子宫颈癌患者血清、盆腔淋巴结HPV检测及其相关性探讨

目的：检测宫颈癌患者原发灶、血清及其盆腔淋巴结中HPVDNA及亚型,探讨其相关性及临床意义。方法：选取16例行广泛全子宫切除术和盆腔淋巴结清扫术宫颈癌患者的原发灶组织、术前静脉血与盆腔淋巴结石蜡组织,运用PCR方法对上述标本进行HPVDNA及亚型的检测。结果：宫颈癌原发灶组织、血清标本中HPVDNA阳性率为50％（8／16）。16例盆腔淋巴结石蜡组织中13例为HPVDNA阳性（13／16,81．25％）,其中总共切除的133个淋巴结中60个为阳性（60／133,45．1％）,8例淋巴结HPVDNA阳性的病例其对应的原发灶组织也为阳性且两者亚型相同。盆腔淋巴结中的HPVDNA阳性率为45．1％（60／133）,显著高于病理证实的淋巴结转移率1．5％（2／133）。6例患者（6／16,37．5％）原发灶、血清、盆腔淋巴结同时均为HPVDNA阳性;2例患者（2／16,12．5％）原发灶、盆腔淋巴结中HPVDNA表达阳性,而血清为阴性;1例患者（1／16,6．25％）淋巴结、血清HPVDNA阳性,而原发灶为阴性;未发现原发灶、血清HPVDNA表达阳性而淋巴结为阴性的病例,而且以上HPVDNA阳性的病例同一个患者对应的HPV亚型也相同。结论：宫颈癌患者血清中HPVDNA检出率与临床分期无关。宫颈癌患者盆腔淋巴结的HPVDNA检测可提高病理诊断淋巴结转移的阳性率,并且淋巴结中HPVDNA的检出率与原发灶的分化程度相关。宫颈癌原发灶、血清、盆腔淋巴结中HPV感染可能存在相关性。     

早期宫颈癌前哨淋巴结中高危型HPV16/18 DNA表达的检测及临床意义

目的：探讨早期宫颈癌患者前哨淋巴结（SLN）中人乳头状瘤病毒（HPV）16/18 DNA表达检测对于微转移的临床意义。方法选取72例早期宫颈癌患者,予患者均行广泛性子宫切除加双侧盆腔淋巴结清扫术,术中采用染料法识别SLN的宫颈癌患者有46例,应用基因检测法（FQ-PCR）检测SLN中HPV16/18 DNA阳性表达情况,并分析其与各种临床病理因素的关系;对SLN病理阴性的33例患者进行长期随访,分析SLN中HPV16/18 DNA阳性与淋巴结转移的关系。结果46例宫颈癌患者SLN中HPV16/18 DNA阳性表达者共22例,其中13例淋巴结病理阳性患者中有10例阳性,而33例淋巴结病理阴性患者中仅12例阳性（P=0.013）;46例患者共检出前哨淋巴结102枚,均用FQ-PCR法检测HPV16/18 DNA,结果13例淋巴结病理阳性患者检出的37枚SLN中有29枚HPV16/18 DNA阳性,而33例淋巴结病理阴性患者检出的65枚SLN中仅有36枚阳性（P=0.033）;分析46例成功检出SLN的早期宫颈癌患者的临床资料,发现SLN中HPV16/18 DNA阳性表达仅与临床分期有关,具有统计学意义（P=0.034）;长期随访33例SLN病理阴性的患者,发现HPV16/18 DNA阳性的患者复发率高于HPV16/18 DNA阴性的患者,具有统计学意义（P=0.02）。结论检测宫颈癌SLN组织中HPV16/18 DNA表达可能是预测早期宫颈癌淋巴结微转移的可行方法。     

