CDH1 Methylation Research Articles

Methylation of HIN-1, RASSF1A, RIL and CDH13 in breast cancer is associated with clinical characteristics, but only RASSF1A methylation is associated with outcome

BackgroundAberrant promoter CpG island hypermethylation is associated with transcriptional silencing. Tumor suppressor genes are the key targets of hypermethylation in breast cancer and therefore may lead to malignancy by deregulation of cell growth and division. Our previous pilot study with pairs of malignant and normal breast tissues identified correlated methylation of two pairs of genes - HIN-1/RASSFIA and RIL/CDH13 - with expression of estrogen receptors (ER), progesterone receptors (PR), and HER2 (HER2). To determine the impact of methylation on clinical outcome, we have conducted a larger study with breast cancers for which time to first recurrence and overall survival are known.MethodsTumors from 193 patients with early stage breast cancer who received no adjuvant systemic therapy were used to analyze methylation levels of RIL, HIN-1, RASSF1A and CDH13 genes for associations with known predictive and prognostic factors and for impact on time to first recurrence and overall survival.ResultsIn this study, we found that ER was associated with RASSF1A methylation (p < 0.001) and HIN-1 methylation (p = 0.002). PR was associated with RIL methylation (p = 0.012), HIN-1 (p = 0.002), and RASSF1A methylation (p = 0.019). Tumor size was associated with RIL and CDH13 methylation (both p = 0.002), and S-phase was associated with RIL methylation (p = 0.036). Only RASSF1A was associated with worse time to first recurrence (p = 0.045) and worse overall survival (p = 0.016) after adjusting for age, tumor size, S-phase, estrogen receptor and progesterone receptor.ConclusionsMethylation of HIN-1, RASSF1A, RIL and CDH13 in breast cancers was associated with clinical characteristics, but only RASSF1A methylation was associated with time to first recurrence and overall survival. Our data suggest that RASSF1A methylation could be a potential prognostic biomarker.

Relationship between Tumor DNA Methylation Status and Patient Characteristics in African-American and European-American Women with Breast Cancer

Aberrant DNA methylation is critical for development and progression of breast cancer. We investigated the association of CpG island methylation in candidate genes and clinicopathological features in 65 African-American (AA) and European-American (EA) breast cancer patients. Quantitative methylation analysis was carried out on bisulfite modified genomic DNA and sequencing (pyrosequencing) for promoter CpG islands of p16, ESR1, RASSF1A, RARβ2, CDH13, HIN1, SFRP1 genes and the LINE1 repetitive element using matched paired non-cancerous and breast tumor specimen (32 AA and 33 EA women). Five of the genes, all known tumor suppressor genes (RASSF1A, RARβ2, CDH13, HIN1 and SFRP1), were found to be frequently hypermethylated in breast tumor tissues but not in the adjacent non-cancerous tissues. Significant differences in the CDH13 methylation status were observed by comparing DNA methylation between AA and EA patients, with more obvious CDH13 methylation differences between the two patient groups in the ER- disease and among young patients (age<50). In addition, we observed associations between CDH13, SFRP1, and RASSF1A methylation and breast cancer subtypes and between SFRP1 methylation and patient's age. Furthermore, tumors that received neoadjuvant therapy tended to have reduced RASSF1A methylation when compared with chemotherapy naïve tumors. Finally, Kaplan Meier survival analysis showed a significant association between methylation at 3 loci (RASSF1A, RARβ2 and CDH13) and reduced overall disease survival. In conclusion, the DNA methylation status of breast tumors was found to be significantly associated with clinicopathological features and race/ethnicity of the patients.

CDH1 methylation in preoperative peritoneal washes is an independent prognostic factor for gastric cancer

Background and ObjectivesTo investigate the clinical value of CDH1 methylation in preoperative peritoneal washes (PPW) from gastric cancer patients.MethodsCDH1 methylation was detected by real-time methylation specific-PCR in tumor tissues and corresponding PPW from 92 gastric cancer patients, gastric mucosa from 40 chronic gastritis patients and 48 normal persons.ResultsCDH1 methylation was found in 75 of 92 (81.5%) gastric cancer tissues, which significantly correlated with size, growth pattern, differentiation, lymphatic invasion, venous invasion, invasion depth, lymph node metastasis, distant metastasis, and TNM stage of tumor (all P < 0.05), but its relationship to age, gender, tumor site, and H. pylori infection was not found (all P > 0.05). The percentage of CDH1 methylation in PPW was 48.9%, of which the Aζ value of ROC curve was 0.8 compared to that in gastric cancer tissues. Kaplan–Meier analysis showed that there was a significant difference in disease-free survival (DFS) between the patients with or without methylated CDH1 in their PPW (χ2 = 109.64, P < 0.000). Cox regression analysis revealed CDH1 methylation in PPW was an independent risk factor for gastric cancer patients, with a remarkable decrease in DFS after postoperative 30 months.ConclusionsMethylated CDH1 in PPW predicts poor prognosis for gastric cancer patients. J. Surg. Oncol. 2012; 106:765–771. © 2012 Wiley Periodicals, Inc.

