Chromosomal imbalances in the colorectal carcinomas with microsatellite instability

Recent molecular genetic studies have revealed that two major types of genomic instabilities, chromosomal instability and microsatellite instability, exist in the colorectal carcinomas. To clarify the relationship between chromosomal abnormalities and mismatch repair gene defects in colorectal carcinomas, we performed a chromosomal analysis on 39 colorectal carcinomas with high-microsatellite instability (MSI-H) and compared the results obtained with those of 20 right-sided microsatellite-stable (MSS) colorectal carcinomas. Chromosomal imbalances (CIs) in MSS colorectal carcinomas were more frequent than in MSI-H colon carcinomas by comparative genomic hybridization analysis (70% versus 31%, P = 0.004). The CI patterns of MSI-H and MSS carcinomas were different. Frequent CIs in MSI-H colon carcinomas were gains of 4q (15%) and 8q (8%), and losses of 9q (21%), 1p (18%), and 11q (18%). In contrast, frequent CIs in right-sided MSS colon carcinomas were gains of 8q (50%), 13q (35%), and 20q (25%), and losses of 18q (55%), 15q (35%), and 17p (30%). We compared the mutation status of 45 target genes and CIs in our MSI-H tumors. Among these 45 target genes, mutation of hRAD50, a member of the DNA repair genes, and FLJ11383 were significantly related to MSI-H colorectal carcinomas with CIs (P = 0.01 and P = 0.02, respectively). Our findings indicate that unique CIs were present in a subset of MSI-H colorectal carcinomas and that these CIs are related to the mutation of several target genes, especially of hRAD50.     

Pathogenesis of non-familial colorectal carcinomas with high microsatellite instability

AIMS: Microsatellite instability (MSI) was first observed in hereditary non-polyposis colorectal carcinoma (HNPCC) and was subsequently seen in non-familial colorectal carcinoma. The relation between MSI and cancer associated genes in non-familial colorectal carcinomas has yet to be evaluated. To clarify this matter, changes in cancer associated genes were examined in non-familial colorectal carcinomas. METHODS: Alterations in the adenomatous polyposis coli (APC), p53, and Ki-ras genes were analysed in 24 MSI high (alterations in four to seven of seven loci), nine MSI low (alterations in one to three of seven loci), and 31 MSI negative non-familial carcinomas. The hMSH2 and hMLH1 genes were also analysed in 24 MSI high carcinomas. RESULTS: Both the frequencies and types of alterations in the APC and p53 genes in MSI high carcinomas were the same as those in MSI low and MSI negative carcinomas; however, they were different from those seen in HNPCC. The frequency of Ki-ras mutation was significantly lower in the MSI high cases (two of 24; 8%) than in the others (15 of 38; 39%). Somatic mutation of hMSH2 or hMLH1 was detected in six of 24 (25%) of the MSI high cases. CONCLUSIONS: These results suggest that APC and p53 alterations occur irrespective of microsatellite instability status in non-familial colorectal carcinomas, and that Ki-ras mutation is not involved in MSI high non-familial colorectal carcinoma. The pathogenesis of these carcinomas may differ from both the usual adenoma-carcinoma sequence and HNPCC carcinogenesis.     

K-ras mutations in endometrial carcinomas with microsatellite instability

K-ras mutations are known to occur in hyperplasias and carcinomas of the endometrium. No clear correlation has been found yet between K-ras mutations and microsatellite instability (MI) in these lesions. Fifty-eight endometrial carcinomas (ECs) and 22 endometrial hyperplasias (EHs) were analysed for K-ras mutation by single-strand conformational polymorphism analysis (SSCP), restriction analysis, and DNA sequencing. MI status had been established previously at five dinucleotide loci and was reconfirmed with markers BAT-25 and BAT-26 by SSCP. K-ras mutations were detected in 11 ECs (18.9%). All 11 tumours were endometrioid carcinomas. K-ras mutations were more frequent in MI-positive (6/14, 42.8%) than in MI-negative tumours (5/44, 11.3%) (p=0.017). Methylation-related transitions were detected in five of the six MI-positive tumours but in only one of the five MI-negative carcinomas. K-ras mutation was identified in only one atypical EH (1/22, 4.5%); in this case, the EH co-existed with EC and both lesions exhibited MI. The results support a close relationship between K-ras mutations and the phenomenon of MI in endometrial carcinomas. The frequent occurrence of methylation-related transitions in these tumours may indicate a cause-effect relationship with the altered methylation status which has been described in association with MI.     