荧光定量PCR检测宫颈癌旁盆腔组织中HPV-DNA的临床意义

目的探讨FQ-PCR检测宫颈癌旁盆腔组织中HPV-DNA对预测早期宫颈癌微小转移的意义。方法采用FQ-PCR方法,检测31例Ia-IIb期宫颈癌患者的原发灶及其癌旁组织(阴道切缘、宫旁切缘、盆腔淋巴结)中的HPV亚型和DNA拷贝数,并与患者术后组织病理学结果比较。结果31例癌旁组织HPV-DNA总检出率为58.1%,其中病理检测发生癌旁转移的7例患者均检测到HPV-DNA。宫颈原发灶、组织病理学阳性和阴性的癌旁组织HPV-DNA拷贝数分别为106.75±0.72,105.70±0.87和103.70±1.22,且它们之间有非常显著性差异(P<0.01)。结论癌旁组织检出HPV早于组织病理学发现转移,检测癌旁组织中HPV对早期微小转移具有提示意义,有助于发现组织病理学不易发现的早期隐匿性转移。     

宫颈癌患者人乳头瘤病毒感染分布特点

目的:探讨人乳头瘤病毒(HPV)各亚型在广西沿海地区宫颈癌患者中的分布情况,HPV感染与宫颈癌患者的年龄、临床分期、病理类型、分化程度、肿瘤盆腔淋巴结转移及肿瘤的复发的关系。方法:通过凯普导流杂交HPV DNA检测法,对76例宫颈癌患者宫颈脱落细胞进行21种HPV亚型的检测。结果:宫颈癌HPV总阳性率为90.8%。宫颈癌患者HPV阳性各亚型出现的频率排序为:HPV16(56.5%),HPV18、33、58各(7.2%),HPV52、53各(5.8%),HPV31(4.3%),HPV45(2.9%),HPV35、51、56、66、68各(1.4%)。HPV6(5.8%),HPV11、44、43各(1.4%)均合并在高危感染中。HPV感染与临床分期、肿瘤分化程度、肿瘤盆腔淋巴结转移及肿瘤的复发关联无显著性(P>0.05),与年龄密切相关,鳞癌HPV阳性率明显高于腺癌及其它癌,差异有统计学意义(P<0.05)。结论:广西沿海地区妇女宫颈癌患者中以HPV16、18、33、58感染为主要型别。HPV感染与宫颈癌的临床分期、肿瘤分化程度、肿瘤盆腔淋巴结转移及肿瘤的复发无明显相关性,与发病年龄、病理类型有关。     

宫颈癌患者人乳头瘤病毒感染分布特点

目的：探讨人乳头瘤病毒（HPV）各亚型在广西沿海地区宫颈癌患者中的分布情况,HPV感染与宫颈癌患者的年龄、临床分期、病理类型、分化程度、肿瘤盆腔淋巴结转移及肿瘤的复发的关系。方法：通过凯普导流杂交HPV DNA检测法,对76例宫颈癌患者宫颈脱落细胞进行21种HPV亚型的检测。结果：宫颈癌HPV总阳性率为90.8%。宫颈癌患者HPV阳性各亚型出现的频率排序为：HPV16（56.5%）,HPV18、33、58各（7.2%）,HPV52、53各（5.8%）,HPV31（4.3%）,HPV45（2.9%）,HPV35、51、56、66、68各（1.4%）。HPV6（5.8%）,HPV11、44、43各（1.4%）均合并在高危感染中。HPV感染与临床分期、肿瘤分化程度、肿瘤盆腔淋巴结转移及肿瘤的复发关联无显著性（P〉0.05）,与年龄密切相关,鳞癌HPV阳性率明显高于腺癌及其它癌,差异有统计学意义（P〈0.05）。结论：广西沿海地区妇女宫颈癌患者中以HPV16、18、33、58感染为主要型别。HPV感染与宫颈癌的临床分期、肿瘤分化程度、肿瘤盆腔淋巴结转移及肿瘤的复发无明显相关性,与发病年龄、病理类型有关。     

