Circulating Endothelial Cells and Procoagulant Microparticles in Patients with Glioblastoma: Prognostic Value

Aim

Circulating endothelial cells and microparticles are prognostic factors in cancer. However, their prognostic and predictive value in patients with glioblastoma is unclear. The objective of this study was to investigate the potential prognostic value of circulating endothelial cells and microparticles in patients with newly diagnosed glioblastoma treated with standard radiotherapy and concomitant temozolomide. In addition, we have analyzed the methylation status of the MGMT promoter.

Methods

Peripheral blood samples were obtained before and at the end of the concomitant treatment. Blood samples from healthy volunteers were also obtained as controls. Endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Microparticles were quantified by flow cytometry. Microparticle-mediated procoagulant activity was measured by endogen thrombin generation and by phospholipid-dependent clotting time. Methylation status of MGMT promoter was determined by multiplex ligation-dependent probe amplification.

Results

Pretreatment levels of circulating endothelial cells and microparticles were higher in patients than in controls (p<0.001). After treatment, levels of microparticles and thrombin generation decreased, and phospholipid-dependent clotting time increased significantly. A high pretreatment endothelial cell count, corresponding to the 99^th percentile in controls, was associated with poor overall survival. MGMT promoter methylation was present in 27% of tumor samples and was associated to a higher overall survival (66 weeks vs 30 weeks, p<0.004).

Conclusion

Levels of circulating endothelial cells may have prognostic value in patients with glioblastoma.     

Comparative Analysis of Matrix Metalloproteinase Family Members Reveals That MMP9 Predicts Survival and Response to Temozolomide in Patients with Primary Glioblastoma

Background

Glioblastoma multiform (GBM) is the most common malignant primary brain tumor in adults. Radiotherapy plus concomitant and adjuvant TMZ chemotherapy is the current standard of care for patients with GBM. Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, are key modulators of tumor invasion and metastasis due to their ECM degradation capacity. The aim of the present study was to identify the most informative MMP member in terms of prognostic and predictive ability for patients with primary GBM.

Method

The mRNA expression profiles of all MMP genes were obtained from the Chinese Glioma Genome Atlas (CGGA), the Repository for Molecular Brain Neoplasia Data (REMBRANDT) and the {"type":"entrez-geo","attrs":{"text":"GSE16011","term_id":"16011"}}GSE16011 dataset. MGMT methylation status was also examined by pyrosequencing. The correlation of MMP9 expression with tumor progression was explored in glioma specimens of all grades. Kaplan–Meier analysis and Cox proportional hazards regression models were used to investigate the association of MMP9 expression with survival and response to temozolomide.

Results

MMP9 was the only significant prognostic factor in three datasets for primary glioblastoma patients. Our results indicated that MMP9 expression is correlated with glioma grade (p<0.0001). Additionally, low expression of MMP9 was correlated with better survival outcome (OS: p = 0.0012 and PFS: p = 0.0066), and MMP9 was an independent prognostic factor in primary GBM (OS: p = 0.027 and PFS: p = 0.032). Additionally, the GBM patients with low MMP9 expression benefited from temozolomide (TMZ) chemotherapy regardless of the MGMT methylation status.

Conclusions

Patients with primary GBMs with low MMP9 expression may have longer survival and may benefit from temozolomide chemotherapy.     

Associations of BDNF Genotype and Promoter Methylation with Acute and Long-Term Stroke Outcomes in an East Asian Cohort

Background

Brain derived neurotrophic factor (BDNF) has been shown to play an important role in poststroke recovery. BDNF secretion is influenced by genetic and epigenetic profiles. This study aimed to investigate whether BDNF val66met polymorphism and promoter methylation status were associated with outcomes at two weeks and one year after stroke.

Methods and Findings

A total of 286 patients were evaluated at the time of admission and two weeks after stroke, and 222 (78%) were followed one year later in order to evaluate consequences of stroke at both acute and chronic stages. Stroke outcomes were dichotomised into good and poor by the modified Rankin Scale. Stroke severity (National Institutes of Health Stroke Scale), physical disability (Barthel Index), and cognitive function (Mini-Mental State Examination) were measured. Associations of BDNF genotype and methylation status on stroke outcomes and assessment scale scores were investigated using logistic regression, repeated measures ANOVA and partial correlation tests. BDNF val66met polymorphism was independently associated with poor outcome at 2 weeks and at 1 year, and with worsening physical disability and cognitive function over that period. Higher BDNF promoter methylation status was independently associated with worse outcomes at 1 year, and with the worsening of physical disability and cognitive function. No significant genotype-methylation interactions were found.

