ALBERT

Description: The proteins of the Fanconi anemia/BRCA pathway are thought to function in specific DNA repair processes. Disruption of these genes results in genomic instability and hypersensitivity to DNA cross-linking agents. As illustrated by patients with Fanconi anemia (FA) [OMIM#227650] this cellular phenotype confers an increased risk for the development of malignancies, in particular acute myeloid leukemia (AML). In the past years several studies have shown that inherited and somatic abnormalities in individual FA genes play a role in the development of sporadic cancers. To study the involvement of the FA genes in sporadic cases of leukemia we analyzed the presence of aberrant promoter methylation of the 11 known FA genes by Methylation Specific-Multiplex Ligation dependent Probe Amplification (MS-MLPA). We studied 119 unselected adult AML bone marrow samples and 20 pediatric AML samples that were selected on the bases of a complex karyotype. A single adult patient sample, diagnosed biphenotypic AML, showed aberrant methylation of the promoter region of the FANCC gene. The aberrant methylation pattern was also present at relapse. Another four biphenotypic AML samples, together with 15 adult and 82 pediatric ALL samples were subsequently analyzed. In this group, one adult ALL sample showed FANCL promoter methylation and three pediatric ALL samples showed FANCC promoter methylation. Phenotypically all four ALL samples were classified as hyperdiploid B-cell precursor ALL. Sodium bisulphite sequencing confirmed hypermethylation of the respective genes in all cases. Colony Forming Unit (CFU) assays were performed to analyze whether hypermethylation of FANCC and FANCL result in an FA-like phenotype of DNA cross-linker sensitivity. Colony formation in the methylated samples as well as control leukemic samples, i.e. without FA-gene methylation, was scored after proliferation in the absence or presence of MMC. Two pediatric ALL samples with FANCC hypermethylation did not show colony formation in the CFU assay. The other four samples with FA gene methylation, including the relapse sample from the biphenotypic AML patient, demonstrated hypersensitivity to MMC in a CFU assay when compared to the controls. In summary, this is the first report showing FANCC and FANCL promoter hypermethylation in acute leukemia with either partial or complete lymphoid differentiation. Silencing of the corresponding FA genes may contribute to the leukemogenic process by imposing an increased level of spontaneous genomic instability. Cross-linkers may thus be of therapeutic use in these selected patients.