宫颈癌患者HPV16型E6蛋白的表达纯化及血清抗体检测

背景与目的:人乳头瘤病毒16型(humanpapillomavirustype16,HPV16)是宫颈癌组织中最常见的高危HPV,其相应蛋白的血清抗体与宫颈癌的发生发展相关。本研究构建HPV16E6重组表达载体并表达纯化获得HPV16E6重组蛋白,用于检测不同人群血清相应抗体,初步探讨本地区HPV16E6血清抗体反应与宫颈癌的相关性。方法:将HPV16E6基因与pRSET-A融合表达载体连接,获得E6表达重组体,转化大肠杆菌BL21(DE3)并用异丙基硫代-$-D-半乳糖苷(isopropylthio-$-D-galactoside,IPTG)诱导表达。表达的包涵体变性后经Ni柱纯化,复性并经活性鉴定后,用以包被ELISA板,检测正常女性、慢性宫颈炎患者和宫颈癌患者血清抗体。同时采用荧光偏振方法分型检测宫颈癌组织HPVDNA。结果:pRSET-16E6表达重组体的工程菌经IPTG诱导后可表达Mr24×103的HPV16E6组氨酸融合蛋白,表达量占菌体蛋白的22.3%。表达形式为包涵体,重组蛋白纯度达95%以上,其活性经ELISA法证实。80例正常女性、46例慢性宫颈炎和32例宫颈癌患者血清抗体阳性率分别为5.0%、6.5%和31.2%,宫颈癌患者HPV16E6血清抗体阳性率显著高于正常人(P<0.002)及慢性宫颈炎患者(P<0.01),而正常人与慢性宫颈炎患者间的差异无显著性。32例宫颈癌患者癌组织中,HPVDNA阳性率90.6%,HPV16DNA阳性率46.9%。HPV16DNA阳性组血清HPV16E6抗体阳性率(46.7%)高于阴性组(17.6%),但两组间的差异无显著性(P>0.05)。结论:在pRSET-A/BL21中表达获得的HPV16E6融合蛋白,可用于宫颈癌相关HPV的血清学研究;宫颈癌患者HPV16E6血清抗体阳性率明显高于正常人和慢性宫颈炎患者。     

环氧合酶-2在宫颈癌中的表达及其与HPV16感染的关系

目的：探讨环氧合酶-2(COX-2)在宫颈癌发生发展中的作用，分析COX-2与宫颈癌中HPV16感染之间的关系。方法：收集46例宫颈癌组织作为实验组，31例健康的宫颈组织作为对照组。用免疫组化方法检测各组的COX-2表达水平。用PCK方法检测宫颈癌组的HPV16DNA。结果：COX-2在宫颈癌组织中有较高表达；COX-2在宫颈癌组织中表达与年龄、临床分期、组织分化程度、浸润深度之间均无相关性。而与淋巴结转移有显著相关性；宫颈癌组织中HPV16DNA阳性率为5652％(26/46)。在COX-2表达阳性的宫颈癌组织中，HPV16DNA阳性组的染色分值明显高于HPV16DNA阴性组。结论：宫颈癌的发生发展中有COX-2的参与。并且COX-2与宫颈癌的转移密切相关。宫颈癌中COX-2高表达与HPV16感染可能有一定的相关性。     

