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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/MLLrearrangements had low rates of deletion (11%, 15%, 13%), whereas patients with t(9;22), t(1;19), TLX3, or TLX1rearrangements had higher frequencies (61%, 42%, 78%, and 89%). In conclusion, CDKN2Adeletion is a significant secondary abnormality in childhood ALL strongly correlated with phenotype and genotype. The variation in the incidence of CDKN2Adeletions by cytogenetic subgroup may explain its inconsistent association with outcome. CNN LOH without apparent CDKN2Ainactivation suggests the presence of other relevant genes in this region.

The assessment of minimal residual disease during therapy is a powerful prognostic marker in childhood acute lymphoblastic leukemia but current techniques, whether molecular analysis of antigen receptor gene rearrangements or flow cytometric analysis of aberrant immunophenotypes, track the blast population(s) that predominate(s) at diagnosis. Recent evidence from diagnostic TEL/AML1 positive cases (Hong et al, 2008) suggests that the candidate leukemic stem cell population may be characterised by the immunophenotype CD34+CD38LowCD19+and thus the identification and quantitation of this cell population at diagnosis and during therapy may have greater clinical significance.

Although balanced translocations often result in the formation of chimaeric fusion genes, the analysis of unbalanced translocations has largely failed to identify target loci. This is due to the heterogeneity of the chromosomal breakpoints and the multiplicity of partner chromosomes. One such example is that of dicentric chromosomes found in patients with acute lymphoblastic leukaemia (ALL). We investigated patients with dic(7;9)(p11;p11~13) [n=13], dic(9;12)(p11~13;p13) [n=38] and dic(9;20)(p11~13;q11) [n=59] to determine the breakpoints of these translocations. FISH with probes for 9p showed that breakpoints were heterogeneous [n=54]. Apart from two cases with dic(9;20), all showed either deletion of the entire PAX5gene [53.7%, n=29] or a breakpoint within the gene [42.6%, n=23]. For those cases with breakpoints within PAX5, FISH identified ETV6as the partner in dic(9;12) cases (18/19 cases tested). Sequencing showed a novel breakpoint within intron 1 of ETV6in a one case of dic(9;12). Molecular copy number counting (MCC) and long distance inverse PCR (LDI-PCR) identified five novel fusion sequences in which PAX5partnered the LOC392027(7p12.1), SLCO1B3(12p12), ASXL1(20q11.1), KIF3B(20q11.21) and C20orf112(20q11.1) loci. The PAX5-ASXL1and PAX5-KIF3Bfusion sequences were in opposing orientation, the remaining three fusions were in the same orientation but out of frame, implicating loss of PAX5 function. To confirm the significance of PAX5disruption, we demonstrated that both the deletion and gene fusion events resulted in the same under-expression of PAX5exons 1–2 (p<0.04), and 4–5 (p<0.04) in the patients with dicentric chromosomes, in association with differential expression of the PAX5target genes, EBF1(p<0.001), ALDH1A1(p<0.001), ATP9A(p<0.01) and FLT3(p<0.006). Genomic quantitative PCR (gqPCR) confirmed the heterozygous loss of PAX5[n=16] and showed homozygous deletions involving the homologous allele in two patients. Mutation analysis [n=20] identified two cases with a single base pair deletion of exon 8 and a single base pair insertion into exon 2, both of which would translate into a truncated PAX5 protein. This is further support that the formation of the dicentric chromosome provided leukemic potential by abrogating normal PAX5function in these cases. The same analysis of chromosome 20 in 26 dic(9;20) cases highlighted ASXL1as the potential target gene in 62% [n=16] patients with the dic(9;20) tested, manifesting as recurrent fusion events and deletions as shown for PAX5. Three additional fusion sequences were identified; involving ASXL1, FRG1B(20q11.1) and LOC149950(20q11.21) fused to sequence centromeric of PAX5. Here, we have shown for the first time, that specific gene loci may be the target of heterogeneous breakpoints in human cancer, acting through a variety of mechanisms. Although several investigations have failed to identify the key molecular events in patients with dicentric chromosomes, we report PAX5and ASXL1as key target genes, as a consequence of their involvement in multiple fusion genes and by deletion. This approach has considerable application to the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent while relatively few genes have been identified. In the absence of a dicentric chromosome, PAX5is also targeted by interstitial deletions and copy number neutral LOH events, further supporting the importance of investigating the underlying molecular basis of unbalanced translocations. From the expansion of this approach into other tumour types, a large number of novel genes will surely emerge, expanding our understanding of carcinogenesis and ultimately leading to improved management of patients with cancer.

