Increased angiogenesis in the uterine cervix associated with human papillomavirus infection

We studied the relationship between angiogenesis (using the CD34 antibody), the presence of human papilloma virus (HPV) infection, HPV E6 protein expression and the accumulation of p53 protein at various phases of tumour progression in the uterine cervix. Expression of CD34, p53 and HPV E6 protein was evaluated by immunocytochemistry. Presence of the mutant p53 was detected using a mutant specific ELISA, and the type of HPV was determined by the Polymerase Chain Reaction. A total of 230 cervical tissue samples were analyzed and included 40 cases of apparently normal cervical epithelium, 37 low grade squamous intraepithelial lesions (SILs), 43 high grade SILs, 36 well-differentiated squamous cell carcinomas (DSCC), 31 moderately differentiated (MDSCC) and 43 poorly differentiated carcinomas (PDSCC). There was an excellent correlation between the extent of angiogenesis and histological abnormality (r = 0.912, p = 0.000004). The least extent of angiogenesis was seen in normal cervical tissue and low grade SILs where the mean (low power) intra lesional vascular density (ILVD) was 12 +/- 1.13 and 25.66 +/- 5.20, respectively. In high grade squamous intraepithelial lesions (SILs), the mean ILVD value was 80.84 +/- 25.57. In well-differentiated squamous cell carcinomas (WDSCC's) the mean value was 144.22 +/- 28.67 while in moderately differentiated squamous cell carcinomas (MDSCC's) the mean value was 166.29 +/- 34.95 and in poorly differentiated tumours (PDSCC's) 192.42 +/- 27.98. The extent of angiogenesis also correlated to presence of HPV (r = 0.505, p = 0.00001). Increased CD34 expression was associated with the presence of HPV types 16 and 18. A similar correlation was also evident in HPV, 16/18 infected cases expressing the E6 protein (r = 0.612, p = 0.000001). CD34 expression also correlated well with p53 accumulation (r = 0.859, p = 0.000002). Presence of HPV infection significantly correlated with the extent of histological abnormality (r = 0.467, p = 0.00001). Expression of E6 also showed this significant correlation (r = 0.644, p = 0.00002). Accumulation of p53 was significantly more elevated in HPV 16-infected lesions (r = 0.518, p = 0.00001) and E6-expressing cells (r = 0.650, p = 0.000004). Only 12 of the 230 cases analyzed showed presence of the mutant p53 protein. Angiogenesis appears to increase with histological abnormality in the uterine cervix. Angiogenesis also appears to be influenced by high risk HPV infection, the expression of the E6 transforming protein and the p53 tumour suppressor protein.     

Pathognomonic signs of human papillomavirus infection in diagnostic material from the uterine cervix

Etiological factors and mechanisms of pretumorous and tumourous processes in the female genitalia are presented in light of current concept of viral cocarcinogenesis. The utility of cytoscreening for the detection of the papilloma-virus infection in the uterine cervix mucosa is founded. Detailed cytological characteristics of cells in the uterine cervix mucosa with a cytopathic effect (coilocytosis, the presence of two or many nuclei, vacuolisation of cytoplasm, nuclear atypia) are presented. A selective localization of the human papilloma virus in the transformation zone of the uterine cervix is determined. The frequency of cells with cytopathic effect is 80.2%.     

Tenascin in human papillomavirus associated lesions of the uterine cervix.

The immunohistochemical expression of tenascin was studied in 80 morphologically diagnosed condylomas and cervical intraepithelial neoplasia (CIN) lesions. The results were compared with the human papillomavirus (HPV) DNA subtype, which was determined by HPV dot blot and in situ hybridisation. Tenascin mRNA synthesis was also determined in 10 selected cases by in situ hybridisation. No statistically significant association was found between tenascin expression and the degree of dysplasia or the HPV subtype. There was, however, a strong correlation between the extent of tenascin immunoreactivity and the degree of inflammation. Synthesis of tenascin mRNA was detected in basal keratinocytes and in fibroblasts by in situ hybridisation. The lack of association between the grade of CIN and tenascin expression precludes its use as a marker of premalignancy in CIN.     

