Microsatellite alterations and p53, TGFβRII,IGFIIR and BAX mutations in sporadic non-small-cell lung cancer

Fifty-two sporadic primary non-small-cell lung carcinomas (NSCLC) were examined for microsatellite instability. Six different microsatellite markers localized on chromosomes 2, 5, 8, 10, 11 and 17 were used. Genomic instability was observed in 35% (18/52) of NSCLC at single or multiple loci. The tumors were also analyzed for p53-gene mutations by PCR-SSCP analysis. Polynucleotide stretch frameshift mutations of TGFβRII (transforming-growth-factor-beta receptor II), IGFIIR (insuline growth-factor II receptor) and BAX genes were also analyzed. RER⁺ (replication-error-positive) tumors appear not to be affected by a higher rate of point mutations in coding sequences: no correlation was found between microsatellite instability and point mutations in the p53 gene, and the RER⁺ tumors showed no alterations in stretches of nucleotide inside TGFβRII, BAX or IGFIIR. Int. J. Cancer 78:606–609, 1998. © 1998 Wiley-Liss, Inc.     

Sporadic gastric carcinomas with microsatellite instability display a particular clinicopathologic profile

Mutations in recently identified genes on chromosomes 2 and 3 seem to be responsible for repair errors (RER⁺) throughout the genome. This novel genetic mechanism was first reported in hereditary non-polyposis colorectal cancer syndrome and in cancers that are characteristic of this syndrome, such as carcinomas of the right colon, stomach and endometrium. We investigated the frequency of RER⁺ phenotype in a series of 34 sporadic gastric carcinomas, in an attempt to see if the RER⁺ cases displayed any particular morphologic features and/or if they showed distinctive clinicopathologic characteristics. Twelve loci were investigated. We found 23 RER^- cases (67.6%) and 11 RER⁺ cases (32.4%). A significant association was found between RER⁺ carcinomas and localization of the tumors: 9 of the 11 RER⁺ carcinomas (81.8%) were located at the antrum whereas all the cardiac tumors were RER^-. The RER⁺ phenotype was also significantly related to the presence of moderate/abundant T-cell lymphoid infiltration within the tumors. The 3-year survival rate of patients with RER⁺ tumors was suggestively longer than that of patients with RER^- tumors. No significant relationship was found between several clinicopathologic characteristics of the cases, including age, sex, staging, histologic type and ploidy, despite a trend towards an association between RER⁺ phenotype and advanced age of the patients and poorly differentiated, intestinal type of the carcinomas. The high frequency of microsatellite instability in sporadic gastric carcinomas supports the involvement of this genetic mechanism in gastric carcinogenesis. Gastric carcinomas with the RER⁺ phenotype tend to occur as poorly differentiated adenocarcinomas in the antrum of elderly patients, display abundant T-cell infiltration and carry a relatively good prognosis. © 1995 Wiley-Liss, Inc.     

Clinicopathological features of RER+ colorectal carcinoma

Objective: Replication errors (RER) is related to initiation and development of colorectal carcinoma (CRC). To investigate the different biological behavior of RER⁺ and RER⁻ CRC. Methods: Silver staining PCR-single strand conformation polymorphism (PCR-SSCP) and denatured polyacrylamide gel electrophoresis methods were used to detect microsatellite instability (MSI) at 4 loci on chromosome 2, 5, 17 in paraffin-embedded specimens of 60 colorectal carcinoma (CRC) and their paired normal tissue. RER⁺ was scored if 2 or more loci behaved as gaining extra bands. Results: The results showed that RER⁺ was found in 19/60 CRC, among which 7 cases had a family history. According to the criteria of Amsterdam, 4 were diagnosed as hereditary nonpolyposis colorectal cancer (HNPCC), and of which 3 cases were RER⁺. The ratio RER⁺ in HNPCC (75%) was significantly higher than that among sporadic CRC (28.5%). Most of the RER⁺ CRC have the feature of poorly differentiated adenocarcinoma (P<0.01), the tendency to involve the right side of the colon (P<0.05), a higher proportion with a family history (P<0.05), Duckes' A and B stage (P<0.05). Conclusion: The results indicated that RER⁺ is a relatively common molecular event in CRC. There are different clinico-pathological features and behavior between RER⁺ and RER⁻ CPC.     

