hMLH1 Mutations Research Articles

A novel germline mutation in hMLH1 in three Korean women with endometrial cancer in a family of Lynch syndrome: case report and literature review

BackgroundEndometrial cancer is often the sentinel cancer in women with Lynch syndrome, among which endometrioid endometrial cancer is the most common. We found a Korean case of uterine carcinosarcoma associated with Lynch syndrome. And we reviewed 27 Korean women with endometrial cancer associated with Lynch syndrome already released in case report so far.Case presentationThe proband, a 45-year-old Korean woman received treatment for endometrioid adenocarcinoma. Her older sister and niece were treated for endometrioid adenocarcinoma and carcinosarcoma, respectively. Family history met the Amsterdam II criteria and immunohistochemical analysis revealed a loss of MLH1 and PMS2. They all harbored a previously unreported germline likely pathogenic variant in c.1367delC in MLH1. They underwent staging operations including total hysterectomy, bilateral salpingo-oophorectomy, pelvic/paraaortic lymph node dissection, and washing cytology. All three women were healthy without evidence of relapse for over 4 years.ConclusionThis report indicates a novel germline c.1367delC variant in MLH1, and presents a Korean case of uterine carcinosarcoma associated with Lynch syndrome. Furthermore, the c.1757_1758insC variant in MLH1 was suggested as a founder mutation in Lynch syndrome in Korean women.

O3-17-1 - Importance to clarify germline hMLH1 K618A missense mutation for clinical surveillance and genetic counseling

Abstract Colorectal cancer (CRC) is the third common cancer and the fourth leading cause of cancer death worldwide. Subproportion CRC with defect function in one of the mismatch repair gene (MMR) are classed as inherited non-polyposis colorectal cancer (HNPCC) later called Lynch syndrome (LS). Disease causing germline mutations have so far been identified in five MMR genes. Among other MMR genes, germline mutations in hMLH1 account up to 50% LS patients and lead to increased risk of cancer development. hMLH1 K618A variant was first identified in our lab and was considered as a disease causing pathogenic alteration, our further investigation also support our previous findings. Although this variant has been investigated intensively during past decade, however the pathogenicity still remain unclear due to controversial data published up to date. In present study, hMLH1 K618A variant were genotyped in 2,743 individuals (1,087 sporadic CRC patients, 239 CRC patients with at least one affected close relative, 69 CRC cases with familial history and 1,348 controls) by TaqMan genotyping assay. Our results demonstrate that 30 individuals were heterozygous for the K618A variant; thirteen normal control had this variants and measurement of allele frequency was 0.96 % which is remarkably higher than previous published data, while eight were in sporadic CRC cases and allele frequency was 0.73 %, six were in CRC patients with at least one affected relative and allele frequency was 2.51 %, three were in CRC patients with familial history and allele frequency was 4.34 %. We conclude that hMLH1 K618A variant may not have effect in sporadic CRC development, in contrast that K618A may significantly increase CRC risk in CRC patients with strong familial history in Swedish population.

Pushing the haystack aside for efficient gene targeting in human cells.

Gene targeting, i.e., the disruption of a specific gene via homologous recombination, is one of the key techniques to study gene function. However, researchers have faced the difficulty that, in human cells, the frequency of correct targeted integration is quite low compared to non-targeted (or random) integration. Adachi and colleagues investigated the factors that influence the ratio of targeted integration to non-targeted integration.

P1-11-1 - Importance to Clarify Germline Hmlh1 K618A Missense Mutation for Clinical Surveillance and Genetic Counseling