P16 overexpression and human papillomavirus infection in small cell carcinoma of the uterine cervix

Carcinogenesis of cervical cancer has been investigated, and p16(INK4a) overexpression in squamous cell carcinoma of the cervix has been reported as a result of infection by human papillomavirus (HPV) (eg, HPV 16), and the consequence of the retinoblastoma (Rb) protein inactivation by HPV E7 protein. However, to our knowledge, there have been no studies on the relation between p16(INK4a) overexpression associated with HPV and small cell carcinoma of the cervix, which behaves more aggressively clinically than squamous cell carcinoma. The purpose of this study was to determine whether p16(INK4a) is overexpressed in small cell carcinoma, and if p16(INK4a) is overexpressed, the types of HPV that are related to this cancer. We reviewed 10 cases of small cell carcinoma and examined them for p16(INK4a) overexpression by immunohistochemistry. We also performed HPV typing with polymerase chain reaction (PCR)-sequencing analysis and in situ hybridization and found that p16(INK4a) was overexpressed in every case. PCR-sequencing analyses revealed that all cases were HPV-positive and that 9 cases were positive for HPV 18. Five of the 9 cases positive for HPV 18 were also positive by in situ hybridization and yielded a punctate signal, considered to represent the integrated form. In conclusion, p16(INK4a) was overexpressed and HPV 18 was frequently detected in an integrated form in small cell carcinoma. Therefore, inactivation of Rb protein by HPV 18 E7 protein may be associated with carcinogenesis of small cell carcinoma the same as inactivation of Rb protein by HPV 16 E7 protein is associated with carcinogenesis of squamous cell carcinoma.     

Human papillomavirus infection of the uterine cervix analyzed by nonisotopic in situ hybridization

Some types of human papillomaviruses (HPVs) have been suggested to be strongly related to uterine cervical carcinoma. An attempt to detect these in formalin-fixed, paraffin-embedded sections was made by either immunohistochemical or by in situ hybridization. Anticapsid protein of bovine papillomavirus antibody labeled with peroxidase was used for immunohistochemistry, and biotin was used instead of radioisotopes to label probes for in situ hybridization, which resulted in low background and a rapid procedure. Condylomatous changes were stained immunochemically with this antibody even in invasive carcinoma, whereas the carcinoma itself was not stained. Direct correlation was demonstrated by in situ hybridization between the HPV genome and histopathological structure, which was impossible by Southern or dot hybridization. HPV DNAs were detected in the nuclei of koilocytes and dyskeratinocytes of condylomata and dysplasias. Furthermore, hybridization signals of HPV DNAs in basal and parabasal cells suggested that HPV infection had already begun in the basal cells. In the case of malignant neoplasia accompanied by dysplasia, the same type of HPV was detected both in the malignant neoplasia and accompanying dysplasia. In one case of intraepithelial carcinoma, the very small focus of carcinoma just arisen in the cells of dysplasia was identified, and both were positive for HPV 18. This fact supports the suggestion that the carcinoma arises in dysplasia. Invasive carcinomas were classified further into keratinized, large-cell nonkeratinized, and small-cell nonkeratinized types, and the positive frequency for HPV 16 decreased as the differentiation of the carcinoma decreased. In the case of keratinized type of invasive carcinoma, strong hybridization signals were prominent around the malignant pearl formation.     

