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Keywords:

  • Amplicor HPV test;
  • cervical intraepithelial neoplasia progression;
  • cervical intraepithelial neoplasia regression;
  • continuous infection;
  • high-risk human papillomavirus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Aim

The aim of this study was to evaluate the clinical performance of the Amplicor HPV test, which detects 13 high-risk human papillomaviruses (HR-HPV), and to determine the association between consistent HR-HPV infection and progression of cervical intraepithelial neoplasia (CIN) 2 to CIN3.

Material and Methods

This multi-institutional prospective study enrolled 122 women diagnosed with CIN2 by central pathological review. Subjects were tested at study entry and every 6 months over a 24-month period by cytology, Amplicor HPV test and colposcopy. Central pathological review was performed at the end of the study or if CIN progression was suspected.

Results

Ninety-three of the 122 participants completed all tests in the study and were included in the analysis. HR-HPV was detected in 87/93 (93.5%) participants at study entry. Twenty-four of the 87 HR-HPV-positive participants progressed to ≥CIN3, compared with none of the six participants who were HR-HPV-negative at study entry. The positive predictive value, negative predictive value, sensitivity and specificity of the Amplicor HPV test at study entry for predicting ≥CIN3 progression were 27.6%, 100%, 100% and 8.7%, respectively. Sixty-two participants were HR-HPV-positive from study entry through to study completion, 24 of whom progressed to ≥CIN3. None of 31 participants without continuous HR-HPV detection progressed to ≥CIN3. For continuous HR-HPV detection, the positive predictive value, negative predictive value, sensitivity and specificity of the Amplicor HPV test were 38.7%, 100%, 100% and 44.9%, respectively.

Conclusions

All participants who progressed to ≥CIN3 were continuously HR-HPV-positive. The Amplicor HPV test thus demonstrated a good performance for predicting CIN3 progression.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Cervical cancer is the second most common gynecologic malignancy worldwide.[1] Human papillomavirus (HPV) is the most common sexually transmitted infection, and a subset of HPV genotypes is now recognized as a single, necessary cause of cervical cancer.[2] Between 13 and 16 HPV genotypes are currently classified as carcinogenic or high-risk (HR) HPV,[3, 4] and their persistent infection has a critical causative function in the development of cervical intraepithelial neoplasia (CIN) and progression of precancerous CIN3 to cervical cancer.[5] Accordingly, HPV DNA testing has become an important part of cervical cancer screening programs.

However, while the majority of sexually active adults will be exposed to HR-HPV during their lifetime, only a small percentage develop CIN3 or carcinoma; the majority of women infected with HR-HPV do not develop CIN3 and eliminate the HR-HPV by an active immune response. Moreover, this immunity also provides lasting protection from re-infection with the same HR-HPV genotype.

HR-HPV testing is more sensitive than cytology for detecting CIN2 or worse pathology in a single screening interval, but shows lower specificity than cytology for CIN3 in a single screening interval. Current cervical cancer screening in Japan is therefore based primarily on cytology to identify women at risk of having or developing CIN3.

CIN3 has been reported to have a high rate of progression to invasive cervical cancer[6] and therefore it is important to investigate the process of progression from CIN2 to ≥CIN3. However, few prospective studies have addressed HR-HPV infection and the timeframe associated with this progression.[7, 8] We conducted a multi-institutional 24-month prospective CIN2 cohort study in Japan designed to examine the development of ≥CIN3 in a cohort of women with histologically confirmed central pathological review (CPR)-diagnosed CIN2.

Cervical cytology is the principal test used to screen for cervical cancer, but it has the disadvantage of low sensitivity for detecting CIN2 or CIN3 in one screening interval.[9] We prospectively evaluated HR-HPV testing using the Amplicor HPV test in combination with cytology for predicting CIN2 progression outcomes over a 2-year period.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Study design

