cDNA array-CGH profiling identifies genomic alterations specific to stage and MYCN-amplification in neuroblastoma

Abstract

Background: Recurrent non-random genomic alterations are the hallmarks of cancer and the characterization of these imbalances is critical to our understanding of tumorigenesis and cancer progression.

Results: We performed array-comparative genomic hybridization (A-CGH) on cDNA microarrays containing 42,000 elements in neuroblastoma (NB). We found that only two chromosomes (2p and 12q) had gene amplifications and all were in the MYCN amplified samples. There were 6 independent non-contiguous amplicons (10.4-69.4 Mb) on chromosome 2, and the largest contiguous region was 1.7 Mb bounded by NAG and an EST (clone: 757451); the smallest region was 27 Kb including an EST (clone: 241343), NCYM, and MYCN. Using a probabilistic approach to identify single copy number changes, we systemically investigated the genomic alterations occurring in Stage 1 and Stage 4 NBs with and without MYCN amplification (stage 1-, 4-, and 4+). We have not found genomic alterations universally present in all (100%) three subgroups of NBs. However we identified both common and unique patterns of genomic imbalance in NB including gain of 7q32, 17q21, 17q23-24 and loss of 3p21 were common to all three categories. Finally we confirm that the most frequent specific changes in Stage 4+ tumors were the loss of 1p36 with gain of 2p24-25 and they had fewer genomic alterations compared to either stage 1 or 4-, indicating that for this subgroup of poor risk NB requires a smaller number of genomic changes are required to develop the malignant phenotype.

Conclusions: cDNA A-CGH analysis is an efficient method for the detection and characterization of amplicons. Furthermore we were able to detect single copy number changes using our probabilistic approach and identified genomic alterations specific to stage and MYCN amplification.

Figure 1

Distribution of cDNA clones in our microarray. Total 23975 unique UniGene clusters remained from the initial 42591 clones after quality filtering. Number of clones in each chromosome was represented in gray bar on the left side. Average spacing (chromosome size/number of clones in the chromosome) was represented in black dot on the right side.

Figure 2

Amplifications in MYCN amplified samples. A. Regression analysis of MYCN ratio (sample vs. normal) obtained from A-CGH and real-time Q-PCR in all of neuroblastoma samples including 13 cell lines and 32 primary tumors. The slope of the regression line is 0.35 indicating that an observed A-CGH ratio of 2 is equivalent to a Q-PCR ratio of 5.7. B. Independent amplicons in chromosome 2p. All amplified genes are listed under each amplicon in genome order. Map position, genome sequence position (Mb) and samples containing the specific amplicon are listed for each amplicon. The percentage of the MYCN amplified samples harboring these amplicons are shown in brackets following the gene name for all clones present in our microarray (gray), the remainder of the clones are predicted genes found in the NCBI database that are mapped between the boundaries of the amplicon. Amplicons were selected based on the criteria of A-CGH ratio ≥2 for at least two contiguous clones in genome sequence order. In cases where a single clone has a ratio <2 but the ratio of its adjacent clones is greater than 2, that single clone was still considered as a part of amplicon. *: previously reported amplification. C. Amplicon in chromosome 12q in tumor NB21.

Figure 3

Sensitivity of A-CGH to detect the low-level copy number alteration. A. Measurement of X-chromosomal copy number. A-CGH was performed to analyze the copy number of genes in the X-chromosome. Female DNA (XX) was used as the reference DNA. Male DNA (XY), female DNA (XX), and DNA samples containing different number of X-chromosome (XXX, XXXX, XXXXX) were used as test DNA, with an expected ratio of test/reference of 0.5, 1.0, 1.5, 2.0, and 2.5 respectively for X-chromosome. Mean fluorescence ratios (±SEM) of autosomal DNAs (blue diamonds) and X-chromosomal DNAs (red circles) from each experiment are shown. The slope of the regression line is 0.3. B. Visualization of p-values derived from the topological statistics as described in the Methods along the X-chromosome from samples containing different X-chromosomal copy numbers. Each column represents a different experiment; and each row represents the p value for the alteration at a given SW-locus (a sliding window of 40 adjacent clones, details in Methods), ordered by genome map position from Xpter to Xqter. Red represents gain and green loss. The intensity of the color shows the level of significance according to the p-value shown in the color scale.

Figure 4

Genome-wide analysis of DNA copy number alteration by A-CGH. Samples were grouped based on sample type, MYCN amplification status and tumor stage. Each column represents a different sample; and each row represents a p-value of a given SW-locus using a sliding window of 40 adjacent clones, ordered in genome order across the whole genome. Black triangles on the right side of image represent centromere positions. Cell-: cell line without MYCN amplification; Cell+: cell line with MYCN amplification; Stage 4-: tumor in stage 4 without MYCN amplification; Stage 4+: tumor in stage 4 with MYCN amplification. On the right is shown an enlarged view of the region around the MYCN gene (2p24) for the amplified NB samples.

Figure 5

Genomic alterations specific to stage and MYCN status. Shown is the graphic depiction of the average p-values (P) of genomic alteration of each SW-locus (using a sliding window of 20 adjacent clones) within each tumor subgroup. All possible pair-wise comparisons among the three subgroups for gains or losses (stage 1 vs 4-, 4+ vs 4- and 1 vs. 4+) are shown. The frequency of alteration is estimated by P such that P = 0.15 is equivalent to a frequency of 70% and the lower the P the higher the frequency (details in Methods). Different colors were used to represent different clusters. Magenta: loci common to all groups; cyan: common to 1 and 4-; blue: 1 specific; green: 4- specific; red: 4+ specific, and black: all remaining loci. Colored dots were enlarged for easier visualization.