In situ detection of HPV DNA in oral mucosal lesions. A comparison of two hybridization kits

The purpose of this study was to compare the sensitivity of ViraType in situ hybridization kit (Life Technologies, Inc. [LT] and PathoGene (Enzo Diagnostics, Inc. [ED]) in situ hybridization kit for human papillomavirus (HPV) DNA detection in oral tissue. Forty benign oral lesions histologically suspicious for HPV infection were analyzed. Specimens were hybridized with DNA probes specific for HPV types 6/11, 16/18, and 31/33/35 [LT] and HPV types 6/11, 16/18, and 31/33/51 [ED]. Positive hybridization reactions were seen for HPV DNA type 6/11 only. Hybridization occurred significantly more often (p less than 0.01, McNemar Exact Test) in LT probed specimens (20/40) than ED assayed sections (12/40). HPV DNA sequences were found in 100% condyloma acuminata (13/13), 100% verruca vulgaris (4/4), and 13% squamous papilloma (3/23) using the LT system. The ED probes yielded positive signals in 77% condyloma acuminata (10/13), 25% verruca vulgaris (1/4), and 4.4% squamous papilloma (1/23). A more intense hybridization signal was exhibited using the LT system. The results indicate that the LT probes and detection reagents are more sensitive for detecting HPV DNA in oral mucosal specimens.     

Focal epithelial hyperplasia arising after delivery of metal-ceramic fixed dental prosthesis

Focal epithelial hyperplasia (FEH) is a human papillomavirus (HPV)-induced alteration of the oral mucosa that presents with a clinically distinct appearance. While other HPV-infected lesions such as squamous papilloma, verruca vulgaris, and condyloma acuminatum involve the skin, oral mucosa, and genital mucosa, FEH occurs only in the oral mucosa. The affected oral mucosa exhibits multiple papules and nodules with each papule/nodule being flat-topped or sessile. The affected region resembles the normal color of oral mucosa rather than appearing as a white color since the epithelial surface is not hyperkeratinized. Almost all cases present with multiple sites of occurrence. This rare, benign epithelial proliferation is related to low-risk HPV, especially HPV-13 and -32, and is not transformed into carcinoma. We report a case of FEH that arose on the attached gingiva of an East Asian male adult related to prosthesis without detection of any HPV subtype in HPV DNA chip and sequencing.     

Oral squamous papillomas: detection of HPV DNA by in situ hybridization

Oral squamous papillomas were segregated from other papillary lesions on the basis of histopathologic features. Twenty representative papillomas were evaluated for the presence of papillomavirus genus-specific antigen with the use of an immunoperoxidase technique. These same tumors were analyzed for human papillomavirus (HPV) types 2, 4, 6, and 11 with biotinylated full-length double-stranded DNA probes by in situ hybridization. Only one case exhibited papillomavirus antigen reactivity. Alternatively, seven of twenty cases (35%) yielded positive results for HPV 6 or 11 DNA; one papilloma exhibited a dual infection with both HPV 2 and 6 when assayed under conditions of high-stringency hybridization. It is concluded that some oral squamous papillomas harbor HPV genotypes akin to those encountered in genital tract condylomas. Viral DNA can be detected in the absence of capsid antigen immunoreactivity, thereby obviating the use of antigen detection assays for determining the presence or absence of virus.     

In situ DNA hybridization analysis of oral papillomas, leukoplakias, and carcinomas for human papillomavirus.

Twenty-one papillomas, 23 ordinary benign keratoses, 13 smokeless tobacco keratoses, 10 verrucous hyperplasias, 10 verrucous carcinomas, 17 squamous cell carcinomas, 3 epithelial dysplasias, and 6 lichen planus lesions were evaluated for human papillomavirus (HPV) types 6/11, 16/18, and 31/33/35, with biotinylated double-stranded DNA probes by in situ hybridization. Sixty-two percent (13/21) of oral squamous papillomas were positive for HPV DNA. HPV DNA types 6 and 11 demonstrated the strongest reactivity. Of the 13 cases, 10 also showed some reactivity with HPV-16/18 and -31/33/35. None of the cases of keratoses, epithelial dysplasia, squamous cell carcinoma, verrucous hyperplasia, verrucous carcinoma, or lichen planus were positive for HPV DNA. This study confirms the consistent and frequent finding of HPV DNA in oral squamous cell papillomas and the inconsistency of being able to identify HPV DNA in keratotic, premalignant, or cancerous lesions of the oral mucous membranes.     

