Sarris A H, Luthra R, Cabanillas F, Morris S W, Pugh W C
Department of Hematology, The University of Texas M.D. Anderson Cancer Center, Houston, USA.
Leuk Lymphoma. 1998 May;29(5-6):507-14. doi: 10.3109/10428199809050910.
Anaplastic large cell lymphoma (ALCL) is an intermediate grade Non-Hodgkin's lymphoma (NHL) characterized by the frequent presence of the t(2;5)(p23;q35). This translocation fuses the nucleophosmin (NPM) gene on 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 genomic DNA, 5'-primers derived from the NPM gene, and 3'-primers derived from the ALK gene. The presence of amplifiable DNA in the samples was established with PCR and oligonucleotide primers designed to amplify a 3,016 bp fragment from the beta-globin locus. The t(2;5) PCR assay was established using DNA isolated from three t(2;5)-positive ALCL cell lines. Its ability to amplify genomic DNA prepared for routine molecular diagnostic use was validated using archival DNA from four ALCL tumors known to be t(2;5)-positive. Its sensitivity was established by serially diluting t(2;5)-positive DNA in normal DNA: amplicons were generated in 100% of reactions diluted 10(4)-fold (6-8 cells per tube) and in 30% of those diluted 10(5)-fold (0.6-0.8 cells per tube.) We subsequently analyzed archival genomic DNA extracted from 38 ALCL, 77 NHLs, 37 Hodgkin's lymphomas, and 9 lymphomatoid papuloses. The t(2;5) was detected in 6 ALCLs (16%, 95% confidence intervals 6%-31%), but not in any other lymphoma, or in lymphomatoid papulosis. By using the published 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 intron that is involved in t(2;5), we established that all breakpoints involve the same introns in the ALK and NPM loci. Detailed analysis demonstrated 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 genomic DNA-PCR is useful for the detection of t(2;5) that in our patient population is restricted to ALCL and is not detectable in other NHL, Hodgkin's disease, or lymphomatoid papulosis. More work is needed to determine the prognostic significance of t(2;5), and to establish the utility of the genomic DNA PCR in monitoring minimal residual disease.
间变性大细胞淋巴瘤（ALCL）是一种中度恶性的非霍奇金淋巴瘤（NHL），其特征是频繁出现t(2;5)(p23;q35)。这种易位将5号染色体q35上的核磷蛋白（NPM）基因与2号染色体p23上的一个蛋白激酶基因（间变性淋巴瘤激酶，ALK）融合。为了确定t(2;5)的频率，我们使用基因组DNA进行DNA聚合酶链反应（PCR）扩增，5'引物来自NPM基因，3'引物来自ALK基因。通过PCR和设计用于扩增β-珠蛋白基因座3,016 bp片段的寡核苷酸引物来确定样品中可扩增DNA的存在。使用从三个t(2;5)阳性ALCL细胞系分离的DNA建立t(2;5) PCR检测方法。使用来自四个已知为t(2;5)阳性的ALCL肿瘤的存档DNA验证其扩增用于常规分子诊断的基因组DNA的能力。通过在正常DNA中连续稀释t(2;5)阳性DNA来确定其灵敏度：在10(4)倍稀释（每管6 - 8个细胞）的所有反应中均产生扩增子，在10(5)倍稀释（每管0.6 - 0.8个细胞）的反应中有30%产生扩增子。随后，我们分析了从38例ALCL、77例NHL、37例霍奇金淋巴瘤和9例淋巴瘤样丘疹病中提取的存档基因组DNA。在6例ALCL中检测到t(2;5)（16%，95%置信区间6% - 31%），但在任何其他淋巴瘤或淋巴瘤样丘疹病中均未检测到。通过使用已发表的参与t(2;5)的第四个NPM内含子序列，并对单个肿瘤扩增子以及参与t(2;5)的正常ALK内含子进行测序，我们确定所有断点均涉及ALK和NPM基因座中的相同内含子。详细分析表明，每个易位产生独特的断点序列，并提示ALK和NPM内含子序列之间的序列同源性可能参与易位。我们得出结论，基因组DNA - PCR可用于检测t(2;5)，在我们的患者群体中，t(2;5)仅限于ALCL，在其他NHL、霍奇金病或淋巴瘤样丘疹病中无法检测到。需要更多工作来确定t(2;5)的预后意义，并确定基因组DNA PCR在监测微小残留病中的效用。
