TY - JOUR AU1 - de Oliveira, Cristina Mendes AU2 - Musselwhite, Laura W. AU3 - de Paula Pantano, Naitielle AU4 - Vazquez, Fabiana Lima AU5 - Smith, Jennifer S. AU6 - Schweizer, Johannes AU7 - Belmares, Michael AU8 - Possati-Resende, Júlio César AU9 - de Andrade Vieira, Marcelo AU1 - Longatto-Filho, Adhemar AU1 - Fregnani, José Humberto Tavares Guerreiro AB - Introduction Cervical cancer is the fourth most common cancer in women worldwide, with an estimated 567,847 new cases and 311,365 deaths occurring annually. Approximately 85% of the global burden is registered in less developed regions, highlighting a major public health problem [1]. In Brazil, cervical cancer is the third most frequent cancer in women, with over 16,000 new cases each year [2]. This high incidence of cervical cancer is especially disturbing when considered that cervical cancer develops over many years through precancerous stages, where treatment can be safely and effectively executed. A key challenge is hence the enablement of screening methods that are most appropriate for use in regions of urgent need. The implementation of organized screening programs based on cervical cytology (Pap test) has led to a significant reduction of cervical cancer incidence and mortality in high-income countries [3–5]. Such reduction, however, has not been achieved in low- and middle-income countries (LMICs). Shortfalls are mainly due to the poor screening program coverage rates, lack of cytology quality control and limited population access to the health care system [6, 7]. Several high-income countries, including the Netherlands [8] and USA [9], are now promoting a paradigm shift in their programs from cervical cytology to high-risk (hr)-HPV testing, which offers increased sensitivity and an improved negative predictive value over cytology [10]. Regardless of the screening method, the success of screening programs depends considerably on a high coverage rate within the target population. It is hence beneficial to not only improve screening test sensitivity and specificity, but to also increase the number of women participating in screening. To improve adherence, particularly of under- and never-screened women, self-collection of vaginal samples has been proposed [11–16]. Some women, however, report pelvic discomfort and/or confusion about how to perform the vaginal self-collection [16]. As an alternative to vaginal self-collection, urine based self-collection has been suggested to be more acceptable by many subjects [17]. Detection of HPV DNA in paired urine and cervical specimens has given inconsistent results with the detection of cervical intraepithelial neoplasia (CIN) 2 or greater (2+), yielding sensitivities ranging from 63 to 95% and specificities of 23 to 89% [18–25]. Recent data derived from use of the Trovagene test has resulted in relatively high sensitivity for high-grade precancerous lesions in higher risk colposcopy referral patients [26, 27]. Urine-based HPV DNA testing remains an attractive alternative to increase screening coverage, mainly among women subgroups that cervical HPV detection is difficult [28]. The presence of HPV E6 oncoprotein is necessary for oncogenic transformation, and its detection in self-collected biological specimens might be an attractive approach in resource-limited settings with a high prevalence of cervical cancer. The detection of the E6 oncoprotein of HPV16/18 from physician-collected cervical samples [29–31] showed promising results in several settings. The objective of this study was to assess the detection of HPV16/18 E6 oncoprotein in urine using the OncoE6 point-of-care test. Materials and methods Study population This study was conducted between January and September of 2017 among 124 non-pregnant women, aged 25–64, who attended the Cancer Prevention or the Gynecologic Oncology Departments of Barretos Cancer Hospital (HCB), Brazil. None of the participants had received HPV vaccination, were hysterectomized or had treated an HPV infection before. Women with invasive cervical cancer were enrolled during the first visit at the outpatient clinic of the Gynecologic Oncology Department, and specimens were collected prior to treatment. Women with histology confirmed CIN2 or 3 were enrolled in the Outpatient Surgical Center, prior to a loop electrosurgical excision (LEEP) procedure, and women with normal cytology were enrolled in the Prevention Department during cervical cancer screening. The local Research Ethics Committee and the Brazilian National Research Ethics Commission (CONEP) approved the study (CAAE 62057316.8.0000.5437). All participants provided written informed consent, and all the personal information was maintained encrypted in a database to ensure participants’ data confidentiality. Specimen collection Prior to a pelvic examination, women provided a random urine sample in a 80 mL polypropylene container, and a self-collected vaginal sample was obtained via the Viba-Brush® (Rovers Medical Devices, Oss, the Netherlands). Women then underwent pelvic examination by a gynecologist, and a physician-collected cervical scraping was obtained via the Cervex-Brush Combi® (Rovers Medical Devices, Oss, the Netherlands). Self-collected vaginal and physician-collected samples were preserved in a methanol-based liquid medium (CellPreserv, Kolplast, Brazil). Within two hours of sample collection, specimens were sent to the Molecular Oncology Research Center of the Barretos Cancer Hospital for processing. Five milliliters of each vaginal self- and physician-collected sample were retrieved from the preservative medium to perform the OncoE6™ Cervical Test and were stored at 4°C. Six milliliters of urine were added to a 50mM solution of EDTA and used to perform HPV DNA testing. The remaining urine was used to perform the OncoE6™ Cervical Test without solution of EDTA. Self-collected vaginal samples, physician-collected samples and urine samples that were designated for HPV DNA testing were stored at 4°C; urine aliquots designed to conduct the OncoE6™ Cervical Test were stored at -20°C. Testing for HPV DNA and E6 oncoprotein were performed on each physician collected sample, vaginal self-collected sample and urine self-collected sample. HPV tests For each participant, HPV DNA testing and OncoE6™ Cervical Test were performed on each of the following specimens: urine sample, self-collected vaginal sample, and physician-collected cervical sample. Detection of HPV DNA was performed using the Cobas® 4800 HPV platform (Roche, Indianapolis, IN, USA) which is a multiplex real-time PCR assay that provides specific genotyping information for HPV16 and 18, while concurrently detecting 12 other high-risk types (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) in a pooled result. Vaginal self-collected and the physician-collected samples were performed according to the manufacturer’s instructions. An aliquot of six milliliters of urine added to a 50mM solution of EDTA was used to perform the HPV DNA testing according to the Cobas® 4800 HPV standard protocol. The detection of the HPV E6 oncoprotein was performed using the OncoE6™ Cervical Test (herein referred as to: “HPV16/18-E6 test”; Arbor Vita Corporation, Fremont, CA, USA), an immunochromatographic assay that detects elevated levels of HPV16/18-E6 oncoproteins using a lateral flow format and high-affinity monoclonal antibodies to capture / detect the E6 oncoprotein from cell lysates generated from cervical specimens. Five milliliters each of the self-collected vaginal and the physician-collected cervical samples were centrifuged. The resulting pellet was suspended in 930μL of Rinse Solution and transferred to a test tube, both provided with the OncoE6™ Cervical Test kit. The E6 test was performed according to the manufacturer’s instructions [32]. Urine specimens were shaken up before aliquots of 7.5mL, 15mL or 30mL were removed and centrifuged. The resulting pellet was also suspended in 930μL of Rinse Solution and then transferred to the test tube. The test was performed according to the manufacturer’s instructions except for the extraction step: upon communication with the manufacturer, volumes for the Lysis Solution and the Conditioning Solution were deceased by 50% with regard to the regular protocol; 416μL and 39μL were used. Statistical analysis Women were divided in two groups: negative (