Two hundred women diagnosed with CIN2 at 24 hospitals geographically distributed throughout Japan were recruited from January 2007 to May 2008 and their biopsies were subjected to CPR for study enrollment. CPR was performed blindly by two pathologists from the Cancer Institute Hospital of the Japanese Foundation for Cancer Research, Department of Pathology. Disagreements between their review results were resolved by discussion. Thirty-eight women with CIN1, 28 women with CIN3 and 12 women with other diagnoses were excluded from the study, and 122 women with CIN2 were finally enrolled as participants. All participants entered the study only after voluntarily signing informed consent. This study was approved by the Institutional Review Boards of each participating hospital. The inclusion criteria for the study, in addition to confirmed CIN2 by CPR, were age 20–50 years and no previous history of cervical abnormality. A cervical sample collected by liquid-based cytology (LBC), HR-HPV test (Amplicor HPV test), and colposcopy were performed at study entry and again at each 6-month visit throughout the 24 months of follow-up. When progression was suspected, cervical biopsy and CPR diagnosis were performed. All participants diagnosed as CIN3 by CPR exited the study for treatment and were scored as CIN3 progression.

At the end of follow-up, 29 participants were excluded from the final analysis for the following reasons: eight participants received treatment before completion of the study period, five became pregnant, three moved out of the study range, four were lost to follow-up, four found it difficult to get to the hospital, and five exited for other reasons. At the end of the 2-year follow-up period, study results from 93 participants were available for analysis.

Cervical sample collection

Cervical samples were taken using a Cervix brush (Roven Medical Devices). The brush with the specimen was suspended in SurePath Preservative Fluid (TriPath Imaging) and stored at 2–8°C. The resulting sample solution was subjected to HR-HPV testing and LBC within 2 weeks.

CPR and LBC

Papanicolaou-stained sample slides were prepared from the SurePath sample using the Autocyte Preparation System. The slides were screened by cytotechnologists and then diagnosed by medical specialists according to the classification of the Japan Association of Obstetricians and Gynecologists, which modified the Papanicolaou classification.[10] This classification system was the most popular cytological assessment system in Japan at the start of this study.

CPR was performed according to the World Health Organization histological typing guidelines using samples acquired by colposcopy-directed punch biopsy at each hospital.

HPV detection

DNA was extracted from 250 μL of the SurePath sample solution and subjected to HR-HPV testing. HR-HPV tests were performed using the Amplicor HPV test, according to the manufacturer's instructions. The Amplicor HPV test involves PCR amplification of target DNA, followed by hybridization in a microwell plate with probes that bind to 13 HR-HPV genotypes, that is, HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Amplified DNA containing biotin bound to immobilized probes was then detected in a colorimetric reaction. An optical density ≥0.2 indicated that the sample was positive for one or more of the 13 HR-HPV genotypes.

Statistical methods

The t-test was used to compare ages between the participants who progressed and those who did not progress to ≥CIN3.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Of the 122 participants enrolled in this study, 29 participants were withdrawn and 93 participants completed the study with multiple HR-HPV test results and biopsy outcomes available for analysis. The mean age of the participants who completed the study was 37.1 ± 6.4 years.

Table 1 shows the results of the Amplicor HPV tests at study entry and the CPR diagnoses at study completion. At the entry-point, 87 of 93 (93.5%) participants tested positive for HR-HPV by the Amplicor HPV test. During the 2-year study period, 24/93 (25.8%) participants progressed to ≥CIN3. CPR outcomes at study completion indicated that CIN2 pathology persisted as CIN2 in 14/93 (15.1%) participants and regressed to CIN1 in 55/93 (59.1%) participants. Age was not a significant factor in predicting CIN2 progression to CIN3 or regression to CIN1; the mean age for CIN3 progressors was 36.5 ± 6.4 years, compared with 37.2 ± 6.5 years for non-progressors. The 24 participants who progressed to ≥CIN3 were all HR-HPV-positive at the study entry-point, whereas no HR-HPV-negative participants progressed to ≥CIN3 within 2 years. A single negative HR-HPV test at the entry-point was highly associated with regression, and the CIN2 pathology in 5/6 participants who tested HR-HPV-negative at the entry-point were confirmed by CPR to regress to CIN1 during the course of the study. However, regression was also observed in some participants who tested HR-HPV-positive at baseline. Of the participants with HR-HPV at the entry-point, 57.5% (50/87) were confirmed by CPR to regress to CIN1 over the 2-year study period. The 24 participants who progressed to ≥CIN3 were all still HR-HPV-positive at 6 months, while none of 11 participants who became HR-HPV-negative at 6 months progressed to ≥CIN3 within 2 years (data not shown).

