Human papillomavirus DNA detection in Papanicolaou-stained cervical smears with a nonradioactive, in situ hybridization assay.

Archived Papanicolaou-stained cervical smears from women with different cervical pathologies were processed for human papillomavirus (HPV) DNA detection and typing with an in situ hybridization (ISH) assay that employed commercial biotinylated HPV DNA probes. Two HPV DNA probes were utilized: one included HPV genotypes 6/11 and the other, 16/18. The method yielded positive results for HPV DNA 6/11 in 5 cases with condylomata acuminata (100%) and in 2 of 47 with flat warty lesions (4.2%), whereas HPV DNA 16/18 was detected in 29/47 of the latter group (61.7%). In cases with cervical intraepithelial III or invasive squamous cell carcinoma the yield was lower: positive results for HPV DNA 16/18 were obtained in only one of the five cases with one or the other cervical pathology (20%). An analysis of the results showed that the sensitivity of the assay correlated with evidence in the Papanicolaou specimens of pathognomonic cell injury from HPV infection. In the presence of such cytologic features, HPV DNA typing was possible in 37/52 cases (65.4%). In view of the modest difficulty and relatively quick execution of the nonradioactive ISH assay, the authors believe that Papanicolaou cervical smears with cytologic changes of HPV infection could be processed by this method in order to acquire information on the HPV type or types involved in the cervical infection.     

应用PCR对尖锐湿疣和外耳道乳头状瘤中HPV DNA的分型检测

从手术切除的24例女性和12例男性尖锐湿疣新鲜标本中,以及42例女性尖锐湿疣、16例男性外耳道乳头状瘤和4例女性假性湿疣的石蜡包埋标本中,提取组织的基因组DNA,用人工合成的人乳头瘤病毒6.11和16型E6区特异性寡聚核苷酸引物,通过PCR进行HPV DNA的分型检测。结果66例女性尖锐湿疣中,感染HPV6型者4例,感染11型者12例,6+11型混合感染者49例;阴性1例,总检出率达98.4％。4例女性假性湿疣中1例为HPV6型感染,阳性率25％。16例男性外耳道乳头状瘤中HPV6+11型感染者5例,6+16型感染者3例,6+11+16型多重感染者8例,阳性率100％。12例男性尖锐湿疣中,HPV11型感染者7例,6+11型4例,阴性1例,总阳性率91.6％。还对细胞学上空泡化和非典型空泡化尖锐湿疣标本的HPV感染做了比较,未发现差异。     

Presence of human papillomavirus genome in human tumor cell lines and cultured cells.

A series of 47 human carcinoma cell lines and their cultured cells were examined for human papillomavirus (HPV) genomes with the use of an HPV detection kit (DNA-RNA hybridization, mixed HPV DNA probe of types 6, 11, 16, 18, 31, 33 and 35). Four of 8 cases of mild dysplasia, 3 of 9 cases of severe dysplasia, 3 of 7 cases of carcinoma in situ, 3 of 15 cases of uterine carcinoma and 5 of 6 cases of condyloma acuminatum were shown to contain the HPV DNA genome in primary cultured cells, while HPV was not detected in the third-passage cells except for the three cases of large cell, nonkeratinizing squamous cell carcinoma. HPV was also not detected in such normal tissues as uterine cervical squamous epithelium, uterine cervical columnar epithelium and endometrium. The presence of HPV DNA genomes was detected consistently in the passages of three lines (SKG-II, HKMUS and HKTUS; large cell nonkeratinizing squamous cell carcinomas of the uterine cervix) with the use of the Southern Blot method (DNA-DNA hybridization, mixed HPV probe of types 6, 11, 16 and 18). HPV type 16 DNA was detected in HKTUS, and HPV type 18 DNA was found in SKG-II and HKMUS. The other 44 cell lines, including ovarian carcinoma, endometrial carcinoma, sarcoma, gastric cancer, pancreatic cancer and rectal cancer, were negative for the HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35 genomes under stringent hybridization conditions.     

应用树状DNA杂交(DDH)对生殖道尖锐湿疣中HPV DNA的分型检测

从手术切除的50例生殖道尖锐湿疣新鲜标本中,以及15例正常人血清中,提取基因组DNA,同时用树状DNA杂交(dendrimer DNA hybridizalion,DDH)技术和PCR进行HPV DNA的分型检测.结果50例尖锐湿疣中,以DDH方法检测,感染HPV6型者20例,感染11型者24例,6/11型混合感染者3例,阴性3例,总检测率达94%;以PCR方法检测,HPV6型感染者21例,11型感染者24例,6/11型混合感染者3例,阴性2例,总检测率为96%.15例正常人血清中,以DDH方法检测,HPV感染的假阳性率为0%;以PCR检测,假阳性率为6.67%.还以HPV阳性标本对DDH方法做了敏感度的测定,结果阳性病例DNA检测最低浓度为97.28pg/ml.研究表明,DDH技术具有较高敏感性和高特异性,且成本较低,操作安全简便,可适用于基层中小医院较大样本量筛查.     

Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P = .017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P = .037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.     

Human papillomavirus prevalence in lung carcinomas in Bulgaria

A possible association between high‐risk human papillomaviruses (HPV) and lung cancer has been investigated for decades with discrepant results. The aim of this study was to determine the prevalence of HPV16 and 18 in Bulgarian patients with lung cancer. Two hundred and nine biopsy specimens from patients with histologically proven lung cancer and without cancer were analyzed. Each sample was subjected to three parallel PCRs using broad spectrum GP5+/6+ primers and type‐specific (TS) primers for HPV types 16 and 18. Of the 132 lung carcinoma samples, 33 (25%) were positive for HPV16 and/or HPV18 by TS PCR whereas only five (3.8%) samples were HPV positive by consensus PCR. All non‐malignant controls were HPV negative. HPV18 was the more prevalent, being found in 11.4% of samples, followed by HPV16 in 9.1% samples; 4.5% of lesions were positive for both HPV16 and HPV18. HPV16/18 were most prevalent in small cell carcinoma (29.2%) and least prevalent in squamous cell carcinoma (23.3%). HPV was only detected in squamous cell carcinoma and adenosquamous carcinoma by consensus PCR. This study revealed a high HPV16/18 prevalence in lung carcinoma samples from Bulgarian patients when TS PCR was used to detect them. The difference between HPV positivity as detected by consensus and by TS PCR was significant, indicating the importance of methodological issues in explaining the discrepancies between previous studies. HPV18 was more common than HPV16. No association between HPV16/18 status and histopathological diagnosis was identified.

     

肛门生殖器区肿瘤组织中HPV的快速检测和分型

迄今已发现的人乳头瘤病毒（ＨＰＶ）型已超过７７型,其中至少有３０个型与泌尿生殖道肿瘤有关。所以ＨＰＶ的检测与分型对于泌尿生殖道肿瘤的病因和预后将是非常重要的。ＨＰＶ有很大的异质性,故用常规的诊断技术来测定未知标本中的ＨＰＶ基因型有一定困难。为此,我们应用了反向点杂交法（ＲＯＢ）来检测ＨＰＶ分型。即用７种序列特异性寡核苷酸探针分别对应７型ＨＰＶ（ＨＰＶ６Ｂ、１１、１６、１８、３１、３３、３５）,这些     

Human papillomavirus prevalence among women with cervical intraepithelial neoplasia III and invasive cervical cancer from Goiânia,Brazil

This study estimated the prevalence and distribution of human papillomavirus (HPV) types among women with cervical intraepithelial neoplasia (CIN) grade III and invasive cervical cancer from Goi s (Brazil Central Region). Seventy-four cases were analyzed and consisted of 18 CIN III, 48 squamous cell carcinomas, 4 adenocarcinomas, 1 adenosquamous carcinoma and 3 undifferentiated carcinomas. HPV-DNA sequences were examined in formalin-fixed and paraffin-embedded tissues using primers from L1 region GP5+/GP6+. Polymerase chain reaction products were typed with dot blot hybridization using probes for HPV 16, 18, 31, 33, 45, 54, 6/11, 42/43/44, 51/52, 56/58. The prevalence of HPV was estimated to be 76% (56/74). HPV 16 was the most frequently found type, followed by HPV 33, 18 and 31. The prevalence of untyped HPV was 6%; 79% percent of the squamous cell carcinoma cases and 61% percent of the CIN III were positive for HPV and the prevalence rate of HPV types was the same for the total number of cases. According to other studies, HPV type 16 is the most prevalent virus in all Brazilian regions, but there is variation regarding to other types. Type 18 is the second most prevalent HPV in North, Southeast and South Brazil regions and types 31 and 33 are the second most prevalent HPV in Northeast and Central Brazil, respectively.     

