人乳头状瘤病毒感染与宫颈癌前病变的关系

目的 探讨人乳头状瘤毒（ＨＰＶ）感染与宫颈湿疣、癌前病变的相关性。方法 对１７９例宫颈细胞涂片异常患者行宫颈多点活检,病理形这观察,其中１２８例同时采用ＰＣＲ方法检测ＨＰＶ－ＤＮＡ,１０例行原位杂交分析。结果（１）形态学观察：宫颈湿疣多为扁平型（９７．３％）;除表现经典的诊断性控空细胞（３９．７％）外,国可见异型性明显的非典型性控空细胞（６０．３％）;宫颈湿疣常伴宫颈上皮内肿瘤（ＣＩＮ,４２．５％     

Detection of human papillomavirus DNA in gynaecological swabs by filter in situ hybridization.

Gynaecological smears from the endo- and ectocervix of women with and without cytological and colposcopic abnormalities of the epithelium were investigated for human papillomavirus (HPV) types 6, 11, 16, and 18 by filter in situ hybridization (FISH). The data were compared with cytological, colposcopic, and histological findings. Of the 266 gynaecological smears, HPV DNA was detected in 84 (32%); of 101 cytologically and colposcopically HPV negative cases, HPV DNA was found in 10%. Of 56 women, cytologically and colposcopically positive for HPV infection, HPV DNA was detected in 68%. The sensitivity of the method was controlled by comparing the results of FISH with those of Southern-blot analysis of five cervical tumour biopsies. The data presented demonstrate the necessity of FISH for identification of the HPV type that might be of prognostic value in cervical pathology. Cytological and colposcopic positivity is a reliable sign in about 70% of the cases where HPV infection was proved by FISH.     

Correlation of the Papanicolaou smear and human papillomavirus type in women with biopsy-proven cervical squamous intraepithelial lesions.

The authors correlated Papanicolaou smear diagnoses with the presence of human papillomavirus (HPV) as determined by in situ hybridization in concurrent biopsy-proven cervical squamous intraepithelial lesions (SILs) in 132 women. Infection by HPV 6 or 11 was associated with a simultaneous normal Papanicolaou smear in 4 of 29 (14%) cases. This result was significantly greater (P less than 0.05) than that found in cases of infection by an oncogenic HPV type (types 16, 31, 33, 35, and others), in which the rate of a concurrent normal Papanicolaou smear was 5 of 88 (5%). Infection by one of these oncogenic types was associated with a Papanicolaou smear diagnostic of SIL in 55 of 88 (63%) cases, whereas infection by HPV 6 or 11 was associated with a Papanicolaou smear diagnostic of SIL significantly (P less than 0.05) less frequently (6 of 29, 18%). It is concluded that, for women with SILs, the likelihood of a Papanicolaou smear diagnostic of the lesion is greater for women with HPV types of known oncogenic potential.     

Correlation of histology, human papillomavirus, and viral load in laryngeal papillomas of childhood.

The purpose of this study was to analyze 47 laryngeal papillomas in children for human papillomavirus (HPV) DNA by in situ hybridization and RT in situ PCR and to correlate these results with the histologic findings. HPV DNA was detected by in situ hybridization in 29 of 47 (62%) of the cases; all positive cases contained HPVs 6 or 11. HPV DNA detection was associated with a statistically significant increase in the presence of keratohyaline granules, nonuniform perinuclear halos, and marked papillomatosis (P<0.02). The viral load was low, defined by less than 20 HPV-positive cells per tissue with a correspondingly weak signal, in 19 of 29 (65%) of the positive cases. In comparison, a high viral load was evident in 19 of 21 (90%) of vulvar condylomas. The laryngeal lesions negative for HPV by in situ hybridization were tested for HPV by RT in situ PCR using primers specific for HPVs 6 and 11. The detection rate of HPV increased to 38 of 47 (81%) after PCR amplification. It is concluded that laryngeal papillomas in childhood are characterized, in general, by a relatively low HPV viral load and that the cases with productive viral infection, as demonstrated by in situ hybridization, are associated with nonuniform keratohyaline granules, nonuniform perinuclear halos, and marked papillomatosis.     

