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.     

Atypical HNPCC owing to MSH6 germline mutations: analysis of a large Dutch pedigree

Hereditary non-polyposis colorectal cancer (HNPCC) is the most common genetic susceptibility syndrome for colorectal cancer. HNPCC is most frequently caused by germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1. Recently, mutations in another MMR gene, MSH6 (also known as GTBP), have also been shown to result in HNPCC. Preliminary data indicate that the phenotype related to MSH6 mutations may differ from the classical HNPCC caused by defects in MSH2 and MLH1. Here, we describe an extended Dutch HNPCC family not fulfilling the Amsterdam criteria II and resulting from a MSH6 mutation. Overall, the penetrance of colorectal cancer appears to be significantly decreased (p<0.001) among the MSH6 mutation carriers in this family when compared with MSH2 and MLH1 carriers (32% by the age of 80 v >80%). Endometrial cancer is a frequent manifestation among female carriers (six out of 13 malignant tumours). Transitional cell carcinoma of the urinary tract is also relatively common in both male and female carriers (10% of the carriers). Moreover, the mean age of onset of both colorectal cancer (MSH6 v MSH2/MLH1 = 55 years v 44/41 years) and endometrial carcinomas (MSH6 v MSH2/MLH1 = 55 years v 49/48 years) is delayed. As previously reported, we confirm that the pattern of microsatellite instability, in combination with immunohistochemical analysis, can predict the presence of a MSH6 germline defect. The detailed characterisation of the clinical phenotype of this kindred contributes to the establishment of genotype-phenotype correlations in HNPCC owing to mutations in specific mismatch repair genes.     

Distinct PTEN mutational spectra in hereditary non-polyposis colon cancer syndrome-related endometrial carcinomas compared to sporadic microsatellite unstable tumors

Germline PTEN mutations cause Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRR), two hamartoma-tumor syndromes with an increased risk of breast, thyroid and endometrial cancers. Somatic genetic and epigenetic inactivation of PTEN is involved in as high as 93% of sporadic endometrial carcinomas (EC), irrespective of microsatellite status, and can occur in the earliest precancers. EC is the most frequent extra-colonic cancer in patients with hereditary non-polyposis colon cancer syndrome (HNPCC), characterized by germline mutations in the mismatch repair (MMR) genes and by microsatellite instability (MSI) in component tumors. To determine whether PTEN is involved in the pathogenesis of EC arising in HNPCC cases, and whether PTEN inactivation precedes MMR deficiency, we obtained 41 ECs from 29 MLH1 or MSH2 mutation positive HNPCC families and subjected them to PTEN expression and mutation analysis. Immunohistochemical analysis revealed 68% (28/41) of the HNPCC-related ECs with absent or weak PTEN expression. The remaining 27% (11/41) of tumors had normal expression and 5% (2/41) with mixed populations showing weak/absent as well as normal expression. Mutation analysis of 20 aberrant PTEN-expressing tumors revealed that 17 (85%) harbored 18 somatic PTEN mutations. All mutations were frameshift, 10 (56%) of which involved the 6(A) tracts in exon 7 or 8. These results suggest that PTEN plays a significant pathogenic role in both HNPCC and sporadic endometrial carcinogenesis, unlike the scenarios for colorectal cancer. Furthermore, we have shown that somatic PTEN mutation, especially frameshift, is a consequence of profound MMR deficiency in HNPCC-related ECs. In contrast, among 60 previously reported MSI+ sporadic ECs with 70 somatic mutations in PTEN, 39 (56%) were frameshift, of which only eight (21%) were affecting the 6(A) tracts in exon 7 or 8 (P = 0.01), suggesting that PTEN mutations may precede MMR deficiency.     

Mutational analysis of MLH1 and MSH2 in 25 prospectively-acquired RER+ endometrial cancers

Mutations in the DNA mismatch repair (MMR) genes MLH1 and MSH2 have been linked to several human cancers which display the replication error (RER) phenotype. Germline mutations in these two genes have been implicated in about 90% of families with hereditary nonpolyposis colorectal cancer (HNPCC). A significant proportion of endometrial cancers, the second most common malignancy of the HNPCC syndrome, also exhibit RER. We screened 125 primary endometrial adenocarcinomas with seven microsatellite markers and identified 25 specimens with RER (20%). We used single-strand conformation variant analysis to search for mutations in MLH1 and MSH2. Direct sequencing of variants revealed only one germline mutation in MLH1 and a single somatic mutation in MSH2. However, six previously unreported sequence polymorphisms in MLH1 were identified. Four of these polymorphisms show clear population-based differences in allele frequency. In addition, a highly informative marker for MLH1 was characterized. The low frequency of mutations in MLH1 and MSH2 in this large series of cancers suggests that other MMR genes are responsible for the RER phenotype in endometrial cancers. Genes Chromosom. Cancer 18:219–227, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Defective mismatch-repair as a minor tumorigenic pathway in Barrett esophagus-associated adenocarcinoma

