Experimental and Molecular Pathology 106 (2019) 149–156

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Experimental and Molecular Pathology

journal homepage: www.elsevier.com/locate/yexmp

Analytical performance evaluation of the HPV OncoCheck assay for T

detection of high-risk HPV infection in liquid-based cervical samples
⁎
Hye-young Wanga, Hyunil Kimb, , Sunyoung Parkc, Kwang Hwa Parkd, Hyeyoung Leec
a
OptipharmM&D, Inc., Wonju Eco Environmental Technology Center, Wonju, Gangwon, South Korea
b
Optipharm, Inc., Cheongju, South Korea
c
Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Gangwon, South Korea
d
Department of Pathology, Wonju College of Medicine, Yonsei University, Wonju, Gangwon, South Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Recent studies have demonstrated low specificity (false positive) of human papillomavirus (HPV) DNA testing
Cervical cancer for the screening and diagnosis of cervical samples. Therefore, we evaluated the performance of the HPV
Human papillomavirus OncoCheck assay, which is an HPV E6/E7 mRNA-based assay, for the detection of 16 high-risk (HR)-HPVs
E6/E7 mRNA including HPV 16 and HPV 18 genotypes in cervical samples using multiplex reverse transcriptase-quantitative
Reverse transcriptase-quantitative PCR
PCR. In the present study, the analytical performance of the assay was evaluated using 16 HPV single strand
Molecular diagnosis
DNAs. Clinical evaluation was performed using 319 Thinprep® liquid-based cytology samples obtained from
women with cervical diseases, and the HPV OncoCheck assay results were compared with those of cytological
diagnosis and sequence analysis.
All 16 types of HPVs were detected with a minimum detection sensitivity of 100 copies per reaction and high
specificity was observed. The sensitivity and specificity of the HPV OncoCheck assay for detecting high-grade
lesions were 94.1% (95% confidence interval (CI), 0.875–0.975; p < .0001) and 95.4% (95% CI, 0.868–0.989;
p < .0001), and sequence analysis were 99.4% (95% CI, 0.965–0.999; p < .0001), and 98% (95% CI,
0.939–0.996; p < .0001), respectively. Moreover, the agreement between the HPV OncoCheck assay and se-
quence analysis for the detection of HR-HPV was 98.8% (κ = 0.98, 95% CI 0.967–0.996; p < .0001). The results
of this study showed high agreement and specificity with cytological diagnoses and sequence analysis. Future
studies with histological follow- up are needed to determine whether use of the HPV OncoCheck assay in cervical
screening may aid detection of the most significant cervical disease while reducing false-positive results.

1. Introduction progression worldwide, accounting for > 70% of the cases (Zeng et al.,
2016).
Cervical cancer is the third most common cancer in females af- In 2009, the American Society for Colposcopy and Cervical
fecting 528,000 women each year and leading to 266,000 deaths Pathology implemented clinical guidelines for individualized ex-
worldwide (Ferlay et al., 2015). Persistent infection with at least one of aminations that focused on the combination of an HPV DNA test and a
the high-risk (HR) oncogenic human papillomavirus (HPV) genotypes is Pap test for cervical cancer screening; HPV DNA testing has been con-
the primary risk factor for the development of cervical cancer (Guan sidered as an adjunctive test for all cervical cytology samples in women
et al., 2012). Therefore, testing for oncogenic HPV infection in cervical over 30 years of age according to the current recommendations
lesions may be an accurate means of identifying women who are at a (Darragh et al., 2012). In 2014, the HPV DNA test was approved as a
risk of developing cervical cancer. > 100 types of HPV have been primary screening method by the Korea Ministry of Food and Drug
identified (Muñoz et al., 2003) and approximately 40 types of HPV Safety (Min et al., 2015). Accordingly, various assays, including probe
infect the genital tract (Bosch et al., 2002). Of these, at least 13 HR-HPV hybridization, PCR, and DNA microarray, have been developed, and are
genotypes—including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, now commercially available for the detection of HPV DNA to facilitate
and 68—are detected in up to 99.7% of all cervical cancer cases and early cancer screening in clinical samples. The current HPV DNA test,
high-grade cervical lesions cases (Arbyn et al., 2014); particularly, HPV mostly targeting the L1 gene, has been reported to have the limitation
16 and 18 are associated with a higher risk of cervical cancer of low specificity (false-positive results) and there is a lack of

⁎
Corresponding author at: Optipharm, Inc., 63 Osongsaengmyeong 6-ro, Osong-eup, Cheongju, Chungcheongbuk-do, 28158, Republic of Korea.
E-mail address: hikim@optipharm.co.kr (H. Kim).

https://doi.org/10.1016/j.yexmp.2018.12.008
Received 8 October 2018; Received in revised form 2 December 2018; Accepted 29 December 2018
Available online 31 December 2018
0014-4800/ © 2019 Elsevier Inc. All rights reserved.
H.-y. Wang et al. Experimental and Molecular Pathology 106 (2019) 149–156

