Application of immunomagnetic cell enrichment in combination with RT‐PCR for the detection of rare circulating head and neck tumor cells in human peripheral blood

Abstract

Detection of rare, circulating tumor cells (CTC's) in human peripheral blood is a potential indicator of prognosis and diagnosis in oncology. Typical methods to detect these CTC's are either by immunocytochemistry (ICCS) or RT-PCR. However without accurate, rapid, and reproducible enrichment processes, these detection techniques are labor intensive and/or unreliable. In this article, a repeatable enrichment process that included a flow-through immunomagnetic cell separation system, the quadrupole magnetic sorter (QMS) was optimized with the aid of a statistical analysis software package. The QMS was operated in a negative mode of operation by immunomagnetically targeting normal human peripheral blood lymphocytes (PBL) through the CD45 surface marker. Three head and neck squamous carcinoma cell lines (HNSCC), Detroit-562, SCC-4, and CAL-27, were used to determine the sensitivity of RT-PCR for the epidermal growth factor receptor (EGFR) in spiked PBL. The detection purity needed for detection was found to be one cell in 10 4 , one cell in 10 3 , and one cell in 10 5 for the Detroit-562, SCC-4, and CAL-27, respectively. The actual number of cancer cells needed for RT-PCR detection ranged from 30 to 1 cell. To mimic the potential concentration of rare CTC present in peripheral blood of cancer patients, the spiking concentration was chosen to be one cancer cell per 10 5 total leukocytes from healthy donors. Using a single step immunomagnetic labeling, the final, optimized enrichment process produced a 57.6 6 30.3-fold (n 5 6) enrichment of the rare cancer cells with a final cancer cell recovery of (77.8 6 6.6)%. q 2007 Clinical Cytometry Society Key terms: immunomagnetic cell separation; circulating tumor cells; reverse transcriptase polymerase chain reaction; head and neck squamous carcinoma cell; Detroit-562; epidermal growth factor receptor; immunocytochemistry staining; quadrupole magnetic sorter Head and Neck cancer comprises *6% of all cancers which results, globally, in *550,000 cases a year. According to the statistics of American Cancer Society, 39,250 new cases of cancer of the oral cavity, oropharynx, pharynx, and larynx are estimated to occur in the US in 2005, and 10,090 deaths were estimated to occur. Greater than 90% of these cases of head and neck cancer are classified as squamous cell carcinomas. For all stages combined, the 5-year survival rate is approximately 58.7%, and this rate has increased slowly in the last 20 years. About 40-50% of the patients will later develop locoregional recurrences, and approximately 80% of these recurrences occur within the first 2 years.Both recent and historical studies have shown that tumor cells are likely to exist in the bone marrow or in the peripheral blood of various types of cancer patients (1-6). It has been presumed that those tumor cells shed from the original tumors and are considered to contribute to the

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