Colorectal cancer (CRC) is the third common cancer and the fourth leading cause of cancer death worldwide. Subproportion CRC with defect function in one of the mismatch repair gene (MMR) are classed as inherited non-polyposis colorectal cancer (HNPCC) later called Lynch syndrome (LS). Disease causing germline mutations have so far been identified in five MMR genes. Among other MMR genes, germline mutations in hMLH1 account up to 50% LS patients and lead to increased risk of cancer development. hMLH1 K618A variant was first identified in our lab and was considered as a disease causing pathogenic alteration, our further investigation also support our previous findings. Although this variant has been investigated intensively during past decade, however the pathogenicity still remain unclear due to controversial data published up to date.In present study, hMLH1 K618A variant were genotyped in 2,743 individuals (1,087 sporadic CRC patients, 239 CRC patients with at least one affected close relative, 69 CRC cases with familial history and 1,348 controls) by TaqMan genotyping assay. Our results demonstrate that 30 individuals were heterozygous for the K618A variant; thirteen normal control had this variants and measurement of allele frequency was 0.96 % which is remarkably higher than previous published data, while eight were in sporadic CRC cases and allele frequency was 0.73 %, six were in CRC patients with at least one affected relative and allele frequency was 2.51 %, three were in CRC patients with familial history and allele frequency was 4.34 %. We conclude that hMLH1 K618A variant may not have effect in sporadic CRC development, in contrast that K618A may significantly increase CRC risk in CRC patients with strong familial history in Swedish population.

Clinicopathological and genetic features of Chinese hereditary nonpolyposis colorectal cancer (HNPCC)

The aim of this study was to investigate the clinical value of different criteria and to understand the relationship between genotype and phenotype in Chinese hereditary nonpolyposis colorectal cancer (HNPCC). A total of 116 unrelated probands of suspected HNPCC families from the Fudan Colorectal Registry were studied. A total of 32, 28, and 56 families fulfilled the Amsterdam criteria, the Fudan criteria and the revised Bethesda guideline, respectively. Direct DNA sequencing of all exons of hMSH2 and hMLH1 genes were performed on all 116 samples. Mutations and clinicopathological features were compared between the groups. Thirty-two pathological germline mutations were identified. Out of 32 mutations, 16 were located at hMLH1 and 16 at hMSH2. The sensitivity of Amsterdam criteria was 50 %, specificity was 81 %, and Youden’s index was 31 %. The sensitivity of Fudan criteria was 75 %, specificity was 58 %, and Youden’s index was 33 %. Among all the 32 families with mutations, families with hMSH2 mutation had a higher ratio of synchronous and metachronous colon cancers than families with hMLH1 mutation (33 vs. 6 %, P = 0.04). Patients with hMSH2 mutation more frequently harbour synchronous and metachronous colon cancers. Fudan criteria had a little higher sensitivity and accuracy than Amsterdam criteria for identification of Chinese HNPCC.

Epithelial-mesenchymal transition in colorectal cancer tissue of patients with Lynch syndrome

To explore the epithelial-mesenchymal transition (EMT) in tissue from patients with Lynch syndrome, and to interpret biological behaviour of Lynch syndrome. Sixty-eight formalin-fixed and paraffin embedded tissue blocks were analyzed in this study, including tissues from Lynch syndrome (n = 30), sporadic colorectal carcinoma (CRC) (n = 30), and tumor-adjacent tissues (n = 8). Tissue sections were stained for human mutS homolog 2 (hMSH2), human mutL homolog 1 (hMLH1), transforming growth factor-β type II receptor (TGFβRII), E-cadherin, β-catenin, matrix metalloproteinase-7 (MMP-7) and tissue inhibitor of metalloproteinase-2 (TIMP-2) by immunohistochemical staining. Furthermore, clinical data such as age, gender and tumor-node-metastasis stage were also collected retrospectively. The positive expression rates of hMSH2, hMLH1, TGFβRII, E-cadherin, β-catenin, MMP-7 and TIMP-2 were significantly related to the depth of invasion and lymph node metastasis, but not to sex or tumour size or location. The differences in the positive expression rates of hMSH2, hMLH1, TGFβRII, E-cadherin, cytomembrane β-catenin, cytoplasmic β-catenin, MMP-7 and TIMP-2 were significant between sporadic CRC and Lynch syndrome. The expression of hMSH2 had a positive correlation with that of hMLH1 in Lynch syndrome and sporadic CRC. The expression of TGFβRII had a positive correlation with that of hMSH2, hMLH1 and MMP-7, and a negative correlation with that of TIMP-2. The expression of MMP-7 had a negative correlation with that of TIMP-2 in Lynch syndrome and sporadic CRC. The expression of E-cadherin was positively correlated with that of cytomembrane β-catenin. However, the expression of cytomembrane β-catenin was negatively correlated with that of cytoplasmic β-catenin, and the expression of cytoplasmic β-catenin was positively correlated with that of MMP-7. EMT may play an important role in the development and progression of Lynch syndrome. Lynch syndrome was caused by the mutations of mismatch repair genes, mainly hMSH2 and hMLH1, which also beget the mutational inactivation of TGFβRII. Therefore, the colorectal cancer of Lynch syndrome can escape the inhibitory effect of TGFβ1. However, TGFβ1 can up-regulate the expression of MMP-7 and down-regulate the expression of TIMP-2 in tumors by disassembling the E-cadherin/β-catenin complex in the cytomembrane.

