Criteria for second generation comparator tests in validation of novel HPV DNA tests for use in cervical cancer screening.

While HC2 and GP5+/6+ PCR-EIA were pivotal in test validation of new HPV assays, they represent the first generation of comparator tests based upon technologies that are not in widespread use anymore. In the current guideline, criteria for second-generation comparator tests are presented that include more detailed resolution of HPV genotypes. Second-generation comparator tests should preferentially target only the 12 genotypes classified as carcinogenic (IARC-group I), and show consistent non-inferior sensitivity for CIN2+ and CIN3+ and specificity for ≤CIN1 compared to one of the first-generations comparators, in at least three validation studies using benchmarks of 0.95 for relative sensitivity and 0.98 for relative specificity. Validation should take into account used storage media and other sample handling procedures. Meta-analyses were conducted to identify the assays that fulfill these stringent criteria. Four tests fulfilled the new criteria: (1) RealTime High-Risk HPV Test (Abbott), (2) Cobas-4800 HPV test (Roche Molecular System), (3) Onclarity HPV Assay (BD Diagnostics), and (4) Anyplex II HPV HR Detection (Seegene), each evaluated in three to six studies. Whereas the four assays target 14 carcinogenic genotypes, the first two identify separately HPV16 and 18, the third assay identifies five types separately and the fourth identifies all the types separately.

Assessment of the possible inhibitory effect of carrageenan in human papillomavirus DNA testing by polymerase chain reaction amplification.

We assessed carrageenan's potential to inhibit human papillomavirus (HPV) DNA extraction and amplification in vaginal swab samples collected in a trial, assessing the efficacy of a carrageenan-based gel against HPV infections. Experiment #1 consisted of adding gel (carrageenan-containing or placebo) to swabs and comparing HPV DNA detection by polymerase chain reaction (PCR) to unmanipulated samples collected from the same participants. For Experiments #2 and #3, we tested vaginal samples for inhibition by addition of an internal control and amplification by real-time PCR. Experiment #4 investigated carrageenan's interference with the extraction process by assessing HPV45 detectability in undiluted and diluted HPV45 positive samples (n = 3) with carrageenan versus no gel. In Experiment #1, there was a loss of HPV positivity with the addition of carrageenan (n = 9), but none with placebo gel (n = 5). In Experiments #2 and #3, the absence of the amplified product was observed in samples from the carrageenan arm: 3.3% (1/30) and 0.5% (1/199) of samples. In Experiment #4, HPV45 was not detected in undiluted carrageenan-containing samples, but after 1/50 dilution, the same HPV45 copy number was detected. Carrageenan does not affect the DNA extraction process, and inhibition of HPV DNA amplification by carrageenan occurs infrequently.

p16 immunostaining in cytology specimens: its application, expression, interpretation, and challenges.

INTRODUCTION: p16 immunostaining is considered as a surrogate marker for human papillomavirus (HPV)-related head and neck squamous cell carcinomas (HNSCC). Herein, the utility of p16 is evaluated in cytology specimens. MATERIAL AND METHODS: The electronic data of a large academic institution was searched for cytology cases accompanied by p16 (2014-2018). Cases were categorized based on body sites. P16 staining was quantified (negative [0%], focal/patchy, or diffusely positive [>70%]). HPV testing was correlated where available. RESULTS: A total of 372 cases were included (male:female, 239:133). The largest differences in application of p16 between men and women were in head/neck cases (209 versus 59) and the abdominal cases (1 versus 33), respectively. p16 diffuse staining is seen in most squamous cell carcinomas, small cell carcinomas, and gynecologic serous carcinomas. p16 expression was patchy or negative in most adenocarcinoma, neuroendocrine carcinoma, spindle cell neoplasms, and benign conditions. HPV testing was done on 217 cases including 138 cases with strong p16 (127 HPV+/11 HPV-), 20 cases with focal/patchy P16 staining (6 HPV+/14 HPV-) and 59 cases with negative p16 staining (3 HPV+/56 HPV-). CONCLUSIONS: Diffuse p16 staining aids in the diagnosis of HPV-related carcinomas, particularly HPV-related HNSCC, across the body and according to sex. In contrast, focal/patchy p16 staining does not correlate with HPV status across various body sites. In conclusion, intensity of p16 matters and should be correlated with cytomorphology, clinical history, and ancillary studies (eg, p40 immunostaining) for an accurate diagnosis and preventing diagnostic pitfalls.

