DAPK Promoter Hypermethylation Research Articles

Promoter hypermethylation of RARβ2, DAPK, hMLH1, p14, and p15 is associated with progression of breast cancer: A PRISMA-compliant meta-analysis.

Background:Numerous studies have investigated the associations between RARβ2, DAPK, hMLH1, p14, and p15 promoter hypermethylation and clinical progression of patients with breast cancer, however the results remained uncertain due to the small sample size. Therefore, we performed a meta-analysis to explore the role of RARβ2, DAPK, hMLH1, p14, and p15 promoter hypermethylation in the susceptibility and clinical progression of breast cancer.Methods:Eligible studies were obtained by searching Medicine, Embase, Web of knowledge, and Chinese National Knowledge Infrastructure (CNKI) databases. The odds ratios (OR) and 95% confidence intervals (CI) were calculated to evaluate the associations of RARβ2, DAPK, hMLH1, p14, and p15 promoter hypermethylation with breast cancer pathogenesis. Trial sequential analysis (TSA) was applied to observe the reliability of pooled results of RARβ2 gene, and obtain a conservative required information size (RIS).Results:In primary screened 445 articles, 39 literatures with 4492 breast cancer patients were finally enrolled in the final meta-analysis. The results indicated that the frequency of RARβ2 promoter hypermethylation in case group was significantly higher than the frequency of control group (OR = 7.21, 95% CI = 1.54–33.80, P < .05). The RARβ2 promoter hypermethylation had a significant association with lymph node metastasis of breast cancer (OR = 2.13, 95% CI = 1.04–4.47, P < .05). And, the RARβ2 promoter hypermethylation was more common in the breast cancer patients of TNM III–IV stage than those patients of TNM I–II stage (OR = 1.85, 95% CI = 1.33–2.57, P < .05). In addition, the promoter hypermethylation of DAPK, hMLH1, and p14 genes were significantly associated with the susceptibility of breast cancer (for DAPK, OR = 4.93, 95% CI = 3.17–7.65; for hMLH1, OR = 1.84, 95% CI = 1.26–1.29; for p14, OR = 22.52, 95% CI = 7.00–72.41; for p15, OR = 2.13, 95% CI = 0.30–15.07).Conclusions:Our findings revealed that the RARβ2 promoter hypermethylation significantly increased the risk of breast cancer. In the meantime, the meta-analysis demonstrated that there were significant associations of RARβ2 promoter hypermethylation with lymph node metastasis and TNM-stage of breast cancer patients. In addition, DAPK, hMLH1, and p14 genes promoter hypermethylation were significantly associated with the susceptibility of breast cancer.

Abstract A36: Clinical and molecular characterization of oropharynx squamous cell carcinoma samples

Abstract Background: Oropharynx squamous cell carcinomas (OPSCC) associated with high-risk human papilloma virus (HR-HPV) infection represent a distinct clinical and epidemiologic entity, with differences in clinical presentation and treatment outcome when compared to tobacco-associated tumors. Objective: The aim of this study was to identify clinical and molecular factors that can differentiate OPSCC patients into subgroups according to their outcome. Materials and Methods: Twenty-five surgically treated OPSCC samples were included in this study. p16 protein expression was used as a surrogate marker for HR-HPV. The methylation status of 5 genes was evaluated by quantitative methylation-specific PCR (qMSP). Exons 4-8 of the P53 gene were evaluated by Sanger sequencing and p53 protein expression was evaluated by immunohistochemistry (clone DO-7). Results: The majority of patients (88%) were male, had advanced disease (80%), and were tobacco users (88%). Twenty percent of the cases were HR-HPV positive and 62.5% presented deleterious P53 mutations, which was moderately concordant with strong p53 protein expression (Kappa value = 0.467; p = 0.022). CCNA1, CDH8, DAPK, PCDH10, and TIMP3 promoter hypermethylation was observed in 16-36%, of the OPSCC evaluated. DAPK hypermethylation was associated with HPV status (p = 0.023), while CCNA1 methylation was inversely related to p53 mutations in primary tumors (p = 0.042) and associated with a better disease-free survival (22.3% vs. 100%; p = 0.028) and overall survival (8% vs. 100%; p = 0.012). Conclusions: Even though all samples analyzed were of the oropharynx, several differences regarding molecular and clinical characteristics were identified. More cases should be evaluated in order to assess whether the differences identified are able to classify these patients according to their outcome and help in a more thorough patient management. Citation Format: Ana Carolina de Carvalho, Bruna Pereira Sorroche, Matias Eliseo Melendez, Cristina da Silva Sabato, Edenir Inez Palmero, Lidia Maria Rebolho Batista Arantes, Cristovam Scapulatempo Neto, André Lopes Carvalho. Clinical and molecular characterization of oropharynx squamous cell carcinoma samples [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A36.