The malignant transformation that characterizes the development of Barrett esophagus-associated adenocarcinomas is a multi-step process in which genetic alterations in various tumor-associated genes accumulate. Defective mismatch repair (MMR) is the cause of microsatellite instability (MSI) pathway that characterizes a subset of gastrointestinal tumors and is specifically associated with tumor development within the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. The few studies that have assessed MMR defects in Barrett-associated adenocarcinomas have reached different results. We therefore assessed the expression of the MMR proteins MLH1 and MSH2 in a series of 59 Barrett adenocarcinomas and found a loss of MMR protein immunostaining in 2/59 (3%) tumors; one tumor showed a loss of MSH2 expression, the other tumor showed a loss of MLH1, and both tumors displayed an MSI-high phenotype. Our findings suggest that only a small subset of Barrett adenocarcinomas develop because of defective MMR, but demonstrate that MLH1 and MSH2 are the primary targets for defective MMR also in this tumor type.     

PAX6 gene variations associated with aniridia in south India

Background

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease with a high risk for colorectal and endometrial cancer caused by germline mutations in DNA mismatch-repair genes (MMR). HNPCC accounts for approximately 2 to 5% of all colorectal cancers. Here we present 6 novel mutations in the DNA mismatch-repair genes MLH1, MSH2 and MSH6.

Methods

Patients with clinical diagnosis of HNPCC were counselled. Tumor specimen were analysed for microsatellite instability and immunohistochemistry for MLH1, MSH2 and MSH6 protein was performed. If one of these proteins was not detectable in the tumor mutation analysis of the corresponding gene was carried out.

Results

We identified 6 frameshift mutations (2 in MLH1, 3 in MSH2, 1 in MSH6) resulting in a premature stop: two mutations in MLH1 (c.2198_2199insAACA [p.N733fsX745], c.2076_2077delTG [p.G693fsX702]), three mutations in MSH2 (c.810_811delGT [p.C271fsX282], c.763_766delAGTGinsTT [p.F255fsX282], c.873_876delGACT [p.L292fsX298]) and one mutation in MSH6 (c.1421_1422dupTG [p.C475fsX480]). All six tumors tested for microsatellite instability showed high levels of microsatellite instability (MSI-H).

Conclusions

HNPCC in families with MSH6 germline mutations may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations.     

Identification of HNPCC by molecular analysis of colorectal and endometrial tumors

Hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) is a dominantly inherited syndrome characterized by the development of colorectal cancer, endometrial cancer and other cancers and the presence of microsatellite instability (MSI) in tumors. The Bethesda guidelines have been proposed for the identification of families suspected of HNPCC that require further molecular analysis. We have evaluated the yield of MSI-analysis in a large series of Dutch families suspected of HNPCC. We also analysed whether the loss of mismatch repair (MMR) protein detected by immunohistochemistry (IHC) of colorectal cancer (CRC) and endometrial cancer correlated with the presence of MSI and/or a MMR gene mutation. The results showed that the Bethesda criteria with a few modifications are appropriate to identify families eligible for genetic testing. In addition, we found that MSI and IHC-analysis of CRC using antibodies against MLH1, MSH2, MSH6 and PMS2 proteins are equally effective for identifying carriers of the known MMR gene defects. However, as long as the role of other putative MMR genes in hereditary CRC has not been elucidated, IHC-analysis cannot completely replace MSI. For this reason, we prefer MSI-analysis as first step in families suspected of HNPCC. On the other hand, in families fulfilling the revised Amsterdam criteria in which the probability of detecting a mutation is relatively high, we would recommend IHC as first diagnostic step because the result might predict the specific underlying MMR gene mutation. MSI or IHC-analysis of endometrial cancer alone was found to be less sensitive compared with these tests performed in colorectal cancer. Therefore, probably the best approach in the analysis of this cancer is to perform both techniques. The identification of HNPCC is important as it makes it possible to target effective preventative measures. Our studies showed that MSI and IHC analysis of colorectal and endometrial cancer, are reliable cost-effective tools that can be used to identify patients with HNPCC.     

