Application of the National Cancer Institute international criteria for determination of microsatellite instability in ovarian cancer

Recently, the National Cancer Institute (NCI) established criteria for determination of microsatellite instability (MSI) in colorectal tumors. Although the best panel of markers for ovarian tumors is not known, we evaluated epithelial ovarian cancers for MSI based on the NCI recommendations. One hundred and nine ovarian tumors were analyzed for MSI by gel analysis of paired germ-line and tumor DNA. PCR amplification was performed using the panel of five microsatellite markers recommended by the NCI (BAT25, BAT26, D5S346, D2S123, and D17S250) and nine additional markers picked based on their genomic location (NME1, D10S197, D11S904, D13S175, DXS981, DXS6800, DXS6807, AR, and D3S1611). Tumors were characterized on the basis of: high-frequency MSI (MSI-H) if two or more of the five NCI markers showed instability or there was instability at 30% or more of all markers tested; or low-frequency MSI (MSI-L) if only one of the five NCI markers showed instability or <30% of all of the markers. All of the other tumors were considered microsatellite stable. On the basis of the NCI markers, 12 (11%) tumors demonstrated MSI-H, and 8 (7%) additional tumors had MSI-L. When all of the 14 markers were considered together, 13 (12%) tumors demonstrated MSI-H (based on 30% or more unstable loci), and 26 (24%) tumors had MSI-L. A single tumor identified to have MSI-H based upon all of the markers tested would have been classified as MSI-L based upon the NCI markers alone. Inclusion of an additional dinucleotide marker (NME1) to the NCI panel allowed detection of all of the tumors with MSI-H using only six markers. MSI-H occurs in approximately 12% of invasive ovarian tumors. For optimal detection of microsatellite instability in ovarian cancer, an additional marker (NME1) may be required, along with the five recommended by the NCI.     

Low-level microsatellite instability in most colorectal carcinomas

Twelve to 16% of colorectal cancers (CRCs) display a high degree of microsatellite instability (MSI-H), whereas most are believed to be microsatellite stable (MSS). The existence of a low degree of instability (MSI-L) group has also been proposed. By using the Bethesda panel of microsatellite markers, the microsatellite instability (MSI) status of CRCs can be determined. This set is recommended to distinguish between MSI-H and MSI-L/MSS. No definition for MSI-L has emerged. Most reports on MSI-L rely on the Bethesda panel, using 5-15markers. Tumors with more than 30% MSI are designated as MSI-H, but the lower limit for MSI-L is ambiguous. We hypothesized that if many markers are studied, almost all CRCs would show some MSI. It would be necessary to establish a cutoff level for MSI-L by showing that, above this cutoff level, tumors display molecular and/or clinical features different from those under the cutoff level. To perform this task, we analyzed 90 BAT26 stable CRC samples with 377 markers. MSI at 1-11 loci was observed in 71 (79%) of the 90 cases. K-RAS mutation, loss of heterozygosity, and MLH1 and MGMT hypermethylation analyses were performed, as well as clinical features being scrutinized, to examine possible differences between MSI-L and MSS tumors using all of the possible cutoff levels for MSI-L. Convincing differences between putative MSI-L and MSS groups were not observed. Our results show that the sensitivity of a typically used marker number to detect MSI-L is very low, and they suggest that MSS and MSI-L tumors have a common molecular background.     

Microsatellite instability, prognosis and metastasis in gastric cancers from a low-risk population