Occurrence of multiple types of human papillomavirus in genital tract lesions. Analysis by in situ hybridization and the polymerase chain reaction.

More than 22 types of human papillomavirus (HPV) have been detected in genital tract squamous cell intraepithelial lesions. Seven of two hundred eighty-six (2.4%) genital tract tissues in which HPV DNA was detected by in situ hybridization contained two or more different HPV types. When analyzed by site, 5 of 204 (2.4%) of cervical intraepithelial lesions were infected by more than one type, compared with 2 of 82 (2.4%) of vulvar lesions. The rate for low-grade lesions was similar (5/218; 2.3%) to that for high-grade lesions (2/68; 2.9%). In contrast, two different HPV types were detected in 6/33 (18%) of tissues by the polymerase chain reaction (PCR) using type-specific primers for eight HPV types. It is concluded that infection by one HPV type is rarely associated with concurrent 'active' infection by a second HPV type, even though DNA of a different viral type can be detected by PCR in about one fifth of such cases. Further study is required to determine if an existing HPV infection can inhibit replication by a different HPV type.     

Detection and localization of human papillomavirus in penile condylomas and squamous cell carcinomas using in situ hybridization with biotinylated DNA viral probes

Human papillomavirus (HPV) has been previously demonstrated in male genital neoplasms using Southern blot hybridization (SBH) and in situ hybridization with radiolabeled probes (ISH-R). In this study we used in situ hybridization with biotinylated DNA viral probes (ISH-B), a technique that can be applied to routinely collected and processed tissue. Thirty cases of exophytic penile condyloma acuminatum and nine cases of invasive squamous cell carcinoma of the penis were examined for the presence of HPV using ISH-B for HPV types 6, 11, 16, 18, 31, and 33. HPV DNA was found in 25 of 30 (83%) penile condylomas; HPV type 6 in 13 (43%); and HPV type 11 in 12 (40%). Slight cross-reactivity between HPV types 6 and 11 was noted. None of the condyloma cases was positive for HPV types 16, 18, 31, or 33. One of the nine patients with squamous cell carcinoma of the penis was positive for HPV 16. In situ hybridization with biotinylated DNA viral probes is a highly sensitive method for detecting and localizing HPV in penile condylomas. This method, however, may not be as sensitive as SBH for detecting HPV in invasive penile squamous cell carcinomas.     

Detection of human papillomavirus DNA in genital lesions by using a modified commercially available in situ hybridization assay.

A modified, commercially available DNA-DNA in situ hybridization test that uses biotinylated probes for the identification of human papillomavirus (HPV) DNA types 6/11, 16/18, and 31/33/35 was evaluated. HPV DNA was detected in 314 of 787 (40%) histologically abnormal genital biopsy specimens by using the ViraType in situ assay (Life Technologies, Gaithersburg, Md.), in which the hybridization time was increased from 2 to 16 h. Ninety percent of positive condyloma acuminata specimens contained HPV type 6/11 DNA. The prevalences of HPV DNA for cervical intraepithelial neoplasia I, II, and III lesions by this in situ hybridization test were 42, 54, and 55%, respectively. The combined prevalence of HPV type 16/18 and 31/33/35 DNAs increased with the severity of the lesion, while the prevalence of type 6/11 DNA decreased. HPV type 6/11 DNA was found only in 1 of 16 (6%) positive cervical intraepithelial neoplasia III specimens. HPV type 16/18 and 31/33/35 DNA was detected in 11 of 16 (69%) and 4 of 16 (25%) in situ hybridization-positive cervical intraepithelial neoplasia III specimens, respectively. Thus, the observation that certain "higher-risk" HPV genotypes are associated with upper-grade cervical precancer lesions was confirmed by this commercial hybridization system. In general, the assay was found to be well suited for use in the clinical laboratory. The ViraType in situ procedure modified for a longer hybridization time may be helpful in identifying lesions containing higher-risk HPV strains.     

