SEARCH

SEARCH BY CITATION

Keywords:

  • APTIMA human papillomavirus (HPV) assay;
  • CINtec PLUS test;
  • dual immunocytochemical stain;
  • low-grade squamous intraepithelial lesion (LSIL);
  • triage

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SUPPORT
  9. REFERENCES

BACKGROUND:

The objective of the current study was to investigate the clinical performance of detecting high-grade lesions with the CINtec PLUS p16INK4a/Ki-67 dual stain and the APTIMA human papillomavirus (HPV) Assay in a cohort of women with low-grade squamous intraepithelial lesion (LSIL) cytology. The authors also assessed the reproducibility of the evaluation of immunocytochemical staining.

METHODS:

The 2 tests were performed on liquid-based residual material from 469 women with LSILs. The samples had at least 5 years of follow-up and the gold standard used was high-grade cervical intraepithelial neoplasia (CIN2+/CIN3+) proven on histology.

RESULTS:

Approximately 69% of all the women included in the study had a positive test for HPV mRNA and 56% was positive for the dual stain. The 2 tests demonstrated high sensitivities. When examining the specificities, the APTIMA HPV Assay performed with significantly lower values than the CINtec PLUS test. For patients with CIN2+, the APTIMA HPV Assay had a specificity of 36.1% versus 51.3% for the CINtec PLUS test, and for women with CIN3+, the specificity was 33.8% versus 48.2%, respectively. The difference was even more pronounced when analyzing women aged < 30 years separately. The kappa values between the 3 observers in scoring the dual stain ranged from 0.43 to 0.49 and improved in a second evaluation round to values ranging from 0.50 to 0.66.

CONCLUSIONS:

The CINtec PLUS p16INK4a/Ki-67 dual-staining test in LSIL cytology samples demonstrated high sensitivity that was similar to that of the APTIMA HPV Assay in the detection of underlying high-grade disease but with enhanced specificity, especially among women aged < 30 years. The kappa value for the evaluation of the CINtec PLUS dual-staining test was moderate but could be improved through training. Cancer (Cancer Cytopathol) 2013. © 2012 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SUPPORT
  9. REFERENCES

Screening with cytology has proven effective in reducing the incidence of cervical cancer.1, 2 However, it has limitations when it comes to the detection of underlying high-grade cervical intraepithelial neoplasia (cervical intraepithelial neoplasia of grade 2 or more [CIN2+]/cervical intraepithelial neoplasia of grade 3 or more [CIN3+]) in cytology samples with atypical squamous cells of undetermined significance (ASCUS) and in low-grade intraepithelial lesions (LSIL).3

In the last decade, the use of human papillomavirus (HPV) tests has been investigated extensively and the usefulness of HPV tests as adjuncts in cytology samples with ASCUS is well documented.4, 5 Triaging women with ASCUS cytology with a HPV test identifies those women at risk of harboring an underlying high-grade neoplasia and distinguishes them from those women who can safely return to the screening program. This reduces the number of women who need to be referred to colposcopy and avoids causing unnecessary anxiety.

Adjunctive testing using an HPV DNA test in women with LSIL is generally not recommended because the specificity is too low.6 The majority of HPV infections are transient infections and testing for HPV DNA does not allow one to discriminate between a transient and a transforming infection. Persistent infection with oncogenic types of HPV is crucial to the development of CIN3 and cervical cancer, and is caused by a shift in viral gene expression, resulting in enhanced expression of the 2 major oncogenes E6 and E7.7 Tests detecting messenger RNA (mRNA) of E6 and E7 have been developed and studies have suggested a role for these tests because as they appear to yield a higher specificity than HPV DNA tests for detecting high-grade disease.8-12

P16INK4a is a cellular kinase inhibitor that has proven to be strongly overexpressed in transforming infections with oncogenic types of HPV by disrupting the retinoblastoma protein-controlled negative feedback loop by E7, and p16 INK4a is believed to be a surrogate marker for active HPV infection.13 Studies have shown that p16INK4a is a specific as well as a sensitive marker for underlying high-grade CIN.14-16 However, immunostaining for p16INK4a is not solely restricted to dysplastic cells but also can be noted in tubal metaplasia and endometrial cells as well as in normal columnar cells from the cervix. This is usually not a problem when dealing with histological material, but is a challenge in examining cytological samples. Different scoring systems have been developed to overcome this problem, but the use of p16INK4a staining is still hampered by the poor reproducibility of the test because of a lack of standardized interpretation of the immunostaining.15, 17

Ki-67 is a nuclear antigen and a cellular proliferation marker that is expressed in all cell cycle phases except G0. A recently commercialized test, the CINtec PLUS test (Roche mtm Laboratories, Heidelberg, Germany) has been developed by combining the immunostaining of p16INK4a with Ki-67. In normal cells, in which p16INK4a functions as a suppressor gene, they should be mutually exclusive and therefore coexpression should identify deregulated cells without having to take the morphology of the cells into consideration. To the best of our knowledge, only a few studies to date have reported high sensitivity and specificity for detecting high-grade CIN.18-21

