Human papillomavirus genotyping and p16INK4a expression in cervical intraepithelial neoplasia of adolescents.

Adolescents have high rates of human papillomavirus (HPV) infection, and persistent high-risk HPV infection can lead to the development of cervical cancer. The cyclin-dependent kinase inhibitor, p16(INK4a) is overexpressed in cervical intraepithelial neoplasia (CIN), probably due to a persistent and integrated HPV infection. This study investigated p16(INK4a) expression, grades of CIN, and high-risk HPV infection in adolescent cervical biopsies. Biopsies were immunohistochemically stained for p16(INK4a). The presence of wide-spectrum, low-risk, or high-risk HPV was determined by amplifying DNA extracted from the cervical biopsies. Biopsies were classified as cervicitis, 15 cases; CIN 1, 48 cases; CIN 2, 46 cases, and CIN 3, 52 cases. The distribution of p16(INK4a) staining was graded as patchy, diffuse basal, and diffuse full thickness. Pearson's chi(2) tests analyzed the relationships between p16(INK4a) staining, HPV infection, and CIN. Biopsies of cervicitis were negative for HPV and for p16(INK4a) expression. High-risk HPV 16, 18, and 31 increased from 18% in CIN 1 to 66% in CIN 2/3 (P<0.001). In CIN 1, p16(INK4a) was positive in 44% of biopsies with 35% showing patchy, 7% diffuse basal, and one case (2%) showing diffuse full thickness staining. In CIN 2/3, p16(INK4a) was positive in 97% of biopsies with 23% showing patchy, 21% diffuse basal, and 53% diffuse full thickness staining. The difference in the proportions of biopsies showing patchy p16(INK4a) staining in CIN 1 and diffuse full thickness staining in CIN 2/3 was significant (P<0.001). In CIN 1, 61% of high-risk HPV-positive biopsies were p16(INK4a) negative, while all high-risk HPV-positive CIN 2/3 biopsies were p16(INK4a) positive. Diffuse, full thickness p16(INK4a) expression discriminated low-grade from high-grade CIN and appears to be a marker of persistent high-risk HPV infection.     

食管鳞状细胞癌中p16基因甲基化及其表达

目的：探讨食管鳞状细胞癌p16／INK4基因启动子区高甲基化和p16、cyclinD1蛋白表达的意义。方法：用甲基化特异性PCR（MSP）法检测p16／INK4基因启动子区的高甲基化，用免疫组织化学方法检测p16、cyclind1蛋白的表达。结果：30例食管鳞状细胞癌中7例p16免疫组织化学阳性，15例cyclinD1阳性，组织学和统计学分析显示p16与cyclinD1的表达呈负相关。4例检出p16／INK4基因启动子区高甲基化，但与p16失表达无统计学意义。结论：（1）在食管鳞状细胞癌可检出p16／INK4基因启动子区的高甲基化，而甲基化不是引起p16失表达的主要原因。（2）p16与cyclinD1的表达呈负相关，提示细胞周期调控因子之间可能存在相互影响表达的反馈机制。     

Epigenetic alteration of p16INK4a gene in dedifferentiation of liposarcoma

The atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WDLPS) is a locally aggressive subtype of liposarcoma unless dedifferentiation occurs. The mechanism driving this progression is not clear. Loss of p16 is believed to be an early and critical event in tumor progression. Gene silencing by methylation of p16INK4a gene promoter has been reported in several soft tissue sarcomas. The aim of this study is to study the role of p16INK4a gene promoter methylation and p16 expression in tumor progression (dedifferentiation) and recurrence of ALT/WDLPS. Four cases of dedifferentiated liposarcomas (DDLPS) and three cases of recurrent well-differentiated liposarcomas (WDLPS) were collected, and methylation status of p16INK4a gene promoter was analyzed using methylation-specific PCR (MSP) on DNA extracted from paraffin blocks. p16 expression was examined by immunohistochemistry on the same blocks. Methylation of p16INK4a gene promoter was seen in the dedifferentiated (DD) components only, in two out of four (2/4, 50%) DDLPS. The other two DDLPS and three recurrent WDLPS were not methylated. Both WD and DD components in all four DDLPS cases showed strong nuclear p16 expression. All three recurrent WDLPS showed positive p16 expression with similar intensity between primary and recurrent tumors. Even though linear correlation between p16 promoter hypermethylation and p16 protein expression was not present, there appears to be a role for p16INK4a gene promoter hypermethylation in DDLPS and not in recurrent WDLPS.     

