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14种高危人乳头瘤状病毒（16和18型分型）检测在宫颈癌筛查中的效果评价
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　　[导读]
　　中日友好医院卞美璐教授为评价在宫颈癌筛查中使用14种高危型人乳头状瘤病毒16和18型分型检测（PCR12+2)的有效性和临床性价值，对在中日友好医院进行宫颈癌筛查的424例妇女进行对比验证实验，并指出：与HC2联合细胞学宫颈癌筛查方案相比，PCR12+2检测用于筛查宫颈癌高度病变及宫颈癌的临床价值无显著性差异，但灵敏性和阴性预测值更高。
　　文章指出，PCR12+2检测可以用于宫颈癌筛查，尤其可以对于HPV16和HPV18进行分型检测，帮助将细胞学阴性者进行分流管理。
　　随着对宫颈癌发病机制了解的逐步深入，目前对于HR-HPV中16和18感染越来越受到重视。但是，HC2检测仅能相对半定量地报告HR-HPV的阳性或阴性，无法鉴别出具体的感染HPV的类型及是否为混合感染，在对HPV阳性的报告进行解读和对HPV阳性患者进行分流时常常给临床医生和HPV感染者带来一定的闲扰，而且其高昂的价格也是妨碍其广泛普及的重要原因之一。
　　而PCR12+2除了能够在进行HR-HPV检测的同时对致癌风险最高的16和18型HPV进行分型检测之外，还具有以下优势：
　　1、能同时对细胞内β-globin进行扩增：应用荧光PCR仪克服了传统PCR需电泳分析和样本间污染等缺点。
　　2、理想的费用效率比：PCR12+2检测试剂盒收费低廉，对于宫颈癌及宫颈高度病变的筛查结果令人满意，而且由于可以对致癌风险最高的16和18型HPV进行分型检测，将更有利于对HR-HPV阳性者的分流管理。
　　中华妇产科杂志2013年8月第48卷第8期
Chin J Obstet Gynecol, August 2013. VoL 48. No.8
　　不同方法检测HPV 的临床效果评价
　　程娇影
马莉 丛笑 陈颖文 刘军
　　[摘要] 目的 探讨宫颈癌筛查中，不同方法检测HPV的临床效果。方法 选择月在中日友好医院行宫颈癌筛查的妇女424例，均行宫颈脱落细胞的液基细胞学检测( LCT)、第二代杂交捕获HPV检测( HC-Ⅱ)和核酸实时荧光PCR同时对HPV16和HPV18进行分型检测( PCR12+2)。检测结果分两组进行评价: HC-Ⅱ组( LCT联合HC-Ⅱ)，共计424例；PCR12+2组( LCT联合PCR12+2)，剔除3例脱落病例.共计421例。两组均对LCT结果在未明确诊断意义的不典型理鳞状上皮细胞(ASCUS)及以上和(或)高危型HPV阳性者行阴道镜活检及组织病理学检查。此外，PCR12+2组同时还对LCT阴性，但HPV16或18阳性者行阴道镜活检及组织病理学检查。结果 ( 1)两组患者中，组织病理学检查结果≥宫颈上皮内瘤变Ⅱ级(CINⅡ)病变的筛查结果比较,差异无统计学意义(χ=3.35. P &0.05 ) 。HC-Ⅱ和PCR12+2用于筛查≥CINⅡ病变的敏感度、特异度、阳性预测值和阴性预测值分别为: 77.8% ,79. 4%，20.4%，98. 1 % 和96.3%，78. 2 %，23.2%，99.7 %。(2)在PCR12+2组中，HPV16阳性34例，HPV18阳性5例(其中l例同时HPV16阳性)，其他高危型HPV阳性74例，阴性309例。其中HPV16和(或)HPV18阳性比其他高危型HPV阳性导致组织病理学≥CINⅡ病变的风险更高，分别为51.3% (20/39 )和8.1% ( 6/74)。(3)在PCR12+2组中，HPV16阳性、HPV18阳性、其他高危型HPV阳性及HPV阴性者的致癌风险比较，差异有统计学意义(χ2=93.98，P&0.01)。结论LCT联合PCR12+2检测用于筛查宫颈癌，可以鉴别出宫颈病变≥CINⅡ风险更高的患者。
　　[关键词] 宫颈肿瘤; 宫颈上皮肉瘤样病变; 乳头状瘤病毒科: 细胞诊断学; 聚合酶链反应
　　宫颈癌是女性第二大常见恶性肿瘤。已经证实，HPV感染是宫颈癌的致病因素之一。HPV 感染导致了95 % - 100%的宫颈癌的发生[1]。大约有15种高危型HPV ( high-risk HPV, HR-HPV )的持续性感染可以导致宫颈癌和癌前病变。过去20年的数据已经证实了HPV检测作为宫颈癌初级筛查辅助手段的作用[2]。美国已将宫颈细胞HPV检测作为宫颈细胞学检查的辅助手段用于宫颈癌初级筛查[3-4]，同际癌症研究中心则认为，可以将HPV 检测作为独立的宫颈癌初级筛查的选择之一[5]。尽管有15种HR-HPV感染与宫颈癌的发病密切相关，但已有研究证实，50%以上的宫颈癌是由HPV16感染导致，10% - 15 %的宫颈癌是由HPV18导致[6-9]。在1项对1739例宫颈鳞癌患者的HPV 检测中发现，HPV16 和HPV 18可以在67. 74 %的宫颈癌病例中被检测到[9]。