Detection of human papillomavirus DNA sequences by in situ DNA-DNA hybridisation in cervical intraepithelial neoplasia and invasive carcinoma: a retrospective study.

Human papillomavirus (HPV) infection of cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma was investigated using in situ DNA-DNA hybridisation on histological sections of formalin fixed, paraffin embedded tissue to assess the technique's sensitivity and to assess retrospectively the association between HPV16 and invasive cervical carcinoma. HPV DNA was detected in 16 of 33 biopsy specimens of CIN. Cells containing viral DNA were more numerous than those positive for viral structural proteins. HPV DNA was also present in less differentiated cells deeper in the epithelium. The detection rate in CIN was lower than that reported for other hybridisation techniques such as Southern blotting. In a retrospective study of biopsy specimens of invasive squamous carcinoma of the cervix HPV16 DNA, the virus most commonly associated with cervical malignant disease, was found in 20 of 25 cases, including those dating from as far back as 1932. The level of sensitivity was similar to that reported for other hybridisation techniques. DNA positive cells were focally distributed in the invasive tumours, and most tumour cells were negative for viral DNA, a result consistent with the low copy number found in malignant cells. It is concluded that HPV16 is not a new virus but that its prevalence is a result of changes in sexual behaviour and that in situ hybridisation is useful in the localisation of HPV DNA replication in CIN and invasive carcinoma.     

Aberrant p53 protein expression in cervical intra-epithelial neoplasia

We investigated aberrant p 53 expression in 81 cases of cervical intra-epithelial neoplasias (CIN) using a polyclonal antibody CM-1. The presence of human papillomavirus (HPV) DNA was evaluated by in situ and dot blot hybridization. Significant (more than 1% of cells positive) p 53 positivity was found in three cases (4%) of which only one contained HPV DNA. In an additional nine cases, occasional p 53 staining was found in basal epithelial cells, frequently associated with epithelial hyperplasia and increased subepithelial inflammation. The results show that aberrant p 53 expression is an infrequent finding in CIN lesions. It can be seen in lesions both with and without HPV infection. Most importantly, there was no p 53 expression in most cases of HPV-negative CIN, suggesting that p 53 inactivation is not an obligatory step in the development of cervical dysplasia. However, our findings do not exclude the possibility that p 53 mutations can occur later in the course of cervical carcinogenesis.     

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.     

Evaluation of p16 immunostaining to predict high‐grade cervical intraepithelial neoplasia in women with Pap results of atypical squamous cells of undetermined significance

p16 immunostaining has been examined to detect high‐grade cervical intraepithelial neoplasia grade (CIN2+) in Pap cytology specimens. However, the utility of p16 in predicting CIN2+ in Pap specimens with atypical squamous cells of undetermined significance (ASC‐US) or atypical squamous cells, cannot exclude high‐grade squamous intraepithelial neoplasm (ASC‐H), is controversial. In this study, we evaluated the utility of p16 immunostaining for predicting CIN2+ in 78 Pap specimens with ASC‐US/ASC‐H and compared the results in high‐risk HPV DNA and the follow‐up biopsies. p16 immunostaining was positive in 47% (37/78) of the Pap specimens. Of the 13 Pap specimens with follow‐up biopsy results of CIN2+, 7 (54%) were positive for p16. p16 positivity in the Pap specimens was not significantly associated with a CIN2+ biopsy result (P = 0.76). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of p16 immunostaining for predicting CIN2+ were 54%, 52%, 19%, and 85%, respectively. High‐risk HPV DNA was detected in 40% (31/78) of the Pap specimens. The sensitivity, specificity, PPV, and NPV of HPV DNA for predicting CIN2+ were 100%, 72%, 42%, and 100%, respectively. High‐risk HPV genotypes were detected in six p16‐negative specimens with follow‐up biopsy results of CIN2+. Our findings suggest that the utility of p16 immunostaining for predicting CIN2+ in Pap specimens with ASC‐US/ASC‐H is limited. Scant abnormal cells in Pap specimens with ASC‐US/ASC‐H may have contributed to the low p16 sensitivity. Diagn. Cytopathol., 2011. © 2010 Wiley‐Liss, Inc.     

Consensus polymerase chain reaction and enzyme-linked immunosorbent assay for human papillomavirus detection and typing in cervical specimens.