Annexin A10 expression correlates with serrated pathway features in colorectal carcinoma with microsatellite instability

Annexin A10 (ANXA10) has recently been identified as a marker of sessile serrated adenomas/polyps of the colorectum. Although the serrated neoplasia pathway is thought to be involved in the majority of microsatellite instability‐high (MSI‐H) sporadic colorectal carcinomas (CRCs), the clinicopathological implications of ANXA10 expression in CRC are unknown. Here, we evaluated ANXA10 expression status in 168 MSI‐H CRCs by immunohistochemistry. Among 168 MSI‐H CRCs, nuclear staining for ANXA10 in tumor cells revealed 28 cases (17%) with ANXA10‐positive (ANXA10+) tumors. Most of the ANXA10+ tumors were located in the proximal colon (96%, p < 0.001). The ANXA10+ phenotype in MSI‐H CRC was significantly associated with female gender (68%, p = 0.016), CpG island methylator phenotype‐high (CIMP‐H) (68%, p < 0.001), MLH1 promoter hypermethylation (61%, p < 0.001), loss of MLH1 expression (82%, p = 0.019), and wild‐type KRAS status (96%, p = 0.023). Survival analysis revealed no prognostic significance of ANXA10 expression in MSI‐H CRC. In conclusion, ANXA10+ MSI‐H colon carcinomas are characterized by serrated pathway features, including proximal location, female predominance, and high frequencies of CIMP‐H status and MLH1 methylation.     

Detection of microsatellite instability in human colorectal carcinomas using a non-radioactive PCR-based screening technique

The aim of the present study was to establish a rapid, non-radioactive screening method for the detection of microsatellite instability (MIN). MIN is the primary characteristic of the mutator phenotype in tumours constituting hereditary non-polyposis colon cancers (HNPCC). We investigated 30 patients suffering from colorectal cancer using a non-radioactive PCR-based technique. MIN was present in 7 of 30 (23%) of the cases. There was a statistically significant correlation between MIN and localization of the tumour. Five of 7 (72%) tumours with MIN but only 4 of 23 (17%) tumours without MIN were localized in the proximal colon (P<0.01). There was a tendency to higher MIN frequency in tumours of patients with familial clustering of cancers. However, this was statistically not significant (P>0.05). In addition, no correlation between MIN and tumour grade and stage was found. For the investigations in the present study we used a non-radioactive PCR-based method followed by denaturating polyacrylamide gel electrophoresis and silver staining. This method is highly sensitive and reproducible. Thus, PCR-based analysis using a non-radioactive staining technique represents a comprehensive tool for MIN screening in diagnostic pathology.     

Somatic mutations in VNTR-Locus D1S7 in human colorectal carcinomas are associated with microsatellite instability

To elucidate mutation mechanisms in hypervariable VNTR loci, we have studied somatic mutation events with the minisatellite probe MSI (VNTR locus D1S7) in 224 colorectal carcinomas (CRC). The D¹ S7 locus consists of a 9-basepair (bp) repeat unit. The copy number varies from about 100 to 2000, and the germline mutation rate is high. Here we demonstrate a high D1S7 somatic mutation rate in CRC (37/224), higher than indicated earlier by others. We atso demonstrate that the most frequent mutational event by far (n = 34) involves small reductions in VNTR fragment size (median loss 22 repeat units, range 2-1.54), furthermore, in one-half of these cases, this event is biallelic. We wanted to test whether these somatic mutations mirror the same genetic instability as seen by RER (replication error), a phenomenon recently described in tumour DNA from both sporadic and familial cases of CRC. All blood/tumour DNA pairs displaying MS1 mutation (n = 37) as well as 37 randomly selected pairs without MS1 mutation were tested with four tetranucleotide short tandem repeats (STRs, microsatellites). There is a strong association between mutations at the D1S7 locus and the occurrence of new STR alleles (P<0.001). This is the first report of the existence of a minisatellite as a marker for genetic instability/RER in colorectal carcinomas. The findings may also cast light upon the mechanism for somatic mutations in this minisatellite. © 1995 Wiley-Liss, Inc.     

