Detection of human papillomavirus (HPV) DNA in oral squamous cell carcinomas by in situ hybridization and polymerase chain reaction

Summary A series of 156 formalin-fixed, paraffin-embedded biopsies from 40 patients with surgically-treated oral squamous cell carcinomas was analysed for the presence of human papillomavirus (HPV) infection by histopathological evaluation, in situ DNA hybridization and polymerase chain reaction (PCR). Epithelial changes suggesting a HPV lesion within, or adjacent to, the carcinoma lesions were found in 16 out of 40 patients (40%). Morphological signs of a flat HPV lesion were found in four cases (10%), those of inverted type in three cases (7.5%), and those of papillary type in nine cases (22.5%). HPV DNA was demonstrated in one of the lesions by in situ hybridization with biotin-labelled DNA cocktail probe containing HPV types 6, 11, 16 and 18. With the PCR technique, samples from 11 (27.5%) of the 40 patients proved to contain HPV DNA. Of these, HPV 6 was demonstrated in one case, HPV 16 in ten cases and HPV 18 in one case. HPV DNA was exclusively detected in the biopsies showing carcinoma tissue or its adjacent precancer lesions. No viral DNA was found in the biopsies derived from the tumour-free resection margins. These results provide further evidence to support the concept of HPV involvement in the aetiology of oral squamous cell carcinomas, most probably acting synergistically with other carcinogens.     

Detection of human papillomavirus DNA sequences in cancer of the urinary bladder by in situ hybridisation and polymerase chain reaction.

OBJECTIVE--To evaluate the prevalence of "high risk" human papillomavirus type 16 (HPV 16) in transitional cell carcinoma of the urinary bladder. MATERIALS AND METHODS--The study included 10 biopsy specimens from male patients of transitional cell carcinoma of the urinary bladder for the detection of HPV DNA sequences. Specimens were collected from the Urology Clinic of the K.G. Medical College Hospital, Lucknow, India. Detection of HPV DNA was carried out by tissue in situ hybridisation (a single copy gene localisation method) using 3H-labelled HPV DNA probe and also by polymerase chain reaction (PCR) techniques using primers to HPV 16 upstream regulatory region (URR). RESULTS--Out of 10 cases of transitional cell carcinoma of the urinary bladder, "high risk" HPV 16 DNA was detected only in one (10%) by using in situ hybridisation whereas two cases (20%) were found to be positive by polymerase chain reaction. CONCLUSION--Our results suggest that the rare occurrence of HPV in bladder carcinoma may not have a causal relation with the viral infection.     

Detection of human papillomavirus in cutaneous extragenital Bowen's disease in immunocompetent patients

INTRODUCTION: A specific link between human papillomavirus (HPV) types 16, 18, 31, and 33 and genital carcinomas and between HPV type 5 and cutaneous extragenital carcinomas in patients with epidermodysplasia verruciformis and renal transplant has been previously found. The aim of this prospective study was to detect HPV in cases of cutaneous extragenital Bowen's disease (BD) from non-immunosuppressed patients. PATIENTS AND METHODS: Twelve cases of cutaneous extragenital BD or Bowen's carcinoma (BC), seen in the period 1994-1996 and confirmed by histologic examination, were included in the study. Tissue sections were studied by in situ hybridization with a mixture of HPV DNA probes and specific HPV DNA probes. In addition, study on fresh materiel from 1995 included: Southern blot hybridization with various usual HPV probes (6, 11, 16, 18, 31, 33, 35, 39, 42), polymerase chain reaction (PCR) with hybridization using consensus HPV probes and probes specific for HPV types 6, 11, 16, 18 and 33. In positive samples with conventional PCR, in situ PCR with probes specific for HPV types 6/11 and 16 was performed on tissue sections. RESULTS: In situ hybridization was negative in all the cases. Southern blot hybridization was negative in our 9 studied cases. Three cases studied by consensus PCR were positive. PCR with specific HPV probes revealed positivity on two of these cases: HPV 6 in one, and HPV 16 in another. In situ PCR was positive with a mixed 6/11 HPV probe in the third positive consensus PCR case. DISCUSSION: Our study revealed the presence of HPV in 3 out of 12 cases of cutaneous extragenital BD and BC. HPV type 16, found in BC of skull, was the most usually found type in the literature. HPV types 6/11, detected in 2 cases, were rarely found in cutaneous extragenital BD and BC and these results are in favor of the oncogenic effect of these virus types. In our study, in situ hybridization and Southern blot hybridization were negative in all the cases; HPV was only found in 3 cases by conventional PCR and in 1 case by in situ PCR. The low range of detection of HPV in cutaneous extragenital BD may be due to the used methods, to difficulties related to sampling and/or to a low number of copies of the HPV genoma.     

