorigenesis in HNPCC and sporadic early-onset colorectal cancer; (b) the mononucleotide repeat sequence of the TGF-beta RII gene is an apparent target of genomic instability but not of germ-line mutation; and (c) the polymorphism of codon 389 (AAC to AAT) is frequent, especially in early-onset colorectal cancer patients, in which it is more frequent than in control group.     

Mutations and Reduced Expression of the Transforming Growth Factor-β Receptor II Gene in Rat Lung Adenocarcinomas Induced by N-Nitrosobis-(2-hydroxypropyl)amine

Mutations and expression of the transforming growth factor-β receptor type II (TGF-βRII) gene were investigated in lung adenocarcinomas induced by N -nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Males of the Wistar strain, 6 weeks old, were given 2000 ppm of BHP in their drinking water for 12 weeks and then maintained without further treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas and mutations in TGF-βRII were investigated by RT-PCR-restriction-SSCP analysis followed by sequencing analysis. Two out of 12 adenocarcinomas showed band shifts, indicative of mutations (16.7%). One was a CTG-to-TTG (Leu to Leu) transition at codon 308 without amino acid alteration and the other a frameshift deletion of one of two guanines at nucleotides 1434 to 1435 (codon 477 to 478). Semi-quantitative RT-PCR analysis demonstrated significantly reduced TGF-βRII expression in adenocarcinomas, as compared with normal lung tissue. These results suggest that TGF-βRII alterations may play a role in the acquisition of growth advantage by lung adenocarcinomas induced by BHP in rats.     

Involvement of APC and K-ras mutation in non-polypoid colorectal tumorigenesis

The aim of this study was to clarify the role of APC and K-ras mutations in non-polypoid colorectal tumorigenesis. DNA from 63 adenomas (31 polypoid, 17 superficial elevated, 15 superficial depressed), 66 submucosally invasive carcinomas (47 polypoid, 19 non-polypoid) and 34 advanced carcinomas were examined for K-ras codon 12 point mutations and APC mutations in the mutation cluster region. K-ras mutation: the frequency in superficial depressed adenomas was lower than that in polypoid adenomas (0% vs 31%: P= 0.018). The frequency in non-polypoid carcinomas was lower than that in polypoid carcinomas (11% vs 56%: P = 0.0008), and was relatively low compared with that in polypoid adenomas (11% vs 31%). APC mutation: the frequency in superficial depressed adenomas was lower than that in polypoid adenomas (7% vs 43%: P = 0.016), and that in polypoid carcinomas was similar to that in non-polypoid carcinomas. Polypoid adenomas, polypoid carcinomas and advanced carcinomas had almost the same frequency. There may be some pathway other than the conventional adenoma-carcinoma sequence in development of non-polypoid carcinomas. The precursors of most non-polypoid carcinomas are considered to be de novo or superficial depressed adenomas. In this non-polypoid pathway, APC mutation seems to be requisite but K-ras mutation not. It is possible that new APC mutations are acquired after the development of superficial depressed adenomas.     

Identification of MARCKS,FLJ11383 and TAF1B as putative novel target genes in colorectal carcinomas with microsatellite instability

Somatic frameshift mutations in some genes containing coding mononucleotide repeats (cMNRs) are well known characteristics of tumors with high microsatellite instability (MSI-H). We identified 22 novel and 11 known target genes containing cMNRs with a length of 10 or more nucleotides by using a systematic database search. Frameshift mutation analysis was performed with these 33 genes in 39 MSI-H and 24 microsatellite stable (MSS) colorectal carcinomas by assessing the mobility shifts of PCR products in gel electrophoresis and by sequencing. All the 39 MSI-H colorectal carcinomas, except one, showed mutations in more than one gene, while no mutations were found in 24 MSS colorectal carcinomas. Of these MSI-H tumors, 11 genes were mutated in more than 40%. The most frequently mutated novel genes were MARCKS (72%), FLJ11383 (74%) and TAF1B (82%). Biallelic inactivation in MARCKS and FLJ11383 was also frequent in MSI-H tumors. The observed mutation frequency of the 11 known target genes was compatible with that found by previous studies. The very high frequency of mutations, biallelic mutations and the predicted truncation of protein products suggests that mutations of MARCKS, FLJ11383 and TAF1B are selected, and play a role in the tumorigenesis of MSI-H colorectal carcinomas.     

