Microsatellite instability and alteration of the expression of hMLH1 and hMSH2 in ovarian clear cell carcinoma

Microsatellite instability (MSI) is commonly seen in tumors associated with the hereditary nonpolyposis colorectal cancer syndrome and is caused by defects in the DNA mismatch repair genes. MSI has also been observed in various sporadic cancers, including colorectal, gastric, and endometrial. The role and incidence of MSI in ovarian clear cell carcinoma remain unknown. This study was conducted to evaluate the frequency of MSI in ovarian clear cell carcinomas and to evaluate the sensitivity and specificity of immunohistochemistry in predicting mismatch-repair gene deficiency. A total of 42 ovarian clear cell carcinomas were analyzed for MSI using a panel of 5 microsatellite markers (BAT25, BAT26, D5S346, D2S123, and D17S250). Alterations in the expression of hMLH1 and hMSH2 proteins in these tumors were examined. Of the 42 ovarian clear cell tumors analyzed, 6 demonstrated a high level of MSI (MSI-H), 3 demonstrated a low level of MSI (MSI-L), and the remaining 33 exhibited microsatellite stability (MSS). No correlation was found between MSI level and patient age or tumor stage or size (P >0.05). Loss of expression of either hMLH1 or hMSH2 was observed in 4 of the 6 (67.7%) MSI-H tumors, whereas 34 of the 36 (94.4%) MSI-L or MSS tumors expressed both the hMLH1 and hMSH2 gene products. Our results indicate that MSI-H is involved in the development of a subset of ovarian clear cell carcinomas. A strong correlation exists between alterations in the expression of hMLH1 and hMSH2 and the presence of MSI-H in these tumors. However, immunohistochemical testing alone may miss a small fraction of cases with MSI-H.     

Microsatellite Instability and hMLH1 and hMSH2 expression analysis in familial and sporadic colorectal cancer

Immunohistochemical expression analysis of mismatch repair gene products has been suggested for the prediction of hereditary nonpolyposis colorectal cancer (HNPCC) carrier status in cancer families and the selection of microsatellite instability (MSI)-positive tumors in sporadic colorectal cancer. In this study, we aimed to evaluate hMSH2 and hMLH1 immunohistochemistry in familial and sporadic colorectal cancer. We found that immunohistochemistry allowed us to identify patients with germline mutations in hMSH2 and many cases with germline mutations in hMLH1. However, some missense and truncating mutations may be missed. In addition, hMLH1 promoter methylation, commonly occurring in familial and sporadic MSI-positive colorectal cancer, can complicate the interpretation of immunohistochemical expression analyses. Our results suggest that immunohistochemistry cannot replace testing for MSI to predict HNPCC carrier status or identify MSI-positive sporadic colorectal cancer.     

Correlation of methylation of the hMLH1 promoter with lack of expression of hMLH1 in sporadic gastric carcinomas with replication error.

Recent studies have demonstrated that the majority of sporadic colorectal carcinomas with replication error (RER) do not harbor mutations of the hMLH1 and hMSH2 genes that account for about 70% of hereditary nonpolyposis colon cancer. Despite the absence of mutations of the hMLH1 gene, the majority of RER-positive sporadic colorectal carcinomas lack hMLH1 protein expression, which have been reported to be related to hypermethylation of the promoter region of hMLH1 gene. High frequency of microsatellite instability (MSI) has been observed in about 15% of sporadic gastric carcinomas. The relationship of tumor MSI, methylation of promoter regions of hMLH1 or hMSH2, and expression of corresponding gene products has not been studied in gastric carcinomas as thoroughly as in colorectal carcinomas. We explored the relationship between methylation of hMLH1 or hMSH2 promoter regions and its protein expression in both RER-positive and RER-negative gastric carcinomas. Of 93 cases, 20 cases comprised the RER+ group (MSI-H tumors) and the remainder comprised the RER- group (7 cases, MSI-L; 66 cases, MSS). By immunohistochemistry absence of hMLH1 protein expression was limited entirely to the RER+ group (20 of 20, 100%). All 93 cases showed hMSH2 protein expression. Nineteen (95%) of 20 RER+ tumors harbored hypermethylation of the hMLH1 promoter region whereas only four cases (5.5%) of the 73 RER- tumors did. Hypermethylation of the hMSH2 promoter region was not observed in either the RER+ group or the RER- group. These results suggest that hypermethylation of the hMLH1 promoter region may be the principal mechanism of gene inactivation in sporadic gastric carcinomas with a high frequency of MSI.     

