Identification of human papillomavirus types in male urethral condylomata acuminata by in situ hybridization

An in situ hybridization technique was applied under stringent conditions to paraffin sections of urethral condylomata from male patients to determine the presence of DNA sequences of human papillomavirus (HPV) types 6, 11, 16, and 18. The material consisted of 15 classical condylomata acuminata, two flat condylomata, and five recurrent lesions. HPV DNA sequences could be identified in all 15 condylomata acuminata; in 13 lesions, two types of viral DNA were observed (types 6 and 11 in 12, types 6 and 18 in one). In the remaining two condylomata acuminata, only HPV type 11 was present. One of the two flat condylomata was negative with all the probes, and one was borderline-positive for HPV 6. Four of five recurrent lesions contained the same types of viral DNA as the primary lesions, albeit with slight differences in the intensity of viral expression. One lesion was negative with all probes. We conclude that urethral condylomata in males contain the same types of HPV as seen in other anogenital lesions of both sexes and that infection with two viral types is common. In situ hybridization with HPV DNA probes is applicable to archival material and therefore may prove to be of value in future epidemiologic studies comparing lesions in sexual partners. The determination of viral type may have therapeutic implications.     

Detection of human papillomavirus deoxyribonucleic acid in the female genital tract

A total of 336 biopsies, scrapes and exfoliated cells from the cervix and from the lower genital tract were screened for human papilloma (HP) viral sequences of types 6, 11, 16 and 18 by Southern blot, dot blot and filter in situ (FISH) hybridizations with cloned ³²P-radiolabeled HPV DNA probes. The specimens included cervical intraepithelial neoplasias (CIN I–III), carcinoma in situ and invasive carcinoma of the cervix and vagina, adenocarcinomas, vulvar and vaginal condylomata acuminata and healthy epithelial samples. The oncogenic HPV 16 was found in 46% of the cervical carcinomas. Most of the type 16 occurences (75%) represented the third stage of inooperable cases. Similarly, HPV 18 was also most frequently present in this stage as well as in carcinoma in situ and in CIN III (25%, 18%). At the same time, in condylomata acuminata, types 6 and 11 were detectable in 88.7% of cares. In all, 13.5% of the normal samples harboured HPV DNA.     

Analysis of human papillomavirus types in exophytic condylomata acuminata by hybrid capture and Southern blot techniques.

Exophytic condylomata acuminata of the external genitalia of 40 patients were analyzed for human papillomavirus (HPV) DNA by the Southern blot and hybrid capture methods. All lesions were initially analyzed by the Southern blot method by using a mixture of HPV type 6, 11, 16, and 18 whole genomic probes. Southern blots demonstrated characteristic PstI restriction patterns of HPV type 6, 11, or 16 in all but one lesion. HPV 6 subtypes accounted for 28 of 39 HPV-positive lesions. Twenty-seven of these 28 lesions contained HPV type 6a, and 1 lesion contained HPV type 6c. Eight lesions contained HPV type 11 and three contained HPV type 16. Two of the three condylomata acuminata containing HPV type 16 were obtained from solid-organ transplant recipients receiving immunosuppressive medications. The third lesion containing HPV type 16 was a typical exophytic condyloma acuminatum from a woman with previously resected vulvar carcinoma. The hybrid capture assay detected HPV DNAs in all lesions except the Southern blot-negative lesion. Twenty-five lesions were positive for the A probe only (HPV types 6 and 11 and related types). All of these lesions were found to contain HPV type 6 or 11 sequences in the Southern blot assay. The remaining 14 lesions were positive for both the A probe and the B probe (HPV types 16 and 18 and related types). The strongest signal in these 14 lesions by the hybrid capture assay was consistent with the result of the Southern blot assay in all but one case. We conclude that (i) HPV type 6a is the most common type found in these lesions, (ii) HPV type 16 may be present more often in exophytic condylomata acuminata from immunosuppressed individuals, (iii) hybrid capture is a useful tool for documenting the presence of HPV sequences in DNAs from exophytic condylomata acuminata, and (iv) in samples containing multiple HPV types, hybrid capture allows detection of minority HPV types.     

