Detection of isolated tumour cells in the blood and bone marrow of patients with gastric cancer by combined sorting, isolation and determination of MAGE-1, -2 mRNA expression

Abstract

Abstract. 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 cytokeratinpositive (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 ß-actinmRNA expression in a single CK + cell. Using ≥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.

Interactions of tumour-derived micro(nano)vesicles with human gastric cancer cells

“…Total RNA was isolated from the GC1415 tumour cells (1 × 10 6 ) and their TMV using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The cDNA synthesis and nested, “real-time” PCR for MAGE-1, -2 and β-actin was performed as previously described [ 16 ]. For the detection of HER-2/neu mRNA “real time” PCR was performed using the following primer pair: for HER-2/neu: sense-5′-CCTCTGACGTCCATCATCTC-3′ and antisense-5′-ATCTTCTCGTGCCGTCGCTT-3′.…”

Detection of Circulating Gastrointestinal Cancer Cells in Conditionally Reprogrammed Cell Culture

“…Though the hTERT mRNA might be expressed in the cells of the CRC culture (15), its expression could differentiate human cancer cells from mouse fibroblast J2 cells. The results regarding MAGE mRNA revealed that this gene is a cancer specific oncogene (17,29).…”

Characterization of human gastric adenocarcinoma cell lines established from peritoneal ascites

“…The isolation of total RNA and qPCR for MAGE-1, −2 and β-actin was performed as previously described ( 13 ). For detection of HER-2/neu mRNA qPCR was performed using the following primers: Sense-5′-CCTCTGACGTCCATCATCTC-3′ and antisense-5′-ATCTTCTCGTGCCGTCGCTT-3′.…”