Prediction of a mismatch repair gene defect by microsatellite instability and immunohistochemical analysis in endometrial tumours from HNPCC patients

Instability of microsatellite repeat sequences has been observed in colorectal carcinomas and in extracolonic malignancies, predominantly endometrial tumours, occurring in the context of hereditary non-polyposis colorectal cancer (HNPCC). Microsatellite instability (MSI) as a feature of human DNA mismatch repair (MMR)-driven tumourigenesis of the uterine mucosa has been studied primarily in sporadic tumours showing predominantly somatic hypermethylation of MLH1. The present study shows that all endometrial carcinomas (n=12) from carriers of MLH1 and MSH2 germline mutations demonstrate an MSI-high phenotype involving all types of repeat markers, while in endometrial carcinomas from MSH6 mutation carriers, only 36% (4 out of 11) demonstrate an MSI-high phenotype. Interestingly, an MSI-high phenotype was found in endometrial hyperplasias from MSH2 mutation carriers, in contrast to hyperplasias from MLH1 mutation carriers, which exhibited an MSI-stable phenotype. Instability of only mononucleotide repeat markers was found in both endometrial carcinomas and hyperplasias from MSH6 mutation carriers. In 29 out of 31 (94%) endometrial tumour foci, combined MSI and immunohistochemical analysis of MLH1, MSH2, and MSH6 could predict the identified germline mutation. The observation of MSI in endometrial hyperplasia and of altered protein staining for the MMR genes supports the idea that inactivation of MMR genes is an early event in endometrial tumourigenesis. A correlation was found between the variation in the extent and level of MSI and the age of onset of carcinoma, suggesting differences in the rate of tumour progression. A high frequency of MSI in hyperplasias, found only in MSH2 mutation carriers, might indicate a more rapid tumour progression, correlating with an earlier age of onset of carcinoma. The present study indicates that assessment of altered protein staining combined with MSI analysis of endometrial tumours might direct the mutational analysis of MMR genes.     

Morphology and microsatellite instability in sporadic serrated and non-serrated colorectal cancer

Colorectal serrated adenocarcinoma originates from serrated adenoma, but definite histological criteria have not yet been established. It presents with frequent DNA microsatellite instability (MSI), but the frequency of low-level (MSI-L) and high-level MSI (MSI-H) and the expression of mismatch-repair (MMR) enzymes in serrated adenocarcinoma are not known. To address these questions, morphological criteria for serrated cancers were established, their validity was tested, and MSI analysis was performed with NIH consensus markers and MMR enzyme immunohistochemistry for hMLH1, hMSH2, and hMSH6 in 35 serrated and 75 non-serrated colorectal carcinomas. Serrated carcinomas frequently showed a serrated, mucinous or trabecular growth pattern; abundant eosinophilic cytoplasm; chromatin condensation; preserved polarity; and the absence of necrosis. With these features, it was possible to distinguish them from non-serrated cancers, with the mean kappa score for five observers being 0.509. MSI analysis was successful in 31 serrated and 73 non-serrated carcinomas. 54.8% of serrated carcinomas were microsatellite-stable (MSS), 29.0% presented with MSI-L, and 16.1% presented with MSI-H, whereas 78.1% of non-serrated carcinomas were MSS, 13.7% were MSI-L, and 8.2% were MSI-H. MSI-L was more common in serrated cancers (p=0.035) and it was associated with patchy immunohistochemical staining (33.3%) of MLH1. MSI-H did not differ between serrated and non-serrated cancers (p=0.14). These results suggest that the biological background of serrated carcinomas differs from sporadic non-serrated colorectal cancer, but is not directly related to MSI.     

