Detection of HPV-16-related DNA sequence in laryngeal squamous cell carcinoma]

The gene libraries of laryngeal squamous cell carcinoma (LSCC) and laryngeal papilloma (LP) were screened by hybridization analysis with alpha-32P-labelled HPV-16 DNA under low stringency (Tm = 40 degrees C). It showed that the relative sequences were detected in LSCCs (11/16, 68.8%), but not in LP. The HPV-16-related DNA harbored in negative hybridization lesions were further analysed by polymerase chain reaction to amplify E6/E7 gene of HPV-16-related DNA. The results showed that 1/5 LSCCs and 2/2 LPs, were positive. The data suggest that development of LSCC should be related to the HPV infection, and HPV may be one of the inducers of LSCC.     

喉癌人乳头瘤病毒感染和p53蛋白表达的研究

对４４例喉鳞癌和１６例声带息肉患者采用原位核酸杂交技术探测其人乳头瘤病毒（ＨＰＶ）６Ｂ，１１，１６，１８型ＤＮＡ同源序列及ＬＳＡＢ免疫组化法探测其Ｐ５３蛋白的表达，结果：（１）喉癌与ＨＰＶ１６／１８杂交阳性率为４３．２％（１９／４４），声带息肉为１２．５％（２／１６）（Ｐ〈０．０５）（２）喉癌ｐ５３蛋白阳性率为５６．８％（２５／４４），声带息肉全部为阴性；（３）喉癌ＨＰＶ１６／１８杂交及ｐ５３蛋白     

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.     

A systematic review of case-control studies of human papillomavirus infection in laryngeal squamous cell carcinoma

A role for human papillomavirus (HPV) has been suggested in laryngeal squamous cell carcinoma (LSCC). In order to quantitate the available evidence, we reviewed studies examining the risk of laryngeal cancer-associated HPV. PubMed was searched for case-control studies conducted worldwide and published in any language since 1966. Relevant papers were hand-searched and cross-referenced. Six studies met the inclusion criteria. The studies are heterogeneous in the methods used to harvest tissue samples and techniques for detecting the virus within the tissue. HPV-16 positivity among cases ranged from 2.7% to 46.9% and 0-5.7% among controls. Two studies showed a significantly increased risk of LSCC if HPV-16 was present (OR 18.5, 95% CI 2.2-154.8, OR 2.6, 95% CI 1.1-6.0). An increased risk was also observed for glottic versus supraglottic cancer in one study (OR 9.69, 95% CI 1.47-64.04). The direction of effect is towards an increase in risk of LSCC in people with evidence of HPV-16 infection. There is marked heterogeneity in the methods used to detect the virus and frequency with which it is detected. An adequately powered study using a reliable detection technique is required to confirm and quantify this risk and to examine effect modification.     

Detection of human papillomavirus in laryngeal squamous dysplasia and carcinoma. An in situ hybridization and signal amplification study

We examined the presence of human papillomavirus (HPV) DNA in 65 cases of laryngeal squamous dysplasia and carcinomas using in situ hybridization with signal amplification in paraffin sections. Hybridization was performed with biotinylated DNA probes for HPV 6/11, 16/18, 31/33 and wide-spectrum HPV (6, 11, 16, 30, 31, 45, 51 and 52). HPV DNA was found in 7 cases of the total sample (10.7%); it was also found in 4 out of 45 (8.8%) cases of invasive carcinoma and in 5 out of 33 (15.5%) cases of squamous dysplasia. Morphological signs suggestive of HPV infection were observed in 35.5% of our sample but they were not related to HPV DNA positivity. In conclusion, HPV probably plays little, if any, role in laryngeal carcinogenesis among the population studied.     

Coinfection of HPV-11 and HPV-16 in a Case of Laryngeal Squamous Papillomas With Severe Dysplasia

Human papillomavirus (HPV) types 6 and 11 have been associated with benign laryngeal papilloma, while HPV-16 is occasionally associated with laryngeal carcinoma. In this study, a case of laryngeal squamous papillomas with severe dysplasia was evaluated for the presence of HPV infection. The biopsy specimens were taken from a 58-year-old female patient at two different time points 3 months apart. Architecturally, the tumor showed papillary configuration reminiscent of squamous papilloma. Cytologically, the lesion showed morphologic features characteristic of severe squamous epithelial dysplasia. HPV infection was determined by DNA in situ hybridization using type-specific HPV-DNA probes. HPV-11 probes demonstrated homogeneous nuclear staining, suggesting productive viral replication. In contrast, HPV-16 probe produced a speckled pattern, suggesting HPV-16 DNA integration. Normal laryngeal epithelium did not yield specific hybridization. The presence of HPV-11 and HPV-16 was confirmed by PCR using HPV type-specific primers. Immunocytochemical staining was performed to detect Ki-67, a proliferation marker, and p53. Ki-67 expression was demonstrated throughout the whole thickness of epithelium. Staining for p53 was negative. This study suggests that multiple HPV infections can occur in the same lesion and that HPV-16 infection and its DNA integration may contribute to the occurrence of severe dysplasia in the lesion described.     