Abstract 5569: DNA methylation status of MLH1, p16, SOCS1, ER and CDH1 in Puerto Rican patients with acute myeloid leukemia

Abstract Background: DNA methylation of gene promoters have been show to play a role in the pathogenesis of acute myeloid leukemia (AML). We present in this study a preliminary report of the methylation status of MLH1, p16, SOCS1, ER and CDH1 in Puerto Rican patients with AML. Methods: Peripheral blood was obtained from 24 patients with diagnosis of acute myeloid leukemia before and after their induction treatments. DNA was isolated and further modified with sodium bisulfite. Methylation-specific polymerase (MSP) chain reaction was performed to detect aberrant promoter methylation of MLH1, p16, SOCS1, ER and CDH1. Results: Samples from a total of 24 patients were analyzed. The mean age was 47 years (range 22-70) with 21/24 patients younger than 60 years (88%). Most of the patients were female (13/24, 54%). Ten patients had diploid cytogenetics (42%), 4 had low risk cytogenetics (17%) and 10 had high risk cytogenetics (42%). Nineteen patients were de Novo AML (79%). Sixteen patients (67%) presented with WBC counts higher than 10 x 109/L. Seventeen patients (71%) achieved a complete response at the end of the induction therapy. Frequencies of gene methylation before treatment were as follows: MLH: 3/24 (13%); p16: 6/24 (25%); SOCS1:13/24 (54%); ER: 16/24 (67%) and CDH1: 4/24 (16.7%). Conclusions: No correlation between methylation status of p16 and MLH1 before treatment and clinicopathological characteristics was noted. Unmethylated CDH1 was more common among the novo AML patients. Methylation of SOCS1 was more common among patients that achieve a CR after induction therapy. This sample is a small one, so any correlation must be further tested in a more ample sample. Collection of more patient's samples and correlation with clinical data and patient's ultimate outcome is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5569. doi:1538-7445.AM2012-5569

Expression and significance of E-cadherin in adenoid cystic carcinoma of salivary glands

To examine the expression of E-cadherin and the methylation status of CDH1 and explore their clinical significance in salivary adenoid cystic carcinoma (SACC). The expression of E-cadherin was detected by the immunohistochemical method. And the methylation of CDH1 gene promoter 5'-CpG island was analyzed by real-time methylation-specific polymerase chain reaction (real-time MSP) in salivary adenoid cystic carcinoma and normal salivary gland tissue respectively. The expression rate of E-cadherin was lower in SACC than that in normal salivary gland tissue (100.0% vs 55.6%, P < 0.05). And the expression of E-cadherin was associated with different histopathological types, T-stage, nerve invasion, lymphatic and distant metastasis (P < 0.05). However, there was no correlation between the expression of E-cadherin and gender, age and tumor location. Partial methylation of CDH1 was detected in 3 of 30 cases with a positive expression of E-cadherin and full methylation of CDH1 in 23 of 24 cases with a negative expression of E-cadherin. There was a negative correlation between the expression of E-cadherin and the methylation of CDH1 in salivary adenoid cystic carcinoma (r = -0.483, P < 0.001). The down-regulation of E-cadherin, as modulated by the methylation of CDH1, may contribute to nerve invasion, lymphatic and distant metastasis in SACC. Thus it may be used as a biological indicator of malignancy and prognosis.

5-aza-2'-deoxycytidine-induced inhibition of CDH13 expression and its inhibitory effect on methylation status in human colon cancer cells in vitro and on growth of xenograft in nude mice