The objective of the current retrospective study was to evaluate the clinical performance of the CINtec PLUS test to detect underlying CIN2+/CIN3+ in a cohort of women with a cytological diagnosis of LSIL and to compare it with the performance of an mRNA test, the APTIMA HPV Assay (Gen-Probe Inc,. San Diego, Calif), which detects E6/E7mRNA from 14 oncogenic HPV types. Furthermore, we aimed to investigate the reproducibility of the evaluation of the immunostaining of p16INK4a/Ki-67 between 3 observers.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SUPPORT
  9. REFERENCES

Study Population

We identified 554 consecutive cervical cytology samples received from January 2006 through February 2007 in the Department of Clinical Pathology at Vejle Hospital in Vejle, Denmark. All 554 samples were diagnosed as LSIL. The residual material was stored in the original vials from the ThinPrep samples (Hologic UK Ltd, Crawley, West Sussex, UK) at room temperature. Forty-seven cases did not have enough residual material for adjunctive testing and 1 sample could not be found. These samples were excluded from the study, leaving a total of 506 specimens for further testing. Twelve samples had cellularity that was too low according to the Bethesda 2001 classification after immunostaining with the CINtec PLUS test and 25 women did not have available follow-up information, leaving a total of 469 women for inclusion in the study.

Follow-up was performed according to the Danish guidelines at the time, which recommended a follow-up with cytology within 3 months. If the cytology control revealed ASCUS or higher, the woman was referred to colposcopy. Conversely, if the cytology control was normal, 1 further control with cytology within 6 months to 12 months was recommended before the patient was returned to the screening program with a recall after 3 years.

Data from the clinical follow-up with histology and/or cytology were retrieved for each patient from the Danish National Pathology Registry (Patobank), which contains all histological and cytological diagnoses from all the pathology departments in Denmark. We were able to identify every woman because of the unique social security number given to every individual in Denmark. The data were collected until 2011, providing at least 5 years of follow-up. For each woman, the most severe diagnoses were recorded. The study was performed according to Danish law.

CINtec PLUS Test

From the residual material of the ThinPrep samples, new slides were prepared using the ThinPrep 3000 (Hologic UK Ltd). Staining was performed using a Dako Autostainer Link 48 (Dako, Glostrup, Denmark) and the CINtec PLUS Kit according to the protocol provided by the manufacturer. The slides were run in batches of 48.

In short, spray fixative was removed from the ThinPrep 3000 to prevent cell loss, and fixation was performed by incubating the slides in 50% ethanol for 10 seconds, followed by 99% ethanol for 1 hour. Slides were then air-dried overnight before staining the next day. Before heat-induced antigen retrieval, slides were rehydrated in distilled water for 10 minutes. Heat-induced antigen retrieval was performed by boiling the slides in epitope retrieval solution in a rice cooker for 10 minutes, and the slides subsequently were cooled in the epitope retrieval solution at room temperature for 20 minutes outside the rice cooker. Slides were then rinsed for at least 5 minutes in wash buffer and placed in the Dako Autostainer Link 48. The reagents for staining were all ready to use with the exception of the chromogens. Endogen peroxidase blocking reagent was applied for 5 minutes, followed by primary antibody solution for 30 minutes. Visualization horseradish peroxidase were applied for 15 minutes each. The chromogens were prepared according to the protocol provided, with the slides incubating in 3,3′ diaminobenzidine chromogen solution for 10 minutes and in Fast Red chromogen solution for 15 minutes. The slides were rinsed with buffer between the steps as described earlier, and underwent a final wash in distilled water. Finally, the slides were counterstained in Mayer hematoxylin for 2 minutes and rinsed in tap water followed by distilled water. The liquid coverslipping CINtec PLUS mount was added and after complete drying overnight the slides were incubated in xylene and mounted in pertex with a conventional glass coverslip. In each run, 1 positive control slide of a high-grade squamous intraepithelial lesion sample and 1 negative control sample of a pooled cytology sample with normal cells were included.

Three observers (2 pathologists and 1 cytotechnician) independently evaluated the dual-stained slide using a standard microscope. A case was considered positive if 1 or more cervical epithelial cells stained with a red nuclear stain for Ki-67 and a brown cytoplasmatic stain for p16INK4a (Figs. 1 and 2), or as negative when no staining or only single staining was observed. Slides that demonstrated too low cellularity according to the Bethesda 2001 classification for cervical cytology were excluded, leaving of total of 469 slides to be included in the study. The observers were unaware of the follow-up data and the results from the mRNA test. Before evaluating the slides, a short training course was provided by the manufacturer. If there was disagreement between any of the 3 observers, a consensus score was made using a multiheaded microscope and this diagnosis was recorded as the final score. After completing the first evaluation, a second evaluation was made on a randomly selected subset of the slides (n = 185) using the same criteria as in the first evaluation round.

thumbnail image

Figure 1. The CINtec PLUS dual-staining test (brown: p16INK4a; red: Ki-67) shows a squamous cell with a reaction for p16INK4a in the cytoplasm and Ki-67 in the nucleus (positive on the CINtec PLUS test) beside a cell demonstrating a reaction for p16INK4a only.