Mutation Rates, Spectra, and Genome-Wide Distribution of Spontaneous Mutations in Mismatch Repair Deficient Yeast

DNA mismatch repair is a highly conserved DNA repair pathway. In humans, germline mutations in hMSH2 or hMLH1, key components of mismatch repair, have been associated with Lynch syndrome, a leading cause of inherited cancer mortality. Current estimates of the mutation rate and the mutational spectra in mismatch repair defective cells are primarily limited to a small number of individual reporter loci. Here we use the yeast Saccharomyces cerevisiae to generate a genome-wide view of the rates, spectra, and distribution of mutation in the absence of mismatch repair. We performed mutation accumulation assays and next generation sequencing on 19 strains, including 16 msh2 missense variants implicated in Lynch cancer syndrome. The mutation rate for DNA mismatch repair null strains was approximately 1 mutation per genome per generation, 225-fold greater than the wild-type rate. The mutations were distributed randomly throughout the genome, independent of replication timing. The mutation spectra included insertions/deletions at homopolymeric runs (87.7%) and at larger microsatellites (5.9%), as well as transitions (4.5%) and transversions (1.9%). Additionally, repeat regions with proximal repeats are more likely to be mutated. A bias toward deletions at homopolymers and insertions at (AT)n microsatellites suggests a different mechanism for mismatch generation at these sites. Interestingly, 5% of the single base pair substitutions might represent double-slippage events that occurred at the junction of immediately adjacent repeats, resulting in a shift in the repeat boundary. These data suggest a closer scrutiny of tumor suppressors with homopolymeric runs with proximal repeats as the potential drivers of oncogenesis in mismatch repair defective cells.

Mismatch repair gene mutation analysis and colonoscopy surveillance in Chinese lynch syndrome families

Lynch syndrome (or HNPCC) is a colorectal cancer syndrome caused by germline mutations in either one of the DNA mismatch repair (MMR) genes hMLH1, hMSH2, hMSH6 or hPMS2. Mutations in hMLH1 and hMSH2 are most prevalent. Here we aimed to determine the cancer risk of MMR gene mutation carriers and, in addition, the efficacy of colonoscopy surveillance in Chinese Lynch syndrome family members with and without MMR gene mutations. A Lynch syndrome family registry encompassing 106 families in Northern China was recently established. Detailed pedigree data for each family were collected and hMLH1 and hMSH2 gene mutation analyses were performed. Germ-line mutations were identified in probands from 42 of these families, and additional genetic analyses were performed in each member of these 42 families to identify mutation and non-mutation carriers. Among the family members included, 180 received colonoscopy and the remaining cases were followed without colonoscopy. Overall 54.8% of the Lynch syndrome family members carried MMR gene mutations, and these mutation carriers exhibited significantly higher colorectal cancer and other Lynch syndrome-associated cancer risks as compared to non-mutation carriers. The cumulative risk for all Lynch syndrome-related cancers at age 70 was 93.8% for both hMLH1 and hMSH2 mutation carriers, and 81.7% and 93.1% for colorectal cancer at this age, respectively. Whereas 43 of 102 (42.2%) mutation carriers exhibited significant colonoscopy findings, including 10 colorectal cancers, none of 78 non-mutation carriers exhibited significant findings, and no cancers were detected. In addition, in the mutation carriers, colonoscopy surveillance led to the detection of more early stage cancers than in the non-surveillance group (70.0% versus 36.5%, p < 0.01). In Lynch syndrome family members, we recommend pre-symptomatic MMR gene mutation analysis in order to identify high risk individuals for colonoscopy surveillance.

Abstract 4229: Study on the germline mutation and protein expression of hMLH1 and hMSH2 gene in Chinese HNPCC probands.

