Detection and typing of human papillomavirus in verrucous carcinoma of the oral cavity using the polymerase chain reaction

OBJECTIVE: This study examined the prevalence and types of human papillomavirus (HPV) DNA in oral cavity verrucous carcinoma. DESIGN: This was of a retrospective screening study. Formalin-fixed, paraffin-embedded tissue samples were examined by the polymerase chain reaction using DNA primers specific for HPV types 6b/11, 16, and 18. SETTING: The majority of patients were seen at referral centers in Ontario, Canada. PATIENTS: This study examined 29 oral cavity verrucous carcinomas occurring in a sample of 25 patients from four institutions between 1966 and 1992. All tumors met standardized histologic diagnostic criteria of verrucous carcinoma. MAIN OUTCOME MEASURE: The prevalence of HPV 6b/11, 16, and 18 DNA was determined by the PCR technique. RESULTS: The HPV DNA was detected in 12 (48%) of 25 patients. The HPV 6b/11 DNA, HPV 16 DNA, HPV 18 DNA, and HPV 16 DNA plus HPV 18 DNA, were detected in one (4%), one (4%), nine (36%), and one (4%) cases, respectively. CONCLUSIONS: The detection of HPV 18 DNA in 40% of oral cavity verrucous carcinomas suggests an association between the presence of HPV 18 DNA and some oral cavity verrucous carcinomas. The etiologic and prognostic significance of HPV 18 for oral cavity verrucous carcinoma remains unanswered and will require further study.     

山东东部57例喉癌人乳头状瘤病毒DNA及其亚型的检测

目的 通过检测山东东部地区喉鳞状细胞癌中人乳头状瘤病毒(HPV)的DNA及其亚型的表达,探讨HPV感染与喉癌的关系及其在喉癌发病中的意义。方法 采用聚合酶链式反应(PCR)扩增和DNA反向点杂交相结合的DNA芯片技术,检测57例喉鳞状细胞瘤各亚型HPV DNA,对照组为同期喉乳头状瘤细胞8例。结果 喉癌组HPV感染率为7.02%(4/57),喉乳头状瘤组75.00%(6/8),差异有统计学意义(χ2=24.906,P＜0.01);喉癌组高危型HPV16与低危型HPV43感染率分别与喉乳头状瘤比较,差异均无统计学意义(χ2= 5.611、0.143,P＞0.05);喉乳头状瘤组低危型HPV11与HPV6感染率较喉癌组高,差异均有统计学意义(χ2=8.611、14.702,P＜0.05)。结论 山东东部地区喉癌患者HPV感染率较低,而低危型HPV6/11与喉乳头状瘤关系较为密切     

Intranasal verrucous carcinoma: relationship to inverting papilloma and human papillomavirus

OBJECTIVES: To establish the incidence, appearance, behavior, and appropriate treatment of intranasal verrucous carcinoma and determine its relationship to inverting papilloma and human papillomavirus (HPV). STUDY DESIGN: Retrospective review of all cases of intranasal verrucous carcinoma seen at the Mayo Clinic from 1960 through May 1996. METHODS: Retrospective chart review and data collection for age, sex, smoking history, location, association with inverting papilloma, treatment, recurrence, and follow-up. Polymerase chain reaction (PCR) testing for the presence of HPV DNA was performed on all specimens. RESULTS: Of the 13 patients identified, most presented with nasal obstruction (10) or a noticeable intranasal lesion (8). The maxillary sinus was the extranasal site most often involved. Five patients had verrucous cancer develop in an inverting papilloma, and one had squamous cell carcinoma with the verrucous component (a hybrid tumor). All but one patient underwent surgery as initial treatment; only one patient had preoperative radiation therapy. Surgical procedures ranged from local excision to a craniofacial resection. Follow-up ranged from 2 months to 32 years (mean, 6.5 y). Four patients had a single recurrence and two tumors recurred a second time. No metastases developed and no one died from the tumor. In seven patients (10 specimens), DNA was successfully amplified for PCR testing, and no HPV DNA was detected. CONCLUSIONS: When verrucous tumors are discovered early, they can be treated effectively with wide local excision. In some cases, a more extensive procedure may be required. A possible role for HPV in the etiology of these tumors was not found.     