Human papillomavirus and oncogenesis (author's transl)

Various types of human papillomavirus (HPV) are responsible for different warts. Characteristic of HPV infections is a lowered cell-mediated immunity (CMI), especially defective in cases of epidermodysplasia verruciformis induced by HPV5. The malignant transformation seems to be related with the type of HPV, and not with the grade of CMI defect and duration of the infection. A life-long infection with HPV3 in cases of EV does not induce a malignant transformation in spite of heavily depressed CMI. The oncogenic potential has seemingly HPV5 responsible for red plaques and pityriasis versicolor-like lesions characteristic of EV. Malignant transformation, although rare, may also occur in long-lasting cases of genital warts (Buschke-Loewenstein-disease) and juvenile larynx-papilloma. The most important problem is an early diagnosis and prophylaxis of genital cancers in women. This was made possible by cytological and virological studies of cervical dysplasia. In the condylomatous and non-condylomatous lesions of the colli uteri virus particles have been found using the immunoperoxidase technique and immunserum against disrupted viral particles, i.e. against the common HPV antigen. However, the type of HPV can not be determined by this method. Using the same immunsera virus particles were found also in the bowenoid genital papules. The best model for human oncogenesis remains EV. The clinical and pathological morphology, virological and immunological studies on the non-specific and HPV-type specific humoral and cellular immunity are presented in detail.     

Multibranched acquired periungual fibrokeratomas with confounding histopathologic findings resembling papillomavirus infection: a report of two cases

Acquired periungual fibrokeratomas are benign fibrous tissue tumors and are considered as the topographical variant of acquired digital fibrokeratoma. They usually present as solitary tumors. In some instance, the entity may appear in multibranched fashion. The main histopathologic features consist of acanthosis, thick collagen bundles mainly oriented in a vertical axis forming a central core, and numerous proliferating fibroblasts. In this article, we present two cases of acquired multibranched periungual fibrokeratoma and depict their varying clinical features over time. Binucleation and perinuclear halos of keratinocytes mimicking human papillomavirus (HPV) infection were detected microscopically, but there was no reactivity with HPV immunostaining. In context, anti‐HPV immunostaining may be helpful in the differentiation of fibrokeratomas from HPV infection. On the other hand, it should be kept in mind that these histopathologic findings may be found in acral biopsies independent of viral effects.     

Oral focal epithelial hyperplasia

Focal epithelial hyperplasia (FEH) or Heck disease, is a rare viral infection of the oral mucosa caused by HPV 13 or HPV 32. In Caucasians there have been only a few cases reported. We present the first case in Greece in a young Caucasian girl in which HPV 13 was detected with PCR analysis. The patient was successfully treated with CO2 laser.     

Detection of human papillomavirus type 56 in Bowen's disease involving the nail matrix

