Human papillomavirus (HPV) cervical lesions: results from 300 Italian women studied with DNA hybridization techniques and morphology

Human papillomavirus cervical infection was investigated in a series of 300 unselected women by comparing morphological diagnoses (cytology and histology) with results of DNA hybridization techniques (filter in situ hybridization of DNA from exfoliated cervical cells and Southern blot analysis of HPV-DNA in cervical biopsy specimens). The prevalence of HPV cervical infection diagnosed by PAP smears was 11.6%. Despite disadvantages, filter in situ hybridization was confirmed to be particularly useful for screening purposes to detect HPV in cervical scrapings. In 3 cases it was the only applicable method for diagnosing "high-risk" HPV infection. Southern blot hybridization of tissue DNA with HPV 16-DNA revealed the presence of this virus in 8 cases, and HPV 31-DNA and HPV 42-DNA in 1 case each.     

Difference in prevalence of human papillomavirus genotypes in cytomorphologically normal cervical smears is associated with a history of cervical intraepithelial neoplasia.

The prevalence of human papillomavirus (HPV) genotypes was investigated by the polymerase chain reaction (PCR) method in cytologically normal and abnormal cervical scrapes obtained from asymptomatic women (n = 1,346), participating in a triennial screening program for cervical cancer, and from a gynecological outpatient population (n = 593). In the symptom-free population oncogenic HPV types 16, 18, 31 and 33 were present in 1.5% of cytologically normal scrapes, while the overall HPV prevalence rate was 3.5%. Significantly, higher HPV prevalence rates of 7% (oncogenic HPV; p less than 0.01) and 14% (all HPV; p less than 0.01), respectively, were found in cytologically normal scrapes of the gynecologic outpatient population. It appeared that in this outpatient group 78% of the smears containing HPV 16 and 18 were associated with a history of cervical pathology, i.e. cervical intraepithelial neoplasia grade I to III. In smears with mild and severe dysplasia and smears suspected of carcinoma in situ from both populations, the overall HPV prevalence was 70%, 84% and 100%, respectively. In all squamous-cell carcinomas of the cervix (n = 50) HPV was detected. Frequencies of HPV 16 and 18 increased from 41% in mild dysplasia to 94% in cervical carcinomas. Since a low prevalence of HPV was found in cytomorphologically normal cervices of women without a clinicopathological history, the findings in this study suggest that HPV detection in population-based screening programs for cervical neoplasia can be an important tool in identifying women who are at risk of developing dysplasia and cervical cancer.     

Increased prevalence of human papillomaviruses in the lower genital tract of pregnant women

In order to evaluate the influence of pregnancy on the presence of human papillomavirus (HPV) in the lower female genital tract, cervical smears of 92 pregnant and 96 non-pregnant women, matched by age, were examined for the presence of HPV-DNA by means of Southern blot hybridization. All patients had negative PAP smears. Twenty-six (28%) of the pregnant women and 12 (12.5%) of the non-pregnant women were positive for HPV. HPV 16 accounted for 42% of all positive pregnant cases and only 25% of the positive non-pregnant cases. Smears of pregnant patients contained more than 10 pg viral DNA in 45% of the cases against 20% in the non-pregnant group. HPV 16 showed the most active replication in both groups. This study demonstrates an increased prevalence of HPV (preferentially of HPV 16) and a higher replication rate of viral DNA during pregnancy.     

采用原位杂交法检测生殖系统尖锐湿疣和鳞状细胞癌内乳头瘤病毒的感染

目的　探讨HPV感染与生殖系统尖锐湿疣和鳞状细胞癌的关系。方法　采用HPV6 /11、16 /18原位杂交试剂盒 ,对 5 5例生殖系统不同病变中乳头瘤病毒感染状况进行分析检测。结果　 2 0例生殖系统尖锐湿疣 19例阳性 ,其中 17例为HPV6 /11型 ,2例为HPV16 /18型。 2 0例生殖系统鳞状细胞癌中 ,15例阳性 ,均为HPV16 /18型。 10例正常生殖系统鳞状上皮组织和 5例外阴白斑组织均呈阴性。结论　HPV6 /11多与生殖系统良性疣状病变有关 ,而HPV16 /18感染多见于生殖系统恶性病变。     