We examined 169 cases of gastric adenocarcinoma for microsatellite instability (MSI), using a panel of 8 microsatellite markers. Of these cases, 142 were from the United States, a country of relatively low risk for gastric cancer. Comparing microdissected tumors to normal cells from the same patient, we classified tumors as being microsatellite-stable (MSS) or having a low frequency of MSI (MSI-L, up to 30% of markers different in the tumor) or a high frequency of MSI (MSI-H, 30% or more of markers different). Among our American cases, we identified 26 (18.2%) showing MSI-H and 15 (10.6%) showing MSI-L. Twenty cases were from Korean patients, and they showed no significant differences in proportions of MSI-H and MSI-L from the American cases. MSI-H tumors in the American patients were characterized by elevated frequencies of band shifts in repeat sequences of the BAX (50%), transforming growth factor-beta receptor type II (TGFbetaRII, 68.9%), beta(2)-microglobulin (21.4%) and E2F4 (51.7%) genes. Alterations in E2F4 in MSI-H tumors were always integral multiples of 3 nucleotides lost or gained, which would not cause a frameshift mutation, and within the range of normal polymorphisms for this sequence. North American patients (n = 127) with MSI-H and MSI-L tumors had a longer median survival of 541 days and 587 days, respectively, compared to 265 days for patients with MSS tumors (p = 0.027). This survival difference may result from a significantly greater tendency for metastases in the MSS group (p = 0.031).     

Loci for efficient detection of microsatellite instability in hereditary non-polyposis colorectal cancer

Most hereditary non-polyposis colorectal cancer (HNPCC) is due to germline mutations in DNA mismatch repair genes. Tumors arising as a result of these mutations display instability in microsatellites, which are short tandem repeats of DNA that are distributed throughout the genome. Although a subset of sporadic colorectal carcinomas also have microsatellite instability (MSI), the phenotype is a useful screening test in identifying patients with HNPCC caused by mutations in mismatch repair (MMR) genes. Studies have shown that some microsatellite markers are more efficient than others in identifying tumors with MSI. Furthermore, the frequency of instability can be assessed by categorizing patients into high (MSI-H, >/= 30-40% positive markers), low (MSI-L), and microsatellite stable (MSS) groups. Using a panel of 28 microsatellite markers, tumor and normal DNA from 10 HNPCC patients was used to identify the five most efficient markers for detecting MSI (BAT26, D2S123, FGA, D18S35, and TP53-DI). Each of the five markers detected MSI in 80-100% of the cases examined. We then expanded the sample size to 17 tumors from HNPCC patients. Each case had evidence for a mutation in either hMSH2 or hMLH1. We compared the efficiency of our panel of five best markers with another panel of five markers (BAT25, BAT26, D2S123, D17S250, and D5S346) identified as being efficient markers for detection of MSI at a recent NCI workshop. Our five selected markers were more efficient (85% vs. 79%) in detecting MSI. However, using either panel, 100% of the cases fell into the MSI-H category and the probability of misclassifying an MSI-H case as MSI-L is very low (0.002-0.008). We also examined four cases meeting the Amsterdam criteria for HNPCC, but with no evidence for mutation in either the hMSH2 or hMLH1 gene. With our panel, three were classified as MSI-H, while only two were classified as such with the NCI reference panel. The probability of misclassifying an MSI-L case as an MSI-H, using a panel of five markers is high (0.263).     

Sporadic colorectal adenocarcinomas with high-frequency microsatellite instability

BACKGROUND: Widespread microsatellite instability (MSI) occurs in nearly 15% of sporadic colorectal cancers. Large bowel carcinomas with high-frequency MSI (MSI-H) (instability at > or = 30% of microsatellite loci) are believed to display distinctive pathologic features and to behave less aggressively than microsatellite-stable (MSS) tumors and carcinomas with low-frequency MSI (MSI-L) (instability at < 30% of microsatellite loci). The aim of the current study was to accurately define the clinicopathologic and biologic features of MSI-H sporadic colorectal carcinomas. METHODS: MSI status was evaluated in 216 large bowel adenocarcinomas using polymerase chain reaction (PCR) and 6 microsatellite markers. Tumors that showed instability with at least two microsatellite markers were classified as MSI-H, whereas the other tumors were classified as MSI-L (instability at one locus) or MSS (no instability). Expression of p53, hMLH1, and hMSH2 gene products was determined by immunohistochemistry, and DNA ploidy pattern was determined by flow cytometry. The prognostic significance of MSI status was assessed by univariate and multivariate survival analyses. RESULTS: The significantly different pathologic features of MSI-H carcinomas were proximal location; large size; mucinous and medullary histotype; poor differentiation; expanding pattern of growth; more frequent Crohn-like conspicuous lymphoid reaction; and low incidence of extramural vein invasion. Most MSI-H tumors were DNA diploid (33 of 40 tumors; 82.5%) and p53 negative (34 of 44 tumors; 77.3%). Conversely, DNA aneuploidy and p53 overexpression were observed in 82.3% (130 of 158 tumors; P < 0.0001) and 54.1% (93 of 172 tumors; P = 0.0002) of MSI-L/MSS tumors, respectively. Loss of hMLH1 or hMSH2 expression was detected in a high fraction of MSI-H carcinomas (86. 0%). Patients with MSI-H tumors showed a better clinical outcome than patients with MSI-L/MSS tumors (P = 0.0017). Furthermore, in multivariate analysis that included conventional clinicopathologic parameters, MSI status, and p53 expression as covariates, MSI status was a significant independent prognostic indicator of disease specific survival. CONCLUSIONS: Assessment of MSI status is an essential step in the genetic characterization of large bowel carcinomas and identifies a subset of tumors with distinct clinical, pathologic, and biologic features.     