Conclusions

A role for BDNF in poststroke recovery was supported, and clinical utility of BDNF genetic and epigenetic profile as prognostic biomarkers and a target for drug development was suggested.     

Comparison of promoter hypermethylation pattern in salivary rinses collected with and without an exfoliating brush from patients with HNSCC

Background

Salivary rinses have been recently proposed as a valuable resource for the development of epigenetic biomarkers for detection and monitoring of head and neck squamous cell carcinoma (HNSCC). Both salivary rinses collected with and without an exfoliating brush from patients with HNSCC are used in detection of promoter hypermethylation, yet their correlation of promoter hypermethylation has not been evaluated. This study was to evaluate the concordance of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush from patients with HNSCC.

Methodolgy

57 paired salivary rinses collected with or without an exfoliating brush from identical HNSCC patients were evaluated for promoter hypermethylation status using Quantitative Methylation-Specific PCR. Target tumor suppressor gene promoter regions were selected based on our previous studies describing a panel for HNSCC screening and surveillance, including P16, CCNA1, DCC, TIMP3, MGMT, DAPK and MINT31.

Principal Findings

In salivary rinses collected with and without brush, frequent methylation was detected in P16 (8.8% vs. 5.2%), CCNA1 (26.3% vs. 22.8%), DCC (33.3% vs. 29.8%), TIMP3 (31.6% vs. 36.8%), MGMT (29.8% vs. 38.6%), DAPK (14.0% vs. 19.2%), and MINT31 (10.5% vs. 8.8%). Spearman''s rank correlation coefficient showed a positive correlation between salivary rinses collected with and without brush for P16 (ρ = 0.79), CCNA1 (ρ = 0.61), DCC (ρ = 0.58), TIMP3 (ρ = 0.10), MGMT (ρ = 0.70), DAPK (ρ = 0.51) and MINT31 (ρ = 0.72) (P<0.01). The percent agreement of promoter methylation between salivary rinses with brush and without brush were 96.5% for P16, 82.5% for CCNA1, 78.9% for DCC, 59.7% for TIMP3, 84.2% for MGMT, 84.2% for DAPK, and 94.7% for MINT31.

Conclusions

Our study demonstrated strong correlations of gene promoter hypermethylation between salivary rinses collected with and without an exfoliating brush. Salivary rinse collection without using an exfoliating brush may offer a cost effective, rapid, non-invasive, and reliable means for development of epigenetic salivary rinse biomarkers.     

Validation of the Memorial Sloan-Kettering Cancer Center Nomogram to Predict Disease-Specific Survival after R0 Resection in a Chinese Gastric Cancer Population

Background

Prediction of disease-specific survival (DSS) for individual patient with gastric cancer after R0 resection remains a clinical concern. Since the clinicopathologic characteristics of gastric cancer vary widely between China and western countries, this study is to evaluate a nomogram from Memorial Sloan-Kettering Cancer Center (MSKCC) for predicting the probability of DSS in patients with gastric cancer from a Chinese cohort.

Methods

From 1998 to 2007, clinical data of 979 patients with gastric cancer who underwent R0 resection were retrospectively collected from Peking University Cancer Hospital & Institute and used for external validation. The performance of the MSKCC nomogram in our population was assessed using concordance index (C-index) and calibration plot.

Results

The C-index for the MSKCC predictive nomogram was 0.74 in the Chinese cohort, compared with 0.69 for American Joint Committee on Cancer (AJCC) staging system (P<0.0001). This suggests that the discriminating value of MSKCC nomogram is superior to AJCC staging system for prognostic prediction in the Chinese population. Calibration plots showed that the actual survival of Chinese patients corresponded closely to the MSKCC nonogram-predicted survival probabilities. Moreover, MSKCC nomogram predictions demonstrated the heterogeneity of survival in stage IIA/IIB/IIIA/IIIB disease of the Chinese patients.

