Article

Lack of prognostic significance of serum DNA methylation of DAPK, MGMT, and GSTPI in patients with non-small cell lung cancer

Authors:
  • KMG Klinik Silbermühle GmbH
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Abstract

To further improve the screening, diagnosis and therapy of patients with non-small cell lung cancer (NSCLC) additional diagnostic tools are desperately warranted. Aim of this study was to investigate the potential of the DNA methylation of DAPK, MGMT, and GSTPI in serum of patients with NSCLC as a prognostic molecular marker in this disease. Seventy-six patients with NSCLC were included in this study. The analysis of DNA methylation in serum of patients was performed on pre-operative samples. Following DNA isolation and bisulfite-treatment, DNA methylation was analyzed by quantitative-methylation-specific real-time PCR with beta-actin as the internal reference gene. DNA methylation was detectable with following frequencies: DAPK 68.4%, MGMT 7.9%, GSTPI 0%. There were no associations between DNA methylation status and histology, tumor stage, grading or gender detectable. With a mean follow-up of 19.7 months the median survival was 26.3 months. There were no associations between the status of DNA methylation in patient's serum and prognosis detectable. The analysis of DNA methylation in serum of patients with NSCLC by quantitative-methylation-specific real-time PCR is technically feasible. Although our results suggest quantification of DNA methylation in serum not of prognostic significance in this disease, further studies are warranted to determine the future potential of this molecular approach.

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... In our study, we detected DAPK hypermethylation in serum DNA of 30.6% of lung cancer patients, which is similar to an earlier published report where 30.8% of lung cancer patients had a methylated DAPK gene in serum (32). In contrast, other studies reported methylation of DAPK in cell-free DNA ranging from 11 (33) to 68.4% (34). The presence of methylated DAPK gene in the serum of cancer-free controls in our study is also supported by another study (33). ...
... In earlier published studies, GSTP1 gene was found to be methylated in 9 (7), 10 (11), and 25% (36) of lung tumors, and in 2% of serum of lung cancer patients (7). In other studies, GSTP1 was found to be rarely methylated in lung cancer tissue (25,29,30,36), in serum of lung cancer patients (34), in matched non-cancerous tissue (29,35), and in blood of lung cancer patients (30). Since in our study we have found almost similar frequency of GSTP1 hypermethylation in tissue biopsy from lung cancer and controls, such hypermethylation may be smoke-induced epigenetic change rather than an early event in carcinogenesis. ...
Article
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Promoter DNA hypermethylation of APC, DAPK, and GSTP1 genes was evaluated in biopsy and matched serum of 160 lung cancer patients and 70 controls. In biopsy, 83.1, 83.1, and 78.1% of lung cancer patients and 72.9, 70, and 70% of controls, while in serum, 52.5, 30.6, and 65.6% of lung cancer patients and 14.3, 18.6, and 30% of controls were positive for APC, DAPK, and GSTP1 hypermethylation respectively. We couldn't find any significant role of DNA hypermethylation in lung cancer. However, long follow-up of methylation positive controls will be required to confirm its role for the prediction of lung cancer.
... Nevertheless, inconsistent data have emerged regarding the ability of MGMT promoter methylation to predict survival in LC. Multiple studies failed to achieve statistical significance on this association in a multivariate analysis [16][17][18][19][20][21][22]; however, two studies have shown that LC patients with MGMT promoter methylation have worse overall survival (OS) [23,24]. ...
... Nine of the studies identified met the inclusion criteria. They were published between 2003 and 2014, and 859 patients were included in the pooled analysis [16][17][18][19][20][21][22][23][24]. Table 1 lists the identified studies and their main characteristics. ...
Article
The role of MGMT promoter methylation in lung cancer (LC) remains controversial. To clarify the association of MGMT promoter methylation with survival in LC, we performed a meta-analysis of the literature with meta-analysis. Trials were selected for further analysis if they provided an independent assessment of MGMT promoter methylation in LC and reported the survival data in the context of MGMT promoter methylation status. Subgroup analyses were conducted according to the study characteristic. A total of 9 trials, which comprised 859 patients, were included in the meta-analysis. The combined hazard ratio (HR) of 1.27 [95% CI 0.88-1.82; test for heterogeneity P = 0.027] suggests that MGMT promoter methylation has none impact on patient survival. In Stage I-III or younger populations, a significant association was found for MGMT promoter methylation in the prognosis of LC. In addition, the heterogeneity disappeared when the analysis was restricted to Stage I-III LC. Our analysis indicates that MGMT promoter methylation in stage I-III or younger patients was significantly correlated with wore survival. Further study is needed to determine these specific subgroups of LC patients.
... 9,10 Several studies indicated that DAPK genes' promoter methylation occurred frequently in NSCLC, and has a tight relationship with occurrence and development of NSCLC. [11][12][13][14][15] However, the results were varied from different studies, and the clinicopathological significance of DAPK promoter hypermethylation remains unclear. In this study, 41 eligible studies were pooled, and we performed a meta-analysis trying to provide a more accurate estimate of the clinicopathological significance of DAPK promoter hypermethylation in NSCLC, including age, gender, smoking status, histological type, differentiation, tumor node metastasis (TNM) stage, and prognostic outcomes. ...