association with the prognosis of the patient (Schiffman and Solomon, a 1-year period (2014–2015) at Yonsei University Wonju Severance
2009; Persson et al., 2012). Therefore, efficient biomarkers and new Christian Hospital, Wonju, Republic of Korea. This study was approved
screening methods for diagnosing cervical lesions are required to im- by the Institutional Ethics Committee at Yonsei University Wonju
prove the treatment of cervical cancer (Fan and Shen, 2018). College of Medicine, and all patients provided written informed con-
Due to the importance of HPV infection in the diagnosis of cervical sent. All clinical samples were collected using ThinPrep® (Hologic, Inc.,
cancer, the upregulation of HPV oncoproteins has been studied as a Bedford, MA, USA) Pap materials containing PreservCyt solution. For
marker for increased risk of cervical cancer (Ratnam et al., 2010). Re- liquid-based cytology slides (Pap smears) were carried out according to
cently, commercialized diagnostic kits, such as APTIMA HPV assay, the manufacturer's recommendations. After preparing the Pap slide,
Hologic; HPV OncoTect 3Dx™, IncellDx; Nuclisens EasyQ HPV E6/E7 1 mL of an exfoliated cervical cell sample was transferred to each of the
mRNA, Biomerieux, for cervical cancer screening based on HPV E6/E7 two 1.5-mL microcentrifuge tubes and stored at −70 °C until use. The
mRNA expression has been introduced (Coquillard et al., 2011; clinical diagnosis was performed by cytopathologists using the 2001
Binnicker et al., 2014; Munkhdelger et al., 2014) and has shown pro- Bethesda System terminology (Solomon et al., 2002). Cytological cases
mise in increasing the specificity of HPV testing (Lie and Dristensen, of squamous cell carcinoma (SCC), high-grade squamous intraepithelial
2008). Previous studies have reported that detection of these mRNA lesion (H-SIL), low-grade SIL, typical squamous cells of undetermined
targets is more specific than detection of corresponding DNA targets significance (ASC-US), and normal (includes reactive change due to
and can predict women who would progress toward high-grade cervical inflammation, fungal infection, and atrophy) were included. The re-
cancer (Dockter et al., 2009). Expression of HPV E6/E7 mRNA is es- maining fluid samples were stored at 4 °C after cytology slide pre-
sential for the development and progression of cervical cancer. The E6 paration, but before RNA extraction.
and E7 proteins stimulate the cell cycle by binding to and inactivating
cellular p53 and pRb, leading to their degradation via the proteasomal 2.4. Total RNA isolation
pathway and, ultimately, to cervical cancer (McLaughlin-Drubin and
Münger, 2009; Brimer and Vande Pol, 2014). These two oncogenes are After cytological slide preparation, a 3 ml of remaining fluid sample
uniformly retained and highly expressed in cervical cancer cells, and was used for RNA isolation. Total cellular RNA was isolated using the
their continuous expression is required for maintaining the tumorigenic High Pure RNA Isolation Kit no. 11828665001 (Roche Diagnostics,
phenotype. Mannheim, Germany) according to the manufacturer’s instructions.
As women infected with HPV 16 and/or HPV 18 are more likely to Briefly, 400 μL of lysis/binding buffer was added to the cell pellet. Cells
develop cervical intraepithelial neoplasia grade 2 or worse lesions than were lysed by vortexing or repeated pipetting and allowed to stand at
women infected with other HR-HPV genotypes (Bulk et al., 2007), the 25 °C for 15 s. The mixture was transferred to a High Pure filter tube
HPV OncoCheck—based on HPV E6/E7 mRNA expression—is an assay and centrifuged at 8,000 × g for 15 s. Add 500 μL of Wash buffer I and
that can distinguish between the HPV 16 and HPV 18 genotypes and II to the upper reservoir of the filter tube assembly and was centrifuged
can detect 16 HR-HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, at 8,000 × g for 15 s, respectively. Finally, 200 μL of Wash buffer II was
56, 58, 59, 66, 68, and 69) added in 12 HR-HPV types classified as IARC added to the upper reservoir of the filter tube, centrifuge 13,000 × g for
carcinogenicity group 1 (Bouvard et al., 2009; IARC, 2012). Further- 2 min to remove any residual Wash buffer, discard the collection tube,
more, the HPV OncoCheck assay includes an internal control (IC) in and was transferred to a new tube. The RNA pellet was dried and eluted
each well to determine whether samples contain PCR inhibitors. In the in 50 μL of elution buffer to the upper reservoir. The purity and con-
present study, we assessed the analytical and clinical performance of centration of total RNA were determined by measuring the absorbance
the HPV OncoCheck assay (Optipharm, Osong, Republic of Korea) as a at 260 and 280 nm using an Infinite 200® spectrophotometer (Tecan,
potential cervical cancer screening method using multiplex reverse Vienna, Austria). All steps in the preparation and handling of total RNA
transcriptase-quantitative PCR (RT-qPCR). were conducted in a laminar flow hood under RNase-free conditions.
The isolated total RNA was stored at −70 °C until use.
2. Materials and methods
2.5. cDNA synthesis
2.1. HPV single strand DNA control
cDNA was synthesized using the PrimeScript™ RT Master Mix
HPV single strand (ss) DNA was prepared as the positive control to (Perfect Real Time; TAKARA Bio, Shiga, Japan), which was designed to
determine the analytical sensitivity and specificity of the HPV perform reverse transcription (RT) optimized for two-step RT-qPCR,
OncoCheck assay. It was synthesized by Integrated DNA Technologies according to the manufacturer's recommendations. Briefly, 16 μL of
(IDT; Coralville, IA) with each of the 16 HPV genotypes at E6 and E7 total RNA was added to the master mix comprising 4 μL of the 5× pre-
regions using GenBank BLAST and was adjusted to 1 × 1012 copies with mixed reagent containing all components needed for RT-qPCR
TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8.0). (PrimeScript RTase, RNase inhibitor, random 6-mers, oligo dT primer,
dNTP mixture, and reaction buffer) in PCR tubes. The cDNA synthesis
2.2. Cell lines reaction was performed at 37 °C for 15 min (reverse transctiption) and
85 °C for 5 s (for heat inactivation of RT).
SiHa, HeLa, and C33A cell lines were purchased from American
Type Culture Collection (ATCC; Manassas, USA) and Korean Cell Line 2.6. HR-HPV E6/E7 mRNA assay
Bank (Seoul, Republic of Korea). SiHa and HeLa cells were cultured in
Dulbecco's modified Eagle medium (DMEM) supplemented with 10% Detection of HPV E6/E7 mRNA in cervical samples was performed
fetal bovine serum (FBS) and 1% Penicillin/Streptomycin. C33A cells with HPV OncoCheck (Optipharm), a quantitative reverse transcriptase-
were cultured in MEM with Earle's BSS and 10% FBS. Cells were PCR (RT-qPCR)-based assay, using the CFX-96 real-time PCR system
counted using the T20™ Automated cell counter (Bio-Rad, Hercules, (Bio-Rad, Hercules, CA, USA) for thermocycling and fluorescence de-
USA) according to the manufacturer's instructions, and each cell was tection. The assay consisted three different sets of HR-HPVs and de-
measured as 106 cells/mL. tected 16 HR-HPV genotypes in three tubes [Group I: HPV16 (FAM),
HPV 31, 33, 35, 52, and 58 (HEX), and internal control (IC) (Cy5);
2.3. Clinical samples and cytological diagnosis Group II: HPV 18 (FAM), HPV 39, 45, 51, and 68 (HEX), and IC (Cy5);
and Group III: HPV 53, 56, 59, 66, and 69 (HEX), and IC (Cy5)] by
Liquid-based cytology samples from 319 women were obtained over incorporating specific TaqMan probes labeled with different