此外，HPV18还引起了超过35%的宫颈腺癌的发生，而宫颈腺癌通过现有的细胞学筛查手段是很难发现的[8]，因此，和2012年美国阴道镜和宫颈病理协会(American Society for Colposcopy and Cervical Pathology, ASCCP)发布的指南建议:年龄≥30岁的妇女，如果宫颈细胞学检查阴性，但HR-HPV阳性可以选择在12个月后重复进行HPV 检测和宫颈细胞学检查，或者直接进行HPV16 和HPV18的分型检测，如果检测结果阳性应立即进行阴道镜检查，而那些HPV16 和(或) 18阴性，但其他型别HR-HPV阳性者，应该在12个月后重复进行细胞学检查和HR-HPV 检测[10-12]。为探讨HPV16和HPV18 分型检测在宫颈癌前病变和宫颈癌筛查中的临床价值，本研究比较了杂交捕获Ⅱ代( hybrid captureⅡ, HC-Ⅱ)方法和实时荧光PCR同时对HPV16和HPV18进行分型检测(PCR12+2)的方法用于筛查宫颈高度病变及宫颈癌的结果，现报道如下。
　　资料与方法
　　所有观察对象均知情同意，而且此项研究遵循本医疗中心的伦理要求。
　　一、资料来源
　　日一11月10日，选择在中日友好医院宫颈癌诊治中心进行宫颈癌筛查和诊治的妇女424例，年龄20 -55岁，平均( 39±12 )岁。观察对象同时进行了宫颈脱落细胞的液基细胞学检测(liquid-based
cytology test , LCT)、PCR12+2检测和HC-Ⅱ检测，将HC-Ⅱ联合LCT检测的424 例定义为HC-Ⅱ组，将PCR12+2检测联合LCT 检测的421例(有3例脱落)定义为PCR12+2组，所有患者均无急性生殖道炎症及其他妇科合并症。
　　二、方法
　　1. LCT检测:用美国TriPath公司生产的AutoCyte Prep型自动制片染色机系列制片，由具备细胞学诊断资质的专业妇科病理医师负责细胞学诊断。细胞学诊断标准采用2001年国际癌症协会的The Bethesda system ( TBS )系统[13]。
　　2. PCR12+2方法检测HR-HPV: 采用广东凯普生物有限公司生产HR-HPV 核酸检测试剂盒检测HR-HPV，同时对HPV16 和HPV18进行分型检测，可在同一反应管中通过仪器的4 种荧光检测通道分别检测: ( 1 )不分型12种HR-HPV ( 31， 33 , 35 , 39 ,45 , 51 , 52 , 56 , 58 , 59 , 66 , 68)；(2) HPV 16；( 3 )HPV 18；( 4 ) 细胞内β-globin 基因。
　　3. HC-Ⅱ系统检测宫颈脱落细胞中HPV : 用美国DIGENE 公司生产的HC-Ⅱ系统检测1 3 种常见的HR-HPV亚型( HPV 1 6、1 8、31、33、35、39、45、51、52、56、58、59、68) 。HC-Ⅱ采用96 孔平版法，1次检测90份临床样本，可检测出待测标本中低至5000拷贝/ml的HPV。
　　4. 病理检查: 对两组LCT结果为未明确诊断意义的不典型鳞状上皮细( ASCUS)及以上和(或)HR- HPV阳性者行阴道镜活检及组织病理学检查。此外， PCR12+2组同时还对细胞学阴性，但HPV 16或18阳性者共计122例行阴道镜活检及组织病理学检查。在阴道镜下对醋酸上皮和腆试验不着色区进行多点活检，正常转化区则取3、6、9 、1 2点共计4点活检。对LCT和HPV检测均正常者定义为组织病理学阴性[14]。
　　三、统计学方法
　　采用SPSS 16.0统计软件，以组织病理学检查结果作为金标准，计算PCR12+2组及HC-Ⅱ组筛查宫颈癌及癌前病变的敏感度、特异度、阳性预测值和阴性预测值。组间比较采用确切概率法χ2检验。
　　424 份标本中有3份由于缺乏。β-globin信号被判定为无效。HPV 1 6、1 8 阴性的122例经过阴道镜和宫颈组织病理学检查，筛查出27例病变程度≥CINⅡ的患者。
　　一、HC-Ⅱ与组织病理学检查结果的关系
　　HC-Ⅱ检测出宫颈病变≥CINⅡ者21 例，组织病理学检测证实HC-Ⅱ检测用于筛查病变程度HC-Ⅱ的敏感性为77.8%，特异性为79.4%，阳性预测值为20.4%，阴性预测值为98. 1 %。见表1。
HC-Ⅱ检测与组织病理学检查结果的关系(例)
　　二、PCR12+2结果
