Dual staining for p16/Ki-67 to detect high-grade cervical lesions: Results from the Screening Triage Ascertaining Intraepithelial Neoplasia by Immunostain Testing study.

We compared clinical performance of p16/Ki-67 dual-stained cytology and human papillomavirus (HPV) genotyping, via different algorithms-alone, or in combination with cytology-to identify cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+) in women referred to as colposcopy. We included 492 cervical specimens (134 normal, 130 CIN1, 99 CIN2, 121 CIN3, 8 cancers) randomly selected from 1158 specimens with valid conventional cytology, HPV (cobas 4800 HPV test) and biopsy results. Dual-stained cytology was retrospectively performed (CINtec PLUS assay) on PreservCyt material; slides were read by a cytologist and confirmed by two pathologists, blinded to cytology, biopsy and genotyping results. Sensitivity and specificity (95% confidence intervals in parentheses) of dual-stained cytology to detect CIN2+ and CIN3+ were compared to other screening tests available for the same women. Positivity rate for dual-stained cytology increased with histological severity: 30.6% in normal, 41.5% in CIN1, 72.7% in CIN2, 86.8% in CIN3 and 87.5% in cancer. Dual-stained cytology alone had lower sensitivity than HPV testing for CIN2+ [80.7% (75.0-85.6) vs 89.9% (85.3-93.5)] and CIN3+ [86.8% (79.7-92.1) vs 92.3% (86.2-96.2)]. However, corresponding specificity values were higher [64.0% (57.9-69.8) vs 56.1% (49.8-62.1) for CIN2+; 54.0% (48.7-59.2) vs 44.4% (39.2-49.6) for CIN3+]. Combining dual-stained cytology with an ASC-US abnormality threshold decreased specificity to 31.4% (25.9-37.4) for CIN2+ and 24.2% (19.9-29.0) for CIN3+. The corresponding values considering low squamous intraepithelial lesion threshold values were 42.8% (36.8-49.0) and 35.0% (30.1-40.1). Dual-stained cytology and HPV testing exhibited similar performance, although the former improved the specificity by 7.9% and 9.6% for CIN2+ and CIN3+, respectively.

Determinants of p16/Ki-67 adequacy and positivity in HPV-positive women from a screening population.

BACKGROUND: The objective of this study was to describe the determinants of adequacy and positivity of the p16/Ki-67 assay in a human papillomavirus (HPV)-positive screening population enrolled within the New Technologies for Cervical Cancer 2 (NTCC2) study. METHODS: ThinPrep slides were immunostained for p16/Ki-67; each slide had 3 reports from different laboratories. The authors included population-related, sampling-related/staining-related, and interpretation-related variables in the analyses. Adequacy and positivity proportions were stratified by variables of interest. Univariate and multivariate logistic models were used to identify determinants of adequacy and positivity. RESULTS: In total, 3100 consecutive HPV-positive cases were analyzed. Because every slide was interpreted by 3 centers, 9300 reports were obtained, including 905 (9.7%) that were inadequate and 2632 (28.3%) that were positive. The percentage of cases in which all 3 reports were inadequate increased with increasing age of the women and with inadequate cytology. The highest percentage of adequacy in all 3 reports and of cases with all 3 reports positive was observed in specimens from women who had grade ≥2 cervical intraepithelial neoplasia (CIN2+), atypical squamous cells of undetermined significance or more severe (ASC-US+) cytology, or mRNA positivity. The number of inadequate reports was significantly associated with increasing age, inadequate cytology, mRNA negativity, and scant cellularity. A positive p16/Ki-67 report was associated with an ASC-US+ result and with a positive mRNA result in cases both with and without CIN2+ but was associated with an HPV type 16 and/or 18 infection only in CIN2+ cases. The presence of CIN2+ was strongly associated with dual staining positivity. CONCLUSIONS: The interpretation of p16/Ki-67 results may be influenced by several different variables, all of which are part of the steps in the procedure, and by the characteristics of the screened population.

Value of CCNA1 promoter methylation in triaging ASC-US cytology.

