N. Fersis

Multimarker Gene Analysis of Circulating Tumor Cells in Pancreatic Cancer Patients: A Feasibility Study

Objective: The aim of this study was to develop an immunomagnetic/real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and assess its clinical value for the molecular detection of circulating tumor cells (CTCs) in peripheral blood of pancreatic cancer patients. Methods: The presence of CTCs was evaluated in 34 pancreatic cancer patients before systemic therapy and in 40 healthy controls, through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 [targeting mucin 1 and epithelial cell adhesion molecule (EpCAM), respectively], followed by real-time RT-PCR analysis of the genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. Results: The developed assay showed high specificity, as none of the healthy controls were found to be positive for the multimarker gene panel. CTCs were detected in 47.1% of the pancreatic cancer patients before the beginning of systemic treatment. Shorter median progression-free survival (PFS) was observed for patients who had at least one detectable tumor-associated transcript, compared with patients who were CTC negative. Median PFS time was 66.0 days [95% confidence interval (CI) 44.8–87.2] for patients with baseline CTC positivity and 138.0 days (95% CI 124.1–151.9) for CTC-negative patients (p = 0.01, log-rank test). Conclusion: Our results suggest that in addition to the current prognostic methods, CTC analysis represents a potential complementary tool for prediction of outcome in pancreatic cancer patients.

Prognostic and predictive value of circulating tumor cell analysis in colorectal cancer patients.

ObjectiveThe aim of this study was to assess the prognostic and predictive values of circulating tumor cell (CTC) analysis in colorectal cancer patients.Patients and methodsPresence of CTCs was evaluated in 60 colorectal cancer patients before systemic therapy - from which 33 patients were also evaluable for CTC analysis during the first 3 months of treatment - through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 (targeting mucin 1 and EpCAM, respectively), followed by real-time RT-PCR analysis of the tumor-associated genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5.ResultsPatients were stratified into groups according to CTC detection (CTC negative, when all marker genes were negative; and CTC positive when at least one of the marker genes was positive). Patients with CTC positivity at baseline had a significant shorter median progression-free survival (median PFS 181.0 days; 95% CI 146.9-215.1) compared with patients with no CTCs (median PFS 329.0 days; 95% CI 299.6-358.4; Log-rank P < .0001). Moreover, a statistically significant correlation was also founded between CTC detection during treatment and radiographic findings at the 6 month staging. This correlation applied to CTC results before therapy (odds ratio (OR), 6.22), 1 to 4 weeks after beginning of treatment (OR, 5.50), 5 to 8 weeks after beginning of treatment (OR, 7.94) 9 to 12 weeks after beginning of treatment (OR, 14.00) and overall CTC fluctuation during the course of treatment (OR, 20.57).ConclusionThe present study provides evidence of a strong correlation between CTC detection and radiographic disease progression in patients receiving chemotherapy for colorectal cancer. Our results suggest that in addition to the current prognostic factors, CTC analysis represent a potential complementary tool for prediction of colorectal cancer patients’ outcome. Moreover, the present test allows for molecular characterization of CTCs, which may be of relevance to the creation of personalized therapies.

Analysis of circulating tumor cells using an optimized composite score for real-time individualized evaluation of disease status in patients with breast cancer.

e21051 Background: Despite advances in breast cancer (BC) treatment many patients with no clinical evidence of systemic dissemination will develop recurrent or metastatic disease. This study aims to evaluate whether the detection of circulating tumor cells (CTCs) in BC patients, using a multimarker panel and an improved cut-off, could be a useful tool for the prediction of disease status. METHODS CTCs were isolated with immunomagnetic separation in blood from BC patients with primary or metastatic disease, using the high affinity antibodies BM7 (MUC1) and VU1D9 (EpCAM) and subsequently analyzed by RT-qPCR for the expression of CK19, MG1, MUC1, EpCAM and Survivin. In parallel, tests using matched calibrator probes containing spiked tumor cells, dilution experiments and negative controls were performed to evaluate the level of expression and define cut-off values. RESULTS We generated individualized C(q) cut-offs that defined the positivity of each marker. After applying the cut-off the specificity of each mRNA marker was elevated to 100.0% (95% CI, 88.4% - 100.0%). The optimized composite score enumerates that a sample is analysed according to marker positivity (1 point per positive marker) and expression level of the markers (1 point if the expression is above the range of 2 cells per 10 ml of PB). In our group of BC patients, 41% were CTCs positive (score ≥1) and 59% of the analysed patients were negative (score = 0). Furthermore, we have selected 2 patients and performed an extended follow-up in order to evaluate the changes in CTCs levels through therapy. During a median follow-up of 12.3 months (range 10.0 months - 14.6 months), we have analyzed CTCs fluctuations and compared it with the disease status. CONCLUSIONS The results of the composite score show an increase of the assay accuracy. CTCs analysis may be of great interest and value in the monitoring of therapy regimens.

Circulating tumor cells: Multimarker gene profile for tailoring chemotherapeutical response in adeno carcinomas.

10638 Background: In this experimental work we present a new immunomagnetic technique by the introduction of 2 antibodies (BM7 and VU1D9) for tumor cell selection and the use of a multimarker panel...

Implementation of a Composite Score System for Evaluation of Circulating Tumor Cells in Blood of Breast Cancer Patients.

Background: We present a highly specific immunomagnetic separation technique in combination with an improved multimarker gene panel for molecular identification and characterization of circulating breast tumor cells in blood. Here we describe the creation of a new composite score in order to manage results and avoid false positives. Methods: Blood from breast cancer patients with primary or metastatic disease was drawn into two 10mL EDTA-tubes. The high affinity antibodies BM7 (MUC1) and VU1D9 (EpCAM) were used for immunomagnetic tumor cell enrichment. Separated cells were lysed and used for mRNA isolation and c-DNA synthesis (Qiagen®). A real-time quantitative RT-PCR approach using MESA FAST SYBR Assay (Eurogentec®) and primers selected from the UniversalProbeLibrary system (Roche AG®) for the epithelial markers cytokeratin 19 (CK19), mammaglobin 1 (MG1), epithelial cell adhesion molecule (EpCAM), baculoviral IAP repeat-containing 5 (Sur), immunosuppressive CD276 (CD276), carcinoembryonic antigen-related cell adhesion molecule 5 (CEA), HER-2, aldehyde dehydrogenase 1 family, member A1 (ALDH1), hypoxia inducible factor (HIF-1alpha) and CD44 molecule (CD44) was used for tumor cell identification and characterization. The s-actin transcript was used for internal control and matched calibrator probes containing 2 or 10 tumor cells were used for quantitative expression analysis of tumor associated genes present in blood.Results: Positivity rate was based on a score that consists of 2 different characteristics: Marker detection (ranking according to the number of positive markers, varies from 0 – negative - to 1 – positive - for each marker) in combination with the marker expression level (ranking according to the C(t) values and varies from 0 – less then 2 tumor cells – to 1 – more then 2 tumor cells). This composite score is based on the expression of CK19, MG1, EpCAM, Sur and CD276 and has a ranking from 0 to 10. Negative cases are classified with score 0 and 1, cases ranked with score 2 need retesting and finally scores above 2 indicate clearly positive patients. The additional surrogate markers CEA, HER-2, ALDH, HIF and CD44 are analysed in positive patients in order to obtain further information. In our group of metastatic breast cancer patients, 53% were classified as positive, 42% of the patients were negative and in 5% a retesting is needed. Conclusion: The results of the composite score clearly show an increase of sensitivity and specificity for this assay. The implementation of this test in the routine monitoring of patients should help us to evaluate treatment response and create individualized treatment schedules. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3015.