Mutational analysis of the hMSH2 gene in a wide variety of tumors

The hMSH2 gene participates in DNA mismatch repair and its mutation can result in genetic instability of the human genome which is an important feature of tumorigenesis. In this study, genetic alterations of the hMSH2 gene were examined in 43 ovarian, 36 non-small cell lung (NSCL), 31 poorly differentiated gastric, 15 endometrial, and 11 colon cancers, nine gastric cancer cell lines, 41 adult T-cell leukemias (ATLs), two ATL cell lines, and 37 non-Hodgkin's lymphomas (NHLs), using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. Microsatellite instability (MSI) was also investigated for ovarian, NSCL, and colon cancers. The incidence of MSI was 1/36 (3%) for NSCL, 2/23 (9%) for ovarian, and 1/11 (9%) for colon cancers. Missense base changes of the hMSH2 gene were identified in two gastric cancer patients (ATG to ATA resulting in Met changing to Ile at codon 688 in exon 13 and ACA to GCA resulting in Thr changing to Ala at codon 803 in exon 14). These mutations were found in samples with no MSI. One ovarian and one gastric cancer, and six ATL samples showed two types of polymorphisms of hMSH2 (CTT to TTT resulting in Leu changing to Phe at codon 390 in exon 7 and CAG to AAG resulting in Gin to Arg at codon 419 in exon 7). Our data suggest that MSI and hMSH2 mutations are uncommon in sporadic tumors.     

Mutations of the human MUT S homologue 6 gene in ampullary carcinoma and gastric cancer

MSH6 has been implicated in repair of single base mispairs and single-base deletion/insertion mutations. Established MSH6-null mice present a frequent occurrence of gastrointestinal tumors without microsatellite instability (MI), suggesting the possibility of the APC gene being a mutational target. Because human ampullary carcinomas and gastric cancers manifest frequent missense or 1-base deletion mutations in cancer-related genes such as p53 and TGFβ-RII, we suspected that the hMSH6 gene mutation might play a role in the carcinogenesis process. Out of the whole coding sequences, hMSH6 (C)₈ (codons 1085–1087) and hMSH3 (A)₈ repeats (codons 381–383) have been shown to be hotspots for frameshift mutations in a certain group of cancers, contributing to an increased genomic instability. We therefore investigated mutations of hMSH6 (C)₈ and hMSH3 (A)₈ in association with microsatellite mutator phenotype (MMP) in 18 ampullary carcinomas and 30 gastric cancers. In addition, overexpression of the P53 protein and mutational status of APC (AG)₅ (codons 1462–1465) and (A)₆ (codons 1554–1556) repeats were also investigated as a potential target of genetic instability secondary to MSH6 dysfunction. Mutation of the hMSH6 gene was not found in ampullary carcinomas and was irrelevant to TGFβ-RII gene mutation. Mutation of the hMSH6 gene was observed in a subset of gastric cancers (4/30, 13.3%), but was not associated with P53 overexpression or APCgene mutation. In contrast to MSH6-null mice that do not show MI, hMSH6gene mutation in human gastric cancers was closely correlated with MMP (3/ 10 MMP vs. 1/ 20 non-MMP). In conclusion, hMSH6 mutation appears only in association with MMP and may underlie augmented MI, resulting in missense or 1-base frameshift mutations in other genes in human gastric cancers. Int. J. Cancer 78:576–580, 1998. © 1998 Wiley-Liss, Inc.     

