Abstract 4967: Array-based genomic resequencing of acute myeloid leukemia

Abstract

Abstract Somatic mutations within cancer-related genes play a central role in the pathogenesis of many human malignancies, as clearly demonstrated for the cases with mutated RAS and EGFR. Although a new generation of sequencing technologies is now available, whole-genome resequencing of many samples remains a demanding task. DNA microarray-based sequencing is suitable for analysis of multiple samples. However, currently available platforms are limited in the number of nucleotides that each array is able to probe. To overcome such limitations, we have now developed extra-large DNA arrays (“wafers”) for resequencing the human genome, in a collaborative study with Perlegen Sciences, Inc. With its capacity to assay ∼9 Mbp, we could place, on the array, oligonucleotides to interrogate sequence alterations at protein-coding exons and exon-intron boundaries for 5648 human genes that are possibly involved in malignant transformation mechanism. To identify transforming mutations in acute myeloid leukemia (AML), hybridization of genomic DNA from CD34-positive blasts of AML (n = 19) or myeloproliferative disorder (MPD) (n = 1) to the arrays revealed a total of 9148 nonsynonymous nucleotide changes on 3403 independent genes in this cohort (Phase I). To discriminate somatic changes from germ-line polymorphisms, we developed a second array for a direct analysis of the 9148 nucleotide changes, which was hybridized with genomic DNA from the leukemic blasts as well as with that from CD4-positive T cell fractions (paired controls) from the same cohort (Phase II). Analysis of CD4-positive fractions revealed that most of these nucleotide changes in Phase I were also present in the paired control genome, leaving only 11 somatic, nonsynonymous mutations among 11 genes. One of these somatic changes results in a Met-to-Ile substitution at amino-acid position 511 of Janus kinase 3 (JAK3), and the JAK3(M511I) protein exhibited transforming potential both in vitro and in vivo. Further screening for JAK3 mutations showed novel and known transforming changes in 9 out of 286 cases with leukemia. Our experiments also showed a somatic change responsible for the Arg-to-His substitution at amino-acid position 882 of DNA methyltransferase 3A, which results in a loss of >50% of the catalytic activity. Our data have thus revealed a unique profile of gene mutations in human leukemias. Given that only a small number of sequence alterations were proved to be “somatic” after Phase II selection, it would be important to use appropriate paired control samples in the search for somatic mutations in the cancer genome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4967. doi:10.1158/1538-7445.AM2011-4967