Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay

BACKGROUND.

The aim of this study was to examine p16^INK4a protein expression in ThinPrep (Cytyc Corporation, Marlborough, Mass) cervical specimens by using the CINtec p16^INK4a Cytology Kit (Dako, Glostrup, Denmark). The ability of this assay to accurately identify underlying high‐grade lesions was assessed by using follow‐up biopsies and comparing these results with Hybrid Capture 2 (Digene, Gaithersburg, Md) high‐risk HPV (hc₂) results.

METHODS.

Three hundred ninety‐eight residual ThinPrep samples were collected, and histological follow‐up data were retrieved for abnormal cytology specimens. After preparation of a Papanicolaou‐stained slide, a second slide was processed in preparation for p16^INK4a immunostaining. High‐risk human papillomavirus testing (hc₂) was also performed.

RESULTS.

Of the 163 cytologically abnormal samples, 6‐month biopsy follow‐up data were available for 45% of the specimens. At initial blinded evaluation, 21 of the 26 cases with cervical intraepithelial neoplasia (CIN) II/III follow‐up were positive for p16^INK4a, yielding an overall diagnostic sensitivity of 81%; 29 of the 47 cases diagnosed as CIN I or less were p16^INK4a negative, yielding a diagnostic specificity of 62%. In comparison, the hc₂ test results indicated a diagnostic sensitivity of 100% with a diagnostic specificity of 15%. After review of selected cases with CIN II/III follow‐up, 25 of 26 slides were deemed to be positive for p16^INK4a, increasing the diagnostic sensitivity to 96%.

CONCLUSIONS.

The CINtec p16^INK4a Cytology Kit, in combination with ThinPrep cervical samples, allowed clear evaluation of p16^INK4a protein overexpression. Diagnostic specificity of the CINtec p16^INK4a assay was significantly improved relative to hc₂. To increase p16^INK4a immunostaining in abnormal cells, a modified kit version with improved staining performance has been developed and is currently being evaluated. Cancer (Cancer Cytopathol) 2007. © 2007 American Cancer Society.     

Evaluation of a new p16(INK4A) ELISA test and a high-risk HPV DNA test for cervical cancer screening: results from proof-of-concept study

p16(INK4a), a cell cycle regulation protein, accumulates in abnormal epithelial cells infected with high-risk human papilloma virus (HPV). In immunostaining studies, p16(INK4a) has shown potential as a marker of high grade cervical intraepithelial neoplasia (CIN) and invasive cervical cancer. To evaluate its potential use in cervical cancer screening, we conducted a feasibility study to compare the performance of a new enzyme linked immunosorbant assay (ELISA) for p16(INK4a) (mtm laboratories, Heidelberg, Germany) to that of the Hybrid Capture 2 (hc2) test for high-risk HPV DNA for the detection of CIN3. Three hundred and nineteen women were referred from Western Washington Planned Parenthood clinics for colposcopy examination and cervical biopsy because of abnormal Pap test results. Cervical samples were obtained from study participants for p16(INK4a) ELISA, liquid-based cytology and hc2. The order (first and second) for obtaining samples for cervical cytology and p16(INK4a) ELISA changed with every other subject. Concentrations of p16(INK4a) protein were higher when the sample was taken before the cytology. The sensitivity of p16(INK4a) ELISA (concentration > or = 8 units/ml) taken as first sample was 90.0% for CIN3, and the sensitivity of HC2 taken as a second sample was 85%. In the same group, the specificity of p16(INK4a) ELISA (46.9%) was slightly better than hc2 (35.4%) Results from this proof-of-concept study suggest that p16(INK4a) ELISA has a similar sensitivity and slightly better specificity for CIN3 compared to hc2. These findings support proceeding with a larger study with samples from a population of women presenting for routine cytology screening.     