To determine the inhibitory effect of 5-aza-2'-deoxycytidine (5-Aza-CdR) on the growth of human colon carcinoma cells and xenografts in nude mice, to observe its effect on CDH13 gene expression and methylation in the xenografts, and to explore the possible mechanisms. Human colon carcinoma cell line HCT116 cells were treated with 5-Aza-CdR, and the cell morphology was observe by phase contrast microscopy. The cell growth was assessed by MTT assay. A tumor-bearing mouse model was generated by subcutaneous inoculation of human colon carcinoma HCT116 cells into nude mice. The tumor growth in the nude mice was observed, the CDH13 gene expression and its methylation status in the tumors were detected using methylation specific PCR (MSP), RT-PCR, Western blotting and immunohistochemistry. After treatment with 5-Aza-CdR, the inhibition rate of the growth of cultured HCT116 cells was increased as the concentration was increasing. The growth of the xenografts in nude mice was significantly inhibited, and the methylated CDH13 gene was reactivated. After 4 weeks of 5-Aza-CdR treatment, no significant difference was found between the body weights of nude mice in the 5-Aza-CdR group [(18.06 ± 1.29) g] and control group [(17.07 ± 0.84) g], (P > 0.10), and the average volume of xenografts of the 5-Aza-CdR group was (907.00 ± 87.29) mm(3), significantly smaller than the (1370.93 ± 130.20) mm(3) in the control group (P < 0.005). No expression of CDH13 gene was found in the control group. The expression of CDH13 gene in the 5-Aza-CdR group was increased along with the increasing concentration of 5-Aza-CdR. 5-Aza-CdR inhibits the growth of human colon cancer cells in culture and in nude mice, and induces the cancer cells to re-express CDH13 in nude mice. Its mechanism may be that demethylation of the methylated CDH13 promoter induced by 5-Aza-CdR restores CDH13 expression and thus inhibits the tumor growth in nude mice.

Aberrant Promoter Methylation of CDH13 and MGMT Genes is Associated With Clinicopathologic Characteristics of Primary Non–Small-Cell Lung Carcinoma

IntroductionSystemic methylation changes may be a diagnostic marker for tumor development or prognosis. Here, we investigate the relationship between gene methylation in lung tumors relative to normal lung tissue and whether DNA methylation changes can be detected in paired blood samples. Material and MethodsSixty-five patients were enrolled in a surgical case series of non–small-cell lung carcinoma at a single institution. By using bisulfite pyrosequencing, CpG methylation was quantified at 5 genes (RASSF1A, CDH13, MGMT, ESR1, and DAPK) in lung tumor, pathologically normal lung tissue, and circulating blood from enrolled cases. ResultsThe analyses of methylation in tumors compared with normal lung tissue identified higher methylation of CDH13, RASSF1A, and DAPK genes, whereas ESR1 and MGMT methylation did not differ significantly between these tissue types. We then examined whether the 3 aberrantly methylated genes could be detected in blood. The difference in methylation observed in tumors was not reflected in methylation status of matching blood samples, which indicated a low feasibility of detecting lung cancer by analyzing these genes in a blood-based test. Lastly, we probed whether tumor methylation was associated with clinical and demographic characteristics. Histology and sex were associated with methylation at the CDH13 gene, whereas, stage was associated with methylation at MGMT. ConclusionOur results showed higher methylation of RASSF1A, CDH13, and DAPK genes in lung tumors compared with normal lung. The lack of reflection of these methylation changes in blood samples from patients with non–small-cell lung carcinoma indicates their poor suitability for a screening test.

Final Report of a Randomized Study of Decitabine Versus Conventional Care (CC) for Maintenance Therapy in Patients with Intermediate and High Risk Acute Myeloid Leukemia (AML) in First or Subsequent Complete Remission (CR)

