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.     

14例鲍温样丘疹病皮损人类乳头瘤病毒DNA原位杂交的检测

应用地高辛标记的 HPV6B/11、16、18型 DNA探针对 14例鲍温样丘疹病进行原位杂交检测。结果显示 14例中 7例 HPV 6B/11和 18型阳性 ,两者反应强度相似 ,即强阳性 3例 ,弱阳性 4例 ,而6B/11、18型阳性同时伴 16型阳性的仅 3例 ,且 16型反应较弱 ,表明有半数鲍温样丘疹病的发病与 6B/11、18型 HPV感染有关     

Detection of human papillomavirus type 2a DNA in verrucae vulgares by electron microscopic in situ hybridization

Electron microscopic in situ hybridization (EMISH) of common warts (verrucae vulgares) of the hands was performed using a biotinylated human papillomavirus type 2a (HPV-2a) DNA probe and immunogold labelling of ultrathin sections of 2% glutaraldehyde-fixed, Lowicryl K4M-embedded tissues. It was first established that the warts contained HPV-2a DNA by light microscopic in situ hybridization. The HPV-2a probe chiefly labelled cells in the horny, granular and upper spinous layers of the epidermis, and labelling decreased towards the basal cell layer. The gold particles were located precisely on the viral particles in the nuclei of granular cells. The lower limit of detection by EMISH was found to be the keratinocytes of the third cellular layer above the basal cells. These keratinocytes showed evidence of a viral cytopathic effect, suggesting that vegetative DNA replication in infected keratinocytes occurs at least as early as this level of the epidermis.Presented in part at the 91st Annual Meeting of the Japanese Dermatological Association, Chiba, April 1992     

Evaluation of a solution chemiluminescent hybridization assay for identification of human papillomavirus from paraffin-embedded tissues

Aims To evaluate the use of the Digene Hybrid Capture^TM system, a chemiluminescent hybridization assay for the analysis of low and intermediate/high risk human papillomavirus (HPV) DNA, on paraffin-embedded tissues and to investigate possible reasons for reaction failure. Methods Fifty cervical biopsies were tested by an in situ hybridization (ISH) method using probes for HPV 6/11. 16/18, 31/33/51 and by the Digene Hybrid Capture^TM system. Results The ISH was able to detect HPV in 23 out of the 50 biopsies. Eight samples were positive with the HPV 6/11 probe, 4 with the 16/18 probe. 6 with the 31/33/51 probe and 5 had mixed infections. With the Hybrid Capture^TM system, having taken the mixed infections into account. 40 samples gave concordant results, while total and partial discordances were observed in 6 and 4 samples respectively. The relative sensitivity of the new assay was 91.6% and specificity 84.2% for low risk HPVs; and 80% and 94.3% respectively for intermediate/high risk HPVs. Agreement rates on HPV positivity for either low risk or intermediate/high risk types were 86% and 94% respectively in comparison with ISH results. Conclusion The Hybrid Capture^TM system is simple, reliable in use and suitable for routine discrimination of HPVs in tissue sections from paraffin-embedded blocks. It might thus be of clinical value in the diagnosis and prognosis of HPV infection.     

HPV不同基因亚型感染与宫颈病变相关性研究

目的 探讨 HPV不同基因亚型感染与宫颈病变间的关系。方法 选取 2015年 1月至 2017年 1月广州市花都区妇幼保健院诊治的 549例宫颈病变患者为研究对象,对患者进行 HPV基因亚型检测及宫颈薄层液基细胞学检测(LCT)。结果 549例研究对象中,HPV总阳性率为 74 86%;阳性率最高 HR HPV为 HPV16;随着疾病严重程度的增加,HR HPV的阳性检出率增高,而 LR HPV降低;除宫颈炎外,其他类型的宫颈病变患者中,HR HPV亚型单一感染的比例组间比较,差异无统计学意义(P>0 05)。结论 宫颈病变患者最常见的 HPV基因型为 HPV16,多重感染与疾病严重程度不相关。     

