Detection of human papillomavirus types 6, 11, 16 and 18 in mucosal and cutaneous lesions by the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) based on the simultaneous amplification of human papillomavirus (HPV) types 6/11, 16 and 18 in a single-step procedure was developed, using primers chosen in the E6-E7 region. The specificity and sensitivity of this technique have been proved by amplifying mixtures or various amounts of plasmid-containing HPV DNA; it allowed the detection of as few as 5-25 HPV DNA copies. Application of the multiplex PCR to 71 clinical samples showed that HPV DNA was detected in 80% (45/57 cases) of mucosal biopsies and 35% (5/14 cases) of cutaneous specimens. HPV 16 was predominant in high-grade CIN whereas HPV 6 and 11 were detected more frequently in genital condylomas and laryngeal papillomas. In cutaneous Bowen's disease HPV 16, 18 or 6/11 + 16 were detected and in squamous cell carcinomas HPV 6/11 or 16 were found. After sequence amplification with primers of one HPV type, the clinical samples displayed the same HPV types but the frequency of positive and coinfected lesions increased. Thus, multiplex PCR is a valuable technique for typing HPV DNA but coinfections may be underestimated.     

Detection of human papillomavirus (HPV) type 16 and 52b in cervical cancer tissues by Southern blot hybridization and polymerase chain reaction (PCR)

DNA samples from cancer tissues diagnosed histologically as squamous cell carcinoma of the uterine cervix were examined for the presence of human papillomavirus (HPV) genome DNA by Southern blot hybridization. Of 63 specimens, 32 were found to hybridize with HPV DNA probes; 23 specimens (35%) with HPV type 16 (HPV16), one (2%) with HPV type 18 (HPV18), four (7%) with HPV type 52b (HPV52b), and four others (7%) weakly with HPV52b. Specimens negative for HPV DNA with Southern blot hybridization were subjected to polymerase chain reaction (PCR) to determine the presence of HPV DNA under more sensitive conditions. After PCR using one set of primers specific for HPV16 and HPV52b, 7 out of 31 specimens were found to have HPV16 DNA. None was positive for HPV52b DNA. Our results indicate that HPV52b, as well as HPV16 and HPV18, is associated with squamous cell carcinoma of uterine cervix, and more sensitive determination of HPV infection can be made by amplification of the viral genome by PCR.     

Human papilloma virus (HPV) is possibly involved in laryngeal but not in lung carcinogenesis

Data on human papilloma virus (HPV) involvement in preneoplastic and neoplastic lesions of the larynx and lung are limited and conflicting. The presence of HPV was investigated in a series of laryngeal specimens and non-small cell lung carcinomas (NSCLCs). The laryngeal samples (154) comprised 14 cases with hyperplasia without dysplasia, 49 with dysplasia, and 91 squamous cell carcinomas (SqCCs). The NSCLCs included 31 SqCCs, 32 adenocarcinomas, and 5 undifferentiated large cell carcinomas. Furthermore, we examined, for HPV DNA sequences, 14 bronchial metaplastic squamous lesions located next to cancerous areas. We used a sensitive nested polymerase chain reaction assay (NPCR), dot blotting, and in situ hybridization. The findings were correlated with clinicopathologic features of the patients. In the laryngeal specimens, NPCR analysis showed HPV DNA in 20 (13%) of the 154 specimens. Notably, 19 of 20 HPV-positive cases were carcinomas and only one was a mild dysplastic lesion. Typing of the carcinomas showed single HPV 6, 16, 18, and 33 infection in 1 (1.1%), 12 (13.2%), 2 (2.2%), and 1 (1.1%) samples, respectively, and HPV 6/33, 16/33, and 6/18 coinfection in three carcinomas. In situ hybridization findings were in agreement with PCR results, with the exception of two cases in which HPV 18 DNA was detected only by PCR. HPV was more frequently observed in heavy smokers than in patients with low daily cigarette consumption and nonsmokers (P = .03). There was no correlation between virus infection and gender, grade, and lymph node status of the carcinomas. None of the NSCLCs or adjacent metaplastic squamous epithelium contained HPV DNA sequences. The presented data suggest a contributory role of HPV in late stages of laryngeal carcinogenesis, because all premalignant lesions were negative but one. This study does not support a potential role of HPV in the development of NSCLCs.     

