THERMOSTABLE DNA-POLYMERASE CHAIN AMPLIFICATION OF T(14-18) CHROMOSOME BREAKPOINTS AND DETECTION OF MINIMAL RESIDUAL DISEASE
THERMOSTABLE DNA-POLYMERASE CHAIN AMPLIFICATION OF T(14-18) CHROMOSOME BREAKPOINTS AND DETECTION OF MINIMAL RESIDUAL DISEASE
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DOI：
10.1073/pnas.85.13.4869
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发表时间：
1988-07-01
影响因子：
11.1
通讯作者：
KORSMEYER, SJ
中科院分区：
文献类型：
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作者：
CRESCENZI, M;SETO, M;KORSMEYER, SJ
实现检测最小数量肿瘤细胞的能力是癌症诊断的主要挑战。在滤泡性和一些非滤泡性淋巴瘤中发现的t(14;18)-(q32;q21)染色体易位提供了肿瘤特异性分子标记。14;18个断点集中在14号染色体上的6个免疫球蛋白重链连接区（JH）之一和18号染色体上BCL2基因的一个小主断点区（MBR）。我们利用BCL2 MBR 5‘区和JH段3’端的通用寡核苷酸引物，启动DNA聚合酶链反应，扩增BCL2-JH连接。使用耐热DNA聚合酶在接近引物熔点的温度下进行退火和合成步骤，提供了一种敏感和特异性的检测方法，能够检测106个正常细胞中的1个淋巴瘤细胞。这项技术确定了所有7名患者的亚临床存在的白血病细胞，包括2名临床缓解。它还评估了从骨髓中清除恶性细胞的方案的有效性。此外，该方法使染色体断点的快速DNA测序无需分子克隆。该试验显著提高了检测最小残留疾病的能力，并应提高确定疾病阶段、分层治疗和评估治疗的能力。
Achieving the capacity to detect minimal numbers of neoplastic cells is a major cancer diagnostic challenge. Chromosomal translocations such as the t(14;18)-(q32;q21) found in follicular and some nonfollicular lymphomas provide a tumor-specific molecular marker. The 14;18 breakpoints are focused at one of six immunoglobulin heavy chain joining (JH) regions on chromosome 14 and a small major breakpoint region (MBR) of the BCL2 gene on chromosome 18. We utilized universal oligonucleotide primers of a region 5'' to the BCL2 MBR and at the 3'' end of JH segments to initiate a DNA polymerase chain reaction that amplified these BCL2-JH junctures. Use of thermostable DNA polymerase enabled annealing and synthesis steps at temperatures approaching the melting point of the primers, providing a sensitive and specific assay capable of detecting 1 lymphoma cell in 106 normal cells. This technique identified the subclinical presence of leukemic cells in all seven patients examined, including two in clinical remission. It also assessed the effectiveness of protocols designed to purge malignant cells cells from marrow. Moreover, this approach enabled the rapid DNA sequencing of chromosomal breakpoints without their molecular cloning. This assay markedly refines the capacity to detect minimal residual disease and should improve the ability to determine the stage of disease, stratify treatment, and evaluate therapy.