Article
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Background Death-associated protein kinase (DAPK) has a strong function of tumor suppression involving apoptosis regulation, autophagy, and metastasis inhibition. Hypermethylation of CpG islands in DAPK gene promoter region is one of the important ways to inactivate this tumor suppressor gene, which might promote lung carcinogenesis. However, the clinicopathological significance of the DAPK promoter hypermethylation in lung cancer remains unclear. In this study, we performed a meta-analysis trying to estimate the clinicopathological significance of DAPK promoter hypermethylation in non-small cell lung cancer (NSCLC). Methods A detailed literature search for publications relevant to DAPK gene promoter methylation and NSCLC was made in PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure, CSTJ, Wanfang databases, and SinoMed (CBM). The random-effects model and fixed-effects model were utilized to pool the relative ratio based on the heterogeneity test in the meta-analysis. Results A total of 41 studies with 3348 patients were included. The frequency of DAPK methylation was significantly higher in NSCLC than in non-malignant control (odds ratio (OR) = 6.88, 95% confidence interval (CI): 4.17–11.35, P < 0.00001). The pooled results also showed that DAPK gene promoter hypermethylation was significantly associated with poor prognosis for overall survival in patients with NSCLC (hazard ratio: 1.23, 95% CI:1.01–1.52, P = 0.04). Moreover, DAPK gene promoter hypermethylation was significantly associated with squamous cell carcinoma (OR: 1.25, 95% CI: 1.01–1.54, P = 0.04) and smoking behavior (OR: 1.42, 95% CI: 1.04–1.93, P = 0.03) but not with TNM stage, tumor differentiation, age, or gender. Conclusion DAPK promoter hypermethylation might be a candidate diagnostic and prognostic tumor marker for NSCLC.
... Primer and probe design for each of the 10 genes was based on previous reports and is detailed in Supplementary Table 1. [10][11][12][13] Promoter methylation was reported as a methylation value percentage (MVP) with tumor suppressor gene levels normalized to ß-Actin in modified DNA. ...
Article
Introduction: While retrospective analyses support an association between early tumor recurrence and tumor suppressor gene promoter methylation in early-stage non-small-cell lung cancers (NSCLCs), few studies have investigated this question prospectively. Methods: Primary tumor tissue from patients with resected pathologic stage I to IIIA NSCLCs was collected at the time of surgery and analyzed for promoter methylation via methylation-specific reverse transcriptase polymerase chain reaction (MethyLight). The primary objective was to determine an association between promoter methylation of 10 individual tumor suppressor genes (CDKN2A, CDH13, RASSF1, APC, MGMT, GSTP1, DAPK1, WIF1, SOCS3, and ADAMTS8) and recurrence-free survival (RFS), with the secondary objectives of determining association with overall survival (OS), and relation to clinical or pathologic features. Results: A total of 107 patients had sufficient tumor tissue for successful promoter methylation analysis. Majority of patients were former/current smokers (88%) with lung adenocarcinoma (78%) and pathologic stage I disease (62%). Median follow-up was 4 years. When controlled for pathologic stage, promoter methylation of the individual genes CDKN2A, CDH13, RASSF1, APC, MGMT, GSTP1, DAPK1, WIF1, and ADAMTS8 was not associated with RFS. Promoter methylation of the same genes was not associated with OS except for DAPK1 which was associated with improved OS (p = 0.03). The total number of genes with methylated promoters did not correlate with RFS (p = 0.89) or OS (p = 0.55). Conclusion: Contrary to data established by previous retrospective series, tumor suppressor gene promoter methylation (CDKN2A, CDH13, RASSF1, APC, MGMT, GSTP1, DAPK1, WIF1, and ADAMTS8) was not prognostic for early tumor recurrence in this prospective study of resected NSCLCs.
... Blood represents the most accessible clinical specimen as it is routinely collected in clinical practice. Both plasma and serum have been examined for DNA methylation abnormalities in lung cancer patients [51,53,[64][65][66] suggesting a number of potential diagnostic targets. A panel of genes (APC, RASSF1A, CDH13, KLK10 and DLEC1) tested in plasma from NSCLC patients demonstrates 84% sensitivity and 74% specificity [67]. ...
Article
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... As a positive regulator of apoptosis (21), the DAPK gene participates in numerous transduction pathways that are mediated by p53, TNF-α, Fas, TNF-β and other apoptotic factors, which also undergo promoter hypermethylation in various types of tumors. An analysis of DNA hypermethylation in the serum of patients with non-small-cell lung carcinoma has shown that the DAPK hypermethylation frequency was 68.4% (22). DAPK is frequently methylated in malignant mesothelioma, which is associated with gene silencing and may be of prognostic significance (23). ...
Article
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Apoptosis protease activating factor-1 (Apaf-1) and death-associated protein kinase (DAPK) are p53 pathway-related genes that play significant roles in the activation of caspases, which are involved in mitochondrial-mediated apoptosis. The present study aimed to confirm the role of hyper-methylation of the Apaf-1 and DAPK gene promoter regions in oral squamous cell carcinoma (OSCC) and the effect of the demethylation drug, 5-aza-2'-deoxycytidine (DAC). mRNA from 53 OSCC samples, 23 normal oral mucosa samples and Tca8113 human tongue carcinoma cell lines was detected using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The DNA from each sample was analyzed using methylation-specific PCR (MSP). The Tca8113 cells were demethylated using DAC and the demethylation and re-expression of Apaf-1 and DAPK were analyzed. The Apaf-1 and DAPK mRNA expression index was decreased in 51 (96.23%) and 50 (94.34%) cases, respectively, in the tumor tissues. Hypermethylation of the Apaf-1 and DAPK promoter regions was detected in 46 (86.79%) and 38 (71.69%) cases, respectively. Promoter hypermethylation of the two genes correlated with a decreased mRNA expression in the tumor tissues. Subsequent to being treated with DAC, Apaf-1 and DAPK were demethylated and re-expressed in the Tca8113 cells. Apaf-1 and DAPK promoter hypermethylation may be associated with low gene expression in OSCC. Furthermore, a loss of Apaf-1 and DAPK expression may recover following demethylation. The data provide evidence that methylation exists in OSCC and may play a role in the development of this disease.
... Over the last few years, research on methylation markers has mainly focused on their usefulness as prognostic, predictive, and monitoring indicators. The results of some recent exemplary studies are given in Table 3 [97,192,193,195,[206][207][208][209][210][211][212][213]. These data illustrate the potential of these markers, especially as tools for the risk stratification of patients and for the prediction of responses to certain types of chemotherapies. ...
