DAPK Methylation Research Articles

Tumor suppressor gene CpG site methylation and outcome in surgically treated stage I-II non-small cell lung cancer patients

11035 Background: Five year survival in completely resected non small cell lung cancer (NSCLC) stage I-II patients approximates 60%. Presently, no adequate prognostic indicators accurately predict outcome. To explore epigenetic influences, promoter methylation (silencing) at multiple CpG dinucleotides of tumor suppressor genes with roles in metastasis and survival was analyzed. Methods: Methylation was quantified at specific CpG sites in p16, MGMT, DAPK, RASSF1, ECAD, LET7–3-a, NORE1(RASSF5) and PTEN promoters in assessable tumor tissue from 88 surgically treated NSCLC patients by pyrosequencing. Results: Overall average promoter methylation ranged from 9.27+6.04% (DAPK) to 71.3+10.5% (LET7–3-a). Of the genes analyzed, RASSF1 showed significant association between overall average promoter methylation and increased recurrence (p=0.024). Upon comparison of methylation at specific CpG sites and any recurrence, MGMT methylation at CpG position 17 in exon1 and DAPK methylation at CpG site -1503 (A of the first ATG codon = +1) were significantly associated with higher recurrence (p=0.030, p=0.031, respectively). NORE1 methylation at -224 and -244 were significantly associated with no recurrence (p=0.024, p=0.030, respectively). Kaplan-Meier survival analysis revealed significantly lower time to regression [TTR; 9.80 vs. 55.2 months (mo), Log Rank p=0.003] and overall survival (OS, p=0.001) with MGMT methylated at exon 1 position 17. Significantly lower TTR and OS were also observed with MGMT methylated at exon 1 position 24 (p=0.020, p=0.017, respectively). In contrast, methylation at -224 in the NORE1 promoter was associated with longer median TTR (p=0.053). Conclusions: These data support an influence of specific CpG site methylation and outcome after surgical resection for stage I and II NSCLC. Methylation was generally associated with shorter TTR and OS, however methylation of particular NORE1 sites was associated with better outcome in this patient group. The effects of methylation of specific promoter sites may reflect accessibility to methylases and/or an interaction of transcription factors required for control of expression of these genes, phenotypically impacting metastatic potential and recurrence. No significant financial relationships to disclose.

Use of Ras effector RASSF1A promoter gene methylation and chromosome 9p loss of heterozygosity (LOH) to predict progression-free survival (PFS) in perioperative chemotherapy (CT) phase III trial IFCT-0002 in resectable non-small cell lung cancer

7500 Background: IFCT-0002 phase 3 trial compared two timings of CT in early lung cancer, all before surgery (PRE) versus PERIoperative, and two CT regimens, CDDP-Gem vs. CBDCA-Pac. 528 patients were randomized. Early results showed no difference between the two CT regimens for response rate or tolerance, but a better compliance in PRE vs. PERI arm. We hypothesized that molecular markers could help to define the subgroups that did not beneficiate from CT. Methods: Molecular alterations in key pathways regulating G1/S transition or apoptosis were screened. TP53, EGFR and K-RAS mutations, RASSF1A, DAPK, ECAD, FHIT, TIMP3 and p16 promoter gene methylation, 3p, 17p and 9p LOH were evaluated in 178 patients with snap-frozen tissue collected at time of surgery. Results: K-ras mutations identified in 16.3% of patients, was the only molecular alteration associated with the absence of tumor response to chemotherapy (crude RR = 2.13 95%CI [1.11–4.55], p=0.019). However, K-ras mutations were not associated with PFS (p=0.89). RASSF1A promoter gene methylation found in 22.2% of patients was not mutually exclusive with K-Ras mutations. Although not associated with tumor response, RASSF1A promoter gene methylation (adjusted HR = 2.34 95%CI [1.46–3.75], p=0.0013) and 9pLOH (adjusted HR = 2.28 95%CI [1.25–4.15], p=0.015) predicted a worse PFS independently of T, Stage, PS and histology, with respectively 14 vs. 64 months and 18 vs. 36 months of median PFS. Alterations of both RASSF1A and 3p LOH, or RASSF1A plus FHIT methylation or 3p LOH, also predicted a worse PFS (respective adjusted HR 2.46 and 2.22, p= 0.024 and 0.031). There only was a non significant trend for alterations of both p53 and 17p, predicting a worse PFS (18 vs. 36 months of median PFS, p=0.13), with a negative impact for disruptive p53 mutations. Conclusions: This study showed a negative prognostic impact for alterations of RASSF1A in early lung cancer. In resectable patients receiving neoadjuvant CT, a limited set of apoptosis or proliferation alterations could represent strong negative prognostic factors. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Eli Lilly France Eli Lilly

