Comparative evaluation of nm23 and p16 expression as biomarkers of high-risk human papillomavirus infection and cervical intraepithelial neoplasia 2(+) lesions of the uterine cervix

Benevolo M, Terrenato I, Mottolese M, Marandino F, Muti P, Carosi M, Rollo F, Ronchetti L, Mariani L, Vocaturo G & Vocaturo A
(2010) Histopathology 57 , 580–586
Comparative evaluation of nm23 and p16 expression as biomarkers of high‐risk human papillomavirus infection and cervical intraepithelial neoplasia 2⁺ lesions of the uterine cervix Aims: To investigate the clinical role of nm23 expression in identifying both high‐risk human papillomavirus (HR‐HPV) and high‐grade cervical lesions or carcinomas [cervical intraepithelial neoplasia 2⁺ (CIN2⁺)], and to compare it with p16 overexpression, as this latter biomarker has already been reported widely in HR‐HPV infected cervical lesions. Methods and results: Immunohistochemical evaluation of nm23 and p16 in 143 cervical biopsy specimens including negative, low‐ and high‐grade lesions and squamous carcinomas (SC). HR‐HPV testing by Digene hybrid capture 2 (HC2) and polymerase chain reaction (PCR) on the cervico‐vaginal samples of the same patients. In detecting CIN2⁺, p16 was significantly more sensitive and specific than nm23 (96.3% versus 81.8% and 66% versus 36.4%, respectively, both P < 0.0001). Concerning HR‐HPV detection by HC2, p16 showed a significantly higher specificity than nm23 (82% versus 47%, P <0.0001), although the sensitivities were comparable (71% versus 76%). We found a significantly direct correlation between nm23 and HC2 findings. However, nm23 expression did not correlate with HPV16/18 infection. In contrast, we observed a significant association between p16 overexpression and HPV16/18 genotypes. Conclusions: We confirm the diagnostic value of p16 overexpression. Moreover, despite in vitro data regarding the interaction with the HPV‐E7 protein, nm23 does not appear to be a more useful biomarker than p16 in identifying CIN2⁺ or HR‐HPV infection.     

p16INK4a在宫颈细胞学鳞状上皮内瘤变中的意义

目的探讨p16^INK4a的表达在宫颈细胞学中鳞状上皮内瘤变中的意义及其与人乳头状瘤病毒（HPV）型别之间的关系。方法对88例经活检证实的液基细胞学标本[包括20例慢性宫颈炎、18例低度鳞状上皮内瘤变（LSIL）、34例高度鳞状上皮内瘤变（HSIL）及16例鳞状上皮细胞癌（SCC）],分别用免疫细胞化学（EnVision方法）检测其p16^INK4a的表达,并对所有标本用聚合酶链反应（PCR）方法检测HPV型别（包括HPV16、18、31、33、35、39、45、51、52、53、56、58、59、66、68、6、11、42、43及44型）。结果p16^INK4a在慢性宫颈炎组呈阴性,在LSIL、HSIL及SCC组的表达率分别为27．8％、100％及100％,LSIL、HSIL及SCC组p16^INK4a的表达均显著高于慢性宫颈炎组（P〈0．01）;p16^INK4a在高危型HPV感染组的表达率（96．4％）显著高于低危型HPV感染组（7．7％）,差异有统计学意义（t=4．32,P〈0．01）。结论p16^INK4a是一种敏感性较高,特异性较好的标记物,可以识别与高危型HPV有关的非典型鳞状上皮细胞。     

p16INK4a expression as a potential marker of low‐grade cervical intraepithelial neoplasia progression

To evaluate p16^INK4a immunoexpression in CIN1 lesions looking for differences between cases that progress to CIN2/3 maintain CIN1 diagnosis, or spontaneously regress. Seventy‐four CIN1 biopsies were studied. In the follow‐up, a second biopsy was performed and 28.7% showed no lesion (regression), 37.9% maintained CIN1, and 33.4% progressed to CIN2/3. Immunostaining for p16^INK4a was performed in the first biopsy and it was considered positive when there was strong and diffuse staining of the basal and parabasal layers. Pearson's chi‐square was used to compare the groups (p ≤ 0.05). The age of the patients was similar. There was no significant difference in p16^INK4a immunoexpression in the groups, however, statistical analyses showed a significant association when only the progression and regression groups were compared (p = 0.042). Considering p16^INK4a positivity and the progression to CIN2/3, the sensitivity, specificity, positive, and negative predictive values in our cohort were 45%, 75%, 47%, and 94%, respectively. We emphasize that CIN1 with p16^INK4a staining was associated with lesion progression, but the sensitivity was not high. However, the negative predictive value was more reliable (94%) and p16^INK4a may represent a useful biomarker that can identify CIN1 lesions that need particular attention, complementing morphology.     