Estimation of prognoses for cervical intraepithelial neoplasia 2 by p16INK4a immunoexpression and high-risk HPV in situ hybridization signal types

The present study used immunohistochemical staining and in situ hybridization (ISH) to examine whether progression of cervical intraepithelial neoplasia, grade 2 (CIN 2) can be predicted by p16INK4a immunoexpression and high-risk human papilloma virus (HPV) ISH signal types. We studied 52 cases histologically diagnosed with CIN 2: dysplasia regressed in 28 cases; 13 cases progressed to CIN 3; and CIN 2 persisted in 11 cases. Expression of p16INK4a and high-risk HPV signal both related to grade of CIN. Stronger p16INK4a immunoexpression and a higher frequency of expression of a punctate nuclear signal were observed in CIN 2 lesions before progression compared with those before regression. CIN 2 cases in which moderate to strong immunoexpression of p16INK4a and a punctate signal were observed simultaneously progressed to CIN 3 in 10 (91%) of 11 cases. CIN 2 cases with moderate to strong immunoexpression of p16INK4a and a high-risk HPV punctate signal should be treated because of the great risk of progression.     

p16INK4A as a marker for cervical dyskaryosis: CIN and cGIN in cervical biopsies and ThinPrep smears

AIM: To examine the potential of p16(INK4A) as a biomarker for dysplastic squamous and glandular cells of the cervix in tissue sections and ThinPrep smears. METHODS: Immunocytochemical analysis of p16(INK4A) expression was performed on 22 normal cervical tissue samples, five cervical glandular intraepithelial neoplasia (cGIN), 38 cervical intraepithelial neoplasia 1 (CIN1), 33 CIN2, 46 CIN3, and 10 invasive cancer cases (eight squamous and two adenocarcinomas). All samples were formalin fixed and paraffin wax embedded, and immunohistochemical analysis was carried out using a mouse monoclonal anti-p16(INK4A) antibody after antigen unmasking. The staining intensity was assessed using a 0 to 3 scoring system. In addition, the expression status of p16(INK4A) was examined in 12 normal ThinPrep smears, one smear exhibiting cGIN, and a total of 20 smears exhibiting mild, moderate, and severe dyskaryosis. Human papillomavirus (HPV) detection was carried out using a modified SYBR green assay system. Fluorogenic polymerase chain reaction (PCR) and solution phase PCR were used for specific HPV typing. RESULTS: p16(INK4A) immunoreactivity was absent in all normal cervical tissues examined. Dysplastic squamous and glandular cells were positive for p16(INK4A) expression in all cases included in this study, except for one CIN3 case. p16(INK4A) expression was mainly nuclear in CIN1 cases, and both nuclear and cytoplasmic in CIN2, CIN3, cGIN, and invasive cases. All cases positive for HPV expressed the p16(INK4A) protein, although not all cases found positive for p16(INK4A) were HPV positive. In general, the p16(INK4A) staining intensity was lower in cases negative for HPV or those containing a low risk HPV type. CONCLUSION: This pattern of overexpression demonstrates the potential use of p16(INK4A) as a diagnostic marker for cervical squamous and also glandular neoplastic lesions. In addition, the technique can be used to identify individual dyskaryotic cells in ThinPrep smears. Thus, p16(INK4A) is a useful marker of cervical dyskaryosis.     

