HPV Detection in Oropharyngeal Cancer: A Narrative Review of Diagnostic and Emerging Molecular Approaches.

There is growing interest in the use of circulating plasma tumor human papillomavirus (HPV) DNA for diagnosis and surveillance of patients with HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). Recent advances in the assays, combining the identification of circulating HPV tumor DNA and tumor DNA fragment analysis (tumor tissue-modified viral [TTMV]-HPV DNA), have been shown to be highly accurate. However, use of these newer techniques has been limited to small cohort studies and clinical trials. To establish the clinical efficacy of plasma TTMV-HPV DNA testing in the diagnosis and surveillance of HPV-associated OPSCC in a contemporary clinical setting. This retrospective observational cohort study included patients with OPSCC who underwent TTMV-HPV DNA testing between April 2020 and September 2022 during the course of routine clinical care. For the diagnosis cohort, patients with at least 1 TTMV-HPV DNA measurement prior to initiation of primary therapy were included. Patients were included in the surveillance cohort if they had at least 1 TTMV-HPV DNA test performed after completion of definitive or salvage therapy. Per-test performance metrics, including sensitivity, specificity, positive predictive value, and negative predictive value, for TTMV-HPV DNA testing. Of 399 patients included in the analysis, 163 were in the diagnostic cohort (median [IQR] age, 63 [56-68.5] years; 142 [87.1%] male), and 290 were in the surveillance cohort (median [IQR] age, 63 [57-70] years; 237 [81.7%] male). Of the 163 patients in the diagnostic cohort, 152 (93.3%) had HPV-associated OPSCC while 11 (6.7%) had HPV-negative OPSCC. The TTMV-HPV DNA sensitivity in pretreatment diagnosis was 91.5% (95% CI, 85.8%-95.4% [139 of 152 tests]), and the specificity was 100% (95% CI, 71.5%-100% [11 of 11 tests]). In the surveillance cohort, 591 tests conducted in 290 patients were evaluated. A total of 23 patients had molecularly confirmed pathologic recurrences. The TTMV-HPV DNA test demonstrated sensitivity of 88.4% (95% CI, 74.9%-96.1% [38 of 43 tests]) and specificity of 100% (95% CI, 99.3%-100% [548 of 548 tests]) in detecting the recurrences. Positive predictive value was 100% (95% CI, 90.7%-100% [38 of 38 tests]), and negative predictive value was 99.1% (95% CI, 97.9%-99.7% [548 of 553 tests]). The median (range) lead time from positive TTMV-HPV DNA test to pathologic confirmation was 47 (0-507) days. This cohort study demonstrated that when evaluated in a clinical setting, the TTMV-HPV DNA assay demonstrated 100% specificity in both diagnosis and surveillance. However, the sensitivity was 91.5% for the diagnosis cohort and 88.4% for the surveillance cohort, signifying that nearly 1 in 10 negative tests among patients with HPV-associated OPSCC was a false negative. Additional research is required to validate the assay's performance and, if validated, then further research into the implementation of this assay into standard clinical practice guidelines will be required.