Molecular detection and typing of human papillomavirus in laryngeal carcinoma specimens

CONCLUSIONS: Human papillomavirus (HPV) DNA was detected in 32% of laryngeal carcinoma biopsy samples studied. The genotypes identified were high-risk types, the most frequent being HPV 16. Viral DNA was integrated into the host genome (genotype HPV 16), providing supporting evidence for a role of HPV in the carcinogenic pathway of laryngeal squamous cell carcinoma. OBJECTIVE: HPV has been detected in laryngeal lesions, both benign and neoplastic, with a variable frequency (8-60%). These viral agents have been proposed as an adjuvant or cofactor in head and neck carcinogenesis because of their oncogenic properties. The aims of this study were to identify HPV in laryngeal carcinoma samples and to describe the physical state of the viral genome, i.e. its integration to the host DNA. MATERIAL AND METHODS: Formalin-fixed, paraffin wax-embedded tumor samples from patients with newly diagnosed laryngeal carcinomas were collected. The HPV genome was identified using polymerase chain reaction (PCR) with primers complementary to the conserved region L1 (MY09-11). Genotyping was accomplished by restriction fragment length polymorphism. Samples positive for HPV 16 were assayed by PCR with primers complementary to region E2, interrupted during viral genome integration. RESULTS: Ten of the 31 samples (32%) were positive for HPV DNA and all of the samples were positive for human beta-globin. The genotypes identified were HPV 16 (n=3), HPV 58 (n=2) and HPV 39, 45, 51, 59, 66 and 69 (n=1 for each). The three samples positive for HPV 16 had lost region E2, meaning that the viral DNA had been integrated into the host genome.     

Molecular detection and typing of human papillomavirus in laryngeal carcinoma specimens

Conclusions. Human papillomavirus (HPV) DNA was detected in 32% of laryngeal carcinoma biopsy samples studied. The genotypes identified were high-risk types, the most frequent being HPV 16. Viral DNA was integrated into the host genome (genotype HPV 16), providing supporting evidence for a role of HPV in the carcinogenic pathway of laryngeal squamous cell carcinoma. Objective. HPV has been detected in laryngeal lesions, both benign and neoplastic, with a variable frequency (8–60%). These viral agents have been proposed as an adjuvant or cofactor in head and neck carcinogenesis because of their oncogenic properties. The aims of this study were to identify HPV in laryngeal carcinoma samples and to describe the physical state of the viral genome, i.e. its integration to the host DNA. Material and methods. Formalin-fixed, paraffin wax-embedded tumor samples from patients with newly diagnosed laryngeal carcinomas were collected. The HPV genome was identified using polymerase chain reaction (PCR) with primers complementary to the conserved region L1 (MY09-11). Genotyping was accomplished by restriction fragment length polymorphism. Samples positive for HPV 16 were assayed by PCR with primers complementary to region E2, interrupted during viral genome integration. Results. Ten of the 31 samples (32%) were positive for HPV DNA and all of the samples were positive for human β-globin. The genotypes identified were HPV 16 (n=3), HPV 58 (n=2) and HPV 39, 45, 51, 59, 66 and 69 (n=1 for each). The three samples positive for HPV 16 had lost region E2, meaning that the viral DNA had been integrated into the host genome.     

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.     

Immunohistochemical demonstration of multiple HPV types in laryngeal squamous cell carcinoma

The aim of this study was to determine the prevalence of human papillomaviruses (HPV) types 6, 11, 16, 18, 31, 33, 42, 51, 52, 56 and 58 in laryngeal squamous cell carcinoma specimens using immunohistochemical reactions and to correlate the presence of HPV with the clinical and pathological characteristics of these patients. Tissue samples were collected from 40 patients with primary laryngeal squamous cell carcinoma (LSCC) and from 33 subjects with non-neoplastic laryngeal lesions or laryngeal nodules, which served as a control group. Human papilloma virus was detected in 6 (15%) of the 40 patients. Five (83.4%) of six patients with HPV positive tumors had G2 (moderately differentiated), one patient (16.6%) had G3 (poorly differentiated), and no patient with HPV positive tumor had a G1 (well-differentiated) tumor. Four (66.6%) of the six HPV positive tumors were in the supraglottic region, one (16.6%) tumor was located in the glottis, and one (16.6%) HPV positive tumor was in the subglotic region. Five (83.4%) of six HPV positive tumors were T3-T4, and one was T2. Three of six HPV positive patients had no clinically evident cervical lymph nodes (N0), and three of the HPV positive patients were N1 or N2. Human papillomavirus was not detected in any of the samples from the control group. The presence of HPV infection in 15% of the cases may suggest a possible role in the etiology of laryngeal squamous cell carcinoma. However, no significant correlation between HPV incidence and histological grading and clinical staging could be demonstrated.     

