宫颈小细胞神经内分泌癌中人乳头瘤病毒的研究

目的探讨宫颈小细胞神经内分泌癌中高危型人乳头瘤病毒（humanpapillomavirus,HPV）感染情况。方法提取1例33岁宫颈小细胞神经内分泌癌患者手术切除组织癌变区域蜡块中的DNA,通过巢式PCR方法检测其中HPV感染情况。结果该患者肿瘤切除组织高危型HPV18型阳性。结论利用巢式PCR方法分型检测宫颈小细胞神经内分泌癌中的高危型HPV型别,其准确性及敏感性均较高。     

宫颈正常细胞和宫颈鳞状细胞癌、腺癌组织中HPV感染基因型的分布

目的 观察宫颈正常细胞、宫颈鳞状细胞癌和腺癌组织标本中人乳头瘤病毒(human papillomavirus,HPV)感染的基因型分布情况及临床意义.方法 采用PCR和基因芯片检测技术对1 047例宫颈正常细胞、161例宫颈鳞状细胞癌和82例宫颈腺癌组织标本进行23种HPV基因分型检测,并对受检者进行相关资料分析.结果 1 047例宫颈细胞正常者检出HPV感染为109例,HPV感染率为10.41％(109/1 047);161例宫颈鳞状细胞癌检出HPV感染者146例,HPV感染率为90.68％(146/161);82例宫颈腺癌检出HPV感染者59例,HPV感染率为71.95％(59/82).结论 PCR与基因芯片检测技术可用于宫颈细胞和组织标本检测,一次可检测23种HPV基因型,对我国女性宫颈HPV感染的分子流行病学调查、宫颈鳞状细胞癌及腺癌的防治及其疫苗的研究具有十分重要的意义.     

细胞图像DNA倍体分析结合高危型HPV、支原体和衣原体检查与宫颈上皮内瘤变相关性研究CSCD

目的 探讨宫颈细胞DNA倍体、高危型HPV、支原体和衣原体检测结果与宫颈上皮内瘤变的相关性,以期结合宫颈感染情况,预测宫颈筛查结果阳性妇女真正罹患宫颈癌前病变的风险,避免宫颈过度治疗.方法 将参加筛查的285名育龄妇女分别采用宫颈细胞图像DNA倍体分析（DNA-ICM）、高危型HPV检查（PCR法）、支原体（UU/Mh）以及衣原体（CT）检查.以阴道镜下宫颈活检的病理学诊断为金标准,分别计算了各种检查方式,对宫颈病变诊断的敏感性、特异性、预测值等.结果 在285个病例的活检结果中,共有慢性炎症179例,CIN Ⅰ级60例,CIN Ⅱ级20例,CINⅢ级25例;癌1例.Logistic回归分析显示,细胞DNA倍体异常与宫颈癌及癌前病变相关性最为显著,其次为高危型HPV感染.UU/Mh和CT感染均无明显相关性.所有存在微生物感染患者中,CINⅠ罹患者中32.43％为双因素感染,而CIN Ⅲ患者为56％.而在CIN Ⅱ中单独出现HR HPV感染的占50.00％,而CIN Ⅲ中占40％.结论 宫颈细胞DNA倍体阳性合并高危型HPV感染者应高度警惕为癌变高危人群,若同时出现UU/Mh感染,将有助于提高诊断的准确性.     

宫颈癌及其前期病变HPV感染与基因型分布

目的了解宫颈癌及宫颈上皮内瘤变CIN患者的HPV的感染和其基因分型及主要感染型别情况。方法应用型特异PCR检测宫颈癌及其前病变的患者的HPV感染及其主要基因分型情况的分析。结果在本研究宫颈癌及宫颈上皮内瘤变患者中,宫颈癌的HPV感染率为91．0％,CINⅠ／Ⅱ／Ⅲ的HPV感染率为73．3％,主要高危型HPV基因型别依次为HPV16、HPV18、HPV58、HPV33。结论在宫颈上皮内瘤变患者中感染主要高危型HPV基因型别依次为HPV16、HPV18、HPV58、HPV33、HPV16在宫颈癌和CIN中的构成比随着宫颈病变的增加而明显增加。     