Download figure to PowerPoint

thumbnail image

Figure 2. The CINtec PLUS dual-staining test (brown: 16INK4a; red: Ki-67) shows a group of cells with cells demonstrating dual staining (positive on the CINtec PLUS test).

Download figure to PowerPoint

APTIMA HPV Assay

Detection of HPV mRNA was performed using the APTIMA HPV Assay. This assay is a qualitative, multiplex nucleic acid amplification test that targets the E6/E7 mRNA of 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The assay is based on 3 main steps (target capture, transcription-mediated amplification, and detection), and the entire procedure is controlled by an internal control, which is incorporated into the assay.

A subset of the samples included in the current study had been previously analyzed, and these data have been published elsewhere.22 The remaining samples were analyzed using the same procedure as described in the earlier study.22 In short, an aliquot of 1 mL of well-mixed ThinPrep liquid-based sample was added to a sample tube containing a buffered detergent solution and loaded onto the fully automated TIGRIS DTS instrument (Gen-Probe Inc). Analysis was performed according to the manufacture's instructions. Positive and negative controls were included in each run to determine the validity of the run and the cutoff value of the internal control as well as the sample.

Linear Array HPV Genotyping Test

For the samples with discordance between the CINtec PLUS test and the APTIMA HPV Assay, an additional analysis was performed using the Linear Array HPV Genotyping Test (Roche Molecular Systems Inc, Alameda, Calif). This is a DNA-based test that is able to detect 37 HPV types, including the 14 high-risk types included in the APTIMA HPV Assay. The linear array test uses polymerase chain reaction amplification of a 450-base pair region of the L1 gene, and detection is performed using hybridization of type-specific oligonucleotide probes. For the linear array test, 1 mL of a well-mixed ThinPrep liquid-based sample was centrifuged and resuspended in 200 μL of phosphate-buffered saline followed by DNA purification using the MagNA Pure LC instrument (Roche Diagnostics, GmbH, Mannheim, Germany). The purified sample was subsequently subjected to the linear array test according to the manufacturer's instructions.

Statistical Analysis

Descriptive statistics were used to evaluate the performance of the 2 tests and to calculate the sensitivities, specificities, and predictive values with corresponding 95% confidence intervals (95% CIs). For this purpose, GraphPad Prism 5.0 software (GraphPad Software Inc, San Diego, Calif) was used. Values obtained without overlapping 95% CIs were considered to be statistically significant. To evaluate the reproducibility of the dual staining between the 3 observers, the kappa statistic was used, using the same software. Kappa values between 0.40 and 0.60 were considered to be moderate and those between 0.60 and 0.80 as good.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SUPPORT
  9. REFERENCES

The mean age of the 469 women included in the current study was 32.3 years, with a median age of 30.0 years (range, 16 years-65 years). Nearly one-half of all the women included (48.2%) underwent follow-up by histology with a punch and/or a cone biopsy. Approximately 52% of the women underwent follow-up by cytology only. Table 1 summarizes the patients' characteristics with the most severe diagnosis specified. In 87 women, the most severe histological diagnosis recorded was CIN2+ and 46 women had CIN3+. Approximately 69% the women had a positive test using the APTIMA HPV Assay and 56% were evaluated as positive by the CINtec PLUS test, when using the consensus score.

Table 1. Test Results of the Women in the Current Study, Including Their Follow-Up Diagnoses
  APTIMA HPV Assay, No.CINtec PLUS, No.
  1. Abbreviations: ASCUS, atypical squamous cells of undetermined significance; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; LSIL, low-grade intraepithelial lesions.

ResultNo. (%)PositiveNegativePositiveNegative
Histology     
Total226 (48.2)1745215472
Normal42 (9.0)28142319
CIN197 (20.7)66315443
CIN241 (8.7)365338
CIN345 (9.6)432432
Carcinoma1 (0.2)1010
Cytology only     
Total243 (51.8)15093109134
Normal217 (46.3)1298893124
ASCUS8 (1.7)7144
LSIL18 (3.8)144126
Totals469 (100)    

The percentage of samples that were positive using the CINtec PLUS test and the APTIMA HPV Assay increased with the severity of the diagnosis, ranging from 43% in samples with normal cytology to 96% in women with CIN3 for the CINtec PLUS test and from 59% to 96% for the APTIMA HPV Assay. The 1 carcinoma included in the current study tested positive in both the mRNA and in the p16/Ki-67 dual-staining tests. The 2 tests demonstrated a concordance of 69.5% (Table 2).

Table 2. Concordance Between the APTIMA HPV Assay and the CINtec PLUS
 APTIMA HPV Assay
 PositiveNegative
  1. Abbreviations: HPV, human papillomavirus; κ, kappa.

