Prevalence of human papillomavirus in sinonasal papillomas: a study using polymerase chain reaction and in situ hybridization.

Inverted and fungiform papillomas of the sinonasal cavity share a common origin from the Schneiderian membrane, but they differ widely in their rates of recurrence and progression to carcinoma. To determine the role of human papillomavirus in the etiology of these lesions, 15 inverted papillomas, five fungiform papillomas, and two squamous cell carcinomas associated with inverted papilloma were examined for the presence of HPV by in situ hybridization (ISH) and polymerase chain reaction (PCR). ISH was carried out on formalin-fixed, paraffin-embedded material using HPV types 6/11, 16/18, and 31/33/35 DNA probes. Tissue DNA was amplified by PCR with HPV L1 consensus primers, and the product was detected by gel electrophoresis, Southern blotting, and hybridization with type specific probes (HPV types 6/11, 16, 18). Three of 15 inverted papillomas and two of five fungiform papillomas were positive for HPV 6/11 by ISH, whereas PCR detected HPV 6/11 sequences in two of 15 inverted and three of five fungiform papillomas. Biopsies from two patients who had serial resections contained HPV 6/11 in the original lesions and all recurrences. No HPV was detected in the carcinomas by ISH, whereas PCR detected HPV 16 in one carcinoma. These findings confirm the presence of HPV DNA sequences in both inverted and fungiform sinonasal papillomas as well as in an associated squamous carcinoma. This would suggest a role for HPV in the pathogenesis of Schneiderian membrane lesions. Furthermore, our data indicate that ISH and PCR are equally sensitive in detecting HPV in sinonasal papillomas.     

Sinonasal Schneiderian papillomas: human papillomavirus typing by polymerase chain reaction.

A series of 35 formalin-fixed, paraffin-embedded Schneiderian papillomas (24 inverted, nine cylindrical cell type, and two fungiform) of the nasal cavity were evaluated for the presence of human papillomavirus (HPV) types 6b/11, 16, and 18 DNA sequences using both a highly sensitive and specific modification of the polymerase chain reaction technique and conventional in situ hybridization. The HPV gene sequences (E6-E7 portions) were not detected in any of the 24 inverted or nine cylindrical cell papillomas. One of the fungiform papillomas was positive for HPV 6b/11. We conclude: (a) the origin of most Schneiderian sinonasal papillomas is not associated with HPV infection of these common types, and (b) fungiform papilloma is a distinctive clinicopathologic subtype of Schneiderian papilloma that may be HPV-related.     

Detection of human papillomavirus (HPV) DNA type 6/11 in a conjunctival papilloma by in situ hybridization with biotinylated probes.

Four cases of conjunctival papilloma in two different patients were examined by in situ hybridization for HPV DNA type 6/11, 16/18 and 31/33/51. Formalin-fixed and paraffin-embedded tissues were hybridized by biotinylated probes. One tumor and one of its recurrences showed nuclear positivity for HPV 6/11 in the superficial cells of the epithelium. The results suggest that HPV type 6/11 may be etiologic agent of conjunctival papillomas. The benign behavior of these neoplasms may be related to the etiologic role of this type of HPV.     

Detection of human papillomavirus in normal and dysplastic tissue by the polymerase chain reaction

Detection of human papilloma virus (HPV) types 16 and 18 in formalin-fixed, paraffin-embedded tissue by a new in vitro DNA amplification method, the polymerase chain reaction, was compared with detection with genomic DNA probes using in situ hybridization. The polymerase chain reaction replicates exponentially HPV DNA sequences present in a single 5- to 10-micron paraffin-embedded tissue section. The amplified sequences are detected with a DNA hybridization probe in a dot blot assay. The HPV polymerase chain reaction was able to detect on the average less than one HPV genome/cell as determined by tests of paraffin sections of cell pellets with known HPV genomic content. Cervical sections from 21 patients with HPV types 16, 18, or 31 as determined by in situ DNA hybridization were analyzed by the polymerase chain reaction. No disagreements between the two methods were detected. The sections comprising normal and dysplastic epithelium were further analyzed by the HPV polymerase chain reaction. The presence of virus correlated with the presence of dysplasia in the sections, though 3 of 10 normal sections contained HPV, and 1 of 21 sections with dysplasia lacked HPV 16 or 18. The polymerase chain reaction can specifically detect HPV 16 or 18 with high sensitivity from paraffin-embedded tissues.     