Microsatellite instability and clinicopathological features in esophageal squamous cell cancer

Since multiple genetic alterations are involved in the molecular pathogenesis of esophageal squamous cell cancer (ESCC), the role of microsatellite instability (MSI) in its carcinogenesis is not well defined. The reported frequency of MSI in ESCC ranges from 2 to 66.7% but the majority of the results are derived from relatively small studies. Therefore, we carried out a precise MSI analysis on a large number of ESCC samples to clarify the significance of MSI in the ESCC tumorigenesis. The MSI status of the DNA extracted from 62 ESCC samples and 62 counterpart-normal esophageal epitheliums were studied with five NCI panel markers and ten microsatellite markers located in 17q24-25. Forty-four paraffin-embedded samples and 18 frozen samples from the ESCC patients who underwent surgery were studied. The MSI status was classified as MSS (microsatellite stable), MSI-L (low-level MSI; <30% of markers examined showed instability) and MSI-H (high-level MSI; >30% of markers reported instability). Among the 62 ESCC cases analyzed by the 15 microsatellite markers, 38 out of 62 cases (61.3%) showed MSS, 19 out of 62 cases (30.6%) showed MSI-L and 5 out of 62 cases (8.1%) showed MSI-H. Although the MSI status was not associated with the status of lymph node metastasis or a histological type of cancer, the depth of cancer invasion was significantly associated with the frequency of MSS status and the levels of MSI-L were inversely correlated with the depth of invasion (T1/T2 vs. T3/4; P=0.0007). However, MSI status was not associated with the prognosis of the ESCC patients. This is the first large scale MSI analysis of the ESCC in comparison with the clinicopathological features. Relatively high frequency of MSI-L was observed in ESCC and the frequency of MSI-L was inversely correlated with the depth of invasion.     

Molecular profiling of sporadic colorectal tumors by microsatellite analysis

To investigate the prognostic value of multiple genetic alterations, individual molecular tumor profiles were established in 79 sporadic colorectal carcinomas (41 stage II and 38 stage III). Tumors were analyzed for allelic loss (LOH) and genetic instability (MSI) using 14 microsatellites intragenic to or associated with tumor suppressor or DNA mismatch repair genes. Molecular profiling identified tumors with LOH at multiple loci without microsatellite instability (MSS), tumors with high levels of LOH and low level microsatellite marker instability (MSI-L), and tumors with high levels of MSI (MSI-H), but rare LOH. K-ras mutations occurred more frequently in MSS/MSI-L carcinomas (26%) than in MSI-H colorectal tumors (10%), the latter showing a high frequency of TGFbeta type II frameshift mutations (82%). Correlation of molecular and clinical data revealed a better prognosis for stage III tumor patients displaying 5q12 loss rather than retention of heterozygosity. Thus, molecular profiling allows the identification of new prognostic markers and might facilitate the stratification of colorectal cancer patients.     