Detection of HPV 16/18 DNA in cervical adenocarcinoma using polymerase chain reaction (PCR) methodology.

Twenty-two tissue samples of primary adenocarcinoma (adenoCA) of the uterine cervix were evaluated for the presence of HPV 16/18 DNA using the polymerase chain reaction (PCR). PCR was used to specifically amplify the E6-E7 gene region of HPV 16/18 DNA. The amplification products were analyzed using gel electrophoresis and Southern dot blotting with 32p labeled type-specific oligonucleotide probes. HPV 18 DNA was identified in 13/22 (59%) and HPV 16 DNA was identified in 5/22 (23%) of the tumors. There were no tumors with mixed infections. In three patients, two different specimens were evaluated, and there was concordance of HPV typing. The presence of squamous carcinoma in situ, koilocytosis and younger patient age were associated with an increased incidence of HPV 16/18 DNA detection. HPV 16/18 DNA was not detected in six metastatic adenoCA to cervix (four endometrial, two ovarian). We conclude that HPV 16/18 DNA is present in a significant proportion of primary adenoCA of the cervix, and we have identified some clinicopathologic associations. The detection of HPV DNA may be useful in distinguishing primary from metastatic adenoCA of the cervix.     

间接法原位PCR检测喉鳞癌组织HPV感染

建立稳定的原位ＰＣＲ方法，并探讨ＨＰＶ感染与喉鳞癌发生的关系。方法采用免疫组化、原位杂变、ＰＣＲ和间接原位ＰＣＲ技术，检测了５０例喉鳞癌中的ＨＰＶ感染情况。结果免疫组化衣壳抗原阳性者６例（１２％），原位杂交阳性者１３例（２６％），ＰＣＲ阳性者１０例（２０％），原位ＰＣＲ阳性者１７例（３４％），综合上述方法的检出率为４２％（２１例）。结论ＨＰＶ感染与喉癌有着明显的关系，间接原位ＰＣＲ在检测ＨＰＶ感染     

HPV DNA in intraepithelial neoplasia and carcinoma of the vulva and penis.

Surgical specimens of 15 patients with early and 12 patients with advanced squamous cell carcinoma of the vulva and the penis were examined for the presence of human papillomavirus (HPV) type 6, 11, 16, and 18 DNA by Southern blotting (SB) and polymerase chain reaction (PCR) analysis. By SB, HPV type 16 DNA was detected in all early carcinomas and 2 of 12 cases of advanced squamous cell carcinoma (ISCC) of the vulva and penis. PCR revealed HPV DNA in four additional cases of vulvar and penile ISCC negative by SB. Three cases contained HPV16 and one HPV18. Two cases of vulvar and penile Buschke-L?wenstein (BL) tumor with malignancy and one case of vulvar verrucous carcinoma were also examined by both techniques. While BL tumors were associated with DNA of HPV6 or 11, no HPV association was found for verrucous carcinoma. Our results confirm that the detection rate of HPV DNA in early vulvar and penile carcinomas is much higher than in invasive carcinomas. In addition, we have shown that in the lower genital tract, 50% of cases of ISCC are HPV16 correlated. The absence of HPV DNA (types 6, 11, 16, and 18) in the remaining 50% of cases of ISCC thus suggests that vulvar and penile ISCC may have more than one pathogenetic pathway.     