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The molecular basis of aberrant hypermethylation of CpG islands observed in a subset of human colorectal tumors is unknown. One potential mechanism is the up-regulation of DNA (cytosine-5)-methyltransferases. Recently, two new mammalian DNA methyltransferase genes have been identified, which are referred to as DNMT3A and DNMT3B. The encoded proteins differ from the predominant mammalian DNA methyltransferase DNMT1 in that they have a substantially higher ratio of de novo to maintenance methyltransferase activity. We have used a highly quantitative 5' nuclease fluorogenic reverse transcription-PCR method (TaqMan) to analyze the expression of all three DNA methyltransferase genes in 25 individual colorectal adenocarcinoma specimens and matched normal mucosa samples. In addition, we examined the methylation patterns of four CpG islands [APC, ESR1 (estrogen receptor), CDKN2A (p16), and MLH1] to determine whether individual tumors show a positive correlation between the level of DNA methyltransferase expression and the frequency of CpG island hypermethylation. All three methyltransferases appear to be up-regulated in tumors when RNA levels are normalized using either ACTB (beta-actin) or POLR2A (RNA pol II large subunit), but not when RNA levels are normalized with proliferation-associated genes, such as H4F2 (histone H4) or PCNA. The frequency or extent of CpG island hypermethylation in individual tumors did not correlate with the expression of any of the three DNA methyltransferases. Our results suggest that deregulation of DNA methyltransferase gene expression does not play a role in establishing tumor-specific abnormal DNA methylation patterns in human colorectal cancer.
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Cytosine-5 DNA methylation occurs in the context of CpG dinucleotides in vertebrates. Aberrant methylation of CpG islands in human tumors has been shown to cause transcriptional silencing of tumor-suppressor genes. Most methods used to analyze cytosine-5 methylation patterns require cumbersome manual techniques that employ gel electrophoresis, restriction enzyme digestion, radiolabeled dNTPs or hybridization probes. The development of high-throughput technology for the analysis of DNA methylation would significantly expand our ability to derive molecular information from clinical specimens. This study describes a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan®) technology that requires no further manipulations after the PCR step. MethyLight is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10 000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation. We show that MethyLight can distinguish between mono-allelic and bi-allelic methylation of the MLH1 mismatch repair gene in human colorectal tumor specimens. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of tumor samples.
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Methylation of 5' CpG islands in promoter and upstream coding regions has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. The purpose of this study was to determine whether hypermethylation of the adenomatous polyposis coli (APC) gene promoter occurs in primary non-small cell lung cancer (NSCLC), and whether hypermethylated APC has any relationship with survival. APC promoter 1A methylation was determined in normal and corresponding tumor tissue from 91 NSCLC patients and in a control group of 10 patients without cancer, using a quantitative fluorogenic real-time PCR (Taqman) system. APC promoter methylation was detectable in 86 (95%) of 91 tumor samples, but also in 80 (88%) of 91 normal samples of NSCLC patients, and in only two (20%) of 10 normal lung tissues of the control group. The median level of APC promoter methylation was 4.75 in tumor compared to 1.57 in normal lung tissue (P<0.001). Patients with low methylation status showed significantly longer survival than did patients with high methylation status (P=0.041). In a multivariate analysis of prognostic factors, APC methylation was a significant independent prognostic factor (P=0.044), as were pT (P=0.050) and pN (P<0.001) classifications. This investigation shows that APC gene promoter methylation occurs in the majority of primary NSCLCs. High APC promoter methylation is significantly associated with inferior survival, showing promise as a biomarker of biologically aggressive disease in NSCLC.
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The serum of cancer patients often harbors increased free DNA levels, which can potentially be used for cancer detection. Because genetic and epigenetic alterations of the adenomatous polyposis coli (APC) gene are common events in gastrointestinal tumor development, we sought to investigate the frequency and level of aberrant APC promoter methylation in primary tumors and paired preoperative serum or plasma samples of lung cancer patients by semiquantitative methylation-specific fluorogenic real-time PCR. We detected methylation of APC in 95 of 99 (96%) primary lung cancer tissues. Forty-two of 89 (47%) available serum and/or plasma samples from these cases carried detectable amounts of methylated APC promoter DNA. In contrast, no methylated APC promoter DNA was detected in serum samples from 50 healthy controls. A highly elevated APC methylation level in lung cancer tissue was the only independent factor predicting inferior survival in this cohort (P = 0.015). APC methylation analysis appears to be promising as a prognostic factor in primary lung cancer and as a noninvasive tumor marker in plasma and/or serum DNA.
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Changes in the status of DNA methylation are one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated whether aberrant DNA methylation in patient pretherapeutic sera is of prognostic significance in breast cancer. Using MethyLight, a high-throughput DNA methylation assay, we analyzed 39 genes in a gene evaluation set, consisting of 10 sera from metastasized patients, 26 patients with primary breast cancer, and 10 control patients. To determine the prognostic value of genes identified within the gene evaluation set, we finally analyzed pretreatment sera of 24 patients having had no adjuvant treatment (training set) to determine their prognostic value. An independent test set consisting of 62 patients was then used to test the validity of genes and combinations of genes, which in the training set were found to be good prognostic markers. In the gene evaluation set we identified five genes (ESR1, APC, HSD17B4, HIC1, and RASSF1A). In the training set, patients with methylated serum DNA for RASSF1A and/or APC had the worst prognosis (P < 0.001). This finding was confirmed by analyzing serum samples from the independent test set (P = 0.007). When analyzing all 86 of the investigated patients, multivariate analysis showed methylated RASSF1A and/or APC serum DNA to be independently associated with poor outcome, with a relative risk for death of 5.7. DNA methylation of particular genes in pretherapeutic sera of breast cancer patients, especially of RASSF1A/APC, is more powerful than standard prognostic parameters.