Abstract 1530 Background:Maintenance therapy in acute myeloid leukemia (AML) is not standard of care. It has been reported that continued therapy with cytotoxic agents similar to those used for induction and consolidation is associated with toxicity but can improve disease free survival (DFS). Methylation status of tumor suppressor genes in clinical remission may predict the relapse in AML, with earlier relapse in patients with methylated genes. Therefore, hypomethylating therapy may be effective in maintaining remission and prolonging survival. Methods: We have conducted a clinical trial comparing decitabine to conventional care (CC, low dose subcutaneous cytarabine, prolonged intensive therapy, or observation) in patients (pts) with AML in their first or subsequent complete remission (CR). Pts with non-favorable risk AML (including intermediate and poor risk) received induction therapy followed by appropriate consolidation. They were then stratified by age (£ 60 vs. > 60) and cytogenetics (intermediate vs. poor risk) and randomized to receive decitabine 20 mg/m2 IV daily × 5 every 4 to 8 weeks for 12 cycles, or CC. Pts in second or subsequent CR were randomized after completion of salvage therapy. Primary endpoint was considered to be no relapse at 1 year. We also monitored minimal residual disease (MRD) by multicolor flow cytometry (MFC) using custom staining panels based on the leukemic phenotypes. Aberrant myeloid blasts were identified by comparison to normal bone marrow CD34+ cells. Serial samples for DNA methylation studies were also collected and methylation analysis was performed. Results: 50 patients have been enrolled. Of those, 45 (19 M, 26 F) pts (including 35 in first CR and 10 in subsequent CR) were evaluable for the primary endpoint. Median age was 57 years (range, 24–77). 27 pts were £ 60 years and 18 > 60. Cytogenetics at diagnosis was intermediate in 34 pts, poor-risk in 10 pts, and favorable [inv(16)] in one patient in second CR. 20 evaluable pts were randomized to decitabine and have received a median of 4.5 cycles (range 1–12). 25 evaluable pts were randomized to CC. With a median duration of follow up for the entire group of 36.3 months (range 6.3–55), 11 pts on the decitabine arm and 10 pts on the CC arm have remained in remission; 9 of 20 (45%) on DAC versus 15 of 25 (60%) on CC have relapsed (p = 0.7). 45 pts including 20 receiving decitabine and 25 receiving CC have been followed for at least 1 year; 9 of 20 (45%) on decitabine versus 9 of 25 (36%) on CC are alive in CR (p=0.9). Toxicity in the decitabine treated pts was limited to 19 episodes of grade 3/4, neutropenia, 14 episodes of grade 3/4 thrombocytopenia, and 1 episode of grade 2 anemia. All were short in duration and reversed without any associated adverse events. Non-hematological grade 3 and 4 adverse events on the decitabine arm include 3 infections, 1 episode each of hypertension and fatigue. There have been no deaths on the study. MFC identified MRD (<5% blasts on smears) in 9 of 19 (47%) patients with subsequent relapse, at levels from 0.04% to 1% of total cells. In 18 pts remaining in CR, no MRD was identified. Relapse occurred from 1.5 to 10 months after the first positive MFC. There was a statistically significant higher LINE methylation at baseline between the pts who relapsed and those who remained in CR (78% vs 73%, P=0.02), whereas no differences were observed for methylation of CDH13 and Mir124a-1 genes. Conclusions: We conclude that administration of decitabine in CR at the above schedule/dose is well tolerated. The study has been terminated early by the data monitoring safety board due to the higher incidence of relapse at 1 year in the decitabine arm. Interestingly, at a median of 36.3 months, decitabine treated patients had lower relapse and higher survival rates than conventionally treated patients, although these differences were not significant. These data suggest the need for larger randomized studies of hypomethylating agents in maintenance therapy in AML. [Display omitted] [Display omitted] Disclosures:Off Label Use: Off label use of decitabine in maintenance therapy in AML. Issa:GSK: Consultancy; SYNDAX: Consultancy; Merck: Research Funding; Eisai: Research Funding; Celgene: Research Funding; Celgene: Honoraria; Novartis: Honoraria; J&J: Honoraria. Faderl:Eisai: Research Funding. Borthakur:Eisai: Research Funding. Cortes:Eisai: Research Funding. Kantarjian:Celgene: Research Funding. Ravandi:Eisai: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Johnson & Johnson: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Targeting the IL-6 Pathway in Multiple Myeloma and its Implications in Cancer-Associated Gene Hypermethylation

Aberrant methylation of tumor suppressor genes (TSG) is an important epigenetic event in cancer, including multiple myeloma (MM). Interleukin-6 (IL-6), which plays a significant role in the pathogenesis of MM, also regulates DNA methylation. However, attempts to bring IL-6 blockade to the clinic have had limited success. We hypothesize that IL-6 regulation of hypermethylation may be an important pathway leading to rational chemotherapeutic/anti-IL-6 combinations. We first studied the correlation of IL-6 expression and dependence in MM cell lines: U266B1, RPMI8226, and KAS6/1. We confirmed that KAS6/1 is IL-6-dependent whereas U266B1 and RPMI8226 cells are IL-6-independent and that blocking IL-6 inhibited the growth of U266B1 (36% inhibition; p<0.05) and KAS6/1 (68% inhibition; p<0.01), but not the RPMI8226 cells. Using RT-PCR, we showed that U266B1 cells express IL-6, but RPMI8226 and KAS6/1 cells do not. This IL-6 expression pattern correlates with the anti-IL-6 inhibition findings. To correlate IL-6 sensitivity with hypermethylation of TSG, we investigated promoter methylation of CDH1 and DcR1. We found that the promoter of DcR1 and CDH1 is methylated in U266B1 cells and un-methylated in RPMI8226 cells. Furthermore, the DcR1 promoter was un-methylated in KAS6/1 cells. These data support our hypothesis that an IL-6-dependent pathway may regulate hypermethylation of TSG in MM. Newer chemotherapeutic agents that affect methylation are being studied in combination with IL-6 blockade.