　　1. PCR12+2与组织病理学检查结果的关系: PCR12+2检测联合细胞学筛查法，在27 例经过组织病理学检查证实为≥CINⅡ的患者中，确定HPV16阳性者共19例，阳性率70% ( 19/ 27)，其中4例为仅HPVI6阳性而细胞学阴性; HPV18阳性者共l例，阳性率4% ( 1/27)，且为其他HR-HPV与HPV18共同感染;非HPV16和HPV18 型的其他HR-HPV阳性者为6例，阳性率为22 % (6/27) ; HR-HPV检测阴性者1例，仅占4% (1/27)。以病变程度≥CINⅡ为病理学阳性，以病变程度运≤CINⅠ为病理学阴性，计算PCR12+2检测用于临床宫颈癌及其癌前病变筛查的敏感度为96.3%，特异度为78.2%，阳性预测值为23.2%，阴性预测值为99.7 %。在34 例HPV16阳性者中，发生≥CINⅡ病变者为19例，其中4 例细胞学检查结果为阴性。而在HPV18 、非HPV16和HPV18型的其他HR-HPV阳性及HR-HPV阴性者中，发生≥CINⅡ病变者分别为1、6、1例，发生率分别为1/5、8 % (6/74) 和0. 3 %(1/309)。HPV16阳性、HPV18阳性、非HPV16和HPV18型的其他HR-HPV 阳性及HR-HPV 阴性者致病风险比较，差异有统计学意义(χ2=93.98 , P &0.01 )。见表2。
　　2. 以年龄30岁分界的PCR12+2检测结果:根据ASCCP指南建议，以30岁为界进一步比较不同年龄段HPV各型别阳性与宫颈癌及癌前病变的关系。& 30岁者HR- HPV感染率为21.7% (26/120) , ≥30岁者为28.6% (86/301 )，两者比较，差异无统计学意义（χ2= 2. 09 , P & 0.05)。HPV16在不同年龄段的感染率分别为6.7% (8/120)和8.6%(26/301)，两者比较，差异也无统计学意义（χ2= 2. 09 ,P & 0. 05 )。不同年龄段人群中，HPV16阳性者中病变程度≥CINⅡ的发生率分别为2/8和65 % (17/26)，两者比较，差异有统计学意义(χ2=4. 123 ,P &0. 05)。由于HPV18检出率过低，未进行相关统计。非HPV16和HPV18型的其他HR-HPV在不同年龄段的感染率分别为15.0% (18/120)和18.6% ( 56/ 301 )，两者比较，差异无统计学意义(χ2= 0. 77 , P & 0. 05 )。不同年龄段人群中，非HPV16 和HPV18型的其他HR-HPV 阳性者中病变程度≥CINⅡ的发生率分别为2/18和7. 1 %(4/56 )，两者比较，差异也无统计学意义(χ2 =0. 00 16 ,P &0. 05 )。见表3。
　　3. LCT、PCR12+2检测和组织病理学结果的关系:LCT结果为正常或炎症者共计378例，其中HPV16、HPV18及非HPV16和HPV18型的其他HR-HPV 阳性者分别为18、5和56例，经过宫颈活检证实，病变程度≥CINⅡ者分别为4、l和0例。在LCT结果为ASCUS的9例中，2例HPV16阳性，均经过宫颈活检病理证实为CINⅡ，其余7例均为HPV16阴性且HPV18阴性，经过活检病理证实为炎症。在LCT结果为低度鳞状上皮内病变( LSIL)的17例中，HPV16阳性者2 例，经过宫颈活检病理证实均为CINⅡ，非HPV 16和HPV18型的其他HR-HPV阳性者11例，经过宫颈活检病理证实为CINⅡ者4例，HR-HPV检测阴性者4例，经过宫颈活检证实为宫颈炎症。见表4。
　　在本研究中HPV16、HPV18、非HPV16和HPV 18型的其他型别HR-HPV阳性和HR-HPV阴性者分别为34、5 、74 和309例，发生≥CINⅡ病变者则分别为19、1、6 和1例，4组人群的致癌风险比较，差异有统计学意义(χ2 =93.98，P & 0.01 )。按照宫颈细胞学结果分类，尤其是对于细胞学检查阴性者、ASCUS者和LSIL者3组人群中，各种型别HPV的致癌风险比较， 差异均有统计学意义（χ2分别为32. 74、9.54和7.65，P值分别= 0. 2和0.022)。
　　三、两组筛查方案的比较
　　HC-Ⅱ组中，阳性定义为需要接受阴道镜和组织病理学检查者( LCT结果& ASCUS者及LCT为ASC US且HR -HPV阳性者)共计39 例，HC-Ⅱ组筛查出21例宫颈病变程度≥CINⅡ者，6例假阴性。PCR12+2组中，阳性定义为需要接受阴道镜和组织病理学检查者[ LCT 结果& ASCUS 者及LCT为ASCUS且HR-HPV 阳性者和无论LCT结果如何，HPV16和(或) HPV18阳性者]共计52例，PCR12+2组筛查出27例宫颈病变程度≥CINⅡ者。两组筛查方案检出结果比较，差异无统计学意义(χ2= 3.35 , P &0.05 )。
　　费用效益比分析显示，PCR12+2检测试剂盒收费250元/例，HC-Ⅱ检测收费350元/例，尽管PCR12+2组需要进行阴道镜检查、宫颈活检及组织病理学检查的例数( 52例)略高于HC-Ⅱ组( 39例)，但PCR12+2组可以以更低廉的价格获得不低于HC-Ⅱ组的筛查结果。
　　一、HPV16、18感染与年龄及宫颈癌发病的关系
　　在HPV感染的自然进程中，大多数的感染是暂时的，尤其是那些年轻的女性，只有很少一部分人存在HPV持续感染，通常在超过10年的持续感染后可能会发展成宫颈癌。因此，美国食品药品管理局(FDA)批准对于年龄≥30岁者才可以在宫颈癌初级筛查中将HPV DNA检测作为细胞学检查的辅助手段[15-16]。本研究中以30岁为界，将观察对象分为& 30岁和≥30岁，两者无论HPV感染率还是HPV16感染率和非HPV16和HPV18型的其他HR-HPV感染率比较，差异均无统计学意义(P &0. 05) ,而HPV18由于感染率过低，在本研究中未进行统计。但是在HPV16阳性妇女中，年龄& 30 岁和≥30岁两个年龄段宫颈病变程度≥CIN Ⅱ患者的比例却有显著性差异(P & 0.05)。可见年龄≥30岁者HPV16感染有发生宫颈病变的更高的风险。