Cytological evaluation using liquid‐based cytology in the male urogenital tract infected with human papillomavirus

This study was performed to determine the associations between human papillomavirus (HPV) infection and cytological changes in the male glans and urethral samples. Two rubbed samples of the glans and distal urethra were collected from 160 patients with urethritis, and the collected cells were placed into preservative solution for liquid‐based cytology. DNA was extracted from all samples, and β‐globin gene amplification, HPV‐DNA test, and HPV genotyping were performed. After papanicolaou staining of each sample, cytological findings were assessed based on nine non‐classic signs, and compared with the results of HPV‐DNA test. Among the β‐globin positive samples, HPV (any type) was detected in 33 and 25% of the glans and urethra samples, respectively. High‐risk HPV‐DNA was detected in 25 and 15% of the glans and urethra samples, respectively. In the glans samples, cytological signs of HPV infection were observed in 17% of low‐risk HPV‐positive samples and in 58% of high‐risk HPV‐positive samples. Cytological atypia suspected to indicate penile intraepithelial neoplasia were observed in 17 and 33%, respectively. On the other hand, abnormal cells were observed in 29% of low‐risk HPV‐positive and 83% of high‐risk HPV‐positive urethral samples. Cytological atypia suspected to be urethral intraepithelial neoplasia was observed in 29 and 65%, respectively. In situ hybridization demonstrated the presence of HPV‐DNA in the morphologically abnormal cells in 31 and 35% of high‐risk HPV‐positive glans and urethral samples, respectively. Cytological changes similar to cervical intraepithelial neoplasia in females could be detected in the HPV‐positive glans and urethral samples. Diagn. Cytopathol. 2014;42:491–497. © 2013 Wiley Periodicals, Inc.     

An analysis on the combination expression of HPV L1 capsid protein and p16INK4a in cervical lesions

Human papillomavirus (HPV) infection in cervix is the most important reason for cervical cancer, but only 2% cervical HPV infection will develop into cervical cancer. So how to identify patients at risk of progressive cervical lesions from those infected with HPV to avoid over treatment is a big issue in clinic. The aims of this study were to detect the expression of HPV L1 capsid protein and p16^INK4a in cervical lesions and to investigate the combination expression of HPV L1 capsid protein and p16^INK4a in cervical lesions and its diagnostic efficiency in clinic. Immunochemical method was used to detect the expression of HPV L1 capsid protein and p16^INK4a in 169 cases of abnormal cytology. Histopathologic test was performed to identify cervical lesions of all the cases. χ² test and spearman's rank correlation were used for statistical analysis. The diagnostic sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), accuracy, and the area under the receive operating characteristic (ROC) curve (denoted by A_Z) were calculated with SPSS 13.0. All the statistical tests were two sided at the 5% level of significance. L1 expression decreased (P < 0.001), but p16^INK4a expression increased (P < 0.001) with histopathologic diagnosis increasing. The expression rates of HPV L1 capsid protein, p16^INK4a, and L1(?)/p16(+) in cervical intraepithelial neoplasia (CIN)2, CIN3, and squamous‐cell carcinoma were statistically different from those in CIN1 (P < 0.001). The expressions of HPV L1 capsid protein, L1(+)/p16(+), L1(+)/p16(?), and L1(?)/p16(?) were negatively correlated with the severity of cervical lesions (P < 0.001), whereas the expressions of p16^INK4a and L1(?)/p16(+) were positively correlated with the severity of cervical lesions (P < 0.001). The specificity and A_Z of combining L1 with p16 ^INK4a were statistically higher than L1 or p16 ^INK4a alone (P < 0.05). L1 and p16^INK4a are useful biomarkers for the early diagnosis of cervical lesions. The combination of L1 and p16^INK4a has a higher diagnostic accuracy than L1 or p16^INK4a alone in diagnosis of cervical lesions. Diagn. Cytopathol. 2010;38:573–578. © 2009 Wiley‐Liss, Inc.     