cAMP/PKA通路在HPV16/18相关宫颈癌中的表达情况及临床意义

目的 探讨cAMP/PKA通路在HPV16/18相关宫颈癌中的表达情况及临床意义。方法 选取86例宫颈癌患者作为研究组，取同期患有妇科其他良性病变行宫颈切除手术患者75例作为对照组。将研究组患者术后切除的宫颈病变组织进行收集，同时收集对照组患者术后切取的宫颈组织标本于液氮中保存。对两组研究对象组织标本中cAMP/PKA通路的表达水平进行比较，采用PCR扩增及膜杂交法对cAMP/PKA阳性组与阴性组标本进行HPV检测。对cAMP/PKA阳性组与阴性组进行免疫组化法检测血管内皮生长因子表达水平情况。采用Spearman相关性分析，对cAMP/PKA通路表达与VEGF表达及宫颈癌HPV16/18感染情况进行相关性分析。结果 研究组标本中cAMP/PKA通路阳性率明显高于对照组(P<0.05)。cAMP/PKA通路阳性组患者HPV16/18总阳性率为93.59%,阴性组HPV16/18总阳性率为50.00%,cAMP/PKA通路阳性组中HPV16/18阳性率明显高于阴性组(P<0.05)。cAMP/PKA通路阳性组中VEGF表达水平呈阳性表达有70例(89.74%),阳性率为89....     

广东省潮汕地区单中心头颈部鳞状细胞癌患者人乳头瘤病毒初步检测分析

目的探讨广东省潮汕地区单中心头颈部鳞状细胞癌(HNSCC)患者人乳头瘤病毒(HPV)感染及亚型状况。方法收集2014年12月至2016年12月汕头大学医学院附属肿瘤医院167例HNSCC患者的肿瘤原发灶标本。采用免疫组织化学(IHC)法检测肿瘤组织中p16蛋白的表达,以肿瘤细胞p16蛋白阳性率≥76%为判断HNSCC存在HPV的依据,分析p16蛋白与患者临床病理因素的关系。采用原位杂交法(ISH)检测肿瘤组织中是否存在HPV 16、18 DNA;应用RNAscope法检测肿瘤组织中18种常见的高危HPV亚型(HPV HR 18)RNA的表达情况,分析p16蛋白阳性细胞比例≥50%的肿瘤组织中HPV HR 18阳性情况。结果患者p16蛋白强表达率为7.2%(12/167);低龄组(<50岁)p16蛋白强表达率高于高龄组(≥50岁)[17.2%(5/29)比5.1%(7/138),χ^2=5.321,P=0.021];口咽癌组p16蛋白强表达率高于非口咽癌组[29.4%(5/17)比4.7%(7/150),χ^2=14.019,P<0.01];性别、烟酒嗜好及肿瘤分期分层患者间p16蛋白强表达率差异均无统计学意义(均P>0.05)。ISH检测示,全部HNSCC原发灶均未发现HPV 16、18 DNA,重复实验结果一致。RNAscope法检测示,肿瘤细胞p16蛋白阳性率≥50%的19例患者中,3例(15.8%)肿瘤组织HPV HR 18 RNA阳性。结论潮汕地区HNSCC患者的HPV阳性率较低,以口咽癌患者最高,且呈年轻化趋势。潮汕地区HNSCC主要致HPV亚型可能并非HPV 16、18,而可能是包括HPV HR 18中的其他致病亚型。     

HPV16/18DNA在原发性胃癌患者肿瘤组织中的表达及意义

目的探讨人乳头瘤病毒(HPV)16/18 DNA在原发性胃癌患者胃癌组织中的表达情况及其临床意义。方法选取行手术切除且经病理确诊的72例原发性胃癌患者的胃癌组织标本,采用荧光定量聚合酶链反应检测胃癌组织中HPV16/18 DNA的表达情况,分析HPV16/18 DNA表达与原发性胃癌患者临床特征及预后的关系。结果72例原发性胃癌患者中,HPV16/18 DNA的阳性表达率为56.94%。不同Lauren分型、TNM分期、淋巴结转移情况胃癌患者胃癌组织中HPV16/18 DNA的阳性表达率比较,差异均有统计学意义(P﹤0.05);其中,Lauren分型为弥漫型和肠型、TNM分期为Ⅲ期和Ⅱ期、有淋巴结转移的胃癌患者胃癌组织中HPV16/18 DNA的阳性表达率较高。HPV16/18 DNA阳性表达胃癌患者的5年累积生存率低于HPV16/18 DNA阴性表达胃癌患者,但差异无统计学意义(P﹥0.05)。结论原发性胃癌患者胃癌组织中HPV16/18 DNA的阳性表达率较高,且HPV16/18 DNA表达与胃癌患者的Lauren分型、TNM分期及淋巴结转移情况有关,并可能影响患者的预后。     