Immunocytochemical colocalization of P16(INK4a) and Ki-67 predicts CIN2/3 and AIS/adenocarcinoma

BACKGROUND:

Although previous studies have shown that p16^INK4a and Ki‐67 are sensitive and specific markers for high‐grade lesions (≥CIN2) on cervical biopsies, limited information is available regarding the performance of a dual‐staining approach as a diagnostic adjunct in cervical cytology. We evaluated a dual p16^INK4a/Ki‐67 immunocytochemistry (ICC) assay to determine its sensitivity and specificity versus that of high‐risk HPV (HR‐HPV) in a US‐based pilot cytology study.

METHODS:

ThinPrep specimens from 122 cervical cytology specimens encompassing 23 negative (NILM), 20 ASC‐US, 22 LSIL, 17 ASCH, 22 HSIL, and 18 AGC cases were processed for multiplexed ICC staining using a CINtec Plus Kit. Dual‐positive assay results were defined based on the detection of 1 or more epithelial cells that were stained for both p16^INK4a and Ki‐67 without regard to cellular morphology. HR‐HPV testing was performed by multiplex PCR with capillary electrophoresis genotyping.

RESULTS:

Dual staining for p16^INK4a and Ki‐67 was frequently detected in HSIL and AGC but was rarely detected in NILM cases. The HR‐HPV assay showed a sensitivity of 76.2% and a specificity of 55.8% for the detection of clinically significant cervical squamous or endometrial lesions. In contrast, the colocalization of p16^INK4a plus Ki‐67 maintained a high sensitivity of 81.8% and improved specificity to 81.8% for biopsy‐confirmed CIN2/3, endocervical adenocarcinoma, or endometrial adenocarcinoma.

CONCLUSIONS:

Dual staining for p16^INK4a/Ki‐67 immunocytochemistry dramatically increased specificity and maintained high‐level sensitivity for the diagnosis of CIN2/3 or glandular lesions compared with PCR‐based testing for HR‐HPV. Cancer (Cancer Cytopathol) 2012. © 2011 American Cancer Society.     

Validation of p16INK4a as a marker of oncogenic human papillomavirus infection in cervical biopsies from a population-based cohort in Costa Rica.

Due to the high prevalence of cancer-associated types of human papillomavirus (HPV) and the poorly reproducible histologic classification of low-grade lesions, identifying infected women at highest risk for cancer prior to neoplastic progression remains a challenge. We therefore explored the utility of p16INK4a immunostaining as a potential diagnostic and prognostic biomarker for cervical neoplasia using paraffin-embedded tissue blocks (punch biopsies and loop electrosurgical excision procedures) obtained from women referred to colposcopy during the enrollment phase of the Guanacaste Project (1993 to 1994). All blocks from 292 women selected by HPV status (HPV negative, nononcogenic HPV positive, or oncogenic HPV positive) and representing the diagnostic spectrum of the population [normal to precancer: cervical intraepithelial neoplasia (CIN) 3] were immunostained for p16INK4a using the p16INK4a research kit based on the monoclonal antibody clone E6H4 (MTM Laboratories, Heidelberg, Germany). For CIN3, the sensitivity of diffuse p16INK4a immunostaining was 100% and the specificity was 95%. For CIN2, the sensitivity and specificity for diffuse staining were 81.1% and 95.4%, respectively. Generalized to the 10,000-woman cohort, this translated to positive predictive value and negative predictive value of 13.9% and 100% for CIN3, respectively, and 20.4% and 99.7% for CIN2 or CIN3, respectively. Of women with an initial diagnosis of less than CIN2 for whom follow-up data for up to 5 to 7 years were available, 44% with diffuse staining developed persistent infection (CIN2 or CIN3). Whereas our data support the diagnostic potential for p16INK4a, further prospective studies with detailed follow-up determining the prognostic capacity of this marker are needed.     