不同型别人乳头瘤病毒感染不孕患者治疗方法的研究

目的 探讨不孕症患者感染不同型别高危型人乳头瘤病毒（HPV）的最佳治疗方法.方法 按宫颈病理学、宫颈状况和不孕患者生育欲望,将144例感染高危型人乳头瘤病毒的门诊不孕患者分为治疗组和不治疗组,以6个月为一复查周期,采用双色实时荧光定量PCR法,检测患者宫颈脱落细胞内的HPV-DNA,观察其阴转情况及受孕情况,并进行比较分析.结果 ①感染不同型别高危型HPV,治疗组的HPV阴转率（56.67%）较高;②次要高危型不治疗组的妊娠率（50.00%）较高,主要高危型治疗组的妊娠率（31.67%）较高,显著高于不治疗组（4.00%）;③主要高危型手术治疗后HPV阴转率和妊娠率（40.00%）均高于药物治疗（6.67%）,手术治疗二种方法中以Leep＋药物最佳.妊娠率为41.18%,高于药物治疗组的6.67%.结论 不孕患者应常规进行宫颈HPV筛查,主要高危型HPV感染是不孕的宫颈性病因,主要高危型HPV感染伴宫颈病变者以LEEP＋药物治疗为最佳.     

宫颈上皮内瘤样病变患者高危型HPV感染基因分型分析

目的了解宫颈上皮内瘤变患者的高危型HPV的感染及其分型和不同程度宫颈病变的主要感染型别情况。方法应用型特异PCR检测宫颈癌前病变的患者的主要高危型HPV-16、18、33、58的感染及其分型情况的相关性研究。结果在本研究宫颈上皮内瘤变患者98例中,4种高危型HPV的总阳性例数为73例,HPV总的感染率为74.5%,存在多重感染。其中HPV-16、18、33、58的总感染率分别为53.1%、38.7%、17.3%和30.6%。CIN的Ⅰ/Ⅱ/Ⅲ3组患者的4种高危型HPV的感染率分别为42.9%、61.1%和93.2%。结论主要高危型HPV在宫颈上皮内瘤变患者中感染的主要型别依次为HPV16、HPV18、HPV58、HPV33,主要为HPV16和HPV18型;不同程度CIN的高危型HPV的总感染率不同,随病变程度的加重而增加。     

高危型HPV阳性患者宫颈支原体和衣原体感染与宫颈组织学改变的关系

目的 探讨高危型HPV阳性不孕患者宫颈支原体和衣原体感染与宫颈组织病变的关系.方法 以HPV阴性不孕患者为对照,回顾分析不同高危型别HPV阳性不孕患者宫颈支原体和衣原体感染与宫颈组织病理分级、炎症分级之间的关系.结果 支原体阳性者HPV感染的发生率与支原体阴性者差异有统计学意义（P＜0.01）,衣原体阳性者HPV感染的发生率与衣原体阴性者差异无统计学意义（P＞0.05）.与HPV阴性组比较,HPV阳性者CIN和宫颈糜烂发生率以及CIN分级均较高（P＜0.01）,HPV阳性者宫颈糜烂程度无差异（P＞0.05）.与单纯HPV阳性组比较,HPV和支原体混合感染者CIN发生率及重度宫颈糜烂无差异（P＞0.05）.结论 支原体感染增加了高危型HPV的感染概率,高危型HPV感染增加了宫颈组织的病理损害程度,支原体感染可能是高危型HPV持续性感染的因素,宫颈组织病理程度是宫颈性不孕不可忽视的因素.     

