METHODS: The qLAMP assay was optimized targeting the HPV-16 E7 gene. The analytical sensitivity and specificity of the assay were determined using HPV-18 (ATCC® 45152D™), HPV-35 (ATCC® 40330™), HPV-43 (ATCC® 40338™) and HPV-56 (ATCC® 40549™) viral strains and oral bacteria. HPV-16 standard curve was constructed for determination of HPV-16 viral load. The diagnostic performance of the assay was evaluated from 63 OSCC patients comprising 63 tissue, 13 saliva and 49 blood samples, in comparison with p16 immunohistochemistry (IHC), in-house PCR and nested PCR assays.
RESULTS: The detection limit of developed LAMP and PCR assays was 4.68 × 101 and 4.68 × 103 copies/μl, respectively. qLAMP assay enabled detection of positive results as early as 23 min at 67 °C. This assay can detect HPV-16 positivity in 23 % (3/13) saliva and 4.8 % (3/63) tissue samples with the viral load ranging from 4.68 × 101 to 4.68 × 104 copies/μl. HPV-16 positivity was not detected in all the blood samples. The sensitivity and specificity of qLAMP were 100 % in comparison with that of p16 IHC and nested PCR.
CONCLUSION: This study reports for the first time on the use of qLAMP assay for detection of HPV-16 in OSCC in both tissue and saliva as the sample matrix which holds promise in improving the diagnostic application owing to its rapidity, simplicity, high sensitivity and specificity.
METHODOLOGY: A total of 102 FFPE cervical tissues were collected from 2 tertiary hospitals and immunohistochemical reactivity staining of E6 and E7 oncoproteins of HPV16 and HPV18 were evaluated using immunoreactive scoring (IRS) system and analysed statistically.
RESULT: The result showed an increased oncoprotein expression with the progression of cervical lesions. There is a statistically significant association between histology grade and HPV16/18-E6 expression (p = 0.028). However, there are no significant association of histological grade to HPV16-E7 immunoreactivity score (p = 0.264) and HPV18-E7 (p=0.080).
CONCLUSION: The immunohistochemical expression of HPV oncoproteins is a potential alternative diagnostic tool applicable in a low-resource laboratory setting. The advantage of the histochemical evaluation is that this method is simpler to apply and less expensive in comparison to in situ mRNA hybridization. Nevertheless, our study also found that antibodies against HPV that are commercially available suffer quite substantial specificity issues such as background staining and inconsistency between different batches. Hence, the utilization of antibody-based staining warrants stringent quality control.
METHODS: Using HPV serology as a marker of HPV-related cancer, we examined the interaction between smoking and HPV16 in 459 oropharyngeal (and 1445 oral cavity and laryngeal) cancer patients and 3024 control participants from two large European multi-centre studies. Odds ratios and credible intervals [CrI], adjusted for potential confounders, were estimated using Bayesian logistic regression.
RESULTS: Both smoking [odds ratio (OR [CrI]: 6.82 [4.52, 10.29]) and HPV seropositivity (OR [CrI]: 235.69 [99.95, 555.74]) were independently associated with oropharyngeal cancer. The joint association of smoking and HPV seropositivity was consistent with that expected on the additive scale (synergy index [CrI]: 1.32 [0.51, 3.45]), suggesting they act as independent risk factors for oropharyngeal cancer.
CONCLUSIONS: Smoking was consistently associated with increase in oropharyngeal cancer risk in models stratified by HPV16 seropositivity. In addition, we report that the prevalence of oropharyngeal cancer increases with smoking for both HPV16-positive and HPV16-negative persons. The impact of smoking on HPV16-positive oropharyngeal cancer highlights the continued need for smoking cessation programmes for primary prevention of head and neck cancer.