Overexpression of p16INK4A in liquid-based specimens (SurePath) as marker of cervical dysplasia and neoplasia

Human papillomavirus (HPV) is recognized as a causal agent for cervical carcinomas. Assimilation of HPV oncogenes E6 and E7 into the host DNA promotes upregulation of cyclin dependent kinase inhibitor (CDKI) p16(INK4A), detectable by monoclonal antibody in the developing cervical cancer cells. The aim of this study was to 1) develop a protocol for p16(INK4A) immunocytochemical staining on SurePath preparations, and 2) determine its utility as an HPV marker on a spectrum of cervical reactive and neoplastic lesions. Seventy-two specimens consisting of 28 nonneoplastic/nondysplastic cases (NN), one reactive glandular cells (RGC), 27 low-grade squamous intraepithelial lesions (LSIL), 10 high-grade squamous intraepithelial lesions (HSIL), one squamous cell carcinoma (SCCA), four atypical glandular cells (AGUS), and two adenocarcinomas (ADCA) were reprepped by SurePath and antibody to p16(INK4A) applied at 1:100 dilution using the Dako Envision + System on the Dako Autostainer. Expression of p16(INK4A) within the nucleus principally and cytoplasm of at least 10-15 cells was considered positive. All initial Papanicolaou-stained discrepant cases (p16(INK4A) positivity of NN and RGC cases and lack of reactivity in LSIL, HSIL, and AGUS) were reviewed. Nine of ten (90%) HSIL, one (100%) SCCA, 21/27 (78%) LSIL, and some reactive and inflammatory specimens demonstrated the presence of p16(INK4A). Reevaluation of discrepant cases revealed that several were underinterpreted (four NN were LSIL, one RGC was AGUS) or overinterpreted (one LSIL was NN). Following reassessment, false-positive staining was present in only 1/25 (1.4%) NN. Six of 30 (20%) LSIL lacked p16(INK4A) positivity. One of 10 (10%) HSIL had no staining. Two of four AGUS did not react with p16(INK4A) antibody. Both SCCA (1) and ADCA (2) had positive expression. This study confirms the intimate relationship between p16(INK4A) and HPV cytopathic effect. The p16(INK4A) immunocytochemical stain can be applied to liquid-based cervical preparations. This technique offers a more objective approach to deciphering "gray areas" of gynecologic cytopathology.     

The retinoblastoma protein/p16 INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma

Stankiewicz E, Prowse D M, Ktori E, Cuzick J, Ambroisine L, Zhang X, Kudahetti S, Watkin N, Corbishley C & Berney D M
(2011) Histopathology 58 , 433–439
The retinoblastoma protein/p16 ^INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma Aims: The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood. Human papillomavirus (HPV) may be involved in carcinogenesis, but few studies have compared cell‐cycle protein expression in HPV positive and negative cancers. The aim was to determine the extent of HPV infection in different histological subtypes of PSCC and its impact on the expression of key cell‐cycle proteins: p53, p21, p16^INK4A and retinoblastoma (RB) protein. Methods and results: One hundred and forty‐eight PSCC samples were examined immunohistochemically for RB, p16^INK4A, p53 and p21 protein expression. One hundred and two cases were typed for HPV by PCR. HPV DNA was detected in 56% of tumours, with HPV16 present in 81%. Basaloid tumours were related strongly to HPV infection (10 of 13), while verrucous were not (three of 13). Fifty‐nine per cent (38 of 64) of usual type SCCs had HPV infection. RB protein correlated negatively (P < 0.0001) and p16^INK4A (P < 0.0001) and p21 (P = 0.0002) correlated positively with HPV infection. p53 did not correlate with HPV infection. Conclusions: HPV infection is present in more than half of penile cancers and it is responsible for RB pathway disruption. However, no link between HPV and p53 immunodetection was found. Only basaloid and half of usual‐type PSSCs correlate with HPV infection, confirming possible separate aetiologies for those tumours.     