CDH13 and FLBN3 Gene Methylation are Associated with Poor Prognosis in Colorectal Cancer

The aim of this study was to identify potential epigenetic prognostic biomarkers for colorectal cancer (CRC) in the Chinese population. The methylation status of five tumor suppressor genes (CDH13, DLEC1, FBLN3, hMHL1 and RUNX3) was determined using manual microdissection followed by methylation-specific PCR in 85 paired CRC specimens and adjacent normal tissue. The results showed that methylation frequencies in cancerous tissues were 31.8% for CDH13, 37.6% for DLEC1, 38.8% for FBLN3, 22.4% for hMHL1 and 27.1% for RUNX3, all of which were significantly higher than in corresponding normal tissue. Furthermore, CDH13 methylation was associated with poor differentiation (P = 0.019) and tended to be predominant in advanced stages (P = 0.084); FBLN3 methylation was associated with advanced stages (P = 0.027) and lymph node metastasis (P = 0.029). Accordingly, the methylation status of CDH13 (P = 0.022), FBLN3 (P = 0.008), CDH13 and/or FBLN3 (P = 0.001) predicted adverse overall survival in CRC, while hMHL1 methylation showed a protective role in survival (P = 0.046). Cox proportional hazard models further indicated that CDH13 and/or FBLN3 methylation, but not that of hMHL1, was an independent prognostic factor for CRC. In conclusion, we found CDH13 and FBLN3 gene methylation are potential biomarkers for poor prognosis in CRC.

CpG island methylation in PTEN, MGMT, and E/H cadherin promoters, mutations of K-ras and B-raf may indicate how progression in the colorectal adenocarcinoma sequence occurs.

3608 Background: The colorectal adenocarcinoma sequence summarizes the epigenetic and genetic modifications, which arise during cancer progression. These modifications have effect on the methylation status of different gene promoters and mutations in the K-ras and B-raf genes. Included in the sequence during cancer progression may be, as we have previously shown (ESMO 2010, abstr.:646P): methylation of the gene promoters of H-cadherin then of MGMT deregulating removal toxic methyl adducts on guanine bases and inducing the activation of K-ras through G to A point mutations, activation of B-raf gene by a V600E mutation and finally methylation of E-cadherin, linked to emergence of metastasis. Methods: DNA extraction was obtained by standard methods from resected tumour samples. In order to add PTEN methylation status in this sequence, shown to co-occur with K-ras and B-raf mutations in colorectal cancer (CRC), we screened for PTEN inactivation by promoter hypermethylation over 200 CRC patients from which MGMT, E- and H-cadherin methylation, K-ras and B-raf status we determined. PTEN methylation was analyzed by methylation-specific PCR, gel electrophoresis after Sybr green staining and UV-photography. From each individual patient we examined germline DNA from white blood cells as described above. Results: Of the 95 out of 222 tumors (43%) with a PTEN hypermethylation, 77 (81%) were also methylated in CDH13, 52 (55%) were MGMT methylated, 35 (37%) had a K-ras gene mutation and were B-raf wild type as expected, their mutations being mutually exclusive. We are analysing the 127 non PTEN hypermethylated patients especially in regards to B-raf mutations, Smad and others. All results were tumor specific as all the sequenced blood controls were unmethylated respectively wild type. Conclusions: PTEN methylation occurs after CDH13 and MGMT methylation and induces mutations in K-ras or B-raf genes. These results further add on and sustain the colorectal adenocarcinoma sequence.

Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population

BackgroundUndifferentiated nasopharyngeal carcinoma (NPC) is strongly related to Epstein-Barr virus (EBV) infection, allowing aberrant antibodies against EBV and viral DNA load as screening tools in high risk populations. Methylation analysis in the promoter of tumor suppressor genes (TSGs) may serve as a complementary marker for identifying early cases. This study determined methylation status of multiple TSGs and evaluated whether it may improve early detection.MethodsNasopharyngeal brushings were taken from 53 NPC patients, 22 high risk subjects and 25 healthy EBV carriers. Corresponding NPC paraffin tissue was included. DNA was bisulfite-modified preceding analysis by methylation-specific PCR (MSP). Ten TSGs were studied.ResultsNPC paraffin and brushing DNA revealed an 81.8% concordance so that MSP analysis was done using either one of both specimens. NPC samples showed methylation for individual TSGs (DAPK1 79.2%, CDH13 77.4%, DLC1 76.9%, RASSF1A 75.5%, CADM1 69.8%, p16 66.0%, WIF1 61.2%, CHFR 58.5%, RIZ1 56.6% and RASSF2A 29.2%). High risk individuals, having elevated EBV IgA and viral load, showed high frequency of methylation of CDH13, DAPK1, DLC1 and CADM1, but low frequency of methylation of p16 and WIF1 and undetectable methylation of RASSF1A, CHFR, RIZ1 and RASSF2A. Healthy subjects showed similar patterns as high risk individuals. A combination of RASSF1A and p16 gave good discrimination between NPC and non-NPC, but best results were combined analysis of five methylation markers (RASSF1A, p16, WIF1, CHFR and RIZ1) with detection rate of 98%.ConclusionMultiple marker MSP is proposed as a complementary test for NPC risk assessment in combination with EBV-based markers.