Missense mutation of the hMSH6 and p53 genes in sporadic urothelial transitional cell carcinoma

Functional defects in DNA mismatch repair genes have been shown to be associated mainly with hereditary human malignancies. We examined genomic DNA from 88 sporadic transitional cell carcinomas (TCCs) of the urinary tract for mutations in hMSH6 gene by polymerase chain reaction and direct sequencing analysis. Mutational status of p53 gene was also studied as a potential target of genetic instability secondary to hMSH6 dysfunction. A total of 5 cases (5.7%: 5/88) displayed hMSH6 mutations all consisted of transition and located in exon 4, including three cases with missense mutation and two without change of corresponding amino acid. These three tumors with hMSH6 missense mutation had no microsatellite instability with five microsatellite markers tested. p53 gene mutations were detected in 22 cases (25.0%: 22/88). No tumors with p53 mutation had any hMSH6 missense mutations. Compared to the cases without hMSH6 alterations, the three patients with hMSH6 alterations had more frequent additional primary cancer (P<0.05). These findings provide the first in vivo evidence for the type of alterations and frequency of possible involvement of the hMSH6 mutations in sporadic type urothelial TCCs.     

Somatically acquired genetic alterations in flat colorectal neoplasias

Somatically acquired mutations in several genes have been reported as playing an important role during colorectal tumorigenesis. Two alternative groups of carcinomas, termed LOH⁺ and RER⁺, have been defined on the basis of their genetic anomalies, a biallelic inactivation of the APC or the TGF-βRII genes, occurring as an alternative, in LOH⁺ or RER⁺ tumors. It is a generally accepted hypothesis that most of colorectal cancers (CRC) develop from a pre-existing adenomatous polyp. Such benign lesions are usually exophytic polyps, a small proportion of adenomas having been described as flat lesions. The latter histological category has thus been proposed to bear specific genetic alterations. In order to examine this hypothesis, we have characterized a series of 44 flat colorectal neoplasias for their RER status and for somatic APC, KRAS and TGF-βRII genes mutations. Flat colorectal neoplasias were found to be of the RER⁺ subtype in 22% of cases, all of them exhibiting a TGF-βRII mutation. A mutation of the APC and KRAS genes has been found in 42% and 4% of tumors, respectively, none of these tumors being of the RER⁺ subtype. With the exception of a low KRAS mutation rate, flat adenomas appear to follow tumorigenesis pathways very similar to those identified in exophytic adenomas and carcinomas. Int. J. Cancer 77:366–369, 1998. © 1998 Wiley-Liss, Inc.     

Microsatellite instability in thyroid tumours and tumour-like lesions

Fifty-one thyroid tumours and tumour-like lesions were analysed for instability at ten dinucleotide microsatellite loci and at two coding mononucleotide repeats within the transforming growth factor β (TGF-β) type II receptor (TβRII) and insulin-like growth factor II (IGF-II) receptor (IGFIIR) genes respectively. Microsatellite instability (MI) was detected in 11 out of 51 cases (21.5%), including six (11.7%) with MI at one or two loci and five (9.8%) with Ml at three or more loci (RER⁺ phenotype). No mutations in the TβRII and IGFIIR repeats were observed. The overall frequency of MI did not significantly vary in relation to age, gender, benign versus malignant status and tumour size. However, widespread MI was significantly more frequent in follicular adenomas and carcinomas than in papillary and Hürthle cell tumours: three out of nine tumours of follicular type (33.3%) resulted in replication error positive (RER⁺), versus 1 out of 29 papillary carcinomas (3.4%, P = 0.01), and zero out of eight Hürthle cell neoplasms. Regional lymph node metastases were present in five MI-negative primary cancers and resulted in MI-positive in two cases. © 1999 Cancer Research Campaign     

Frameshift mutations and a length polymorphism in the hMSH3 gene and the spectrum of microsatellite instability in sporadic colon cancer.

Mutations in the hMVSH3 gene in sporadic colon cancer with microsatellite instability (MSI) were investigated, since several mismatch repair genes were known to be mutated in cancers with MSI, but only deletions in the (A)8 region in the hMSH3 gene have been reported. We also analyzed the relationships between hMSH3 mutations and the spectrum of MSI. We screened MSI in 79 sporadic colon cancer samples using mono- and dinucleotide repeat markers and the samples with MSI were further analyzed for tri- and tetranucleotide repeat instability and mutations in the hMSH3 gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Five (6%) out of 79 tumors were MSI-H and 15 (19%) were MSI-L. Two MSI-H tumors showed insertion in the (C)8 region in the hMSH6 gene and one tumor showed insertion and deletion in the (A)8 region in the hMSH3 gene, and two of the three above tumors showed MSI in tri-and tetranucleotide repeats. One MSI-L tumor showed somatic alteration in a 9-bp repeat sequence in hMSH3. No frameshift mutations were found in the (A)7 and (A)6 regions in hMSH3. Thus, we confirmed that the (A)8 region in hMSH3 is a hot spot and mutations in the (A)7 and (A)6 regions in hMSH3 are not common. The hMSH3 mutation may enhance genomic instability in some colorectal cancers.     