Causal association between human papilloma virus infection and head and neck and esophageal squamous cell carcinoma

HPVs commonly cause proliferative lesions of squamous epithelium, and infection with certain HPV types carries a high risk of malignant transformation. We used molecular techniques to detect and type HPV in papillomas and carcinomas in the oral cavity and esophagus. DNA was extracted from 150 fresh or paraffin embedded biopsy specimens, and analyzed for HPV by PCR with 15 sets of consensus primers directed to conserved regions of L1 gene, three sets of HPV16E6 primers (specific for the HPV 16 prototype and L83V variant), and sets of primers specific for the E6 gene of other mucosa type HPVs including HPV 6, 11, 16, 18, 52, 58, 66 and 73. Overall, HPV sequences were detected in 61 of 150 specimens. HPV DNA sequences were detected in 16/32 specimens in the oropharyngeal region, in 13/36 specimens in larynx and 32/82 specimens in esophagus. Papillomas contained only the episomal form of HPV 16. In the esophagus, the most common type was HPV 73. In all specimens examined, HPV 6/11 (4/150), HPV 16 (23/150), HPV 35 (1/150), HPV 45 (1/150), HPV 54 (1/150), HPV 58 (1/150), HPV 61 (1/150), HPV 66 (1/150), HPV 68 (2/150), HPV 70 (3/150), HPV 72 (1/150), HPV 73 (16/150), double HPV infection (2/150), and unidentified HPV type (4/150) was detected. Interestingly, HPV was found in all verrucous carcinomas and in 18/22 basaloid squamous cell carcinomas. HPV16E6 T350G mutant were observed only in two of eight carcinomas. Using correspondence analysis, a segregation of specific virus types in specific clinico-pathologic lesions (verrucous carcinoma and basaloid squamous cell carcinoma) was proved. It was shown that the relative rates of the HPV positive tumors were significantly higher in women than in men. The synergic action of mucosal irritation and HPV infection may be necessary for the development of the papillomas and the specific types of carcinomas in the oral cavity and in the esophagus.     

DNA sequences of human papillomavirus types 11, 16, and 18 in lesions of the uterine cervix in the west of Scotland.

Punch biopsy specimens of the cervix were examined both histologically and for the presence of human papillomavirus (HPV) DNA sequences. The presence of HPV DNA sequences was sought with the Southern blot technique using radioactively labelled HPV-6, 11, 16, and 18 DNA probes, both together and separately. Twenty six biopsy specimens were examined. Histological examination showed cervical intraepithelial neoplasia grade 2 or 3 in 16 specimens, viral changes (koilocytosis) in four, and inflammation or a normal appearance in three. Eleven specimens were negative for HPV DNA sequences, 10 contained HPV-16 DNA, four contained HPV-18 DNA, and one contained both HPV-18 and HPV-11 DNA. Episomal HPV-16 DNA was detected in one case of cervical intraepithelial neoplasia grade 3 and in five cases of cervical intraepithelial neoplasia grade 2/3 with koilocytosis; and episomal HPV-18 DNA was found in two specimens classed as cervical intraepithelial neoplasia grade 2/3, one of which also contained HPV-11 DNA, and in one specimen that showed viral changes alone. Integrated HPV DNA was found in six specimens (four with HPV-16 DNA and two with HPV-18 DNA), including two cases of chronically inflamed cervix with no histological evidence of viral infection or cervical intraepithelial neoplasia. Detection of viral DNA in early lesions may identify patients at risk of malignant progression. This is the first report of HPV-18 DNA in cervical intraepithelial neoplasia in Scotland.     

Human papillomaviruses and DNA ploidy in anal condylomata acuminata

Previous studies have emphasized the usefulness of DNA ploidy measurement and Human Papillomavirus (HPV) detection as prognostic markers in low grade cervical lesions. We addressed the eventual relationship between HPV type, DNA profile, and p53 tumor suppressor protein expression in anal condylomata acuminata to eventually determine parameters which may be considered as predictive risk factors for the development of cancer. DNA ploidy was assessed by image cytometry after Feulgen staining of contiguous serial sections of 45 anal condylomata acuminata without atypia containing HPV detected by in situ hybridization and Polymerase Chain Reaction (PCR). p53 expression was detected by immunohistochemistry. DNA aneuploidy was found in 53.3% of these lesions, 48.9% containing non oncogenic HPV types 6 and/or 11 and 4.4% harbouring HPV types 11 and 18. The DNA diploid lesions were all associated with non oncogenic HPV types 6 and/or 11 and one case also contained HPV type 33. There was no significant correlation between the detection of DNA aneuploidy and the presence of immuno-detected p53. DNA aneuploidy was not related to the presence of oncogenic HPV in anal condylomata acuminata. The DNA aneuploid profile frequently observed, especially in lesions associated with non oncogenic HPV types, is not yet well explained and cannot be considered as a prognostic factor. In contrast, a more intensive clinical follow-up should be proposed in patients with oncogenic HPV associated to DNA aneuploidy.     