Promoter hypermethylation of PTPL1, PTPN6, DAPK, p16 and 5-azacitidine inhibits growth in DLBCL.

Aberrant hypermethylation of CpG islands of tumor suppressor is one of the mechanisms for epigenetic loss of gene function. In the present study, the methylation status of the promoter regions of protein tyrosine phosphatase (PTPN) 6, DAPK, and p16 were studied using methylation-specific polymerase chain reaction (MSP) in 26 diffuse large B cell lymphoma (DLBCL) lymphomas. In OCI-LY1 cell line, gene methylation status, expression of PTPL1 and its reactivation by DNA demethylation was determined by PCR and on the protein level by western blotting. ELISA-like reaction was used to detect global DNA methylation measurement. Induction of apoptosis by 5-azacitidine was analyzed by Annexin V/PI staining and flow cytometry. Our results show that hypermethylation of the PTPN6 gene promoter region was found in 15.4% (4/26), the DAPK gene promoter region in 30.8% (8/26), the p16 gene promoter region in 7.7% (2/26). Notably, we identified that PTPL1 was hypermethylated and transcriptionally silenced in OCI-LY1 cell line. The expression of PTPL1 was re-inducible by 5-azacytidine. 5-azacytidine also inhibits the proliferation and decreases the global methylation level of the OCI-LY1 cell line. We can conclude from our study that a higher prevalence of methylation of PTPL1, PTPN6, DAPK and p16 occur in DLBCL. Our data also highlights 5-azacytidine as a potential therapeutic candidate for DLBCL. Further studies are required to substantiate the role of methylation of PTPL1, PTPN6, DAPK and p16 as a marker in diffuse large B cell lymphoma.

Clinical significance of DAPK promoter hypermethylation in lung cancer: a meta-analysis.

Death-associated protein kinase 1 (DAPK) is an important serine/threonine kinase involved in various cellular processes, including apoptosis, autophagy, and inflammation. DAPK expression and activity are deregulated in a variety of diseases including cancer. Methylation of the DAPK gene is common in many types of cancer and can lead to loss of DAPK expression. However, the association between DAPK promoter hypermethylation and the clinicopathological significance of lung cancer remains unclear. In this study, we searched the MEDLINE, PubMed, Web of Science, and Scopus databases, systematically investigated the studies of DAPK promoter hypermethylation in lung cancer and quantified the association between DAPK promoter hypermethylation and its clinicopathological significance by meta-analysis. We observed that the frequency of DAPK methylation was significantly higher in lung cancer than in non-malignant lung tissues (odds ratio 6.02, 95% confidence interval 3.17–11.42, P<0.00001). The pooled results also showed the presence of a prognostic impact of DAPK gene methylation in lung cancer patients (odds ratio 3.63, 95% confidence interval 1.09–12.06, P=0.04). In addition, we summarized these findings and discuss tumor suppressor function, clinicopathological significance, and potential drug targeting of DAPK in lung cancer.

Unexpected close surgical margin in resected buccal cancer: Very close margin and DAPK promoter hypermethylation predict poor clinical outcomes

In resected buccal cancer patients, an unexpected close surgical margin has been observed to correlate with poor clinical outcomes. However, close surgical margin alone does not independently guide post-operative therapies, revealing a clinical debate. Hence, the present study intended to explore epigenetic-based bio-predictors for further stratifying this debating patient population. Between 2000 and 2008, we retrospectively recruited 44 resected buccal cancer patients with a close surgical margin of ≤5 mm. All patients had post-operative radiotherapy. Genomic DNA was extracted from tumor-enrich areas that contained cancer cells of >70%. Methylation-specific PCR was performed to detect promoter methylation of four tumor suppressor genes, including RASSF1A, DAPK, IRF8, and SFRP1. Post-irradiation locoregional control was defined as the primary end point. There were 40 males and 4 females, with a median age of 53.5 years (range, 32-82 years). Multivariate analysis identified two independent predictors for locoregional recurrence: very close margin of ≤1 mm (HR: 4.96; 95% CI, 1.63-15.09; P=0.018) and promoter hypermethylation of DAPK (HR: 2.83; 95% CI, 1.05-7.63; P=0.042). The highest risk of locoregional recurrence was observed in patients with both of the two factors (HR, 8.05; 95% CI, 2.56-25.82; P=0.002) when compared with patients with none. Shorter disease-free survival, but not overall survival, was also observed. More aggressive managements should be considered in resected buccal cancer patients with both very close margin and DAPK promoter hypermethylation rather than post-operative observation or radiotherapy alone.

