A comprehensive analysis of the CDKN2A gene in childhood acute lymphoblastic leukemia reveals genomic deletion, copy number neutral loss of heterozygosity, and association with specific cytogenetic subgroups.

Inactivation of the tumor suppressor gene, CDKN2A, can occur by deletion, methylation, or mutation. We assessed the principal mode of inactivation in childhood acute lymphoblastic leukemia (ALL) and frequency in biologically relevant subgroups. Mutation or methylation was rare, whereas genomic deletion occurred in 21% of B-cell precursor ALL and 50% of T-ALL patients. Single nucleotide polymorphism arrays revealed copy number neutral (CNN) loss of heterozygosity (LOH) in 8% of patients. Array-based comparative genomic hybridization demonstrated that the mean size of deletions was 14.8 Mb and biallelic deletions composed a large and small deletion (mean sizes, 23.3 Mb and 1.4 Mb). Among 86 patients, only 2 small deletions were below the resolution of detection by fluorescence in situ hybridization. Patients with high hyperdiploidy, ETV6-RUNX1, or 11q23/MLL rearrangements had low rates of deletion (11%, 15%, 13%), whereas patients with t(9;22), t(1;19), TLX3, or TLX1 rearrangements had higher frequencies (61%, 42%, 78%, and 89%). In conclusion, CDKN2A deletion is a significant secondary abnormality in childhood ALL strongly correlated with phenotype and genotype. The variation in the incidence of CDKN2A deletions by cytogenetic subgroup may explain its inconsistent association with outcome. CNN LOH without apparent CDKN2A inactivation suggests the presence of other relevant genes in this region.

Ploidy and karyotype complexity are powerful prognostic indicators in the Ewing's sarcoma family of tumors: a study by the United Kingdom Cancer Cytogenetics and the Children's Cancer and Leukaemia Group.

Ewing's sarcoma family tumors (ESFT) are characterized by the presence of EWSR1-ETS fusion genes. Secondary chromosome changes are frequently described, although their clinical significance is not clear. In this study, we have collected and reviewed abnormal karyotypes from 88 patients with primary ESFT and a rearrangement of 22q12. Secondary changes were identified in 80% (70/88) of tumors at diagnosis. Multivariate analysis showed a worse overall and relapse free survival (RFS) for those with a complex karyotype (overall survival, P = 0.005; RFS, P = 0.04), independent of metastatic disease. Univariate survival analysis showed that a chromosome number above 50 or a complex karyotype was associated with a worse overall survival (>50 chromosomes, P = 0.05; complex karyotype, P = 0.04). There was no association between type of cytogenetic abnormality and the presence of metastatic disease at diagnosis. Univariate and multivariate survival analysis of a small subgroup with trisomy 20 indicated that trisomy 20 was associated with a worse overall and RFS. There was no difference in outcome associated with other recurrent trisomies (2, 5, 7, 8, or 12) or the common recurrent secondary structural rearrangements (deletions of 1p36, 9p12, 17p13, and 16q, and gain of 1q), although numbers were small. These data demonstrate the continued value of cytogenetics as a genome-wide screen in ESFT and illustrates the potential importance of secondary chromosome changes for stratification of patients for risk. Specifically, karyotype complexity appears to be a powerful predictor of prognosis, and the presence of trisomy 20 may be a marker of a more aggressive subset of this group.

High-resolution analysis of allelic imbalance in neuroblastoma cell lines by single nucleotide polymorphism arrays.

Genomic copy number changes are detectable in many malignancies, including neuroblastoma, using techniques such as comparative genomic hybridization (CGH), microsatellite analysis, conventional karyotyping, and fluorescence in situ hybridization (FISH). We report the use of 10K single nucleotide polymorphism (SNP) microarrays to detect copy number changes and allelic imbalance in six neuroblastoma cell lines (IMR32, SHEP, NBL-S, SJNB-1, LS, and SKNBE2c). SNP data were generated using the GeneChip DNA Analysis and GeneChip chromosome copy number software (Affymetrix). SNP arrays confirmed the presence of all previously reported cytogenetic abnormalities in the cell lines, including chromosome 1p deletion, MYCN amplification, gain of 17q and 11q, and 14q deletions. In addition, the SNP arrays revealed several chromosome gains and losses not detected by CGH or karyotyping; these included gain of 8q21.1 approximately 24.3 and gain of chromosome 12 in IMR-32 cells; loss at 4p15.3 approximately 16.1 and loss at 16p12.3 approximately 13.2, 11q loss with loss of heterozygosity (LOH) at 11q14.3 approximately 23.3 in SJNB-1 cells; and loss at 8p21.2 approximately 23.3 and 9p21.3 approximately 22.1 with corresponding LOH in SHEP cells. The SNP arrays refined the mapping of the 2p amplicons in LS, BE2c, and IMR-32 cell lines, the 12q amplicon in LS cells, and also identified an 11q13 amplicon in LS cells. There was good concordance among SNP arrays, CGH, and karyotyping. SNP array analysis is a powerful tool for the detection of allelic imbalance in neuroblastoma and also allows identification of LOH without changes in copy number (uniparental disomy).

Loss of heterozygosity in childhood acute lymphoblastic leukemia detected by genome-wide microarray single nucleotide polymorphism analysis.

Loss of heterozygosity (LOH) is detectable in many forms of malignancy, including leukemia, using techniques such as microsatellite analysis and comparative genomic hybridization. However, these techniques are laborious and require the use of relatively large amounts of DNA if the whole genome is to be examined. Here we describe the use of oligonucleotide microarrays to characterize single nucleotide polymorphisms (SNPs) in lymphoblasts isolated from children with acute lymphoblastic leukemia for the pan-genomic mapping of LOH with a resolution of 100 to 200 kb. Results were compared with DNA obtained during remission and on relapse. Abnormalities were seen in 8 of 10 cases. The two cases with no abnormalities and one case that showed identical changes at relapse and presentation remain in remission 1 to 9 years following retreatment. The remaining seven patients died following relapse. In four cases, LOH was only detectable at relapse suggesting that progressive LOH may be a cause of disease progression and/or drug resistance. This was supported by detailed analysis of one case in which LOH involving the glucocorticoid receptor was associated with mutation of the remaining allele. The most frequent abnormality detected involved chromosome 9p. In each of the four cases where this was observed LOH included the INK4 locus. In three of the four cases, INK4 loss was only observed at relapse, suggesting that this abnormality may be commonly associated with treatment failure. These observations show that SNP array analysis is a powerful new tool for the analysis of allelic imbalance in leukemic blasts.