Oral wart associated with human papillomavirus type 2

More than 100 human papillomavirus (HPV) types have been identified to date. Of these, 24 types have been described as being associated with oral lesions. HPV-2 has been frequently associated with skin lesions, but the reports of oral lesions as features of mucosal infection are limited. A biopsy specimen of an oral wart on the right palate was taken from a 48-year-old man and examined for the presence of HPV The sections showed papillary growth of the epithelium with hyperkeratosis and parakeratosis, and koilocytotic changes of the cells located in the upper layers of the oral squamous cell epithelium. These histological features corresponded well to those of verruca vulgaris on the skin. Immunohistochemically, papillomavirus genus-specific capsid antigen was detected in most of the koilocytotic cells. In addition, Southern blot hybridization analysis revealed that the lesion harbored HPV-2 DNA. In situ hybridization with a biotinylated HPV-2 DNA probe clearly demonstrated viral DNA in the nuclei of squamous cells, which were located in a deeper layer of the epithelium than viral antigen-positive cells.     

Condyloma acuminatum and human papilloma virus infection in the oral mucosa of children

PURPOSE: The purpose of this study was to investigate the clinicopathological features of oral condylomas in children and condylomatous lesions of their mothers. Moreover, the authors sought to determine the mode of transmission of this disease and to find the genotype of human papilloma virus (HPV) in the children's oral condyloma. METHODS: Nine instances of oral condyloma acuminatum in children and lesions in their mothers were reviewed. Their HPV genotypes were evaluated by in situ hybridization (ISH). RESULTS: This study revealed that the lesions appeared during 3 years of age and the most common location was the hard and soft palate. Seven of the 9 mothers had experienced vulva and/or oral cavity condylomata during pregnancy. Social evaluation confirmed sexual abuse in 1 girl, and probable sexual abuse in another girl. The results of ISH demonstrated HPV 16/18 DNA being positive in 5 of the 9 cases, and HPV 6 and HPV 11, HPV 6 and HPV 16/18, HPV 6, and HPV 11 DNA being positive, respectively, in 1 case. HPV DNA types in mother-child pairs were not concordant. CONCLUSIONS: Oral condyloma acuminatum in children is probably induced by HPV 16/18. The mode of transmission by sexual abuse is the most likely route. Prenatal transmission of HPV to children is rare. This study provides further confirmation of possible different genotype and transmission in oral CA of children and adults.     

Identification of human papillomavirus types in oral verruca vulgaris

Eleven oral verruca vulgaris specimens were examined for the presence of papillomavirus structural antigens by reaction with antibody to type-common antigens and detection by the avidin-biotin-peroxidase complex method. The specimens were also examined by in situ hybridization with biotin-labelled human papillomavirus (HPV) DNA to determine the specific HPV types present in the lesions. Six of the 11 specimens were positive for papillomavirus structural antigens. Of these 6, 5 hybridized to the HPV Type 2 (HPV2) probe and one to the HPV4 probe.     

Human papillomavirus (HPV) DNA in focal epithelial hyperplasia by in situ hybridization

Eighteen cases of focal epithelial hyperplasia (FEH) were investigated for the presence of human papillomavirus (HPV) group specific antigen by immunocytochemistry and HPV types 1, 6, 11, 13, 16, 18 and 32 by DNA in situ hybridization employing biotinylated probes. Seven (39%) specimens demonstrated the presence of HPV group specific antigen. Fifteen (83%) specimens were positive for HPV DNA: 9 (60%) showed HPV 32, of which 6 were on non-keratinized mucosa and 3 on border of keratinized and non-keratinized mucosa; 5 (33%) showed HPV 13, 4 lesions on keratinized mucosa and 1 on non-keratinized mucosa; 1 (7%) specimen on non-keratinized mucosa showed HPV-11 related. Two specimens on different sites from one patient showed the same HPV type and one patient had, in addition to FEH, a squamous papilloma also demonstrating the same HPV type. Results show a specific HPV distribution pattern in the epithelium indicating areas of high viral concentration adjacent to areas of low or no viral concentration. This study also indicates the possibility of tissue-site specificity or a latent infection and the possibility of a yet unidentified HPV type associated with FEH. It is suggested that future monitoring of patients be carried out with special reference to HPV type and anatomical distribution pattern for FEH lesions.     