Clinical and molecular characteristics of hereditary non-polyposis colorectal cancer families in Southeast Asia

Lee S-C, Guo J-Y, Lim R, Soo R, Koay E, Salto-Tellez M, Leong A, Goh B-C. Clinical and molecular characteristics of hereditary non-polyposis colorectal cancer families in Southeast Asia.Hereditary non-polyposis colorectal cancer (HNPCC), predominantly due to germline MLH1/MSH2 mutations, is the commonest form of hereditary colorectal cancer (CRC), but data in Asians are sparse. We sequenced the MLH1/MSH2 coding and promoter core regions in CRC patients diagnosed below age 40, and/or with multiple primary cancers or familial cancer clustering suggestive of HNPCC, and correlated deleterious mutations with clinical and tumour features. Forty-six Chinese, Malay and Indian kindreds participated. Of the 153 cancers reported in the 46 kindreds, stomach (14%) and urogenital cancers (13%) were the most common extracolonic cancers, whereas endometrial cancer comprised only 7%. Eleven different MLH1 and 12 MSH2 mutations were identified, including nine novel and four recurring mutations in the Chinese. One Indian was a compound heterozygote for an MLH1 and MSH2 mutation. The MLH1/MSH2 mutation data in the Malays and the Indians represents the first in these ethnic groups. Factors strongly associated with deleterious mutations were the Amsterdam criteria, family history of stomach or multiple primary cancers, and MSI-high tumours, whereas family history of endometrial cancer and young cancer age alone correlated poorly. Distinct clinical and molecular characteristics were identified among Asian HNPCC kindreds and may have important clinical implications.     

Epigenetic mechanisms in the pathogenesis of Lynch syndrome

Inherited defects in the DNA mismatch repair (MMR) system, MLH1, MSH2, MSH6, and PMS2 genes, underlie Lynch syndrome, one of the most prevalent cancer syndromes in man. The syndrome offers a model for cancers arising through MMR defects and microsatellite instability, which applies to ?15% of all colorectal, endometrial, and other cancers. Lynch syndrome also illustrates the significance of the epigenetic component in cancer development. Inactivation of tumor suppressor genes by epigenetic mechanisms is an acquired property of many tumors developing in Lynch syndrome. Furthermore, constitutional epimutations of MMR genes may explain a proportion of mutation‐negative families lacking MLH1 or MSH2 protein expression in tumor tissue. This review provides an update of the molecular basis of Lynch syndrome by focusing on the role of epigenetic mechanisms in the pathogenesis of the disease.     

Germline and somatic mutation analysis of MLH3 in MSI-positive colorectal cancer

Microsatellite instability (MSI) is characteristic of hereditary nonpolyposis colorectal cancer, and occurs in a subset (10 to 15%) of unselected colorectal cancer cases. In hereditary nonpolyposis colorectal cancer, MSI is caused by defects in five mismatch repair genes, and in sporadic cases the main cause seems to be somatic MLH1 promoter methylation. Most likely additional hereditary nonpolyposis colorectal cancer genes remain to be discovered. Genes with simple repeats in their coding region are often targets for deletions in MSI-positive tumors. Several genes (TGFbeta RII, IGFIIR, MSH3, MSH6, BAX, MBD4) with significance in tumorigenesis harbor repeats in their coding regions and are often somatically inactivated because of deletions causing frameshifts. Recently, a novel human mismatch repair gene, MLH3, was cloned and shown to be involved in mammalian mismatch repair. To evaluate the possible role of MLH3 in hereditary cancer, we performed germline single-strand conformation polymorphism-analysis for 52 patients displaying features of inherited colorectal cancer. Forty-six of these had been diagnosed with MSI-positive tumors. No germline mutations were found. Similar to MSH3 and MSH6, MLH3 harbors mononucleotide repeats, ie, (A(6))-(A(9)), in its coding region, which makes it a putative target for somatic mutations in MSI-positive tumors. To evaluate its somatic inactivation we performed a deletion search focusing on eight exonic MLH3 mononucleotide repeats in a series of 93 MSI-positive tumors. Somatic deletions were found in 8.6% of the samples, a frequency similar to one detected in neutral noncoding mononucleotide repeats. No evidence of involvement of MLH3 in MSI tumorigenesis was obtained.     