Correlation between Load of HPV 16 DNA in Cervical Cancer and HPV 16 DNA in Lymph Nodes

OBJECTIVE To determine the association between viral load of human papillomavirus 16 (HPV16) DNA in the primary focus of cervical carcinoma and HPV16 DNA in pelvic lymph nodes. METHODS The HPV16 DNA load was measured by fluorescent quantisation polymerase chain reaction (FQ-PCR) in 17 primary foci. HPV16 DNA was detected by polymerase chain reaction (PCR) using HPV16 type-specific primers in 296 pelvic lymph nodes which were from 17 cases of cervical cancer. RESULTS The viral load of HPV16 DNA showed statistically significant differences between tumors with a diameter of < 4 cm and > 4 cm (P < 0.05). Seven of 17 cervical cancer cases had HPV16 DNA positive lymph nodes, designated as the positive group, while the remaining 10 without positive lymph nodes was designated the negative group. The average load of HPV16 DNA showed no significant difference between the 2 groups (P > 0.05). The load of HPV16 in the primary lesion was not associated with that in the lymph nodes. There were 38 HPV16 DNA positive nodes in the total 296 nodes. The rate of positivity of HPV16 DNA in lymph nodes showed statistically significant differences in consideration of maximum tumor diameter, tumor differentiation, histologic type, depth of myometial infiltration and the metastatic status of the nodes, respectively (P < 0.05).CONCLUSION Viral load of HPV16 in the primary cancer focus correlated with the quantity of tumor cells in the primary focus but not with the existence of HPV DNA positive lymph nodes. Detection of HPV DNA may help to find the early metastases that cannot be evaluated histopathologically but the prognostic value of HPV positive lymph nodes needs further examination.     

Prevalence and distribution of human papillomavirus type-16 DNA in pelvic lymph nodes of patients with cervical cancer and in women with no history of cervical abnormality.

The polymerase chain reaction (PCR) was used to investigate the prevalence and distribution of human papillomavirus (HPV)-16 DNA in paraffin sections of all pelvic lymph nodes removed from 14 patients with Stage Ib-cervical cancer at the time of resection of their primary tumours. The results were compared with those obtained from 8 women with no known history of cervical abnormality. In all, 22 cervical biopsies and 40 I lymph nodes (296 paraffin blocks) were examined. Nine of the 14 cervical cancer patients had primary tumours that were positive for HPV-16 DNA: only 3 of these had lymph nodes with histological evidence of metastasis, and HPV 16 DNA was detected in each of the corresponding paraffin blocks. HPV 16 DNA was also detected in varying proportions (8%-92%) of the histologically-negative lymph nodes from these women. There was no correlation between the HPV DNA-positive lymph nodes and their proximity to the primary tumour. HPV-16 DNA was not identified in any of the lymph nodes from the 5 women whose cancers were not HPV-16-related, or in those of women with no evidence of cervical abnormality. This preliminary survey suggests that HPV DNA is frequently transported from HPV-16-related cervical tumours to regional lymph nodes. However, its practical significance will not be clear until sufficient time has elapsed for correlation of the results with the clinical outcome.     