Human papillomavirus (HPV) infection is common in cervical intraepithelial neoplasia (CIN). This study investigates HPV detection and typing assay based on polymerase chain reaction amplification of L1 open reading frame with general primers GP5/GP6, followed by enzyme-linked immunosorbent assay detection with type-specific DNA probes. To determine the sensitivity of this assay, formalin-fixed CaSki cells were used as reference cell lines. Fifty copies of viral DNA diluted in DNA from 100,000 noninfected cells could be detected. This assay was also investigated for HPV detection and typing of 67 cervical specimens diagnosed with with CIN III or carcinoma in situ (CIS) and their adjacent squamous epithelium. The CIN III lesions were infected in approximately 80% of the samples, 81% in the neighboring CIN II, and 68% in CIN I. The HPV infection was even detectable in 54% of nondysplastic epithelium located near a CIN III lesion.     

Distribution of HPV infection and tumour markers in cervical intraepithelial neoplasia from cone biopsies of Mozambican women

AIMS: To evaluate human papillomavirus (HPV) infection in whole cervical cone specimens with cervical intraepithelial neoplasia (CIN). In addition, to evaluate the relation between the presence of CIN lesions and HPV infection and the expression of Ki-67, p53, cytokeratins, Gp230 glycoprotein, and simple mucin-type carbohydrates. METHODS: Cervical cone specimens from five patients with CIN were studied. For each specimen, serial sections encompassing the whole cone were collected (52 samples). HPV infection and HPV types were detected by the polymerase chain reaction and enzyme immunoassay. The expression of Ki-67, p53, cytokeratins, Gp230, and simple mucin-type carbohydrates was examined immunohistochemically. RESULTS: All cases showed high risk HPV types, namely types 16, 33, 35, and 58. Four of the five patients were infected by multiple viral types. HPV-58 was always seen in CIN III, whereas HPV-35 was more frequent in CIN I. The expression of Ki-67 and p53 was higher in CIN III lesions. The expression of cytokeratins 8 and 17 showed complete or almost complete overlap with CIN III. Altered expression of Gp230, Tn, and sialyl-T was often seen in all grades of CIN. CONCLUSIONS: When whole cervical cone specimens are evaluated the rate of multiple HPV infection is very high. The expression of cytokeratins 8 and 17 is a useful marker of CIN III.     

子宫颈病变中HPV L1蛋白和p16的表达

目的 研究HPV L1蛋白和p16在子宫颈各种病变中的表达情况,探讨它们在子宫颈病变进展中的预测价值.方法 应用免疫组化方法检测41例各种子宫颈病变(CIN1级18例、CIN2级9例、CIN3级8例和浸润性鳞状细胞癌6例)中HPV L1蛋白和p16的表达.结果 HPV L1蛋白在各种子宫颈病变中的阳性率为26.8%.其中HPV L1在CIN1中的阳性表达率为38.9%,CIN2为44.4%,CIN3和浸润性鳞状细胞癌均无表达.p16在各种子宫颈病变中的阳性率为68.3%,其在CIN1中的阳性表达率为38.9%,CIN2为77.8%,CIN3和浸润性鳞状细胞癌均表达阳性.100%CIN3和浸润性鳞状细胞癌为p16+/HPV L1-,而61.1% CIN1中为p16-/HPV L1+或p16-/HPV L1-.结论 随着子宫颈病变的进展,HPV L1阳性表达率降低而p16阳性表达率增高.p16+/HPV L1-提示子宫颈鳞状上皮内瘤变有进展的可能,而p16-/HPV L1+和p16-/HPV L1-可能为无进展的或潜在消退的子宫颈病变.     

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.     

Cyclin dependent kinase inhibitor p27Kip1 expression in normal and neoplastic cervical epithelium

AIM: To investigate whether there is loss of the p27Kip1 protein in developing cervical cancer and whether p27Kip1 immunoreactivity has any relation to the proliferative indicator Ki-67. METHODS: The expression of p27Kip1 and Ki-67 was assessed by immunohistochemistry in serial sections from normal epithelium (13), low grade (27) and high grade (19) squamous intraepithelial lesions (LSIL, HSIL), and invasive cervical cancer (23). In the SIL cases the presence of human papillomavirus (HPV) genomic sequences was assessed by in situ hybridisation. The results were evaluated by image analysis, and reported as mean score of the percentage of p27Kip1 and of Ki-67 positive cells in each histological group. RESULTS: In general, p27Kip1 immunostaining was related to squamous differentation, and was intense in normal epithelium (47%), while it was reduced in SIL lesions as an effect of the decreased number of differentiating cells. However, decrease in the p27Kip1 expression was more evident in LSIL (36%) than in HSIL (39%); in the latter, p27Kip1 had a different intraepithelial distribution in that the staining extended to the basal cells. The average levels of p27Kip1 were similar in SIL lesions associated to low, intermediate, and high risk HPV types. Compared with normal epithelium and dysplasia, invasive cancer showed significantly lower p27Kip1 levels (23%). There was no relation between p27Kip1 and Ki-67 labelling indices in any of the histological groups examined. CONCLUSIONS: A reduction in p27Kip1 protein occurs in cervical cancer independently of the proliferative status. The changes in p27Kip1 expression may be related to the unregulated kinetics of developing cervical cancer.     