　　Song[17]等研究已经证实了HPV DNA检测的作用，包括宫颈癌的筛查计划，宫颈细胞学检查结果可疑患者的分流，以及高度宫颈病变患者治疗后的随访。将HPV DNA检测联合宫颈细胞学检查作为宫颈癌初筛方案，在临床上产生了一定的困境，即对HPV DNA阳性而宫颈细胞学检查阴性的妇女该如何管理？本研究中HPV16、18、非HPV16和HPV18的其他HR-HPV阳性和HR-HPV阴性者宫颈病变≥CINⅡ的发生率比较，差异有统计学意义。按照宫颈细胞学结果分类，尤其是在细胞学检查阴性者、ASCUS者和LSIL者中，各种型别的HPV的致癌风险排列如下: HPV16 & HPV18 &非HPV16和HPV18型的其他HR-HPV阳性& HR-HPV阴性。已有研究将这组人群进一步分流的解决方案是检测其感染的HPV型别是否为HPV16和HPV18，HPV16和HPV18阳性者应该立即进行阴道镜检查，而HPV16和HPV18均为阴性者则可以随诊，1年后复查[5,18]。本研究结果显示，在进行HR-HPV总体检测的同时，将HPV16和HPV18进行分型检测将会进一步提高阳性预测值。年龄≥30岁的妇女在进行宫颈癌初级筛查时应该进行细胞学检查、HR-HPV检测及HPV16和HPV18的分型检测。HPV16和HPV18分型检测将有利于对这组人群进行风险分类和管理。HR-HPV阴性者同时细胞学结果也为阴性者可以间隔3年再行复诊。虽然本研究用两个不同方案进行宫颈高度病变及宫颈癌筛查的价值相近，但ASCCP指南和西班牙宫颈癌筛查与预防计划对HPV16和(或) HPV18阳性与其他HR-HPV阳性者的处理截然不同[10]，认为细胞学检查阴性但HR-HPV阳性者可以进行HPV16和HPV18的分型检测，如果HPV16或HPV18为阳性仍需要进行阴道镜检查。本研究所采用的PCR12+2检测，可以在进行HR-HPV检测的同时单独对HPV16和HPV18进行分型检测，既节省了患者就诊的时间也减少了复诊的次数。另外，HPV18分型检测对筛查宫颈高度病变尤为重要，因为HPV18与一些细胞学检查难以发现的宫颈病变(如腺癌)有关[19]。在本研究中HPV18阳性者共计5例，其中l例宫颈细胞学检查为炎症，而经过宫颈活检却证实为宫颈腺癌。因此，在进行宫颈癌筛查时，仅仅报告有无HR-HPV感染(至少不能分别检测出HPV16 和HPV18 )是明显不够的。
　　二、传统的HC-Ⅱ检测的特点
　　HC-Ⅱ检测由于有着高度的敏感性和阴性预测值而得到全世界范围内的广泛应用。但是，随着对宫颈癌发病机制了解的逐步深入，目前对于HR-HPV中16和18感染越来越受到重视。但是，HC-Ⅱ检测仅能相对半定量地报告HR-HPV的阳性或阴性，无法鉴别出具体的感染HPV的类型及是否为混合感染，在对HPV阳性的报告进行解读和对HPV阳性患者进行分流时常常给临床医生和HPV感染者带来一定的闲扰，而且其高昂的价格也是妨碍其广泛普及的重要原因之一。
　　三、PCR12+2检测的优势及其临床意义
　　PCR12+2检测除了能够在进行HR-HPV检测的同时对致癌风险最高的16和18型HPV进行分型检测之外，还具有以下两个优势：
　　1.同时对细胞内β-globin进行扩增:应用荧光PCR仪克服了传统PCR需电泳分析和样本间污染等缺点[20]。PCR的扩增和检测在同一个反应管中进行，在这个多重反应管中有4种不同报告染色基团可以对不同目标进行示踪的探针，其中报告染色基团4可以对细胞内β-globin进行检测和示踪，以评估采集样本质量及PCR抑制因素，从而对检测过程进行质量控制[21]。在本研究的424 份标本中出现了3份β-globin阴性的标本，考虑原因可能为采集到的宫颈脱落细胞过少，HPV 拷贝数过少从而导致检测结果出现假阴性，或者标本中含有较多的PCR抑制物，如血液中含有的可以严重抑制PCR的复合物如来自亚铁血红素族的卟啉和抗凝血剂如拧朦酸盐和肝素等。如果没有此项质控，势必增加HPV DNA检测的假阴性率，因此在进行HPV 检测的同时加入质控是必要的。
　　2. 理想的费用效益比: PCR12+2检测试剂盒收费低廉，费用效益分析显示，其对于宫颈癌及宫颈高度病变的筛查结果令人满意，而且由于可以对致癌风险最高的16和18型HPV进行分型检测，将更有利于对HR-HPV阳性者的分流管理。
　　参考文献
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　　Department of Gynecology and Obstetrics,China-Japan Friendship Hospital, Beijing