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.     

Detection of Human Papillomavirus in Laryngeal Squamous Dysplasia and Carcinoma. An In Situ Hybridization and Signal Amplification Study

We examined the presence of human papillomavirus (HPV) DNA in 65 cases of laryngeal squamous dysplasia and carcinomas using in situ hybridization with signal amplification in paraffin sections. Hybridization was performed with biotynilated DNA probes for HPV 6/11, 16/18, 31/33 and wide-spectrum HPV (6, 11, 16, 30, 31, 45, 51 and 52). HPV DNA was found in 7 cases of the total sample (10.7%); it was also found in 4 out of 45 (8.8%) cases of invasive carcinoma and in 5 out of 33 (15.5%) cases of squamous dysplasia. Morphological signs suggestive of HPV infection were observed in 35.5% of our sample but they were not related to HPV DNA positivity. In conclusion, HPV probably plays little, if any, role in laryngeal carcinogenesis among the population studied.     

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.     

Identification of HPV DNA in patients with juvenile-onset recurrent respiratory papillomatosis using SYBR Green real-time PCR

OBJECTIVE: Recurrent respiratory papillomatosis (RRP) is the most common benign neoplasm affecting the larynx and upper respiratory tract in children. Human papillomavirus (HPV) has been implicated as the cause of RRP, most commonly types 6 and 11. The present study was undertaken to evaluate the occurrence of HPV types in a group of patients with juvenile-onset RRP (JORRP). METHODS: The study group consists of 23 patients with JORRP. The clinical records of the patients were reviewed, and JORRP was classified as non-aggressive or aggressive. The laryngeal biopsies were taken and investigated for HPV DNA presence using real-time polymerase chain reaction (PCR) with a set of consensus primers (MY09/11). Viral typing was subsequently performed by real-time PCR with type-specific primers for HPV types 6, 11, 16, 18, 31, and 33. RESULTS: HPV presence was detected in all samples with amplifiable DNA. HPV-11 was revealed in 61.9% of the patients and HPV-6 in 23.8%. Double positivity for HPV types 6 and 11 was identified in 14.3%. Our findings suggest that RRP runs a more aggressive clinical course when HPV-11 infection is present (p=0.0265). CONCLUSIONS: Our results suggest a high frequency of HPV infection in the upper respiratory tract of the studied patients. We believe that the routine application of molecular techniques such as PCR for detection and analysis of HPVs in patients with RRP has diagnostic and prognostic significance.     

Detection of human papillomavirus type 16 and 18 in laryngeal cancer using PCR methods

Human papillomavirus (HPV) is a small DNA virus. HPV is can be divided into two groups: mucosal and cutaneous. HPV have various oncogenic potential. The correlation between presence of high oncogenic type of HPV and carcinogenesis was confirmed in human anogenital tract and cervical carcinoma. The latest studies suggest HPV infection involvement in benign and malignant lesions of aerodigestive tract. The aim of our study was to determine the presence of the HPV genes E6/E7. The specimens were taken after total laryngectomy from 20 patients with squamous cell carcinoma. The fresh tissue specimens were frozen at -70 degrees C until DNA extraction and HPV detection. The presence of HPV DNA was assessed by polymerase chain reaction using specific primers for HPV 16 and 18 E6/E7. There were 7 (35%) HPV positive specimens in our group. 5 (25%) specimens were HPV 16 positive, 4 (20%) specimens were HPV-18 positive. In two cases HPV 16 and HPV 18 was present. The results suggest that high oncogenic types of HPV may play a role in pathogenesis of laryngeal carcinoma.     