Immunohistochemical detection of p53 in cervical epithelial lesions with or without infection of human papillomavirus types 16 and 18

Using formalin-fixed and paraffin-embedded cervical tissues, we examined infection with human papillomavirus (HPV) types 16 and 18 by Southern blot analysis following polymerase chain reaction (PCR), and the accumulation of p53 protein by immunohistochemistry in 30 cases of normal or metaplastic cervix, 17 cases of cervical intraepithelial neoplasia grade I (CIN I), 20 cases of CIN II, 37 cases of CIN III and 23 cases of invasive squamous cell carcinoma (ISCC). In addition, we examined the ratio of HPV-infected cells by in situ hybridization (ISH) and the alteration of p53 gene using PCR followed by single-strand conformation polymorphism (PCR-SSCP) in 2 cases of CIN III and 12 cases of ISCC, in which overexpression of p53 was immunohistochemically detected. HPV DNA was detected in 5 cases (16.7%) of normal or metaplastic cervix, 5 cases (29.4%) of CIN I, 9 cases (45.0%) of CIN II, 26 cases (70.3%) of CIN III and 15 cases (65.2%) of ISCC. Positivity for HPV in the groups of CIN III and ISCC was significantly higher than in the normal or metaplastic cervix (P<0.05). The accumulation of p53 was not detected in the normal or metaplastic cervix, CIN I and CIN II. High-level p53 accumulation was identified in basal and suprabasal atypical cells in 27.0% (10/37) of CIN III and in carcinoma cells in 43.5% (10/23) of ISCC cases, and low-level accumulation was identified in atypical cells of 35.1% (13/37) of CIN III and in carcinoma cells in 30.4% (7/23) of ISCC cases. The accumulation of p53 was found to coexist with infection by HPV in 17 (46.0%) of 37 CIN III cases and 12 (52.2%) of 23 ISCC cases, and high-level p53 accumulation was more frequently detected in HPV-positive ISCC cases. Either HPV infection or accumulation of p53 was found in 16.7% (5/30) of the cases of normal or metaplastic cervix, 29.4% (5/17) of CIN I, 45.0% (9/20) of CIN II, 86.5% (32/37) of CIN III and 87.0% (20/23) of ISCC cases. These results suggest that the inactivation of p53 function by HPV infection or alteration of p53 protein itself precedes the development of tumours with a fully malignant and invasive phenotype and plays an important role in tumorigenesis in the uterine cervix. ISH study provided no correlation between the degree of immunohistochemical positivity for p53 and the ratio of HPV-positive cells in the same lesions. PCR-SSCP detected the alteration of p53 gene in at least 4 cases of ISCC, 2 of which were accompanied by HPV infection.     

Human papillomavirus localization in cervical adenocarcinoma and adenosquamous carcinoma using in situ polymerase chain reaction: review of the literature of human papillomavirus detection in these carcinomas

Many studies have suggested that human papillomavirus (HPV) infection plays an important role in the carcinogenesis of the cervical adenocarcinoma. However, the prevalence of HPV infection in cervical adenocarcinoma and adenosquamous carcinoma varies among the studies. Cervical adenocarcinoma (24 cases) and adenosquamous carcinoma (16 cases), including the underlying non-neoplastic epithelium were examined for HPV-DNA using in situ polymerase chain reaction (PCR), which enabled visualization of the localization on a glass slide. In adenocarcinoma, HPV-DNA was found in 13 cases (54%) and in eight cases in underlying non-neoplastic epithelium, resulting in a total of 21 positive cases (88%). In adenosquamous carcinoma, HPV-DNA was detected in 12 cases (75%) and and the HPV-DNA localization of each component was pure adenocarcinoma, 28.6%; mixed, 54.5%; and pure squamous cell carcinoma, 83.3%. In the underlying non-neoplastic epithelium, HPV-DNA was found more frequently in the squamous epithelium (73.3%) than the cervical glands (6.3%). In conclusion, HPV-DNA was detected in 54% of adenocarcinoma, and the rate was elevated by HPV localization in the underlying non-neoplastic epithelium. HPV infection in the underlying squamous epithelium might be related to the carcinogenesis, even in cervical adenocarcinoma. HPV-DNA localization was different in each component of adenosquamous carcinoma.     