Prognostic significance of tumor-related genes hypermethylation detected in cancer-free surgical margins of oral squamous cell carcinomas

Oral squamous cell carcinoma (OSCC) is characterized by high mortality rates. High incidence of local recurrences in the normal-appearing surgical margins of OSCC patients indicates the existence molecular alterations, including DNA methylation, which could not be detectable by histopathologic analysis. The objective of our study was to determine correlation of tumor-related genes hypermethylation detected in histopathologically negative surgical margins with clinical and prognostic parameters. The genes selected for methylation analysis covered a wide range cellular processes including cell cycle control (p16), apoptosis (DAPK and RASSF1A), Wnt signaling (APC, WIF1 and RUNX3), cell-cell adhesion (E-cad), and DNA repair (MGMT and hMLH1). All of 47 patients had histologically confirmed negative surgical margins. For each of patient, samples from primary malignant tissue and the two consecutive surgical margins were taken at the time of surgery. DNA methylation was determined by multiplex nested methylation-specific PCR. Twenty-seven patients were margin-positive for promoter hypermethylation of at least one gene under study. The presence of DAPK promoter hypermethylation detected in surgical margins was associated with the decreased overall survival (p=0.004, log rank test). Multivariate analysis revealed that DAPK promoter hypermethylation in surgical margins is an independent prognostic factor for overall survival, HR=4.105 (1.458-11.555, 95% CI, p=0.007). Hypermethylation of other tumor-related genes under study did not have prognostic significance. These results demonstrate that DNA hypermethylation in histologically negative surgical margins is a frequent event. Promoter hypermethylation of DAPK gene detected in surgical margins may be a useful molecular marker for the poor survival in OSCC patients. Further investigation into the therapeutic potential of these findings in OSCC is warranted.

Link of Metabolic Phenotypes of Gastric Cancer Tissue and Blood Plasma

G A A b st ra ct s were 0.674, 0.727, 0.680, 0.739, respectively. Two peaks were found in the autofluorescence spectrum of gastric juice from all patients. The first peak (P1FI) was distinctively higher in gastric cancer than that in benign lesion (P<0.01). The AUC from ROC for P1FI was 0.872, the sensitivity, specificity, and accuracy of P1FI were 83.8%, 75.2%, and 78.8%, respectively, while the false positive rate was 24.8% (41/165). Furthermore, all of the four candidate genes and P1FI were evaluated in univariate and multivariate logistic regression (LR) models. The multivariate model included logarithmic transformations of E-cadherin, DAPK and P1FI, and the AUC from ROC was 0.933, which was statistically superior to either parameter alone (P<0.01), the sensitivity, specificity and accuracy were 89.7%, 86.7% and 87.9%, respectively, the false positive rate was 13.3% (22/165). Additionally, CART (Classification and Regression Trees) (Salford Systems) was carried out to set up the diagnostic model for gastric cancer and similar to the results of LR, P1FI, E-cadherin and DAPK were identified as tree nodes(Fig.1). The sensitivity, specificity, and accuracy were 81.2 %, 91.5%, and 87.2%, respectively. It is noticeable that the false positive rate was 8.5% (14/165). Conclusions The complementary use of both LR and CART models could be a promising approach to analyze multiple markers. The model, which combining the autofluorescene spectrum with the DNA promoter hypermethylation of E-cadherin and DAPK of gastric juice, could be a potential promising diagnostic model for better decision making for gastric cancer screening.