Detection of human papillomavirus DNA in squamous bronchial metaplasia and squamous cell carcinomas of the lung by in situ hybridization using biotinylated probes in paraffin-embedded specimens

We examined a series of paraffin-embedded tissue specimens from 10 cases of squamous bronchial metaplasia and 33 cases of squamous cell carcinoma of the lung for histologic characteristics and for the presence and typing of human papillomavirus (HPV) by molecular in situ hybridization with biotinylated probes types 6, 11, 16 and 18 under stringent conditions (temperature, 19 degrees C). Fourteen of these lesions (32.5%) showed typical condylomatous histologic changes. Human papillomavirus DNA was present in seven (16%) specimens. Type 6 HPV DNA was detected in one of the squamous bronchial metaplasia cases. In six of the squamous cell carcinomas cases (18%), HPV DNA was identified (type 18, three cases; type 16, one case; type 11, one case; and type 6, one case); one of the squamous cell carcinoma specimens contained both HPV types 16 and 18. Our data confirm the presence of HPV DNA in squamous metaplastic bronchial mucosa and epidermoid lung carcinoma on paraffin-embedded tissues. This suggests that an HPV infection with benign or potentially oncogenic HPV types could be associated not only with genital tumors, but also with bronchial and lung tumors. The role of HPV DNA in the process of malignancy conversion is not yet known; HPV DNA could possibly be a cocarcinogenic factor. In situ hybridization with biotinylated probes is a useful and appropriate method of retrospective analysis of HPV DNA sequences in routinely paraffin-embedded lesions. It may be used to identify patients at risk of more serious or possibly malignant progression.     

Penile/anal condylomas and squamous cell cancer

Summary Acuminate condylomas from the penis (n=17) and anus (six cases), three anal/penile giant condylomas, anal Bowen's disease (four cases), and intraanal squamous cell carcinomas with associated condylomatous changes (10 cases) including two verrucous carcinoma were studied for human papillomavirus (HPV) infections with nick translated, biotinylated cDNA probes for HPV 6, 11, 16 and 18. In addition, six cases of flat white penile lesions designated as lichen sclerosus et atrophicus were examined.Reannealed complementary DNA strands were detected in situ with either immunoenzyme or immunogold protocols.The in situ hybridizations resulted in 1/6 positive penile lichenoid lesions, 12/17 positive penile acuminate condylomas, 6/6 positive anal acuminate condylomas (including two condylomas with cellular atypias), 2/3 positive giant condylomas, 1/4 positive anal bowenoid lesions, and 4/10 positive keratinized squamous cell carcinomas, two of them being verrucous carcinomas. All penile/anal condylomas and two giant condylomas harboured HPV 6 and/or 11 DNA.The five positive carcinomas (carcinoma in situ/invasive cancer) contained HPV 6 and/or 11 in two cases (including the verrucous carcinomas), and HPV 16 and/or 18 in three cases (one carcinoma in situ, two invasive carcinomas).Recurrent malignancies were seen in one case to harbour the same HPV type as the primary lesions (HPV 16). In one particular patient, a double infection with HPV 16 and HPV 18 was demonstrated in distantly located malignant tumours. Our study confirms the restrictions and the value of non-isotopic hybridization methods applied to archival tissues, and extends the knowledge on the presence and distribution of HPV infections at anogenital sites.This study was supported by the Deutsche Forschungsgemeinschaft (Lo 285/2-4) and the Hamburger Stiftung zur Förderung der Krebsbekämpfung     

Human papillomavirus DNA in respiratory papillomatosis detected by in situ hybridization and the polymerase chain reaction.

The authors have demonstrated the presence of human papillomavirus (HPV) types 6 and 11 in 10 of 13 (77%) juvenile laryngeal papillomatosis by in situ DNA hybridization using as probes the radiolabeled DNAs of HPVs 6, 11, 16, and 18. Of six specimens from adult laryngeal papillomatosis assayed by the same technique, only 33% were positive. Immunohistochemistry to detect HPV capsid antigens performed on serial sections gave positive signals in 44% (8 of 18) of the specimens, all from juvenile lesions. These results were in agreement with in situ hybridization, except in two cases. When both series (juvenile and adult) were analysed by amplification of a 450-bp fragment corresponding to the L1 ORF of the HPV genomes directed by the polymerase chain reaction, the frequency of positive specimens rose to 100%. Our data agree with the concept that HPV is implicated in the etiology of laryngeal papillomatosis.     

In situ hybridization study on human papillomavirus DNA expression in benign and malignant squamous lesions of the esophagus.