Utility of p16 and HPV testing in oropharyngeal squamous cell carcinoma: An institutional review.

In the US, 60% to 80% of oropharyngeal squamous cell carcinomas (OPSCCs) are associated with human papillomavirus (HPV). However, until recently, no consensus existed about when and how to test for HPV in patients with head and neck cancers. We aimed to evaluate the use of p16 and HPV testing at our institution because p16 immunohistochemistry is reportedly a reliable surrogate marker for HPV detection in OPSCCs. METHODS: We identified all cases at our institution of primary or metastatic squamous cell carcinoma (SCC) of the head and neck with a concurrent p16 immunostain analysis from January 1, 2013, through August 31, 2018. Patient demographic data, tumor characteristics, p16 result, and any HPV result (in situ hybridization and E6 and E7 RNA test) were captured. RESULTS: We identified 104 patients. Most primary tumors (53/57 [93.0%]) and metastases (40/47 [85.1%]) were positive for p16. Thirty-seven cases (35.6%) had reflex high-risk HPV (HR HPV) testing performed. Of the 35 p16-positive cases, 6 had discrepant HR HPV results (p16+ /HPV- ). We identified 47 p16 immunostains that were performed on lymph nodes with primary tumors of unknown origin. Most were cytology cases (34/47 [72.3%]), and most were p16 positive (40/47 [85.1%]). Neither tumor differentiation nor tumor keratinization was predictive of p16 positivity. Tumors with basaloid differentiation were universally p16 positive. CONCLUSION: p16 immunohistochemistry accurately identifies HPV-positive OPSCC. Cytology specimens have an important role in characterizing SCC of unknown origin. HR HPV testing is not routinely required, and results may be discrepant with p16 findings.

Self-obtained vaginal samples for HPV DNA testing to detect HPV-related cervical disease.

OBJECTIVE: To investigate if a self-obtained vaginal sample (SOVAS) contains sufficient DNA for a human papillomavirus (HPV) test and if the results are comparable to those obtained via cervical samples (CS) collected by a physician. METHODS: One hundred and fifty-one women who had abnormal cervical smears or who were HPV-positive were enrolled. Self-sampling was done after reading instructions and watching a 2-min-long video, whereas CS was obtained with a cervical cytobrush during a gynecologic examination. RESULTS: A multiplex real-time polymerase chain reaction-based assay detected the prevalence of any type of HPV to be 67.5% in the SOVAS and 57.4% in the CS, and that of high-risk (HR-) HPV to be 58.7% in the SOVAS and 48.6% in the CS. The sensitivity of detection of HR-HPV in the SOVAS was 100% (95% confidence interval [CI] -0.09 to 0.32) for high-grade squamous intraepithelial lesion, 78% (95% CI -0.09 to 0.13) for atypical squamous cells of undetermined significance or worse, and 95% (95% CI -0.01 to 0.25) for low-grade squamous intraepithelial lesion or worse, which was statistically within the non-inferiority margin compared with that of CS. CONCLUSION: Our study shows that the collection of a SOVAS is feasible and it is comparable to a CS for HPV DNA testing. Future studies are required to investigate the feasibility and cost-effectiveness of a mail-delivered SOVAS for cervical cancer screening.

Application of the Roche Cobas® HPV 4800 in Formalin-Fixed, Paraffin-Embedded Samples for Head and Neck Squamous Cell Carcinomas.