宫颈高危型人乳头瘤病毒感染与宫颈病变相关性研究

目的:研究宫颈高危型人乳头瘤病毒(high-risk human papillomavirus,HR-HPV)与宫颈病变患者相关性,为宫颈病变患者诊断及防治提供参考。方法:选取2014年6月至2015年12月在余姚市人民医院和浙江大学医学院附属妇产科医院共同收治的宫颈病变病人282例,其中包含55位慢性宫颈炎病人、65位宫颈上皮瘤样病变(CIN)Ⅰ期病人、91位CINⅡ期病人和71位CINⅢ期病人,荧光定量PCR方法对HR-HPV-DNA分型及和病毒载量进行检测,同时使用SPSS20.0统计软件进行数据分析。结果:282例研究对象中发现有196例病人感染HR-HPV,阳性率为69.50%,HR-HPV亚型主要有HPV16和HPV18,其所占比例为30.61%和21.43%;CINⅠ~Ⅲ期的病人HR-HPV阳性率和病毒载量比慢性宫颈炎病人的都高(P0.05);但是CINⅠ~Ⅲ期病人HR-HPV阳性率的对比,差异没有统计学意义(P0.05),同时发现病毒载量病人的病变严重情况呈正相关。结论:HR-HPV和病毒载量是影响宫颈病变的重要高危因素,本研究发现HR-HPV和病毒载量和宫颈病变相关紧密,病毒载量和宫颈病变严重情况为正相关性,宫颈病变的高发度和HR-HPV感染紧密相关。     

Human papillomavirus 25-related DNA in solitary keratoacanthoma

Summary Solitary keratoacanthomas of 32 patients were screened for the presence of human papillomavirus (HPV) 25 DNA, which was originally isolated and molecularly cloned from warts of an epidermodysplasia verruciformis (Ev) patient. Biotinylated virus DNA was hybridized in situ to thin sections obtained from paraffin-embedded material. HPV DNA was detected in 12 of 32 tumors under stringent conditions, and in 2 additional tumors under relaxed conditions.Abbreviations ev Epidermodysplasia verruciformis - HPV human papillomavirus Dedicated to Professor Miklós Simon in honour of his 70th birthday     

No evidence of human papillomavirus DNA in actinic keratosis

Human papillomaviruses (HPVs) are involved in premalignant and malignant skin diseases as well as in a variety of benign cutaneous and mucosal lesions. Actinic keratosis (AK) is a common premalignant disease. Its association with HPV infection has recently been evaluated in a few studies, but the results are contradictory. For further assessment of the role of HPVs in AK, a series of 100 paraffin-embedded biopsy specimens taken from subjects with AK were studied for the presence of HPV types 1, 2, 3, 4, 5, 7, 12, 15, 26, 36, 37 and 59 DNA using in situ hybridization (ISH) under high stringency conditions (Tm -10°C). All specimens were definitely negative for all biotinylated HPV DNA probes tested. One-fifth of the specimens were studied using the polymerase chain reaction (PCR) with general primers to confirm the negative results. All cases were also negative in the PCR. Our results suggest that HPVs are not directly involved in the aetiology of AK.     

原位杂交法检测尖锐湿疣中人乳头瘤病毒DNA型别

以非放射性地高辛配基标记人乳头瘤病毒（ＨＰＶ）６ｂ、１１、１６、１８型探针，用原位杂交法检测了重庆地区５０例经临床病理学诊断为尖锐湿疣的石蜡组织切片中ＨＰＶＤＮＡ型别，杂交条件分别采用严格条件（Ｔｍ－１２℃）和非严格条件（Ｔｍ－３５℃）进行。结果：尖锐湿疣中ＨＰＶＤＮＡ阳性率为８２％，其中ＨＰＶ６ｂ、１１、１６型分别为６２％、２４％、１０％，ＨＰＶ１８型未检出。杂交信号主要分布于表皮浅中层，且许多分布于空泡细胞内，但并非所有杂交信号均在空泡细胞内。     

核酸分子原位杂交检测人乳头瘤病毒与光镜诊断尖锐湿疣的比较及其图像定量分析

采用原位杂交方法对 50例尖锐湿疣组织进行 HPV6B、11型检测。其中光镜诊断为尖锐湿疣的 2 3例中 14例阳性 ,阳性率 60 .9% ;光镜诊断符合尖锐湿疣的 2 7例中 7例阳性 ,阳性率 2 5.9%。结果提示 :原位杂交方法对尖锐湿疣的诊断、鉴别诊断、防止误诊、漏诊 ,提高确诊率 ,可以提供直接可靠的依据。图像定量分析对于判断 HPV感染的程度及其 DNA含量有一定意义。     