人乳头瘤病毒阴性的喉鳞癌细胞系的建立

目的 建立1株人乳头状瘤病毒（HPV）阴性的喉鳞状细胞癌细胞系,为体外研究喉癌提供理想的实验模型。方法 以逆转录聚合酶链反应（RT-PCR）证实为HPV阴性的高分化喉鳞癌手术切除标本接种于裸鼠皮下,取连续传代2次的裸鼠皮下移植瘤进行体外原代培养。通过光镜、电镜、生长曲线、细胞周期时相、软琼脂克隆实验、异种移植成瘤实验、角蛋白、癌胚抗原及HPV检测,对其生物学特性进行初步分析。结果 经裸鼠过渡所建立的高分化喉鳞癌细胞系（Lscc-02）目前已传至86代,细胞生长增殖稳定。该细胞系呈单层形式生长,群体倍增时间为39．1h。透射电镜下见胞质内典型的张力原纤维,细胞问以桥粒方式连接。染色体为人类核型,呈亚三倍体,众数分布在63～72。该细胞系具有恶性肿瘤细胞生长特征：软琼脂中形成克隆,裸鼠接种成瘤且形态结构、分化程度与原发瘤相似。免疫组织化学显示高分子量细胞角蛋白及癌胚抗原阳性,PCR显示HPV阴性。结论 建立Lscc-02细胞系为研究无HPV感染喉癌的发生、发展规律及HPV与喉癌演进的关系提供了有价值的体外模型。     

Human papillomavirus detection in dysplastic and malignant oral verrucous lesions

The role of human papillomaviruses (HPV) in dysplastic and malignant oral verrucous lesions is controversial since there is a wide range in the incidence of virus detection. This study used a multi-tiered method of HPV detection using DNA in-situ hybridisation (ISH) for low- and high-risk subtypes, consensus PCR, and HPV genotype analysis in archival tissue from 20 cases of dysplastic and malignant oral verrucous lesions. The biological significance of HPV DNA detection was assessed by p16 immunohistochemistry (IHC). While 1/7 carcinomas and 5/13 dysplasias contained HPV DNA by consensus PCR and genotype analysis, all specimens were negative for low- and high-risk HPV ISH and negative for p16 IHC. Results show that although high-risk HPV DNA is detectable in a subset of these lesions, the lack of p16 overexpression suggests that the oncogenic process is not driven by HPV oncoproteins.     

用聚合酶链反应检测食管癌组织中人乳头瘤病毒DNA

应用聚合酶链反应（ＰＣＲ）技术对汕头市区６８例食管癌的石蜡包埋标本进行人乳头瘤病毒（ＨＰＶ）ＤＮＡ序列检测，结果显示，ＨＰＶＤＮＡ总阳性率为６６．１８％（４５／６８），检出型别主要为ＨＰＶ６、１１、１６，检出率分别为２７．９４％、３６．７６％和２７．９４％，经统计学处理三型间无显著性差异；ＨＰＶ－１８及未定型别各占８．８２％。值得注意的是ＨＰＶ感染中多重感染占阳性病例的５３．３３％（２４／４５）。初步结果表明，汕头市食管癌高发区有较高的ＨＰＶ感染率，此与食管癌的发生，可能有密切关系。     

Human papillomavirus (HPV) genotypes associated with laryngeal papilloma.

Biopsy specimens from 14 patients treated for laryngeal papillomatosis were tested for the presence of human papillomavirus (HPV) genome by the technique of DNA-DNA hybridisation. According to the age of initial presentation, cases were subdivided into juvenile (less than 16 years) and adult onset (older than 16 years) groups. Histological investigation confirmed that it was impossible to distinguish the groups on this basis. Molecular virology using both dot blot and Southern transfer techniques showed that 10 cases carried the HPV type 6 genome, three cases HPV type 11, and in one case no HPV DNA was detected. All six adult onset cases carried HPV 6 sequences while the juvenile onset group comprised four HPV 6 and three HPV 11 cases. In the juvenile onset group more females were affected; in the adult onset group more males were affected. Two of the patients shown to have HPV type 11 sequences in their biopsy material were the most resistant to treatment. One of the adult onset cases subsequently developed a squamous cell carcinoma of the larynx in which HPV 6 DNA was detected. As far as we know this is first time that HPV-DNA has been confirmed in laryngeal papilloma undergoing malignant change.     

