Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting

G Model

ARTICLE IN PRESS

JCV-3492; No. of Pages 11

Journal of Clinical Virology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv

Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting a ˇ Mario Poljak a,∗ , Anja Oˇstrbenk a , Katja Seme a , Anja Sterbenc , Nina Janˇcar b , b Eda Vrtaˇcnik Bokal a b

Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Department of Obstetrics and Gynecology, University Medical Center, Ljubljana, Slovenia

a r t i c l e

i n f o

Article history: Received 26 October 2015 Received in revised form 9 November 2015 Accepted 14 November 2015 Keywords: Human papillomaviruses Cervical cancer Screening HPV testing Abbott RealTime

a b s t r a c t Background: Testing cervical smears for the presence of high-risk human papillomaviruses (hrHPV) increases the sensitivity for detecting women with underlying high-grade cervical intraepithelial neoplasia (CIN) and provides better and longer protection against invasive cervical cancer compared to cytology testing alone. The Abbott RealTime High Risk HPV test (RealTime) is a hrHPV DNA test with concurrent partial genotyping for HPV16 and HPV18 and aggregate detection of 12 other hrHPV types that have been extensively analytically and clinically evaluated over the last 6 years. Objectives and study design: To provide the first 3-year longitudinal data regarding the clinical performance of RealTime, the risk of CIN2+ according to various negative baseline characteristics, and baseline and future risk for CIN2+ at 3 years for women with baseline infection with various hrHPV types were assessed in a cohort of 3,920 Slovenian women that had hrHPV DNA and/or cytology in 36- to 48-month follow-up results after a baseline screening round in 2009/2010. Results: A total of 36 CIN2+ cases were identified in the second screening round. Of these, 17 CIN2+ cases were identified passively through questionnaires/data registries and 19 cases actively as the result of actions triggered by second-round cytology and/or HPV test results. Accumulation of CIN2+ cases during follow-up occurred predominantly in woman with normal cytology at baseline. Among women >30 years old, significantly better protection against CIN2+ at 3 years was associated with a negative hrHPV DNA result at baseline (risk for CIN2+ 0.04% [95 CI, 0.00–0.22%]) than by normal cytology at baseline (risk for CIN2+ 0.68% [95 CI, 0.40–1.08%]). Women with baseline HPV16 infection had a significantly higher risk of CIN2+ at baseline (21.9% [95 CI, 15.2–30.4%]) and baseline plus future risk at 3 years for CIN2+ (33.3% [95 CI, 24.7–44.0%]) in comparison to women with baseline non-HPV16/18 hrHPV infection (7.0% [95 CI, 4.6–10.2%]) or those that were hrHPV-positive (11.7% [95 CI, 9.1–14.9%]). Conclusions: 3-year longitudinal data reinforce evidence from previous studies that RealTime can be safely used in primary HPV-based cervical cancer screening. Concurrent partial genotyping for HPV16/18 should be strongly considered as a triage method for HPV screen-positive women. © 2015 Elsevier B.V. All rights reserved.

1. Background The development of cervical cancer and its immediate precursors is inevitably associated with persistent infection with high-risk human papillomavirus (hrHPV) types, mainly HPV16 and HPV18 [1,2]. These findings led to a recent paradigm change in cervical cancer screening so that several countries are now in the process of switching from cytology-based to hrHPV-based cervical cancer

∗ Corresponding author. Tel.: +386 1 543 7453; fax: +386 1 543 7401. E-mail address: [email protected] (M. Poljak).

screening [3–10]. Primary hrHPV-based cervical cancer screening is an important scientific and clinical advance because it offers better reassurance of low cancer risk compared to cytology-only cervical cancer screening conducted at the same interval [11]. The effectiveness and safety of HPV-based primary cervical cancer screening has been assessed in large-scale randomized clinical trials performed in several European countries, Canada, and India [12–25] and in screening cohorts with longitudinal follow-up data from Europe and the United States [26–39]. According to their results, hrHPV testing as a stand-alone test or in combination with cytology (co-testing) increases the sensitivity for detecting women with underlying cervical intraepithelial neoplasia (CIN)

http://dx.doi.org/10.1016/j.jcv.2015.11.021 1386-6532/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021

G Model JCV-3492; No. of Pages 11

ARTICLE IN PRESS M. Poljak et al. / Journal of Clinical Virology xxx (2015) xxx–xxx

2

grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+), provides better and longer protection against invasive cervical cancer, and reduces cervical cancer mortality compared to cytology testing alone [12,13,15,18,19,26,32,33,40,41]. A recent inventory of commercial HPV tests currently available on the market identified 193 distinct commercial HPV tests and at least 127 test variants [42]; however, only a handful of them can be recommended at present as reliable tools in HPV-based primary cervical cancer screening using clinician-collected cervical samples [43], based on criteria defined in 2009 by an international expert team [44]. The Abbott RealTime High Risk HPV test (RealTime; Abbott, Wiesbaden, Germany) [45,46] is now considered one of the eight HPV assays that fully matches all cross-sectional criteria for primary cervical cancer screening defined by this expert team [43] due to its non-inferior clinical sensitivity and clinical specificity compared to the two clinically validated HPV tests: Hybrid Capture-2 (hc2; Qiagen, Gaithersburg, MD, USA) [47,48] and GP5+/6+ PCR-EIA [49], as well as its high intra-laboratory reproducibility and inter-laboratory agreement for detecting targeted hrHPV assessed in three independent studies [47–49]. However, for hrHPV DNA assays such as RealTime, for which equivalent cross-sectional accuracy as hc2 or GP5+/6+ PCR-EIA are accepted as sufficient evidence to allow their use in primary cervical cancer screening, the generation of longitudinal data remains useful, provides further reassurance, and corroborates the evidence level [43]. Thus, here we provide for the first time 3-year longitudinal data on the clinical performance of RealTime assessed in a cohort of 3,920 Slovenian women 20–64 years old attending the routine organized national cervical cancer screening program, with more than 70% coverage. The three-year longitudinal data of RealTime showed better protection against CIN2+ and CIN3+ associated with a negative hrHPV DNA baseline result than by normal baseline cytology and additionally strengthened the evidence that RealTime is a test that can be safely used in primary HPV-based cervical cancer screening. 2. Objectives To provide the first 3-year longitudinal data regarding the clinical performance of RealTime, the risk of CIN2+ according to various negative baseline characteristics and current (baseline) and future risk for CIN2+ and CIN3+ at 3 years for women with baseline infection with various hrHPV types were assessed in a cohort of 3,920 Slovenian women that had hrHPV DNA and/or cytology at 36–48 months in follow-up results after baseline testing with cytology, hc2, and RealTime in 2009/2010. 3. Study design 3.1. Study population and protocol A total of 4,432 women 20–64 years old, who attended the routine organized national cervical cancer screening program in Slovenia between December 2009 and August 2010, were enrolled in the baseline screening round in 16 outpatient gynecology services [48]. The baseline study was provisionally closed in August 2010 to allow publication of data [48]. All of the women that participated in the baseline screening round were invited after 36 months to participate in the second screening round, which started in December 2012 and finished in late October 2014. According to the study protocol, we excluded from the second screening round all women with CIN2+ detected in the baseline screening round before provisional closure of the study in August 2010, all pregnant women, and all those with diseases or other clinical conditions in which a cervical smear was impossible to obtain (Fig. 1). The same gynecologists that participated in the baseline study were

responsible for patient recruitment and management in the second screening round. Written standardized informed consent was obtained from all of the women by the participating gynecologists, and patient identities were kept secret from all study participants except the participating gynecologists. The second screening round study was approved by the Medical Ethics Committee of the Republic of Slovenia (consent number: 109/08/12). A similar approach as in the baseline screening round [48] was also used in the second screening round. Briefly, after visual inspection of the cervix, two cervical specimens from as many eligible women (Fig. 1) as possible were collected for routine traditional cervical cytology and HPV DNA testing. A sample for HPV DNA testing was obtained with either a Cervex-Brush (Rovers Medical Devices, Oss, Netherlands) or a Pap Perfect Plastic Spatula and Cytobrash Plus GT Gentle Touch (Medscand sample collection kit; Medscand Medical, Berlin, Germany) and placed into ThinPrep PreservCyt solution (Hologic, Marlborough, MA, USA). In contrast to the baseline screening round, the presence of 14 hrHPV types was determined using RealTime only, which allows concurrent partial genotyping for HPV16 and HPV18 and aggregate detection of 12 HPV types: HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68 [45]. To determine the HPV type(s) present in the samples, all RealTime-positive specimens were tested using the Linear Array HPV Genotyping Test (Linear Array; Roche Molecular Diagnostics, Branchburg, NJ, USA) and if necessary with additional genotyping tests. The detailed algorithm of HPV testing, three-step genotyping strategy, discordant analysis, cytological examination, and colposcopic referral were described previously [48]. Briefly, all cervical smears were examined under routine conditions by certified cytologists normally used by each participating gynecology practice and blinded to HPV results. The estimated cross-sectional sensitivity of traditional cytology for CIN2+ and CIN3+ in Slovenia is 66.2% and 80.6%, respectively [48]. Women were referred for colposcopy at the cytology threshold of “atypical squamous cells, cannot exclude high-grade lesion (ASC-H)” or worse in accordance with the national screening standards [50]. In addition, according to the study protocol, immediate colposcopy for all HPV16- and/or HPV18-positive women regardless of their cytology result was strongly recommended and, for those positive for hrHPV other than HPV16 and HPV18, colposcopy was performed at the physician’s discretion. Colposcopically directed punch biopsies obtained from the suspicious areas were histopathologically assessed by certified pathologists, who were blinded to the HPV status. To document any clinically relevant events (e.g., cytology, colposcopy, histology, treatment, or HPV testing) that occurred between two screening rounds, detailed patient- and physicianbased questionnaires were designed and data were collected from all participating women. In addition, for non-responders, data regarding all recorded cytology smear and HPV DNA testing results were obtained passively from the national centralized cervical cancer screening registry through three search rounds, the last one being performed on October 3rd, 2014. The final disease status after 3-year follow-up was determined for all enrolled women. To be included in the final second screening round analysis, a woman must have had either (i) a consensus CIN2+ result, or (ii) at least one valid follow-up hrHPV DNA and/or cytology result on cervical smear collected 36–48 months after the baseline testing with cytology, hc2, and RealTime, including all required colposcopies for those with ASC-H or more severe cytology and/or those HPV16- and/or HPV18-positive regardless of their cytology result. Women vaccinated against HPV between two screening rounds and those with a high probability of therapeutic procedure or known therapeutic procedures between two screening rounds were excluded from the final analysis (Fig. 1).

