GENOMIC DNA AMPLIFICATION AND THE DETECTION OF T(2-5)(P23-Q35) IN LYMPHOID NEOPLASMS

Anaplastic large cell lymphoma (ALCL) is an intermediate grade Non-Hod gkin's lymphoma (NHL) characterized by the frequent presence of the t( 2;5)(p23;q35). This translocation fuses the nucleophosmin (NPM) gene o n chromosome 5q35 to a protein kinase gene (Anaplastic Lymphoma Kinase ALK) on chromosome 2p23. In order to determine the frequency of t(2;5 ) we used a DNA polymerase chain reaction (PCR) amplification using ge nomic DNA, 5'-primers derived from the NPM gene, and 3'-primers derive d from the ALK gene. The presence of amplifiable DNA in the samples wa s established with PCR and oligonucleotide primers designed to amplify a 3,016 bp fragment from the P-globin locus. The t(2;5) PCR assay was established using DNA isolated from three t(2;5)-positive ALCL cell l ines. Its ability to amplify genomic DNA prepared for routine molecula r diagnostic use was validated using archival DNA from four ALCL tumor s known to be t(2;5)-positive. Its sensitivity was established by seri ally diluting t(2;5)-positive DNA in normal DNA: amplicons were genera ted in 100% of reactions diluted 10(4)-fold (6-8 cells per tube) and i n 30% of those diluted 10(5)-fold (0.6-0.8 cells per tube.) We subsequ ently analyzed archival genomic DNA extracted from 38 ALCL, 77 NHLs, 3 7 Hodgkin's lymphomas, and 9 lymphomatoid papuloses. The t(2;5) was de tected in 6 ALCLs (16%, 95% confidence intervals 6%-31%), but not in a ny other lymphoma, or in lymphomatoid papulosis. By using the publishe d sequence of the fourth NPM intron that is involved in t(2;5) and by sequencing the individual tumor amplicons and also the normal ALK intr on that is involved in t(2;5), we established that all breakpoints inv olve the same introns in the ALK and NPM loci. Detailed analysis demon strated that each translocation generates a unique breakpoint sequence , and suggested that sequence homology between the ALK and NPM intron sequences may be involved in the translocation. We conclude that genom ic DNA-PCR is useful for the detection of t(2;5) that in our patient p opulation is restricted to ALCL and is not detectable in other NHL, Ho dgkin's disease, or lymphomatoid papulosis. More work is needed to det ermine the prognostic significance of t(2;5), and to establish the uti lity of the genomic DNA PCR in monitoring minimal residual disease.