Testing for high risk human papillomavirus (HR-HPV) status is standard of care in squamous cell carcinomas of the oropharynx as well as cervical lymph node squamous cell carcinomas of unknown primary origin. DNA or RNA in-situ hybridization (ISH) and p16 immunohistochemistry, widely used currently for HPV detection are operator-dependent. In addition, DNA ISH has a relatively low sensitivity, and p16 is not entirely specific for HR-HPV infection. In this study, we examined the performance of the cobas® HPV genotyping assay in formalin-fixed, paraffin-embedded (FFPE) samples of head and neck squamous cell carcinoma. FFPE samples from head neck and other anatomic sites tested by ISH and p16 for HR-HPV at ARUP Laboratories were selected for this study. Samples were deparaffinized, stained and micro-dissected for tumor contents followed by tissue lysis, then tested with cobas® for HR-HPV. All the samples were also tested by HPV Linear Array for confirmation. All (N = 18) high risk HPV positive specimens tested by cobas® were confirmed as positive by the Linear Array test. All the specimens tested as negative by cobas® were tested as negative (N = 5) or positive only for low risk HPV (N = 3) by Linear Array, as cobas® only detects HR HPV. Limits of detection for HPV16 and 18 were established at 160-320 and 320-1600 copies, respectively. Our data suggest that cobas® HR-HPV genotyping is a viable option for detection of HR-HPV in formalin-fixed, paraffin-embedded samples from head and neck and other anatomic sites and has been validated for clinical use.

CyclinA1 Promoter Methylation in Self-Sampling Test.

BACKGROUND: Self sampled HPV testing is a cervical cancer screening method . However, cytology in self-sampled specimen cannot be used as a triage test.  Therefore, other methods for triage should be considered. CyclinA1 (CCNA1) promoter methylation has strong association with cervical precancerous and cancerous lesion. The objective of this study was to compare the diagnostic value of CCNA1 and self-sampled specimen for detecting high-grade cervical intraepithelial lesions or worse (CIN2+). MATERIALS AND METHODS: A cross sectional study was conducted. Women with abnormal cytology or positive for high risk HPV (hrHPV) indicated for colposcopic examination were enrolled.  Self-collected sampling for hrHPV DNA (SS-HPV) and CCNA1 were performed. hrHPV DNA testing was done by Cobas 4800 method. CCNA1 promoter methylation was detected by CCNA1 duplex methylation specific PCR. Histopathologic result as CIN2+ obtaining from colposcopic directed biopsy or excisional procedure  was considered as positive a gold standard. The results of hrHPV and CCNA1 were reported as positive or negative. Sensitivity specificity, positive predictive value, and negative predictive value of SS-HPV and CCNA1 were calculated by comparing the results with the gold standard. RESULTS: Two hundreds and eighty women were recruited. High-grade cervical lesions and cervical cancer (CIN2+) were diagnosed in 21.8% (61 cases) of the patients. The most common type of hrHPV was non 16, 18 subtype, followed by HPV16 and 18. CCNA1 was positive in 13 patients out of whom, twelve were CIN2+. Sensitivity of CCNA1 was 19.7 % and its  specificity and accuracy were 99.5% and 82.14%, respectively.  The sensitivity of SS-HPV was 70.5%, and its  specificity and accuracy were 39.2% and 43.3%, respectively. CONCLUSION:   Due to high specificity and positive predictive value of CCNA1, it can be used as alarming sign of having high-grade cervical intraepithelial lesions, especially in patient who has positive hrHPV DNA test based on self-collected sampling.
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HrHPV testing vs liquid-based cytology in cervical cancer screening among women aged 50 and older: a prospective study.

INTRODUCTION: Evidence supports high-risk human papilloma virus (HPV) testing as the primary cervical cancer screening tool. However, benefits and harms should be carefully considered before replacing liquid-based cytology. In women age 50 and older, we evaluated how a commercially available DNA amplification HPV test compares with routine liquid-based cytology. METHODS: This prospective study included 4043 patients who had a cervical sample analyzed from September 2011 to September 2012. Patients were followed between 64 and 76 months (median: 70 months). Samples were analyzed using both liquid-based cytology and the Cobas 4800 HPV DNA test. We calculated the diagnostic efficacy of liquid-based cytology and HPV, with or without the opposite test as triage, using cervical intraepithelial neoplasia (CIN2+/CIN3+) as reference. RESULTS: The patients had a median age of 58 years, (range; 50-90). At baseline, HPV prevalence was 8.0%: a total of 3.7% of patients had atypical squamous cells of undetermined significance or worse (ASCUS+). Positive test results were 1.9% for liquid-based cytology with HPV triage and 3.0% for HPV with liquid-based cytology triage. The cumulative incidence of CIN3+ was 1.0% (40/4043). Sensitivities for CIN3+ were: liquid-based cytology 47.5% (31.5%-63.9%); liquid-based cytology with HPV triage 45.0% (29.3%-61.5%); HPV 90.0% (76.3%-97.2%); and HPV with liquid-based cytology triage 67.5% (50.9%-81.4%). Corresponding specificities were: liquid-based cytology 96.6% (96.0%-97.2%); liquid-based cytology with HPV triage 98.5% (98.0%-98.8%); HPV 92.8% (92.0%-93.6%); and HPV with liquid-based cytology triage 97.7% (97.2%-98.1%). At baseline, HPV testing overlooked five cases of gynecological cancer other than cervical cancer. Five cervical cancers were detected, two had been overlooked at baseline by liquid-based cytology and two by HPV testing CONCLUSION: HPV screening using DNA amplification is a promising alternative to liquid-based cytology in women age 50 and older, but evaluation of alternative triage methods is warranted. The risk of overlooking cancers needs consideration when replacing liquid-based cytology with HPV testing as a method for primary screening.