女性下生殖道癌临床病理特征与人乳头状瘤病毒型别间的相关性研究

目的：探讨女性下生殖道癌的临床病理与人乳头状瘤病毒（HPV）型别之间的关系。方法：回顾性研究100例下生殖道癌（宫颈癌63例,外阴癌37例）的临床病理特征,并应用PCR技术检测每份标本的HPV状态。结果：在模板内参照阳性的87份中,宫颈癌54份,外阴癌33份,HPV阳性率在宫颈癌中为88．3％,以HPV16型（55．6％）和HPV18型（24．4％）为主,在33例外阴鳞癌中,HPV阳性仅见于基底细胞癌和混疣样癌,阳性率均为83．3％,以HPV16型为主（70．0％）;6例基底细胞样癌中有3例合并宫颈鳞状上皮肿瘤,其中2例宫颈与外阴的肿瘤均为HPV16型阳性;21例角化鳞癌则未检测出HPV－DNA,但有发病年龄高（平均63．3岁）。形态学上角化明显和预后较差等特征。结论：HPV16型和18型在宫颈癌中性阳率较高,而在外阴癌中HPV阳性的意义则因组织学类型而不相同。     

Is p16 immunohistochemistry a more cost-effective method for identification of human papilloma virus–associated head and neck squamous cell carcinoma?

Tobacco and alcohol use are established risk factors for head and neck squamous cell carcinoma (HNSCC). However, patients with a unique subset of human papilloma virus (HPV)-associated HNSCC have been documented to have a better survival outcome. These tumors occur more frequently in the tonsils and oropharyngeal sites, among younger patients, higher socioeconomic group, and those exposed to more sexual partners and oral sex compared with HPV-negative HNSCC. Although tobacco- and alcohol-related HNSCCs appear to be on the decline, tonsillar and oropharyngeal HPV-associated tumors seem to be on the rise, and their prevalence varies widely in published reports, ranging from 20% to 60%. Human papilloma virus detection methods in tumor tissue vary and include polymerase chain reaction, in situ hybridization (ISH) technique for HPV DNA, and E6/E7 messenger RNA, with p16 immunohistochemistry (IHC) as a surrogate marker. The sensitivity and specificity of the different methods used are likely contributory factors to this wide variation. This study compares the p16 IHC staining patterns in HNSCC and laryngeal papillomas and assesses the concordance of p16 and high-risk HPV-ISH to determine the usefulness of p16 as a first-line marker. Using an objective criterion of diffuse intense confluent staining pattern as definite positive (akin to the 3+ of HER2/neu in breast cancer) and focal scattered staining pattern as equivocal reaction requiring confirmatory HPV assay, p16 IHC expression shows good concordance with high-risk HPV-ISH and can be used as a first-line marker. We propose p16 overexpression as a suitable surrogate and screening marker, with high potential of impacting the standard of care.     

人喉癌组织中人乳头瘤病毒DNA的检测

目的为探讨喉癌与人乳头瘤病毒（ＨＰＶ）感染的关系和ＨＰＶ在喉癌中基因组型的分布与表达。方法应用聚合酶链反应技术（ＰＣＲ）制备非放射性探针标记物－地高辛标记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ＨＰＶＤＮＡ感染的检测;阳性者用多重引物ＰＣＲ方法分型。结果喉癌ＨＰＶ感染阳性率４５．６％（３１／６８）,喉癌颈转移淋巴结组织阳性率２０．０％（３／１５）,喉癌前病变阳性率１１．８％（２／１７）,声带息肉阳性率６．３％（１／１６）,１５例癌旁及１５例癌周正常喉组织均为ＨＰＶＤＮＡ阴性。ＨＰＶＤＮＡ型别分布在喉癌中以ＨＰＶ１６、１８型为主,喉良性病变中以ＨＰＶ６、１１型为主。结论喉癌发生与ＨＰＶ感染有关。     