Evaluation of p16INK4a, minichromosome maintenance protein 2, DNA topoisomerase IIalpha, ProEX C, and p16INK4a/ProEX C in cervical squamous intraepithelial lesions

p16INK4a has been shown to be overexpressed in nearly all high-grade squamous intraepithelial lesions (HSILs). Other cell-cycle regulators, such as minichromosome maintenance protein 2 (MCM2), DNA topoisomerase IIalpha (TOP IIA), and ProE(X) C (a cocktail of MCM2 and TOP IIA), have also demonstrated some value in identifying squamous intraepithelial lesions. Data on direct comparison of those cell regulatory proteins in the detection of squamous intraepithelial lesions, with a focus on low-grade squamous intraepithelial lesions (LSILs), are limited. We immunohistochemically evaluated the diagnostic value of p16, MCM2, TOP IIA, ProE(X) C, and a cocktail of p16 and ProE(X) C in 62 cervical biopsy specimens, including 14 cases of benign squamous mucosa (group 1), 34 cases of LSILs (group 2), and 14 cases of HSILs (group 3). The staining intensity and distribution were recorded. The results demonstrated that positive staining for p16 and the p16/ProE(X) C was observed in 100% of cases in group 3, whereas 79%, 86%, and 79% of cases were positive for CM2, TOP IIA, and ProE(X) C, respectively. ProE(X) C and the p16/ProE(X) C showed positive staining in 94% and 100% of cases in group 2, respectively. In contrast, immunoreactivity for p16, MCM2, and TOP IIA was detected in only 76% of cases in group 2. Importantly, all 8 p16-negative cases in group 2 were positive for p16/ProE(X) C (P = .003). Our data indicate that (1) p16 is a more sensitive and specific marker for identifying HSILs; (2) ProE(X) C is a better marker for the detection of LSILs; and (3) p16/ProE(X) C provides the highest diagnostic value for the detection of both HSILs and LSILs.     

宫颈上皮内瘤变中p16INK4A和Ki-67的表达

摘要：目的探讨p16^INK4A、Ki-67和HPV抗原表达及高危型HPV（HR—HPV）检测在宫颈上皮内瘤变（CIN）病理诊断中的意义。方法选取宫颈活检病理确诊为CIN的组织蜡块101例,重新切片,应用免疫组化两步法检测p16^INK4A、Ki-67和HPV抗原表达,并取正常宫颈组织50例进行对比研究。同时,应用第二代杂交捕获法对其中25例CIN组织样品进行HR—HPV检测。结果p16^INK4A蛋白表达水平在CINI、CINⅡ、CINⅢ级之间差异有非常显著性（P〈0．001）,其表达水平随着CIN级别的增高而增加,呈现良好的线性相关性（P〈0．001）;Ki-67蛋白表达阳性细胞多少与CIN分级之间无显著相关性（P〉0．05）,但其在宫颈鳞状上皮中的位置分布与CIN级别之间却有显著相关性（P〈0．05）;HPV抗原免疫组化染色阳性反应仅呈现于CIN鳞状上皮表层挖空细胞内,其阳性率在不同级别CIN之间的差异无统计学意义（P〉0．05）;HR—HPV在CINI、CINⅡ和CINⅢ级的检出率分别为81．8％、80．0％和100．0％,但其检出率在不同级别CIN之间差异也无显著性（P〉0．05）。结论p16^INK4A和Ki-67染色对CIN的病理诊断和分级具有一定诊断价值,对于CINI级形态结构不典型的病例,HR—HPV的检测结果对病理诊断有辅助意义。     

Immunocytochemical staining of p16(ink4a) protein as an adjunct test in equivocal liquid-based cytology

For cervical cancer screening, HPV-DNA test is expensive and is not easily available in all clinical situations. Thus, we investigated the role of p16(ink4a) immunostaining as another adjunct test to diagnose cervical neoplasia in equivocal liquid based cytology. Eighty-seven patients were randomly selected for this study (3 patients with normal, 84 patients with abnormal including 24 ASCUS, 30 LSIL, and 30 HSIL). We performed p16(ink4a) immunostaining on ThinPrep slide and on each case from the corresponding cervical biopsy tissues. High-risk HPV-DNA testing was also performed on all the subjects. We found that the immunoreactivity of p16(ink4a) is strongly correlated with the grade of cytologic and histologic diagnoses as well as with Hybrid Capture 2. In comparing the p16(ink4a) immunostaining with the Hybrid Capture 2 for accuracy of the diagnosis of CIN II/III or a higher-grade disease in the case of ASCUS/LSIL on ThinPrep, no significant differences were observed. Our data implies that p16(ink4a) immunocytochemical staining in liquid-based cytology specimens might be used as a good adjunct test to predict cervical histology in equivocal ThinPrep tests.     

