Yeh K H, Chen Y C, Yeh S H, Chen C P, Lin J T, Cheng A L
Department of Oncology, National Taiwan University Hospital, Taipei, R.O.C.
Anticancer Res. 1998 Mar-Apr;18(2B):1283-6.
Intermediate filament cytokeratin-19 (K19) protein is expressed in normal and malignant gastrointestinal epithelial cells, but not in peripheral blood (PB). Small amount of circulating gastric cancer cells can be detected by a sensitive nested reverse transcription-polymerase chain reaction (RTPCR) with primers specific for K19 mRNA. Thirty-four PB samples obtained from patients with inoperable/metastatic gastric cancer were examined. The mononuclear cell (MNC) fraction was collected by Ficoll centrifugation, and followed by total RNA extraction by acid guanidinium thiocyanate-phenol-chloroform method. RNA from 8 gastric cancer cell lines and the mononuclear cells of 33 healthy adults were used as positive and negative controls, respectively. DNA fragment of 774 bp amplified by the internal primers was found to be a highly reliable marker for K19 mRNA expression. The sensitivity of detection was between 1 and 10 cells/10(6) normal MNCs. The K19 transcripts were detected in 20.6% (7/34; 8-37%, 95% C.I.) of PB samples. None of the other pertinent clinicopathological features, including the disease extent and the histopathologic types of the tumors, were related to the expression of K19 in PB. All 34 patients had been treated by systemic chemotherapy. Among the 17 non-responders to chemotherapy, the survival of the 4 patients with detectable K19 was significantly shorter than that of 13 patients without detectable K19 in their circulating blood (p = 0.014). However, the survival impact of K19 was less significant in the other 17 patients whose tumors had responded to systemic chemotherapy. Of the whole group of patients, the median survival of the 7 and 27 patients with and without detectable K19 in their circulating blood was 1 and 3.5 months, respectively (p = 0.368). We concluded that detecting circulating cancer cells by K19 nested RT-PCR is associated with poor prognosis of gastric cancer, particularly in those patients who are not responsive to systemic chemotherapy.
中间丝细胞角蛋白19（K19）蛋白在正常和恶性胃肠道上皮细胞中表达，但在外周血（PB）中不表达。通过对K19 mRNA特异的引物进行敏感的巢式逆转录-聚合酶链反应（RTPCR），可以检测到少量循环胃癌细胞。对34例无法手术/转移性胃癌患者的外周血样本进行了检测。通过Ficoll离心收集单个核细胞（MNC）部分，然后用酸性异硫氰酸胍-苯酚-氯仿法提取总RNA。分别以8种胃癌细胞系的RNA和33名健康成年人的单个核细胞作为阳性和阴性对照。内部引物扩增出的774 bp DNA片段被发现是K19 mRNA表达的高度可靠标志物。检测灵敏度为1至10个细胞/10(6)个正常MNC。在20.6%（7/34；8 - 37%，95%置信区间）的外周血样本中检测到K19转录本。其他相关临床病理特征，包括疾病范围和肿瘤组织学类型，均与外周血中K19的表达无关。所有34例患者均接受了全身化疗。在17例化疗无反应者中，循环血中可检测到K19的4例患者的生存期明显短于13例循环血中未检测到K19的患者（p = 0.014）。然而，在其他17例肿瘤对全身化疗有反应的患者中，K19对生存期的影响较小。在整个患者组中，循环血中可检测到K19和未检测到K19的7例和27例患者的中位生存期分别为1个月和3.5个月（p = 0.368）。我们得出结论，通过K19巢式RT-PCR检测循环癌细胞与胃癌预后不良相关，尤其是在那些对全身化疗无反应的患者中。