Abstract BACKGROUND&amp;AIMS: Compare with sporadic colorectal cancer (SCRC), Hereditary nonpolyposis colorectal cancer (HNPCC) shows its own characteristcs associated with the molecular mechanism,clinical features,the methods for treatment and management of HNPCC kindreds. The molecular genetics basis of HNPCC was the mutation of mismatch repair (MMR) gene. Some HNPCC families which can found the mutation of MMR gene didn't fulfil any clinical criteria while more than fifty percent suspected HNPCC patients can't found mutation of MMR gene. Therefore, the tumorigenesis of HNPCC must be associated with other disease genes which have not been credited. The expression of hMLH1 and hMSH2 protein in 50 HNPCC probands fulfilling defferent clinical criteria and the germline mutation of hMLH1 and hMSH2 gene in 26 probands were analysed. Methods: The peripheral blood was collected from the probands of 26 HNPCC families fulfilling different clinical criterial, of which 7 families fulfilled AC, 19 kindreds fit BG. Genomic DNA was extracted following the manafacturer's protocol and used as the template to amplify 35 exons of the 2 genes by PCR. PCR products were purified and used as the template for direct DNA sequencing. The results of sequencing were analysed by different bioanalysis software. To further investigate the pathological effects of detected missense mutations, we analyse the same exon of hPMS2 gene using PCR-based sequencing in 130 healthy persons without family history. IHC envision two step method was performed.Results: 8 germline mutation of hMLH1 and hMSH2 was found in 26 HNPCC probands families. 4 fulfilled AC and 4 fulfilled BG..17 probands was found to be hMLH1 and hMSH2 protein deficient. Conclusion: The rate of germline mutation and protein expression was 30.8% and 34%. AC are the most sensitve clinical creteria to predict mutations. Immunohistochemical staining are reliable screening method with high predictive value for the detection of mutation. Some suspected HNPCC patients can't found mutation of MMR gene. Citation Format: Xia Sheng. Study on the germline mutation and protein expression of hMLH1 and hMSH2 gene in Chinese HNPCC probands. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4229. doi:10.1158/1538-7445.AM2013-4229

Novel DNA Variants and Mutation Frequencies of hMLH1 and hMSH2 Genes in Colorectal Cancer in the Northeast China Population

Research on hMLH1 and hMSH2 mutations tend to focus on Lynch syndrome (LS) and LS-like colorectal cancer (CRC). No studies to date have assessed the role of hMLH1 and hMSH2 genes in mass sporadic CRC (without preselection by MSI or early age of onset). We aimed to identify novel hMLH1 and hMSH2 DNA variants, to determine the mutation frequencies and sites in both sporadic and LS CRC and their relationships with clinicopathological characteristics of CRC in Northeast of China. 452 sporadic and 21 LS CRC patients were screened for germline and somatic mutations in hMLH1 and hMSH2 genes with PCR–SSCP sequencing. We identified 11 hMLH1 and seven hMSH2 DNA variants in our study cohort. Six of them were novel: four in hMLH1 gene (IVS8-16 A>T, c.644 GAT>GTT, c.1529 CAG>CGG and c.1831 ATT>TTT) and two in hMSH2 gene (−39 C>T, insertion AACAACA at c.1127 and deletion AAG at c.1129). In sporadic CRC, germline and somatic mutation frequencies of hMLH1/hMSH2 gene were 15.59% and 17.54%, respectively (p = 0.52). Germline mutations present in hMLH1 and hMSH2 genes were 5.28% and 10.78%, respectively (p<0.01). Somatic mutations in hMLH1 and hMSH2 genes were 6.73% and 11.70%, respectively (p = 0.02). In LS CRC, both germline and somatic mutation frequencies of hMLH1/hMSH2 gene were 28.57%. The most prevalent germline mutation site in hMSH2 gene was c.1168 CTT>TTT (3.90%), a polymorphism. Somatic mutation frequency of hMLH1/hMSH2 gene was significantly different in proximal, distal colon and rectal cancer (p = 0.03). Our findings elucidate the mutation spectrum and frequency of hMLH1 and hMSH2 genes in sporadic and LS CRC, and their relationships with clinicopathological characteristics of CRC.

