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Objectives  Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) is increasing in incidence. Although HPV+ OPSCC has favorable prognosis, 10 to 25% of HPV+ OPSCCs eventually recur. We sought to evaluate the feasibility of detection of HPV16  E6/E7  expression in Circulating Tumor Cells (CTCs) and its utility as a prognostic tool in HPV16-associated OPSCC.    Materials and methods  We developed a highly sensitive RT-qPCR assay for HPV mRNA expression in EpCAM(+) CTCs. In 22 patients with early stage and locally advanced OPSCC we evaluated HPV16  E6/E7  expression in the EpCAM(+) CTC fraction at baseline and at the end of concurrent chemoradiotherapy. HPV status in pre-therapy formalin-fixed paraffin-embedded (FFPE) tumor biopsies was assessed by p16 immunohistochemistry and polymerase chain reaction (PCR) and double positives were subjected to Real-time qPCR assay for detection of HPV16, 18 and 31 types.    Results  Fourteen of 22 OPSCC (63.6%) were HPV DNA+/p16+. Among HPV+/p16+ patients, 10 patients (71.4%) were HPV16 DNA+. HPV16  E6/E7 (+) CTCs were detected in 3 of 10 patients (30%) at baseline and 4 of 9 patients (44.4%) at the end-of-treatment, all of which were p16+/HPV16 DNA+. Survival analysis showed a significantly higher risk for disease relapse (p = 0.001) and death (p = 0.005) in patients with HPV16  E6/E7 (+) baseline CTCs.    Conclusion  Detection of HPV  E6/E7 (+) CTCs might be a useful noninvasive test in liquid biopsy samples for determination of a clinically relevant HPV infection in HPV+ OPSCC. Combined interpretation of HPV  E6/E7 (+) CTCs with UICC staging data may lead to alteration of risk definition of patient subsets, with improved risk discrimination in early-stage disease.

HPV16 E6/E7 expression in circulating tumor cells in oropharyngeal squamous cell cancers: A pilot study