Review of the current medical literature and assessment of current utilization patterns regarding human papillomavirus in situ hybridization and immunohistochemistry in dermatopathology

Human papillomaviruses have been implicated in many cutaneous diseases. Practicing dermatopathologists often consider using immunohistochemistry and in situ hybridization to help clarify the histologic diagnosis, particularly in cases with borderline or nondiagnostic features. We reviewed the current evidence behind the use of these two techniques in dermatopathology. We identified only two studies utilizing the currently available immunohistochemical antibodies. We found more evidence regarding the use of in situ hybridization; however, the majority of this evidence focuses on diagnosing condylomas and other lesions of the genital skin. We also assessed current utilization patterns of attendees of the American Society of Dermatopathology annual meeting (Chicago, 2016) which revealed a wide spectrum of current utilization ranging from no use to regular use more than once per month. Two‐thirds of respondents utilized these tests primarily when requested by the submitting clinician and one‐third of the respondents utilize these tests reflexively in specific clinical scenarios.     

尖锐湿疣皮损中高危型和低危型人乳头瘤病毒感染情况研究

目的:研究尖锐湿疣皮损中高危型和低危型人乳头瘤病毒(Human papillomavirus,HPV)感染情况。方法:研究纳入235例人乳头瘤病毒检查结果阳性的患者,对其型别、各型感染率及病毒拷贝数进行统计分析。结果:235例人乳头瘤病毒感染阳性患者,存在348人次人乳头瘤病毒感染,统计结果如下:HPV6和HPV11(36.7%,128/348)、HPV16(7.5%,26/348)、HPVl8(3.5%,12/348)、HPV31(1.7%,6/348)、HPV33(2.9%,10/348)、HPV35(2%,7/348)、HPV39(5.2%,18/348)、HPV45(1.1%,4/348)、HPV51(5.7%,20/348)、HPV52(11.5%,40/348)、HPV56(2.9%,10/348)、HPV58(10.1%,35/348)、HPV59(5.5%,,19/348)、HPV68(3.7%,13/348)。235例患者中128例为尖锐湿疣低危型HPV6及HPV11感染,高危型HPV中HPV52感染率最高,其次为HPV58,然后依次为HPV16,HPV51,HPV59,HPV39,HPV68,HPV18,HPV33,HPV56,HPV33,HPV31,感染率最低的是HPV45。感染低危型HPV6及HPV11患者为128例,只感染低危型HPV6及HPV11的患者为82例;感染高危型HPV患者为153例,只感染高危型HPV的患者为107例。低危型和高危型混合感染患者为46例。HPV感染拷贝数主要集中在107-103/104细胞。结论:尖锐湿疣中HPV6和11阳性率最高,高危型HPV可以和低位型HPV合并感染,也可以单独感染,发病率呈增高趋势,因此需要加强尖锐湿疣患者的治疗至关重要。     

尖锐湿疣及湿疣样病变中HPV6／11DNA原位杂交的观察

对４８例生殖道尖锐湿疣（ＣＡ）及湿疣样病变常规石蜡包埋组织切片进行人乳头瘤病毒（ＨＰＶ）６／１１型ＤＮＡ原位分子杂交及病理组织学观察，在１２例ＣＡ中９例（７５％）和湿疣样病变中１例（２．７％）检出ＨＰＶ６／１１ＤＮＡ，阳性细胞位于鳞状上皮中表层的凹空细胞及其周围细胞核内。在ＣＡ与湿疣样病变的鉴别诊断中，ＨＰＶ６／１１ＤＮＡ原位杂交检测具有重要的价值。     