Prevalence of Pathological Germline Mutations of hMLH1 and hMSH2 Genes in Colorectal Cancer

The prevalence of pathological germline mutations in colorectal cancer has been widely studied, as germline mutations in the DNA mismatch repair genes hMLH1 and hMSH2 confer a high risk of colorectal cancer. However, because the sample size and population of previous studies are very different from each other, the conclusions still remain controversial. In this paper, Databases such as PubMed were applied to search for related papers. The data were imported into Comprehensive Meta-Analysis V2, which was used to estimate the weighted prevalence of hMLH1 and hMSH2 pathological mutations and compare the differences of prevalence among different family histories, ethnicities and related factors. This study collected and utilized data from 102 papers. In the Amsterdam-criteria positive group, the prevalence of pathological germline mutations of the hMLH1 and hMSH2 genes was 28.55% (95%CI 26.04%–31.19%) and 19.41% (95%CI 15.88%–23.51%), respectively, and the prevalence of germline mutations in hMLH1/hMSH2 was 15.44%/10.02%, 20.43%/13.26% and 15.43%/11.70% in Asian, American multiethnic and European/Australian populations, respectively. Substitution mutations accounted for the largest proportion of germline mutations (hMLH1: 52.34%, hMSH2: 43.25%). The total prevalence of mutations of hMLH1 and hMSH2 in Amsterdam-criteria positive, Amsterdam-criteria negative and sporadic colorectal cancers was around 45%, 25% and 15%, respectively, and there were no obvious differences in the prevalence of germline mutations among different ethnicities.

Irrelevance of microsatellite instability in the epidemiology of sporadic pancreatic ductal adenocarcinoma.

Background and AimsPancreatic cancer risk is increased in Lynch syndrome (LS) patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI). However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS.MethodsMS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27) in 338 consecutive pancreatic ductal adenocarcinoma (PDAC), resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers.ResultsOnly one PDAC (0.3%) showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5%) LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one.ConclusionsMSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers.

Relationship of lower uterine segment cancer with Lynch syndrome: A novel case with an hMLH1 germline mutation

Lynch syndrome is a genetic disease that often develops in patients with endometrial cancer and is caused by abnormal DNA mismatch repair (MMR) genes. In the United States, it was recently reported that the prevalence of Lynch syndrome with an hMSH2 mutation in patients with endometrial cancer in the lower uterine segment (LUS) is much greater than that in patients with endometrial cancer, although no such reports have been published in Asia. In this study, we examined the correlation between endometrial cancer in LUS and abnormalities in MMR genes. We examined 625 patients, who were diagnosed with endometrial cancer and underwent a hysterectomy. Nine patients (1.4%) had cancer based on pathological confirmation of a tumor in the lower part of the uterus and no cancer in the upper part. These cases were compared with 27 cases of sporadic endometrial (non-LUS) cancer. The age and BMI of the patients with LUS cancer were significantly lower than those of the patients with non-LUS cancer. No differences were observed in the pathological characteristics. The microsatellite instability (MSI)-positive rates were similar. Immunohistochemistry showed a decreased expression of hMLH1 and hMSH6 in patients with LUS cancer. In contrast with earlier reports from the United States, hMSH2 was expressed in all the cases. Of the 2 patients with LUS cancer who exhibited high MSI, 1 patient showed abnormal methylation of hMLH1, while the other patient was diagnosed with Lynch syndrome with a mutation in the hMLH1 gene. This is the second report on the relationship of LUS cancer and Lynch syndrome, and the first to describe an Asian patient with LUS cancer with Lynch syndrome induced by an hMLH1 mutation.

Microsatellite instability in young patients with sporadic colorectal adenomas.

This study was designed to determine the prevalence of microsatellite instability (MSI) among colorectal adenomas detected in patients ≤ 40 years of age and to compare the prevalence of MSI in young (≤ 40 years) and older (>40 years) patients with colorectal adenomas. Additionally, we attempted to identify the underlying cause of MSI in these patients. We prospectively tested for the presence of MSI using five NCI markers in samples from the two patient groups. The frequency of MSI was compared and the underlying causes of MSI were determined by methylation specific PCR and germ-line mutation analysis for mismatch repair genes. The frequency of MSI was higher in the ≤ 40 group than the >40 group (31.4% and 6.4%, respectively, p=0.0004). The MSI-high pattern was also more prevalent in the ≤ 40 group than the >40 group (15.7% and 2.5%, respectively, p=0.014). The hypermethylated hMLH1 gene was demonstrated in 7/8 (87.5%) patients with MSI-high in the ≤ 40 group and in 1/2 (50.0%) patients with MSI-high in the >40 group. No study subject showed a germline mutation of hMLH1 or hMSH2. MSI-high was more frequent in young (≤ 40 years) patients with colorectal adenoma than in older (>40 years) patients. Hypermethylation of the hMLH1 gene appears to be an important cause of MSI-high in these patients.