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021

G Model JCV-3492; No. of Pages 11

ARTICLE IN PRESS M. Poljak et al. / Journal of Clinical Virology xxx (2015) xxx–xxx

3

Fig. 1. Overview of the enrollment procedure of the second screening round study participants. In 2009/2010, 4,432 Slovenian women attending the routine national cervical cancer screening program were enrolled in the baseline screening round in 16 outpatient gynecology services. Of these, 4,004 were enrolled in the second screening round, which started in December 2012. Final analysis of the 3-year longitudinal clinical performance of the Abbott RealTime High Risk HPV test (RealTime) was performed on 3,920 women.

3.2. Data analysis As the outcome measure, histologically confirmed CIN2+ or the end of the follow-up, whichever was first, was used for statistical analysis. CIN2+ cases detected 0–12 months after baseline testing and identified due to colposcopy referral on the basis of baseline cytology and/or HPV results were allocated to the baseline screening round, whereas CIN2+ cases identified at a later time (up to 48 months after baseline testing) and not triggered by baseline cytology and/or HPV result were allocated to the second screening round. Because additional CIN2+ cases were identified and allocated to the baseline screening round, the corrected baseline cross-sectional clinical sensitivity and specificity, negative predictive values, positive predictive values, and corresponding 95% confidence intervals (95% CI) of both RealTime and hc2 at relative light unit per cutoff (RLU/CO) ratio values of 1.00 and 2.50 were calculated.

The risk for CIN2+ at 3 years for women with various negative baseline results with corresponding 95% CIs were computed by dividing the number of CIN2+ cases identified in the second screening round by the number of women at risk (i.e., women included in the final second screening round analysis) (Fig. 1) for different baseline characteristics, separately for women >30 years old and for the total study population. Analyses were performed separately for women with normal cytology at baseline, women with negative hrHPV DNA at baseline using RealTime and hc2, and women with negative hrHPV DNA and normal cytology at baseline. To predict the risk of baseline infection with particular hrHPV types for the development of CIN2+ and CIN3+ at 3 years, women were stratified according to the baseline HPV result and two separate analyses were performed. The risk of CIN2+ and CIN3+ at baseline (risk for cervical disease at the time of baseline cytology and HPV testing) with corresponding 95% CIs was assessed in the total study population by dividing the number of CIN2+/CIN3+

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021

G Model JCV-3492; No. of Pages 11

ARTICLE IN PRESS M. Poljak et al. / Journal of Clinical Virology xxx (2015) xxx–xxx

4

Fig. 2. Distribution of 104 cervical intraepithelial neoplasia grade 2 or worse (CIN2+) cases identified in both screening rounds. In the baseline screening round, we identified a total of 57 CIN2+ cases before provisional closure of the study in August 2010. An additional 11 CIN2+ cases were identified in this study due to colposcopy referral on the basis of baseline cytology and/or HPV results and allocated to the baseline screening round. In the second screening round, we identified a total of 36 CIN2+ cases; of these, 17 CIN2+ cases were identified passively through questionnaires/data registries and 19 CIN2+ cases actively as the result of actions triggered by second-round cytology and/or HPV test results.

cases identified in the baseline screening round by the number of women enrolled in the baseline screening round. The baseline plus future risk for CIN2+ and CIN3+ at 3 years (risk for cervical disease in the future with regard to the time of hrHPV and cytology testing) with corresponding 95% CIs in the total study population was computed by dividing the CIN2+/CIN3+ cases identified in both screening rounds by the number of women at risk (i.e., women included in the final second screening round analysis). Because not all women had a colposcopy at baseline, the individual estimated risk at 3 years may reflect either incident or progressive disease, or disease that was present but not detected at baseline. All data analyses were performed using R software version 3.1.3 (Free Software Foundation, Boston, MA, USA). 4. Results 4.1. Main characteristics of the study population As shown in Fig. 1, 4,432 women were enrolled in the baseline screening round and a total of 57 CIN2+ cases were identified in the total study population before provisional closure of the study in August 2010, thus leaving 4,375 women eligible for the second screening round. Of these, 371 women were excluded due to various reasons detailed in Fig. 1 and, consequently, 4,004 women were enrolled in the second screening round performed between December 2012 and October 2014. Three-year follow-up cervical samples for both cytology and hrHPV testing were actively collected for 3,195 women. In addition, 809 women were passively enrolled

based on availability of at least one cytology follow-up result on a cervical smear collected 36–48 months after baseline testing in the national centralized cervical cancer screening registry. Overall, 84 women were excluded due to various reasons listed in Fig. 1. The final second screening round data analysis was performed on a total of 3,920 women that completed the 3-year follow-up with a known disease status (88.5% of those participating in baseline screening round). The mean follow-up time of 3,920 women included in the final second screening round data analysis was 38.2 months. RealTime showed the presence of hrHPV in 276 out of 3,117 (8.9%) samples tested in the second screening round with a valid RealTime result. Using the applied three-step genotyping strategy [48], we were able to identify the presence of at least one RealTimetargeted HPV type in 274/276 (99.3%) of RealTime-positive samples. Of two RealTime-false positive samples, one sample tested positive for HPV43 only and one sample tested negative for all HPV types covered by the three genotyping methods. At baseline, the overall prevalence of hrHPV infection among the participating women assessed by RealTime was 11.6% (515/4,431; 95% CI, 10.7–12.6%) [48] and in the second screening round among those included in the final second screening round data analysis 8.8% (274/3,117; 95% CI, 7.8–9.9%). Although, the overall prevalence of hrHPV infection among the women included in the final second screening round analysis was significantly lower than among the baseline screening round participants, the difference in prevalence rates of hrHPV DNA positivity in all 5-year age subgroups was not significant (data not shown). The greatest difference in overall hrHPV prevalence between baseline vs. second-round par-

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021

G Model JCV-3492; No. of Pages 11

ARTICLE IN PRESS M. Poljak et al. / Journal of Clinical Virology xxx (2015) xxx–xxx

5

Fig. 3. Time trends of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) identified in women >30 years old during the baseline screening round (month 0) and during the 36- to 48-month follow-up according to various negative baseline characteristics: (i) normal cytology at baseline (marked with diamonds), (ii) negative hrHPV DNA result using the Abbott RealTime High Risk HPV test (RealTime) at baseline (marked with triangles), and (iii) negative hrHPV DNA result using the Qiagen Hybrid Capture 2 HPV DNA test (hc2) at baseline (marked with squares).

