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

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

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

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

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

High-Resolution Array CGH Profiling Identifies Na/K Transporting ATPase Interacting 2 (NKAIN2) as a Predisposing Candidate Gene in Neuroblastoma

<div><p>Neuroblastoma (NB), the most common solid cancer in early childhood, usually occurs sporadically but also its familial occurance is known in 1-2% of NB patients. Germline mutations in the <i>ALK</i> and <i>PHOX2B</i> genes have been found in a subset of familial NBs. However, because some individuals harbouring mutations in these genes do not develop this tumor, additional genetic alterations appear to be required for NB pathogenesis. Herein, we studied an Italian family with three NB patients, two siblings and a first cousin, carrying an <i>ALK</i> germline-activating mutation R1192P, that was inherited from their unaffected mothers and with no mutations in the <i>PHOX2B</i> gene. A comparison between somatic and germline DNA copy number changes in the two affected siblings by a high resolution array-based Comparative Genomic Hybridization (CGH) analysis revealed a germline gain at <i>NKAIN2</i> (Na/K transporting ATPase interacting 2) locus in one of the sibling, that was inherited from the parent who does not carry the ALK mutation. Surprisingly, NKAIN2 was expressed at high levels also in the affected sibling that lacks the genomic gain at this locus, clearly suggesting the existance of other regulatory mechanisms. High levels of NKAIN2 were detected in the MYCN-amplified NB cell lines and in the most aggressive NB lesions as well as in the peripheral blood of a large cohort of NB patients. Consistent with a role of NKAIN2 in NB development, NKAIN2 was down-regulated during all-trans retinoic acid differentiation in two NB cell lines. Taken together, these data indicate a potential role of NKAIN2 gene in NB growth and differentiation.</p></div

Clinical-pathologic characteristics of NB patients.

<p>INSS, International Neuroblastoma Staging System; histology is based on International Neuroblastoma Pathology Classification (INPC).</p>a<p>Age at diagnosis; months.</p

Expression of <i>NKAIN2, PCNX</i> and <i>CASC4</i> in the affected siblings and their parents.

<p>qPCR analysis of mRNAs from age-matched healthy donors (HD, n = 6), the affected sibling (III-2 and III-3) and their parents (II-2 and II-3). β-actin was used for normalization. The error bars represent the mean ± SD (n = 3).</p

Array-CGH results in tumor and peripheral blood DNA samples.

<p>Array-CGH results in tumor and peripheral blood DNA samples.</p