Relation between HPV-DNA and expression of p53, bcl-2, p21WAF-1, MIB-1, HER-2/neu and DNA ploidy in early cervical carcinoma: correlation with clinical outcome

The purpose of the present study was to analyze the relation between the expression of p53, bcl-2, p21WAF1, MIB-1, HER-2/neu, DNA ploidy and HPV16 or 18 infections with clinical parameters. HPV-DNA was evaluated in 171 early cervical carcinomas treated from 1965 to 1990 and detected by PCR (polymerase chain reaction) on paraffin specimens obtained before therapy was started. HPV-DNA of any type was detected in 78% (86/110) of all tumors, HPV16 was the predominant type and was seen in 56% (62/110), HPV18 in 8% (9/110) and HPV35 in 21% (23/110). Patients with HPV16 or 18 were significantly (P=0.011) younger than patients with tumors not containing these two HPV subtypes. Lymph node metastases were seen more frequently (P=0.047) in tumors expressing HPV16 or 18. Tumor size was associated with the HPV-type. The frequency of DNA aneuploidy was lower in high-risk HPV tumors than in tumors with other HPV subtypes (P=0.014). MIB-1 expression was highly significantly (P=0.00007) associated with presence of HPV16 or 18. The cancer-specific survival rate was lower for patients with HPV16 and 18 positive tumors, but the difference was not statistically significant. The overall 5-year survival rate of the complete series was 91%. In conclusion, the HPV DNA subtype was a prognostic factor in early stage cervical cancer and it was associated with age, positive lymph nodes, tumor size, DNA ploidy and the proliferation marker MIB-1.     

Human papilloma virus and lymphatic metastasis in squamous cell carcinoma of the cervix

Our study included 112 patients with squamous cell cervical carcinoma la-lib stages (FIGO). All of them were restaged on the basis of histological evidence after surgical treatment. Ninety-eight were staged at final analysis. Human papilloma virus (HPV) was detected in endocervical smears and paraffin blocks of lymph nodes by PCR and real-time PCR. Oncogenic HPV in primary tumor was detected in 86 (87.8%); two or more genotypes--65 (75.58%): still more--21 (24.4%). In the latter group, the frequency of regional lymph node metastases was significantly higher. HPV DNA was identified in iliac lymph nodes in 29 (26.6%); 27 of them had metastases to those nodes. HPV in DNA which was significantly more frequent in involved lymphocytes may be used as marker. Since our method for early detection of metastases to regional lymph nodes is highly specific, it may be recommended as a diagnostic procedure.     

Correlation between load of HPV 16 DNA in cervical cancer and HPV 16 DNA in lymph nodes

OBJECTIVE To determine the association between viral load of human papillomavirus 16 （HPV16） DNA in the primary focus of cervical carcinoma and HPV16 DNA in pelvic lymph nodes. METHODS The HPV16 DNA load was measured by fluorescent quantitation polymerase chain reaction （FQ-PCR） in 17 primary foci. HPV16 DNA was detected by polymerase chain reaction （PCR） using HPV16 type-specific primers in 296 pelvic lymph nodes which were from 17 cases of cervical cancer. RESULTS The viral load of HPV16 DNA showed statistically significant differences between tumors with a diameter of 〈 4 cm and ≥ 4 cm （P 〈 0.05）. Seven of 17 cervical cancer cases had HPV16 DNA positive lymph nodes, designated as the positive group, while the remaining 10 without positive lymph nodes was designated the negative group. The average load of HPV16 DNA showed no significant difference between the 2 groups （P 〉 0.05）. The load of HPV16 in the primary lesion was not associated with that in the lymph nodes. There were 38 HPV16 DNA positive nodes in the total 296 nodes. The rate of positivity of HPV16 DNA in lymph nodes showed statistically significant differences in consideration of maximum tumor diameter, tumor differentiation, histologic type, depth of myometial infiltration and the metastatic status of the nodes, respectively （P 〈 0.05）. CONCLUSION Viral load of HPV16 in the primary cancer focus correlated with the quantity of tumor cells in the primary focus but not with the existence of HPV DNA positive lymph nodes. Detection of HPV DNA may help to find the early metastases that cannot be evaluated histopathologically, but the prognostic value of HPV positive lymph nodes needs further examination.