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American Head & Neck Society
Annual Meeting, April 10-11, 2013
JW Marriott Grande Lakes
Orlando, Florida

During the
Combined Otolaryngology Spring Meeting
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ROLE OF HPV DNA DETECTION IN PLASMA AND SALIVA IN THE EARLY DETECTION AND PREDICTION OF RECURRENCE IN HPV POSITIVE OROPHARYNGEAL CARCINOMA

Presentation: S031
Topic: Basic Science - Molecular Markers
Type: Oral Presentation
Date: Thursday, April 11, 2013
Session: 03:45 PM - 04:45 PM Basic Science
Authors: Sun M Ahn, MD, Jason Y Chan, MBBS, Daria Gaykalova, PhD, Joseph A Califano, MD
Institution(s): Department of Otolaryngology, Head and Neck Surgery, Johns Hopkins Medical Institutions & Milton J Dance Head and Neck Center, Greater Baltimore Medical Center, Baltimore, Maryland

Background:
Human papilloma virus (HPV) 16 is a major causative factor in squamous cell carcinoma (SCC) of the oropharynx. Previous studies have demonstrated that HPV-16 DNA can be detected in the pre-treatment plasma and salivary rinses from these patients. Here we investigated the feasibility of HPV-16 DNA detection in both pre and post-treatment plasma and salivary rinses and its potential role as a marker of recurrent disease.

Methods:
A cohort of 54 patients with oropharyngeal and unknown primary SCC with known HPV-16 tumor status, pre- and post-treatment plasma and salivary samples was assembled. Real time quantitative polymerase chain reaction was utilized and the CaSki (American Type Culture Collection, Manassas, VA) cell line, with 600 copies/ genome HPV was used to develop standard curves for the HPV viral copy number. Standard curves for HPV-16 E6 and E7 were developed using DNA extracted from CaSki cells serially diluted to 5ng, 0.5ng, 0.05ng, 0.005ng and 0.0005ng. A standard curve was also developed for the housekeeping gene β-actin (2 copies/ genome). HPV-16 E6 and E7 DNA copy numbers were determined in the plasma and salivary samples and considered positive if >0.001 copy/genome. Simple sensitivity and specificity analyses were performed.

Results:
Forty (74%) patients had HPV-16 detected in their primary tumors prior to treatment, and of these HPV positive patients, 5 (12.5%) recurred (Figure 1). Of the 40 patients with HPV positive tumor, 30 (75%) patients had HPV-16 DNA detected in pre-treatment samples and one (2.5%) patient had HPV-16 DNA detected in post-treatment surveillance samples. None of the patients with HPV negative tumor had detectable HPV-16 DNA in the pre-treatment samples. The one (100%) patient with HPV positive post-treatment sample ultimately developed recurrence. Of the 14 patients with HPV negative tumors and 29 patients with HPV positive tumors who did not recur, none were HPV-16 positive in post-treatment plasma and saliva. The sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of pre-treatment combined salivary-based and plasma-based HPV-16 DNA status are 75%, 100%, 58% and 100% respectively (Table 1). The sensitivities of saliva or plasma alone were 64.86% and 63.64%, respectively. The presence of HPV-16 DNA in post-treatment plasma and salivary rinse was 100% specific and 25% sensitive in detecting loco-regional or metastatic recurrence (Table 2) .

Conclusion:
Using a combination of pre-treatment plasma and salivary rinses can increase the sensitivity of pre-treatment HPV-16 status as a tool for screening patients with HPV positive oropharyngeal HNSCC. In addition, patients with the presence of HPV-16 DNA in surveillance plasma or salivary rinse may be at a significant risk of developing recurrence. Quantitative analysis of HPV-16 DNA in salivary rinses after primary treatment may allow for early detection of recurrence in patients with HPV positive oropharyngeal HNSCC.Figure 1.  Layout of patient groups by HPV status using combined saliva and plasma pre-treatment and post-treatment samples
Table 1. Sensitivity, specificity, and predictive values of pre-treatment combined saliva and plasma HPV-16 status
Table 2. Sensitivity, specificity, and predictive values of post-treatment combined saliva and plasma HPV-16 status in HPV-16 positive tumor

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