Triage of high-risk human papillomavirus-positive women by methylated POU4F3
© Pun et al. 2015
Received: 4 May 2015
Accepted: 5 August 2015
Published: 21 August 2015
Insufficient specificity of the high-risk human papillomavirus (hrHPV) assay in primary cervical cancer screening results in unnecessary referral. Additional assays to triage hrHPV-positive women are needed to improve molecular cervical cancer screening. DNA methylation is a promising biomarker in cervical cancer. We evaluated the clinical performance of potentially methylated genes as a triage assay for hrHPV-positive women.
We conducted a retrospective hospital-based case–control study in Taiwan. Cervical scrapings were collected before colposcopy for hrHPV testing and quantitative methylation-specific PCR (QMSP) of 16 genes. Five genes, POU4F3, HS3ST2, AJAP1, PAX1, and SOX1, were prioritized for the clinical performance to triage hrHPV-positive women. Two hundred cervical scrapings were randomly classified into a training set (n = 111) and testing set (n = 89). All samples were tested for hrHPV using a Hybrid Capture II (HCII) assay. HrHPV-positive women were subjected to DNA methylation analysis by QMSP. In the training set, the receiver operating characteristic (ROC) curves defined the optimal methylation index (M-index) cutoff values for discriminating CIN3+ from CIN1/normal, which then were applied to the testing set. Among the five genes, POU4F3 revealed the highest area under the ROC curve (AUC) (0.86; 95 % CI, 0.78–0.95) in detecting CIN3+. In the testing set, POU4F3 revealed the best clinical performance in triage of hrHPV-positive women with a sensitivity of 74 % and specificity of 89 % for detecting CIN3+.
POU4F3 methylation analysis is a potential molecular tool for triage in detecting CIN3+ in hrHPV-positive women. The combined use of broad-spectrum HPV assay and POU4F3 methylation analysis as a new generation of molecular cervical cancer screening warrants further population-based study.
KeywordsDNA methylation HrHPV test QMSP Biomarker Cervical intraepithelial neoplasia (CIN) Cervical cancer screening
Cervical cancer is a common medical problem in women, with 528,000 new cases and 266,000 deaths globally in 2012 indicating the need to develop and implement an effective cancer screening strategy . The Papanicolaou (Pap) smear for cytological examination has been used for the detection of precancerous cellular abnormalities of cervical cells for decades and has lessened the disease burden by reducing the mortality and morbidity of cervical cancer . The Pap smear or cytology test has high specificity for cervical intraepithelial neoplasia; however, it has many drawbacks such as suboptimal sensitivity  and moderate accuracy to detect relevant lesions and subjective diagnosis of cervical abnormalities with poor reproducibility . Oncogenic high-risk human papillomavirus (hrHPV) infection is a well-known etiology of cervical cancer . Because the duration of the initial hrHPV infection until the development of invasive cancer is long, assay of HPV DNA as a screening tool is appealing [6, 7]. However, HPV infection is transient in nature, and only few infected lesions further progress as invasive cancer . Insufficient specificity of the HPV DNA assay results in a high false-positive rate and extra medical burden because of the consequent high colposcopy referral rate . Findings of HPV-positive assay results also cause adverse psychosocial impact . Therefore, an additional triage assay is required to improve HPV-based molecular cervical cancer screening [10, 11].
Persistent oncogenic hrHPV infection causes genetic and epigenetic changes . Promoter hypermethylation-mediated silencing of tumor suppressor genes is common in cervical carcinogenesis [12, 13]. Because DNA methylation can be easily quantitated using molecular methods, it is gaining attraction as a molecular assay for detecting cervical cancer . Several studies including our group have revealed that numerous aberrantly DNA-methylated cervical cancer-related genes could be potential biomarkers to improve cervical cancer detection [12, 15–17], to triage women with atypical squamous cells [18, 19] and low-grade squamous intraepithelial lesions (LSILs) in Pap smears . Methylated genes could be potential markers for the triage of hrHPV-positive women [21–27]. However, the sensitivity and specificity are not satisfactory even if combining two or more genes [23, 24, 26], highlighting the need for novel methylation biomarkers.
