Human papillomavirus in laryngeal papillomas and in adjacent normal epithelium

Eleven adults with laryngeal papillomas were studied for the presence of human papillomavirus (HPV) DNA by in situ hybridization. As well as from the papillomas, three additional biopsies were taken from the normal-appearing mucosa as follows: the involved vocal cord, the opposite vocal cord (when the papilloma was unilateral), and from the ventricular fold on the side of the lesion. These normal tissues were analysed by polymerase chain reaction (PCR) to detect HPV DNA. All except one of the 11 papillomas contained HPV DNA; nine were HPV 6/11 DNA positive and one positive for HPV 16 DNA. The normal-appearing laryngeal mucosa harboured HPV DNA in eight out of 11 patients. The present results strongly support the concept that the adult-type laryngeal papilloma is an HPV-induced lesion, mostly due to HPV types 6 and 11. The persistence of HPV DNA in the adjacent normal epithelium is consistent with the frequent recurrence of these lesions.     

Lead article

Human papillomaviruses (HPV) have been detected in squamous cell carcinoma (SCC) of the aerodigestive tract with varying frequency of 10%-100% mainly due to detection methods and primer pairs used. Polymerase chain reaction (PCR) is the most sensitive and Southern blot hybridization (SBH) the most specific detection method of HPV DNA. Both methods achieve the most reliable results. 22 SCC DNA samples of the hypopharynx were analyzed by type specific and consensus primer PCR and Southern blot analysis. HPV was detected in 5/22 (22.6%) hypopharyngeal SCC specimens. HPV 18 and HPV 45 were identified in one case each. An HPV prevalence of 23% is a realistic approximation in hypopharyngeal SCC. The high rate of HPV positive only detected by non type specific detection methods indicates the presence of previously undescribed HPV types.     

婴幼儿咽喉乳头瘤组织人乳头瘤病毒感染的探讨

目的　探讨温州地区人乳头瘤病毒感染和婴幼儿咽喉乳头状瘤的关系。方法　应用聚合酶链反应和核酸斑点杂交技术检测 35例婴幼儿咽喉乳头瘤组织和 10例对照组组织 (小儿声带小结 )HPV6、11、16、18、3 3 5个型别的DNA。结果　乳头瘤组织HPV感染率为 91 4% (30 / 35 ) ,其中HPV6型检出率为 5 4 2 % (19/ 35 ) ,HPV11型感染率为 2 5 7% (9/ 35 ) ,多重型别HPV6 11感染率为 11 4% (4/ 35 ) ;HPV16、18、3 3 型 ,均为阴性。对照组各型检测结果均为阴性 ,两者对比差异有高度显著性 (P <0 0 0 1)。结论　温州地区婴幼儿咽喉乳头瘤的发生与HPV感染密切相关 ,尤以HPV6感染为主。PCR结合核酸斑点杂交技术检测HPV具有敏感性高、特异性强的优点 ,值得推广。     

Mucosal swabs detect HPV in laryngeal papillomatosis patients but not family members

Seven patients, aged 2-7 years, with active recurrent respiratory papillomatosis (RRP) attending the University of Michigan Pediatric Otolaryngology Clinic were studied to determine if human papillomavirus (HPV) is harbored in sites of the upper aerodigestive tract other than in the laryngeal papilloma itself. We also determined if close family members had detectable virus in their oral cavities. Noninvasive swabs of buccal mucosa, posterior pharynx, nasal vestibule, and tonsillar pillar of patients, as well as buccal mucosa and posterior pharyngeal swabs of family members were studied. Swabs of the patients' papillomas served as the positive controls. HPV was detected using polymerase chain reaction (PCR) amplification and Southern hybridization techniques. Six of seven patients had detectable HPV in papilloma and endolaryngeal swabs. Four were HPV type 6, and two were HPV type 11. The patient whose swab was negative for HPV was found to be biopsy negative for papilloma 3 weeks after a single laser excision which was performed 6 months prior to the endolaryngeal swab. HPV types 16, 18 and 31 were not found in any of the patients. No swabs from other sites in patients or family members were HPV positive despite the presence of adequate DNA in the swabbed material for successful amplification of beta-actin sequences. The absence of HPV (other than in the papilloma itself) in the upper aerodigestive tract of patients and caregivers is consistent with the absence of reported cases of horizontal transmission to siblings or other family members. The findings are also consistent with the conventional view that juvenile respiratory HPV is transmitted vertically from vaginal condylomas in the mother.     