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Fig. 1. Scheme of the HPV OncoCheck assay.

fluorophores (Fig. 1). The results can be also visualized separately for negative predictive value (NPV), and 95% confidence interval (CI) of
each group. Each real-time PCR kit contained IC DNA, which was used predictive ability were estimated. The Agreement between the HPV
to indicate successful nucleic acid extraction, the quality of the sample, OncoCheck assay and sequence analysis results was calculated using
and to check for the presence of PCR inhibitors in the reaction. IC DNA Cohen's kappa coefficients (k), which were classified as follows:
was designed to have minimal sequence similarity to the target gene 0.00–0.19, poor; 0.20–0.39, fair; 0.40–0.59, moderate; 0.60–0.79,
and served to detect false-negatives. Therefore, it did not directly strong; 0.80–1.00, excellent (Estrade and Sahli, 2014). Significance
compete with the amplification of the species-specific target in multi- between proportions was assessed with McNemar's test. p-values < .05
plex real-time PCR. When no target was detected, positive detection of were statistically significant.
IC DNA indicated the absence of PCR inhibition and that the con-
centration of target in the sample was below the detection limit. The IC
signal showed a constant value under the following conditions: suc- 3. Results
cessful nucleic acid extraction, satisfactory sample quality, absence of
PCR inhibition. Real-time PCR amplification of HPV E6/E7 mRNA was 3.1. Analytical sensitivity of the HPV OncoCheck assay
performed in a reaction mixture containing 10 μL of 2× Thunderbird
probe qPCR mix (Toyobo, Osaka, Japan), 5 μL of primer and TaqMan To determine the analytical sensitivity of the assay, cervical cancer
probe mixture, 2 μL of template cDNA, and distilled water (DW) to a cell lines—HPV 16-infected SiHa, HPV 18-infected HeLa, and HPV-ne-
final volume of 20 μL for each sample. Positive and negative controls gative C33A—were serially diluted 10-fold from 1 × 104 to 1 × 100
were included throughout the procedure. No-template controls con- cells/mL. The analytical sensitivity was estimated as the last serial
taining sterile DW rather than template DNA were incorporated in each linear concentration that yielded positive results in all five replicates,
run. Each sample was tested in duplicate, and all PCR runs were per- and the corresponding Ct value was selected as the analytical cutoff.
formed in duplicate. PCR cycling conditions were as follows: 95 °C for The Cq values for GAPDH with SiHa and Hela cell concentrates
3 min, 40 cycles of 95 °C for 3 s, and 55 °C for 30 s. The mRNA expres- (104–100 cells/mL) ranged from 23.4 ± 0.3 (95%, CI 23–23.8) to
sion level was quantified by determining the cycle threshold (CT), 30.2 ± 0.6 (95%, CI 29.5–30.9) and 21.9 ± 0.1 (95% CI, 21.7–22) to
which is the number of PCR cycles required for the fluorescence to 37.4 ± 0.04 (95% CI, 37.3–37.5), respectively. The detection limit of
exceed a value significantly higher than that of background fluores- the assay for SiHa and HeLa cell lines was 102 cells/mL and 101 cells/
cence. To avoid false-negatives because of mRNA degradation, glycer- mL, respectively (Table 1). For the C33A cell line, all tests of each group
aldehyde-3-phosphate dehydrogenase (GAPDH) was used as the con- of the HPV OncoCheck assay showed negative results. In addition, the
trol, and CT values of ≥33 were excluded. A positive result for Group I, analytical sensitivity of the assay for detecting HPV E6/E7 mRNA was
II, and III was indicated when the CT value was < 35 after observing determined using 10-fold serially diluted (103 copies to 1 copy) control
signal formation of wavelength from each channel. Target gene mRNA HPV ssDNA obtained from all 16 HR-HPV genotypes. The analytical
expression levels relative to GAPDH were automatically calculated sensitivity was estimated as the last serial linear concentration that
using the comparative quantification cycle values (Cq) method using yielded positive results in all 20 replicates, and the corresponding Ct
the CFX Manager Software v3.0 (Bio-Rad). value was selected as the analytical cutoff. HPV 51, 53, and 68 were
detected at a concentration of 1 copy per reaction; HPV 56 and 58, at
2.7. Sequence analysis 100 copies per reaction; and other HPV-type ssDNA, at 10 copies per
reaction. The CV of this assay was below 3%, and the mean Cq value
To confirm HR-HPVs identified by the HPV OncoCheck assay, HPV ranged from 25 ± 0.4 (95% CI, 24.8–25.2) to 34.6 ± 0.5 (95% CI,
E6/E7 gene regions were sequenced. The primer sets used to amplify 34.4–34.8) (Table 2).
the target HPV E6/E7 gene were 60F-5′-CCGAAAMCGGTKVRTATAA-
AAGCA-3′ and 970R-5′-GTACCTKCWGGATCAGCCAT-3′. Amplified
PCR products were sequenced using the ABI Prism BigDye Terminator 3.2. Analytical specificity of the HPV OncoCheck assay
and ABI 3730 automated DNA sequencer (Cosmo Genetech, Seoul,
Republic of Korea). The obtained sequences were compared with se- To assess the potential cross-reactivity with other bacteria and
quences of the National Center for Biotechnology Information GenBank viruses, which include normal flora or sexually-transmitted infectious
database for species assignment. agents present in the female genitalia, analytical specificity was per-
formed with 23 bacteria and 27 viruses exhibiting 16 HR-HPV geno-
2.8. Statistical analysis types. At medically significant concentrations, 106 colony forming units
(CFU)/mL for bacteria and 105 plaque forming units (PFU)/mL for virus
Statistical analysis was performed using GraphPad Prism software were mixed with HPV C33A cell line at 106 cells/mL. Specificity test
version 5.02 (GraphPad, Inc., La Jolla, CA, USA) and SPSS software results of the assay showed that groups I, II, and III, which allowed
version 18.0 (SPSS, Inc., Chicago, IL, USA). For the HPV OncoCheck HPV-specific probes in three tubes, were detected accurately without
assay, the sensitivity, specificity, positive predictive value (PPV), cross-reactivity (Table 3).