Histologic changes suggesting HPV infection are occasionally found adjacent to squamous cell carcinoma or in squamous papilloma of the esophagus, but the relationship between HPV infection and benign and malignant squamous lesions of the esophagus is not yet dear. The aim of this study was to examine the role of HPV in squamous lesions of the esophagus. Microscopic examination with emphasis on HPV infection was done on 15 cases of squamous cell carcinoma and 26 cases of squamous papilloma. In situ hybridization technique for wide-spectrum HPV probe was performed on 35 endoscopically biopsied esophageal tissues. Among the histologic parameters suggesting HPV infection, acanthosis was the most frequent finding: 100.0% in benign and malignant esophageal lesions, and koilocytosis and intraepithelial capillary loops were the second (92.7%).: Dyskeratosis, basal cell hyperplasia and bi- or multinucleation were 52.3%, 44.0% and 34.1% in frequency, respectively. On in situ hybridization study, the HPV DNA expression rates of 10 squamous cell carcinomas with evidence of HPV infection and 15 carcinomas without evidence of HPV infection were 60.0% and 33.3%, respectively. In contrast to the carcinoma cases, only one (10.0%) of 10 squamous papillomas revealed positive signal. In conclusion, HPV infection is strongly associated with squamous cell carcinoma, but the causal relation of HPV to squamous papilloma is inconspicous.     

Human papillomavirus (HPV) genotyping using paired exfoliated cervicovaginal cells and paraffin-embedded tissues to highlight difficulties in attributing HPV types to specific lesions

Defining type-specific human papillomavirus (HPV) infections within cervical tissues is important for understanding the pathogenesis of cervical neoplasia and assessing the effectiveness of prophylactic vaccines with limited type-specific spectra. We compared HPV DNA-testing results from 146 matched exfoliated-cell and formalin-fixed-tissue specimens collected by cervicovaginal lavage (CVL) within 90 days of each other from women with histologically confirmed cervical intraepithelial lesions (CIN). The CVL specimens were HPV typed using a MY09/11 L1 consensus primer PCR method followed by dot blot hybridization. The tissue specimens were HPV typed using an SPF(10) line probe assay HPV detection system. Of the 146 specimen pairs with evidence of CIN in the tissue, 91.8% were positive for one or more HPV types in both the tissue and cellular specimens. Tissue sections were more likely to be HPV negative (P < 0.01). Typing directly from tissue sections resolved multiple infections detected in exfoliated cells to a single HPV type in only 46.9% of cases. Combined use of both specimen types to attribute lesions to HPV type 16 (HPV-16) and/or -18 led to 43.1% attributed to HPV-16 and/or -18 by both specimen types and 19.9% attributed to HPV-16 and/or -18 by one, but not both, specimen types. Unambiguous attribution of cervical lesions to a single, specific HPV type remains a difficult proposition. Use of multiple specimen types or the development of highly sensitive and robust in situ hybridization HPV-testing methods to evaluate the certainty of attribution of lesions to HPV types might provide insights in future efforts, including HPV vaccine trials.     

Detection of human papillomavirus deoxyribonucleic acid by filter in situ hybridization during pregnancy

Samples taken from 101 healthy pregnant women (49 over and 52 under the 20-week gestational period) and 108 healthy nonpregnant women were tested for human papillomavirus (HPV) types. Using 6, 11, 16, and 18 HPV DNA probes, 3-5 x 10(5) exfoliated cells scraped from the cervix were tested by filter in situ hybridization (FISH). Thirty-five of the pregnant women (34.6%) had evidence of the presence of HPV DNA: with 11.8% (12/101) HPV 6; 7.9% (8/101) HPV 11; 8.9% (9/101) HPV 16; and 5.9% (6/101) HPV 18 positivity. HPV DNA was detected in 20.4% (22/108) of the non-pregnant women. Compared with the healthy, nonpregnant group, the higher level of asymptomatic cervical HPV infection was mainly due to the accumulation of HPV 16 and 18 nucleic acids during the gestational period: with detection of HPV 16 in 8/49 cases (16.3%) and of HPV 18 DNA sequences in 4/49 (7.6%) cases. Screening 6-8 weeks after delivery indicated a decline of HPV positivity. Of the 4/12 HPV type 16 positive mothers, only one retained the presence of HPV 16 DNA, whereas neither of the 2/12 type 18 positive women reacted after birth with the type 18 radioactive probe.     

