Outcome prediction and risk assessment by quantitative pyrosequencing methylation analysis of the SFN gene 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: 5.09). 08/2009; 126(3):656-68. DOI: 10.1002/ijc.24768
Source: PubMed


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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Available from: Stefano Bonassi, Sep 05, 2014
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    • "Similarly to what we have observed for the SFN gene [10], the overall distribution of the PCDHB methylation levels observed in our series of high risk stage 4 patients did not follow the bimodal distribution described by Abe et al [14] for a population of patients with neuroblastoma representative of all stages of the disease ( Figure S1). "
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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.
    PLoS ONE 05/2013; 8(5):e63253. DOI:10.1371/journal.pone.0063253 · 3.23 Impact Factor
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    • "SFN has been found to be fully methylated in MYCN-amplified neuroblastoma and partially methylated in non-amplified tumors (Banelli et al., 2005b). More recently, quantitative pyrosequencing analysis has identified that a methylation threshold level of 85% for the SFN gene distinguishes neuroblastoma patients presenting with progressive disease from those with a more favorable outcome, independent of other prognostic markers (Banelli et al., 2010). The HOXA9 (homeobox A9) gene encodes a sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis of an organism (The NCBI handbook, 2002). "

    Neuroblastoma - Present and Future, 02/2012; , ISBN: 978-953-307-016-2
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    • "Moreover, we speculate that a further plausible explanation for the directionality of expression of T-UCRs in long- versus short-term survivors may be due to the contribution of an epigenetic regulation of T-UCR expression. This is supported by the fact that a CpG island methylator phenotype is present specifically in neuroblastoma with poor prognosis [24,25], and that epigenetic mechanisms not only regulate coding genes but also non-coding RNAs, such as miRNAs [26]. According to these information, we retrieved the promoter sequences of T-UCR-host genes from DataBase of Transcriptional Start Sites (DBTSS, release 7.0,, "
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    ABSTRACT: Neuroblastoma is the most common, pediatric, extra-cranial, malignant solid tumor. Despite multimodal therapeutic protocols, outcome for children with a high-risk clinical phenotype remains poor, with long-term survival still less than 40%. Hereby, we evaluated the potential of non-coding RNA expression to predict outcome in high-risk, stage 4 neuroblastoma. We analyzed expression of 481 Ultra Conserved Regions (UCRs) by reverse transcription-quantitative real-time PCR and of 723 microRNAs by microarrays in 34 high-risk, stage 4 neuroblastoma patients. First, the comparison of 8 short- versus 12 long-term survivors showed that 54 UCRs were significantly (P < 0.0491) over-expressed in the former group. For 48 Ultra Conserved Region (UCRs) the expression levels above the cut-off values defined by ROC curves were strongly associated with good-outcome (OS: 0.0001 <P < 0.0185, EFS: 0.0001 <P < 0.0491). Then we tested the Transcribed-UCR (T-UCR) threshold risk-prediction model on an independent cohort of 14 patients. The expression profile of 28 T-UCRs was significantly associated to prognosis and at least 15 up-regulated T-UCRs are needed to discriminate (P < 0.0001) short- from long-survivors at the highest sensitivity and specificity (94.12%). We also identified a signature of 13 microRNAs differently expressed between long- and short-surviving patients. The comparative analysis of the two classes of non-coding RNAs disclosed that 9 T-UCRs display their expression level that are inversely correlated with expression of 5 complementary microRNAs of the signature, indicating a negative regulation of T-UCRs by direct interaction with microRNAs. Moreover, 4 microRNAs down-regulated in tumors of long-survivors target 3 genes implicated in neuronal differentiation, that are known to be over-expressed in low-risk tumors. Our pilot study suggests that a deregulation of the microRNA/T-UCR network may play an important role in the pathogenesis of neuroblastoma. After further validation on a larger independent set of samples, such findings may be applied as the first T-UCR prognostic signature for high-risk neuroblastoma patients.
    BMC Cancer 12/2009; 9(1):441. DOI:10.1186/1471-2407-9-441 · 3.36 Impact Factor
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