BACKGROUND:
Using HPV testing to triage ASC-US still has some problems of unnecessary colposcopy in many cases. A previous study reported that methylation of CCNA1, a tumor suppressor gene, can differentiate between low and high grade lesions. This study was designed to evaluate the diagnostic values and application of CCNA1 methylation in the patients with ASC-US group.
Materials and methods:
Cross sectional analytic study was conducted in the patients with
ASC-US cytology. HPV DNA testing and CCNA1 promoter methylation testing were performed. The patients were sent for colposcopic examination and biopsy. Biopsy results were considered as gold standard. Diagnostic test of HPV test and CCNA1 methylation test were calculated for sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), likelihood ratio for test positive and negative and 95% confidence interval.
Results:
One hundred and seventy patients were enrolled. Mean age was 39.7 years old. HR-HPV was positive in 70% of the patients. HPV type 16, type 18 and non-16,18 were 12.4%, 4.7% and 42.4%, respectively. CIN2+ were found in 12.4% (21 cases). CCNA1 promoter methylation was positive in 5 cases. CCNA1 had high specificity 99.3%, NPV 89.2% and PPV 80% in detection of CIN2+ but sensitivity was 19%. Likelihood ratio for positive test was 28.4 and likelihood ratio for negative test was 0.8. HPV test had sensitivity of 90.5% and NPV of 95.9% but low specificity and PPV as 31.5% and 15.7%, respectively.
Conclusion:
CCNA1 promoter methylation testing had very high specificity, likelihood ratio for the positive test and PPV (99.3%, 28.4 and 80.0, respectively). Therefore, CCNA1 promoter methylation test may be used in the HPV DNA positive cases to classify the urgency of colposcopy and the colposcopist should pay more attention to CCNA1 positive patients because of their higher chance to identify the significant lesions.

Sensitivity and specificity values of high-risk HPV DNA, p16/ki-67 and HPV mRNA in young women with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL).

OBJECTIVE: The aim of the study was to evaluate the sensitivity and specificity values of high-risk HPV DNA test, p16/ki-67, and HPV mRNA in histologically high-grade cervical intraepithelial lesions (CIN2-CIN3) in women aged 21-24 years with diagnosis of atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL) at pap smear. PATIENTS AND METHODS: 342 patients between 21-24 years old, attending spontaneously our clinics, 118 with ASCUS and 224 with LSIL, were enrolled in the study. All patients underwent colposcopy and biopsies were performed in the areas with major changes. All patients were tested at the same time for p16/ki-67, high-risk HPV DNA and HPV mRNA. RESULTS: Nineteen out of 118 women with ASCUS showed a high-grade cervical intraepithelial lesion, 11 out of 118 (9.32%) CIN2, and 8 out of 118 (6.78%) CIN3. The sensitivity of high-risk HPV DNA was 99.9%, and the specificity 23.2%; p16/ki-67 pointed out a sensitivity of 90.9%, and a specificity of 81.8%; HPV mRNA showed a sensitivity of 81.8%, and specificity of 87.9% in CIN2 lesions. In CIN3 lesions, the sensitivity of high-risk HPV DNA was 99.9%, while the specificity was 19.1%; p16/ki-67 showed a sensitivity of 99.9%, and a specificity of 73.7%; HPV mRNA relived a sensitivity of 87.5%, and a specificity of 80.8%. In women with LSIL, a total of 42/224 (18.75%) of CIN2 were found at the histopathological examination, while 17/224 (7.59%) women presented a CIN3. No case of invasive cancer was identified. High-risk HPV DNA was positive in 190/224 (84.8%), p16/ki-67 in 119/224 (53.1%), and HPV mRNA in 104/224 (46.4%). In women with CIN2, the sensitivity of high-risk HPV DNA was of 92.8%, and the specificity 17.5%, the sensitivity of p16/ki-67 was 95.2%, and specificity 61.8%. HPV mRNA showed a sensitivity of 88.8% and a specificity of 87.8%. In women with CIN3, the sensitivity of high-risk HPV DNA was 88.2%, and the specificity 29.7%; p16/ki-67 pointed out a sensitivity of 94.1%, and a specificity of 49%; HPV mRNA showed a sensitivity of 88.2% and a specificity of 80.6. CONCLUSIONS: Taking into account the high rate of spontaneous regression of high-grade lesions in young women, these tests, in particular, the HPV mRNA test, used as a triage test for ASCUS or LSIL, can modify follow-up triage strategy. In fact, this biomarker, due to its high specificity, could lead to a cytology repetition instead of an immediate colposcopy, avoiding over diagnosis and potential overtreatment in this category of women.

