Epidermal growth factor receptor expression in colorectal tumors with and without microsatellite instability.
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Colorectal Neoplasms↗
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Results for “DNA MICROSATELLITE INSTABILITY”
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[Microsatellite alterations of circulating DNA in the plasma of patients with hepatocellular carcinoma].
OBJECTIVE: To explore the features of microsatellite alterations of circulating DNA in the plasma of patients with hepatocellular carcinoma (HCC) and whether they are in concordance with those in the carcinoma tissues. METHODS: Peripheral blood samples were collected from 62 HCC patients and the corresponding tumor tissues were obtained during operation. Three high-polymorphic microsatellite markers located at chromosome 8p, D8S277, D8S298, and D8S1771, were selected to be used to detect the loss heterozygosity (LOH) and microsatellite instability (MSI) by PCR and sequencing. RESULTS: Joint detection showed that 39 out of the 62 tissue samples showed LOH at all the 3 loci, and the same alterations at the same loci were seen in 33 matched plasma samples with a concordance rate of 84.6%. Nineteen tissue samples showed MSI at all 3 loci, and the same alterations were shown in 14 matched plasma samples with a concordance rate of 73.3%. The rate of LOH for at least one locus in the plasma DNA was 58.1% (36/62), significantly higher than the rate of MSI at at least one locus in the plasma samples (29.0%, 18/62, P < 0.01). The MSI positive rate in the loci D8S277 of the plasma DNA was 22.6%, significantly higher than those of the other 2 loci (4.8% and 4.8% respectively, both P < 0.05). The MSI positive rate at the loci D8S277 of the cancer tissue was 46.8%, significantly higher than those of the other 2 loci (38.7% for D8S298 and 37.1% for D8S1771, both P < 0.05). CONCLUSION: Microsatellite alterations show a high concordant pattern between the tissue and plasma DNA in HCC, which indicates that the microsatellite alterations of tumor tissue are reflected by plasma DNA, LOH may play an important role in hepatocarcinogenesis whereas MSI may also contribute to this progress in a less significant way, and D8S277 is a sensitive locus to MSI in HCC.
Carcinoma, Hepatocellular↗
Bracken fern-induced malignant tumors in rats: absence of mutations in p53, H-ras and K-ras and no microsatellite instability.
Bracken fern (genus Pteridium) has been shown to induce tumors in domestic and experimental animals. Epidemiological studies have also shown an association between human exposure to bracken toxins and increased risk for the development of upper gastrointestinal tract tumors. Our aim in this study was to investigate possible genomic alterations in bracken fern-induced tumors of experimental animals searching for molecular markers that might be used for human epidemiological studies. Using human colorectal carcinogenesis as a molecular model, we examined eight malignant bracken fern-induced tumors of rats for mutations in the genes associated with the "classic pathway" of colorectal cancer, i.e. p53 and ras, and also in the "mutator pathway" by evaluating microsatellite instability. Exons 5-9 of the p53 gene and exons 1 and 2 of the K-ras and H-ras genes were examined by DNA sequencing and no mutations were found in any of the eight tumors. Amplification of five previously validated microsatellite loci (one with mono-, three with di- and one with tetra-nucleotide repeat motifs) in the malignant tumors and in the surrounding normal tissue did not reveal any instability. The involvement of epigenetic alterations or of mutations in other tumor suppressor genes or oncogenes should be further investigated in the search for human epidemiological markers.
Animals↗
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 beta (TGF-beta) 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-beta 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.
Colorectal Neoplasms↗
Detection of plasma tumor DNA in head and neck squamous cell carcinoma by microsatellite typing and p53 mutation analysis.