　　10029,P.R.China Received May 21,2013;Accepted September 11,2013
　　DOI:10.3892/etm.
　　Evaluation of the detection of 14 high-risk human
　　papillomaviruses with HPV 16 and HPV 18 genotyping for cervical cancer screening
　　MEI-LU BIAN,JIAO-YING CHENG,LI MA,XIAO CONG,JUN LIU,YING CHEN and
　　XI CHEN
　　Abstract.
　　The American Society for Colposcopy and Cervical Pathology (ASCCP) suggests that women ≥30 years old, with a negative cytopathological test but a positive high-risk (HR) human papillomavirus (HPV) test should undergo HPV 16 and HPV 18 genotyping. If this test is positive, immediate cervical pathology is required. Therefore, the aim of this study was to evaluate the effectiveness and clinical value of testing for 14 HR HPVs with HPV 16 and HPV 18 genotyping for cervical cancer (CC) screening. A total of 424 females from the China-Japan Friendship Hospital were selected and randomly divided into two groups (A and B). All participants underwent two different testing methods: the liquid-based cytology test (LCT) and a HPV DNA test. For the HPV DNA test, participants in group A underwent the hybrid capture II (HC-II) testing method while participants in group B were tested using the quantitative polymerase chain reaction (qPCR; HBRT-H14) method. The sensitivity, specificity, positive predictive value and negative predictive value for the detec?tion of cervical intraepithelial neoplasia (CIN) grade II or greater using HBRT-H14 were 96.30, 78.17, 23.21 and 99.68%, respectively. In Group B, compared with other HR HPV types, HPV 16 and HPV 18 infection led to the increased possibility of cervical lesions graded CIN II or higher (8.11 and 51.28%, respectively). A significant difference in the rates of CC and CIN II or higher was observed among women who were i) infected with HPV 16 and/or HPV 18, ii) infected with other HR HPV types and iii) diagnosed as negative for HR HPV infection (χ2=93.976, P=0.0001). In conclusion, HBRT-H14 is applicable for CC screening with the advantage of genotyping for HPV 16 and HPV 18, which may help to improve triage management for women with negative cytology.