喉鳞状细胞癌组织中Survivin基因与p15和p16基因表达的关系

OBJECTIVE:: To investigate the expression of Survivin (SVV) and its relationship with expression of p15, p16 in laryngeal squamous cell carcinoma (LSCC). METHODS: Using strep avidin-biotin complex (SABC) method, we examined the expression of SVV, p15 and p16 gene in 48 LSCC tissus samples, 24 normal laryngeal mucosa tissus samples and 24 normal laryngeal tissus adjacent to the tumors samples. RESULTS: SVV was expression in 27 of 48 (56.3%) samples of LSCC and expression in 6 of 24 (25.0%) samples of normal laryngeal tissus adjacent to the tumors. Normal laryngeal mocosa tissus samples did not expressed SVV. Overexpression of SVV was related to the tumor site, grade, clinical stage and tumor prognosis (P < 0.05). The expression of SVV in LSCC was positive correlated with p16 expression( C = 0.52 P < 0.001), but not with p15 expression. CONCLUSION: SVV may play a role in the pathway of carcinogenesis and tumor progress. It is feasible for early diagnosis and prognosis estimation. Overexpression of SVV gene and de-activation of antioncogene p16 may be play synergetic roles in the carcinogenesis of LSCC.     

喉鳞状细胞癌组织中Survivin基因与p15和p16基因表达的关系

目的　探讨抑凋亡基因Survivin(SVV)在喉鳞状细胞癌 (laryngealsquamouscellcarcinomas,LSCC)中的表达意义及与抑癌基因p15、p16表达的关系。 方法　采用免疫组化链酶卵白素 生物素复合体 (strepavidin biotincomplex ,SABC)方法检测了 4 8例LSCC组织及 2 4例癌旁组织、2 4例正常喉黏膜组织SVV、p15、p16蛋白的表达并进行统计分析。 结果　SVV蛋白在正常喉黏膜中不表达 ,4 8例LSCC组织中 2 7例SVV蛋白阳性表达 ,占 5 6 3% ,2 4例癌旁组织中 6例SVV蛋白阳性表达 ,占 2 5 0 % SVV蛋白在LSCC组织与癌旁组织中的表达以及SVV在癌旁组织与正常喉黏膜中的表达差异均有显著性 (P <0 0 5 )。SVV蛋白表达与LSCC的发生部位、TNM分期、病理分级、淋巴结转移及预后有关 (P <0 0 5 )。p16蛋白强阳性组、p16蛋白弱阳性组及 p16蛋白阴性组的SVV蛋白阳性率差异有显著性 (P <0 0 0 1) ,且表达呈正相关 (C =0 5 2 )。p15蛋白阳性组与 p15蛋白阴性组的SVV蛋白阳性表达差异无显著性。结论SVV基因在LSCC的发生发展中起重要作用 ,并对LSCC的早期诊断、预后判断有意义。SVV的过度表达与 p16抑癌作用的失去可能在LSCC的发病机制中有协同作用。     

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.     

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.     

垂体肿瘤转移基因在喉鳞状细胞癌中的表达及临床意义

目的探讨喉鳞状细胞癌中垂体肿瘤转移基因(pituitary tumor transforming gene,PTTG)蛋白表达及其临床意义。方法采用免疫组织化学S-P法检测56例喉鳞癌及13例喉正常黏膜中PTTG的表达,分析PTTG表达与喉鳞癌临床病理指标之间的关系。结果 PTTG蛋白在喉鳞癌组织中的阳性表达率为64.3%,在喉正常黏膜细胞中的阳性表达率为23.1%(P<0.05);PTTG的阳性表达及其强度与喉鳞癌临床病理指标之间差异具有统计学意义(P<0.05)。结论 PTTG基因蛋白阳性表达及其强度可做为判定喉鳞癌生物学行为及其预后的参考指标之一。     

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。     

In situ hybridization and immunohistochemical study of human papillomavirus infection in adult laryngeal papillomas

Routinely processed paraffin sections from 20 patients with adult laryngeal papillomas were examined for the presence of human papillomavirus type 11 (HPV-11) DNA and its specific mRNA by in situ hybridization methods using 35S-labeled RNA probes. Immunohistochemical techniques were also used to identify papillomavirus genus-specific common antigen (pgs-antigen). HPV-11 DNA signals and/or papillomavirus genus-specific common antigen were detected in all eight samples of multiple laryngeal papilloma. On the other hand, in 12 samples of single laryngeal papilloma, neither papillomavirus genus-specific common antigen nor HPV-11 DNA were detected. Four patients were positive for both HPV-11 DNA and pgs-antigen. In three of these four patients, HPV-11 mRNA signals were also detected. These results provided direct evidence of the association of HPV and adult multiple laryngeal papilloma.     

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)