Using methylomic approaches, many methylated candidate genes have been revealed, including ADRA1D, AJAP1, COL6A2, EDN3, EPO, HS3ST2, MAGI2, POU4F3, PTGDR, SOX8, SOX17, ST6GAL2, SYT9, ZNF614 , SOX1, and PAX1 . The performance of these methylated genes to triage hrHPV-positive women remains unexplored.
Selection of potential candidate genes in hrHPV-positive women
Sensitivities and specificities of candidate genes in hrHPV-positive women (N = 67) in the selection set
Generation of methylation cutoff values for triage of hrHPV-positive women in the training set
Histopathology, mean age, and HPV percentage of the patients
Mean ± SD
46.8 ± 13.5
44.2 ± 13.3
Result of pathology
Performance of methylation biomarkers to detect CIN3+ in hrHPV-positive women at training and testing sets
M-index cutoff value
Training set (N = 68)
Testing set (N = 55)
Validation of the clinical performance of methylated genes in the testing set
Clinical performance of methylation biomarker in hrHPV-positive women of testing set stratified by histology
Total number of detectable
Methylation positive/total number (%)
Result of pathology
0/5 (0 %)
0/5 (0 %)
0/5 (0 %)
2/13 (15.4 %)
0/14 (0 %)
5/14 (35.7 %)
15/24 (62.5 %)
8/22 (36.3 %)
17/24 (70.8 %)
11/11 (100 %)
10/11 (90.9 %)
11/11 (100 %)
Previous studies support the concept that DNA methylation could be a potential molecular biomarker for detection of cervical lesions [12, 15, 16, 28, 30]. An ideal methylation biomarker should have better specificity than HPV testing and better sensitivity than cytology when applied as a primary screening tool. Recent studies proposed the alternative role of methylation biomarkers as a triage method for hrHPV-positive women [22–24, 26, 27]. More high-risk HPV genotype detection may have a better chance to include more women at risk for triage in the primary screening. In addition, the distribution of HPV type varies across continents because 16, 31, 33, and 18 are prevalent in Europe, and 16, 58, 52 and 18 are prevalent in the Asia–Pacific region [31, 32]. We used the Hybrid Capture II (HCII) assay for hrHPV testing, which assays 13 high-risk genotypes simultaneously [7, 32, 33]. The present study demonstrated that DNA methylation analysis as a triage for hrHPV-positive women is feasible. The POU4F3 methylation analysis confers the best clinical performance when combined with the HCII assay. In this study, the primary objective was to use broad-spectrum hrHPV testing capable of detecting more susceptible women for further triage with POU4F3 methylation to achieve a better sensitivity. Further hrHPV subtype analysis may clarify type-specific correlation with POU4F3 methylation, which may be useful in estimating the impact of molecular screening strategy using HPV detection followed by methylation triage in post-vaccination era.
POU4F3 is located on chromosome 5q32 and plays various biological functions, such as regulation of transcription, cellular and metabolic processes, organ development, cellular differentiation, nervous system development, neurogenesis, and generation of neurons . The function of POU4F3 in cancer biology remains largely unknown. POU4F3 hypermethylation in cervical cancer and glioma suggests its suppressor role in cancer [28, 34]. This study supports the concept that POU4F3 could be a potential triage biomarker for hrHPV-positive women.
In the present study, we adapted histopathologically diagnosed CIN3+ as the end point because CIN2 is equivocal in nature with a tendency to regress to normal instead of progressing to CIN3+, where the likelihood of CIN2 progression to invasive cancer is only 5 % . Further, diagnosis of CIN2 is much less reproducible than CIN3 because of the difference in the natural history of CIN2 from that of CIN3 . However, CIN3 has a higher tendency to progress to invasive cancer because it is an immediate precursor with a similar virological profile and has better reproducibility . Therefore, it is more appropriate to adapt CIN3+ as a surrogate end point for early diagnosis of cervical cancer.