Utility of human papillomavirus L1 capsid protein and HPV test as prognostic markers for cervical intraepithelial neoplasia 2+ in women with persistent ASCUS /LSIL cervical cytology.

Objective: Efficient and highly predictive biomarkers reflecting the prognosis of persistent atypical squamous cells of unknown significance(ASCUS) and low grade squamous intraepithelial lesion(LSIL)s are unavailable and need to be developed urgently. We aimed to develop a predictive model for diagnosis of cervical intraepithelial neoplasia(CIN)2+ by analyzing the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS and LSIL with a high risk of HPV infection. Methods: Cervical cytology samples comprising (70 ASCUS and 215 LSIL Pap smears) were analyzed. Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed. Expression levels of HPV L1 capsid protein in cervical cytology samples were measured, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. The risk for CIN2+ was calculated using the results of immunocytochemistry and the HPV DNA test. Results: Negative results for HPV L1 immunochemistry test were more frequently observed in CIN2+, and expression of the HPV L1 capsid protein was higher in CIN1 or cervicitis (Fisher's exact test, p<0.05). Diagnosis rates for CIN2+ were highest for the combination of HPV L1 capsid protein immunocytochemistry, cytology and HPV test when compared with other combinations (Akaike information criterion (AIC): 191.7, Schwarz criterion(SC): 206.3, p<0.001). Conclusion: Absence of HPV L1 capsid expression and presence of HPV type 16 or 18 infection are reliable predictors of progression to CIN2+ in patients showing persistent ASCUS and LSIL.

Performance of p16/Ki67 immunostaining, HPV E6/E7 mRNA testing, and HPV DNA assay to detect high-grade cervical dysplasia in women with ASCUS.

BACKGROUND: Atypical squamous cell of undetermined significance (ASCUS) is a common cervical cytological diagnosis. At present, HPV DNA assay is used to triage these patients, but its lower specificity brings a series of problems. The purpose of this study was to evaluated the value of p16/Ki67 immunostaining, HPV E6/E7 mRNA testing in triaging women with ASCUS by comparing HPV DNA assay. METHODS: Liquid based cytology specimens were collected from 300 patients. P16/Ki67 immunocytochemistry using the CINtec® Plus Kit and HPV E6/E7 mRNA testing by QuantiVirus®HPV E6/E7 mRNA assay used the same cytology sample. Detection rates of each test were evaluated against histopathology. RESULTS: All assays yielded a high sensitivity for the detection of CIN3+ (100% (86.7-100) for HPV DNA assay, 88.0% (70.0-95.8) for HPV E6/E7 mRNA testing and 100% (86.7-100) for p16/Ki67 immunocytochemistry) and CIN2+ (98.2% (90.2-99.7) for HPV DNA assay, 87.0% (75.6-93.6) for HPV E6/E7 mRNA testing, 98.2% (90.2-99.7) for p16/Ki67 immunocytochemistry). The specificity to detect high grade dysplasia was highest for p16/Ki67 immunocytochemistry (74.2% (68.7-79.0) in CIN3+ and 82.5% (77.3-86.8) in CIN2+), followed by HPV E6/E7 mRNA testing (39.6% (34.0-45.5) in CIN3+ and 42.7% (36.7-48.9) in CIN2+) and HPV DNA assay (16.0% (12.1-20.8) in CIN3+ and 17.5% (13.2-22.7) in CIN2+). CONCLUSIONS: p16/Ki67 immunostaining and HPV E6/E7 mRNA testing, especially the former, may be promising tools in triage of ASCUS.

Meta-analysis of the accuracy of p16 or p16/Ki-67 immunocytochemistry versus HPV testing for the detection of CIN2+/CIN3+ in triage of women with minor abnormal cytology.

