Aberrant Gene Promoter Methylation Research Articles

P-667 Prognostic value of aberrant promoter methylation of DAPK andRASSF1A genes in early stage of non-small cell lung cancer (NSCLC)

P-667 Prognostic value of aberrant promoter methylation of DAPK andRASSF1A genes in early stage of non-small cell lung cancer (NSCLC)

Detection of promoter hypermethylation of RARβ, RASSF1A, DAPK and P16/ INK4a genes in cell-free DNA from serum (s) and pleural liquid (pl) of patients (p) with pleural effusion

9637 Background: Aberrant gene promoter methylation (AM) is a major mechanism for tumour suppressor gene (TSG) silencing in all tumour types and affects their development and aggressiveness.AM of RARβ,RASSF1A, DAPK,P16/INK4a genes have shown to be involved in most tumors. Objectives: 1) to assess the concordance of gene promoter hypermethylation in paired s and pl samples of cancer p with malignant pleural effusion 2) to investigate their role as a marker of pleural effusion malignancy. Methods: We evaluated 53 consecutive p presenting pleural effusion and malignancy(24 lung,11 digestive,8 breast,4 ovary,6 others) at the General Hospital of Alicante (Spain).Hypermethylation of TSG were investigated by using methylation-specific PCR(MSP)after sodium bisulphite treatment. Results: Conventional cytology of pleural effusion samples revealed neoplastic cells in 17 of 48 p (35,5%). AM of at least one of the genes tested was detected in 24 out of 53 p (45,3%) in s and in 31 out of 53 p (58,6%) in pl. In pl 34% had only 1 gene methylated,17, 0% had 2 genes methylated, 5,7% had 3 genes methylated and only 1,9% had 4 genes methylated. In s,14% had only 1 gene methylated,8% had 2 genes methylated and only 2% had 3 genes methylated. Concordance in the pattern of aberrant methylation was found in paired s and pl samples of 13 p. In 11 p, methylation was found only in pl and not in s; only in 3 patients was found in serum and not in pleural liquid. AM of DAPK was found in 7 p (13,2%) in s and 10 p (18,9%) in pl; in 10 p (18,9%) in s and 20 p (37,7%) in pl for P16/INK4a; in 9 p (17%) in s and 8 p (15,1%) in pl for RASSF1A and in 10 p (18,9%) in s and 11 p (20,8%) in pl for RARβ. Correlation coefficient between s and pl methylated genes was r=0,6 p<,0001. Median survival time of p with at least one gene methylated in pl was 12,63±5,95 mo; for p with none of genes methylated median survival has not yet reached. Conclusions: 1) Aberrant gene promoter methylation of TSG can be detected in pleural liquid 2) In pleural liquid, 58,6% of p were found to have at least one TSG methylated vs 35,5% of p with neoplastic cells by conventional cytology 3) Aberrant methylation showed a negative prognostic effect. No significant financial relationships to disclose.

Correction: Article on the Aberrant Promoter Methylation of Cancer-Related Genes in Sporadic Breast Tumors

Correction: Article on the Aberrant Promoter Methylation of Cancer-Related Genes in Sporadic Breast Tumors

Nonrandom distribution of aberrant promoter methylation of cancer-related genes in sporadic breast tumors.