Recent arguments have suggested that tumor DNA in cancer patients could be found in plasma, but different points remain unclear. Using a series of 117 head and neck squamous cell carcinoma tumors, our goals for this study were: (a) to quantify the amount of plasma DNA; (b) to evaluate the presence of plasma tumor DNA; and (c) to analyze the clinical relevance of tests based on plasma DNA analyses. Low levels of plasma DNA were found in most samples, but all were successfully amplified. Two different methods were used to detect tumor-specific genetic alterations: (a) microsatellite instability at UT5085 with an established sensitivity of 1:500; and (b) p53 mutation screening. Of the 117 tumors typed at UT5085, 65 demonstrated bandshifts (55%). Plasma and tumor DNA a showed similar alteration in only one case among these samples, and the prevalence of tumor DNA in plasma was estimated to be <2% using microsatellite analysis. Tumor DNA was detected in plasma at a higher prevalence (2 of 11 cases) when using p53 mutant allele-specific amplification. These results showed that in plasma, tumor DNA is largely diluted by normal DNA. By comparison with previously published studies, the prevalence of microsatellite alterations in plasma in this series of head and neck squamous cell carcinomas is very low, despite the fact that a large series of tumors was analyzed. To explain this discrepancy, we analyzed the possibility of PCR artifacts as suspected by the presence of loss of heterozygosity in two plasma DNA samples without a similar tumor DNA alteration. When DNA concentrations were under the threshold of detection (<100 ng/ml), we demonstrated that PCR artifacts could occur at random, and, if misinterpreted, these false genetic alterations could artificially enhance the frequency of plasma DNA alterations. This may have been suspected in previously published series, but it has never been discussed before. Microsatellite analysis on plasma DNA is difficult to interpret and can frequently be misleading. Plasma DNA should be analyzed with very sensitive and specific methods such as mutant allele-specific amplification, which excludes artifacts but requires specific optimization that is probably not compatible with routine and clinical use.
Carcinoma, Squamous Cell↗
High incidence of microsatellite instability in colorectal cancer from African Americans.
PURPOSE: Colorectal carcinoma (CRC) is the second most common cause of cancer death in the United States, and the rate of CRC is nearly 1.5 times higher in African-Americans (AA) than in Caucasians. Microsatellite instability (MSI) is observed in sporadic CRC reflecting promoter hypermethylation of the DNA mismatch repair gene hMLH1, and anecdotal evidence suggests an increased incidence of MSI among AAs. Additionally, p16 can be inactivated by hypermethylation of the promoter region, abrogating its ability to regulate cell proliferation. The objective of this study is to determine the frequency of MSI and p16 gene methylation in CRC from AA patients. EXPERIMENTAL DESIGN: Experiments were conducted on serially collected archival samples of colon cancer and adjacent normal tissue (n = 22). Five microsatellite markers were used to measure MSI in tumors with direct comparison to normal tissue from the same patient. p16 promoter methylation status was determined by methylation-specific PCR. RESULTS: Ten cancers (45%) demonstrated high MSI (MSI-H), 1 demonstrated low MSI, and the remaining 11 tumors were microsatellite stable. Most of the MSI-H tumors were proximal, well differentiated, and showed high levels of mucin production. Most patients in the MSI-H group were female (70%), whereas most of the microsatellite-stable group (81%) were male. Five of the 22 tumors (22%) had methylation of the p16 promoter. CONCLUSION: Data provided here demonstrated that the incidence of MSI-H tumors was 3-fold higher in our study group of AA patients compared with data reported in nonracially selected but serially collected studies. Odds ratio analysis indicates that the chance of female patients having MSI-H was 11.7 times more than male patients (P < 0.03). The reason for this gender difference is unknown. These findings might reflect dietary differences or genetic polymorphisms that may be common in the AA population. Additional investigation in a larger patient population is needed before strong conclusion can be drawn.
Adaptor Proteins, Signal Transducing↗
Unilateral aneuploid dedifferentiated acinic cell carcinoma associated with bilateral-low grade diploid acinic cell carcinoma of the parotid gland.
A dedifferentiated acinic cell carcinoma (AciCC) of the right parotid gland with lymph node metastases occurred in a 36-year-old woman. The tumour was associated with a bilateral well-differentiated AciCC. The two components of this tumour had different (high and low) proliferative activity measured by Mib-1 and different (aneuploid and diploid) DNA content. Despite the presence of a high-grade component, TP53 mutations, microsatellite instability (MSI) and/or loss of heterozygosity (LOH) at the p53 locus were not detected. Although the follow-up of the patient is very short, the aggressiveness of the tumour is shown by a recurrence in the right parotid within 4 months and by the rapid development of regional metastases.
Adult↗
BRAF/K-ras mutation, microsatellite instability, and promoter hypermethylation of hMLH1/MGMT in human gastric carcinomas.