Detection of human papillomavirus DNA in oral inverted ductal papillomas

AIMS: To determine the presence or absence of human papillomavirus (HPV) DNA in oral inverted ductal papillomas (IDPs) using in situ hybridisation (ISH), and to analyse all cases for histological features of HPV infection. METHODS: Six cases were retrieved from archival material and paraffin wax blocks were submitted for the detection of HPV DNA by means of ISH. A wide spectrum probe for HPV subtypes 6, 11, 16, 18, 31, 33, 45, 51, and 52 was used initially. Cases that were positive using this wide spectrum probe were further subtyped using HPV type specific probes (6/11, 16/18, and 31/33). The histological features of all tumours were analysed using routine microscopy. RESULTS: Of the six cases of oral IDP identified, three were positive for HPV subtypes 6/11. All positive cases showed histological features of HPV infection (koilocytosis, papillomatosis, binucleated keratinocytes, and abnormal mitosis) in both the surface and the inverted epithelium. The three cases that tested negative for HPV DNA also exhibited focal histological features of HPV infection (two in the surface epithelium and one in the endophytic epithelium). CONCLUSIONS: These are the first documented cases of oral IDP to demonstrate positivity for HPV DNA and also to show histological features of HPV infection.     

Human papillomavirus 6, 11, and 16 in laryngeal papillomas.

Twenty-seven cases of benign laryngeal papillomas, both single and multiple variants, were analysed for human papillomavirus (HPV) by DNA slot-blot hybridization chiefly to determine the pattern of infection in Hong Kong Chinese. DNA was extracted from paraffin blocks of formalin-fixed tissue and probed separately for HPV 6, 11, 16, and 18. Sixteen cases (59 per cent) showed the presence of at least one of these four HPV genomes. Thirteen cases (48 per cent) were positive for HPV 11 only. Three other cases (11 per cent) showed triple positivity for HPV 6, 11, and 16. None were positive for HPV 18. The predominance of HPV 11 infection contrasts with other series which have shown either an almost equal distribution of HPV 6 and 11 or a predominance of HPV 6. The finding of HPV 16 in three cases was unexpected. Using the polymerase chain reaction (PCR) with primers complementary to the upstream regulatory region of the HPV 16 viral DNA, the presence of HPV 16 genome was confirmed in all three cases. As the number of HPV 16-positive cases in this study is small, analysis of more cases using fresh biopsy material and a wider range of HPV type-specific PCR primers is warranted to determine the relative incidence of HPV subtypes in these benign laryngeal papillomas.     

Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes

Twenty-three conjunctival papillomas and 28 conjunctival dysplasias were examined for human papillomavirus (HPV)-DNA sequences by in situ hybridization with nick-translated 35S-labeled HPV probes. Adjacent paraffin sections were hybridized with HPV type 2, 6, 16, and 18 probes at Tm - 17 degrees C. Fifteen tissues, all papillomas, displayed positive hybridization with the HPV-6 probe. Infection with HPV-6 (or the closely related HPV-11) appeared to be responsible for most of the conjunctival papillomas of children and young adults. The presence of genital tract HPV-6 in these lesions suggests that some of the infections were acquired during passage through an infected birth canal. The lack of hybridization in adult conjunctival dysplasias indicates either that HPVs are not associated with this condition or that the probes and the technique utilized were not adequate for demonstration of this association.     

Detection and localization of human papillomavirus DNA in human genital condylomas by in situ hybridization with biotinylated probes

We have examined the distribution of human papillomavirus (HPV) DNA in paraffin sections of humans warts by in situ hybridization with biotin-labeled DNA probes. Recombinant plasmid DNAs (HPV-1, -6, -11, -16) were labeled by nick translation with biotinylated deoxyuridine triphosphate. Paraffin sections were hybridized with the probes for 18 h in stringent or non-stringent conditions, and DNA-DNA hybrids were detected by immunocytochemistry. Paraffin sections of warts were also examined for the presence of HPV capsid antigen with the avidin-biotin peroxidase complex method for immunocytochemistry. HPV DNA was detected and localized in paraffin sections from a plantar wart, a laryngeal papilloma, and seven anogenital condylomas. The specific HPV type present in each lesion was determined by hybridization under stringent conditions with the homologous DNA probe. The papillomas were found to contain many more cells with replicating virus DNA, as demonstrated by in situ hybridization, than was apparent from the number of cells containing detectable virus antigen. In situ hybridization with biotin-labeled probes is an effective technique for the identification of HPV infection in routinely collected and processed tissue specimens.     

Laryngeal papillomatosis: molecular,histopathological, and clinical evaluation

Molecular, histopathological, and clinical studies were carried out on a series of 79 laryngeal papillomas (LP) from 36 patients in order to investigate the hypothesis that juvenile and adult LP may represent a biological entity causally related to Human papilloma virus (HPV) infection. Using in situ hybridization with biotin-labelled probes and polymerase chain reaction, we detected human papilloma virus (HPV) 6/11 in 28 of 29 juvenile LP, in 26 of 30 adult multiple, and in 17 of 20 adult solitary LP. None of LP was found to harbour HPV types 16, 18, 31, 33, and 51. There were no clear-cut histological differences between juvenile and adult LP, the presence of koilocytosis was equally observed in both, and there was no prevalent type of epithelial hyperplasia in either form, except that all three cases of atypical hyperplasias (precancerous lesions) were found among adult patients. During a 14 year follow-up, no carcinomatous transformation of LP was observed. All juvenile LP in our study had frequent recurrences of the disease, however, numerous surgical procedures were also required in 16 of 27 adult patients. Our study supports Lindeberg's hypothesis of a similar pathogenesis for all forms of LP caused by the HPV types 6/11.     