Frameshift Mutations and a Length Polymorphism in the hMSH3 Gene and the Spectrum of Microsatellite Instability in Sporadic Colon Cancer

Mutations in the hMSH3 gene in sporadic colon cancer with microsatellite instability (MSI) were investigated, since several mismatch repair genes were known to be mutated in cancers with MSI, but only deletions in the (A)₈ region in the hMSH3 gene have been reported. We also analyzed the relationships between hMSH3 mutations and the spectrum of MSI. We screened MSI in 79 sporadic colon cancer samples using mono- and dinucleotide repeat markers and the samples with MSI were further analyzed for tri- and tetranucleotide repeat instability and mutations in the hMSH3 gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Five (6%) out of 79 tumors were MSI-H and 15 (19%) were MSI-L. Two MSI-H tumors showed insertion in the (C)₈ region in the hMSH6 gene and one tumor showed insertion and deletion in the (A)₈ region in the hMSH3 gene, and two of the three above tumors showed MSI in tri-and tetranucleotide repeats. One MSI-L tumor showed somatic alteration in a 9-bp repeat sequence in hMSH3. No frameshift mutations were found in the (A)₇ and (A)₆ regions in hMSH3. Thus, we confirmed that the (A)₈ region in hMSH3 is a hot spot and mutations in the (A)₇ and (A)₆ regions in hMSH3 are not common. The hMSH3 mutation may enhance genomic instability in some colorectal cancers.     

Microsatellite instability, MLH-1 promoter hypermethylation, and frameshift mutations at coding mononucleotide repeat microsatellites in ovarian tumors.

BACKGROUND: Microsatellite instability (MI) is frequent in endometrial carcinomas (ECs), but its occurrence in ovarian tumors is uncertain. Microsatellite instability positive ECs frequently are associated with frameshift mutations in coding mononucleotide tracts in IGFIIR, BAX, hMSH6, and hMSH3. METHODS: DNA from 52 consecutive patients with ovarian tumors (10 benign, 7 borderline, and 35 malignant) was obtained from neoplastic and normal tissue. After preliminary results, the series was expanded by including 41 additional, previously selected, endometrioid and clear cell carcinomas. Microsatellite instability analysis was assessed by evaluating three (CA)n dinucleotide repeats (D2S123, D5S346, D17S250) and two mononucleotide tracts (BAT 25 and BAT 26). Frameshift mutations at coding mononucleotide repeats (IGFIIR, TGF beta II, BAX, hMSH6, and hMSH3) were investigated by single-strand conformation polymorphism analysis and DNA sequencing. MLH-1 methylation was assessed by methylation specific PCR. RESULTS: Microsatellite instability was identified in only 2 of the 52 (3.8%) tumors of the initial series (1 endometrioid and 1 clear cell carcinoma). After expanding the initial series of 15 endometrioid and clear cell carcinomas with 41 additional endometrioid and clear cell carcinomas, MI was found in 7 of the total series of 56 endometrioid and clear cell carcinomas (12.5%). Frameshift mutations in coding mononucleotide tracts were detected in BAX (6 of 7), IGFIIR (1 of 7), and MSH3 (2 of 7). MLH-1 promoter hypermethylation was identified in three of six MI positive tumors. CONCLUSIONS: Microsatellite instability was infrequent in this series of ovarian tumors, and it was limited to endometrioid and clear cell carcinomas. Like EC, many ovarian carcinomas with MI follow the same process of MLH-1 promoter methylation and accumulation of mutations in coding mononucleotide tracts.     