子宫颈病变中p16INK4A 蛋白表达及其与HPV16/18感染的关系

目的 探讨p16INK4A 蛋白在子宫颈鳞癌(SCC)和子宫颈上皮内肿瘤(CIN)中的表达及其与HPV16/18感染的关系.方法 用原位杂交法检测HPV16/18在25例子宫颈癌、45例CIN及10例慢性子宫颈炎中的表达,同时用免疫组化EliVision法检测p16INK4A 蛋白的表达.结果 (1)与慢性子宫颈炎相比,CIN Ⅱ级、CIN Ⅲ级、浸润癌HPV16/18杂交信号阳性率显著增高(P<0.01);(2)子宫颈鳞癌组织、CIN Ⅰ级、CIN Ⅱ级、Ⅲ级及慢性子官颈炎标本中p16INK4A 蛋白阳性率分别为100.0%、20.0%、46.7%、100.0%和10.0%;(3)在子宫颈鳞癌及CIN HPV16/18感染的标本中p16INK4A 蛋白表达均是阳性.结论 子宫颈鳞癌的形成与HPV感染、p16INK4A 蛋白过表达是呈正相关关系,p16INK4A蛋白可能作为子宫颈鳞癌及CIN的标志物,对子宫颈癌筛查和预防有重要意义.     

宫颈鳞癌中p16、p21WAF1、Rb、cyclinE蛋白的表达

目的通过观察p16、p21WAF1、Rb、cyclinE 4种细胞周期相关蛋白在宫颈鳞状细胞癌中的表达,探讨它们在宫颈癌的细胞周期调控中的作用.方法采用免疫组化Eli Vision二步法对88例宫颈鳞癌组织,16例宫颈上皮内病变(CIN)组织,15例宫颈炎组织进行p16、p21WAF1、Rb、cyclinE 4种蛋白表达的检测.结果p16、p21WAA1、cyclinE在宫颈癌中的表达高于宫颈炎(P＜0.05);Rb在宫颈癌的表达少于宫颈炎(P＜0.05);Rb与p16在宫颈癌中的表达呈负相关(r=-0.675,P＜0.05).结论宫颈癌细胞周期G1期中,由于Rb蛋白的缺失,使cyclinE表达升高,致使癌细胞增生;同时,p16、p21WAF1蛋白在宫颈癌中的表达增高,并失去抑制作用.     

Aberrant methylation of p14(ARF), p15(INK4b) and p16(INK4a) genes and location of the primary site in pulmonary squamous cell carcinoma

Aberrant methylation of cytosines in CpG islands of the promoter regions of tumor suppressor genes is found in human tumors as a common mechanism of gene silencing. We investigated the methylation status of the chromosome 9p21 gene cluster (p14(ARF), p15(INK4b) and p16(INK4a) genes) by methylation-specific polymerase chain reaction in 20 central and 40 peripheral types of pulmonary squamous cell carcinoma (SqCC) in order to determine the differences between the pathogeneses of the central and peripheral types of SqCC. The frequencies of methylation were 30% for the p14(ARF) gene, 20% for the p15(INK4b) gene and 40% for the p16(INK4a) gene in the central type and 25% for the p14(ARF) gene, 10% for the p15(INK4b) gene and 38% for the p16(INK4a) gene in the peripheral type. Cases in which there was methylation of the p16(INK4a) gene had a higher smoking index in the peripheral type (P = 0.007). This trend was not detected in the central type. Methylation of two or three genes was observed in 55% of methylation in at least one gene of the central type but in only 17% of the peripheral type. This overlap methylation of the chromosome 9p21 gene cluster was found more frequently in the central type (P = 0.02). These findings suggest one of the epigenetic differences between the central and peripheral types of SqCC.     

