hMLH1 Protein Expression Research Articles

Abstract 5227: Suppressive role of elevated estrogen on endometrial carcinogenesis

Abstract Background: Although estrogen has been regarded as a risk factor for endometrial carcinoma, its concentration-dependent carcinogenetic effects remain unclear, because most endometrial carcinomas occur in post-menopausal women, whose serum estrogen levels are relatively low. Because impaired mismatch repair (MMR) was reportedly crucial in the early stages of endometrial carcinogenesis, we focused on the relationship between various estrogen concentration and MMR activity in normal and malignant endometrial glandular cells. In those cells, we reported that the protein and mRNA expression of both hMLH1 and hMSH2, the key factors of MMR, were up-regulated by estrogen stimulation. In addition, we demonstrated that the estrogen stimulation enhanced the MMR activity in those cells using in vitro MMR assay with the M13mp2 phage heteroduplex DNAs and that the proliferating cells without hMLH1/hMSH2 expressions implying “high risk” cells were more frequently observed under relative-low estrogen concentrations. In this study, to further confirm the role of E2 in vivo, we employed a mouse endometrial carcinoma model induced by intra-uterine injection of a carcinogen, N-methyl-N-nitrosourea (MNU) under various E2 concentrations. Methods: MNU was injected into the uterine cavity of 29 CD-1 mice at 6 weeks of age, and E2 was administered by pellets or orally. Uteri were removed for histological examinations 24 weeks later, and serum E2 levels were measured. The immunohistochemical expression of MMR proteins in uterine epithelia was investigated. Results: Of 29 mice, 8, 8, 8, and 5 showed atrophic, normal, hyperplastic, and carcinomatous endometria, respectively. The mean E2 levels of each group were 0.2pg/ml, 3.8pg/ml, 190.0pg/ml, and 6.7pg/ml, with significant differences. The expression of the MMR proteins was stronger in mice with elevated E2. Conclusions: Elevated E2 levels preferentially induced endometrial hyperplasia rather than carcinoma, and this may be mediated by MMR proteins. These results indicate that modest E2 is needed, whereas elevated E2 levels are not necessarily advantageous for carcinogenesis, suggesting the importance of low-chronic (un-opposed) estrogen in human endometrial carcinogenesis. Citation Format: Ryoichi Asaka, Tsutomu Miyamoto, Koichi Ida, Hodaka Takeuchi, Hirofumi Ando, Yasushi Yamada, David Hamisi Mvunta, Hisanori Kobara, Hiroyasu Kashima, Tanri Shiozawa. Suppressive role of elevated estrogen on endometrial carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5227.

Association Between hMLH1 Promoter Methylation and Risk of Gastric Cancer: A Meta-Analysis.

Background: Human mutL homolog 1 (hMLH1) is located on chromosome 3q21-23. As a classic tumor suppressor gene, many researchers have studied the association between hMLH1 promoter methylation and gastric cancer, but their conclusions were not always consistent. Therefore, we performed a meta-analysis to make a more integrated and precise estimate of the associations.Method: PubMed, EMBASE, and Cochrane Library were retrieved without language restrictions. Data were analyzed by Review Manager 5.2 and Stata 12.0 software. Odds ratio (OR) with 95% confidence interval (95%CI) was used to assess the statistical associations.Result: A total of 39 studies published before January 20, 2018 were included in this study. The results indicated that the frequency of hMLH1 promoter methylation in gastric cancers was substantially higher than that in non-cancer controls (OR = 7.94, 95%CI = 4.32–14.58, P < 0.001). Furthermore, hMLH1 promoter methylation had considerable associations with lymph node metastasis, microsatellite instability (MSI), and low expression of hMLH1 protein (OR = 1.53, 95%CI = 1.04–2.26, P = 0.03; OR = 15.33, 95%CI = 9.26–25.36, P < 0.001; OR = 37.86, 95%CI = 18.03–79.50, P < 0.001, respectively). No association was found between hMLH1 promoter methylation and Lauren classification or Helicobacter pylori (HP) infection status.Conclusion: The present study provides evidence that promoter methylation of hMLH1 is a major causative event in the occurrence and development of human gastric cancer.

