Identification of Candidate Oncogenes in Acute Megakaryoblastic Leukemias with Gain of Chromosome 19.

Acute Megakaryocytic leukemia is a rare subtype of AML that is often difficult to diagnose. Although GATA1 mutations are always found in AML-M7 associated with Down’s syndrome, such mutations are a rare event in adult AML-M7 leukemia. In 2001, we reported that Comparative Genomic Hybridization (CGH) identifies gain of chromosome 19 as part of the spectrum of nonrandom abnormalities characterizing megakaryoblastic proliferations, being its frequency underestimated when only conventional cytogenetics is used (1,2).

To identify candidate genes amplified and overexpressed on chromosome 19, we selected 10 cell lines derived from AML-M7 patients, that were previously characterized and classified by CGH according to their chr. 19 gain status. For each cell line, two hybridizations were performed: one with total RNA and one with DNA. Both hybridizations were done against the same c-DNA CNIO Oncochip, which allowed us to compare the results. As controls, 2 hybridizations with RNA obtained from CD34 cells selected from different leukapheresis were used to normalize the expression pattern from the cell lines.

First, we analyzed the expression values (normalized by the CD34+ cells) of the 201 c-DNA clones localized on chr. 19. The total number of overexpressed clones was higher in cell lines with gain or high level amplification of chr. 19 ( 38 to 79 of c-DNAs up) than in cell lines with normal chr. 19 or a discrete gain of 19q13.2 (29 to 31 of c-DNAs up). Second, examining the copy number changes and expression data allowed us to exclude some functionally interesting genes as MLL4 and to identify 36 clones that were recurrent and simultaneously gained and overexpressed. Eight out of these 36 clones showed at least three times greater expression in at least two cell lines. Six of these genes (KCNK6, APOC1, CD33, CEACAM5, CCNE1, and PPF1A3) are located on the q arm and two of them (NR2F6 and ASF1B) on the p arm. Interestingly, 80% of these 8 genes have at least one Gene Ontology annotated term relative to cell growth and/or maintenance. The dendogram of the unsupervised analysis of these 8 genes gives three arms that discriminate between 1) the cell lines with gain of chr. 19, 2) the cell lines with high level amplification of 19q, and 3) cell lines with normal chromosome19 or a discrete gain of 19q13.2. Moreover, the supervised analyses of the expression pattern of these 8 genes could identify significant differences among the four different states of chr.19 in the cell lines: high level amplification of 19q, gain of 19q13.2, gain of the whole 19, or normal 19, based on the expression of three genes: KCNK6, NR2F6, and APOC1. Further analyses, to identify the function of these genes in acute megakaryocytic leukemia cells are in progress.