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).     

Role for p16(INK4a) in progression of gastrointestinal stromal tumors of the stomach: alteration of p16(INK4a) network members

Gastrointestinal stromal tumors feature a wide spectrum of biologic behavior, ranging from benign to extremely malignant. To determine the role of p16^INK4a alteration in progression of gastrointestinal stromal tumors of the stomach, we have investigated protein expression and gene methylation in correlation with clinicopathologic factors and survival. In addition to immunohistochemical analysis of p16^INK4a in a series of 95 cases, real-time quantitative methylation specific polymerase chain reaction for p16^INK4a and immunostaining for cyclin D1, cyclin E, pRb, DP-1, E2F-1, and Ki-67 were also evaluated in randomly selected samples. The p16^INK4a labeling indices ranged from 0% to 74% (median, 21%), demonstrating a significant inverse correlation with size (P = .046). On univariate (P = .003) and multivariate (P = .067) analyses, loss of p16^INK4a expression increased the likelihood of a poor tumor-related survival. In addition, size (P = .036) and the mitotic index (P = .005) had independent prognostic influence. The p16^INK4a methylation index, which ranged from 0% to 100% (median, 17%), was significantly higher in larger tumors (P < .001) and in high-risk category lesions (P = .001) and inversely correlated with protein expression. Hierarchical cluster analysis based on expression of p16^INK4a network members identified 2 clusters in 27 randomly selected tumor samples, containing 11 and 16 tumors each. Former cluster samples demonstrated higher risk category (P = .022), higher p16^INK4a methylation (P < .001), and more reduced pRb expression (P < .018). In addition, p16^INK4a network members clustered into 2 groups: (1) showing down-regulated p16^INK4a protein and up-regulating of both cyclin D1 and DP-1 and (2) down-regulated pRb and up-regulated E2F-1. We conclude that p16^INK4a alteration has an important role in progression of gastrointestinal stromal tumors of the stomach. Furthermore, the study provides a possible link between regulation of p16^INK4a network members and gastrointestinal stromal tumors.     

p16(INK4a) promoter methylation and 9p21 allelic loss in colorectal carcinomas: relation with immunohistochemical p16(INK4a) expression and with tumor budding

In colorectal carcinomas, p16(INK4a) inactivation is known to occur by allelic loss and by promoter methylation, but mutations are rare. p16(INK4a) is up-regulated in tumor buds, and the consequent shutdown of proliferation may be a prerequisite for tumor budding. Fifty-seven colorectal carcinomas from a consecutive series were investigated. Using DNA from tissue homogenates, p16(INK4a) promoter methylation was seen in 17 of 57 tumors by methylation-specific polymerase chain reaction, and this could be confirmed using DNA from laser-capture microdissected material in 16 of these cases. A total loss of immunohistochemical p16(INK4a) expression was seen in 6 of 17 tumors with promoter methylation. Quantification of immunohistochemical p16(INK4a) expression for the remaining 11 cases revealed statistically lower frequencies of expression as compared with cases without p16(INK4a) promoter methylation. 9p21 allelic loss was observed in 9 cases, but p16(INK4a) expression in these carcinomas was not reduced. Attempted linear regression of p16(INK4a) expression in tumor buds on the degree of tumor budding, as counted on pan-cytokeratin immunostains, did not show a correlation. p16(INK4a) promoter methylation can completely abrogate p16(INK4a) expression in colorectal carcinomas. In many cases, however, it has an appreciable but only modulatory influence on p16(INK4a) expression. Possibly, methylations are heterozygous, and/or mosaic in colorectal carcinomas and/or methylations are not totally stable but can be lost between carcinoma cell replication cycles. Up-regulation of p16(INK4a) does not seem to be a strict requirement for tumor budding, hence, the absence of a correlation.     