BACKGROUND: Women with atypical squamous cells of undetermined significance (ASC-US) can be triaged accurately with a high-risk human papillomavirus (hrHPV) test to identify those who need a referral. However, the triage of low-grade squamous intraepithelial lesion (LSIL) with hrHPV testing has very low specificity. Overexpression of p16, with or without Ki-67, indicates neoplastic transformation of human papillomavirus-infected cervical cells and may more accurately predict underlying cervical intraepithelial neoplasia of grade 3 or worse (CIN3+). METHODS: A literature search was conducted in 3 bibliographic databases. Studies were selected if they included women with ASC-US or LSIL who were triaged with dual staining (p16/Ki-67) and/or p16 staining and, if available, with a comparator hrHPV test to detect cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) or CIN3+. RESULTS: Thirty-eight studies were eligible. The sensitivity of p16 staining for CIN3+ was significantly lower than that of hrHPV DNA testing (ratio for ASC-US, 0.87; 95% confidence interval [CI], 0.78-0.97; ratio for LSIL, 0.86; 95% CI, 0.80-0.93). In contrast, the specificity of p16 staining was substantially higher with relative specificities of 1.60 (95% CI, 1.35-1.88) and 2.29 (95% CI, 2.05-2.56) for ASC-US and LSIL respectively. Dual staining was as sensitive as hrHPV DNA testing but was more specific (ratio for ASC-US, 1.65; 95% CI, 1.42-1.92; ratio for LSIL, 2.45; 95% CI, 2.17-2.77). CONCLUSIONS: This meta-analysis confirms that p16 staining and p16/Ki-67 staining are more specific for CIN2+/CIN3+ than hrHPV DNA testing. Although p16 staining is less sensitive for CIN3+ than hrHPV DNA testing, dual staining has similar sensitivity.

[Significance of p16/Ki-67 double immunocytochemical staining in cervical cytology ASCUS, LSIL, and ASC-H].

Objective: To investigate the application value of p16/cell proliferation associated nuclear antigen (Ki-67) double-staining and human papillomavirus mRNA in the cytological screening. Methods: Two hundred and fifty-one cases who suffered from atypical squamous cell of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), atypical squamous cell-cannot exclude high-grade squamous intraepithelial lesion (ASC-H) in ThinPrep cytologic test (TCT) were collected in Peking University First Hospital between October 2015 and March 2016. And p16/Ki-67 double-staining and hybrid capture Ⅱ (HC-Ⅱ) detection were performed on the cervical cells. The result was compared with the pathological result of colposcope guided biopsy. All statistical analysis was completed by Stata 12.0 statistical software analysis. The results of diagnostic tests were described by using the sensitivity, specificity, positive predictive value,negative predictive value, and the area under the receiver operating characteristic (ROC) curve. Results: (1) One hundred and eight cases of liquid based cytology diagnosis of ASCUS patients, the positive rate of p16/Ki-67 was 13.9% (15/108), 102 cases of liquid based cytology diagnosis of LSIL patients, the positive rate of p16/Ki-67 was 21.6% (22/102), 41 cases of liquid based cytology diagnosis of ASC-H patients, the positive rate of p16/Ki-67 was 39.0% (16/41), compared amongthree groups, the difference was statistically significant (χ(2)=78.516, P<0.05); cervical exfoliated cells p16/Ki-67 expression rate was 13.0%(28/215) in cervical low-grade lesions [cervical intraepithelial neoplasia (CIN) Ⅰ], which was 69.4%(25/36) in high level lesions (CIN Ⅱ-Ⅲ), the difference was statistically significant (χ(2)=7.932, P<0.05). (2) The specificity of p16/Ki-67 detection and diagnosis were higher than those of HC-Ⅱ in ASCUS, LSIL, and ASC-H (89.8% vs 71.4%, 83.3% vs 15.6%, 88.9% vs 40.7%; all P<0.05), meanwhile, the positive predictive value of p16/Ki-67 detection and diagnosis exceed those of HC-Ⅱ in ASCUS, LSIL, and ASC-H (33.3% vs 26.3%, 31.8% vs 12.6%, 81.3% vs 38.5%; all P<0.05). Moreover, the ROC curve of p16/Ki-67 were bigger than those of HC-Ⅱ in ASCUS, LSIL, and ASC-H (0.799 vs 0.696, 0.708 vs 0.531, 0.909 vs 0.561; all P<0.05). Conclusion: For patients with cytological diagnosis of ASCUS, LSIL, and ASC-H, p16/Ki-67 double staining method could be used as an effective method to assist in the diagnosis of high-grade cervical lesions, and the screening efficiency is superior to that of high-rist HPV.