Comparison of Virapap filter hybridization with polymerase chain reaction and Southern blot hybridization methods for detection of human papillomavirus in tonsillar and pharyngeal cancers

Summary A method using a commercial dot filter hybridization kit, Virapap, was compared with Southern blot hybridization and polymerase chain reaction (PCR) for the detection of human papillomavirus (HPV) types 16 and 18 in pharyngeal and tonsillar cancers of 12 patients as well as tonsillar biopsies from 28 patients with chronic tonsillitis. Concordant results between Virapap and PCR, Virapap and Southern hybridization, and PCR and Southern hybridization methods were obtained respectively in 41.7%, 58.3% and 83.3% of the cancer cases, and 67.9%, 67,9% and 85.7% of the control (tonsillitis) cases. Virapap false-positive results were found in 5 cancer cases and 5 control cases. Although the Virapap method is reported to be useful for detecting HPV in gynecological tissues, this method cannot be recommended for the detection of HPV in pharyngeal and tonsillar cancers.     

婴幼儿咽喉乳头瘤组织人乳头瘤病毒感染的探讨

目的 探讨温州地区人乳头瘤病毒感染和婴幼儿咽喉乳头状瘤的关系。方法 应用聚合酶链反应和核酸斑点杂交技术检测３５例婴幼儿咽喉乳头瘤组织和１０例对照组组织（小儿声带小结）ＨＰＶ６、１１、１６、１８、３３５个型别的ＤＮＡ。结果 乳头瘤组织ＨＰＶ感染率为９１．４％（３０／３５）,其中ＨＰＶ６型检出率为５４．２％（１９／３５）,ＨＰＶ１１型感染率为２５．７％（９／３５）,多重型别ＨＰＶ６＋１１感染率为１１．     

Presence of human papillomavirus type-6-related sequences in inverted nasal papillomas

Summary Twenty DNA samples obtained from seven cases of inverted papillomas, eight cases of nasal polyps and five cases of chronic sinusitis were investigated by Southern blot hybridization for the possible presence of sequences homologous to human papillomavirus (HPV) types 6, 11, 16 and 18. HPV type-6-related DNA was identified in one of the seven inverted papillomas. The restriction endonuclease cleavage patterns showed that this latter DNA is a new subtype of HPV type 6 DNA. In the other six papillomas and in all cases of nasal polyps and chronic sinusitis, no HPV sequence could be demonstrated, even under low stringent conditions (T _m–40°C). These results indicate that HPV infection might be one of the possible causative factors in the pathogenesis of inverted papillomas but is not essential for the induction of the tumor.     

Prevalence of Human Papillomavirus in Squamous Cell Carcinoma of the Head and Neck Determined by Polymerase Chain Reaction and Southern Blot Hybridization: Proposal for Optimized Diagnostic Requirements

Human papillomaviruses (HPV) are discussed as cofactors in the carcinogenesis of squamous cell carcinoma of the head and neck (SCCHN). The prevalence of HPV infection in SCCHN is the subject of controversy since reported HPV prevalences range from less than 10% to almost 100%, depending mainly on the detection method employed. This study presents a realistic approximation to the real prevalence of HPV in SSCHN by applying polymerase chain reaction (PCR) and Southern blot hybridization (SBH), which are the most sensitive and specific HPV detection methods. Diagnostic procedures were optimized by applying a ''hot-start'' PCR protocol followed by a confirmatory SBH of the PCR products to reactions using both type-specific and consensus primers and probes. DNA of 75 tumour samples and 22 normal mucosa samples of the same patients were investigated. In 14 cases genomic SBH using complete HPV genomes as probes was performed additionally. HPV DNA was detected in 17/75 (22.7%) SCCHN specimens. HPV 16 was identified in four cases, HPV 45 in three cases, and HPV 6 and 18 in one case each. Hot-start PCR and SBH are the most reliable HPV detection methods, as they minimize both false-positive and false-negative results. With these methods, a HPV prevalence of 23% was achieved, which can be assumed to be representative for comparable study populations. The significant number of positives detected only by consensus primer PCR, along with the identification of HPV 45, indicate that further HPV types may play an important role in the genesis of SCCHN.     

