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113 records · Page 7Linked to original sources
PubMed2002

Detection of circulating cytokeratin-positive cells in the blood of breast cancer patients using immunomagnetic enrichment and digital microscopy.

PURPOSE: To examine the feasibility for identifying and enumerating cytokeratin positive (CK+) cells in the peripheral blood of breast cancer patients. EXPERIMENTAL DESIGN: Blood specimens from 34 normal donors (negative controls), 15 samples to which carcinoma cells were added (positive controls), and 84 breast cancer patients [27 node-negative (N-), 29 node-positive (N+), and 28 metastatic] were studied. RBCs were lysed with ammonium chloride and the resulting cell suspension incubated with anti-EpCAM-conjugated immunomagnetic beads for carcinoma cell enrichment. Immunomagnetically selected cells were placed on slides; stained for CKs 8, 18, and 19; and evaluated with an automated digital microscopy system that rapidly scanned the slide and collected images of cells meeting predefined staining and cytomorphological criteria. A montage of the CK+ cells was reviewed to confirm tumor cell morphology. RESULTS: Eighteen specimens (9 normal, 2 N-, 4 N+, and 3 metastatic) were excluded because of poor cytomorphology or staining artifact. All 15 of the positive controls [95% confidence interval (CI), 78-100%] and none of the 25 negative controls (95% CI, 0-14%) demonstrated CK+ cells. Twenty-one of the 75 (28%; 95% CI, 18-40%) samples from breast cancer patients demonstrated CK+ cells including 76% of patients with metastatic disease (95% CI, 55-91%), 8% with N+ disease (95% CI, 1-26%), and none of those with N- disease (95% CI, 0-14). The mean number of CK+ cells detected in the 21 CK+ patients was 18.4 (range, 1-120). CONCLUSIONS: Breast carcinoma cells can be detected in the blood from a significant fraction of metastatic breast cancer patients using immunomagnetic cell enrichment and digital microscopy. The incidence of CK+ cells was low in those with resected N+ disease (at most 26%) and those with resected N- breast cancer (at most 14%). This technique could be used in large prospective studies of patients with breast cancer to learn whether the detection of rare carcinoma cells is a useful predictive or prognostic factor.

Breast Neoplasms↗
PubMed2001

Minimal costimulatory requirements for T cell priming and TH1 differentiation: activation of naive human T lymphocytes by tumor cells armed with bifunctional antibody constructs.

Direct priming of naive human CD8+ and CD4+ T cells by tumor cells devoid of any intrinsic antigen presentation properties, but passively armed with recombinant proteins mediating primary and costimulatory T cell signals, was investigated. Bifunctional antibody constructs were used to specifically target costimulatory molecules such as B7-1, B7-2 and LFA-3 to the epithelial cell adhesion molecule (EpCAM), a surface antigen successfully used as target for antibody therapy of minimal residual colorectal cancer. T cell priming was monitored by flow cytometric analysis of CD45 isoform expression and confirmed by measuring typical effector functions of primed T cells known to be absent from naive T lymphocytes. Accordingly, CD8+ T cells were tested for cytotoxic activity and secretion of TNF-alpha, while secretion of IFN-gamma, IL-5 and IL-4 was determined for CD4+ T cells. B7, known to be required for the initial activation of naive T cells, also proved to be sufficient for T cell priming when present as the only costimulatory molecule together with an appropriate primary signal. The requirement of dendritic and other antigen presenting cells (APCs) for T cell priming through non-APCs such as tumor cells could be ruled out. Under minimal priming conditions, naive CD4+ T cells were found to exclusively enter the TH1 developmental pathway, while several factors thought to favor TH2 polarization, like weak primary signals and B7-2 versus B7-1 costimulation, could be excluded as dominant TH2 promoters.

Animals↗
PubMed2003

Rare expression of target antigens for immunotherapy on disseminated tumor cells in breast cancer patients without overt metastases.

