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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 10  |  Page : 587-593

Meta-analysis on the performance of p16/Ki-67 dual immunostaining in detecting high-grade cervical intraepithelial neoplasm


1 Department of Obstetrics and Gynecology, School of Medicine, Zhongda Hospital, Southeast University, Nanjing 210009, China
2 Department of Pathology, University of Arizona College of Medicine, Tucson, AZ 85724, USA

Date of Web Publication24-Sep-2018

Correspondence Address:
Yang Shen
Department of Obstetrics and Gynecology, School of Medicine, Zhongda Hospital, Southeast University, Nanjing 210009
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.183216

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 > Abstract 


Aim: Although routine screening contributes to substantial reductions in cervical cancer morbidity and mortality, the low specificity of HPV detection and limited sensitivity of cervical cytology necessitates the application of more optimized markers, such as the newly-introduced p16/Ki-67 dual-staining method. Here we reviewed several studies to evaluate the performance of this method in cervical cancer screening.
Methods: An electronic database search was performed on PubMed, Web of Science, CNKI and Wanfang Database for studies assessing p16/Ki-67 dual immunostaining in the diagnosis of high-grade cervical intraepithelial neoplasm (HGCIN) with abnormal cytological morphologies. Two reviewers screened literatures, extracted data and assessed the quality of the included studies independently. Meta-analysis was performed using ReV. Man 5.2 and Meta-DiSc 1.2 software packages.
Results: The absolute sensitivity of p16/Ki-67 dual staining for diagnosing HGCIN ranged from 80% to 94%, while the sensitivity of triage method with hrHPV testing ranged from 78% to 96%. The specificity of p16/Ki-67 testing and hrHPV detection for predicting absence of CIN2+ ranged from 39% to 79% and 15% to 44%, respectively. Quantitative meta-analysis showed that the pooled sensitivity of p16/ki-67 dual staining is 0.88 [95'CI (0.86-0.90)], the pooled specificity is 0.58 [95'CI (0.56-0.60)]. For hrHPV testing, the pooled sensitivity and pooled specificity is 0.94 [95'CI (0.93-0.96)] and 0.32 [95'CI (0.29-0.34)], respectively.
Conclusions: p16/Ki-67 dual immunostaining had comparable sensitivity and improved specificity in screening HGCIN or CC when compared with hrHPV detection. Further studies may be beneficial to assess the efficacy of this novel biomarker, which can be potentially used as one of the initial screening assays.

Keywords: Diagnostic accuracy, high-grade cervical intraepithelial, high-risk human papillomavirus, p16/Ki-67 dual stain


How to cite this article:
Sun M, Shen Y, Ren ML, Dong YM. Meta-analysis on the performance of p16/Ki-67 dual immunostaining in detecting high-grade cervical intraepithelial neoplasm. J Can Res Ther 2018;14, Suppl S3:587-93

How to cite this URL:
Sun M, Shen Y, Ren ML, Dong YM. Meta-analysis on the performance of p16/Ki-67 dual immunostaining in detecting high-grade cervical intraepithelial neoplasm. J Can Res Ther [serial online] 2018 [cited 2020 Oct 24];14:587-93. Available from: https://www.cancerjournal.net/text.asp?2018/14/10/587/183216




 > Introduction Top


Cervical cancer (CC) is the second most common cancer among women worldwide and the major cause of female tumor morbidity in developing countries. Routine  Pap smear More Details screening contributes to substantial reductions in CC morbidity and mortality. Nevertheless, papanicolaou cytology has the pitfall of low reproducibility, and a single Pap test has limited sensitivity (SEN) to detect CC precursors.[1] Therefore, the development of new molecular assays to improve CC screening is necessary. Among these newly developed assays, human papillomavirus (HPV)-DNA testing, surpasses with high SEN to identify high-grade cervical intraepithelial neoplasm (HGCIN).[2],[3] However, since there is high proportion of clinically irrelevant HPV infections, the HPV-DNA test has low specificity (SPE), which makes the search for specific makers compelling.[4],[5]