Distinct mutations in MLH1 and MSH2 genes in Hereditary Non-polyposis Colorectal Cancer (HNPCC) families from China

Hereditary non-polyposis Colorectal Cancer (HNPCC) is an autosomal dominant inheritance syndrome. HNPCC is the most common hereditary variant of colorectal cancer (CRC), which accounts for 2-5% CRCs, mainly due to hMLH1 and hMSH2 mutations that impair DNA repair functions. Our study aimed to identify the patterns of hMSH2 and hMLH1 mutations in Chinese HNPCC patients. Ninety-eight unrelated families from China meeting Amsterdam or Bethesda criteria were included in our study. Germline mutations in MLH1 and MSH2 genes, located in the exons and the splice-site junctions, were screened in the 98 probands by direct sequencing. Eleven mutations were found in ten patients (11%), with six in MLH1 (54.5%) and five in MSH2 (45.5%) genes. One patient had mutations in both MLH1 and MSH2 genes. Three novel mutations in MLH1 gene (c.157_160delGAGG, c.2157dupT and c.-64G>T) were found for the first time, and one suspected hotspot in MSH2 (c.1168C>T) was revealed.

P.1.12: EUS-GUIDED PARTIALLY COVERED WALLFLEX(tm) BILIARY STENT PLACEMENT FOR PALLIATION OF MALIGNANT BILIARY OBSTRUCTION: A PROSPECTIVE STUDY

In 145 CRCs, by multivariate correspondence analysis, we found an association between Vangl2 methylation and BRAF mutation (p=0.0001), functional p53 (p<0.0001), and low IHC expression of hMLH1 (p=0.0141), Cox-2 (p=0.002), E-cadherin (p=0.0019) and cytoplasmic β-catenin (p=0.0019). No association was found with KRAS mutation. Conclusions: Epigenetic loss of Vangl2 occurs in MSI CRC and is associated with other molecular features of this subset of tumors. The association with BRAF mutation suggests that Vangl2 methylation might contribute to Wnt derangement in sporadic MSI CRC.

P.1.13: ASYMPTOMATIC UPPER GASTROINTESTINAL ORGANIC LESIONS IN THE GENERAL POPULATION

In 145 CRCs, by multivariate correspondence analysis, we found an association between Vangl2 methylation and BRAF mutation (p=0.0001), functional p53 (p<0.0001), and low IHC expression of hMLH1 (p=0.0141), Cox-2 (p=0.002), E-cadherin (p=0.0019) and cytoplasmic β-catenin (p=0.0019). No association was found with KRAS mutation. Conclusions: Epigenetic loss of Vangl2 occurs in MSI CRC and is associated with other molecular features of this subset of tumors. The association with BRAF mutation suggests that Vangl2 methylation might contribute to Wnt derangement in sporadic MSI CRC.

Identification and surveillance of 19 Lynch syndrome families in southern Italy: report of six novel germline mutations and a common founder mutation

Lynch syndrome (LS), or hereditary non-polyposis colorectal cancer (HNPCC), is an autosomal dominant condition responsible for early onset cancer mostly in the colonrectum and endometrium as well as in other organ sites. Lynch syndrome is caused by germline mutations in mismatch repair genes, prevalently in hMSH2, hMLH1, and less frequently in hMSH6 and hPMS2. Twenty-nine non-related index cases with colorectal cancer (CRC) were collected from a region in southeast Italy (Apulia). Among this set of patients, fifteen fulfilled the Amsterdam criteria II. The presence of tumor microsatellite instability (MSI) was assessed in all index cases and 19 (15 AC+/4 AC-) were classified as MSI-H. Mutation analysis performed on all patients, identified 15 pathogenic mutations in hMLH1 and 4 in hMSH2. 4/15 mutations in hMLH1 and 2/4 hMSH2 mutations have not been previously reported. Three previously reported mutations were further investigated for the possibility of a common founder effect. Genetic counseling was offered to all probands and extended to 183 relatives after molecular testing and 85 (46%) mutation carriers were identified. Eighty mutation carriers underwent an accurate clinical and instrumental surveillance protocol. Our results confirm that the identification of LS patients based exclusively on family history may miss patients carrying germline mutations in the MMR genes. Moreover, our results demonstrated that molecular screening and subsequent instrumental surveillance are very effective in identifying CRCs at earlier stages and reducing the number of deaths from secondary cancers in HNPCC patients.