Evidence for an hMSH3 defect in familial hamartomatous polyps

BACKGROUND:

Patients with hamartomatous polyposis syndromes have increased risk for colorectal cancer (CRC). Although progression of polyps to carcinoma is observed, pathogenic mechanisms remain unknown. The authors examined whether familial hamartomatous polyps harbor defects in DNA mismatch repair (MMR), and assayed for somatic mutation of PTEN, a gene inactivated in the germline of some hamartomatous polyposis syndrome patients.

METHODS:

Ten hamartomatous polyposis syndrome patients were genotyped for germline mutations. Epithelial and nonepithelial polyp DNA were assayed for microsatellite instability (MSI) and PTEN frameshift mutation. DNA MMR and PTEN protein expression were assessed in all polyps by immunohistochemistry. In addition, 99 MSI‐high sporadic CRCs and 50 each of hMLH1^?/? and hMSH3^?/? cell clones were examined for PTEN frameshifts.

RESULTS:

Twenty‐five (58%) of 43 hamartomatous polyposis syndrome polyps demonstrated dinucleotide or greater MSI in polyp epithelium, consistent with hMSH3 deficiency. MSI domains lost hMSH3 expression, and PTEN expression was lost in polyps from germline PTEN patients; sporadic hamartomatous polyps did not show any of these findings. PTEN analysis revealed wild type exon 7 and 8 sequences suggestive of nonexistent or rare events for PTEN frameshifts; however, MSI‐high sporadic CRC showed 11 (11%) of 99 frameshifts within PTEN, with 4 tumors having complete loss of PTEN expression. Subcloning hMLH1^?/? and hMSH3^?/? cells revealed somatic PTEN frameshifts in 4% and 12% of clones, respectively.

CONCLUSIONS:

Nondysplastic epithelium from hamartomatous polyposis syndrome polyps harbors hMSH3 defects, which may prime neoplastic transformation. Polyps from PTEN^+/? patients lose PTEN expression, but loss is not a universal early feature of all hamartomatous polyposis syndrome. However, PTEN frameshifts can occur in hMSH3‐deficient cells, suggesting that hMSH3 deficiency could drive hamartomatous polyposis syndrome tumorigenesis. Cancer 2011. © 2010 American Cancer Society.     

Lack of frameshift mutations at coding mononucleotide repeats in hepatocellular carcinoma in Japanese patients

BACKGROUND: Microsatellite instability occurs frequently in hereditary nonpolyposis colorectal carcinoma, in sporadic gastrointestinal carcinoma, and in other tumors. In these tumors, slippage-related frameshift mutations have been detected at coding mononucleotide repeats in genes such as those for transforming growth factor-beta receptor type II (TGFbetaRII), mannose 6-phosphate/insulinlike growth factor II receptor (M6P/IGFIIR), hMSH3, hMSH6, and Bcl-2-associated X protein (BAX). Because these genes regulate cell growth or repair DNA mismatches, loss of their function is thought to promote tumor development. The authors screened for these frameshift mutations and investigated the incidence of microsatellite instability (MI) in hepatocellular carcinoma (HCC) in Japan. METHODS: Fifty HCC samples were analyzed in this study. The authors used polymerase chain reactions to screen for frameshift mutation at the TGFbetaRII (A)(10) tract, the M6P/IGFIIR (G)(8) tract, the hMSH3 (A)(8) tract, the hMSH6 (C)(8) tract, and the BAX (G)(8) tract. For MI analysis, matched tumor and nontumor liver DNA were investigated with respect to 10 microsatellite loci. RESULTS: No frameshift mutation was detected in any case, and only 4% of these cancers exhibited MI in comparisons between tumor and nontumor liver specimens. CONCLUSIONS: This study suggests that frameshift mutation at coding mononucleotide repeats within TGFbetaRII, M6P/IGFIIR, hMSH3, hMSH6, and BAX genes did not seem to be involved in hepatocarcinogenesis in the Japanese population studied.     

