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.     

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     

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.     

Detection of human papillomavirus DNA in an ameloblastoma using the in situ hybridization technique

HPV type 18 DNA was identified in an intrabony ameloblastoma using radio-labelled in situ hybridization. The viral DNA was found in a verrucous lesion in a cystic area of the tumor. The absence of HPV DNA in other epithelial areas of the ameloblastoma is suggestive of a secondary infection. HPV is not considered to be an etiological factor in the pathogenesis of this ameloblastoma.     

Risk of oral cancer associated with human papillomavirus infection, betel quid chewing, and cigarette smoking in Taiwan — an integrated molecular and epidemiological study of 58 cases

BACKGROUND: The association between oral squamous cell carcinoma (OSCC) and human papillomavirus (HPV) 6, 11, 16 and 18 is uncertain. Past reports varied in the methodology and results. We conducted this study using in situ PCR in situ hybridization (ISH) assay which was considered as the most sensitive method for detection of viral DNA. We undertook an epidemiologic survey about the history of betel quid chewing and cigarette smoking, since these habits are common in Taiwan. METHODS: In situ PCR ISH was performed on the tumor specimens from 29 patients with OSCC and the oral mucosal specimens from 29 patients without OSCC. Their betel quid chewing and cigarette smoking histories were also reviewed. RESULTS: HPV16, HPV18, betel quid chewing and cigarette smoking were statistically significant risk factors in univariate analysis. HPV6 and 11 were not. Multivariate analysis showed that HPV16 infection (adjusted Odds ratio = 11.20) and betel quid chewing (adjusted Odds ratio = 17.06) remained to be independent factors for OSCC. CONCLUSIONS: Our results showed that HPV16 and betel quid chewing were two major risk factors for OSCC in Taiwan, indicating that they act through different mechanisms in the pathogenesis of OSCC.     

Highly sensitive detection of HPV-DNA in paraffin sections of human oral carcinomas

BACKGROUND: Although human papillomavirus (HPV) infection has been shown to be a significant carcinogen in cervical squamous cell carcinoma (SCC), its significance in oral SCC remains unclear. METHODS: We developed highly sensitive detection methods for HPV to elucidate the prevalence and localization of HPV in paraffin sections from human oral SCC using modified in situ polymerase chain reaction (PCR) and in situ hybridization AT tailing (ISH-AT). Analyses revealed a high prevalence of several HPV types (HPV-16, -18, -22, -38 and -70) under optimal conditions. The ISH-AT method can be used as an alternative to in situ PCR. RESULTS: Various staining patterns were observed in the 20 cases examined, and HPV-positive cells were localized within the surface epithelium as well as in neoplastic cells. We demonstrated that HPV-DNA could be detected in paraffin sections using either the method of in situ PCR or ISH, providing an appropriate primer and probe are used. CONCLUSION: These results suggest that HPV infection could be one of several risk factors being involved in oral SCC.     

High risk human papillomavirus in oral squamous carcinoma: evidence of risk factors in a Venezuelan rural population. Preliminary report

In a search for the presence of human papillomavirus (HPV) and some etiologic cofactors in oral squamous cell carcinoma (OSCC), 50 women diagnosed as OSCC were analyzed by a multiplex polymerase chain reaction (PCR) assay specific for HPV types 6, 11, 16, and 18. This study revealed that 60% (30/50) of the OSCC patients were positive for HPV-DNA sequences. This group was analyzed according to smoking, alcohol consumption, number of pregnancies, poor oral health and low social economic status. The current results indicate an increased incidence of HPV malignant types in the oral cavity in women with OSCC. Also, they support a multifactorial model of oral cancer causation.     

Use of quantum dots to detect human papillomavirus in oral squamous cell carcinoma

Objective:  To examine the association of oral squamous cell carcinoma with human papillomavirus (HPV) using quantum dots (QD) in situ hybridization (ISH).
Methods:  Expression of HPV16/18 was analyzed in a representative collection of 21 oral squamous cell carcinomas by tissue microarrays. The presence of HPV16/18 high risk was detected by applying QDISH which is compared with conventional ISH.
Results:  Seven cases out of 21 (33.3%) were positive for QDISH while 1 out of 21 (4.8%) was positive for ISH, although all of HPV DNA were localized in the nuclei in the spinous and basal cell layer of the epithelium. The difference between these two methods was significant ( P  < 0.05).
Conclusions:  These results demonstrate that the QD might be an efficient method for determination of HPV infection and HPV-associated oral squamous cell carcinoma.     