Methylated TMS1 and DAPK genes predict prognosis and response to chemotherapy in gastric cancer

Gastric cancer is the second most common cause of cancer deaths worldwide. The identification of molecular genetic parameters that are associated with response to chemotherapy and prognosis is of utmost interest. We examined methylation of the apoptosis-related genes, TMS1 and DAPK, in 81 primary gastric cancers using methylation-specific PCR and compared their methylation status with clinicopathological findings. Aberrant methylation of TMS1 and DAPK genes was detected in 26 (32.1%) tumors and in 18 (22.2%) tumors, respectively. The overall survival of patients with both methylated genes was significantly shorter compared with those with only one methylated gene or no methylated genes (p = 0.0003). Neither gene methylation had any relation to other clinicopathological findings. Next, we examined 43 patients treated by 5-fluorouracil-based chemotherapy, who had distant metastasis or recurrence after radical resection, to determine the relation between chemosensitivity and methylation. The response rate was lower in patients with either methylation than without (TMS1: 22.2% vs. 48.0%; DAPK: 21.4% vs. 44.8%). Overall survival tended to be shorter in the patients with both methylations compared with either or no methylations (p = 0.0806). The time to progression of patients with methylation of TMS1 or DAPK was significantly shorter than patients without methylation (TMS1: p = 0.0123; DAPK: p = 0.0464). Furthermore, the time to progression of patients with both methylated genes was significantly shorter than patients with one methylation or no methylation (p = 0.0082). In conclusion, TMS1 and DAPK methylation might predict the prognosis and response to chemotherapy in gastric cancer.

Quantitative hypermethylation of a small panel of genes augments the diagnostic accuracy in fine-needle aspirate washings of breast lesions

We hypothesized that comprehensive breast cancer methylation profiling might provide biomarkers for diagnostic assessment of suspicious breast lesions using fine needle aspiration biopsy (FNA). Twenty-three gene promoters were surveyed by quantitative methylation-specific PCR in bisulfite-modified DNA from 66 breast carcinomas (BCa), 31 fibroadenomas (FB) and 12 normal breast (NT) samples to define a set of genes differentially methylated in malignant and non-malignant tissues. This set was tested in 78 FNA washings obtained pre-operatively (66 malignant, 12 benign), with histopathological diagnosis. Receiver operator characteristic (ROC) curve analysis identified a gene panel which might distinguish cancer from non-cancerous lesions. Finally, this panel was validated in an independent series of FNA washings (45 cases) in which cytomorphology did not reach definitive diagnosis. In tissue samples, 14-3-3-sigma, DAPK, CCND2, RASSF1A, CALCA, APC, HIN1, RARbeta2, TIG1, and GSTP1 methylation levels differed significantly among BCa, FB, and NT. ROC curve analysis identified a panel of four gene loci (CCND2, RASSF1A, APC, and HIN1) that discriminated BCa from benign lesions in a set of 78 FNA washings from histologically characterized breast lesions. When this panel was tested in the validation dataset of 45 FNA washings, breast cancer was identified with perfect specificity (100%) when 3 of 4 gene loci tested positive, providing estimated added information of 91% over cytomorphologic evaluation alone. Our data provide evidence that multigene methylation analysis augments diagnostic accuracy of cytological assessment of suspicious breast lesions, and might be a valuable ancillary tool for breast cancer diagnosis.