Promoter methylation of p16, Runx3, DAPK and CHFR genes is frequent in gastric carcinoma

Transcriptional silencing induced by hypermethylation of CpG islands in the promoter regions of genes is believed to be an important mechanism of carcinogenesis in human cancers including gastric cancer. A number of reports on methylation of various genes in gastric cancer have been published, but most of these studies focused on cancer tissues or only a single gene. In this study, we determined the promoter hypermethylation status and mRNA expression of 4 genes: p16, Runx3, DAPK and CHFR. Methylation-specific polymerase chain reaction (MSP) was used to determine the methylation status of p16, Runx3, DAPK and CHFR gene promoters in cancer and adjacent normal gastric mucosa specimens from 70 patients with gastric cancer, as well as normal gastric biopsy samples from 30 people without cancer serving as controls. In addition, the mRNA expression of p16, Runx3, DAPK and CHFR was investigated in 34 gastric cancer patients by RT-PCR. Bisulfite DNA sequence analysis was applied to check the positive samples detected by MSP. When carcinoma specimens were compared with adjacent normal gastric mucosa samples, a significant increase in promoter methylation of p16, Runx3, DAPK and CHFR was observed, while all 30 histologically normal gastric specimens were methylation free for all 4 genes. The methylation rate of the 4 genes increased from normal stomach tissue to tumor-adjacent gastric mucosa to gastric cancer tissue. Concurrent methylation in 2 or more genes was found in 22.9% of tumor-adjacent normal gastric mucosa and 75.7% of cancer tissues. No correlation was found between hypermethylation and other clinicopathological parameters such as sex, age, and tumor location. However, the frequency of DAPK and CHFR methylation in cancer tissues was significantly associated with the extent of differentiation and lymph node metastasis (P < 0.05) and the frequency of Runx3 methylation was significantly associated with tumor size (P < 0.05). Weak expression and loss of expression of the 4 genes was observed in cancer tissues and was significantly associated with promoter hypermethylation (P < 0.05). Promoter hypermethylation of p16, Runx3, DAPK and CHFR is frequent in gastric cancer. DAPK and CHFR promoter hypermethylation may be an important help in evaluating the differentiation grade and lymph node status of gastric cancer. Weak gene expression and loss of gene expression due to promoter hypermethylation may be a cancer-specific event.

PTEN, RASSF1 and DAPK site‐specific hypermethylation and outcome in surgically treated stage I and II nonsmall cell lung cancer patients

The primary objective of this study is to identify prognostic site-specific epigenetic changes in surgically treated Stage I and II nonsmall cell lung cancer (NSCLC) patients by quantifying methylation levels at multiple CpG sites within each gene promoter. Paraffin-embedded tumors from stage Ib, IIa and IIb in training and validation groups of 75 and 57 surgically treated NSCLC patients, respectively, were analyzed for p16, MGMT, RASSF1, RASSF5, CDH1, LET7, DAPK and PTEN promoter hypermethylation. Hypermethylation status was quantified individually at multiple CpG sites within each promoter by pyrosequencing. Molecular and clinical characteristics with time to recurrence (TTR) and overall survival (OS) were evaluated. Overall average promoter methylation levels of MGMT and RASSF1 were significantly higher in smokers than in nonsmokers (p = 0.006 and p = 0.029, respectively). Methylation levels of the p16 promoter were significantly higher in squamous cell carcinoma than in adenocarcinoma (p = 0.020). In univariate analysis, hypermethylation of RASSF1 at CpG sites -53 and -48 and PTEN at CpG site -1310 were the significantly associated with shorter TTR (p = 0.002 and p < 0.000, respectively). Hypermethylation of PTEN at -1310 and DAPK at -1482 were most significantly associated with outcome in multivariate analysis. These results show that methylation of specific promoter CpG sites in PTEN, RASSF1 and DAPK is associated with outcome in early stage surgically treated NSCLC.

Prognostic significance of DAPK and RASSF1A promoter hypermethylation in non-small cell lung cancer (NSCLC).

Prognostic significance of DAPK and RASSF1A promoter hypermethylation in non-small cell lung cancer (NSCLC).

Prognostic Significance of Promoter Methylation of Multiple Genes in Germinal Center Hyperplasia (GCH) and Lymphomas of Germinal Center Origin.