Human papillomavirus infection and anal carcinoma. Retrospective analysis by in situ hybridization and the polymerase chain reaction.

To examine the association of human papillomavirus (HPV) infection with anal squamous cell carcinoma, the authors applied the highly sensitive polymerase chain reaction (PCR) and in situ hybridization (ISH) techniques to detect HPV DNA in formalin-fixed, paraffin-embedded tissues from 18 patients. The presence of HPV types 16/18 in 3 (16.7%) of 18 patients with anal carcinoma was found, using a colorimetric ISH technique for HPV types 6, 11, 16, 18, 31, 35, and 51. Results from one of these three patients were also positive for HPV 31, 35, 51 by ISH techniques. When the same series was analyzed using the PCR and consensus primers to the L1 open reading frame of the HPV genomes, the frequency of positive patients rose to 14 (77.8%) of 18. PCR analysis of the 14 lesions containing HPV DNA, using type-specific primers and probes for HPV 6, 11, 16, 18, and 33, showed that 1 contained HPV 6, 1 contained HPV 11, 4 contained HPV 16, 1 contained HPV 18, 1 contained HPV 33, 5 contained HPV of unclassified type(s), and 1 contained a mixture of three HPV types. There was concordance between typing of cases that were positive by ISH and PCR methods. These data agree with the concept that HPV, in particular type 16, is implicated in the pathogenesis of anal cancer.     

The predictive value of human papilloma virus (HPV) typing in the prognosis of bronchial squamous cell papillomas

Five solitary squamous papillomas of bronchus with variable degrees of dysplasia, one combined with a laryngeal papilloma and with a neuroendocrine carcinoma in the contralateral lung, and five papillomas combined with invasive squamous cell carcinomas were investigated for their expression of human papilloma virus DNA by in situ hybridization. Benign squamous cell papillomas showed an association with papilloma virus type 11 and rarely type 6, whereas types 16 or 18, sometimes in combination with types 31/33/35 were found in papillomas associated with carcinomas. In one patient a benign papilloma containing human papilloma virus type 18 and 31/33/35-positive preceded a recurrence combined with carcinoma by 2 years; this recurrent papilloma and the carcinoma were also positive for human papilloma virus 18 DNA. We suggest that human papilloma virus typing should be performed in every squamous cell papilloma of bronchus. Patients with papillomas exhibiting human papilloma virus 16 or 18 positivity are at high risk for the development of squamous cell carcinoma. Furthermore, virus typing may be of prognostic importance in relation to the development of squamous carcinoma.     

Failure to detect human papillomavirus DNA in malignant epithelial neoplasms of conjunctiva by polymerase chain reaction

To elucidate the putative role of human papillomavirus (HPV) infection in the etiology of conjunctival tumors, 44 formalin-fixed, paraffin-embedded specimens of conjunctival tumors (24 patients with papillomas and 20 patients with dysplastic and/or malignant tumors) were screened for HPV infection using 4 different polymerase chain reactions (PCRs). Of the 24 samples of papilloma, 14 (58%) displayed positive results by applying nested PCR using primer sets of HPV consensus L1 region. HPV type 6 or 11 was detected in 9 cases of papilloma by type-specific primer sets, but none of them were positive for HPV type 16 or 18. However, by using the highly sensitive PCR technique, we failed to demonstrate the HPV DNA of HPV types 6, 11, 16, and 18 in any of the 20 malignant epithelial tumors of conjunctiva. We conclude that HPV-6 or HPV-11 is present in a substantial percentage of conjunctival papillomas, which is in accordance with findings of previously reported studies. In contrast, malignant conjunctival carcinomas are not associated with HPV infection; other pathogenic mechanisms, such as UV light, probably are more important in the cause of these malignant lesions.     