Four novel MSH2 and MLH1 frameshift mutations and occurrence of a breast cancer phenocopy in hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations of genes encoding for proteins of the mismatch repair (MMR) machinery. The majority of mutations occur in the MLH1 and MSH2 genes, and consist of splice-site, frameshift and nonsense changes, leading to loss of protein function. In this study, we screened 7 HNPCC families for MLH1/MSH2 mutations. Sequence changes were identified in 5 families. Four alterations were novel 1- or 2-bp deletions or insertions causing a frameshift and appearance of premature stop codons (MLH1: c.597-598delGA, c.1520-1521insT; MSH2: c.1444delA, c.119delG). The four small insertions/ deletions were located within stretches of simple repeated sequences. By reviewing the HNPCC mutation database, we found that the majority of 1-2 bp frameshift mutations similarly affects simple repetitive stretches, pointing to DNA polymerase slippage during replication as the most likely source of such errors. We also evaluated microsatellite instability (MSI) in a breast carcinoma (BC) from an MLH1 mutation carrier. While a colon cancer from the same individual showed MSI, the BC specimen was MSI-negative, indicating that development of the latter tumor was unrelated to MMR impairment, despite presence of a constitutional MLH1 mutation. Hum Mutat 17:521, 2001.     

Somatic frameshift alterations in mononucleotide repeat-containing genes in different tumor types from an HNPCC family with germline MSH2 mutation

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by a germline mutation in one of several DNA repair genes, which in the tumors is reflected as microsatellite instability (MSI). MSI+ tumors have been found to carry somatic frameshift mutations in mononucleotide repeats within the coding regions of several genes involved in growth control, apoptosis, and DNA repair, e.g., TGFBRII, BAX, IGFIIR, TCF4, MSH3, and MSH6. We have studied the occurrence of somatic frameshift alterations in these mononucleotide repeat-containing genes in 24 tumors (15 colorectal cancers, 1 colon adenoma, 4 endometrial cancers, 1 ovarian cancer, 1 gastric cancer, 1 urothelial cancer, and 1 duodenal cancer) from 14 individuals in an HNPCC family with germline hMSH2 mutation. Such somatic frameshift mutations occurred at a variable frequency; the long mononucleotide repeats that characterize intronic MSI markers were mutated in the majority of tumors, 13 of the tumors displayed alterations in the (A)(10) tract of TGFBII, eight tumors (all of gastrointestinal origin) had alterations in the (A)(9) repeat of TCF4, and one to five tumors had somatic frameshift alterations in the shorter mononucleotide repeats of IGFIIR, BAX, MSH3, and MSH6. Thus, longer mononucleotide repeats were more frequently affected by somatic frameshift mutations. The pattern of alterations varied between the tumors from different family members as well as between different tumors from the same individual. To what extent this variable pattern depends on the widespread mismatch repair deficiency induced by the underlying MSH2 mutation, or represents alternative ways whereby the tumors can achieve a tumorigenic phenotype, is unknown. We suggest, however, that the accumulation of somatic frameshifts, rather than the specific loci in which these occur, drives the development of the tumorigenic phenotype in HNPCC.     

Role of endometrial cancer abnormal MMR protein in screening Lynch-syndrome families

Objective: To identify patients with endometrial cancer with potential Lynch-related DNA mismatch repair (MMR) protein expression defects and to explore the role of these defects in screening for LS. Methods: Endometrial cancers from 173 patients recruited to the Nanchong Central Hospital were tested for MMR (MLH1, MSH2, PMS2, and MSH6) protein expression using immunohistochemistry (IHC). Results: In the 173 tumor tissue samples, the expression loss rates of MSH6, MSH2, PMS2 and MLH1 protein were 16.18% (28/173), 12.14% (21/173), 7.51% (13/173) and 5.78% (10/173), respectively. The total loss rate of MMR protein was 29.89% (27/87). There were 19 patients with a family history of cancer, of which 18 patients demonstrated loss of expression of MMR protein. In the 22 abnormal MMR patients without family history, five families were found to have Lynch-associated cancer (colorectal cancer, endometrial cancer, ovarian cancer, stomach cancer) after follow-up for two years. Conclusion: MMR proteins play an important role in the progress of endometrial cancer. The routine testing of MMR proteins in endometrial cancer can contribute to the screening of LS families, especially small families.     

Mononucleotide microsatellite instability and germline MSH6 mutation analysis in early onset colorectal cancer.

