New route for fast detection of antibodies against zoonotic pathogens in sera of slaughtered pigs by means of flow-through chemiluminescence immunochips.

The research on fast screening methods for antibodies against zoonotic pathogens in slaughter animals is important for food safety in farming and meat-processing industries. As a proof-of-concept study, antibodies against the emerging zoonotic pathogen hepatitis E virus (HEV) and enteropathogenic Yersinia spp. were analyzed in parallel using immobilized recombinant antigens (rAgs) of HEV genotypes 1 and 3 and Yersinia outer protein D (YopD) on a flow-through chemiluminescence immunochip. These rAgs are usually part of commercially available line immunoassays (LIAs) used for human diagnostics. In this study, sera from slaughtered pigs were tested on the microarray analysis platform MCR 3 to detect anti-HEV and anti-Yersinia IgG. The new method was characterized regarding signal reproducibility and specificity. The analytical performance was compared with in-house enzyme-linked immunosorbent assay (ELISA) and a LIA based on recomLine HEV (Mikrogen) or the ELISA test kit pigtype Yersinia Ab (Qiagen), respectively. The immunochip revealed the highest analytical sensitivity and was processed in 9 min automatically on the MCR 3. A comparative screening of swine serum samples from Bavarian slaughterhouses regarding anti-HEV and anti-Yersinia IgG seroprevalence was conducted. By using the LIA, 78% of the sera were tested positive for HEV antibodies. The immunochip and the ELISA identified anti-HEV IgG in 96% and 93% of the tested samples using the O2C-gt1 and O2C-gt3 rAg, respectively. The screening for anti-Yersinia IgG resulted in 86% positive findings using the immunochip and 57% and 48% for the ELISA methods, respectively, indicating a higher detection capability of the new method. Serum samples of slaughtered pigs could be analyzed faster and in an automated way on the microarray analysis platform MCR 3 which shows the great potential of the new immunochip assay format for multiplexed serum screening purposes.

Heterogeneous expression of tumor-associated genes in disseminated breast cancer cells.

BACKGROUND: Gene expression profiles were determined to demonstrate heterogeneity of viable disseminated tumor cells (DTC) in the blood of breast cancer patients. PATIENTS AND METHODS: All patients (n = 48) suffered from metastatic disease (M1) and were treated with chemotherapy and/or Herceptin, respectively. Blood samples were analyzed by a DTC detection assay consisting of immunomagnetic tumor cell selection combined with expression profiling of the tumor-associated transcripts GA733-2, MUC-1, HER-2 and Claudin-7. In addition, the correlation of HER-2 expression in DTC with histopathologically determined HER-2 status in distant metastases and primary tumors in selected cases was investigated. RESULTS: DTC were detected in 69% (p < 0.0001) of breast cancer patients. The expression profiles were shown to be heterogeneous within different patients and even within the follow-up period of patients, reflecting the expected heterogeneity of DTC. Furthermore, preliminary results showed a correlation between HER-2 gene expression in DTC and HER-2 overexpression in tumor tissue of distant metastases. CONCLUSION: The results suggest a clinical value of the DTC detection assay with respect to a more precise characterization of individual cancer disease and selection for therapy. More emphasis should be placed on HER-2 expression in DTC as a possible precursor of distant metastases.

The clinical significance of circulating tumour cells in breast cancer and colorectal cancer patients.

BACKGROUND: Circulating tumour cells (CTC) in the blood of cancer patients indicate disease progression. Their presence reflects a relapse or metastasising process since CTC survive only a short time in the circulation. MATERIALS AND METHODS: Test systems developed by AdnaGen have been used for the sensitive and specific analysis of CTC. RESULTS: Case reports of 2 breast cancer patients demonstrate the successful detection of CTC for therapy monitoring purposes. The disappearance of CTC reflects therapy success. The patient that responded towards therapy was characterized by the disappearance of CTC from the first therapeutic unit (TU) onwards. In contrast, CTC remained detectable in the other patient during the whole therapy pointing to only limited therapeutic efficacy and a progressive disease. Furthermore, systematic changes in the expression profile of CTC in colorectal patients at different stages of disease could be observed. Whereas EGFR was expressed in 90% of the patients with CTC during primary disease the expression level decreased to 15% in CTC of metastatic patients. On the other hand the expression of CEA was low in CTC found after primary surgery (15%) and dominant in CTC of metastatic patients (80%). CONCLUSION: The analysis of CTC is a useful tool for therapy monitoring of breast cancer and colorectal cancer patients in the adjuvant and palliative situation. The molecular profiling of CTC may be used to identify therapeutic targets such as HER2 or EGFR for personalised treatment that is likely to have an important impact on the therapeutic efficacy of drugs like Herceptin or Erbitux.