60例喉乳头状瘤人类乳头状瘤病毒感染分析

[目的]研究人类乳头状瘤病毒感染与喉乳头状瘤发生的关系。[方法]利用免疫组织化学、原位分子杂交及PCR检测60例喉乳头状瘤、15例喉鳞状非典型增生上皮和10例正常喉黏膜上皮中人类乳头状瘤病毒(HPV)16/18E6蛋白的表达。[结果]免疫组化显示在正常喉黏膜、非典型增生上皮及喉乳头状瘤组织中HPV16/18E6蛋白的阳性率分别为10.0%(1/10)、33.3%(5/15)和80.0%(48/60);瘤组织中HPV16/18E6蛋白的阳性率明显高于非典型增生上皮和正常喉黏膜(P<0.05),非典型增生上皮中HPV16/18E6蛋白的阳性率亦明显高于正常喉黏膜(P<0.05)。原位杂交显示在正常喉黏膜、非典型增生上皮及喉乳头状瘤组织中HPV16/18E6DNA的阳性率分别为10.0%(1/10)、40.0%(6/15)和83.3%(50/60);瘤组织中HPV16/18E6DNA的阳性率明显高于非典型增生上皮和正常喉黏膜(P<0.05),非典型增生上皮中HPV16/18E6DNA的阳性率亦明显高于正常喉黏膜(P<0.05)。PCR显示在正常喉黏膜、非典型增生上皮及喉乳头状瘤组织中HPV16的阳性率分别为10.0%(1/10)、46.7%(7/15)和86.7%(52/60);瘤组织中HPV16的阳性率明显高于非典型增生上皮和正常喉黏膜(P<0.05),非典型增生上皮中HPV16的阳性率亦明显高于正常喉黏膜(P<0.05)。[结论]高危型HPV16/18型感染可能在喉乳头状瘤的发生中起重要作用。     

Prevalence of cervical dysplasia and HPV infection according to sexual behavior

A total of 165 uterine cervix smears from Venezuelan women were examined by cytological techniques to identify malignant and pre-malignant cervical changes, as well as to identify Human Papilloma Virus (HPV) DNA types 6, 11, 16, 18, 31, 33 and 35. Of these smears, 119 were from nonmonogamous women who participated in a cervical carcinoma screening program. In this group, HPV-DNA was detected by hybridization in 42 samples (35%) and cervical intraepithelial neoplasia (CIN) in 13 (11%). Forty-six monogamous aboriginal women were similarly studied and no evidence of abnormal cytology or HPV-DNA of the types studied here was found in any of them. In the non-monogamous group, age at first sexual intercourse and index of parity were not associated with cervical HPV infection and/or CIN. The rates of HPV infection, however, were significantly different between the two populations, confirming that sexual behavior involving multiple partners is associated with HPV infection.     

Implication of human papillomavirus in postirradiation dysplasia.

The presence of human papillomavirus (HPV) DNA and association of condylomata acuminata (CA) in the biopsy tissues of postirradiation dysplasia (PRD) of the cervix and/or vagina from 17 patients who previously had radiation therapy for malignancies of the uterine cervix, vagina, and endometrium were evaluated with DNA in situ hybridization. Eight of 17 patients (47.1%) had HPV DNA identified in the lesions of postirradiation dysplasia (PRD). Five of eight cases (62.5%) contained HPV DNA of more than one type. Type 16 HPV DNA (HPV-16) was the most frequently identified type. Several PRD lesions also contained HPV-6, HPV-18, HPV-31, and/or HPV-33 DNA. Eleven patients (64.7%) showed CA in the vicinity of PRD. In two cases, different types of HPV were found in the lesions of PRD and contiguous CA. The frequency of the cases containing HPV DNA, the types of HPV, and the distribution pattern of silver grains in the preparations of in situ hybridization over the nuclei of cells of PRD were very similar to those found in naturally occurring dysplasia. Based on these findings, persistent or repeat HPV infection was the most likely etiologic factor of PRD, which might be facilitated by immunosuppression due to pelvic irradiation.     

Human papillomavirus type 31 DNA detected in part of the dysplasia but in no part of the squamous metaplasia in a specimen taken from one patient.

