Comprehensive Analysis of p16^INK4a in Childhood Acute Lymphoblastic Leukemia Reveals Homozygous Deletion, Haploinsufficiency and Acquired Isodisomy at the 9p Locus with Intact p16^INK4a.

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 p16^INK4a (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 p16^INK4a in ALL can occur by deletion, promoter hypermethylation or somatic mutation. However, published reports are inconsistent in terms of both incidence and route of p16^INK4a inactivation 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 p16^INK4a in 115 DNA samples with childhood ALL (86 cases at presentation and 29 cases at relapse) in which a combination of techniques including, fluorescence in situ hybridization (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 p16^INK4a locus 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 p16^INK4a homozygous (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 p16^INK4a allele or retention of both alleles. Partial methylation of p16^INK4a was 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 p16^INK4a are rare events in childhood ALL but that homozygous and hemizygous deletion is relatively common. The loss of only one p16^INK4a allele in this latter group, without evidence for mutation or hypermethylation of the remaining one suggests that p16^INK4a may 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 p16^INK4a, suggests the existence of another tumor suppressor gene or oncogene in this region, which may have importance in leukemogenesis.