Association between promoter methylation and protein expression of tumor suppressor genes with clinical characteristics in gastric carcinoma and its effect on the biological function of gastric cancer.

e15509 Background: Studies showed that the gene promoter methylation may be the third important mechanisms of gene expression regulation. Abnormal methylation of tumor suppressor gene can be detected in the early stage of tumor, by screening the methylation profile of gastric cancer (GC), it can provide theoretical and experimental evidence for early diagnosis, treatment and prognosis. Methods: Detecte p16, hMLH1 and CDH1 promoter methylation, the p16, hMLH1 and E-cadher expression in GC tissues and its correlation with clinicopathological features and prognosis. Detect methylation of hMLH1 promoter and mRNA expression in GC cell line and normal gastric mucosa cell line. Clone formation assay and Transwell test. Results: The methylation of hMLH1 and CDH1 in GC and adjacent tissues were statistically significant . The methylation of hMLH1 was related to alcohol , CA199 level and Borrman. The methylation of CDH1 was related to the CEA level. The methylation of hMLH1 promoter is related to OS in GC patients. The methylation of p16 and CDH1 was not related to the OS ofGC patients. The GC patients with 0 or 1 gene methylation had a better prognosis than those with 3 or 2 gene methylation. The p16, hMLH1 and E-cadherin protein expression in GC and adjacent tissues were statistically significant. The p16 protein expression was related to tumor size. The hMLH1 protein expression was related to the CA199 level and tumor size. The E-cadherin expression in GC patients with drinking history was lower than ones without drinking history. The p16, hMLH1 and E-cadherin protein expression was not related to the OS of GC patients. There was a negative correlation between hMLH1 gene methylation and its protein expression in GC. The number of clones in the control group and the experimental group were 180 ± 3 and 169 ± 6. The number of cells in the control group and the experimental group were 135 ± 4.08 and 125 ± 4.18. Conclusions: The methylation of multiple genes is associated with the prognosis, which can provide theoretical basis for accurate treatment and prognosis for GC patients.

Prognostic biomarkers for metastatic colorectal cancer

In colorectal cancer (CRC) distant metastases essentially determine the overall survival and prognosis. Biomarkers that correlate with the presence of distant metastases are therefore of great clinical importance for risk stratification and clinical treatment decisions. As part of the habilitation project various prognostic biomarkers were analyzed and correlated with the occurrence of distant metastases and different patterns of distant spread in CRC. It could be demonstrated that CRC with microsatellite instability (MSI), as detected by a loss of hMLH1 protein expression, have a very low risk of distant metastases. In contrast, microsatellite stable (MSS) CRC (with positive hMLH1 expression) with concurrent activation of the Wnt-beta catenin signaling pathway and strong expression of the cancer stem cell marker CD133, exhibit a very high risk for liver metastases. From these observations a two-step immunohistochemical algorithm based on three immunohistochemical stains could be derived, allowing CRC to be classified according to the risk of distant metastases. Further studies demonstrated that a deregulation of the Wnt-beta catenin signaling pathway and the expression of cancer stem cell markers, such as CD133 and CD44 correlated with hematogenous metastasis to the liver but not with peritoneal carcinomatosis or hematogenous metastasis to the central nervous system (CNS). Finally, in CRC patients with CNS metastases, increased rates of mutations in the mitogen-activated protein kinases (MAPK) pathway (KRAS and BRAF mutations) in combination with a low beta catenin expression could be detected. It can be concluded from these results that for CRC with different patterns of distant spread alternative molecular mechanisms must play a role.

Link between immunoexpression of hMLH1 and hMSH2 proteins and clinical-epidemiological aspects of actinic cheilitis.

BackgroundThe studies found in the literature associate the immunoexpression of hMLH1 and hMSH2 proteins with histologic aspects, but do not correlate it with clinical and epidemiological data.ObjectiveTo evaluate the immunoexpression of hMLH1 and hMSH2 in actinic cheilitis, correlating it with clinical characteristics.MethodsWe analyzed 40 cases. Histological and immunohistochemical analyses were performed. The following clinical variables were evaluated: gender, age range, ethnicity, clinical aspect and occupational sunlight exposure. Statistical evaluation included the Student t-test, while the significance level was set at 5%.ResultsGreater immunoexpression of hMLH1 and hMSH2 was observed in females, individuals aged over 40, and mixed-race/black patients. Furthermore, the immunoexpression of these proteins was greater in actinic cheilitis with a white-colored appearance and in patients without occupational sunlight exposure. No statistical differences were observed for the variables studied.ConclusionThis study uncovered variations of hMLH1 and hMSH2 protein expression upon evaluation of clinical aspects in actinic cheilitis.