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Table 1
Analytical sensitivity of the HPV OncoCheck assay using cervical cell lines.
Cell lines Cells/mL⁎, Cq indicates (95% CI)

104 cells 103 cells 102 cells 101 cells 1 cell

SiHa GAPDH 23.4 (23.0–23.8) 25.8 (25.6–26.4) 27.8 (27.5–28.1) 30.2 (29.5–30.9) N/A
(n = 10) CV (%) 1.4 0.8 0.8 2
HPV 16 27.9 (27.2–28.7) 29.9 (29.3–30.5) 32.4 (31.7–33.0) 38.1 (37.5–38.7) N/A
CV (%) 2.8 2.1 2.2 1.6
HeLa GAPDH 21.9 (21.7–22.0) 25 (24.9–25.1) 28.7 (28.2–29.2) 30.4 (30.0–31.4) 37.4 (37.3–37.5)
(n = 10) CV (%) 0.6 0.3 1.5 1.5 0.1
HPV 18 22.5 (22.2–22.9) 25.5 (25.3–25.7) 28.5 (28.3–28.7) 33.4 (32.9–33.8) 37.2 (37–37.4)
CV (%) 1.4 0.7 0.5 1.2 0.5

Abbreviations: *, assumes that each SiHa and HeLa cell contains approximately 1–2 copies and 10–50 copies, respectively, of integrated HPV 16 and 18 DNA [19];
Cq, mean of the quantification cycle in five repeated tests; 95% CI, 95% confidence interval; CV, coefficient of variation; N/A, not amplified.

3.3. Results of the HPV OncoCheck assay with cervical samples (24.8 ± 2.5, 95% CI, 24.5–25.1). The positive Cq values for each group
detected by the HPV OncoCheck assay ranged from 19.1 to 34.1
The age of the women who participated in the study ranged from 21 (28.5 ± 4.4, 95% CI, 27.4–29.6) for HPV 16, 17.6 to 33.5 (26.9 ± 4.4,
to 87 years with a mean age of 44 ± 12.5 years (95% CI, 42.5–45.5). 95% CI, 24.4–29.4) for HPV 18, 17.9 to 33.8 (28.6 ± 4.5, 95% CI,
To evaluate the HPV OncoCheck assay performance, 319 cervical 27.4–29.8) for groups I, 19.8 to 31.5 (25.9 ± 3.3, 95% CI, 23.9–27.9)
samples were used, and the results were compared with those of cyto- for group II, 13.6 to 30.2 (22.4 ± 5.2, 95% CI, 20.5–24.3) for group III,
logical diagnosis. Of the 319 cytologically diagnosed cases, the HR-HPV and 15.0 to 32.0 (23.3 ± 5.0, 95% CI, 20.9–25.6) for multiple infec-
positive rates determined by the HPV OncoCheck assay were 94.7% (36 tion, respectively (Table 4).
of 38) in SCC cases, 93.8% (60 of 64) in HSIL cases, 61.4% (35 of 57) in
LSIL cases, 38.9% (37 of 95) in ASC-US cases, and 4.6% (3 of 65) in
normal cases. Nine cases of multiple infections were detected among 3.4. Comparison of the results between the HPV OncoCheck assay and
the positive samples, with eight LSIL cases and one ASCUS case sequence analysis for HR-HPV genotype identification of the cervical
(Table 4). All 319 ThinPrep® Pap samples were positive for GAPDH samples
mRNA expression, and the Cq values ranged from 19.4 to 31.7
In order to confirm the results obtained from the HPV OncoCheck

Table 2
Detection limits analysis for the 16 high-risk HPV types.
HPV type Copy number of HPV ssDNA per reaction

