Genetic changes in childhood acute lymphoblastic leukaemia and other lymphoid malignancies

Sammanfattning: Malignant transformation of normal cells is the result of defects in cell growth control, differentiation and programmed cell death. It has been convincingly shown that malignant cells carry mutations in the genes controlling these cellular processes. Acute lymphoblastic leukaemia (ALL) is the most frequent malignant disease of childhood, constituting approximately 25% of all childhood malignancies. Despite the great improvement in the outcome of these children, approximately 20% of the patients still die from their disease, due to recurrence, to side effects of the treatment or, rarely, to resistant disease. Clonal chromosomal abnormalities can be shown by karyotyping in 80% of the ALL cases, the genetic alterations in the remaining 20% can only be detected by molecular techniques. Deletions in 6q, 9p, 13q and the translocation t(12;21) frequently resulting in loss of heterozygosity (LOH) in 12p, are examples of mutations found in ALL. Our screening of primary ALL-clones for genetic alterations showed LOH in 6q in 24% of the cases, deletion in chromosome 9 in 3 1 %, LOH or deletion in chromosome 13 in 10% and LOH in 12p in 12% of the cases. An inverse correlation between LOH in 12p and deletions in the Ink4-locus on 9p was found. This may suggest that the mutations on the respective chromosomes are affecting the same molecular pathway, alternatively it reflects the immunophenotypic groups commonly affected by these mutations. Deletions in the long arm of chromosome 6 are frequently found in lymphoid malignancies. Expanded LOH studies revealed that LOH in 6q occurred in 32% of the ALL cases studied: a higher rate than previously reported. A minimally deleted region of less than 1 centiMorgan (cM) around the marker D6S283 was identified. Our results suggest that this region may harbour a tumour suppressor gene relevant for the development of ALL. Our results also indicate a second less well defined region on 6q telomeric of the region lost in ALL, in which LOH can be detected in non-Hodgkin's lymphoma (NHL) of adulthood. The Ink4-locus in 9p21 contains three genes involved in the regulation of the G1 phase of the cell cycle (p16ink4a,p15ink4b and p14ARF). Malignant cells from children with ALL were analysed for mutations within the Ink4-locus, using Southern blot hybridisation, single-strand conformation polymorphism (SSCP) analysis and nucleotide sequencing. The results showed that inactivation of the p16ink4a and p14ARF genes within the Ink4-locus is associated with an inferior prognosis. Although this genetic marker co-varied with other high-risk features such as high white blood cell count and T-cell phenotype, it was identified as an independent prognostic factor by multivariate analysis. Hyperdiploid karyotype has been associated with a subgroup of childhood ALL with favourable clinical features and a low probability of relapse. DNA-hyperdiploidy, measured as an increased DNA-index by image-DNA-cytometry (ICM), and bi-allelic deletions within the Ink4-locus in ALL, showed a significant inverse correlation. Ink4-locus deletion had a stronger impact on outcome than DNA-hyperdiploidy. H9 is a malignant T-cell line that has bi-allelic deletions of the Ink4-locus as well as mutated Rb and p53. H9 is deficient in P21 protein expression although p21 Waf1/Cip1 mRNA was detected. A stretch of approximately 400bp missing from the coding exon 2 was found in the cDNA, possibly explaining the lack 1of P21 protein expression in H9 cells.