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.     

Comparison of p16INK4A and Hybrid Capture 2 human papillomavirus testing as adjunctive tests in liquid-based gynecologic SurePath preparations

p16(INK4a), cyclin-dependent kinase inhibitor, is functionally inactivated in many tumors, including cervical cancer. We compared p16(INK4A) immunocytochemical staining and Hybrid Capture 2 (HCII) on SurePath specimens using tissue biopsies (as the gold standard). Their utility in a spectrum of atypical and preneoplastic lesions, and their ability to accurately identify underlying lesions of CIN II or greater was assessed using biopsy follow-up data. One-hundred and seventeen residual SurePath samples were collected: 43 atypical squamous cells of undetermined significance (ASCUS), 47 low-grade (LGSIL), and 27 high-grade (HGSIL) squamous intraepithelial lesions. Two slides were prepared from each sample; one stained with the SurePath autocyte stain and one immunostained using the CINtec p16(INK4a) Cytology Kit (Dakocytomation). High-risk HPV testing was performed using the HCII DNA test (Digene, Gaithersburg, MD). Available tissue biopsy follow-up data was retrieved. p16(INK4a) was positive in 32.6% (14/43) ASCUS, 46.8% (22/47) LGSIL, and 48.1% (13/27) HGSIL specimens. HCII DNA test was positive in 41.9% (18/43) ASCUS, 78.7% (37/47) LGSIL, and 96.3% (26/27) HGSIL samples. The sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of p16(INK4a) and HCII were: 58.7% and 89.8%, 58.6% and 34.6%, 69.2% and 72.1%, 47.2% and 64.3%, respectively. In patients with cervical biopsies, the PPV of HCII (92.3%) results for a biopsy with CINII/III was significantly higher than the PPV of p16(INK4a) (52%) (P=0.001). Using liquid-based cytology specimens, HCII is a more sensitive test than p16(INK4a) for detection of abnormal cytology. HCII has a higher PPV than p16(INK4a) for identifying CIN II/III.     

p16INK4A在动情周期和早孕期小鼠子宫内膜的表达

目的 检测肿瘤抑制基因p16INK4A在小鼠动情周期和早孕期子宫内膜中的表达,初步探讨p16^INK4A在小鼠胚胎植入过程中的生物学作用。方法 运用反转录-聚合酶链反应(RT-PCR)技术检测动情周期和早孕期小鼠子宫内膜p16^INK4AmRNA的表达水平;用免疫组织化学和Western blotting方法检测p16INK4A蛋白的表达。结果 RT-PCR显示,早孕期比动情周期p16^INK4AmRNA的表达有显著增强。动情周期中动情期比其他3期表达明显增强;早孕期中,随妊娠天数的增加,p16^INK4AmRNA的表达也逐渐增强,孕5d达峰值,之后又逐渐减弱。免疫组织化学和Western blotting的结果也都显示,p16^INK4A蛋白在子宫内膜的表达及规律与RT-PCR的结果一致。结论 p16^INK4A在小鼠的胚泡植入过程中起重要作用。     

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。     

An evaluation of the diagnostic and prognostic significance of p16INK4a/p21WAF1/Cip1 immunostaining in squamous intraepithelial lesions of the uterine cervix using liquid‐based cytology specimens

Human papillomavirus (HPV) infection frequently causes squamous intraepithelial lesions (SIL) of the uterine cervix and consequently gives rise to squamous cell carcinoma. It is therefore important to identify cases that potentially develop higher grades of SIL at an early stage of the disease. In this study, we thus investigated whether immunocytochemistry for p21^WAF1/Cip1 and p16^INK4a could be applicable in the diagnosis and the prognostic prediction of SIL in combination with genomic analyses of HPV. The genomic analysis of high‐risk HPV (hrHPV), which was done by reversed dot blotting and by in situ hybridization, and immunocytochemistry were performed on liquid‐based cytological specimens. A cross‐sectional study comprising 145 cases of NILM, ASC‐US, LSIL, and HSIL indicated that the incidence of the positive cases for p16^INK4a and p21^WAF1/Cip1 and hrHPV increased with the grade of SIL. A double positive status for p16^INK4a and p21^WAF1/Cip1 was a significant discriminator between HSIL and LSIL/NILM, even when applied in conjunction with the genomic test for hrHPV (P = 0.006 by logistic regression analysis). However, a prospective study employing 61 NILM/ASC‐US cases, revealed that the p16^INK4a/p21^WAF1/Cip1 immunostaining was not a significant predictor for the progression of SIL, whereas the cytological diagnosis (NILM vs. ASC‐US) and the infection status of hrHPV conferred significant effects on the prognosis. Immunostaining of p16^INK4a and p21^WAF1/Cip1 provides additional information on the cytological diagnosis of SIL. A further analysis using a larger population is warranted to obtain a conclusive result regarding the prognostic significance of p16^INK4a/p21^WAF1/Cip1 immunocytochemistry in the diagnosis of SIL. Diagn. Cytopathol. 2014;42: 125–133. © 2013 Wiley Periodicals, Inc.     