103 copies 102 copies 10 copies 1 copy

HPV 16 Cq avg. (95% CI) 25 (24.8–25.2) 28.4 (28.3–28.5) 32 (31.9–32.1) 35.5 (35.3–35.7)
CV (%) 1.4 0.7 0.7 1
HPV 18 Cq avg. (95% CI) 25.9 (25.8–25.9) 30.5 (30.2–30.8) 34.1 (33.9–34.3) 38 (37.7–38.3)
CV (%) 0.0 2 1.3 1.8
HPV 31 Cq avg. (95% CI) 27.7 (27.5–27.9) 31.4 (31.3–31.5) 34.3 (34.2–34.4) 37.6 (37.4–37.8)
CV (%) 1.7 0.7 0.9 1.3
HPV 33 Cq avg. (95% CI) 26.2 (26.1–26.3) 30.6 (30.5–30.7) 34 (33.9–34.1) 38.7 (38.5–38.8)
CV (%) 0.9 0.8 0.7 1.0
HPV 35 Cq avg. (95% CI) 25.8 (25.7–25.9) 29.3 (29.2–29.4) 32.7 (32.6–32.8) 38.3 (37.7–38.9)
CV (%) 1 0.9 0.6 3.7
HPV 39 Cq avg. (95% CI) 26.8 (26.7–26.9) 31.1 (30.9–31.3) 34.6 (34.4–34.8) N/A
CV (%) 1.2 1.5 1.4
HPV 45 Cq avg. (95% CI) 26.1 (25.8–26.3) 28.8 (28.4–29.2) 33.9 (33.7–34.1) 36.4 (36.2–36.6)
CV (%) 0.8 2.6 1.5 1.4
HPV 51 Cq avg. (95% CI) 25.5 (24.7–26.4) 27.9 (26.3–27.6) 30.2 (29.9–30.5) 33.4 (33.1–33.8)
CV (%) 2.3 1.8 2.1 1.4
HPV 52 Cq avg. (95% CI) 27.4 (27.3–27.5) 30.5(30.4–30.5) 33.7 (33.6–33.8) 37.1 (36.9–37.3)
CV (%) 0.7 0.5 0.8 1.1
HPV 53 Cq avg. (95% CI) 24.7 (24.4–24.9) 27.9 (27.6–28.2) 31.3 (31.2–31.4) 34.8 (34.6–35.0)
CV (%) 0.8 2.1 1 0.0
HPV 56 Cq avg. (95% CI) 30.5 (30.4–30.6) 33.9 (33.8–34.0) 37.5 (37.2–37.8) N/A
CV (%) 0.8 0.7 1.5
HPV 58 Cq avg. (95% CI) 28.7 (28.6–28.8) 32.1 (32.0–32.2) 36.1 (35.9–36.3) 35.4 (33.5–37.2)
CV (%) 0.7 0.6 1 2.7
HPV 59 Cq avg. (95% CI) 24.4 (24.3–24.4) 26.8(26.5–27.0) 30.1 (30.0–30.2) 36.0 (35.8–36.2)
CV (%) 0.1 0.7 0.1 1.3
HPV 66 Cq avg. (95% CI) 25.2 (25.1–25.3) 28.6 (28.5–28.7) 32 (31.9–32.1) 36.1 (35.8–36.4)
CV (%) 1.2 1.2 0.6 1.8
HPV 68 Cq avg. (95% CI) 26.8 (26.3–27.3) 27.9 (27.7–28.2) 29.3 (29.3–29.4) 33.2 (32.7–33.6)
CV (%) 1.4 0.7 0.1 1.0
HPV 69 Cq avg. (95% CI) 24.7 (24.6–24.8) 27.0 (26.4–27.7) 31.4 (31.4–31.6) 35.4 (35.2–35.6)
CV (%) 0.3 1.7 0.3 1.2

Abbreviations: HPV ssDNA, HPV single strand DNA; Cq avg., average of the quantification cycle in 20 repeated tests; 95% CI, 95% confidence interval; CV, coefficient
of variation; N/A, not amplified.

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Table 3
Analytical specificity of the HPV OncoCheck assay.
Bacteria (106 CFU/mL) ATCC no. Positive % (n/total) Viruses (105 PFU/mL) ATCC no. Positive % (n/total)

