Chinese Medical Journal 2011;124(16):2443-2447
Value of P16 expression in the triage of liquid-based cervical cytology with atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesions

MA Yuan-ying,  CHENG Xiao-dong,  ZHOU Cai-yun,  QIU Li-qian,  CHEN Xiao-duan,  LÜ Wei-guo,  XIE Xing

MA Yuan-ying (Department of Women’s Health Care, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China)

CHENG Xiao-dong (Department of Gynecologic Oncology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China)

ZHOU Cai-yun (Department of Pathology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China)

QIU Li-qian (Department of Women’s Health Care, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China)

CHEN Xiao-duan (Department of Pathology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China)

LÜ Wei-guo (Department of Gynecologic Oncology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China)

XIE Xing (Department of Gynecologic Oncology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China)

Correspondence to:CHENG Xiao-dong,Department of Gynecologic Oncology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China (Tel: 86-571-87913404. Fax:86-571-87913404. E-mail:chengxd@zju.edu.cn)
Keywords
cervical intraepithelial neoplasia; cervical cancer; P16INK4α; human papillomavirus; cytology
Abstract
Background  The management of atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesions (ASCUS/LSIL) is still controversial and it is advisable to make a triage for these two cytological abnormalities. P16INK4 (P16) has been shown to be a potential biomarker for predicting high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. The aim of the study was to determine the value of P16 expression by immunostaining method compared with high-risk human papillomavirus (HR-HPV) DNA test in the triage of ASCUS/LSIL women.
Methods  Totally 86 eligible residual liquid-based cytological specimens with ASCUS and 45 with LSIL were obtained. All specimens were submitted to HR-HPV DNA test (HC2) and P16 immunocytochemical staining simultaneously. And all women underwent colposcopy and biopsy after cytology.
Results  The positive rate of P16 staining was 32.6% in ASCUS and 42.2% in LSIL, which was significantly lower than that of HR-HPV test in both ASCUS (P <0.05) and LSIL (P <0.05). Moreover, the positive rate of P16 staining was 12.7% in normal histology, 61.5% in CIN 1, 87.0% in CIN 2-3, and 100.0% in cancer, in which P16 positive rate was significantly lower than HR-HPV positive rate in normal group. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of P16 staining for predicting CIN 2 or more were 87.5%, 68.6%, 38.9%, 96.0%, and 72.1%, respectively in the ASCUS; while 90.0%, 71.4%, 47.4%, 96.2% and 54.7%, respectively in the LSIL, in which the specificity and accuracy of P16 staining were significantly higher than those of HR-HPV test in both ASCUS and LSIL (P <0.05).
Conclusion  P16 immunostaining had significantly higher specificity and accuracy than HR-HPV DNA test for predicting for high-grade CIN and cervical cancer in ASCUS and LSIL and can be used for the triage of women with ASCUS/LSIL cytological abnormality.
Cytology is one of the most frequent means in cervical cancer screening. About 5% of cytologic abnormalities are defined as atypical squamous cells of undetermined significance (ASCUS) and 2%–3% as low-grade squamous intraepithelial lesions (LSIL).1 It is still controversial to manage these two cytological abnormalities. The evidence has shown that only 5%–17% of ASCUS and 25% of LSIL women are finally diagnosed as cervical intraepithelial neoplaisa (CIN) 2–3 or more in biopsied tissues.2  Thus, it is advisable to make a triage for ASCUS and LISL to avoid clinical over-management. High-risk human papillomavirus (HR-HPV) is a causal factor in the pathogenesis of cervical cancer,3 and HR-HPV test has been widely employed in cervical cancer screening. Because high sensitivity plus easy and reproducibility of analysis, HR-HPV test is also used for the triage of ASCUS/ LISL.4 However, the effectiveness of this triage is still uncertain because of its low specificity and low positive predictive value (PPV) for predicting high-grade CIN and cervical cancer.5,6  Therefore, to search a more available triage method is imperative for ASCUS/LSIL women.
 