　　Introduction
　　It has been identified that ~95-100% of the incidences of cervical cancer (CC) are due to infection with the human papillomavirus (HPV) (1-3). There are 15 types of high-risk (HR) HPV that may lead to the development of cervical intraepithelial neoplasia (CIN) and CC (4,5). Over the past 20 years, various studies have stated that the detection of HPV may be used as an auxiliary tool for primary screening in CC prevention (6-10). Furthermore, the International Agency for Research on Cancer (IARC) has proposed the use of HPV detec?tion as a screening option (11). Though 15 types of HR HPV have been linked to CC, studies have shown that &50% of CC cases are caused by HPV 16, and 10-15% are caused by HPV 18 (12,13). Thus HPV 16 and HPV 18 may be detected in ~70% of CC cases (14). In addition, HVP 18 causes &35% of cervical adenocarcinomas, which are difficult to detect using cytological tests (12). Therefore the manual of the American Society for Colposcopy and Cervical Pathology (ASCCP;
and 2012) suggests that women ≥30 years old, with a negative cytopathological test but a positive HR HPV test should undergo HPV 16 and HPV 18 genotyping. If this test is positive, immediate cervical pathology is required. Patients who are HPV 16/18 negative, but who are HR HPV positive should undergo cytopathology and HR HPV testing again after a further 12 months (15,16). In this study, we evaluated the effectiveness of a real-time PCR method from Hybribio to detect 14 high-risk HPV with HPV 16 and HPV 18 genotyping kit (qPCR; HBRT-H14) for CC screening.
　　Materials and methods
　　Study population.
　　Between August 15, 2011 and November 10, 2011, a total of 424 females, with a mean age of 39.02±12.00 years (range, 20-55 years), underwent CC screening and treatment in the China-Japan Friendship Hospital cervical cancer treatment center (Beijing, China). None of the recruited patients had exhibited acute inflammation of the reproductive tract. Written informed consent was obtained from all patients. In group A, hybrid capture II (HC-II) and liquid-based cytology testing (LCT) in group B, 14 patients underwent HR HPV with HPV 16 and HPV 18 genotyping using the quantitative polymerase chain reaction (qPCR; HBRT-H14) test and LCT. For subjects who were HR HPV positive and had an LCT result that was at least as severe as atypical squamous cells-undetermined significance (ASC-US); HPV 16 and/or HPV 18 positive with negative or had a cytological test result that was at least as severe as ASC-US and were HPV negative, colposcopies were performed.
　　The thin-layer cytology specimens were obtained using SurePath? (TriPath Imaging, Becton Dickinson, Burlington, NC, USA), and an experienced gynecologist evaluated the histopathological slides. The Bethesda system (TBS) protocol, revised by the International Cancer Association in 2001, was used as the standard for cytological diagnosis (17). Squamous epithelial abnormalities include ASC [including ASC-US and ASC but excluding high-grade squamous intraepithelial lesions (ASC-H)], low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC).
　　Detection of 14HR HPVs with HPV 16 and HPV 18 genotyping and qPCR(HBRT-H14).
　　HBRT-H14 qPCR is a new PCR assay that detects 12 HR HPV genotypes (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) with simultaneous differentiation between HPV 16 and HPV 18. This method was based on the multiplex qPCR assay which utilizes 14 sets of specific primers and four probe sets, designed in the E region of the HPV genome, with varying fluorophores in order to obtain fluorescent detection signals. The channels of reporter dyes FAM, HEX, ROX and Cy5 represent 12 HR HPV genotypes, HPV 16, HPV 18 and the β-globin gene (internal control), respectively. Amplification was performed according to the manufacturer’s instructions (Hybribio Biotechnology Ltd. Corp., Chaozhou, China).
　　HC-Ⅱ for HPV detection .
　　The HC-II test system (Qiagen, Gaithersburg, MD, USA) was used to detect HPV in the obtained samples (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) as previously described (18). The 96-well HC-II is able to detect up to 90 clinical samples. Samples require ≥5,000 copies/ml of HR HPV in order to be detected.
　　Pathological diagnosis.
　　A cohort that were: HBRT-H14 diagnosed with ASC-US or HPV 16 and/or HPV 18 positive in the HBRT-H14 assay but cytologically negative (n=122) underwent colposcopy testing and cervical biopsies. Multi-point biopsies were performed on the area of epithelial cells to which acetic acid had been applied and on the unstained iodine test point. For the normal transformation zone, four points (2,4,8,10) in total were bioposies. Sherman et al (19) reported that the cumulative incidence of CC was 0.79% with negative cytological and HPV test results, and all samples were not deemed to be CIN or CC according to the histological tests. It was assumed that 300 subjects with negative results in the cytological and HPV tests were normal without CIN or CC.