Group I Group II Group III Group I Group II Group III

Chlamydia trachomatis VR-573 100 (0/15) 100 (0/15) 100 (0/15) Herpes simplex virus 1 VR-1493 100 (0/15) 100 (0/15) 100 (0/15)
Gardnerella vaginalis 14018 100 (0/15) 100 (0/15) 100 (0/15) Herpes simplex virus 2 VR-734 100 (0/15) 100 (0/15) 100 (0/15)
Ureaplasma urealyticum 27618 100 (0/15) 100 (0/15) 100 (0/15) Cytomegalovirus VR-677 100 (0/15) 100 (0/15) 100 (0/15)
Neisseria gonorrhoeae 49226 100 (0/15) 100 (0/15) 100 (0/15) Adenovirus 2 VR-846 100 (0/15) 100 (0/15) 100 (0/15)
Mycoplasma hominis 23114 100 (0/15) 100 (0/15) 100 (0/15) Adenovirus 5 VR-1516 100 (0/15) 100 (0/15) 100 (0/15)
Bacteroides fragilis 25285 100 (0/15) 100 (0/15) 100 (0/15) HPV 16 45113D 0 (15/15) 100 (0/15) 100 (0/15)
Bifidobacterium breve 15700 100 (0/15) 100 (0/15) 100 (0/15) HPV 31 65446 0 (15/15) 100 (0/15) 100 (0/15)
Fusobacterium mortiferum 25557 100 (0/15) 100 (0/15) 100 (0/15) HPV 33 Clinical isolate 0 (15/15) 100 (0/15) 100 (0/15)
Staphylococcus aureus 29213 100 (0/15) 100 (0/15) 100 (0/15) HPV 35 40330 0 (15/15) 100 (0/15) 100 (0/15)
Staphylococcus epidermidis 12228 100 (0/15) 100 (0/15) 100 (0/15) HPV 52 Clinical isolate 0 (15/15) 100 (0/15) 100 (0/15)
Streptococcus faecalis 29221 100 (0/15) 100 (0/15) 100 (0/15) HPV 58 Clinical isolate 0 (15/15) 100 (0/15) 100 (0/15)
Streptococcus pyogenes 12344 100 (0/15) 100 (0/15) 100 (0/15) HPV 18 Clinical isolate 100 (0/15) 0 (15/15) 100 (0/15)
Streptococcus agalactiae 13813 100 (0/15) 100 (0/15) 100 (0/15) HPV 39 Clinical isolate 100 (0/15) 0 (15/15) 100 (0/15)
Lactobacillus acidophilus 4356 100 (0/15) 100 (0/15) 100 (0/15) HPV 45 Clinical isolate 100 (0/15) 0 (15/15) 100 (0/15)
Corynebacterium diphtheriae 11913 100 (0/15) 100 (0/15) 100 (0/15) HPV 59 Clinical isolate 100 (0/15) 0 (15/15) 100 (0/15)
Enterococcus faecalis 29212 100 (0/15) 100 (0/15) 100 (0/15) HPV 68 Clinical isolate 100 (0/15) 0 (15/15) 100 (0/15)
Peptostreptococcus anaerobius 27337 100 (0/15) 100 (0/15) 100 (0/15) HPV 51 Clinical isolate 100 (0/15) 100 (0/15) 0 (15/15)
Escherichia coli 25922 100 (0/15) 100 (0/15) 100 (0/15) HPV 53 Clinical isolate 100 (0/15) 100 (0/15) 0 (15/15)
Enterobacter cloaceae 13047 100 (0/15) 100 (0/15) 100 (0/15) HPV 56 40549 100 (0/15) 100 (0/15) 0 (15/15)
Pseudomonas aeruginosa 27853 100 (0/15) 100 (0/15) 100 (0/15) HPV 66 Clinical isolate 100 (0/15) 100 (0/15) 0 (15/15)
Klebsiella oxytoca 700324 100 (0/15) 100 (0/15) 100 (0/15) HPV 69 Clinical isolate 100 (0/15) 100 (0/15) 0 (15/15)
Proteus vulgaris 49132 100 (0/15) 100 (0/15) 100 (0/15) HPV 6 Clinical isolate 100 (0/15) 100 (0/15) 100 (0/15)
Candida albicans 36802 100 (0/15) 100 (0/15) 100 (0/15) HPV 11 Clinical isolate 100 (0/15) 100 (0/15) 100 (0/15)
HPV 40 Clinical isolate 100 (0/15) 100 (0/15) 100 (0/15)
HPV 54 Clinical isolate 100 (0/15) 100 (0/15) 100 (0/15)
HPV 84 Clinical isolate 100 (0/15) 100 (0/15) 100 (0/15)
HPV 87 Clinical isolate 100 (0/15) 100 (0/15) 100 (0/15)

Abbreviations: CFU, Colony forming units; ATCC, American type culture collection; PFU, Plaque forming units.

assay, sequence analysis was performed using the same cervical sam- could be included in the cervical cytology fluid. In nine substances,
ples (Table 5). The results of the HPV OncoCheck and sequence analysis except for blood and EDTA, no interference was observed at the highest
methods were all concordant for all but four cases. Of the four dis- tested concentration (10%), but interference was observed at 10% with
crepant cases, three cases (group I, II, and III) detected by the HPV blood and 20 mM EDTA. (ii) Repeatability and reproducibility of this
OncoCheck assay were not identified by sequencing, and the case that assay was performed with a total of 144 intra-laboratory test
was not detected by the HPV OncoCheck assay was identified as HPV 16 (12 days × 2 runs/day × 2 replicates × 3 lots). These results showed
by sequence analysis (Table 5). that the intra-laboratory reproducibility over time was 95.1% (κ = 0.9,
95% CI, 0.832–0.973), and CV of the assay was below 3% (data not
shown). These results suggested that the assay might yield stable re-
4. Discussion
sults.
This study evaluated the positive rates of the cervical lesions that
The purpose of this study was to evaluate the analytical and clinical
were cytologically diagnosed based on the Bethesda system using the
performance of the HPV OncoCheck assay for the detection of HPV 16
HPV OncoCheck assay; the overall positive rates were the highest for
and 18 genotypes as well as 14 HR-HPVs (HPV 16, 18, 31, 33, 35, 39,
high-grade lesions (SCC and HSIL), followed by low-grade lesion (LSIL,
45, 51, 52, 53, 56, 58, 59, 66, 68, and 69) in bulks in cervical cancer
ASCUS, and normal). During comparative analysis of HR-HPV, a good
screening and describe HPV E6/E7 mRNA as an early biomarker with
agreement (94.6%, κ = 0.89, 95% CI, 0.816–0.959) was observed be-
cytological diagnosis. To assess the performance and accuracy of the
tween the results of the HPV OncoCheck assay and high-grade cytology
HPV OncoCheck assay, a synthesized HPV ssDNA was used as the re-
diagnosis. The clinical sensitivity, specificity, PPV, and NPV of the HPV
ference standard and Cq values were determined in 20 replicate mea-
OncoCheck assay for detecting high-grade lesions were 94.1% (95% CI,
surements. As all of the 16 HR-HPV genotypes were detected at 10–100
0.875–0.975, p < .0001), 95.4% (95% CI, 0.868–0.989, p < .0001),
copies per reaction, the detection limit of the assay was determined to
97% (95% CI, 0.911–0.993, p < .0001), and 91.2% (95% CI,
be 100 copies per reaction. These results are similar to those obtained
0.817–962, p < .0001), respectively. These results are similar to a
for the cervical cell lines (10–100 cells) as SiHa and HeLa cell lines
previously reported sensitivity of 91.9–92.6% and specificity of
contain 1–2 and 10–50 copies, respectively, of HPV 16 and 18 DNA/cell
98.5–98.6% with the APTIMA HPV assay (Dockter et al., 2009) and
(Mincheva et al., 1987). The assay was tested using bacterial and viral
sensitivity of 84–91% and specificity of 85–96% with the InCellDx
specimens in combination with HPV C33A cell lines. Consequently, no
Oncotect E6/E7 mRNA assay (Coquillard et al., 2011). However, the
cross-reaction was observed between the specimens, demonstrating that
presently reported sensitivity and specificity were higher than the
the presence of other samples in HPV-infected endocervical specimens
previously reported sensitivity of 91.8–92.1% and specificity of
did not affect the performance of the assay. In addition to sensitivity
39–45.6% with the Luminex XMAP (Zhao et al., 2017). Previous studies
and specificity, we also conducted analytical performance tests of in-
have reported an upregulation in E6 and E7 mRNA levels with lesion
terference and reproducibility: i) For interfering reactions, we used the
severity; therefore, the detection of HPV E6/E7 mRNA may be of higher
following 11 substances—PreservCyt Solution, five vaginal tablets
prognostic value and may improve specificity and PPV compared with
(Nowon Vaginal Suppositories, Ovestin Vaginal Suppositories, Gynoflor
those by cytological analysis and HPV DNA testing, which are used in
Vaginal Tab, Gynobetadine Vaginal Suppositories, and Crinone vag
traditional cervical cancer screening (Castle et al., 2007; Benevolo
gel), two antifungal agents (Canesten Cream and Polygynax), a lu-
et al., 2011; Duvlis et al., 2015). Our results also showed that the HPV
bricant (Surgytube; Clear Transparent Jelly), blood, and EDTA that

