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 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.     

Molecular cloning and characterization of the DNA of two papillomaviruses from monkeys

Benign and malignant lesions from monkeys were analyzed for the presence of papillomavirus (PV) DNA. By hybridization with different PV DNA probes under conditions of lowered stringency, two tumors were found to contain PV-specific DNA sequences: (1) a cutaneous papilloma from a Colobus monkey; and, (2) a lymph node metastasis of a squamous cell carcinoma of the penis from a Rhesus monkey. Analysis of the DNA of the papilloma from the Colobus monkey indicated the presence of extrachromosomal DNA whereas analysis of DNA from the Rhesus tumor suggested the presence of integrated viral DNA. The physical size (7.8 and 8.1 kb), colinear alignment to HPV-5, and cross-hybridization with other PV types under low stringency indicate that the two genomic DNA clones represent new PV types that have been tentatively designated as Rhesus papillomavirus type 1 (RhPV 1) and Colobus guereza papillomavirus type 2 (CgPV 2). A putative viral-host DNA junction fragment was also isolated from the Rhesus genomic library. Nucleotide sequences very closely related to RhPV 1 were observed by in situ hybridization in a laryngeal carcinoma from the Colobus guereza monkey. This report communicates the finding of novel papillomaviruses associated with a benign cutaneous tumor and genital and laryngeal malignancies in non-human primates which may have significance as a putative system for the study of papillomavirus-induced genital and laryngeal malignancies in humans.     

Detection of human papillomavirus DNA in prostate gland tissue by using the polymerase chain reaction amplification assay.

Human papillomavirus (HPV) is associated with specific benign and malignant lesions of the epithelial and mucosal surfaces. Of the sexually transmitted types, HPV type 16 (HPV 16) and HPV 18 are commonly associated with severe dysplasia and carcinoma of the uterine cervix. In men, genital HPV infections which have been studied are manifest as external lesions usually involving types other than 16 and 18. The nature of HPV 16 and 18 infections in men has not been explored. Since the most common neoplasias of the male genital tract involve the prostate gland, we assayed benign hyperplastic and cancerous prostate tumors for the presence of HPV DNA, using type-specific primers in polymerase chain reaction amplifications. Normal prostatic tissue obtained at autopsy was included in the survey. Amplified sequences specific for HPV 16 were found in 14 of 15 benign prostatic hyperplasias and in all of four carcinomas tested. In contrast, HPV 18 was identified in only three benign hyperplasias, which also contained HPV 16 DNA. Four of five normal prostates demonstrated no HPV infection; one contained HPV 16 sequences. The presence of these oncogenic HPV types in prostate tissues suggests a reservoir for sexual transmission; a potential role for the virus in the etiology of prostatic neoplasia remains to be demonstrated.     

A comparison of biotin- and 35S-based in situ hybridization methodologies for detection of human papillomavirus DNA

In situ hybridization is commonly used for the detection of human papillomavirus (HPV) DNA in genital tract lesions. Systems based on biotin complexes are quicker and easier to use than 35S-based systems, although reportedly less sensitive. We compared three in situ hybridization systems for HPV DNA detection: two biotin [PathoGene DNA probe assay [Enzo Diagnostics] and Viratype in situ HPV probes and HPV tissue hybridization kit [Life Technologies Inc. (LTI)] and one 35S based. By using serial sections from 80 female genital tract lesions with the histologic features of an HPV infection, sequences homologous to HPV DNA were detected in 59 cases (74%) with the LTI system and 25 cases (31%) with the Enzo system. The Enzo system uses a streptavidinbiotinylated horseradish peroxidase complex and 2% 3-amino-9-ethylcarbazole as the chromogen. The LTI system uses a streptavidin alkaline phosphatase conjugate in which the chromogen is 5-bromo-4-chloro-3-indolylphosphate in the presence of nitroblue tetrazolium. Replacing the Enzo detection system with the LTI detection system increased the sensitivity of the Enzo kit. The LTI biotin system was equally sensitive when compared against 35S-labeled HPV probes. The sensitivity with the biotin probes, reported to be less than the 35S probes in a previous study (Lab Invest 58:354, 1988), was increased if the current LTI detection system replaced the detection system used in that study. It is concluded that biotin-labeled DNA probes can be equally sensitive to 35S-labeled DNA probes for the detection of sequences homologous to HPV DNA. The enhanced sensitivity for the biotin system is primarily due to improved detection of the probe/target complex.     