Genetic classification of ovarian carcinoma based on microsatellite analysis: relationship to clinicopathological features and patient survival

Ovarian carcinomas can progress through two pathways of genomic instability: chromosomal instability (CIN) and microsatellite instability (MSI). However, it is unknown whether these two mechanisms could be distinguished from each other in the molecular characteristics in ovarian carcinomas. We hypothesized that these two pathways are not always independent in ovarian carcinomas. We classified 51 ovarian carcinomas based on their MSI and CIN status using microsatellite analysis and assessed whether these carcinogenic pathways affect the clinicopathological features and patient survival. Of the 51 cases, 77.4% of the tumors were microsatellite stable (MSS), 5.9% were MSI-Low (MSI-L) whilst, 16.7% were MSI-High (MSI-H). Overall, 56.8% of the tumors had at least one loss of heterozygosity (LOH) event, i.e., 56.8% CIN. Notably, we identified a significant degree of overlap between the MSI and CIN pathways. Of the 34 tumors with LOH events (CIN), 5 (14.7%) were MSI-H. In addition, of the 7 tumors that were MSI-H, 5 (71.4%) had one or more LOH events (CIN). We also identified a group of 29.4% of all tumors that did not demonstrate any evidence of either of the two pathways of genomic instability as they were MSS/MSI-L with no evidence of LOH events (CIN negative). Furthermore, patients with CIN with MSS/MSI-L have a significantly shorter overall survival compared to those in other genetic categories (P=0.019). Cox regression analysis revealed that tumors with CIN with MSS/MSI-L exhibit a poor prognostic outcome after adjustment for FIGO stage and grade. These findings suggest that some ovarian carcinomas have a significant degree of overlap between the two pathways of genomic instability and that the genetic classification using microsatellite markers may represent a potential new biomarker of risk prediction in ovarian carcinoma.     

Frequent alterations of the beta-catenin and TCF-4 genes, but not of the APC gene, in colon cancers with high-frequency microsatellite instability

High-frequency microsatellite instability (MSI-H) due to defective DNA mismatch repair (MMR) is a characteristic of the majority of tumors from kindreds with hereditary nonpolyposis colorectal cancer (HNPCC) and a subset of sporadic cancers. To better understand the molecular characteristics of colon cancers with MSI-H, we analyzed these cancers for alterations of genes, such as APC, beta-catenin, and TCF-4 genes, involved in the Wnt signaling pathway. Following the National Cancer Institute (NCI) criteria, 385 unselected colon cancers were classified as follows: 50 (13%) MSI-H tumors, 36 (9%) low-frequency MSI (MSI-L) tumors, and 299 (78%) microsatellite stable (MSS) tumors. The frequency of APC mutations was significantly lower in MSI-H tumors (9 out of 50) than in MSI-L (12 out of 20) and MSS (66 out of 100) tumors (P = 0.0005 and P < 0.0001, respectively). In contrast, the frequency of exon 3 mutations in the beta-catenin gene was higher in MSI-H tumors (10 out of 50) than in MSI-L tumors (0 out of 30; P = 0.0110) and MSS tumors (3 out of 100; P = 0.0010). Frameshift mutations in a (A)9 tract of the TCF-4 gene were detected in 44% (22 out of 50) of MSI-H tumors, but not in any of the 20 MSI-L tumors or 40 MSS tumors. In total, 78% of MSI-H tumors and 84% of the remaining tumors had at least one alteration in APC, beta-catenin, or the TCF-4 genes. Although further analysis is needed to functionally characterize the consequences of each of these alterations on beta-catenin/TCF target gene expression, our results suggest that the activation of the Wnt signaling pathway plays a pivotal role in colon tumorigenesis, irrespective of MSI status.     

Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors.