Conclusion

In this study, we externally validated MSKCC nomogram for predicting the probability of 5- and 9-year DSS after R0 resection for gastric cancer in a Chinese population. The MSKCC nomogram performed well with good discrimination and calibration. The MSKCC nomogram improved individualized predictions of survival, and may assist Chinese clinicians and patients in individual follow-up scheduling, and decision making with regard to various treatment options.     

Investigating the potential role of genetic and epigenetic variation of DNA methyltransferase genes in hyperplastic polyposis syndrome

Background

Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS.

Methods

We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters.

Results

No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01) compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053). BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps.

Conclusions

Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.     

Aberrant Promoter Methylation of Caveolin-1 Is Associated with Favorable Response to Taxane-Platinum Combination Chemotherapy in Advanced NSCLC

Purpose

Aberrant promoter DNA methylation can serve as a predictive biomarker for improved clinical responses to certain chemotherapeutics. One of the major advantages of methylation biomarkers is the ease of detection and clinical application. In order to identify methylation biomarkers predictive of a response to a taxane-platinum based chemotherapy regimen in advanced NSCLC we performed an unbiased methylation analysis of 1,536 CpG dinucleotides in cancer-associated gene loci and correlated results with clinical outcomes.

Methods

We studied a cohort of 49 patients (median age 62 years) with advanced NSCLC treated at the Atlanta VAMC between 1999 and 2010. Methylation analysis was done on the Illumina GoldenGate Cancer panel 1 methylation microarray platform. Methylation data were correlated with clinical response and adjusted for false discovery rates.

Results

Cav1 methylation emerged as a powerful predictor for achieving disease stabilization following platinum taxane based chemotherapy (p = 1.21E-05, FDR significance  = 0.018176). In Cox regression analysis after multivariate adjustment for age, performance status, gender, histology and the use of bevacizumab, CAV1 methylation was significantly associated with improved overall survival (HR 0.18 (95%CI: 0.03–0.94)). Silencing of CAV1 expression in lung cancer cell lines(A549, EKVX)by shRNA led to alterations in taxane retention.

Conclusions

CAV1 methylation is a predictor of disease stabilization and improved overall survival following chemotherapy with a taxane-platinum combination regimen in advanced NSCLC. CAV1 methylation may predict improved outcomes for other chemotherapeutic agents which are subject to cellular clearance mediated by caveolae.     

The Clinical Significance of MiR-148a as a Predictive Biomarker in Patients with Advanced Colorectal Cancer

Aim

Development of robust prognostic and/or predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of certain miRNAs might have prognostic/predictive value in CRC patients treated with conventional cytotoxic chemotherapies.

Methods

We studied a cohort of 273 CRC specimens from stage II/III patients treated with 5-fluorouracil-based adjuvant chemotherapy and stage IV patients subjected to 5-fluorouracil and oxaliplatin-based chemotherapy. In a screening set (n = 44), 13 of 21 candidate miRNAs were successfully quantified by multiplex quantitative RT-PCR. In the validation set comprising of the entire patient cohort, miR-148a expression status was assessed by quantitative RT-PCR, and its promoter methylation was quantified by bisulfite pyrosequencing. Lastly, we analyzed the associations between miR-148a expression and patient survival.

Results

Among the candidate miRNAs studied, miR-148a expression was most significantly down-regulated in advanced CRC tissues. In stage III and IV CRC, low miR-148a expression was associated with significantly shorter disease free-survival (DFS), a worse therapeutic response, and poor overall survival (OS). Furthermore, miR-148a methylation status correlated inversely with its expression, and was associated with worse survival in stage IV CRC. In multivariate analysis, miR-148a expression was an independent prognostic/predictive biomarker for advanced CRC patients (DFS in stage III, low vs. high expression, HR 2.11; OS in stage IV, HR 1.93).

Discussion

MiR-148a status has a prognostic/predictive value in advanced CRC patients treated with conventional chemotherapy, which has important clinical implications in improving therapeutic strategies and personalized management of this malignancy.     

SULF2 Methylation Is Associated with In Vitro Cisplatin Sensitivity and Clinical Efficacy for Gastric Cancer Patients Treated with a Modified FOLFOX Regimen

Objective

Biomarkers capable of discriminating the patients who are likely to respond to certain chemotherapeutic agents could improve the clinical efficiency. The sulfatases(SULFs) play a critical role in the pathogenesis of a variety of human cancers. Here, we focused our investigation on the prognostic and predictive impact of SULF2 methylation in gastric cancer.

