Cervical Intraepithelial Neoplasia Specimens Research Articles

Transcriptional Inactivation of p53, Bax, Bcl-2 and Mdm2 Correlates with Malignant Transformation of the Uterine Cervix

Deregulation of the apoptotic machinery plays a major role in cell death, cellular transformation and cancer. p53, Bcl-2, Bcl-XL, Bax and Mdm2 mRNA expression patterns were evaluated in tissue samples with cervical intraepithelial neoplasia (CIN) and cervical cancer compared to those of normal cervical tissues, and correlated with the underlying cervical lesions. Transcript levels of the above genes were assessed by RT-PCR analysis in a total of 44 cervical specimens. p53, Bcl-2, Bax and Mdm2 transcript levels were significantly different in the normal, CIN and cancer specimen groups (p=0.003, p=0.009, p=0.040 and p=0.001, respectively). Specifically, p53, Bax and Bcl-2 exhibited substantially lower transcript levels in CIN lesions compared to controls, whereas Bax mRNA levels showed a significant decrease in cancer compared to normal specimens. Mdm2 mRNA expression was considerably lower in cancer than in CIN lesions or normal cervix. High-grade squamous intraepithelial lesions exhibited lower p53 and Bcl-2 mRNA levels than controls (p=0.002, p=0.016). Coexpression analysis revealed more correlations between the above apoptosis-related molecules in normal tissues compared to CIN or cancer specimens. p53 showed significant coexpression with Bax, Bcl-2 and Mdm2 (p=0.040, p=0.013 and p=0.015, respectively) in normal cervical specimens. Bax and Bcl-XL mRNA expression was negatively correlated. Mdm2 transcriptional levels correlated significantly with those of Bax, Bcl-XL and Bcl-2. Our findings show that p53, Bax, Bcl-2 and Mdm2 mRNA expression levels correlate with the malignant transformation of the uterine cervix. mRNA coexpression patterns of the members of the pro- and anti-apoptotic family examined in cervical carcinogenesis were found to be disrupted in CIN and cancer, as already demonstrated at the protein level.

Transcriptional inactivation of p53, Bax, Bcl-2 and Mdm2 correlates with malignant transformation of the uterine cervix.

Deregulation of the apoptotic machinery plays a major role in cell death, cellular transformation and cancer. p53, Bcl-2, Bcl-XL, Bax and Mdm2 mRNA expression patterns were evaluated in tissue samples with cervical intraepithelial neoplasia (CIN) and cervical cancer compared to those of normal cervical tissues, and correlated with the underlying cervical lesions. Transcript levels of the above genes were assessed by RT-PCR analysis in a total of 44 cervical specimens. p53, Bcl-2, Bax and Mdm2 transcript levels were significantly different in the normal, CIN and cancer specimen groups (p=0.003, p=0.009, p=0.040 and p=0.001, respectively). Specifically, p53, Bax and Bcl-2 exhibited substantially lower transcript levels in CIN lesions compared to controls, whereas Bax mRNA levels showed a significant decrease in cancer compared to normal specimens. Mdm2 mRNA expression was considerably lower in cancer than in CIN lesions or normal cervix. High-grade squamous intraepithelial lesions exhibited lower p53 and Bcl-2 mRNA levels than controls (p=0.002, p=0.016). Coexpression analysis revealed more correlations between the above apoptosis-related molecules in normal tissues compared to CIN or cancer specimens. p53 showed significant coexpression with Bax, Bcl-2 and Mdm2 (p=0.040, p=0.013 and p=0.015, respectively) in normal cervical specimens. Bax and Bcl-XL mRNA expression was negatively correlated. Mdm2 transcriptional levels correlated significantly with those of Bax, Bcl-XL and Bcl-2. Our findings show that p53, Bax, Bcl-2 and Mdm2 mRNA expression levels correlate with the malignant transformation of the uterine cervix. mRNA coexpression patterns of the members of the pro- and anti-apoptotic family examined in cervical carcinogenesis were found to be disrupted in CIN and cancer, as already demonstrated at the protein level. (Int J Biol Markers 2005; 20: 18-27).