PURPOSE: To compare microsatellite instability (MSI) testing with immunohistochemical (IHC) detection of hMLH1 and hMSH2 in colorectal cancer. PATIENTS AND METHODS: Colorectal cancers from 1,144 patients were assessed for DNA mismatch repair deficiency by two methods: MSI testing and IHC detection of hMLH1 and hMSH2 gene products. High-frequency MSI (MSI-H) was defined as more than 30% instability of at least five markers; low-level MSI (MSI-L) was defined as 1% to 29% of loci unstable. RESULTS: Of 1,144 tumors tested, 818 showed intact expression of hMLH1 and hMSH2. Of these, 680 were microsatellite stable (MSS), 27 were MSI-H, and 111 were MSI-L. In all, 228 tumors showed absence of hMLH1 expression and 98 showed absence of hMSH2 expression: all were MSI-H. CONCLUSION: IHC in colorectal tumors for protein products hMLH1 and hMSH2 provides a rapid, cost-effective, sensitive (92.3%), and extremely specific (100%) method for screening for DNA mismatch repair defects. The predictive value of normal IHC for an MSS/MSI-L phenotype was 96.7%, and the predictive value of abnormal IHC was 100% for an MSI-H phenotype. Testing strategies must take into account acceptability of missing some cases of MSI-H tumors if only IHC is performed.     

High thymidylate synthase expression in colorectal cancer with microsatellite instability: implications for chemotherapeutic strategies.

Colon cancers displaying microsatellite instability (MSI) are clinically less aggressive. Based on in vitro studies and recent clinical data, cancers displaying MSI do not respond to 5-fluorouracil (5-FU). The reasons why MSI tumors are clinically less aggressive and do not respond to 5-FU-based therapies have not been fully elucidated. PURPOSE: We investigated biomolecular markers in an attempt to explain the different clinical behavior and chemotherapeutic responses of MSI and non-MSI colon cancers. EXPERIMENTAL DESIGN: One hundred ninety-two sporadic colon cancers were tested for MSI with five mononucleotide markers and methylation of the hMLH1 promoter. Slides were stained for thymidylate synthase (TS), p53, MDM2, p21(WAF1/CIP1), beta-catenin, vascular endothelial growth factor, hMLH1, hMSH2, and hMSH6. Tumors were regarded as having wild-type, functional p53 (Fp53) if reduced expression of p53 and positive MDM2 and p21(WAF1/CIP1) expressions were found. RESULTS: Of the cases, 12.5% were MSI-H (at least two markers mutated). Of MSI-H cases, 83.3% were characterized by a complete loss of at least one of the mismatch repair proteins, in particular loss of hMLH1 by promoter hypermethylation. MSI-H colon cancers showed higher expression of TS compared with MSS (no mutated markers)/MSI-L (one mutated marker) colon cancers (66.6% for MSI-H versus 14.8% MSS/MSI-L; P < 0.0001); 20.8% of MSI-H cases showed high expression of the vascular endothelial growth factor, compared with 45.8% MSS/MSI-L colon cancers (P = 0.0005); 45.8% MSI-H cases had Fp53 compared 11.9% MSS/MSI-L cases (P < 0.0001). CONCLUSIONS: About 12% of colon cancers display MSI mostly due to lack of hMLH1 resulting from promoter hypermethylation. These tumors have high expression of TS and retain fully functional p53 system. Thus, these data suggest why sporadic hMLH1-defective colon cancers often do not respond to 5-FU.     

Inverse association between cyclooxygenase-2 overexpression and microsatellite instability in gastric cancer

This study examined the association between cyclooxygenase-2 (COX-2) overexpression and microsatellite instability (MSI) in gastric cancer. COX-2 expression was assessed by immunohistochemistry and scored in a semi-quantitative manner whereas MSI status was characterized by nine microsatellite markers. The clinicopathological features of cancers including survival data were analyzed. Of the 109 gastric cancers studied, COX-2 overexpression and high level of MSI (MSI-H) was detected in 64.2 and 22.0% cases respectively. Gastric tumors with MSI-H phenotypes had significantly lower level of COX-2 expression levels when compared to MSI-L and MSS tumors (P=0.002). Moreover, COX-2 overexpression was associated with tumor invasion beyond submucosa (P=0.045) and there was a trend favoring better survival in gastric cancers without COX-2 overexpression (P=0.07). The results from this study suggest that gastric cancer with microsatellite instability or COX-2 overexpression present with diverse clinicopathological features.     