Assessment of precancerous lesions of the uterine cervix for evidence of human papillomavirus infection: a histological and immunohistochemical study

Cervical biopsies obtained by colposcopic direction from 358 women were histologically examined for squamous dysplasia (cervical intra-epithelial neoplasia; CIN) and human papillomavirus (HPV) infection. Of the 358 biopsies, 136 were stained by an immunoperoxidase method using an antiserum against genus-specific (common) antigen of bovine papillomavirus. HPV antigens were detected in 40% of biopsies showing definite histological evidence of HPV effect, and in 7.9% and 2.6% of those with possible or no HPV effect, respectively. HPV effect was commonly seen in association with CIN. The frequency of histological evidence of HPV effect and positive immunoperoxidase staining decreased with increasing grades of CIN. HPV antigen was found in 57% of areas of HPV change with minor atypia, 34% of zones of CIN I and in only 8% of zones of CIN II. No antigenic staining or definite histological evidence of HPV effect was observed within areas of CIN III. Antigen was generally confined to the nuclei of superficial koilocytes, cells with lesser degrees of perinuclear clearing and parakeratotic cells. These results how a strong association between HPV infection and precancerous lesions of the uterine cervix and are consistent with the hypothesis that production of the HPV structural antigen requires a high degree of squamous cell maturation. The immunoperoxidase findings and the histopathological observations support the view that HPV change and dysplasia are part of a morphological continuum in which the cytopathic effect of HPV is expressed mainly in lower grades of dysplasia.     

Apoptosis in woman uterine cervix in pathologies associated with human papillomavirus

The level of apoptosis in uterine cervical tissue was evaluated in healthy women and in patients with various pathologies. No signs of apoptosis were found in unchanged stratified epithelium, condyloma latum, and condyloma acuminatum. The level of apoptosis decreased with progression of neoplastic epithelial transformations, usually no apoptosis was observed in samples of stage III cervical intraepithelial neoplasms. The development of preinvasive carcinoma was accompanied by activation of apoptotic processes most pronounced in the upper third of the epithelium. In some stage I and stage I-II cervical intraepithelial neoplasms, apoptosis and elimination of the basal layer cells caused rejection of the epithelium which can explain regression of this pathology at the initial stages. The prevalence of human papillomavirus infection directly correlated with neoplastic changes in the cervical epithelium.     

Human papillomavirus DNA in glandular lesions of the uterine cervix.

AIMS--To assess the role of human papillomavirus in the pathogenesis of adenocarcinoma in situ, endocervical glandular dysplasia (a presumed precursor of adenocarcinoma) and endocervical glandular epithelial giant cell change. METHODS--Viral detection was carried out using an in situ hybridisation technique on paraffin wax sections. Biotinylated probes for human papillomavirus types 6/11, 16/18, 31/33/35 were used with a colorimetric detection system. RESULTS--Nine out of 21 (43%) cases of adenocarcinoma in situ contained human papillomavirus types 16/18, one of which was also positive for 31/33/35. Ten cases of glandular dysplasia and four cases of glandular epithelial multinucleation did not react with the probes used. CONCLUSIONS--These results indicate that while adenocarcinoma in situ is strongly associated with human papillomavirus infection, endocervical glandular dysplasia and glandular epithelial multinucleation are probably not associated with the virus.     

宫颈小细胞神经内分泌癌中人乳头瘤病毒的研究

目的探讨宫颈小细胞神经内分泌癌中高危型人乳头瘤病毒（humanpapillomavirus,HPV）感染情况。方法提取1例33岁宫颈小细胞神经内分泌癌患者手术切除组织癌变区域蜡块中的DNA,通过巢式PCR方法检测其中HPV感染情况。结果该患者肿瘤切除组织高危型HPV18型阳性。结论利用巢式PCR方法分型检测宫颈小细胞神经内分泌癌中的高危型HPV型别,其准确性及敏感性均较高。     

Detection and typing of human papillomavirus infection of the uterine cervix by dot blot hybridisation: comparison of scrapes and biopsies

Paired scrapes and biopsies from 100 women attending a routine colposcopy clinic were examined by dot blot DNA hybridisation for infection with human papillomavirus (HPV) types 6/11, 16, 18, and 31; 51% of the scrapes and 50% of the biopsies were positive for HPV infection. Scrapes detected more HPV 18 (10% vs. 2%, P = less than 0.05) and HPV 31 (7% vs. 3%, not significant) than did the biopsies, but biopsies detected more HPV 16 (42% vs. 33%, not significant). Comparison of the results for each patient revealed that the correlation between scrapes and biopsies was not very close: only 34 patients were HPV-positive by both sampling methods, whereas 67 were positive if the results were combined. This report discusses the implications of these findings.     