ticipants was observed in the age groups 20–24 years old (25.8% [95% CI, 22.3–29.5%] vs. 21.0% [95% CI, 15.2–28.2%]), and 25–29 years old (20.0% [95% CI, 17.2–23.1%] vs. 18.8% [95% CI, 15.1–23.1%]), although the difference in the prevalence did not show a statistical significance in either age group. 4.2. Final results of the baseline screening round As shown in Fig. 2, in addition to 57 CIN2+ cases identified in the total study population in the baseline screening round before provisional closure of the study in August 2010, 11 CIN2+ cases were additionally identified and allocated to the baseline screening round. Corrected clinical sensitivity, clinical specificity, negative predictive values, positive predictive values, and corresponding 95% CIs of RealTime and hc2 at RLU/CO cutoff values of 1.00 and 2.50 of figures published previously [48] are shown in Table 1. An additional 11 CIN2+ cases allocated to the baseline screening increased the number of CIN2+ cases to 68 cases (44 cases in women >30 years old) (Fig. 2), but newly identified CIN2+ cases in this study did not significantly change any originally published values; only the corresponding 95% CI changed slightly for some values (Table 1). 4.3. The risk of CIN2+ at 3 years according to various negative baseline characteristics As shown in Fig. 2, a total of 36 CIN2+ cases were identified in the second screening round. Of these, 17 CIN2+ cases were identified passively through questionnaires/data registries and 19 CIN2+ cases were identified actively as the result of actions triggered by second-round cytology and/or HPV test results. Time trends of CIN2+ identified during the baseline screening round (month 0) and during the 36- to 48-month follow-up according to various negative

baseline characteristics for women >30 years old and for the total study population are shown in Figs. 3 and 4, respectively. In both study populations, accumulation of CIN2+ cases during follow-up occurred predominantly in woman with normal cytology at baseline (Figs. 3 and 4). As shown in Table 2, in both study populations the risk for CIN2+ at 3 years was significantly lower for women tested hrHPV DNA-negative at baseline using either RealTime or hc2 in comparison to those with normal cytology at baseline. The risk for CIN2+ at 3 years in women tested negative for hrHPV DNA and with normal cytology at baseline (double-negative women) was not significantly different from the risk for CIN2+ at 3 years among those tested hrHPV DNA-negative at baseline using either RealTime or hc2 (Table 2). 4.4. The risk for CIN2+ and CIN3+ of baseline infection with different hrHPV results The risk at baseline (risk for cervical disease at the time of baseline cytology and HPV testing) and baseline plus future risk at 3 years (risk for cervical disease in the future with regard to the time of hrHPV and cytology testing) for CIN2+ and CIN3+ for women with baseline infection with various hrHPV types are presented in Table 3. As shown in Table 3, women with baseline HPV16 and HPV16/18 infection had a significantly higher risk at baseline and baseline plus future risk at 3 years for CIN2+ in comparison to women with baseline non-HPV16/18 hrHPV infection or those that were hrHPV-positive. The difference between baseline risks connected with baseline HPV16 and HPV16/18 infection vs. baseline non-HPV16/18 hrHPV infection was also statistically significant for CIN3+ (Table 3). The same also applied for baseline plus future risk at 3 years for CIN3+ for women with baseline HPV16 infection (Table 3). These results indicate that information about baseline

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021

G Model JCV-3492; No. of Pages 11

ARTICLE IN PRESS M. Poljak et al. / Journal of Clinical Virology xxx (2015) xxx–xxx

6

Table 1 Final results of baseline screening round: cross-sectional clinical sensitivity for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+), clinical specificity for detecting lesions less than CIN2, positive predictive value, and negative predictive value of the Abbott RealTime High Risk HPV test (RealTime) and Qiagen Hybrid Capture 2 HPV DNA test (hc2) at relative light unit per cutoff (RLU/CO) ratio values of 1.00 and 2.50 in women >30 years old and in the total study population with corresponding 95% confidence intervals (95 CI). The final data presented are an update of results originally presented in Table 2 in the reference Poljak et al., 2011 [48]. Study group and HPV test Women >30 years RealTime

hc2 RLU/CO ratio of 1.00

RLU/CO ratio of 2.50

Total study population RealTime

hc2 RLU/CO ratio of 1.00

RLU/CO ratio of 2.50

Clinical sensitivity (%)

Clinical specificity (%)

Positive predictive value (%)

Negative predictive value (%)

100.0 (44/44; 95 CI, 90.0–100.0)

93.5 (2,885/3,085; 95 CI, 92.6–94.3)

18.0 (44/244; 95 CI, 13.5–23.6)

100.0 (2,885/2,885; 95 CI, 99.8–100.0)

95.5 (42/44; 95 CI, 83.3–99.2) 90.9 (40/44; 95 CI, 77.4–97.0)

91.9 (2,836/3,085; 95 CI, 90.9–92.9) 93.1 (2,871/3,085; 95 CI, 92.1–93.9)

14.4 (42/291; 95 CI, 10.7–19.1) 15.4 (39/254; 95 CI, 11.3–20.5)

99.9 (2,836/2,838; 95 CI, 99.7–100.0) 99.9 (2,871/2,875; 95 CI, 99.6–100.0)

98.5 (67/68; 95 CI, 91.0–99.9)

89.7 (3,915/4,364; 95 CI, 88.8–90.6)

13.0 (67/516; 95 CI, 10.3–16.3)

100.0 (3,915/3,916; 95 CI, 99.9–100.0)

94.1 (64/68; 95 CI, 84.9–98.1) 89.7 (61/68; 95 CI, 79.3–95.4)

87.9 (3,838/4,364; 95 CI, 86.9–88.9) 89.3 (3,899/4,364; 95 CI, 88.4–90.2)

10.8 (64/590; 95 CI, 8.5–13.7) 11.6 (61/526; 95 CI, 9.1–14.7)

99.9 (3,838/3,842; 95 CI, 99.7–100.0) 99.8 (3,899/3,906; 95 CI, 99.6–99.9)

HPV16 and/or HPV18 status is indeed highly predictive for the presence of underlying disease and/or short-term high risk for the development of both CIN2+ and CIN3+ because, for example, one in five women positive for HPV16 at baseline will have underlying CIN2+ and one in ten underlying CIN3+ (Table 3). In addition, one in three and one in six women positive for HPV16 at baseline will be diagnosed with CIN2+ and CIN3+, respectively, at baseline and/or within 36–48 months after baseline testing (Table 3). 5. Discussion RealTime is a hrHPV DNA test with concurrent partial genotyping for HPV16 and HPV18 and aggregate detection of 12 other hrHPV types. It was launched in 2009 and has been extensively analytically and clinically evaluated over the last 6 years [45,47–49,51–77]. Probit analysis showed that the analytical sensitivity of RealTime is between 500 and 5,000 copies of HPV DNA per assay, depending on the HPV type [58]. The high analytical specificity of RealTime and virtually no cross-reactivity with non-targeted HPV types in clinical specimens (including all relevant low-risk HPV types) has been confirmed in all studies performed to date, which have focused on its analytical performance [47,48,51–56,78,79]. Regarding clinical performance, RealTime has been successfully evaluated for all three major agreed-upon indications for HPV testing in current clinical practice: (i) management and triage of women with borderline cytological findings; (ii) primary cervical cancer screening; and (iii) post-treatment follow-up or test of a cure. Ten validation studies of RealTime in referral settings have shown its consistently high absolute clinical sensitivity for both CIN2+ (range 88.3–100%) and CIN3+ (range 93.0–100%), as well as comparative clinical sensitivity relative to hc2 [52–54,57,59–62,64,65]. Due to the significantly different composition of the referral populations, RealTime absolute clinical specificity for CIN2+ and CIN3+ varied greatly across studies, but was comparable relative to hc2 [46]. Four validation studies of RealTime performance in primary cervical cancer screening settings, including a Slovenian baseline screening round study, have shown its consistently high absolute clinical sensitivity for both CIN2+ and CIN3+, as well as comparative clinical sensitivity and

specificity relative to hc2 [47,48,66] and GP5+/6+ PCR-EIA [49]. A single study that evaluated RealTime in post-treatment follow-up or test of a cure context showed 100% sensitivity and 78% specificity for detecting CIN2+ at 6 months post treatment [78]. RealTime also showed high clinical sensitivity for cervical cancer and CIN3 in two well-characterized series of clinical samples [45,63]. RealTime was also successfully evaluated and shown to be useful for detecting hrHPV in alternative clinical samples such as self-collected cervicovaginal lavage samples [72], vaginal self-sampling of dry cell material [68], and formalin-fixed, paraffin-embedded tissue of cervical cancer, CIN2+ lesions, and head and neck cancers [69–71]. Recent studies showed that glacial acetic acid pre-treatment of cervical liquid-based cytology specimens that are heavily contaminated with blood had little effect on the performance of RealTime [77] and that automation of pre-analytic processing of liquid-based cytology specimens improves laboratory efficiency of the standard RealTime procedure [79]. Finally, RealTime was comparatively evaluated head-to-head with various clinically validated and nonvalidated HPV tests in seven studies on clinically relatively poorly defined populations [55,56,67,73–76]. This study presents the results of a 3-year longitudinal evaluation of clinical performance of RealTime. The original cohort for evaluating the clinical performance of RealTime was established between December 2009 and August 2010, when cervical specimens from a total of 4,432 women 20–64 years old that attended the routine organized national cervical screening program in Slovenia were tested in the baseline screening round with cytology, hc2, and RealTime [48]. Because the cohort fairly represented women participating in the national cervical cancer screening program, in addition to evaluation of the clinical performance of RealTime, we were also able to generate data of high public relevance from the baseline screening round. Thus, the overall and age-specific pre-vaccination prevalence of cervical infections with any of the 12 hrHPV types and 25 non-hrHPV types were assessed [80,81], providing the first baseline data for monitoring of the impact of the HPV vaccination program in central Europe. In addition to cervical specimens, a total of 3,321 participants in the baseline screening round also contributed a blood specimen for estimating overall and age-specific anti-HPV seropositivity for 11 selected

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021

G Model JCV-3492; No. of Pages 11

ARTICLE IN PRESS M. Poljak et al. / Journal of Clinical Virology xxx (2015) xxx–xxx

7

Fig. 4. Time trends of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) identified in the total study population during baseline screening round (month 0) and during the 36- to 48-month follow-up according to various negative baseline characteristics: (i) normal cytology at baseline (marked with diamonds), (ii) negative hrHPV DNA result using the Abbott RealTime High Risk HPV test (RealTime) at baseline (marked with triangles), and (iii) negative hrHPV DNA result using the Qiagen Hybrid Capture 2 HPV DNA test (hc2) at baseline (marked with squares).