Alterations of DNA mismatch repair proteins and microsatellite instability levels in gastric cancer cell lines

Alterations in DNA mismatch repair (MMR) proteins result in microsatellite instability (MSI), increased mutation accumulation at target genes and cancer development. About one-third of gastric cancers display high-level microsatellite instability (MSI-High) and low-level microsatellite instability (MSI-Low) is frequently detected. To determine whether variations in the levels of MMR proteins or mutations in the main DNA MMR genes are associated with MSI-Low and MSI-High in gastric cancer cell lines, the MSI status (MSI-High, MSI-Low or MS-Stable (MSS)) of 14 gastric cancer lines was determined using multiple clone analysis with a panel of five microsatellite markers. Protein levels of hMLH1, hMSH2, hMSH6, hPMS2 and hPMS1 were determined by Western blot. Sequence analysis of hMLH1 and hMSH2 was performed and the methylation status of the hMLH1 promoter was examined. The cell lines SNU1 and SNU638 showed MSI-High, decreased to essentially absent hMLH1 and hPMS2 and reduced hPMS1 and hMSH6 protein levels. The hMLH1 promoter region was hypermethylated in SNU638 cells. The MKN28, MKN87, KATOIII and SNU601 cell lines showed MSI-Low. The MMR protein levels of cells with MSI-Low status was similar to the levels detected in MSS cells. A marked decrease in the expression levels of MutL MMR proteins (hMLH1, hPMS2 and hPMS1) is associated with high levels of MSI mutations in gastric cancer cells. Gastric cancer cell lines with MSI-Low status do not show significant changes in the levels of the main DNA MMR proteins or mutations in the DNA mismatch repair genes hMSH2 and hMLH1. These well-characterized gastric cancer cell lines are a valuable resource to further our understanding of DNA MMR deficiency in cancer development, progression and prognosis.     

Tissue microarray immunohistochemical expression analysis of mismatch repair (hMLH1 and hMSH2 genes) in endometrial carcinoma and atypical endometrial hyperplasia: relationship with microsatellite instability.

Alterations in the mismatch repair genes (hMLH1 and hMSH2) play an important role in the development of microsatellite instability in sporadic endometrial cancer. Tissue microarray technology allows molecular profiling of tumor samples at the DNA, RNA, and protein levels. We analyzed hMLH1 and hMSH2 expression by immunohistochemistry in a group of atypical endometrial hyperplasias (n = 10), endometrioid endometrial carcinomas (n = 58), and nonendometrioid endometrial carcinomas (n = 27) on tissue microarray. The results were correlated with microsatellite instability status as evaluated by BAT-25 and BAT-26. Overall, 29.4% of lesions showed microsatellite instability. Loss of nuclear hMLH1 and hMSH2 protein expression was seen in 22.3% and 6.5% of cases, respectively. Immunohistochemistry for hMLH1 and hMSH2 showed lack of protein expression in 64% and 16.6% of microsatellite instability-positive endometrial lesions, respectively. Taken together, hMLH1 or hMSH2 protein expression was absent in 18 of 24 microsatellite instability-positive cases (75% sensitivity). A high level of concordance was found between immunohistochemistry for hMLH1 and hMSH2 and microsatellite instability status evaluated by BAT-25 and BAT-26 (kappa value of 0.7). Of the 57 cases found to be microsatellite instability negative, 53 showed normal expression of both proteins (93% specificity). The observed predictive value of absence of expression of hMLH1 for predicting microsatellite instability-positive status was 82%. The predictive value of normal expression of both proteins for predicting microsatellite instability-negative status was 90%. These results are consistent with those previously reported in whole tissue sections. Therefore, immunohistochemical analysis of hMLH1 and hMSH2 expression on tissue microarray provides an accurate technique for screening for tumors with microsatellite instability. Tissue microarrays represent an ideal approach for comparing different diagnostic or predictive markers with one another in consecutive tissue microarray sections.     

Mismatch repair proteins and microsatellites hit clinical practice

Hereditary nonpolyposis colon cancer (HNPCC) is one of the most common familial cancers with characteristic molecular changes that are different from those found in familial adenomatous polyposis (FAP) coli. Genetic mutations in the germline and somatic cells lead to loss of expression of one of the two most commonly involved mismatch repair genes, hMSH2 or hMLH1, and consequently, to expansion of certain repetitive DNA sequences (microsatellite instability (MSI)). The paper describes a distinct subtype of "HNPCC-like" sporadic colonic carcinoma that can easily be identified by immunohistochemistry. Recognition of this subtype of colonic cancer is important because it occurs in the younger age group and is associated with better survival, but also a five-fold chance of developing a second colorectal carcinoma compared to "conventional" colorectal carcinomas.     