Tracing human papillomavirus DNA in nasal polyps by polymerase chain reaction

Human papillomavirus (HPV) infections are related to the genesis of various benign and malignant human neoplasias. The HPV types 16 and 18 seem to be causally related to the development of most squamous cell carcinoma of the anogenital tract and a proportion of carcinomas of the upper aerodigestive tract. The near 100% positivity of the HPV types 6 and 11 in laryngeal papillomatosis is well established. We investigated whether HPV also plays a role in non-neoplastic mucosal entities such as sinunasal polyposis, the genesis of which has been discussed as being triggered by viral infections. On DNA from 39 sinunasal polyps (33 patients), polymerase chain reaction (PCR) was performed using beta-globin primers for demonstration of amplifiable DNA in the tissue extracts. Consensus primers for the detection of several different HPV types were applied to the beta-globin-positive samples. The results were confirmed by Southern blot hybridization using consensus probes. Cycle sequencing was performed on the positive cases. All 39 samples showed positive signals for beta-globin. HPV-DNA investigations showed a slight positive signal in only 1 of the 39 investigated cases (2.6%). Further molecular investigations of this sample, including cycle sequencing, could not confirm this result. All the other tissue samples remained HPV-DNA-negative. Therefore, those HPV types readily detectable with the PCR primers and probes used are not frequently associated with sinunasal polyposis. The data confirm the hypothesis that HPV is correlated to a lesser extent to infectious mucosal lesions than to proliferative lesions. Furthermore, the results emphasize that the presence of HPV in specific lesions does not occur by chance, but represents a specific infection of the mucosa leading to proliferation and even to malignancy.     

Human papillomavirus type 16 DNA detected by the polymerase chain reaction in non-cancer tissues of the head and neck

Cancer-free tissues from various anatomical subsites in the head and neck were examined by the polymerase chain reaction (PCR) for the incidence of human papillomavirus (HPV) types 16 and 18. We detected HPV 16 DNA in 9 of 103 samples (8.7%), including specimens from the paranasal sinuses, tonsil, hypopharynx and larynx. However, no HPV-16/18 DNA was detected by Southern hybridization in these 9 samples. The significance of the presence of HPV 16 DNA in non-cancer tissues is still unknown, but PCR detection only of high-risk HPV DNA in head and neck cancer should be evaluated cautiously because of its ubiquity in this region.     

General primer-mediated polymerase chain reaction for simultaneous detection and typing of human papillomavirus DNA in laryngeal squamous cell carcinomas

The aim of this study was to investigate the prevalence of different human papillomavirus (HPV) types in laryngeal squamous cell carcinomas using general primer-mediated polymerase chain reaction (PCR). Tumour sections from 42 patients with laryngeal carcinomas were investigated. For HPV DNA amplification, consensus primers were used which were directed to the LI coding region of the HPV genome. Analysis of the PCR products was done using 2% agarose gel electrophoresis followed by restriction enzyme analysis to identify different HPV types. Amplification of the human TGF-β DNA was successfully performed in 36/42 (85.7%) of samples confirming the presence of sufficient DNA for viral amplification. HPV DNA was detected in 8/36 (22.2%) of the tumours examined (three HPV-6, two HPV-16, one HPV-11, two unknown HPV types). HPV DNA was not detected in any of the non-neoplastic laryngeal mucosa which was used as control (n = 15). Fifty per cent of women had HPV-positive tumours compared with 8% of men (x²= 5.8, P<0.05). Our data indicate that while the overall prevalence of HPV in laryngeal carcinomas is fairly high (22.2%), the frequency of high-risk types (HPV-16 & HPV-18) is low (5.5%). HPV probably acts as a promoter in the multistep process of carcinogenesis in squamous mucosal cells of the larynx.     