Table 2 Risk for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) at 3 years with corresponding 95% confidence intervals (95% CI) according to various negative baseline characteristics calculated separately for women >30 years old and for the total study population. Only women that had valid follow-up results of hrHPV DNA testing and/or cytology performed on samples collected between 36 and 48 months after baseline testing with cytology, the Abbott RealTime High Risk HPV test (RealTime), and the Qiagen Hybrid Capture 2 HPV DNA test (hc2) in 2009/2010, as well as all requested post-testing procedures according to the protocol (e.g., colposcopy), were considered for this analysis. Only CIN2+ cases allocated to the second screening round were counted for this analysis. Women >30 years old

Normal cytology at baseline Negative RealTime at baseline Negative hc2 at baseline Negative hrHPV DNA and normal cytology at baseline

Total study population

n

% (95% CI)

n

% (95% CI)

18/2,641 1/2,546 2/2,517 1/2,453

0.68% (0.40–1.08%) 0.04% (0.00–0.22%) 0.08% (0.01–0.29%) 0.04% (0.00–0.23%)

31/3,753 3/3,464 4/3,413 2/3,344

0.83% (0.56–1.17%) 0.09% (0.02–0.25%) 0.12% (0.03–0.30%) 0.06% (0.01–0.22%)

hrHPV types and 4 selected non-hrHPV types, generating the first reliable data about the cumulative lifetime of HPV infection prevalence in central/eastern Europe [82,83]. With the second screening round described here and further follow-up planned, our cohort will provide important data for future studies of the natural course of HPV infection among Slovenian women and women in the region with the highest incidence of cervical cancer in Europe [84,85]. An important limitation of our study is that cytology and hrHPV testing were in both screening rounds performed from two different cervical swabs instead of one cervical sample being taken for both screening modalities. However, this was unavoidable, due to the fact that Slovenia, as great majority of the countries in cen-

tral/eastern Europe is not using liquid, but traditional cytology in the national cervical cancer screening program [84,85]. Thus, we cannot exclude the possibility that discrepant cytology/hrHPV testing results in some women are the consequence of screening tests being performed from two different cervical swabs, although we deem that the likelihood is low. Here we present the final results of the baseline screening round with corrected RealTime and hc2 clinical performance values [48]. The final values of the baseline screening round additionally confirmed that RealTime is cross-sectionally non-inferior to hc2 in a primary cervical cancer screening setting. In the baseline screening round, a total of 68 CIN2+ cases were finally identified (all 68

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021

G Model

ARTICLE IN PRESS

JCV-3492; No. of Pages 11

M. Poljak et al. / Journal of Clinical Virology xxx (2015) xxx–xxx

8

Table 3 Risk at baseline and baseline plus future risk at 3 years for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+) with corresponding 95% confidence intervals (95% CI) for women with different baseline characteristics in the total study population. The risk for CIN2+/CIN3+ at baseline was assessed in women enrolled in the baseline screening round based on CIN2+/CIN3+ cases identified in the baseline screening round. The baseline plus future risk for CIN2+/CIN3+ at 3 years was assessed in women included in the second screening round final analysis based on CIN2+/CIN3+ cases identified in both screening rounds. CIN2+

HPV16-positive

CIN3+

Risk at baseline

Baseline plus future risk at 3 years Risk at baseline

n

n

35/160

% (95% CI)

21.9% (15.2–30.4%) 41/201 20.4% HPV16/18-positive (14.6–27.7%) non-16/18 hrHPV-positive 26/372 7.0% (4.6–10.2%) 67/573 11.7% hrHPV-positive (9.1–14.9%) 1/3,937 0.03% hrHPV-negative (0.00–0.14%)

% (95% CI)

50/150

33.3% (24.7–44.0%) 57/187 30.5% (23.1–39.5%) 43/327 13.2% (9.5–17.7%) 100/514 19.5% (15.8–23.7%) 4/3,474 0.12% (0.03–0.29%)

women were RealTime HPV DNA-positive), and of these 14 (20.6%) CIN2+ cases occurred among women with normal cytology (Fig. 4). Thus, the final results of the baseline screening round confirmed significantly higher cross-sectional sensitivity for CIN2+ of hrHPV testing in comparison to cytology, which is in line with the results of similar studies worldwide and recent meta-analyses in the field [4,86–88]. Our data suggest that primary hrHPV-based cervical cancer screening merits consideration as valid alternative for current cytology-based screening in Slovenia, particularly giving the decent HPV vaccine coverage for three doses ranging from 45.5% to 55.3% in school year 2013/2014 and 2010/2011, respectively [84,85]. In this study, we generated the first 3-year clinical performance longitudinal data for RealTime by assessing the risk of CIN2+ according to various negative baseline characteristics of women included in the second screening round. In the second screening round, a total of 36 CIN2+ cases were identified: 17 cases through questionnaires/data registries and 19 cases actively due to secondround cytology and/or HPV test results. In the second screening round, we detected significantly less disease than in the baseline screening round (36 vs. 68 CIN2+ cases, respectively). This figure is in line with the results of published trials that reported data from the second round of screening [13,14,16–18] because prevalent disease is predominately detected in the baseline round of screening. During the 36- to 48-month follow-up in both our study populations (women >30 years old and the total study population), accumulation of CIN2+ cases predominantly occurred in woman with normal cytology at baseline. In addition, in both study populations the risk of CIN2+ at 3 years was significantly lower for women tested hrHPV DNA-negative at baseline using either RealTime or hc2 in comparison to those with normal cytology at baseline. With the generation of RealTime 3-year longitudinal data, to the best of our knowledge, RealTime become the fifth hrHPV test with at least 3-year longitudinal data generated in primary cervical cancer screening settings [43]. Two hrHPV tests—hc2 and GP5+/6+ PCREIA—were fully clinically and epidemiologically validated in four large randomized clinical trials with at least an 8-year follow-up period and thus represent standard comparator tests for assessing the clinical performance of other HPV tests [4,12,27,30–32,43]. In addition, two hrHPV tests have published longitudinal data regarding their performance in primary cervical cancer screening settings: the Cobas 4800 HPV Test (Cobas; Roche Molecular Systems, Alameda, CA, USA) [89] and the APTIMA HPV assay (Hologic, Madison, WI, USA) [90,91]. Longitudinal data from Cobas were generated in the ATHENA clinical trial, which enrolled 42,209 women ≥25 years from the United States and was the basis for the US Food and Drug Administration approval of Cobas as the first HPV DNA stand-alone cervical cancer screening test. In the final report of the

n 17/160

% (95% CI)

10.6% (6.2–17.0%) 20/201 10.0% (6.1–15.4%) 11/372 3.0% (1.5–5.3%) 31/573 5.4% (3.7–7.7%) 0/3,937 0.0%

Baseline plus future risk at 3 years n

% (95% CI)

24/150

16.0% (10.3–23.8%) 27/187 14.4% (9.5–21.0%) 22/327 6.7% (4.2–10.2%) 49/514 9.5% (7.1–12.6%) 0/3,474 0.0%

ATHENA trial, the cumulative 3-year risk of CIN3+ after a negative Cobas test result was 0.3% (95% CI, 0.1–0.7%) in comparison with the 3-year risk of CIN3+ of 0.8% (95% CI, 0.5–1.1%) and 0.3% (95% CI, 0.1–0.6%), after negative cytology and after a co-negative cytology Cobas test result, respectively [89]. The longitudinal clinical performance of the APTIMA HPV assay was assessed in the CLEAR study on a total of 10,860 US women >30 years old [90]. Their results demonstrate that women with normal cytology and with a negative APTIMA HPV assay or hc2 test result at baseline have a very similar low risk (<0.3%) of developing CIN2+ after 3 years of followup, compared to 6.3% and 5.1% 3-year CIN2+ risk in women with positive results on the APTIMA HPV assay and hc2 test, respectively [90]. In our study, the risk for CIN2+ at 3 years in women tested RealTime-negative and with normal cytology at baseline (doublenegative women) was not significantly different from the risk for CIN2+ at 3 years among those tested RealTime-negative at baseline, indicating no added protective value against CIN2+ of a negative cytology result over a negative RealTime result in both of our study populations. Our results are similar to those obtained in other European randomized clinical trials [12–18,20–23], which represented the foundation on which evidence for the latest European guidelines for cervical cancer screening were built [92] and which clearly recommend avoiding co-testing (hrHPV and cytology primary testing) at any age. Although in all longitudinal studies, including our study, women with a positive hrHPV result at baseline had a substantial risk of developing CIN2+ over 3 years of follow-up due to the fact that the prevalence of hrHPV is relatively high in the screened population and that the great majority of HPV infections are merely transient, efficient triage strategies for hrHPV-positive women (especially those with negative cytology) are needed to avoid over-screening and overtreatment of hrHPV-positive women [93,94]. Several studies have been conducted to determine the most effective triage approach to identify women with an underlying CIN2+ among those that are hrHPV-positive. To date, the most frequently evaluated and used triage strategies include reflex cytology, partial genotyping for HPV types with the highest oncogenic potential (e.g., HPV16/18, p16, p16/Ki-67), host and viral methylation, and HPV E6 protein determination [93–99]. The decision on which triage strategy to use is not straightforward because the acceptance of risks varies between different countries and the choice of preferred triage strategy depends on the resources available. The ideal triage strategy should be simple since complex triage management pathways are more prone to misinterpretation and mismanagement of patients. According to the latest European guidelines for cervical cancer screening [92] and protocols announced in European coun-