Immunoreactivity of p16 in anal cytology specimens: histologic correlation

BACKGROUND: Cytology has been proposed as a potential screening tool in the evaluation of squamous anorectal disease in view of the morphologic similarities between anal and cervical squamous lesions. Previous studies have demonstrated that p16 overexpression correlates with the degree of dysplasia in the uterine cervix with promising results. Due to potential diagnostic pitfalls in anal cytology, p16 overexpression in these specimens was studied. METHODS: Patients with anorectal cytology who underwent follow-up biopsy within 1 year were selected. Forty-three anorectal cytologic specimens from 29 patients were selected. One slide of each case was destained. Avidin-biotin immunocytochemical studies with the monoclonal antibody CINtec p16(INK4a) were performed. The results of the p16 immunostaining were correlated with the histologic findings. RESULTS: Twenty-eight of the 43 cases demonstrated the presence of squamous cells immunoreactive for p16 in cytology specimens. The p16-positive cells were identified in cases of low-grade squamous intraepithelial lesion (LSIL) (n = 3 cases), high-grade squamous intraepithelial lesion (HSIL) (n = 22 cases), and invasive squamous carcinoma (n = 1 case), and in 2 cases with negative follow-up biopsies. No cell immunoreactive for p16 was found in 15 cases (5 benign cases and 10 cases with either LSIL or HSIL). The sensitivity and specificity of p16 immunoreactivity in the detection of anal intraepithelial neoplasia or carcinoma were 72% and 71%, respectively. The positive and negative predictive values were 93% and 33%, respectively. CONCLUSIONS: The presence of p16 immunoreactivity is a good predictor of dysplasia in anal specimens. However, the sensitivity and specificity of this marker are not high.     

The study of the combination detection of HPV-DNA and p16INK4a in cervical lesions

The objective of this study is to detect the infection of human papillomavirus (HPV) and the expression of p16(INK4a) in cervical lesions and to investigate the interaction between hrHPV and p16(INK4a) for cervical lesions and its diagnostic efficiency. hrHPV-DNA was detected by the hybrid capture II (HC-II) system. Immunochemical method was used to detect the expression of p16(INK4a), and histopathologic test was performed to identify cervical lesions. χ(2) test and Spearman's rank correlation were used for statistical analysis. Additive effects model was used to analyze the interaction. The diagnostic sensitivity, specificity, positive predictive values, negative predictive values, accuracy, and the area under the receiver operating characteristic curve were calculated with SPSS 13.0. hrHPV and p16(INK4a) positive rate increased (P < 0.05) with histopathologic diagnosis increasing. The positive rates of hrHPV and p16(INK4a) in negative or chronic inflammation were statistically lower than that in cervical intraepithelial neoplasia (CIN)1, CIN2, CIN3, and squamous-cell carcinoma (SCC) (P < 0.05), respectively. There was a positive interaction between hrHPV and p16(INK4a), relative excess risk of interaction (RERI) was 52.49, attributable proportions of interaction (API) were 72.34%, and the synergy index (S) was 3.75. The specificity and AUC of combining hrHPV with p16(INK4a) were statistically higher than hrHPV or p16(INK4a) alone (P < 0.05). hrHPV and p16(INK4a) are useful markers for the early diagnosis of cervical lesions. A positive interaction between hrHPV and p16(INK4a) is seen. The combination of hrHPV and p16(INK4a) has a higher diagnostic accuracy than hrHPV or p16(INK4a) alone in diagnosis of cervical lesions.     

Combined p16INK4a and human papillomavirus testing improves the prediction of cervical intraepithelial neoplasia (CIN II-III) in Thai patients with low-grade cytological abnormalities