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.     

p16INK4a expression in basal-like breast carcinoma

BLBC represents a distinctive group of invasive breast carcinomas with specific genotype and immunopro-file. BLBC is usually defined by gene expression profiling and is currently associated with poor outcome. BLBCs are estrogen receptor (ER) negative, progesterone receptor (PgR) negative, HER2 negative, and usually show a variable expression of basal cytokeratins (CKs), EGFR and CD117. p16 ^INK4a is a tumor suppressor protein, encoded by the CDKN2A gene, which regulates cell cycle. The reported association of abnormalities in the p16/Rb pathway with increased risk of malignancy prompted us to determine the expression of p16^INK4a in a group of BLBC; the results were compared with a group of high-grade invasive carcinoma (HG-IC) of breast. Tissue microarrays (TMA) were constructed in triplicate including 18 BLBC and 18 HG-IC. All BLBC cases were ER-/PgR-/HER2-. Seventeen (94%) BLBC were CK 5/6+/CK 14+; 14 (78%) BLCB showed EGFR expression and 13 (72%) were CD117 positive. BLBCs showed a strong positive reaction with p16 ^INK4a antibody in 16 of 18 (89%) cases. Although the significance of p16 ^INK4a expression in breast cancer is not fully understood, we have shown that p16^INK4a is strongly expressed in breast cancers with basal-like phenotype. Since it is known that p16^INK4a is associated with aggressive behavior in human carcinomas, these data suggest that p16^INK4a play a role in the poor prognosis of BLBC.     

p16INK4A overexpression and HPV infection in uterine cervix adenocarcinoma

Human papillomaviruses (HPVs) are causally involved in the genesis of cervical carcinomas and their precursors, and there is a strong relationship between the cyclin-dependant kinase inhibitor p16^INK4A and HPV infection. This study was carried out to assess the correlations between p16^INK4A expression as an early biomarker of the endocervical adenocarcinoma and HPV infection. p16^INK4A expression and HPV typing were performed on 46 samples including 5 normal endocervix, 9 benign lesions of the endocervix, 25 endocervical adenocarcinomas, and 7 endometrioid adenocarcinomas of the uterine corpus. A semiquantification of the p16^INK4A immunostaining was realized (using both the staining intensity and the percentage of positive cells) and was graded from 0 to 15. All of the 25 endocervical adenocarcinomas overexpressed p16^INK4A; the adjacent epithelium and the connective tissue were strictly negative. No p16^INK4A was detected in nine benign endocervical lesions and in five normal endocervix. Few endometrioid adenocarcinomas of the uterine corpus that infiltrate the endocervix exhibited a low immunoreactivity (score 0/15 or 1/15). This pattern of expression is significantly associated with HPV infection (p<10 ⁻³), mainly high-risk HPV types (p=0.02). Our results suggest that p16^INK4A is a putative molecular biomarker that consistently discriminates uterine cervix adenocarcinomas from benign lesions and from endometrioid adenocarcinomas of the uterine corpus .     

Combined detection of p16INK4a and IMP3 increase the concordance rate between cervical cytologic and histologic diagnosis

Currently, there are discrepancies in the interpretation between cervical liquid-based cytology (LBC) and histologic diagnoses. The aim of our study was to evaluate the utility of p16^INK4a (p16) and IMP3 staining of LBC specimens to increase the concordance rate. A total of 98 cell blocks with biopsy results, including 37 low-grade squamous intraepithelial lesions (LSIL), 36 high-grade squamous intraepithelial lesions (HSIL), and 25 squamous cell carcinomas (SCC), were selected for the immunocytochemical analysis of p16 and IMP3. The LBC diagnoses corresponded with histological diagnoses for 59.5% (22/37), 63.9% (23/36), and 88.0% (22/25) of LSIL, HSIL, and SCC lesions, respectively. We found a high frequency of p16 positivity in HSIL (72.2%) and SCC (100%), but not LSIL (29.7%). IMP3 was frequently expressed in SCC (84.0%), but rarely in LSIL (8.1%) and HSIL (25.0%). Cervical intraepithelial neoplasia 1 (CIN1) was negative for both p16 and IMP3, CIN2/3 tended to be positive for p16 and negative for IMP3, and SCC was positive for both p16 and IMP3. The combination of p16 and IMP3 immunostaining had a higher sensitivity and specificity for detecting CIN1 and CIN2/3 than cytology. For detecting SCC, p16/IMP3 had a higher sensitivity than cytology, but a lower specificity. IMP3 is a useful diagnostic immunomarker that can be used to identify SCC and the combination of p16/IMP3 expression was found to improve the discrepant results between cytologic and histologic diagnoses.     