p14ARF deletion and methylation in genetic pathways to glioblastomas

The CDKN2A locus on chromosome 9p21 contains the p14ARF and p16INK4a genes, and is frequently deleted in human neoplasms, including brain tumors. In this study, we screened 34 primary (de novo) glioblastomas and 16 secondary glioblastomas that had progressed from low-grade diffuse astrocytomas for alterations of the p14ARF and p16INK4a genes, including homozygous deletion by differential PCR, promoter hypermethylation by methylation-specific PCR, and protein expression by immunohistochemistry. A total of 29 glioblastomas (58%) had a p14ARF homozygous deletion or methylation, and 17 (34%) showed p16INK4a homozygous deletion or methylation. Thirteen glioblastomas showed both p14ARF and p16INK4a homozygous deletion, while nine showed only a p14ARF deletion. Immunohistochemistry revealed loss of p14ARF expression in the majority of glioblastomas (38/50, 76%), and this correlated with the gene status, i.e. homozygous deletion or promoter hypermethylation. There was no significant difference in the overall frequency of p14ARF and p16INK4a alterations between primary and secondary glioblastomas. The analysis of multiple biopsies from the same patients revealed hypermethylation of p14ARF (5/15 cases) and p16INK4a (1/15 cases) already at the stage of low-grade diffuse astrocytoma but consistent absence of homozygous deletions. These results suggest that aberrant p14ARF expression due to homozygous deletion or promoter hypermethylation is associated with the evolution of both primary and secondary glioblastomas, and that p14ARF promoter methylation is an early event in subset of astrocytomas that undergo malignant progression to secondary glioblastoma.     

Alterations in the tumor suppressor gene p16(INK4A) are associated with aggressive behavior of penile carcinomas

Alterations in the p16/cyclinD1/Rb and ARF/Mdm2/p53 pathways are frequent events in the pathogenesis of squamous cell carcinomas. Different mechanisms of p16 regulation have been described for penile carcinomas so far. Therefore, expression of p16 and p53 was immunohistochemically detected with monoclonal antibodies in 52 primary invasive penile squamous cell carcinomas. The carcinomas were analyzed for allelic loss (LOH) in p16 ^INK4A and p53, as well as for mutations in the p16 ^INK4A and the p53 gene. In addition, we examined the promoter status of p16 ^INK4A by methylation-specific PCR. The presence of human papilloma virus (HPV) 6/11, HPV 16 and HPV 18 DNA was analyzed by PCR. Data were compared to clinical data. Concerning p16, 26 (50%) tumors showed positive immunohistochemistry, 32 (62%) tumors showed allelic loss and 22 tumors (42%) showed promoter hypermethylation. All tumors with negative p16 immunohistochemistry showed LOH near the p16 ^INK4A locus and/or hypermethylation of the p16 ^INK4A promoter. HPV 16 DNA was detected in 17 tumors, ten of them with positive p16 immunostaining. The remaining seven tumors with negative p16 staining showed allelic loss and/or promoter hypermethylation. Evidence of lymph node metastasis was significantly associated with negative p16 immunohistochemistry as well as with combined LOH and promoter hypermethylation (p = 0.003 and p = 0.018, respectively). Allelic loss around p53 was found in 22 tumors (42%), and seven mutations of the p53 gene could be demonstrated in our tumors. No correlations could be found between any p53 alteration and clinical parameters.     

脑膜瘤P16^INK48、RB基因的甲基化分析及对基因表达的影响

目的 检测脑膜瘤中发生P16^INK48和RB基因甲基化的情况及对蛋白表达的影响。方法 用甲基化特异性聚合酶链反应对50例脑膜瘤进行了P16^INK48和RB的甲基化分析；并对其中的25例检测了P16^INK48蛋白的表达。结果 良性脑膜瘤中没有检测到甲基化，分别有6例ⅡⅢ级(37．5％)和4例Ⅲ级(28．6％)肿瘤发生至少一种基因的甲基化，其中有1例不典型脑膜瘤同时发生了两种基因的甲基化。全部13例P16^INK48阳性表达的肿瘤都是没有检测到甲基化者。结论 P16^INK48或RB的甲基化与不典型和间变性脑膜瘤的发生发展有关，其机制可能是甲基化使蛋白表达丢失并导致P16^INK48细胞周期蛋白D1／CDK4／RB途径功能障碍。     