Germline mutations in the MSH2 and MLH1 mismatch repair genes account for most cases of hereditary non-polyposis colon cancer syndrome (HNPCC). In addition, germline MSH2 and MLH1 mutations have been detected in patients with non-HNPCC early onset colorectal cancer. Germline MSH6 mutations appear to be rare in classical HNPCC families, but their frequency in young colorectal cancer cases has not been studied previously. In a population based study of early onset colorectal cancer (<50 years) investigated for tumour microsatellite instability (MSI), we identified a subgroup of tumours with MSI for mono- but not dinucleotide repeat markers (m-MSI+ group). In contrast to tumours with classical MSI for dinucleotide markers (d-MSI+), the m-MSI+ group cancers were mainly left sided (6/7). As MSH6 mutations in yeast and human cell lines are associated with weak (and preferential mononucleotide) MSI, the complete MSH6 gene coding region was sequenced in blood DNA from the five m-MSI+ cases available for analysis. A germline nonsense mutation was identified in an isolated case of early onset colorectal cancer (age 43 years). These results support previous findings that germline MSH6 mutations may not be associated with classical MSI and suggest a role for germline MSH6 mutations in isolated early onset colorectal cancer.     

Frequency of constitutional MSH6 mutations in a consecutive series of families with clinical suspicion of HNPCC

A large majority of constitutional mutations in hereditary non-polyposis colorectal cancer (HNPCC) are because of the MHL 1 or MSH 2 genes. In a lower fraction of cases, another gene of the mismatch repair (MMR) machinery, MSH6, may be responsible. Families with MSH6 mutations are difficult to recognize, as microsatellite instability (MSI) may not be detectable and immunohistochemistry (IHC) may give ambiguous results. In the present study, we proposed (i) to determine the frequency of MSH6 mutations in a selected population of colorectal cancer patients obtained from a tumor registry, (ii) to assess whether IHC is a suitable tool for selecting and identifying MSH6 mutation carriers. One hundred neoplasms of the large bowel from suspected HNPCC families were analyzed for MSI (BAT 25 and BAT 26 markers) and immunohistochemical expression of the MSH6 protein. We found on 12 tumors (from different families) showing instability or lack of MSH6 expression. Among these, four potentially pathogenic MSH6 mutations were detected (del A at 2984; del TT at 3119; del AGG cod 385; and del CGT cod 1242) by direct gene sequencing. These represented 12.9% of all families with constitutional mutations of the DNA MMR genes. Thus, some 5% of all HNPCC families are featured by constitutional mutation of the MSH6 gene. This appears, however, as a minimum estimate; routine use of IHC and the study of large numbers of individuals and families with little or no evidence of Lynch syndrome might reveal that mutation of this gene account for a large fraction of HNPCC.     

Protein expression profiles of deoxyribonucleic acid mismatch repair genes: Association with clinicopathological characteristics of Malaysian Lynch syndrome patients

Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer, accounts for approximately 1–5% of all colorectal cancers. Germline mutations in a group of deoxyribonucleic acid (DNA) mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS1, and PMS2) are responsible for Lynch syndrome cases. This study focuses on the determination of MMR (MLH1, MSH2, MSH6, and PMS2) protein expression profile by immunohistochemical analysis and its association with clinicopathological characteristics in clinically diagnosed Malaysian Lynch syndrome patients. Fifty patients who fulfilled any of the revised Bethesda Guidelines criteria were recruited from four collaborating centers in Malaysia. Clinicopathological information of clinically diagnosed Lynch syndrome cases that underwent bowel resection was reviewed. Immunohistochemical analysis for MLH1, MSH2, MSH6, and PMS2 proteins were performed on paraffin-embedded carcinomatous tissues. Colorectal cancer protein expression analysis for MLH1, MSH2, MSH6, and PMS2 antigens showed absence of expression of any MMR proteins in 18 out of 50 clinically diagnosed Lynch syndrome patients (36.0%). There was a significant association between abnormal MMR protein expression with tumor size (p = 0.012), histological differentiation of cancers (p = 0.012), and growth pattern of tumor (p = 0.01). Abnormal expression of MMR protein in colorectal cancers in clinically diagnosed Lynch syndrome patients was associated with specific clinicopathological characteristics such as tumor size, histological differentiation of cancers, and growth pattern of tumor. Immunohistochemical analysis proved to be an advantageous pre-screening tool for Lynch syndrome in Malaysian patients and highly predictive of a germline mutation in DNA MMR genes.     