用聚合酶链反应技术制备地高辛素标记核酸探针检测国人喉癌人类乳头瘤病毒DNA

应用聚合酶链反应（PCR）技术制备非放射性探针标记物——地高辛素标记人类乳头瘤病毒（HPV）共有引物探针，对146例喉不同病变的新鲜组织标本（喉癌68例，喉其它病变48例，正常喉组织30例）进行HPV6，11，16，18，31，33，35，42，58共9型HPVSDNAS感染的检测。结果表明，喉癌组HPV感染阳性率45．6％（31／68），喉癌颈转移淋巴结组阳性率21．0％（3／15），喉癌前病变阳性率11．8％（2／17），声带息肉组阳性率6．3％（1／16），15例癌旁及15例癌周正常喉组织均为HPVDNA阴性。文中还对HPV在喉癌中的致病作用及应用PCR技术制备地高李标记HPV共有引物探针的敏感性及特异性进行了讨论。     

喉乳头状瘤临床行为与细胞增殖活性关系的研究

利用ISH方法以及免疫病理手段对不同型的LP组织的标本进行PCNA、P53检测，探讨不同型HPV与LP的发生发展的相关机制，以寻求有效的检测方法帮助临床对LP的预后进行评估。标本选自1994年1月～1995年12月间我院收治的LP共36例。ISH法HPV6b／11阳性率为75％，明显高于HPV16和／或HPV18的表达。10例喉鳞癌各有1例HPV16、18阳性，无HPV6b／11阳性。ABC法行P53检测，36例LP标本中仅1例恶变组织阳性表达Ⅱ级（2．8％）；10例喉鳞癌中9例阳性表达（90．0％），其中Ⅱ级以上阳性表达6例（60．0％）。PCNA阳性表达27／36例（75％）；其中JOP组与AOP组阳性率有显著差异（P＜0．05）。本研究表明LP与HPV感染有极为密切的关系，认为HPV分型的检测在判断LP转归中有意义。PCNA阳性表达程度在预测LP肿瘤的活跃程度方面是一个很有意义的指标。P53蛋白表达在喉鳞癌与LP中有显著差异。     

Laryngeal verrucous carcinoma: A clinicopathologic study and detection of human papillomavirus using polymerase chain reaction

Laryngeal verrucous carcinoma (LVC) is a rare, well-differentiated variant of squamous carcinoma with a low malignant potential. Human papillomavirus (HPV)–16 DNA has been identified in a small number of LVC and an etiologic relationship has been suggested. A correlative clinical and molecular pathological study was performed in order to determine the prevalence and typing of HPV DNA in LVC. Possible associations between patient and tumor subsets, and the presence of HPV DNA were also investigated. Formalin-fixed, paraffin-embedded tissue samples from 29 patients with LVC were examined by polymerase chain reaction (PCR) using DNA primers specific for HPV types 6b/11, 16, and 18. Overall, HPVDNA was detected in 13 (45%) of the cases. Of these, HPV-16 DNA, HPV-18 DNA, and both HPV-16 DNA and HPV-18 DNA were detected in 4 (14% overall; 31% of positive cases), 4, and 5 (17% overall; 38% of positive cases), respectively. HPV-6b/11 DNA was not detected in any LVCs. In 16 cases, no HPV DNA was detected. There was a trend toward HPV DNA detection in higher stage tumors. HPV DNA detection was unrelated to patient age, tumor site, or radiotherapeutic responsiveness. The detection of HPV DNA in 45% of LVCs suggests an association between the presence of HPV-16 DNA and HPV-18 DNA, and some LVCs.     

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.     

Detection and Typing of Human Papillomavirus DNA in Benign and Malignant Tumours of Laryngeal Epithelium

The role of human papillomaviruses (HPV) in laryngeal squamous cell carcinoma has not yet been established. Thirty-three cases of laryngeal squamous cell carcinoma were analysed for the presence of HPV DNA and compared with 25 cases of normal larynx and 29 cases of laryngeal squamous papilloma in their positivity index. The presence of HPV DNA was analysed by using L1 consensus primers and also by primers specific for the E7 gene of HPV types 16 and 18. Four normal laryngeal samples (16%) were positive for HPV DNA against the 24 samples (82%) (p&lt;0.001) found for laryngeal papilloma and 16 (48.5%) (p&lt;0.05) found for laryngeal squamous cell carcinoma. HPV 16 was the type most frequently found in laryngeal carcinoma samples. Our results support an etiologic role for this type of HPV in the pathogenesis of laryngeal carcinoma.     