The clinicopathological significance of &lt;em&gt;hMLH1&lt;/em&gt; hypermethylation in non-small-cell lung cancer: a meta-analysis and literature review

The hMLH1 gene plays an essential role in DNA repair. Methylation of the hMLH1 gene is common in many types of cancer and can lead to the loss of hMLH1 expression. However, the association and clinicopathological significance between hMLH1 promoter hypermethylation and non-small-cell lung cancer (NSCLC) is elusive. Here, we investigated the correlation of hMLH1 promoter hypermethylation and NSCLC using 13 studies by comprising 1,056 lung cancer patients via a meta-analysis. We observed that 1) loss of hMLH1 protein expression was significantly associated with its promoter hypermethylation, 2) hMLH1 gene inactivation through hypermethylation contributed to the tumorigenesis of NSCLC, which could be a decisive factor for the pathogenesis of NSCLC due to its high occurrence in NSCLC tissues compared to normal lung tissues, 3) a correlation exists between histologic subtypes/disease stages (TNM I+II vs III+IV) and hypermethylation status of hMLH1 gene, and 4) NSCLC patients with hMLH1 hypermethylation and subsequent low expression levels of hMLH1 have a short overall survival period than those patients with normal expression of hMLH1 gene. hMLH1 mRNA predicts patient survival in lung cancer, and this was confirmed by using a public database. We then discussed the tumor suppressor function of hMLH1 and the clinicopathological significance of hMLH1 in NSCLC. We concluded that hMLH1 hypermethylation should be an early diagnostic marker for NSCLC and also a prognostic index for NSCLC. hMLH1 is an interesting therapeutic target in human lung cancers.

Reappraisal of hMLH1 promoter methylation and protein expression status in the serrated neoplasia pathway.

The aim of this study was to determine whether human mutL homologue 1 (hMLH1) inactivation precedes the progression of sessile serrated lesion (SSL) into SSL with cytological dysplasia (SSL/D) and to define the histological stage at which promoter methylation and inactivation of hMLH1 occur. Using the MassARRAY EpiTYPER assay and immunohistochemistry, we examined methylation levels and the protein expression status of hMLH1 in 33 SSL/Ds with conventional epithelial dysplasia and compared the results with those of control hyperplastic polyps (HPs) and SSLs. The methylation level of hMLH1 was higher in the dysplastic component than in the non-dysplastic component of SSL/Ds (P = 0.005), and differed significantly with regard to the degree of dysplasia (P = 0.002). The methylation levels of hMLH1 in the dysplastic component of SSL/Ds tended to be higher than those of control SSLs and HPs (P = 0.063 and P = 0.017, respectively). The loss of hMLH1 protein expression was identified in only 13 of 33 (39.39%) dysplastic components of SSL/Ds. Promoter methylation and loss of protein expression of hMLH1 are not parallel processes that occur concurrently. hMLH1 methylation is an early molecular event which occurs even in HP. However, the loss of hMLH1 expression is a much later step, found in approximately 40% of SSL/Ds at various histological stages. Notably, the loss of hMLH1 protein expression does not necessarily precede the development of cytological dysplasia in SSL.

In vitro study of human mutL homolog 1 hypermethylation in inducing drug resistance of esophageal carcinoma.

Aberrant promoter methylation of tumor suppressor gene can inhibit corresponding protein expression and promote carcinogenesis. Many studies have demonstrated that human mutL homolog 1(hMLH1) promoter methylation is correlated with occurrence and progression of multiple types of tumors. However, its correlation with esophageal carcinoma drug resistance is still unknown. To confirm methylation status of hMLH1 promoter in drug-resistance cell line of esophageal carcinoma, further confirm whether hMLH1 promoter methylation is responsible for drug resistance. Two stable esophageal carcinoma drug-resistance cell lines were successfully established by Cisplatin (DDP) concentration increment method; methylation status of hMLH1 promoter, mRNA and protein expression of hMLH1 were detected by methylation-specific PCR (MSP), RT-PCR and western blot, respectively; Drug-resistance ability assay was used to detect drug-resistance ability. Stronger methylation status of hMLH1 promoter, lower hMLH1 mRNA and protein expression were found in both drug-resistance cell lines; after removing methylated bands using 5-aza-2'-deoxycytidine(5-Aza-CdR) in drug-resistance cell lines, hMLH1 mRNA and protein expression were restored and drug-resistance abilities declined nearly by half. hMLH1 promoter hypermethylation plays important roles in esophageal carcinoma drug-resistance and show us the prospect that combination of demethylation treatment with conventional chemotherapy drugs may bring better therapy effect.

