MAPPING OF GENOMIC T(2-5)(P23-Q35) BREAK POINTS IN PATIENTS WITH ANAPLASTIC LARGE-CELL LYMPHOMA BY SEQUENCING LONG-RANGE PCR PRODUCTS

The t(2;5)(p23;q35) that is frequently detected in anaplastic large ce ll lymphoma (ALCL) fuses the nucleophosmin (NPM) gene on chromosome 5 to a novel tyrosine kinase gene designated anaplastic lymphoma kinase (ALK) on chromosome 2. The fusion of NPM and ALK genes results in the production of chimeric transcripts containing NPM amino-terminal seque nces fused to the ALK carboxyterminal catalytic domain. Because fusion transcripts and proteins in almost all t(2;5)-positive cell lines and tumors are identical, it is likely that the chromosomal breaks involv e the same introns of NPM and ALK genes. We have previously developed a long-range genomic DNA-PCR assay to amplify the genomic NPM-ALK brea kpoints. Using high-molecular-weight DNA extracted from 2 ALCL cell li nes and from 9 primary ALCLs known to be t(2;5)-positive, we have demo nstrated that all 11 amplicons were of different sizes, suggesting tha t the t(2;5) breakpoints were unique and involved the same inrrons on both chromosomes. We decided to confirm this and map the t(2;5) breakp oints by genomic DNA sequencing. Using the same long-range DNA-PCR tec hnique, primers from the ALK locus, and normal genomic DNA, we sequenc ed the ALK intron involved in t(2;5). We subsequently sequenced all 11 amplicons from t(2;5)-positive ALCL cell lines and rumors. Comparison of the sequences derived from ALCL amplicons with the published seque nces of intron 4 from the NPM locus (910 bp) and with the newly sequen ced intron from the ALK locus (1935 bp) accurately mapped all break po ints and demonstrated that their nucleotide sequences,were unique. We conclude that the genomic t(2;5) breakpoints can be easily mapped by s equencing the amplicons generated from genomic DNA with long-range PCR and that they are unique for each patient The sequences of the break points and of the newly identified ALK intron may be useful in the con struction of patient-specific primers for monitoring and determination of the clinical relevance of minimal residual disease.