CINtec PLUS positive22241
CINtec PLUS negative102104
Concordance = 69.5% (κ = 0.361) 

Using CIN2+ and CIN3+ lesions as the gold standard, the sensitivities, specificities, and predictive values for both tests were calculated and are listed in Table 3. The sensitivity for the CINtec PLUS test for the detection of underlying CIN2+ was 88.5% and was 95.7% for detecting CIN3+. The corresponding specificities were 51.3% for CIN2+ and 48.2% for CIN3+. The APTIMA HPV Assay demonstrated similar sensitivities for detecting CIN2+ (92.0%) and CIN3+ (95.7%). When examining the specificities, the APTIMA HPV Assay performed with significantly lower values than the CINtec PLUS test. Accordingly, the specificity for identifying CIN2+ was 36.1% (95% CI, 31.3%-41.2%) and was 33.8% (95% CI, 29.3%-38.5%) for CIN3+ using the APTIMA Assay versus 51.3% (95% CI, 46.2%-56.4%) for CIN2+ and 48.2% (95% CI, 43.3%-53.1%) for CIN3+ when the CINtec PLUS test was used. The positive and negative predictive values for detecting an underlying CIN3+ lesion using the CINtec PLUS test were 16.7% and 99.0%, respectively, and were 13.6% and 98.6% respectively, for the APTIMA HPV Assay.

Table 3. Sensitivity, Specificity, and Predictive Values Including 95% CI for Detecting CIN2+ and CIN3+ for the APTIMA HPV and the CINtec PLUS Assays
 CIN2+    
 PositiveNegativeSensitivitySpecificityPPVNPV
  1. Abbreviations: 95% CI, 95% confidence interval; CIN2+, cervical intraepithelial neoplasia of grade 2 or more; CIN3+, cervical intraepithelial neoplasia of grade 3 or more; HPV, human papillomavirus; NPV, negative predictive value; PPV, positive predictive value.

APTIMA HPV assay      
Positive8024492.0% (84.1%-96.7%)36.1% (31.3%-41.2%)24.7% (20.1%-29.8%)95.2% (90.3%-98.0%)
Negative7138    
CINtec PLUS      
Positive7718688.5% (79.9%-94.4%)51.3% (46.2%-56.4%)29.3% (23.9%-35.2%)95.2% (91.3%-97.7%)
Negative10196    
 CIN3+    
APTIMA HPV assay      
Positive4428095.7% (85.2%-99.5%)33.8% (29.3%-38.5%)13.6% (10.0%-17.8%)98.6% (95.1%-99.8%)
Negative2143    
CINtec PLUS      
Positive4421995.7% (85.2%-99.5%)48.2% (43.4%-53.1%)16.7% (12.4%-21.8%)99.0% (96.5%-99.9%)
Negative2204    

When analyzing women aged < 30 years and women aged ≥ 30 years separately, the CINtec PLUS test had a specificity of 42.9% (95% CI, 35.9%-50.1%) for women aged < 30 years and a significantly higher specificity of 52.9% (95% CI, 46.1%-59.6%) for those women aged ≥ 30 years for the detection of CIN3+ (Table 4 and Table 5). This finding was even more pronounced for the APTIMA HPV Assay, with a specificity of 22.2% (95% CI, 16.6%-28.7%) for the younger women and a significantly higher specificity of 44.0% (95% CI, 37.4%-50.8%) for the older women when using CIN3+ as the gold standard. The sensitivities for the 2 tests were not found to be significantly different between the 2 age groups.

Table 4. Sensitivity, Specificity, and Predictive Values Including 95% CIs Calculated for the APTIMA HPV Assay and the CINtec PLUS for Women Age <30 Years
Women Age <30 Years (n = 219)CIN2+    
PositiveNegativeSensitivitySpecificityPPVNPV
  1. Abbreviations: 95% CI, 95% confidence interval; CIN2+, cervical intraepithelial neoplasia of grade 2 or more; CIN3+, cervical intraepithelial neoplasia of grade 3 or more; HPV, human papillomavirus; NPV, negative predictive value; PPV, positive predictive value.

APTIMA HPV assay      
Positive4413093.6% (82.5%-98.7%)24.4% (18.2%-31.5%)25.3% (19.0%-32.4%)93.3% (81.7%-98.6%)
Negative342    
CINtec PLUS      
Positive429289.4% (76.9%-96.5%)46.5% (38.9%-54.3%)31.3% (23.6%-33.9%)94.1% (86.8%-98.1%)
Negative580    
 CIN3+    
APTIMA HPV assay      
Positive2015495.2% (76.2%-99.9%)22.2% (16.6%-28.7%)11.5% (7.2%-17.2%)97.8% (88.2%-99.9%)
Negative144    
CINtec PLUS      
Positive21113100.0% (83.9%-100.0%)42.9% (35.9%-50.1%)15.7% (10.0%-23.0%)100.0% (95.8%-100.0%)
Negative085    
Table 5. Sensitivity, Specificity, and Predictive Values Including 95% CIs Calculated for the APTIMA HPV Assay and the CINtec PLUS Assay for Women Age ≥30 Years
Women Age ≥30 Years (n = 250)CIN2+    
PositiveNegativeSensitivitySpecificityPPVNPV
  1. Abbreviations: 95% CI, 95% confidence interval; CIN2+, cervical intraepithelial neoplasia of grade 2 or more; CIN3+, cervical intraepithelial neoplasia of grade 3 or more; HPV, human papillomavirus; NPV, negative predictive value; PPV, positive predictive value.