Background As Bowen’s disease of the nail apparatus is quite rare, there have been only a few reports on the prevalence of human papillomavirus (HPV) infection in this condition. Objectives The purpose of this study was to clarify the association of HPV with this disease involving the nail apparatus. Methods Five patients with Bowen’s disease of the nail apparatus were investigated clinically, virologically and histologically. Total DNAs extracted from excised skin lesions were analysed using polymerase chain reaction (PCR) for the presence of HPV DNA and the amplified products were subjected to DNA sequence analyses. Histological localization of HPV DNA was examined by in situ hybridization. Results In three of five patients, HPV was detected by PCR amplification, and subsequent sequence analyses of the PCR products showed the sequences of HPV type 56. A common clinical feature of the three HPV‐positive patients was longitudinal melanonychia. In contrast, the two HPV‐negative patients presented with a convex nail deformity and a periungual ulcerative lesion. In two of three positive cases, there was a silent point mutation in the L1 gene of each HPV. In the remaining one case, the nucleotide sequence was consistent with the consensus sequence of HPV 56. Sequence analyses of the E6 gene revealed the infection of different variants of HPV 56 among the three cases. The viral genomes were located in keratinocyte nuclei upon in situ hybridization. Conclusions HPV 56 may be involved in the carcinogenesis of Bowen’s disease affecting the nail matrix with longitudinal pigmentation.     

性病门诊378例女性宫颈分泌物的HPV基因型分析

目的检测和分析性病门诊女性患者宫颈分泌物中人乳头瘤病毒(HPV)的基因型别。方法收集我院性病门诊就诊的女性患者378例,按患者有无高危接触和宫颈口有无尖锐湿疣症状划分为3组,即有接触有症状组(161例)、有接触无症状组(117例)和无接触无症状组(100例)。取3组患者脱落细胞,采用反向分子杂交法进行HPV基因分型检测。结果性病门诊378例女性宫颈分泌物HPV阳性率为56.61%(214/378),有接触有症状组、有接触无症状组和无接触无症状组的阳性率分别为98.75%(159/161)、40.17%(47/117)和8.00%(8/100),3组分别检出22种、14种和5种HPV基因型别,检出的主要型别是HPV6、11、16、58、56、18、33、68。有接触有症状组和有接触无症状组单一感染和复合感染的感染率差异无统计学意义(P0.05),在多重复合感染中,以二重和三重感染为主,最多发现八重感染。有接触有症状组低危型121例次(76.10%),高危型99例次(62.26%),有接触无症状组低危型35人次(74.46%),高危型26例次(55.32%),两组感染率较高,但差异无统计学意义(P0.05)。结论 HPV6、11、16、58、56、18、33、68是性病门诊女性患者宫颈感染HPV的主要型别,健康体检者中亦能查出,临床应加强对性病门诊女性就诊者宫颈HPV基因的检测,及对HPV阳性患者进行定期的跟踪随访,尤其是高危型和复合感染患者。     

直接原位聚合酶链反应检测尖锐湿疣中的人乳头瘤病毒

评价用直接原位聚合酶链反应(PCR)技术辅助诊断尖锐湿疣(CA)的价值及观察人乳头瘤病毒(HPV)在CA中的组织学分布模式。25例CA中,10例具典型组织学特征的CA均有HPV分布,主要位于表皮浅层,另11例缺乏典型空泡细胞的CA在颗粒层或棘细胞全层测得HPV.直接原位PCR通过标记生物素的寡核苷酸在扩增时直接掺入结合,再用免疫组织化学测定,能在石蜡切片上将病毒检测结果与组织学变化直接相比,有助于病理学家对可疑损害作HPV感染的分析。     

Characterization of skin lesions induced by skin‐tropic α‐ and β‐papillomaviruses in a patient with epidermodysplasia verruciformis

Epidermodysplasia verruciformis (EV) is a rare, lifelong, autosomal recessive skin disease associated with an unusual susceptibility to infections with ubiquitous β‐human papillomaviruses (β‐HPVs), and in some cases also skin‐tropic α genotypes. In this case report, HPV infection patterns were correlated with pathology and clinical manifestations of skin lesions from a patient with EV, without loss‐of‐function mutations in the EVER genes. HPV infection was investigated by both polymerase chain reaction (PCR) and laser capture microdissection (LCM) PCR, alongside immunofluorescence for the viral proteins E4 and L1. Analysis of eyebrow hair bulbs revealed multiple β‐genus HPV infections, including HPV20 and HPV24, which were consistently found in all 11 skin lesions on the patient. Six lesions were also positive for the skin tropic α‐genotype, HPV27. Clear‐cut differences between two wart‐like lesions, one caused by a skin‐tropic α‐genotype and the other by β‐genotypes (as detected by LCM PCR) are shown, including the high cellular proliferation rate in β‐HPV‐induced lesions. Widespread expression of the early protein E4 was also evident in skin lesions positive for HPV20 by LCM PCR in both tumours and nearby intraepidermal proliferative areas. L1 expression was restricted to areas of intraepidermal proliferation showing productive infection. The patient's inability to control HPV infections is conclusive to the uncontrolled replication of few genotypes from both α and β genera, which cause proliferative lesions with clear‐cut clinical and histological features.     