Genetic alterations of sporadic colorectal cancer with microsatellite instability, especially characteristics of primary multiple colorectal cancers

BACKGROUND AND OBJECTIVES: The purpose of this study was to elucidate genetic alterations of sporadic colorectal cancers with Microsatellite instability (MSI). METHODS: One hundred and ten patients with sporadic colorectal cancer were examined. The MSI was assessed using eight microsatellite markers. In addition, mutation analysis was performed for Transforming growth-beta type II receptor (TGF beta R II), bcl-2 associated X protein (BAX), Insulin-like growth factor II receptor (IGF II R), human MutS homolog 3 (hMSH3), human MutS homolog 6 (hMSH6), human MutS homolog 2 (hMSH2), and human MutL homolog 1 (hMLH1) genes. Tumors with three or more positive loci have been determined to be MSI-H (high-frequency MSI), tumors with one or two positive loci were designated as MSI-L (low-frequency MSI) and tumors lacking apparent instability were designated as MSS (microsatellite stable). RESULTS: There were 11 cases with MSI-H (MSI-H group) and 99 cases with MSI-L/MSS (MSI-L/MSS group). The frequency of cases with multiple colorectal cancer in the MSI-H group was significantly higher than that of MSI-L and MSS group (81.8% vs. 34.3%, p = 0.0022). The frequency of cases with multiple cancers increased as the number of locus with microsatellite instability increased. Among 11 tumors with MSI-H, 7 indicated mutation of the TGF beta R II (63.6%), whereas no tumor had a mutation of the TGF beta R II among 20 tumors with MSI-L (p = 0.0001). Regarding the BAX, hMSH3, hMSH6 gene, the same trend was obtained as the TGF beta R II gene (18.2% vs. 0%, p = 0.0487, 36.4% vs. 0%, p = 0.0039, 27.3% vs. 0%, p = 0.0140, respectively). Only two cases indicated a somatic point mutation of the hMSH2 gene. CONCLUSIONS: The multiple occurrence of the colorectal carcinoma may be related to MSI-H as well as the mutation of genes possessing repetitive mononucleotide tracts. Furthermore, we recommend strict follow-up in sporadic colorectal patients that indicate MSI-H.     

The role of hMSH3 and hMSH6 in ovarian endometrioid carcinoma and relationship with microsatellite instability phenotype

Frequent frameshift mutations in the DNA mismatch repair genes hMSH3, and hMSH6, have been reported in colorectal and endometrial cancers with microsatellite instability, however, it is unclear whether they are similarly altered in ovarian endometrioid carcinoma. In this study, we examined frequency of frameshift mutation and protein expression in hMSH3 and hMSH6 in ovarian endometrioid carcinoma with or without microsatellite instability. Only 1 frameshift mutation of the 16 cases with microsatellite instability-high phenotype (6%) was detected in poly(A)8 tract of the hMSH3, but not in poly(C)8 tract of hMSH6 genes. In addition, none of the 6 microsatellite instability-low or 20 microsatellite-stable tumors showed mutations in these regions in either gene. These results indicate that mutations in the mono-nucleotide tracts of hMSH3 and hMSH6 are infrequent in ovarian endometrioid adenocarcinomas, other mechanisms may play a more important role in the development of these tumors.     

Mutational analysis of transforming growth factor beta receptor type II and DNA mismatch repair genes in sporadic endometrial carcinomas with microsatellite instability

To clarify how microsatellite instability (MI) is involved in carcinogenesis of sporadic endometrial carcinoma, we examined mutations of the transforming growth factor beta receptor type II (TGF beta RII) gene in 32 patients with MI-positive sporadic endometrial carcinoma. Moreover, mutations of 4 DNA mismatch repair (MMR) genes (hPMS1, hPMS2, hMLH1, hMSH2), which are considered to cause MI, were investigated as well. With respect to the TGF beta RII gene, mutations in the 10-bp polyadenine repeat sequence were observed in 7 of 29 informative cases (24%). Concerning MMR genes, a T to C point mutation at the -6 intronic splice acceptor site of exon 13 of hMSH2 was detected in 43% (6/14). However, there was no mutation in any exon of these 4 MMR genes. These results suggest that there is a carcinogenic mechanism via mutation of the TGF beta RII gene in some cases of MI-positive sporadic endometrial carcinoma. It seems unlikely that the unknown MMR genes are responsible for MI. The implication of the mutation at the intronic splice acceptor site in hMSH2 remains to be clarified.     