Germline mutation analysis, microsatellite instability and clinical pathology in Hazakh in China hereditary nonpolyposis colorectal cancer pedigree

Objective To study the significance of mismatch repair (MMR) gene and microsatellite instability (MSI) in Hazakh in China hereditary nonpolyposis colorectal cancer (HNPCC) pedigree.Methods The tumor tissues and normal intestine mucosa of the 105 members of this family were immunohistochemically examined for hMLH1 and hMSH2.The mutation of BAT25,BAT26,D2S123,D5S346 and D17S250 were analyzed with polymerase chain reaction-single-stmded conformation polymorphism (PCR-SSCP) in tissue paraffin imbedding in all cases of the family.Results MSI-positive rate of 17tumor in HNPCC families group was 100 ％,in which there are 16 patients (94.1％) with high frequency microsatellite instability (MSI-H),1 patients with low frequency microsatellite instability (MSI-L)(5.9％).The negative expression of hMLH1 or hMSH2 rate of 17 tumor in HNPCC families group was 100％.There are 44 patients (50.0％) in the other 88 members of the HNPCC family with MSI-H.There are 40 patients with hMLH1 or hMSH2 MMR protein expression absence in the 44 patients with MSI-H.Conclusion HNPCC patients with MSI-H and hMLH1 or hMSH2 MMR protein expression absence have a better consistency with earlier age of onset,right colon cancer incidence and the occurrence of poorly differentiated carcinoma. Key words: Hereditary nonpolyposis colorectal cancer; Microsatellite instability; Mismatch repair gene; Hazakh

Promoter methylation and immunohistochemical expression of hMLH1 and hMSH2 in sporadic colorectal cancer: a study from India

To determine the etiological factors of human colorectal cancer (CRC) we assessed the frequency and prognostic significance of hMLH1 and hMSH2 genes in conjunction with hMLH1 and hMSH2 protein expression in 30 Indian CRC patients. The protein expression and promoter methylation of hMLH1 and hMSH2; Mismatch Repair genes (MMR) were analyzed by immunohistochemistry and methylation-specific PCR (MSP), respectively. A loss of hMLH1 expression was recognized in 4(13.3%) and loss of hMSH2 expression was recognized in 2(6.6%) of 30 CRC cases whereas 50% tumors showed reduced expression of hMLH1 and 33.3% showed reduced expression of hMSH2 protein. One tumor showed a loss of both hMLH1 and hMSH2 expression. Normal nuclear staining pattern of hMLH1 and hMSH2 was observed in almost all the adjoining and normal mucosa. Promoter hypermethylation of the hMLH1 gene was detected in 15 of 30 CRC cases (50%) and of hMSH2 gene was only in 3 of 30 CRC cases (10%). No promoter methylation of hMLH1 and hMSH2 genes was observed in adjoining and normal mucosa. Combination of methylation of hMLH1 and hMSH2 gene was observed in two tumors (6.6%). A significant correlation between histological grade of the tumor, methylation and expression of hMLH1 gene (p < 0.05) was observed. Normal expression of hMLH1 and hMSH2 was seen in all of the unmethylated tumors (100%). Nuclear staining and promoter methylation of hMLH1 and hMSH2 did not significantly influence survival. hMLH1 methylation was common and was significantly correlated with loss of hMLH1 protein expression. In contrast, hMSH2 methylation was infrequent. These findings suggest that the inactivation of MMR gene expression probably via hypermethylation may lead to inactivation of their functions which finally leads to tumor aggressiveness and the immunostaining of hMLH1 protein can be used as a prognostic factor for determining the grade of the tumor.

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

MicroRNA-31-5p modulates cell cycle by targeting human mutL homolog 1 in human cancer cells

MicroRNAs (miRNAs) and DNA mismatch repair (MMR) have been linked to human cancer progression. Human mutL homolog 1 (hMLH1), one of the core MMR genes, defects in lung cancer development. However, the interaction between miRNAs and MMR genes and their regulatory effect on cell cycle remain poorly understood. In this study, we investigated the role of miR-31-5p in hMLH1 gene expression and the effect of miR-31-5p on cell cycle in non-small cell lung cancer (NSCLC). We found that miR-31-5p was inversely correlated with hMLH1 expression in NSCLC cell lines and hMLH1 was a direct target of miR-31-5p. Knockdown of miR-31-5p induced a cell cycle arrest at G2/M phase and increased hMLH1 protein expression in NSCLC cells. Conversely, overexpression of miR-31-5p significantly induced cell cycle arrest at S phase and decreased hMLH1 protein expression. Furthermore, knockdown of hMLH1 upregulated miR-31-5p expression and caused cell cycle arrest at S phase. Data from this study revealed that miR-31-5p modulates cell cycle by targeting hMLH1 protein at the posttranscriptional level in NSCLC, which may represent a novel therapy strategy for lung cancer by targeting miR-31-5p.

Expression of hMLH1 in rectal intraepithelial neoplasm and early rectal carcinoma

To explore whether the abnormality of hMLH1 gene may be an early event of carcinogenesis in rectal carcinoma, and to evaluate the diagnostic value in differentiation between intraepithelial neoplasm and early stage of colorectal carcinoma. The expression of hMLH1 protein in 28 cases with early invasive rectal carcinoma(EIRC), 36 cases with rectal intraepithelial neoplasm(RIEN), and 30 cases with normal rectal mucosa(NRM) which were collected through surgical operations were detected by PV-9000 immunohistochemical method. The positive expression rates of hMLH1 protein were 100%(30/30), 77.8%(28/36), and 39.3%(11/28) in NRM, RIEN, and EIRC respectively. The difference was statistically significant between RIEN and EIRC(P=0.002), and the difference was also statistically significant between RIEN and NRM(P=0.006). The positive expression of hMLH1 was not related to age, gender, tumor maximum diameter, dysplasia, tumor types, and distance from the anal verge in RIEV group(P>0.05). In EIRC group, hMLH1 was associated with tumor differentiation(P<0.05). hMLH1 gene deletion may be an early event during carcinogenesis of rectal carcinoma, which may be useful in differentiation of intraepithelial neoplasm from early rectal carcinoma.

HMLH1 gene promoter methylation in joint cartilage in patients with osteoarthritis

To investigate the role of Human MutL homologue 1 (hMLH1) gene promoter methylation in the occurrence and development of osteoarthritis (OA). General DNA was dealt with sodium bisulfite. The methylation of hMLH1 promoter was detected by methylation-specific PCR (MSP). hMLH1 protein expression in joint cartilage was detected by immunohistochemical method. The positive percent of hMLH1 promoter methylation in OA patients was higher than that in healthy persons (χ(2)=30.634, P<0.001); the positive percent of hMLH1 protein in OA patients was significantly lower than that in healthy persons (χ(2)=37.724, P<0.001); prmoter methylation and protein expression level of hMLH1 gene showed negative correlation (rs=-0.554, P<0.001). hMLH1 promoter is hypermethlated in joint cartilage cells of OA patients. Hypermethylation may affect the protein expression of hMLH1, which might play a role in the occurrence and development of OA.

Case-Case Study of Factors Associated to hMLH1, hMSH2, and hMSH6 Protein Expression Among Endometrial Cancer Patients of the University District Hospital of San Juan, Puerto Rico

Lynch syndrome (LS) is an autosomal dominant disorder caused by DNA mismatch repair (MMR) system deficiencies. Women affected by LS present a 40% to 60% lifetime risk of endometrial cancer (EC). This case-case study aims to determine the frequency of the hMLH1, hMSH2, and hMSH6 MMR proteins and the factors (age, family history of cancer [FHC] related to LS, and body mass index [BMI]) associated to their absence in EC patients attending the University District Hospital of San Juan, Puerto Rico. Twenty cases were preliminary evaluated for the MMR protein expression by immunohistochemistry testing and classified as positive cases (presence of protein) or negative cases (absence of protein). The statistical analysis was based on the logistic regression model using the maximum likelihood estimation (MLE). The Bayesian approach was used to determine the posterior probability (posterior Pr[odds ratio {OR} > 1]). Results showed absence for at least 1 MMR protein in 25% of the cases, 15% for hMLH1, and 10% for hMSH2. None of the cases showed an absence for hMSH6. The MLE demonstrated that women diagnosed with EC before the age of 50 (OR: 12.4; 95% confidence interval [CI] = 0.5-322.7), having FHC related to LS (OR: 17.7; 95% CI = 0.6-534.6), and having lower BMI (OR: 2.38; 95% CI = 0.39-14.28) present higher odds than their counterparts of lacking an MMR protein, once adjusted for potential predictors (P > 0.05). The posterior probability that an excess risk of lacking an MMR protein occurs was 95% or greater for each predictor. Our study in this Hispanic population supports previous studies in that younger age, FHC, and lower BMI are associated with increased odds of having an absence of MMR protein expression. Further studies with larger sample sizes should be performed.

