Immunohistochemical identification of human papillomavirus infection in tissues from cervical precancerous and early cancerous lesions among Indian women

Paraffin sections of 50 cervical condyloma biopsies from patients with precancerous and early cancerous lesions of the uterine cervix were screened for human papillomavirus (HPV) antigen by peroxidase-antiperoxidase (PAP) staining using genus specific anti-bovine papillomavirus serum (rabbit). The intra-nuclear HPV positivity was observed in 20 percent (10/50) of biopsies studied. Further, proportionately larger number of cases with moderate dysplasia (CIN II) associated with koilocytotic changes were found to be positive for HPV antigen (40%). No HPV positivity was detected from inflammation and mild dysplasia (CIN I) cases. The study strongly supports the association of HPV infection in the process of neoplastic transformation of the uterine cervix.     

The Detection of Human Papillomaviruses in Cervical Biopsies by Immunohistochemistry and In Situ Hybridization

The presence of human papillomavirus (HPV) types 6, 16 and 8 in cervical biopsies can be detected by an immunoperoxidase technique using type-restricted monoclonal antibodies raised against fusion proteins representing the L1 major capsid proteins of these three HPV types. In a retrospective study ( n = 54) we have used these antibodies and biotinylated DNA probes of HPV 6.16 and 18 lo detect and type HPV in formalin-fixed material from the cervix. The biopsies were classified histologically into normals, wart infections without dysplasia, cervical intraepithelial neoplasia (CIN)and squamous cell carcinomas. Antibody staining showed that 22% of all CIN was positive for HPV 16and 40% of cervical warts were positive for HPV 6, 16 and 18, There was no HPV capsid protein detected in the normals and squamous cell carcinomas using these antibodies, whereas 25% of the tumours were positive for HPV 16 by in situ hybridization. Sections of cervical warts and CIN positive for HPV types by in situ hybridization were also positive by antibody staining which suggests that both techniques are detecting replicating virus. We feel these two techniques complement each other in detection and typing of HPV in cervical biopsies from patients with active disease.     

Routine papillomavirus antigen staining of cervical punch biopsy specimens.

Immunocytochemical staining for papillomavirus antigen was carried out on 1147 consecutive cervical punch biopsy specimens over 12 months. Of 876 cases with cervical intraepithelial neoplasia (CIN) 351, were antigen positive and of 49 cases with histological evidence of human papillomavirus (HPV) infection but no CIN, 14 were positive. There were 204 cases reported to be normal on routine histological examination and 12 cases reported to show features suggestive but not diagnostic of HPV infection. Of the normal group, 24 (12%) were antigen positive and of the equivocal group, two were positive. In 122 of the normal or equivocal groups cytological examination was repeated at the time of colposcopy, and dyskaryosis was reported in 36. In only four cases was disease shown by HPV antigen staining when there was no diagnostic histological or cytological abnormality. HPV antigen staining assists in the recognition of the range of histological changes associated with productive HPV infection but is an insensitive test and has only limited value in supplementing histological and cytological examinations as a diagnostic aid in routine colposcopic pathology.     

p53 immunoreactivity in cervical intraepithelial neoplasia and non-neoplastic cervical squamous epithelium.

