Luthra R, Pugh W C, Waasdorp M, Morris W, Cabanillas F, Chan P K, Sarris A H
Department of Pathology, M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Hematopathol Mol Hematol. 1998;11(3-4):173-83.
The t(2;5) (p23;q35) that is frequently detected in anaplastic large cell 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 sequences fused to the ALK carboxy-terminal 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 involve 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 break points. Using high-molecular-weight DNA extracted from 2 ALCL cell lines and from 9 primary ALCLs known to be t(2;5)-positive, we have demonstrated that all 11 amplicons were of different sizes, suggesting that the t(2;5) break points were unique and involved the same introns on both chromosomes. We decided to confirm this and map the t(2;5) break points by genomic DNA sequencing. Using the same long-range DNA-PCR technique, primers from the ALK locus, and normal genomic DNA, we sequenced the ALK intron involved in t(2;5). We subsequently sequenced all 11 amplicons from t(2;5)-positive ALCL cell lines and tumors. Comparison of the sequences derived from ALCL amplicons with the published sequences of intron 4 from the NPM locus (910 bp) and with the newly sequenced intron from the ALK locus (1935 bp) accurately mapped all break points and demonstrated that their nucleotide sequences were unique. We conclude that the genomic t(2;5) break points can be easily mapped by sequencing 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 construction of patient-specific primers for monitoring and determination of the clinical relevance of minimal residual disease.
在间变性大细胞淋巴瘤（ALCL）中经常检测到的t(2;5)（p23;q35），将5号染色体上的核仁磷酸蛋白（NPM）基因与2号染色体上一个名为间变性淋巴瘤激酶（ALK）的新型酪氨酸激酶基因融合。NPM和ALK基因的融合导致产生嵌合转录本，其包含与ALK羧基末端催化结构域融合的NPM氨基末端序列。由于几乎所有t(2;5)阳性细胞系和肿瘤中的融合转录本和蛋白质都是相同的，所以染色体断裂可能涉及NPM和ALK基因的相同内含子。我们之前开发了一种长程基因组DNA-PCR检测方法来扩增基因组NPM-ALK断裂点。使用从2个ALCL细胞系和9个已知为t(2;5)阳性的原发性ALCL中提取的高分子量DNA，我们证明所有11个扩增子大小不同，这表明t(2;5)断裂点是独特的，并且涉及两条染色体上的相同内含子。我们决定通过基因组DNA测序来证实这一点并绘制t(2;5)断裂点图谱。使用相同的长程DNA-PCR技术、来自ALK基因座的引物和正常基因组DNA，我们对涉及t(2;5)的ALK内含子进行了测序。随后，我们对来自t(2;5)阳性ALCL细胞系和肿瘤的所有11个扩增子进行了测序。将来自ALCL扩增子的序列与已发表的NPM基因座内含子4序列（910 bp）以及新测序的ALK基因座内含子序列（1935 bp）进行比较，准确地绘制了所有断裂点，并证明它们的核苷酸序列是独特的。我们得出结论，通过对长程PCR从基因组DNA产生的扩增子进行测序，可以轻松绘制基因组t(2;5)断裂点图谱，并且它们对每个患者都是独特的。断裂点序列和新鉴定的ALK内含子序列可能有助于构建患者特异性引物，用于监测和确定微小残留病的临床相关性。