HMLH1 promoter hypermethylation and MSI status in human endometrial carcinomas with and without metastases

We investigated the methylation status of mismatch repair gene hMLH1 in 80 primary human endometrial carcinomas (ECs) and in 30 metastatic lesions. It was correlated to the expression of hMLH1 protein, microsatellite instability (MSI) of ECs and to the well-known clinico-pathological variables of cancer. The hMLH1 promoter methylation was detected in 24 out of 64 (37.5%) primary ECs but only in one out of 18 (5.6%) metastatic lesions investigated. Promoter hMLH1 hypermethylation was found more often in early stage ECs and was associated with a decrease of hMLH1 protein expression immunohistochemically. An inverse relationship between hMLH1 expression and clinical stage of the disease was found (p=0.048). Interestingly, there was a significant correlation between MSI and hMLH1 protein expression level (p=0.042). MSI phenotype was found more often in EC metastases compared to the primary tumors (66.7% vs 29.3%; p=0.039). However, neither hMLH1 promoter hypermethylation nor MSI was independent predictive factors for patient's outcome. Using an in vitro model we showed that hMLH1 methylation is reversible. These data showed that hMLH1 methylation with a consequent protein decrease occurred early during EC tumorigenesis and may cause a MSI phenotype, which occurs relatively late. MSI may be an important mechanism supporting further the tumor progression. These findings may have importance for the specific chemosensitization of the primary tumors/metastases and can improve our understanding of endometrial carcinogenesis in humans.

Predictive value of MGMT, hMLH1, hMSH2 and BRCA1 protein expression for pathological complete response to neoadjuvant chemotherapy in basal-like breast cancer patients

To evaluate the importance of biological markers to predict pathologic complete response (pCR) to neoadjuvant chemotherapy (NACT) in patients with locally advanced basal-like breast cancers (BLBCs). Thirty-two BLBC patients receiving NACT with an anthracycline-based regimen plus taxane were included in this study. The immunoreactivities of MGMT, MLH1, MSH2 and BRCA1 before and after NACT were evaluated. A pCR was obtained in 10 of 32 cases (31%). Cancer-related (P = 0.013) and disease-free (P = 0.023) survival rates were significantly higher in the pCR group than in the non-pCR group. In biopsy samples before NACT, attenuated expression of MGMT, MLH1, MSH2 and BRCA1 was observed in 12/32 (38%), 0/32 (0%), 5/32 (16%) and 28/32 (88%) cases, respectively. On evaluation of pCR, patients' characteristics (patients' age, menopausal status, or clinical and pathological stages) and immunohistochemical patterns, attenuated expression of MGMT was only found to be significantly predictive of a pCR (P = 0.018). Paired biopsy sample before NACT and a surgical tumor material after NACT were available for 19 cases of non-pCR. In these cases, decrease in expression during NACT were more frequently observed for MGMT as compared to MLH1, MSH2 or BRCA1 (P = 0.021). MGMT status is a predictive factor for pCR with neoadjuvant anthracycline-based plus taxane combination chemotherapy, which may be helpful in the selection of appropriate NACT for Japanese patients with BLBC.