Table 1. Results of Amplicor HPV testing at entry-point and CPR at completion point
Amplicor HPV test result at entry-pointTotalCPR result at completion point
 ≥CIN3≤CIN2CIN2CIN1
nnnnn
  1. CIN, cervical intraepithelial neoplasia; CPR, central pathological review; HPV, human papillomavirus; HR, high-risk.

HR-HPV-positive8724631350
HR-HPV-negative60615
Total9324691455

Table 2 shows the results of continuous Amplicor HPV testing and CPR at the study completion point. Twenty-five of 87 participants who were HR-HPV-positive at the entry-point became HR-HPV-negative within 2 years, and a total of 31 (33.3%) participants were not continuously HR-HPV-positive for the duration of the study. None of the 31 participants who were not continuously HR-HPV-positive progressed to ≥CIN3, and 96.7% (30/31) of them regressed to CIN1 within 2 years. A total of 62 (66.7%) participants were continuously HR-HPV-positive from study entry to completion, including all participants who progressed to ≥CIN3. Thus, continuous HR-HPV-positivity appears to be necessary not only for progression to ≥CIN3, but also for persistence of CIN2.

Table 2. Results of continuous Amplicor HPV testing and CPR at completion point
Continuous HR-HPV infection result with Amplicor HPV testTotalCPR result at completion point
 ≥CIN3≤CIN2CIN2CIN1
nnnnn
  1. CIN, cervical intraepithelial neoplasia; CPR, central pathological review; HPV, human papillomavirus; HR, high-risk.

Continuous HR-HPV-positive6224381325
Not continuous HR-HPV-positive31031130
Total9324691455

Table 3 shows the performance characteristics of the Amplicor HPV test. The positive predictive values (PPV), negative predictive values (NPV), sensitivities and specificities of the Amplicor HPV test at the entry-point, at 6 months, and on continuous testing from study entry to completion are summarized. Amplicor HPV tests showed good performance in terms of NPV and sensitivity, which were 100% in all cases. This means that participants who were HR-HPV-negative at any point did not progress to ≥CIN3 within 2 years, and all participants who did progress to ≥CIN3 were HR-HPV-positive at all points. When HR-HPV testing at only the entry-point was analyzed, the PPV and specificity of the Amplicor HPV test were low (27.6 and 8.7%, respectively). In contrast, repeated HR-HPV testing and continuous investigation of HR-HPV infection increased the PPV and specificity to 38.7% and 44.9%, respectively.

Table 3. Performance characteristics of Amplicor HPV test for prediction of progression to ≥CIN3
 Amplicor HPV test at entry-pointAmplicor HPV test at 6 monthsContinuous Amplicor HPV testing
  1. CI, confidence interval; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; NPV, negative predictive value; PPV, positive predictive value.

PPV(95%CI)27.6(18.5–38.2)29.3(19.7–40.4)38.7(26.6–51.9)
NPV(95%CI)100(54.1–100)100(71.5–100)100(88.8–100)
Sensitivity(95%CI)100(85.7–100)100(85.7–100)100(85.7–100)
Specificity(95%CI)8.7(3.3–18.0)15.9(8.2–26.7)44.9(32.9–57.4)

In this study, LBC was performed every 6 months, in addition to the Amplicor HPV test. The LBC sample taken at study entry was collected close to the time of biopsy taken for pathology at each hospital, and the cytological outcome could thus have been influenced by the biopsy collection. The cytological results from LBC samples collected at 6 months were therefore evaluated, instead of the LBC results at the entry-point. Table 4 shows the results of LBC at 6 months and CPR at the completion point. There were no LBC results at 6 months for one participant, and the total number in Table 4 is therefore 92. Twenty-seven participants were diagnosed by LBC with ≥class IIIb, and 65 participants were diagnosed with ≤class IIIa at 6 months, based on the cytological classification of the Japan Association of Obstetricians and Gynecologists. This Japanese classification system divides class III into IIIa and IIIb, defining cases with mild and moderate dysplasia, and with severe dysplasia, respectively. Fourteen (51.8%) out of 27 participants with ≥class IIIb cytology and 10 (15.4%) out of 65 participants with ≤class IIIa cytology progressed to ≥CIN3 within 2 years. Ten (90.0%) of the 11 HR-HPV-negative participants at 6 months and 44 (67.7%) of the 65 ≤class IIIa participants regressed to CIN1 within 2 years (Table 4). LBC cytology thus seems to predict regression less accurately than the Amplicor HPV test.