Recently, based on the broaden understanding of HPV carcinogenesis; numerous markers have been identified to detect HGCIN or CC. The progression from initial HPV infection to CC is characterized by substantial changes in the viral gene expression. The initial transient infection is remarkable with overexpression of structural genes while the advanced stage is usually accompanied with overexpression of viral oncogenes to induce the host cell cycle deregulation.[2],[5],[6] The HPV oncogenes also affect the expression of some host genes, most of which are involved in cell cycle regulation and cellular proliferation as represented by p16 and Ki-67.

p16 is a cyclin-dependent kinase inhibitor which regulates the G1 checkpoint and has proven to be overexpressed in infections with oncogenic HPVs. The mechanism by which p16 mutation deregulates the cell cycle is correlated to the increased expression of the viral oncoprotein E7 that disrupts a key cell cycle regulator, pRb, in transforming HPV infections. The disturbance of the Rb pathway leads to a compensatory over expression of p16ink4a through a negative feedback loop.[7],[8] p16 is an effective biomarker of CC and has been widely used to improve the reproducibility of cervical biopsy interpretations and detect CC precursors accurately.[9],[10] However, in addition, to overexpress in nearly all cases of HGCIN (CIN2/3), squamous cell carcinoma (SCC), adenocarcinoma in situ (AIS), and adenocarcinoma, it is also sporadically positive in some benign glandular and squamous epithelial cells, and even in squamous metaplasia. The unspecific staining requires additional subjective morphological evaluation, which may compromise the accuracy, request more experienced cytologist, and burden the undeveloped countries.

Ki-67 is a cell proliferation marker and a strong predictor of progression in early intraepithelial lesions because it regulates RNA transcription of ribosomes during nuclein formation. Similar to p16, Ki-67 is generally overexpressed under wide spectrum, from normal basal layer and benign proliferative lesions to malignant lesions such as CIN 2/3, SCC, AIS, and adenocarcinoma. However, Ki-67 is not expressed in benign endocervical cells, tubo-endometrial metaplasia, or squamous metaplasia.[11]

A novel system was introduced to assess the expression of p16 and Ki-67 in cervical cytology simultaneously in 2011.[12],[13] The co-expression of these two proteins does not occur in normal cervical cells since these two genes typically function as cell cycle regulators with reverse functions. Therefore, co-staining of the two markers in the same cell may interpreted as an indicator of a deregulated cell cycle caused by HPV oncoproteins, potentially obviating the necessity for morphological interpretation.[13],[14]

There are currently limited data available for p16 and Ki-67 cytology dual staining to detect CC precursors.[12],[13],[15] To evaluate the performance of this novel biomarker test in diagnosing HGCIN, we summarized and discussed the clinical utility of p16/Ki-67 dual staining in this paper, and made a comparison of this novel method with high-risk HPV (hrHPV) testing on diagnostic accuracy.


 > Materials and Methods Top


Search strategy

We searched the PubMed, Web of Science, CNKI, and Wanfang Data for all investigations that assessed p16/Ki-67 co-expression using dual immunohistochemical staining method in the diagnosis of HGCIN till June 2015. The key words used for searching are composed of “p16/Ki-67 dual stain,” “cervical cancer/CIN,” and “dual stain cervical cancer/CIN.” Only English publications are included. Comments, letters, nonsystematic reviews, and conference abstracts were excluded from the searching results. The search was complemented by a manual search in PubMed using names of authors and/or in references for relevant studies.