Microsatellite instability in esophageal adenocarcinoma

The frequency of microsatellite instability (MSI), a result of defective mismatch repair during DNA replication, has been reported inconsistently in primary esophageal adenocarcinoma (EADC). Using a panel of 15 markers, the primary aim of this study was to analyze the frequency of MSI in a well-characterized series of 27 primary EADCs, defined according to strict clinicopathologic criteria. Polymerase chain reaction was used to amplify the following microsatellite repeat loci: D2S123, D10S197, D2S119, D11S904, D2S147, D3S1764, D7S1830, D7S1805, D2S434, D9S299, BAT25, BAT26, D5S346, D17S250, and TGF-beta-RII. Tumors were classified as microsatellite-stable (MSS) when no alterations were seen in tumor DNA compared to matched normal tissues, low-level MSI (MSI-L) when 1-5 of 15 markers were altered, and high-level MSI (MSI-H) when more than five markers were altered. Using these stringent criteria, 9/27 (33%) tumors were MSS, 18/27 (67%) tumors were MSI-L, and no tumor was MSI-H. Immunohistochemistry demonstrated cell nuclear expression of DNA mismatch repair proteins (both hMLH1 and hMSH2) in 78% (21/27) of tumors. No associations were seen between MSI and immunohistochemical expression of hMLH1, hMSH2, alterations in p53 or MBD4, tumor grade, pathologic stage, or patient survival. In conclusion, the finding of low levels of MSI in most tumors suggests an inherent baseline genomic instability, and potentially increased susceptibility to mutations during the progression of esophageal adenocarcinoma.     

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.     

Microsatellite instability testing in colorectal carcinoma: choice of markers affects sensitivity of detection of mismatch repair-deficient tumors.

PURPOSE: Microsatellite instability (MSI) is found in 10% to 15% of sporadic colorectal tumors and is usually caused by defects in DNA mismatch repair (MMR). In 1997, a panel of microsatellite markers including mononucleotide and dinucleotide repeats was recommended by a National Cancer Institute workshop on MSI. We investigated the relationship between instability of these markers and MMR protein expression in a cohort of sporadic colorectal cancer patients. EXPERIMENTAL DESIGN: Paraffin sections of normal and tumor tissue from 262 colorectal cancer patients were examined for MSI status by PCR amplification and for MMR protein expression using antibodies against hMLH1, hPMS2, hMSH2, and hMSH6. RESULTS: Twenty-six (10%) of the patients studied had tumors with a high level of MSI (MSI-H). The frequencies of MSI were the same in African-American and Caucasian patients. Each of the MSI-H tumors had mutations in both mononucleotide and dinucleotide repeats and had loss of MMR protein expression, as did two tumors that had low levels of MSI (MSI-L). These two MSI-L tumors exhibited mutations in mononucleotide repeats only, whereas eight of the other nine MSI-L tumors had mutations in just a single dinucleotide repeat. There was not a statistically significant difference in outcomes between patients whose tumors were MMR-positive or MMR-negative, although there was a slight trend toward improved survival among those with MMR-deficient tumors. CONCLUSIONS: The choice of microsatellite markers is important for MSI testing. Examination of mononucleotide repeats is sufficient for detection of tumors with MMR defects, whereas instability only in dinucleotides is characteristic of MSI-L/MMR-positive tumors.     

Assessment of markers for the identification of microsatellite instability phenotype in gastric neoplasms

We tested three mononucleotide, 45 dinucleotide, and five tetranucleotide repeats in 30 gastric adenomas and 30 gastric carcinomas for microsatellite instability (MSI) in order to evaluate which microsatellites might indicate the MSI status in gastric neoplasms. Along with the increase in tested markers, the proportion of low-frequency MSI (MSI-L) tumors increased. On immunohistochemistry, MSI-L gastric neoplasms did not show any alteration in hMLH1 or hMSH2 protein expression, while most of the high-frequency MSI (MSI-H) tumors did show alterations in the above mismatch repair proteins. The above findings suggested that MSI-L tumors cannot be distinguished from microsatellite stable tumors. Two mononucleotides, BAT25 and BAT26, were sufficient for the screening of MSI. An additional three dinucleotides, D17S786, D6S105 and D19S188, were also highly sensitive and specific in identifying MSI phenotype tumors.     