Human Papillomavirus (HPV) DNA Detection Using Self-Sampling Devices in Women Undergoing Long Term Immunosuppressive Therapy.

Immunosuppression is a risk factor of persistent human papillomavirus (HPV) infections, which might lead to development of (pre)malignant lesions of the cervix and lower anogenital tract. Results of HPV DNA testing using cervicovaginal self-samples are comparable to those that are clinician-obtained and therefore might be used in cervical screening. The aim of this study was to assess the prevalence of high-risk HPV (hrHPV) infections, their risk factors and the genotypes distribution among women undergoing immunosuppressive therapy. Women undergoing immunosuppressive therapy for at least three months due to solid organ transplantation or autoimmune disorders were asked to self-collect samples for HPV testing using cervicovaginal brushes and complete questionnaires regarding cervical cancer risk factors. HPV DNA detection and genotyping were performed using Genotyping kit HPV GP version 2. hrHPV was detected in 26/90 (28.9%) specimens. Genotyping revealed a broad range of hrHPV, with type 16 being the most common genotype (11/26). The components of bivalent/quadrivalent or nonavalent vaccines cover all genotypes present in 4.4% and 17.8% women, respectively, and occur as a co-infection with other types in 12.2% and 23.3% of women, respectively. The only feature significantly associated with being hrHPV-positive was having at least two lifetime sexual partners. The high prevalence of hrHPV infections among immunosuppressed women emphasizes the need for regular cervical cancer screening with HPV DNA testing, which might be performed on self-collected specimen.

Consistency evaluation of Liferiver, Yaneng, Darui, and the Cobas 4800 test for high-risk human papillomavirus screening.

BACKGROUND: In recent years, several high-risk human papillomavirus (HR-HPV) tests have been developed. The assay capabilities need to be systematically reviewed. Here, we compared the clinical sample performance of three novel HR-HPV assays (Liferiver, Yaneng, and Darui) based on different platforms with the widely adopted cobas4800 test. METHODS: A total of 346 cervical swabs from women who were screened for cervical cancer were analyzed for the presence of 14 HR-HPV genotypes. The distinction between the four assays was investigated by the genotyping and direct sequencing. RESULTS: The positive rates of the four assays ranged from 61.56% to 64.16%. The overall concordance was 88.15%. The Yaneng assays displayed the best sensitivity (100%) and specificity (98.43%). The sensitivity (98.17%) and specificity (98.43%) of the Darui assay were superior to those of the cobas4800 test (97.72% and 93.70%, respectively). The Liferiver assay displayed comparable sensitivity with the cobas4800 test (95.89% and 97.72%, respectively). The specificity of the cobas4800 was lower than that of the Liferiver assay (93.70% vs. 97.64%). CONCLUSIONS: The three novel HR-HPV assays displayed good agreement with the cobas4800 test. The analytical performance of all four fulfilled the requirements of sensitivity and specificity for HR-HPV detection.

Performance analysis of high-throughput HPV testing on three automated workflows.