人乳头状瘤病毒与鼻内翻性乳头状瘤的关系

为探讨人乳头状瘤病毒（ＨＰＶ）感染与鼻内翻性乳头状瘤（ＩＰ）发病及其恶变的关系，研究选取组织病理确认为ＩＰ（３６例）和ＩＰ癌变（１６例）的石蜡包埋标本，采用多聚酶链反应（ＰＣＲ）方法对标本进行ＨＰＶ相关ＤＮＡ序列扩增。结果显示：３６例ＩＰ组织有２１例（５８．３％）为ＨＰＶ阳性；１６例ＩＰ癌变组织中有１１例（６８．８％）阳性。经统计学处理，二者间无显著性差异（Ｐ〉０．０５）．提示ＨＰＶ感染在ＩＰ的发     

Short-fragment PCR assay for highly sensitive broad-spectrum detection of human papillomaviruses in laryngeal squamous cell carcinoma and normal mucosa: clinico-pathological evaluation

The aim of the study was to determine the prevalence and genotypes of HPV infection in laryngeal cancer specimens, normal mucosa obtained from the surgical margin and laryngeal nodules using a novel high sensitive and specific SPF₁₀ HPV DNA test, PCR/DEIA method and INNO-LiPA genotyping assay. The correlation between HPV presence and clinico-pathological features was analyzed. Tissue samples were collected from 93 primary laryngeal squamous cell carcinoma (LSCC), 49 specimens of normal mucosa and from 22 specimens of laryngeal nodules serving as control group. HPV DNA was amplified by the short PCR fragment (SPF₁₀) primer set using HPV DNA enzyme immunoassay (DNA/DEIA) method and INNO-LiPA HPV genotyping assay. Human papillomavirus was detected in 33 (35.5%) of the 93 samples from LSCC, in 4 (8.2%) of 49 samples of the normal mucosa and it was not detected in any of the sample from the control group. Twenty-eight of 33 (81.8%) were positive for HPV-16, 6 of 33 (18.2%) were positive for HPV-18 and 5 of 33 (15.1%) were positive for HPV-33. Multiple infection was found in 5 of 33 (15.1%); 3 samples were positive for HPV-16 and HPV-33, 2 samples for HPV-16 and HPV-18. There was a statistically significant correlation between the presence of HPV in LSCC tumors and in control group samples and between the presence of HPV in the tumors and normal mucosa from the free surgical margin. The presence of HPV infection in 35.5% of the cases suggests a possible role in the etiology of laryngeal cancer and supports the role of high-risk types of HPV (16, 18 and 33) in LSCC. HPV infection is not likely to influence survival rates as an independent prognostic factor in patients with laryngeal cancer.     

Verrucous carcinoma--histological and molecular analysis

Carcinoma verrucosum is a histologic variant of a well differentiated squamous cell carcinoma, applied to 1-4% of all cases of laryngeal cancer. These lesions rarely cause deep invasion or metastasis to cervical lymph nodes and distant metastases. Among etiologic factors are smoking and purely oral hygiene. Last studies suggest HPV infection etiology in laryngeal cancer, mainly HPV 16 and and HPV 18. The aim of the study was to determine morphologic assessment of verruous carcinoma and the presence of HPV infection in patients suffered from verrucous carcinoma of laeynxd in the Department of Otolaryngology Medical Academy of Bialystok in years 2000-2002. The primers pU-1M/pU-2R were used to detect the high risk HPV which are HPV 16,18,31,33,35. The pair of pU-31B/pU-2R were used to detect low risk HPV which are HPV 6,11. In 2 cases (40%) HPV of high risk was determined. The histological assessment as well as molecular may play an important role in the clinical and diagnostic estimation in order to make a proper tumor classification and decide on the most effective treatment.     

Thymidine labeling index in laryngeal squamous cell carcinoma in correlation with pTNM,age, histological grade and recurrence

In this study, our aim was to investigate the prognostic features of the thymidine labeling index (TLI) in laryngeal squamous cell carcinoma (SCC). The TLI values in tumor tissues and adjacent healthy tissues were assessed in 40 patients who had a pathological diagnosis of laryngeal SCC. The data were correlated with age, pTNM, histological grade and recurrence. The tissues (tumor and adjacent tumor-free tissue) obtained during surgery were labeled with (3)H-thymidine. A statistically significant difference was observed between tumoral tissue and adjacent tumor-free tissue (p < 0.05), but we could not confirm any statistically significant correlation between TLI and age, pTNM, histological grade and recurrence. In conclusion, TLI in laryngeal SCC had no significant relation with the clinicopathological features, probably due to the variation in tissue sampling and for tumor-dependent reasons. TLI may assist in differentiating malignant from benign lesions.     