Thailand is in the process of developing a national cervical screening program. This study examined p16INK4a staining and HPV prevalence in abnormal cervical samples with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL), to evaluate the efficacy of combined HPV and p16INK4a detection to predict CIN II-III. Totals of 125 ASCUS and 87 LSIL cases were re-evaluated by Pap test and cervical cells of ASCUS and LSIL cases were prepared on slides for p16INK4a detection by immunocytochemistry. HPV genotyping of DNA extracts was performed by GP5+/6+ PCR and reverse line blot hybridization. Histopathologic tests were performed to identify cervical lesion. Total of 212 cases were diagnosed to normal (20), ASCUS (112), LSIL (78) and HSIL (2). HPV was detected in ASCUS (49/112, 43.8%), LSIL (60/78, 76.9%) and HSIL (2/2, 100%) cases. The majority of HPV positive samples typed for high-risk HPV. 55.7% (107/192) of abnormal cases (ASCUS, LSIL and HSIL) were positive p16INK4a. For the 111 HPV DNA positive cases, 34 of 49 (69.4%) ASCUS cases and 49 of 60 (81.7%) LSIL cases were p16INK4a positive. 140 biopsies were taken and histological classified: CIN negative (65 cases), CIN I (56 cases) and CIN II-III (19 cases). HPV DNA detection predicted CIN II-III with sensitivity and specificity of 84% and 49%, whereas p16INK4a staining showed higher sensitivity (89.5%) and specificity (56.2%). The prediction of CIN II-III was significantly better by combination of positive HPV DNA and p16INK4a with 93.8% sensitivity and 59.2% specificity. Detection of HPV DNA combined with p16INK4a in cervical cells can predict CIN II-III and may improve the screening diagnosis of Thai women at risk for CIN II-III or cancer.     

p16INK4a immunocytochemistry versus human papillomavirus testing for triage of women with minor cytologic abnormalities

The best method for identifying women who have minor cervical lesions that require diagnostic workup remains unclear. The authors of this report performed a meta‐analysis to assess the accuracy of cyclin‐dependent kinase inhibitor 2A (p16^INK4a) immunocytochemistry compared with high‐risk human papillomavirus DNA testing with Hybrid Capture 2 (HC2) to detect grade 2 or greater cervical intraepithelial neoplasia (CIN2+) and CIN3+ among women who had cervical cytology indicating atypical squamous cells of undetermined significance (ASC‐US) or low‐grade cervical lesions (LSIL). A literature search was performed in 3 electronic databases to identify studies that were eligible for this meta‐analysis. Seventeen studies were included in the meta‐analysis. The pooled sensitivity of p16^INK4a to detect CIN2+ was 83.2% (95% confidence interval [CI], 76.8%‐88.2%) and 83.8% (95% CI, 73.5%‐90.6%) in ASC‐US and LSIL cervical cytology, respectively, and the pooled specificities were 71% (95% CI, 65%‐76.4%) and 65.7% (95% CI, 54.2%‐75.6%), respectively. Eight studies provided both HC2 and p16^INK4a triage data. p16^INK4a and HC2 had similar sensitivity, and p16^INK4a has significantly higher specificity in the triage of women with ASC‐US (relative sensitivity, 0.95 [95% CI, 0.89‐1.01]; relative specificity, 1.82 [95% CI, 1.57‐2.12]). In the triage of LSIL, p16^INK4a had significantly lower sensitivity but higher specificity compared with HC2 (relative sensitivity, 0.87 [95% CI, 0.81‐0.94]; relative specificity, 2.74 [95% CI, 1.99‐3.76]). The published literature indicated the improved accuracy of p16^INK4a compared with HC2 testing in the triage of women with ASC‐US. In LSIL triage, p16^INK4a was more specific but less sensitive. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.     

Comparison of p16INK4a Immunocytochemistry with the HPV Polymerase Chain Reaction in Predicting High Grade Cervical Squamous Intraepithelial Lesions