AIMS--To determine the pattern of p53 immunoreactivity in cervical squamous epithelium and to investigate the relation between p53 immunostaining and human papillomavirus (HPV) infection. METHODS--Immunocytochemistry for p53 was performed in 65 specimens of formalin fixed, paraffin wax embedded cervical tissue using a polyclonal antibody against recombinant p53. Microwave oven heating was used for antigen retrieval. Eight normal biopsy specimens, eight cases with histological features of HPV infection, and 49 cases of cervical intraepithelial neoplasia (CIN) were examined. Thirty one cases of CIN were examined. Thirty one cases of CIN were examined for evidence of HPV infection using in situ hybridisation with probes directed against wide spectrum HPV, HPV 16 and HPV 18. RESULTS--p53 immunoreactivity was seen in seven of eight (87%) of specimens with histological features of HPV infection, five of eight (62%) normal specimens, 13 of 22 (59%) CIN III, three of 14 (21%) CIN II and five of 13 (38%) CIN I specimens. The numbers of positive nuclei were small in cases of CIN and the location of positive nuclei within the epithelium paralleled the degree of dysplasia. Eleven of 15 (73%) CIN specimens which were immunoreactive for p53 yielded a positive signal for HPV by in situ hybridisation. A positive signal for HPV was also seen in 10 of 16 (63%) of CIN specimens in which p53 staining was absent. CONCLUSIONS--p53 immunoreactivity can be demonstrated in a small proportion of cells in the cervical squamous epithelium in a significant proportion of cases of CIN. This immunoreactivity seems to be independent of the presence of HPV, as assessed by in situ hybridisation. p53 immunoreactivity also occurs in non-neoplastic cervical squamous epithelium with a pattern of distribution within the epithelium which differs from that seen in CIN. Antigen retrieval by microwave oven heating enhances p53 immunostaining and may result in visualisation of cellular p53 in the absence of mutation.     

Human papillomavirus in dysplasia and carcinoma of the cervix in Singapore

A prospective study was conducted in Singapore in 1985 where 107 women with abnormal cervical smears were studied for cervical neoplasia and its association with the human papillomaviruses (HPV), using HPV 11, 16 and 18 DNA as probes. Cervical biopsies were performed for histology as well as for DNA Southern Blot hybridization studies to detect the presence of HPV 11, 16 or 18 genome. The prevalence of the various types of papillomavirus DNA in cervical tissue samples from cervical carcinoma and dysplasias is presented. HPV types 16 and 18 were found in 5 of 8 (63%) cases of invasive squamous cell carcinoma (SCC), in 12 of 41 (29%) cases of squamous carcinoma-in-situ (CIN 3) and in 3 of 48 (6%) cases of lower grade dysplasia. None of our cases of SCC were associated with unknown HPV types detected using a mixture of HPV 16 or 18 DNA under conditions of low stringency. These unknown HPV types were present in approximately one quarter of our cases of dysplasia of all grades.     

Immunohistochemical localization of cdc6 in squamous and glandular neoplasia of the uterine cervix

CONTEXT: Cdc6 has been extensively studied as a marker for cellular proliferation that is expressed during the normal cell cycle. Recent studies indicate that Cdc6 may be a marker for cervical intraepithelial neoplasia (CIN) and carcinoma; however, the histologic distribution of Cdc6 has not been explicitly defined. Expression of Cdc6 in the endocervical mucosa also remains unexplored. OBJECTIVE: The goal of the current study was to evaluate the distribution of Cdc6 protein, MIB-1 protein, and human papillomavirus (HPV) DNA in a broad range of cervical tissues, including normal, potentially premalignant, and malignant lesions of the ectocervical and endocervical mucosa. METHODS: We used an indirect immunoperoxidase method to stain formalin-fixed, paraffin-embedded tissues and frozen tissues, including biopsy and hysterectomy specimens, for Cdc6 and MIB-1 proteins, and we used in situ hybridization to detect HPV DNA in a subset of cases. RESULTS: Cdc6 staining was exclusively nuclear and was present in both squamous and glandular epithelial cells of histologic sections. Cdc6 staining was rarely present in specimens of normal cervical squamous mucosa (2/84, 2.4%) or in specimens with squamous metaplasia (3/59, 5.1%) and was not detected in normal endocervical glands (0/84). Staining was present in most cases of CIN I (31/48, 65%). Staining was present in the majority of cases of CIN II (25/28, 89%) and in all cases of CIN III (36/36) and squamous cell carcinomas (34/34). The proportion of cells staining for Cdc6 increased with the grade of dysplasia, and the proportion of stained cells in squamous cell carcinomas was similar to that in lesions of high-grade dysplasia. Cdc6 staining was present in the majority of cases in glandular lesions including adenocarcinoma in situ (11/14, 79%) and adenocarcinoma (8/10, 80%). The histologic distribution of Cdc6-immunoreactive cells was similar to that of cells with a strong signal for HPV DNA, but Cdc6 protein and HPV DNA did not colocalize at the level of individual cells. CONCLUSION: Cdc6 expression is a marker for high-grade cervical squamous and glandular dysplasia and carcinoma and is associated with HPV infection. The mechanistic basis of the association between HPV infection and Cdc6 immunopositivity remains to be determined but may represent either up-regulation of Cdc6 expression or stabilization of the Cdc6 protein.     