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Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of human cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the urine and serum samples of renal cancer patients. We examined the tumor and the matched urine and serum DNA for aberrant methylation of nine gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-beta2, and ARF) from 17 patients with primary kidney cancer by quantitative fluorogenic real-time PCR. An additional 9 urine samples (total, 26) and 1 serum sample (total, 18) also were tested from renal cancer patients. Urine from 91 patients without genitourinary cancer and serum from 30 age-matched noncancer individuals were used as controls. Promoter hypermethylation of at least two of the genes studied was detected in 16 (94%) of 17 primary tumors. Aberrant methylation in urine and serum DNA generally was accompanied by methylation in the matched tumor samples. Urine samples from 91 control subjects without evidence of genitourinary cancer revealed no methylation of the MGMT, GSTP1, p16, and ARF genes, whereas methylation of RAR-beta2, RASSF1A, CDH1, APC, and TIMP3 was detected at low levels in a few control subjects. Overall, 23 (88%) of 26 urine samples and 12 (67%) of 18 serum samples from cancer patients were methylation positive for at least one of the genes tested. By combination of urine or serum analysis of renal cancer patients, hypermethylation was detected in 16 of 17 patients (94% sensitivity) with high specificity. Our findings suggest that promoter hypermethylation in urine or serum can be detected in the majority of renal cancer patients. This noninvasive high-throughput approach needs to be evaluated in large studies to assess its value in the early detection and surveillance of renal cancer.
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Enhanced prognostication power is becoming more desirable in clinical oncology. In this study, we explored the prognostic potential of multigene hypermethylation profiling in non-small-cell lung cancer. We evaluated a panel of eight genes (p16, APC, ATM, hMLH1, MGMT, DAPK, ECAD, and RASSF1A) using methylation-specific PCR in 105 archived specimens of non-small-cell lung cancer representing all stages of the illness. We analyzed the effect of gene methylation status on outcome individually in a cumulative manner and in a combinatorial approach using recursive partitioning to identify methylation profiles, which affect overall survival. In this data set, tumors harboring promoter hypermethylation at two or more genes exhibit similar survival trends to others in the cohort. Using recursive partitioning, three genes (APC, ATM, and RASSF1A) emerged as determinants of prognostic groups. This designation retained its statistical significance even when disease stage and age were entered into a multivariate analysis. Using this approach, patients whose tumors were hypermethylated at APC and those hypermethylated at only ATM (not also at APC or RASSF1A) enjoyed substantially longer 1- and 2-year survival than patients in the remaining groups. In 32 adjacent histologically normal lung tissue specimens, we detected similar methylation abnormalities. Assessment of promoter hypermethylation aberrations may facilitate prognostic profiling of lung tumors, but validation in independent data sets is needed to verify these profiles. This system uses material that is abundantly available with linked outcome data and can be used to generate reliable epigenetic determinants.
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The p16(INK4A) tumor suppressor gene is inactivated in many solid tumors, including non-small cell lung cancers (NSCLCs), through promoter hypermethylation. Presence of p16(INK4A) hypermethylation in precursor lesions of NSCLC and in body fluids of individuals at risk makes it a potential candidate for early disease detection. However, the current low sensitivity of p16(INK4A) hypermethylation detection in plasma limits its consideration in a diagnostic grid. A fluorescent methylation-specific PCR assay (F-MSP) was established to evaluate p16(INK4A) promoter hypermethylation in 35 NSCLC and paired plasma samples and in 15 plasmas from healthy donors. F-MSP sensitivity was investigated in combination with microsatellite alterations on 3p (evaluated by fluorescent PCR), K-ras mutations (determined by a mutant-enriched PCR), and quantification of circulating DNA. Assay results were analyzed by two-sided chi(2) or Fisher's exact tests. p16(INK4A) promoter hypermethylation, detectable by F-MSP in 22 of 35 NSLCs (63%) and in 12 of 22 (55%) plasmas from patients with methylated tumors, was independent of microsatellite alterations (detectable in 57% of tumors and 50% of paired plasmas), K-ras mutations (detectable in 31% of tumors but in no paired plasma), or amount of circulating DNA. p16(INK4A) methylation in association with microsatellite alterations identified 62% (18 of 29) of plasma samples from patients presenting the same alteration in their tumors, and its sensitivity increased to 80% when combined with the amount of circulating DNA. The establishment of F-MSP remarkably improved p16(INK4A) promoter hypermethylation detection in plasmas from NSCLC patients. Microsatellite alterations, circulating DNA quantification, and p16(INK4A) hypermethylation might contribute to a diagnostic grid for NSCLC.
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Context.— Even among cases of non–small cell lung cancer (NSCLC) in the most favorable stage (IA), the disease-specific mortality is 25% or greater. One plausible explanation implicates the simplistic standard pathologic procedures used to designate lymph node involvement. A more sensitive assessment of the nodal status may improve staging. Objective.—To determine the prognostic impact of detecting an abnormal molecular event (promoter hypermethylation in a set of relevant genes) in histologically uninvolved lymph nodes in resected NSCLC. Design.—In this retrospective analysis of archived material, we examined DNA extracted from lymph nodes of stage I NSCLC (n = 180). Patients underwent surgery between 1991 and 1995 in a single institution. Methylation-specific polymerase chain reaction was used to detect promoter hypermethylation in a panel of 8 genes. Survival data were extracted from the computerized database at the Tumor Registry. Results.—Evidence of promoter hypermethylation in at least 1 gene was detected in 67% of these N0 nodes. The most commonly hypermethylated gene was E-cadherin (53%). The hypermethylation frequency for the remaining genes were as follows: APC, 5%; p16, 9%; MGMT, 11%; hMLH1, 15%; RASSF1A, 4%; DAP kinase, 9%; and ATM, 19%. The presence of promoter hypermethylation in 2 or more genes did not influence the overall, median, or 5-year survival rates. Conclusions.—Identifying promoter hypermethylation (in our panel) in N0 lymph nodes in stage I NSCLC cannot be recommended for clinical decision making. Molecular abnormalities, including those found in cancer by qualitative methylation-specific polymerase chain reaction, are not synonymous with established, histologically detectable metastasis.