Butchers' warts: no evidence for person to person transmission of HPV7

Summary The distribution of warts due to HPV7 in workers in six abattoirs and 103 retail and wholesale butcheries has been studied to determine whether the high prevalence of HPV7 in the meat trade is the result of enhanced person to person transmission, or whether it is a ubiquitous virus which is activated by an unknown factor in meat. Warts were detected in 164 of 486 men. Scrapings were taken from 156 men, and HPV DNA was found in 112 samples, 74 of which contained HPV7. HPV7 was found in 36 workplaces, and there was no evidence of clustering of cases, as would be expected if person to person transmission was occurring in the workplace. This suggests that HPV7 is widely distributed in the community, but only causes clinical disease under specific conditions. We suggest that some unknown factor in meat enhances viral replication.     

Common warts from immunocompetent patients show the same distribution of human papillomavirus types as common warts from immunocompromised patients

We studied the papillomfiviruses (HPV) found in 131 common warts Irom 111 immunocompetent patients by amplification of viral DNA sequences with the general-primer-mediatcd polymerase chain reaction (PCR). The virus types were determined by restriction-enzyme cleavage and reverse-hlot analysis. Results were confirmed by using the Southern blot technique. Forty patients harboured HPV 2a. 25 individuals showed HPV 2c and 13 yielded HPV 57. Common warts from 16 patients were induced by a variant of HPV 57. HPV 7 was found in four patients. HPV 1 was identified in two patients, and there was evidence for HPV 4 in only one case. One individual yielded an HPV type wbich was only weakly related to HPV 2. Three patients were infected by more than one HPV type. PCR did not demonstrate HPV-DNA in warts from six individuals. The distribution and variation of HPV types found in the common warts of immunocompetent patients were similar to the findings in immunocompromised patients reported by other authors.     

Analysis of oral papillomas, leukoplakias, and invasive carcinomas for human papillomavirus type related DNA

Five papillomas, five leukoplakias, and six carcinomas were investigated for the presence of papillomavirus group-specific antigens and viral DNA. Viral proteins were identified with genus-specific papillomavirus antibodies. Cloned human papillomavirus (HPV) 11 and 16 DNA were used as probes in Southern blot hybridization at conditions of different stringency in order to determine viral DNA. Four of five papillomas, four of five leukoplakias, and three of six carcinomas reacted with HPV DNA probes and revealed some stained cells after exposure to HPV antibodies. HPV type 16 was found in one carcinoma and HPV type 11 was demonstrated in another case of carcinoma.     

In situ DNA hybridization analysis of human papillomavirus (HPV) sequences in benign oral mucosal lesions