乳腺浸润性导管癌中HPV18、HPV16感染的研究

目的 :了解乳腺浸润性导管癌中HPV18、HPV16的感染情况 ,分析其是否是乳腺癌发生的危险因素及与临床病理的相关性。方法 :根据HPV16、HPV18的DNA序列 ,合成相应特异的寡核苷酸片段 ,用加尾标记法制备地高辛标记探针 ,用原位杂交法检测 5 1例乳腺浸润性导管癌、10例相应正常乳腺上皮及 15例良性乳腺病变中HPV18、HPV16的感染 ,并分析其与患者发病年龄、肿块大小及淋巴结转移的相关性。结果 :浸润性导管癌中HPV18或 16的总阳性率达 70 6 % ,其中HPV18与HPV16的阳性率分别为 5 8 8%、4 5 1% ,均明显高于正常乳腺上皮的感染率 (30 0 %、10 0 % ;P <0 0 5 ) ;乳腺良性病变的HPV18、16阳性率分别是 6 0 0 %、6 0 % ,其中HPV18的阳性率亦显著高于正常乳腺上皮 (P <0 0 5 )。结论 :(1)HPV16和18可能是乳腺浸润性导管癌发生的致病因子 ,HPV18尚可能与乳腺良性病变的发生有关。 (2 )HPV的感染与患者年龄、肿块大小及淋巴结转移无相关性。     

A new nonisotopic detection of human papillomavirus DNA using polymerase chain reaction.

A novel technique using a two-step polymerase chain reaction (PCR) with specific primers detecting human papillomavirus (HPV) DNA of types 6/11, 16, and 18 and a final nonisotopic colorimetric detection has been developed. Sixty formalin-fixed and paraffin-embedded sections were treated with this methodology and the results compared with those obtained with in situ hybridization (ISH). Twenty cases displaying HPV DNA with ISH were positive with PCR. Seven (35%) of 20 cases negative for ISH but evocative of HPV infection with classic histology displayed HPV DNA with the two-step PCR. Only one case (5%) of 20 normal tissues and/or inflammatory lesions not evocative of HPV infection and negative upon ISH showed HPV DNA. This original technique allows rapid, highly sensitive, and specific detection of HPV DNA and is suitable for most laboratories.     

Evaluation of methods for detecting human papillomavirus deoxyribonucleotide sequences in clinical specimens.

Specimens from 26 condylomatous lesions, 24 invasive cancer cells, and 33 cervices, without evidence of the diseases, were tested for the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 by Southern blot hybridization, in situ filter hybridization, or in situ tissue hybridization methods. A total of 89% (23 of 26) of the condylomatous lesions contained HPV DNAs, as determined by one or more of the methods. The positive rates for the detection of HPV DNA in condylomas by the different methods were 82% for Southern blot hybridization, 62% for in situ filter hybridization, and 72% for in situ tissue hybridization. Among the specimens from patients with cancer, HPV DNA was found in 83% (19 of 23) by one or more of the methods. Positive rates of 89 and 70%, respectively, were obtained for cancer lesions tested by the filter in situ and Southern blot hybridization methods; however, only 30% of those lesions were positive by the in situ tissue hybridization method. Thirteen percent of the control cervices were positive for HPV DNA by one or more of the assays. With respect to all disease categories, the methods had comparable sensitivities and specificities, except for the in situ tissue hybridization method, which revealed a specificity of 72% for condylomatous lesions and 30% for invasive cancer cells.     