Connective Tissue Activating Peptide III Expression Disappears Progressively with Increased Dysplasia in Human Cervical Epithelium

Objective. The aim of this study was to explore the possible involvement of CTAP-III in the development of cervical cancer as it progresses through several cervical intraepithelial neoplasia (CIN) stages.Material and methods. Twenty-four cervical specimens were obtained by direct punch biopsy, conization, or hysterectomy. Diagnosis of CIN I to CIN III was based on standard morphological criteria in 12 specimens. Tissue specimens were also obtained from 4 normal uteri and 8 cases of invasive squamous cell cervical carcinoma. RT-PCR, using CTAP-III-specific primers, was used to identify CTAP-III mRNA and polyclonal antibodies, directed against the N-terminus of CTAP-III, for immunostaining of the CTAP-III protein.Results. RT-PCR yielded amplified fragments in RNA derived from normal cervical tissue, while no PCR product was detected in the invasive cervical carcinoma tissue. The PCR product corresponded to a CTAP-III plasmid PCR product. Both tissues expressed the same amounts of GAPDH mRNA as the control for the integrity and equal amounts of the isolated RNA. In each of the 16 specimens of normal cervices and of CIN tissues, epithelial cells were stained with the anti-CTAP-III antibodies. In normal epithelium, CTAP-III staining was homogeneously distributed in all epithelial layers, except in the highly active and proliferating basal cells. CTAP-III was localized at the epithelial cell membrane or between adjacent epithelial cells in a granular, chain-like pattern of staining. In the CIN specimens, CTAP-III staining was no longer seen in the deep epithelial layers, consistent with the dysplastic appearance of the cells, and remained in the seemingly normal superficial epithelial layers. Cells of invasive cervical carcinoma did not stain for CTAP-III and were detected in endothelial cells of capillary blood vessels.Conclusion. The specific localization of CTAP-III between adjacent epithelial cells suggests a possible role of this chemokine in maintaining the normal architecture of epithelial tissues. Its progressive disappearance in increasingly severe CIN may be applied to distinguish between specific stages in the progression of cervical carcinoma.

Fragile histidine triad transcription abnormalities and human papillomavirus E6–E7 mRNA expression in the development of cervical carcinoma

BACKGROUND. Human papillomaviruses (HPV) are the most important etiologic factor for cervical carcinoma. However, additional cellular events may be necessary for malignant progression in the cervix. Recently, the fragile histidine triad (FHIT) gene, which is frequently lost in many cancers, was identified as a candidate tumor suppressor gene at chromosome 3p locus 14.2. METHODS. Thirty-five invasive cervical carcinomas (ICC), 13 Grade 2-3 cervical intraepithelial neoplasia specimens (high grade CIN), and 21 normal cervices were examined for expression of the FHIT gene and oncogenic HPV E6-E7 mRNA by reverse transcription-polymerase chain reaction (RT-PCR), and all of these FHIT cDNA were directly sequenced. RESULTS. Fourteen of 35 ICC (40%) showed abnormal FHIT transcription, whereas none of the high grade CIN or normal cervices had FHIT abnormalities. FHIT abnormalities included 13 short transcripts that were variously truncated between exons 4 and 8, 3 short transcripts with unknown sequences inserted, and 1 with no transcription. FHIT abnormalities were observed in all the small cell carcinomas, 4 of 7 adenocarcinomas and adenosquamous cell carcinomas (57%), and 7 of 25 squamous cell carcinomas (28%). Small cell carcinoma appeared to express the aberrant transcripts more frequently than squamous cell carcinoma (P = 0.037). Immunohistochemical analysis showed that four ICCs with abnormal FHIT transcription did not express FHIT protein, whereas five of six CIN and ICC with no FHIT abnormality did express it. When expression of HPV E6-E7 mRNA was examined in the samples positive for HPV-DNA, E6-E7 transcripts were expressed in 7 of 9 high grade CIN (78%) and 18 of 28 ICC (64%). There was no difference in the frequency of HPV E6-E7 expression between CIN and ICC. In contrast, HPV E6-E7 expression was repressed in 8 of 10 (80%) ICC with FHIT abnormalities and in 2 of 18 (11%) in ICC with no FHIT abnormalities. CONCLUSIONS. Although E6-E7 genes of oncogenic HPV are important for the development of CIN and ICC, they may not be required for the development of carcinoma cells that have abnormal FHIT genes.

Fragile histidine triad transcription abnormalities and human papillomavirus E6-E7 mRNA expression in the development of cervical carcinoma