Aim: To compare p16INK4a immunocytochemistry with the HPV polymerase chain reaction in predictinghigh grade cervical squamous intraepithelial lesions. Materials and Methods: This diagnostic case-control studywas conducted from January 2010 until December 2010. We obtained 30 samples, classified according to thedegree of cervical intraepithelial neoplasia (CIN): 11 samples for CIN 1, 9 samples for CIN 2, and 10 samples forCIN 3. HPV PCR, p16INK4a immunocytochemistry, and histopathological examination were performed on allsamples. Statistical analysis was conducted using SPSS 20.0. Results: In predicting CIN 2-3, we found p16INK4ato have similar specificity and positive predictive value as HPV PCR (95%, 97.2% vs 96.7%), but better sensitivity(87.5% vs 72.5%) and negative predictive value (82.1% vs 67.6%). The most prevalent types of high-risk HPVin our study were HPV 33, 35, 58, 52, and 16. Conclusions: p16INK4a has better diagnostic values than HPVPCR and may be incorporated in the triage of ASCUS and LSIL to replace HPV PCR. Genotype distribution ofHPV differs in each region, providing a challenge to develop HPV vaccines based on the epidemiology of HPVin that particular region.     

The utility of p16INK4a and Ki-67 staining on cell blocks prepared from residual thin-layer cervicovaginal material

BACKGROUND: Cell blocks can be prepared from residual thin-layer cervicovaginal (ThinPrep) material and can be used in immunohistochemical staining assays for p16INK4a and Ki-67, which are surrogate markers related to human papillomavirus infection and cell proliferation, respectively. The objectives of the current study were 1) to investigate the feasibility and the role of cell block preparations in identifying significant neoplastic and preneoplastic lesions of the uterine cervix and 2) to assess the feasibility of using p16INK4a and Ki-67 immunohistochemical staining patterns on cell blocks to identify significant preneoplastic cervical lesions. METHODS: Cervicovaginal cytology specimens from 85 patients were analyzed. Cytologic diagnoses based on ThinPrep Papanicolaou test results were as follows: squamous cell carcinoma was diagnosed in 3 specimens, high-grade squamous intraepithelial lesions (HSIL) were diagnosed in 27 specimens, low-grade squamous intraepithelial lesions (LSIL) were diagnosed in 20 specimens, and atypical squamous cells of uncertain significance (ASCUS) were diagnosed in 11 specimens. Diagnoses of negativity for intraepithelial lesions or malignancy (NILM) were made in 24 specimens. Cell block sections were stained with hematoxylin and eosin and were immunostained with antibodies against p16INK4a protein and Ki-67 antigen. RESULTS: The cytomorphologic diagnoses made using cell block preparations were as follows: SCC in 2 specimens, HSIL in 20 specimens, LSIL in 30 specimens, NILM in 32 specimens, and no diagnosis in 1 specimen. In 62 cases (73%), the diagnoses made using cell block preparations were in agreement with the ThinPrep diagnoses. Immunostaining of cell blocks for p16INK4a and Ki-67 exhibited a statistically significant association (P < 0.05) with the presence of significant lesions on either cell block or ThinPrep analysis. CONCLUSIONS: To the authors' knowledge, p16INK4a has not been analyzed previously in ThinPrep cell blocks, and the correlation between Ki-67 expression and cell block diagnoses also has not been reported previously. The current results indicate that cell blocks prepared from residual ThinPrep material represent an additional reliable diagnostic tool in the evaluation of cervical samples. Furthermore, immunohistochemical studies may be helpful in differentiating significant preneoplastic changes from other cervical lesions, such as atrophy.     

p16(INK4a) is a useful marker of human papillomavirus integration allowing risk stratification for cervical malignancies

The present study was conducted to assess utility of p16(INK4a) immunopositivity as a surrogate marker for genomic integration of high-risk human papillomavirus infection (hrHPV). A total of 29 formalin-fixed, paraffin-embedded cervical low-grade squamous intraepithelial lesions (LSILs), 27 high-grade squamous intraepithelial lesions (HSILs) and 53 invasive squamous cell carcinomas (SCCs), histologically-diagnosed between 1st January 2006 to 31st December 2008 at the University of Malaya Medical Centre were stained for p16(INK4a) (CINtec Histology Kit (REF 9511, mtm laboratories AG, Heidelberg, Germany). Immunopositvity was defined as diffuse staining of the squamous cell cytoplasm and or nucleus (involving > 75% of the intraepithelial lesions or SCCs). Staining of basal and parabasal layers of intraepithelial lesions was pre-requisite. One (3.4%) LSIL, 24 (88.9%) HSIL and 46 (86.8%) SCC were p16(INK4a) immunopositive. All normal squamous epithelium did not express p16(INK4). p16(INK4a) expression was significantly lower (p<0.05) in LSIL compared with HSIL and SCC with no difference in expression between HSIL and SCC.The increased p16(INK4a) immunopositivity in HSIL and SCC appears in line with the integrated existence of the hrHPV and may provide more insightful information on risk of malignant transformation of cervical squamous intraepithelial lesions than mere hrHPV detection.     