Abstract 3950Poster Board III-886 INTRODUCTIONGerminal centers (GC) are unique sites in peripheral lymphoid tissue where clonal selection of B cells takes place. GC have been known to be a major source of B-cell lymphomas, including follicular (FL) and diffuse large cell (DLCL). DNA methylation of tumor-suppressor genes is a mechanism of gene silencing involved in the pathogenesis of FL and DLCBLs. Much less is known about the role of methylation in GCH. We determined the methylation status of 5 tumor-suppressor genes in 50 patients with lymphoma of GC origin and 50 GCH in order to find any differences between the pathological and the physiological state as well as its prognostic significance. MATERIAL AND METHODS. Genomic DNA extracted from paraffin-embedded samples of 30 DLCL, 20 FL and 50 GCH were analyzed by methylation-specific polymerase chain-reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, p14 and MGMT. Methylation status of each gene was correlated with clinicopathological status. Overall survival (OS) rates were calculated by the Kaplan-Meier method and differences were compared with the log-rank test. RESULTS. Median age was 65 in patients with lymphoma and 19 for GCH. Sex distribution was similar in all entities (60% females). Both lymphoma groups were balanced with respect to the presence of B symptoms, bulky disease, bone marrow infiltration, advanced stage and high IPI/FLIPI. DAP-k promoter methylation was present in more patients with lymphoma (89 and 87%) than with BFH (37%) p<0.0001. RaRB was methylated with higher frequency in FL (60%) than in DLCL (23%) and FH (12%) p<0.0001. SHP1 was more frequently methylated in FL (67%) and GCH (58%) than in DLCL (20%) p=0.01. Promoter hypermethylation of SHP1 was significantly associated with longer OS (p=0.021). Methylation of RaRB, p14 and MGMT were associated with shortened OS but the differences were not statistically significant. Those patients with DAPK methylated live longer but not significantly. In multivariate analysis hypermethylation of none of the genes studied remained an independent prognostic factor. CONCLUSIONS. Inactivation of DAP-K, and Rarβ is present in GC lymphomas with significantly higher frequency than in BFH. Thus, it may have pathogenic significance. SHP1 is methylated more frequently if FL and BFH than in DLCL, therefore that gene may be associated with aggressive disease. Methylation of DAP-k, SHP1, Rarβ, p14 and MGMT has no significant impact on overall survival. Markers for aberrant methylation may represent a promising way to monitor the onset and progression of malignancies but more extensive and prospective trials are needed to precisely define its role. Disclosures:No relevant conflicts of interest to declare.

Expression of EBV Latent Antigens, Mammalian Target of Rapamycin, and Tumor Suppression Genes in EBV-Positive Smooth Muscle Tumors: Clinical and Therapeutic Implications

EBV-positive smooth muscle tumor (EBV+SMT) is a rare disease with no established therapy. We describe the largest single institution analysis in renal transplant recipients. It aims to define its clinical features and determine the expression of EBV latent genes as well as key molecular pathways. Patients with EBV+SMT were identified from the Singapore General Hospital Renal Transplant Registry database. These tumors were investigated for expression of EBV latent genes with Southern blots, EBV latent antigens, mammalian target of rapamycin (mTOR), Akt, p70 S6 kinase, and vascular endothelial growth factor using immunohistochemistry, as well as methylation status of cancer-related genes using methylation-specific PCR. Eight were found to be EBV+SMT in 1,123 transplant patients. All displayed indolent clinical courses and were unresponsive to immunosuppression reduction. Complete tumor regression was seen in one patient following administration of sirolimus. These tumors display the full range of known EBV latent genes. Immunohistochemistry with total and phosphorylated mTOR and Akt were positive for all patients, and vascular endothelial growth factor was positive in 25% of patients, suggesting activation of the mTOR/Akt pathway. Methylation of RASSF1A was found in all tissue samples, whereas promoter hypermethylation of RARbeta, GSTP1, DAPK, and p14 was observed in some samples. Our results suggest that these tumors display a EBV type III latency pattern. The mTOR pathway is also activated. EBV may play a role in silencing RASSF1A. EBV-specific immunotherapy, mTOR inhibitors, and demethylating agents are possible therapeutic options in this disease.

Promoter methylation of TIMP3 and CDH1 predicts better outcome in head and neck squamous cell carcinoma treated by radiotherapy only