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.     

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.     

p16INK4A expression in cervical premalignant and malignant lesions

p16^INK4a is a cyclin-dependent kinase (CDK) inhibitor which decelerates cell cycle by inactivating CDKs that phosphorylate pRb. Human Papillomavirus persistent infection plays an important role on cervical carcinogenesis, mainly by the action of two viral oncoproteins, E6 and E7, which interact with p53 and pRb, respectively. Increasing expression of E6 and E7 in dysplastic cervical cells might thus be reflected by increased expression of p16^INK4a. Recent studies revealed that p16^INK4a expression could be a marker for dysplastic and neoplastic cervical cells. The aim of this study was to analyze p16^INK4a expression in cervical preneoplastic and neoplastic lesions and correlate with lesion grade. Expression of p16^INK4a was analyzed by immunohistochemistry. A total of 6 low-grade squamous intraepithelial lesion (LSIL), 21 high-grade squamous intraepithelial lesions (HSIL) and 27 cancer samples were studied. In HPV-positive cervical samples (n = 48), p16^INK4a expression was observed in 1 of 3 LSIL, in 18 of 19 HSIL and in all 26 cancer cases. These results are in accordance with the hypothesis that functional inactivation of pRb by HPV-E7 protein induces p16^INK4a expression in cervical lesions. In our study, a statistically significant association was observed between cervical lesion grade and p16^INK4a expression (P < 0.001).     

p16INK4A和 p15INK4B对人肝癌细胞增殖和凋亡影响的研究

目的　为探讨抑癌基因 p16 INK4 A和 p15 INK4 B对 Rb基因状态不同的人肝癌细胞系增殖和凋亡的影响。方法　在分析细胞系遗传背景鉴定基础上采用脂质体将构建的 p XJ- p16、p XJ- p15重组质粒转染人肝癌细胞系 BEL74 0 2 (p16 / p15 Rb )和 SMMC772 1(p16 / p15 / Rb- )。应用 PCR、RNA斑点印迹、MTT、集落形成、流式细胞仪等技术检测外源性 p16和 p15基因、m RNA表达及其对肝癌细胞增殖、凋亡的影响。结果　经转染的 BEL74 0 2 - p16 ,BEL74 0 2 - p15细胞 ,分别存在外源性 p16、p15基因 ,在含外源基因细胞中相应的 m RNA表达增强 ;BEL74 0 2 - p15细胞生长速度、集落形成率显著低于对照细胞 BEL 74 0 2 (P<0 .0 1) ;细胞周期分析观察到与亲本细胞比较 ,BEL 74 0 2 - p15的 G1期细胞由 37.7%增高到 4 3.6 % ,S期细胞由 2 2 %下降到 13% (P<0 .0 5 ) ,并出现 G1亚峰 (凋亡峰 )。与此相反 ,BEL74 0 2 - p16细胞增殖未见抑制 ,细胞周期分布无明显差异 ,集落形成率也未见减少。此外 ,SMMC772 1- p16细胞增殖也无抑制。结论　外源性 p15 INK4 B具有抑制人肝癌细胞生长 ,诱导细胞凋亡的作用 ,其作用不受内源性p15影响。而 p16 INK4 A对肝癌细胞生长抑制的作用可能依赖于 RB途径的完整性。     

P16(INK4a) expression as a potential prognostic marker in cervical pre-neoplastic and neoplastic lesions