Aberrant Methylation of Different DNA Repair Genes Demonstrates Distinct Prognostic Value for Esophageal Cancer

DNA mismatch repair (MMR) deficiency results in a strong mutator phenotype and high-frequency microsatellite instability (MSI-H), which are the hallmarks of many tumors. The objective of this study is to investigate the promoter CpG island methylation status of mismatch repair genes human mutL homolog 1 (hMLH1), human mutS homolog 2 (hMSH2), and O(6)-methylguanine-DNA methyltransferase (MGMT) in esophageal squamous cell carcinoma (ESCC) and its roles in alkylating agents chemotherapy. Real-time methylation-specific polymerase chain reaction (PCR) (real-time MSP) was employed to detect promoter CpG island methylation of the hMLH1, hMSH2, as well as MGMT genes in 235 surgical tumor tissue samples from ESCC patients and their corresponding normal tissue samples. Promoter CpG island methylation of hMLH1, hMSH2, and MGMT were detectable in 43.4, 28.9, and 40.4% of ESCC tumor DNA, respectively, and the loss rates of hMLH1, hMSH2, and MGMT protein expression were 48.6, 34.5, and 40.9% in tumor tissues, respectively. For the entire population of 235 ESCC patients who were enrolled in operating treatment combined with radiotherapy and chemotherapy with alkylating agents, there was a significant difference in the overall survival between patients with methylated MGMT promoter and those with an unmethylated MGMT promoter (P<0.05). Promoter CpG island methylation may be a frequent event in ESCC carcinogenesis. Detection of the methylated sequences of hMLH1, hMSH2, and MGMT appears to be promising as a predictive factor in primary ESCC.

Clinical implications of mismatched repair gene promoter methylation in pancreatic cancer

To investigate the relationship between the hypermethylation statuses of the mismatch repair genes (hMLH1 and hMSH2) promoters and explore the correlation between it and the development of pancreatic cancer and the biological behavior of pancreatic cancer. We selected 90 patients who were diagnosed with pancreatic cancer and underwent radical operations in the First Affiliated Hospital of the Dalian Medical University between January 2002 and June 2008. The methylation status of the hMLH1 and hMSH2 promoters was detected by methylation-specific polymerase chain reaction (MSP). Meanwhile, the expression of hMLH1 and hMSH2 protein was detected by Western blot and immunohistochemistry, and its correlation with biological behavior of pancreatic cancer was explored. Of the 90 cases, hMLH1 promoter methylation was detected in 54 (60.0%) patients, while none of the paracancerous tissues indicated methylation. In the study, the hMLH1-methylated tumors lost hMLH1 protein expression, but the non-hMLH1-methylated tumors were not seen to have a loss of hMLH1 protein expression (P < 0.001). On the other hand, there were four cases of hMSH2 promoter methylation. However, no significant difference was found between hMSH2 promoter methylation and non-hMSH2 promoter methylation cases (P > 0.05). After universal analysis, hMLH1 expression was significantly related to tumor differentiation, lymph node metastasis, and tumor location (P < 0.05) while not related to the age, sex, and tumor size (P > 0.05). Our study suggests that the mismatch repair genes play an important role in pancreatic cancer carcinogenesis and progression through epigenetic modification, and it may be regarded as a potential target for the management of pancreatic cancer.

Epigenetic inactivation of hMLH1 in the malignant transformation of ovarian endometriosis

To investigate the role of epigenetic inactivation of hMLH1 during the malignant transformation of ovarian endometriosis (EMs), and to explore the relationship between the epigenetic inactivation of hMLH1 in eutopic endometria and the malignant transformation of ovarian EMs. The target tissue from 29 cases of the endometriosis-associated ovarian carcinoma (EAOC) group, 20 cases of EMs group and 16 cases of control endometrium (CEs) group was obtained by laser capture microdissection (LCM). The methylation statue of hMLH1 promoter was determined by methylation-specific PCR (MSP) and the protein expression of hMLH1 was analysed by immunohistochemistry. The frequency of promoter hypermethylation of hMLH1 in the neoplastic tissue or ectopic endometria of the EAOC group was higher than that of the EMs group (p<0.05), and the frequency of promoter hypermethylation of hMLH1 in eutopic endometria of the EAOC group was higher than that of the EMs and CEs groups (p<0.05). In addition, the protein expression of hMLH1 in eutopic endometria of the EAOC group was lower than that of the EMs and CEs group (p<0.05), and absence of hMLH1 protein expression was significantly correlated with promoter hypermethylation of the gene. Epigenetic inactivation of hMLH1 was an early event in the malignant transformation of ovarian EMs. Epigenetic inactivation of hMLH1 in eutopic endometria was synchronous with that in ectopic endometria and the epigenetic inactivation of hMLH1 in eutopic endometria of EMs might be a potential molecular tool for early diagnosis of the malignant transformation of ovarian EMs.