Primary high-risk human papillomavirus (hrHPV) DNA testing has been introduced in several countries worldwide, including The Netherlands. The objective of this study was to compare three automated workflow procedures for hrHPV testing of which the hrHPV detection assays meet the international guidelines for HPV testing. To mimic a realistic screening situation, we aimed to process 15 000 residual PreservCyt cervical samples in a period of 3 months. During a 3 months period, four technicians were involved in processing 5000 specimens per month on three automated platforms, (1) Qiagen Digene® HC2 HPV DNA test (HC2, signal amplification); (2) Roche Cobas® HPV test (DNA amplification), and (3) Hologic Aptima® HPV test (RNA amplification). We measured and scored general aspects (time-to-results, hands-on-time (HOT)), maintenance, pre-run, run and post-run aspects, inventory (orders, storage), and number of errors on a scale from 1 to 10. As determined for one complete workflow each, maximum processing capacity and HOT were 296 samples and 2 h:55 m, 282 samples and 3 h:20 m, and 264 samples and 4 h:15 m for Aptima, Cobas, and HC2, respectively. The mean throughput time per run was 5 h:51 m for Cobas in which 94 samples could be processed. For Aptima, the mean throughput time per run was 6 h:30 m for 60 samples. Mean throughput time for HC2 is longer since results were provided on day 2. In this study, the fully automated Aptima workflow scores best with a 7.2, followed by Cobas with a score of 7.1 and HC2 with a score of 5.8. Although all HPV tests used in this comparison meet the international test guidelines, the performance (workflow) characteristics of the assays vary widely. A specific choice of a laboratory for high-throughput testing can be different based on the laboratory's demands, but also hands-on-time, time-to-results/ # samples, maintenance, pre-run, run and post-run parameters, consumables, technical support, and number of errors are important operational factors for the selection of a fully automated workflow for hrHPV testing.

A comparative study of extracellular vesicle-associated and cell-free DNA and RNA for HPV detection in oropharyngeal squamous cell carcinoma.

PURPOSE: This study compares the detection sensitivity of two separate liquid biopsy sources, cell-free (cf) DNA/RNA and extracellular vesicle (EV)-associated DNA/RNA (EV-DNA/RNA), to identify circulating Human Papilloma Virus (HPV) DNA/RNA in plasma obtained from patients with oropharyngeal squamous cell carcinoma (OPCSCC). We also report on the longitudinal changes observed in HPV-DNA levels in response to treatment. EXPERIMENTAL DESIGN: A prospective study was conducted that included 22 patients with locally advanced disease and six patients with metastatic OPCSCC. Twenty-three patients had HPV-related OPCSCC defined by p16 immunohistochemistry. Levels of circulating HPV-DNA and HPV-RNA from plasma-derived cf-DNA/RNA and EV-DNA/RNA were quantified using digital droplet PCR. RESULTS: Circulating HPV-DNA was detected with higher sensitivity in cf-DNA compared to EV-DNA at 91% vs. 42% (p = <0.001). Similarly, circulating tumoral HPV-RNA was detected at a higher sensitivity in cf-RNA compared to EV-RNA, at 83% vs. 50% (p = 0.0019). In the locally advanced cohort, 100% (n = 16) of HPV-OPCSCC patients demonstrated a reduction in circulating HPV-DNA levels in cf-DNA following curative treatment, with 81% of patients demonstrating complete clearance to undetectable levels. However, in metastatic HPV-OPCSCC patients (n = 4), HPV-DNA levels did not correlate with treatment response. CONCLUSION: Our study demonstrates that although HPV-DNA/RNA can be detected in EV associated DNA/RNA, cf-DNA/RNA is the more sensitive liquid biopsy medium. As circulating HPV-DNA levels were found to only correlate with treatment response in the locally advanced but not metastatic setting in our small cohort of patients, the use of HPV-DNA as a dynamic biomarker to monitor treatment response requires further evaluation.

Human papillomavirus prevalence and distribution in self-collected samples from female university students in Maputo.