Human papillomaviruses (HPV) are the most important etiologic factor for cervical carcinoma. However, additional cellular events may be necessary for malignant progression in the cervix. Recently, the fragile histidine triad (FHIT) gene, which is frequently lost in many cancers, was identified as a candidate tumor suppressor gene at chromosome 3p locus 14.2. Thirty-five invasive cervical carcinomas (ICC), 13 Grade 2-3 cervical intraepithelial neoplasia specimens (high grade CIN), and 21 normal cervices were examined for expression of the FHIT gene and oncogenic HPV E6-E7 mRNA by reverse transcription-polymerase chain reaction (RT-PCR), and all of these FHIT cDNA were directly sequenced. Fourteen of 35 ICC (40%) showed abnormal FHIT transcription, whereas none of the high grade CIN or normal cervices had FHIT abnormalities. FHIT abnormalities included 13 short transcripts that were variously truncated between exons 4 and 8, 3 short transcripts with unknown sequences inserted, and 1 with no transcription. FHIT abnormalities were observed in all the small cell carcinomas, 4 of 7 adenocarcinomas and adenosquamous cell carcinomas (57%), and 7 of 25 squamous cell carcinomas (28%). Small cell carcinoma appeared to express the aberrant transcripts more frequently than squamous cell carcinoma (P=0.037). Immunohistochemical analysis showed that four ICCs with abnormal FHIT transcription did not express FHIT protein, whereas five of six CIN and ICC with no FHIT abnormality did express it. When expression of HPV E6-E7 mRNA was examined in the samples positive for HPV-DNA, E6-E7 transcripts were expressed in 7 of 9 high grade CIN (78%) and 18 of 28 ICC (64%). There was no difference in the frequency of HPV E6-E7 expression between CIN and ICC. In contrast, HPV E6-E7 expression was repressed in 8 of 10 (80%) ICC with FHIT abnormalities and in 2 of 18 (11%) in ICC with no FHIT abnormalities. Although E6-E7 genes of oncogenic HPV are important for the development of CIN and ICC, they may not be required for the development of carcinoma cells that have abnormal FHIT genes.

Correlation of vascular endothelial growth factor expression and microvessel density in cervical intraepithelial neoplasia.

Angiogenesis (the formation of new blood vessels) appears to be required for the growth of invasive tumors, but little information exists about its role in the development of preinvasive lesions. We examined microvessel density and expression of vascular endothelial growth factor in specimens of cervical intraepithelial neoplasia (CIN), a preinvasive lesion of the uterine cervix, to determine whether a connection could be established between these parameters of angiogenesis and the grade of dysplasia (i.e., tissue abnormality). Sections of biopsy specimens from 83 patients with grades I-III CIN were examined retrospectively. Microvessels were localized by use of a polyclonal antibody directed against factor VIII-related antigen; vascular endothelial growth factor was detected by means of a monoclonal antibody. Reported P values are two-sided. Highest microvessel densities and highest expression of vascular endothelial growth factor were found in a narrow border region between CIN lesions and the underlying stroma. A significant correlation was observed between high vascular endothelial growth factor expression and high microvessel density (Kendall's tau = 0.27; 95% confidence interval [CI] = 0.03-0.50; P = .018). Mean microvessel density values +/- standard deviations for CIN I, CIN II, and CIN III lesions were 19.4 +/- 5.8, 21.9 +/- 7.0, and 34.1 +/- 14.8, respectively (Kendall's tau = 0.46; 95% CI = 0.30-0.61; P<.0001). Corresponding values for vascular endothelial growth factor expression were 8.3 +/- 3.5, 8.4 +/- 2.0, and 12.2 +/- 3.6, respectively (Kendall's tau = 0.41; 95% CI = 0.20-0.60; P<.0001). Our results are consistent with the idea that progression of cervical dysplasia is dependent on angiogenesis.

P53 immunoreactivity in cervical intraepithelial neoplasia and non-neoplastic cervical squamous epithelium.

To determine the pattern of p53 immunoreactivity in cervical squamous epithelium and to investigate the relation between p53 immunostaining and human papillomavirus (HPV) infection. 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. 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. 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.

Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm

The integration of human papillomavirus (HPV) DNA into the human genome has been generally accepted as a characteristic of malignant lesions. To gain a better understanding of this phenomenon, genomic DNA from 181 cervical biopsy specimens was isolated and analyzed for HPV type and physical state of the HPV genome. These specimens represented the full spectrum of cervical disease, from condyloma to invasive carcinoma. Discrimination between integrated and episomal HPV DNA was accomplished by the detection of HPV-human DNA junction fragments on Southern blots. In most cases in which ambiguous Southern blot results were obtained, the specimens were reanalyzed by two-dimensional gel electrophoresis. Of the 100 biopsy specimens of cervical intraepithelial neoplasia analyzed, only 3 showed integrated HPV DNA, in contrast to 56 (81%) of 69 cervical carcinomas (P less than 0.001) showing integrated HPV DNA. Of the 40 carcinomas containing HPV 16 DNA, 29 (72%) had integrated HPV DNA, of which 8 (20%) also had episomal HPV DNA. In 11 (27%) cancers, only episomal HPV 16 DNA was detected. All 23 HPV 18-containing carcinomas had integrated HPV DNA, and 1 also had episomal HPV 18 DNA. The difference between HPV types 16 and 18 with respect to frequency of integration was statistically significant (P less than 0.01). The results of this study indicate that detectable integration of HPV DNA, regardless of type, occurs infrequently in cervical intraepithelial neoplasia. The absence of HPV 16 DNA integration in some carcinomas implies that integration is not always required for malignant progression. In contrast, the consistent integration of HPV 18 DNA in all cervical cancers examined may be related to its greater transforming efficiency in vitro and its reported clinical association with more aggressive cervical cancers.