Beta-catenin mutations are more frequent in small colorectal adenomas than in larger adenomas and invasive carcinomas

Loss of serine or threonine phosphorylation sites from exon 3 of beta-catenin has been identified in approximately half of colorectal tumors which lack adenomatous polyposis coli (APC) mutations, but the overall contribution of beta-catenin mutations to sporadic colorectal tumorigenesis is unclear. We therefore used PCR to amplify and sequence exon 3 of beta-catenin from 202 sporadic colorectal tumors. Exon 3 beta-catenin mutations were identified in 6 of 48 small (< 1 cm) adenomas, 2 of 82 large (> or =1 cm) adenomas, and 1 of 72 invasive carcinomas. Eight of the nine mutations, including all of those in the small adenomas and the invasive cancer, involved loss of serine or threonine phosphorylation sites. The percentage of beta-catenin mutations in small adenomas (12.5%) was significantly greater than that in large adenomas (2.4%) and invasive cancers (1.4%; P = 0.05 and P = 0.02, respectively). We conclude that mutation of beta-catenin can be an early, perhaps initiating, event in colorectal tumorigenesis. Small adenomas with beta-catenin mutations do not appear to be as likely to progress to larger adenomas and invasive carcinomas as other adenomas, however, with the result that beta-catenin mutations are only rarely seen in invasive cancers. This suggests that APC and beta-catenin mutations are not functionally equivalent, and that the APC gene may have other tumor suppressor functions besides the degradation of beta-catenin.     

Damage to the transforming growth factor TGF-beta type II receptor gene and microsatellite instability in carcinoma cells of the gastrointestinal tract

In order to compare the frequency of damage to the transforming growth factor TGF-beta receptor type II gene (RII gene) and microsatellite instability (MIN) in oncogenesis of sporadic and hereditary cancer of gastrointestinal tract (GIT), 4 groups of carcinomas were analyzed. They included sporadic gastric (GC), family gastric (FGC), sporadic colorectal (CC) and hereditary nonpolyposis colorectal (HNPCC) carcinomas having appropriate clinical and pathological characteristics. Each group consisted of two types of carcinomas, one of them showing MIN. The RII gene damage occurred in 89% of GC (8 cases out of 9), 86% of CC (6 out of 7), 71% of FGC (5 out of 7), 50% of HNPCC (3 out of 6) for carcinomas coupled with MIN, whereas only in 6% (1 out of 18) of GC and 5% (1 out of 22) of CC for carcinomas without MIN. No damage to RII gene was found in the cases of hereditary carcinomas which did not show MIN though the number of cases analyzed was not sufficient for final conclusions (3 cases of FGC and 3 HNPCC). The data revealed a correlation between the MIN phenotype and mutations in RII gene both for sporadic (p < 0.001) and for hereditary (p < 0.02) cases. For all 4 groups the frequency of RII gene damage was found for early and advanced carcinomas. This suggests that the deficiency of TGF-beta receptor complex in both sporadic and hereditary carcinomas of GIT is revealed at early stages of tumor development and consequently may be responsible for tumor progression. The correlation between RII gene damages and MIN in GIT carcinoma cells suggests that genetic change predetermined the neoplasia of colorectal and gastric epithelium and partially overlapped for both sporadic and hereditary cases.     

Active TGF-β1 correlates with myofibroblasts and malignancy in the colorectal adenoma-carcinoma sequence

Transforming growth factor-β1 (TGF-β1), a cytokine involved in various stages of cancer, is produced as a latent complex and requires processing to become active. We have determined total and active TGF-β1 levels in homogenates of colorectal neoplasia. In contrast to total TGF-β levels, showing a stepwise increase in the mucosa-adenoma-carcinoma sequence, active TGF-β1 levels are increased only in carcinomas but not in premalignant adenomas. Furthermore, solely active TGF-β1 levels are associated with the stage of the carcinomas and worse patient prognosis. Active TGF-β1 levels correlated significantly with plasminogen activator inhibitor (PAI)-1, α-smooth muscle actin (SMA) and several matrix-remodeling proteinases. Interestingly, SMA levels are also significantly increased in colorectal carcinomas but not in adenomas, suggesting that despite the enhanced total TGF-β1 levels, myofibroblast accumulation is not (yet) occurring in these premalignant neoplasias. The correlation between active TGF-β1 and SMA expression in tumors indicates that tumor-promoting myofibroblasts might arise as a result of increased TGF-β1 activation. These data underline the significance of the interaction between malignant cells and (myo)-fibroblasts in the tumor microenvironment, modulating the biologic behavior of colorectal cancer. ( Cancer Sci 2009; 100: 663–670)     

Genetic and epigenetic changes of components affecting the WNT pathway in colorectal carcinomas stratified by microsatellite instability

