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

Date Published: May 9, 2019

Publisher: Public Library of Science

Author(s): Panagiota Economopoulou, George Koutsodontis, Margaritis Avgeris, Areti Strati, Christos Kroupis, Ioannis Pateras, Euthymios Kirodimos, Evangelos Giotakis, Ioannis Kotsantis, Pavlos Maragoudakis, Vassilis Gorgoulis, Andreas Scorilas, Evi Lianidou, Amanda Psyrri, Jeffrey Chalmers.


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.

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.

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.

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.

Partial Text

Human papillomavirus (HPV) is a well-recognized etiologic factor in oropharyngeal squamous cell carcinoma (OPSCC), and it has been demonstrated that seropositivity for antibodies against HPV16 oncogenic proteins E6 /E7 is present in prediagnostic samples of patients with OPSCC many years before diagnosis [1]. HPV16 is the subtype most commonly implicated in the pathogenesis of OPSCC, with a prevalence over 90%, followed by HPV18 (3%) [2]. During the past few years, the global burden of the disease has steadily increased, and it has been predicted to surpass cervical cancer in some developed countries [3]. It is commonly accepted that HPV-associated oropharyngeal tumors (HPV+ OPSCC) comprise a distinct disease entity, showing a distinct clinical behavior and better survival outcomes [1]. However, approximately 10–25% of HPV+ OPSCCs have bad prognosis and eventually recur [4]; this population needs to be identified.

A total of 22 FFPE samples from patients with OPSCC were obtained and assessed for p16/HPV markers. Median follow-up was 31 months. Stage was determined according to American Joint Committee on Cancer (AJCC) 7th edition TNM staging system. Baseline clinicopathological characteristics of patients are shown in Table 1.

In the present report, we sought to evaluate whether HPV16 E6/E7 oncogene expressing CTCs could be detected before and after treatment in a cohort of patients with OPSCC. Importantly, we show for the first time a significant association between HPV16 E6/E7(+) CTCs at baseline and both PFS (p = 0.001) and OS (p = 0.005). Although HPV+ OPSCC has favorable prognosis, 10% to 25% of HPV+ OPSCCs eventually recur. The identification of poor prognosis HPV16+ OPSCC has important treatment implications. The favorable prognosis of HPV+ OPSCC has fueled the development of deintensification strategies aiming to spare these patients the devastating consequences of aggressive treatment. HPV16 E6/E7(+) CTC detection could be used for better selection of deintensification candidates.