Crescenzi M, Seto M, Herzig G P, Weiss P D, Griffith R C, Korsmeyer S J
Department of Medicine, Howard Hughes Medical Institute, Washington University School of Medicine, Saint Louis, MO 63110.
Proc Natl Acad Sci U S A. 1988 Jul;85(13):4869-73. doi: 10.1073/pnas.85.13.4869.
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 10(6) 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 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.
实现检测极少量肿瘤细胞的能力是癌症诊断面临的一项重大挑战。染色体易位，如在滤泡性和一些非滤泡性淋巴瘤中发现的t(14;18)(q32;q21)，提供了一种肿瘤特异性分子标记。14号和18号染色体的断点集中在14号染色体上六个免疫球蛋白重链连接（JH）区域之一以及18号染色体上BCL2基因的一个小的主要断点区域（MBR）。我们利用位于BCL2 MBR 5'端和JH片段3'端区域的通用寡核苷酸引物启动DNA聚合酶链反应，扩增这些BCL2-JH连接点。使用耐热DNA聚合酶可在接近引物熔点的温度下进行退火和合成步骤，提供一种灵敏且特异的检测方法，能够在10(6)个正常细胞中检测到1个淋巴瘤细胞。该技术在所有7例接受检查的患者中均检测到白血病细胞的亚临床存在，包括2例临床缓解患者。它还评估了旨在清除骨髓中恶性细胞的方案的有效性。此外，这种方法能够在不进行分子克隆的情况下对染色体断点进行快速DNA测序。该检测方法显著提高了检测微小残留病的能力，应能改善确定疾病分期、分层治疗和评估治疗效果的能力。