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021

G Model JCV-3492; No. of Pages 11

ARTICLE IN PRESS M. Poljak et al. / Journal of Clinical Virology xxx (2015) xxx–xxx

tries moving toward HPV primary screening, cytology will be used as the main reflex method for hrHPV screen-positive women in Europe, in contrast to the United States, where recent interim clinical guidelines for cervical cancer screening favor triage of hrHPV screen-positive women using a combination of HPV16/18 typing and reflex cytology for women positive for the 12 other hrHPV types [11]. Similarly, Australian HPV-based primary screening, which will start in May 2017, will triage with partial HPV genotyping and reflex cytology [100]. As a result of these new triage initiatives, a range of novel HPV tests with concurrent or reflex partial genotyping capabilities to detect hrHPV, such as RealTime, have been designed in the last few years [42]. Because RealTime includes genotyping for HPV16 and HPV18 in two separate channels, thereby providing an integrated screening and immediate triage for HPV screen-positive women, it is perfectly suited for countries that use or will use partial genotyping as a main or ancillary triage method. Although our study was not designed and powered to assess partial genotyping triage capability of RealTime, this worked well in our study. In agreement with other similar studies, women with baseline HPV16 and HPV16/18 infection had significantly higher risk at baseline and baseline plus future risk at 3 years for CIN2+ and CIN3+ in comparison to women with baseline non-HPV16/18 hrHPV infection or those that are hrHPV-positive. Information about HPV16 status is indeed of great clinical value because, for example, in our study one in three women positive for HPV16 at baseline were diagnosed with CIN2+ at baseline and/or developed CIN2+ within 36–48 months after baseline testing. The fact that HPV16 status corresponded to the greatest type-specific risk stratification, followed by HPV18 and/or HPV33 and/or HPV31, has been reinforced in all of the most recently published long-term follow-up studies [95,101,102]. However, it should be stressed that partial genotyping for HPV16/18 is mainly useful in non-vaccinated populations. Because HPV-vaccinated women form an increasing proportion of women in the screened population, the utility of partial genotyping for HPV16/18 and immediate referral will be reduced as their prevalence decreases [94].

9

Funding This study was funded by Abbott Molecular and the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana. Abbott Molecular was not involved in the study design, data collection and analysis, interpretation of the results, or writing the manuscript. Mario Poljak and Anja Oˇstrbenk are supported by the seventh framework program of DG Research of the European Commission, through the COHEAHR Network (grant no. 603019). Ethical approval This study was approved by the National Medical Ethics Committee at the Ministry of Health of the Republic of Slovenia (consent numbers: 83/11/09 and 109/08/12). Acknowledgements We thank our clinical colleagues Lara Beseniˇcar Pregelj, Gabriˇ ˇ Martina Buˇcar, Tanja Burnik Papler, Simona Copi, jela Brˇzan Simenc, Irena Grahelj, Mojca Jemec, Joˇzefa Keˇzar, Tatjana Kodriˇc, Zdravka Koman, Sara Koroˇsec, Jasna Kostanjˇsek, Jasna Kuhelj Recer, Mili Lomˇsek, Petra Megliˇc, Aleksander Merlo, Anamarija Petek, Andreja ´ Kaja Rebek, Urˇsula Peterlin, Suzana Peternelj Marinˇsek, Miˇso Rajic, Reˇs Muravec, Filip Simoniti, Tinkara Srnovrˇsnik, Mateja Darija ˇ ˇ Strah, Vesna Salamun, Ksenija Selih Martinec, and Andrej Zore for patient recruitment and management; Robert Kroˇselj for excellent laboratory assistance; Marja Lenart and Margareta Strojan Fleˇzar for cytology and histology review; Veronika Uˇcakar and Irena Klavs for drafting the study protocol and proposal for the ethics committee; Urˇska Ivanuˇs and Maja Primic Zˇ akelj for retrieval of data from the national cervical cancer screening registry and critical discussion; Vanja Erˇculj for help with statistical analysis; and Miha Pirc for sample transportation. References

6. Conclusion The first 3-year longitudinal study of clinical performance of RealTime performed in a cohort of 3,920 Slovenian women that had hrHPV DNA and/or cytology in the 36- to 48-month followup results after baseline testing with cytology, hc2, and RealTime showed significantly better protection against CIN2+ associated with a negative hrHPV DNA baseline result than with normal baseline cytology. Our findings reinforce evidence from previous studies that RealTime can safely be used in primary HPV-based cervical cancer screening. Women with baseline HPV16 and HPV16/18 infection had significantly higher risk at baseline and baseline plus future risk at 3 years for CIN2+ and CIN3+ in comparison to women with baseline non-HPV16/18 hrHPV infection, supporting the evidence that concurrent partial genotyping for HPV types with exceptionally high oncogenic potential should be strongly considered as a triage method for HPV screen-positive women.

Conflict of interest The authors’ institution received several research grants from Abbott Molecular. Mario Poljak received reimbursement of travel expenses for attending meetings and conferences from Abbott Molecular, and honoraria for speaking and consultancy from Abbott Molecular until mid-2014. Mario Poljak declares no competing interest since September 2014. The other authors declare no competing interest.

[1] H. Zur Hausen, Papillomaviruses in the causation of human cancers—a brief historical account, Virology 384 (2009) 260–265. [2] V. Bouvard, R. Baan, K. Straif, Y. Grosse, B. Secretan, F. El Ghissassi, et al., A review of human carcinogens— Part B: biological agents, Lancet Oncol. 10 (2009) 321–322. [3] J. Cuzick, M. Arbyn, R. Sankaranarayanan, V. Tsu, G. Ronco, M.H. Mayrand, et al., Overview of human papillomavirus-based and other novel options for cervical cancer screening in developed and developing countries, Vaccine 26 (Suppl. 10) (2008) K29–K41. [4] M. Arbyn, G. Ronco, A. Anttila, C.J. Meijer, M. Poljak, G. Ogilvie, et al., Evidence regarding human papillomavirus testing in secondary prevention of cervical cancer, Vaccine 30 (Suppl. 5) (2012) F88–F99. [5] M. Poljak, Towards cervical cancer eradication: joint force of HPV vaccination and HPV-based cervical cancer screening, Clin. Microbiol. Infect. 21 (2015) 806–807. [6] F.X. Bosch, C. Robles, M. Diaz, M. Arbyn, I. Baussano, C. Clavel, et al., HPV-FASTER broadening the scope for prevention of HPV-related cancer, Nat. Rev. Clin. Oncol. (2015) [Epub ahead of print]. [7] J.T. Cox, History of the use of HPV testing in cervical screening and in the management of abnormal cervical screening results, J. Clin. Virol. 45 (Suppl. 1) (2009) S3–12. [8] K.S. Cuschieri, H.A. Cubie, The role of human papillomavirus testing in cervical screening, J. Clin. Virol. 32 (Suppl. 1) (2005) S34–S42. [9] M.S. Desai, H.A. Cubie, The HPV test in cervical screening: a brave new world? Cytopathology 16 (2005) 3–6. [10] E.P. Whitlock, K.K. Vesco, M. Eder, J.S. Lin, C.A. Senger, B.U. Burda, Liquid-based cytology and human papillomavirus testing to screen for cervical cancer: a systematic review for the U.S. preventive services task force, Ann. Intern. Med. 155 (2011) 687–697. [11] W.K. Huh, K.A. Ault, D. Chelmow, D.D. Davey, R.A. Goulart, F.A. Garcia, et al., Use of primary high-risk human papillomavirus testing for cervical cancer screening: interim clinical guidance, Gynecol. Oncol. 136 (2015) 178–182. [12] K.M. Elfström, V. Smelov, A.L. Johansson, C. Eklund, P. Nauclér, L. Arnheim-Dahlström, et al., Long term duration of protective effect for HPV negative women: follow-up of primary HPV screening randomised controlled trial, BMJ 348 (2014) g130.