POU4F3 methylation testing is a potential molecular biomarker for the triage of hrHPV-positive women for CIN3+ lesions. We envision an era of molecular screening for cervical cancer.
We conducted a retrospective case–control study using hospital-based patient samples in the Tri-Service General Hospital, Taiwan, from December 2009 to November 2010. Patients aged ≥20 years referred for a colposcopy and cervical biopsy and who were managed with conization or surgery after biopsy revealing CIN3+ were enrolled in this study. Cervical scrapings for laboratory analysis were collected in sterile phosphate-buffered saline before biopsy using a cervical brush and were stored at 4 °C until DNA extraction for HPV testing using a HCII hrHPV DNA assay (Digene, Silver Spring, MD, USA) and quantitative DNA methylation analysis of potential candidate genes using QMSP. Healthy women undergoing routine Pap screening were selected as controls, only when their Pap smears showed normal pattern. Women with positive or suspicious Pap smears were excluded from control. Before the study, all the subjects were informed about the study and were enrolled after obtaining documented full consent. Final diagnosis regarding different stages of cancer was performed by tissue-proven histopathological examination, except in healthy control women. Exclusion criteria applied in this study were compromised quality of Pap smears, patients previously vaccinated with anti-HPV vaccine, cervical neoplasia or existence of other malignancies, surgery related to the uterine cervix, an immunocompromised state, genital warts, or pregnancy. Further, all specimens were delinked from clinical information after numbering each of them until data analysis. In this study, all the women were tested for HPV infection and only samples from hrHPV-positive women underwent DNA methylation analysis. Cervical scrapings of 67 hrHPV-positive women among 100 recruited women underwent DNA methylation analysis to prioritize candidate genes for further analysis of clinical performance. Cervical scrapings from 200 women recruited for analysis of clinical performance were randomly classified using a random number table as a 1:1 ratio into a training set (n = 111) and a testing set (n = 89). The training set included 46 women with histopathologically confirmed CIN3+ and 65 women with normal/CIN1. Cervical scrapings from 68 hrHPV-positive women of the 111 underwent DNA methylation analysis. Methylation levels in the training set were used to generate optimal M-index cutoff values of candidate genes that can distinguish relevant cancerous cases from control. The clinical performance of candidate genes was validated using the optimal cutoff values in the testing set. The testing set comprised 89 women including 39 women with CIN3+ and 50 women with normal/CIN1. Of the 89 women, cervical scrapings from 55 hrHPV-positive women were analyzed further for quantitative assay of DNA methylation. This study was approved by the Institutional Review Board of the Tri-Service General Hospital, National Defense Medical Center.
Extraction of DNA followed by bisulfite modification
Genomic DNA was extracted as previously described using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations . Those samples with a DNA yield as low as 500 ng or more (>500 ng) as measured by NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE, USA) were considered for further analysis in this study. Bisulfite modification of genomic DNA samples was performed using a CpGenome DNA Modification Kit (Millipore, Temecula, CA, USA) according to the manufacturer’s recommendations, and the samples were dissolved in 70 μL of nuclease-free water . Bisulfite-converted DNA was stored at −80 °C until further use.