Cytology of metastatic cervical squamous cell carcinoma in pleural fluid: Report of a case confirmed by human papillomavirus typing

Cervical squamous cell carcinomas are rarely the cause of malignant effusions. Their identification can be relatively easy when keratinizing atypical squamous cells are present, but may be very difficult when only nonkeratinizing malignant cells are present. We present the case of a 47‐year‐old woman who presented with a large left pleural effusion after having recently completed chemoradiation therapy for stage IIB cervical squamous cell carcinoma. Cytologic examination of the fluid showed a uniform population of single atypical cells with finely vacuolated cytoplasm, ectoendoplasmic demarcation, cell‐in‐cell arrangements, and short rows of cells with intervening “windows,” all features reminiscent of mesothelial cells. No keratinization or three‐dimensional cell clusters were identified. A panel of immunohistochemical stains was performed on the cell block material, and the atypical cells were positive for cytokeratin 5/6, p63, and p16 but not for cytokeratin 7, calretinin, WT1, or Ber‐EP4 or TTF1. These findings were consistent with metastatic squamous cell carcinoma. HPV DNA determination and typing by PCR confirmed the presence of HPV16 in an aliquot of pleural fluid. This is to our knowledge the first reported case of pleural fluid involved by metastatic squamous cell carcinoma where HPV DNA testing was used to confirm the origin of the metastasis. Despite its rarity, metastatic nonkeratinizing squamous cell carcinoma should be considered when a single cell population of large atypical cells is found in effusions. Immunoperoxidase stains and HPV testing can be performed to establish the diagnosis and confirm the origin from a cervical primary. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Detection of human papillomavirus DNA in squamous bronchial metaplasia and squamous cell carcinomas of the lung by in situ hybridization using biotinylated probes in paraffin-embedded specimens

We examined a series of paraffin-embedded tissue specimens from 10 cases of squamous bronchial metaplasia and 33 cases of squamous cell carcinoma of the lung for histologic characteristics and for the presence and typing of human papillomavirus (HPV) by molecular in situ hybridization with biotinylated probes types 6, 11, 16 and 18 under stringent conditions (temperature, 19 degrees C). Fourteen of these lesions (32.5%) showed typical condylomatous histologic changes. Human papillomavirus DNA was present in seven (16%) specimens. Type 6 HPV DNA was detected in one of the squamous bronchial metaplasia cases. In six of the squamous cell carcinomas cases (18%), HPV DNA was identified (type 18, three cases; type 16, one case; type 11, one case; and type 6, one case); one of the squamous cell carcinoma specimens contained both HPV types 16 and 18. Our data confirm the presence of HPV DNA in squamous metaplastic bronchial mucosa and epidermoid lung carcinoma on paraffin-embedded tissues. This suggests that an HPV infection with benign or potentially oncogenic HPV types could be associated not only with genital tumors, but also with bronchial and lung tumors. The role of HPV DNA in the process of malignancy conversion is not yet known; HPV DNA could possibly be a cocarcinogenic factor. In situ hybridization with biotinylated probes is a useful and appropriate method of retrospective analysis of HPV DNA sequences in routinely paraffin-embedded lesions. It may be used to identify patients at risk of more serious or possibly malignant progression.     

Prevalence of human papillomavirus in sinonasal papillomas: a study using polymerase chain reaction and in situ hybridization.