Selection of studies

Two researchers screened each record to evaluate the eligibility independently. The consensus was achieved by group discussion with the third party when disagreement appeared. References were selected if they fulfilled the following criteria:

  1. Participants: All women had a colposcopy performed and at least one biopsy was taken because of abnormal cytology and/or infection of hrHPV, the patients also should have undergone p16/Ki-67 dual staining, hrHPV subtype detection and subsequent colposcopy-directed biopsies and histologic analysis
  2. Index test: p16/Ki-67 dual immunocytostaining as triage test in screening CC was compared with hrHPV detection
  3. Target condition: HGCIN (CIN2+), includes CIN2, CIN 3, or CC
  4. Reference standard: Worst histologic diagnosis of colposcopy-guided biopsy and loop electrosurgical excision procedure outcomes by hematoxylin-eosin staining
  5. The diagnosis results had to be achieved by p16/Ki-67 dual staining and hrHPV testing directly or computing them from the diagnostic accuracy parameters described in the method section from the published study
  6. Screening results of the hrHPV detection and p16/Ki-67 dual staining have to be reported separately.


Methodological quality and validity assessment

Using published guidelines to report the diagnostic studies (Standards for Reporting of Diagnostic Accuracy [STARD]) and quality metrics (QUADAS-2), the methodological quality and validity assessment of diagnostic accuracy from all studies enrolled in this paper (diagnostic accuracy) were performed.[16],[17] The methodological quality items were adapted for the review question, and two researchers from our group judged each quality item independently. In case of discrepancies, consensus was achieved by group discussion with the third party. The results of the methodological quality assessment are summarized in [Appendix S1].



Data extraction and analysis

Extracted data were composed of study characteristics including study population characteristics, inclusion/exclusion criteria, cytological results from initial diagnosis, and follow-up visited. Methodological properties composed of sample preparation, blinding, review procedures, and positivity criteria were extracted to conduct index and reference tests.

For each study enrolled into this paper, we separated the data from triage groups with abnormal cytology. Moreover, we obtained the absolute values of true-positives, false-positives, true-negatives, and false-negatives for CIN2+ (CIN2, CIN3, or higher) for each study.

Diagnostic accuracy statistical analysis was achieved by calculation/computation using Meta-DiSc 1.2 (UK) and Rev. Man 5.2.


 > Results Top


Search results

The literature search identified 135 reports concerning the review question after the initial screening. Thirty-three reports were considered potentially relevant after subsequent screening with keywords in titles and abstracts. Four of the 33 publications were excluded from our study for the following reasons: two of them did not address the review question, and the other two were conference abstracts and comments. In addition, 21 of the remainder 29 studies were excluded because they did not meet the eligibility criteria. Therefore, the above selection process left eight eligible studies that met all the inclusion criteria described in the methods section[15],[18],[19],[20],[21],[22],[23],[24] [Appendix S2].



Among these eight articles, two were conducted by Wentzensen et al. using different objectives; therefore, contained samples from the study of Kaiser Permanente Northern California.[18],[24] The remainder six studies were conducted by different groups from hospital-based screening centers in America, Italy, Spain, Belgium, France, and Germany. Study characteristics of all eight articles are summarized in [Appendix S3].



Based on the results of these articles, 3667 samples were analyzed for the performance of p16/Ki-67 dual staining in diagnosing HGCIN as comparison with previous abnormal Pap cytology (atypical squamous cells of undetermined significance (ASCUS), atypical squamous cells of undetermined significance, cannot exclude HSIL (ASC-H), low grade squamous intraepithelial lesion (LSIL), or high grade squamous intraepithelial lesion (HSIL)) results and/or positive hrHPV. In six enrolled studies, 2851 samples were used to compare the dual staining results with hrHPV testing results directly.[15],[19],[21],[22],[23],[24] One of these six studies described a diagnostic case–control study (double-gate study), the other five studies achieved their conclusions from cross-sectional diagnostic cohort studies (single-gate studies).[15],[19],[21],[22],[23],[24] All these eight studies used colposcopy-guided biopsy as the only diagnostic standard. Moreover, liquid-based Pap tests had to be applied before colposcopic biopsy for dual staining and hrHPV test. In all enrolled studies, p16/Ki-67 dual staining of cervical cytology slides was performed using the CINtec® Plus Kit (Roche MTM Laboratories) according to the manufacturer's instructions. A sample was considered positive if one or more cervical epithelial cell(s) showed positive with both brown cytoplasmic stain (p16) and a red nuclear (Ki-67), and this evaluation should be irrelevant with the interpretation of morphologic abnormalities. Slides without any double-stained cells were defined as negative for p16/Ki-67 dual staining. As a result, we got the eligible screening results and tried to carry out the meta-analysis.