DNA mismatch repair protein expression and microsatellite instability in primary mucosal melanomas of the head and neck

AIMS: To examine the expression of DNA mismatch repair (MMR) proteins and the presence of microsatellite instability (MSI) in seven primary mucosal melanomas of the head and neck (MMHN). METHODS AND RESULTS: Haematoxylin and eosin staining and immunohistochemical analysis for routine diagnostic markers and for MMR proteins were performed. Six cases were examined for MSI. Four cases were monomorphous and three cases were pleomorphic type MMHN. Melanocytic markers were positive in all cases. Immunoreactivity for MMR proteins was weak in normal epithelium. The neoplastic tissue in six cases showed positivity for all MMR proteins with different percentages. One case showed weak positivity for hMSH2 and hMSH6 and no immunoreactivity for hMLH1 or hPMS2. Staining intensity was higher in tumour cells than in matched normal mucosa in three cases for hMSH2 and hMLH1 and in two cases for hPMS2. None of the examined cases showed MSI. CONCLUSIONS: Expression of hMSH2 and hMLH1 proteins was up-regulated in three cases, whereas in two cases that of hPMS2 was increased. hMSH6 expression was comparable to that of normal cells in all cases. The percentage of positive neoplastic cells and the intensity of staining seemed to be greater in pleomorphic melanomas. Six cases were MMR-proficient and microsatellite stable.     

Characterization of mutator pathway in younger-age-onset colorectal adenocarcinomas

The high-frequency microsatellite instability (MSI-H) phenotype, frequently identified in hereditary nonpolyposis colorectal cancer (HNPCC), also accounts for approximately 15% of sporadic colorectal cancers. Microsatellite instability (MSI) occurs from the mutational inactivation of the DNA mismatch repair genes, i.e. hMSH2 and hMLH1 in HNPCC, as well as from epigenetic inactivation of hMLH1 in sporadic colorectal tumors. The mutator pathway including microsatellite instability, hMLH1 promoter methylation, and hMSH2 and hMLH1 mutation patterns were identified in 21 sporadic colorectal adenocarcinoma patients younger than 30 yr excluding HNPCC. More than half of tumors showed MSI, with five MSI-H and six MSI-L (low-frequency microsatellite instability). Three of six MSI-H tumors showed the hMLH1 promoter methylation and did not express the hMLH1 protein. On the other hand, all MSI-L and all MSS (microsatellite stable) tumors expressed both hMSH2 and hMLH1 proteins. Two novel mutations, i.e. a missense mutation in hMLH1 and a splice-site alteration in hMSH2, were identified in two patients respectively. Although mutator pathway was implicated in younger-age-onset colorectal carcinogenesis, many tumors appeared to evolve from different genetic events other than hMSH2 and hMLH1 mutations frequently identified in HNPCC.     

Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status

In sporadic colorectal tumours the BRAFV600E is associated with microsatellite instability (MSI-H) and inversely associated to KRAS mutations. Tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients carrying germline mutations in hMSH2 or hMLH1 do not show BRAFV600E, however no consistent data exist regarding KRAS mutation frequency and spectrum in HNPCC tumours. We investigated KRAS in 158 HNPCC tumours from patients with germline hMLH1, hMSH2 or hMSH6 mutations, 166 MSI-H and 688 microsatellite stable (MSS) sporadic carcinomas. All tumours were characterized for MSI and 81 of 166 sporadic MSI-H colorectal cancer (CRCs) were analysed for hMLH1 promoter hypermethylation. KRAS mutations were observed in 40% of HNPCC tumours, and the mutation frequency varied upon the mismatch repair gene affected: 48% (29/61) in hMSH2, 32% (29/91) in hMLH1 and 83% (5/6) in hMSH6 (P = 0.01). KRAS mutation frequency was different between HNPCC, MSS and MSI-H CRCs (P = 0.002), and MSI-H with hMLH1 hypermethylation (P = 0.005). Furthermore, HNPCC CRCs had more G13D mutations than MSS (P < 0.0001), MSI-H (P = 0.02) or MSI-H tumours with hMLH1 hypermethylation (P = 0.03). HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. In conclusion, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs, may target distinct kinases within the RAS/RAF/MAPK pathway.     

