Genome-wide analysis of neuroblastomas using high-density single nucleotide polymorphism arrays

Abstract

Background: Neuroblastomas are characterized by chromosomal alterations with biological and clinical significance. We analyzed paired blood and primary tumor samples from 22 children with high-risk neuroblastoma for loss of heterozygosity (LOH) and DNA copy number change using the Affymetrix 10K single nucleotide polymorphism (SNP) array.

Findings: Multiple areas of LOH and copy number gain were seen. The most commonly observed area of LOH was on chromosome arm 11q (15/22 samples; 68%). Chromosome 11q LOH was highly associated with occurrence of chromosome 3p LOH: 9 of the 15 samples with 11q LOH had concomitant 3p LOH (P = 0.016). Chromosome 1p LOH was seen in one-third of cases. LOH events on chromosomes 11q and 1p were generally accompanied by copy number loss, indicating hemizygous deletion within these regions. The one exception was on chromosome 11p, where LOH in all four cases was accompanied by normal copy number or diploidy, implying uniparental disomy. Gain of copy number was most frequently observed on chromosome arm 17q (21/22 samples; 95%) and was associated with allelic imbalance in six samples. Amplification of MYCN was also noted, and also amplification of a second gene, ALK, in a single case.

Conclusions: This analysis demonstrates the power of SNP arrays for high-resolution determination of LOH and DNA copy number change in neuroblastoma, a tumor in which specific allelic changes drive clinical outcome and selection of therapy.

Figure 1

Frequency of chromosomal aberrations. Fraction of samples with copy number >2.8 (red bars above baseline), copy number <1.2 (grey bars below baseline), and LOH (blue bars below baseline). SNPs are mapped according to their chromosomal position, from chromosome 1 on the left to chromosome X on the right. (Copy number alterations for chromosome X in samples derived from males were counted if copy number was >1.4 or <0.6.)

Figure 2

LOH and copy number changes on chromosome 11. SNP array analysis of neuroblastoma tumor samples and matched constitutional DNA showing LOH on the left and copy number on the right of the chromosome 11 ideogram. For each sample, chromosome 11 is depicted as a vertical bar in both the LOH and copy number panels. Blue areas represent regions of LOH, while yellow denotes retention of heterozygosity. Copy number is marked by shades of red, with ≤1 copy in light red and ≥3 copies in dark red (see scale at the bottom of the panel). Both chromosome 11q and 11p LOH, as well as gain of 11p, are shown.

Figure 3

LOH and copy number changes on chromosomes 11, 3, 4, 1, 17, and 7. Global view of common areas of LOH (left) and copy number change (right) in 22 primary neuroblastomas. Each sample is depicted as a series of vertical bars in both the LOH and copy number panels. Blue areas represent regions of LOH, while yellow denotes retention of heterozygosity. Copy number is marked by shades of red, with ≤1 copy in light red and ≥3 copies in dark red (see scale at the bottom of the panel).

Figure 4

Copy number and LOH analysis of chromosome 17. SNP array analysis of neuroblastoma tumor samples and matched constitutional DNA showing LOH on the left and copy number on the right of the chromosome 17 ideogram. For each sample, chromosome 17 is depicted as a vertical bar in both the LOH and copy number panels. Blue areas represent regions of LOH, while yellow denotes retention of heterozygosity. Copy number is marked by shades of red, with ≤1 copy in light red and ≥3 copies in dark red (see scale at the bottom of the panel). This figure depicts copy number gain of chromosome 17q in 21 out of 22 samples.

Figure 5

Amplifications on chromosome 2p. (A) SNP copy number analysis of a portion of chromosome arm 2p demonstrating amplification (represented by the darker red SNPs) at the MYCN gene locus (2p24.3) in three tumor samples and at the ALK gene locus (2p23.2) in one sample. (B) Individual SNP copy number assessment of the SNPs surrounding the ALK locus. The blue curve in the graph on the right indicates the degree of amplification of each SNP from 0 on the left to 40 on the right. The red vertical line indicates a copy number of 2. (C) FISH of neuroblastoma with the MYCN probe showing multiple copies of MYCN (left panel), and FISH of the same tumor using the ALK break apart probe showing amplification of ALK (right panel).