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.     

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.     

"HPV score", a scoring system for histological diagnosis of human papillomavirus infection in dysplasia of the uterine cervix

We devised a scoring system, "HPV score", for histological diagnosis of human papillomavirus (HPV) infection in dysplasia of the uterine cervix. Four hundred and sixty cases of cervical dysplasia were screened for the presence of HPV infection using this system, and 116 cases (25%) were judged to be HPV infection. The results showed good correlation with those obtained using the immunoperoxidase (IMPO) method, but 42 of the 116 cases (36%) were negative for HPV antigen by the IMPO method. Severe stromal inflammation was noted in about half of such cases. Among the four histological types of cervical condyloma, the flat type was most common among the cases with HPV infection judged according to HPV score. It was concluded that this scoring system is simple and accurate for the detection of HPV infection in cervical lesions.     

Negative colposcopic biopsy after positive human papilloma virus (HPV) DNA testing: false-positive HPV results or false-negative histologic findings?

We studied histologic examination-related factors contributing to false-negative colposcopic biopsy results. Patients positive for high-risk human papillomavirus (HPV) DNA with negative cervical histologic findings were identified between January 2002 and December 2003. Three additional H&E-stained levels were obtained when the original diagnosis was confirmed on review. Patients with atypical squamous cells of undetermined significance (ASC) Papanicolaou test results, positive HPV DNA results, and negative cervical histologic findings accounted for 4.5% of all ASC smears submitted for HPV DNA testing. Slides and tissue blocks were available for 95 cases; 4% had focal HPV infection or mild dysplasia. When deeper levels were examined, 31% had clinically significant lesions: HPV infection or cervical intraepithelial neoplasia (CIN) 1, 19%; CIN 2/3, 8%; and dysplasia, not otherwise specified, 3%. Of the remaining patients, follow-up revealed squamous abnormalities in 25%. About 5% of patients with positive HPV DNA results had a negative follow-up biopsy result. "False-negative" biopsies accounted for one third of cases. Additional levels should be obtained for discrepant results. Close follow-up is crucial when the initial biopsy result is negative because a small number of patients will have squamous abnormalities in subsequent studies.     

p16INK4A, CDC6, and MCM5: predictive biomarkers in cervical preinvasive neoplasia and cervical cancer

AIM: To analyse and compare expression patterns of three potential biomarkers-p16(INK4A), CDC6, and MCM5-and evaluate their use as predictive biomarkers in squamous and glandular cervical preinvasive neoplasia. METHODS: Immunocytochemical analysis of p16(INK4A), MCM5, and CDC6 expression was performed on 20 normal, 38 cervical intraepithelial neoplasia 1 (CIN1), 33 CIN2, 46 CIN3, 10 squamous cell carcinoma, 19 cervical glandular intraepithelial neoplasia (cGIN), and 10 adenocarcinoma samples. Staining intensity was assessed using a 0-3 scoring system. p16(INK4A), MCM5, and CDC6 expression was also examined in ThinPrep slides exhibiting mild, moderate, and severe dyskaryosis. Human papillomavirus (HPV) was detected using a modified SYBR green assay. Fluorogenic polymerase chain reaction (PCR) and solution phase PCR were used for specific HPV typing. RESULTS: All three markers showed a linear correlation between expression and grade of dysplasia. p16(INK4A) and MCM5 protein expression was upregulated in all grades of squamous and glandular dysplasia. CDC6 protein was preferentially expressed in high grade lesions and in invasive squamous cell carcinoma. CONCLUSION: p16(INK4A) expression was closely associated with high risk HPV infection-all grades of squamous and glandular cervical lesions were immunohistochemically positive. MCM5 staining intensity was independent of high risk HPV infection, highlighting its potential as a biomarker in both HPV dependent and independent cervical dysplasia. CDC6 may be a biomarker of high grade and invasive lesions of the cervix, with limited use in low grade dysplasia. p16(INK4A) was the most reliable marker of cervical dysplasia. Combinations of dysplastic biomarkers may be useful in difficult diagnostic cases.     

