In 2009, According to the National Cancer Institute, 48 010 new cases of oral cavity, pharyngeal, and laryngeal cancer were estimated to occur in the United States; approximately 11 260 deaths were attributed to these cases. It has been established that squamous cell carcinomas make up greater than 95% of these cases.1,2 For all stages combined, the 5-year survival rate is approximately 50%,3 and this rate has not changed significantly in the last 3 decades. Treatment failure in patients with squamous cell carcinoma of the head and neck (SCCHN) can include local recurrence, regional recurrence (cervical lymph nodes), distant metastasis, or development of a second primary cancer. Despite intensive therapeutic treatment of the primary lesion, approximately 50% to 60% of patients will later develop locoregional recurrence, and 20% of these patients will go on to develop distant metastasis. Despite surgical resection with negative histopathological margins, approximately 20% of patients will have local recurrence. There are various theories with respect to the cause of local cancer recurrence including field cancerization4 or that clinically relevant micrometastatic spread is not detectable by histopathological margins alone.5 In oral cavity cancer, between 23% and 27% of patients with SCCHN with a clinically negative neck on physical examination will have micrometastatic disease on elective neck dissection.6,7 The presence of lymphatic metastasis is currently the most significant prognostic factor affecting survival; the presence of this alone can reduce survival by 50%.8–10 These current statistics indicate that there is a significant need for a reliable blood marker to determine prognosis in patients with SCCHN, specifically those who may be at higher risk of locoregional or distant recurrence. The concept of the development of metastasis involves a tumor cell or microembolus that dissociates from the primary tumor, circulates within lymphatic or vascular channels, and ultimately resides in a regional or distant site. The ability to detect such cells, known as circulating tumor cells (CTCs), may have an impact on the prognosis and treatment of patients with cancer by providing (1) definitive evidence for early occult spread of the primary cancer, (2) a risk factor for the development of future metastasis, and (3) a peripheral marker for treatment susceptibility and cancer surveillance. Furthermore, genetic analysis of CTCs may lead to targeted treatment strategies.11 Studies have linked circulating/ disseminated tumor cells to poor prognosis in breast,12 lung,13,14 prostate,15 and colorectal cancers.16 It should be noted, however, that the role of CTCs in patients with SCCHN is yet to be conclusively elucidated, and the mechanism between CTCs and cancer recurrence or metastasis is not yet known. Nevertheless, the presence of CTCs could be an independent marker of more aggressive cancers. It is a diagnostic challenge to identify CTCs from a patient’s blood specimen when 1 mL of blood contains an average of 5 billion red blood cells (RBCs), 7 million white blood cells (WBCs), and 295 million platelets.17 A negative depletion method involves removal of normal, unwanted cells, such as RBCs through lysis, and normal lymphocytes through magnetic depletion of CD45+-labeled cells. In contrast, positive selection of tumor cells can be performed by magnetic selection of epithelial cells using antibodies binding to surface markers such as epithelial cell adhesion molecule (EpCAM). At present, there are 3 general types of CTC detection methods: (1) immunocytochemistry, which implies visual observations, (2) flow cytometry and/or image cytometry, and (3) reverse transcriptase–polymerase chain reaction. Prior to the use of 1 of these 3 detection methods, an enrichment method (using negative depletion or positive selection) can greatly increase both the sensitivity and specificity of the test. A majority of the published studies on the detection of CTCs in the blood of patients with cancer uses a combination of a negative depletion step (removal of RBCs) and a positive selection of the CTCs using an antiepithelial antibody bound to a magnetic particle. The most well known of these methods available is the CellSearch System by Veridex LLC, Raritan, New Jersey.18,19 In addition, the “CTC Chip” technology uses a positive selection method to initially isolate cells expressing EpCAM and then allow staining with secondary antibody markers.20 However, the use of a positive selection technique for the CTCs introduces a potential, significant bias into the final detection analysis: the CTCs must express the surface marker to which the antibodies are specific. This bias has been recently experimentally demonstrated by a study from Sieuwerts et al.21 Specifically, these researchers demonstrated that the effectiveness of the CellSearch System varied significantly with respect to its ability to detect subtypes of human breast cancer cells spiked into healthy human blood. As expected, the CellSearch System was able to recover 85% of the breast cancer cells exhibiting the typical epithelial characteristics. However, as the cell types expressed a different phenotype characteristic of tumor-initiating stem cells (CD24 low and CD44 high), the recovery of the spiked cells drops to only 2%.21 EpCAM may be downregulated during epithelial-to-mesenchymal transition, in which cancer cells may acquire a more invasive, migratory phenotype.20 Therefore, using a positive selection technique may not identify CTCs in some patients. In contrast to the positive selection approaches, which introduce significant bias into the type of CTCs detected, we have been developing an enrichment method that is based only on negative depletion of normal cells. We recently demonstrated that this optimized method is able to obtain an average 5.66 log10 enrichment of CTCs in the blood of patients with head and neck cancer.22 In this initial study, we sought to determine the clinical significance of CTCs with regard to disease-free survival in patients with SCCHN and present our prospective clinical follow-up to this point.