Expression of the cytokeratin marker CAM 5.2 in cervical neoplasia

It has been suggested that cytokeratin CAM 5.2 is a useful marker to indicate malignant transformation and invasive potential in cervical neoplasia. In this study we examined normal ectocervical epithelium, endocervical squamous metaplasia, cervical intra-epithelial neoplasia (CIN) and invasive carcinoma by the indirect immunoperoxidase method using commercially available CAM 5.2. Positive staining was seen in 12 of 42 (28%) invasive carcinomas and in 2 of 26 specimens of CIN III. No positive staining was observed in any case of CIN II (22 specimens), CIN I (19), squamous metaplasia (21) or normal ectocervical epithelium (16). These results suggest that although CAM 5.2 expression is found in only 28% of cervical squamous carcinoma, it is highly specific for malignant transformation of cervical squamous epithelium. In view of its potential diagnostic value in doubtful cases of CIN III and squamous cell carcinoma, the specificity and sensitivity of CAM 5.2 expression in cervical neoplasia need to be examined in other laboratories under various processing schedules.     

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.     

Absence of human papilloma virus in cervical adenocarcinoma determined by in situ hybridisation.

A few studies using DNA technology have suggested that human papillomavirus (HPV) may be an aetiological factor for adenocarcinoma of the uterine cervix. Twenty one cases of cervical adenocarcinoma were studied by in situ hybridisation using biotinylated DNA probes for HPV types 6, 11, 16 and 18 and a streptavidin, biotinylated alkaline phosphatase detection system. Intranuclear HPV DNA was detected in none of the adenocarcinomas, while positive controls gave a clear intranuclear signal. Adjacent areas of normal, koilocytic, and dysplastic squamous epithelium also gave positive results. It may be that squamous epithelium contaminates adenocarcinomas reported as positive by Southern blotting. Our results showing absence of detectable HPV DNA within adenocarcinomas suggest that HPV infection may not have a major role in the aetiology of adenocarcinoma of the uterine cervix.     

HPV感染和p53异常表达与宫颈鳞癌的关系

目的研究宫颈鳞癌与p53蛋白表达和HPV感染的关系,探讨宫颈鳞癌的形成机制。方法采用RT-PCR法分别检测p53基因在39例宫颈鳞癌组织和39例正常宫颈黏膜组织中的表达情况以及PCR方法检测HPV在这些组织中的感染情况。结果 HPV在宫颈鳞癌组中阳性率为48.72%(19/39),正常组织中为14.81%(4/27),p53基因表达在宫颈鳞癌组中阳性率为53.85%(21/39),正常组织中为18.52%(5/27),宫颈癌组的HPV感染和p53表达均高于正常组(P0.05)。结论 HPV感染与p53基因的异常表达与宫颈鳞癌发生密切相关,联合检测能提高准确性。     

P53 immunohistochemical expression: messages in cervical carcinogenesis

AIMS: The pattern of p53 expression was studied in pre-invasive and invasive cervical carcinoma in an attempt to clarify its role in cervical carcinogenesis. METHODS: A total of 234 invasive cervical carcinomas (152 squamous cell carcinomas, 61 adenocarcinomas and 21 adenosquamous carcinomas) and 16 cervical intraepithelial neoplasia (CIN) I, six CIN II and 25 CIN III were immunohistochemically studied for p53. RESULTS: p53 was detected more frequently in CIN and invasive carcinoma (100% of CIN I, 74.2% CIN II + III and 70.1% invasive carcinoma) compared with benign cervices (P< 0.001); however, only three squamous cell carcinomas, 11 adenocarcinomas and two adenosquamous carcinomas exhibited p53 expression in >75% of tumour nuclei. Six of the 11 adenocarcinomas and both adenosquamous carcinomas were poorly differentiated compared with one of the three squamous carcinomas. p53 immunoreactive cells were randomly distributed in invasive carcinoma, confined to the lower third of the epithelium in CIN I, reached the middle third in 20% of CIN II and upper third in 16.6% of CIN III. CONCLUSIONS: Assuming that p53 immunoreactivity indicates gene mutation when the majority (> 75%) of neoplastic cells express p53, p53 mutations would seem uncommon in cervical carcinogenesis. Nonetheless, glandular malignancies, in particular poorly differentiated variants, may show a higher frequency of mutation. p53 was detected more frequently in CIN I compared with CIN II/III and invasive carcinoma which may be due to p53 protein degradation following interaction with high risk human papillomavirus E6 protein in CIN II/III and invasive carcinoma.     