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.     

hMLH1 Promoter Hypermethylation Is an Early Event in Human Endometrial Tumorigenesis

It has recently been suggested that silencing of the hMLH1 gene by promoter hypermethylation is the mechanism underlying the presence of the microsatellite instability (MSI) phenotype in sporadic colon and endometrial carcinomas. To determine whether hMLH1 promoter hypermethylation is a relatively early event in endometrial tumorigenesis we evaluated endometrial hyperplasia (EH) characterized as simple, complex, and atypical (the direct precursor of endometrial carcinoma) for hMLH1 aberrant methylation. In addition, we studied the hMLH1, hMSH2, hMSH3, and hMSH6 promoter methylation and MSI status of those endometrial carcinomas with synchronous hyperplasias and those without them. We found that 11 of 12 (91%) cases of endometrial carcinoma (EC) displaying MSI had hMLH1 promoter hypermethylation, whereas aberrant methylation of any of the other mismatch repair genes was not observed. All 15 cases of EC without MSI were unmethylated at hMLH1. Abnormal methylation of hMLH1 was also present in 8 of 116 (7%) cases of EH and was restricted primarily to the atypical endometrial hyperplasia (AEH) type with coexisting endometrial carcinoma. In this set, half of EH methylated at hMLH1 displayed MSI, whereas none of the unmethylated EH had MSI. Our data suggest that hypermethylation of hMLH1 can be an early event in the pathogenesis of EC, preceding the development of an apparent MSI phenotype in a subset of cases.     

Microsatellite instability and expression of MLH1 and MSH2 in normal and malignant endometrial and ovarian epithelium in hereditary nonpolyposis colorectal cancer family members.

Mismatch repair deficiency is a characteristic molecular finding in hereditary nonpolyposis colorectal cancer (HNPCC), and has been demonstrated in both colorectal cancers and benign adenomas. Endometrial and ovarian cancers are common extracolonic tumors in this syndrome; however, few studies have investigated whether genetic changes occur in histologically normal endometrial and ovarian epithelia from HNPCC family members. If early genetic changes exist, they might be used as molecular markers to detect susceptibility to endometrial and ovarian cancers. In this study, we analyzed microsatellite instability (MSI) and MLH1 and MSH2 immunohistochemical expression in 20 histologically normal epithelia (12 endometrial and 8 ovarian) and 8 cancers (4 endometrial and 4 ovarian) obtained from 20 individuals representing 7 unrelated HNPCC families. While MSI was observed in endometrial (75%) and ovarian (100%) cancers, no case was determined to exhibit MSI in histologically normal epithelia of the endometrium or ovary. Similarly, in immunohistochemical expressions for MLH1 and MSH2, histologically normal epithelia had no genetic changes predisposing to malignancy. In cancer cases, a correlation existed between the expression of MLH1 and MSH2, the presence of germline mutations in the hMLH1 and hMSH2 genes, and the presence of tumor MSI. These data suggest that MSI and MLH1 and MSH2 expression are not useful biomarkers for the early detection of endometrial and ovarian malignancy in cancer-unaffected HNPCC germline mutation carriers. Further studies of other genetic changes in normal and premalignant precursor lesions are needed.     

Kolorektale Karzinome unter Berücksichtigung der neuen S3-Leitlinie von 2013

In the current German S3 guidelines for colorectal carcinoma (CRC), morphologically based tumor grading is extended by molecular grading for poorly differentiated and undifferentiated carcinomas, as well as for special morphological subtypes. These CRC are classified as low-grade when microsatellite instability (MSI) is found. In routine diagnostics, immunohistochemistry for hMLH1 and hMSH2, capturing MSI-CRC with high sensitivity and specificity, can be used as an inexpensive substitute for molecular MSI-testing. In patients with positive Bethesda criteria, a stepwise immunohistochemical and molecular diagnostic scheme is proposed. The detection of a BRAF mutation in tumors with hMLH1 loss allows distinguishing between sporadic and HNPCC-associated MSI-CRC. For rectal cancer the residual tumor classification (R-status) is completed by the circumferential resection margin classification (CRM).     