Methods

Promoter CpG island methylation of SULF2 was analyzed in 100 gastric cancer samples. The in vitro sensitivity to cisplatin, docetaxel, gemcitabine, irinotecan and pemetrexed were determined by histoculture drug response assay(HDRA). Additionally, 56 gastric cancer patients treated with a modified FOLFOX regimen(biweekly oxaliplatin plus 5-FU and folinic acid) were retrospectively analyzed to further evaluate the prognostic and predictive impact of SULF2 methylation in gastric cancer.

Results

Methylated SULF2(SULF2M) was detected in 28 patients, while the remaining 72 patients showed unmethylated SULF2(SULF2U, methylation rate: 28%). Samples with SULF2U were more sensitive to cisplatin than those with SULF2M(inhibition rate: 48.80% vs. 38.15%, P = 0.02), while samples with SULF2M were more sensitive to irinotecan than SULF2U(inhibition rate: 53.61% vs. 40.92%, P = 0.01). There were no association between SULF2 methylation and in vitro sensitivity to docetaxel, gemcitabine and pemetrexed. SULF2 methylation was found to have a significant association with cisplatin efficacy(SULF2M: 57.14%, SULF2U: 80.56%, P = 0.02) and irinotecan efficacy(SULF2M: 89.29%, SULF2U: 62.50%, P = 0.01). Among the 56 patients receiving the modified FOLFOX regimen, a significant association was observed between survival and SULF2 methylation status(SULF2M: 309 days, 95% CI = 236 to 382 days; SULF2U: 481 days, 95% CI = 418 to 490 days; P = 0.02). Multivariate analysis revealed that SULF2 methylation was an independent prognostic factor of overall survival in gastric cancer patients treated with platinum-based chemotherapy.

Conclusion

SULF2 methylation is negatively associated with cisplatin sensitivity in vitro. SULF2 methylation may be a novel prognostic biomarker for gastric cancer patients treated with platinum-based chemotherapy.     

The prognostic significance of whole blood global and specific DNA methylation levels in gastric adenocarcinoma

Background

Epigenetics, particularly DNA methylation, has recently been elucidated as important in gastric cancer (GC) initiation and progression. We investigated the clinical and prognostic importance of whole blood global and site-specific DNA methylation in GC.

Methods

Genomic DNA was extracted from the peripheral blood of 105 Omani GC patients at diagnosis. DNA methylation was quantified by pyrosequencing of global DNA and specific gene promoter regions at 5 CpG sites for CDH1, 7 CpG sites for p16, 4 CpG sites for p53, and 3 CpG sites for RUNX3. DNA methylation levels in patients were categorized into low, medium, and high tertiles. Associations between methylation level category and clinicopathological features were evaluated using χ² tests. Survival analyses were carried out using the Kaplan-Meier method and log rank test. A backward conditional Cox proportional hazards regression model was used to identify independent predictors of survival.

Results

Older GC patients had increased methylation levels at specific CpG sites within the CDH1, p53, and RUNX-3 promoters. Male gender was significantly associated with reduced global and increased site-specific DNA methylation levels in CDH1, p16, and p53 promoters. Global DNA low methylation level was associated with better survival on univariate analysis. Patients with high and medium methylation vs. low methylation levels across p16 promoter CpG sites, site 2 in particular, had better survival. Multivariate analysis showed that global DNA hypermethylation was a significant independent predictor of worse survival (hazard ratio (HR) = 2.0, 95% CI: 1.1–3.8; p = 0.02) and high methylation mean values across p16 promoter sites 1–7 were associated with better survival with HR of 0.3 (95% CI, 0.1–0.8; p = 0.02) respectively.

Conclusions

Analysis of global and site-specific DNA methylation in peripheral blood by pyrosequencing provides quantitative DNA methylation values that may serve as important prognostic indicators.     

IGFBP3 Methylation Is a Novel Diagnostic and Predictive Biomarker in Colorectal Cancer

Background and Aim

Aberrant hypermethylation of cancer-related genes has emerged as a promising strategy for the development of diagnostic, prognostic and predictive biomarkers in human cancer, including colorectal cancer (CRC). The aim of this study was to perform a systematic and comprehensive analysis of a panel of CRC-specific genes as potential diagnostic, prognostic and predictive biomarkers in a large, population-based CRC cohort.

