Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay: correlation with biopsy follow-up results

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 (hc(2)) 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 (hc(2)) 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 hc(2) 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 hc(2). To increase p16(INK4a) immunostaining in abnormal cells, a modified kit version with improved staining performance has been developed and is currently being evaluated.     

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.     

高危型HPV负荷量和p16INK4A蛋白监测评估宫颈锥切术治疗CIN

[目的]评价高危型人乳头状瘤病毒HPV负荷量的检测和p16INK4A蛋白的表达在预测宫颈上皮内瘤变(CIN)宫颈锥切术后残存病变或复发中的意义.[方法]回顾性分析142例2008年10月至2010年12月因CIN行宫颈锥形切除术治疗患者的临床资料.所有患者均于宫颈锥形切除术前6个月以内和术后6～12个月进行HPV负荷量检测,并采用免疫组化方法检测HPV DNA阳性患者宫颈细胞中p16INK4A蛋白表达.[结果]宫颈锥切术前,随着CIN级别的上升,HPV负荷量以及p16INK4A蛋白表达均明显增强(P＜0.05).但在宫颈锥切术后,HPV负荷量和p16INK4A蛋白表达明显降低,宫颈锥切术前和术后两者之间差异有统计学意义(P＜0.05).[结论] HPV负荷量持续增高和p16INK4A蛋白持续呈强阳性是宫颈锥切术后发生残存病变或复发的高危因素,在监测HPV负荷量的同时检测p16INK4A蛋白的表达,对判断宫颈锥切术后发生残存病变或复发有重要意义.     

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.     

Impact of utilizing p16INK4A immunohistochemistry on estimated performance of three cervical cancer screening tests

The histopathological diagnosis of cervical intraepithelial neoplasia grade 2,3 (CIN 2,3) is subjective and prone to variability. In our study, we analyzed the impact of utilizing a biomarker (p16(INK4A)) together with histopathology to refine the "gold standard" utilized for evaluating the performance of 3 different cervical cancer screening tests: cervical cytology, human papillomavirus (HPV) DNA testing and visual inspection with acetic acid (VIA). Cervical biopsies from 2 South African cervical cancer screening studies originally diagnosed by a single pathologist were reevaluated by a second pathologist and a consensus pathology diagnosis obtained. Immunohistochemical staining for p16(INK4A) was then performed. The estimated sensitivity of some cervical cancer screening tests was markedly impacted by the criteria utilized to define CIN 2,3. Use of routine histopathology markedly underestimated the sensitivity of both conventional cytology and HPV DNA testing compared to an improved gold standard of consensus pathology and p16(INK4A) positivity. In contrast, routine histopathology overestimated the sensitivity of VIA. Our results demonstrate that refining the diagnosis of CIN 2,3 through the use of consensus pathology and immunohistochemical staining for p16(INK4A) has an important impact on measurement of the performance of cervical cancer screening tests. The sensitivity of screening tests such as HPV DNA testing and conventional cytology may be underestimated when an imperfect gold standard (routine histopathology) is used. In contrast, the sensitivity of other tests, such as VIA, may be overestimated with an imperfect gold standard.     

Identification of high-grade cervical dysplasia by the detection of p16INK4a in cell lysates obtained from cervical samples

BACKGROUND: Current cervical cancer screening approaches are based on cytology supplemented by human papillomavirus (HPV) testing in some settings. Whereas cytology is laborious and depends on the cytologists' experience, HPV testing has limited specificity when it is used to detect high-grade lesions. A dichotomous test to identify high-grade lesions with greater specificity may be a useful tool for cervical cancer screening. p16(INK4a) is a cell-cycle regulator that has demonstrated strong overexpression in cervical precancer cells and cervical cancer induced by the deregulated expression of HPV oncogenes. METHODS: The authors used a sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the amount of solubilized p16(INK4a) protein in lysates that were prepared from cervical samples to detect high-grade cervical lesions. In total, 187 specimens that were obtained after sampling for conventional cytology in women who attended a cervical colposcopy clinic were analyzed. Seventy-six women underwent a biopsy, and 45 of those women showed histologically confirmed, high-grade cervical intraepithelial neoplasia. RESULTS: For 76 women with biopsy-proven diagnoses, receiver operating characteristic (ROC) analysis of different cutoff values showed an area under the ROC curve of 0.89 for the detection of high-grade cervical dysplasia. At a cutoff value of 8 U/mL, the sensitivity of the p16(INK4a) ELISA for detecting high-grade dysplastic cervical lesions was 96%. CONCLUSIONS: The data obtained in this study suggested that ELISA-based quantification of solubilized p16(INK4a) protein may have high sensitivity for detecting cervical precancer. Further population-based studies will be necessary to analyze the specificity and predictive values of p16(INK4a) protein quantification in cervical samples.     