Highly frequent promoter methylation and PIK3CA amplification in non-small cell lung cancer (NSCLC)

BackgroundLung cancer is the leading cause of cancer-related death worldwide. Genetic and epigenetic alterations have been identified frequently in lung cancer, such as promoter methylation, gene mutations and genomic amplification. However, the interaction between genetic and epigenetic events and their significance in lung tumorigenesis remains poorly understood.MethodsWe determined the promoter methylation of 6 genes and PIK3CA amplification using quantitative methylation-specific PCR (Q-MSP) and real-time quantitative PCR, respectively, and explore the association of promoter methylation with PIK3CA amplification in a large cohort of clinically well-characterized non-small cell lung cancer (NSCLC).ResultsHighly frequent promoter methylation was observed in NSCLC. With 100% diagnostic specificity, excellent sensitivity, ranging from 45.8 to 84.1%, was found for each of the 6 genes. The promoter methylation was associated with histologic type. Methylation of CALCA, CDH1, DAPK1, and EVX2 was more common in squamous cell carcinomas (SCC) compared to adenocarcinomas (ADC). Conversely, there was a trend toward a higher frequency of RASSF1A methylation in ADC than SCC. In addition, PIK3CA amplification was frequently found in NSCLC, and was associated with certain clinicopathologic features, such as smoking history, histologic type and pleural indentation. Importantly, aberrant promoter methylation of certain genes was significantly associated with PIK3CA amplification.ConclusionsOur data showed highly frequent promoter methylation and PIK3CA amplification in Chinese NSCLC population, and first demonstrated the associations of gene methylation with PIK3CA amplification, suggesting that these epigenetic events may be a consequence of overactivation of PI3K/Akt pathway.

Abstract 321: Aberrant methylation of multiple tumor suppressor genes and clinicopathological parameters as predictors for genotype-4 hepatitis C virus-associated hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the few tumors in which the incidence is on the rise worldwide. The increasing incidence of HCC is associated with the rise in hepatitis C virus (HCV) infection. It is estimated that 2.0% of the US general population are infected with HCV. Egypt has the highest prevalence of HCV/genotype 4 infection in the world, with 14% of the population infected. Little is known about the role(s) of epigenetic changes that may participate in HCV-induced HCC? Epigenetic studies directed at mapping the etiology of HCV disease progression to HCC and how they correlate with patients clinicopathological parameters are expected to provide new insights that will aid in the design of effective strategies for earlier detection and management of the disease, and therefore of great public/global health interest. The aim of the current study was to investigate the aberrant methylation of 11 genes in liver tissue samples obtained from Egyptian patients infected with HCV/genotype 4, and to explore possible relationships between aberrant methylation and clinicopathological features in HCC. Thirty-eight HCC and matching non-tumor tissues, thirty-one chronic hepatitis liver tissues, and thirteen normal liver tissues were analyzed for the methylation status of APC, FHIT, p15, p73, p14, p16, DAPK1, CDH1, RARβ, RASSF1A and O6MGMT genes by quantitative methylation-specific PCR. Based on methylation profiles using McNemar's test for paired binary data, a methylation of only 7 genes was found to be highly significant, which is dependent on disease state. For chronic hepatitis tissues, promoter methylation of p14 was highly significant in both liver tissues and peripheral blood leukocytes (PBL) (P &amp;lt; 0.0047 and &amp;lt;0.003, respectively), whereas methylation of RASSF1A was highly significant in normal liver tissues (P &amp;lt; 0.0005). For HCC, promoter methylation of p15, p14 and APC was highly significant in tumor tissues, non-tumor tissues, and PBL, respectively (P &amp;lt; 0.0001, &amp;lt; 0.0076 and &amp;lt; 0.0001, respectively). To account for the clinical variables of patients in the methylation status of a gene and disease state, a logistic regression analysis was performed. For HCC, promoter methylation of APC, DAPK1, and p14 was highly significant in addition to the blood profiles of patients (e.g., platelets, hemoglobin, WBCs levels). For patients with chronic hepatitis, methylation of CDH1 (E-Cadherin) was highly significant in addition to the patient's liver enzymes level (e.g., AST, ALT). Our data indicate that DNA methylation may serve as a marker for HCV-associated HCC, and demonstrate the importance of considering the clinicopathological variables as predicators of disease state. Quantitative analysis of methylated p14 may serve as a powerful molecular marker in detecting HCV disease progression to HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 321. doi:10.1158/1538-7445.AM2011-321

DNA methylation profile during multistage progression of pulmonary adenocarcinomas