Risk of bias and applicability

We then assessed the risk of bias and applicability of all eight studies using the QUADAS-2 tool (www.quadas.org).[17] The criteria were established that low risk of patient selection was accounts for 82%, unclear risk of the index test in applicability concerns was 20%, and unclear risk of bias in flow and time was approximately 35%. Using these criteria, the overall quality of enrolled studies was comparatively high, according to our evaluation for each domain presented as percentages across the studies [summarized in [Appendix S1]].

Almost all enrolled studies are prospective.[15],[18],[19],[21],[23],[24] The material for index test was obtained <3 months before colposcopy biopsy procedures in all studies. The short intervals between index test and reference test guarantee the same reference standards of diagnosis. Therefore, these enrolled studies are more optimized with a low risk of verification bias. Furthermore, the reference standard result was interpreted independently from the index test result. It contributed to the reduction of publication bias as well. Last but not least, withdrawals were listed form each study, however, without details. In summary, we concluded that meta-analysis based on these studies would be informative and provide potentially effective guidance for clinical application.

Test for heterogeneity

Heterogeneity across all enrolled studies was evaluated using Meta-DiSc software (http://www.hrc.es/investigacion/metadisc _en.htm, UK). Based on the heterogeneity analysis using Meta-DiSc, we concluded that there was no threshold effect as shown with receiver operating characteristic (ROC) curve plotting and diagnostic threshold analysis of p16/Ki-67 immunostaining [Figure 1] and Spearman correlation coefficient: 0.190, P = 0.651]. However, the heterogeneity caused by nonthreshold effect existed when analyzed using diagnostic odds ratio [OR] [Q = 34.88, P = 0.00, [Figure 2]. We did not continue to search the origin of heterogeneity because of the limited numbers of enrolled studies.
Figure 1: The receiver operating characteristic curve plotting of included studies *the receiver operating characteristic curve plotting form Meta-DiSc 1.2 showed no threshold effects

Click here to view
Figure 2: Analysis of diagnostic threshold *the diagnostic odds ratio analysis form Meta-DiSc 1.2 showed that the Q = 34.88, P = 0.00, means the heterogeneity exit which is caused by nonthreshold effect. The value of DOR is 10.89. When DOR>1, the greater value means better diagnostic test effect

Click here to view


Test performance of p16/Ki-67 dual staining in eligible studies

The diagnostic accuracies measured in the eight studies are summarized in [Table 1] by Rev. Man 5.2. The absolute SEN of triage using p16/Ki-67 dual staining ranged from 80% to 94%,[15],[19] and the SEN of triage with hrHPV testing ranged from 78% to 96%.[21],[22],[23] The SPE of p16/Ki-67 testing ranged from 39% to 79%,[20],[23] whereas the accuracy of hrHPV testing for predicting the absence of CIN2+ ranged from 15% to 44%.[19],[23] Based on the forest plot and summary ROC (SROC) curve, we concluded that the test performance of p16/Ki-67 dual staining is superior to that of hrHPV testing.
Table 1: True positives (TP), false positives (FP), true negatives (TN), false negatives (PN), sensitivity, specificity and diagnostic odd ratio (DOR) for all studies with 95% confidence intervals (CI) for p16/Ki-67 and hrHPV detection in the enrolled studies at cut-off CIN2+