Colposcopic evaluation of human immunodeficiency virus seropositive women

To determine the effect of human immunodeficiency virus (HIV) infection on cervical histology, 32 known HIV-seropositive women underwent cervical colposcopic evaluation. All had cervical cytology, colposcopically directed biopsy, and T-cell studies performed. Thirteen of 32 patients (41%) had cervical intraepithelial neoplasia (CIN). Another 14 of 32 patients (44%) had histologic evidence of cervicitis. Abnormal cytology, noted in only three women, suggested CIN in one and inflammatory atypia in two. All (five of five) patients with a clinical diagnosis of AIDS had CIN, compared with 30% (eight of 27) of non-AIDS HIV-positive patients (P less than .05). Patients diagnosed with CIN had significantly lower CD4 cell counts (221/mm3 versus 408/mm3; P less than .06) and CD4:CD8 ratios (0.33 versus 0.62; P less than .02) than those without CIN. Patients with cervicitis had greater T-cell immunosuppression than did those with normal histology. In addition, patients with AIDS were more likely to have higher-grade lesions than were non-AIDS HIV-seropositive patients. Seven of 12 CIN specimens available for analysis by polymerase chain reaction using consensus sequence primers detected human papillomavirus (HPV) DNA, including three patients with three or more HPV types. Our data suggest that abnormal cervical pathology is common among HIV-positive women and that cytologic screening is not predictive of CIN in this population. In addition, the presence and severity of cervical dysplasia correlates with quantitative T-cell function. We strongly recommend that cervical colposcopy be part of the routine management of HIV-seropositive women.

Molecular cloning of a novel human papillomarvirus (type 58) from an invasive cervical carcinoma

A novel human papillomavirus type (HPV) was cloned from an invasive cervical carcinoma. The viral clone showed no homology with other known prototypes of HPV (HPV-1 through HPV-57), except HPV-33 by Southern blot analysis under stringent conditions. It showed less than 20% homology to HPV-33 by reassociation kinetic analysis. The restriction endonuclease map of the clone was different from those of other HPV types and its predicted genome organization surmised by hybridization with subgenomic fragment probes of HPV-33 DNA showed the typical HPV genome organization. The results indicate that this clone is a new type of HPV, designated as HPV-58, distinct from the other known types of HPV. HPV-58 was detected in none of 6 specimens of cervical condylomata acuminata, in 7 of 58 specimens of cervical intraepithelial neoplasia, and in 4 of 50 specimens of invasive cervical carcinoma studied in Nagano prefecture, Japan.

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.

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.

Sexually transmitted papillomaviral infections: I. The anatomic distribution and pathologic grade of neoplastic lesions associated with different viral types

Multiple colposcopic biopsy specimens were collected from 160 women, with sampling of principal cervical and vulvar lesions as well as secondary areas of either minor acetowhitening or normal epitheluim. Papillomaviral deoxyribonucleic acid was detected by Southern blot hybridization in 197 (90%) of the 218 principal biopsy specimens and 93 (46%) of 198 secondary biopsy specimens. Although different papillomaviruses were found at different sites in 31 women, only six of 416 specimens contained multiple types within the same sample. Specific viral types were associated with specific disease patterns. Only one of 80 type 6 or 11 infections had a diagnosis greater than cervical intraepithelial neoplasia, grade 2. In contrast, 42 of 48 (90%) biopsy specimens of cervical intraepithelial neoplasia, grade 3, or invasive cancer contained type 16, 18, or 31. Nonetheless, 12 of 124 (10%) cases of condyloma and cervical intraepithelial neoplasia, grade 1, were associated with types 16, 18, and 31 infections. Of 58 women with multicentric disease, 46 had positive hybridizations for both cervical and vulvar lesions (32 showing the same type in both samples and 14 showing different viruses). Differing patterns of papillomavirus-induced disease arise partly from the predilection of specific viral types for certain anatomic sites and partly through variations in host response. Detection of viral deoxyribonucleic acid in 46% of the secondary biopsy specimens suggests that disease expression may represent focal breakdown of host surveillance within a field of latent papillomaviral infection.