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021

G Model JCV-3492; No. of Pages 11 10

ARTICLE IN PRESS M. Poljak et al. / Journal of Clinical Virology xxx (2015) xxx–xxx

[13] G. Ronco, J. Dillner, K.M. Elfström, S. Tunesi, P.J. Snijders, M. Arbyn, et al., Efficacy of HPV-based screening for prevention of invasive cervical cancer: follow-up of four European randomised controlled trials, Lancet 383 (2014) 524–532. [14] P. Naucler, W. Ryd, S. Törnberg, A. Strand, G. Wadell, K. Elfgren, et al., Human papillomavirus and papanicolaou tests to screen for cervical cancer, N. Engl. J. Med. 357 (2007) 1589–1597. [15] N.W. Bulkmans, J. Berkhof, L. Rozendaal, F.J. van Kemenade, A.J. Boeke, S. Bulk, et al., Human papillomavirus DNA testing for the detection of cervical intraepithelial neoplasia grade 3 and cancer: 5-year follow-up of a randomised controlled implementation trial, Lancet 370 (2007) 1764–1772. [16] D.C. Rijkaart, J. Berkhof, F.J. van Kemenade, V.M. Coupe, A.T. Hesselink, L. Rozendaal, et al., Evaluation of 14 triage strategies for HPV DNA-positive women in population-based cervical screening, Int. J. Cancer 130 (2012) 602–610. [17] H.C. Kitchener, M. Almonte, C. Thomson, P. Wheeler, A. Sargent, B. Stoykova, et al., HPV testing in combination with liquid-based cytology in primary cervical screening (ARTISTIC): a randomised controlled trial, Lancet Oncol. 10 (2009) 672–682. [18] G. Ronco, P. Giorgi-Rossi, F. Carozzi, M. Confortini, P. Dalla Palma, A. Del Mistro, et al., Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia: a randomised controlled trial, Lancet Oncol. 11 (2010) 249–257. [19] R. Sankaranarayanan, B.M. Nene, S.S. Shastri, K. Jayant, R. Muwonge, A.M. Budukh, et al., HPV screening for cervical cancer in rural India, N. Engl. J. Med. 360 (2009) 1385–1394. [20] G. Ronco, N. Segnan, P. Giorgi-Rossi, M. Zappa, G.P. Casadei, F. Carozzi, et al., Human papillomavirus testing and liquid-based cytology in primary cervical screening: results at recruitment from the new technologies for cervical cancer randomized controlled trial, J. Natl. Cancer Inst. 98 (2006) 765–774. [21] G. Ronco, P. Giorgi-Rossi, F. Carozzi, P.P. Dalla, A. Del Mistro, L. De Marco, et al., Human papillomavirus testing and liquid-based cytology in primary screening of women younger than 35 years: results at recruitment for a randomised controlled trial, Lancet Oncol. 7 (2006) 547–555. [22] M. Leinonen, P. Nieminen, L. Kotaniemi-Talonen, N. Malila, J. Tarkkanen, P. Laurila, et al., Age-specific evaluation of primary human papillomavirus screening vs conventional cytology in a randomized setting, J. Natl. Cancer Inst. 101 (2009) 1612–1623. [23] N.W. Bulkmans, L. Rozendaal, P.J. Snijders, F.J. Voorhorst, A.J. Boeke, G.R. Zandwijken, POBASCAM, a population-based randomized controlled trial for implementation of high-risk HPV testing in cervical screening: design, methods and baseline data of 44,102 women, Int. J. Cancer 110 (2004) 94–101. [24] G.S. Ogilvie, M. Krajden, D.J. van Niekerk, R.E. Martin, T.G. Ehlen, K. Ceballos, et al., Primary cervical cancer screening with HPV testing compared with liquid-based cytology: results of round 1 of a randomised controlled trial—the HPV FOCAL study, Br. J. Cancer 107 (2012) 1917–1924. [25] M.H. Mayrand, E. Duarte-Franco, I. Rodrigues, S.D. Walter, J. Hanley, A. Ferenczy, et al., Human papillomavirus DNA versus papanicolaou screening tests for cervical cancer, N. Engl. J. Med. 357 (2007) 1579–1588. [26] M.H. Uijterwaal, N.J. Polman, F.J. Van Kemenade, S. Van Den Haselkamp, B.I. Witte, D. Rijkaart, et al., Five-year cervical (Pre) cancer risk of women screened by HPV and cytology testing, Cancer Prev. Res. (Phila) 8 (2015) 502–508. [27] J. Dillner, M. Rebolj, P. Birembaut, K.U. Petry, A. Szarewski, C. Munk, et al., Long term predictive values of cytology and human papillomavirus testing in cervical cancer screening: joint European cohort study, BMJ 337 (2008) a1754. [28] H.A. Katki, W.K. Kinney, B. Fetterman, T. Lorey, N.E. Poitras, L. Cheung, et al., Cervical cancer risk for women undergoing concurrent testing for human papillomavirus and cervical cytology: a population-based study in routine clinical practice, Lancet Oncol. 12 (2011) 663–672. [29] M.E. Sherman, A.T. Lorincz, D.R. Scott, S. Wacholder, P.E. Castle, A.G. Glass, et al., Baseline cytology, human papillomavirus testing, and risk for cervical neoplasia: acohort 10-year analysis, J. Natl. Cancer Inst. 95 (2003) 46–52. [30] H.A. Katki, M. Schiffman, P.E. Castle, B. Fetterman, N.E. Poitras, T. Lorey, et al., Benchmarking CIN 3+ risk as the basis for incorporating HPV and Pap cotesting into cervical screening and management guidelines, J. Low Genit. Tract. Dis. 17 (Suppl. 1) (2013) S28–35. [31] P.E. Castle, A.G. Glass, B.B. Rush, D.R. Scott, N. Wentzensen, J.C. Gage, et al., Clinical human papillomavirus detection forecasts cervical cancer risk in women over 18 years of follow-up, J. Clin. Oncol. 30 (2012) 3044–3050. [32] M.A. Vink, J.A. Bogaards, C.J. Meijer, J. Berkhof, Primary human papillomavirus DNA screening for cervical cancer prevention: can the screening interval be safely extended? Int. J. Cancer 137 (2015) 420–427. [33] J.C. Gage, M. Schiffman, H.A. Katki, P.E. Castle, B. Fetterman, N. Wentzensen, et al., Reassurance against future risk of precancer and cancer conferred by a negative human papillomavirus test, J. Natl. Cancer Inst. 106 (2014), http:// dx.doi.org/10.1093/jnci/dju153. [34] K.U. Petry, S. Menton, M. Menton, F. van Loenen-Frosch, H. de Carvalho Gomes, B. Holz, et al., Inclusion of HPV testing in routine cervical cancer screening for women above 29 years in Germany: results for 8466 patients, Br. J. Cancer 88 (2003) 1570–1577. [35] S.K. Kjaer, A.J. van den Brule, G. Paull, E.I. Svare, M.E. Sherman, B.L. Thomsen, et al., Type specific persistence of high risk human papillomavirus (HPV) as

[36]

[37]

[38]

[39]

[40]