人乳头瘤病毒检测在宫颈病变中的应用价值

目的了解宫颈细胞学异常患者中人乳头瘤“毒（HPV）的感染状况,评估HPV检测在宫颈病变筛查中的价值。方法随机选取宫颈薄层液基细胞学检测异常的101例患者进行了HPV检测,同时行组织病理学检查。结果（1）101例宫颈细胞学异常患者中,细胞学为ASCUS、LSIL、HSIL、鳞状细胞癌时高危型HPV阳性率分别为84．2％、88．6％、100．0％和2／2;（2）10l例细胞学异常患者中20例为CINI,81例为CINⅡ及以上级别,高危型HPV阳性率存CINI、CINⅡ及以上级别分别为60％、97．5％;（3）ASCUS组中,高危型HPV阳性患者中CINⅡ及以上病变的发生率为87．5％,HPV阴性患者中CINⅡ及以上病变的发生率为16．7％;（4）高危型HPV型别分布由高到低分别为HPVl6型39．6％（40／101）,HPV58型17．8％（18／101）,HPV52型16．8％（17／101）,HPVl8型9．9％（10／101）以及HPV33型9．9％（10／101）。结论高危型HPV感染率与宫颈病变级别呈正相关;HPV检测可作为ASCUS患者的有效分流手段。宫颈病变患者中高危型HPV感染以16、58、52、18、33型为主。     

许昌地区妇女宫颈人乳头瘤病毒感染型别分布特征分析

目的了解许昌地区妇女宫颈人乳头瘤病毒（HPV）感染的分布情况及高危因素,确定该地区的优势型别。方法采用PCR加导流杂交技术对3100例已婚妇女宫颈脱落细胞进行21种HPV基因检测,分析HPV感染高危因素及宫颈病变中HPV亚型感染分布特点。结论在检测的3100例妇女中感染总阳性率为18.8%,HPV感染阳性率排在前5位的亚型从高到低依次为HPV16、HPV58、HPV52、HPV11、HPV6,最低为亚型HPV43,占0.1%。HPV感染的高峰年龄为31～40岁,各年龄段HPV感染检出率差异在统计学上有显著意义（P〈0.05）。结论许昌地区HPV亚型感染分布以HPV16、HPV58、HPV52型为主,且存在明显的地域差异,HPV感染高发于中青年妇女。     

宫颈细胞学筛查及高危型HPV检测与宫颈组织学改变的相关性研究

目的 探讨宫颈细胞学及高危型HPV检测结果与宫颈组织病变的相关性,提高宫颈病变早期诊断的敏感度和特异度.方法 将254例不孕患者宫颈细胞学及高危型HPV检测结果分为四组,A组:宫颈细胞学阳性、高危型HPV阳性:B组:宫颈细胞学阳性、高危型HPV阴性;C组:宫颈细胞学阴性、高危型HPV阳性;D组:宫颈细胞学阴性、高危型HPV阴性,回顾分析此四组检测数据与宫颈组织病理之间的关系;并分别回顾分析宫颈细胞学阳性及高危型HPV阳性与宫颈组织病理之间的关系,比较其敏感度和特异度.结果 A组宫颈CINⅡ级及以上的发现率显著高于B组(P＜0.01),A组、B组和C组的CIN Ⅰ级发现率无差异(P＞0.05);单一宫颈细胞学检测CINⅡ级及以上阳性的敏感度100.0％、特异度46.74％,串行高危型HPV检测后其敏感度97.22％、特异度87.16％.结论 不孕症患者乃至日常体检宫颈筛查仍首选宫颈细胞学检查,宫颈细胞学串行高危型HPV检查明显降低了宫颈病变的误诊率.     

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.     

Comparison of real-time PCR signal-amplified in situ hybridization and conventional PCR for detection and quantification of human papillomavirus in archival cervical cancer tissue

Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (P = 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 (kappa = 0.661) and HPV18 (kappa = 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification.     