Genetic alterations play a key role in the leukemogenesis of childhood ALL. Inactivation of CDKN2A (p16), a tumour suppressor gene located at 9p21, can occur by deletion, methylation or mutation. Published reports are inconsistent in terms of incidence and mode of inactivation. We report a comprehensive analysis of CDKN2Ainactivation in 1230 diagnostic and 101 relapse samples, including 46 matched diagnostic and relapse pairs, from 1285 children with ALL. Using data from cytogenetics (CC) (n=1088), FISH (n=1209), SNP arrays (SNPA) (n=106), CGH arrays (aCGH) (n=106), dHPLC (n=48) and methylation specific-PCR (MSP) (n=96) we have assessed the mode and frequency of CDKN2Ainactivation. Mutation or methylation of CDKN2Awas rare occurring in 1 patient each (2% and 1% respectively). In contrast, CDKN2Adeletion was highly prevalent. The frequencies of deletion detected by the different methodologies were: CC 166 (15%), FISH 335 (28%), SNPA 17 (16%) and aCGH 35 (33%). The proportion of biallelic deletions also varied: CC 15 (9%), FISH 174 (52%) and aCGH 15 (65%). This variation was directly related to the resolution of each technique with a high degree of concordance across samples investigated by >1 method. Analysis of 50 deletions by aCGH showed that the size of the deletion ranged from 0.03Mb to 39.1Mb with a mean of 14.8Mb. Furthermore, analysis of 15 biallelic deletions demonstrated that they comprised one large deletion (mean size 23.3Mb) and a second much smaller deletion (mean size 1.4Mb). In addition, SNPA revealed copy number neutral LOH in 8 (8%) cases, but only once in association with a CDKN2Amutation. At diagnosis CDKN2Ainactivation by any method was noted in 329 (27%) patients which was not different from that observed at relapse [25 (25%)]. However, the frequencies of CDKN2Ainactivation and biallelic deletion were significantly greater in T-ALL compared with B cell precursor (BCP) ALL: 135/269 (50%) v 190/918 (21%) (p<0.001) and 83/135 (61%) v 82/190 (43%) (p=0.001), respectively. Within BCP-ALL, older patients (10+ yrs) were more likely to have CDKN2Ainactivation compared to younger patients (<10 yrs) whereas the reverse was true in T-ALL: 52/196 (27%) v 138/722 (19%) (p=0.023) and 53/126 (42%) v 82/143 (57%) (p=0.012). Among 46 matched samples CDKN2Ainactivation was retained (n=8), lost (n=3) or gained (n=6). The frequency of CDKN2Ainactivation was strongly correlated with cytogenetics. Lower frequencies were observed among high hyperdiploid [31/302 (10%) p<0.001] and ETV6-RUNX1patients [36/236 (15%) p=0.02] with higher frequencies among those with t(9;22) [11/19 (58%) p<0.001], t(1;19) [10/25 (40%) p=0.019] and other abnormalities [92/226 (41%) p<0.001]. In conclusion, we have confirmed the importance of CDKN2Ainactivation in childhood ALL and demonstrated that by far the most prevalent method of inactivation is deletion. While it is clear that loss of CDKN2Aacts as a cooperating mutation in childhood ALL it is strongly correlated with age, phenotype and genotype. The observation that it is negatively correlated with good risk cytogenetic subgroups may explain why it has been inconsistently associated with a poor outcome. The discovery of copy number neutral LOH at 9p with no evidence of CDKN2Ainactivation suggests the presence of another tumour suppressor gene or oncogene in this region.

Genetic alterations including chromosomal translocation, promoter hypermethylation, somatic mutation and gene deletion are believed to play a key role in the leukemogenic process in childhood acute lymphoblastic leukemia (ALL). The p16INK4a(CDKN2A/MTS1/p16/INK4a) gene located on chromosome 9p21 is a tumor suppressor gene whose product can block cell division during the G1/S phase of the cell cycle. Inactivation of p16INK4ain ALL can occur by deletion, promoter hypermethylation or somatic mutation. However, published reports are inconsistent in terms of both incidence and route of p16INK4ainactivation suggesting that a detailed analysis of all possible modes of inactivation in a large cohort is essential to clarify the status of this gene in leukemogenesis. In this study, we report the findings of a comprehensive analysis of p16INK4ain 115 DNA samples with childhood ALL (86 cases at presentation and 29 cases at relapse) in which a combination of techniques including, fluorescence in situhybridization (FISH), mapping arrays, denaturing high performance liquid chromatography (dHPLC) and methylation specific-PCR (MSP) were used to assess the mode of inactivation of this gene. Data from a genome-wide screening in 86 presentation cases and 20 of 29 relapse cases using Affymetrix Mapping 10K and/or 50K single nucleotide polymorphism (SNP) microarray technique showed loss of heterozygosity (LOH) at the p16INK4alocus in 21% (22/106) of cases (14 at presentation and 8 at relapse), 14 (8 at presentation and 6 at relapse) with an associated loss of copy number and 8 (6 at presentation and 2 at relapse) with a normal copy number, indicative of acquired isodisomy (AID). FISH analysis on 19 of the 22 confirmed that those cases with LOH and copy number loss had either p16INK4ahomozygous (n=6) or hemizygous (n=6) deletion and those with LOH associated with AID (n=7) retained 2 copies. Mutation and methylation analyses were performed on those cases identified to have one p16INK4aallele or retention of both alleles. Partial methylation of p16INK4awas found in only 1 case. Mutational screening by dHPLC of the coding region revealed a somatic mutation, H83Y, in a subpopulation of leukemic blasts in one patient at relapse. Three common SNPs were identified including A148T in exon 2 and 500C>G and 540 C>T in the 3′ UTR. These data show that mutation and hypermethylation of p16INK4aare rare events in childhood ALL but that homozygous and hemizygous deletion is relatively common. The loss of only one p16INK4aallele in this latter group, without evidence for mutation or hypermethylation of the remaining one suggests that p16INK4amay be haploinsufficient in ALL. The finding that LOH on 9p locus is common but in nearly 40% of these cases is associated with AID with intact p16INK4a, suggests the existence of another tumor suppressor gene or oncogene in this region, which may have importance in leukemogenesis.