Methylation of CpG islands of p16(INK4a) and cyclinD1 overexpression associated with progression of intraductal proliferative lesions of the breast

P16(INK4a) is a tumor suppressor gene frequently inactivated by aberrant promoter hypermethylation. In this study, p16(INK4a) methylation was evaluated in intraductal proliferative lesions of the breast, using real-time quantitative polymerase chain reaction (MethyLight) and methylation-sensitive restriction endonuclease polymerase chain reaction. Immunohistochemistry was performed to compare and validate the methylation analysis. P16(INK4a) methylation associated with oncogene cyclinD1 expression, detected through the use of in situ hybridization and immunohistochemistry, was likewise characterized. P16(INK4a) methylation displayed varying significance among different types of intraductal proliferative lesions. Both the positive rate and the median quantitative methylation value increased with the evolution of intraductal proliferative lesions through the use of quantitative and qualitative assays. P16(INK4a) methylation was positively correlated to cyclinD1 overexpression. This study demonstrated that p16(INK4a) methylation served as the silencing mechanism of p16(INK4a) protein expression and played a crucial role in the intraductal proliferative lesions' progression. In the differential diagnosis of intraductal proliferative lesions, quantitative DNA methylation analysis of p16(INK4a) by MethyLight may be used as a surrogate, especially to distinguish atypical ductal hyperplasia from usual ductal hyperplasia and low-grade ductal carcinoma in situ. Furthermore, this study discovered that flat epithelial atypia do not share similar molecular profiles of p16(INK4a) epigenetic modification with atypical ductal hyperplasia and low-grade ductal carcinoma in situ.     

Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescence

Endothelial cells (ECs) damage is an initial and pivotal step in the formation of atherosclerosis. Endothelial progenitor cells (EPCs), which have been considered as the precursor of ECs, can migrate and home to the site of injured ECs to divide into mature ECs and keep the integrity of the endothelial monolayer. It has been shown that the number and function of EPCs are negatively correlated with various atherosclerotic risk factors. This finding may be explained partly by accelerated senescence of EPCs induced by telomere attrition or shortening owning to oxidative stress and accumulative ROS. However, elevated telomerase activity which extends the telomere cannot lead to cellular immortal in the presence of the cyclin-dependent kinase inhibitor p16(INK4a). Researchers have the opinion that senescence is the balance between the regeneration and cancer. High expression of phosphorylated p16(INK4a), which is caused by oxidative stress and accumulative ROS, can prevent tumor cells from unlimited division and becoming malignant ones by accelerating premalignant cells premature senescence. It has been demonstrated that the expression of p16(INK4a) increases remarkably with age due to oxidative stress and accumulative ROS in some stem and progenitor cells, and regulates these cells age-dependent senescence. It is observed that telomeres shortening exists in these cells. Therefore, it can be hypothesized that p16(INK4a), together with telomerase, may co-modulate EPCs senescence.     

Transition of cervical carcinoma in situ to invasive cancer: Role of p16 expression in progression and in recurrence

To investigate the expression of p16^INK4a in cervical carcinoma and its relation to the transition of carcinoma in situ to invasive carcinoma, and its role in recurrence of cervical lesions as well, a series of 90 patients with cervical carcinoma (49 with in situ lesion and 41 with invasive lesion) were selected from July 2001 and September 2002. Groups with in situ and invasive lesions were paired for a series of risk variables for cervical cancer and followed up for 60 months. The follow-up visits occurred every 6 months in the first three years and annually up to the fifth year. It was observed that 87.9% of the patients with invasive lesion showed overexpression of p16^INK4a, in comparison with 37.6% of those with in situ lesion (X²: 13.68; 2 df; p = 0.0002; OR: 12.08), demonstrating overexpression of p16^INK4a as a risk of invasion of the basal layer by dysplastic cells. We also observed an association between overexpression of p16^INK4a and staging of cancer (X²: 18.38; 6 df; p = 0.0003). A prospective analysis, when controlled for interaction with cervical lesion groups (by Cox regression), demonstrated a risk of recurrence of 4.83 times attributed to overexpression of p16^INK4a, albeit not statistically significant (p = 0.14).     