Study of multiple human papillomavirus-related lesions of the lower female genital tract by in situ hybridization

Twenty-six women with multiple human papillomavirus (HPV)-related lesions of the lower genital tract were investigated by immunohistochemistry for the internal genus-specific capsid antigen of HPV and by DNA-DNA in situ hybridization with 35S-radiolabeled probes for sequences of HPV-6/11, HPV-16, and HPV-18. The vulva was the most frequently affected site in all of these cases; the cervix was the second most frequently affected site. The lesions displayed the features of papillomavirus infection in 14 patients, and there was also histologic evidence of early neoplasia in 12 patients. The mean age of patients with and without neoplasia was 40 and 30 years, respectively. Evidence of HPV association was found in 73% of the vulvar lesions and in 40% of the other synchronous lesions by one or both methods. Viral DNA was found in 67% of patients with neoplasia and in 71% of patients without neoplasia. Eleven of 12 HPV-positive patients with neoplasia revealed the presence of HPV-16 in their tissues by in situ hybridization. On the other hand, 50% of those without neoplasia had HPV-16 DNA, whereas the presence of HPV-6/11 was found in the other 50%. The clinical course of the disease, the distribution of HPV type, and the type of antigen in patients with and without neoplasia suggest that progression to neoplasia was associated with HPV-16. These results stress the practical value of the in situ hybridization method for the identification of those patients with HPV infection who are at risk for progression to malignancy.     

乳头瘤病毒16型E6,P53,RB,PCNA在宫颈癌中的表达…

应用核酸原位杂交和免疫组织化学技术，检测人子宫颈癌中人乳头瘤病毒（ＨＰＶ）１６型Ｅ６ＲＦ与抑癌基因产物Ｐ５３，ＲＢ和增殖细胞核抗原（ＰＣＮＡ）。在４４例宫颈癌石蜡切片中，原位杂交检测出ＨＰＶ１６Ｅ６ＯＲＦ阳性２７例（６１．３６％），其中免疫组化检测出Ｐ５３、ＲＢ、ＰＣＮＡ一分别为８例（２９．６３＃），１４例（５２．８５％）、２０例（７４．０７％），而在１７例ＨＰＶ１６Ｅ６阴性 本中Ｐ５３、ＲＢ、Ｐ     

A comparison of the detection of biomarkers in infections due to low risk versus high-risk human papillomavirus types

Adjunctive immunohistochemistry tests for human papillomavirus (HPV) infection include p16 and Ki67 as well as the more recently discovered biomarkers importin-β, exportin-5, Mcl1, and PDL1. The purpose of this study was to compare the expression of these biomarkers in HPV infection due to the high-risk types such as HPVs 16, 18, 31, 33, 35, and 51 versus lesions that contain the low risk types HPV 2, 6 or 11. We studied 35 lesions with low risk HPV types (verruca vulgaris = 10 cases, condyloma acuminatum = 15 cases, CIN 1 with HPV 6/11 = 10 cases) and 25 CIN 1 or 2 lesions with a high-risk HPV type. The 25 high-risk positive CIN 1–2 cases had strong expression of the panel p16, Ki67, importin-β, exportin-5, Mcl1, and PDL1 where each protein localized to the cells in the parabasal aspect of the lesion. In comparison, neither p16, importin-β, exportin-5, Mcl1, nor PDL1 were increased in the epithelia of the lesions with the low risk HPV types; Ki67 showed variable expression. HPV viral capsid L1 protein and viral DNA were excellent markers of infection in the lesions with low risk types. Thus, p16, importin-β, exportin-5, Mcl1, and PDL1 are not only biomarkers of high-risk HPV infection but can also differentiate such lesions from those that contain low risk HPV types. Low risk HPV infections can be best differentiated from their mimics by viral L1 capsid detection and/or HPV DNA by in situ hybridization.     

Human papillomavirus type 13 and focal epithelial hyperplasia of the oral mucosa: DNA hybridization on paraffin-embedded specimens

Summary 16 cases of focal epithelial hyperplasia (Heck's disease) were studied for the presence of human papillomavirus DNA by means of nucleic acid hybridization. Hybridization was carried out in situ with biotin-labelled probes of HPV 1, 6, 11, 13, 16, and 18 DNA under stringent and non-stringent conditions. Under non-stringent conditions, 6 of 16 cases (38%) hybridized to a mixture of HPV 1, 6, 11, 16, and 18 DNA. When these probes were applied under stringent conditions, only one case could be shown to be weakly positive for HPV 6/11 DNA. Further stringent hybridizations, which were conducted with a HPV 13 probe on 12 of our 16 cases, revealed a positive result in 9 of 12 cases (75%). The results of our study strongly substantiate the concept that HPV 13 or a closely related HPV type is associated with lesions morphologically presenting as focal epithelial hyperplasia.