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. 2016 May 1;6(5):973-80.
eCollection 2016.

Molecular characterization of circulating tumor cells in ovarian cancer

Katarina Kolostova  1 , Michael Pinkas  1 , Anna Jakabova  1 , Eliska Pospisilova  1 , Pavla Svobodova  1 , Jan Spicka  1 , Martin Cegan  2 , Rafal Matkowski  3 , Vladimir Bobek  4
Affiliations

Molecular characterization of circulating tumor cells in ovarian cancer

Katarina Kolostova et al. Am J Cancer Res. .

Abstract

The main focus of the study was to detect circulating tumor cells (CTCs) in ovarian cancer (OC) patients using a new methodological approach (MetaCell(TM)) which is based on size-dependent separation of CTCs and subsequent cytomorphological evaluation. Cytomorphological evaluation using vital fluorescence microscopy approach enables to use the captured cells for further RNA/DNA analysis. The cytomorphological analysis is then completed by gene expression analysis (GEA). GEA showed that relative expression of EPCAM is elevated in CTC-enriched fractions in comparison to the whole peripheral blood sample and that the expression grows with in vitro cultivation time. Comparison of the relative gene expression level in the group of peripheral blood samples and CTC-fraction samples confirmed a statistically significant difference for the following genes (p < 0.02): KRT7, WT1, EPCAM, MUC16, MUC1, KRT18 and KRT19. Thus, we suggest that the combination of the above listed genes could confirm CTCs presence in OC patients with higher specificity than when GEA tests are performed for one marker only. The GEA revealed two separate clusters identifying patients with or without CTCs.

Keywords: CTCs; MetaCell; circulating tumor cells; cultivation; gene expression; in vitro; ovarian cancer.

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Figures

Figure 1
Figure 1
CTCs isolated from patients with ovarian carcinoma, captured on the separation membrane (vital fluorescent staining - NucBlue® and Celltracker®). Bar represents 10 µm.
Figure 2
Figure 2
CTCs captured on the separation membrane after immunohistochemistry staining (WT-1 antibody) on Benchmark Ultra automat (Ventana, Roche). Bar represents 10 µm.
Figure 3
Figure 3
Relative expression of EPCAM RNA in peripheral blood and CTC fractions compared after qPCR analysis.
Figure 4
Figure 4
Comparison of averaged relative RNA expression for the genes, shown for all sample types (1-4). Sample type 1 (Peripheral Blood), Sample type 2 (CTC fraction stored immediately after separation process), Sample type 3 (CTC fraction after in vitro culture), Sample type 4 (bottom fraction - cells overgrowing the membrane).
Figure 5
Figure 5
Comparison of the relative gene expression level for the listed genes in the group of peripheral blood (Sample type 1) and CTC-fraction (after 3 days of in vitro culture - Sample type 3). Gene expression levels are relative to the whole peripheral blood data averaged for the patients group. A significant difference was noted for the following genes (p < 0.02): KRT7, WT1, EPCAM, CD68, MUC16, MUC1, KRT18 and KRT19. Thus, we suggest that the combination of the above listed genes should confirm CTCs presence in OC patients with higher specificity than when tests are performed for one marker only.
Figure 6
Figure 6
Cluster analysis for patient - RNA relative amount (log2) of tested genes in all of the tested fracions is shown in colours (please refer to the colour scale on the left). Tested fractions (peripheral blood PK), CTC after isolation (PK IZO), CTC after culture (PK SK), CTC overgrowing the membrane (PK DK). The highest expression of EPCAM was detected in PK SK fraction CTCs on the separation membrane.
Figure 7
Figure 7
Cluster analysis of gene expression data RNA relative amount (log2) for CTC-membrane fractions (PK SK) of all tested patients clearly identifies CTC-positive and CTC-negative group of patients (Higher expression is shown in red).
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