Implications of Methylation Patterns of Cancer Genes in Salivary Gland Tumors

We investigated the methylation status and protein expression of four tumor suppressor genes to determine their role in salivary gland tumorigenesis. We performed methylation-specific PCR and protein analyses of 29 normal salivary glands, 23 benign, and 79 malignant salivary gland neoplasms to determine the pattern and potential diagnostic and/or biological role of the RASSF1, RARbeta2, DAPK, and MGMT tumor suppressor gene methylation in these tumors. No methylation was detected in the normal tissues. Methylation occurred in 9 of 23 (39.1%) benign tumors; 3 (25.0%) pleomorphic adenomas and 6 (66.7%) Warthin's tumors at the MGMT, DAPK, or RASSF1 genes. Methylation occurred in 33 of 79 (41.8%) malignant tumors; 8 (30.8%) adenoid cystic carcinomas, 6 (33.3%) mucoepidermoid carcinomas, 6 (42.9%) acinic cell carcinomas, and 13 (62.0%) salivary duct carcinomas. RASSF1 and RARbeta2 represented 75.8% of methylation events occurring most frequently in salivary duct and acinic cell carcinomas. Overall, we found no significant correlation between protein expression and methylation status of individual genes, but observed low or absent protein expression in several methylated tumors. Significant correlations were found between methylation and aggressive malignant phenotypes (P = 0.0004) and age (P = 0.05). (a) Benign and malignant salivary tumors differed in the frequency and pattern of gene methylation; (b) high-grade carcinomas were significantly methylated compared with low-grade phenotypes; (c) RASSF1 and RARbeta2 were highly methylated in malignant tumors and can be targeted for therapy; and (d) methylation pattern may serve as a diagnostic and biological marker in assessing these tumors.

APC gene methylation is inversely correlated with features of the CpG island methylator phenotype in colorectal cancer

The notion of a CpG island methylator phenotype (CIMP) was proposed to describe a subset of colorectal cancers (CRC) displaying frequent and concordant methylation of CpG islands located within gene promoter regions. Some workers have failed to observe associations between CIMP and specific clinicopathological features of CRC, possibly because of the choice of genes used to define this phenotype. The aim of the current study was to determine whether the aberrant methylation of 6 genes implicated in CRC development was associated with the same phenotypic features of this tumour type. The MethyLight assay was used to provide quantitative estimates of MLH1, P16, TIMP3, P14, DAPK and APC methylation levels in 199 unselected colorectal tumours. The methylation of MLH1, P16, TIMP3 and P14 was highly concordant (p < 0.0001 for each pair) but that of DAPK and APC was not. An inverse association was observed between the methylation of APC and TIMP3 (p = 0.004). Methylation of the MLH1, P16, TIMP3 and P14 genes was associated with tumour infiltrating lymphocytes (p < 0.05), microsatellite instability (p < 0.001), BRAF mutation (p < 0.0001) and elevated concentrations of the methyl group carriers tetrahydrofolate (THF) and 5,10-methylene THF (p < 0.05). In contrast, APC methylation was associated with wildtype BRAF (p = 0.003) and with lower concentrations of methyl group carriers (p < 0.05). These findings highlight the importance of gene selection in studies that aim to characterize the biological features and clinical behaviour of CIMP+ tumours.

DNA Methylation Changes in Plasma Cell Discrasias.