Detection of persistent oral human papillomavirus (HPV) DNA may be associated with recurrence of HPV-positive head and neck squamous cell carcinoma (HNSCC). To evaluate the dynamics of oral HPV DNA detection and associations with disease outcomes in patients with HPV-positive and HPV-negative HNSCC. This prospective, 2-institution, tertiary referral center study of 396 patients with newly diagnosed oral cavity or oropharyngeal HNSCC was performed from July 11, 2011, to May 7, 2016. Oral rinse samples were prospectively collected at diagnosis and at completion of primary therapy. Weekly oral rinse samples were collected during radiotherapy. Purified tumor and oral rinse sample DNA were evaluated for 37 HPV types, and viral load was quantified by type-specific real-time polymerase chain reaction. Cancers were stratified by tumor HPV status, and HPV was classified as tumor type if identical to that detected in the tumor or nontumor type. Prevalence of HPV DNA before, during, and after therapy. Associations between tumor-type and nontumor-type oral HPV DNA detection and recurrence-free and overall survival were evaluated. Of the 396 patients (median age, 59 years [range, 19-96 years]; 295 [74.5%] men; and 354 [89.4%] white race/ethnicity), 217 had oropharyngeal cancer; 170, oral cavity cancer; and 9, unknown primary HNSCC. The prevalence of oral HPV detection at diagnosis was higher among patients with HPV-positive compared with HPV-negative HNSCC (24 of 194 [84.2%] vs 170 of 202 [12.4%]; P < .001). Oral HPV-16 DNA had an 81% sensitivity and 100% specificity for HPV-16-positive HNSCC. The prevalence and load of tumor-type HPV decreased significantly during primary therapy with odds ratio for probability of infection with each increasing month after diagnosis (0.41; 95% CI, 0.33-0.52; P < .001), whereas those of nontumor types did not (1.01; 95% CI, 0.97-1.06; P = .62). Current smoking was significantly associated with a reduced clearance of tumor-type HPV DNA (hazard ratio [HR], 0.54; 95% CI, 0.32-0.93). Two-year overall survival was significantly lower among the HPV-positive patients with persistent detection of tumor-type HPV after therapy than among those without detectable tumor-type DNA after therapy (68% vs 95%; adjusted HR, 6.61; 95% CI, 1.86-23.44; P = .003), as was recurrence-free survival (55% vs 88%; adjusted HR, 3.72; 95% CI, 1.71-8.09; P < .001). No associations were observed for nontumor type HPV DNA among patients with HPV-positive or HPV-negative HNSCC. Prevalence and viral load of tumor-type HPV DNA decreased rapidly with therapy, and persistent detection was associated with increased risk of recurrence and death. Analysis of tumor type HPV DNA has considerable promise as a biomarker for treatment response and risk of progression.

Introduction:Oral cancer ranks third among all cancers in the Indian population with approximately 45% of call cancer cases in India being diagnosed as oral cancer, among which 20%–50% of the cases are observed to be associated with human papillomavirus (HPV) infection.Aim:This study aims to detect and evaluate the presence of p16 in oropharyngeal squamous cell carcinoma (OPSCC) patients using immunohistochemistry (IHC).Materials and Methods:This study was based on samples collected from 21 patients with primary OPSCC who were diagnosed and treated during the period of December 2017–March 2018. Inclusion criteria were complete clinicopathologic data, adequate clinical follow-up, and availability of sufficient paraffin-embedded tumor material. HPV immunoreactivity was further investigated by means of IHC using p16 as a marker.Results:IHC results revealed p16 positivity in six OPSCC cases. There was no statistically significant association of the p16 positivity of HPV with the age, gender, or site.Conclusion:Our results suggest that IHC-based detection of p16 provides a suboptimal prognostic information if not combined with detection of HPV DNA. Although p16 expression and HPV DNA infection are correlated with HPV-associated OPSCCs, neither of the tests alone is the optimal method for HPV status detection.

BackgroundOncogenic human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (SCC) is a subtype of head-and-neck cancer with a distinct clinical and prognostic profile. While there are calls to undertake HPV testing...

The role of human papillomaviruses (HPV) in oropharyngeal squamous papillomas remains incompletely defined, in contrast to their established involvement in oropharyngeal squamous cell carcinoma and laryngeal papillomatosis. This study aimed to assess HPV prevalence, genotype distribution, and associated biomarker expression in oropharyngeal papillomas, with particular emphasis on p16 and viral E4 protein immunoexpression. We retrospectively analyzed 51 formalin-fixed paraffin-embedded oropharyngeal squamous papillomas diagnosed between 2015 and 2023. HPV DNA detection and genotyping were performed using the INNO-LiPA assay. Immunohistochemistry for p16 and E4 was correlated with HPV status. For comparison, 20 oropharyngeal squamous cell carcinomas were included as controls, comprising 10 HPV16-positive and 10 HPV-negative cases. HPV DNA was detected in 57% of papillomas, predominantly high-risk genotypes, most frequently HPV16. E4 immunoexpression was observed in 55.5% of evaluable cases and was significantly associated with HPV DNA positivity (p < 0.0001). In contrast diffuse p16 overexpression was uncommon and showed no significant association with HPV detection. The prevailing immunophenotype in HPV-positive papillomas was E4 + /p16-, whereas HPV16-positive carcinomas uniformly exhibited a p16 + /E4- profile. No statistically significant differences in recurrence, dysplasia, or malignant transformation were observed between HPV-positive and HPV-negative papillomas, although adverse events occurred exclusively in high-risk HPV-associated cases. These findings suggest that a substantial subset of oropharyngeal squamous papillomas is associated with productive high-risk HPV infection, biologically distinct from transforming infections seen in carcinoma. While combined E4/p16 immunoprofiling may contributed to lesion characterization and prognostic stratification, its clinical utility warrants validation in larger, prospective cohorts.