BACKGROUND: The BRAF and K-ras genes are the most frequently mutated oncogenes in various human malignancies. We examined BRAF and K-ras mutations in human gastric cancer, and investigated their relationship with microsatellite instability (MSI) and the hypermethylation of promoter regions in hMLH1 and O6-methylguanine DNA methyltransferase (MGMT). METHODS: Sixteen gastric cancer cell lines and 62 gastric cancer tissue samples were screened for BRAF and K-ras mutations by direct sequencing. We also performed a microsatellite assay and investigated methylation status in the promoter regions of hMLH1 and MGMT. RESULTS: mutation was not found in any of the cancer cell lines examined. One (1.6%) cancer tissue sample showed a point mutation in the BRAF gene (GTG --> GAG; V599E). K-ras mutation (GGT --> GAT, G12D) was detected in five (31%) gastric cancer cell lines and in 1 (1.6%) gastric cancer tissue sample. In the gastric cancer tissue samples examined, MSI was detected in 23 (37%) samples. Hypermethylated promoter regions in hMLH1 and MGMT, respectively, were detected in 6 (10%) and 13 (21%) gastric cancer tissue samples. Microsatellite stable (MSS) tumors showed frequent lymphatic invasion (P = 0.050). CONCLUSION: Although BRAF mutation has been reported in a variety of other human cancers, it is a rare event in the carcinogenesis and progression/development of gastric cancer.
Adaptor Proteins, Signal Transducing↗
Stepwise deletions of polyA sequences in mismatch repair-deficient colorectal cancers.
PolyA simple repeat sequence deletions are common in tumors with microsatellite instability (MSI+). Such deletions occur one base at a time in DNA mismatch repair (MMR)-deficient yeast suggesting larger deletions in human MSI+ tumors represent multiple sequential stepwise losses. Sum total deletions in four polyA repeats were variable (between -17 to -45 bp) in 20 sporadic MSI+ colorectal cancers. Progressive but less extensive total deletions (maximum of -12 bp) occurred in similar polyA sequences in MMR-deficient mice (mlh1-/-) up to 478 days old. PolyA repeat lengths were relatively stable but already shortened in the MMR-deficient cell line HCT116. A transgene with 26 A's transfected into HCT116 shortened an average of 3.8 bases pairs after 469 days in culture, less than average deletions of BAT25 (-5.3) or BAT26 (-9.0) in MSI+ cancers. These findings further suggest that extensive polyA deletions common in MSI+ tumors likely reflect multiple stepwise smaller deletions that accumulate more than hundreds of divisions after loss of MMR.
Adaptor Proteins, Signal Transducing↗
Clinicopathologic features in colorectal cancer patients with microsatellite instability.
The microsatellite instability (MSI) mutational pathway is critical to carcinogenesis in a small but significant proportion of colorectal cancers. While MSI is identified in most cancers in individuals with hereditary non-polyposis colorectal cancer, the majority of MSI tumors are found in individuals with sporadic disease. Colorectal cancers arising as a result of MSI have distinct clinicopathologic features distinguishing them from those with microsatellite stability. MSI colorectal cancers affect a larger percentage of women, are usually localized proximal to the splenic flexure, and have a higher incidence of synchronous and metachronous tumors. They are associated with a mucinous histology, tumor-infiltrating lymphocytes, a Crohn's-like inflammatory response, and a higher grade but lower stage. Overall survival is better in individuals with MSI. The benefit of chemotherapy in MSI colorectal cancers, with and without lymph node metastases, remains unclear.
Base Pair Mismatch↗
Mutation of hMSH3 and hMSH6 mismatch repair genes in genetically unstable human colorectal and gastric carcinomas.
Mutations within microsatellite sequences, consisting of additions or deletions of repeat units, are known as the replication/repair error positive (RER+) phenotype or micorsatellite instability (MI). Microsatellite instability has been demonstrated in hereditary and sporadic colorectal carcinomas and is usually observed in noncoding regions of genomic DNA. However, relatively few coding region targets of MI have been identified thus far. Using PCR, we amplified regions encompassing (A)8 and (C)8 microsatellite tracts within hMSH3 and hMSH6 from 31 RER+ sporadic colorectal tumors, 8 hereditary colon cancers, 23 RER+ gastric carcinomas, and 32 RER- gastric tumors. Mutations were found in 11 (36%) of 31 sporadic colon carcinomas, 4 (50%) of 8 hereditary colorectal cancers, and 5 (22%) of 23 RER+ gastric carcinomas, but in only 2 (6%) of 32 RER- gastric carcinomas. These frameshift mutations cause premature stop codons downstream that are predicted to abolish normal protein function. Our results and those of others suggest that DNA mismatch repair genes, such as hMSH3 and hMSH6, are targets for the mutagenic activity of upstream mismatch repair gene mutations and that this enhanced genomic instability may accelerate the accumulation of mutations in RER+ tumors.