The identification of markers that link HR-HPV infection with molecular changes during the neoplastic process could theoretically increase sensitivity and specificity for detection of cervical disease.7,8 Based on studies published so far, P16INK4 (P16) could be potentially used as such a marker. Several studies have revealed that the utility of P16 can improve ascertainment of difficult histological interpretations and identification of the risk for CIN 2 or more in ASCUS/LSIL.9,10 However, it is still needed to evaluate whether P16 expression is more effective than HR-HPV to predict high-grade CIN and cervical cancer in ASCUS/LSIL. The aim of the study is to determine the value of P16 expression by immunostaining method, compared with HR-HPV DNA test, in the triage of ASCUS/LSIL women.
 
METHODS
 
A total of 156 residual liquid specimens cytologically diagnosed as ASCUS or LSIL (the 2001 Bethesda System) were collected in the cervical disease clinic of Women’s Hospital, Zhejiang University School of Medicine, China, during May 2008 to June 2009. These women had no history of diagnosis and treatment of cervical lesion and acute pelvic inflammation previously, and no pregnancy in past one year. The residual liquid-based cytological specimens from all women were submitted to the HR-HPV DNA test (HC2) and P16 immunocytochemical staining simultaneously. All women underwent colposcopy and biopsy performed by a senior gynecologist within one month after cytology. The study was approved by the Hospital Ethical Committee and the informed consent was obtained from each woman before the study initiation. Women aged from 19–79 years with a mean of 41.3, including 30 postmenopause women, all live as long residenters in Zhejiang Province. Totally 131 eligible residual liquid-based cytological specimens, including 86 of ASCUS and 45 of LSIL, were finally obtained.
 
HR-HPV DNA test and histological diagnosis
The liquid-based specimens were collected according to the Cytyc T2000 processing system. HR-HPV DNA was detected by HC-2 method (Digene, Gaithersburg, MD, USA), using relative light units/cutoff ≥1.0 as a positive criterion. This method detected 13 types of HR-HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68).11 The criteria for histological diagnosis of CIN and invasive cancer were abided by WHO Tumor Classification (2000).12 A histological diagnosis of CIN 2 or more was regarded as a criterion for HR-HPV test and P16 staining positive. Complied with Richare’s criteria,13 a higher grade diagnosis was made in the simultaneous existence of multi-grade cervical lesions.
 
P16 immunocytochemistry and outcome interpretation
Residual liquid-based Thin Prep specimens were fixed for 30 minutes in 95% alcohol, rinsed with distilled water, heat repaired, and rinsed three times for 5 minutes with distilled water. They were immersed in PBS (pH=7.2–7.4) for 5 minutes, heat repaired, incubated for 10 minutes in a 3% hydrogen peroxide solution at room temperature (RT), rinsed three times for 2 minutes with PBS. Samples were then exposed to dropwise primary mouse anti-human P16 protein antibodies (GT 201329, Gene Company Limited USA) diluted 25-fold at RT for 2 hours, rinsed three times for 2 minutes with PBS. They were then exposed to dropwise Envision + HRP (secondary antibodies) at RT for 1 hour, rinsed three times with distilled water, and rinsed three times for 2 minutes with PBS. Staining was developed using diaminobenzidine (DAB) while controlling the chromogenic process under a microscope. Samples were then re-dyed using hematoxylin and dehydrated using a series of graded alcohols (70%–100%). Vitrification was achieved with dimethylbenzene and neutral plastics sealed the specimens.
 
P16 staining showed brown or yellow-brown staining in the nucleus or cytoplasm (Figure). The score of P16 were ranked five grades (score 0, 1, 2, 3, and 4), which was performed applying criterion defined by Wentzensen et al.14 That was, score 0: no staining in any squamous epithelial cell (Figure A); score 1: staining in cells with no nuclear abnormality (Figure B); score 2: staining in cells having increased nucleocytoplasmic (n/c) ratio or altered chromatin or altered nuclear shape or anisonucleosis (Figure C); score 3: staining in cells with increased n/c ratio and altered chromatin or altered nuclear shape or anisonucleosis (Figure D); score 4: increased n/c ratio and more altered chromatin, and/or altered nuclear shape and/or anisonucleosis (Figure E). The P16 stained slides were reviewed and scored independently by two authors. Slides with discrepant diagnosis were reviewed by the third author who did not know two original P16 scores, and final P16 score was determined by consistent scoring by two of three reviewers: the score ≥2 were recognized as positive.
 