Using in situ hybridization, human papillomavirus (HPV 6, 16, 18, 31, 33) DNAs were detected in a cervical severe dysplasia accompanied by squamous metaplasia. It was found that, only HPV 31 DNA was harbored in the cervical severe dysplasia, but HPV DNAs were not identified in a lesion of squamous metaplasia. The in situ hybridization method will be of use, therefore, when dysplasia with squamous metaplasia or other lesions are examined for HPV DNA. In a cervical smear, HPV 31 DNA could be detected on the nuclei of dysplastic cells, so this method is applicable to cervical smears. If squamous metaplasia is to be considered as a precursor lesion to cervical dysplasia, the HPV DNA harbored in the dysplasia must also be detected in the accompanying squamous metaplasia. Our results suggested that not all squamous metaplasias were involved with HPV, as far as we were able to detect using five types of HPV DNA probe.     

In situ hybridization for the detection of human papillomavirus (HPV) in gynaecological biopsies. A study of two commercial kits.

Human papillomavirus (HPV) detection in biopsies from the lower genital tract may be requested by clinicians as a complement to ordinary histopathological diagnosis. In the present study, two commercial kits, (Enzo Diagnostics Inc., New York, USA and Biohit, Helsinki, Finland) used for in situ hybridization with biotinylated c-DNA probes were compared and the HPV-expression was evaluated in relation to histopathological findings. The Enzo kit identifies HPV-types 6/11, 16/18, 18 and 31/33/51, whereas the Biohit kit has separate probes for HPV 6, 11, 16, 18, 31 and 33, but none for HPV 51. The usefulness of a general probe (probemix) for the visualization of HPV irrespective of type (Enzo Diagnostics Inc.) was also studied Altogether 226 biopsies from the lower female genital tract were formalin-fixed, paraffin-embedded and processed for routine histopathological grading. Consecutive sections were employed for in situ hybridization. 50 biopsies were subject to double-testing with Enzo and Biohit, whereas 176 were tested with Enzo only. Of the double-tested biopsies, 30% displayed a nuclear staining with the Enzo kit and 28% with the Biohit kit. It is concluded that the probes of these two kits have the same sensitivity in detecting HPV in tissue sections. Condylomata acuminata were HPV-positive in 81%, mostly for types 6/11. Flat condylomas were HPV-positive in 35%. The HPV-positivity of biopsies with low grade SIL (I) was 50% and that of high grade SIL (II and III) was 37%. High grade SIL contained either HPV-types 16/18 or 31/33/51. A correlation was found between the occurrence of koilocytosis and the presence of HPV-DNA. HPV-expression was most easily visualized in condylomata acuminata. In epithelium of normal appearance or with inflammatory alterations HPV-DNA was not seen.     

Relation between human papillomavirus (HPV) and cancer of uterine cervix

HPV-DNA fragments were detected in biopsy specimens (29 cases of cancer of uterine cervix, 2 cervical dysplasia and 9 normal cervix) using DNA hybridization technique. It was demonstrated that 52% of biopsy specimens of the cancer of uterine cervix was positive for HPV 16 DNA probe, while 9% was positive for HPV 18 DNA probe. 11% of non-cancerous biopsy specimens had a positive result for HPV 16 DNA probe. It was also demonstrated that the positive rate of HPV 16 DNA was 75% in grossly cauliflower and nodular type tumors but only 25% in erosion type. It seems that the positive rate of HPV 16 DNA is correlated to gross appearance of the tumor. The positive rates of HPV 16 DNA were different in 6 provinces in China. It was 64% in Shanxi Province, a high incidence area but 36% in Sichuan Province, a low incidence area. These results suggest that the carcinogenesis of cancer of the uterine cervix, be related to HPV infection.     