Transitional cell carcinoma of the bladder: low incidence of human papillomavirus DNA detected by the polymerase chain reaction and in situ hybridization

Viral studies on mammalian urothelium have shown an association between the bovine papillomavirus and cancer of the bladder in cattle. However, the evidence for human papillomavirus (HPV) involvement in urinary bladder in man is less clear. The aim of this study was to investigate the association between HPV DNA and transitional cell carcinoma of the bladder, using the highly sensitive polymerase chain reaction (PCR) and non-isotopic DNA in situ hybridization on formalin-fixed paraffin-embedded tissues from 76 patients. An HPV type specific set of primers was localized on the E6-gene for HPV 16/18 DNA. The second and third set of primers were specific for HPV 6/11 DNA. A biotinylated DNA probe which recognizes HPV 6/11, 16/18, and 31/33/35 was used for in situ hybridization. Of the 76 cases investigated, PCR analysis showed positive signals in seven (9.2%) of cases–six for HPV 16 DNA, and one for HPV 16 DNA and HPV 6 DNA. Four (5.2%) were also reactive for HPV 16/18 DNA using in situ hybridization. Most transitional cell carcinomas (71.4%) associated with HPV DNA were of high pathological grade/stage. One case had koilocytosis. Our results suggest that HPV DNA in transitional cell carcinoma is probably a rare occurrence, although the finding of the high risk HPV 16 DNA may indicate a role for it in this tumour's aetiology.     

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.     

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.     

The characteristics of human papillomavirus DNA in head and neck cancers and papillomas

AIM: To determine the prevalence, type, physical state, and viral load of human papillomavirus (HPV) DNA in cases of head and neck cancer and recurrent respiratory papillomatosis (RRP). METHODS: The prevalence and type of HPV DNA was determined in 27 fresh frozen tissue specimens from patients with head and neck cancers and 16 specimens from 10 patients with RRP by MY09/MY11 and GP5+/GP6+ nested polymerase chain reaction (PCR) and subsequent restriction enzyme cleavage. The physical state of HPV DNA was analysed by E1, E2, and E1E2 specific PCRs and Southern blot hybridisation (SBH). RESULTS: HPV DNA was detected in 13 of 27 cancers and 10 of 10 papillomas. Both low risk HPV-6 and HPV-11 and high risk HPV-16 were present in cancers in low copy numbers, whereas papillomas exclusively harboured low risk HPV-6 and HPV-11. E1E2 PCRs failed to determine the physical state of HPV in cancers except one case where HPV-6 DNA was integrated. In contrast to cancers, all papillomas showed the episomal state of HPV DNA and a relatively higher viral load. CONCLUSIONS: Based on the prevalence, type, physical state, and copy number of HPV DNA, cancers and papillomas tend to show a different HPV DNA profile. The 100% positivity rate of low risk HPV types confirms the role of HPV-6 and HPV-11 in the aetiology of RRP.     

Prevalence of six types of human papillomavirus in inverted papilloma and papillary transitional cell carcinoma of the bladder: an evaluation by polymerase chain reaction.

AIMS: To study the prevalence of high risk oncogenic human papillomaviruses (HPV) in inverted papilloma and papillary transitional cell carcinoma of the bladder. METHODS: Ten cases of inverted papilloma and 20 cases of papillary transitional cell carcinoma of the bladder from Chinese patients in Hong Kong were examined for the presence of HPV type 6, 11, 16, 18, 31, and 33 genomes using the polymerase chain reaction and HPV type specific primer probe combinations on paraffin wax embedded biopsy specimens. RESULTS: Of the 10 cases of inverted papilloma, cases 1 and 6 showed the presence of HPV types 16 and 18, respectively. Six of the 20 papillary transitional cell carcinomas were positive for HPV type 18. The other HPV types were not detected. CONCLUSIONS: HPV type 18 was found in 60% and 30% of cases of inverted papilloma and papillary transitional cell carcinoma of the bladder, respectively. These tumours were rarely associated with HPV types 6, 11, 16, 31, and 33. The role of HPV type 18 in oncogenesis of inverted papilloma and transitional cell carcinoma of the bladder requires further studies.     

Does HPV play a role in the etiopathogenesis of ameloblastoma? An immunohistochemical, in situ hybridization and polymerase chain reaction study of 18 cases using laser capture microdissection.

Ameloblastomas are epithelial tumors of odontogenic origin, biologically characterized by local recurrence. Among different etiologic factors, HPV infection has been recently postulated to be somehow involved in ameloblastoma etiopathogenesis. To address this issue, we studied 18 ameloblastomas by means of immunohistochemistry, in situ hybridization (conventional and amplified), polymerase chain reaction and nested-polymerase chain reaction analyses using laser capture microdissection in order to detect the occurrence of HPV in this setting. No evidence of HPV infection was detected by morphological examination, immunohistochemistry, in situ hybridization and conventional polymerase chain reaction, while nested-polymerase chain reaction showed a weak positive band in two cases. However, the subsequent restriction enzyme analysis carried out from the nested-polymerase chain reaction amplification products of these two samples excluded the presence of HPV subtypes 16, 18, 31, 33, 35, 52, and 58. The search for HPV 6 and 11 in the same specimens was also negative. In conclusion, our data do not support an etiopathogenetic evidence for HPV in ameloblastoma.     

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

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