Kaplan-Meier curves for OS rates.

<p>OS rates were compared between CC and any G (GC or GG) for the SNP <i>IL-6</i>–174 in (A) all patients, (B) in not <i>MYCN</i> amplified patients, (C) in high-risk patients and (D) not <i>MYCN</i> amplified high-risk patients.</p

Characteristics of NB patients stratified per <i>IL-6</i> -176 (G>C) SNP genotype.

<p>N.A.  =  not available.</p>a<p>p-values and ORs from comparison of allelic frequencies.</p>b<p>p-values and ORs from comparison of genotype frequencies (GG/GC vs CC).</p>c<p>p-values and ORs from comparison of stage 1+2 patients vs stage 3+4 patients.</p

Impact of Interleukin-6 –174 G>C Gene Promoter Polymorphism on Neuroblastoma

<div><p>Background</p><p>Common variants in DNA may predispose to onset and progression of neuroblastoma (NB). The genotype GG of single nucleotide polymorphism (SNP) rs1800795 (−174 G>C) in interleukin (IL)-6 promoter has been associated with lower survival of high-risk NB.</p><p>Result</p><p>To evaluate the impact of <i>IL-6</i> SNP rs1800795 on disease risk and phenotype, we analyzed 326 Italian NB patients and 511 controls. Moreover, we performed <i>in silico</i> and quantitative Real Time (qRT)-PCR analyses to evaluate the influence of the SNP on gene expression in 198 lymphoblastoid cell lines (LCLs) and in 31 NB tumors, respectively. Kaplan-Meier analysis was used to verify the association between <i>IL-6</i> gene expression and patient survival. We found that <i>IL-6</i> SNP is not involved in susceptibility to NB development. However, our results show that a low frequency of genotype CC is significantly associated with a low overall survival, advanced stage, and high-risk phenotype. The <i>in silico</i> (<i>p</i> = 2.61×10⁻⁵) and qRT-PCR (<i>p</i> = 0.03) analyses showed similar trend indicating that the CC genotype is correlated with increased level of <i>IL-6</i> expression. In report gene assay, we showed that the −174 C variant had a significantly increased transcriptional activity compared with G allele (<i>p</i> = 0.0006). Moreover, Kaplan-Meier analysis demonstrated that high levels of <i>IL-6</i> are associated with poor outcome in children with NB in two independent gene expression array datasets.</p><p>Conclusions</p><p>The biological effect of SNP <i>IL-6</i>–174 G>C in relation to promotion of cancer progression is consistent with the observed decreased survival time. The present study suggests that SNP <i>IL-6</i>–174 G>C may be a useful marker for NB prognosis.</p></div

Case-Control study of <i>IL-6</i>–176 (G>C) SNP.

a<p>P-value and OR obtained by Armitage's trend test.</p

Test for independent statistical significance of −174 <i>IL-6</i> SNP after adjustment for NB risk factors.

<p>HR, hazard ratio; CI, confidence interval; C-index, concordance index.</p>a<p>C-index calculated including in the model only the NB risk factor (Age or MYCN or INSS stage 4).</p>b<p>C-index calculated including in the model the NB risk factor and <i>IL-6</i> SNP.</p

SNP genotype correlation to <i>IL-6</i> gene expression and NB outcome.

<p><i>In silico</i> and qRT-PCR analysis of <i>IL-6</i> mRNA expression in (A) 198 LCLs and (B) 31 NB tumors, respectively, stratified according to the SNP <i>IL-6</i>–174. (C) Luciferase report gene assay carried out in HEK293 cells. Data shown in percentage are the mean ± SD from three independent transfection experiments, each done in triplicate and compared with promoter less control. Kaplan-Meier analysis is shown, with individuals grouped by median of expression of <i>IL-6</i> for OS and Progression Free Survival (PFS) rates in (D and E) 88 NB patients and (F) only PFS for 102 INSS stage 4 patients with <i>MYCN</i> not amplified. OS data of Seeger dataset were not available.</p

The four-gene signature predict neuroblastoma outcome.

<p>Kaplan–Meier and log-rank analysis for progression-free (PFS) and overall (OS) survival in discovery set 1 (A), set 3 (B), and set 4 (C) based on the four-gene signature score. (D, E) Comparison of signature performance between all 25 and the 4 selected genes. Results from discovery and validation study strategy. (D) Gene predictors made in discovery set 4 and predictions made in discovery set 1. (E) Gene predictors made in validation set 4 and predictions made in discovery set 5. X axis indicates clinical factors: death event (DE), relapse event (RE), and INSS stage; Y axis indicates Area Under Curve (AUC).</p

Correlation of p53 repressed genes and poor prognosis.

<p>(A) Heat map of the gene set significantly down-regulated by Nutlin-3a but not Nutlin-3b. (B) Heat map with hierarchical clustering of the gene set with respect to neuroblastoma prognosis (discovery set 5). (C) Kaplan–Meier and log-rank analysis for progression-free (PFS) and overall (OS) survival of discovery set 2 based on 25 gene set: survival of 101 neuroblastoma patients in the 4 quartiles of the signature score. Numbers in parentheses refer to number of patients who experienced an event. (D) Multivariate logistic regression analysis: Odds Ratio and p-values are shown for disease stage (stage 4 vs. other), age (< or > 12 months), <i>MYCN</i> status (amplified vs. non-amplified), and the 25-gene signature score according to methods previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079843#pone.0079843-DePreter1" target="_blank">[19]</a>.</p

The four-gene signature is regulated by p53 activity.

<p>Activation of p53 represses the four-gene signature. (A, B) Quantitative PCR demonstrates marked decrease of mRNA levels of the four genes after Nutlin-3a treatment (10 uM for 8 hours) in multiple p53 wild-type neuroblastoma lines (shown here LAN5 and IMR32). However, this effect is completely abrogated after p53 silencing or when the effect of Nutlin-3a is tested in a p53 mutant neuroblastoma cell line (LAN1) (C). Each error bar represents two biological replicates.</p

Independent validation of the four-gene signature in a large cohort of neuroblastoma patients (SIOPEN/GPOH).

<p>(A) Kaplan–Meier and log-rank analysis for progression-free (PFS) and overall (OS) survival of the SIOPEN/GPOH cohort (discovery set 6): survival of 348 neuroblastoma patients in the 4 quartiles of the signature score. (B) Validation of the signature based on the four-gene PAM classifier. (C) Classification of the SIOPEN stage 4 tumors with <i>MYCN</i> single copy based on the four-gene PAM classifier. (D) Multivariate logistic regression analysis: Odds Ratio and p-values are shown for disease stage (stage 4 vs. other), age (<> 1y), <i>MYCN</i> status, and the four-gene signature score.</p