(182b) Application of Immunomagnetic Cell Enrichment in Combination with Rt- Pcr for the Detection of Rare Circulating Cancer Cells in Peripheral Blood and Bone Marrow from Patients with Head and Neck Squamous Cell Carcinoma (Hnscc)

Authors: 
Yang, L., The Ohio State University
Tong, X., The Ohio State Unversity
Lang, J. C., The Ohio State University
Zborowski, M., Cleveland Clinic Foundation
Chalmers, J. J., The Ohio State University


Detection of rare, circulating tumor cells, CTC's, or disseminated tumor cells, DTC's, in human peripheral blood and/or bone marrow is of significant importance in cancer clinics. Presence of CTC's is a potential indicator of prognosis and diagnosis in oncology (Braun et al. NEJM 342:525-533; 2000). Studies have shown that tumor cells have their own mechanism to disseminate into body fluids, including peripheral blood and bone marrow of various types of cancer patients (Braun et al. Engl J Med. 25;353(8):793-802; 2005; Berois et al., Int. J. Can. 103: 550-555; 2003; Hardingham et al., Int. J. Can. 89: 8-13;2000; O'Hara et al., Clin. Chem. 50(5): 826-835; 2004). Existence of those cells is believed to contribute to cancer recurrence and metastasis.

Traditional methods to detect CTC's or DTC's include immunocytochemistry and RT-PCR, both of which can be unreliable without an efficient enrichment step before the detection. In our lab, we have developed an efficient and high performance immunomagnetic cell separation method to isolate those rare cancer cells prior to RT-PCR detection. Specifically, rare cancer cells in peripheral blood and bone marrow are enriched by depletion of normal cells using red cell lysis followed by a flow-through magnetic cell separation. The cell product can the be analyized microscopically or with RT-PCR. We have optimized the operation parameters to achieve high cancer cell recovery and depletion of ?normal' leukocytes. We have validated this protocol by doing spiking experiments. On average, we get a 78% cancer cell recovery and a 5 log depletion of normal blood cells. We are able to detect rare cancer cells at a concentration of 1 cancer cell per 105 nucleated blood cells.

Most recently, this methodology is being applied to the peripheral blood of cancer patients, specifically, from patients of Head and Neck Squamous Cell Carcinoma (HNSCC) and Breast Cancer patients. Correlations between the actual presence/quantity of circulating tumor cells, as well as the presence of certain biomarkers in these isolated cells, to the HNSCC patient diagnosis and prognosis are being explored. Molecular characterization of these cells could improve our understanding of the metastatic process, greatly facilitating patient monitoring and management.