The histologic differentiation of oral condyloma acuminatum from its mimics

OBJECTIVE: The purpose of this study was to determine which histologic features could enable one to distinguish oral condyloma, as defined by the detection of human papillomavirus (HPV) DNA through in situ hybridization, from its mimics. STUDY DESIGN: Thirty-two paraffin-embedded specimens from 28 patients that were clinically suggestive of oral condyloma were analyzed histologically and through in situ hybridization with a consensus HPV probe. RESULTS: HPV DNA was detected in 17/32 (53%) of the lesions; no additional positive cases were detected after polymerase chain reaction amplification. Only 5 of the 17 virus-positive cases were considered to be histopathologically unequivocal for condyloma. The histologic features significantly associated with HPV detection were nonuniform perinuclear halos, often in association with epithelial crevices (P =.02), and papillomatosis (P =.02). Each of the 17 patients who were HPV-positive had either HPV-6 or HPV-11, a finding that is similar to those for condyloma involving the penis and vulva/vagina. CONCLUSION: Differentiation between oral condyloma and its mimics is best accomplished by using a combination of histologic, clinical, and in situ viral studies.     

HPV in oral squamous cell carcinomas of a Brazilian population: amplification by PCR

Human Papilomaviruses (HPV) are a group of viruses associated with benign and malignant lesions of cutaneous and mucosal epithelia. Some "high risk" HPV types, especially HPV 16 and 18, are strongly correlated with cervical and anogenital cancers and are also related to the genesis of oral squamous cell carcinomas (OSCC). The aim of this work was to investigate the incidence of HPV infection in 40 paraffin-embedded or fresh specimens of OSCC, using PCR amplification of the viral DNA. Literature based primers (GP5+/GP6+) were used in order to amplify HPV DNA from the L1 gene, present in more than 22 types of HPV. A condyloma case with HPV 16 and 18 detected by in situ hybridization was used as a positive control. Amplification of HPV was observed only in the positive control. No squamous cell carcinoma cases showed DNA viral amplification. Absence of HPV DNA amplification by PCR in the analyzed specimens of OSCCs suggests that this virus not always plays a role in the carcinogenesis process. Discrepancy with some studies found in the literature may be related to methodology or population differences.     

Identification of human papillomavirus types in oral verruca vulgaris

Abstract Eleven oral verruca vulgaris specimens were examined for the presence of papillomavirus structural antigens by reaction with antibody to type-common antigens and detection by the avidin-biotin-peroxidase complex method. The specimens were also examined by in situ hybridization with biotin-labelled human papillomavirus (HPV) DNA to determine the specific HPV types present in the lesions. Six of the 11 specimens were positive for papillomavirus structural antigens. Of these 6, 5 hybridized to the HPV Type 2 (HPV2) probe and one to the HPV4 probe.     

Human papillomavirus (HPV) types 2 and 57 in oral verrucae demonstrated by in situ hybridization

Twenty-one cases of verrucae vulgaris (oral warts) were investigated for human papillomavirus (HPV)-group specific antigen by immunocytochemistry and for HPV types 1, 2, 4, 6, 11, 16, 18 and 57 by DNA in situ hybridization with biotinylated probes. Twelve (57%) cases demonstrated the presence of HPV-group specific antigen. Fifteen (71%) cases showed the presence of HPV DNA, 13 of which (87%) demonstrated both HPV types 2 and 57 in the same cells and 2 of which (13%) demonstrated only HPV 2. Six cases were negative for HPV 2 and 57 and all 21 cases (100%) were negative for HPV types 1, 4, 6, 11, 16 and 18. Results indicate the association of a new and as yet unidentified HPV type, closely related to HPV 2 and 57, with oral warts. The identification of both cutaneous type HPV 2 and another type closely related to HPV 2 and 57 in oral verrucae on keratinized and non-keratinized mucosal surfaces indicates the possibility of a latent infection; three patients had a history of warts on their hands, suggesting autoinoculation. This study indicated that future investigations of oral warts, based on a correlation of clinical and histological features with HPV types by DNA in situ hybridization, are called for.     