Summary A series of 144 surgically treated benign oral mucosal lesions were analysed using an in situ DNA hybridization technique with ³⁵S-labeled human papillomavirus (HPV) DNA probes to demonstrate the DNA of HPV types 6, 11, 13, and 16, in routinely processed, paraffin-embedded biopsy specimens. These lesions and an additional 62 benign oral mucosal biopsy specimens (total, 206 specimens) were also assessed by the indirect immunoperoxidase (IP-PAP) technique to detect the expression of HPV structural proteins (viral antigens). A total of 54/206 (26.2%) lesions were observed to express HPV antigens, being found in 45/92 (48.9%) of the squamous cell papillomas/condylomas, in 1/54 fibrous hyperplasias, in 1/8 true fibromas, and in 7/8 (87.5%) of the focal epithelial hyperplasia (FEH) lesions. Of the HPV DNA-positive lesions, 15/45 (33.3%) expressed HPV antigens, the expression not being related to any particular HPV type. HPV DNA sequences were found in 45/144 (31.3%) of the lesions. HPV DNA was present with the highest frequency in FEH (83.3%), papillary hyperplasia (28.6%), fibrous hyperplasia (24.4%), and true fibromas (14.3%). The most frequent HPV type was HPV 11, representing 37.8% of the DNA-positive lesions. HPV 13 DNA, previously regarded as specific to FEH, was disclosed as a single HPV type in seven cases, and as a double infection by HPV 11 and 13 in an additional three cases, including all five morphologically distinct entities. Noteworthy is the discovery, of the high-risk HPV type 16 DNA in 17.8% of the DNA-positive lesions, four papilloma/condyloma lesions, three fibrous hyperplasias, and one FEH. The results confirm the previously reported evidence regarding HPV involvement in oral mucosal lesions. The implications of these findings are discussed in terms of the epidemiology, HPV etiology, and proper classification of the oral mucosal lesions, with special emphasis on the discovery of the high-risk HPV type 16 in the benign lesions as well as in oral cancer. The use of the in situ DNA hybridization as a powerful tool in detecting the specific HPV DNA sequences in routinely processed oral biopsy specimens is strongly recommended.     

人乳头瘤病毒与皮肤Bowen病的相关性

目的 探讨人乳头瘤病毒(HPV)与皮肤Bowen病发病的相关性.方法 41例皮肤Bowen病患者皮损以及48例健康对照皮肤,采用多对引物,应用巢式PCR进行HPV DNA检测,同时应用半定量PCR进行病毒定量,对HPV DNA阳性标本进一步采用原位杂交方法分析组织内病毒的分布状况.结果 41例皮肤Bowen病患者皮损HPV DNA阳性检出率为12%(5例),其中黏膜型3例(2例HPV16,1例HPV33),病毒定量相当于101～103拷贝,原位杂交显示在多数肿瘤细胞核中有广泛阳性表达,而肿瘤邻近的正常组织无信号表达;皮肤型2例(HPV27和HPV76各1例),原位杂交均无阳性表达.此外,对照组中有1例检出HPV DNA,属于疣状表皮发育不良相关型HPV23,检出率为2.1%,与皮肤Bowen病皮损中HPV检出率比较,差异无统计学意义.2例患者皮肤型病毒定量较低,与正常对照组中检出的1例HPV23相似,均相当于10-2～10-3拷贝.结论 某些皮肤Bowen病的发病与黏膜型HPV密切相关.     

Diagnosis of genital human papillomavirus (HPV) lesions in the male: correlation of peniscopy, histology and in situ hybridisation.

OBJECTIVE--To assess the diagnostic criteria of genital HPV lesions in male sexual partners of HPV infected women. METHODS--Peniscopically directed biopsy specimens (from 693 lesions in 300 men) were examined on light microscopy and in situ hybridisation (ISH) for HPV types 6,11,16,18,31,33 and 42. The predictive value of different histological criteria for ISH positivity was also evaluated using stepwise logistic regression analysis. RESULTS--Flat HPV lesions were most accurately predicted by the punctuation pattern on peniscopy, giving the concordance between peniscopy and histology between peniscopy and histology of 79.5% (66/83) and that between peniscopy and ISH of 56.6% (47/83). Diffuse acetowhite pattern disclosed a typical HPV lesion in only 17.8% (13/73), and HPV DNA was found in 11.0% (8/73) of cases. Of the 114 biopsy specimens from peniscopically healthy areas adjacent (0.5-1 cm) to the lesions, 93.0% (106/114) were normal on light microscopy, and HPV DNA was found in only 2.6%. Penile intraepithelial neoplasia (PIN) lesions were most frequently ISH positive, 81.1% (30/37), 50% showing HPV 16 and/or 18 DNA. Lesions classified as HPV-suspicious or nonspecific on light microscopy were HPV DNA-positive in 16.9% (11/65) and 8.1% (13/160), the frequency of high-risk HPV types being 3.1% and 1.3%, respectively. In logistic regression analysis, koilocytosis was the most powerful predictor of ISH-positivity in the flat lesions (without PIN), the risk ratio being 3.7. CONCLUSION--No conclusive peniscopic criteria for male HPV infections could be established, making histological evaluation mandatory. Care should be exercised in interpreting as HPV lesions the cases devoid of koilocytosis, HPV typing being essential in confirming the diagnosis in doubtful cases.     