Adenocarcinoma↗
Microsatellite instability and the clinicopathological features of sporadic colorectal cancer.
BACKGROUND AND AIMS: In this study, we prospectively examined the clinical significance of the microsatellite instability (MSI) phenotype in sporadic colorectal cancer, and investigated methods for effective identification of these tumours in routine pathology practice. METHODS: DNA was extracted from 310 tumours collected from 302 consecutive individuals undergoing curative surgery for sporadic colorectal cancer. Microsatellite status was determined by polymerase chain reaction amplification using standard markers, while immunostaining was used to examine expression of MLH1, MSH2, and p53. RESULTS: Eleven per cent of tumours showed high level instability (MSI-H), 6.8% had low level instability (MSI-L), and the remainder were stable. MSI-H tumours were significantly more likely to be of high histopathological grade, have a mucinous phenotype, and to harbour increased numbers of intraepithelial lymphocytes. They were also more likely to be right sided, occur in women, and be associated with improved overall survival. In total, 25 (8%) tumours showed loss of staining for MLH1 and a further three tumours showed absence of staining for MSH2. The positive and negative predictive value of immunohistochemistry in the detection of MSI-H tumours was greater than 95%. CONCLUSIONS: We conclude that the MSI-H phenotype constitutes a pathologically and clinically distinct subtype of sporadic colorectal cancer. Immunohistochemical staining for MLH1 and MSH2 represents an inexpensive and accurate means of identifying such tumours.
Adult↗
Mutation analysis of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic human colon cancer using genomic DNA and intron primers.
Mutations in the transforming growth factor beta type II receptor (TGFbeta RII) gene have been detected in several human cancers exhibiting microsatellite instability. To extend analyses of this gene, we previously investigated the exon-intron organization of the TGFbeta RII gene and defined seven exons and flanking intron sequences. In this study, we further determined the nucleotide sequences surrounding these seven exons and designed eight sets of intron-based primers to examine the entire coding region of the TGFbeta RII gene. Using these primers, we screened genomic DNA sequences from 30 sporadic colorectal cancers for mutations of the TGFbeta RII gene. TGFbeta RII mutations were detected in two of 30 tumors and both displayed microsatellite instability. One had a deletion in a polyadenine tract in exon 3 and the other had a point mutation in the kinase domain located in exon 7. There were no mutations in exons 1, 2, 4, 5 and 6. These results further implicate the polyadenine tract and kinase domain as mutational hotspots in the TGFbeta RII gene in cells with genomic instability and suggest that TGFbeta RII gene mutations occur rarely in cells lacking genomic instability.
Base Sequence↗
Microsatellite instability in germ cell tumors of the testis and ovary.
OBJECTIVES: Widespread replication-type error (RER) is a genetic alteration that has been observed in many different neoplasms and has been associated with defective DNA repair activity. There are conflicting data regarding the role that this type of genetic instability plays in the development and progression of adult germ cell tumors. METHODS: Universal amplification was performed on 104 paired specimens of tumor and constitutional DNA isolated from adult male and female germ cell tumors, in addition to subpopulations of carcinoma in situ (CIS), the precursor of testicular germ cell tumors (TGCTs). Preamplified DNA samples of TGCTs and ovarian germ cell tumors (OGCTs) were assayed for the presence of RER at 78 and 64 microsatellite loci, respectively. RESULTS: RER was observed at a single microsatellite locus in 7 of 24 individual testicular germ cell tumors, including subpopulations of CIS isolated from one of these patients. There was some evidence of RER clustering for microsatellite loci mapping to the short arm of chromosome 12. Genetic instability was more frequent in OGCTs, with widespread RER observed at 38 of 64 microsatellite loci. These alterations were noted in 12 of 36 malignant OGCTs showing RER at 1 or more loci, including 3 OGCTs demonstrating RER at more than 6 separate microsatellite loci. CONCLUSIONS: The pathogenetic significance of genetic instability in germ cell tumors remains uncertain, although the results of this study suggest a lesser role in TGCTs compared to that in OGCTs.
Adolescent↗
Differences in patterns of TP53 and KRAS2 mutations in a large series of endometrial carcinomas with or without microsatellite instability.