Significance of human papillomavirus in sinonasal papillomas

Recent investigations have suggested human papillomavirus (HPV) to be involved in the development of sinonasal papillomas (SNP). Forty-three patients operated for SNP were studied to determine the prevalence of HPV-DNA sequences in these tumours and to evaluate their value as a prognostic parameter. The original sections of all cases were reviewed and reclassified according to the WHO. Paraffin blocks available from 37 patients were subjected to in situ hybridization (ISH) and polymerase chain reaction (PCR). Histology revealed 34 cases of inverted papilloma (IP) (79 per cent), five cases of exophytic papilloma (EP) (12 per cent) and four cases of columnar cell papilloma (CCP) (nine per cent). Recurrences developed in seven of 41 patients (17 per cent), and malignancy occurred in four of 43 patients (nine per cent). HPV was detected in four of 37 specimens (11 per cent) both by ISH and PCR. In particular, HPV-11 was found in three lesions (two EP, one IP) (eight per cent), and HPV-6b was detected in one lesion (one EP) (three per cent). Our findings suggest a possible role for HPV in the pathogenesis of exophytic papillomas. As no correlation was found to malignancy and recurrence of disease, screening for HPV seems not to be useful as a prognostic parameter.     

Detection of human papillomavirus type 6f genome in nasal papillomatosis.

Five cases of nasal papillomatosis were studied clinicopathologically and virologically. In a case of recurrent papillomatosis of non-inverted type located on the nasal septum, human papillomavirus (HPV) DNA was detected by dot blot hybridization with an RNA cocktail probe of mucosal HPVs. In Southern blot hybridization, the DNA hybridized with that of HPV types 6 and 11 but not with those of types 16 and 18. Its restriction endonuclease-cleavage patterns corresponded well to those of HPV type 6f. These results suggested that HPV type 6 would also be associated with nasal non-inverted papillomatosis.     

Human papillomavirus DNA in squamous cell carcinoma of the upper aerodigestive tract

Nineteen samples of DNA from squamous cell carcinoma of the upper aerodigestive tract were analyzed by Southern blot hybridization for the presence of human papillomavirus (HPV) DNA. Two samples of DNA contained HPV 16 DNA or its homologous sequence. In one maxillary carcinoma, the sequences homologous to HPV 16 were detected. In one tonsillar carcinoma, HPV 16 sequence was also shown to be present. The patients positive for HPV DNA were female and had neither smoking nor drinking habits. These results indicate that HPV infections may play a role in the development of some types of squamous cell carcinomas of the upper aerodigestive tract.     

Human papillomaviruses in lymph node neck metastases of head and neck cancers

CONCLUSION: The results of this study corroborate earlier findings that human papillomavirus (HPV)16 is the most prevalent type of HPV in squamous cell carcinomas of the head and neck (SCCHNs) and reinforce a possible influence of HPV on SCCHN progression by showing that the majority of HPV-positive patients harbor HPV16 (or HPV33) both in their primary tumors and in lymph node neck metastases (LNNMs). OBJECTIVE: HPVs are causally associated with carcinomas of the uterine cervix and have also been linked to a subset of SCCHNs. In order to further investigate the predicted causative role of HPV in SCCHNs, we analyzed pairs of primary tumors and LNNMs or LNNMs alone for the presence of HPV DNA using polymerase chain reaction (PCR). MATERIAL AND METHODS: DNA was extracted from fresh frozen tissue samples of primary tumors and the corresponding LNNMs of 18 patients and from LNNMs alone in 17 patients. For the detection and typing of HPV, PCR was performed using both type-specific and consensus primer pairs, followed by Southern hybridization and, in selected cases, sequencing of the PCR products. RESULTS: Of the 35 patients investigated, 22 (63%) were found to have HPV DNA in their tumors: HPV16 DNA in 21 cases and HPV33 in 1. The highest HPV prevalence was detected in tumors of Waldeyer's tonsillar ring (8/9 patients; 89%). Of the 18 patients in whom primary tumors and LNNMs were analyzed, 7 (39%) were HPV-positive in both samples (HPV16, n = 6; HPV33, n = 1), in 3 (17%) the primary tumors were HPV-negative and the LNNMs HPV16-positive and in 1 (5.5%) the primary tumor contained HPV16 and the LNNM was negative. Interestingly, of the 7 patients in whom LNNMs had been detected only several months after diagnosis and treatment of the primary tumors, only 1 showed infection with HPV (HPV33).     