OBJECTIVE: To characterize human papillomavirus (HPV) prevalence and distribution among female university students in Maputo, Mozambique, and evaluate the determinants of HPV infection. METHODS: A cross-sectional study among 504 female university students between February and April 2017. Cervicovaginal self-collected samples were analyzed for HPV genotypes by polymerase chain reaction-restriction fragment length polymorphism and AnyplexTM II HPV28 Detection kit (Seegene® ). RESULTS: The prevalence of any HPV genotype was 28.6% (144/504). Single and multiple HPV infections were detected in 76 (15.1%) and 68 (13.5%) participants, respectively. Prevalence of high-risk HPV was significantly higher than that of low-risk HPV (P<0.001). HPV16 was the most frequent genotype, followed by HPV58, HPV66, HPV52, HPV18, HPV56, HPV61, and HPV70. The prevalence of genotypes covered by the bivalent, quadrivalent, and nonavalent vaccine was 14.3%, 15.9%, and 23.4%, respectively. Number of sexual partners over lifetime and in the past 12 months was associated with HPV infection (P<0.001 and P=0.039, respectively). CONCLUSIONS: Knowledge of HPV genotype-specific prevalence among young women is important to set up strategies for HPV vaccination. The findings suggest that introduction of the nonavalent HPV vaccine might be the way forward in the present low-resource setting. In addition, self-sampling was useful for HPV detection and genotyping.

Diagnostic accuracy of self-collected vaginal samples for HPV DNA detection in women from South India.

OBJECTIVES: To assess the efficacy of self-collected vaginal samples compared with physician-collected cervical samples for the detection of HPVDNA. METHODS: A hospital-based cross-sectional study was carried out among patients with newly diagnosed cervical cancer attending the Gynecologic Oncology Division, Department of Obstetrics and Gynecology and Radiation Oncology Department at Government Medical College, Kozhikode, Kerala between March 2017 and April 2019. Consenting patients collected their vaginal samples, followed by cervical sample collection by the clinician. The paired samples were transported at 4-8 °C to the laboratory. Amplification of LCR/E6/E7 regions of the HPV genome was done by polymerase chain reaction (PCR). The agreement level between paired samples was assessed by the Kappa index. RESULTS: Among the 114 cervical cancer patients enrolled in the present cross-sectional study, the prevalence of HPV DNA was 78.1% (95% confidence interval [CI] 69.2%-85%) in cervical samples and 77.2% in vaginal samples (95% CI 68.7%-83.9%). The overall agreement between the two sampling methods was 93.9% and the kappa value was 0.82 (P<0.001). The sensitivity of HPV detection using vaginal samples was 98.9% (95% CI 93.9%-99.8%) and the specificity was 100% (95% CI 86.7%-100%) with cervical sampling as the gold standard. By Kappa index, an almost perfect agreement for HPV DNA detection between self-collected and physician-collected samples was observed. CONCLUSION: Self-collection of vaginal samples ensures equity of cervical cancer screening in low-income countries such as India.

Molecular Diagnostics in Human Papillomavirus-Related Head and Neck Squamous Cell Carcinoma.

The incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma continues to increase. Accurate diagnosis of the HPV status of a tumor is vital, as HPV+ versus HPV- tumors represent two unique biological and clinical entities with different treatment strategies. High-risk HPV subtypes encode oncoproteins E6 and E7 that disrupt cellular senescence and ultimately drive tumorigenesis. Current methods for detection of HPV take advantage of this established oncogenic pathway and detect HPV at various biological stages. This review article provides an overview of the existing technologies employed for the detection of HPV and their current or potential future role in management and prognostication.

Evaluation of the intraoperative human papillomavirus test as a marker of early cure at 12 months after electrosurgical excision procedure in women with cervical high-grade squamous intraepithelial lesion: a prospective cohort study.

OBJECTIVE: To evaluate if the intraoperative human papillomavirus (IOP-HPV) test has the same prognostic value as the HPV test performed at 6 months after treatment of high-grade squamous intraepithelial lesion (HSIL) to predict treatment failure. DESIGN: Prospective cohort study. SETTING: Barcelona, Spain. POPULATION: A cohort of 216 women diagnosed with HSIL and treated with loop electrosurgical excision procedure (LEEP). METHODS: After LEEP, an HPV test was performed using the Hybrid Capture 2 system. If this was positive, genotyping was performed with the CLART HPV2 technique. The IOP-HPV test was compared with HPV test at 6 months and with surgical margins. MAIN OUTCOME MEASURE: Treatment failure. RESULTS: Recurrence rate of HSIL was 6%. There was a strong association between a positive IOP-HPV test, a positive 6-month HPV test, positive HPV 16 genotype, positive surgical margins and HSIL recurrence. Sensitivity, specificity, and positive and negative predictive values of the IOP-HPV test were 85.7, 80.8,24.0 and 98.8% and of the HPV test at 6 months were 76.9, 75.8, 17.2 and 98.0%. CONCLUSION: Intraoperative HPV test accurately predicts treatment failure in women with cervical intraepithelial neoplasia grade 2/3. This new approach may allow early identification of patients with recurrent disease, which will not delay the treatment. Genotyping could be useful in detecting high-risk patients. TWEETABLE ABSTRACT: IOP-HPV test accurately predicts treatment failure in women with CIN 2/3.