Detection of human papillomavirus genome in nasolaryngeal papillomas using digoxigenin labeled DNA probes

It is being reported that human papillomavirus (HPV) has been implicated in the pathogenesis of various neoplastic lesions of the genital organs. To investigate the etiological role of HPV and its types in nasolaryngeal papillomas, we retrospectively analyzed HPV genomes by nucleic acid hybridization methods; for detecting DNA and mRNA, we employed the recently developed nonradioactive (digoxigenin labeled) DNA probes and compared the results by radioisotope methods. In total, 43 cases of papillomatous lesions were examined. They were verruca vulgaris of the nasal vestibule (Nr = 2), nasal inverted papilloma (IP, Nr = 26), and laryngeal papilloma (Nr = 15). HPV types examined were type 2, 6, 11, 16 and 18. Two cases of verruca vulgaris were shown to contain HPV-2 DNA and its mRNA by in situ hybridization. HPV-11 DNA was detected in 3 cases (12%) of nasal inverted papilloma whereas HPV-16 was detected in 1 case (4%); the latter case was associated with squamous cell carcinoma. These results suggest that HPV may be implicated in the development of IP, and HPV-16 may play an important role in the malignant transformation of IP. In the cases of multiple laryngeal papilloma (Nr = 8, one juvenile type and 7 adult type), either HPV-6 or HPV-11 was detected at the high rate (6/8, 75%). The presence of the HPV genomes provides strong evidence for the HPV etiology of these laryngeal papillomas. Whereas in the cases of adult single laryngeal papilloma (Nr = 7), HPV was not detected. Technically, the sensitivity of digoxigenin (DIG) labeled DNA probe was almost same as 35S labeled probe by dot blot hybridization, thus we applied DIG labeled probe to Southern blot hybridization with low background. By in situ hybridization using digoxigenin labeled probes, the rates of HPV detection were almost equal to those by 35S labeled probes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Is human papillomavirus involved in laryngeal neuroendocrine carcinoma?

The purpose of this study was to detect human papillomavirus (HPV) infection in laryngeal neuroendocrine carcinoma (LNEC) and to explore the possible relationship between HPV-induced malignant transformation and prognosis in LNEC. Ten cases of LNEC from a tertiary referral hospital were retrospectively analyzed. Clinical data were subtracted from patients’ files. Pretreatment biopsy material was tested for the presence of HPV6, 11, 16, and 18 using a PCR-based detection method. Immunohistochemical staining was performed for Ki-67, p16^INK4A, and p53 expression. All cases were negative for the low-risk HPV types HPV6 and HPV11 that are associated with laryngeal papillomatosis. High-risk HPV was detected in two cases; an atypical carcinoid was positive for HPV16 and a large-cell neuroendocrine carcinoma for HPV18. Both HPV-positive tumors had a high Ki-67 labeling index. Two of the four cases with a good response to therapy were hrHPV-positive (both HPV DNA positive) compared with none of the five poor responders. Our findings show that HPV may play a role in the pathogenesis of LNEC. The relationship between HPV, improved prognosis and good response to therapy for squamous cell carcinoma of the head and neck may also be true for a subset of LNEC.     

Human papillomavirus and risk of laryngeal cancer

We determined the relationship between human papillomavirus (HPV) infection and the HPV types detected in 44 patients with squamous cell carcinoma, 10 laryngeal leukoplakia patients, and 12 patients evaluated for benign laryngeal conditions (controls). The sources of HPV DNA were from brushings from the upper respiratory tract and lesion (benign or malignant), oral rinses, and biopsies of patient lesions. Polymerase chain reaction (PCR) and DNA sequencing were used to identify and type HPV. We detected HPV in 25.0% (11/44) of patients with laryngeal cancer, in 30.0% (3/10) of patients with laryngeal leukoplakia, and in 16.7% (2/12) of noncancer controls. Patients with cancer were not more likely to be identified with oncogenic HPV types ( 18.2%) than either the leukoplakia group (20%) or the control group (16.7%). An increased risk of disease was associated with current tobacco use and former alcohol drinking in cancer patients versus controls and in leukoplakia patients versus controls (all p < .05). After we controlled for tobacco and alcohol effects on the risk of disease, exposure to oncogenic HPV types was associated with an increased risk of laryngeal cancer (odds ratio = 3.0) and of laryngeal leukoplakia (odds ratio = 6.0) compared to controls, although the results were not statistically significant. This study suggests that although HPV infection and HPV oncogenic types are not found at a higher frequency in laryngeal cancer or laryngeal leukoplakia as compared to controls, infection is associated with an increased risk of disease after controlling for the effects of alcohol and tobacco use.