Clinicopathologic characteristics of colorectal cancer with microsatellite instability

Colorectal cancer (CRC) can be classified according to the level of microsatellite instability (MSI) exhibited by the tumor. The aim of this study was to determine MSI status in CRC from Tunisia and to identify clinical and pathological characteristics of MSI-H tumors.     

There is no increase in frequency of somatic mutations in metastases compared with primary colorectal carcinomas with microsatellite instability

This study investigates the molecular features of metastasis in sporadic colon carcinomas with high-level microsatellite instability (MSI-H). DNA from 51 regions from 10 MSI-H metastatic carcinomas and 26 corresponding metastases was analyzed for mutations in TGFBRII, IGFIIR, BAX, MSH3, MSH6, and TCF4, which are associated with MSI-H carcinomas. In addition, 10 metastatic and 10 non-metastatic MSI-H carcinomas and 10 metastatic microsatellite-stable (MSS) carcinomas were examined for expression of vascular endothelial growth factor (VEGF) and mutant TP53. The frequency of microsatellite instability and somatic mutations was not significantly increased in the metastases compared with the that of primary carcinomas. Although significantly fewer MSI-H carcinomas expressed VEGF (P < 0.01) and mutant TP53 (P < 0.005) than MSS carcinomas, there was no difference in VEGF and mutant TP53 expression in metastatic and non-metastatic MSI-H carcinomas. In conclusion, metastasis does not appear to be associated with an increase in somatic mutation rate in any of the genes examined in MSI-H colon carcinomas. Furthermore, VEGF and TP53 expression did not appear to be involved in metastasis in MSI-H colon carcinomas.     

Absence of microsatellite instability in mucinous carcinomas of the breast

Microsatellite instability (MSI) is a form of genetic instability that results from defects in DNA mismatch repair. MSI is reported to be rare in unselected breast cancers, however it is a common feature in subsets of colorectal, ovarian and endometrial cancers. In these anatomical sites, MSI-high carcinomas often display a mucinous histology. The aim of this study was to determine whether mucinous carcinomas of the breast would more frequently display MSI-high than invasive ductal carcinomas of no special type (IDC-NSTs). The expression of four MSI markers (i.e. MSH2, MSH6, MLH1 and PMS2) was immunohistochemically assessed in 35 mucinous breast carcinomas and 35 histological grade- and oestrogen receptor (ER) status-matched IDC-NSTs, and in a series of 245 invasive breast cancers. Cases were considered as potentially MSI-high if tumour cells lacked expression of at least two MSI markers and internal controls displayed nuclear staining. Nine mucinous carcinomas were microdissected and subjected to MSI analysis by PCR using the MSI markers BAT26 and BAT40. No immunohistochemical evidence of MSI-high was found in the 35 mucinous carcinomas and 35 grade- and ER-matched IDC-NSTs, and in the cohort of 245 invasive breast cancers. In addition, no evidence of MSI-high was observed by PCR analysis using the BAT26 and BAT40 markers in the nine mucinous carcinomas tested. Our results demonstrate that MSI-high phenotype is remarkably rare in invasive breast cancer, and that, in contrast to mucinous carcinomas of other anatomical sites, MSI is not a common event in mucinous carcinomas of the breast.     

A novel logistic model based on clinicopathological features predicts microsatellite instability in colorectal carcinomas.

