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

Abstract

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.

Diagnostic Accuracy of HPV Detection in Patients with Oropharyngeal Squamous Cell Carcinomas: A Systematic Review and Meta-Analysis

“…Nine studies ( n = 1353, 77.6% HPV+) examined the diagnostic accuracy of HPV detection by liquid biopsy using blood samples. One study collected the blood samples both at time of diagnosis and during treatment and evaluated the accuracy of the test regardless of the time of blood collection [ 35 ], the other eight studies collected blood samples before treatment initiation [ 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ]. Five studies tested HPV in plasma [ 36 , 37 , 38 , 39 , 40 ], two studies reported detection in blood [ 35 , 41 ] and two studies investigated HPV in serum [ 42 , 43 ].…”

“…Studies predominantly evaluated detection of circulating HPV DNA in the blood. However, one study examined the accuracy of detecting HPV16 E6/E7 expression in circulating tumour cells (CtCs) [ 41 ]. The study addressing diagnostic accuracy of HPV expression in CtCs had a significantly lower sensitivity compared to the remaining studies ( Figure 4 ).…”

“…Three studies used p16 IHC combined with HPV DNA as reference [44,46,47], one study only used p16 IHC as reference [34] and one study used mRNA E6 and E7 as reference [45]. In the pooled analysis of sensitivity and specificity, the overall sensitivity was 81.4% (95% CI 62.9-91.9), and the overall specificity was 94.8% (95% CI 91.4-96.9) covering all studies regardless of detection method and reference [35,36,38,[40][41][42][43] (excluding the two studies with non-OPSCC patients [37,39]).…”

“…The study addressing diagnostic accuracy of HPV expression in CtCs had a significantly lower sensitivity compared to the remaining studies (Figure 4). In the pooled analysis of sensitivity and specificity, the overall sensitivity was 81.4% (95% CI 62.9-91.9), and the overall specificity was 94.8% (95% CI 91.4-96.9) covering all studies regardless of detection method and reference [35,36,38,[40][41][42][43] (excluding the two studies with non-OPSCC patients [37,39]).…”

Molecular Diagnostics in Human Papillomavirus-Related Head and Neck Squamous Cell Carcinoma

“…Furthermore, the data for post-treatment follow-up blood samples were mixed as not all patients with HPV+ tumors had detectable levels of serum HPV DNA prior to locoregional recurrence. In contrast, when using quantitative reverse transcription PCR to detect HPV16 E6/E7 expression, the presence of circulating HPV16 E6/E7 mRNA at baseline, prior to treatment, was associated with worse progression-free survival and overall survival [56]. This was similarly true of different individual studies, which found that increased levels of pre-treatment HPV16 ctDNA were correlated with worse prognosis [57], and in one study, worse TNM staging as well [58].…”