Oral contraceptive pills are associated with artifacts in ThinPrep Pap smears that mimic low-grade squamous intraepithelial lesions

BACKGROUND: The authors noted a significant increase in the diagnosis of atypical squamous cells of undetermined significance (ASCUS) in premenopausal women using oral contraceptive pills (OCP) (9%) versus women not on OCP (4%) using ThinPrep Pap smears (P = 0.02). The purpose of this study was to correlate these morphologic changes with human papillomavirus (HPV) DNA detection by in situ hybridization and clinical follow-up. METHODS: Eighty-four ThinPrep Pap smears diagnosed as ASCUS or ASCUS favor low-grade squamous intraepithelial lesions (LGSIL) in young women receiving OCP were studied by HPV in situ hybridization. This information was correlated with colposcopic follow-up and AutoCyte Pap smear results. The authors also studied 80 ThinPreps from young women with a Pap smear diagnosis of unequivocal LGSIL where there was a corresponding biopsy (positive controls) and 40 ThinPreps diagnosed as within normal limits and that rescreened as such (negative controls). RESULTS: The detection rate of HPV DNA by in situ hybridization was 33% (28 of 84) in ASCUS cases versus 90% (72 of 80) for cases of unequivocal LGSIL. A blinded review of the 29 ASCUS Pap smears performed by AutoCyte showed that 66% (19 of 29) were diagnosed as within normal limits. The majority of the cervical biopsies in the women with ASCUS by ThinPrep Pap smears were negative for dysplasia (29 of 39 [74%]) whereas 71 of 80 biopsies (88%) showed dysplasia in women with Pap smears diagnosed as unequivocal LGSIL. The periodic acid-Schiff (PAS) stain demonstrated that many of the cells with halos in the ThinPrep Pap smears that mimicked LGSIL contained glycogen. CONCLUSION: The ThinPrep induces changes that mimic LGSIL in young women on OCP. These artifacts may reflect pressure-induced alteration of glycogen. PAS analysis and, more directly, HPV testing by in situ hybridization can help differentiate this mimicking from actual LGSILs.     

肺鳞癌与人乳头瘤病毒相关性研究

目的:了解肺鳞癌组织中人乳头瘤病毒存在情况,并分析人乳头瘤病毒与肺癌发生部位、性别因素、角化程度、吸烟因素的关系.方法:采用地高辛标记的DNA探针对肺癌组织进行原位杂交检测,同时结合临床资料分析多种因素的相关性.结果:肺鳞癌组织,鳞状上皮生化组织、粘膜慢性炎组织HPV检出率分别为48.4%,27.4%,5.6%,各组比较差异显著(P＜0.01),中心型肺癌与周围型肺癌组织HPV检出率分别为50.6%和4.4%,两组比较差异显著(P＜0.01).男女两组HPV检出率分别为46.1%和54.3%,差异无显著性(P＞0.05).吸烟组HPV检出率为50.6%,非吸烟组为43.9%,差异不显著(P＞0.05).结论:肺癌组织中存在一定比例HPV感染,HPV感染与肺鳞癌关系密切,中心型肺癌与HPV感染密切相关,HPV感染与性别及吸烟因素未发现明显相关性.人乳头瘤病毒感染可能与肺癌发生发展有关.     

Human papillomavirus 6/11 and 16/18 in Schneiderian inverted papillomas. In situ hybridization with human papillomavirus RNA probes

Schneiderian inverted papillomas may be troublesome lesions for clinicians with propensity for recurrences. Dysplasia is not uncommonly seen, and some of these lesions do progress to develop squamous carcinoma. The authors hybridized in situ seven inverted papillomas with RNA probes to human papillomavirus (HPV) 6, 11, 16, and 18. Four of these contained dysplasia, two were without dysplasia, and one contained invasive squamous cell carcinoma. Five inverted papillomas showed evidence of HPV infection based on hybridization. One with mild to moderate and one with severe dysplasia, and one without dysplasia hybridized with mixed probe HPV 6/11. One with mild dysplasia and one associated with invasive squamous cell carcinoma hybridized with mixed probe HPV 16/18. One inverted papilloma without dysplasia and one with severe dysplasia did not definitively hybridize with either mixed probe. These findings raise interesting questions as to role of HPV 6/11 and 16/18 in the development of inverted papillomas, and probably on the progression to and carcinoma.     