Prevalence,viral load,and physical status of HPV 16 and 18 in cervical adenosquamous carcinoma

Adenosquamous carcinoma of the uterine cervix is a rare mixture of malignant squamous and glandular epithelial elements and accounts for approximately 10% of cervical carcinomas. The aims of the present study were to evaluate the prevalence, physical status, and viral load of HPV 16 and 18 in adenosquamous carcinoma. Formalin-fixed paraffin-embedded tissue samples from 20 cases of histologically diagnosed adenosquamous carcinoma were examined. The squamous and glandular components were separately microdissected and analyzed for their HPV DNA subtype, viral load, and physical status using real-time polymerase chain reaction (PCR). The percentages of HPV 16- and 18-positive cases among all the HPV-positive cases were 36.8% (7/19) and 57.9% (11/19) in the squamous epithelial elements and 33.3% (6/18) and 61.1% (11/18) in the glandular elements, respectively. PCR analysis with E2 primers revealed that seven of eleven (63.6%) HPV 18-positive cases had the pure integrated form in both elements. The mean HPV 16 DNA copy numbers/cell was 7.22 in the squamous elements and 1.33 in the glandular elements (p = 0.04) while the corresponding mean HPV 18 DNA copy numbers/cell was 1.50 and 0.89, respectively. The prevalence of HPV 18 in adenosquamous carcinoma was high and many HPV 18-positive cases were the pure integrated form resulting in very low copy numbers/cell. It is possible that more aggressive transformation with early integration of HPV 18 results in cases with greater chromosomal instabilities, higher growth rates, and rapid progression.     

Failure to detect human papillomavirus DNA in malignant epithelial neoplasms of conjunctiva by polymerase chain reaction

To elucidate the putative role of human papillomavirus (HPV) infection in the etiology of conjunctival tumors, 44 formalin-fixed, paraffin-embedded specimens of conjunctival tumors (24 patients with papillomas and 20 patients with dysplastic and/or malignant tumors) were screened for HPV infection using 4 different polymerase chain reactions (PCRs). Of the 24 samples of papilloma, 14 (58%) displayed positive results by applying nested PCR using primer sets of HPV consensus L1 region. HPV type 6 or 11 was detected in 9 cases of papilloma by type-specific primer sets, but none of them were positive for HPV type 16 or 18. However, by using the highly sensitive PCR technique, we failed to demonstrate the HPV DNA of HPV types 6, 11, 16, and 18 in any of the 20 malignant epithelial tumors of conjunctiva. We conclude that HPV-6 or HPV-11 is present in a substantial percentage of conjunctival papillomas, which is in accordance with findings of previously reported studies. In contrast, malignant conjunctival carcinomas are not associated with HPV infection; other pathogenic mechanisms, such as UV light, probably are more important in the cause of these malignant lesions.     

The biomarkers of human papillomavirus infection in tonsillar squamous cell carcinoma-molecular basis and predicting favorable outcome.

Presence of human papillomavirus (HPV) in variable proportions in tonsillar squamous cell carcinoma tissues has been demonstrated by several worldwide studies. Some reports emphasized the significance of HPV in predicting a better prognosis, as well as ethnic differences between Chinese and Caucasians. In order to understand the biological role of HPV and find out clinically accessible methods to determine its prognostic significance in primary tonsillar squamous cell carcinoma, we collected 92 patients with primary tonsillar squamous cell carcinoma diagnosed or treated in National Taiwan University Hospital, for whom archival tumor tissue were available. Immunohistochemical stains of p16(INK4A), high-risk HPV in situ hybridization, and nested polymerase chain reaction (PCR)-based genechips were performed to detect HPV infection and determine its genotype. Clinical data were compared with HPV infection detected by the different methods mentioned above. Real-time PCR was also performed on the HPV16-positive [HPV16(+)] lesions to understand viral integration status. The positive rates of nested PCR-based genechips, overexpression of p16(INK4A), and high-risk HPV in situ hybridization were 75% (69/92), 53% (49/92), and 44% (40/92), respectively. Both overexpression of P16(INK4A) and high-risk HPV in situ hybridization positivity were associated with favorable prognoses (P=0.004 and 0.001, respectively) and also independent prognostic factors in multivariate analyses (P=0.01 and 0.01, respectively). The positivity of nested PCR-based genechips was not statistically significant. From our data, primary tonsillar squamous cell carcinoma with positive immunohistochemical stains of p16(INK4A) and/or high-risk HPV in situ hybridization is associated with a better outcome, and both methods may serve as clinically accessible markers.     