High-risk human papillomavirus (HPV) is not associated with p53 and bcl-2 expression in oral squamous cell carcinomas

Objective

To determine the frequency and type of human papillomavirus (HPV) in oral squamous cell carcinomas (OSCC), as well as to identify a possible association between HPV infection and the expression pattern of p53 and bcl-2, and identify whether the oral HPV infection is a characteristic finding in our sample.

Methods

We performed polymerase chain reaction and dot blot hybridization for the detection of HPV DNA in paraffin sections as well as immunohistochemical analysis of p53 and bcl-2 in our sample.

Results

Twenty-six cases (29.5%) were positive for the virus by PCR. Dot blot hybridization identified HPV 18 in 21 (80.8%) cases, HPV 16 in one (3.8%) case and a combination of the two types in the four (15.4%) remaining cases. No other type of HPV was detected in the sample. Immunohistochemistry showed p53 in 26 (60.4%) cases and bcl-2 in 17 (39.5%) ones. No significant association was observed between the presence of HPV and the expression of the proteins studied (p = 0.988 and p = 0.748, respectively).

Conclusions

Although this investigation have detected only 29.5% of HR-HPV DNA in OSCC, it is possible that this virus contribute to the development of some case of this tumor. Furthermore, it seems that the immunohistochemical expression of p53 and bcl-2 and the presence of HPV DNA are independent events in OSCC.     

PCR analysis of nasal polyps,chronic sinusitis,and hypertrophied turbinates for DNA encoding bacterial 16S rRNA

BACKGROUND: Nasal polyps are considered to result from chronic inflammation, but the initial or persisting stimulus for the inflammation is not known. A variety of bacteria and fungi have been cultured from nasal polyps, but approximately 35% have sterile cultures. Previously, Mycoplasma pneumoniae-specific DNA was detected in human nasal polyps using polymerase chain reaction (PCR) techniques, suggesting M. pneumoniae as a causative agent in the etiology of nasal polyps. METHODS: In this study, we tested for the presence of bacterial DNA in nasal polyps resected from 40 patients, in nasal mucosa membrane from 9 patients undergoing turbinectomy for hypertrophy, and in sinus mucosa membrane from 6 patients undergoing endoscopic surgery for chronic sinusitis. Tissue DNA was extracted and analyzed by PCR using M. pneumoniae specific primers for DNA that encode the 16S rRNA gene in 41 specimens (31 polyps, 6 turbinates, and 4 sinus), and by consensus sequence-based PCR using broad range primers for most eubacterial DNA encoding the 16S rRNA gene in 38 specimens (26 polyps, 7 turbinates, and 5 sinuses). RESULTS: Only two samples were positive for bacterial DNA encoding 16S rRNA: Streptococcus sp. DNA was isolatedfrom one polyp specimen and Pseudomonas aeruginosa DNA was isolated in one maxillary sinusitis specimen. No evidence of M. pneumoniae-specific DNA encoding 16S rRNA was found in any of the tissues. CONCLUSIONS: This study suggests that chronic bacterial infection is not a major component of nasal polyp etiology.     

Persistence and expression of human papillomavirus during interferon therapy

Alpha interferon did not prevent the persistence or expression of human papillomavirus (HPV) types 6 or 11 in respiratory tract papillomas. Seventeen patients receiving interferon therapy for a minimum of six months and a maximum of 39 months still had one to ten copies per cell genome of HPV DNA in their laryngeal tissues, seen by Southern blot hybridization. In papillomas that recurred during treatment, HPV RNA could be detected by in situ hybridization, and capsid protein could be detected by immunoperoxidase staining. Persistence of the HPV DNA, and recurrences that occurred during therapy, are attributed to the failure of interferon therapy to eliminate latent virus.