Multiple genetic and epigenetic alterations are known to be involved in the carcinogenesis of peripheral pulmonary adenocarcinoma (ADC). However, epigenetic abnormalities have not been extensively investigated in the following multistage progression sequence: atypical adenomatous hyperplasia (AAH) to adenocarcinoma in situ (AIS), to invasive ADC. To determine the potential role of promoter methylation during ADC development of the lung, we examined methylation status in 20 normal, 20 AAH, 30 AIS, and 60 ADC lung tissues and compared methylation status among the lesions. The MethyLight assay was used to determine the methylation status of 18 CpG island loci, which were hypermethylated in ADC compared to noncancerous lung tissues. The mean number of methylated CpG island loci was significantly higher in ADC than in AAH and AIS, (p < 0.003 between ADC and AAH, p < 0.005 between ADC and AIS). Aberrant methylation of HOXA1, TMEFF2, and RARB was frequently observed in preinvasive lesions, including AAH and AIS. Furthermore, methylation of PENK, BCL2, RUNX3, DLEC1, MT1G, GRIN2B, CDH13, CCND2, and HOXA10 was significantly more frequent in invasive ADC than AAH or AIS. Our results indicate that epigenetic alterations are involved in the multistep progression of pulmonary ADC development, and aberrant CpG island methylation accumulates during multistep carcinogenesis. In addition, aberrant methylation of HOXA1, TMEFF2, and RARB occurred in preinvasive lesions, which indicates that epigenetic alterations of these genes are involved in the early stages of pulmonary ADC development. In contrast, hypermethylation of PENK, BCL2, RUNX3, DLEC1, MT1G, GRIN2B, CDH13, CCND2, and HOXA10 was more frequent in invasive ADC than in preinvasive lesions, which indicates that methylation of these genes occurs later during tumor invasion in the AAH-AIS-ADC sequence.

Abstract 4793: Aberrant methylation of CDH1 correlates with poor prognosis in patients with head and neck squamous cell carcinoma

Abstract Objective: CDH1 codes for E-cadherin, a key adhesion molecule of epithelial cells, whose loss of expression has been associated with carcinogenesis in several types of cancer. Promoter hypermethylation of CDH1 results in loss of expression of E-cadherin and may be involved in the development of metastasis and a poorer prognosis. The purpose of this study was to determine the methylation profile of CDH1 in a cohort of HNSCC patients and evaluated its association with clinicopathological parameters. Methods: CDH1 methylation status was determined using the Methylight assay in a cohort of 59 patients that had CDKNA (p16) methylation status assessed previously. Correlations between clinical parameters and methylation profiles were statistically analyzed using Fisher's exact test and chi-square test when appropriate. Results: Aberrant methylation of CDH1 was detected in 78% of the cases analyzed. In 33% of the patients had also CDKN2A aberrantly methylated. Most patients (69%) with CDH1 aberrantly methylated were in advanced stages of the disease (III/IV). They also had a poorer survival rate. No statistically significant association was found between CDH1 hypermethylation and gender, age, risk factors and staging. Conclusion: CDH1 aberrantly methylated was detected at early and late stages of the disease. These results suggest that CDH1 hypermethylation may have a role in the initiation and in progression of HNSCC and potentially reduce the survival of HNSCC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4793. doi:10.1158/1538-7445.AM2011-4793

The expression of CDH1 gene methylation in patients with esophagus and stomach double primary carcinoma and its significance

Objective To study the changes of CDH1 gene promoter CpG island methylation and its clinical significance in patients with esophagus and stomach double primary carcinoma(ESDC).Methods The expression of CDH1 gene methylation in cancerous tissues and adjacent cancerous tissues in 18 cases of ESDC were detected using methylation-specific PCR method. Results Eighteen patients were endoscopically diagnosed as ESDC between Jan. 2007 and Sep. 2009 in the 4th Hospital Affiliated to Hebei Medical University. The positive methylation of CDH1 gene in tissues of esophageal squamous cell carcinoma (ESCC)and adjacent cancer were 66.7% and 33. 3%, respectively, with significant difference (χ2= 4. 167, P = 0. 031). Whereas the positive methylation of CDH1 gene in tissues of gastric carcinoma (GA) and adjacent cancer were 77.8% and 44.4%, respectively, without statistical difference (χ2=1.786, P= 0. 180). There was no significant difference (P=0. 500) in positive rate of CDH1 gene methylation between ESCC tissues and GA tissues in same individual with ESDC. For 18 patients with ESDC, consistent change of CDH1 methylation in tissues of two kinds of cancers was found in 16 patients with a total agreement of 88.9 % (positive agreement of 66.7 % and negative agreement of 22. 2%). Statistical analysis showed a significant correlation between two groups (P = 0. 005). Conclusion In patients with ESDC, there is a high consistency of CDH1 methylation change, between ESCC and GA,which suggests that two kinds of cancer may have similar risk factors and molecular mechanisms. Key words: Stomach neoplasms; Esophasus neoplas double primary carcinoma; Esophageal squmaous cell carcinoma; CDH1; Methylatlion