As with other solid tumor types, head and neck squamous cell carcinoma (HNSCC) has been identified as an epigenetic, as well as genetic, disease. Consequently, promoter hypermethylation, being the most important aberrant epigenetic characteristic, has been intensively investigated for its biomarker potential in this cancer type. As many of these evaluations are obscured by a heterogeneity of treatments, the current study aimed to evaluate the incidence and prognostic value of the promoter hypermethylation of TIMP3, CDH1, DAPK, RASSF1A, p16INK4A and MGMT in HNSCC treated solely by radiotherapy. In 46 patients with advanced HNSCC treated with a hybrid accelerated fractionation radiotherapy schedule, DNA extracted from pretreatment paraffin-embedded tumor biopsies was used to determine the methylation status of the genes of interest by methylation-specific PCR (MSP). The detected epigenetic silencing was related with outcome in terms of locoregional control (LRC), and overall (OS), disease-free (DFS) and disease-specific survival (DSS). Tumor biopsies revealed the epigenetic silencing of MGMT in 42.5% (17 of 40) of patients and of TIMP3 in 40.5% (17 of 42) of cases. For the remaining investigated genes, a lower methylation percentage was detected: 13.2% (5 of 38) for CDH1, 11.4% (4 of 44) for DAPK, 4.8% (2 of 42) for p16INK4A and 2.4% (1 of 41) for RASSF1A. The promoter hypermethylation of TIMP3 and CDH1 was significantly related with better LRC (p=0.009 and p=0.02, respectively), OS (p=0.005 and p=0.002, respectively), DFS (p=0.02 and p=0.004, respectively) and DSS (p=0.12 and p=0.007, respectively). In conclusion, in this representative group of 46 patients with advanced HNSCC treated by radiotherapy only, the epigenetic silencing of TIMP3 and CDH1 predicted a better outcome.

Promoter Methylation of Multiple Genes in Germinal Center Hyperplasia and Lymphomas of Germinal Center Origin

INTRODUCTION. Germinal centers (GC) are unique sites in peripheral lymphoid tissue where clonal selection of B cells takes place. This occurs as a response to stimulation by various antigens originating, sometimes, follicular hyperplasia (FH). GC have been known as a major source of B-cell lymphomas including follicular (FL) and diffuse large cell (DLCL). DNA methylation of tumor-suppressor genes is a mechanism of gene silencing involved in the pathogenesis of FL and DLCL. Much less is known about the role of methylation in FH. We determined the methylation status of 6 tumor-suppressor genes in 43 patients with FH, 18 patients with FL and 49 patients with DLCL in order to see the differential implication of this epigenetic mechanism in the pathological and the physiological development of GC.MATERIAL AND METHODS. Genomic DNA extracted from paraffin-embedded samples of 43 FH, 18 FL and 49 DLCL after being treated with EZ DNA-Methylation Kit (Zymo Research) with the manufacturer's instructions, were analyzed by methylation-specific polymerase chain-reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, p14, SHP1, MGMT and PDRM1. All samples were obtained mostly from lymph nodes and tonsils. Diagnosis was based on morphology and immunohistochemistry analysis. All cases were matched for age, sex and ethnic origin.RESULTS: DAP-k promoter methylation occurred with higher frequency in FL(89%) than in DLCL(78%) and FH(40%). SHP1 was methylated in 61% of FL, 58% of FH and 23% of DLCL. RARb was methylated in 67% of FL patients, 30% of DLCL and only 12% of FH. Eight (44%) FL, seventeen (35%) DLCL and four (10%) BFH patients showed MGMT methylation. Promoter hypermethylation of p14 was detected only in 5 (12%) FH, 2 (4%) DLCL and none FL patients. Methylation of PRMD1 was present only in 1 (6%) FL, 2 (6%) DLCL and 1 (4%) FH samples.CONCLUSIONS. Inactivacion of DAP-K and SHP1 is present in B-cell malignancies, DLCL and FL, and BFH. Therefore, it may represent a physiologic event conferring a temporal survival advantage necessary for a GC hyperplastic response. Inactivation of the retinoic acid response through the methylation of Rarâ is significantly more frequent in lymphomas than in FH. As reported in other tumors methylation of MGMT is more frequent in lymphomas than in FH. With our data methylation of Cyclin dependent kinase inhibitors p14 is not a differential pathogenic event in lymphomas of GC origin, in fact it is more frequent in FH. Promoter Methylation of PDRM1 is not the mechanism involved in lymphomagenesis in FL and DLCL, the two FH positive deserve further follow-up to determine its significance.