APTIMA HPV assay      
Positive3611490.0% (76.3%-97.2%)45.7% (38.8%-52.7%)24.0% (17.4%-31.7%)96.0% (90.1%-98.9%)
Negative496    
CINtec PLUS      
Positive359487.5% (73.2%-95.8%)55.2% (48.2%-62.1%)27.1% (19.7%-35.7%)95.9% (90.6%-98.6%)
Negative5116    
 CIN3+    
APTIMA HPV assay      
Positive2412696.0% (79.7%-99.9%)44.0% (37.4%-50.8%)16.0% (10.5%-22.9%)99.0% (94.6%-99.97%)
Negative199    
CINtec PLUS      
Positive2310692.0% (74.0%-99.0%)52.9% (46.1%-59.6%)17.8% (11.7%-25.5%)98.4% (94.2%-99.8%)
Negative2119    

A total of 13 samples with CIN2+ tested negative for 1 (n = 9) or both tests (n = 4) in the study, and these samples were tested for HPV DNA using the Linear Array HPV Genotyping Test (Table 6). One women diagnosed as having CIN2 in the follow-up and whose sample was negative using both the CINtec PLUS test and APTIMA HPV Assay tested negative for HPV DNA. One woman who had CIN3 and was found to be negative for both tests demonstrated HPV type 54, which was considered to be a low-risk HPV type. An additional 2 women with CIN2 only demonstrated low-risk HPV types (types 6, 40, and 42) using the Linear Array HPV Genotyping Test. In the remaining 9 women, high-risk HPV types could be detected.

Table 6. Linear Array Results for the Samples That Were Negative for the APTIMA HPV Assay and/or CINtec PLUS
Case No.Age, YearsHistological DiagnosisAPTIMA HPV AssayCINtec PLUSHPV Types Found by Linear Arraya
  • Abbreviations: CIN2, cervical intraepithelial neoplasia of grade 2; CIN3, cervical intraepithelial neoplasia of grade 3; HPV, human papillomavirus.

  • a

    Linear array was only considered positive when positive for ≥1 of the 14 oncogenic HPV types included in the APTIMA HPV mRNA assay (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68).

141CIN2NegativeNegative16, 51, 52, 59, 61, 62, 66, 84, CP6108
236CIN2NegativeNegative42
336CIN2NegativeNegativeNegative
425CIN2NegativePositive6, 39, 68
521CIN2NegativePositive6, 40
626CIN2PositiveNegative18, 51
723CIN2PositiveNegative16, 33
816CIN2PositiveNegative6, 16, 18, 82, 84
926CIN2PositiveNegative6, 39, 66
1027CIN2PositiveNegative18, 51, 52, 56, 62, 73, 82, CP6108
1136CIN3NegativeNegative54
1221CIN3NegativePositive31
1337CIN3PositiveNegative16

In the current study, we also examined the interobserver and intraobserver variability for the evaluation of the CINtec PLUS p16INK4a/Ki-67 dual stain between 3 observers during a first and a second evaluation round. In the first evaluation, all 469 slides were scored by the 3 observers and in the second evaluation round 185 randomly selected cases were scored. The results are listed in Table 7. In the first evaluation of the dual-stained slides, the kappa values were moderate and varied between 0.43 and 0.49. In the second round, the agreement was found to be improved, with kappa values ranging from 0.50 to 0.66, and for 2 of the evaluations the agreement was now considered as good. The intraobserver agreement between the 2 evaluations demonstrated kappa values of 0.42, 0.46, and 0.78, respectively, for the 3 observers.

Table 7. Concordance Between the 3 Observers and Between the First and Second Evaluation Rounds
Observer and RoundaConcordance, %Kappa
  • a

    The 3 observers were named A, B and C, and the first and second evaluation rounds were designated as 1 and 2, respectively.

A1/A289.20.784
B1/B271.90.461
C1/C271.90.425
A1/B171.60.432
A1/C174.60.493
B1/C171.40.441
A2/B282.70.657
A2/C274.60.499
B2/C282.20.615

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SUPPORT
  9. REFERENCES

It is difficult to effectively triage women with LSIL cytology because transient infection is very frequent in this group of women and HPV DNA testing is generally not recommended.4, 6 This is especially true for younger women, who tend to have a very high frequency of HPV infection.23 Newer tests have been developed and investigated, and tests for HPV mRNA9, 10, 12, 22 and p16INKk4a12, 15, 16, 24 have shown higher specificities for detecting high-grade lesions compared with HPV DNA testing. Accordingly, unnecessary procedures can be avoided.

In the current study, we evaluated the clinical performance of the CINtec PLUS test, a recently released commercially available test that combines the immunostaining of p16INK4a with the immunostaining of Ki-67. We performed the test on newly prepared slides from the residual material of old archived ThinPrep samples, all of which had a cytological diagnosis of LSIL. This approach allowed for the long-term follow-up of the women of at least 5 years. The 469 samples were also tested for mRNA from 14 high-risk types of HPV using the APTIMA HPV Assay.