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Table 4
HPV OncoCheck assay results for the discrimination of the 16 HR-HPV genotypes, including HPV 16 and 18, in 172 cytologically diagnosed samples.
HPV OncoCheck assay Range of IC SCC (n = 36) HSIL (n = 60) LSIL ASCUS (n = 37) Normal (n = 3) Total
Cq range (mean ± SD) (n = 35) (n = 172)

n (%) Cq range n (%) Cq range n (%) Cq range n (%) Cq range n (%) Cq range
(mean ± SD) (mean ± SD) (mean ± SD) (mean ± SD)

HPV 16 21.3–31.7 (25.4 ± 2.9) 22 (61.1) 24.3–34.1 22 (36.7) 19.1–33.9 3 (8.6) 22.9–31.6 9 (24.3) 20.2–28.5 1 (33.3) 27.73 57 (33.1)
(28.7 ± 3.3) (28.7 ± 4.1) (28.1 ± 4.6) (27.3 ± 6.7)
HPV 18 22.0–27.9 (26.1 ± 2.1) 3 (8.3) 26.1–29.2 3 (5.0) 21.8–33.5 2 (5.7) 17.6–31.5 3 (8.1) 27.2–31.8 1 (33.3) 25.02 12 97.0)
(27.3 ± 1.7) (29.6 ± 6.2) (24.5 ± 9.8) (28.9 ± 2.4)
Group I 19.4–30.3 (25.3 ± 2.5) 11 (30.6) 25.7–33.2 32 (53.3) 17.9–33.8 5 (14.3) 16.4–27.7 5 (13.5) 23.1–32.6 53 (30.8)
(29.8 ± 2.1) (29.5 ± 4.5) (22.6 ± 4.5) (27.6 ± 3.9)
Group II 21.2–25.7 (22.9 ± 1.6) 4 (11.4) 19.8–31.5 7 (18.9) 23.5–30.0 11 (6.4)

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(25.2 ± 4.9) (26.3 ± 2.3)
Group III 21.1–26.4(24.1 ± 2.4) 3 (5.0) 26.3–30.2 13 (37.1) 13.6–27.1 13 (35.1) 13.2–27.1 1 (33.3) 27.46 30 (17.4)
(28.9 ± 2.2) (20.7 ± 4.6) (22.3 ± 5.5)
Multiple infection 21.7–25.5 (23.2 ± 1.3) 9 (5.2)
(n = 9)
HPV 16 + group III 21.7–24.9 (23.6 ± 1.6) 3 (8.6) 21.3–32.0 3 (1.7)
(25.8 ± 3.9)
HPV 18 + group III 21.9–25.5(23.1 ± 1.6) 4 (11.4) 15.0–31.3 4 (2.3)
(21.7 ± 5.6)
Group I + group III 22.7–23.3 (22.9 ± 0.4) 1 (2.9) 16.4–22.5 1 (12.5) 26.6–26.9 1 (1.2)
(19.4 ± 4.3) (26.8 ± 0.2)