Case-control study of human papillomaviruses and cervical cancer in Latin America

Human papillomavirus (HPV) types 16 and 18 have been implicated as risk factors for cervical dysplasia and neoplasia. However, most studies have been observational, uncontrolled and conducted in populations at low risk for invasive cancer. We report a pilot case-control study of incident invasive cervical cancer in Panama, Costa Rica and Bogota, Colombia. Between July and September 1985 we enrolled 46 consecutive newly diagnosed invasive cervical cancer cases and 51 age-matched control women. Subjects were interviewed and samples collected for HPV DNA assays. HPV infection was defined by a filter in situ DNA hybridization technique under non-stringent and stringent conditions against HPV-6/11, 16 and 18 DNA probes. More cases (91%) than controls (63%) had HPV DNA detected (non-stringent) and more cases than controls had HPV-16 or 18 DNA (67% vs. 43%, p = 0.02). Age at first intercourse was the most significant risk factor for HPV 16/18 infection in all subjects. Smoking was significantly associated with cervical cancer (52% of cases vs. 27% controls) but was not associated with HPV infection.     

Human Papillomavirus Genotyping among Different Cervical Smears in Duhok/Iraq

Background/Objective: Cervical cancer ranks the second among the most common gynecologic cancers. This studywas established to determine the distribution of cervical HPV genotypes among different Pap readings in Duhok/Iraq.Methods: Between January and September-2016, HPV-DNA was tested in 64 women. Genotyping was carried out bythe hybridization reverse blot technique. Cervical smears were taken, performed by ThinPrep technique and stained byPap stain. Results: Twenty six (40.6%) cases were positive for HPV, 12 (46.2%) in normal and 14 (53.8%) in abnormalPap smears. 39 (19 high-risk and 16 low-risk) genotypes were identified. The high risk group comprised 6 HPV16, 4HPV18, 2 HPV66, 2 HPV52, 2 HPV39, 1 HPV56, 1 HPV31 and 1 HPV45. The 16 low risk strains encompassed 4HPV6 strains, 4 HPV71, 2 HPV54 and 2 HPV83, HPV11, HPV61 HPV84, and HPV62. Mixed infections were describedin 4 women (6.25%), limited to the NILM, ASC-US and LSIL smears. They included variable admixtures of 7 highrisk genotypes, HPV39 (both copies), HPV66 (both copies), HPV52, HPV31, HPV45 and 6 low risk strains: HPV83,HPV6, HPV11, HPV54, HPV62 and HPV71. Conclusions: The higher frequency of HR-HPV than the LR-HPV withidentification of 4 mixed cases indicates that our women are at risk of developing cervical cancer. Detection of HR-HPVin NILM and ASC-US smears with restriction of some strains to these 2 categories highlights the great value of HPVgenotyping as a surrogate test to pick up unscreened women at risk of developing cervical malignancy particularlywhen a proper screening program is absent.     

Prevalence of human papillomavirus types 16 and 18 in cervical carcinomas: a study by dot and Southern blot hybridization and the polymerase chain reaction

Histologically classified biopsies from 83 women with invasive cervical carcinoma were analyzed by dot blot hybridization for human papillomavirus (HPV) types 16 and 18 infection. Sixty of the 83 (72.3%) were found to contain HPV DNA, of which 43 (51.8%) contained HPV 16 DNA, 12 (14.5%) contained HPV 18 DNA and 5 (6.0%) contained both HPV 16 and 18 DNAs. Southern blot analysis on 65 specimens gave similar results. Of 23 specimens negative by dot blot, 21 were tested by the polymerase chain reaction. Seventeen of the 21 were positive for HPV DNA, of which 13 contained HPV 16 DNA and 4 contained both HPV 16 and 18 DNAs. In all, 95.1% (77/81) were positive for HPV 16 and/or 18 DNA sequences.     

Prevalence of Human Papillomavirus Types 16 and 18 in Cervical Carcinomas: A Study by Dot and Southern Blot Hybridization and the Polymerase Chain Reaction

Histologically classified biopsies from 83 women with invasive cervical carcinoma were analyzed by dot blot hybridization for human papillomavirus (HPV) types 16 and 18 infection. Sixty of the 83 (72.3%) were found to contain HPV DNA, of which 43 (51.8%) contained HPV 16 DNA, 12 (14.5%) contained HPV 18 DNA and 5 (6.0%) contained both HPV 16 and 18 DNAs. Southern blot analysis on 65 specimens gave similar results. Of 23 specimens negative by dot blot, 21 were tested by the polymerase chain reaction. Seventeen of the 21 were positive for HPV DNA, of which 13 contained HPV 16 DNA and 4 contained both HPV 16 and 18 DNAs. In all, 95.1% (77/81) were positive for HPV 16 and/or 18 DNA sequences.     

