High-resolution molecular allelokaryotyping identifies novel genomic alterations in acute promyelocytic leukemia (APL)

Introduction: 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 its development. Aim: To define whether additional submicroscopic genomic alterations may characterize APL and may be used to better classify the disease by dissection of genomic subsets. Methods: 105 adult patients with acute myeloid leukemia were analyzed. These cases included all French-American-British subtypes, miscellaneous cytogenetic abnormalities and normal karyotype subgroups. Among these, the M3 subtype included 28 patients, representing the 33% of the whole study population. Genomic DNA was isolated from blast cells and applied to Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA) following the manufacturer's instructions. Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results: A wide spectrum of different copy number alterations (CNAs) were identified in all cases and no significant difference in the average number of alterations was detected among different leukemia cytogenetic subgroups except for the complex subgroup, which had an average of 55 CNA/patient. In APL cases an average of 8 CNAs per case (range, 1-24) was found. The macroscopic alterations were rare, confirmed conventional cytogenetics and involved trisomy of chromosome 8 in 3 cases, loss of chromosome 6, loss of chromosome 20 and deletions on chromosome 9 and 7. Microscopic CNAs (< 1.5 Mbps) involved every chromosome at least once and predominantly chromosomes 1, 2, 9, 15 and 17. Genetic gains were more common than losses and their median size was 300 kb (range 0.2- 1.4 Mb). The majority of lesions were not recurrent, being identified in only a single patient. Focal genetic alterations were detected at the breakpoints of t(15;17)(q22;q21) in PML and RARA genes. Hemizygous deletions were identified at at 2q33.3-q34 involving ERBB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4 avian), at 3p11.2 (EPHA3) and at 12p13 (ETV6). 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 oncogenes MYC and PVT1 at 8q24 (4.33 Mb), PRAME at the 22q11.22. Copy neutral loss of heterozygosity events affected 1p34.2-1p32.3, 10p11.2 (MLLT10), 11p11.2 (WT1, CDKN1C, HRAS). Patients with more than 10 CNAs were associated with a worse prognosis. Conclusions: 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: AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, Strategico di Ateneo.