Click here to view


Because of heterogeneity caused by nonthreshold effect, we performed the meta-analysis using Random Effect Model [Table 2]. The pooled SEN of p16/Ki-67 dual staining was 0.88 (95% confidence interval [CI] [0.86–0.90]), the pooled SPE was 0.58 (95% CI [0.56–0.60]), the pooled positive likelihood ratio (LR+) was 2.26 (95% CI [1.77–2.89]), the pooled negative likelihood ratio (LR−) was 0.22 (95% CI [0.16–0.29]), and the pooled diagnostic OR was 10.89 (95% CI [6.43–18.44]). As comparison, the analysis on hrHPV detection showed that the pooled SEN was 0.94 (95% CI [0.93–0.96]), the pooled SPE was 0.32 (95% CI [0.29–0.34]), the pooled LR + was 1.35 (95% CI [1.17–1.56]), the pooled LR − was 0.22 (95% CI [0.13–0.38]), and the pooled diagnostic OR was 6.38 (95% CI [3.48–11.70]). The higher diagnostic OR means better test power. We concluded that the accuracy of p16/Ki-67 dual staining was higher than hrHPV detection in diagnosing HGCIN. This conclusion was also supported using the SROC curves.
Table 2: The sensitivity, specificity, Positive Likelihood Ratio (LR+), Negative Likelihood Ratio (LR-) and diagnostic odd ratio (DOR) for enrolled studies with 95% confidence intervals (CI) for p16/Ki-67 dual staining and hrHPV detection in the enrolled studies at cut-off CIN2+

Click here to view


The area under the ROC curve (AUC) of p16/Ki-67 dual staining in the SROC curve was 0.88, Q = 0.81, almost equaled to that of hrHPV detection (AUC is 0.88, Q = 0.81) when analyzing all eight enrolled studies [Figure 3]a and [Figure 3]c. The p16/Ki-67 AUC was higher than that calculated from six enrolled studies using direct data [AUCs were 0.86 and 0.70, Q = 0.79 and 0.65 for dual staining and hrHPV, respectively as shown in [Figure 3]b and 3d]. We concluded that the dual-staining method showed a better performance in diagnostic accuracy compared with hrHPV testing in general screening people. The comparable efficiency was observed on computation from all the enrolled samples. In summary, we concluded that p16/Ki-67 immunostaining had comparable SEN, improved SPE, and therefore is more advantageous in diagnosing CC when compared to hrHPV.
Figure 3: (a-d) The SROC curve of p16/Ki-67 dual staining and high-risk HPV detection. *SROC curve of p16/Ki-67 dual staining and high-risk HPV testing in identifying high-grade cervical intraepithelial neoplasm or cervical cancer. In the four pictures, a/b were for p16/Ki-67 dual staining, and c/d were for high-risk HPV test. The area under curves were 0.88, 0.86, 0.88, 0.70, respectively. SROC: Summary receiver operating characteristic. Accuracy is measured by the area under the ROC curve. An area of one represents a perfect test; an area of 0.5 represents a worthless test. A rough guide for classifying the accuracy of a diagnostic test is the traditional academic point system: 0.90–1 = excellent, 0.80–0.90 = good, 0.70–0.80 = fair, 0.60–0.70 = poor, 0.50–0.60 = fail

Click here to view



 > Discussion Top


Our meta-analysis concluded that p16/Ki-67 dual staining outperformed hrHPV as a triage tool for Pap smears analysis based on its comparable SEN and higher SPE in the identification of CC precursors and was helpful for the initial screening test.

Six enrolled studies compared p16/Ki-67 test performance with hrHPV-DNA detection. In addition of high SEN, the p16/Ki-67 testing also demonstrated a higher SPE for diagnosing CIN2+.[15],[19],[20],[21],[22],[23],[24]

The Pap test as the most common method to screen CC has limited SEN to detect HGCIN. Moreover, it does detect a significant number of patients with ASCUS and LSIL. These aspects compromised the application of Pap test in screening CC. The recognition of hrHPV infection as the etiology for CC has led to a paradigm transition in CC screening programs, and HPV-DNA detection test becomes the primary tool. Although hrHPV detection is highly sensitive, it cannot identify common transient infections from premalignant lesions. Consequently, additional colposcopy examination is required to confirm the diagnosis.

