Slade M J, Smith B M, Sinnett H D, Cross N C, Coombes R C
Department of Cancer Medicine, Imperial College School of Medicine, London, United Kingdom.
J Clin Oncol. 1999 Mar;17(3):870-9. doi: 10.1200/JCO.1999.17.3.870.
Previous reports have indicated that reverse transcriptase polymerase chain reaction (RT-PCR) for cytokeratin 19 (CK-19) may be useful in the management of patients with breast cancer. However, the specificity of this technique is low, principally because of a high rate of false-positive results. To improve the specificity of this assay, we developed a quantitative RT-PCR methodology that enables an estimate to be made of the number of CK-19 transcripts in blood and bone marrow samples.
We examined 45 peripheral-blood samples and 30 bone marrow samples from patients with a variety of nonneoplastic conditions using nested RT-PCR for CK-19. We also examined bone marrow and peripheral-blood samples from 23 patients with primary breast cancer and peripheral-blood samples from 37 patients with metastatic breast cancer. The number of CK-19 transcripts was estimated in positive specimens by competitive PCR and normalized to the number of ABL transcripts as an internal control for the quality and quantity of cDNA. RT-PCR results were compared with the numbers of CK-19-positive cells detected by immunocytochemistry.
Analysis of samples from patients without cancer enabled us to define an upper limit for the background ratio of CK-19 to ABL transcripts (1:1,000 for blood samples and 1:1,600 for bone marrow samples). Using these figures as cut-off points, elevated CK-19: ABL ratios were detected in peripheral-blood samples of 20 of 37 (54%) patients with metastatic breast cancer and in bone marrow samples of 14 of 23 (61%) patients with primary breast cancer. Only three of 23 (13%) primary breast cancer peripheral-blood samples and none of the control samples were positive by these criteria. Only two of 23 patients (9%) with primary breast cancer showed immunocytochemically detectable cells in the blood; 10 of 23 (43%) showed immunocytochemically detectable cells in the bone marrow. Of 36 patients with metastatic breast cancer, eight (22%) showed positive events.
Quantitative RT-PCR for CK-19 detects a percentage of patients with breast cancer and may enable the progression or regression of the disease to be monitored.
先前的报告表明，细胞角蛋白19（CK-19）的逆转录聚合酶链反应（RT-PCR）可能有助于乳腺癌患者的管理。然而，该技术的特异性较低，主要原因是假阳性结果率较高。为了提高该检测方法的特异性，我们开发了一种定量RT-PCR方法，能够估计血液和骨髓样本中CK-19转录本的数量。
我们使用针对CK-19的巢式RT-PCR检测了45份来自患有各种非肿瘤性疾病患者的外周血样本和30份骨髓样本。我们还检测了23例原发性乳腺癌患者的骨髓和外周血样本以及37例转移性乳腺癌患者的外周血样本。通过竞争性PCR估计阳性标本中CK-19转录本的数量，并将其标准化为ABL转录本的数量，作为cDNA质量和数量的内部对照。将RT-PCR结果与通过免疫细胞化学检测到的CK-19阳性细胞数量进行比较。
对无癌患者样本的分析使我们能够确定CK-19与ABL转录本背景比率的上限（血液样本为1:1000，骨髓样本为1:1600）。以这些数字作为截断点，在37例转移性乳腺癌患者中的20例（54%）外周血样本和23例原发性乳腺癌患者中的14例（61%）骨髓样本中检测到CK-19:ABL比率升高。根据这些标准，23例原发性乳腺癌外周血样本中只有3例（13%）呈阳性，对照样本均无阳性。23例原发性乳腺癌患者中只有2例（9%）血液中显示免疫细胞化学可检测到的细胞；23例中有10例（43%）骨髓中显示免疫细胞化学可检测到的细胞。在36例转移性乳腺癌患者中，8例（22%）出现阳性事件。
CK-19的定量RT-PCR可检测出一定比例的乳腺癌患者，并可能有助于监测疾病的进展或消退。
