Rapid and ultrasensitive detection of circulating human papillomavirus E7 cell-free DNA as a cervical cancer biomarker.

Circulating cell-free DNA (cfDNA) has attracted attention as a non-invasive biomarker for diagnosing and monitoring various cancers. Given that human papillomavirus (HPV) DNA integration and overexpression of E6/E7 oncogenes are pivotal events for carcinogenesis, we sought to determine if HPV E7 cfDNA could serve as a specific biomarker for cervical cancer detection. We applied droplet digital PCR (ddPCR) to quantify HPV16/18 E7 cfDNA from the serum of patients with cervical cancer, cervical intraepithelial neoplasia, and controls. HPV16/18 E7 cfDNA was highly specific for cervical cancer, displaying 30.77% sensitivity, 100% specificity, and an area under the curve of 0.65. Furthermore, we developed a sensitive isothermal detection of HPV16/18 E7 and the PIK3CA WT reference gene based on recombinase polymerase amplification combined with a lateral flow strip (RPA-LF). The assay took less than 30 min and the detection limit was 5-10 copies. RPA-LF exhibited 100% sensitivity and 88.24% specificity towards HPV16/18 E7 cfDNA in clinical samples. The agreement between RPA-LF and ddPCR was 83.33% (κ = 0.67) for HPV16 E7 and 100% (κ = 1.0) for HPV18 E7, indicating a good correlation between both tests. Therefore, we conclude that HPV E7 cfDNA represents a potential tumor marker with excellent specificity and moderate sensitivity for minimally invasive cervical cancer monitoring. Moreover, the RPA-LF assay provides an affordable, rapid, and ultrasensitive tool for detecting HPV cfDNA in resource-limited settings.

E6 and E7 Antibody Levels Are Potential Biomarkers of Recurrence in Patients with Advanced-Stage Human Papillomavirus-Positive Oropharyngeal Squamous Cell Carcinoma.

Purpose: There is a paucity of biomarkers to predict failure in human papillomavirus-positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) following curative therapy. E6/E7 viral oncoproteins are constitutively expressed in HPV+ tumors and highly immunogenic, resulting in readily detected serum antibodies. The purpose of this study is to determine whether serum E6 and E7 antibody levels can potentially serve as a biomarker of recurrence in patients with HPV+OPSCC.Experimental Design: We evaluated E6/E7 antibody levels in patients with previously untreated, advanced stage (III, IVa-b), HPV+OPSCC receiving definitive chemoradiation under a uniform protocol from 2003 to 2010. Baseline and longitudinal serum samples were obtained from our archived repository. E6/E7 serum levels were measured using a glutathione-S-transferase capture ELISA and quantified by approximating the area under the dilution curve, and were analyzed using ANOVA and linear mixed model for longitudinal analysis.Results: We compared 22 HPV+OPSCC patients who developed recurrence with 30 patients who remained disease-free. There were no differences in T classification, N classification, disease subsite, or smoking status between the groups. In a longitudinal analysis, recurrent patients had significantly higher E6 and E7 serum antibody levels than the nonrecurrent patients over the follow-up period (P = 0.02 and P = 0.002, respectively). Patients who recurred had a lower clearance of E7 antibody than patients who remained disease-free (P = 0.0016).Conclusions: Patients with HPV+OPSCC whose disease recurs have a lower clearance of E6 and E7 antibodies than patients who do not have recurrence. The ratio of E7 antibody at disease recurrence compared with baseline is potentially a clinically significant measurement of disease status in HPV+OPSCC. Clin Cancer Res; 23(11); 2723-9. ©2016 AACR.

Application of flat panel OLED display technology for the point-of-care detection of circulating cancer biomarkers.

Point-of-care molecular diagnostics can provide efficient and cost-effective medical care, and they have the potential to fundamentally change our approach to global health. However, most existing approaches are not scalable to include multiple biomarkers. As a solution, we have combined commercial flat panel OLED display technology with protein microarray technology to enable high-density fluorescent, programmable, multiplexed biorecognition in a compact and disposable configuration with clinical-level sensitivity. Our approach leverages advances in commercial display technology to reduce pre-functionalized biosensor substrate costs to pennies per cm(2). Here, we demonstrate quantitative detection of IgG antibodies to multiple viral antigens in patient serum samples with detection limits for human IgG in the 10 pg/mL range. We also demonstrate multiplexed detection of antibodies to the HPV16 proteins E2, E6, and E7, which are circulating biomarkers for cervical as well as head and neck cancers.