In an effort to additionally determine the global patterns of CpG island hypermethylation in sporadic breast cancer, we searched for aberrant promoter methylation at 10 gene loci in 54 primary breast cancer and 10 breast benign lesions. Genomic DNA sodium bisulfate converted from benign and malignant tissues was used as template in methyl-specific PCR for BRCA1, p16, ESR1, GSTP1, TRbeta1, RARbeta2, HIC1, APC, CCND2, and CDH1 genes. The majority of the breast cancer (85%) showed aberrant methylation in at least 1 of the loci tested with half of them displaying 3 or more methylated genes. The highest frequency of aberrant promoter methylation was found for HIC1 (48%) followed by ESR1 (46%), and CDH1 (39%). Similar methylation frequencies were detected for breast benign lesions with the exception of the CDH1 gene (P = 0.02). The analysis of methylation distribution indicates a statistically significant association between methylation of the ESR1 promoter, and methylation at CDH1, TRbeta1, GSTP1, and CCND2 loci (P < 0.03). Methylated status of the BRCA1 promoter was inversely correlated with methylation at the RARbeta2 locus (P < 0.03). Our results suggest a nonrandom distribution for promoter hypermethylation in sporadic breast cancer, with tumor subsets characterized by aberrant methylation of specific cancer-related genes. These breast cancer subgroups may represent separate biological entities with potential differences in sensitivity to therapy, occurrence of metastasis, and overall prognosis.

Presence of simian virus 40 DNA sequences in human lymphoid and hematopoietic malignancies and their relationship to aberrant promoter methylation of multiple genes.

The simian polyoma virus SV40 has been detected in specific human tumors including non-Hodgkin's lymphomas, although a causative role for the virus has not been convincingly demonstrated. Aberrant methylation of CpG islands in promoter regions is a frequent method of silencing tumor suppressor genes (TSGs) in cancers and may be induced by oncogenic viruses. We investigated the relationship between the presence of SV40 or EBV DNA sequences and the methylation profiles for 10 TSGs in 90 cases of non-Hodgkin's lymphomas/leukemias and 56 control tissues. SV40 sequences were present in 33/90 (37%) non-Hodgkin's lymphomas/leukemias, and EBV was present in 11/42 (26%) of non-Hodgkin's lymphomas. We found a highly significant correlation between the presence of SV40 and methylation of seven genes (P values, 0.006 to <0.0001). In lymphomas, there was no relationship between EBV and methylation. Oncogenic viruses and methylation were rarely present in control tissues. We investigated methylation of the same 10 TSGs in peripheral blood mononuclear cells (PBMC) from a healthy volunteer infected with EBV or EBV and SV40. Promoter methylation of CDH1 and CDH13 were noted in dual SV40- and EBV-infected PBMC, and these two genes were also highly significantly correlated to the presence of SV40 sequences in tumors. SV40 infection also resulted in appearance of the lymphoma/leukemia-specific marker, methylated SHP1. Methylation was completely absent in uninfected and EBV-infected PBMC. Our results demonstrate that the presence of SV40 in hematological malignancies is associated with promoter methylation of TSGs and that in all probability, the virus plays a role in tumor pathogenesis.

Hypermethylation of FHIT as a prognostic marker in nonsmall cell lung carcinoma.

Methylation of CpG islands in the promoter and upstream coding regions has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. The purpose of the current study was to determine the correlation between the aberrant promoter methylation of multiple genes and survival in patients with nonsmall cell lung carcinoma (NSCLC). The methylation status of nine genes was determined in 124 surgically resected NSCLC cases using methylation-specific polymerase chain reaction. The methylation frequencies of the genes tested in NSCLC specimens were 52% for E-cadherin (CDH1), 41% for RAS association domain family protein (RASSF1A), 38% for fragile histidine triad (FHIT) and adenomatous polyposis coli (APC), 27% for retinoic acid receptor beta (RARbeta) and H-cadherin (CDH13), 20% for p16INK4A, 0.8% for O6-methylguanine-DNA-methyltransferase (MGMT), and 0% for glutathione S-transferase P1 (GSTP1). The survival of the patients with FHIT methylation-positive tumors was found to be significantly shorter than that for those patients with methylation-negative tumors (P=0.03), even in those patients with International Union Against Cancer TNM Stage I or Stage II disease (P=0.007). In contrast, there were no significant survival differences noted between the methylation-positive and methylation-negative tumors for the other genes tested. In addition, based on multivariate analyses, FHIT methylation-positive status was found to be independently associated with poor survival (P=0.046) and disease stage (P<0.0001). The results of the current study suggest that methylation of FHIT is a useful biomarker of biologically aggressive disease in patients with NSCLC.