It has been reported that p16/Ki-67 dual staining positivity was associated with hrHPV genotypes, which was responsible for the majority of the CC and its precursors.[25] On the other hand, the widespread utilization of p16/Ki-67 dual-staining triage could result in significant health care cost savings while accurately detecting CC precursors. The satisfying dual-staining method would lead to fewer office visits and procedures.[19] It has also been reported that the combination of p16/Ki-67 dual staining and cytomorphologic evaluation can achieve SPE more than 90%. Conclusively, p16/Ki-67 dual staining is a potential initial method to triage HPV-positive women.[26]

There reproducibility of dual-staining interpretations was high, and this test interpretation can be achieved even by nonexpertise. These advantages have remarkable effects in developing countries, where skilled cytologists are insufficient.[20] The most common application of p16/Ki-67 dual staining is to select cytology samples preliminarily with suspicious morphological abnormalities. The potential role of this method of initial screening can thus been established for CC and its precursors.[27] The SPE and SEN of p16/Ki-67 dual staining are related to the age of the patients. Both values are higher in patients younger than 30-year-old. The dual- staining method shows more promising than Hybrid Capture II (HC2) assay, in which samples are considered positive if the relative light units cutoff is >1 and only hrHPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) are tested. In the group of women who are referred to colposcopy with abnormal cytology including ASC, AGC, or LSIL, or those with normal cytology but positive hrHPV, the application of p16/Ki-67 dual staining maintained improve SPE compared with HC2.[13],[21] These findings support that p16/Ki-67 is a feasible option of triage test for women with mild cytological abnormalities, particularly useful in women under 30-year-old or with a pap result of LSIL, who are usually positive for hrHPV.

After correlating the dual-staining results with the cytological and histological diagnosis, we found that value of predicted positivity of the dual staining was significantly increased when the lesion is more severe cytologically and/or histologically. Moreover, none of the cytologically negative cases displayed positive p16/Ki-67 staining. The high SPE is more advantageous than p16 single staining assay which was 23% positive among negative for intraepithelial lesion or malignancy samples.[28] Eventually, the fact that the dual staining result is evaluated independently from cell morphologic evaluation makes the dual staining more objective in diagnosing equivocal cases.[27]

Among the limited number of diagnostic accuracy studies included in this review, most reports complied with reporting standards (e.g., STARD). Details of each study such as participants, blinding and quality control of index test and reference standards, estimates measures of statistical uncertainty, were listed in [Appendix S1]. In addition, most studies largely fulfilled the quality standards (QUADAS-2).[17] The same criterion on the diagnosis of index test and reference test is critical in reducing quality bias. The indeterminate results were excluded from final analysis in all enrolled studies. These strict criterion/optimized characteristics make our conclusions convincible.

There are always insufficient samples with available biopsies. Therefore, more hrHPV positive samples are included in various studies, and the selection bias may exist.[18],[20] A strict inclusion criterion for study participants leads to less confounding bias, but may add selection bias and overvalue the test performance on the general population. There was a diagnostic case–control study enrolled in our meta-analysis, which may possibly inflate test performance characteristics. Another pitfall of these studies came from the reproducibility and accuracy of the index test evaluators, which may lead to publication bias. Eventually, follow-up periods were too short to track the reproducibility the diagnostic marker, and sometimes inconsistent within studies.

Despite these disadvantages, the short interval between index test and reference test is appropriate and reasonable under target condition. In addition, the “blind principle” was adopted by all studies to obtain the objective results. Based on these two advantages, the enrolled studies provided qualified information to our analysis.

Studies with more samples, more experienced evaluators, and long-term follow-ups may provide more convincing evidence. Future studies might also investigate the potential for this dual staining as a primary screening test and whether the initial evaluation of stained slides could be adapted to computerized image analysis techniques.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.





 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

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