Patients and Methods

Methylation status of the SEPT9, TWIST1, IGFBP3, GAS7, ALX4 and miR137 genes was studied by quantitative bisulfite pyrosequencing in a population-based cohort of 425 CRC patients.

Results

Methylation levels of all genes analyzed were significantly higher in tumor tissues compared to normal mucosa (p<0.0001); however, cancer-associated hypermethylation was most frequently observed for miR137 (86.7%) and IGFBP3 (83%) in CRC patients. Methylation analysis using the combination of these two genes demonstrated greatest accuracy for the identification of colonic tumors (sensitivity 95.5%; specificity 90.5%). Low levels of IGFBP3 promoter methylation emerged as an independent risk factor for predicting poor disease free survival in stage II and III CRC patients (HR = 0.49, 95% CI: 0.28–0.85, p = 0.01). Our results also suggest that stage II & III CRC patients with high levels of IGFBP3 methylation do not benefit from adjuvant 5FU-based chemotherapy.

Conclusion

By analyzing a large, population-based CRC cohort, we demonstrate the potential clinical significance of miR137 and IGFBP3 hypermethylation as promising diagnostic biomarkers in CRC. Our data also revealed that IGFBP3 hypermethylation may serve as an independent prognostic and predictive biomarker in stage II and III CRC patients.     

Hypermethylation of Genes Detected in Urine from Ghanaian Adults with Bladder Pathology Associated with Schistosoma haematobium Infection

Purpose

Schistosoma haematobium is associated with chronic bladder damage and may subsequently induce bladder cancer in humans, thus posing a serious threat where the parasite is endemic. Here we evaluated aberrant promoter DNA methylation as a potential biomarker to detect severe bladder damage that is associated with schistosomiasis by analyzing urine specimens.

Materials and Methods

A quantitative methylation-specific PCR (QMSP) assay was used to examine the methylation status of seven genes (RASSF1A, RARβ2, RUNX3, TIMP3, MGMT, P16, ARF) in 57 urine samples obtained from volunteers that include infected and uninfected by S. haematobium from an endemic region. The Fishers Exact Test and Logistic Regression analysis were used to evaluate the methylation status with bladder damage (as assessed by ultrasound examination) in subjects with S. haematobium infection.

Results

RASSF1A and TIMP3 were significant to predict severe bladder damage both in univariate (p = 0.015 and 0.023 respectively) and in multivariate (p = 0.022 and 0.032 respectively) logistic regression analysis. Area under the receiver operator characteristic curves (AUC-ROC) for RASSF1A and TIMP3 to predict severe bladder damage were 67.84% and 63.73% respectively. The combined model, which used both RASSF1A and TIMP3 promoter methylation, resulted in significant increase in AUC-ROC compared to that of TIMP3 (77.55% vs. 63.73%.29; p = 0.023).

Conclusions

In this pilot study, we showed that aberrant promoter methylation of RASSF1A and TIMP3 are present in urine sediments of patients with severe bladder damage associated with S. haematobium infection and that may be used to develop non-invasive biomarker of S. haematobium exposure and early molecular risk assessmentof neoplastic transformation.     

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2''-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.     

Clinical Significance of MLH1 Methylation and CpG Island Methylator Phenotype as Prognostic Markers in Patients with Gastric Cancer

Background

To improve the outcome of patients suffering from gastric cancer, a better understanding of underlying genetic and epigenetic events in this malignancy is required. Although CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) have been shown to play pivotal roles in gastric cancer pathogenesis, the clinical significance of these events on survival outcomes in patients with gastric cancer remains unknown.

Methods

This study included a patient cohort with pathologically confirmed gastric cancer who had surgical resections. A cohort of 68 gastric cancers was analyzed. CIMP and MSI statuses were determined by analyzing promoter CpG island methylation status of 28 genes/loci, and genomic instability at 10 microsatellite markers, respectively. A Cox’s proportional hazards model was performed for multivariate analysis including age, stage, tumor differentiation, KRAS mutation status, and combined CIMP/MLH1 methylation status in relation to overall survival (OS).

Results

By multivariate analysis, longer OS was significantly correlated with lower pathologic stage (P = 0.0088), better tumor differentiation (P = 0.0267) and CIMP-high and MLH1 3'' methylated status (P = 0.0312). Stratification of CIMP status with regards to MLH1 methylation status further enabled prediction of gastric cancer prognosis.