The histopathology of biopsies taken from women attending a New Zealand colposcopy clinic.

Analysis of the histopathology of biopsies taken from 1371 women attending the Dunedin Hospital Colposcopy Clinic during the period 1982-1988 showed that CIN3, either alone or in conjunction with human papillomavirus (HPV) infection, was the most commonly encountered abnormality. Women under the age of 29 years accounted for 58.3% of the CIN3 lesions. There was also a dramatic increase in the number of women with evidence of HPV infection. Several cases of invasive carcinoma were encountered, 3 in very young women. However, there was no obvious age predilection for the various grades of CIN and for HPV lesions. This raises the question of whether or not HPVs in cervical cancer are chance associations rather than being actively involved in lesion information.     

DNA hybridization for human papillomavirus (HPV) in cervical lesions. Relationship of the presence of various viral subtypes to expression of HPV structural proteins, involucrin, and carcinoembryonic antigen

We studied cervical tissue from 20 patients with a variety of condylomatous, preneoplastic, and neoplastic lesions to detect various subtypes of human papillomavirus (HPV) at the molecular level by DNA hybridization. In addition, mirror image biopsy specimens were studied by an immunoperoxidase technique for the presence of HPV structural antigens, carcinoembryonic antigen (CEA), and involucrin, as markers of disturbed maturation and/or neoplasia. Of the 20 patients studied, we were able to demonstrate the presence of either HPV 16 or 18, or both, in three of seven squamous carcinomas tested, and three of five dysplasias. Interestingly, two cases of squamous carcinoma and one moderate dysplasia demonstrated HPV types 6 and 11 concurrently with HPV 16 and/or 18. Four of six condylomas showed HPV subtypes 6/11, and only one condyloma showed weak hybridization with HPV 18. Human papillomavirus structural antigen was seen in three of eight condylomas, and three of five dysplasias, but not in any carcinoma. All condylomas showed intense staining for involucrin in full-thickness of the epithelium, but the high-grade dysplasias and carcinomas showed only focal or absence of staining for involucrin. Carcinoembryonic antigen was expressed in 50% of the carcinomas with a pattern similar to that seen with involucrin, but did not correlate with any particular subtype of HPV. The molecular hybridization method seems to be superior for the detection of HPV lesions, and possibly for the prediction of their biologic behavior.     

Immunohistochemical localization of survivin in benign cervical mucosa,cervical dysplasia,and invasive squamous cell carcinoma

Survivin is an inhibitor of apoptosis protein (IAP) that is expressed in fetal development and in cancer Survivin expression in premalignant lesions remains undefined. We obtained 73 samples of cervical squamous tissue, including 31 normal, 17 low- and 15 high-grade squamous intraepithelial lesions (LSILs, HSILs), and 10 squamous cell carcinomas (SCCs)from cone biopsy and hysterectomy specimens, and stained for survivin using an immunoperoxidase method. Nuclear staining was detected in normal mucosa, LSILs, and HSILs; staining intensity was greatest in cases with morphologic evidence of human papillomavirus (HPV) infection. In situ hybridization of serial sections demonstrated colocalization of HPV DNA and survivin. Cytoplasmic staining was observed in immature squamous metaplasia and in SCCs. Survivin expression in immature metaplastic squamous mucosa may reflect a rolefor survivin in normal squamous differentiation. However, the histologic correlation between nuclear staining and HPV infection suggests involvement of survivin in HPV-mediated disruption of normal cellular maturation.     

In-situ hybridization using biotinylated DNA probes to human papillomavirus in adenocarcinoma-in-situ and endocervical glandular dysplasia of the uterine cervix.