Relation between human papilloma virus DNA and expressions of p53 and p21 proteins in cervical lesions

Oncogenic types of human papilloma virus (HPV) are known to be closely associated with cervical carcinoma. On the other hand, the oncogenic process is associated with various abnormalities in the mechanisms of cellular regulation. In this study, we detected the expressions of p53 and p21 proteins in cervical lesions by immunohistochemical techniques, and examined the relationship with HPV infection as well as the clinical usefulness of the results. Cervical biopsy specimens from 107 cases of cervical lesions were studied. HPV-DNA was detected by the hybrid capture method using probe A for low oncogenic types and probe B for high oncogenic types. Anti p21, anti-p53 antibodies were used to perform immunostaining. Point mutation in the p53 gene was analyzed by the DGGE method. High oncogenic HPV types were detected at high frequencies in CIN and SCC. In lesions associated with high oncogenic HPV, p53 protein was detected in 33.4% of the lesions and p21 protein in 36.3%. The p53 gene was analyzed in all cases, and point mutation was not detected. No relation was detected between HPV infection and p53/p21 protein expression. Since mutation was not found in the p53 gene, the p53 protein expressed was considered to be wild-type, which is suspected to play a role in inhibiting disease progression in some cases.     

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.     

人乳头状瘤病毒不同型别与宫颈病变的相关性研究

目的探讨人乳头状瘤病毒（ＨＰＶ）不同型别与宫颈病变性质的关系。方法应用ＰＣＲ技术和原位杂交方法对６１例宫颈上皮内瘤（ＣｅｒｖｉｃａｌｉｎｔｒａｅｐｉｔｈｅｌｉａｌＮｅｏｐｌａｓｉａＣＩＮ）和１２例宫颈鳞癌（ＳＣＣ）进行ＨＰＶ６Ｂ／１１、１６、１８ＤＮＡ检测。结果ＰＣＲ检测结果显示ＨＰＶ６、１１主要分布于低度鳞状上皮内病变（６１９％）和一部分ＣＩＮⅡ中（２０％），而在ＣＩＮⅢ和ＳＣＣ中检测不到；ＨＰＶ１６、１８的检出率随ＣＩＮ级别增高而增加，在ＳＣＣ中高达８３３％。原位杂交结果显示在低度鳞状上皮内病变中，地高辛（Ｄｉｇ）标记的ＨＰＶ６Ｂ／１１、１６、１８ＤＮＡ杂交物质在核中均呈细颗粒状，为“游离型”。上述杂交阳性信号形态亦出现于ＣＩＮⅡ的所有ＨＰＶ６Ｂ／１１及部分ＨＰＶ１６、１８型感染中，而ＣＩＮⅢ和宫颈鳞癌及部分ＣＩＮⅡ中，其杂交阳性信号均为非颗粒状的“整合型”。结论低度鳞状上皮内病变是以ＨＰＶ６、１１低危型为主的多型别病毒的繁殖性感染，ＣＩＮⅢ和宫颈鳞癌为ＨＰＶ１６、１８高危型病毒的整合型感染，而在ＣＩＮⅡ中存在着ＨＰＶ６，１１和ＨＰＶ１６，１８的繁殖性感染及ＨＰＶ１６，１８的整合型感染     

Episomal and integrated human papillomavirus in cervical neoplasia shown by non-isotopic in situ hybridisation.

It was postulated that non-isotopic in situ hybridisation (NISH) signal types 1-3 for human papillomavirus in cervical biopsy specimens represent episomal or integrated virus. The aim of this study was to validate this hypothesis by independent molecular techniques. Fresh cervical intraepithelial neoplasia (CIN) and squamous cell cancer (SCC) tissue were examined for NISH signal pattern by hybridising with digoxigenin labelled HPV 16. DNA was extracted from the same samples and analysed by restriction endonuclease digestion and Southern blotting to determine the physical state of the viral genome. Six CIN biopsy specimens showed a type 1 NISH signal for HPV 16. On Southern analysis these biopsy specimens contained only episomal HPV 16. Three SCC with a type 2 NISH signal contained integrated HPV 16 by Southern analysis. Two specimens, a CIN 3 and an SCC with a type 3 NISH signal for HPV 16, showed the presence of both episomal and integrated HPV 16 with conventional Southern analysis and two dimensional gel electrophoresis. These results show that episomal HPV can be reliably determined by NISH type 1 signal, integrated HPV by type 2, and a combination of both episomal and integrated HPV, by a type 3 signal in archival paraffin wax embedded cervical biopsy specimens. This will add another variable to the epidemiological studies of HPV infection. In particular, it will now allow retrospective studies to be done to define the role of episomal and integrated HPV in the evolution of cervical intraepithelial neoplasia and other cervical disease associated with this virus.