Differences in genomic instability between intestinal- and diffuse-type gastric cancer

Background Microsatellite instability (MSI) and loss of heterozygosity (LOH) are lesions in the genome found with different frequencies in gastric carcinomas (GCAs). Despite a great body of studies, no systematic approach to the detailed classification of MSI and LOH in the two major types of GCA has been published. Methods Thirty-seven advanced GCAs, 25 intestinal-type (IGCAs) and 12 diffuse-type (DGCAs), were assayed with 15 autosomal tetranucleotide markers on 14 chromosomal arms. The observed frequencies and types of microsatellite alterations allowed stratification into subgroups, i.e., high- and low-grade MSI (MSI-H, MSI-L) or microsatellite-stable (MSS), and high- or low-grade, or non-detectable LOH (LOH-H, LOH-L, LOH-N). Results Collectively, the markers detected MSI-H tumors with sensitivity equal to that of BAT-26 (a single marker highly specific for MSI-H). Likewise, the markers detected LOH at chromosomal arms 5q, 18q, and 21q with a sensitivity equal to markers used previously. Seven (19%) MSI-H and six (16%) LOH-H tumors were found, with a significant association (P = 0.027) with IGCA: 92% of MSI-H and LOH-H occurred in IGCA patients only. Conversely, in DGCA, a significantly higher prevalence of a stable (LOH-N/MSS) phenotype was found as compared with IGCA (75.1% vs 28.0%; P = 0.035). The MSI-L phenotype was found in 57.9% of non-MSI-H IGCA tumors and was associated significantly (P = 0.015) with LOH-H. Conclusion A clear difference in genomic instability between IGCA and DGCA was found. In IGCA, the MSI and LOH pathways were more commonly involved, whereas in DGCA, a stable phenotype was predominant. As a novel finding, MSI-L as a true phenomenon and its association with LOH was observed in IGCA.     

Incidence of microsatellite instability in synchronous tumors of the ovary and endometrium.

PURPOSE: Families with hereditary nonpolyposis colorectal cancer (HNPCC) have an increased lifetime risk of endometrial (40%) and ovarian (10%) carcinomas. Endometrial and ovarian carcinomas from members of these families frequently display a mutator phenotype as manifest by high levels of microsatellite instability (MSI-H). Microsatellite instability (MSI) occurs in 17-32% of sporadic endometrial carcinomas and 3-17% of sporadic ovarian carcinomas. We hypothesized that there might be a higher rate of MSI in tumors from women with synchronous primary carcinomas of the ovary and endometrium. EXPERIMENTAL DESIGN: We identified 52 cases of synchronous tumors of the ovary and endometrium from the databases of four gynecological oncology units. Archival material and clinical data were available on 45 of these patients. We examined DNA extracted from ovarian and endometrial tumor tissue for MSI using DNA extracted from normal tissue of that patient as a germ-line DNA control. MSI was assessed using a panel of five standard microsatellite markers: D2S123, D5S346, D17S250, BAT25, and BAT26. MSI-H was defined by more than two markers being positive. Low-level MSI (MSI-L) was defined as one or two markers positive and microsatellite stable (MSS) was defined as no markers positive. RESULTS: The 45 patients had a median age at diagnosis of 53 years. Of a total of 134 samples analyzed, only three samples (3.3%) were MSI-H. No patient had high levels of MSI in both ovarian and endometrial tumors. One ovarian carcinoma had five of five markers positive with the corresponding endometrial carcinoma being MSI-L. Two endometrial carcinomas were MSI-H, and the corresponding ovarian carcinomas were MSI-L and MSS, respectively. Seven ovarian tumors and seven endometrial tumors were MSI-L. The majority of patients had early-stage ovarian carcinoma [International Federation of Gynecology and Obstetrics (FIGO) stage I, 44.4%; stage II, 26.7%; and stage III, 26.6%]. Eighty-two % of the endometrial primaries were FIGO stage I. Progression-free survival was significantly better for patients with synchronous primaries than those presenting with ovarian carcinoma alone [adjusted hazards ratio, 1.94; P = 0.023; 95% confidence interval, 1.096-3.44]. CONCLUSION: Synchronous primary carcinomas of the ovary and endometrium are unlikely to be part of the HNPCC syndrome unless the family history is in keeping with the modified Amsterdam criteria.     