In-situ hybridization using biotinylated probes to human papillomavirus (HPV) DNA was performed on formalin fixed paraffin embedded tissue in 30 patients with histologically confirmed adenocarcinoma-in-situ (AIS). Thirteen of the 30 cases contained areas of endocervical glandular dysplasia (EGD) admixed with AIS. Twenty one patients showed positive staining of the AIS nuclei for HPV DNA. Ten cases (33%) were positive for HPV 16 DNA and 11 cases (37%) were positive for HPV 18 DNA. No case showed synchronous expression of HPV 16 and 18 DNA. All cases of AIS were negative for HPV 6b and 11 DNA. Four cases of EGD were positive for HPV 18 DNA and 2 cases were positive for HPV 16 DNA. Four of 6 cases of intestinal dysplasia/AIS were positive for HPV 18 DNA. Associated squamous abnormalities (HPV +/- CIN +/- SCC) were noted in 15 cases. Of these, 7 showed positive staining for HPV DNA in the squamous lesion. Moreover, 5 of these were positive in both the AIS and squamous lesion. In-situ hybridization using biotinylated DNA probes is a sensitive and safe technique readily adaptable to routine histopathology.     

Expression of major histocompatibility class II antigens by Langerhans'' cells in cervical intraepithelial neoplasia.

Cervical biopsy samples from 67 patients who had various grades of cervical intraepithelial neoplasia (CIN) or who showed evidence, in the form of koilocytosis, of human papillomavirus (HPV) infection of the uterine cervix, and from 10 women with normal cervices were examined. Cryostat sections from the biopsy samples were stained using monoclonal antibodies to T6, a Langerhans' cell marker, and to major histocompatibility complex (MHC) class II antigens (HLA-DP, DQ, and DR). Epithelial Langerhans' cells were reduced in number and showed changed morphology and distribution in koilocytic lesions and in all grades of CIN (p less than 0.01) except CIN I. HLA-DR expression by Langerhans' cells was significantly increased in koilocytic lesions and in CIN grades I and II (p less than 0.05); HLA-DQ expression was significantly increased in all grades of CIN (p less than 0.05) with the increase being most pronounced in CIN I (p less than 0.01). Columnar epithelium expressed MHC class II antigens in all samples tested and squamous epithelium in four of 29 cases of CIN III. These findings support the view that there is a localised disturbance of immune function in both neoplastic cervical epithelium and that infected with papillomavirus.     

Current concepts in the relationship of human papillomavirus infection to the pathogenesis and classification of precancerous squamous lesions of the uterine cervix

Pathologic and epidemiologic studies performed over the past three decades have provided evidence that the development of squamous cell carcinoma of the cervix is a multistep process involving a precursor preinvasive stage. The results of recent molecular analyses now suggest that the human papillomavirus (HPV) plays a role in this process and is an important but insufficient factor in the development of invasive carcinoma. Infection by a variety of HPV types may result in active viral intranuclear replication without integration into the cellular genome. This episomal form of infection is manifested morphologically by the development of mild dysplasia, cervical intraepithelial neoplasia (CIN) 1 with koilocytosis and acanthosis. Approximately 20 different HPV types have been associated with CIN 1 lesions, whereas high-grade dysplasia and carcinoma in situ (CIN 2 and 3) are associated with only a few viral types (mainly HPVs 16, 31, 33, and 35). Low-grade lesions are differentiated and have a low risk of progression to cancer, whereas high-grade lesions are characterized by nearly complete or complete loss of squamous maturation and a higher risk of progression to invasive cancer. Based on the biologic dichotomy of an infectious and a neoplastic process and the segregation of HPV types into two groups, a modification of the CIN classification into low-grade and high-grade squamous intraepithelial lesions in accordance with the Bethesda System is proposed. Although HPV plays a significant role in the development of cervical neoplasia, the value of identifying HPV DNA by such molecular techniques as Southern blot analysis, in situ hybridization, and the polymerase chain reaction in the early detection of preinvasive lesions has not been determined and their routine use is not at present recommended.     

