Szatanek Rafal, Drabik Grazyna, Baran Jaroslaw, Kolodziejczyk Piotr, Kulig Jan, Stachura Jerzy, Zembala Marek
Department of Clinical Immunology and Transplantation, Jagiellonian University Medical College, PL-30663 Cracow, Poland.
Oncol Rep. 2008 Apr;19(4):1055-60.
The detection of isolated (circulating or disseminated) tumour cells (ITC) in patients with cancer requires very sensitive methods, as such cells are very rare. In the present study, the method that combines the negative isolation of CD45- leukocytes from the blood and bone marrow of patients with gastric cancer by flow cytometry, followed by the positive isolation of single cytokeratin-positive (CK+) cells by a Laser Capture Microdissection System for the determination of MAGE-1, -2 mRNA expression was used to detect ITC. This study shows that this method is highly sensitive as it allows to determine beta-actin-mRNA expression in a single CK+ cell. Using > or =5 CK+ cells as a cut-off level, the MAGE-1 mRNA expression was detected in 100% of CK+ cells in the peripheral blood and in 75% of bone marrow samples of patients with gastric cancer. The MAGE-2 mRNA expression was observed in 40 and 58% of samples, respectively. Furthermore, an analysis of primary tumours and locoreginal lymph nodes with respect to the mRNA expression of the two genes showed that MAGE-1 mRNA expression was detected in 88% of the primary tumours and in 67% of the lymph node samples, whereas the MAGE-2 mRNA expression was observed in 72 and 67% of the cases, respectively. Thus, the method described here allows the precise and sensitive determination of tumour-associated gene expression in single ITC present in the blood and bone marrow of patients with gastric cancer.
由于癌症患者体内的孤立(循环或播散)肿瘤细胞(ITC)非常罕见,因此对其进行检测需要非常灵敏的方法。在本研究中,采用了一种方法,即通过流式细胞术从胃癌患者的血液和骨髓中阴性分离CD45⁻白细胞,然后用激光捕获显微切割系统阳性分离单个细胞角蛋白阳性(CK⁺)细胞,以测定MAGE-1、-2 mRNA表达,从而检测ITC。本研究表明,该方法具有高度灵敏性,因为它能够在单个CK⁺细胞中测定β-肌动蛋白mRNA表达。以≥5个CK⁺细胞为临界值,在胃癌患者外周血100%的CK⁺细胞以及75%的骨髓样本中检测到了MAGE-1 mRNA表达。在样本中分别观察到MAGE-2 mRNA表达的比例为40%和58%。此外,对原发性肿瘤和局部区域淋巴结进行的这两个基因mRNA表达分析表明,在88%的原发性肿瘤和67%的淋巴结样本中检测到了MAGE-1 mRNA表达,而分别在72%和67%的病例中观察到了MAGE-2 mRNA表达。因此,本文所述方法能够精确且灵敏地测定胃癌患者血液和骨髓中单个ITC内肿瘤相关基因的表达。