Methylation assays of potential candidate genes
QMSP used for analysis of the methylation status of the candidate genes was based on the principle of fluorescence-based real-time PCR. TaqMan-based QMSP amplification was performed on the bisulfite-treated DNA . The type II collagen gene (COL2A) was used as an internal reference. In vitro methylated genomic DNA treated with CpG methyltransferase (M.SssI; New England Biolabs, Beverly, MA, USA) was used as a positive control. While prioritizing potential candidate methylated genes for further performance analysis, QMSP was performed in a TaqMan probe system using an Applied Biosystems 7900HT Fast Real-Time PCR System in a total volume of 20 μL reaction mixture containing 2 μL of bisulfite template DNA, 250 nM of each primer, 225 nM TaqMan probe, and 10 μL of FastStart Universal Probe Master (ROX) (Roche Diagnostics, Roche Applied Science, Mannheim, Germany) . 6-Carboxy-fluorescein was used to label the 5′ end of probes, while a quencher dye (MGB by Applied Biosystems, or BHQ1 by TIB) was used to label the 3′ end of the probes (Additional file 1: Table S1). However, for analysis of clinical performance of candidate genes, QMSP for AJAP1, HS3ST2, and POU4F3 and multiplex QMSP for PAX1 and SOX1 were performed in a TaqMan probe system using a LightCycler 480 Real-Time PCR System (Roche Diagnostics, Roche Applied Science) . Briefly, the total reaction volume of 20 μL contained 2 μL of modified template DNA, 1 μL of 20× Custom TaqMan reagent, and 10 μL LightCycler 480 Probes Master (Roche Diagnostics, Roche Applied Science). A mixture of primers and probes was used for PAX1 and SOX1. The reactions were conducted using an initial incubation at 95 °C for 10 min, followed by 50 cycles of 95 °C for 10 s, and annealing and extension for 40 s at 60 °C (using the thermal cycler protocol in the standard mode). The level of DNA methylation was measured in terms of M-index . Results showing the very high Cp values of COL2A (>36) were defined as detection failures.
HPV DNA assay
The HCII hrHPV DNA assay (Digene) was used as the primary assay in this study following the manufacturer’s protocol to detect hrHPV infection. This HCII assay can detect 13 high-risk HPV subtypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Samples with a ratio of relative light units (RLU)/cutoff value higher than 1.0 were recorded as positive.
ROC curves for each of the candidate genes were calculated using the data from the training set. Optimal M-index cutoff values of the candidate genes were generated from ROC curves and were used to further analyze the clinical performance of the candidate methylated genes in the testing set. Sensitivities, specificities, positive predictive values (PPV), and negative predictive values (NPV) of AJAP1, HS3ST2, POU4F3, PAX1, and SOX1 for detecting CIN3+ were calculated. IBM SPSS Statistics for Windows, version 20.0 (Armonk, NY, USA) was used for all statistical analyses.
- ADRA1D :
adrenoceptor alpha 1D
- AJAP1 :
adherens junctions-associated protein 1
area under the receiver operating characteristic curve
cervical intraepithelial neoplasia
- CIN3+ :
CIN grade 3 or worse
- COL6A2 :
collagen, type VI, alpha 2
- EDN3 :
- EPO :
high-risk human papillomavirus
- HS3ST2 :
heparan sulfate (glucosamine) 3-O-sulfotransferase 2
- MAGI2 :
membrane-associated guanylate kinase, WW and PDZ domain-containing 2
- PAX1 :
paired box gene 1
- POU4F3 :
POU class 4 homeobox 3
- PTGDR :
prostaglandin DP receptor
quantitative methylation-specific PCR
- SOX1 :
sex-determining region Y box 1
- SOX17 :
- SOX8 :
SRY (sex-determining region Y)-box 8
- ST6GAL2 :
ST6 betagalactosamide alpha-2,6-sialyltranferase 2
- SYT9 :
- ZNF614 :
zinc finger protein 614
This work was supported in part by the following grants: grant numbers NSC 102-2628-B-038-010-MY3 from the Ministry of Science and Technology; 103TMU-SHH-11, TMU103-AE1-B06, TMUTOP103005-1, and 104TMU-SHH-07 from Taipei Medical University; and TMU-NDMC-104-05 from Taipei Medical University and National Defense Medical Center. This work was also supported by the Teh-Tzer Study Group for Human Medical Research Foundation. PBP is thankful to Taiwan International Graduate program (TIGP) for the PhD Fellowship.
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