Detection of Multiple Human Papillomavirus Types in Condylomata Acuminata Lesions from Otherwise Healthy and Immunosuppressed Patients

Condylomata acuminata, or genital warts, are proliferative lesions of genital epithelium caused by human papillomavirus (HPV) infection. HPV types 6 and 11 are most often detected in these lesions. Genital lesions consistent with exophytic condylomata acuminata were removed by excision biopsy from 65 patients, 41 of whom were otherwise healthy individuals (control group) and 24 of whom had conditions known to cause immunosuppression. Histologically, the majority of the lesions were typical condylomata acuminata. Three lesions removed from immunosuppressed individuals also contained foci of moderate to severe dysplasia (intraepithelial neoplasia grade II/III). A recently developed PCR and reverse blot strip assay was used to determine the specific HPV types present in the genital lesions. With a set of oligonucleotide primers based on the same primer binding regions used for the MY09 and MY11 primer pair, this PCR assay detects the presence of 27 HPV types known to infect the genital tract. All but two condylomata acuminata contained either HPV type 6 or 11. The predominant type in the lesions from control patients was HPV 6, while lesions from immunosuppressed types most often contained HPV 11. Condylomata acuminata from immunosuppressed patients contained significantly more overall HPV types than lesions from the control group. HPV types associated with an increased risk of dysplasia (high-risk types) were detected in 42 (64.6%) of the total of 65 specimens; 18 (43.9%) specimens were detected in the 41 otherwise healthy individuals, and 24 (100%) specimens were detected in the 24 immunosuppressed patients. HPV 16 was the most common high-risk type detected, found in 21 of 65 (32.3%) specimens. After HPV types 6 and 11, HPV types 53 and 54 were the most frequently detected low-risk HPV types. This study demonstrates that a high percentage of condylomata acuminata lesions contain multiple HPV types, including types associated with a high risk of dysplastic abnormalities. Further studies are needed to determine the influence these additional HPV types have on the epidemiology of genital tract HPV infections and the natural history of condylomata acuminata, especially in immunosuppressed patients.     

Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections.

A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and 35S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.     

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.     

Successful application of indirect in-situ polymerase chain reaction to tissues fixed in Bouin''s solution

AIMS: To evaluate the value of polymerase chain reaction-in situ hybridization (PCR-ISH) for the detection of human papillomaviruses (HPV) in paraffin sections of cervical biopsies fixed either in 10% formalin or in Bouin's solution. METHODS AND RESULTS: We analysed 40 biopsies from Italian women infected with the human immunodeficiency virus type 1 (HIV 1). In-situ hybridization techniques were performed with commercial biotinylated probes. The PCR-ISH was carried out by the 'hot start modification'. Cervical intraepithelial neoplasia (CIN) was found in 23 of 40 patients (57. 5%); eight cases showed condylomatous features. Human papillomavirus was detected in 42.5% by ISH and in 65% by PCR-ISH. Sixty-nine per cent of positive biopsies contained HPV 16, 18, 31 and 33. HPV 6 and 11 were found only in condylomata acuminata samples. CONCLUSIONS: The results point to a high incidence of HPV infection as well as of CIN in HIV-positive patients. Human papillomavirus type 16 appears to be most frequently associated with CIN. Polymerase chain reaction-ISH is more sensitive than ISH in the detection and typing of HPV DNA both in clinical and in 'latent' infections. The two techniques yielded the same results with either formalin- or Bouin's-fixed material.     

Detection of EBV and HPV in nasopharyngeal carcinoma by in situ hybridization

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia and is frequently associated with Epstein-Barr virus (EBV). Human papilloma virus (HPV) is an epitheliotrophic oncogenic virus that has been detected in a variety of head and neck tumors including NPC. This retrospective study was undertaken to investigate the prevalence of EBV and HPV infection subtypes 6/11 and 16/18 in 20 patients with NPC. METHODS: In situ hybridization for EBV-encoded RNA (EBER) and tyramid signal amplification of ISH for HPV DNA subtypes 6/11 and 16/18 was performed to evaluate the prevalence of EBV and HPV latency infection among Iranian Patients with NPC. RESULTS: 16 cases were classified as WHO type III (undifferentiated carcinoma) and 4 as WHO type II (non-keratinizing SCC). EBER-ISH was positive in 19 (95%) of NPCs evaluated and in one metastases from cervical primary, included in this series. Two of 20 NPC (10%) contained HPV 6/11 sequences and two of 20 NPC (10%) contained HPV 16/18 sequences, and combined EBV and HPV infection was detected in 3 of the 20 (15%) patients. CONCLUSION: Our data indicated that EBV is closely associated with NPC in Iran. In addition, a low percentage of EBV positive NPC contained HPV sequences. The significance of coexistence of EBV and HPV in NPC requires further study.     

