Genomic Amplifications and Distal 6q Loss: Novel Markers for Poor Survival in High-risk Neuroblastoma Patients

Abstract

Background: Neuroblastoma is characterized by substantial clinical heterogeneity. Despite intensive treatment, the survival rates of high-risk neuroblastoma patients are still disappointingly low. Somatic chromosomal copy number aberrations have been shown to be associated with patient outcome, particularly in low- and intermediate-risk neuroblastoma patients. To improve outcome prediction in high-risk neuroblastoma, we aimed to design a prognostic classification method based on copy number aberrations.

Methods: In an international collaboration, normalized high-resolution DNA copy number data (arrayCGH and SNP arrays) from 556 high-risk neuroblastomas obtained at diagnosis were collected from nine collaborative groups and segmented using the same method. We applied logistic and Cox proportional hazard regression to identify genomic aberrations associated with poor outcome.

Results: In this study, we identified two types of copy number aberrations that are associated with extremely poor outcome. Distal 6q losses were detected in 5.9% of patients and were associated with a 10-year survival probability of only 3.4% (95% confidence interval [CI] = 0.5% to 23.3%, two-sided P = .002). Amplifications of regions not encompassing the MYCN locus were detected in 18.1% of patients and were associated with a 10-year survival probability of only 5.8% (95% CI = 1.5% to 22.2%, two-sided P < .001).

Conclusions: Using a unique large copy number data set of high-risk neuroblastoma cases, we identified a small subset of high-risk neuroblastoma patients with extremely low survival probability that might be eligible for inclusion in clinical trials of new therapeutics. The amplicons may also nominate alternative treatments that target the amplified genes.

Figure 1.

Exploratory analysis of DNA copy number aberrations. A) Overall survival of high-risk patients with numerical DNA copy number profiles compared with patients with segmental profiles, showing two-sided P value of log-rank test. Curve labels represent the number of samples with number of events between brackets. B) Frequency of copy number gains/amplifications (upper part) and losses (lower part) for chromosomes 1 to 22 in 542 high-risk neuroblastoma samples with segmental copy number aberrations. OS = overall survival.

Figure 2.

Impact of number of breakpoints on patient survival. Comparison of overall survival (A and C) and event-free survival (B and D) of cases with many breakpoints (9–61, above or equal to median) vs cases with a lower number of breakpoints (2–8, below median), within both the subgroup of MYCN-amplified cases (A and B) and the subgroup of MYCN-nonamplified cases (C and D). P values represent two-sided log-rank tests. Curve labels represent the number of samples with the number of events between brackets. EFS = event-free survival; MNA = MYCN-amplified; non-MNA = MYCN-nonamplified; OS = overall survival.

Figure 3.

Association of patient survival with the presence of amplicons (MYCN locus and other loci). A and B) Comparison of survival of patients with and without MYCN amplification, for overall survival (A) and event-free survival (B). C and D) Within the subgroup of patients with MYCN-amplified tumors, comparison of overall (C) and event-free survival (D) of patients with an additional amplicon (not encompassing the MYCN locus) and patients with only the MYCN amplification. E and F) Within the subgroup of patients with MYCN-nonamplified tumors, comparison of overall (E) and event-free (F) survival for patients with an amplicon (not encompassing MYCN) and patients without an amplicon. P values represent two-sided log-rank tests. Curve labels represent the number of samples with the number of events between brackets. EFS = event-free survival; MNA = MYCN-amplified; non-MNA = MYCN-nonamplified; OS = overall survival.

Figure 4.

Association of distal 6q loss with patient survival. Overall survival of patients with a distal 6q loss compared with patients without a distal 6q loss in the training set (cohort 1 + 2) (A) and the two validation sets, cohort 3 (B) and cohort 4 (C). P values represent two-sided log-rank tests. Curve labels represent the number of samples with the number of events between brackets. OS = overall survival.

Figure 5.

Detailed view of distal 6q losses. Top: Exact location of the distal 6q deletions found in 32 patients (all cohorts). Censored patients are indicated with a lighter shade. Middle: -log10 of P values (uncorrected) of Cox regression for chromosome 6 in the training set (cohort 1 + 2). Bottom: Genes in the statistically significant region (based on Cox regression) that met at least one of the following criteria: statistically significantly lower expression of the gene in neuroblastoma cell lines with vs without a 6q deletion (squares), at least one mutation in the gene as described in primary tumors (circles), and statistically significantly worse survival outcome when gene expression is low (lowest 10th percentile) in high-risk neuroblastoma tumors (triangles). SRO = smallest region of overlap.

Figure 6.

Survival of a small subset of high-risk patients characterized by the presence of a 6q loss and/or an amplification not encompassing the MYCN locus. Kaplan-Meier and log-rank analyses of the cases with and without these genomic aberrations are depicted for overall survival (A) and event-free survival (B). Only samples analyzed on Agilent arrays are taken into account. P values represent two-sided log-rank tests. Curve labels represent the number of samples with the number of events between brackets. EFS = event-free survival; OS = overall survival.