Outcome prediction and risk assessment by quantitative pyrosequencing methylation analysis of the SFN gene in advanced stage, high-risk, neuroblastic tumor patients.

Laboratory of Tumor Genetics, Istituto Nazionale per la Ricerca sul Cancro, IST, Genova, Italy.
International Journal of Cancer (Impact Factor: 6.2). 08/2009; 126(3):656-68. DOI: 10.1002/ijc.24768
Source: PubMed

ABSTRACT The aim of our study was to identify threshold levels of DNA methylation predictive of the outcome to better define the risk group of stage 4 neuroblastic tumor patients. Quantitative pyrosequencing analysis was applied to a training set of 50 stage 4, high risk patients and to a validation cohort of 72 consecutive patients. Stage 4 patients at lower risk and ganglioneuroma patients were included as control groups. Predictive thresholds of methylation were identified by ROC curve analysis. The prognostic end points of the study were the overall and progression-free survival at 60 months. Data were analyzed with the Cox proportional hazard model. In a multivariate model the methylation threshold identified for the SFN gene (14.3.3sigma) distinguished the patients presenting favorable outcome from those with progressing disease, independently from all known predictors (Training set: Overall Survival HR 8.53, p = 0.001; Validation set: HR 4.07, p = 0.008). The level of methylation in the tumors of high-risk patients surviving more than 60 months was comparable to that of tumors derived from lower risk patients and to that of benign ganglioneuroma. Methylation above the threshold level was associated with reduced SFN expression in comparison with samples below the threshold. Quantitative methylation is a promising tool to predict survival in neuroblastic tumor patients. Our results lead to the hypothesis that a subset of patients considered at high risk-but displaying low levels of methylation-could be assigned at a lower risk group.

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    ABSTRACT: Approximately 20% of stage 4 high-risk neuroblastoma patients are alive and disease-free 5 years after disease onset while the remaining experience rapid and fatal progression. Numerous findings underline the prognostic role of methylation of defined target genes in neuroblastoma without taking into account the clinical and biological heterogeneity of this disease. In this report we have investigated the methylation of the PCDHB cluster, the most informative member of the "Methylator Phenotype" in neuroblastoma, hypothesizing that if this epigenetic mark can predict overall and progression free survival in high-risk stage 4 neuroblastoma, it could be utilized to improve the risk stratification of the patients, alone or in conjunction with the previously identified methylation of the SFN gene (14.3.3sigma) that can accurately predict outcome in these patients. We have utilized univariate and multivariate models to compare the prognostic power of PCDHB methylation in terms of overall and progression free survival, quantitatively determined by pyrosequencing, with that of other markers utilized for the patients' stratification utilizing methylation thresholds calculated on neuroblastoma at stage 1-4 and only on stage 4, high-risk patients. Our results indicate that PCDHB accurately distinguishes between high- and intermediate/low risk stage 4 neuroblastoma in agreement with the established risk stratification criteria. However PCDHB cannot predict outcome in the subgroup of stage 4 patients at high-risk whereas methylation levels of SFN are suggestive of a "methylation gradient" associated with tumor aggressiveness as suggested by the finding of a higher threshold that defines a subset of patients with an extremely severe disease (OS <24 months). Because of the heterogeneity of neuroblastoma we believe that clinically relevant methylation markers should be selected and tested on homogeneous groups of patients rather than on patients at all stages.
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    ABSTRACT: We have addressed whether inter-individual methylation variation in somatic (white blood cells, WBCs) DNA of ovarian cancer patients provides potential for prognostic and/or pharmacoepigenetic stratification. WBC DNA methylation was analysed by bisulphite pyrosequencing at ataxia telangiectasia mutated (ATM), estrogen receptor 1 (ESR1), progesterone receptor (PGR), mutL homologue 1 (MLH1), breast cancer susceptibility gene (BRCA1), secreted frizzled-related protein 1 (SFRP1), stratifin (SFN), retinoic acid receptor beta (RARB) loci and the repetitive element LINE1 in 880 SCOTROC1 trial patients [paclitaxel (Taxol)-carboplatin versus docetaxel (Taxotere)-carboplatin as primary chemotherapy for stage Ic-IV epithelial ovarian cancer]. We observed no significant associations (P < 0.005, after correction for multiple testing) for progression-free survival (PFS) using test and validation sets. However, we did identify mean SFN methylation associated with PFS (hazard ratio, HR = 1.01 per 1% increase in methylation, q = 0.028); particularly in the paclitaxel (HR = 1.01, q = 0.006), but not in the docetaxel arm in stratified analyses. Furthermore, higher methylation within the ESR1 gene was associated with CA125 response (odds ratio, OR = 1.06, q = 0.04) and with neuropathy (HR = 0.95, q = 0.002), but only in the paclitaxel arm of the trial. This is the first study linking DNA methylation variability in WBC to clinical outcomes for any tumour type; the data generated on novel prognostic and pharmacoepigenetic DNA methylation biomarkers in the circulation now need independent further evaluation.
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