Introduction: p16 immunohistochemistry (IHC) is the most commonly used test for diagnosing Human Papillomavirus (HPV) related oropharyngeal cancers. HPV DNA or RNA tests have been used increasingly more recently. However, the significance of the intratumoral HPV expression levels is not established yet. Here, we analyzed the intratumoral HPV expression levels using The Cancer Genome Atlas (TCGA) database. Methods: HPV levels were analyzed for oropharyngeal and cervical cancers using the TCGA database at cBioPortal.1,2 Alphapapilloma virus genus was included in the analysis to capture all high-risk HPV types. The association of HPV levels with genomic and clinical features was assessed using either a Spearman correlation coefficient (continuous variables) or a rank-sum test (categorical variables). Results: In oropharyngeal cancer patients, the genomic analysis revealed that CYLD-mutated HPV-positive tumors had significantly higher HPV levels (p=0.0162). There was no difference in virus levels depending on PIK3CA mutation (p=0.370). Gene expression analysis showed a significant correlation of virus levels with MAP3K7 (TAK1) expression (r=0.37, p&amp;lt;0.01). There was no difference in clinical features or survival depending on HPV levels. There was no significant difference among the common oncogenic mutations depending on HPV levels in cervical cancer patients. However, patients with low virus levels had shorter overall survival (PS) and disease-specific survival (DSS) (p=0.01 and 0.027, respectively). Patients with adenocarcinoma had higher levels of the virus when compared to squamous cell carcinoma. Discussion: Our study showed that intratumoral HPV level was correlated with CYLD mutations and TAK1 expression in oropharyngeal cancer patients. CYLD mutations are common in HPV-related head&amp;neck cancers, occurring in up to 20% of tumors. CYLD is known to negatively regulate the NF-κB pathway by inactivating TAK1 and IKK complexes.3 Clinical outcomes were not different depending on the HPV levels in patients with oropharyngeal cancers in our study; however, there were a small number of patients in this study (n=48). Another HPV-related cancer, cervical cancer, was more commonly represented in the TCGA database (n=297). The patients with low HPV levels had poor prognosis in this larger cohort. HPV-directed therapies are emerging for HPV-related cancers. Understanding the role of the intratumoral HPV levels can help to improve viral targeted therapies.

Prognosis of P16 and HPV Discordant Oropharyngeal Cancers: Natural Language Processing to Extract Data from Free-Text Pathology Reports

Human papillomavirus (HPV) DNA in situ hybridization (ISH) assay and p16 immunohistochemistry (IHC) are used to determine high-risk HPV status in formalin-fixed, paraffin-embedded (FFPE) tissues in oropharyngeal squamous cell carcinoma (SCC). Although high sensitivity and specificity for HPV can be obtained by combined p16 IHC and HPV DNA ISH, the occasional discrepancy between these assays has prompted evaluation of Cervista HPV assays in FFPE tissue from patients with oropharyngeal SCC. To compare the efficacy of Cervista HPV 16/18 and Cervista HPV HR assay to that of HPV DNA ISH assay and p16 IHC in FFPE tissue in head and neck squamous cell carcinoma of oropharyngeal origin. Archived FFPE tissue from 84 patients with SCC of oropharyngeal origin and available HPV DNA ISH and p16 IHC test results were tested with the Cervista HPV 16/18 assay and further verified by polymerase chain reaction (PCR)-based HPV16/18 genotyping tests in cases with discrepancy. Of the 84 specimens, 75% (63 of 84) were positive and 16% (13 of 84) had discrepant or equivocal findings by p16 IHC and HPV DNA ISH testing. Use of Cervista HPV assays, either to clarify discrepant/equivocal findings or as confirmation after initial p16 IHC/HPV DNA ISH tests, identified 81% (68 of 84) of HPV-positive cases without equivocal HPV results. Five of 13 cases with discrepancy or equivocal HPV DNA ISH results tested positive for HPV16 or HPV18 by Cervista HPV 16/18 assay, which was further confirmed by PCR-based HPV 16/18 genotyping. The Cervista HPV assays are a reasonable alternative to HPV DNA ISH in determining HPV status in FFPE tissue specimens from patients with oropharyngeal SCC.