Molecular characterization of the spectrum of genomic deletions in the mismatch repair genes MSH2, MLH1, MSH6, and PMS2 responsible for hereditary nonpolyposis colorectal cancer (HNPCC)

A systematic search by Southern blot analysis in a cohort of 439 hereditary nonpolyposis colorectal cancer (HNPCC) families for genomic rearrangements in the main mismatch repair (MMR) genes, namely, MSH2, MLH1, MSH6, and PMS2, identified 48 genomic rearrangements causative of this inherited predisposition to colorectal cancer in 68 unrelated kindreds. Twenty-nine of the 48 rearrangements were found in MSH2, 13 in MLH1, 2 in MSH6, and 4 in PMS2. The vast majority were deletions, although one previously described large inversion, an intronic insertion, and a more complex rearrangement also were found. Twenty-four deletion breakpoints have been identified and sequenced in order to determine the underlying recombination mechanisms. Most fall within repetitive sequences, mainly Alu repeats, in agreement with the differential distribution of deletions between the MSH2 and MLH1 genes: the higher number and density of Alu repeats in MSH2 corresponded with a higher incidence of genomic rearrangement at this disease locus when compared with other MMR genes. Long interspersed nuclear element (LINE) repeats, relatively abundant in, for example, MLH1, did not seem to contribute to the genesis of the deletions, presumably because of their older evolutionary age and divergence among individual repeat units when compared with short interspersed nuclear element (SINE) repeats, including Alu repeats. Moreover, Southern blot analysis of the introns and the genomic regions flanking the MMR genes allowed us to detect 6 novel genomic rearrangements that left the coding region of the disease-causing gene intact. These rearrangements comprised 4 deletions upstream of the coding region of MSH2 (3 cases) and MSH6 (1 case), a 2-kb insertion in intron 7 of PMS2, and a small (459-bp) deletion in intron 13 of MLH1. The characterization of these genomic rearrangements underlines the importance of genomic deletions in the etiology of HNPCC and will facilitate the development of PCR-based tests for their detection in diagnostic laboratories.     

PTEN mutational spectra,expression levels,and subcellular localization in microsatellite stable and unstable colorectal cancers

PTEN on 10q23.3 encodes a dual-specificity phosphatase that negatively regulates the phosphoinositol-3-kinase/Akt pathway and mediates cell-cycle arrest and apoptosis. Germline PTEN mutations cause Cowden syndrome and a range of several different hamartoma-tumor syndromes. Hereditary nonpolyposis colon cancer (HNPCC) syndrome is characterized by germline mutations in the mismatch repair (MMR) genes and by microsatellite instability (MSI) in component tumors. Although both colorectal carcinoma and endometrial carcinoma are the most frequent component cancers in HNPCC, only endometrial cancer has been shown to be a minor component of Cowden syndrome. We have demonstrated that somatic inactivation of PTEN is involved in both sporadic endometrial cancers and HNPCC-related endometrial cancers but with different mutational spectra and different relationships to MSI. In the current study, we sought to determine the relationship of PTEN mutation, 10q23 loss of heterozygosity, PTEN expression, and MSI status in colorectal cancers (CRCs). Among 11 HNPCC CRCs, 32 MSI+ sporadic cancers, and 39 MSI- tumors, loss of heterozygosity at 10q23.3 was found in 0%, 8%, and 19%, respectively. Somatic mutations were found in 18% (2 of 11) of the HNPCC CRCs and 13% (4 of 32) of the MSI+ sporadic tumors, but not in MSI- cancers (P = 0.015). All somatic mutations occurred in the two 6(A) coding mononucleotide tracts in PTEN, suggestive of the etiological role of the deficient MMR. Immunohistochemical analysis revealed 31% (14 of 45) of the HNPCC CRCs and 41% (9 of 22) of the MSI+ sporadic tumors with absent or depressed PTEN expression. Approximately 17% (4 of 23) of the MSI- CRCs had decreased PTEN expression, and no MSI- tumor had complete loss of PTEN expression. Among the five HNPCC or MSI+ sporadic CRCs carrying frameshift somatic mutations with immunohistochemistry data, three had lost all PTEN expression, one showed weak PTEN expression levels, and one had mixed tumor cell populations with weak and moderate expression levels. These results suggest that PTEN frameshift mutations in HNPCC and sporadic MSI+ tumors are a consequence of mismatch repair deficiency. Further, hemizygous deletions in MSI- CRCs lead to loss or reduction of PTEN protein levels and contribute to tumor progression. Finally, our data also suggest that epigenetic inactivation of PTEN, including differential subcellular compartmentalization, occurs in CRCs.