Detection of human papillomavirus in cervical smears. A comparison of in situ hybridization, immunocytochemistry and cytopathology

A comparison of different methods for the detection of cervical human papillomavirus (HPV) infection was made on patients attending the cervical dysplasia clinic. Cytomorphology, immunocytochemistry and in situ hybridization were compared for their ability to detect HPV. Separate cervicovaginal smears from 50 patients were tested for HPV types 6/11, 16 and 18 by in situ hybridization using 35S-labeled DNA probes. Duplicate smears from the same patients were Papanicolaou stained and evaluated for evidence of condylomatous and dysplastic changes. Twenty-five matching cervical biopsies were immunostained for HPV capsid antigen and tested by in situ hybridization for HPV DNA. The cytologic smears of 20 patients (40%) were positive for HPV DNA. Six patients had HPV 6/11, ten had HPV 16, three had HPV 18, and one had both HPV 6/11 and HPV 16. There was a high correlation between condylomatous cytopathology and antigen and DNA detection. One-third of the specimens with condylomatous changes were DNA negative by the tested probes, suggesting the presence of other HPV types in the genital tract.     

Comparative study of in situ hybridization and immunohistochemical techniques for the detection of human papillomavirus in lesions of the uterine cervix.

Among the techniques currently used for the detection of human papillomavirus (HPV) in genital lesions, only two correlate HPV with the histopathological findings of the lesion: immunohistochemistry and in situ hybridization. Consequently, we were prompted to carry out a comparative study on both techniques to check their utility and efficacy as routine diagnostic methods. 52 biopsy specimens of uterine cervix diagnosed histopathologically as condylomas and cervical intraepithelial neoplasia+koilocytosis were studied by immunohistochemical and in situ hybridization techniques using a polyclonal antibody against the common antigen of the HPV capsid and three biotinylated DNA probes specific to HPV types 6/11, 16/18 and 31/35/51. Immunohistochemistry detected 21 positive cases (40.38%), whereas in situ hybridization detected 40 positive cases (76.92%); of the latter, 30 were positive for HPV types 6/11, 3 for HPV types 16/18 and 11 for HPV types 31/35/51. The results suggest that in situ hybridization is a more sensitive technique than immunohistochemistry. However, we recommend the use of both techniques in the case of potentially malignant lesions since better prognostic information can be obtained from joint analysis of both results.     

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

This paper aimed at studying the transplacental transmission of HPV and looking at the epidemiological factors involved in maternal viral infection. The following sampling methods were used: (1) in the pregnant woman, (a) genital; (b) peripheral blood; (2) in the newborn, (a) oral cavity, axillary and inguinal regions; (b) nasopharyngeal aspirate, and (c) cord blood; (3) in the placenta. The HPV DNA was identified using two methods: multiplex PCR of human β-globin and of HPV using the PGMY09 and PGMY11 primers; and nested-PCR, which combines degenerated primers of the E6/E7 regions of the HPV virus, that allowed the identification of genotypes 6/11, 16, 18, 31, 33, 42, 52 and 58. Transplacental transmission was considered when type-specific HPV concordance was found between the mother, the placenta and the newborn or the mother and cord blood. The study included 49 HPV DNA-positive pregnant women at delivery. Twelve placentas (24.5%, n = 12/49) had a positive result for HPV DNA. Eleven newborn were HPV DNA positive in samples from the nasopharyngeal or buccal and body or cord blood. In 5 cases (10.2%, n = 5/49) there was HPV type-specific agreement between genital/placenta/newborn samples. In one case (2%, n = 1/49) there was type specific HPV concordance between genital/cord blood and also suggested transplacental transmission. A positive and significant correlation was observed between transplacental transmission of HPV infection and the maternal variables of immunodepression history (HIV, p = 0.011). In conclusion the study suggests placental infection in 23.3% of the cases studied and transplacental transmission in 12.2%. It is suggested that in future HPV DNA be researched in the normal endometrium of women of reproductive age. The possible consequence of fetal exposure to HPV should be observed.     

