Detection of Survivin-expressing circulating cancer cells in the peripheral blood of breast cancer patients by a RT-PCR ELISA

Survivin mRNA expression was detected in 69.2%–93.8% of primary breast carcinomas, but is rarely expressed in normal breast tissues and hematopoietic cells. The objective of this study was to investigate the significance that the detection of Survinin-expressing circulating breast cancer cells in the peripheral blood has on clinical outcomes. The detection method was based on a RT-PCR ELISA technique developed in our laboratory. Sixty-seven breast cancer patients in various stages and 135 normal healthy women were investigated. Survivin-expressing circulating cancer cells were detected in the peripheral blood samples from 34 (50.7%) out of 67 breast cancer patients, but not in the healthy women that were used as controls. The presence of Survivin-expressing circulating breast cancer cells was found to be significantly associated with various clinicopathological parameters such as vessel infiltration, histological grade, tumor size, nodal status, ER/PgR status, Her-2 status and clinical stages of the disease (P < 0.01). During a follow-up period of 36 months, 9 out of 11 (81.8%) breast cancer patients that had a positive Survivin-expressing at the time of the initial assay test suffered a relapse of the disease, whereas recurrence was only found in 2 out of 6 (33.3%) breast cancer patients that had a negative Survivin-expression. Thus, the detection of circulating cancer cells expressing Survivin mRNA could provide valuable information for the prediction of metastasis and recurrence of breast cancer.     

Comparative analysis of innate immune system function in metastatic breast,colorectal, and prostate cancer patients with circulating tumor cells

In recent years, circulating tumor cells (CTCs) in metastatic cancer patients have been found to be a promising biomarker to predict overall survival and tumor progression in these patients. A relatively high number of CTCs has been correlated with disease progression and poorer prognosis. This study was designed to assess innate immune system function, known to be responsible for the immune defense against developing neoplasms, in metastatic cancer patients with CTCs. Our aim is to provide a link between indication of poorer prognosis, represented by the number of CTCs to the cytotoxic activity of natural killer cells, an important component of the innate immune system, and to represent a promising expanded approach to management of metastatic cancer patients with CTCs. Seventy-four patients, with metastatic breast, colorectal, or prostate cancer, were recruited for this study. Using a flow cytometric assay, we measured natural killer (NK) cell cytotoxicity against K562 target cells; and CTCs were enumerated using the CellSearch System. Toll-like receptors 2 and 4 expression was also determined by flow cytometry.     

Immunomagnetic enrichment of disseminated tumor cells in bone marrow and blood of breast cancer patients by the Thomsen-Friedenreich-Antigen

Purpose The presence of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has shown independent prognostic impact. Immunomagnetic enrichment of such cells is an approach to increase the number of detected cells with limited sample volume, especially for circulating tumor cells (CTCs) in blood. The Thomsen-Friedenreich (TF) antigen (CD 176) is a specific oncofetal carbohydrate epitope (Galβ1-3GalNAcα-O) expressed on the surface of various carcinomas. Own studies demonstrated a nearly complete TF expression on DTC-BM, indicating its suitability as marker for immunomagnetic enrichment. Methods BM samples of 65 and peripheral blood samples of 11 breast cancer patients were examined immunocytochemically by staining with the anti-Cytokeratin antibody A45-B/B3 before and after immunomagnetic enrichment. Enrichment was done by incubation with the primary antibody TF 2 (IgM), followed by secondary magnetically labelled rat-anti mouse IgM. Cytospin slides were screened manually by bright-field microscopy. Results 15/65 pts (23%) showed DTC-BM in primary screening with a median of 2/2 mio cells (range 1–10). By enrichment, a median of 23.3 mio cells (0.8–218) could be analysed, increasing positivity to 72% (47/65 pts) with a med. of 4 DTCs (1–105, P < .0001). Blood from 1/11 pts before and 5/11 (45%) after enrichment showed CTCs (med. 2, 1–20), at a med. of 12.4 mio (2.6–38.5) cells analysed. Comparing BM and blood of the same patients after enrichment, 5 were positive in both compartments, 4 showed DTC-BM without presence of CTCs. Conclusion The positive immunomagnetic enrichment technique with TF-antibodies enables to analyse larger sample volumes and increase tumor cell detection rate. This could allow monitoring and characterisation of CTCs as targets for therapies.