山东省女性HPV6/11和HPV16/18感染情况分析

目的:对山东省女性人乳头状瘤病毒(human papillomavirus,HPV)6/11和HPV16/18感染的检测结果进行回顾分析,以了解山东省各年龄段、各地区女性HPV的感染状况。方法:采用实时荧光定量PCR技术,对2012年1月至2016年6月间济南金域医学检验中心收集的3995例和7508例样本,分别进行HPV6/11或HPV16/18检测,并对检测结果进行回顾分析。结果:HPV6/11检测的阳性率为27.2%(1085/3995),明显高于HPV16/18检测的5.1%(384/7508)。两种HPV检测的阳性率呈现不对称双峰分布,均以20岁组阳性率最高,分别为35.5%和21.7%。随后HPV6/11和HPV16/18的阳性率随年龄增长而逐渐下降,后HPV6/11的阳性率又逐渐升高至51~60岁组的34.3%和60岁组的43.8%;HPV16/18检测的阳性率在60岁组也略有上升,为3.4%。各地区中鲁中(31.6%)和鲁西(30.6%)HPV6/11的阳性率要高于鲁东(22.1%)和鲁北(21.7%);HPV16/18的检出率以鲁中(16.3%)最高,而鲁东最低(4.1%)。结论:本研究结果反映了山东省女性HPV6/11、HPV16/18感染的最新分布状况,对掌握山东省女性HPV感染的流行病学特征,为子宫颈癌筛查和HPV疫苗的接种提供科学依据。     

TCT、HPV-DNA联合阴道镜检查在宫颈病变诊断中的应用价值

目的:探讨TCT、HPV-DNA联合阴道镜检查在宫颈病变诊断中的应用价值。方法:选取2013年3月至2015年5月在我院同时行TCT、HPV-DNA及阴道镜检查的650例患者作为研究对象,对其中1项或以上检查异常者行病理活检,以病理学分级为标准分析诊断结果。结果:650例受检者中TCT阳性102例,其中ASCUS 80例,LSIL 15例,HSIL 6例,SCC 1例,HBV-DNA阳性40例,阴道镜阳性58例;TCT阳性病理活检结果为CINⅠ16例,CINⅡ10例,CINⅢ4例,浸润癌2例;TCT、HPV-DNA联合阴道镜检查诊断CINⅠ、Ⅱ、Ⅲ准确率明显高于TCT联合HPV-DNA,差异有统计学意义(P<0.05)。结论:TCT、HPVDNA联合阴道镜检查是一种高效、快速的宫颈癌筛查方式,可为宫颈癌的临床诊断提供依据,在临床上具有重要应用价值。     

妇科门诊妇女人乳头瘤病毒感染情况分析

目的:调查我院妇科门诊就诊患者人乳头瘤病毒(HPV)的感染状况、基因分型,为预防HPV感染和宫颈癌防治提供理论依据。方法:采用HPV核酸扩增分型检测试剂盒对我院980例门诊女性进行HPV检测,并对其亚型感染情况进行分析。结果:980例受试者中,220例(22.45%)HPV阳性,均按各基因型HPV阳性率计算,单一基因型感染率为16.12%(158/980),多重感染率为6.33%(62/980);其中单纯高危型感染率为17.53%(170/980),单纯低危型感染率为3.57%(35/980),高危型/低危型感染率为1.53%(15/980)。高危型最高为HPV-16(6.12%),其次为HPV-52(3.57%)、HPV-58(2.96%)、HPV-56(2.14%),低危型主要为HPV-6(2.65%)与HPV-11(2.04%)。对220例HPV阳性感染者进行液基细胞学检测(LCT)检查,LCT阳性率为38.18%,同期102例HPV阴性的LCT阳性率为1.96%,HPV阳性与同期HPV阴性妇女的LCT结果的阳性率有显著差别(χ~2=20.343,P=0.01)。结论:本地区妇科门诊就诊患者HPV阳性率较高,以高危型为主,且HPV阳性率与宫颈癌变发生率有相关性。     

Assessment of the presence of mucosal human papillomaviruses in malignant melanomas using combined fluorescent in situ hybridization and chemiluminescent immunohistochemistry