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Background: Death-associated protein (DAP) kinase is a serine/threonine kinase that is important in ligandinduced programmed cell death and plays an important role in lung cancer metastasis in animal models. Hypermethylation of the promoter represses the expression of the DAP kinase gene. Our purpose was to determine whether the hypermethylation status of the DAP kinase promoter influences the prognosis of non-small-cell lung cancer (NSCLC). Methods: We retrospectively studied 135 patients with pathologic stage I NSCLC who had undergone curative surgery. Methylation-specific polymerase chain reaction was used to determine the methylation status of the DAP kinase promoter in resected specimens from patients with primary NSCLC. Statistical analyses, all twosided, were performed to determine the prognostic effect of methylation status on various clinical parameters. Results: Hypermethylation of the DAP kinase promoter was found in 59 (44%) of the 135 tumors. Patients whose tumors exhibited such hypermethylation had a statistically significantly poorer probability of overall survival at 5 years after surgery than those without such hypermethylation (.46 versus .68; P = .007). Moreover, the groups with and without hypermethylation of the DAP kinase promoter showed a striking difference in the probability of diseasespecific survival; i.e., among people who died of lung cancer-related causes specifically, the probability of 5-year survival was .56 for those with such hypermethylation and .92 for those without it (P<.001). Multivariate analysis indicated that hypermethylation of the DAP kinase promoter is the only independent predictor for disease-specific survival among clinical and histologic parameters tested. Conclusions: Hypermethylation of the DAP kinase promoter is a common abnormality in early-stage NSCLC. This abnormality is strongly associated with survival, suggesting that DAP kinase plays an important role in determining the biologic aggressiveness of early-stage NSCLC. [J Natl Cancer Inst 2000;92: 1511–6]
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Background: The adenomatous polyposis coli (APC) locus on chromosome 5q21-22 shows frequent loss of heterozygosity (LOH) in esophageal carcinomas. However, the prevalence of truncating mutations in the APC gene in esophageal carcinomas is low. Because hypermethylation of promoter regions is known to affect several other tumor suppressor genes, we investigated whether the APC promoter region is hypermethylated in esophageal cancer patients and whether this abnormality could serve as a prognostic plasma biomarker. Methods: We assayed DNA from tumor tissue and matched plasma from esophageal cancer patients for hypermethylation of the promoter region of the APC gene. We used the maximal chi-square statistic to identify a discriminatory cutoff value for hypermethylated APC DNA levels in plasma and used bootstrap-like simulations to determine the P value to test for the strength of this association. This cutoff value was used to generate Kaplan-Meier survival curves. All P values were based on two-sided tests. Results: Hypermethylation of the promoter region of the APC gene occurred in abnormal esophageal tissue in 48 (92%) of 52 patients with esophageal adenocarcinoma, in 16 (50%) of 32 patients with esophageal squamous cell carcinoma, and in 17 (39.5%) of 43 patients with Barrett's metaplasia but not in matching normal esophageal tissues. Hypermethylated APC DNA was observed in the plasma of 13 (25%) of 52 adenocarcinoma patients and in two (6.3%) of 32 squamous carcinoma patients. High plasma levels of methylated APC DNA were statistically significantly associated with reduced patient survival (P = .016). Conclusion: The APC promoter region was hypermethylated in tumors of the majority of patients with primary esophageal adenocarcinomas. Levels of hypermethylated APC gene DNA in the plasma may be a useful biomarker of biologically aggressive disease in esophageal adenocarcinoma patients and should be evaluated as a potential biomarker in additional tumor types.
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Two samples can be compared by selecting a cut point and forming a 2 X 2 table of the numbers of observations above and below the cut point in each sample. Different cut points correspond to the maximum chi square statistic, the maximum squared log odds ratio, and the generalized Kolmogorov-Smirnov statistic. We simulated the small-sample null distribution of these statistics. The distributions of the maximally selected chi square statistic and the squared log odds ratio statistic are compared to each other and to the large-sample results of Miller and Siegmund (1982, Biometrics 38, 1011-1016. The distributions of and agreement among the three cut points are also considered.
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A method is presented for quantitative evaluation of observer detection performance data based on elementary principles of information therapy. The resulting index of detectability, average information content per observation, is compared with previously proposed measures of observer performance both on theoretical grounds and for the practical problem of evaluating radiographic screen film systems.
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Loss of heterozygosity on chromosome 9p21 is one of the most frequent genetic alterations identified in human cancer. The rate of point mutations of p16, a candidate suppressor gene of this area, is low in most primary tumours with allelic loss of 9p21. Monosomic cell lines with structurally unaltered p16 show methylation of the 5' CpG island of p16. This distinct methylation pattern was associated with a complete transcriptional block that was reversible upon treatment with 5-deoxyazacytidine. Moreover, de novo methylation of the 5' CpG island of p16 was also found in approximately 20% of different primary neoplasms, but not in normal tissues, potentially representing a common pathway of tumour suppressor gene inactivation in human cancers.