The role of oxidative stress in premalignant lesions.

PURPOSE OF INVESTIGATION: The authors aimed to evaluate serum total oxidant status (TOS), total antioxidant status (TAS), and oxida- tive stress index (OSI) in women with abnormal cervical cytology, to determine the association between serum oxidant and antioxidant status of these women, and the progression of abnormal cervical cytology. MATERIALS AND METHODS: A total of 75 women enrolled in the study: 20 women with a determination of atypical squamous cells of undetermined significance (ASCUS), 20 women with low squamous intraepithelial lesions (LSIL), 15 women with high squamous intraepithelial lesions (HSIL) and 20 healthy controls. Serum TOS and TAS were determined and OSI was calculated as the indicator of degree of oxidative stress. RESULTS: Serum TOS levels and OSI were highest in the HSIL group and there was a trend toward increasing serum TOS levels and OSI from ASCUS to HSIL group. CONCLUSION: The authors demonstrated that increased oxidative stress with altered antioxidant level is associated with abnormal cervical cytology. Serum oxidant and antioxidant status may provide guidance as a simple and cost-effective method for follow-up, treatment, and recommendation in all stages of lesions.

Evaluation of p16/Ki-67 dual staining in detection of cervical precancer and cancers: a multicenter study in China.

PURPOSE: To analyze the clinical performance of p16/Ki-67 dual-stained cytology identifying high-grade cervical intraepithelial neoplasia (CIN2+) in Chinese women. METHODS: 1079 women attending ongoing cervical cancer screening and 211 "enriched" women aged ≥30yrs with biopsy-confirmed CIN2+ from five Chinese hospitals were enrolled during year 2014-2015. Cervical specimens were collected for high-risk human papillomavirus (HR-HPV) DNA analysis, Liquid-based cytology (LBC) and p16/Ki-67 dual staining. Colposcopy and biopsy were performed on women with any abnormal result. RESULTS: p16/Ki-67 positivity increased with histologic severity. It was 18.4%(183/996) in normal histology, 54.0%(34/63) in CIN1, 81.0%(34/42) in CIN2, 93.3%(111/119) in CIN3, 71.4% (5/7) in adenocarcinoma and 95.2%(60/63) in squamous cell carcinoma. Compared with the HR-HPV negatives, p16/Ki-67 expression was significantly higher in the HPV16/18 positive (OR: 35.45(95%CI: 23.35-53.84)) and other 12 HR-HPV types positive group (OR: 8.01(95%CI: 5.81-11.05). The sensitivity and specificity of p16/Ki-67 to detect CIN2+ in the entire population were 90.9% and 79.5%, respectively. In women with ASC-US and LSIL, sensitivity and specificity for detection of CIN2+ were 87.5% and 66.4%, respectively, with a referral rate of 43.8%. In women who tested positive for HR-HPV, sensitivity and specificity of dual-staining for detection of CIN2+ were 92.7% and 52.7%, respectively, and the referral rate was 68.7%. CONCLUSIONS: p16/Ki-67 dual-stained cytology provided a high sensitivity and moderate specificity to detect underlying cervical precancer and cancers in various settings, and might be considered as an efficient screening tool in China.

Expression of p16/Ki-67 in ASC-US/LSIL or Normal Cytology with Presence of Oncogenic HPV DNA.