Aberrant methylation of trail decoy receptor genes is frequent in multiple tumor types.

TNF-related apoptosis-inducing ligand (TRAIL) selectively induces programmed cell death (apoptosis) in various cancer cells but not in normal cells. TRAIL is known to bind to 4 different receptors, 2 proapoptotic (DR4 and DR5), and 2 potentially antiapoptotic receptors lacking death domains (DcR1 and DcR2). Aberrant promoter methylation and resultant silencing of tumor suppressor genes play an important role in the pathogenesis of many tumor types. Recently aberrant methylation of TRAIL decoy receptors was reported in pediatric tumor cell lines and neuroblastomas. We examined the methylation and expression status of TRAIL receptor genes in cancers of breast, lung, mesothelioma, prostate, bladder, cervix, ovary, brain and in hematopoietic malignancies. Aberrant methylation of DcR1 or DcR2 was present in 70% of primary breast cancers, 31% of primary lung cancers, in 63% of primary malignant mesothelioma (MM), in 60% of prostate cancer, in 42% of bladder cancer, in 100% of cervical cancer, in 43% of ovarian cancer, in 41% of lymphoma, in 26% of leukemia and in 56% of multiple myeloma. Methylation of DR4 and DR5 was rare in all the tumor types examined. Methylation of all the 4 receptors was rare in non malignant tissues. In cell lines, aberrant methylation of DcR1 was present in 11 of 23 (48%) breast, 10 of 27 (37%) lung and 3 of 7 (43%) MM, whereas aberrant methylation of DcR2 was present in 17 of 23 (74%) breast, 13 of 27 (48%) lung and 5 of 7 (71%) MM. The concordance between loss of gene expression and aberrant methylation ranged from 70-100%. Treatment with 5-aza-2'-deoxycytidine restored DcR1 and DcR2 expression in 9 methylated cell lines confirming that aberrant methylation was the cause for silencing of DcR1 and DcR2 expression. Our results demonstrate that DcR1 and DcR2 genes are frequently methylated in various tumor types, and that the role of decoy receptors in tumor pathogenesis needs to be re-evaluated.

Aberrant CBFA2T3B gene promoter methylation in breast tumors

BackgroundThe CBFA2T3 locus located on the human chromosome region 16q24.3 is frequently deleted in breast tumors. CBFA2T3 gene expression levels are aberrant in breast tumor cell lines and the CBFA2T3B isoform is a potential tumor suppressor gene. In the absence of identified mutations to further support a role for this gene in tumorigenesis, we explored whether the CBFA2T3B promoter region is aberrantly methylated and whether this correlates with expression.ResultsAberrant hypo and hypermethylation of the CBFA2T3B promoter was detected in breast tumor cell lines and primary breast tumor samples relative to methylation index interquartile ranges in normal breast counterpart and normal whole blood samples. A statistically significant inverse correlation between aberrant CBFA2T3B promoter methylation and gene expression was established.ConclusionCBFA2T3B is a potential breast tumor suppressor gene affected by aberrant promoter methylation and gene expression. The methylation levels were quantitated using a second-round real-time methylation-specific PCR assay. The detection of both hypo and hypermethylation is a technicality regarding the methylation methodology.

Tumor suppressor gene hypermethylation as a predictor of gastric stromal tumor behavior.

