The Effectiveness of Molecular Assays and Sampling Methods: An Application on HPV Detection in Oropharyngeal and Oral Cavity Cancers

Purpose:

In recent years, the oropharyngeal (OP) squamous cell carcinoma (SCC) demographic has changed drastically.  While OPSCC once afflicted older men who were heavy smokers and drinkers, the new generation of OPSCC patient tends to be a younger affluent male who is a non-smoker and non-drinker, with more sexual partners, greater oral sex exposure, and an HPV positive tumor.  Currently, there are no FDA approved screening tests for the detection of HPV infections specifically targeting the oropharynx.  This study aims to a) identify differences in the sensitivity and specificity across molecular and cytological assays and sampling methods, and b) explore how well molecular and cytological assays predict the presence of HPV infection of the oropharynx.     

Methods:

Using a cross-sectional design, this exploratory pilot study involved 41 individuals with a sore, irritation, lump, and/or thick, red or white patch in their mouth or on their tonsils, who presented to a Midwestern Head & Neck clinic.  Informed consent was valid for collection of: 1) data from the Sexual History and Social Behavior Survey, 2) exfoliated cells from the lesion, 3) saliva samples, and 4) tissue (routine biopsy).  The analytic methods explored included Hologic Cervista^TM HR HPV Test (Third Wave Invader Chemistry-DNA®) and cytology.  The two sampling methods explored included the Non-GYN Thin Prep®³ and Oragene®·DNA Saliva containers.  Analytic and sampling methods and biological samples were tested in various combinations and compared to the gold standard (biopsy tissue): 1) histology for clinical diagnosis, 2) DNA analysis for HR HPV status, and 3) p16 immunohistochemistry (IHC) staining (a biomarker of HPV infection) (Please refer to Table 1).     

Results:

The sensitivities for the Oragene-Saliva-CervistaHR, ThinPrep-Saliva-CervistaHR, ThinPrep-Scraping-CervistaHR, and ThinPrep-Scraping-Cytology were 100%, 50%, 73.68%, and 36.84%, in order, and their respective specificities were 23.81%, 66.67%, 80.95%, and 71.43%.  There were no statistically significant differences between their performance when testing oropharyngeal and oral cavity lesions (p > 0.05).

Conclusion:

The high sensitivity (100%) and low specificity (23.81) of Oragene-Saliva-CervistaHR shows that it has a tendency to produce a large number of false positive results.  Since biopsy diagnosis is both expensive and invasive, a test like the ThinPrep-Scraping-CervistaHR with a more balanced sensitivity (73.68%) and specificity (80.95%) is preferred.  Of the four combinations tested, we recommend further investigation of the ThinPrep-Scraping-CervistaHR test.  If proven to be effective, this mildly invasive, readily available, and cost effective method can promote early detection and improve prognosis for the new generation of OPSCC patients.

TABLE 1.  METHODS
Sexual History & Social     Behavior Questionnaire	Demographic & Descriptives
Saliva, Exfoliated Cells   and Biopsy Tissue	1.  Gold Standard    Biopsy tissue Histology:  Routine Evaluation & Diagnosis DNA Analysis for HR HPV Status p16 IHC Staining for HPV Detection   2.  Combinations (Biologic Sample, Collection Container, and Assay HPV Positive & Negative Saliva and Exfoliated Cells Oragene®-DNA/Saliva Container AND Cytology Oragene®-DNA/Saliva Container AND Hologic Cervista™ HR HPV Test (Third Wave Invader Chemistry-DNA®) Non-GYN ThinPrep® Liquid Based Container/Saliva AND Cytology Non-GYN ThinPrep® Liquid Based Container /Saliva AND Hologic Cervista™ HR HPV Test (Third Wave Invader Chemistry-DNA®)