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Figure. The score of P16 immunostaning with hematoxylin counterstain (HE, original magnification ×400). A: Score 0 showed no nuclear aberration with no staining in a squamous cell. B: Score 1 showed no nuclear aberration with staining in a squamous cell. C: Score 2 showed altered chromatin and increasing n/c ratio with staining in a squamous cell. D: Score 3 showed increasing n/c ratio and altered chromatin with strong nuclear staining in a squamous cell. E: Score 4 showed markedly increased n/c ratio, strong nuclear staining, altered nuclear shape and anisonucleosis.
 
Combination of P16 test and HR-HPV test
As for estimating the value of combination of P16 staining and HR-HPV test on predicting CIN 2 or more, both positive were regarded as a positive criterion, while either negative was recognized as a negative criterion.
 
Statistical analysis
SPSS for Windows 16.0 (SPSS Inc., USA) was used as the statistical software. Comparison among P16 staining, HR-HPV test, and combination of both methods used χ2 test, and if it is necessary, Ficher’s exact test was used. Statistical significance was set for 0.05.
 
RESULTS
 
In 86 ASCUS women, 53 (61.6%) were HR-HPV test positive and 28 (32.6 %) got a gain of P16; Similarly in 45 LSIL women, 35 (77.8%) were HR-HPV positive and 19 (42.2 %) had a gain of P16. There were significant differences of positive rate between HR-HPV test and P16 immunostaining in both ASCUS (χ2=16.584, P <0.001) and LSIL (χ2=11.852, P=0.001). Totally 26 women were histologically diagnosed as CIN1, 23 as CIN2-3, and 3 as invasive cancer by biopsied tissues. HR-HPV positive rate was significantly higher than P16 positive rate in normal group (Table 1).
 

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Table 1. Distributions of P16 staining and HR-HPV test positive according to histology
 
The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of P16 staining, HR-HPV test, and combined two methods for predicting CIN 2 or more were shown in Tables 2 and 3. P16 staining showed significantly higher specificity and accuracy than HR-HPV DNA for predicting high-grade CIN and cervical cancer in both ASCUS and LSIL (P <0.05), but there were no significant differences in sensitivity, PPV, and NPV. When predictive value of combination of P16 staining and HR-HPV test was estimated, significantly higher specificity, PPV, and accuracy using combined methods were observed in ASCUS (P <0.05) and significantly higher specificity in LSIL (P=0.006), compared with HR-HPV test alone. However, no improved predictive values of combined methods were found in both ASCUS and LSIL compared with P16 staining alone (all P >0.05).
 

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Table 2. Comparison of three triage methods in ASCUS
 

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Table 3. Comparison of three triage methods in LSIL
 
DISCUSSION
 
The management of ASCUS/LSIL cytology is still a controversial task in cervical cancer screening programs. Three strategies have been recommended by America Society of Colposcopy and Pathology (ASCCP) in 2006: (1) Repeat cytology 3–6 months after initial cytology, (2) Direct referral for colposcopy with or without biopsy, and (3) HR-HPV DNA test.15 Accumulated evidenced showed that each of these approaches has own efficacy and limits. For instance, HR-HPV DNA test presents a higher sensitivity than repeat cytology in ASCUS triage, but fails in the triage of LSIL.16,17 The fact that 83% of LSIL women are HPV positive limits the utility of HR-HPV DNA test in LSIL triage.18 In contrast, repeat cytology test is limited by lower sensitivity as it is affected by cellular shape, menstrual cycle, ages, drug effect and others.19 Although colposcopy is an option for ASCUS/ LSIL triage, evidences showed that only 5%–17% in ASCUS and 25% in LSIL women were finally diagnosed as CIN 2 or more in biopsied tissues,2 thus over-management is possible if all women are referred for colposcopy.
 