[41] [42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

indicator of high grade cervical squamous intraepithelial lesions in young women: population based prospective follow up study, BMJ 325 (2002) 572. J. Cuzick, A. Szarewski, D. Mesher, L. Cadman, J. Austin, K. Perryman, et al., Long-term follow-up of cervical abnormalities among women screened by HPV testing and cytology-results from the Hammersmith study, Int. J. Cancer 122 (2008) 2294–2300. C. Clavel, M. Masure, J.P. Bory, I. Putaud, C. Mangeonjean, M. Lorenzato, et al., Human papillomavirus testing in primary screening for the detection of high-grade cervical lesions: a study of 7932 women, Br. J. Cancer 84 (2001) 1616–1623. ˜ S. de Sanjose, R. Almirall, B. Lloveras, R. Font, M. Diaz, N. Munoz, et al., Cervical human papillomavirus infection in the female population in Barcelona, Spain, Sex Transm. Dis. 30 (2003) 788–793. K. Elfgren, E. Rylander, T. Rådberg, B. Strander, A. Strand, K. Paajanen, et al., Colposcopic and histopathologic evaluation of women participating in population-based screening for human papillomavirus deoxyribonucleic acid persistence, Am. J. Obstet. Gynecol. 193 (2005) 650–657. K.B. Roland, A. Soman, V.B. Benard, M. Saraiya, Human papillomavirus and papanicolaou tests screening interval recommendations in the United States, Am. J. Obstet. Gynecol. 205 (447) (2011) e1–e8. J. Dillner, Primary human papillomavirus testing in organized cervical screening, Curr. Opin. Obstet. Gynecol. 25 (2013) 11–16. M. Poljak, B.J. Kocjan, A. Oˇstrbenk, K. Seme, Commercially available molecular tests for human papillomaviruses (HPV)-2015 update, J. Clin. Virol. (2015), HPV Suppl; in this issue. M. Arbyn, P.J. Snijders, C.J. Meijer, J. Berkhof, K. Cuschieri, B.J. Kocjan, et al., Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening? Clin. Microbiol. Infect. 21 (2015) 817–826. C.J.L.M. Meijer, P.E. Castle, A.T. Hesselink, E.L. Franco, G. Ronco, M. Arbyn, et al., Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older, Int. J. Cancer 124 (2009) 516–520. N. Tang, S. Huang, B. Erickson, W.B. Mak, J. Salituro, J. Robinson, et al., HPV High-risk detection and concurrent HPV 16 and 18 typing with Abbott RealTime High Risk HPV test, J. Clin. Virol. 45 (Suppl. 1) (2009) S25–S28. M. Poljak, A. Oˇstrbenk, HPV The Abbott RealTime High Risk test is a clinically validated human papillomavirus assay for triage in the referral population and use in primary cervical cancer screening in women 30 years and older: a review of validation studies, Acta Dermatovenerol. Alp. Pannonica Adriat. 22 (2013) 43–47. F.M. Carozzi, E. Burroni, S. Bisanzi, D. Puliti, M. Confortini, P. Giorgi Rossi, et al., Comparison of clinical performance of Abbott RealTime High Risk HPV test with that of Hybrid Capture 2 assay in a screening setting, J. Clin. Microbiol. 49 (2011) 1446–1451. M. Poljak, A. Oˇstrbenk, K. Seme, V. Uˇcakar, P. Hillemanns, E. Vrtaˇcnik Bokal, et al., Comparison of clinical and analytical performance of the Abbott RealTime High Risk HPV test and Hybrid Capture 2 in population-based cervical cancer screening, J. Clin. Microbiol. 49 (2011) 1721–1729. A.T. Hesselink, C.J. Meijer, M. Poljak, J. Berkhof, F.J. van Kemenade, M.L. van der Salm, et al., Clinical validation of the Abbott RealTime High Risk HPV assay according to the guidelines for human papillomavirus DNA test requirements for cervical screening, J. Clin. Microbiol. 51 (2013) 2409–2410. Society for gynecological oncology, colposcopy and cervical pathology, Slovenian guidelines for management of pathological cervical smears, M. Urˇsiˇc Vrˇscˇ aj (Ed.), in Slovene: Zdruˇzenje za ginekoloˇsko onkologijo, kolposkopijo in cervikalno patologijo. Smernice za celostno obravnavo zˇ ensk s predrakavimi spremembami materniˇcnega vratu. Available at: http://zora.onko-i.si/fileadmin/user upload/dokumenti/strokovna priporocila/2011 Smernice web.pdf (accessed 25.9.15). M. Poljak, A. Kovanda, B.J. Kocjan, K. Seme, N. Janˇcar, E. Vrtaˇcnik-Bokal, The Abbott RealTime High Risk HPV test: comparative evaluation of analytical specificity and clinical sensitivity for cervical carcinoma and CIN 3 lesions with the Hybrid Capture 2HPV DNA test, Acta Dermatovenerol. Alp. Panonica Adriat. 18 (2009) 94–103. P. Halfon, D. Benmoura, A. Agostini, H. Khiri, G. Penaranda, A. Martineau, et al., Evaluation of the clinical performance of the Abbott RealTime high-Risk HPV for carcinogenic HPV detection, J. Clin. Virol. 48 (2010) 246–250. S. Venturoli, E. Leo, M. Nocera, D. Barbieri, M. Cricca, S. Costa, et al., Comparison of Abbott RealTime High Risk HPV and Hybrid Capture 2 for the detection of high-risk HPV DNA in a referral population setting, J. Clin. Virol. 53 (2012) 121–124. M. Jentschke, P. Soergel, V. Lange, B. Kocjan, T. Doerk, A. Luyten, et al., Evaluation of a new multiplex real-time polymerase chain reaction assay for the detection of human papillomavirus infections in a referral population, Int. J. Gynecol. Cancer 22 (2012) 1050–1056. Y. Park, E. Lee, J. Choi, S. Jeong, H.S. Kim, Comparison of the Abbott RealTime High-Risk human papillomavirus (HPV), Roche Cobas HPV, and Hybrid Capture 2 assays to direct sequencing and genotyping of HPV DNA, J. Clin. Microbiol. 50 (2012) 2359–2365. Y. Hwang, M. Lee, Comparison of the AdvanSure human papillomavirus screening real-time PCR, the Abbott RealTime High Risk human papillomavirus test, and the Hybrid Capture human papillomavirus DNA test for the detection of human papillomavirus, Ann. Lab. Med. 32 (2012) 201–205.

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021

G Model JCV-3492; No. of Pages 11

ARTICLE IN PRESS M. Poljak et al. / Journal of Clinical Virology xxx (2015) xxx–xxx

[57] P. Halfon, D. Benmoura, A. Agostini, H. Khiri, G. Pénaranda, A. Martineau, et al., Stepwise algorithm combining HPV high-risk DNA-based assays and RNA-based assay for high grade CIN in women with abnormal smears referred to colposcopy, Cancer Biomark 7 (2010) 133–139. [58] S. Huang, N. Tang, W.B. Mak, B. Erickson, J. Salituro, Y. Li, et al., Principles and analytical performance of Abbott RealTime High Risk HPV test, J. Clin. Virol. 45 (Suppl. 1) (2009) S13–S17. [59] S. Huang, B. Erickson, N. Tang, W.B. Mak, J. Salituro, J. Robinson, et al., Clinical performance of Abbott RealTime High Risk HPV test for detection of high-grade cervical intraepithelial neoplasia in women with abnormal cytology, J. Clin. Virol. 45 (Suppl. 1) (2009) S19–S23. [60] J. Cuzick, L. Ambroisine, L. Cadman, J. Austin, L. Ho, G. Terry, et al., Performance of the Abbott RealTime high-risk HPV test in women with abnormal cervical cytology smears, J. Med. Virol. 82 (2010) 1186–1191. [61] O.G. Wong, C.K. Lo, E. Szeto, A.N. Cheung, Efficacy of Abbott RealTime High Risk HPV test in evaluation of atypical squamous cells of undetermined significance from an Asian screening population, J. Clin. Virol. 51 (2011) 136–138. [62] A. Szarewski, D. Mesher, L. Cadman, J. Austin, L. Ashdown-Barr, L. Ho, et al., Comparison of seven tests for high-grade cervical intraepithelial neoplasia in women with abnormal smears: the predictors 2 study, J. Clin. Microbiol. 50 (2012) 1867–1873. [63] A. Kovanda, U. Juvan, A. Sterbenc, B.J. Kocjan, K. Seme, N. Jancar, et al., Pre-vaccination distribution of human papillomavirus (HPV) genotypes in women with cervical intraepithelial neoplasia grade 3 (CIN 3) lesions in Slovenia, Acta Dermatovenerol. Alp. Panonica Adriat. 18 (2009) 47–52. [64] O. Veijalainen, S. Tuomisaari, T. Luukkaala, J. Mäenpää, High risk HPV testing in the triage of repeat ASC-US and LSIL, Acta Obstet. Gynecol. Scand. 94 (2015) 931–936. ˇ ca, A. Smailji, M. Hukic, ´ ´ A. Tomic´ Ciˇ ´ Comparison of the [65] I. Salimovic-Beˇ sic, detection of HPV-16, 18, 31, 33, and 45 by type-specific DNA- and E6/E7 mRNA-based assays of HPV DNA positive women with abnormal Pap smears, J. Virol. Methods 194 (2013) 222–228. [66] J. Cuzick, L. Cadman, D. Mesher, J. Austin, L. Ashdown-Barr, L. Ho, et al., Comparing the performance of six human papillomavirus tests in a screening population, Br. J. Cancer 108 (2013) 908–913. [67] H.S. Chung, C. Hahm, M. Lee, Comparison of the clinical performances of the AdvanSure HPV screening Real-Time PCR, the Abbott Real-Time High-Risk HPV Test, and the Hybrid Capture High-Risk HPV DNA Test for cervical cancer screening, J. Virol. Methods 205C (2014) 57–60. [68] M. Jentschke, K. Chen, M. Arbyn, B. Hertel, M. Noskowicz, P. Soergel, et al., Comparative evaluation of two vaginal self-sampling devices for the detection of human papillomavirus infections, J. Clin. Virol. (2015), HPV Suppl; in this issue. [69] A.C. Bateman, K. Katundu, P. Polepole, A. Shibemba, M. Mwanahamuntu, D.P. Dittmer, et al., Identification of human papillomaviruses from formalin-fixed, paraffin-embedded pre-cancer and invasive cervical cancer specimens in Zambia: a cross-sectional study, Virol. J. 12 (2015) 2. [70] B.J. Kocjan, K. Seme, M. Poljak, Comparison of the Abbott RealTime High Risk HPV test and INNO-LiPA HPV genotyping extra test for the detection of human papillomaviruses in formalin-fixed, paraffin-embedded cervical cancer specimens, J. Virol. Methods 175 (2011) 117–119. [71] B.J. Kocjan, P.J. Maver, L. Hosnjak, N. Zidar, K. Odar, N. Gale, et al., Comparative evaluation of the Abbott RealTime High Risk HPV test and INNO-LiPA HPV genotyping extra test for detecting and identifying human papillomaviruses in archival tissue specimens of head and neck cancers, Acta Dermatovenerol. Alp. Panonica Adriat. 21 (2012) 73–75. [72] M. Jentschke, P. Soergel, P. Hillemanns, Evaluation of a multiplex real time PCR assay for the detection of human papillomavirus infections on self-collected cervicovaginal lavage samples, J. Virol. Methods 193 (2013) 131–134. [73] E. Padalko, C. Ali-Risasi, S. Mesmaekers, I. Ryckaert, L. Van Renterghem, K. Lambein, et al., Comparative analysis of cervical cytology and human papillomavirus genotyping by three different methods in a routine diagnostic setting, Eur. J. Cancer Prev. 24 (2015) 447–453. [74] S. Park, Y. Kang, D.G. Kim, E.C. Kim, S.S. Park, M.W. Seong, Comparison of the analytical and clinical performances of Abbott RealTime High Risk HPV, Hybrid Capture 2, and DNA chip assays in gynecology patients, Diagn. Microbiol. Infect. Dis. 76 (2013) 432–436. [75] J. Choi, Y. Park, E.H. Lee, S. Kim, J.H. Kim, H.S. Kim, Detection and genotyping of human papillomavirus by five assays according to cytologic results, J. Virol. Methods 187 (2013) 79–84. [76] C. Sias, A.R. Garbuglia, P. Piselli, C. Cimaglia, D. Lapa, F. Del Nonno, et al., Comparison of the Abbott RealTime High Risk HPV with genomica HPV clinical array for the detection of human papillomavirus DNA, APMIS 121 (2013) 1054–1063. [77] C. Moore, K. Cuschieri, F. McQueen, E. Duvall, C. Graham, H.A. Cubie, Effect of glacial acetic acid pre-treatment of cervical liquid-based cytology specimens on the molecular detection of human papillomavirus, Cytopathology 24 (2013) 314–320. [78] H.A. Cubie, M. Canham, C. Moore, J. Pedraza, C. Graham, K. Cuschieri, Evaluation of commercial HPV assays in the context of post-treatment follow-up: Scottish Test of Cure Study (STOCS-H), J. Clin. Pathol. 67 (2014) 458–463. [79] D. Barbieri, S. Venturoli, S. Costa, M.P. Landini, Improving laboratory efficiency by automation of preanalytic processing of ThinPrep specimens