Introduction: Aberrant methylation of the 5′ gene promoter regions is an epigenetic phenomenon that is the one of the major mechanism of inactivation of tumor supressor genes. DNA methylation of the promoter region has been described for several genes in various malignant diseases, and each tumour type may have its own pattern of methylation. To determine the methylation status and expression of cell cycle inhibitors genes (p14, p15, p16), repair genes (MGMT and hMLH1) and the apoptosis regulator gene (DAPKinase) and the possible role of this epigenetic phenomenon in tumour progression of plasma cell disorders, we analyzed the methylation profile of MM, MGUS, and plasmacytomas comparing them with their protein expression.Patients and Methods: A total of 51 cases: 30 MM, 13 MGUS, and 8 plasmacytomas (3 Solitary Bone Plasmacytoma, 5 Extramedullary Plasmacytoma) were included in the study. Bone marrow plasma cells were purified using magnetic microbeads labeled with CD138 in samples with MM and MGUS. Methylation-specific polymerase chain reaction (MSP) for p14, p15, p16, MGMT, hMLH1 and DAPKinase was performed. MSP results were matched to protein expression studies by immunohistochemistry for p15, p16, MGMT and hMLH1.Results: The frequency of aberrant methylation among the MM samples was: 50% for p16, 16.7% for p15, 10% for hMLH1, 23.3% for MGMT, 30% for DAPK. In MGUS samples we found 38.5% for p16, 15.4% for p15, 0% for hMLH1, 7.7% for MGMT and 15.4% for DAPK methylation. The frequency of methylation in plasmacytomas was 62.5% for p16, 25% for p15, 0% for hMLH1, 62.5% for MGMT and 50% for DAPK. p14 was unmethylated in all cases (n=51). The correlation between gene methylation status and protein expression was assessed in 17 MM, 11 MGUS, and 8 plasmacytomas and we found that promoter methylation was strongly associated with gene silencing. All the samples methylated had lost the protein expression. In summary these findings demonstrate that aberrant methylation is an important mechanism of gene silencing in plasma cell disorders: 83.3% of MM, 46.1 of MGUS, and 75% of plasmacytomas have at least one hypermethylated gene (figure 1). We also show, that hypermethylation of p16 is a common phenomenon in plasma cell discrasias while there was no methylation of p14. Although the size of sample is small, we found that hMLH1 hypermethylation was found only in MM, while in plasmacytomas hypermethylation of MGMT and DAPK were frequent events. Moreover in survival studies of MM patients, a trend was observed between simultaneous aberrant methylation of hMLH1 and MGMT and poorer survival, but the number of cases studied limits the statistical analysis and the clinical implications of these results. To better define the clinical impact of methylation markers in plasma cell discrasias, it is therefore necessary to analyze a large number of patient samples. [Display omitted]

Promoter methylation of genes in bronchial lavages: A marker for early diagnosis of primary and relapsing non-small cell lung cancer?

A prospective screening program, including CT, autofluorescent bronchoscopy, biopsies and bronchial lavage (BL) collection, was initiated with the specific goal of identifying biomarkers for the early detection of non-small cell lung cancer. We report and discuss the results of p16, DAPK, MGMT, FHIT and APC methylation analysis in the 126 first patients: 77 at high risk of cancer and 49 followed up after primary cancer resection. Positive results were found in 49% of BLs, 53% in current smokers and 43% in former smokers. In presence of peripheral tumours, only 38% of BLs were abnormal versus 73% in presence of central tumours, 50% in presence of preneoplasic lesions and 47% in absence of lesions. FHIT methylation was an early event, observed in one-third of the BLs from patients with or without lesions as well as in tumours. APC methylation was a late event observed in 33% of tumours but rarely in BLs. p16 was methylated in 17% of BLs but in 48% of tumours; DAPK in 15% of BL and 22% of tumours. MGMT methylation was rare. Among patients followed up after cancer surgery, 14 were in remission with normalised BL, whereas three had positive BLs and relapsed with a central tumour. Thus, gene methylation in BL might help to detect central tumours but a CT is crucial for peripheral cancer detection.