Abbreviations: HR-HPV, high-risk human papillomavirus; SCC, squamous cell carcinoma; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; ASCUS, atypical squamous
cells of undetermined significance; Cq, quantification cycles; The assay consisted three different sets of HR-HPVs [Group I: HPV16 (FAM), HPV 31, 33, 35, 52, and 58 (HEX), and internal control (IC) (Cy5); Group II: HPV
18 (FAM), HPV 39, 45, 51, and 68 (HEX), and IC (Cy5); and Group III: HPV 53, 56, 59, 66, and 69 (HEX), and IC (Cy5)] by incorporating specific TaqMan probes labeled with different fluorophores.
Experimental and Molecular Pathology 106 (2019) 149–156
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Table 5
Comparison of the HPV OncoCheck assay and sequence analysis for the detection of the HR-HPV genotypes in 319 cervical samples.
HPV OncoCheck assay No (%) of Sequence analysis, no. (%) Sensitivity (%) 95% CI Specificity (%) 95% CI
samples
HPV Consistent Not detected Discrepant
genotypes results result

HR-HPV positive 172 (53.9) 169 (98.3) 3 (1.7) 98.3 0.948–0.996 100 0.978–1.000
Single infection 163 (94.8) 160 (98.2) 3 (1.8) 98.3 0.945–0.996 100 0.979–1.000
HPV 16 57 (35.0) HPV 16 57 (35.0) 100 0.946–1.000 100 0.970–1.000
HPV 18 12 (7.4) HPV 18 12 (7.4) 100 0.784–1.000 100 0.979–1.000
Group I 53 (32.5) 52 (98.1) 1 (1.9)* 98.1 0.891–1.000 100 0.971–1.000
HPV 33 20 (37.7)
HPV 58 13 (24.5)
HPV 52 9 (17.0)
HPV 35 6 (11.3)
HPV 31 4 (7.5)
Group II 12 (7.4) 11 (91.7) 1 (8.3)* 91.7 0.625–0.999 100 0.979–1.000
HPV 68 8 (66.7)
HPV 39 3 (25.0)
Group III 29 (17.8) 28 (99.6) 1 (3.4)* 99.6 0.814–0.999 100 0.976–1.000
HPV 53 12 (41.4)
HPV 51 7 (24.1)
HPV 56 7 (24.1)
HPV 69 2 (6.9)
Multiple infection 9 (5.2) 100 0.731–1.000 100 0.979–1.000
HPV 16 + group III 3 (33.3) HPV 16, 53 2 (66.7)
HPV 16, 66 1 (33.3)
HPV 18 + group III 4 (44.4) HPV 18, 53 3 (75.0)
HPV 18, 66 1 (25.0)
Group I + group III 2 (22.2) HPV 35, 53 1 (50.0)
HPV 52, 53 1 (50.0)
HR-HPV negative 147 (46.1) HPV 16 146 (99.3) 1 (0.7)† 99.3 0.959–0.999 99.3 0.959–0.999
Total 319 (100) 315 (98.8) 3 (0.9) 1 (0.3) 98.8 0.967–0.996 99.7 0.981–0.999

Abbreviations: HR-HPV, high-risk human papillomavirus; 95% CI, 95% confidence interval; *, three cases detected by the HPV OncoCheck assay were not identified
by sequencing; †, the one case not detected by the HPV OncoCheck assay was identified as HPV 16 by sequence analysis; The assay consisted three different sets of
HR-HPVs [Group I: HPV16 (FAM), HPV 31, 33, 35, 52, and 58 (HEX), and internal control (IC) (Cy5); Group II: HPV 18 (FAM), HPV 39, 45, 51, and 68 (HEX), and IC
(Cy5); and Group III: HPV 53, 56, 59, 66, and 69 (HEX), and IC (Cy5)] by incorporating specific TaqMan probes labeled with different fluorophores.

OncoCheck assay yielded a significantly lower rate of positive results rapid method with a turnaround time that usually requires 3 h, in-
(4.6% for HPV OncoCheck vs. 27.7% for HPV microarray or 10.8% for cluding 1.5 h for RNA preparation and cDNA synthesis and 1.5 h for
REBA HPV-ID; p < .05) in patients with normal cytology than those target amplification. It allowed for the rapid identification of 16 HR-
with the HPV DNA test (data not shown). These results suggest that the HPV genotypes, including HPV 16 and 18, without post-PCR proces-
selective targeting used in the HPV OncoCheck assay can reduce the sing.
positive detection rate of HPV infections in women with normal or low- The potential limitations of our study were the relatively small
grade cytology lesions. Because HPV OncoCheck is an assay based on sample size and no information about the histological diagnosis of the
E6/E7 mRNA, the accuracy of the assay was compared with sequence samples. Therefore, it will be necessary to determine whether the HPV
analysis. Sensitivity and specificity between the two assays were 99.4% OncoCheck assay shows similar analytical performance and efficacy in
(95% CI, 0.965–0.999, p < .0001) and 98% (95% CI, 0.939–0.996, detecting HPV with a larger number of samples confirmed by histolo-
p < .0001), respectively. We also found that the agreement between gical diagnosis.
the HPV OncoCheck assay and sequence analysis for the detection of In conclusion, the HPV OncoCheck assay generally showed high
HR-HPV was 98.8% (κ = 0.98, 95% CI, 0.967–0.996, p < .0001). Of agreement and specificity with cytological diagnosis and sequence
the four discrepant cases, three were not detected by sequencing results analysis. When similar results are obtained by histological diagnosis,
because of low yields of the PCR product, and lack of matching nu- use of this newly developed molecular diagnostic assay in cervical
cleotide sequences. Additionally, group I (HPV 31, 33, 35, 52, and 58) screening may help to detect the most significant cervical disease while
including HPV 16 genotype was detected in approximately 36% of the reducing false-positive results and has the potential to serve as a sen-
total samples, whereas HPV 18 was detected in approximately 5% of sitive and specific tool for the early detection and diagnosis of cervical
the total samples. The results of the HPV OncoCheck assay were con- cancer.
firmed by sequence analysis. The HPV OncoCheck assay may offer ac-
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