Both the CINtec PLUS test and the APTIMA HPV Assay demonstrated high sensitivities for the detection of underlying high-grade lesions (CIN2+/CIN3+) in women with LSIL cytology. The sensitivity for detecting CIN2+ was higher for the APTIMA HPV Assay than for the CINtec PLUS test (92.0% [95% CI, 84.1%-96.7%] vs 88.5% [95% CI, 79.9%-94.4%]), although this was not statistically significant. The sensitivity for detecting CIN3+ for the 2 tests was identical.

To the best of our knowledge, only a few studies have been published to date investigating the CINtec PLUS test. In a study by Schmidt el al, in which 810 samples with ASCUS and LSIL were evaluated, the authors reported a high sensitivity for the CINtec PLUS test for the detection of CIN3 in LSIL samples of 95.8% (95% CI, 88.3%-99.1%), which is similar to our results.18 In the ASCUS patients included in their study, they demonstrated a similar sensitivity,18 which is in contrast to the study by Edgerton el al, who also examined women with ASCUS using the CINtec PLUS test.21 Edgerton el al reported a moderate sensitivity of 64%, but only a small number of patients (n = 64) were included in the study.21 Furthermore, they performed the immunocytological staining on previously prepared SurePath (BD, Franklin Lakes, NJ) Papanicolaou-stained slides, in contrast to the newly prepared slides from ThinPrep vials used in the current study.

As previous described, we performed the tests on the residual material from old ThinPrep specimens that had been stored at room temperature. This is a limitation to the current study, and could have hampered the sensitivities for 1 or both tests because of the loss of antigenicity and degradation of mRNA. However, the high sensitivity of the APTIMA HPV Assay for triage demonstrated in the current study is not statistically different from the results reported by others.9-12, 25

The high sensitivities for detecting CIN3+ for both tests included in the current study, with negative predictive values as high as 98.6% for the APTIMA HPV Assay and 99.0% for the CINtec PLUS test, indicate that both tests could be safe to use in a triage setting. However, to reduce the cost of the program and avoid unnecessary referral to colposcopy and overtreatment, the specificity of the tests becomes important. The specificity of the CINtec PLUS test for detecting CIN2+ was 51.5% (95% CI, 46.2%-56.4%) and was 48.2% (95% CI, 43.4%-53.1%) for CIN3+. This is similar to the results reported by Edgerton et al,21 but lower than in the study by Schmidt et al.18 They reported a specificity for detecting CIN2+ in the patients with LSIL of 68.0% (95% CI, 62.2%-73.4%). The samples in their study were collected from 5 cytology laboratories and only included those women with a histological follow-up within 6 months. Moreover, the study cohort had been enriched with disease cases to meet a minimum number of CIN2+ cases. The histological diagnoses were adjudicated, which they were not in our study, and this is indeed a limitation to the current study because it is well recognized that the reproducibility of CIN2 especially is difficult.3 In contrast, the current study was a single-institution experiment and approximately one-half of the women included had a follow-up by cytology only. However, the strength of the current study was that it was population-based and included consecutive samples with LSIL cytology, and that we were able to identify all the women because of the unique social security numbers given to each Danish citizen and the Danish National Pathology Registry, which contains all histological as well as cytological data from all the pathology departments in Denmark.

When examining specificity, the CINtec PLUS test demonstrated significantly higher specificity for the detection of CIN2+ than the APTIMA HPV Assay. Part of the APTIMA results have been included in a prior study.22 The specificity of the APTIMA HPV Assay in the current study was lower than in the first predictor study by Szarewski el al,25 but similar to the data from the second study, which reported a specificity for the detection of CIN2+ of 28.8% (95% CI, 26.9%-32.2%) for all the women included, and 29.1% (95% CI, 25.4%-33.1%) for the group of women with a referral smear of less or equal to mild dyskaryosis.9, 25 The current study data are also comparable to data from other recent studies examining women with LSIL.10, 26 Higher specificities have been reported when evaluating the APTIMA HPV Assay in a primary screening setting, but this cannot be compared with the results of the current study because of the different study populations.27

We evaluated women aged < 30 years and those aged ≥ 30 years separately. As reported by others, the sensitivities were not statistically significantly different between the 2 age groups for the 2 tests.9, 18 For the APTIMA HPV Assay, the specificity was significantly lower for the younger women compared with the older women (24.4% [95% CI, 18.2%-31.5%] vs 45.7% [95% CI, 38.8%-52.7%] for CIN2+ and 22.2% [95% CI, 16.6%-28.7%] vs 44.0% [95% CI, 37.4%-50.8%] for CIN3+). Decreased specificity to detect high-grade CIN in the younger women using the APTIMA HPV Assay has also been reported by others.9 A decrease in the specificity for the CINtec PLUS test in the younger women was also noted in the current study, although it was less pronounced than for the APTIMA HPV Assay (46.5% [95% CI, 38.9%-54.3%] vs 55.2% [95% CI, 48.2%-62.1%] for CIN2+ and 42.9% [95% CI, 35.9%-50.1%] vs 52.9% [95% CI, 46.1%-59.6%] for CIN3+). In the study by Schmidt et al,18 the specificity to reveal underlying CIN2+ among women aged < 30 years was 62.1% and was 70.7% for those aged ≥ 30 years. The difference was not significant in their study, indicating retained specificity for the younger women. Although we did observe a significant decrease in specificity for the CINtec PLUS test in the current study among the younger women, this was less pronounced than for the APTIMA HPV Assay.

