Purpose: A negative selection method for the enumeration and characterization of circulating epithelial/cancer cells (CCC) in Breast Cancer (BC) patients is described. This manual procedure yields reproducible results of high sensitivity and selectivity suitable for research laboratories.
Patients and methods: We conducted a prospective blood sampling study in 105 women with stage 1-4 BC attending clinics at the University of Maryland Greenebaum Cancer Center to define the prevalence of CCC utilizing our sensitive double gradient centrifugation and magnetic cell sorting CCC detection and enumeration method. CCC were isolated and enumerated from 15 to 20 ml of venous blood drawn before the start of systemic therapy and periodically thereafter for up to 24 months. One or more CCC/sample was considered a positive result.
Results: We analyzed 487 samples for the presence of CCC; the median number of samples/patient was 4 (range 1-8). CCC were detected in 56% of patients, 19%-stage 1; 43%-stage 2; 46%-stage 3; 83%-stage 4. The probability of being positive for the presence of CCC is significantly associated with the stage of cancer (P < 0.0001). The frequency of CCC positive patients and samples increased with the advancing stage of disease. Presence of more than 10 CCC/sample was associated with the decreased survival and increased probability of having metastatic disease P = 0.001.
Conclusions: Increasing number of CCC/sample correlates with the adverse outcome and poorer survival (P < 0.0001). Our CCC test based on the negative selection procedure may provide valuable prognostic information.