Human papillomavirus E7 serology and association with p16 immunohistochemistry in squamous cell carcinoma of the head and neck.

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal cancer is associated with improved survival and treatment response as compared to HPV-negative cancers. P16 overexpression is widely accepted as a surrogate marker for HPV positivity. METHODS: A total of 92 serum samples from 75 head and neck squamous cell carcinoma (HNSCC) patients were examined for HPV16 and 18 E7 antibodies by ELISA. Available tissue was tested for HPV-DNA by PCR, and p16 immunohistochemistry was obtained from a deidentified database. RESULTS: Of 75 HNSCC patients, 25 were HPV E7 seropositive. Seropositivity was strongly associated with cancers of the oropharynx, and correlated with positive p16 immunohistochemistry (IHC) and HPV-DNA. Post-treatment serum was available in a limited subset of patients, revealing a decrease in antibody titers following response to treatment. CONCLUSIONS: HPV E7 seropositivity correlated with positive tumor HPV-DNA and p16 expression, and was strongly associated with cancers of the oropharynx. E7 serology warrants further study as a potential biomarker in HPV-positive HNSCC.

Seropositivity of HPV 16 E6 and E7 and the risk of oral cancer.

OBJECTIVES: The objective of the study was to determine the prevalence of HPV seropositivity among patients with oral squamous cell carcinoma (OSCC) and healthy individuals and to correlate the association between HPV 16 seropositivity and risk of OSCC. MATERIALS AND METHODS: HPV 16 E6 and E7 plasmids were constructed for the production of recombinant protein, which was used as the antigen in ELISA. HPV ELISA was performed on serum samples from 50 healthy individuals and 50 patients with OSCC. RESULTS: Using the HPV ELISA, 30% (OR = 2.25, 95% CI = 0.85-5.93) and 18% (OR = 1.61, 95% CI = 0.53-4.92) of patients with oral cancer were found to be HPV 16 E6 and E7 seropositive, respectively. Significant association was found between HPV 16 seropositivity and increased risk of OSCC in men, but not in male subjects. A similar trend was observed in non-betel quid chewers. CONCLUSIONS: Potential associations between HPV 16 E6/E7 seropositivity and oral cancer were revealed in men and non-betel quid chewer subjects, suggesting a possible etiological role of HPV 16 in subgroup of patients with OSCC in Malaysia.

Biomarkers of HPV in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) is an accepted cause of head and neck squamous cell carcinoma (HNSCC), and patients with HPV-associated HNSCC have a favorable prognosis. Currently, there is no general guidance on the most appropriate biomarkers for clinical assessment of HPV in these malignancies. We compared PCR-based and serologic HPV assays, as well as p16 immunohistochemistry, individually and in combination in a single population-based study to assess their associations with overall survival among patients with HNSCC, and thus their potential value as biomarkers. HPV16 serology was determined for 488 patients; immunohistochemical detection of p16 expression in tumors was conducted in a subset of 233 cases, and PCR-based methods to assess the presence of HPV16 DNA in a subset of 179 cases of tumors. Considering each biomarker individually in the subset of patients studied for all endpoints, seropositivity for the E6 and E7 proteins was significantly associated with enhanced all-cause survival in oropharyngeal disease [HR(E6/E7+) = 0.1, 95% confidence interval (CI) = 0.02-0.3]. Neither the presence of HPV16 DNA nor p16 immunostaining was associated with significant enhanced overall survival in oropharyngeal disease (HR(DNA) = 0.9, 95% CI = 0.3-2.9; HR(p16) = 0.3, 95% CI = 0.1-1.1). However, the combination of HPV-positive DNA and E6 or E7 serology was associated with enhanced overall survival in oropharyngeal disease (HR(DNA+/E6/E7+) = 0.1, 95% CI = 0.02-1.0), whereas E6/E7 seronegative patients with evidence of HPV in tumor DNA did not show any evidence of favorable survival (HR(DNA+/E6-/E7-) = 3.4, 95% CI = 0.6-18.1). Furthermore, patients with p16 staining and E6 or E7 seropositivity had favorable survival from oropharyngeal disease (HR(p16+/E6/E7+) = 0.1, 95% CI = 0.02-0.4), whereas patients who were p16 positive and E6/E7 seronegative had significantly increased hazard of all causes of death (HR(p16+/E6-/E7-) = 3.1, 95% CI = 1.2-7.7). A stronger association of HPV presence with prognosis (assessed by all-cause survival) is observed when "HPV-associated" HNSCC is defined using tumor status (HPV DNA status or P16) and HPV E6/E7 serology in combination rather using tumor HPV status alone.