Several studies have previously investigated p16ink4a cytology tests and have shown good specificity rates, but the test has been hampered by poor reproducibility because of a lack of standardization, primarily in the interpretation of the immunocytochemical staining.15, 17 When evaluating single immunocytochemical staining for p16ink4a, the morphology has to be taken into consideration. This should not be the case for the CINtec PLUS test. One dual-stained cell should indicate the presence of an underlying high-grade CIN, and the test should be evaluated as positive. In the current study, we investigated the interobserver and intraobserver reproducibility among 3 observers in a first and second evaluation round. The reproducibility evaluated by the kappa statistic was only moderate by the first evaluation. The interpretation of the slides were in our experience not always easy and could be rather time-consuming. The immunocytochemical staining, especially for p16ink4a, could sometimes be rather weak; the dual-stained metaplastic cells occasionally were missed at screening magnification because of the scant cytoplasm; and, not rarely, the dual-stained cells were few and sometimes only a single cell was present on the slide. We also frequently found it challenging to interpret groups of cells. However, we did manage to increase reproducibility after training and obtained better kappa values at the time of the second evaluation, categorizing the kappa values except 1 as good.

In the current retrospective study, the CINtec PLUS p16INK4a/Ki-67 dual-staining test demonstrated high sensitivity similar to the APTIMA HPV Assay in the detection of underlying high-grade disease, suggesting that both tests may be used in triage of patients with LSIL. The specificity for the CINtec PLUS test was significantly higher than that of the APTIMA HPV Assay, indicating that referral to colposcopy might be reduced when using this test. However, the reproducibility of the evaluation of the dual-staining test was only acceptable but this could be improved by training. The high and relatively preserved specificity among women aged < 30 years could make the CINtec PLUS test an attractive option for the triage of women with LSIL, particularly in this age group.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SUPPORT
  9. REFERENCES

We thank biotechnicians Tinna Herløv Jensen, Martin Nielsen, and Nina Mogensen for their careful and skilled work.

FUNDING SUPPORT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SUPPORT
  9. REFERENCES

The authors thank Professor Flemming Brandt Sørensen for providing the financial resources to perform the current study.