BACKGROUND: The correlation between tumor microsatellite instability (MSI) and the accumulation of mutations in KRAS2 and TP53 is uncertain. The authors evaluated the TP53 and KRAS2 genes for mutations in sporadic endometrial carcinomas with microsatellite instability (MSI) and matched MSI negative controls to determine whether defective DNA mismatch repair impacts the patterns of mutations in two genes known to be involved in endometrial tumorigenesis. METHODS: Twenty-five MSI positive endometrial tumors were matched for prognostic factors with 25 MSI negative tumors. Mutations in codon 12 and 13 of KRAS2 were assessed using a polymerase chain reaction (PCR) restriction assay. Mutations in codon 61 of KRAS2 and exons 5-8 of TP53 were evaluated using PCR amplification and single strand conformation variant (SSCV) analysis. All variants were subjected to direct DNA sequencing. RESULTS: KRAS2 and TP53 mutations were identified with equal frequency in the MSI positive and MSI negative groups. For TP53, the authors identified 5 mutations (20%) in the MSI positive specimens compared with 8 (32%) in the MSI negative group. For KRAS2, there were identified 8 mutations (32%) in the MSI positive specimens compared with 7 (28%) in the MSI negative tumors. The mutational spectra evident from sequence analysis of TP53 and KRAS2 variants were similar between MSI negative and MSI positive tumors. MSI negative tumors were more likely to have mutations in both KRAS2 and TP53 than MSI positive tumors, which were rarely mutant in both genes (P=0.046). CONCLUSIONS: Although the overall frequency of mutations in TP53 and KRAS2 is similar, MSI positive tumors are less likely to have mutations in both genes than MSI negative sporadic endometrial carcinomas. MSI positive and MSI negative endometrial carcinomas may arise through distinct genetic pathways.
DNA Repair↗
Instability of interstitial telomeric sequences in the human genome.
The length variability of four human interstitial telomeric sequences (ITs) is described. Three of the ITs contain short telomeric stretches ranging between 53 and 84 bp and are localized in 21q22, 2q31, and 7q36; the fourth IT derives from the subtelomeric domain of chromosome 6p and contains a tract of a few hundred basepairs of exact and degenerate repeats. Using primers flanking the repeats, we amplified the genomic DNA from unrelated individuals and from family members, and we found that all the loci are polymorphic. At the 21q22 IT locus, two equally frequent alleles were found, while the number of alleles at the 2q31, 7q36, and 6pter IT loci was 8, 6, and 4, respectively. Sequence analysis revealed that in the three loci containing short ITs the alleles differ from one another for multiples of the hexanucleotide; it is likely that the mechanism leading to the polymorphism is DNA polymerase slippage. These loci were also unstable in gastric tumor cells characterized by microsatellite instability. At the 6pter IT locus, the four alleles range in length from about 500 to about 700 bp; this variability is probably due to unequal exchange or gene conversion. Our data indicate that stretches of exact internal telomeric repeats can be highly unstable, like microsatellites with shorter units, and that they can be useful polymorphic markers for linkage analysis, for forensic applications, and for the detection of genetic instability in tumors.
Alleles↗
The impact of microsatellite instability on the molecular phenotype of colorectal tumors.
Frequent microsatellite instability MSI (MSI-H) occurring in human tumors is characterized by defective DNA mismatch repair and unique clinical features. However, infrequent MSI (MSI-L) has not been attributable to any other defined molecular pathway, and its existence as a biologically distinct category has been challenged. Moreover, the global molecular phenotypes (GMPs) underlying MSI-H, MSI-L, or microsatellite-stable (MSS) tumors have never been evaluated. To evaluate the impact of MSI status on GMP and to determine the importance of MSI relative to other molecular and clinical features, cDNA microarray-derived data from 41 colon cancers were interpreted using principal components analysis. The clinically relevant principal component with the greatest impact on GMP was component 3, which distinguished MSI-H from non-MSI-H (i.e., MSI-L and microsatellite stable) tumors and was designated the MSI-H separator. Notably, MSI-L cancers were also clearly distinguished from non-MSI-L tumors by another principle component, component 10 (the "MSI-L separator"). This second finding validates the existence of MSI-L tumors as a distinct molecular phenotypic category. Thus, both components 3 and 10 reflected different aspects of MSI and helped to establish principal components analysis as a useful tool to identify and characterize distinct biological features of human malignancy.
Aged↗
[Microsatellite instability in hereditary gastrointestinal polyposis].
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Base Pair Mismatch↗