Molecular Characterization of Genomic Structural Alterations through CGH Arrays in Acute Myeloid Leukemia with Normal Cytogenetic Findings.

Acute myeloid leukemia (AML) is an heterogeneous disease. Cytogenetic findings are one of the main prognosis criteria beside age, leucocytosis, and antecedent of previous hemopathy. More than 50% of AML patients, mainly with normal cytogenetic, belong to the intermediate cytogenetic risk group. Even in this group, cryptic molecular abnormalities as mutation are frequent with prognosis value. In this work we have studied 31 AML patients (Pts) with normal caryotype by high resolution comparative genomic hybridization array CGH using agilent 185k chips to detect gene copy number alterations (CNA). Tumor DNA was obtained from bone marrow diagnosis samples (>60% of blasts) and control DNA from pooled blood samples obtained from 30 healthy donors or from AML patients in CR during the follow up. The design of the study was first to hybridize tumor DNA from the 31 patients against pooled control DNA to detect both copy number polymorphisms (CNP) and acquired CNA in AML and in a second time to hybridize tumor DNA from the 10 pts with the greater number of abnormalities against autologous DNA obtained at CR time to distinguish acquired CNA from CNP. Briefly DNA labelling was performed using Cy3dUTP and Cy5 dUTP respectively for control and tumor samples. Fluorescence ratios were normalized and spots with value that deviated significantly from background were retained as significant evidence of DNA copy number alterations after statistical analysis using Feature Extraction and CGH Analytics AGILENT software. BAC from the Human Genome Center (Wellcome Trust Sanger) were used to obtain FISH probes to confirm abnormalities observed by CGH arrays on metaphase spreads obtained from bone marrow diagnosis sample when available. In each case, two colour FISH was performed by the simultaneous hybridization on a green BAC probe located in the CNA regions and a red control BAC probe located in the vicinity of the CNA regions obtained from the UCSC genome browser data.

Results

98 DNA copy number alterations were observed with 56 losses and 42 gains cases. Those CNA were variable in size from 8,6 Mb to <100 Kb. 8 CNA regions were recurrent (5 losses in 7q31 (12pts), 4q13 (3pts), 1q31 (4pts), 2q37 (2pts) et 22q13 (3pts) and 3 gains in 1q23 (3pts), 2p11(2pts) et 17q21(2pts). Non recurrent CNA were located in 1p36.2, 1p34.3, 1p21.1, 1p22, 1q31.2, 3q12.2, 4p13, 5p15.3, 5q14.1, 6p22.1, 7p22.1, 7q11.23, 10q26.3, 11q13.2, 15q11.2, 15q15.3, 15q25.3, 15q26.1, 16q24.2, 17p13.2, 17p11.2, 22q13,33 for gains and in 1p33, 1q32.1, 1q32.2, 3p14.1, 3p13-p12.3, 4q26, 7p11.2, 7p14.3, 7q35, 8p23.2, 8q23.1, 9p24.1, 9p13.1-p12, 9q34.11-q34.12, 12p13.31, 13q14.2, 14q21.2, 14q22.1, 14q23.2, 15q22.31, 16q22.1, 21q21.2, 21q22.12 for losses. No one of the patients had a genome without CNA and 6 patients (20%) had more than 5 CNAs. The CGH data showed clearly that 80% of the CNA observed with pool control DNA were not present when tumor DNA was hybridized against the autologous CR DNA confirming the high frequency of CNP as reported on http://projects.tcag.ca/variation website. All of the acquired CNA regions observed were concordant with the FISH results involving genes known to be crucial in leukemia as RUNX1, Abl, CEBPa but also many other genes. In conclusion cryptic abnormalities are frequent in normal caryotype AML and 20% of the patients studied had more than 5 CNA traducing a great instability of the genome of leukemic cells.