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A prospective study was performed in patients with non-small cell lung cancer (NSCLC) to evaluate the prognostic importance of multiple molecular marker (p53, c-Ki-ras, c-erbB-2) testing. 103 patients with potentially curative resections (RO resection) for NSCLC in histopathological stages I-IIIA were included. SSCP analysis and DNA sequencing for p53 and c-Ki-ras genes were performed on paired tumour and normal lung tissue samples and immunohistochemistry (c-erbB-2) was done on frozen tissue sections with a specific anti-c-erbB-2 monoclonal antibody. 46/103 (44.6%) NSCLC showed p53 mutations and 17/103 (16.5%) c-Ki-ras mutations including 12/37 (32.4%) adenocarcinomas. Overexpression of c-erbB-2 (p185) was detected in 56/103 (54.4%) tumours. 24/103 (23.3%) NSCLC were negative for alterations in all 3 parameters (c-Ki-ras, p53 and p185) whereas 79/103 (76.7%) were positive for at least one of the 3 parameters. In a regression model including a multiple molecular marker parameter (negative for all 3 markers versus positive for at least one marker), histopathological stage (P<0.00001), respectively the pT (P<0.01) and pN (P<0.00001) categories and the multiple molecular marker parameter (P<0.01) were of significant prognostic importance. This study demonstrates that testing 3 molecular markers (c-Ki-ras, p53 and c-erbB-2) improves estimation of prognosis compared to single marker testing and appears to define low (82.6%+/-7.9% 5-year survival) and high risk (40.2%+/-5.5% 5-year survival) groups for treatment failure in potentially curative (RO) resected NSCLC.
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The adenomatous polyposis coli (APC) locus on chromosome 5q21-22 shows frequent loss of heterozygosity (LOH) in esophageal carcinomas. However, the prevalence of truncating mutations in the APC gene in esophageal carcinomas is low. Because hypermethylation of promoter regions is known to affect several other tumor suppressor genes, we investigated whether the APC promoter region is hypermethylated in esophageal cancer patients and whether this abnormality could serve as a prognostic plasma biomarker. We assayed DNA from tumor tissue and matched plasma from esophageal cancer patients for hypermethylation of the promoter region of the APC gene. We used the maximal chi-square statistic to identify a discriminatory cutoff value for hypermethylated APC DNA levels in plasma and used bootstrap-like simulations to determine the P: value to test for the strength of this association. This cutoff value was used to generate Kaplan-Meier survival curves. All P values were based on two-sided tests. Hypermethylation of the promoter region of the APC gene occurred in abnormal esophageal tissue in 48 (92%) of 52 patients with esophageal adenocarcinoma, in 16 (50%) of 32 patients with esophageal squamous cell carcinoma, and in 17 (39.5%) of 43 patients with Barrett's metaplasia but not in matching normal esophageal tissues. Hypermethylated APC DNA was observed in the plasma of 13 (25%) of 52 adenocarcinoma patients and in two (6.3%) of 32 squamous carcinoma patients. High plasma levels of methylated APC DNA were statistically significantly associated with reduced patient survival (P =.016). The APC promoter region was hypermethylated in tumors of the majority of patients with primary esophageal adenocarcinomas. Levels of hypermethylated APC gene DNA in the plasma may be a useful biomarker of biologically aggressive disease in esophageal adenocarcinoma patients and should be evaluated as a potential biomarker in additional tumor types.
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To provide an introduction to the concept of DNA methylation and its function in normal cells, and to explain the possible mechanisms as to how abnormalities in this phenomenon can relate to carcinogenesis. The clinical implications with reference to common malignancies encountered in surgical practice are discussed. Methylation of DNA is a heritable, enzyme-induced modification to DNA structure without alteration of the specific sequence of the base pairs responsible for encoding the genome. DNA methylation can both directly inhibit the expression of genes and also increase the probability that affected genes undergo a mutational event. Although DNA methylation plays an essential role in normal biologic processes, distinct and abnormal patterns of methylation are observed in cancers. In particular, there has been increased documentation that methylation of the promoter regions of several genes, including known tumor suppressor genes, results in the subsequent failure to express their functional proteins. Consequently, DNA methylation may represent an early and fundamental step in the pathway by which normal tissue undergoes neoplastic transformation. Further, an assessment of the methylation profiles within neoplastic tissues may provide key information in enhancing the diagnosis, predicting the clinical behavior, and designing specific treatment plans for individual patients. Published literature from 1925 to 2000 contributing to an understanding of the purpose of DNA methylation and how pathology of this phenomenon could contribute to cancer are reviewed. Theories on these issues and the evidence that led to them are described. The present status of the subject in a clinical context is discussed. Gene expression can be significantly modulated by alterations in DNA methylation patterns. Methylation within the promoter regions of tumor suppressor genes causes their silencing, and methylation within the gene itself can induce mutational events. These mechanisms may play a fundamental role in precipitating the development of a large and diverse number of human cancers. DNA methylation is an important factor in the development of cancer. A greater understanding of the relationship between DNA methylation events at the molecular level and its interaction in the clinical context may provide the basis for future advances in the surgical and pharmacologic management of malignant diseases.
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Retinoid X receptors (RXRs) have inhibitory effects on non-small cell lung cancer (NSCLC) cell growth, and RXRbeta expression is reduced in NSCLC specimens compared with normal lung tissue. We hypothesized that suppressed RXR expression might be a prognostic factor of worse clinical outcome in patients with NSCLC. Using a quantitative real-time reverse transcription-PCR (TaqMan) method, we analyzed RXRalpha, RXRbeta, and RXRgamma mRNA expression in normal lung tissue and matching tumor samples from 88 patients with NSCLC. The median mRNA expression levels of all three RXR subtypes were frequently decreased in tumor tissues compared with matching normal lung tissue (RXRalpha, 67%; RXRbeta, 55%; RXRgamma, 89%). The RXRalpha(P = 0.001) and RXRgamma(P < 0.001) median expression levels were significantly lower in the tumors. Patients whose tumors exhibited low RXRbeta expression levels had a statistically significant worse overall survival (P = 0.0005), whereas a trend toward worse survival was observed for patients with low RXRalpha expression. Multivariate analysis indicated that low RXRbeta expression is an independent predictor of worse survival in patients with NSCLC (P = 0.017). Suppressed mRNA expression of all three RXR subtypes is a frequent event in NSCLC. Reduced RXRbeta expression might be an important biomarker for more aggressive disease in patients with NSCLC.
