Integrative genomics identifies distinct molecular classes of neuroblastoma and shows that multiple genes are targeted by regional alterations in DNA copy number.

Neuroblastoma is remarkable for its clinical heterogeneity and is characterized by genomic alterations that are strongly correlated with tumor behavior. The specific genes that influence neuroblastoma biology and are targeted by genomic alterations remain largely unknown. We quantified mRNA expression in a highly annotated series of 101 prospectively collected diagnostic neuroblastoma primary tumors using an oligonucleotide-based microarray. Genomic copy number status at the prognostically relevant loci 1p36, 2p24 (MYCN), 11q23, and 17q23 was determined by PCR and was aberrant in 26, 20, 40, and 38 cases, respectively. In addition, 72 diagnostic neuroblastoma primary tumors assayed in a different laboratory were used as an independent validation set. Unsupervised hierarchical clustering showed that gene expression was highly correlated with genomic alterations and clinical markers of tumor behavior. The vast majority of samples with MYCN amplification and 1p36 loss of heterozygosity (LOH) clustered together on a terminal node of the sample dendrogram, whereas the majority of samples with 11q deletion clustered separately and both of these were largely distinct from the copy number neutral group of tumors. Genes involved in neurodevelopment were broadly overrepresented in the more benign tumors, whereas genes involved in RNA processing and cellular proliferation were highly represented in the most malignant cases. By combining transcriptomic and genomic data, we showed that LOH at 1p and 11q was associated with significantly decreased expression of 122 (61%) and 88 (27%) of the genes mapping to 1p35-36 and all of 11q, respectively, suggesting that multiple genes may be targeted by LOH events. A total of 71 of the 1p35-36 genes were also differentially expressed in the independent validation data set, providing a prioritized list of candidate neuroblastoma suppressor genes. Taken together, these data are consistent with the hypotheses that the neuroblastoma transcriptome is a sensitive marker of underlying tumor biology and that chromosomal deletion events in this cancer likely target multiple genes through alteration in mRNA dosage. Lead positional candidates for neuroblastoma suppressor genes can be inferred from these data, but the potential multiplicity of transcripts involved has significant implications for ongoing gene discovery strategies.

Chromosome 1p and 11q deletions and outcome in neuroblastoma.

BACKGROUND: Neuroblastoma is a childhood cancer with considerable morbidity and mortality. Tumor-derived biomarkers may improve risk stratification. METHODS: We screened 915 samples of neuroblastoma for loss of heterozygosity (LOH) at chromosome bands 1p36 and 11q23. Additional analyses identified a subgroup of cases of 11q23 LOH with unbalanced 11q LOH (unb11q LOH; defined as loss of 11q with retention of 11p). The associations of LOH with relapse and survival were determined. RESULTS: LOH at 1p36 was identified in 209 of 898 tumors (23 percent) and LOH at 11q23 in 307 of 913 (34 percent). Unb11q LOH was found in 151 of 307 tumors with 11q23 LOH (17 percent of the total cohort). There was a strong association of 1p36 LOH, 11q23 LOH, and unb11q LOH with most high-risk disease features (P<0.001). LOH at 1p36 was associated with amplification of the MYCN oncogene (P<0.001), but 11q23 LOH and unb11q LOH were not (P<0.001 and P=0.002, respectively). Cases with unb11q LOH were associated with three-year event-free and overall survival rates (+/-SE) of 50+/-5 percent and 66+/-5 percent, respectively, as compared with 74+/-2 percent and 83+/-2 percent among cases without unb11q LOH (P<0.001 for both comparisons). In a multivariate model, unb11q LOH was independently associated with decreased event-free survival (P=0.009) in the entire cohort, and both 1p36 LOH and unb11q LOH were independently associated with decreased progression-free survival in the subgroup of patients with features of low-risk and intermediate-risk disease (P=0.002 and P=0.02, respectively). CONCLUSIONS: Unb11q LOH and 1p36 LOH are independently associated with a worse outcome in patients with neuroblastoma.

High-resolution detection and mapping of genomic DNA alterations in neuroblastoma.

We used array-based comparative genomic hybridization (aCGH) to measure genomic copy number alterations (CNAs) in 42 neuroblastoma cell lines with known 1p36.3, 2p24 (MYCN), 11q23, and 17q23 allelic status. All cell lines showed CNAs, with an average of 22.0% of the genome of each sample showing evidence of gain (11.6%) or loss (10.4%). MYCN amplification was detected in 81% of cell lines, but other regions with high-level genomic amplification were observed only rarely. Gain of 17q material was present in 75% of the samples, and four discrete genomic regions at 17q23.2-17q25.3 were defined. Novel regions of gain were identified, including a 2.6-Mb subtelomeric region at 5p that includes the telomerase reverse transcriptase gene (TERT), which was found in 45% of the cell lines. Hemizygous deletions were noted at 1p36.23-1p36.32 and 11q23.3-11q25 in 60% and 36%, respectively, of the samples, with other frequent (>25%) regions of deletion localized to 1p32.1, 3p21.31-3p22.1, 5q35.2-5q35.3, 7q31.2, 7q34, 9q22.3-9q24.1, 10q26.11-10q26.12, 16q23.1-16q24.3, 18q21.32-18q23, and 20p11.21-20p11.23. A smallest region of overlap (SRO) for CNAs was mapped across all experiments and in each case was consistent with or refined the published data. A single cell line showed a homozygous deletion at 3p22.3, which was verified, and this location was refined by FISH and PCR. There was outstanding concordance of aCGH with PCR-based CNA detection methods. Several potential cooperating loci were identified, including deletion of 11q23-25, which was highly associated with both regional gain and loss at multiple chromosomal loci but was inversely correlated with the deletion of 1p36. Taking all of this together indicates that aCGH can accurately measure CNAs in the neuroblastoma genome and facilitate gene discovery efforts by high-throughput refinement of candidate loci.

Detection of single-copy chromosome 17q gain in human neuroblastomas using real-time quantitative polymerase chain reaction.

Regional genomic alterations resulting from single-copy allelic loss or gain have been well characterized in many human cancers and are often of prognostic relevance. Unbalanced gain of 17q material is common in malignant human neuroblastomas and typically results from unbalanced translocations. Unbalanced 17q gain may be an independent predictor of disease outcome, but technical difficulties with quantifying such gain using fluorescent in situ hybridization gives this method limited clinical applicability. We now describe a duplex genomic DNA-based quantitative polymerase chain reaction assay to determine the presence or absence of unbalanced gain of chromosome 17q in primary neuroblastoma specimens. The technique was first refined and validated in a panel of nine human neuroblastoma-derived cell lines by direct comparison with dual-color fluorescent in situ hybridization. Prospective blinded comparison of quantitative polymerase chain reaction and fluorescence in situ hybridization in 40 human neuroblastoma primary tumor samples showed a sensitivity of 96% and 100% specificity for detecting unbalanced 17q gain when a relative 17q copy number ratio of 1.3 was used to define unbalanced gain. Tumors with ratios >1.3 were highly associated with malignant tumor phenotypic features such as metastatic disease (P <.0001) and tumor MYCN amplification (P =.008). These data suggest that quantitative polymerase chain reaction determination of 17q status is feasible and highly specific in primary tumor samples. Sensitivity may be limited because of the inherent complexity of both the chromosomal rearrangements and heterogeneity of some tumor samples. Taken together, quantitative polymerase chain reaction can be used as a high-throughput screening tool for 17q aberrations, but a subset of samples may also require fluorescence in situ hybridization analysis in an attempt to conclusively determine 17q allelic status.