Mutational Analysis of Mismatch Repair Genes, hMLH1 and hMSH2, in Sporadic Endometrial Carcinomas with Microsatellite Instability

Microsatellite instability, monitored by replication error (RER), bas been observed in both sporadic and hereditary types of endometrial carcinoma. In the hereditary tumors, this instability is considered to be caused by a germline defect in the DNA mismatch-repair system. We previously reported that nearly one-quarter of sporadic endometrial carcinomas examined revealed an RER-positive phenotype at multiple microsatellite loci. To investigate the role of genetic alterations of DNA mismatch-repair genes in sporadic endometrial carcinomas, we screened 18 RER(+) endometrial carcinomas for mutations of hMLH1 and hMSH2 . Although we found no germline mutations, we detected two somatic mutations of hMLH1 in a single endometrial cancer; these two mutations had occurred on different alleles, suggesting that two separate mutational events had affected both copies of hMLH1 in this particular tumor. These data implied that mutations of hMLH1 or hMSH2 play limited roles in the development of sporadic endometrial carcinomas, and that the tumors with genetic instability might have alterations of other mismatch-repair genes, such as hPMS1 and hPMS2 , or of unknown genes related to the mismatch-repair system.     

Comparative genomic hybridization and mutation analyses of sporadic schwannomas

Summary Schwannomas of the vestibular nerve are the striking characteristics of neurofibromatosis type 2 (NF2), an autosomal dominant hereditary disease. The NF2 gene on 22q12 has been isolated as the gene responsible for NF2. Previous studies have reported that 60% of sporadic schwannomas showed inactivation of the NF2 gene, but genetic alterations of remaining 40% tumors remain elusive. Moreover, detailed genetic alterations of this tumor remain an open question. In this study, we analyzed genomic alterations in 17 sporadic schwannomas using comparative genomic hybridization (CGH). Loss of chromosome 22q, including the NF2 locus, was the only notable abnormality (5/17, 29%). Further, we performed fluorescence in situ hybridization analysis with a genomic BAC clone harboring the NF2 gene and found that the 5 tumors with loss detected by CGH as well as three cases without such a detectable loss by CGH, or a total, 8/17 (47%), showed loss of the NF2 locus. Mutation search by PCR-SSCP followed by direct sequencing revealed that 71% (12/17) of the tumors had one or two mutations in the NF2 gene. Our analyses disclosed that 14 (82%) of 17 tumors had structural alteration of NF2; among these 14 cases, 9 (64%) had two inactivating mutations in the NF2 gene, either a somatic mutation in one allele coupled with loss of the other allele or two independent somatic mutations. Our present results suggested that (i) most of the sporadic schwannomas have two-hit mutations in the NF2 gene, and (ii) NF2 is the only major causative gene in the genesis of schwannomas that is activated or inactivated by copy number alterations.     

Genetic and epigenetic modification of mismatch repair genes hMSH2 and hMLH1 in sporadic breast cancer with microsatellite instability

Breast cancer is the most common cancer in women, but its pathogenesis is still unclear. Microsatellite instability (MSI) has been identified in breast cancer cells, suggesting an association with mismatch repair defects. To test this hypothesis, we investigated MSI, protein expression of hMSH2 and hMLH1, as well as genetic and epigenetic modifications of these two genes in 32 sporadic breast tumors. MSI was identified in 15 cases. Immunohistochemistry analysis revealed that all MSI cases but one had lower than normal expression of hMSH2 (nine cases), hMLH1 (12 cases), or both (seven cases). In tumors with MSI, both genetic and epigenetic modifications of these mismatch repair genes were also identified. Eight cases harbored mutations or polymorphisms in hMSH2 and hMLH1, and 10 exhibited hypermethylation in the promoter region of hMLH1. These results suggest that both genetic and epigenetic alterations of hMSH2 and especially of hMLH1 contribute to genomic instability and tumorigenesis in sporadic breast cancer.