Genome-Wide Analysis by High-Resolution SNP Array Identifies Novel Genomic Alterations in Acute Promyelocytic Leukemia (APL).

Introduction: Acute promyelocytic leukemia (APL) is characterized by the chromosomal translocation t(15;17), resulting in the fusion of the promyelocytic leukemia (PML) gene and retinoic acid receptor {alpha} (RARA) gene (PML-RARA). Experimental evidence obtained in transgenic mice revealed that PML-RARA is necessary but not sufficient for the development of APL, suggesting that additional genetic mutations are also required for the development of APL. Aim: To define whether additional submicroscopic genomic alterations may characterize APL and be used to better classify the disease by dissection of genomic subsets. Methods: At the time of writing, DNA from the bone marrow of 21 cases of t(15;17) APL at diagnosis were examined. Genomic DNA was isolated from mononuclear APL cells and applied to Genome-Wide Human SNP 6.0 array microarrays (Affymetrix, Santa Clara, CA) following the manufacturer's instructions. Copy number aberrations were scored using the Hidden Markov Model and the segmentation approach available within the Partek software package as well as by visual inspection. All aberrations were calculated with respect to a set of 270 Hapmap normal individuals and a set of samples obtained from acute leukaemia cases in remission in order to reduce the noise of raw copy number data. When available, in order to exclude inherited copy number variants a comparison to paired constitutional DNA and to paired remission DNA was performed. Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results: In all patients we identified one or more genomic abnormalities ranging from loss or gain of complete chromosome arms (trisomy 8, loss of 20q and loss of 6q) to submicroscopic genomic intervals. Focal genetic alterations were detected at the breakpoints of t(15;17)(q22;q21) in PML and RARA genes. Hemizygous deletions were identified at 1p13.3 affecting the glutathione S-transferase mu 1 (GSTM1), at 2q33.3–q34 involving ERBB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4 avian), at 3p11.2 (EPHA3), at 12p13 (ETV6), and at 17q12 (NF1). Deletions also affected genes involved in cell regulations as CDKN2A (9p21) and RB1 (13q14.2). Most frequent gains affected the TP73 gene at 1p36.3, the oncogene MYC at 8q24 (4.33 Mb), the oncogene ETS1 (11q23.3), the RAS p21 protein activator 3 (RASA3) at 13q34 and PRAME at the 22q11.22. Interestingly, in 2 cases we observed a gain of the region (5p15.33) containing the telomerase reverse transcriptase (TERT) which is important for maintenance of telomere ends. Other recurring genetic lesions were uncommon and were identified only in single cases. Some lesions affected regions lacking annotated genes and they are under investigation for miRNAs. For each alteration we interrogated a collated library of copy-number variants (CNVs, Database of Genomics Variants and USCS Genome Browser) to assure that these regions were not known as CNVs. Conclusion: These data demonstrate that different cooperating events may be involved in the generation of APL. These novel findings may be used to stratify patients according to genomic changes and to facilitate the screening for novel therapeutic targets. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, PIO project 2007, Strategico di Ateneo.