The growing understanding of the epigenetic changes associated with cancer, including aberrant promoter methylation of tumor suppressor genes that afford selective growth advantages to human neoplasms, suggests that the characterization of gene methylation patterns among gastrointestinal stromal tumors (GISTs) may be useful for predicting tumor behavior. Thirty-eight c-kit-positive gastric stromal tumors were subjected to methylation-specific polymerase chain reaction (MSP) to detect promoter methylation associated with 11 candidate tumor suppressor genes (p16/INK4a, APC, MGMT, hMLH1, p73, E-cadherin, RAR-beta, RASSF1A, RB, ER, and DAPK), established to have a role in tumorigenesis of several solid human organs. Aberrant methylation of any of the 11 candidate tumor suppressor genes was detected in 84% of all GISTs. In decreasing order of frequency, the six most commonly methylated genes were: MGMT (47%), p16 (45%), RASSF1A (40%), E-cadherin (37%), hMLH1 (34%), and APC (31%). For all of the GISTs, promoter methylation was less reliable than tumor mitotic rate in predicting 5-year tumor-free survival for the GISTs; however, E-cadherin methylation was a multivariate prognostic factor for early recurrence of GISTs (50% at 2 years; P=0.030). Among the mitotically active (>5 per 50 high-power field), histologically indistinguishable GISTs, E-cadherin methylation was an independent predictor of tumor-related mortality: 5-year disease-free survival was worse for the E-cadherin methylated GISTs (19%) compared to the E-cadherin unmethylated tumors (71%; P=0.010). Detection of methylation within selected genes may afford a reliable and accurate molecular marker system for predicting neoplastic behavior among GISTs. This study supports the methylation status of E-cadherin as a prognostic marker for early GIST recurrence and survival.

Aberrant gene promoter methylation in acute promyelocytic leukaemia: profile and prognostic significance.

Acute promyelocytic leukaemia (APL) has distinct clinicopathological and molecular features. However, the profile of aberrant gene promoter methylation is undefined. In this study, methylation-specific polymerase chain reaction (MSP) was used to define the methylation status of a panel of nine genes, comprising p15, p16, RARbeta, oestrogen receptor (ER), E-cadherin (E-CAD), p73, caspase 8 (CASP8), VHL and MGMT, in 29 patients with APL. Aberrant methylation of p15, ER, RARbeta, p16 and E-CAD occurred, respectively, in 23 (79%), 14 (48%), six (21%), six (21%) and two (7%) patients at diagnosis, but p73, VHL, CASP8 and MGMT were not methylated in any patients. There was methylation of one gene in 13 patients (45%), two genes in four patients (14%), three genes in six patients (21%) and four genes in three patients (10%). Concurrent methylation of two or more genes occurred in 13 patients (45%). No association was identified between gene methylation and presenting clinicopathological features. However, p15 methylation was significantly associated with an inferior disease-free survival (DFS, P = 0.008), and remained the only poor prognostic factor in multivariate analysis (P = 0.019). In APL, p15, p16, ER and RARbeta were most frequently methylated. This profile is distinct from other types of myeloid leukaemias. p15 methylation has a poor prognostic impact on DFS.

Aberrant p15 gene promoter methylation in therapy-related myelodysplastic syndrome and acute myeloid leukaemia: clinicopathological and karyotypic associations.

Seventeen patients with therapy-related myelodysplastic syndrome/acute myeloid leukaemia (t-MDS/AML) were examined for aberrant p15 gene methylation by methylation-specific polymerase chain reaction. Ten patients (58%) showed p15 methylation, which was significantly related to monosomy/deletion of chromosome 7q, but not to antecedent chemotherapy, blast count, leukaemic evolution or survival. In three of six patients with marrow samples obtained prior to the diagnosis of t-MDS/AML, p15 methylation predated disease development by up to 2 years. Bone marrow transplantation led to the disappearance of p15 methylation in one patient. These results showed that p15 methylation was an early event in the evolution of some t-MDS/AML patients.

Colorectal cancers with both p16 and p14 methylation show invasive characteristics.