In situ detection of human papillomavirus Types 13 and 32 in focal epithelial hyperplasia of the oral mucosa

17 cases of focal epithelial hyperplasia of the oral mucosa (FEH, Heck's disease) were investigated for the presence of human papillomavirus (HPV) nucleic acid sequences by means of in situ DNA hybridization using biotinylated DNA probes of HPV types 1, 6, 11, 13, 16, 18, and 32. Ten of 17 cases were positive for HPV 13 DNA in contrast to 6 of 17 positive cases obtained after application of the HPV 32 probe, with a double infection in one case. The results of our study suggest, that HPV 13 and HPV 32 are very specifically found in lesions of FEH and can be detected in a high percentage of cases using in situ hybridization.     

Immunohistochemical demonstration of human papilloma virus (HPV) antigens in oral squamous cell lesions

Six oral squamous cell tumours classified as focal epithelial hyperplasia (FEH), Condyloma acuminatum (CA) and squamous cell papilloma (SQP) were subjected to indirect immunoperoxidase staining with anti-human papillomavirus (anti-HPV) antiserum to demonstrate the possible presence of HPV antigens in these lesions. The results are discussed in the light of the observations on HPV-lesions elsewhere in the body (in uterine cervix), and a suggestion is made to adopt the name condyloma for all those tumours where HPV aetiology can be established by ultrastructural or immunohistochemical means.     

Detection of human papillomavirus in oral lesions using commercially developed typing kits

Biopsy material from 20 oral lesions (19 condylomas and 1 squamous papilloma) previously shown to contain human papillomavirus (HPV) 6 and HPV 11 sequences by in situ hybridization were examined using 3 commercially available HPV typing kits. Sensitivity and specificity were compared with in-house methods. Previous in situ hybridization had detected HPV 6b in 11 (55%) of the biopsies, HPV 6 and 11 in 7 (53%) and HPV 11 alone in 1 biopsy. Only one of the commercial assays (assay 1) detected HPV in all 20 biopsies (11 positive for HPV 6b only, 1 for HPV 11 only and 7 for HPV 6b and 11). The wide spectrum probe of assay 2 detected HPV in only 10 (50%) of the biopsies, and in a further 2 biopsies the hybridization results were difficult to interpret because of background staining. Assay 3 used a combined HPV 6/11 probe and detected HPV in 15 (75%) of the biopsies. Clear hybridization signals were demonstrated in the intermediate and upper layers only of squamous epithelium, as expected from the known association of HPV replication with epithelium differentiation. In most specimens background levels were not a problem, and all commercial assays were easy to use. The findings are discussed in the context of the digestion procedures, sensitivity of the probes provided and the conditions of hybridization, all of which would influence the detection of HPV     

Papillary lesions of the oral cavity: relationship to human papillomaviruses

Human papillomaviruses are a group of genetically related organisms that infect stratified squamous epithelium. Unlike many other viruses that infect oral epithelium and induce lysis of the cells they penetrate, HPVs induce proliferative changes in these cells that result in both benign and malignant tumors. The common skin wart (verruca vulgaris) is induced by HPV 2 and 4. Genital warts (condylomas) and the common solitary oral papilloma are associated with HPV 6 and 11. Either HPV 13 or 32 causes focal epithelial hyperplasia. All of these wart-like lesions are benign growths of the stratified squamous lining of the oral cavity and lips and can be treated by surgical excision or laser ablation. HPV 16 and other less frequently encountered genotypes are associated with uterine cervix cancer in 95 percent to 98 percent of cases, and the evidence for a causal role is robust. There are emerging data that implicate HPV in certain subsets of oral cancer, particularly those that arise in the oropharynx/tonsillar region. Some instances of the various histologic subtypes subsumed under proliferative verrucous leukoplakia and verrucous carcinoma also harbor HPV.     

Human papillomavirus as a risk factor in oral carcinogenesis: a study using in situ hybridization with signal amplification

Introduction:  It is still controversial whether human papillomavirus (HPV) can be considered a risk factor in oral carcinogenesis. The aim of this study was to detect HPV DNA in 50 cases diagnosed as oral leukoplakias, with different degrees of epithelial dysplasia, and as oral squamous cell carcinomas, using in situ hybridization with signal amplification (CSA-ISH).
Methods:  HPV DNA was assessed in paraffin sections using CSA-ISH with a wide-spectrum biotinylated DNA probe. In HPV-positive cases, genotyping with specific probes to HPV types 6/11, 16/18 and 31/33 was performed.
Results:  The overall prevalence of HPV infection was 24%, markedly higher than that found in the control group. Results showed a discrete proportional relationship in the indices found in leukoplakia with no dysplasia, leukoplakia with dysplasia, and squamous cell carcinoma, but this was not statistically significant. When separating the group of leukoplakia by degrees of dysplasia, this relation of proportion was not observed. In genotyping, HPV types 16/18 were the most prevalent, and types 6/11 were only found in groups of mild or no dysplasia.
Conclusion:  The results suggest that HPV is not likely to play a role in the progression of malignant transformation in oral lesions. Nevertheless, the increased prevalence of HPV infection compared to normal oral mucosa and the fact that high-risk HPV types were the most frequently identified do not allow the exclusion of HPV as a risk factor in oral carcinogenesis.     