Prognostic significance of aberrant promoter hypermethylation of CpG islands in patients with diffuse large B-cell lymphomas

BackgroundDiffuse large B-cell lymphoma (DLBCL) exhibits heterogeneous clinical features and a marked variable response to treatment. Patients and methodsWe investigated the prognostic significance of the methylation status of DAPK, GSTP1, P14, P15, P16, P33, RB1, SHP1, CDH1, APC, BLU, VHL, TIMP3, and RASSF1A genes in 46 DLBCL specimens from Tunisian patients. Methylation status of each gene was correlated with clinicopathological parameters including the International Prognostic Index (IPI), the germinal center immunophenotype, and response to treatment and survival. Overall survival (OS) and disease-free survival (DFS) rates were calculated by the Kaplan–Meier method and differences were compared with the log-rank test. ResultsHypermethylation of SHP1 was associated with elevated lactate dehydrogenase level (P = 0.031). P16 and VHL were frequently hypermethylated in patients with high IPI scores (P = 0.006 and 0.004) and a performance status of two or more (P = 0.007 and 0.047). In addition, hypermethylation of P16 was significantly associated with advanced clinical stages and B symptoms (P = 0.041 and 0.012). Interestingly, hypermethylation of DAPK was significantly correlated with resistance to treatment (P = 0.023). With regard to survival rates, promoter hypermethylation of DAPK, P16, and VHL were significantly associated with shortened OS (P = 0.003, 0.001, and 0.017, respectively) and DFS (P = 0.006, 0.003, and 0.046, respectively). In multivariate analysis, hypermethylation of DAPK remains an independent prognostic factor in predicting shortened OS (P = 0.001) and DFS (P = 0.024), as well as the IPI and the germinal center status. ConclusionsThis study demonstrates that DLBCLs with hypermethylated P16, VHL, DAPK, and SHP1 commonly show a biologically aggressive phenotype and worse prognosis. Interestingly, hypermethylation of DAPK was found to be an independent prognostic factor that may be used in conjunction with the conventional prognostic factors such as the IPI and the germinal center status.

Promoter Methylation of Multiple Genes in Diffuse Large Cell Lymphoma (DLCL), Follicular Lymphoma (FL) and Bening Follicular Hyperplasia (BFH).

Transcriptional silencing of tumor suppressor genes, associated with DNA methylation, is a common epigenetic event in leukemias and myelodisplastic syndromes. Much less is known about the specific methylation changes that occur in DCLC, FL and their normal counterpart BFH. Methylation of cyclin dependent kinase inhibitors is more common in high grade lymphomas and may be an important step in the progression and transformation of follicular lymphoma. We determined the methylation status of 7 tumor suppressor genes in 31 patients with B-cell malignancies. Seven patients with benign follicular hyperplasia were used as normal controls. The target genes chosen are potentially involved in B-cells malignancies and encoding proteins implicated in apoptosis regulation (death associated protein kinase, DAP-K), Jak/STAT3 signalling pathway (SHP1), hormonal response (RARb), DNA repair (O6-methilguanine-DNA methyltranferase, MGMT), cell cycle control (p14ARF and p15INK4b) and detoxification of environmental xenobiotics such as doxorubicin (glutathione S-transferase P1, GSTP1). Genomic DNA extracted from paraffin-embedded samples of thirty one B-cell malignancies (twenty six DLCL and five FL) were analyzed by methylation-specific polymerase chain reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, MGMT, p14, p15 and GSTP1. Samples were obtained mostly from lymph nodes; spleen, stomach, skin, and parathyroid gland biopsies were also included. Specimens were collected at diagnosis before specific therapy. Diagnosis was based on morphology and immunohistochemistry analysis. All cases were matched for age, sex and ethnic origin. DAP-k promoter methylation occurred with similar frequency in DLCL (100%), FL (100%) and BFH (86%). SHP1 was methylated in 75% of DLCL, all FL and the 7 patients with BFH. RARb was methylated in all DLCL and FL patients and 85% of the BFH. Sixteen (50%) DLCL, three (60%) FL and none BFH patients showed MGMT methylation. Promoter hypermethylation of p14 and p15 was detected in 11 (42%) and 13 (50%) DLCL, 2 (40%) and 3(60%) FL, 3(42%) and 5(71%) BFH patients respectively. Methylation of GSTP1 was absent in all samples. Inactivacion of DAP-K, SHP1 and Rarβ is present in B-cell malignancies, DLCL and FL, and BFH. Therefore, it may represent a physiologic event conferring a temporal survival advantage necessary for a BFH response. With our data methylation of Cyclin dependent kinase inhibitors such as p14 and p15 is not a differential pathogenic event in DLCL with respect to FL or BFH. Finally, the absence of GSTP1 methylation in DLCL and FL questions its relevance in B-cell neoplasia development.