尖锐湿疣样本中HPV病毒的分子检测

调查男性和女性尖锐湿疣样本中人乳头瘤病毒（HPV）的检出率及病毒类型,为研发相关防治疫苗提供依据,以HPV 外壳蛋白DNA序列为模板设计特异引物,SSP－PCR扩增检测样本中HPV的感染率和病毒类型。收集了北京及邯郸市医院门诊尖锐湿疣样本22例,其中男性13例,女性9例。检测发现所有样本中存在着高浓度的HPV病毒DNA。男性样本中有5例感染HPV6型,6例感染HPV11型,2例为HPV6＋HPV11混合感染。女性样本中有3例感染HPV6型,2例感染HPV11型,4例为 HPV6＋HPV11混合感染。被诊断为宫颈湿疣的4位女性还在其含宫颈粘膜脱落细胞的样本中检出了HPV16、HPV18、HPV33、HPV35、HPV45、HPV54、HPV56或HPV58等高危险型病毒类型。所有检测到的HPV病毒DNA片段均TA克隆并将测定的DNA序列存入了国际基因数据库GenBank（DQ003066－DQ003079）。调查没有在单纯的男女尖锐湿疣组织块中检测到除HPV6和HPV11以外的其他HPV类型。该研究建立了灵敏可靠的HPV分子检测及分型方法,尖锐湿疣中HPV的检出率达100％。 本研究初步结果显示导致男女尖锐湿疣的HPV病毒类型没有显著差异,主要为HPV6及HPV11型。     

Detection of human papillomavirus DNA in skin warts from immunocompromised patients but not in semen from men whose wives have abnormal cervical cytology.

The epidemiology of human papillomaviruses (HPV) was studied in 61 immunocompromised patients (e.g. renal and cardiac transplants; Bowen's disease; genital cancer) undergoing therapy at the University Hospital of Wales at Cardiff U.K. Warts from various sites of these patients were studied for the presence of HPV types 6, 11, 16 and 18 using the dot-blot DNA hybridization technique. Four HPV-16 and one HPV-11 was detected. The presence of HPV-16 in our study is quite significant since it suggests the potential occurrence of genital HPV types in skin warts in immunocompromised patients and hence the need for screening such patients against HPV types. HPV, mainly types 16 and 18 are usually associated with genital cancer, cervical malignancies and cervical intraepithelial neoplasia. The semen of the husband of 30 women with cervical abnormalities and the semen of 30 husbands (control) of wives with normal cervix were tested for HPV-6, 11, 16 and 18. No HPV-DNA could be detected in all of the 60 specimen. This suggests that specimens were either truly negative for any of those types or because virus DNA could present in a small amount less than 5 pg/microliters in some patients. Whether semen plays a role in transmitting HPV is still controversial.     

Uterine cervical carcinoma and human papillomaviruses]

For many years it has been thought that a significant proportion of cervical cancer could be attributed to sexually transmitted agents, such as sperm, smegma, Treponema pallidum, Gonococcus and herpes simplexvirus type 2. Recent advances of molecular biology, however, have revealed that human papillomavirus (HPV) might be the most causative virus of the disease. Since HPV type 16 DNA was found in a patient with cervical cancer in 1983, many HPV types have been cloned from cervical cancers, also from premalignant lesions (intraepithelial neoplasias). In Japan, we have found 6 new types of HPV (HPV 58, 59, 61, 62, 64, 67) in the female genital tract so far. Especially, HPV 58, which was cloned from a patient with cervical squamous cell carcinoma and was already fully sequenced, is thought to be an important agent for the development of cervical cancer as well as HPV 16. Now we are investigating extensively to clarify the real relationship between genital HPV infection and cervical cancer.     

用昆虫-杆状病毒系统表达的HPV6型L1蛋白检测尖锐湿疣患者血清中抗体

用杆状病毒表达系统表达人乳头瘤病毒6型（human papillomavirus type 6,HPV6）主要衣壳蛋白（major capsid protein,L1）作为抗原,对尖锐湿疣（condyloma acuminata,CA）患者抗体进行检测。采用昆虫杆状病毒系统表达HPV6L1蛋白,通过镍柱亲和层析法获得纯化抗原;以酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）检测30例CA、20例献血员和10例儿童血清中的HPV6 LI IgG抗体。感染重组杆状病毒的昆虫细胞经SDS-PAGE和Western blot检测,在大约55kD处有明显的外源蛋白表达条带。ELISA结果显示,CA组的血清阳性率为66．7％（20／30）,献血员组的阳性率为15％（3／20）,两组之间有显著性差异（P〈0．05）。22例HPV6型感染的CA患者有15例血清阳性（68．2％）,6例HPV11型CA患者4例阳性（66．7％）,1例混合感染者为阳性,1例HPV16型患者为阴性。女性CA患者的血清抗体阳性率高于男性（P=0．0052）。本研究建立的ELISA体系具有敏感性和针对低危型HPV感染的特异性。这不仅对于HPV血清流行病学研究是有价值的,而且对于临床诊断HPV感染可能具有一定的应用价值。