p16INK4a免疫细胞化学检测在筛查宫颈癌中的作用

目的 探讨p16^INK4a免疫细胞化学检测在筛查宫颈癌及其癌前病变中的作用。方法 选择220例宫颈液基细胞学剩余标本,制作液基薄片进行p16^INK4a 免疫细胞化学检测,随访组织活检结果,并与高危人乳头瘤病毒（HR—HPV）DNA检测结果进行对照。结果 p16^INK4a在宫颈细胞学诊断的鳞状细胞癌（SCC）、鳞状上皮内高度病变（HSIL）、鳞状上皮内低度病变（LSIL）、非典型鳞状细胞-小除外上皮内高度病变（ASC—H）和非典型鳞状细胞-不能明确意义（ASC—US）病例的阳性表达率分别为100．0％（7／7）、92．2％（107／116）、24．3％（17／70）、100．0％（14／14）和36．4％（4／11）。150例p16^INK4a阳性者中,111例有组织活检诊断,其中宫颈上皮内瘤变（CIN）2级及以上病变者97例（87．4％）;70例p16^INK4a阴性者中,18例有组织活检诊断,无一例CIN2及以上病变。p16^INK4a在CIN2及以上病变与在CIN1之间的阳性表达率差异有统计学意义（P〈0.01）,而HR-HPV DNA的阳性率在两者之间差异无统计学意义。结论 p16^INK4a在宫颈HSIL及以上病变中高表达,有利于高危病例的筛选。     

A comparison of the clinical utility of p16(INK4a) immunolocalization with the presence of human papillomavirus by hybrid capture 2 for the detection of cervical dysplasia/neoplasia

BACKGROUND: Evidence suggests that overexpression of p16(INK4a) protein indicates infection and genomic integration of high-risk human papillomavirus (HR HPV) and predicts progression to cervical high-grade squamous intraepithelial lesions (HSILs) and carcinoma. The authors compared the ability of p16(INK4a) and HR HPV detection by Hybrid Capture 2 (HC2) to detect the presence of significant cervical disease. METHODS.: Four hundred ThinPrep specimens (100 each in 4 categories: 100 specimens that were negative for intraepithelial lesions, 100 specimens of atypical squamous cells of undetermined significance [ASC-US], 100 specimens of low-grade squamous intraepithelial lesions [LSILs], and 100 specimens of HSILs) were analyzed. p16(INK4a) protein was immunolocalized using a specific monoclonal antibody, and the detection of HR HPV in all 400 specimens was determined using HC2. RESULTS: p16(INK4a) was found to be positive in 78% of HSIL specimens, 42% of LSIL specimens, and 36% of ASC-US specimens; whereas HC2 was positive in 92% of HSIL specimens, 81% of LSIL specimens, and 45% of ASC-US specimens. In the HSIL category, the sensitivity, which was calculated using Grade 2 or greater cervical intraepithelial neoplasia as the endpoint, was 78% (50 of 66 specimens) for p16(INK4a) and 91% (60 of 66 specimens) for HC2. For LSIL, the sensitivity was 75% (3 of 4 specimens) for p16(INK4a) and 100% (4 of 4 specimens) for HC2. In the ASC-US category, the sensitivity was 89% (8 of 9 specimens) for p16(INK4a) and 100% (9 of 9 specimens) for HC2. Overall, the sensitivity for HSIL was 92% for HC2 and 78% for p16(INK4a). The specificity for HC2 was 8.3% for HSIL, 16.9% for LSIL, and 48.7% for ASC-US; whereas the specificity for p16(INK4a) was 25% in HSIL, 59.1% in LSIL, and 68.4% in ASC-US. The overall specificity was 25% for HC2 and 56% for p16(INK4a). CONCLUSIONS: Although both p16(INK4a) and HC2 may aid in the clinical management of patients with clinically significant lesions, HC2 was found to have greater sensitivity, and p16(INK4a) greater specificity. The labeling of normal cells and bacteria may preclude the use of p16(INK4a) in automated screening or nonmorphologic assays.     