Tumor-associated gene expression in disseminated tumor cells correlates with disease progression and tumor stage in colorectal cancer.

BACKGROUND: A possible correlation of disease progression and tumor stage in colorectal cancer patients with tumor-associated gene expression in disseminated tumor cells (DTC) was evaluated. Detection of DTC and expression of tumor-associated genes might be of clinical value with respect to individual patient prognosis, monitoring of therapy and as a surrogate tumor staging parameter. PATIENTS AND METHODS: In a multicenter study, a total of 196 peripheral blood samples were collected from 76 patients with tumor stage Dukes' A to D and analyzed using a DTC detection assay consisting of immunomagnetic selection and expression analysis of the tumor-associated genes CEA, EGFR and GA733-2. DTC detection rates were assessed prior to surgery and post surgery in patients with tumor stage Dukes' A, B and C, and compared with results in metastatic patients. CEA serum protein levels were determined and compared with DTC and CEA expression, respectively. RESULTS: In a comparison analysis, EGFR and CEA expression was detected in 88% (p = 0.001) and 0% (p = 0.002) prior to surgery, in 66% (p = 0.001) and 20% (p = 0.002) post surgery, as well as in 15% (p < 0.0001) and 66% (p < 0.0001) of blood samples collected from metastatic patients, respectively. Expression of tumor-associated genes in DTC prior to surgery and in follow-up samples indicated an ongoing metastatic process. DTC detection rates in patients with Dukes' A (14%), Dukes' B (13%) and Dukes' C (40%) prior to surgery correlated statistically with the expected recurrence rate. There was no correlation between DTC expressing CEA and elevation of CEA serum protein levels. CONCLUSION: EGFR and CEA gene expression correlated with disease progression and tumor stage. Detection of CEA expression in DTC might have a predictive value in colorectal cancer and may help to identify patients at a greater risk of relapse. DTC in peripheral blood collected prior to surgery as well as in follow-up samples have a prognostic clinical value.

Quantitative real-time RT-PCR of disseminated tumor cells in combination with immunomagnetic cell enrichment.

Detection of disseminated tumor cells in the blood circulation is important in assessing tumor progression. The objective of this examination was to develop a highly specific and sensitive quantitative realtime reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for the detection of relevant tumor-associated transcripts in patients' blood. The qRT-PCR assays detect the human epidermal growth factor receptor 2 (HER2) and CK20 transcripts of two tumor cells spiked into 5 mL of blood after an immunomagnetic tumor cell enrichment. Furthermore, the HER2 assay is only specific when enrichment is included. This procedure is a useful alternative to fluorescence in situ hybridization and immunocytochemistry for gene alteration analysis in human tumors. The analysis of the studied molecular markers of tumor cells in blood may be useful in the detection of disseminated tumor cells as well as for monitoring treatment response, early detection of relapse, and for stratification of patients with carcinoma.

Combination of immunomagnetic enrichment with multiplex RT-PCR analysis for the detection of disseminated tumor cells.

BACKGROUND: A highly specific and sensitive tumor cell detection assay is reported, which combines immunomagnetic enrichment with multiplex RT-PCR analysis. MATERIALS AND METHODS: The effect on the recovery rate of breast, testicular and colorectal cancer cells using single antibodies and combinations of them for IMS was examined by fluorescence microscopy and multiplex RT-PCR. The clinical utility of a tumor cell detection assay using IMS with multiplex RT-PCR was tested by examination of colorectal cancer blood samples and by comparing the results with CEA serum protein levels. RESULTS: A combination of antibodies for IMS and multiplex RT-PCR analysis proved to be the most sensitive approach for detection of tumor cells in peripheral blood with a detection limit of two tumor cells. The examination of blood of colorectal cancer patients by using a multiplex RT-PCR assay in comparison with CEA serum protein levels indicated a distinct advantage of the former over the latter with respect to a more reliable prediction of an ongoing metastatic process. CONCLUSION: The results indicate that a combination of antibodies for immunomagnetic enrichment with multiplex RT-PCR analysis detects disseminated tumor cells with high sensitivity and specificity, thus indicating a metastatic process several months earlier compared to CEA serum protein level measurements. This assay might be valuable for prognosis in cancer.