Evaluation of methylation pattern in promoter region of E-cadherin gene and its relation to tumor grade and stage in breast cancer

The epithelial cadherin gene (CDH1) has been identified as a tumor suppressor gene located within the 16q22.1 region. The CDH1 gene encodes a transmembrane glycoprotein involved in cell to cell adhesion and loss of CDH1 expression contributes to increased proliferation, invasion and metastasis in breast carcinoma. No mutation in CDH1 have been identified in invasive ductal carcinoma (IDC), suggesting that, other inactivation mechanisms are responsible for IDC oncogenesis. In order to analyze the role of promoter methylation in CDH1 gene inactivation in breast cancer, the CpG methylation status of Ecadherin promoter region by bisulfite sequencing PCR (BSP) was investigated. 10 CpG sites [nucleotide (nt) 863, 865, 873, 879, 887, 892, 901, 918, 920 and 940] in the promoter region were screened for methylation. The CDH1 methylation was detected in 94% (47 to 50) of breast tumors which was associated with higher tumor grade (p = 0.035), tumor stage (p = 0.000) and tumor metastasis (p = 0.000). There was also a significant correlation between tumor stage, grade and metastatic status with sites of methylation (p = 0.000). The data indicate that CDH1 promoter methylation might be a potential mechanism for epigenetic silencing of CDH1 in primary breast cancer suggesting a valuable molecular marker for detection of breast cancer progression. Key words: Breast cancer, E-cadherin, methylation pattern, tumor stage, tumor grade.

Hypermethylation-modulated down-regulation of CDH1 expression contributes to the progression of esophageal cancer

CDH1, a cell adhesion molecule, which plays a key role in maintaining the epithelial phenotype, is regarded as an invasion-suppressor gene in light of accumulating evidence from in vitro experiments and clinical observations. In an attempt to clarify the mechanism responsible for inactivation of this gene in carcinomas, we investigated the methylation status of the CDH1 gene 5'-CpG islands and its regulatory mechanism in the progression of esophageal squamous cell carcinoma. Real-time methylation-specific polymerase chain reaction (qMSP) and treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) were conducted to analyze the methylation status at the CDH1 promoter region in the human esophageal carcinoma cell lines, EC1 and EC9706. A total of 235 invasive esophageal squamous cell carcinomas (ESCC) at stages I-IV and their corresponding normal tissue samples, were included in an immunohistochemistry study and methylation analysis of CDH1. The results demonstrate that in EC1 and EC9706 cells, the CDH1 promoter is methylated and treatment with 5-Aza-CdR restored CDH1 expression. Enhanced CDH1 expression decreased cell migration, invasion ability and increased adhesion ability. Decreased CDH1 expression was detected in 59.6% of ESCC tissues, compared with their adjacent non-neoplastic epithelia, which had a close correlation with the primary tumor status, lymph node status, distant metatasis and clinicopathologic stage. Hypermethylation at the CDH1 promoter was detected in 97.9% of 140 cases of ESCC with low CDH1 expression. The methylation of CDH1 promoters (P=0.929) was closely correlated with the lack of expression of their corresponding proteins. The Cox regression model for survival analysis showed that increases in CDH1 methylation had a greater impact on the prognosis than tumor clinical stage. These findings suggest that CDH1 gene silencing by promoter hypermethylation and the resultant reduction of CDH1 expression may play an important role in the progression of ESCC. CDH1 methylation was a significant predictor of survival in ESCC patients after surgery.

Clinical Significance ofCDH13Promoter Methylation in Serum Samples from Patients with Bladder Transitional Cell Carcinoma

H-cadherin (CDH13; also known as T-cadherin), which functions as a tumour suppressor, is frequently silenced by promoter methylation in human cancers including bladder transitional cell carcinoma (TCC). This study investigated the clinical significance of methylation of the CDH13 gene promoter in serum from patients with bladder TCC. Methylation status of CDH13 in serum samples from 127 patients with primary bladder TCC and 41 healthy volunteers (controls) was examined by methylation-specific polymerase chain reaction. CDH13 methylation was found in 39 patients with bladder TCC (30.7%) but in no controls. CDH13 methylation was significantly associated with advanced tumour stage, high-grade tumour, large tumour size, tumour recurrence and poor prognosis. The results suggested that CDH13 methylation in serum may be a potential predictive biomarker for malignancy in bladder TCC, and an independent pretherapeutic predictor of outcome. Demonstration of CDH13 methylation in serum may facilitate in the prediction of which patients require more aggressive additional post-operative systemic therapy.