HPV、HSV和CMV感染与宫颈癌的关系

背景与目的： 探讨宫颈癌前病变和宫颈癌的发生发展与人乳头状瘤病毒及单纯疱疹病毒(Herpes Simplex Virus,HSV)、巨细胞病毒(Cytomegalovirus,CMV)的关系。 材料与方法： 对81例不同宫颈病变组织进行HPV16/18和HPV6/11原位杂交,同时对103例不同宫颈病变组织用DNA扩增法检测HPV、HSV和CMV。 结果: 病毒DNA原位杂交信号的分布与HE染色中挖空细胞的分布一致。 HPV16/18与不同宫颈病变组织原位杂交阳性率平均为51.1 ％,HPV6/11的则为64.7 ％。经PCR检测,HPV16/18 、HPV6/11、HSV、CMV在不同宫颈病变组织中的阳性率分别为21 ％、4 ％、23 ％、0 ％。 结论： HPV感染具有特定的组织学部位,HSV可协同HPV16/18恶性转化宫颈上皮细胞。     

Human papillomavirus (HPV) DNA in esophageal precancer lesions and squamous cell carcinomas from China

A series of 51 biopsies derived from the same number of patients with established invasive squamous-cell carcinoma of the esophagus in Linxian, a high-risk area for esophageal cancer in China, were analyzed histologically and by in situ DNA hybridization to demonstrate human papillomavirus (HPV) infection. Epithelial changes suggesting HPV infection within or adjacent to the carcinoma lesions were found in 25 cases (49.0%). Esophageal lesions with HPV morphology showed both flat (25 cases) and inverted condylomas (2 cases) resembling those found in the genital tract. HPV 6, 11, 16 or 18 DNA sequences were detected in 22/51 (43.1%) of the esophageal specimens. HPV DNA was most frequently localized in epithelium adjacent to carcinomas in areas showing either epithelial hyperplasia (36.1%) or dysplasia (22.2%). Of the lesions with morphological HPV changes, 64% (16/22) were shown to contain HPV DNA. In 2 specimens, HPV DNA was found in frankly malignant cells. High-risk types HPV 16 and/or 18 DNA sequences were found in 16 of the 22 HPV DNA-positive cases (72.7%). Our results confirm previously reported HPV involvement in esophageal squamous-cell lesions, and support the hypothesis of HPV as a possible etiological agent in esophageal carcinogenesis.     

Analytical methods for evaluation on whole cells of human papillomavirus infection

Analytical methods for evaluation on whole cells of human papillomavirus infection. Human papillomavirus (HPV) infection is currently identified by the presence of viral DNA using molecular biology. As in situ hybridization is valuable for HPV-DNA detection mainly with non-isotopic probes, we evaluated the sensitivity of various techniques, using as models three cell lines containing different copy numbers of HPV DNA/cell (CaSki with 600 copies of HPV 16, SiHa with 1-2 copies of HPV 16, HeLa with 10-50 copies of HPV 18). Epifluorescence microscopy and flow cytometry allowed detection of 600 copies in CaSki cells; in addition, cell fixation was found to influence the fluorescent intensity. Several procedures were assayed to increase the sensitivity of in situ hybridization. The use of biotinylated HPV-16 oligonucleotides as probes was not effective, because only CaSki cells were positive. After amplification of HPV-16 or -18 DNA sequences with polymerase chain reaction (PCR) on whole cells in suspension and hybridization with plasmid probes, fluorescent hybridization spots were found in CaSki and HeLa cells by both epifluorescence microscopy and flow cytometry. The various procedures applied for revelation of DNA-DNA hybrids (use of phycoerythrin or cyanine instead of fluorescein, Pinkel's 3-step amplified system of fluorescein) did not enhance the sensitivity of in situ hybridization. HPV DNA was very effectively detected by cell examination under a laser-scanning confocal microscope, since 1-2 copies of HPV 16 were observed in SiHa cells without previous PCR amplification. Thus, the efficacy of in situ hybridization for HPV detection may be conditioned by different factors. Laser-scanning microscopy represents an alternative to the use of PCR amplification. These techniques are potentially useful to study single genes.