High-frequency microsatellite instability has been reported to be associated with good prognosis in colorectal adenocarcinoma. However, methods to assess microsatellite instability (MIN) are based on genetic assays and are not ideally suited to most histopathology laboratories. The aim of the present study was to develop a model for prediction of MIN status in colorectal cancer based on phenotypic characteristics. Clinicopathological features of a cohort of 204 patients with primary colon cancer were retrospectively reviewed following predetermined criteria. Genetic assessment of MIN status was performed on DNA extracted from sections of formalin-fixed, paraffin-embedded specimens by testing a panel of 11 microsatellite markers. Logistic regression analysis generated a mathematical tool capable of identifying colorectal tumors displaying MIN status with a sensitivity of 77.8% and a specificity of 96.8%. Features associated with instability included the proximal location of the lesions, occurrence of solid and/or mucinous differentiation, absence of cribriform structures, presence of peritumoral Crohn-like reaction, expansive growth pattern, high Ki67 proliferative index, and p53-negative phenotype. This approach predicts microsatellite instability in colorectal carcinoma with an overall assigned accuracy of 95.1% and a negative predictive value of 97.8%. Implementation of this tool to routine histopathological studies could improve the management of patients with colorectal cancer, especially those presenting with stage II and III of the disease. It will also assist in identifying a subset of patients likely to benefit from adjuvant chemotherapy.     

Absence of microsatellite instability in mucinous carcinomas of the breast

Microsatellite instability (MSI) is a form of genetic instability that results from defects in DNA mismatch repair. MSI is reported to be rare in unselected breast cancers, however it is a common feature in subsets of colorectal, ovarian and endometrial cancers. In these anatomical sites, MSI-high carcinomas often display a mucinous histology. The aim of this study was to determine whether mucinous carcinomas of the breast would more frequently display MSI-high than invasive ductal carcinomas of no special type (IDC-NSTs). The expression of four MSI markers (i.e. MSH2, MSH6, MLH1 and PMS2) was immunohistochemically assessed in 35 mucinous breast carcinomas and 35 histological grade- and oestrogen receptor (ER) status-matched IDC-NSTs, and in a series of 245 invasive breast cancers. Cases were considered as potentially MSI-high if tumour cells lacked expression of at least two MSI markers and internal controls displayed nuclear staining. Nine mucinous carcinomas were microdissected and subjected to MSI analysis by PCR using the MSI markers BAT26 and BAT40. No immunohistochemical evidence of MSI-high was found in the 35 mucinous carcinomas and 35 grade- and ER-matched IDC-NSTs, and in the cohort of 245 invasive breast cancers. In addition, no evidence of MSI-high was observed by PCR analysis using the BAT26 and BAT40 markers in the nine mucinous carcinomas tested. Our results demonstrate that MSI-high phenotype is remarkably rare in invasive breast cancer, and that, in contrast to mucinous carcinomas of other anatomical sites, MSI is not a common event in mucinous carcinomas of the breast.     

MBD4 mutations are rare in gastric carcinomas with microsatellite instability

MBD4 encodes a protein that interacts with the mismatch repair protein hMLH1. Therefore, it has been postulated that mutations in MBD4 may result in mismatch repair deficiency. Furthermore, it was shown that MBD4 is a target gene in mismatch repair-deficient tumors. We searched for mutations of MBD4 in sporadic gastric carcinomas (SGC) and compared them with the MSI status of the tumors by screening a series of 42 SGC [22 high microsatellte instability (MSI-H) and 20 microsatellite stable (MSS)] for mutations in both the poly(A)10 and (A)6 tracts of the gene as well as in its entire coding sequence. Mutations of the poly(A)10 tract of MBD4 were also screened in 126 MSI-H colon carcinomas. One of the 22 MSI-H gastric carcinomas and 12 of 126 MSI-H colon carcinomas showed MBD4 mutations in the poly(A)10 tract of exon 3. In addition, seven sequence variants were found in SGC, all of them having a polymorphic nature. We conclude that MBD4 mutations in the entire coding sequence, including the two poly(A) tracts, are rare in MSI gastric carcinomas. MBD4 is likely to be a bystander target gene in MSI-H tumors.     