Biological features of sporadic colorectal carcinoma with high-frequency microsatellite instability: special reference to tumor proliferation and apoptosis

Background We evaluated the relationship between biological behavior and microsatellite instability (MSI) status, with or without p53 status, in sporadic colorectal carcinoma.Methods MSI was analyzed with regard to biological features such as cellular proliferation and apoptotic cell death, in addition to clinicopathological features, in 87 patients with sporadic colorectal carcinoma.Results Fourteen (16.1%) of 87 tumors showed instability at two or more of the five loci examined (high-frequency MSI [MSI-H]). Four demonstrated instability at one locus (low-frequency MSI [MSI-L]), and 69 showed no instability (microsatellite-stable [MSS]). The MSI-H tumors tended to be located in the proximal colon and more often were mucinous carcinoma. The MSI-H tumors also tended to be in patients with multiple colorectal carcinomas and to demonstrate, rarely, an infiltrating growth pattern or venous invasion. The incidence of p53 protein overexpression in the MSI-H tumors was significantly lower than that in the MSI-L/MSS tumors (21% vs 54%). There was no significant difference in the proliferating-cell nuclear antigen (PCNA) labeling index (PI) or apoptotic index (AI) between the MSI-H and MSI-L/MSS tumors. The AI in the MSI-H tumors with p53 overexpression was significantly lower than that in the MSI-H tumors without p53 overexpression, and was also significantly lower than that in the MSI-L/MSS tumors with p53 overexpression. In the MSI-H tumors with p53 overexpression, no expression of BAX protein was found, and there was high expression of bcl-2 protein, resulting in a low BAX/bcl-2 ratio.Conclusion In sporadic colorectal carcinoma, an MSI-H tumor with p53 protein overexpression may display aggressive biological features.     

Genetic and clinical features of human pancreatic ductal adenocarcinomas with widespread microsatellite instability

The incidences of microsatellite instability (MSI) and underlying DNA mismatch repair (MMR) defects in pancreatic carcinogenesis have not been well established. We analyzed 100 sporadic and 3 hereditary pancreatic ductal adenocarcinomas for MSI, and high-frequency MSI (MSI-H) and low-frequency MSI (MSI-L) tumors were further analyzed for frameshift mutations of possible target genes and for promoter methylation and mutation of DNA MMR genes, including hMLH1, hMSH2, hMSH3, and hMSH6 genes. Among the 100 sporadic tumors, 13 (13%) were MSI-H, 13 (13%) were MSI-L, and 74 (74%) were microsatellite stable (MSS) tumors. All of the three hereditary tumors from hereditary nonpolyposis colorectal cancer (HNPCC) patients were MSI-H. MSI-H tumors were significantly associated with poor differentiation and the presence of wild-type K-RAS and p53 genes. Patients with MSI-H tumors had a significantly longer overall survival time than did those with MSI-L or MSS tumors (P = 0.0057). Frameshift mutations of hMSH3, hMLH3, BRCA-2, TGF-beta type II receptor, and BAX genes were detected in MSI-H tumors. Hypermethylation of the hMLH1 promoter was observed in 6 (46%) of the 13 sporadic MSI-H tumors but not in any of the 3 hereditary MSI-H tumors or 13 MSI-L tumors. All of the 3 HNPCC cases had germ-line hMLH1 mutation accompanied by loss of heterogeneity or other mutation in the tumor. Our results suggest that pancreatic carcinomas with MSI-H represent a distinctive oncogenic pathway because they exhibit peculiar clinical, pathological, and molecular characteristics. Our results also suggest the principal involvement of epigenetic or genetic inactivation of the hMLH1 gene in the pathogenesis of pancreatic carcinoma with MSI-H.