High prevalence of human papillomaviruses in the normal oral cavity of adults

Human papillomavirus (HPV) infection in the normal oral cavity was studied by the sensitive polymerase chain reaction (PCR) using primers for the L1 region of human papillomavirus DNA and high fidelity amplification system. Cells were scraped from the oral mucosae of 7 (mean age; 42 years) and 30 (mean age; 32 years) volunteers with and without skin warts, respectively. Human papillomavirus DNA was detected in 30/37 (81.1%) specimens and their copy numbers per cell were 10(-1) to 10(-4) (mean, 10(-3)). The human papillomavirus types determined by PCR-based sequencing analysis were HPV-18 (26/30; 86.7%), -61 (18/30; 60%), -59 (7/30; 23.3%), -16 (2/30; 6.7%), -6 (1/30; 3.3%) and an unknown type (HPV-X71) (1/30; 3.3%). Multiple human papillomavirus types were present in 17/30 (56.7%) specimens. HPV-6 was detected in 2 of 7 skin warts and differed from the human papillomavirus types of the corresponding oral specimens. These data suggest that human papillomavirus infection in the oral mucosa occurs much more frequently than previously considered.     

The prevalence and distribution of human papillomavirus genotypes in oral epithelial hyperplasia: proposal of a concept

Background:  Evidence is accumulating for the aetiological role of human papillomavirus (HPV) in the pathogenesis of potentially malignant oral mucosal lesions and squamous cell carcinomas.
Methods:  Paraffin tissue sections from 49 patients with 'white patches' of the oral mucosa were investigated histologically, by broad-spectrum PCR followed by genotyping and chromogenic in situ hybridisation (CISH).
Results:  Histologically, 33 flat hyperplasias and 16 papillary hyperplasias were diagnosed. Twenty-two of 28 samples studied (78.6%) were positive for HPV DNA by PCR and six were negative. The following HPV types were detected in decreasing order of prevalence: HPV 35, HPV 6, HPV16, HPV 53, HPV 18, HPV 51 and HPV 55. Seventeen samples (60.7%) contained high-risk HPV DNA. Using CISH, ≥ 1 HPV signals were detected at least in a few epithelial cells in 95% of cases studied. All but one case were positive with the high-risk HPV probe and all HPV infections contained low viral load. Concordant positive results both by PCR and CISH were detected in 14 of 19 cases (73.7%) analysed.
Conclusions:  The high prevalence of HPV infection in hyperplastic 'white patches' of the oral mucosa supports the putative role of HPV at an early stage of oral carcinogenesis. These results further indicate that the majority of white oral mucosal lesions – flat, exophytic, wart-like or papillary proliferations – could be considered as the clinical manifestations of oral HPV infection. This finding has clinical relevance regarding therapy and patient management and may help in elucidating the role of HPV infection in oral carcinogenesis.     

Detection of human papillomavirus DNA in benign oral squamous epithelial lesions in Venezuela

The polymerase chain reaction (PCR) was applied to the detection of human papillomavirus (HPV) infection in biopsies taken from clinically normal oral mucosa of 20 subjects and clinical lesions of 40 patients. PCR for HPV-DNA amplification was performed using consensus primers MYO9/MYO11 and subsequent typing for HPV of high and low oncogenic risk HPV types were identified by restriction enzyme analysis (restriction fragment length polymorphism, RFLP). The HPV viral genome was present in 55% (22/40) of the oral benign lesions (OBL) and in 10% (2/20) of the control samples. In the PCR+ OBL, we observed 90.9% of low oncogenic risk types (HPV-6 -13 and -32) and 9.1% of the samples had a mixed infection with low and high oncogenic types (HPV-6 and -16). In the control samples, we observed one patient with HPV-6 and another with HPV-6 and -16 in the same sample. All of the eight focal epithelial hyperplasia cases were positive for low risk HPV types (88% HPV-13 and 12.5% HPV-32). In conclusion, this study demonstrates a high incidence of HPV in oral benign lesions from Venezuelan patients.     

In situ hybridization study on tissue inhibitors of metalloproteinases (TIMPs) mRNA-expressing cells in human inflamed gingival tissue

This study presents the exact cell types and localization of tissue inhibitors of metalloproteinases (TIMPs) production sites in periodontal diseased gingiva by means of in situ hybridization. Gingival tissue specimens were fixed, embedded and hybridized in situ with specific digoxigenin-labeled cRNA probes (386 and 496 bp). TIMP-1 and -2 mRNAs were expressed on macrophages, mononuclear cells, capillary endothelial cells and some fibroblasts throughout the gingival tissue. In periodontitis, TIMP-1 and -2 mRNA-expressing cells showed significantly different localization. TIMP-1 mRNA was broadly observed in the gingival connective tissue while TIMP-2 mRNA was predominantly expressed in the connective tissue adjacent to the pocket epithelium (p < 0.01). Fewer TIMPs mRNA were observed in minimal gingivitis than in periodontitis, especially in the middle zone of gingival tissue. Thus, TIMP-1 and TIMP-2 mRNA was detected differentially and site-specifically in periodontal diseased gingival tissue.     