Human papillomavirus DNA determination of anal condylomata, dysplasias, and squamous carcinomas with in situ hybridization

Infection with types 6, 11, 16, and 18 of the human papillomavirus (HPV) is associated with condylomatous, dysplastic, or carcinomatous changes in the genital tract. Emerging evidence suggests that a similar series of lesions develops in the anal canal after exposure to the same HPV types. In situ hybridization was performed with the use of biotinylated DNA probes to HPV 6, 11, 16, and 18, so as to determine the frequency of HPV DNA in 45 perianal and/or anal condylomata, 6 anal intraepithelial neoplasias, and 13 anal squamous cell carcinomas. Of the 33 perianal and/or anal condylomata in which HPV DNA was detected, 13 contained HPV 6 and 11, 12 HPV 6, 7 HPV 11, and 1 HPV 6, 11, and 18. Two of four severe anal dysplasias contained HPV 16, whereas one case each of mild and moderate anal dysplasia contained HPV 6. No HPV DNA was detected in the anal squamous cell carcinomas. The study demonstrated the presence of HPV DNA in 73% of condylomata and 67% of anal dysplasias. The observations suggest that the cloacogenically derived anal epithelium is susceptible to infection by the same HPV types as infect the similarly derived epithelium of the lower female genital tract and that these HPV types result in some similar lesions, i.e., condylomata and dysplasias in both sites. A role in the genesis of anal cancer was not found in this study.     

Influence of fixation on human papillomavirus DNA detection in frozen and embedded paraffin lesions by in situ hybridization with biotinylated probes.

Series of frozen or paraffin-embedded tissues from various body sites, taken from non-immunosuppressed or immunosuppressed patients with persistent papilloma lesions were examined for the presence of group specific antigen from human papillomavirus (HPV) by indirect immunofluorescence or HPV DNA by in situ hybridization with biotinylated probes. We have shown that it is possible to detect HPV DNA after fixation of tissues in neutral formalin, Bouin's or Baker's solution. However, the sensitivity was reduced as compared to frozen tissues. The HPV DNA was detected in nuclei of heavily infected epithelial cells such as plantar or hand warts or in dispersed cells containing high copy numbers of HPV DNA from lesions such as squamous cell carcinomas or keratoacanthomas. In premalignant or malignant lesions of both immunosuppressed or non-immunosuppressed patients, HPV DNA was rarely detected after fixation. HPV types commonly described for skin and genital samples were identified in non-immunosuppressed patients, whereas in transplant recipients oncogenic HPV type 16 was identified in benign warts as well as in premalignant or malignant lesions. Positive reactions with several HPV types were more frequent in lesions from grafted patients than from the normal population. Virus antigen was detectable more frequently in frozen sections than in fixed tissues. Our findings indicate that in situ hybridization is an appropriate and rapid technique to study the presence of HPV infection. However, numerous controls are needed to avoid misinterpretations.     

Detection of human papillomavirus in skin and genital lesions of renal allograft recipients by in situ hybridization

Renal allograft recipients have an increased incidence of malignancy including squamous carcinoma of cervix and skin. There is growing evidence that human papillomavirus (HPV) has a part to play in malignant transformation at these sites. We have previously identified HPV DNA in the skin and genital lesions of such patients by dot and Southern blotting. In situ hybridization studies, using biotinylated DNA probes for HPV 4, 5 and 8 in skin lesions and 6, 11. 16 and 18 in genital lesions, were performed on tissues derived from the same group of patients. In the cutaneous lesions, only 25% of the specimens probed were found to contain virus by in situ hybridization; 60% of these specimens were found to harbour virus by dot and Southern blotting. In situ hybridization revealed HPV 16 and/or 18 in 86% of the genital lesions probed.     

Detection of human papillomavirus 16 and 18 DNA in epithelial lesions of the lower genital tract by in situ hybridization and polymerase chain reaction: cervical scrapes are not substitutes for biopsies.

Human papillomavirus (HPV) types 16 and 18 in 66 women with histologically documented lesions of the genital tract and 64 control cohorts were investigated. The efficacies of in situ hybridization and polymerase chain reaction (PCR) in detecting HPV 16 and 18 DNA were analyzed. In order to assess the usefulness of replacing biopsies with cervical scrapes, the two samples were compared by PCR. The prevalence rates of HPV infection by PCR were 59.1 and 10.9% in patients and controls, respectively. PCR was three times more sensitive than in situ hybridization (52.6 versus 17.8%). However, the need to improve PCR sensitivity by subsequent dot blot hybridization reduced one of the main advantages of PCR, i.e., expeditious diagnosis. Cervical scrapes were less sensitive than biopsies (13.6 versus 53%), although with four (6.1%) patients with intraepithelial neoplasias, HPV DNA was identified only by means of cervical scraping. We conclude that obtaining biopsy specimens and cervical scraping are complementary sampling procedures.     