Genital human papilloma virus infection in Oslo studied by dot blot DNA hybridization and the polymerase chain reaction.

Samples from patients with genital condyloma acuminata or with cervical condylomas and/or dysplasia and from women without cytological/clinical evidence of cervical affection were examined by dot blot DNA hybridization or the polymerase chain reaction (PCR). The PCR was much more sensitive than dot blot, more than doubling the human papilloma virus (HPV) findings. HPV DNA, mainly HPV 6/11, was detected in 18 of 19 biopsies of condyloma acuminata, whereas HPV 16 was most frequently detected in the 21 cervices (76%) with condyloma and/or dysplasia. HPV 16 was detected in eight of 103 cervical smears with no signs of infection. The prevalence of HPV 16 in cervical samples was somewhat higher than expected. This suggests that, in Oslo, HPV 16 is a common HPV type in women with cytologically normal cervices. HPV 18 was relatively rare and was detected only in combination with other HPVs.     

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.     

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.     

Demonstration of multiple HPV types in normal cervix and in cervical squamous cell carcinoma using the polymerase chain reaction on paraffin wax embedded material.

The prevalence of human papilloma virus (HPV) types 6, 11, 16 and 18 was investigated using the polymerase chain reaction on formalin fixed, paraffin wax embedded material in 19 cases of cervical squamous cell carcinoma and in 10 normal cervices. HPV DNA was detected in 16 of 19 carcinomas, with multiple types present in 11 of these. HPV 16 or 18, or both, were present in all cases in which HPV was shown. Six of 10 cases of normal cervix contained HPV; five of these contained two or more HPV types, including HPV 16 or 18, or both. This study shows the feasibility of using the PCR on paraffin wax embedded material and indicates a high rate of carriage of multiple HPV types in both normal and neoplastic cervix.     

High prevalence of human papillomavirus infection and possible association with betel quid chewing and smoking in oral epidermoid carcinomas in Taiwan

Seventeen oral epidermoid carcinomas, three oral papillomas, and 17 normal gingival tissues were tested for the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 sequences by Southern blot hybridization. Episomal HPV-16 sequences in various amounts were detected in 76.4% of the oral carcinomas and in all three cases of papilloma. However, only one of the 17 normal tissues was HPV positive with an unknown type. None of the samples contained HPV-6, -11, or -18 sequences. Examination of the habits of the patients showed that 59% of the patients were betel quid chewers and 82% were smokers. Thus, the concurrent incidence of HPV infection and betal quid chewing and/or smoking habits in oral carcinoma patients observed in Taiwan is consistent with the view that both viral and chemical factors may be involved in the process of carcinogenesis.     

Intraepithelial neoplasia of the anal canal in hemorrhoidal tissue: a study of 19 cases.

The investigators report the clinical and pathologic features of 19 cases of intraepithelial neoplasia occurring in the anal canal mucosa of routinely excised hemorrhoidal tissue, a condition that has been infrequently described. The patients were 12 women and seven men having an age range of 21 to 74 years (mean, 48 years). Two patients had coexistent anogenital condylomata acuminata. Leukoplakia of the hemorrhoidal surface was noted in two patients. Intraepithelial neoplasia arose in the transition zone of the anal canal of 11 cases, in the squamous zone of three cases, and in both sites of five cases. All were high-grade intraepithelial neoplasms; one was classified moderate to severe dysplasia, 17 exhibited severe dysplasia/carcinoma in situ, and one contained microinvasive carcinoma. Both keratinizing and cloacogenic type neoplasms were observed. Associated koilocytotic atypia was identified in 16 cases (84%). In situ hybridization for human papillomavirus (HPV) messenger RNA demonstrated HPV RNA sequences in seven of nine neoplasms (78%) studied by that technique (five HPV type 16, one HPV type 18, and one coinfection with HPV types 6 and 18). Eighteen patients had no clinically evident recurrent or progressive disease at mean follow-up of 6.6 years. Residual/recurrent intraepithelial neoplasia was noted in one patient at 1, 2, 5, and 49 months posthemorrhoidectomy. Our data indicate that incidentally discovered high-grade intraepithelial neoplasia present in hemorroidal tissue is a clinically nonaggressive lesion frequently associated with HPV infection. Hemorrhoidectomy alone is curative in most cases.     