Assessment of immunohistochemistry for p16INK4 and high-risk HPV DNA by in situ hybridization in esophageal squamous cell carcinoma

The role of high-risk human papillomavirus (HPV) in the pathogenesis of esophageal squamous cell carcinoma (ESCC) remains unclear. p16(INK4) is used as a surrogate marker to detect HPV-related tumors but has had discrepant results in ESCC. In this study, 32 cases of ESCC were examined to determine the relationship between p16(INK4) expression and high-risk HPV. All the tumors were stained by immunohistochemistry for p16(INK4). Tumors having p16(INK4) nuclear and/or nuclear and cytoplasmic expression were considered positive. Tumors positive for p16(INK4) expression were tested for high-risk HPV by in situ hybridization (ISH). In all, 20 cases of ESCC (63%) showed only cytoplasmic staining for p16(INK4), and 11 cases (34%) showed both cytoplasmic and nuclear staining for p16(INK4); 4 cases (13%) showed no staining for p16(INK4). None of the p16(INK4) -positive cases were positive for high-risk HPV by ISH. These results indicate that p16(INK4) expression in ESCC does not correlate with the presence of high-risk HPV DNA by ISH. High-risk HPV does not seem to play a major role in the carcinogenesis of ESCC in low-risk areas.     

Prognostic value of p16INK4a and p14ARF gene hypermethylation in human colon cancer

The INK4a/ARF locus (9p21) encodes two unique and unrelated cell cycle regulators, p16INK4a and p14ARF. This study was performed to evaluate the methylation status of p16INK4a and p14ARF genes, as well as its association with p16 and p53 expression, microsatellite instability (MI) status, and various clinicopathologic parameters in sporadic colorectal cancer. Sixty-five cases of primary colorectal adenocarcinoma with a series of clinicopathological parameters were obtained. We performed methylation-specific PCR of p16INK4a and p14ARF genes in colorectal cancer paraffin blocks with its paired normal samples, as well as immunohistochemical stainings for p16 and p53, and MI analysis. Aberrant methylations of p16INK4a and p14ARF gene were present in 21 (32.3%) and 33 (50.8%) out of 65 cases, respectively. p16INK4a aberrant methylation was correlated with p16 negativity (P=0.021) and p53 overexpression (P=0.007). p16INK4a aberrant methylation was more frequently present in poorly differentiated adenocarcinomas (P=0.002). Aberrant methylation of p14ARF gene occurred more frequently in patients under 50 years of age and in left-sided colon cancers, and was not statistically significant. Compared with the group with simultaneous absence of methylation in both promoters, the group showing concomitant alterations in both p16INK4a and p14ARF genes (n=10) more frequently presented lymph node metastasis (P=0.020) and higher tumor grade (P=0.014). There was no correlation between p16INK4a and p14ARF gene hypermethylation or MI status. This study suggests that simultaneous hypermethylation of both p16INK4a and p14ARF genes is greater prognostic value in sporadic human colorectal cancer.     

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.     

Human papillomavirus genotyping by oligonucleotide microarray and p16 expression in uterine cervical intraepithelial neoplasm and in invasive carcinoma in Korean women