Prognostic Factors in Resected Stage I Non–Small-Cell Lung Cancer: A Multivariate Analysis of Six Molecular Markers

To analyze the prognostic significance of six molecular biomarkers (death-associated protein kinase [DAPK] promoter methylation, interleukin-10 [IL-10] protein expression, cyclooxygenase-2 [COX-2] mRNA expression, human telomerase reverse transcriptase catalytic subunit [hTERT] mRNA expression, retinoic acid receptor-beta [RAR-beta] mRNA expression, and K-ras mutational status) in stage I non-small-cell lung cancer (NSCLC) patients. Biomarker analyses were performed on tumors from 94 patients with stage I NSCLC who underwent surgical resection at our institution. A minimum follow-up period of 5 years was required. DAPK methylation was assessed by methylation-specific polymerase chain reaction (PCR). RAR-beta, COX-2, and hTERT mRNA levels were determined by in situ hybridization with digoxigenin-labeled antisense riboprobes. K-ras mutation status was determined by the PCR-primer introduced restriction with enrichment for mutant alleles method. IL-10 protein expression was analyzed by immunohistochemistry using a polyclonal antihuman IL-10 antibody. Cancer-specific survival was analyzed with a Cox proportional hazards model. To identify independent prognostic factors, a stepwise selection method was used. DAPK methylation, IL-10 lack of expression, COX-2 expression, hTERT expression, RAR-beta expression, and K-ras mutations were observed in 46.8%, 29.8%, 59.6%, 34.0%, 23.4%, and 34.0% of patients, respectively. In the final model, DAPK methylation and IL-10 lack of expression were significant negative prognostic factors for cancer-specific survival, whereas COX-2 expression was of borderline significance. In this cohort of resected stage I NSCLC patients, molecular markers that independently predict cancer-specific survival have been identified. The prognostic roles of DAPK methylation, IL-10, and other biomarkers in NSCLC merit further investigation.

Multigene methylation analysis of Wilms' tumour and adult renal cell carcinoma.

To investigate the role of epigenetic gene silencing in the pathogenesis of Wilms' tumour and renal cell carcinoma (RCC), we determined their methylation profile using a candidate gene approach. Thus, 40 Wilms' tumours and up to 49 adult RCC were analysed by methylation-specific PCR for promoter methylation at CASP8, CDH1, CDH13, DAPK, MGMT, NORE1A, p14ARF and RARB2 in primary Wilms' tumours and CASP8, CDH1, CDH13, CRBP1, DAPK, MGMT, MT1G, NORE1A, p16INK4a, SDHB and RARB2 in primary RCC. Both tumour sample sets had previously been analysed for RASSF1A promoter methylation, and p16INK4a methylation results were also available for the Wilms' tumour samples. Wilms' tumours demonstrated a high incidence of methylation at CASP8 (43%) and MGMT (30%), intermediate frequencies at NORE1A (15%), p14ARF (15%), p16INK4a (10%), DAPK (11%) and CRBP1 (9%), but promoter methylation was rare or absent at RARB2 (0%), CDH13 (0%) and CDH1 (3%). No association was detected between methylation of RASSF1A, CASP8 or MGMT in individual tumours. The frequency of MGMT methylation was higher in stage 1 and 2 tumours (50%) than in stage 3 and 4 tumours (17%) but this did not reach statistical significance (P=0.06). RCC were most frequently methylated at DAPK (24%), MT1G (20%), NORE1A (19%), CDH1 (16%) and MGMT (9%) and not or rarely at SDHB (4%), RARB2 (0%), p16INK4a (0%) and CDH13 (3%). There were no associations between methylation of RASSF1A, DAPK and CDH1 in individual tumours. Papillary RCC demonstrated a higher frequency of DAPK methylation (43%) than clear cell tumours (19%) (P=0.14). We have demonstrated that de novo promoter methylation is frequent in Wilms' tumour and RCC, and these data enable methylation profiles to be constructed for each tumour type. Thus, combining our results with data published previously, it appears that promoter methylation occurs frequently (> or =20% of primary tumours) at CASP8, SLIT2 and RASSF1A in Wilms' tumour and at RASSF1A, TIMP3, DAPK, SLIT2, MT1G and GSTP1 in RCC.