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To investigate cyclooxygenase-2 (COX-2) mRNA expression in curatively resected non-small cell lung cancer (NSCLC) and to determine its association with prognosis. Lung cancer is one of the most common malignancies in the world. Despite improvements in the diagnosis and treatment of NSCLC, the 5-year survival rate remains less than 15%. Identification of prognostic predictors based on molecular alterations could lead to additional diagnostic tools and eventually to more effective therapeutic options. Overexpression of COX-2 has been reported in several human malignancies, including lung cancer, but the prognostic importance of this overexpression has not been elucidated. COX-2 mRNA expression was analyzed using a quantitative real-time polymerase chain reaction (Taqman) method in surgically resected tumor specimens from 89 patients with curatively resected NSCLC. COX-2 mRNA was detectable in all 89 (100%) tumor tissues. High COX-2 expression in tumors was significantly associated with inferior survival. Multivariate analysis showed that high COX-2 expression is an independent predictor of worse survival in patients with NSCLC. High COX-2 mRNA expression is an important biomarker for biologically aggressive disease in NSCLC and might be helpful in identifying patients who would benefit from additional therapies for controlling their disease.
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Hypermethylation of the O(6)-methylguanine DNA methyltransferase (MGMT) promoter region leads to transcriptional repression of the MGMT gene and is a common event in primary human neoplasia. The purpose of this study was to determine the frequency and clinical relevance of MGMT gene promoter hypermethylation in curatively resected non-small cell lung cancer (NSCLC). MGMT hypermethylation, expressed as the ratio between methylated MGMT to unmethylated MYOD1 in genomic DNA, was analyzed in normal and matching tumor tissue from 90 patients with NSCLC, and a control group of 10 patients without cancer using a methylation-specific fluorogenic Real-Time PCR (Taqman) system. Hypermethylation of the MGMT promoter was detected in 34 of 90 (38%) tumor specimens and 16 of 90 (18%) matching normal lung tissues of patients with NSCLC, and in 0 (0%) cases of the control group without lung cancer. The mean MGMT methylation level was significantly higher in tumor than in matching normal tissue (P < 0.001). MGMT methylation in normal tissue was always accompanied with MGMT methylation in matching tumor tissue. Patients without MGMT promoter hypermethylation showed a significantly better survival than patients with MGMT promoter hypermethylation (P = 0.017). Multivariate analysis revealed MGMT promoter methylation as an independent unfavorable prognostic factor (P = 0.030). MGMT promoter hypermethylation is a common event in patients with primary NSCLC. This epigenetic alteration is associated with inferior survival, suggesting that MGMT promoter hypermethylation might be an important biomarker for a biological aggressive disease in NSCLC.
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Promoter hypermethylation is an important pathway for repression of gene transcription in cancer cells and a promising marker for cancer detection. We tested five gene promoters [CDKN2A (p16), O(6)-methylguanine-DNA-methyltransferase, glutathione S-transferase P1 (GSTP1), adenomatous polyposis coli (APC), and death-associated protein kinase (DAPK)] by real-time methylation-specific PCR in primary tumors from 90 stage I lung cancer patients for aberrant DNA methylation. We then used the presence of tumor methylation as a marker to investigate the presence of occult metastasis in corresponding histologically negative lymph nodes. Of the primary tumors, 73 of 90 (81%) displayed promoter hypermethylation in at least one of the genes studied: 17% (15 of 90) at p16 (CDKN2A); 16% (14 of 90) at O(6)-methylguanine-DNA-methyltransferase; 8% (7 of 90) at GSTP1; 72% (65 of 90) at APC; and 17% (15 of 90) at DAPK. Squamous histology was predictive of worse overall survival (P = 0.074, log-rank test). APC methylation and GSTP1 methylation in the primary tumor were both correlated with nonsquamous histology (P = 0.02 and P = 0.01 likelihood ratio respectively). The presence of both APC methylation and DAPK methylation in the primary tumor predicted a worse outcome, with 7 of 13 (54%) deaths in this group compared with 21 of 77 (27%) deaths in cases without both genes methylated (P = 0.229, log-rank test). The same methylation pattern was detected in DNA from at least one of the corresponding lymph nodes in 11 of 73 (15%) cases. Five of 11 (45%) patients with occult metastasis detected by methylation analysis have died compared with 17 of 62 (27%) patients with negative lymph nodes, although survival analysis did not reach statistical significance (P = 0.632, log-rank test). Promoter hypermethylation is common in lung cancer and represents a promising marker for the molecular staging of lung cancer patients. Although this study showed important trends, a larger prospective study is required to better understand the value of methylation analysis in detecting occult metastasis.
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Silencing of genes by aberrant promoter hypermethylation is now recognized as a crucial component in cancer initiation and progression. Highly sensitive assays have been developed to assess gene-promoter methylation in biological fluids. The detection of methylated genes in sputum could lead to the development of a screening test to non-invasively identify early cancer in high-risk people.
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The purpose of this study is to evaluate the usefulness of serum DNA methylation of five tumor suppressor genes for early detection of lung cancer. Methylation status in serum DNA from 200 patients undergoing bronchofiberscopic examination for abnormal findings on chest radiograph detected by lung cancer screening or surveillance was examined using methylation-specific PCR. Ninety-one patients were given a pathologic diagnosis of lung cancer, 9 other malignant diseases, and 100 nonmalignant pulmonary diseases. In patients with lung cancer, methylation was detected in 18.7% for MGMT, 15.4% for p16(INK4a), 12.1% for RASSF1A, 11.0% for DAPK, and 6.6% for RAR-beta, which was higher compared with that in patients with nonmalignant diseases. Age and smoking status seemed to associate with methylation status. Sensitivity, specificity, and predictive value of methylation in at least one gene for diagnosis of lung cancer were 49.5%, 85.0%, and 75.0%, respectively. Adjusted odds ratio (95% confidence interval) for having lung cancer was 5.28 (2.39-11.7) for patients with methylation in one gene and 5.89 (1.53-22.7) for those with methylation in two or more genes. It is of note that methylation was identified in 50.9% of stage I lung cancer patients, whereas serum protein tumor markers were positive in 11.3% of them. These results suggest that identification of promoter methylation of tumor suppressor genes in serum DNA could be useful for early detection of lung cancer.