For evaluating the diagnostic significance of p16(INK4A) over-expression in the uterine cervical intraepithelial neoplasm and in invasive carcinoma, human papillomavirus (HPV) was detected and genotyped by oligonucleotide microarray in archival tissues of 117 cervical specimens, including 47 invasive squamous cell carcinomas (SCC), 30 cases of cervical intraepithelial neoplasia (CIN), 20 adenocarcinomas, and 20 cases of non-neoplastic cervix. The expression of p16(INK4A) protein was immunohistochemically studied in these cases and in five HPV-positive and one HPV-negative cervical cancer cell lines. HPV was detected in 50% of CIN, 61.7% of SCC, and 45.5% of adenocarcinomas. p16(INK4A) expression was seen in all 20 cases of adenocarcinoma, 78.7% (37/47) of SCC, and 96.7% (29/30) of CIN, but not in any cases of the non-neoplastic cervix. There was no difference in p16(INK4A) expression between the HPV-positive and HPV-negative cervical lesions. All HPV-positive and -negative cervical cancer cell lines expressed p16(INK4A) protein. In conclusion, the presence of p16(INK4A) expression in cervical squamous and glandular epithelium indicates the existence of dysplasia or malignancy in the uterine cervix, regardless of HPV infection.     

High‐risk human papillomavirus load and biomarkers in cervical intraepithelial neoplasia and cancer

The purpose of this study was to examine the implication of high‐risk human papillomavirus (HPV) load in cervical intraepithelial neoplasia (CIN) and cancer, and to detect biomarkers in cervical disease. We conducted high‐risk HPV DNA load and cervical cytology tests in 343 women, cervical tissue biopsy in 143 women, and immunohistochemistry for p16^INK4A, cyclin D1, p53, cyclooxygenase‐2, Ki‐67, GLUT1, hPygopus2, and beta‐catenin. As a result, HPV load [relative light units (RLU) value] was correlated with the histological severity of cervical disease (p < 0.05). In the ‘atypical squamous cells of undetermined significance’ cytology group, 2.385 of HPV load seemed to be the cut‐off value at which ‘benign’ or CIN I can be differentiated from ‘CIN II or more severe’ (AUC = 0.712), but not statistically significant. The relative risk (odds ratio) of p16^INK4A and GLUT1 overexpression increased gradually according to the histological severity of cervical disease. The p16^INK4A showed statistically significant odds ratios in CIN II, CIN III, and cancer; GLUT1, in CIN II and CIN III; hPygopus2, in CIN III; and beta‐catenin, in CIN III and cancer. Conclusively, HPV load, p16^INK4A, and GLUT1 can be instrumental in predicting the severity of HPV‐related cervical disease. The beta‐catenin/hPygopus2 signaling may be involved in proceeding to CIN III.     

Hypermethylation of the CDKN2/p16INK4A promotor in thyroid carcinogenesis

Functional inactivation of the p16INK4A gene has been reported to be involved in the development of a variety of human malignancies. In thyroid carcinomas, mutations of the p16INK4A gene or homozygous deletions of the gene locus 9p21 are rare. This study investigated whether p16INK4A promotor methylation is an alternative mechanism for p16INK4A gene inactivation during thyroid carcinogenesis. A methylation-specific polymerase chain reaction protocol was applied. A total of 77 thyroid tumor specimens, including 18 follicular adenomas, 18 follicular carcinomas, 16 papillary carcinomas, 12 poorly differentiated carcinomas, and 13 undifferentiated carcinomas were analyzed longitudinally. In addition, 15 tumor-free thyroid tissues were investigated. The p16INK4A promotor status was compared with p16INK4A protein expression and patient-specific data. p16INK4A promotor hypermethylation was detected in 13% of non-tumorous tissue; in 33% of follicular adenomas; in 44% of papillary carcinomas; in 50% of follicular carcinomas; in 75% of poorly differentiated carcinomas; and in 85% of undifferentiated carcinomas. With the exception of two cases, the p16INK4A protein was lost as a result of promotor hypermethylation. Comparing the methylation status with tumor stage, no correlation was found. However, lymph node and distant metastasis status showed a statistically significant prevalence for the p16INK4A promotor methylation (p = 0.035). There was no association between p16INK4A promotor methylation and age and sex. These results suggest that hypermethylation of the p16INK4A promotor region is a frequent and an early event during thyroid carcinogenesis and is associated with tumor progression and dedifferentiation.     

Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions.