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. FUNDING SUPPORT
  9. REFERENCES
  • 1
    Bray F, Loos AH, McCarron P, et al. Trends in cervical squamous cell carcinoma incidence in 13 European countries: changing risk and the effects of screening. Cancer Epidemiol Biomarkers Prev. 2005; 14: 677-686.
  • 2
    Arbyn M, Raifu AO, Weiderpass E, Bray F, Anttila A. Trends of cervical cancer mortality in the member states of the European Union. Eur J Cancer. 2009; 45: 2640-2648.
  • 3
    Stoler MH, Schiffman M; Atypical Squamous Cells of Undetermined Significance-Low-grade Squamous Intraepithelial Lesion Triage Study (ALTS) Group. Interobserver reproducibility of cervical cytologic and histologic interpretations: realistic estimates from the ASCUS-LSIL Triage Study. JAMA. 2001; 285: 1500-1505.
  • 4
    Human papillomavirus testing for triage of women with cytologic evidence of low-grade squamous intraepithelial lesions: baseline data from a randomized trial. The Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesions Triage Study (ALTS) Group. J Natl Cancer Inst. 2000; 92: 397-402.
  • 5
    Arbyn M, Sasieni P, Meijer CJ, Clavel C, Koliopoulos G, Dillner J. Chapter 9: Clinical applications of HPV testing: a summary of meta-analyses. Vaccine. 2006; 24(suppl 3): S3/78-S3/89.
  • 6
    Arbyn M, Martin-Hirsch P, Buntinx F, Van Ranst M, Paraskevaidis E, Dillner J. Triage of women with equivocal or low-grade cervical cytology results: a meta-analysis of the HPV test positivity rate. J Cell Mol Med. 2009; 13: 648-659.
  • 7
    Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer. Clin Sci (Lond). 2006; 110: 525-541.
  • 8
    Molden T, Nygard JF, Kraus I, et al. Predicting CIN2+ when detecting HPV mRNA and DNA by PreTect HPV-proofer and consensus PCR: a 2-year follow-up of women with ASCUS or LSIL Pap smear. Int J Cancer. 2005; 114: 973-976.
  • 9
    Szarewski A, Ambroisine L, Cadman L, et al. Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears. Cancer Epidemiol Biomarkers Prev. 2008; 17: 3033-3042.
  • 10
    Ovestad IT, Vennestrom U, Andersen L, et al. Comparison of different commercial methods for HPV detection in follow-up cytology after ASCUS/LSIL, prediction of CIN2-3 in follow up biopsies and spontaneous regression of CIN2-3. Gynecol Oncol. 2011; 123: 278-283.
  • 11
    Waldstrom M, Ornskov D. Comparison of the clinical performance of an HPV mRNA test and an HPV DNA test in triage of atypical squamous cells of undetermined significance (ASC-US) [published online ahead of print September 20, 2011]. Cytopathology. doi: 10.1111/j. 1365-2303.2011.00923.x.
  • 12
    Reuschenbach M, Clad A, von Knebel Doeberitz C, et al. Performance of p16INK4a-cytology, HPV mRNA, and HPV DNA testing to identify high grade cervical dysplasia in women with abnormal screening results. Gynecol Oncol. 2010; 119: 98-105.
  • 13
    Wentzensen N, von Knebel Doeberitz M. Biomarkers in cervical cancer screening. Dis Markers. 2007; 23: 315-330.
  • 14
    Wentzensen N, Bergeron C, Cas F, Vinokurova S, von Knebel Doeberitz M. Triage of women with ASCUS and LSIL cytology: use of qualitative assessment of p16INK4a positive cells to identify patients with high-grade cervical intraepithelial neoplasia. Cancer. 2007; 111: 58-66.
  • 15
    Tsoumpou I, Arbyn M, Kyrgiou M, et al. p16(INK4a) immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis. Cancer Treat Rev. 2009; 35: 210-220.
  • 16
    Roelens J, Reuschenbach M, von Knebel Doeberitz M, Wentzensen N, Bergeron C, Arbyn M. p16(INK4a) immunocytochemistry versus human papilloma virus testing for triage of women with minor cytologic abnormalities: a systematic review and meta-analysis [published online ahead of print June 14, 2012]. Cancer (Cancer Cytopathol). doi: 10.1002/cncy. 21205.
  • 17
    Schledermann D, Andersen BT, Bisgaard K, et al. Are adjunctive markers useful in routine cervical cancer screening? Application of p16(INK4a) and HPV-PCR on ThinPrep samples with histological follow-up. Diagn Cytopathol. 2008; 36: 453-459.
  • 18
    Schmidt D, Bergeron C, Denton KJ, Ridder R; European CINtec Cytology Study Group. p16/ki-67 dual-stain cytology in the triage of ASCUS and LSIL papanicolaou cytology: results from the European equivocal or mildly abnormal Papanicolaou cytology study. Cancer (Cancer Cytopathol). 2011; 119: 158-166.
  • 19
    Singh M, Mockler D, Akalin A, Burke S, Shroyer A, Shroyer KR. Immunocytochemical colocalization of P16(INK4a) and Ki-67 predicts CIN2/3 and AIS/adenocarcinoma. Cancer (Cancer Cytopathol). 2012; 120: 26-34.
  • 20
    Yoshida T, Sano T, Kanuma T, et al. Usefulness of CINtec® PLUS p16/Ki-67 double-staining in cytological screening of cervical cancer. Acta Cytol. 2011; 55: 413-420.
  • 21
    Edgerton N, Cohen C, Siddiqui MT. Evaluation of CINtec PLUS® testing as an adjunctive test in ASC-US diagnosed SurePath® preparations [published online ahead of print June 27, 2011]. Diagn Cytopathol. doi: 10.1002/dc. 21757.
  • 22
    Waldstrom M, Ornskov D. Clinical performance of a human papillomavirus messenger RNA test (Aptima HPV Assay) on residual material from archived 3-year-old PreservCyt samples with low-grade squamous intraepithelial lesion. Arch Pathol Lab Med. 2011; 135: 1052-1056.
  • 23
    Kjaer SK, Breugelmans G, Munk C, Junge J, Watson M, Iftner T. Population-based prevalence, type- and age-specific distribution of HPV in women before introduction of an HPV-vaccination program in Denmark. Int J Cancer. 2008; 123: 1864-1870.
  • 24
    Denton KJ, Bergeron C, Klement P, Trunk MJ, Keller T, Ridder R; European CINtec Cytology Study Group. The sensitivity and specificity of p16(INK4a) cytology vs HPV testing for detecting high-grade cervical disease in the triage of ASC-US and LSIL pap cytology results. Am J Clin Pathol. 2010; 134: 12-21.
  • 25
    Szarewski A, Mesher D, Cadman L, et al. Comparison of seven tests for high-grade cervical intraepithelial neoplasia in women with abnormal smears: the Predictors 2 study. J Clin Microbiol. 2012; 50: 1867-1873.
  • 26
    Ratnam S, Coutlee F, Fontaine D, et al. Aptima HPV E6/E7 mRNA test is as sensitive as Hybrid Capture 2 Assay but more specific at detecting cervical precancer and cancer. J Clin Microbiol. 2011; 49: 557-564.
  • 27
    Monsonego J, Hudgens MG, Zerat L, Zerat JC, Syrjanen K, Smith JS. Risk assessment and clinical impact of liquid-based cytology, oncogenic human papillomavirus (HPV) DNA and mRNA testing in primary cervical cancer screening (the FASE study). Gynecol Oncol. 2012; 125: 175-180.