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88] [89]

[90]

[91] [92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

11

for real-time PCR high-risk HPV testing, J. Lab. Autom. (2015) [Epub ahead of print]. V. Uˇcakar, M. Poljak, I. Klavs, Pre-vaccination prevalence and distribution of high-risk human papillomavirus (HPV) types in Slovenian women: a cervical cancer screening based study, Vaccine 30 (2012) 116–120. V. Uˇcakar, M. Poljak, A. Oˇstrbenk, I. Klavs, Pre-vaccination prevalence of infections with 25 non-high-risk human papillomavirus types among 1000 Slovenian women in cervical cancer screening, J. Med. Virol. 86 (2014) 1772–1779. V. Uˇcakar, M.M. Jelen, H. Faust, M. Poljak, J. Dillner, I. Klavs, Pre-vaccination seroprevalence of 15 human papillomavirus (HPV) types among women in the population-based Slovenian cervical screening program, Vaccine 31 (2013) 4935–4939. H. Faust, M.M. Jelen, M. Poljak, I. Klavs, V. Uˇcakar, J. Dillner, Serum antibodies to human papillomaviruses (HPV) pseudovirions correlate with natural infection for 13 genital HPV types, J. Clin. Virol. 56 (2013) 336–341. ´ F. Bray, J. Berkhof, K. Seme, et al., M. Poljak, S.I. Rogovskaya, V. Kesic, Recommendations for cervical cancer prevention in Central and Eastern Europe and Central Asia, Vaccine 31 (Suppl. 7) (2013) H80–H82. V. Kesic, M. Poljak, S. Rogovskaya, Cervical cancer burden and prevention activities in Europe, Cancer Epidemiol. Biomarkers Prev. 21 (2012) 1423–1433. J. Cuzick, C. Clavel, K.U. Petry, C.J. Meijer, H. Hoyer, S. Ratnam, et al., Overview of the European and North American studies on HPV testing in primary cervical cancer screening, Int. J. Cancer 119 (2006) 1095–1101. M. Arbyn, P. Sasieni, C.J. Meijer, C. Clavel, G. Koliopoulos, J. Dillner, Chapter 9: Clinical applications of HPV testing: a summary of meta-analyses, Vaccine 24 (Suppl. 3) (2006), S3/78-89. E.L. Franco, Chapter 13: Primary screening of cervical cancer with human papillomavirus tests, J. Natl. Cancer Inst. Monogr. 31 (2003) 89–96. T.C. Wright, M.H. Stoler, C.M. Behrens, A. Sharma, G. Zhang, T.L. Wright, Primary cervical cancer screening with human papillomavirus: end of study results from the ATHENA study using HPV as the first-line screening test, Gynecol. Oncol. 136 (2015) 189–197. J.L. Reid, T.C. Wright Jr., M.H. Stoler, J. Cuzick, P.E. Castle, J. Dockter, et al., Human papillomavirus oncogenic mRNA testing for cervical cancer screening: baseline and longitudinal results from the CLEAR study, Am. J. Clin. Pathol. 144 (2015) 473–483. J. Haedicke, T. Iftner, A review of the clinical performance of the Aptima HPV assay, J. Clin. Virol. (2015), HPV Suppl; in this issue. L. von Karsa, M. Arbyn, H. De Vuyst, J. Dillner, L. Dillner, S. Franceschi, et al., European guidelines for quality assurance in cervical cancers screening. Summary of the supplements on HPV screening and vaccination, Papillomavirus Res. (2015) [Epub ahead of print]. J. Cuzick, C. Bergeron, M. von Knebel Doeberitz, et al., New technologies and procedures for cervical cancer screening, Vaccine 30 (Suppl. 5) (2012) F107–F116. N. Wentzensen, M. Schiffman, T. Palmer, M. Arbyn, Triage of HPV positive women in cervical cancer screening, J. Clin. Virol. (2015), HPV Suppl; in this issue. M. Schiffman, R.D. Burk, S. Boyle, T. Raine-Bennett, H.A. Katki, J.C. Gage, et al., A study of genotyping for management of human papillomavirus-positive, cytology-negative cervical screening results, J. Clin. Microbiol. 53 (2015) 52–59. J.T. Cox, P.E. Castle, C.M. Behrens, A. Sharma, T.C. Wright Jr., J. Cuzick, et al., Comparison of cervical cancer screening strategies incorporating different combinations of cytology, HPV testing, and genotyping for HPV 16/18: results from the ATHENA HPV study, Am. J. Obstet. Gynecol. 208 (184) (2013) e1–11. V.M. Verhoef, R.P. Bosgraaf, F.J. van Kemenade, L. Rozendaal, D.A. Heideman, A.T. Hesselink, et al., Triage by methylation-marker testing versus cytology in women who test HPV-positive on self-collected cervicovaginal specimens (PROHTECT-3): a randomised controlled non-inferiority trial, Lancet Oncol. 15 (2014) 315–322. F. Carozzi, A. Gillio-Tos, M. Confortini, et al., Risk of high-grade cervical intraepithelial neoplasia during follow-up in HPV-positive women according to baseline p16-INK4A results: a prospective analysis of a nested substudy of the NTCC randomised controlled trial, Lancet Oncol. 14 (2013) 168–176. K.U. Petry, D. Schmidt, S. Scherbring, et al., Triaging Pap cytology negative, HPV positive cervical cancer screening results with p16/Ki-67 dual-stained cytology, Gynecol. Oncol. 121 (2011) 505–509. O. Denham, S.M. Garland, A. Gorelik, G. Ogilvie, J. Tan, D. Mazza, et al., Attitudes of changes in cervical screening: views of Australian general practitioners and nurses, J. Clin. Virol (2015), HPV Suppl; in this issue. L.T. Thomsen, K. Frederiksen, C. Munk, J. Junge, T. Iftner, S.K. Kjaer, Long-term risk of cervical intraepithelial neoplasia grade 3 or worse according to high-risk human papillomavirus genotype and semi-quantitative viral load among 33,288 women with normal cervical cytology, Int. J. Cancer 137 (2015) 193–203. V. Smelov, K.M. Elfström, A.L. Johansson, C. Eklund, P. Naucler, L. Arnheim-Dahlström, et al., HPV long-term type-specific risks of high-grade cervical intraepithelial lesions: a 14-year follow-up of a randomized primary HPV screening trial, Int. J. Cancer 136 (2015) 1171–1180.

Please cite this article in press as: M. Poljak, et al., Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting, J Clin Virol (2015), http://dx.doi.org/10.1016/j.jcv.2015.11.021