Conclusions

CIMP and/or MLH1 methylation status may have a potential to be prognostic biomarkers for patients with gastric cancer.     

DNA Methylation Profiles and Their Relationship with Cytogenetic Status in Adult Acute Myeloid Leukemia

Background

Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required.

Methodology/Principal Findings

We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients.

Conclusions/Significance

Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.     

Gene promoter methylation assayed in exhaled breath,with differences in smokers and lung cancer patients

Background

There is a need for new, noninvasive risk assessment tools for use in lung cancer population screening and prevention programs.

Methods

To investigate the technical feasibility of determining DNA methylation in exhaled breath condensate, we applied our previously-developed method for tag-adapted bisulfite genomic DNA sequencing (tBGS) for mapping of DNA methylation, and adapted it to exhaled breath condensate (EBC) from lung cancer cases and non-cancer controls. Promoter methylation patterns were analyzed in DAPK, RASSF1A and PAX5β promoters in EBC samples from 54 individuals, comprised of 37 controls [current- (n = 19), former- (n = 10), and never-smokers (n = 8)] and 17 lung cancer cases [current- (n = 5), former- (n = 11), and never-smokers (n = 1)].

Results

We found: (1) Wide inter-individual variability in methylation density and spatial distribution for DAPK, PAX5β and RASSF1A. (2) Methylation patterns from paired exhaled breath condensate and mouth rinse specimens were completely divergent. (3) For smoking status, the methylation density of RASSF1A was statistically different (p = 0.0285); pair-wise comparisons showed that the former smokers had higher methylation density versus never smokers and current smokers (p = 0.019 and p = 0.031). For DAPK and PAX5β, there was no such significant smoking-related difference. Underlying lung disease did not impact on methylation density for this geneset. (4) In case-control comparisons, CpG at -63 of DAPK promoter and +52 of PAX5β promoter were significantly associated with lung cancer status (p = 0.0042 and 0.0093, respectively). After adjusting for multiple testing, both loci were of borderline significance (p_adj = 0.054 and 0.031). (5) The DAPK gene had a regional methylation pattern with two blocks (1)~-215~-113 and (2) -84 ~+26); while similar in block 1, there was a significant case-control difference in methylation density in block 2 (p = 0.045); (6)Tumor stage and histology did not impact on the methylation density among the cases. (7) The results of qMSP applied to EBC correlated with the corresponding tBGS sequencing map loci.

Conclusion

Our results show that DNA methylation in exhaled breath condensate is detectable and is likely of lung origin. Suggestive correlations with smoking and lung cancer case-control status depend on individual gene and CpG site examined.     

A Meta-Analysis of the Relationship Between RARβ Gene Promoter Methylation and Non-Small Cell Lung Cancer

Background

Hypermethylation of CpG islands in tumor suppressor gene plays an important role in carcinogenesis. Many studies have demonstrated that hypermethylation in promoter region of RARβ gene could be found with high prevalence in tumor tissue and autologous controls such as corresponding non-tumor lung tissue, sputum and plasma of the NSCLC patients. But with the small number subjects included in the individual studie, the statistical power is limited. Accordingly, we performed this meta-analysis to further asses the relationship of methylation prevalence between the cancer tissue and atuologous controls (corresponding non-tumor lung tissue, sputum and plasma).

Methods

The published articles about RARβ gene promoter hypermethyltion were identified using a systematic search strategy in PubMed, EMBASE and CNKI databases. The pooled odds ratio (OR) of RARβ promoter methylation in lung cancer tissue versus autologous controls were calculated.

Results

Finally, eleven articles, including 1347 tumor tissue samples and 1137 autologous controls were included in this meta-analysis. The pooled odds ratio of RARβ promoter methylation in cancer tissue was 3.60 (95%CI: 2.46–5.27) compared to autologous controls with random-effect model. Strong and significant correlation between tumor tissue and autologous controls of RARβ gene promoter hypermethylation prevalence across studies (Correlation coefficient 0.53) was found.

Conclusion

RARβ promoter methylation may play an important role in carcinogenesis of the NSCLC. With significant methylation prevalence correlation between tumor tissue and autologous of this gene, methylation detection may be a potential method for searching biomarker for NSCLC.