Table 4. Results of LBC at 6 months and CPR at completion point
LBC result at 6 monthsTotalCPR result at completion point
 ≥CIN3≤CIN2CIN2CIN1
nnnnn
  1. †Severe dysplasia. ‡Mild and moderate dysplasia. §No LBC data for one participant. CIN, cervical intraepithelial neoplasia; CPR, central pathological review; LBC, liquid-based cytology.

≥Class IIIb271413310
≤Class IIIa6510551144
Total92§24681455

Table 5 shows the results of Amplicor HPV tests at 6 months and CPR at the completion point in two groups divided on the basis of their LBC results at 6 months. Ten (15.4%) of the 65 participants with ≤class IIIa progressed to ≥CIN3 in HR-HPV-positive cases, whereas six (22.2%) of the 27 participants with ≥class IIIb did not progress in HR-HPV-negative cases.

Table 5. Results of Amplicor HPV tests at 6 months and CPR at completion point in ≥class IIIb and ≤class IIIa participants by LBC at 6 months
Amplicor HPV test result at 6 months≤class IIIa≥class IIIb
TotalCPR result at completion pointTotalCPR result at completion point
 ≥CIN3≤CIN2 ≥CIN3≤CIN2
nnnnnn
  1. CIN, cervical intraepithelial neoplasia; CPR, central pathological review; HPV, human papillomavirus; HR, high-risk; LBC, liquid-based cytology.

HR-HPV-positive60105021147
HR-HPV-negative505606
Total651055271413

Table 6 shows the performance characteristics of LBC at 6 months, the Amplicor HPV test at 6 months, and the Amplicor HPV test in combination with LBC. The results of the Amplicor HPV test at 6 months were recalculated based on a total number of 92, because one participant had no LBC data at 6 months. Compared with the Amplicor HPV test at 6 months alone, the LBC results showed a higher PPV (29.6 vs 51.9%) and specificity (16.2 vs 80.9%), but a lower NPV (100 vs 84.6%) and sensitivity (100 vs 58.3%) for the prediction of progression.

Table 6. Performance characteristics of LBC alone, Amplicor HPV test alone, and Amplicor HPV in combination with LBC for prediction of progression to ≥CIN3
 LBC aloneAmplicor HPV test alone≥class IIIb and Amplicor HPV test
  1. CI, confidence interval; CIN, cervical intraepithelial neoplasia; LBC, liquid-based cytology; NPV, negative predictive value; PPV, positive predictive value.

PPV(95%CI)51.9(31.9–71.3)29.6(12.9–51.5)66.7(43.0–85.4)
NPV(95%CI)84.6(73.5–92.4)100(71.5–100)100(76.8–100)
Sensitivity(95%CI)58.3(36.6–77.9)100(85.7–100)100(54.1–100)
Specificity(95%CI)80.9(69.5–89.4)16.2(4.5–36.8)46.2(19.2–74.9)

In the case of ≥class IIIb, the PPV increased from 51.9% to 66.7% for LBC alone, and from 29.6% for the Amplicor HPV test alone. The participants who progressed to ≥CIN3 could be predicted more accurately by a combination of LBC and the Amplicor HPV test. The NPV and sensitivity increased from 84.6% and 58.3%, respectively, for LBC alone, to 100% for LBC in combination with the Amplicor HPV test. Participants giving false-negative results with LBC could be detected when LBC was combined with the Amplicor HPV test. The low specificity of the Amplicor HPV test increased from 16.2% to 46.2% in combination with LBC.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Cervical cancer is now recognized to be caused by persistent infection with carcinogenic HR-HPV, which develop primarily as precancerous CIN lesions and then progress to invasive cancer.[2, 3, 5, 6] It is therefore desirable to screen and treat patients at the CIN stage, before progression to cancer. The 2006 consensus guidelines of the American Society for Colposcopy and Cervical Pathology (ASCCP) recommend ablation and excision for CIN2 and 3, with a high evidence level.[11]

In this prospective cohort study, we recruited women diagnosed with CIN2 by pathologists at each hospital, and only women further diagnosed with CIN2 according to CPR were enrolled. The CPR was performed by two pathologists, as in a recent report from Japan.[8] The diagnosis of CIN2 has been reported to be less reproducible than CIN3.[12] In this study, however, CIN2 was diagnosed by at least three pathologists (a pathologist from the hospital in addition to two CPR pathologists) making the chance of misdiagnosis of CIN2 at study entry unlikely.