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.     

Triage options to manage high-risk human papillomavirus-positive women: A population-based cross-sectional study from rural China

Improvement in managing HPV-positive women is urgently needed. Based on a population-based study which included 2112 women aged 49 to 69 from Shanxi, China, we aimed to evaluate the clinical performance of multiple triage strategies based on liquid-based cytology (LBC), p16^INK4a, viral load and partial genotyping, as a single or combined strategy for detecting cervical intraepithelial neoplasia grade 2/3 or higher (CIN2+/CIN3+) in women who tested positive by Hybrid Capture 2 (HC2). Among 452 HC2-positive women, the test positivity of LBC (ASC-US+), p16^INK4a, HPV16/18 and HPV16/18/31/33/45 were 39.6%, 38.5%, 18.0% and 40.0%, respectively. Compared to LBC (ASC-US+) triage, a single triage strategies using p16^INK4a or extended genotyping (SureX HPV16/18/31/33/45) achieved comparable sensitivity (relative sensitivity: 1.08, 95% confidence interval [CI]: 0.93-1.26 and 0.96, 95% CI: 0.76-1.22) and specificity (relative specificity: 1.05, 95% CI: 0.96-1.14 and 1.02, 95% CI: 0.92-1.14) for CIN3+. Viral load triage using a ≥50 RLU/CO cut-point also yielded similar results with LBC (ASC-US+). Among combined triage strategies, HPV16/18 genotyping with reflex p16^INK4a showed higher sensitivity and slightly lower specificity than LBC (ASC-US+) for CIN3+ detection, however, the differences were not statistically significant. Of note, after a negative result by p16^INK4a or LBC among HPV16/18 negative women, the posttest probability of CIN3+ was lower than 1%. Our study suggested that p16^INK4a, extended genotyping and increased viral load cut-point could be promising alternatives to cytology triage. Combined triage algorithms of HPV16/18 with reflex p16^INK4a or cytology, if negative, are associated with the substantial low posttest risk sufficient to release women to next screening round.     

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.     

Role of P16(INK4a) expression in identifying CIN2 or more severe lesions among HPV-positive patients referred for colposcopy after abnormal cytology

BACKGROUND: p16 is strongly overexpressed in dysplastic cervical cells because of the transforming activity of the E7 oncogene of all high-risk human papillomavirus (HR-HPV) types and may be easily revealed by immunochemistry: p16 may, therefore, be considered a surrogate marker for the activated oncogene expression of HR-HPV in dysplastic cervical cells. METHODS: HPV and p16(INK4a) testing were performed in a consecutive series of 283 patients with abnormal cytology referred to colposcopy assessment or follow-up. Triage of patients to colposcopy by HPV or HPV and p16 testing was simulated, and the relative sensitivity, specificity, and positive predictive value (PPV) of HPV and p16 testing for > CIN2 lesions was determined as well as the cost balance of the two triage types. RESULTS: Compared to current protocol, triage by HPV testing reduced the number of colposcopies by 44.2%, but also reduced the > CIN2 detection rate by 10.7%, and was associated with a cost of euro 54.16 per assessed woman and of euro 613.20 per > CIN2 detected. Compared with current protocol, triage by HPV and p16 testing combined reduced the number of colposcopies by 73.1%, but reduced > CIN2 detection rate by 21.5%, and was associated with a cost of euro 54.73 per woman assessed and of euro 704.09 per > CIN2 detected. CONCLUSIONS: Triage by HPV and p16 improves considerably the PPV of diagnostic assessment, but decreases > CIN2 detection rate, and is associated with substantially higher costs. Further decrease of molecular immunochemistry testing due to technological progress may allow HPV and p16 testing to become a cost effective procedure in the future.     

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。     

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.     