An unselected series of 310 colorectal carcinomas, stratified according to microsatellite instability (MSI) and DNA ploidy, was examined for mutations and/or promoter hypermethylation of five components of the WNT signaling cascade [APC, CTNNB1 (encoding beta-catenin), AXIN2, TCF4, and WISP3] and three genes indirectly affecting this pathway [CDH1 (encoding E-cadherin), PTEN, and TP53]. APC and TP53 mutations were each present more often in microsatellite-stable (MSS) tumors than in those with MSI (P < .001 for both). We confirmed that the aneuploid MSS tumors frequently contained TP53 mutations (P < .001), whereas tumors with APC mutations and/or promoter hypermethylation revealed no associations to ploidy. Mutations in APC upstream of codons 1020 to 1169, encoding the beta-catenin binding site, were found in 15/144 mutated tumors and these patients seemed to have poor clinical outcome (P = .096). Frameshift mutations in AXIN2, PTEN, TCF4, and WISP3 were found in 20%, 17%, 46%, and 28% of the MSI tumors, respectively. More than half of the tumors with heterozygote mutations in AXIN2 were concurrently mutated in APC. The present study showed that more than 90% of all samples had alteration in one or more of the genes investigated, adding further evidence to the vital importance of activated WNT signaling in colorectal carcinogenesis.     

Mutations of the adenomatous polyposis coli gene in the mutation cluster region: Comparison of human pancreatic and colorectal cancers

We analyzed the adenomatous polyposis coli (APC) tumorsuppressor gene as one of the possible genes mutated in human pancreatic carcinomas. DNAs from 39 surgical specimens were subjected to single-strand conformation polymorphism analysis of polymerase chain reaction products and the mutation cluster region (MCR) of the gene was examined. We also examined the same region of DNAs from 27 surgical specimens of sporadic colon carcinomas and detected mutations in 11 carcinomas (41%). This mutation frequency in colon carcinomas was similar to those reported previously. Using this system, we detected a mutated APC gene in one of 39 pancreatic carcinomas. The results indicated that mutation of the MCR in the APC gene is involved in genesis of some of human pancreatic carcinomas, but its frequency is much lower than in colorectal carcinomas.     

Genetic Alterations in Ulcerative Colitis-associated Neoplasia Focusing on APC, K-ras Gene and Microsatellite Instability

The status of genetic alterations in ulcerative colitis (UC)-associated neoplasia (UCAN) was investigated focusing on microsatellite instability (MSI) which is seen in a certain fraction of colorectal carcinomas, and adenomatous polyposis coli (APC) gene and K-ras gene, in which mutations occur in the early stage of sporadic colorectal tumorigenesis. Thirty-one UCAN from 15 UC patients who had undergone colorectal resection at our institution were investigated. There were 8 lesions of invasive carcinoma, 15 high-grade dysplasia (HGD) and 8 low-grade dysplasia (LGD). DNA was extracted from each neoplastic lesion and corresponding non-neoplastic tissue by a microdissection method. MSI status at 9 microsatellite loci, loss of heterozygosity (LOH) at the APC locus, and K-ras codon 12 point mutation were examined. As for MSI, 4/31 (13%) UCAN (carcinoma: 1/8 (13%), HGD: 2/15 (13%), LGD: 1/8 (13%)) were MSI-high (3 or more unstable loci) and 12/31 (39%) UCAN (carcinoma: 3/8 (38%), HGD: 6/15 (40%), LGD: 3/8 (38%)) were MSI-low (1 or 2 unstable loci). LOH at the APC locus was not found in 9 UCAN from 6 informative (heterozygous) cases. The K-ras mutation rate of UCAN was 3/31 (9.7%) (carcinoma: 2/8 (25%), HGD: 1/15 (7%) and LGD: 0/8). MSI is relatively common in UCAN and is present at the early stage of tumorigenesis of UCAN, while the involvement of genetic alterations of the APC gene and K-ras gene is small. MSI may be one of the mechanisms of the increased neoplastic risk in UC, and UCAN may develop through a different carcinogenic pathway from sporadic carcinomas.     

Genetic alterations in ulcerative colitis-associated neoplasia focusing on APC, K-ras gene and microsatellite instability.

The status of genetic alterations in ulcerative colitis (UC)-associated neoplasia (UCAN) was investigated focusing on microsatellite instability (MSI) which is seen in a certain fraction of colorectal carcinomas, and adenomatous polyposis coli (APC) gene and K-ras gene, in which mutations occur in the early stage of sporadic colorectal tumorigenesis. Thirty-one UCAN from 15 UC patients who had undergone colorectal resection at our institution were investigated. There were 8 lesions of invasive carcinoma, 15 high-grade dysplasia (HGD) and 8 low-grade dysplasia (LGD). DNA was extracted from each neoplastic lesion and corresponding non-neoplastic tissue by a microdissection method. MSI status at 9 microsatellite loci, loss of heterozygosity (LOH) at the APC locus, and K-ras codon 12 point mutation were examined. As for MSI, 4/31 (13%) UCAN (carcinoma: 1/8 (13%), HGD: 2/15 (13%), LGD: 1/8 (13%)) were MSI-high (3 or more unstable loci) and 12/31 (39%) UCAN (carcinoma: 3/8 (38%), HGD: 6/15 (40%), LGD: 3/8 (38%)) were MSI-low (1 or 2 unstable loci). LOH at the APC locus was not found in 9 UCAN from 6 informative (heterozygous) cases. The K-ras mutation rate of UCAN was 3/31 (9.7%) (carcinoma: 2/8 (25%), HGD: 1/15 (7%) and LGD: 0/8). MSI is relatively common in UCAN and is present at the early stage of tumorigenesis of UCAN, while the involvement of genetic alterations of the APC gene and K-ras gene is small. MSI may be one of the mechanisms of the increased neoplastic risk in UC, and UCAN may develop through a different carcinogenic pathway from sporadic carcinomas.     