Human papillomavirus infection of the uterine cervix. Tissue sampling and laboratory methods affect correlations between infection rates and dysplasia.

Two common tissue sampling techniques--colposcopic biopsy and cervical scrape--and two common human papillomavirus (HPV) detection techniques--Southern blot and dot blot (SB and ViraPap [VP])--were compared to determine whether differences in these techniques alter correlations between "oncogenic" HPVs and cervical neoplasia. In 87 women with persistently abnormal Papanicolaou (Pap) smears, concurrent biopsy and scrape specimens contained HPV in 21 (24%) and contained no HPV in 26 (30%); 30 scrape specimens (34.5%) tested positive when the biopsy tested negative and 10 (11.5%) scrape specimens tested negative when the biopsy tested positive (overall concordance, 54%). Concordance for the most prevalent HPVs (16/18) was 59%. In carcinoma in situ, HPV was found in biopsy samples significantly more frequently than in scrape specimens: 17 of 23 (75%) biopsy samples versus 9 of 23 (39%) scrape specimens (P = 0.018). Conversely, in mild or no dysplasia, 0 of 42 biopsy samples tested positive for HPV 16/18 compared with 12 of 42 scrape specimens (29%; P = 0.0001). Of 229 specimens analyzed by SB and VP, 43 (19%) tested positive and 148 (65%) tested negative for HPV by both methods (concordance, 84%). Corroborative results indicated that 29 of 35 (83%) VP-positive SB-negative results were truly positive compared with none of three SB-positive VP-negative results. Both the cervical sampling technique and the method for HPV detection can significantly affect statistical correlations between cervical dysplasia and HPV type.     

IL-10 promoter nt -1082A/G polymorphism and human papillomavirus infection in cytologic abnormalities of the uterine cervix.

The role of A/G polymorphism at nucleotide -1082 in the interleukin-10 (IL-10) promoter was assessed by following the disease course of 253 patients who had had a routine diagnostic Hybrid Capture human papillomavirus (HPV) test because of cytologic or colposcopic abnormalities of the uterine cervix. At baseline, 97 (78%) of the 125 high-risk HPV-positive and 83 (65%) of the 128 HPV-negative patients had equivocal cytologic atypia classified as P3 by the Papanicolaou classification, and the rest of the patients had mild colposcopic atypia with cytologic results of no oncogenic significance. In the high-risk HPV-infected patients, the frequency distribution of the nt -1082 genotypes (A/A: 28%; A/G: 52%; G/G: 20%) did not differ significantly from that in the controls (A/A: 25%; A/G: 51%; G/G: 24%; p = 0.70). On the other hand, the nt -1082 G allele tended to decrease susceptibility to equivocal cytologic atypia unrelated to HPV infection (A/G: OR = 0.56 [95% CI: 0.31-1.02], G/G: OR = 0.27 [95% CI: 0.11-0.63], p for trend = 0.05). With respect to the development of high-grade cervical intraepithelial neoplasia (CIN), the established risk factors, such as high-risk HPV infection (RR = 104.6, 95% CI: 14.2-769.9) and cytologic atypia (RR = 9.6, 95% CI: 2.34-39.7) but not the various nt -1082 genotypes (A/A: reference; A/G: RR = 1.11 [95% CI: 0.59-2.08]; G/G: RR = 0.62 [95% CI: 0.25-1.50]) were found to increase the risk for high-grade CIN. In conclusion, the nt -1082 polymorphism had no influence on the early phase of cervical carcinogenesis but may determine different susceptibilities to cervical abnormalities unrelated to HPV infection.     

Detection of integrated high risk human papillomavirus in adenoid cystic carcinoma of the uterine cervix.

