High Frequency of P53 Mutations Detected by a Microarray Resequencing Assay, the AmpliChip P53 Test, in AML with a Complex Aberrant Karyotype.

Acute myeloid leukemia (AML) is heterogeneous in morphology, cytogenetics, and prognosis. On the basis of cytogenetics it can be subdivided into three groups with favorable, intermediate, or unfavorable prognosis. Accounting for 10%–20% of all AML cases, AML with complex aberrant karyotypes is more frequent at higher ages and is the subgroup with the worst prognosis. AML with complex aberrant karyotype is generally defined by the presence of three or more clonal chromosome abnormalities. Genetically, it has a characteristic pattern of chromosomal gains and losses, resulting also in a unique gene expression pattern including upregulation of genes involved in DNA repair. One of the important characteristics of AML with complex aberrant karyotype is the high incidence of deletions and/or mutations in the tumor suppressor p53 gene (60%–80%) which occur very unfrequently in other AML subgroups. Here we present data on 119 AML cases with complex aberrant karyotypes that were analyzed with the new AmpliChip p53 Test, a microarray-based resequencing test that is designed to detect single nucleotide changes in the entire coding region of the p53 gene. The AmpliChip p53 Test queries for the presence of sequence alterations through comparative analysis of the microarray hybridization pattern of a series of probes from sample DNA to wild type (wt) reference DNA. Starting from genomic DNA, the 10-hour assay includes PCR amplification, fragmentation and labeling, hybridization to the microarray, washing and staining, and mutation data analysis. By use of this technique we have detected p53 mutations located in exons 4–10 in 89/119 (74.8%) samples. These mutations included missense mutations, frameshift mutations, splice site mutations, silent mutations, and nonsense mutations. All 119 samples had also been analyzed in parallel for p53 mutations by a combination of several alternative methods including the predecessor Affymetrix p53 GeneChip microarray, denaturing high performance liquid chromatography (DHPLC), as well as conventional sequencing. The specificity and sensitivity of the AmpliChip p53 Test were compared to the results obtained with these alternative assays. Discordant results were observed in 27 samples when comparing the AmpliChip p53 Test to the alternative assays. Among these 27 samples the AmpliChip p53 Test detected mutations in Exon 10 in 2 samples which had not been tested by DHPLC and further exclusively detected the R72P polymorphism in 9 samples. The remaining group of discordant samples contained:

• 5 samples with a I03 dup 16bp +40 (acctggagggctgggg);

• 3 samples containing a > 2 bp deletion;

• 2 samples with insertion mutations; and

• one with a mutation in intron 3.

Due to technical reasons, all of these aberrations noted above are out of the specifications of the AmpliChip p53 Test. In 5 samples the AmpliChip p53 Test detected additional mutations that were not identified by conventional methods. In conclusion, the AmpliChip p53 Test is a robust and rapid diagnostic tool that allows to identify a poor prognostic subset of AML. Unraveling the biology of this disease may provide new pathophysiologic insights applicable to therapeutic intervention, especially for the targeted therapy of AML with complex aberrant karyotype.