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.     

Sinonasal papillomas and human papillomavirus: human papillomavirus 11 detected in fungiform Schneiderian papillomas by in situ hybridization and the polymerase chain reaction.

A series of 19 paraffin-embedded sinonasal papillomas (four squamous papillomas, three fungiform papillomas, nine inverted papillomas, and three cylindrical cell papillomas) were investigated for evidence of human papillomavirus (HPV) infection using immunohistochemistry (polyclonal antibody to HPV capsid antigen), in situ hybridization (DNA probes for HPV 6/11, 16/18, and 31/33/35), and the polymerase chain reaction (primers and probes for HPV 6, 11, 16, 18, and 33). All three fungiform papillomas were positive by all three techniques: immunohistochemistry, in situ hybridization for HPV 6/11, and the polymerase chain reaction for HPV 11. None of the other lesions contained detectable HPV using the specific probes included in this study. These results support the continued classification of fungiform papilloma as a distinctive variant of schneiderian papilloma characterized by a predominantly exophytic growth pattern and an association with HPV 11.     

Ultrastructural localization of human papilloma virus by nonradioactive in situ hybridization on tissue of human cervical intraepithelial neoplasia.

BACKGROUND: A nonradioactive in situ hybridization was developed to localize human papilloma virus (HPV) at the ultrastructural level. EXPERIMENTAL DESIGN: Cervical biopsies from human uterine cervices clinically suspicious of condyloma were embedded in Lowicryl K4M at low temperature. Postembedding in situ hybridization was performed with DNA probes specific for HPV types 6/11, 16, and 18. The hybrids were detected by anti-horseradish peroxidase antibodies conjugated with 10 nm colloidal gold particles. RESULTS: Localization for HPV 16 and 18 both was to intranuclear and cytoplasmic sites. Cytoplasmic detected HPV signals were between masses of intermediate filaments and in vacuoles; other organelles were devoid of positive signal. Within the nucleus the precise localization of the viral nucleic acid was episomal, vacuolar, and chromosomal. In situ hybridization with plasmid control DNA confirmed the specificity of the HPV positive signals. CONCLUSIONS: This study helps define the subcellular compartmentalization of HPV DNA in infected human cells.     

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.     

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.     

Filaggrin expression in cutaneous and mucosal human papillomavirus induced lesions

A series of 32 human papillomavirus induced lesions derived from epidermis and mucosa was studied for the modulation of filaggrin-profilaggrin (F-PF) expression according to the degree of virus infection as compared to normal skin and mucosa biopsies. This investigation was carried out on frozen sections using indirect immunofluorescence for filaggrin detection and group specific viral antigen and by in situ hybridization with biotinylated probes for viral DNA detection and typing. The 9 cutaneous warts showed an increase of F-PF expression in upper layer cells as compared to normal epidermis, which could be related to the high production of virus (viral antigen and HPV types 1 or 2). The 5 condyloma acuminata displayed also an enhanced expression of these components which was located in several upper layers but virus infection was confirmed in 2 of them with HPV types 6, 11 or 16. The 6 laryngeal papillomas exhibited a granular reactivity pattern for F-PF in suprabasal cell layers with an increase in the upper layers; viral antigen was found in 4 cases and HPV DNA types 6, 11 or 16 were detected in 4 specimens. Conversely among 12 cervical intraepithelial neoplasia, F-PF was expressed only in very superficial layers in few cases, without any correlation with the DNA detection (6, 11 or 16, 18). Taken together these data are suggestive of an intense expression of F-PF in benign lesions which can replicate the virus and a discrete or an absent expression of these components in premalignant or malignant lesions.