In this study, 122 women with CIN2 were enrolled and cervical samples were analyzed every 6 months for cytology and by the Amplicor HPV test, which detects 13 HR-HPV, during a 2-year follow-up period. Twenty-nine participants exited the study for various reasons, and 93 participants were thus finally analyzed.

The HR-HPV infection rate at the entry-point was 93.5%. This was higher than in previous studies in Japan, which reported that 54 (80.6%) out of 67 participants with CIN2, 231 (79.4%) of 291 participants with CIN 2 and 3, and 60 (89.5%) of 67 participants with CIN2 were HR-HPV-positive.[7, 8, 13] The progression rate of 25.8% in 2 years was also high compared with previous studies, which found that 24.2% of 91 participants with CIN2 progressed to CIN3 in 5 years[8] and 15.4% of 71 participants with CIN2 progressed to CIN3 in 3.1–57 months.[7] The high infection and progression rates in this study may reflect the fact that CPR diagnosed CIN2 very strictly with a special criterion[14] at the entry-point.

The 24 participants who progressed to ≥CIN3 were all HR-HPV-positive at the entry-point, while none of the participants who were HR-HPV-negative at the entry-point progressed to ≥CIN3. The NPV and sensitivity of the Amplicor HPV test performed at the entry-point were thus both 100%; however, its PPV and specificity were low. These results are consistent with previous studies.[8, 15, 16]

When the Amplicor HPV test was performed periodically, its PPV and specificity increased, and its NPV and sensitivity remained at 100%. Thus the 24 participants who progressed to ≥CIN3 were all continuously HR-HPV-positive throughout the study, while none of the 31 participants without continuous HR-HPV infection progressed to ≥CIN3. This shows that continuous HR-HPV infection is important for CIN progression.

From the viewpoint of regression, five (83.3%) of six participants without HR-HPV infection at the entry-point and 30 (96.8%) of 31 participants without continuous HR-HPV infection regressed to CIN1 within 2 years, indicating that the Amplicor HPV test could predict CIN regression in negative cases. The Amplicor HPV test is thus useful for predicting not only CIN progression, but also CIN regression.

Cytological tests have been reported to be insufficient for cervical cancer screening, mainly because of their low PPV and sensitivity.[9] When ≤class IIIa and ≥class IIIb cases were divided into HR-HPV-positive and -negative cases, the LBC at 6 months showed an NPV of 84.6% and a sensitivity of 58.3%. The Amplicor HPV test in combination with LBC compensated for the disadvantage of LBC and identified the false negatives and false positives picked out by LBC alone. The combination of the Amplicor HPV test and LBC increased the relatively low PPV and specificity. These results thus confirmed the practical usefulness of HR-HPV testing for triage of LBC-diagnosed participants with CIN2, as previously reported.[9, 15, 16]

The number of patients with CIN3 has increased dramatically in Japan, from 2679 in 2002 to 7116 in 2009,[17, 18] suggesting that many women might be newly diagnosed with CIN2 every year. Management at the CIN2 stage is thus an urgent issue in Japan, and the detection of persistent HR-HPV infection appears to be essential for adequate treatment of CIN2.

In conclusion, the results of this study suggest that the Amplicor HPV test is useful for the prediction of not only CIN progression, but also CIN regression. In addition, the Amplicor HPV test in combination with LBC can increase the detection of high-risk cases and exclude low-risk cases in participants with CIN2.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The authors thank Roche Diagnostics K.K., Tokyo, Japan for financial and practical support in this study. They would also like to thank Osaka City General Hospital, Fujita Health University Hospital, Keio University Hospital, Jikei University Hospital, Tohoku University Hospital, University of Ryukyu Hospital, Nagoya University Hospital, Kurume University Hospital, Niigata University Medical & Dental Hospital, Teikyo University Hospital, Kagoshima City Hospital, Hokkaido University Hospital, Iwate Medical University Hospital, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Tottori University Hospital, Kitasato University Hospital and Sapporo Medical University Hospital for patient enrollment.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

H. Tanaka and K. Watanabe are employees of Roche Diagnostics K.K. The other authors declare that they have no conflicts of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
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