An immunohistochemical analysis with monoclonal antibody p16(INK4a) was performed in formalin-fixed, paraffin-embedded samples of 60 cases. The aim was to investigate in biopsies the expression of p16(INK4a) of normal uterine cervical tissue, pre-cancerous and cancerous lesions, and their relation with human papilloma virus (HPV) and HIV status. Three parameters were evaluated: percentage of p16(INK4a) positive cells, reaction intensity, and cell staining pattern. All of these parameters were statistically different when compared among different histological groups. However, logistic regression model showed that the reaction intensity was the best indicator of the expression of p16(INK4a). This expression increases from normal to invasive squamous carcinoma. Sixty-six percent of the patients with CIN grade 1 (CIN1) expressed p16(INK4a) (all these cases were infected with high risk HPV). Our study supports the hypothesis that p16(INK4a) expression in pre-cancerous lesions and cancers can be used to identify HPV-transformed cells. Of great interest for routine diagnostic use is the fact that immunohistochemical testing for p16(INK4a) seems to be capable of identifying HPV-positive cells and potentially recognizing those lesions with an increased risk of progression to high-grade lesions.     

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 and pRb expression in cervical squamous and glandular neoplasia

p16(INK4a) is known to play a critical role as a negative regulator of cell cycle progression and differentiation by controlling the activity of the tumor-suppressor protein pRb. The present study evaluated the expression of p16(INK4a) and pRb in cervical squamous and glandular neoplasia. Immunohistochemical staining was performed for p16(INK4a) and pRb in formalin-fixed, paraffin-embedded tissue sections of the uterine cervix using an indirect immunoperoxidase method. p16(INK4a) staining was detected in 7 of 108 sections (6.5%) of normal squamous mucosa, in scattered ciliated columnar cells in 33 of 88 sections (37.5%) of normal endocervical glands, in 9 of 30 sections (30%) with Nabothian cysts, and in 4 of 4 areas (100%) of tubal metaplasia. In contrast, strong p16(INK4a) staining was found in 13 of 18 cases (72.2%) of cervical intraepithelial neoplasia (CIN) I and in all cases of CIN II/III (n = 46), squamous cell carcinoma (n = 18), endocervical glandular dysplasia (n = 10), adenocarcinoma in situ (n = 23), and invasive adenocarcinoma (n = 12). pRb expression was detected in each diagnostic category; however, the proportion of pRb-positive cells was relatively decreased in high-grade premalignant and malignant lesions of the squamous and endocervical mucosa and showed a generally inverse correlation with the expression of p16(INK4a) at the tissue level. These findings confirm a correlation between the expression of p16(INK4a) and pRb in cervical neoplasias and indicate that p16(INK4a) is a specific marker for premalignant and malignant lesions of the squamous and endocervical mucosa.     

Immunohistochemical expression of p16(INK4A) protein as a helpful marker of a subset of potentially malignant oral epithelial lesions: study on a series with long-term follow-up

Montebugnoli L, Cervellati F, Cocchi R, Farnedi A, Pennesi M G, Flamminio F & Foschini M P
(2010) Histopathology 57 , 528–534
Immunohistochemical expression of p16^INK4A protein as a helpful marker of a subset of potentially malignant oral epithelial lesions: study on a series with long‐term follow‐up Aim: To examine a group of lesions that progressed to oral squamous cell carcinoma (OSCC) to determine whether p16^INK4A expression is an early finding during malignant transformation, and whether immunohistochemical evaluation of p16^INK4A is an appropriate prognostic marker. Methods and results: Twenty cases of OSCC were investigated. All cases had had a biopsy on the same site as OSCC performed at least 1 year before OSCC (range 1–11 years; mean 3.15 ± 3.1 years). Twenty specimens from normal oral mucosa served as controls. p16^INK4A expression was evaluated by immunohistochemical analysis and cases showing >5% of stained cells were defined as ‘positive’. All 20 control cases were negative for p16^INK4A. Oral lesions were p16^INK4A‐positive in nine cases and negative in 11. No significant relationship was found between p16^INK4A positivity and the presence/absence of dysplasia. Among OSCC, nine tumours showed p16^INK4A positivity and 11 showed negativity. A significant relationship (χ² = 7.1; P < 0.01) was found between the presence/absence of p16^INK4A staining in OSCC and the presence/absence of p16^INK4A staining in lesions preceding OSCC. Conclusions: p16^INK4A immunohistochemistry has a potential role in detecting a subset of p16^INK4A‐positive lesions with malignant potential.     

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.     

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.     

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.