Other Methods for Isolation & Investigation of CTCs
CTCs could be isolated from blood based on its buoyant property. A common method is by use of Ficoll-Paque Plus (GE Healthcare, Pittsburgh, PA, USA), which separates red blood cells, mononuclear cells and plasma into three separate layers. Both blood and bone marrow could be used for isolation of CTCs or DTCs, where they co-layer with mononuclear cells after centrifugation. However, both recovery and enrichment are poor and requires further rounds of purification using other methods. A modification of this method is provided by OncoQuick® (Greiner Bio-One, Germany), which uses a porous membrane to reduce contaminating mononuclear cells without reducing CTC recovery rate.
Size Tumor cells are larger than blood cells and this allows isolation of CTCs based on size. Examples of this method include ISET® (Isolation by Size of Epithelial Tumor cells, Rarecells, France) and ScreenCell® (ScreenCell, France), which enrich CTCs by filtration of blood through a porous membrane.[121,122] Since this method is not limited by the heterogeneity of expression of cell surface markers, a wider range of CTCs could be captured.[121,123,124] Recovery rates of up to 90% have been achieved for cancer cells spiked into blood samples. In fact, it was reported that 5.5-times more CTCs were recovered by a filtration device than by CellSearch. The procedure is simple and quick to perform and isolated cells could be further used for downstream analyses. However, the purity of the CTC fraction obtained by these methods is generally poor and could contain up to 2000 contaminating leucocytes per ml of blood filtered.
Reverse Transcription Polymerase Chain Reaction
Besides cell enumeration, gene expression analyses of CTCs are also performed to gain a better understanding of their molecular and biological characteristics. In this regard, the high specificity, sensitivity and multiplexing capabilities offered by RT-PCR make this technique one of the most commonly used methods for CTC investigations.
A recent study has compared the performance of the CellSearch System, the AdnaTest and RT-PCR amplification of CK19 and mammaglobin (MGB1) transcripts in detecting CTCs. AdnaTest identifies CTCs by first performing immunomagnetic separation followed by multiplex RT-PCR detection of HER2, Muc1 and GA773-2 transcripts from patient blood. The test result is considered positive if at least one of the transcripts was detected. It was found that among the three methods, RT-PCR was more likely to detect presence of CTCs in patients with metastatic breast cancer than CellSearch or AdnaTest, demonstrating superior sensitivity among the three methods. Concordant results were observed between RT-PCR and CellSearch in 72% (CK19) and 60% (MGB1) of cases, respectively. Pitfalls of the RT-PCR method include its inability to enumerate CTCs and that morphological analysis of detected cells is not possible.
In recent reports, CK19 and MGB1 transcripts were detected in 41–75% and 8–38% of blood from patients with breast cancer. The detection of MGB1 transcript was associated with worse disease-free survival while that for CK19 expression was associated with shorter overall survival and worse disease-free survival.[127,128]
Expert Rev Proteomics. 2013;10(6):579-589. © 2013 Expert Reviews Ltd.