Genetic progression in sporadic endometrial and gastrointestinal cancers with high microsatellite instability

This study selected a series of 136 MSI-H (microsatellite instability at high frequency) gastric, colorectal, and endometrial carcinomas combining immunohistochemical analysis for hMLH1 or hMSH2 gene products and microsatellite study. The clinico-pathological profile of all tumours was correlated with the overall instability rates at coding and non-coding repeats, in order to clarify the role and the mutation timing of seven target genes (TGFbetaRII, IGFIIR, BAX, hMSH3, hMSH6, CHK1, and BRCA2) in the progression of an MSI-H neoplasm. Regardless of the primary site, the results confirm a model of oncogenesis in which inactivation of hMLH1, or less frequently hMSH2, may initiate a pathway culminating in a progressive accumulation of frameshifts in coding region (CDR) microsatellites. Comparing gastrointestinal and endometrial tumours, significantly lower levels of microsatellite instability at both coding and non-coding repeats were observed. Among gastric and colorectal tumours, the detection of small shortening within Bat-26 and Bat-25 markers defines a subgroup of MSI-H gastrointestinal tumours invariably characterized by early stage at diagnosis. In these tumours, mutations of TGFbetaRII or BAX genes precede frameshifts in the other tested genes. The analysis of correlations between the mutational and clinico-pathological profiles of advanced gastrointestinal tumours revealed that the higher levels of microsatellite instability at both coding and non-coding repeats were not associated with a more advanced clinico-pathological stage or a less favourable outcome. A significant association was observed between a low number of CDR frameshifts and the presence of lymph-node metastasis in advanced gastrointestinal tumours. The existence of advanced MSI-H tumours with more aggressive behaviour and a 'mild mutator phenotype' could be explained by hypothesizing an overlapping of different mechanisms of tumourigenesis, including both the mutator and the suppressor pathways; this should be tested by further studies.     

Causes of microsatellite instability in colorectal tumors: implications for hereditary non-polyposis colorectal cancer screening

Microsatellite instability (MSI) analysis was performed using a "reference panel" of microsatellite markers in 345 unselected primary colorectal cancers (CRC). Thirty-five (10%) tumors were classified as high MSI (MSI-H). We identified 6 (17%) MSI-H tumors with germline mutations in mismatch repair (MMR) genes (tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome) and 29 (83%) MSI-H tumors without germline MMR mutations (sporadic MSI-H tumors). Hypermethylation of the hMLH1 promoter was found in 26/29 (90%) sporadic MSI-H tumors but only in 1/6 (17%) HNPCC tumors (P<.001). Somatic alterations were identified in both MMR genes in HNPCC tumors but mainly in the hMSH2 gene in sporadic MSI-H tumors. LOH at MMR loci was detected in 3/6 (50%) HNPCC tumors and in 4/26 (15%) informative sporadic MSI-H tumors. These results together indicate different mode of inactivation of MMR genes in sporadic MSI-H tumors versus MSI-H tumors in HNPCC patients. We therefore propose that MSI analysis of newly diagnosed primary CRC followed by methylation analysis of hMLH1 promoter in MSI-H tumors and mutational analysis of MMR genes in MSI-H tumors lacking hMLH1 promoter methylation might be an efficient molecular genetic approach for HNPCC screening.     

A mononucleotide markers panel to identify hMLH1/hMSH2 germline mutations

Hereditary NonPolyposis Colorectal Cancer (Lynch syndrome) is an autosomal dominant disease caused by germline mutations in a class of genes deputed to maintain genomic integrity during cell replication, mutations result in a generalized genomic instability, particularly evident at microsatellite loci (Microsatellite Instability, MSI). MSI is present in 85-90% of colorectal cancers that occur in Lynch Syndrome. To standardize the molecular diagnosis of MSI, a panel of 5 microsatellite markers was proposed (known as the "Bethesda panel"). Aim of our study is to evaluate if MSI testing with two mononucleotide markers, such as BAT25 and BAT26, was sufficient to identify patients with hMLH1/hMSH2 germline mutations. We tested 105 tumours for MSI using both the Bethesda markers and the two mononucleotide markers BAT25 and BAT26. Moreover, immunohistochemical evaluation of MLH1 and MSH2 proteins was executed on the tumours with at least one unstable microsatellite, whereas germline hMLH1/hMSH2 mutations were searched for all cases showing two or more unstable microsatellites. The Bethesda panel detected more MSI(+) tumors than the mononucleotide panel (49.5% and 28.6%, respectively). However, the mononucleotide panel was more efficient to detect MSI(+) tumours with lack of expression of Mismatch Repair proteins (93% vs 54%). Germline mutations were detected in almost all patients whose tumours showed MSI and no expression of MLH1/MSH2 proteins. No germline mutations were found in patients with MSI(+) tumour defined only through dinucleotide markers. In conclusion, the proposed mononucleotide markers panel seems to have a higher predictive value to identify hMLH1 and hMSH2 mutation-positive patients with Lynch syndrome. Moreover, this panel showed increased specificity, thus improving the cost/effectiveness ratio of the biomolecular analyses.     

