Quantitative Real-Time RT-PCR of Disseminated Tumor Cells in Combination With Immunomagnetic Cell Enrichment

Abstract

Detection of disseminated tumor cells in the blood circulation is important in assessing tumor progression. The objective of this examination was to develop a highly specific and sensitive quantitative realtime reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for the detection of relevant tumor-associated transcripts in patients' blood. The qRT-PCR assays detect the human epidermal growth factor receptor 2 (HER2) and CK20 transcripts of two tumor cells spiked into 5 mL of blood after an immunomagnetic tumor cell enrichment. Furthermore, the HER2 assay is only specific when enrichment is included. This procedure is a useful alternative to fluorescence in situ hybridization and immunocytochemistry for gene alteration analysis in human tumors. The analysis of the studied molecular markers of tumor cells in blood may be useful in the detection of disseminated tumor cells as well as for monitoring treatment response, early detection of relapse, and for stratification of patients with carcinoma.

Molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer: an option for monitoring response to breast cancer related therapies

“…Reverse transcription resulted in cDNA, which was the template for tumor cell detection and characterization by multiplex RT-PCR. Sensiscript Ò Reverse Transcriptase (QIAGEN GmbH, Hilden, Germany) was used for the reverse transcription because of its high sensitivity (recommended for amounts of \50 ng RNA) in combination with oligo(dT) coupled Dynabeads of the mRNA DIRECT TM Micro Kit (Dynal Biotech GmbH) according to the manufacturer's instructions [28]. cDNA was synthesized in a thermocycler under the following conditions: Reverse transcription was performed at 37°C for 60 min followed by 3 min at 93°C for inactivation of the reaction.…”

Towards an optimized platform for the detection, enrichment, and semi-quantitation circulating tumor cells

“…While automated immunohistochemical methods are beginning to show promise, traditional manual methods are time-consuming, and the results can be subjective [6]. We and others [7][8][9][10][11][12] have used QPCR as an alternate method for the detection of tumor cell-specific mRNA in patients as an estimate of tumor cell load, although different studies have invariably used a variety of different procedures to achieve this. The aim of this study was therefore to test various processing and analytical methodologies in an attempt to find the optimal procedure for the detection of CTCs by QPCR.…”

“…Moreover, these methods can be prone to false-positives and can lack sensitivity when the cells of interest are at very low frequency, as is invariably the case in peripheral blood [6]. In an attempt to increase sensitivity and concurrently decrease subjectivity in the enumeration of CTCs in cancer patients, increasing numbers of studies have investigated QPCR as a detection platform [7][8][9][10][11][12]. Despite this work, however, there has been little consensus as to the optimal analytical procedures to use to achieve this goal.…”

Circulating tumor cells: utility for predicting response to anti-EGFR therapies?

“…While the RT-PCR technology allows evaluation of a wider range of markers, it reduces the ability to quantify CTCs or analyze other molecular features [13,14].…”