Comparison of human papillomavirus testing strategies for triage of women referred with low-grade cytological abnormalities

AimTo compare triage strategies using different human papillomavirus (HPV) consensus and genotyping tests and a p16^INK4a test.Methods1228 women referred with a borderline or single mildly dyskaryotic smear. Samples were taken at colposcopy using PreservCyt. Tests included Hybrid Capture 2, Abbott RealTime PCR, BD HPV, Cobas 4800, PreTect HPV-Proofer, APTIMA and p16^INK4a. Results were based on the worst histology within 9 months.Results97/1228 (7.9%) women had CIN3+ (203/1228 (17%) CIN2+). HPV testing alone using Hybrid Capture 2, Abbott RealTime PCR, BD HPV, Cobas 4800 or APTIMA had a sensitivity for CIN3+ ranging from 99.0% to 100.0% and specificity for <CIN2 from 23.3% to 34.7%. p16^INK4a had a sensitivity of 86.8% and specificity of 50.7%. PreTect HPV-Proofer had a sensitivity of 85.1% and specificity of 73.2%. Testing for HPV type 16 only had sensitivities ranging from 66.0% to 75.5% and specificities from 81.3% to 87.6%. Dual testing with HPV type 16 combined with p16^INK4a gave a high sensitivity for CIN3+ (78.7% to 98.0%) and specificity for <CIN2 of 58.6% to 81.5%.ConclusionsTriage with sensitive HPV testing assays can substantially reduce the number of unnecessary referrals in women with low grade cytology with virtually no loss of sensitivity. Even greater gains can be made if p16 and type 16 are used, but some cases of CIN2 will be missed. In both cases short term surveillance will be needed.     

Combined use of cytology,p16 immunostaining and genotyping for triage of women positive for high-risk human papillomavirus at primary screening

Human papillomavirus (HPV) testing is very sensitive for primary cervical screening but has low specificity. Triage tests that improve specificity but maintain high sensitivity are needed. Women enrolled in the experimental arm of Phase 2 of the New Technologies for Cervical Cancer randomized controlled cervical screening trial were tested for high-risk HPV (hrHPV) and referred to colposcopy if positive. hrHPV-positive women also had HPV genotyping (by polymerase chain reaction with GP5+/GP6+ primers and reverse line blotting), immunostaining for p16 overexpression and cytology. We computed sensitivity, specificity and positive predictive value (PPV) for different combinations of tests and determined potential hierarchical ordering of triage tests. A number of 1,091 HPV-positive women had valid tests for cytology, p16 and genotyping. Ninety-two of them had cervical intraepithelial neoplasia grade 2+ (CIN2+) histology and 40 of them had CIN grade 3+ (CIN3+) histology. The PPV for CIN2+ was >10% in hrHPV-positive women with positive high-grade squamous intraepithelial lesion (61.3%), positive low-grade squamous intraepithelial lesion (LSIL+) (18.3%) and positive atypical squamous cells of undetermined significance (14.8%) cytology, p16 positive (16.7%) and, hierarchically, for infections by HPV33, 16, 35, 59, 31 and 52 (in decreasing order). Referral of women positive for either p16 or LSIL+ cytology had 97.8% sensitivity for CIN2+ and women negative for both of these had a 3-year CIN3+ risk of 0.2%. Similar results were seen for women being either p16 or HPV16/33 positive. hrHPV-positive women who were negative for p16 and cytology (LSIL threshold) had a very low CIN3+ rate in the following 3 years. Recalling them after that interval and referring those positive for either test to immediate colposcopy seem to be an efficient triage strategy. The same applies to p16 and HPV16.     

Expression of p16(INK4a) in relation to histopathology and viral load of 'high-risk' HPV types in cervical neoplastic lesions

A total of 91 cervical archival biopsy series were analysed for the presence and viral load of 'high-risk' types of human papillomavirus (HR-HPV), and p16(INK4a) expression. The women had various degrees of CIN (cervical intraepithelial neoplasia). HPV 16 was the most prevalent type found, at 47% frequency. The frequency of HPV 16 increased with increasing immunoreactivity to p16(INK4a), from 39% to 44% at cases scored low to medium, to 65% at high reactivity. Thirty (33%) of the samples had negative p16(INK4a) analysis results, but were positive for HR-HPV. There was no significant correlation between viral load and the level of p16(INK4a) expression, while the grade of CIN correlated to such expressions. Thus, p16(INK4a) expression analysis yielded information which is consistent with results from the histopathology and might complement the HPV analysis in a clinical prognostic procedure in order to find women at risk for cervical cancer.     

p16INK4a is superior to high‐risk human papillomavirus testing in cervical cytology for the prediction of underlying high‐grade dysplasia

BACKGROUND

The primary goal of this study was to compare the clinical performance of an optimized and rigorously controlled immunocytochemical (ICC) assay for p16^INK4a to high‐risk (HR) human papillomavirus (HPV) detection by polymerase chain reaction (PCR) as diagnostic adjuncts for cytology specimens from colposcopy patients.