More Frequent β-Catenin Gene Mutations in Adenomas than in Aberrant Crypt Foci or Adenocarcinomas in the Large Intestines of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-treated Rats

Alteration of adenomatous polyposis coli (APC) is known to be an early event in neoplasia, causing activation of the β-catenin/Tcf pathway. Although it is thought that alterations in APC and β- catenin may complement one another, the contribution of β- catenin mutations to colorectal carcino-genesis remains unclear. We therefore performed PCR-single strand conformation polymorphism analysis and direct sequencing of exon 3 of β- catenin gene in adenomas, adenocarcinomas, and aberrant crypt foci (ACF), considered to be putative precursor lesions of colorectal neoplasias, in 2-amino-1-methyl-6-phenylimidazo[4,5 b ]pyridine (PhIP) treated F344 rats. β- Catenin mutations were identified in all of 7 adenomas (100%) and 6 of 12 (50%) adenocarcinomas. All of the mutations were found in codons 32 through 34, the serine encoded by codon 33 being an important phosphorylation site by glycogen synthase kinase-3β. Regarding ACF, 14 of 46 (30.4%) were found to be mutated, eleven (78%) in codon 34, and the others in codon 45 (frequently altered in human colon cancer), and codons 47 and 56 (which have not been previously reported). The frequency of β- catenin mutations in adenomas was significantly higher than in ACF ( P < 0.001) and adenocarcinomas ( P < 0.05). Thus, β- catenin mutations may have more importance in the genesis of adenomas than ACF or adenocarcinomas in rat colon carcinogens by PhIP.     

Pathological features and genetic alterations in colorectal carcinomas with characteristics of nonpolypoid growth

We sought to clarify pathological features and genetic alterations in colorectal carcinomas with characteristics of nonpolypoid growth. Colorectal carcinomas resected at Showa University Hospital in Tokyo included 86 with characteristics of polypoid growth (PG) and 21 with those of nonpolypoid growth (NPG). Mutations of APC, Ki-ras, and p53 genes, as well as microsatellite instability (MSI), were analysed using fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Carcinomas with an NPG pattern were smaller than PG tumours (P<0.0001). Carcinomas with a PG pattern were more likely to harbour Ki-ras mutations (36%) than NPG tumours (0%; P<0.0001). Mutation types in the APC gene differed significantly between PG and NPG carcinomas (P=0.0189), including frameshift mutations in 66% of PG carcinomas but no NPG carcinomas. Presence of a p53 mutation at a 'hot spot' also was more likely in PG carcinomas (37%) than in NPG carcinomas (0%; P=0.0124). No significant difference in presence of MSI was evident between carcinomas with PG and NPG patterns. In conclusion, significant genetic differences were evident between carcinomas with PG and NPG patterns. Genetic changes in NPG carcinomas differed from those of the conventional adenoma-carcinoma sequence. Assuming that some nonpolypoid growth lesions transform rapidly into advanced carcinomas, 20% of all colorectal carcinomas may progress in this manner.     

Genetic instability and mutation of the TGF-β-receptor-II gene in ampullary carcinomas

Ampullary carcinomas are relatively rare cancers of which very little is known in terms of carcinogenetic mechanisms at the molecular level. Genetic instability caused by mutations of mismatch-repair genes has been demonstrated to be responsible for hereditary non-polyposis colorectal cancers and a sub-set of sporadic colorectal cancers. In some of those tumors showing genetic instability, the transforming-growth-factor-β(TGF-β)-receptor-II gene has been found to be mutated in repetitive sequences and considered to be a target of replication error. We studied the role of genetic instability and associated TGF-β-receptor-II-gene mutations in a series of 18 sporadic cases by analyzing 5 microsatellite loci (D2S123, D3S1029, D5S409, TP53 and BAT26) and by sequencing a poly-A repeat (nucleotides 709-718) in the TGF-β-receptor-II gene. Microsatellite instability was observed in 4 (22.2%) and gene mutations in 14 (77.8%) cases. These data indicate that the TGF-β-receptor-II gene might be a preferential target of genetic instability whose alteration might be specifically advantageous and constitute a common step in the development of ampullary carcinomas. Int. J. Cancer 76:407–411, 1998.© 1998 Wiley-Liss, Inc.