P2-089: Prognostic significance of p16INK4A, DAPK and RASSF1A promoter hypermethylation in non-small cell lung cancer (NSCLC)

P2-089: Prognostic significance of p16INK4A, DAPK and RASSF1A promoter hypermethylation in non-small cell lung cancer (NSCLC)

Promoter hypermethylation of multiple genes in primary gastric lymphoma

Aberrant hypermethylation of CpG islands in the promoter region of tumor suppressor and other important genes in neoplastic cells of lymphoma has been demonstrated to be one of the mechanisms for epigenetic loss of gene function. In this study, we analyzed promoter hypermethylation of the following genes in 49 cases of primary gastric lymphoma (PGL): ATM, p16INK4a(CDKN2A), hMLH1, MGMT, DAPK, and CDH1(ECAD). The PGL cases studied included 26 (53%) cases of diffuse large B-cell lymphoma (DLBCL), 12 (25%) cases of extranodal marginal zone lymphoma (MZL), 7 (14%) cases of MZL with large cell transformation (MZL/DLBCL), 1 (2%) case of follicular lymphoma (FL), one (2%) case of Burkitt-like lymphoma (BL), one case (2%) of lymphoplasmacytic lymphoma (LPL) and one case (2%) of peripheral T-cell lymphoma. Available pathologic data regarding to extragastric involvement at the time of resection of the PGLs were reviewed and correlated. Promoter hypermethylation was detected in 6 of 49 (12.2%) cases for ATM; 13 of 49 (26.5%) for p16INK4a, 19 of 49 (38.8%) for hMLH1; 22 of 49 (44.9%) for MGMT; 27 of 49 (55.1%) for DAPK and 16 of 49 (32.7%) for CDH1. A total of 85% of the PGLs had promoter hypermethylation in at least one of these genes. With different histologic subtypes, promoter hypermethylation of DAPK, hMLH1, and CDH1 genes occurred in 70%, 42%, and 42% respectively for DLBCL, which appeared to be higher than combined MZL and MZL/DLBCL subgroup. Approximately 81% PGLs demonstrated H. pylori infection by immunohistochemistry. H. pylori status did not appear to be statistically correlated with promoter hypermethylation of the genes. Of 37 PGL cases, 19 cases had extragastric involvement at the time of resection, indicating relatively higher stage disease. The frequencies of promoter methylation in those cases were 58% for DAPK, 42% for hMLH1, 37% for CDH1, 26% for p16INK4a and 11% for ATM respectively. The promoter methylation at MGMT gene was significantly higher in the PGLs without extragastric involvement (61%) as compared to those with extragastric involvement (26%).

Hypermethylation of the proapoptotic gene TMS1/ASC: prognostic importance in glioblastoma multiforme

The identification of clinical subsets of glioblastomas (GBM) associated with different molecular genetic profiles had opened the possibility to design tailored therapies to individual patients. One of the most intrigued subtypes is the long-term survival (LTS) GBM, which responds better to current therapies. The present investigation on GBM from 50 consecutive GBM displaying classic survival and seven LTS GBM is based on molecular epigenetic, clinical and histopathological analyses. Our aim was to recognize biomarkers useful to distinguish LTS from classic GBM. We analyzed the promoter methylation status of key regulator genes implicated in tumor invasion (TIMP2, TIMP3), apoptosis and inflammation (TMS1/ASC, DAPK) as well as overall survival, therapy status and tumor pathological features. For the first purpose a methylation-specific PCR approach was performed to analyze the CpG island promoter methylation status of each gene. The overall TMS1/ASC methylation rate in the 57 analyzed tumors was 21.05%. Hypermethylation of TMS1/ASC was significantly more frequent in LTS GBM (57.1% vs. 16%, P=0.029, Fisher's exact test). DAPK promoter hypermethylation was only observed in the LTS subset (14.3%) whereas TIMP2 and TIMP3 were unmethylated in both GBM collectives. Our results strongly suggest that, compared to classic GBM, LTS GBM display distinct epigenetic characteristics which might provide additional prognostic biomarkers for the assessment of this malignancy.

PD-017 Detection of promoter hypermethylation of RARβ, RASSF1A,DAPK and P16/ INK4a genes in cell-free DNA from serum and pleural liquid of patients with pleural effusion

PD-017 Detection of promoter hypermethylation of RARβ, RASSF1A,DAPK and P16/ INK4a genes in cell-free DNA from serum and pleural liquid of patients with pleural effusion