AIM: To investigate the role of human papillomavirus (HPV) in adenoid cystic carcinoma of the uterine cervix. METHODS: Eleven archival, paraffin wax embedded specimens were analysed by non-isotopic in situ hybridisation (NISH) for HPV types 6, 11, 16, 18, 31, and 33 using digoxigenin labelled probes. The polymerase chain reaction (PCR) was carried out on each of the cases using consensus primers to HPV. RESULTS: A total of eight adenoid cystic carcinomas harboured the HPV genome by NISH, of which five were PCR positive. Integrated HPV 16 DNA was demonstrated in seven of the eight NISH positive cases. One adenoid cystic carcinoma showed integrated HPV 31. HPV DNA was not detected in the three remaining cases. CONCLUSIONS: Integrated high risk HPV genome, in particular type 16, is associated with this uncommon type of primary cervical cancer.     

Characterization of human papillomavirus type 66 from an invasive carcinoma of the uterine cervix.

Human papillomavirus (HPV) DNA sequences coexisting with HPV16 and HPV45 were cloned from an invasive cervical carcinoma. The cloned HPV was shown to be a novel type, named HPV66, and is related to HPV56 (an HPV detected in cervical cancer). After screening 160 anogenital biopsies, four specimens exhibited histological features of intraepithelial neoplasia and contained HPV66 sequences. Of these, three were found to be associated with another HPV type.     

Human papillomavirus coinfections of the vulva and uterine cervix

DNA filter in situ hybridisation (FISH) was used to determine the presence of human papillomavirus (HPV) genotypes 6/11, 16/18, and 31/33 in cell scrapes of the cervix and vulva of 128 women who had precancerous lesions and/or HPV infection of the cervix diagnosed by cytology, colposcopy, and histology. HPV-DNA was found in 87 (68%) vulval and 95 (74%) cervical cell scrapes, and in both the vulval and cervical scrapes of 73 (57%) women, but not in either the vulva or the cervix of 19 women (15%). Of the HPV-DNA-positive smears, the prevalence of the HPV types was 61% HPV 16/18, 14% HPV 6/11, 3% HPV 31/33, and 22% HPV 6/11 and 16/18. By contrast, HPV-DNA was not detected in the cervical smears of a control group of 35 women who were assessed to be free of cervical abnormalities by colposcopy and cytology. The epithelial response of the vulva and the cervix to application of 5% acetic acid was assessed by colposcopy and the results correlated with the presence of HPV genotypes. A possible or definite disorder of the cervix and vulva was detected by colposcopy in 95 (74%) and 96 (75%) of the 128 cases, respectively. The colposcopic assessment of the vulva was inconclusive in ten cases (8%), and only eight women (6%) were found to be free of both a vulval and cervical disorder. This study shows subclinical papillomavirus infections of the vulva frequently coexist with HPV infections and precancerous lesions of the cervix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human papillomavirus infection of the uterine cervix: histological appearances in 28 cases identified by immunohistochemical techniques

Twenty eight biopsy specimens of the cervix showed positive immunohistochemical staining when treated with an antiserum raised against an internal capsid antigen of human papillomavirus (HPV). Histological examination of adjoining sections from the same blocks showed a much wider range of abnormalities than those already described in association with HPV infection. The picture was usually diagnostic. It rested chiefly on identifying the koilocyte--the cell with the perinuclear halo that carries the viral antigen in its nucleus--but abnormal keratinisation was also a feature. The accompanying epithelial findings ranged from normal to CIN III (cervical intraepithelial neoplasia). The latter was of an unusual but distinct appearance, in which cytoplasmic maturation was preserved to some degree but in which gross nuclear atypia was seen in all layers of the epithelium.     

Adenocarcinoma of the uterine cervix of probable Wolffian origin

An unusual type of adenocarcinoma involving the endocervix and the lower segment of the uterus resulting in a vaginal vault recurrence 7 years after hysterectomy is discussed. Features of the tumour which suggest a probable Wolffian duct (or mesonephric) origin are outlined.     

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.