The p16(INK4a) is a cyclin-dependent kinase inhibitor that decelerates the cell cycle by inactivating the cyclin-dependent kinases involved in the phosphorylation of the retinoblastoma protein (RB). Expression of E6 and E7 oncogenes of high-risk (HR) human papillomavirus (HPV), affecting the RB-p16 pathway, leads to p16 upregulation. Although it is widely reported that p16 is overexpressed in a high percentage of preneoplastic lesions and in almost all carcinomas of the uterine cervix, protein upregulation and its correlation with HPV infection in low-grade lesions is still being debated. In this study, we investigated in parallel, p16 expression and HPV infection in 100 cervical biopsies (17 normal tissues, 54 CIN1, 10 CIN2, 11 CIN3, eight invasive squamous cancers). Results obtained demonstrated that none of the 17 normal cervical tissues, evaluated by immunohistochemistry, presented p16 positivity whereas, starting from CIN1 (31%) to CIN2 (90%), CIN3 (100%) and carcinomas (100%), a constant and significant increase of protein overexpression (P<0.0001) was observed. In addition, p16 overexpression consistently showed elevated sensitivity (84%) and specificity (98%) in detecting HR-HPV infection with a high positive predictive value (97%) and negative predictive value (86%). Of interest, 93% of the p16-positive CIN1 were also HR-HPV infected. Our findings confirmed that p16 overexpression is associated to high-grade precancerous lesions and cervical carcinomas, and further demonstrated that immunohistochemical evaluation of p16 may be a useful biomarker in identifying HR-HPV-infected low-grade lesions.     

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.     

p16(INK4a) as a complementary marker of high-grade intraepithelial lesions of the uterine cervix. I: Experience with squamous lesions in 189 consecutive cervical biopsies

AIM: To test the usefulness of p16(INK4a) immunostaining for improving the diagnostic accuracy of cervical punch biopsies referred to a routine laboratory setting during the investigation of women with abnormal Papanicolaou smears. METHODS: A total of 188 consecutive and unselected colposcopically directed cervical biopsies and a single contemporaneous cervical polyp were accessioned prospectively over a 3-month period, step-serially sectioned and examined by H&E and immunostained for p16(INK4a). The clinical context, results of concurrent Papanicolaou smears/ThinPrep slides and Digene hybrid capture tests for high-risk human papillomavirus (HPV) subtypes, as well as follow-up cervical smears/ThinPrep, biopsies and loop excisions of transformation zones or cone biopsies were all correlated with the morphological and immunohistochemical findings. RESULTS: Seventy-seven biopsies (40.7%) displayed a high-grade squamous intraepithelial lesion (HGSIL; cervical intraepithelial neoplasia [CIN] 2-3), 27 (14.3%) showed a low grade squamous intraepithelial lesion (HPV +/- CIN1) and 85 (45%) showed a range of non-dysplastic (inflammatory or reactive) changes. Diffuse strong parabasal immunostaining for p16(INK4a), suggestive of integrated high-risk HPV DNA into the host genome, was observed in 81 biopsies (42.9%, including the cervical polyp) and correlated (>90%) with HGSIL in the H&E sections. Only one case revealed irreconcilable discordance between the histological features and this strong parabasal immunostaining pattern. Focal and weaker midzonal or superficial p16(INK4a) immunostaining, suggestive of episomal HPV infection, was noted in 19 biopsies (10%) and these biopsies exhibited a range of histological changes but predominantly low grade squamous intraepithelial lesion (LGSIL). No staining of the squamous epithelium was seen in 89 biopsies (47.1%). Again, only one case revealed irreconcilable discordance between the histological features and this negative immunostaining pattern. On review of all cases where discordant results were noted between the H&E appearances and expected p16(INK4a) immunostaining, we found 26 cases (13.7%) in which this discordance prompted justifiable modification of the original diagnosis. CONCLUSIONS: Thus, within a routine diagnostic laboratory, p16(INK4a) immunostaining appears to be a very useful adjunctive test in the examination of colposcopically directed cervical biopsies, in the diagnostic cascade of women investigated for abnormal Papanicolaou smears. It is possible, as further data accumulate concerning the importance of integration of high-risk HPV DNA into the host cell genome and the reliability with which this can be identified by p16(INK4a) immunostaining, that this will become the diagnostic 'lesion of interest', replacing the subjective histological grading of cervical dysplasia, in the management of such patients; i.e., the discriminatory watershed between continued surveillance and active intervention.     