Prognostic significance of p16INK4a/p53 in Tunisian patients with breast carcinoma

Infiltrating ductal carcinoma (IDC) of the breast is a result of genetic alterations that affect the regulation of the cell cycle check-point and apoptosis. The aim of the present study was analysis using immunohistochemical localization of mouse double minute-2 (mdm2), p16INK4a, p53, bax and bcl-2 markers in Tunisian patients with breast IDC and to determine if there was correlation with the major clinico-pathological parameters and with survival of patients. We showed that the expression of p53, p16INK4a, mdm2, bcl-2, and bax was observed in 46.3%, 20.7%, 38%, 50% and 11.9% of cases, respectively. Statistical analysis revealed that positive expression of mdm2 was associated with larger tumors (P = 0.013), whereas bax positivity was more prevalent in younger patients and in tumors of smaller size (P = 0.008 and P = 0.012 respectively). Furthermore, the expression of p16INK4a correlated with advanced grade (P < 0.0001), triple negative tumors (ER-/PR-/HER2-, P = 0.001) and mdm2 expression (P = 0.017). The absence of nuclear p53 accumulation was predictive of good prognosis as well as when it was associated with negative expression of p16INK4a. Our findings suggest that among the biomarkers tested, p16INK4a might have a useful clinical and prognostic significance in infiltrating ductal carcinoma of the breast.     

人脑神经胶质瘤中p16、p21~(WAF1/CIP1)蛋白表达的研究

目的　探讨 p16、p2 1WAF1/CIP1两种抑癌基因与人脑神经胶质瘤恶性程度的关系。方法　采用SABC免疫组织化学方法对 6 4例人脑胶质瘤组织及 8例正常脑组织标本中p16和 p2 1WAF1/CIP1表达情况进行检测 ,并进行相关分析。结果　①p16和 p2 1WAF1/CIP1阳性表达率在人脑胶质瘤中分别为 4 5 .3%和 6 4 .1%与正常脑组织中的表达情况差异显著 (P <0 0 5 ) ;②p16蛋白和 p2 1WAF1/CIP1蛋白阳性表达率均随着胶质瘤的恶性程度的增高而降低 ,差异显著 (P <0 0 1) ,且呈负相关关系 ;③p16蛋白和 p2 1WAF1/CIP1蛋白可协同表达 ,且在正常脑组织和脑胶质瘤各分级中 p16蛋白和 p2 1WAF1/CIP1蛋白协同表达率差异显著 (P <0 0 5 ) ,并呈负相关关系。结论　p16与p2 1WAF1/CIP1蛋白的阳性表达率及协同表达率可在一定程度上反映胶质瘤细胞的恶性程度 ,可作为判断其恶性程度的有效指标     

Immunocytochemical staining of p16INK4a protein from conventional Pap test and its association with human papillomavirus infection

The p16INK4a protein is immunocytochemically detected in liquid-based (LB) specimens as a diagnostic marker of cervical dysplasia and neoplasia. Its up-regulation is promoted by high-risk human papillomavirus (HR-HPV) infection. We aimed to detect p16INK4a on conventional Papanicolaou (Pap) test (CPT) slides and to determine the relationship between its overexpression and HR-HPV infection. CPT and LB Pap test (LBPT) slides (165 samples of each) were examined by immunocytochemical staining for p16INK4a. After polymerase chain reaction (PCR), HPV-DNA was genotyped by dot blot hybridization. The CPT slides displayed more numerous dispersed squamous cells and LBPT slides had a clearer background. Positive p16INK4a on CPT occurred in 0% (0/30), 52.5% (21/40), 54.3% (19/35), 100% (30/30), and 100% (30/30) in normal, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesions (LSILs), high-grade SILs (HSILs), and squamous cell carcinomas (SCCs) cases, respectively. LBPT slides showed comparable results but were less sensitive. HPV-DNA was detected in 86.7, 70, 45, 57.14, and 10% in SCCs, HSILs, ASCUS, LSILs, and normal cervical cells, respectively. Because HR-HPV was identified in all HPV+ samples of high-grade dysplasia (HSILs and SCCs) and all positive p16INK4a samples infected with HR-HPV, the association of p16INK4a overexpression with HR-HPV infection was confirmed. This study suggests that immunocytochemical staining of p16INK4a on CPT slides is convenient and cost-effective for cervical cancer screening by the detection of dysplastic cells infected with HR-HPV.