　　Statistical analysis.
　　Histopathological results were considered to be the gold standard. SPSS 16.0 (SPSS, Inc., Chicago, IL, USA) was used to calculate the sensitivity, specificity, positive predictive value and negative predictive value of HBRT-H14 for CC screening. Comparisons among groups were made using the χ2 test. P&0.05 was considered to indicate a statistically significant difference.
　　Results
　　Correlation between HBRT-H14 HPV and histopathological test results
　　Using the HBRT-H14 HPV test and the LCT screening method, 19 samples in 27 subjects with CIN II or above were HPV 16 positive according to the HPV DNA test, accounting for 70.37% (19/27), among which four samples were cytologically negative. One sample (3.70%, 1/27) was identified to be HPV 18 positive with other HR HPV six subjects tested positive for other HR HPV (non-HPV 16 and non-HPV 18), accounting for 22.22% (6/27); and one sample (3.70%, 1/27) gave a negative HR HPV result (Table I). CIN II and above was considered to be pathologically positive with CIN I and below considered to be pathologically negative. For the screening of CIN and CC, the sensitivity of the HBRT-H14 HPV test was 96.30%, the specificity was 78.17%, the positive predictive value was 23.21% and the negative predictive value was 99.68%. Nineteen samples in 34 subjects with HPV 16 positive were CIN II or above (histopathology). However, four samples were HPV positive and cytopathologically negative. The subjects who were graded as CIN II or above comprised one (20%, 1/5) who was HPV 18 positive, six (8.11%, 6/74) who were other HR (non-HPV16 and non-HPV 18) positive and one (0.32%, 1/309) who was HR HPV negative. The risk of cervical precancer and cancer demonstrated a significant difference between HPV 16 positive, HPV 18 positive, other HR HPV (non-HPV 16 and non-HPV 18) positive and HR HPV negative subjects (χ2=93.976, P=0.0001).
　　Evaluation of the clinical value of the HBRT-H14 HPV test
　　In order to examine whether the HBRT-H14 HPV test is only required in the CC screening of females ≥30 years old, the subjects were divided into two groups with 30 years of age as the borderline. Table II shows that the HR HPV infection rate was 21.76% (26/120) in group 1 (females &30 years old), and 28.57% (86/301) in group 2 (females ≥30 years old). There was no statistical significance between the two groups (χ2=2.09, P&0.05). The HPV 16 infection rates [6.67%, (8/120) and 8.64% (26/301)] also showed no statistically significant difference between the two groups (χ2=2.09, P&0.05). In HPV 16 positive subjects, the percentage of patients graded as CIN II or above was 25% (2/8) for group 1 and 65.38% (17/26) for group 2; the difference was deemed to be statistically significant (χ2=4.123, P&0.05). Due to the low detection rate of HPV 18, no statistical analysis was performed for this virus. The infection rates of other HR HPV (non-HPV 16 and non-HPV 18) infections were 15.00% (18/120) and 18.60% (56/301) in groups 1 and 2, respectively (χ2=0.77, P&0.05); there was no statistically significant difference between the two groups. The rates of other HR HPV (non-HPV 16 and non-HPV 18) infection with CIN II or above are 11.11% (2/18) and 7.14% (4/56) in groups 1 and 2, respectively (χ2=0.0016, P&0.05); the difference between groups was considered to have no statistical significance
　　Analysis of cervical cytological, HBRT-H14 and histopathological test results
　　Of the 379 subjects considered to be normal or inflamed by the cervical cytological method, 18, 5, and 56 subjects, respectively, were tested to be HPV 16, HPV 18 and other HR HPV (non-HPV 16 and non-HPV 18) positive. Histological results following the cervical biopsy were that samples from four of the HPV 16 positive patients, one of the HPV 18 positive patients and none of the patients positive for other HR HPV were graded as CIN II or above. In the nine subjects diagnosed with ASC-US using the cervical cytological method, two were HPV 16 positive and were graded CIN II according to the other seven subjects were both HPV 16 and HPV 18 negative, and evaluated as having inflammation according to the biopsy. Using the cervical cytological test, 17 subjects were diagnosed with LSIL, of which two were HPV 16 positive and graded CIN II by biopsy, and 11 subjects were other HR HPV (non-HPV 16 and non-HPV 18) positive, among which four were graded CIN II. The remaining four subjects were HR HPV negative and one was confirmed to have inflammation, three were confirmed to have CIN I according to the biopsy results (Table III). A grading of CIN II or above was made in 19 of the 34 HPV 16 positive patients, one of the 5 HPV 18 positive patients, six of the 74 other HRHPV (non-HPV 16 and non-HPV 18) positive patients and one of the 309 HR HPV negative subjects. The differences between groups were considered to be statistically significant (χ2=93.976, P=0.0001). In cytologically negative subjects diagnosed with ASC-US and LSIL, the differences between the various types of HPV associated with the CC were deemed to be statistically significant (χ2=32.742, 9.535
　　and 7.654, P=0. and 0.022, respectively).