Occult disseminated tumor cells are the major cause of relapse in patients with primary operable breast cancer but detection and characterization of these few cells is difficult. Applying immunohistochemistry, an immunomagnetic enrichment technique (IET) and immunocytochemistry (IC), we studied 58 breast cancer patients without overt metastases for the frequency of cytokeratin-positive (CK+) bone marrow (BM) cells coexpressing the epithelial adhesion molecule 17-1A (EpCAM) and c-erbB-2 and analyzed the primary tumor for these antigens as a strategy for additional immunotherapy. The primary tumors were analyzed for the target antigens by a pathologist. Dissemination of CK+ cells was studied in 4-6 x 10(6) BM cells by IC alone. For characterization of CK+ cells, 10-15 x 10(6) BM cells were incubated with microbeads coupled to antibodies detecting the target antigens, labelled cells were separated on selection columns and the positively (BM cells carrying the target antigen) and negatively (BM cells without target antigen) selected fractions were stained for CK+ cells. The effectiveness of these methods was confirmed in cell culture models. 17-1A was detected in all primary tumors and c-erbB-2 overexpression (2+, 3+) was found in 25/58 tissue samples. In total, analyzing 15-20 x 10(6) BM cells in each patient, the detection rate for CK+ cells in the BM was 69% (40/58 patients). Interestingly, analysis of the positive and negative enrichment fractions showed that the 17-1A antigen was coexpressed on CK+ cells in only 6 patients and c-erbB-2/CK+ cells were found in only one patient. Although 17-1A and c-erbB-2 were frequently detected in the primary tumor, these antigens were rarely expressed on CK+ BM cells. Whether the applied IET is not able to detect low amounts of these target antigens has to be clarified. Nevertheless, applying cell-cycle independent protocols in clinical trials requires careful elucidation of those patients who might benefit from these therapies.

Antigens, Neoplasm↗
PubMed2004

Minimal residual disease detection in breast cancer: improved sensitivity using cytokeratin 19 and epidermal growth factor receptor RT-PCR.

Breast cancer cells pose a difficult target for detection due to their lack of tumor specific markers. Small numbers of metastatic cells circulate through the blood and reach target organs, but current methods are presently not sensitive enough to allow for early detection. Our goal is to develop a method for specific and sensitive detection for breast cancer tumor cells by using cell enrichment coupled to RT-PCR with selective primer pairs. Fifty million normal donor mononuclear cells (NDMNC) were spiked with serial dilutions of a breast cancer cell line, positively selected for surface expression of the epithelial cell adhesion molecule (EPCAM) with anti-Ber-EP4 mAb. Total RNA was isolated and the cDNA was transcribed and measured by quantitative PCR for cytokeratin 19 (CK19), epidermal growth factor receptor (EGF-R), and beta-2-microglobulin (beta2M) transcripts using specific hybridization probes with the LightCycler System. A sensitivity of 1 tumor cell in 5 million NDMNC was consistently achieved with a metastatic breast cancer cell line with target primer sets for CK19 and EGF-R. Less, but appreciable sensitivity was achieved by spiking NDMNC with other breast cancer cells. Detection of both gene targets ranged between 1 in 5,000 to 5 million cells. The method described, a cell enrichment procedure and RT-PCR using specific hybridization probes (spanning introns and hybridizing to areas that discriminate transcribed pseudogenes), results in increased sensitivity while decreasing false positives above those methods previously reported. This general approach may have wide applicability to increasing sensitivity of minimal residual disease detection in other cancers.

Biomarkers, Tumor↗
PubMedDate not supplied

Combined treatment of metastatic breast cancer (MBC) by high-dose chemotherapy (HDCT) and bispecific antibodies: a pilot study.

UNLABELLED: This pilot study analyzed the efficacy and toxicity of high-dose chemotherapy (HDCT), autologous stem-cell transplantation (ASCT) and subsequent immunotherapy with T-cell reinfusion and trifunctional antibodies (trAbs) in chemotherapy-sensitive patients with metastatic breast cancer (MBC). PATIENTS AND METHODS: After leukapheresis and cryopreservation of T-cells, patients received 2 cycles of induction chemotherapy ET (epirubicin/paclitaxel) and 1 cycle of El (epirubicin/ifosfamide), followed by G-CSF and stem-cell harvest. After a final cycle of ET, responders (CR/PR) underwent HDCT (thiotepa 600 mg/m2/melphalan 140-180 mg/m2) and ASCT. Once reconstitution was achieved, T-cells were reinfused, followed by application of trifunctional antibodies with specificities anti-EpCAM X anti-CD3 and anti-Her2/neu X anti-CD3. RESULTS: Thirty-three patients were recruited into the study and 19, who had responded to initial chemotherapy, underwent HDCT and ASCT (4 CR, 15 PR, OR = 57.6%; 95% CI: 40-75%). Two early deaths were observed (1 toxic, 1 early progression). T-cell reinfusion and trAbs were given to 17 patients. TrAbs treatment resulted in intermittent fever, chills and elevated liver enzymes, which were seen in all patients. The median overall survival was 27.7 months (range: 5.9-82.6+). Patients who received 3 trAbs doses showed a trend towards an improved overall survival (47.2 vs. 22.4 months, p = 0.08 log rank). CONCLUSION: This pilot study has shown the feasibility of combining HDCT with immunotherapy in MBC. Further investigation of this approach is indicated.

Adult↗