Evaluation of p16INK4a/Ki‐67 dual stain in comparison with an mRNA human papillomavirus test on liquid‐based cytology samples with low‐grade squamous intraepithelial lesion

BACKGROUND:

The objective of the current study was to investigate the clinical performance of detecting high‐grade lesions with the CINtec PLUS p16^INK4a/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 p16^INK4a/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.     

p16INK4A immunohistochemical staining and predictive value for progression of cervical intraepithelial neoplasia grade 1: A prospective study in China

p16^INK4A is strongly expressed in tissues diagnosed as cervical intraepithelial neoplasia (CIN) and cancer in women infected with human papillomavirus (HPV), but few prospective studies have evaluated p16^INK4A as a marker for the risk of low‐grade CIN (CIN1) progression. We investigated the prevalence of p16^INK4A immunostaining by CIN grade and whether overexpression of p16^INK4A in CIN1 predicts future risk for high‐grade CIN in Chinese women. 6,557 Chinese women aged 30–49 years were screened from 2003 to 2005 using cytology and carcinogenic HPV test. Colposcopy was performed on women with any abnormal result. p16^INK4A Immunostaining was performed on biopsies from all women with CIN1, as well as randomly selected women with normal or CIN grade 2 and worse (CIN2+) biopsies. Women with CIN1 were followed up without treatment. Colposcopy was performed on all untreated women at a 2‐year interval. The prevalence of p16^INK4A staining was 2.7%, 42.7%, 75.5%, 79.6% and 100% among women with normal, CIN1, 2, 3 and cancer biopsies, respectively (p < 0.001). HPV positivity was strongly associated with p16^INK4A staining [odds ratios (OR) = 12.8; 95% confidence intervals (CI): 5.2–31.6]. p16^INK4A staining of CIN1 biopsies at baseline was associated with an increased risk of finding high‐grade CIN over 2 years of follow‐up (OR = 1.43; 95% CI: 0.52–3.91). The two‐year cumulative incidence of CIN2+ for p16^INK4A positive women was higher at 10.71% than for p16^INK4A negative women at 1.30% (crude RR = 8.25, 95% CI: 1.02–66.62). p16^INK4A overexpression is strongly associated with grade of CIN and risk of progression to high‐grade CIN in women with low‐grade lesions.     

p16INK4a在宫颈液基细胞学辅助筛查中的标记意义

目的 评价p16INK4a在宫颈液基细胞学检查中的标记意义.方法 收集74例宫颈外口和颈管的脱落细胞标本,分别进行液基细胞学检测和p16INK4a免疫细胞化学染色,并应用杂交捕获二代法检测高危人乳头瘤病毒(HR-HPV)感染.结果 74例标本中,细胞学诊断未见癌细胞或癌前病变细胞(阴性)10例,意义不明的不典型鳞状细胞(ASC-US)15例,鳞状上皮内低度病变(LSIL)28例,不除外上皮内高度病变的不典型鳞状细胞(ASC-H)5例,鳞状上皮内高度病变(HSIL)11例,鳞状细胞癌(SCC)5例.各级别病变中,HR-HPV阳性者分别为1、4、3、9、7和5例,p16INK4a免疫细胞化学染色阳性者分别为2、5、3、8、9和5例.随着宫颈病变级别的上升,HR-HPV和p16INK4a免疫细胞化学染色阳性率均增高.结论 p16INK4a免疫细胞化学染色增强了对不典型细胞的区分能力,可以提高宫颈癌筛查的准确性.     