Distinct promoter hypermethylation of p16INK4a,CDH1, and RAR-beta in intestinal,diffuse-adherent,and diffuse-scattered type gastric carcinomas

Hypermethylation of CpG islands in gene promoters is associated with silencing of various tumour suppressor genes. Recent studies of colorectal and gastric carcinomas have defined a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. In this study, methylation-specific polymerase chain reaction (PCR) was performed to study methylation of CpG islands in the promoters of the p16(INK4a), cadherin 1 (CDH1), and retinoic acid receptor-beta (RAR-beta) genes in 45 gastric carcinomas and to investigate whether CDH1 and RAR-beta promoter hypermethylation is associated with CIMP-positive gastric carcinoma. CpG island hypermethylation of the p16(INK4a), CDH1, and RAR-beta promoters was detected in 12 (27%), 26 (58%), and 24 (53%) of the 45 gastric carcinomas, respectively. Hypermethylation of the p16(INK4a) promoter was more common in intestinal type than in diffuse type gastric carcinomas (p = 0.0023; Fisher's exact test) and was inversely associated with p53 mutations (p = 0.0225; Fisher's exact test). However, CDH1 and RAR-beta promoter hypermethylation was observed more frequently in diffuse-scattered type gastric carcinoma than in other types (intestinal and diffuse-adherent types) (p = 0.0175 and p = 0.0335, respectively; Fisher's exact test) and was not associated with p53 mutation status. Moreover, hypermethylation of the CDH1 and RAR-beta promoters occurred concordantly (p < 0.0001; Fisher's exact test). These results suggest that at least two types of promoter methylation status are involved in the development of the intestinal (p16(INK4a) promoter hypermethylation) and diffuse-scattered types (CDH1 and RAR-beta promoter hypermethylation) of gastric carcinoma.     

Frequent p16ink4a inactivation is an early and frequent event of intraductal papillary neoplasm of the liver arising in hepatolithiasis

Intraductal papillary neoplasm of the liver (IPNL) is a precursor lesion of intrahepatic cholangiocarcinoma (ICC) arising in hepatolithiasis. In this study, 98 foci of IPNL identified in 39 surgically resected hepatolithiatic livers were investigated for expression of p16INK4a, cyclin D1, p21WAF1/CIP1, p53, mouse double-minute 2 (MDM2), and pRb. In addition, methylation-specific polymerase chain reaction (MSP) for p16 INK4a promoter region was performed in these foci. Nonneoplastic bile ducts from 11 hepatolithiatic livers, 5 histologically normal livers, and 9 cases of nonpapillary conventional ICC were used as controls. Decreased expression of p16INK4A was seen in IPNL group 1 with mild dysplasia and continued along the progression of IPNL to ICC. The expression of cyclin D1, p21WAF1/CIP1,and pRb gradually increased along the progression of IPNL to ICC and became significantly high in IPNL of group 3 (carcinoma in situ). The expression of p53 and MDM2 was increased in IPNL group 3 and group 4 with evident invasive carcinoma. MSP revealed that 54.6% of 44 IPNL foci harbored p16INK4a promoter hypermethylation, and such foci were significantly correlated with decreased expression of p16INK4a protein. Ki-67 labeling index exhibited a stepwise increase from IPNL group 1 to group 4. We conclude that p16INK4a inactivation, due mainly to its promoter hypermethylation, is a frequent and early event of IPNL and may be responsible for genetic and epigenetic alterations of other cell cycle regulators in IPNL.