More testing, more questions: Screening tests for oral human papillomavirus infection

Although the global incidence of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is increasing, there is little information on southern Chinese population available. We analyzed 207 patients which constituted 63.5% of all newly diagnosed OPSCC in Hong Kong during a 5-year period from 2005 to 2009. We used E6/7 mRNA as a marker of oncogenic involvement and found 20.8% (43/207) of OPSCC and 29.0% (36/124) of tonsillar SCC was associated with HPV. HPV-16 was identified in all cases except one (HPV-18). Patients with HPV-associated OPSCCs were significantly younger than HPV-negative patients (mean age: 59.8 vs. 63.9 years, P = 0.05). Multivariate analysis showed that HPV-associated OPSCC was more likely to occur in nonsmokers (39.5% vs. 15.1%, OR: 2.89, P = 0.05), nondrinkers (52.5% vs. 25.6%, OR: 2.72, P = 0.04), originate from the palatine tonsils (83.7% vs. 53.7%, OR: 3.88, P = 0.01), present with an early primary tumor (T1/2; 79.1% vs. 47.6%, OR: 3.81, P = 0.004), and exhibit basaloid differentiation (33.3% vs. 7.3%, OR: 19.74, P = 0.006). HPV positivity was an independent predictor for better prognosis for both 5-year overall and 5-year disease-specific survivals (DSS; 63.0% vs. 29.7%, HR: 0.33, P < 0.001, and 87.8% vs. 42.6%, HR: 0.16, P < 0.001, respectively). The estimated age-standardized incidence of OPSCC in Hong Kong during the period 2005-2009 was 0.12/100,000/year. This study has provided the most comprehensive clinical and pathologic information to date about this newly recognized disease in southern Chinese. In view of the global trend, we should anticipate and prepare for an increase in HPV-related OPSCC in southern China.

BackgroundThe incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been rising in Western countries and this has been attributed to human papillomavirus (HPV) infection. p16 expression is a marker for HPV infection and p16 positive OPSCC is now recognized as a separate disease entity. There are only limited data available regarding HPV-related OPSCC in Asian countries and no data from Malaysia.MethodsWe identified 60 Malaysian patients with OPSCC over a 12-year period (2004–2015) from four different hospitals in two major cities, Kuala Lumpur and Penang. The detection of HPV was carried out using p16 immunohistochemistry and high risk HPV DNA in situ hybridisation.ResultsOverall, 15 (25%) tumours were p16 positive by immunohistochemistry, 10 of which were also positive for high risk HPV DNA by in situ hybridisation. By comparison, a matched cohort of UK patients had a p16 positive rate of 49%. However, between 2009 and 2015, where cases were available from all four hospitals, 13 of 37 (35%) cases were p16 positive. In our Malaysian cohort, 53% of patients were of Chinese ethnicity and 80% of the p16 positive cases were found in these patients; no Indian patients had p16 positive disease, despite representing 35% of the total cohort.ConclusionThe proportion of OPSCCs associated with HPV in Malaysia appears to be lower than in European and American cohorts and could possibly be more prevalent amongst Malaysians of Chinese ethnicity. Further, our data suggests that the burden of HPV-related OPSCC could be increasing in Malaysia. Larger cross-sectional studies of Malaysian patients are required to determine the public health implications of these preliminary findings.