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.     

p16 and Ki67 expression in normal,dysplastic and neoplastic uterine cervical epithelium and human papillomavirus (HPV) infection

Cellular cycle proteins like the p16^INK4a and the Ki67 proliferation nuclear antigen have been used as oncogenicity cellular markers. The E6 and E7 oncoproteins interact with tumor suppressor genes p53 and pRb, culminating with the p16^INK4a overexpression.     

Role of INK4a locus in normal eye development and cataract genesis

The murine INK4a locus encodes the critical tumor suppressor proteins, p16(INK4a) and p19(ARF). Mice lacking both p16(INK4a) and p19(ARF) (INK4a-/-) in their FVB/NJ genetic backgrounds developed cataracts and microophthalmia. Histopathologically, INK4a-/- mice showed defects in the developmental regression of the hyaloid vascular system (HVS), retinal dysplasia, and cataracts with numerous vacuolations, closely resembling human persistent hyperplastic primary vitreous (PHPV). Ocular defects, such as retinal fold and abnormal migration of lens fiber cells, were observed as early as embryonic day (E) 15.5, thereby resulting in the abnormal differentiation of the lens. We also found that ectopic expression of p16(INK4a) resulted in the induction of gammaF-crystallin, suggesting an important role of INK4a locus during mouse eye development, and also providing insights into the potential genetic basis of human cataract genesis.     

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.     

Human papilloma virus (HPV) status, p16INK4a, and p53 overexpression in epithelial malignant and borderline ovarian neoplasms

This investigation is the first to evaluate simultaneously human papilloma virus (HPV) status, p16(INK4a), and p53 immunoreactivity in epithelial ovarian neoplasms. The results were analyzed and correlated with histological type, histological grade, and survival of patients. Subtypes considered are papillary serous and mucinous. Polymerase chain reaction (PCR) analysis, performed in our previous study, had already demonstrated a small number of HPV-positive epithelial ovarian neoplasms. No significant correlation was found between the presence of HPV DNA and subtypes of ovarian neoplasms; thus, HPV cannot be considered responsible for epithelial ovarian neoplasm. Since p16 immunoreactivity was present in many other HPV-negative cases of epithelial ovarian neoplasms, this study suggests that p16 overexpression in some neoplasms of the female genital tract is not related to HPV carcinogenesis. A higher p53 expression rate observed between borderline and malignant serous tumors and between serous and mucinous neoplasms can confirm a recent dualistic model of ovarian carcinogenesis. According to this theory, low-grade serous carcinomas (serous intraepithelial carcinomas, serous borderline neoplasm, and ovarian mucinous neoplasms) (type I tumors) develop from mutations of KAS and BRAF, while high-grade serous carcinomas (type II tumors) develop from mutation of p53. In malignant neoplasms, for univariate analysis, patient survival seems to be related to p53, strong and diffuse p16 overexpression, and the stage of development of neoplasms at the diagnosis. In multinomial logistic regression, used to evaluate the role of staging, grading, p16 and p53 immunopositivity as predictor variables of unfavorable outcome of the disease, only p16 positivity was significantly related to the poor prognosis of the cancer.     

P16INK4a overexpression and survival in osteosarcoma patients: a meta analysis

Osteosarcoma is one of the most common primary bone malignancies. Although there is a significant improvement of survival on osteosarcoma patients in the past decades, treatment of osteosarcoma is still unsatisfactory for the development of pulmonary metastasis. The potential prognostic value of p16^INK4a in osteosarcoma has been investigated, however, the results from different studies were somewhat controversial. To elucidate whether p16^INK4a is indeed a prognostic factor of osteosarcoma, we conducted a meta-analysis of the published literatures to provide a comprehensive evaluation of the significance of p16^INK4a expression in patients with osteosarcoma. Eight studies with a total of 354 patients with osteosarcoma were examined. The pooled odds ratio (OR) with corresponding 95% confidence interval (95% CI) was calculated to evaluate the effect of p16^INK4a expression on overall survival. Meta-analysis showed that patients with high p16^INK4a expression were significantly associated with favourable overall survival when compared to their counterparts with low or undetectable p16^INK4a expression (OR = 0.270, 95% CI 0.162-0.451, P < 0.001). Sensitivity analysis suggested the pooled OR was stable and not significantly changed when a single study was removed. In conclusion, the results from this meta-analysis highlight that p16^INK4a is an effective biomarker of survival in patients with osteosarcoma.     

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。     

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.