METHODS:

The study included 403 cervical cytology specimens collected within 3 months of colposcopy. The colposcopic impression and cervical biopsy diagnosis served as the standards for correlation with cytological, p16^INK4a, and HPV data. p16^INK4a was evaluated using an immunoperoxidase‐based assay that was linear over 4 logs for the detection of HeLa‐spiked positive control cytology specimens, using a threshold for positive test results that was based on receiver operating characteristic curve analysis. HR‐HPV was detected by multiplex PCR using genotype‐specific primers.

RESULTS:

In all combined diagnostic categories (negative for intraepithelial lesion and malignancy, atypical glandular cells, atypical squamous cells of undetermined significance, atypical squamous cells cannot exclude high‐grade squamous intraepithelial lesion, low‐grade squamous intraepithelial lesion, and high‐grade squamous intraepithelial lesion), the p16^INK4a ICC and HR‐HPV assays, respectively, had sensitivity of 81.7% and 83.3% (P = .81) and specificity of 78.1% and 50.9% (P < .001) for the detection of underlying ≥grade 2 cervical intraepithelial neoplasia (CIN) lesions on biopsy. Furthermore, the positive predictive value of p16^INK4a ICC was greater than that of HR‐HPV for patients with biopsies ≥CIN‐2 (41.2% and 24.2%, respectively, P = .001).

CONCLUSIONS:

This p16^INK4a immunocytochemical assay has superior specificity but similar sensitivity to HR‐HPV testing to predict underlying high‐grade dysplastic lesions in patients who are referred for colposcopy. The determination of the overall performance characteristics of p16^INK4a immunocytochemistry, as an independent test or in combination with HPV testing in low‐risk screening populations, however, will require subsequent large‐scale prospective clinical trials. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.     

p16INK4a immunohistochemistry is a promising biomarker to predict the outcome of low grade cervical intraepithelial neoplasia: comparison study with HPV genotyping

Objective

In cervical intraepithelial neoplasia (CIN), p16^INK4a immunohistochemistry has been reported to be a useful diagnostic biomarker. However, limited information is available about the association between the p16^INK4a immunohistochemistry and the outcomes of CIN. Here, we report p16^INK4a immunohistochemistry as an effective biomarker to predict the outcomes of CIN.

Methods

p16^INK4a immunohistochemistry was performed in patients with CIN from January 2000 to August 2009. Among these patients, we have performed a retrospective analysis of the medical records to evaluate the outcome of CIN 1-2 and performed statistical analysis to determine the correlation between p16^INK4a expression and the outcomes. We also performed HPV genotyping and analyzed the relation between the infecting human papillomavirus (HPV) genotype and the outcomes.

Results

A total of 244 patients, including 82 with CIN 1, 60 with CIN 2, and 102 with CIN 3, were examined. The rate of p16^INK4a overexpression increased with increasing CIN grade, 20.7% for CIN 1, 80.0% for CIN 2, and 89.2% for CIN 3, with significant differences between CIN 1 and CIN 2-3 group. In the 131 CIN 1-2 patients, the progression rate was significantly higher for the patients showing p16^INK4a overexpression than for those not showing p16^INK4a overexpression (p=0.005); the regression rate was also found to be significantly lower for the patients showing p16^INK4a overexpression (p=0.003). High-risk HPV genotypes were detected in 73 patients (73.7%). Both progression and regression rates were not significantly different between the high-risk HPV-positive and HPV-negative groups (p=0.401 and p=0.381, respectively).

Conclusion

p16^INK4a overexpression was correlated with the outcome of CIN 1-2, and p16^INK4a is considered to be a superior biomarker for predicting the outcome of CIN 1-2 compared with HPV genotyping.     

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.