Table 1.

Summary of HPV detection methods for OPSCC

Method	Mechanism	Strength	Weakness	Sensitivity	Specificity
				(%)	(%)
HPV DNA PCR	Use Taq DNA polymerase and specific primers in a standard PCR reaction to amplify the target DNA fragment in the sample	Highly sensitive and specific	Unable to distinguish “clinically significant” HPV infections (i.e., HPV transcriptional active infection in tumor cells with expression of E6 and E7)	94–100	74–92
qPCR	The amount of the nucleic acid present in the sample is quantified using: (1) fluorescent dyes that non-specifically intercalate with double-stranded DNA, and (2) sequence-specific DNA probes consisting of fluorescently labelled reports (Abreu et al., 2012)	Able to detect transcriptionally active HPV; providing an estimation of HPV viral load; highly sensitive and specific; no post-PCR processing	Laborious; instrument is too costly; multiplexing is still limited	>95	100
Droplet digital PCR	Based on water-oil emulsion droplet technology for measuring the absolute copy number of nucleic acid targets without external standards	Highly specific and sensitive; requiring minimal amounts of nucleic acids for detection; providing absolute target quantification without reference to the standard/calibration curve; able to detect rare mutations and quantify gene mutations; technically feasible; lower sensitivity to PCR inhibitors; more precise and reproducible data compared to qPCR; cost-effective	Need further research	100	100
LAMP	Rely on auto-cycling strand displacement DNA synthesis Bst DNA polymerase enzyme with high strand displacement activity and a set of two specially designed inner and outer primers (Notomi et al., 2000)	Highly specific and sensitive; cost-effective; rapid; robust	False-positive results in subsequent reactions	99	>90
p16 IHC	Involve alteration of p16 mRNA expression resulting from p16 gene methylation of the promoter region. Methylation results in cascade events of pRb and E2F followed by p16 upregulation, which is highly associated and is unique for HPV-positive oropharyngeal carcinoma (Lewis et al., 2012)	Highly sensitive; cost-effective; widely available	Lack of specificity (false positive results); time-consuming; result interpretation requires a pathologist for confirmation; different p16ʏINK4aһ cut-offs to detect HPV-transformed OPSCC	91–97	78–88
DNA ISH	Labeled probes hybridize to target HPV DNA sequences in the tumor cell nucleus, and the probes may be either HPV-specific or cocktail-specific to detect different high-risk HPV types simultaneously	Highly specific; cost-effective; allowing a reliable identification of viral physical state	Limited sensitivity (false negative results are particularly likely with low HPV copy numbers); signal interpretation can be subjective due to lack of clean, clear staining signals; time-consuming; requiring a large amount of purified DNA; uncertain performance in detection of less common high-risk HPV subtypes	76–92	78–96
HPV E6/E7 mRNA ISH	Involve reverse transcription of mRNA into cDNA and subsequent amplification of target DNA sequences of interest using PCR	Highly specific and sensitive; allowing detection of transcriptionally active HPV	Require dedicated equipment and workflow; cost-prohibitive for some clinical laboratories; require technical expertise that is not routinely available in pathology laboratories; interpretation is somewhat subjective based on amplification curve analysis	>93	>90

cDNA: complementary DNA; E2F: E2 factor; HPV: human papillomavirus; IHC: immunohistochemistry; ISH: in situ hybridization; LAMP: loop-mediated isothermal amplification; OPSCC: oropharyngeal squamous cell carcinoma; PCR: polymerase chain reaction; qPCR: quantitative PCR; pRb: retinoblastoma protein