Papillomavirus infection and squamous neoplasia of the cervix

In the past, the lack of a tissue culture system or an animal model suitable for inducing HPV lesions precluded the study of the role of PV in human disease. The development of a genus-specific cross reacting PV antiserum and molecular hybridization techniques now enables pathologists and molecular virologists to assay tissues for the presence of PV structural antigens and DNA sequences using highly specific and sensitive PV probes. The data that has been generated with these new research tools has far reaching implications. For example, the multifocality of squamous carcinoma of the vulva, vagina and cervix together with their similar histology, epidemiology and relationship to precursor lesions suggests that all of these neoplasms as well as related tumors in males may be caused by HPV. Since the immunoperoxidase technique using the PV antiserum can be performed on formalin-fixed paraffin-embedded tissue, systematic evaluation of these lesions can be done retrospectively. After identifying PV-associated lesions by the immunocytochemical method, prospective studies can be undertaken utilizing molecular hybridization in an effort to detect PV-DNA sequences and thereby to more clearly define the role of HPV in squamous neoplasia of the female genital tract.     

A high frequency of human papillomavirus DNA sequences in cervical carcinomas of Indian women as revealed by Southern blot hybridization and polymerase chain reaction.

Ninety-six colposcopically directed biopsies from squamous epithelial carcinoma of the uterine cervix and 22 age-matched normal control biopsy specimens were examined by both Southern blot hybridization and polymerase chain reaction (PCR) for the presence of different human papillomavirus (HPV) DNA types. Cancer of the uterine cervix, which is the most common malignant disease in Indian women, showed a high frequency (98%) of HPV as compared to those reported from other parts of the world. HPV type 16 was found to be the dominant (64%) type while the frequency of HPV type 18 was very low (3%). On individual typing of HPV, no biopsy was found to contain any other known HPV types under stringent conditions of hybridization except a single case of HPV type 11. Only one case of double infection with HPV types 16 and 18 was recorded. Under low stringency conditions of hybridization with a mixed probe of HPV types 16 and 18, 29 additional biopsies were found to be positive. Southern blot hybridization alone detected HPV DNA in 92% of the cases but none in the controls. By PCR, six (6.25%) more cases and four (18.18%) healthy women were found to be positive for HPVs. Analysis of the physical state of HPV 16 indicated integration in about 70% of carcinoma cases while 30% of them were in episomal form. The findings suggest that infection with HPV is an important etiologic factor for the development of cervical cancer, that a number of such tumours may arise without HPV infection, and that integration of the viral DNA into host genome is not always essential for malignant progression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays

Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities.     

Human papillomavirus type 6 detected by the polymerase chain reaction in invasive sinonasal papillary squamous cell carcinoma.

Eight sinonasal carcinomas (one adenocarcinoma, two undifferentiated nasopharyngeal carcinomas, and five squamous cell carcinomas) were investigated for evidence of human papillomavirus (HPV) infection using in situ hybridization and the polymerase chain reaction for HPV types 6, 11, 16, 18, and 33. All eight cases were negative for HPV infection by in situ hybridization, while a single HPV-6-positive case was identified by the polymerase chain reaction. The HPV-positive case was an invasive papillary squamous cell carcinoma of the maxillary sinus. Although HPV-6 is usually associated with benign anogenital condylomata, it has been identified in malignant lesions of the upper respiratory tract. This may reflect exposure of the upper aerodigestive tract to additional carcinogens, such as smoke and alcohol, superimposed on the background proliferative stimulus of the HPV infection.     

Human papillomavirus segregation patterns in genital and nongenital warts in prepubertal children and adults.

This study compared the segregation patterns of human papillomavirus (HPV) in genital and nongenital warts in prepubertal children and adults. HPV 2 was detected in most nongenital warts in children and adults, whereas neither HPV 6 or 11 was detected at nongenital sites in either group with the use of in situ or Southern blot hybridization analyses. Of nine genital tract lesions in children. HPV 2 was detected in two and HPV 6 or 11 in six. More than 90% of cases of regional tract condylomata in adults contained HPV 6 or 11. HPV 2 was not detected in any of 99 genital tract lesions in adults. It is concluded that HPV 6/11 cannot proliferate at nongenital cutaneous sites and HPV 2 can proliferate in the genital tract of children but not adults. Thus, the detection of HPV 6 or 11 in a genital wart in a child implies, assuming cutaneous transmission, infection from a genital site, whereas the detection of HPV 2 presumes nongenital transmission.     