Comparison of Molecular Assays for HPV Testing in Oropharyngeal Squamous Cell Carcinomas: A Population-Based Study in Northern Ireland.

BACKGROUND: Determination of human papillomavirus (HPV) status has become clinically relevant for patient stratification under UICC TNM8 staging. Within the United Kingdom, a combination of p16 IHC and HPV DNA-ISH is recommended for classifying HPV status. This study will assess a series of clinically applicable second-line molecular tests to run in combination with p16 IHC to optimally determine HPV status. METHODS: The ability of HPV RNA-ISH, HPV DNA-ISH, and HPV DNA-PCR to identify p16-positive/HPV-positive patients was investigated in a population-based oropharyngeal squamous cell carcinoma (OPSCC) cohort of patients diagnosed in Northern Ireland from 2000 to 2011. RESULTS: Only 41% of the Northern Irish OPSCC patient population was associated with HPV-driven carcinogenesis. Both ISH assays were more specific than the DNA-PCR assay (100% and 95% vs. 67%) and were less likely to be affected by preanalytic factors such as increasing block age. A pooled HPV genotype probe for RNA-ISH was found to be the most accurate molecular assay assessed (95% accuracy) when compared with p16 positivity. CONCLUSIONS: Our study demonstrates the advantage of tissue-based molecular assays when determining HPV status in retrospective samples. Specifically, we demonstrate the enhanced sensitivity and specificity of ISH techniques compared with PCR-based methodology when working with formalin-fixed paraffin-embedded tissue, and found HPV RNA-ISH to be the most effective assay for determining HPV status. IMPACT: As p16 IHC is a relatively inexpensive, accessible, and sensitive test for stratifying patients by HPV status, this study finds that more patients would benefit from first-line p16 IHC followed by specific HPV testing using HPV RNA-ISH to confirm HPV status.

Economic evaluation of HPV DNA test as primary screening method for cervical cancer: A health policy discussion in Greece.

BACKGROUND: HPV test appears to be more effective in cervical cancer (CC) screening. However, the decision of its adoption as a primary screening method by substituting the established cytology lies in the evaluation of multiple criteria. Aim of this study is to evaluate the economic and clinical impact of HPV test as primary screening method for CC. METHODS: A decision tree and a Markov model were developed to simulate the screening algorithm and the natural history of CC. Fourteen different screening strategies were evaluated, for women 25-65 years old. Clinical inputs were drawn from the HERMES study and cost inputs from the official price lists. In the absence of CC treatment cost data, the respective Spanish costs were used after being converted to 2017 Greek values. One-way and probabilistic sensitivity analyses were conducted. RESULTS: All screening strategies, that offer as primary screening method triennial HPV genotyping (simultaneous or reflex) alone or as co-testing with cytology appear to be more effective than all other strategies, with regards to both annual CC mortality, due to missed disease (-10.1), and CC incidence(-7.5) versus annual cytology (current practice). Of those, the strategy with HPV test with simultaneous 16/18 genotyping is the strategy that provides savings of 1.050 million euros annually. However, when the above strategy is offered quinquennially despite the fact that outcomes are decreased it remains more effective than current practice (-7.7 deaths and -1.3 incidence) and more savings per death averted (1.323 million) or incidence reduced (7.837 million) are realized. CONCLUSIONS: HPV 16/18 genotyping as a primary screening method for CC appears to be one of the most effective strategies and dominates current practice in respect to both cost and outcomes. Even when compared with all other strategies, the outcomes that it generates justify the cost that it requires, representing a good value for money alternative.