Detection of human papillomavirus-related oral verruca vulgaris among Venezuelans

A sensitive in situ hybridization test under low stringency conditions (LCS) with a set of digoxigenin-labeled human papillomavirus mixed probes (D-L HPV MP) revelaed a positive reaction in 8 of 10 cases of oral verruca vulgaris (OVV), Ages ranged from 5 to 37 years with a mean of 14.5 years. 50% of all cases were located intraorally on the hard palate, followed in frequency by the commissures. These preliminary findings provide evidence of the role of HPV in OVV from a sample of the Venezuelan population. We show that in situ hybridization conducted under LSC is useful in HPV detection (regardless of the type) and the digoxigenin-labeling system is a rapid, relatively easy and specific method. In addition, this technique permits the retrospective evaluation of routinely processed material, thus widening the investigative spectrum for HPV.     

Human papillomavirus (HPV) DNA sequences in oral precancerous lesions and squamous cell carcinoma demonstrated by in situ hybridization

A series of routinely processed, paraffin-embedded biopsies from 73 surgically treated oral precancerous lesions (OPL) (22 cases), and oral squamous cell carcinomas (SCC) (51 cases), was first screened using an in situ DNA hybridization technique with a human papillomavirus (HPV) DNA probe cocktail containing the 35S-labelled DNA of HPV types 6, 11, 13, 16, 18 and 30. The specific HPV types in lesions shown to contain HPV DNA in this procedure were further analysed by using in situ hybridization and the 6 HPV DNA probes separately. A total of 12/73 (16.4%) of the lesions proved to contain HPV DNA; 6/51 (11.8%) carcinomas and 6/21 (28.6%) dysplasias. The most frequent sites of HPV DNA-positive lesions were palate (4/7; 57%), followed by the floor of the mouth (2/8; 25%), the tongue and gingiva (11.8%). HPV 13 or HPV 30 were not found in any of the lesions studied. HPV 11 DNA was demonstrated in 2 mild dysplasia lesions, but not in carcinomas. One additional mild dysplasia proved to contain HPV 6 DNA. HPV 16 DNA was present in 5 biopsies; 3 carcinomas and 2 dysplasias. In one of the HPV 16-positive carcinomas, HPV 18 DNA was simultaneously present. HPV 18 alone was found in 3 additional carcinomas and in one moderate dysplasia lesion. The results confirm the recently reported evidence on HPV involvement in OPL and oral cancer. The implications of these findings are discussed in terms of the possible HPV etiology of oral SCC. The use of the in situ DNA hybridization as a powerful tool (enabling the localization of specific HPV DNA sequences and the proper classification of the lesion at the same site) in the study of routinely processed oral biopsies is strongly advocated.     

Study of human papillomavirus in oral epithelial dysplasia and epidermoid carcinoma in the absence of tobacco and alcohol use

Human papillomaviruses (HPVs) are implicated in the etiology of benign and malignant mucosal lesions in both human beings and animals. The oncogenic role of HPV in malignant cervical lesions is well supported by DNA hybridization techniques and epidemiologic studies. However, the role of HPV in oral epithelial dysplasia and epidermoid carcinoma remains speculative. In this study the in situ hybridization technique was used to detect HPV genotypes 6 and 11; 16 and 18; and 31, 33, and 35 in tissue specimens from a study group consisting of 18 patients who were non-tobacco users, or non-tobacco and non-alcohol users, in whom oral epithelial dysplasia and epidermoid carcinoma developed. The hybridization findings were compared with those of a comparable control group of patients with similar lesions who had a history of tobacco and alcohol use. None of the study group cases was reactive with any of the DNA probes. Two cases of the control group showed positive hybridization with HPV DNA probe types 6/11 and 16/18. The implications of these findings are presented and discussed in an attempt to clarify the role of HPV in HPV-associated oral epidermoid carcinomas.