PCR detection of human papillomavirus of the mucosa: comparison between MY09/11 and GP5+/6+ primer sets

BACKGROUND AND OBJECTIVES: Human papillomaviruses (HPV) are the causal agent for the development of carcinomas in the cervix uteri and further pathological changes of the skin including mucosa, particularly warts, condylomas and dysplasias. Therefore, we investigated the efficacy of different consensus primers pairs for HPV detection by PCR using brushed samples from the oral cavity in comparison with samples from the cervix uteri. STUDY DESIGN: In the present study, we used two well-established sets of PCR primers in different combinations for the detection of HPV DNA in 106 non-invasive brush biopsy samples of the oral mucosa and 56 samples from the cervix uteri. Direct sequencing of PCR products in all cases determined HPV genotype and specificity. RESULTS: Overall, HPV was detected in 69 of 106 oral mucosa samples. HPV specific amplicons were obtained in 35.8% (N = 38) when using GP5+/6+ primers. The positivity rate was increased to 65.1% in a GP5+/6+ auto-nested PCR approach. In contrast, MY9/11 PCR and nested PCR with MY9/11 outer followed by GP5+/6+ inner primers yielded 2.2% and 16.1%, respectively. In gynaecological samples, PCR results were similar independent of the primer combination used. Thus, DNA quality and DNA content could be additional factors influencing the rate of positivity. CONCLUSION: For oral mucosa samples, auto-nested GP5+/6+ PCR is in our hands the most suitable approach for epidemiological studies because of its high sensitivity, high reliability and reproducibility as well as its relatively simple laboratory procedure.     

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。     

Improved detection of cutaneous human papillomavirus DNA by single tube nested 'hanging droplet' PCR

A single tube nested ‘hanging droplet’ PCR was developed for detection of cutaneous human papillomavirus (HPV) DNA of the phylogenetic group B1. The nested PCR was compared with a single round PCR method by testing 56 fresh biopsies from Australian skin tumour patients. HPV DNA was detected in 64% (36/56) of the biopsies by nested PCR and in 30% (17/56) by single round PCR (P<0.001). HPV DNA was more often detected by nested PCR than by single round PCR in basal cell carcinoma [62% (16/26) vs. 19%; (5/26); P=0.003], squamous cell carcinoma [43% (7/16) vs. 25% (4/16)] and in solar keratosis [93% (13/14) vs. 57% (8/14); P=0.038]. The nested PCR and the single round PCR system detected 26 and 11 different HPV types/putative types/subtypes, respectively. Multiple types were found in eight samples by the nested PCR and two samples by single round PCR. The nested HPV PCR is more sensitive and capable of amplifying a broad spectrum of HPV types from skin tumours, but further improvements are needed before all HPV infections in skin can be detected by a single assay.     

Detection of specific human papillomavirus types in paraffin-embedded sections of cervical carcinomas

Human papillomaviruses (HPV) are the causative agents of most cervical carcinomas. A complete understanding of the HPV types that cause cervical carcinoma is needed as vaccines are designed. Fresh tissues are not always available for such studies. We therefore sought to determine the feasibility of HPV studies using formalin-fixed, paraffin-embedded sections of 56 cervical carcinomas, correlating typing information with the pathology and physical state of the HPV sequences within cells. Sections from each specimen were used to extract and purify DNA. Specific HPV types were identified using a PCR/reverse blot strip assay. Tyramide signal-amplified, fluorescent DNA in situ hybridization (FISH) was used to localize HPV within cells. Human beta-globin sequences were amplified in DNA from all specimens. HPV sequences from oncogenic types were identified in 52 of 56 (92.9%) by PCR/reverse blot strip assay, and in one additional case using an HPV 16 multiplex PCR assay. HPV 16 was the most commonly detected type, present in most cases as a solitary isolate. Thirty- five of 42 HPV 16 or HPV 18 PCR-positive specimens were also positive in the FISH assay, in most cases in a pattern consistent with viral integration. We conclude that HPV typing from formalin-fixed, paraffin-embedded sections of cervical carcinomas is possible, with a sensitivity that is similar to that found in studies using fresh tissue.