Methylation of the hMLH1 promoter in multiple gastric carcinomas with microsatellite instability

Hypermethylation of the hMLH1 promoter is observed in the majority of sporadic gastric carcinomas with high frequency microsatellite instability (MSI), and it contributes to the genesis of MSI-positive gastric carcinoma. Multiple gastric carcinoma is known to have a higher frequency of MSI positivity than single gastric carcinoma. However, the molecular basis of MSI in these tumors remains obscure. We investigated the role of hMLH1 promoter hypermethylation in the genesis of multiple gastric carcinoma with MSI. We analyzed 33 tumors from 15 patients with multiple gastric carcinoma (12 double tumors and three triple tumors) for MSI, expression of hMLH1 and hMSH2, and hypermethylation of hMLH1 and hMSH2 promoters. High frequency MSI was found in seven out of 33 tumors (21%) in five out of 15 patients (33%). All of the tumors with high frequency MSI had a lack of hMLH1 expression, with the presence of hMSH2 expression, while all the tumors with no MSI or low frequency MSI were positive for both hMLH1 and hMSH2. All of the tumors with no expression of hMLH1 had hMLH1 hypermethylation, whereas hMLH1 hypermethylation was observed in two out of 26 (8%) tumors with no or low frequency MSI. None of the tumors showed hMSH2 hypermethylation. These results suggest that epigenetic changes in the hMLH1 promoter account for the genesis of multiple gastric carcinoma with high frequency MSI.     

Standardized approach for microsatellite instability detection in gastric carcinomas

Microsatellite instability (MSI) defines a specific type of genetic instability. Although consensus diagnostic criteria for MSI definition in colorectal cancer have been established, their utility in other tumor types remain to be proven. Previously we developed a mathematical model for MSI definition in colorectal cancer. The aim of this study was to establish diagnostic criteria for MSI evaluation in human gastric cancer. We designed an algorithm for the efficient characterization of MSI and used it to analyze data on 7 microsatellite markers in 35 gastric carcinomas. Theoretical models considering 1, 2, or 3 populations were tested against the data collected. Also, hypermethylation of hMLH1 gene promoter and hMLH1 protein expression were studied. The observed frequencies of MSI in our series of samples best fit a 2-population model: stable and unstable, defined by instability in 2 or more of a minimum of 7 markers analyzed. MSI was observed in 29% of the tumors. Misclassification rate was <4% when any 7 loci were analyzed. MSI(+) tumors inversely associated with p53 protein overexpression. A good correlation between hMLH1 status (either protein or promoter hypermethylation) and MSI classification was observed. We have developed a simple, sensitive, and specific approach to assess the presence of MSI in gastric cancer that may have clinical applications.     

Transforming growth factor-beta receptor type 2 mutations and microsatellite instability in sporadic colorectal adenomas and carcinomas.

Frame-shift mutations in a run of 10 adenines (A10) of the transforming growth factor-beta receptor type 2 gene (TGF-beta RII) are commonly seen in inherited and sporadic colonic cancers that exhibit microsatellite instability. A10 mutations and instability also are commonly seen in hereditary nonpolyposis colon cancer-associated adenomas. However, instability is quite rare in sporadic adenomas, and the timing of acquisition of A10 mutations with respect to the sporadic adenoma-carcinoma sequence has not been reported. We evaluated 100 sporadic colorectal cancers and 164 sporadic adenomas for microsatellite instability with a set of 10 tetranucleotide polymerase chain reaction primer sets and for A10 frame-shift mutations. A10 mutations were significantly associated with microsatellite instability in colorectal cancers, being seen in 9 of 11 cancers with 50% or greater instability and in 0 of 89 tumors with less than 50% instability (P < 0.0001). A10 mutations were not detected in any adenomas, only three of which (1.8%) exhibited significant (30% or greater) instability. We conclude that both TGF-beta RII frame-shift mutations and microsatellite instability occur at a relatively late stage of sporadic colorectal tumorigenesis. A10 frame-shift mutations appear to be restricted to sporadic colorectal cancers with extensive microsatellite instability.