Recent studies indicated that p16 and p14 inactivation owing to promoter methylation was important for colorectal tumorigenesis. In this study, we examined the methylation status of these genes in 86 primary colorectal cancers using methylation‐specific PCR (MSP) and correlated the results with the clinicopathological features of the patients. Aberrant promoter methylation of p16 and p14 genes was detected in 43 of 86 (50%) and 25 of 86 (29%) colorectal cancers, respectively. Next, we examined the correlation of methylation status with the clinicopathological features. We found a significant difference in maximal tumor size (P=0.022) when patients with both p16 and p14 methylation were compared to other patients. On the other hand, there was no significant difference in other factors, such as the extent of tumor and Dukes stage. These results suggested that colorectal cancer with both p16 and p14 methylation has the same invasiveness at a smaller size compared to that of the cancer with neither p16 nor p14 methylation. Inactivation of both p16 and p14 genes may result in a malignant change in colorectal cancer cells, leading to advanced cancers with a smaller size than those with p16 or p14 activity.

Surfactant protein DNA methylation: A new entrant in the field of lung cancer diagnostics? (Review)

Lung cancer is a major cause of cancer-related mortality in both men and women. A 5-year survival of lung cancer patients is only 15% with a negative correlation between progressively advanced lung cancer stage and a 5-year survival period. The only chance for cure is surgical resection if done at the early stage of the disease. Therefore, an early diagnosis and a better prediction of prognosis could decrease mortality. An early diagnosis could provide the opportunity for a therapeutic intervention early in the course of the disease. Genetic alterations in the cancer genome include aneuploidy, deletions and amplifications of chromosomal regions, loss of heterozygosity (LOH), microsatellite alterations, point mutations and aberrant promoter methylation. Of the various types of genetic alterations (i.e. gene amplifications, allele deletions, point mutations or deletions and methylation) reported in different tumor types, aberrant promoter methylation of genes is recent and is the focus of the present review. Specifically, we will briefly review the role of promoter methylation in various malignancies and then focus on lung cancer diagnosis and promoter gene methylation with emphasis on the methylation status of genes of the innate host defense, namely the surfactant proteins A and D.

The flip side of DNA methylation

Gregory L. Verdine Department of Chemistry Harvard University Cambridge, Massachusetts 02138 DNA methylation has come of age. For more than 40 years, it has been known that eukaryotes tag their DNA by the covalent addition of a methyl group to cytosine residues; however, until very recently, the functional significance of this modification has remained on a precariously specula- tive footing. A series of discoveries over the last few years has thrust DNA methylation firmly into the mainstream of biology and medicine, thereby invigorating the field with afirmlyestablished sense of mission. Fragile X syndrome, the leading cause of inherited mental retardation, has been traced to expansion and abnormal methylation of a triplet repeat, through which transcription of the FMR-7 gene becomes silenced (Trottier et al., 1993). Aberrant promoter methylation of tumor suppressor genes has now emerged as an epigenetic inactivation pathway contribut- ing to tumorigenesis; evidence increasingly supports the notion that ectopic methylation may play a broad role in gene inactivation and mutation in mammals (reviewed by Bestor and Coxon, 1993). Much excitement has revolved around the roleof DNA methylation in genomic imprinting, a phenomenon in which parental alleles of the same gene are expressed at unequal dosages (Barlow, 1993). Abnor- rnal expression of imprinted loci is now implicated in sev- eral human disorders (Ogawa et al., 1993; Rainier et al., 1993; Davies, 1992), and more instances seem likely to be discovered. The entire field of DNA methylation, with its foundations resting largelyon the strength of correlative clata, took both a collective breath of relief and a bold step forward with the direct demonstration of Li et al. (1992) that the DNA MTase gene is essential for development in mice. A molecular mechanism for spatiotemporal coupling of DNA replication and methylation has been suggested by the finding that DNA MTase interacts directly with the replication apparatus (Leonhardt et al., 1992). Just as these advances represent the dawning of a new era in the biology of DNA methylation, so does remarkable progress in the alter ego of this scientific problem-its chemistry-also foretell a bright future. A turning point in the chemistry of DNA methylation is ushered in by the cocrystal structure of a DNA methyltransferase bound co- valently to DNA, which is reported by Klimasauskas et al. (1994 [this issue of CeIj). More generally speaking, this