Copy Number Variations (CNV)
As no paired DNA had been obtained, Copy Number Variations (CNV) were distinguished from Copy Number Abnormalities (CNA) based on various criteria: i) sequence size below 2 Mb; ii) the presence of repetitive identical breakpoints between patients; iii) the genes involved such as olfactory receptor genes, the NF1P1 locus or GSTT1 (chromosome 22) and iv) consultation of the Database of Genomic Variants 
. Many Mendelian CNVs, that are present throughout the entire genome 
, were seen in the two AML samples (). Together with classic genes, four microRNA embedded in CNV regions were found: mir-570 in 3q29 within MUC20, mir-1268 in 15q11 within NF1P1, BCL8 and olfactory receptors, mir-1233 in 15q14 within GOLGCABB, and mir-1826 in 16p11.2 within TP53TG3 HERC2P4 ().
Germinal and immunoglobulin genes related CNVs.
Deletions inside immunoglobulin gene (IG) clusters, consecutive to VDJ rearrangement were observed mainly in t-AML. These illegitimate recombinations were considered as acquired CNVs 
, characteristic of the malignant clone. In t-AML, four losses were located in 2p11 in IGLK and 9 in 14q32.3 in IGH. Two t-AML were bi-phenotypic acute leukemia (cases 2 and 7). In three cases (t-15, t-17 and t-25), both genes were rearranged (). No relationships were observed with MLL or other translocations. In p-AML (), only 2 rearrangements were observed: on IGLK (case p-21) and on IGH (case p-24). No rearrangement of IGLL on 22q11 was observed. The difference in the proportion of IG rearrangements in t-AML compared to p-AML was statistically significant with a p<0.002.
Gains and losses CNA in t-AML and revised karyotype after aCGH.
Gains and losses CNA in p-AML and revised karyotype.
Karyotypes and CNA
Karyotypes and aCGH were well correlated, with few exceptions. Ninety-six unbalanced chromosomal abnormalities previously undetected by mitotic karyotypes, were detected after high resolution aCGH (, , and ). Most of the additional abnormalities were too small to be detected by karyotypes. Some revealed masked rearrangements (). In case t-1, the discrepancy between the karyotype and aCGH was probably due to a combination of hyperploidy, genetic heterogeneity and the presence of normal cells resulting in aCGH detection of only the del(5q).
Details of critical rearrangements concerning MCRs.
In both groups of AML, 25 cytogenetically balanced translocations (, and ) did not show any cryptic rearrangement (gain or loss) at their breakpoint locations. In 5 cases (t-3, t-5, t-12, p-18, p-13), a CNA was found near the breakpoints. Patient t-3 was considered as having multiple balanced translocations that could not be further analyzed due to insufficient material. Patient t-5 had a poorly defined translocation implicating 12p and according to aCGH analysis, a small loss began in the middle of ETV6, suggesting a rearrangement of this gene. Patient t-12, had a standard t(8;21) and following “good practice” rules, no further investigation was initially performed. aCGH revealed a duplication of 21q22.1qter that was confirmed by FISH as another translocation or insertion on an unidentified chromosome. The unbalanced translocation breakpoint was 30 Kb centromeric to the 3′ end of RUNX1 suggesting a possible double event. Patient p-18, with a t(8;21) p-AML, had a 1Mb loss that included the first two exons of RUNXT1 on 8q21.3. Patient p13 had a t (15.17) accompanied by a small duplication of the telomeric part of RARA and of the TOP2A genes ().
In some cases the confrontation between aCGH and the karyotype allowed us to better define the previously diagnosed rearrangements. In patient t-13, an add(1)(q3?) was found in the morphological karyotype. With aCGH, it was concluded that it resulted from an unbalanced translocation der(1)t(1;2)(q42.3;p16.1). The breakpoint was virtually cloned from the arrays: on 1q42.3<231.374>, it was sitting in the middle of PCNXL2, between exons 11 and 12. On 2p16.1<54.943>, it was lying in an EML6 gene in the vicinity of exon 12 of the gene. P-22, a primary AML with a t(3;5), had a loss of 10Mb of 12p13 secondary to a additional complex translocation encompassing ETV6 and CDKN1B ().
All patients (excepted three who had received radiotherapy alone) had been treated with multi-agent chemotherapy (AA and ATII) combined or not with radiotherapy. Thus, the type of induction mechanism was most of the time, deduced from the observed chromosomal abnormalities (): 9 cases were probably AA induced, 9 were ATII-induced and in 12, the mechanism could not be determined because CNA categorization was indistinguishable between t-AML induced by AA or by ATII. The medium time elapsed between cancer therapy and the diagnosis of t-AML was 5.1 years, 2.3 years and 4.7 years respectively.
Twenty-five patients (80%) with 104 CNA exhibited 41 gains and 63 losses (). The mean number per total case number was 3.46. Six patients with a normal karyotype had at least one CNA. Six patients (20%) had no CNA, 3 had a normal karyotype and the 2 others had a balanced translocation (one a t(15;17) and one a MLL rearrangement). The mean CNA length was 4.1Mb for the t-AML patients with at least an unbalanced rearrangement.
The twelve losses in the immunoglobulin genes (IG) on 2p11 and 14q32.3 were considered as a special category of CNA. Among the 8 patients with IG rearrangements, 2 had bi-phenotypic leukemia and the others had various forms of AML.
Among 36 patients, 12 had a normal karyotype, 4 exhibited an anomaly on chromosomes 5 or 7. A t(15;17)(q22;q21), characterizing AML-M3, was present in 9 cases ().
In p-AML, 64 CNAs were observed () with 30 losses and 34 gains while the mean number was 1.78. Twenty-two patients had no CNA, 11 had a recurrent balanced translocation while the 11 others had a normal karyotype. The mean number of CNA among p-AML patients with unbalanced rearrangements was 2.66. This value was due to 4 patients with very complex genomic rearrangements (>8 chromosomal abnormalities).
Minimal Critical Regions (MCR)
A MCR was defined as such if 2 cases or more shared a common genomic location. Twelve MCRs were observed in t-AML and eight in p-AML ().
Minimal Critical Region in the two groups of patients.
The size of MCRs was smaller in t-AML, with a minimum of 0.04 Mb compared to p-AML with a minimum of 0.95 Mb. The longest MCR in t-AML was 36.44 Mb, of the same order of magnitude as in p-AML where it was 45.69 Mb.
These single cases were of various sizes, from 0.04Mb to 26.89 Mb. Most of them were small and not known as polymorphisms. They are detailed in .