Evaluation of cervical cone biopsies for coexpression of p16INK4a and Ki-67 in epithelial cells

Diffuse overexpression of p16(INK4a) in basal and parabasal cells of cervical epithelium is a hallmark of human papillomavirus-mediated transformation. Focal p16(INK4a) expression is occasionally observed in nondysplastic epithelium. In normal cells, expression of p16(INK4a) triggers cell cycle arrest. However, cells undergoing transformation in intraepithelial lesions actively proliferate. To prove that the different expression patterns of p16(INK4a) , i.e., focal versus diffuse, reflect biologically different entities, we hypothesized that p16(INK4a) -positive cells in epithelia displaying focal p16(INK4a) expression pattern do not coexpress proliferation-associated Ki-67 protein, while p16(INK4a) -positive cells in lesions with diffuse p16(INK4a) expression may do. A total of 138 cervical cone biopsies were stained for the expression of p16(INK4a) and Ki-67 using a primary antibody cocktail. All metaplastic lesions (n = 21) displayed focal staining for p16(INK4a) , and in all of these lesions p16(INK4a) -positive cells were found to be negative for Ki-67 expression. Diffuse expression of p16(INK4a) was observed in 12/21 (57.1%) cervical intraepithelial neoplasia (CIN) 1 lesions, all of them simultaneously showed Ki-67 immunoreactivity in a large proportion of p16(INK4a) -positive cells. Seventeen of 23 (73.9%) CIN2 lesions and all 27 (100%) CIN3/carcinoma in situ (CIS) as well as all 46 (100%) carcinoma cases displayed diffuse and combined expression of p16(INK4a) and Ki-67. Coexpression of Ki-67 and p16(INK4a) in the same cell is entirely restricted to cervical lesions displaying diffuse p16(INK4a) expression, whereas in lesions with focal p16(INK4a) expression, p16(INK4a) -expressing cells are negative for Ki-67. Thus, diffuse expression of p16(INK4a) reflects lesions with proliferation-competent cells, while p16(INK4a) -expressing cells associated with focal expression patterns are cell cycle arrested.     

Evaluation of a nuclear score for p16INK4a-stained cervical squamous cells in liquid-based cytology samples

BACKGROUND: The p16INK4a gene product is overexpressed strongly in abnormal cervical epithelia and may serve as a valuable biomarker to identify abnormal cells in cervical smears or liquid-based cytology samples. METHODS: The authors performed p16INK4a immunocytochemistry to locate cells that expressed p16INK4a in liquid-based cytology samples and used a nuclear scoring system based on several morphologic criteria to interpret the degree of abnormality of these cells. RESULTS: Among 108 samples that were scored as normal in Papanicolaou-stained, parallel slides, any p16INK4a-positive cells were observed in 13 samples (12%), but only 1 of 108 samples (1%) was scored abnormal after applying nuclear scoring criteria. In the group of 52 low-grade squamous intraepithelial lesion (LSIL) samples, 19 samples (37%) were positive for any p16INK4a reactivity, but only 5 of those samples (10%) were scored abnormal after applying the nuclear score. Among the 50 high-grade squamous intraepithelial lesion (HSIL) samples, 49 samples (98%) were positive for p16INK4a and were scored as abnormal. Comparison of the scoring results of independent observers revealed good reproducibility of the nuclear score. CONCLUSIONS: The current results suggested that p16INK4a enables the location of potentially abnormal cells on liquid-based cytology samples. The nuclear score facilitated interpretation of the degree of abnormality of p16INK4a-stained cells. Thus, locating potentially abnormal cells by p16INK4a immunocytochemistry and their interpretation based on the nuclear score described here may help to identify patients with HSIL in cytologic screening programs and may represent a new approach for reducing the number of equivocal or misinterpreted cytologic specimens.