Detection of circulating tumor cells in blood of metastatic breast cancer patients using a combination of cytokeratin and EpCAM antibodies.

BACKGROUND: Circulating tumor cells (CTCs) are detectable in peripheral blood of metastatic breast cancer patients (MBC). In this paper we evaluate a new CTC separation method based on a combination of anti-EpCAM- and anti-cytokeratin magnetic cell separation with the aim to improve CTC detection with low target antigen densities. METHODS: Blood samples of healthy donors spiked with breast cancer cell line HCC1937 were used to determine accuracy and precision of the method. 10 healthy subjects were examined to evaluate specificity. CTC counts in 59 patients with MBC were measured to evaluate the prognostic value on overall survival. RESULTS: Regression analysis of numbers of recovered vs. spiked HCC1937 cells yielded a coefficient of determination of R(2) = 0.957. The average percentage of cell recovery was 84%. The average within-run coefficient of variation for spiking of 185, 85 and 30 cells was 14%. For spiking of 10 cells the within-run CV was 30%. No CTCs were detected in blood of 10 healthy subjects examined. A standard threshold of 5 CTC/7.5 ml blood as a cut-off point between risk groups led to a highly significant prognostic marker (p < 0.001). To assess the prognostic value of medium CTC levels we additionally considered a low (CTC-L: 0 CTC), a medium (CTC-M: 1-4 CTC) and a high risk group (CTC-H: ≥5 CTC). The effect of this CTC-LMH marker on overall survival was significant as well (p < 0.001). A log-ratio test performed to compare the model with 3 vs. the model with 2 risk groups rejected the model with 2 risk groups (p = 0.026). For CTC as a count variable, we propose an offset reciprocal transformation 1/(1 + x) for overall survival prediction (p < 0.001). CONCLUSIONS: We show that our CTC detection method is feasible and leads to accurate and reliable results. Our data suggest that a refined differentiation between patients with different CTC levels is reasonable.

Comparison of Viscum album QuFrF extract with vincristine in an in vitro model of human B cell lymphoma WSU-1.

UNLABELLED: Viscum album L. extract (Iscador) with different preparations is used either alone or in combination with chemo/radiotherapy in the treatment of patients with various tumours.Vincristine (CAS 57-22-7) as a chemotherapeutic agent is used mainly in combination with other chemotherapeutic substances in the therapy of B cell lymphoma. In this study, the effects of Viscum album (VA) QuFrF extract and vincristine were compared on B cell lymphoma cell line WSU-1 in an in vitro model. As parameters were measured: proliferation, viability, apoptosis/necrosis, IL-6 production, DNA synthesis and the cell cycle phases of the lymphoma cells at various time points. RESULTS: The cytostatic (inhibition of proliferation) effects ofVAQuFrF and vincristine at 10 microg/10(5) cells were the same at 48 h and 72 h. This means that the anti-proliferative effect of 2 ng lectin (in 10 microg extract) is equivalent to 10 microg of vincristine. There was a relationship between the cytostatic and cytocidal (killing of viable cells) effects for both substances. This means that both substances first inhibit the proliferation of the tumour cells, and then the cells die by apoptosis or necrosis. VAQuFrF and vincristine reduced the DNA synthesis markedly and arrested the G2/M cell cycle phase. Both substances led to a clearly dose-dependent apoptosis at 12 h and 24 h. Neither VAQuFrF nor vincristine led to IL-6 production of the lymphoma cells. CONCLUSION: The effects of VAQuFrF on the B cell lymphoma cell line WSU-1 were comparable to those of vincristine in all parameters. The effective dose range lay between 50 and 100 microg/10(6) cells for VAQuFrF, for vincristine it was somewhat lower.

Cytostatic and cytocidal effects of mistletoe (Viscum album L.) quercus extract Iscador.

Mistletoe (Viscum album) extract (Iscador) is used either alone or in combination with chemotherapy or radiotherapy in the treatment of tumor patients. In this study the effects of Viscum album Quercus (VA Qu) extract (1000 ng lectin and 6 microg viscotoxin/ml) in various doses were investigated in an in vitro model with tumor cells: three multiple myeloma (MM) cell lines (OPM-2, RPMI-8226, U-266) and three B cell lymphoma cell lines (U-698, DOHH-2, WSU-1). None of the three B lymphoma cell lines and none of the three multiple myeloma cell lines produced interleukin (IL)-6 spontaneously or after treatment with VA Qu extract. All three multiple myeloma cell lines expressed surface IL-6R and gp130, the B cell lymphomas expressed only gp130. Viscum album/Qu extract markedly downregulated the membrane expression of IL-6R and gp130 in RPMI-8226 (down to 29% and 32%) and the expression of gp130 in WSU-1 (down to 22%). There was a marked reduction of viable cells of RPMI-8226 (down to 28%) and WSU-1 (down to 8 %) at 100 microg/10(6) cells /ml. There was a clear relationship between the inhibition of proliferation and viability: the percentage reductions of the viable cells at 48 and 72 h were similar to those of proliferation at 24 and 48 h, respectively. This means that firstly the proliferation of the tumor cell is inhibited and then afterwards these cells die by apoptosis or necrosis. The inhibitory effect of VA Qu on the proliferation can be termed cytostatic, on the survival cytocidal. VA Qu was more effective in cells having a high proliferation rate than in those with a low proliferation rate. The effective dose range lay between 25 and 100 microg/10(6) cells/ml (5-20 ng lectin/10(6) cells/ml) for all parameters.