　　Analysis of HC-II HPV and the cytological method of testing
　　Table IV shows the correlation between the HC-II HPV and histological results. CIN II and above was considered to be pathologically positive and CIN I and below was considered to be pathologically negative. The sensitivity, specificity, positive predictive value and negative predictive value for the detection of CIN II and above using HC-II HPV and cytological testing were 77.78, 79.35, 20.39 and 98.13%, respectively. In terms of clinical value for the detection of CIN and CC, there was no significant difference between HBRT-H14 and HC-II when used with cytological examination (P&0.05). Furthermore, the sensitivity and the negative predictive value were improved using HBRT-H14.
　　Discussion
　　Analysis of 14 HR HPVs with the 16/18 genotyping real-time PCR kit is based on a multiplex qPCR assay utilizing specific sets of amplification primers with varying fluorophores in order to obtain fluorescent detection signals. qPCR is capable of dispensing with the problems associated with electrophoresis and the probability of contamination from traditional PCR (20). PCR amplification and detection are conducted in th multiple reaction tubes use four different reporter dyes capable of tracking different targets. The reaction mixture for each sample includes a cellular β-globin gene which is capable of fully monitoring the entire testing process from DNA extraction to signal detection.
　　In this study, three β-globin negative samples were reported among 424 samples (0.7%), rendering these three subjects invalid. It was possible that the HPV copy number was too low to be amplified due to too few cells being obtained. False negative results in HPV DNA detection are likely to increase if this quality monitoring option is lacking. Thus it is critical to include quality monitoring in HPV detection.
　　In the natural history of HPV infection, the majority of infections are temporary, particularly among young women. Only a small proportion are persistently infected by HR HPV, and usually CC develops following 10 years of continuous infection (21). Thus, the Food and Drug Administration (FDA) approved that the testing of women ≥30 years old may include HPV DNA detection as a supplementary method of diagnosis (22-24). In the present study, no statistically significant differences were observed in the incidence of HPV 16 infection and of other HR HPV (excluding HPV 16 and 18) infection between the age groups (P&0.05). Statistical analysis was not performed for HPV 18 due to its low infection rate. However, there was a statistically significant difference between the two age groups of HPV 16 positive females (&30 and ≥30 years old), graded CIN II and above (P&0.05). Thus for women ≥30 years old, HPV 16 infection has a very high risk.
　　Song et al (25) considered the HPV DNA test to be a valuable resource for CC screening, triage and the management of patients with ASC-US and LSIL, and post-surgical follow-up for CIN and CC. The clinical dilemma is how to manage female patients who are HR HPV positive but cytologically negative. In the present study, statistically significant differences were identified among the HPV 16 positive, HPV 18 positive, other HR HPV (excluding HPV 16 and 18) positive and HR HPV negative females with regard to a grading of CIN II and above. Thus, the risk factors for CC may be listed as: HPV 16 positive & HPV 18 positive & other HR HPV type positive & HR HPV negative. Our study has shown that the patients may also divided according to whether the type of HPV infection is HPV 16 or HPV 18. Colposcopies should be performed if the females are demonstrated to be HVP 16 and/or HPV 18 positive, whereas subjects who are HPV 16 and HPV 18 negative should be tested again one year later (10,11).
　　The guidelines from the ASCCP and those of the Spanish CC screening and prevention programs suggest that cytologically negative but HR HPV positive subjects should continue to undergo HPV 16 and HPV 18 genotyping (26). If the HPV 16 or HPV 18 tests are positive, examination by colposcopy is required. Therefore, the HBRT-H14 screening method that detects 14 HR HPV types, including the specific detection of HPV 16/18 genotypes, is likely to save time. Furthermore, HPV 18 infection related to certain cervical lesions is difficult to detect using cytological methods (for example cervical adenocarcinoma). In our study, five cases were HPV 18 positive. These cases included one that was diagnosed as inflammation by cervical cytological testing but was demonstrated to be cervical adenocarcinoma by biopsy. Therefore, it is not sufficient to report the infection of HR HPVs wi HPV 16 and HPV 18 genotyping should also be performed. Our study indicated with other HR-HPV, HPV 16 and HPV 18 had a higher risk of causing CC. The results of the current study were similar to those of Wong et al (27).
　　Acknowledgments
　　The authors would like to thank Longxu Xie and Liejun Li for critically reviewing the manuscript.
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作者：程娇影 卞美璐
马莉 丛笑 陈颖文 刘军
来源：中华妇产科杂志
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