Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction

Abstract

BACKGROUND:The incidence of oropharyngeal squamous cell carcinoma caused by oncogenic HPV (HPV-OPSCC) is rising worldwide. HPV-OPSCC is commonly diagnosed by RT-qPCR of HPV-16 E6 and E7 oncoproteins or by cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1 (p16) immunohistochemistry (IHC). Droplet digital PCR (ddPCR) has been recently reported as ultrasensitive and highly precise method of nucleic acid quantification for biomarker analysis. We aimed to validate this method for the detection of HPV-16 E6 and E7 in HPV-OPSCC. METHODS: Participants were recruited from January 2015-November 2015 at initial presentation to the University of Alberta Head and Neck Oncology Clinic. RNA was extracted, purified and quantified from prospectively collected participant tissues, and ddPCR was performed with fluorescent probes detecting HPV-16 E6 and E7. Results from ddPCR were compared with p16 IHC performed by clinical pathology as standard of care. RESULTS: Head and neck tissues were prospectively obtained from 68 participants including 29 patients with OPSCC, 29 patients with non-OPSCC and 10 patients without carcinoma. 79.2% of patients with OPSCC were p16 positive. The sensitivity and specificity of ddPCR HPV E6/E7 compared with p16 IHC in OPSCC was 91.3 and 100%, respectively. The amount of target RNA used was 1 ng, 20-50 times lower than reported by other for RT-qPCR HPV E6/E7. CONCLUSIONS: The ddPCR of HPV E6/E7 is a novel and highly specific method of detecting HPV-16 in OPSCC.

HPV DNA in saliva from patients with SCC of the head and neck is specific for p16-positive oropharyngeal tumours

“…Finally, several recent studies have shown that a saliva-based assay can be used as a post-treatment surveillance tool because the presence of HPV DNA after treatment is associated with a higher risk of recurrence [22–24]. While the sensitivity of our assay was similar to these studies, it was significantly lower than studies which measured HPV DNA directly in tumoral tissue [25]. As a result of its modest sensitivity but high specificity, a saliva based assay is best suited for confirming the presence of a clinically occult oropharyngeal tumour and monitoring for recurrence of an HPV positive tumour after treatment.…”

Transoral robotic surgery with radial forearm free flap reconstruction: Case control analysis

“…Over the past decade, the incidence of HPV-related OPSCC has been rising worldwide [1–7]. When compared to non-HPV related OPSCC, HPV-OPSCC is molecularly distinct and patients with these tumors have improved survival outcomes regardless of treatment modality [8–20].…”

Quantitative polymerase chain reaction‐based detection of HPV 16 E6 and E7 DNA in oral squamous cell carcinoma

“…Several studies have demonstrated the use of HPV 16 E6/E7 qPCR as an analytical and surveillance tool for cervical, head and neck, and oropharyngeal and cancers . However, there is lack of data to detect the HPV 16 E6/E7 DNA expression in OSCC samples using the qPCR technique.…”