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Recent studies imply that HER2/neu is a potential prognostic factor in patients with non-small-cell lung cancer (NSCLC). Whereas considerable evidence indicates sex differences in epidemiologic, hormonal, biologic, and genetic factors in this disease, it has remained unknown whether HER2/neu has a diverse function as a prognostic factor in men and women. We investigated the association between gene expression levels of HER2/neu in the primary tumors of 90 patients with curable resected NSCLC and survival, especially analyzing whether there is a different potential of this molecular factor in its prognostic impact between men and women. High HER2/neu gene expression levels were found in 62 patients (68.9%), and low HER2/neu gene expression levels were found in 28 patients (31.1%). High HER2/neu messenger RNA expression levels were associated with inferior survival (P = 0.09) compared with lower HER2/neu expression. Survival analysis was then carried out separately for men and women in this group of patients. An HER2/neu gene expression cutoff point was identified that separated women, but not men, into good and poor prognostic groups. These findings suggest that HER2/neu as a prognostic factor is strongly sex specific, indicating that it is not useful for men but highly predictive for women.
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A significant association between aberrant methylation in regulatory regions of tumor suppressor genes and clinical outcome in various different cancer types has been described. The molecular events for this epigenetic alteration still remain unknown. Evidence suggests that overexpression of DNA methyltransferases (DNMTs) is one potential mechanism for hypermethylation. Therefore, we investigated the influence of gene expression levels of the 3 DNMT isoforms (DNMT1, DNMT3a, and DNMT3b) and the hypermethylation of adenomatous polyposis coli (APC), the death-associated protein kinase (DAPK), glutathione S-transferase Pi (GSTPI), and the DNA repair gene O6-methylguanine DNA transferase (MGMT) in the pathogenesis and prognosis of patients with non-small cell lung cancer and determined their association to each other. Using a quantitative real-time reverse-transcriptase polymerase chain reaction, we measured messenger RNA expression of DNMT1, DNMT3a, and DNMT3b and DNA hypermethylation of APC, DAPK, GSTPI, and MGMT in 91 matching tumor and nonmalignant lung tissue samples from patients with curatively resected non-small-cell lung cancer. In tumor tissue, the expression of all 3 DNMT isoforms was significantly higher compared with matched normal-appearing tissue (P < 0.001). Hypermethylation in tumor tissue was found in 95% for APC, in 92% for DAPK, in 18% for GSTPI, and in 38% for MGMT. No correlation was found between the DNMT messenger RNA expression and DNA hypermethylation status in tumor tissues. Multivariate analysis revealed DNA hypermethylation status and TNM stage as independent prognostic factors.
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Even among cases of non-small cell lung cancer (NSCLC) in the most favorable stage (IA), the disease-specific mortality is 25% or greater. One plausible explanation implicates the simplistic standard pathologic procedures used to designate lymph node involvement. A more sensitive assessment of the nodal status may improve staging. To determine the prognostic impact of detecting an abnormal molecular event (promoter hypermethylation in a set of relevant genes) in histologically uninvolved lymph nodes in resected NSCLC. In this retrospective analysis of archived material, we examined DNA extracted from lymph nodes of stage I NSCLC (n = 180). Patients underwent surgery between 1991 and 1995 in a single institution. Methylation-specific polymerase chain reaction was used to detect promoter hypermethylation in a panel of 8 genes. Survival data were extracted from the computerized database at the Tumor Registry. Evidence of promoter hypermethylation in at least 1 gene was detected in 67% of these N0 nodes. The most commonly hypermethylated gene was E-cadherin (53%). The hypermethylation frequency for the remaining genes were as follows: APC, 5%; p16, 9%; MGMT, 11%; hMLH1, 15%; RASSF1A, 4%; DAP kinase, 9%; and ATM, 19%. The presence of promoter hypermethylation in 2 or more genes did not influence the overall, median, or 5-year survival rates. Identifying promoter hypermethylation (in our panel) in N0 lymph nodes in stage I NSCLC cannot be recommended for clinical decision making. Molecular abnormalities, including those found in cancer by qualitative methylation-specific polymerase chain reaction, are not synonymous with established, histologically detectable metastasis.
Article
Each year, the American Cancer Society estimates the number of new cancer cases and deaths expected in the United States in the current year and compiles the most recent data on cancer incidence, mortality, and survival based on incidence data from the National Cancer Institute, Centers for Disease Control and Prevention, and the North American Association of Central Cancer Registries and mortality data from the National Center for Health Statistics. Incidence and death rates are age-standardized to the 2000 US standard million population. A total of 1,437,180 new cancer cases and 565,650 deaths from cancer are projected to occur in the United States in 2008. Notable trends in cancer incidence and mortality include stabilization of incidence rates for all cancer sites combined in men from 1995 through 2004 and in women from 1999 through 2004 and a continued decrease in the cancer death rate since 1990 in men and since 1991 in women. Overall cancer death rates in 2004 compared with 1990 in men and 1991 in women decreased by 18.4% and 10.5%, respectively, resulting in the avoidance of over a half million deaths from cancer during this time interval. This report also examines cancer incidence, mortality, and survival by site, sex, race/ethnicity, education, geographic area, and calendar year, as well as the proportionate contribution of selected sites to the overall trends. Although much progress has been made in reducing mortality rates, stabilizing incidence rates, and improving survival, cancer still accounts for more deaths than heart disease in persons under age 85 years. Further progress can be accelerated by supporting new discoveries and by applying existing cancer control knowledge across all segments of the population.
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