A polymerase chain reaction (PCR) investigation of oral verrucae which contain HPV types 2 and 57 by in situ hybridization

The polymerase chain reaction (PCR) and direct DNA sequencing have been used to identify strain variants of HPV types 2a/57 in formalin-fixed sections of human oral verrucae, where the virus had previously been detected by both immunofluorescence and in situ hybridization. By employing type-specific and type-common PCR primers we show that these lesions contain a mixture of viral DNAs which vary by up to 27% in DNA sequence, in a region where the variation between HPV types 2a and 57 is only 4%. The extra discriminatory power of fluorescent sequencing indicates that the lesions may also contain wild-type HPV2a/57 DNA which could provide a helper function for defective viral DNA molecules or indicate a mosaic origin for the lesions.     

Detection of Epstein-Barr virus DNA in tongue tissues from AIDS autopsies without clinical evidence of oral hairy leukoplakia

Epstein-Barr virus (EBV) DNA was detected by in situ hybridization at 3 sites of 30 samples taken from clinically normal lateral border of tongue mucosa from 15 AIDS autopsies and in none of 20 samples from 10 controls. The first positive case showed a thin layer of parakeratosis correlated with positive signals for EBV in one area and an adjacent area without obvious parakeratosis was also positive for EBV. These findings were present on both sides of the tongue. The second case was unilaterally positive for EBV and parakeratosis was absent. The hybridization signals were localised to koilocyte-like cells in the stratum spinosum, as in oral hairy leukoplakia (OHL). These observations suggest that the in situ hybridization technique can detect very early or subclinical OHL, and supports the role of EBV in the pathogenesis of this lesion.     

儿童口腔尖锐湿疣的临床表现及HPV检测

目的:探讨儿童口腔粘膜尖锐湿疣的病毒类型、传播途径、临床病理特点及预后等。方法:回顾6 例被确诊为口腔粘膜尖锐湿疣患儿的临床特点及HE 切片,并对其中5 例采用免疫组化染色及原位杂交检测人乳头状瘤病毒(HPV)DNA。结果:儿童口腔尖锐湿疣多发生在2 岁左右,发病部位多位于腭部,并且多数有家族感染史,镜下见棘层上部或角化层常出现灶性凹空细胞,免疫组化检测结果显示5 例HPV 共同抗原全部阳性,5 例中有4 例HPV16P18- E6 阳性;原位杂交结果显示仅1 例HPV6 和HPV11 同时阳性,另1 例初发时HPV6 阳性而复发后呈阴性。结论:儿童口腔尖锐湿疣的病毒类型、传播途径可能与成人不同。     

Trends in incidence of oral and pharyngeal carcinoma in Florida: 1981-2008

Objective: While the overall incidence rates of oral and pharyngeal squamous cell carcinoma (SCC) have decreased in the United States, there is evidence of increasing incidence at selected anatomic sites, particularly among younger adults. The objective of this study was to examine trends in incidence rates of oral and pharyngeal cancers in Florida. Methods: Using data from the Florida Cancer Data System, we examined the incidence of oral and pharyngeal carcinomas in Florida from 1981 through 2008. Factors of interest included sex, race, and trends over time. Percent change (PC) and annual percent change (APC) were computed to characterize trends over time. Results: A total of 53,648 cases of oral or pharyngeal cancer were identified from 1981 through 2008. Significant increasing trends were observed only for pharyngeal cancers in males, with significant decreasing trends for pharyngeal cancer in females and oral cancer for both sexes. For tonsil and base of tongue cancers, increasing trends were detected for white males only. Further investigation among white males showed that, except for base of tongue cancer in the 20‐44 age group, the incidence of both cancers increased across all age groups, with the largest increase for both sites found in the 45‐64 age group. Conclusions: This study supports the finding of increasing incidence of SCC of the tonsil and base of tongue in males, in contrast to decreasing trends for most oral and pharyngeal carcinomas. However, we observed that this increase occurred in white males only and the most dramatic increase occurred in the 45‐64 age group.     

牙本质磷蛋白在人牙胚发育中的原位杂交研究

目的：观察牙本质磷蛋白（DPP）基因在人牙胚组织发育过程中的表达，探讨其在牙齿发育中的作用。方法：采用原位杂交的方法，检测帽状期、钟状期牙胚组织DPPmRNA的表达。结果：人牙胚帽状期、钟状早期均未检测到DPPmRNA的表达，在钟状晚期前期牙本质形成时成牙本质细胞、前成釉细胞中呈阳性表达。结论：牙本质磷蛋白基因的转录具有明显的的时空特异性，它可能在牙体硬组织的形成中起重要的作用。