OBJECTIVES--To investigate the relationship between clinical findings and the detection of human papillomavirus (HPV) DNA in a range of anatomical sites in patients with and without anogenital warts. SUBJECTS--Men and women with a clinical diagnosis of anogenital warts, or a current partner with anogenital warts. SETTING--A department of genitourinary medicine in central London. METHODS--The anogenital areas of the patients were thoroughly examined using a colposcope before and after application of acetic acid. Different types of specimens were taken from a variety of anatomical sites. Superficial skin sampling was performed by the application of slides covered with "Superglue" (SG) to clinically normal and abnormal areas of anogenital skin. The presence of human cells in the SG samples was confirmed by detection of the beta-globin gene using the polymerase chain reaction (PCR). HPV DNA was extracted from the specimens and amplified by using consensus primers with the PCR. HPV types 6, 11, 16, 18, 31 and 33 were identified by Southern blotting followed by hybridisation. RESULTS--In women, HPV DNA was detected in 83% of wart biopsies, 29% of cervical biopsies, 36% of cervical scrapes, 25% of urethral loop specimens, 37% of vaginal washes and 33% of rectal swab specimens. In men, HPV DNA was detected in 67% of wart biopsies, 37% of urethral loop specimens and 12% of rectal swab specimens. Of the SG samples containing the beta-globin gene, 49% from women and 50% from men contained HPV DNA. HPV DNA was not detected in buccal scrapes and serum samples from women or men. Of all specimens with detectable HPV DNA, there was evidence of a single HPV type in 41%, multiple types in 48% and undetermined types in 11%. Samples taken from different sites of a patient tended to have HPV types in common. Sexual partners, however, did not consistently have HPV types in common. CONCLUSIONS--HPV DNA was distributed widely in the anogenital area, in warts, acetowhite areas and clinically normal skin. The SG technique was well tolerated by patients and produced results consistent with other findings. Sampling from a single site of the genitalia on one occasion may significantly underestimate the infection rate with HPV. Multifocal infection of the anogenital area with HPV should be taken into consideration when interpreting epidemiological studies and management strategies.

At least 14 genotypes of human papillomavirus (HPV) have been identified to have high oncogenic potential. While molecular diagnostic testing for HPV is widely available for liquid cytologic cervical samples, testing is limited for other sample types, including liquid biopsy samples, such as platelet-poor plasma (PPP). With the rising incidence of HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC), laboratory testing is an essential part of patient diagnosis, management, and surveillance. Here, we summarize the development and analytical performance validation of a multiplexed, droplet digital PCR (ddPCR) assay for the detection and quantitation of HPV circulating tumor DNA (ctDNA) in PPP. This assay may provide clinicians with a tool to address minimal residual disease for patients with an HPV-associated cancer.

The De-escalated Adjuvant Radiation Therapy (DART) phase 3 randomized clinical trial (RCT) showed that in patients with human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma, postoperative minimal residual disease (MRD), detected through circulating tumor HPV DNA (ctHPVDNA), was associated with a higher risk of disease progression. When considered along with pathologic factors, postoperative ctHPVDNA assessment may improve patient selection for adjuvant treatment de-escalation; however, more data are needed to demonstrate how it may be used in personalizing treatment intensity. To determine the association of postoperative MRD status with progression-free survival (PFS) after surgery for HPV-associated oropharyngeal squamous cell carcinoma. This was a secondary analysis of the DART RCT, which was conducted from October 2016 to August 2020 in multiple sites in the US. Participants from the de-escalated adjuvant radiation therapy group and the standard of care group with available blood specimen data were included. Reports from 3-month posttreatment surveillance visits were used to assess associations and outcomes. Data analyses were performed from March 2023 to March of 2025. The DART group received 30 to 36 Gy of radiation therapy in 1.5 to 1.8 Gy twice daily, plus docetaxel, 15 mg/m2, on days 1 and 8. The standard of care group received 60 Gy with or without weekly cisplatin, 40 mg/m2. PFS. The analysis included 140 patients (mean [SD] age, 59.1 [8.4] years; 12 [8.6%] females and 128 [91.4%] males; 97 [69.3%] with no smoking history); characteristics were similar to the overall DART RCT population. Of these, 17 patients (12.1%) had postoperative MRD (13 of 96 [13.5%] receiving DART and 4 of 44 [9.1%] receiving standard of care). For all patients, postoperative MRD positivity was strongly associated with worsened PFS at 24 months (MRD positivity, 69.5%; MRD negativity, 95.9%; hazard ratio [HR], 0.19; 95% CI, 0.06-0.59). MRD positivity was associated with PFS when evaluating only those patients in the DART group, where 24-month PFS was 68.4% compared to 92.6% for MRD-negative patients (HR, 0.28; 95% CI, 0.08-0.93). Three months after completion of all treatment, 8 of 117 patients (6.8%) had detectable ctHPVDNA, whereas 109 of 117 (93.2%) did not, and detection was highly associated with PFS (HR, 20.48; 95% CI, 6.91-60.67). This secondary analysis of the DART RCT found that patients with detectable ctHPVDNA after surgery had a higher risk of disease progression. When added to the pathologic factors considered, ctHPVDNA assessment may improve selection of patients for treatment de-escalation. In addition, the 3-month posttreatment time point, early in surveillance, may identify a sizable portion of patients with progression and may guide intervention and surveillance after surgery for HPV-associated oropharyngeal squamous cell carcinoma. ClinicalTrials.gov Identifier: NCT02908477.