Immunohistochemical detection of mismatch repair gene proteins as a useful tool for the identification of colorectal carcinoma with the mutator phenotype

There are two well-defined pathways for colorectal carcinogenesis, the suppressor and the mutator pathways. The latter is characteristic of hereditary non-polyposis colorectal cancer (HNPCC), but can also be found in a subset of sporadic colorectal cancer (SCC) possessing distinctive clinical and pathological features, namely early age of onset, location in the right colon, poor differentiation, and a predominant mucinous component. This mutator pathway results from inactivation of mismatch repair (MMR) genes, namely MSH2 and MLH1. The aim of this study was to ascertain if abnormal MMR protein gene expression is a good indicator for identifying tumours from the mutator pathway. Seventy-six cases of SCC were studied by immunohistochemistry using two monoclonal mouse antibodies that react against MSH2 and MLH1 protein gene products. Immunoexpression was assessed both in tumour and in non-neoplastic, adjacent and distant mucosa. Microsatellite instability (MSI) was detected by evaluating the length of poly(CA) repeated sequences at seven loci, or by the detection of small unstable alleles in a poly(A) repeat - BAT-26. Except for BAT-26, in which only tumour DNA was used, MSI analysis was performed in both tumour and normal mucosal DNA. MSI was classified as high (MSI-H), low (MSI-L) or stable (MSS). Abnormal protein expression was found in 9/76 (12%) tumours. Immunohistochemistry for hmlh1 and hmsh2 detected 75% of MSI-H. There was also a highly significant correlation between the observed immunoexpression and several clinical and pathological characteristics described as the phenotypic profile of the mutator pathway, such as right-sided location (p=0.003), mucin production (p=0.008), and a peritumoural lymphoid infiltrate (p=0.009). Non-neoplastic adjacent mucosa showed normal hMSH2 expression in all cases, but in ten cases there was no hMLH1 expression in this transitional mucosa, which is known to display an alterated mucin pattern and a high proliferative rate. These results demonstrated a good correlation between hMLH1 and hMSH2 gene immunoexpression and the clinico-pathological features characteristic of the mutator phenotype and support the use of this method as a rapid and efficient way to detect tumours arising from this pathway.     

Diagnostic application of hMLH1 methylation in hereditary non-polyposis colorectal cancer

Colorectal cancer (CRC) due to mismatch repair (MMR) defect has distinct characteristics among unselected CRCs. These CRCs are biologically less aggressive and, thus, showing better prognosis but less sensitive to the 5FU-based chemotherapy. CRCs with MMR defect derive from both hereditary and sporadic reasons. Germline inactivation of MMR genes (hMLH1, hMSH2, hMSH6, and hPMS2) underlies the hereditary CRC with MMR defect (Lynch syndrome) and epigenetic silencing of hMLH1 gene causes the sporadic CRC with MMR defect. Hereditary and sporadic CRC with MMR defect can be detectable by microsatellite instability (MSI) test or immunohistochemical analysis among general CRCs. Lynch syndrome can be diagnosed by the clinical criteria or by genetic test to detect pathogenic germline mutations in MMR genes. However, both clinical criteria and genetic test are inadequate for the diagnosis of Lynch syndrome. Since genetic test for the diagnosis of the Lynch syndrome is expensive and not always identify pathogenic germline mutations, effective and inexpensive screening program is desirable. Here we propose a possible application of methylation test combined with MSI or pathological analysis as an effective and a cost-saving new strategy for screening of Lynch syndrome.