Molecular Characterization of AML1 (RUNX1) Amplification: A Poor Risk Chromosomal Marker in Acute Lymphoblastic Leukaemia (ALL).

We have recently defined a new recurrent chromosomal abnormality in patients with acute lymphoblastic leukemia (ALL) involving the duplication of chromosomal band 21q22 and amplification of the AML1 (RUNX1) gene. Currently, fluorescence in situ hybridization (FISH) with probes directed to the AML1 gene is the only reliable detection method. AML1 signals are seen as clusters in interphase cells and in tandem duplication on the long arm of chromosome 21 in metaphase. To date, we have identified 46 patients with this abnormality: 26 males and 20 females. The median age of the 44 children was 9 years (range 4–16 years): two adults were 20 and 30 years old. Their median white cell count was 3.7x10⁹/L (range 1−80x10⁹/L) and all had a common/pre-B immunophenotype. The 3 year event free survival for the 24 children treated on MRC ALL97 was 51% (95% confidence interval 27%–71%). Hence, this abnormality has been associated with older children with a low white cell count and, most significantly, a poor prognosis. The NCRI UK Childhood Leukaemia Working Party has categorized these children into a high-risk group and recommended that they be treated on a more intensive protocol, making their accurate detection vital. To achieve this goal, we have attempted to define the amplified region on chromosome 21 and to identify the genes involved, employing a range of molecular techniques. Microarray analysis (genomic and expression) in conjunction with FISH and quantitative PCR were used to investigate DNA and/or RNA samples from 11 patients. Comparative genomic hybridization (Spectral Genomics 1Mb) (aCGH) showed variable regions of amplification (common minimal region ~10Mb DNA).

Complementary FISH, using a range of specific probes corresponding to the aCGH clones, confirmed the extent of the amplified region on chromosome 21, while indicating the precise copy number changes for each clone. FISH also revealed unexpected chromosomal rearrangements of the amplified chromosome, including loss of the 21q telomeric region. These observations confirm the chromosomal instability associated with this abnormality, as previously indicated from the variable chromosome morphology seen by cytogenetic analysis. Expression profiling (Affymetrix HG-U133) detected 60 over-expressed genes in these patients, of which six were located within the chromosome 21 amplicon. This study has defined the extent of the chromosome 21 amplified region surrounding AML1, and highlighted the over-expression of specific genes within the amplicon. This combined molecular approach will help to identify the most appropriate test for the detection of this high-risk abnormality.