Papillomavirus and cervical cancer: a clinical and laboratory study

It is now widely accepted that HPV types 16, 18, 31, and 33 are associated with the development of high grade intraepithelial neoplasia and malignant lesions in the cervix. On this basis, the identification of HPV types in cervical scrape samples has been advocated as a supplement to cytological screening tests. However, little is known of the distribution of the virus at different sites in the lower female genital tract or of how this distribution may change during the natural course of HPV infection. In this survey, HPV DNA dot hybridizations and, in some instances, Southern blot hybridizations with mixed HPV 6/11 and 16/18 probes were undertaken to detect HPV DNA in cervical scrapes and biopsies of the cervix, vagina, and vulva. A total of 92 women attending a Sydney hospital were screened: 59 of these patients had cervical disease, either invasive cervical carcinoma (CaCx) or cervical intraepithelial neoplasia (CIN), grades I-III. A group of 33 women who lacked evidence of cervical abnormalities served as controls. HPV DNA, predominantly type 16/18, was detected in the cervical biopsies of 96% of the CaCx patients, 80% of the CIN III patients, and 65% of the CIN I-II patients. In contrast only 9% of the cervical biopsies from the control group contained detectable HPV 6, 11, 16, or 18 DNA. A high proportion of the women with cervical abnormalities had evidence of concurrent vaginal and/or vulval papillomavirus involvement. The significance of these findings for routine screening and subsequent management of patients with HPV-associated cervical disease is discussed.     

Simultaneous detection and typing of human papillomavirus in cervical biopsies using PCR-reverse hybridization

INTRODUCTION: Genotyping of Human papillomavirus (HPV) is an important step in the clinical evaluation of the oncogenic risk associated with HPV infection of cervical mucosa. The purpose of this work was to develop a fast PCR-reverse-hybridization assay (PCR-RH) for the simultaneous detection and genotyping of anogenital HPVs. METHODS: HPV DNA from cervical biopsies was amplified by consensus primer-PCR. Digoxigenin-labeled PCR products were hybridized to type-specific probes anchored to the surface of plastic microwells and revealed by an ELISA system. RESULTS: The method was tested on 115 clinical samples (81 koilocytic atypias, 11 CIN1, 10 CIN2, 12 CIN3 and 1 squamous carcinoma). HPV DNA was found in 56.7% koilocytic atypias, in 90.9% of CIN1 and in 100% of CIN2 and higher-grade lesions. Thus, PCR-RH is sensitive, rapid, easy-to-perform and readily applicable to the routine analysis of a large number of samples.     

Cytopathic effects of human papillomavirus infection and the severity of cervical intraepithelial neoplasia : A frequency study

Cytopathic effects related to the human papillomavirus (HPV) infection are more frequently found in cervical intraepithelial neoplasia (CIN) 1; however, there are indications that at least half the histological diagnoses of CIN2 and CIN3 include koilocytosis areas. The objective of this study was to evaluate the frequency of the cytological criteria suggestive of HPV infection in the cervical smears of women with a histological diagnosis of CIN. One hundred and sixty-two women with abnormal cervical smears and a diagnosis of CIN confirmed by histopathology were selected, including 46 cases of CIN 1, 42 of CIN 2 and 74 cases of CIN 3. Koilocytosis was found in 63% of the smears from women with a histopathological diagnosis of CIN 1. This sign was observed in 26.2% and 25.7% of smears of women with a diagnosis of CIN 2 and CIN 3, respectively. Cytomegaly also was frequent in cervical smears of women with histopathological diagnosis of CIN 1 (71.8%). On the other hand, spindle cells and atypical metaplasia were more frequent in women with CIN 2 and CIN 3. Atypical parakeratosis showed similar frequency in all grades of CIN diagnosis. Koilocytois and cytomegaly were inversely correlated with the diagnosis of CIN2 or CIN 3, with OR values respectively of 0.30 (95%CI 0.13-0.68) and 0.26 (95%CI 0.11-0.58). The others signs analyzed did not show any significant association. Koilocitosis and cytomegaly can provides good reassurance that a patient with atypical cervical smear have CIN 1.