尖锐湿疣组织中HPV型别检测及HPV11型L1基因的克隆与测序

目的分析黑龙江地区尖锐湿疣组织感染的HPV型别分布情况,并对HPV11型流行株L1基因进行克隆和测序,检测序列变异情况。方法提取30例尖锐湿疣组织标本中的DNA,用HPVL1区通用引物及特异性HPV6b,11,16,18型引物进行PCR扩增,分析尖锐湿疣组织中感染的HPV型别分布。对其中4株HPV11型阳性标本L1基因,经酶切、连接,分别构建pSP73-HPV11L1重组质粒,并对其L1片段进行测序。结果30例标本中,HPV6b和11型感染数分别为11例,各占36.7%;有2例合并HPV6b/11/16型的混合感染,占总病例数的6.7%;1例HPV16型单独感染,未鉴定出HPV18型感染,其他型别5例。pSP73HPV11L1重组质粒测序后,4株HPV11L1基因序列完全一致,可认为是黑龙江省流行株。其序列与原始株序列相比,仅发现了两处同义点突变。结论黑龙江地区尖锐湿疣组织中HPVDNA检出率为100%,以6b和11型为主,偶见高危型别16型感染。克隆测序表明黑龙江省流行株HPV11型L1基因高度保守。     

Detection of human papillomavirus type 16 in Bowen's carcinoma of the toe

Background Human papillomavirus (HPV) is known to cause cervical cancer. Because it has been detected in lesions of Bowenoid papulosis, Bowen’s disease, and Bowen’s carcinoma, HPV infection has been implicated in the pathogenesis of these diseases. Methods A 44‐year‐old man was diagnosed clinicopathologically with Bowen’s carcinoma of the right great toe. He developed multiple organ metastases and died. We examined HPV DNA in skin biopsy specimens from the primary and skin metastatic lesions by polymerase chain reaction (PCR) and in situ hybridization (ISH). The PCR assay was carried out using primer sets specifically designed for detecting the E6 and E7 genes of the HPV types associated with malignancy. Purified and cloned PCR products were subjected to DNA sequence analysis. The ISH studies used INFORM^® HPV III probes. Results We found HPV DNA in specimens from both the primary and the skin metastatic lesions. DNA sequencing detected HPV type 16. We compared the base sequences of viral DNA from the primary and metastatic lesions. Point mutations of the base sequences of the E6 and E7 genes were observed in viral DNA from metastases but not in that from primary lesions. The E6 gene had mutated from G to A in the 383rd base sequence, causing a Glu‐to‐Lys amino acid change. Results of ISH showed punctuate signals in the nuclei of tumor cells. Conclusions We suspect an association between HPV 16 infection and the development of this malignant occurrence.     

人乳头瘤病毒型别及载量与寻常疣临床特征相关性研究

目的 探讨人乳头瘤病毒型别、载量与寻常疣临床特征的相关性。 方法 提取48例寻常疣组织DNA;正反向测序通用引物PCR扩增片段,通过blast比对分型,并通过型别特异性引物PCR来验证和补充测序结果;实时荧光定量PCR相对定量ΔCt法检测HPV病毒载量;HE染色法观察病理变化。 结果 35例HPV PCR检测阳性的标本中HPV 7型32例;HPV 57型1例,为四肢末端多发;HPV 2和HPV 7混合感染2例,为头面部、躯干多发病例。单发组与多发组、病程 ＜ 6个月组与病程6个月至1年组病毒载量、空泡化细胞数目无明显差异,1年后病毒载量有下降趋势,病程 ＞ 2年的皮损,角化过度更明显,空泡化细胞减少。 结论 寻常疣的HPV感染型别主要是HPV 7;HPV 7型感染好发于头面部;对于病程1年内的寻常疣,HPV病毒载量及空泡化细胞数目与其数目、病程长短无明显相关性。