LABELLING PATTERN OBTAINED BY NON-ISOTOPIC IN SITU HYBRIDIZATION AS A PROGNOSTIC FACTOR IN HPV-ASSOCIATED LESIONS

In the study of infection of the lower female genital tract caused by human papillomavirus (HPV), one of the main concerns is the search for prognostic factors to predict the evolution of premalignant low- and high-grade squamous intraepithelial lesions. This study has evaluated the prognostic usefulness of the patterns of positive reaction obtained by non-isotopic in situ hybridization (NISH), referred to as diffuse, punctate, or mixed ‘labelling patterns’. The study examined 141 vulvar and uterine cervical biopsy specimens that were positive for HPV by a NISH screening technique and that had the following histological diagnoses: low-grade squamous intraepithelial lesion (LSIL; n =87); high-grade squamous intraepithelial lesion (HSIL, n =40); and squamous cell carcinoma (SCC n =14). Typing of all the specimens was carried out by NISH with DNA probes specific for HPV types 6/11 (low risk), 16/18 (high risk), and 31/33/51 (intermediate risk), and the labelling pattern observed in each specimen was recorded. Statistical analysis of the results showed that there was a significant difference in the distribution of labelling patterns, both by lesion diagnosis ( P ≤0·004) and by infecting viral type ( P ≤10⁻⁶). Lesions with a punctate or mixed pattern are considered more likely to undergo malignant evolution and consequently have a worse prognosis than lesions with a diffuse pattern.     

Buffered formalin is the superior fixative for the detection of HPV DNA by in situ hybridization analysis.

In situ hybridization is used commonly for detection of human papillomavirus (HPV) DNA. There is little information, however, on whether the detection of HPV DNA by in situ hybridization can be affected by the way in which the tissue is fixed. To address this question, the authors compared the hybridization signal using this technique under low stringency conditions for several genital condylomata containing HPV 6 or 11 that were randomly subdivided and fixed in various fixatives for 16 hours. In all cases, the largest proportion of cells with koilocytotic atypia that had detectable HPV DNA was in buffered formalin-fixed tissue (80%), followed by tissue fixed in unbuffered formalin (70%), Hartman's solution (40%), and Bouin's solution (10%). After a high stringency wash, the greatest decrease in the overall hybridization signal was with tissue fixed in Bouin's solution; a minimal decrease was noted with tissue fixed in buffered formalin. Fixation in Bouin's solution for 2 hours gave in situ hybridization results comparable with buffered formalin fixation but with poorer cytologic detail. It is concluded that, of the fixatives studied, buffered formalin is superior for the detection of HPV DNA by in situ hybridization analysis.     

生殖器疣活检标本中HPV多重检测技术的建立

目的探索生殖器疣活检标本中人乳头瘤病毒（HPV）的多重检测分型技术,为我国生殖器疣感染HPV的检测和分型研究的开展提供有效手段。方法针对病毒L1区基因序列自行设计通用引物MY09、MY11、GP5＋和GP6＋,并对反应条件进行优化,建立基于聚合酶链式反应（PCR）及序列分析的检测生殖器疣活检标本中HPV感染型别的方法。结果采用巢氏PCR方法能够特异扩出140bp的目的条带,实现HPV感染的定性检测;通过对目的基因片段的测序分析能够实现单型别感染/多型别复合感染HPV的分型和鉴定,并可以检测到HPV少见的特殊类型,同时也能提供已知的HPV型别的突变信息。结论基于HPV病毒L1区基因序列建立的PCR和序列分析检测技术是快速检测和分型生殖器疣活检标本中HPV的一种有效方法。     

Human papillomavirus type 11DNA in papillary squamous cell lung carcinoma

Summary We report a case of papillary squamous cell carcinoma of the lung developing in relation to a condylomatous papilloma and related to human papillomavirus (HPV) infection. The viral origin of the bronchial papillomatous lesion is strongly suggested by cytological and histological features with marked condylomatous changes. No viral capsid antigen was detected by immunohistochemistry. Transmission electron microscopy failed to reveal intranuclear viral-like particles in the papillary part of the carcinoma, but typical ultrastructural koilocytotic cells with irregular nucleus and coarse chromatin were observed. HPV DNA type 11 was detected by in situ hybridization using biotinylated probes on paraffin-embedded specimens, under stringent conditions (T _m }-12, 50% formamide). Papillary squamous cell carcinoma may result from the malignant conversion of benign squamous papilloma of the bronchus. HPV type 11 may be associated with malignant conversion of benign papilloma of the pulmonary tract, as in the upper respiratory tract. In situ hybridization with biotinylated probes is a relatively simple and appropriate method for retrospective analysis of HPV DNA sequences in surgical specimens.     

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.