The development of objective biomarkers may provide more available methods for ASCUS/LSIL triage. P16 is a cyclin-dependent kinasein inhibitor that negatively regulates cell proliferation by inhibiting hyper- phosphorylation of pRb via the cdk4/6. Overexpression of P16 protein is a consequence of pRb targeted inactivation from HR-HPV E7 protein and has been well documented in cervical cancer.20-22 Our previous study has revealed that the amplification of p16 using FISH is a potential alternative triage method for HR-HPV positive in cervical cancer screening.23 Here, we explored the value of P16 as a biomarker in the triage of ASCUS /LSIL. Compared with positive rate of HPV test, positive rate of P16 staining was significantly lower in both ASCUS (61.6% vs. 32.6%) and LSIL (77.8% vs. 42.2%). Similarly in histological diagnosis, 12.7% of P16 positive rate were showed in normal tissues, 61.5% in CIN 1, 87.0% in CIN 2-3, and 100.0% in carcinoma, respectively, most of which were lower than HR-HPV test. Especially, we found that P16 had a remarkably lower positive rate than HPV test in cytologically ASCUS/LSIL but histologocally normal women (12.7% vs. 54.4%, P <0.01). Tsoumpou et al24 previously reported in a meta-analysis that average positive rate of P16 was 2% in normal biopsies (95% CI: 0.4%–30%), 38% in CIN1 (95% CI: 23%–53%), 68% in CIN2 (95% CI: 44%–92%), and 82% in CIN3 (95% CI: 72%–92%). Thus, our and previous results suggest that P16 immunostaining presents lower positive rate than HPV test and probably has lower false-positive rate in the triage of ASCUS/LSIL cytology.
 
The potential advantage of P16 immunostaining implies its value in predicting high-grade CIN or more in ASCUS/LSIL cytology. Wentzensen et al25 reported that the sensitivity, specificity, PPV, and NPV were 94.7%, 83.9%, 48.6%, and 99.0%, respectively, in the ASCUS were 100.0%, 81.7%, 28.6%, and 100.0%, respectively, in the LSIL. However, it is still controversial whether P16 staining is superior to HR-HPV test in the triage of ASCUS/LSIL. Some studies showed that P16 detection was more sensitive and specific than the HR-HPV test in finding high-grade CIN and invasive cancers in cervical cancer screening,15, 26-28 while others did not revealed that P16 staining was superior to HPV DNA test and repeated cytology in the triage for ASCUS.25,29 These discordant results may be due to different evaluation criteria for P16 positivity and varied observers’ experiences.27,30,31 Our study used a method of P16 scoring by combining immunotaining intensity and morphological abnormalities of cells. We uncovered that P16 staining had a significantly higher specificity and accuracy than HR-HPV test in both ASCUS and LSIL women, while sensitivity, PPV, and NPV were similar between P16 staining and HR-HPV test. Furthermore, we estimated whether combined P16 staining and HR-HPV test were superior to either one alone. We found that combined two methods elevated both specificity and accuracy in ASCUS and improved specificity in LSIL compared with HR-HPV test alone, suggesting that combination of P16 staining and HR-HPV test can elevate higher value in triage of low-grade cytologic abnormalities than HR-HPV alone, especially in ASCUS women. However, we did not find that combination of P16 staining and HR-HPV test was able to elevate further the predictive value of P16 staining alone. Therefore, our results suggest that P16 immunostaining is a useful triage, superior to HR-HPV test, for ASCUS/LSIL women and can reduce the possibility of overtreatment for ASCUS/LSIL abnormality.
 
Our data also showed that NPV of P16 staining reached as high as 99.0% for predicting high-grade CIN or more, which suggests that negative P16 means a very low possibility of the existence of high-grade CIN and cervical cancer in women with ASCUS/ LISL. In contrast, The PPV of P16 staining was only 38.9% in ASCUS and 47.4% in LSIL in the study, as similar as that of HR-HPV test, but we found that in 26 false-positive P16 tests, 16 were in CIN1. Tsoumpou et al24 previously reported in a meta-analysis that p16 immunostaining in CIN1 could be a predictor of disease progress that need more intensive follow-up, but the number in this study was limited and further research is needed to estimate the value of P16 gain in CIN 1 for predicting the likelihood for progress to high-grade CIN.
 
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  1. Ministry of Health Research Fund–a Major Medical and Health, Zhejiang Province Science and Technology Projects,No. WKJ 2007-2-014;