Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells

Abstract

BackgroundImmunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and sufficiently sensitive to enable detection of single tumor cells. We have developed a method to test RT-PCR assays for any cancer. This has been investigated using a panel of RT-PCR markers suitable for the detection of breast cancer cells.MethodsIn the assay, a single cell line-derived tumor cell is added to 100 peripheral blood mononuclear cells (PBMNCs) after which mRNA is isolated and reverse transcribed for RT-PCR analysis. PBMNCs without added tumor cells are used as specificity controls. The previously studied markers epidermal growth factor receptor (EGFR), mammaglobin 1 (MGB1), epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA) were tested. Two new epithelial-specific markers ELF3 and EphB4 were also tested.ResultsMUC1 was unsuitable as strong amplification was detected in 100 cell PBMNC controls. Expression of ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 was found to be both specific for the tumor cell, as demonstrated by the absence of a signal in most 100 cell PBMNC controls, and sensitive enough to detect a single tumor cell in 100 PBMNCs using a single round of RT-PCR.ConclusionsELF3, EphB4, EpCAM, EGFR, CEA and MGB1 are appropriate RT-PCR markers for use in a marker panel to detect disseminated breast cancer cells after immunomagnetic enrichment.

Real‐time RT‐PCR detection of CK19, CK7 and MUC1 mRNA for diagnosis of lymph node micrometastases in non small cell lung carcinoma

“…Moreover, although LNs from patients with benign disease were negative for MUC1, 3 out of 4 PBMNs from healthy volunteers gave positive results demonstrating a crossreaction with noncarcinomatous cells. These results, already reported by others, 24,25 could be due to expression of MUC1 mRNA by skin sebaceous and sweat glands contaminating blood samples collected by transcutaneous venipuncture 34 or by monocytes. 35 Therefore, MUC1 mRNA detection was not considered to be a reliable marker for the detection of micrometastatic cells.…”

“…21 The 3 markers were mRNAs from cytokeratin 19 (CK19), cytokeratin 7 (CK7) and mucin type 1 (MUC1), as CK19 22,23 and CK7 23 have been demonstrated to be components of the cancer cell cytoskeleton in NSCLC and MUC1 is a cell surface glycoprotein expressed in lung tissue. Although cross-reaction with noncarcinomatous cells has been previously reported, 24,25 MUC1 has been shown to be expressed in most NSCLCs (approximately 80-100%) 16 and is used for the detection of occult cancer cells. The aim of this study was therefore to test the accuracy of each of these markers as well as their combined use.…”

Identification of Early-Stage Colorectal Cancer Patients at Risk of Relapse Post-Resection by Immunobead Reverse Transcription-PCR Analysis of Peritoneal Lavage Fluid for Malignant Cells

“…Use of the immunobead technique for the prior isolation of epithelial cells has two advantages over RT-PCR using RNA from whole samples: first, the possibility of detecting free mRNA that may be present in the plasma or attached to blood cells of some cancer patients is avoided (33); second, the possibility of detecting ''illegitimate'' transcription from other cells (34) is substantially reduced. The tumor specificity of the CEA marker has been shown to improve when used in the immunobead RT-PCR method (12,35) compared with the use of RNA from density gradientseparated blood cells in RT-PCR (9). As different molecular markers were expressed at different levels in any individual tumor, a panel of markers that we found to be overexpressed in most tumors was used to minimize the possibility of falsenegative results.…”

Epidermal growth factor receptor downregulation in cultured bovine cumulus cells: reconstitution of calcium signaling and stimulated membrane permeabilization

“…PCR was accomplished following the protocol of Raynor et al (2002). In brief, CCs were collected after hyaluronidase treatment of bovine COCs and a fraction of the cells were lysed and stored at 2 80 8C.…”