Characterisation of chromosomal aberrations in childhood leukaemia

Sammanfattning: Acute lymphoblastic leukaemia (ALL) is the most common malignancy in childhood, accounting for approximately 25% of all paediatric malignancies. Based on clinical risk criteria and modem laboratory investigations including immunophenotyping, cytogenetics, and molecular genetics, patients can be divided into prognostic groups and assigned to risk- adjusted treatment protocols. Despite the great improvement in the outcome, still approximately 15% of the children in the Nordic countries die from ALL. Cytogenetic analysis in ALL is often hampered by poor chromosome morphology and few malignant metaphases, and sometimes only normal metaphases derived from normal cells are found after cell culture. Structural as well as numerical aberrations may therefore remain undetected using conventional G-banding. In order to investigate the resolving power of new molecular cytogenetic techniques, we first applied spectral karyotyping (SKY) and microdissection in combination with fluorescence in situ hybridisation (FISH) on metaphases from a pilot case, a patient with primary plasma cell leukaemia with a severely hypodiploid karyotype with 12 marker chromosomes. We were able to resolve very complex chromosomal changes in a way that has not been previously possible. Fortythree breakpoints were found, and 25 could be identified at the band level, among others 14q32, where the immunoglobulin heavy chain locus is situated. SKY was then performed on metaphase spreads from 19 high-hyperdiploid (>51 chromosomes) childhood ALL with a poor chromosome morphology. In all cases, the numerical changes could be fully characterised, and a non-random pattern of chromosomal gain was identified with chromosomes X, 21, 14, 17, 6, 18, 4, and 10 being most frequently gained. Structural aberrations were identified in 47% of the cases and the chromosomal origin of all marker chromosomes and of eleven structural rearrangements could be identified. Twenty-two cases of childhood ALL with normal G or Q-banded karyotypes were studied by interphase FISH and SKY. Probes detecting MLL, BCR/ABL, and TEL/AML1 rearrangements were used for the interphase studies along with centromere specific probes from chromosomes 17 and X. In ten patients (45%), previously undetected aberrations were demonstrable. Specific gene rearrangements and structural changes were found in six cases and numerical changes in five. Five of these aberrations have previously been reported to have an impact on prognosis, A consecutive series of seventy children with ALL who were all treated according to the NOPHO ALL-92 treatment protocols were analysed for chromosomal abnormalities with conventional G-banding, SKY and interphase FISH using probes to detect MLL, BCR/ABL, TEL/AML1 rearrangements and INK4 locus deletions. Numerical and/or structural changes could be identified in 80% by use of the molecular cytogenetic techniques, whereas abnormalities could be detected in only 60% of the patients using G-banding alone. Altogether, 106 structural aberrations were defined by FISH compared to 34 using G-banding. Seventy-four percent of the patients had numerical aberrations, 54% had structural aberrations and 20% had no identified aberrations. Twelve cases had prognostically unfavourable chromosomal aberrations that had not been detected by G-banding. We identified three novel TEL partner breakpoints on 1q41, 8q24 and 21p12, and a recurrent translocation t(1;12)(p32;p13). In addition, two cases displayed amplification (7-15 copies) of AML1. The results of this thesis emphasises the value of SKY and interphase FISH in identifying prognostically important and novel chromosomal rearrangements as a complement to conventional cytogenetic banding analysis in childhood ALL.