Clinicopathologic and genetic features of Chinese hereditary nonpolyposis colorectal cancer (HNPCC).

382 Background: Hereditary non-polyposis colorectal cancer (HNPCC) is the most common hereditary colorectal cancer (CRC). At least five mismatch repair genes, majorly hMLH1and hMSH2 gene mutations, are associated with this syndrome. Given the geographical and racial differences, Chinese HNPCC patients have some special characteristics compared with patients in West countries. Based on our former studies, we developed a criteria, the Fudan Criteria, for clinical diagnosis of Chinese HNPCC. The aim of this study is (1) to understand the relationship between genotype and phenotype in Chinese HNPCC; and (2) to investigate the clinical value of Fudan Criteria. Methods: 116 unrelated probands of HNPCC or suspected HNPCC families from the Fudan Colorectal Registry were studied. 32 families fulfilled the Amsterdam criteria (AC group), 28 families fulfilled the Fudan Criteria (FD group). 56 families met the revised Bethesda guideline (BG Group). Direct DNA sequencing of all exons of hMSH2 and hMLH1 genes were performed. Mutation and clinicalpathological features were compared between the groups. Results: 32 pathological germline mutations were identified. 16/32 mutations were located at hMLH1 and 16/32 located at hMSH2. The sensitivity of Amsterdam Criteria was 50%, specificity was 81% and Youden's index was 31%. The sensitivity of Fudan Criteria was 75%, specificity was 58% and Youden's index was 33%. In AC group, there were no significant clinicopathological differences in patients with or without mutation. However, families with hMSH2 mutation had a higher ratio of synchronous and metachronous CRC than families with hMLH1 mutation (33% vs. 6%; P=0.04). Families in AC group share the same clinicopathological features with families in FD group, except multiple relative tumors (21.6% vs.6%; P=0.001). Conclusions: There seems no close relation between genotype and phenotype, except patients with hMSH2 mutation more frequently harbor synchronous and metachronous CRC. Fudan Criteria had a little higher sensitivity and accuracy than Amsterdam Criteria for identification of Chinese HNPCC. No significant financial relationships to disclose.

788 Investigation of the differentially expressed C-FABP & FABP-pm in human prostate tissues and cell lines: histopathological and molecular biology study

Gastric cancer (GC) remains a leading cause of cancer mortality worldwide. Genetic factors are implicated, including DNA mismatch repair (MMR) deficiency manifested as tumor microsatellite instability (MSI). However, a standardized panel of markers and a definition of low-versus-high level MSI in GC are lacking. We examined a population-based cohort of early onset (≤50 yrs) gastric cancer. We identified 211 cases of early onset gastric cancer in Central-East Ontario from 1989 to 1993, with archival material available for 139 cases. Testing included a six-mononucleotide marker panel and a three-MMR immunohistochemical panel. Overall, 30% (41 of 139) of GC were MSI+, with allelic shifts at one to eight markers. An unexpected discordance between the BAT-25, BAT-26, and BAT-40 markers was observed in the MSI+ cases. Six cases showing multiple loci instability (≥3 markers MSI+/MSI-high) demonstrated MMR protein deficiency. Three novel hMLH1 mutations (two germline frameshift and one somatic nonsense) were also found. The only significant clinicopathological associations were increased tumor size in MSI+ cases (P = 0.04) and Lauren histotype (P = 0.006) and tumor grade (P = 0.007) in MSI-high cases. Tumor size, location, depth, nodal status, and Ming subtype were significant prognostic variables. Therefore, we propose a new definition of high-level MSI based on unifying characteristics of instability of more than or equal to three of six mononucleotide markers and loss of MMR protein expression.