Background: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) carries a distinct clinical behavior. The c-Met oncogene is an important driver for tumorigenesis, however, its relationship with HPV oncoproteins remains unclear. The present study aims to uncover the role of HPV in regulation of c-Met in OPSCC. Methods: We first knocked down the HPV oncogene E6 by siRNA to check alteration of c-Met expression through western blot and qRT-PCR in several HPV-positive HNSCC cell lines and the cervical cancer cell line CaSki. Next, we tested if E6 regulated c-Met expression is p53 dependent. Simultaneous knockdown of p53 and E6 was compared to E6 knockdown alone for the impact of p53 on c-Met expression using western blot analysis. In addition, the effects of c-Met inhibition by both siRNA and a c-Met specific inhibitor SU11274 on cell proliferation, migration, and colony formation were tested by sulforhodamine B colorimetric assay (SRB), wound healing assay, and colony formation assay, respectively, in HPV-positive cancer cell lines. Using retrospectively collected tissue specimens from 78 patients with OPSCC (N = 58 for HPV-positive vs. N = 20 for HPV-negative), we stained for c-Met and p53 by immunohistochemistry and correlated their expression with HPV status determined by HPV DNA in situ hybridization as well as other clinical characteristics. Results: E6 knockdown significantly decreased c-Met protein and mRNA expression in HPV-associated HNSCC and CaSki cell lines. E6 regulated c-Met expression was partially blocked by the elimination of p53. c-Met inhibition significantly decreased cell proliferation, migration, and colony formation. In patient samples, multivariate analysis revealed a significant association of c-Met expression with HPV status (positive vs. negative, OR = 4.11, 95%CI: 1.16-14.55, P = 0.028) as well as tumor stage (T1/2 vs. T3/4, OR = 0.27, 95%CI: 0.08-0.93, P = 0.039). In T3/T4 stage patients (N = 21), c-Met expression was significantly higher in HPV-positive patients and inversely related with p53 levels, supporting an axis of E6-p53-c-Met regulation. Conclusion: Our results show that c-Met expression is upregulated by HPV E6, which is partially mediated by p53. The data suggest that targeting c-Met may serve as a novel approach for treating HPV-associated OPSCC. (This study was supported by grants from Small Business Innovation Research (SBIR) Program (HHSN261201200097C), National Institutes of Health (R33 CA161873), and National Cancer Institute (NCI P50 CA 128613, Head and Neck SPORE). Citation Format: Guoqing Qian, Dongsheng Wang, Kelly R. Magliocca, Praveen Duggal, Sreenivas Nannapaneni, Sungjin Kim, Zhengjia Chen, Dong M. Shin, Nabil F. Saba, Zhuo G. Chen. Human papillomavirus 16 oncoprotein E6 upregulates c-Met partially through p53 in squamous cell carcinoma of the head and neck. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 813. doi:10.1158/1538-7445.AM2015-813