Inverted and fungiform papillomas of the sinonasal cavity share a common origin from the Schneiderian membrane, but they differ widely in their rates of recurrence and progression to carcinoma. To determine the role of human papillomavirus in the etiology of these lesions, 15 inverted papillomas, five fungiform papillomas, and two squamous cell carcinomas associated with inverted papilloma were examined for the presence of HPV by in situ hybridization (ISH) and polymerase chain reaction (PCR). ISH was carried out on formalin-fixed, paraffin-embedded material using HPV types 6/11, 16/18, and 31/33/35 DNA probes. Tissue DNA was amplified by PCR with HPV L1 consensus primers, and the product was detected by gel electrophoresis, Southern blotting, and hybridization with type specific probes (HPV types 6/11, 16, 18). Three of 15 inverted papillomas and two of five fungiform papillomas were positive for HPV 6/11 by ISH, whereas PCR detected HPV 6/11 sequences in two of 15 inverted and three of five fungiform papillomas. Biopsies from two patients who had serial resections contained HPV 6/11 in the original lesions and all recurrences. No HPV was detected in the carcinomas by ISH, whereas PCR detected HPV 16 in one carcinoma. These findings confirm the presence of HPV DNA sequences in both inverted and fungiform sinonasal papillomas as well as in an associated squamous carcinoma. This would suggest a role for HPV in the pathogenesis of Schneiderian membrane lesions. Furthermore, our data indicate that ISH and PCR are equally sensitive in detecting HPV in sinonasal papillomas.     

Human papillomavirus was not detected by PCR using multiple consensus primer sets in esophageal adenocarcinomas in Chinese patients

The role of human papillomavirus (HPV) infection in the development of esophageal squamous cell carcinoma is well established; however, there are few reports on the role of HPV in esophageal adenocarcinoma. To evaluate the putative role of HPV infection in esophageal adenocarcinoma, 57 formalin‐fixed, paraffin‐embedded esophageal adenocarcinoma specimens were collected from four hospitals in Shanghai and Anyang, China, between 1999 and 2008. HPV DNA was analyzed using PCR with multiple sets of consensus primers for HPV, GP5+/6+, CPI/CPIIG, SPF₁₀, pU‐1M/pU2R, and pU31B/pU2R. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), the internal control, was amplified successfully in all 57 specimens. However, HPV amplification was not detected in any specimens with any of the consensus primer sets used. The present study indicates that HPV infection is not likely to be a major factor in the etiology of esophageal adenocarcinoma in the Chinese population. J. Med. Virol. 85: 1053–1057, 2013. © 2013 Wiley Periodicals, Inc.     

Detection of papilloma virus of the human uterine cervix by in situ hybridization method--comparison with immunohistochemistry

The usual methods for pathological diagnosis of HPV infection of the uterine cervix include screening in cytodiagnosis and histodiagnosis and confirmation by immunohistochemistry (IHC) method. However, some institutes have recently begun to use in situ hybridization (ISH) method for definitive diagnosis using a DNA probe. We compared IHC with ISH with regards to the localization and rate of detection of HPV in lesions of the uterine cervix such as dysplasia and squamous cell carcinoma in the present study. The cases found positive by IHC showed brownish nuclei of the epithelium and those positive in ISH showed purple to purplish-black nuclei. The comparison of cases positive by both methods revealed that the number of cells positive by IHC was smaller than that by ISH, and the cells positive by IHC were localized in the superficial layer. HPV was detected by the IHC various lesions of the uterine cervix in 13 (12.3%) of 106 patients, while it was detected by the ISH in 39 (36.8%) of 106 patients. The results of both methods were in accordance in 66.0% (77 patients; positively in 8 and negatively in 62). The detection sensitivity of IHC is lower than that of ISH. IHC cannot be used to identify the type of HPV, and it is impossible to confirm the presence or absence of virus by this method in cases of malignant changes. ISH is therefore necessary for identification of HPV and investigation of a histopathological relationship between HPV type and malignant change.     

Human papillomavirus in oesophageal squamous cell carcinoma.

Thirty seven cases of oesophageal squamous cell carcinoma were studied by applying DNA slot blot analysis and in situ hybridisation using type specific probes for HPV 6, 11, 16 and 18. Cases of condyloma accuminata, cervical carcinoma, and laryngeal papilloma were used as controls. Blocks including areas of invasive carcinoma, intraepithelial neoplasia, and normal epithelium were studied in each case. No HPV genome was detectable in any of the oesophageal cases. It is concluded that these types of HPV do not have an association with oesophageal squamous cell carcinoma.