BACKGROUND: The vast majority of studies aimed at detecting human papillomavirus (HPV) DNA in skin cancer have used sensitive polymerase chain reaction (PCR) methods but the PCR technique, despite its high sensitivity, is not suitable to ascertain whether (i) the presence of HPV can be related only to few cells harbouring the virus, (ii) the presence of HPV is due to a tumour surface contamination and (iii) the presence of HPV is localized in cancer cells, rather than in normal keratinocytes present in the tumour biopsy. In a recent work we have found mucosal high-risk (HR) HPV genotypes in primary melanoma by PCR. OBJECTIVES: To localize mucosal HR-HPV nucleic acids and tumoural melanocytic marker in the same sections of primary melanoma samples in order to understand the relationship between HPVs and melanoma cells. METHODS: We have developed a very sensitive method that combines an enzyme-amplified fluorescent in situ hybridization (ISH) for the detection of HPV nucleic acids (types 16 and 18) with a chemiluminescent immunohistochemistry (IHC) method for the detection of the tumoural melanocytic marker HMB-45 sequentially in the same section. Digital images of fluorescent ISH and chemiluminescent IHC were separately recorded, assigned different colours and merged using specific software for image analysis. RESULTS: The combined fluorescent ISH and chemiluminescent IHC demonstrated a sharp colocalization (in the range 60-80%) of HPV nucleic acids and melanoma marker inside the same sections of melanoma biopsies, with a strong specificity and sensitivity. CONCLUSIONS: The strong colocalization of mucosal HR-HPV nucleic acids and HMB-45 melanocytic marker emphasized that viral nucleic acids were specifically present in melanoma cells and supported a possible active role of HPV in malignant melanoma.     

尖锐湿疣患者皮损中HPV的检测分型及HPV6b L1变异分析

目的 探讨温州地区尖锐湿疣（CA）患者人乳头瘤病毒（HPV）感染型别及HPV6基因多态性。 方法 采用基因芯片检测73例CA患者皮损组织HPV 21种基因型的感染率,对所获得的11例患者的HPV6阳性者行HPV6b L1基因测序分析。 结果 73例CA患者标本中检出HPV阳性61例,总的阳性检出率为83.56％。在61例HPV阳性患者,其中单一型别阳性检出率为93.44％,单一型别感染中以HPV11（29.51％）型为主,其次为HPV6 （18.03％）型;混合型感染阳性检出率为6.56％,以HPV6+11型感染为主。此外,CA患者HPV 6b L1基因测序结果表明,6个位点的核苷酸存在多态性,其中810位点A/T变异存在于所有标本,可能为温州地方株突变。结论 HPV11、6型感染为CA的主要致病型别,HPV 6b L1基因存在多处突变。     

端粒酶逆转录酶在皮肤肿瘤原位表达水平的比较

目的　研究端粒酶在皮肤恶性肿瘤发病机制中的作用。方法　采用人端粒酶逆转录酶 (hTERT)cRNA探针与石蜡标本进行原位杂交的方法检测 3 0例皮肤基底细胞癌、15例皮肤鳞状细胞癌、19例脂溢性角化、14例正常皮肤中hTERTmRNA的表达水平 ,并进行比较。结果　hTERT阳性率基底细胞癌为 73 .3 5 %(2 2 /3 0 ) ,鳞状细胞癌为80 .0 0 %(12 /15 ) ,均明显高于脂溢性角化 3 6.84%(7/19)和正常皮肤 2 8.5 7%(4 /14 ) ,并具有统计学意义。结论　hTERT在恶性皮肤肿瘤中的阳性表达率明显高于良性肿瘤和正常皮肤 ,提示端粒酶在皮肤恶性肿瘤发病机制中起着重要作用。原位杂交检测hTERT表达水平的方法有可能成为鉴别皮肤良恶性肿瘤的辅助检查手段。     

高危型人乳头瘤病毒检测在子宫颈癌筛查中的临床应用效果研究

目的:探讨人乳头瘤病毒及行为危险因素与子宫颈癌的临床关系。方法:通过分析2014年6月至2015年6月医院收治的203例HPV感染患者的临床资料,对患者临床资料进行整合,探讨HPV感染及相关行为危险因素及其同子宫颈癌的相关关系。结果:HPV感染并发宫颈癌患者中高危HPV患者占比显著高于低危HPV感染患者,比较差异有统计学意义(P0.05);两组患者行为危险因素中吸烟、首次性行为年龄、性伴侣个数、性卫生意识、孕次和避孕方式相关因素指标存在显著差异,有统计学意义(P0.05)。结论:高危型HPV感染是诱发子宫颈癌的高危因素,性行为及吸烟等行为危险因素同子宫颈癌发病有一定的相关性。