AIM: To determine if positive dual staining of p16/Ki-67 in cytology samples from women with atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL) or normal cytological reports with presence of high-risk human papillomavirus (HPV), helps in predicting the risk of developing high-grade cervical lesions during one-year of follow-up after a normal initial colposcopy. MATERIALS AND METHODS: One-hundred and sixty women with ASC-US, LSIL or otherwise normal cytology, but with the presence of high-risk HPV, were referred to the colposcopy Unit of our Hospital. Cytology and HPV testing were repeated and dual staining of p16/Ki-67 performed on a new cytological specimen, and subsequently patients were colposcopically assessed and prospectively followed-up for one year, after which the colposcopy was repeated. An optional intermediate colposcopical assessment after six months was also offered. RESULTS: Out of 143/160 women with a normal initial colposcopy, 13 were ultimately lost to follow-up. Out of the remaining 130, nine developed histologically verified cervical intraepithelial neoplasia or higher grade (CIN2+) lesions during the one-year follow-up period. Two thirds of them (6/9) were initially p16/Ki-67-positive. CONCLUSION: Biomarker detection may identify women at higher risk of CIN2+, and these women may benefit from early colposcopic assessment. Women who test negatively for the biomarkers could eventually follow a less aggressive protocol.

Value of PAX1 Methylation Analysis by MS-HRM in the Triage of Atypical Squamous Cells of Undetermined Significance.

BACKGROUND: Detection of cervical high grade lesions in patients with atypical squamous cells of undetermined significance (ASCUS) is still a challenge. Our study tested the efficacy of the paired boxed gene 1 (PAX1) methylation analysis by methylation-sensitive high-resolution melting (MS-HRM) in the detection of high grade lesions in ASCUS and compared performance with the hybrid capture 2 (HC2) human papillomavirus (HPV) test. MATERIALS AND METHODS: A total of 463 consecutive ASCUS women from primary screening were selected. Their cervical scrapings were collected and assessed by PAX1 methylation analysis (MS-HRM) and high-risk HPV-DNA test (HC2). All patients with ASCUS were admitted to colposcopy and cervical biopsies. The Chi- square test was used to test the differences of PAX1 methylation or HPV infection between groups. RESULTS: The specificity, sensitivity, and accuracy for detecting CIN2 + lesions were: 95.6%, 82.4%, and 94.6%, respectively, for the PAX1 MS-HRM test; and 59.7%, 64.7%, and 60.0% for the HC2 HPV test. CONCLUSIONS: The PAX1 methylation analysis by MS-HRM demonstrated a better performance than the high-risk HPV-DNA test for the detection of high grade lesions (CIN2 +) in ASCUS cases. This approach could screen out the majority of low grade cases of ASCUS, and thus reduce the referral rate to colposcopy.

Comparative analysis of cervical cytology and human papillomavirus genotyping by three different methods in a routine diagnostic setting.

Application of Bethesda guidelines on cervical cytology involves human papillomavirus (HPV) determinations on all ASC-US and ASC-H results. We compared HPV DNA results in view of the eventual development of a cervical intraepithelial neoplasia lesion determined either on cytology or histology. A total of 214 liquid-based cytology samples were analysed. Three different HPV DNA methods were applied: the Abbott RealTime High Risk HPV test, INNO-Lipa HPV Genotyping Extra and Full Spectrum PCR HPV Amplification and Detection/Genotyping System by Lab2Lab Diagnostic Service. A comparison of these three methods showed full concordance only for 49 samples (23%), and 27 (13%) of the samples were discordant in indicating the presence of the high-risk HPV type. Out of 214 patients, 88 were selected who presented with a cervical intraepithelial neoplasia or a VAIN lesion at follow-up cytology or histology. In this group, full concordance with HPV genotyping was present only in 19 (22%) follow-up samples. Nine (10%) follow-up samples showed discordant results for the presence of a high-risk genotype between the three genotyping methods tested either by negativity for high-risk HPV by one of the methods (n=6) or by failure to genotype HPV (n=2), or by a combination of both (n=1). Moreover, discordance for the detection of HPV16 or HPV18 was observed between the three HPV DNA genotyping methods used in 9 (10%) follow-up samples. In addition, the performance of genotyping methods on 20 external quality samples was assessed, showing discordant results for HPV16 and HPV18. Major differences were found in the genotyping results according to the HPV DNA method. Our findings highlight the importance of careful interpretation of data from studies using different HPV genotyping methods and underline the need for standardization by method validation in clinical laboratories, especially in the setting of primary HPV screening.