Detection of the NPM-ALK genomic rearrangement of Ki-1 lymphoma and isolation of the involved NPM and ALK introns.
The breakpoints of the translocation t(2;5)(p23;q35) associated with Ki-1-positive anaplastic large-cell lymphoma (Ki-1 ALCL) involve a novel tyrosine kinase gene, ALK, at 2p23 and the nucleophosmin gene, NPM, at 5q35. Reverse transcriptase-polymerase chain reaction (RT-PCR) using NPM and ALK primers detects a consistent fusion product in Ki-1 ALCL cases with the translocation, resulting from genomic breakpoints within the same respective introns of NPM and ALK. To examine the feasibility of long-range DNA PCR with the same exonic NPM and ALK primers for the detection of the genomic NPM-ALK rearrangement, we examined 20 cases of Ki-1 ALCL previously characterized by NPM-ALK RT-PCR. Ten cases were positive for the NPM-ALK fusion RNA and 10 were negative. We first confirmed that both the NPM and ALK normal introns are relatively short, approximately 1 and 2 kb, respectively, suggesting that the largest possible size for the chimeric NPM-ALK intron would be about 3 kb. All 10 cases positive by RT-PCR were also positive by long-range DNA PCR. The DNA PCR products ranged, as expected, from the sizes of the normal introns, between 0.5 and 2.5 kb. All 10 RT-PCR-negative cases were also negative by long-range DNA PCR, and control templates for RT-PCR and long-range DNA PCR were successfully amplified. Thus, we have shown that the introns involved by the NPM-ALK rearrangement seen in some Ki-1 lymphomas are relatively short, making the genomic rearrangement amenable to reliable detection by long-range DNA PCR. Furthermore, the variability observed in the sizes of chimeric introns in evidence against clustering of the genomic breakpoints within these introns.
Ladanyi
M
M
Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.
Cavalchire
G
G
D000077548:Anaplastic Lymphoma Kinase; D015321:Gene Rearrangement; D006801:Humans; D007438:Introns; D017728:Lymphoma, Large-Cell, Anaplastic / Q000235:genetics* / Q000473:pathology*; D009687:Nuclear Proteins / Q000235:genetics*; D009697:Nucleolus Organizer Region / Q000235:genetics; D000090243:Nucleophosmin; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D020794:Receptor Protein-Tyrosine Kinases; D014178:Translocation, Genetic / Q000276:immunology
C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases