CN103562722A - 一种区分甲状腺癌变的方法、实施该方法的试剂盒以及金属硫蛋白在区分甲状腺癌变中的用途 - Google Patents
一种区分甲状腺癌变的方法、实施该方法的试剂盒以及金属硫蛋白在区分甲状腺癌变中的用途 Download PDFInfo
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Abstract
本发明是关于区分包含滤泡状腺瘤和滤泡状癌的甲状腺癌变的方法,其特征在于采用抗金属硫蛋白单克隆抗体,利用免疫组织化学方法评估患者的癌组织样本中的金属硫蛋白(MT)的表达,根据Remmele的半定量免疫反应积分法在光学显微镜下评估所述金属硫蛋白表达的强度,其中若所述金属硫蛋白的表达强度的半定量免疫反应积分值等于或大于8,确定具有滤泡状癌。
Description
技术领域
本发明的主题是一种区分包含滤泡状腺瘤和滤泡状癌的甲状腺癌变的方法、实施该方法的试剂盒以及金属硫蛋白在区分上述甲状腺癌变类型中的用途。本发明的主题属于诊断领域以及通过免疫组织化学方法区分甲状腺肿瘤领域。
背景技术
甲状腺癌是最常见的内分泌系统肿瘤。甲状腺滤泡细胞发展成常见于年轻人群的高分化型癌症(乳头状和滤泡状)以及常见于六十岁以上人群的低分化型退行发育性癌症。乳头状癌占甲状腺癌的70-80%,并被认为是该器官最轻微的一种癌症。滤泡状癌占甲状腺癌的5-15%。由于滤泡状癌和滤泡状腺瘤只有微小区别,难以区分二者,因此它们均称为滤泡状肿瘤。仅可通过术后离析区分二者,却无法通过细针穿刺活检进行区分。
最普遍的诊断增殖标记物之一是Ki-67。在其他诸如乳腺癌、肺癌、前列腺癌、软组织癌和脑瘤中,Ki-67表达均有上升,但是还未揭示它的生物学作用。迄今为止,许多已公开的报道比较了各种类型的肿瘤中金属硫蛋白(MT)和抗原Ki-67的表达。至今,人们已对位于结肠肿瘤、肾上腺皮质肿瘤、口腔肿瘤、前列腺肿瘤、乳腺肿瘤、卵巢肿瘤、软组织肉瘤以及非小细胞肺癌均展开研究。在大多数情况下,金属硫蛋白和Ki-67抗原的表达呈明显的相关性。然而,研究对金属硫蛋白及其亚型在甲状腺良性及恶性病变中的表达尚未得到一致结果。
现有技术包括Ferrario等人所著的公开文献(作者:C·Ferrario、P·Lavagni、M·Gariboldi、C·Miranda、M·Losa、L·Cleris、F·Formelli、S·Pilotti、MA·Pierotti和A·Greco;名称:金属硫蛋白1G在乳头状甲状腺癌中用作抑癌物;Lab Invest88:474-481,2008),其中作者揭示了乳头状癌中的金属硫蛋白的功能亚型(MT1E、MT1G、MT1F和MT2A)的表达下降,并用细胞模型证明了MT1G可能发挥了致癌抑制物的作用。该文献仅涉及区分乳头状癌和滤泡状癌中恶性类型病变的问题,而并未触及滤泡状腺瘤和滤泡状癌诊断的问题。Y·Huang发表的文章揭示了乳头状甲状腺癌中MT-1G的表达下降是由其启动子高甲基化所导致的(Y·Huang、A·de laChapelle和NS·Pellegata;名称:高甲基化(而不是杂合性缺失)与乳头状甲状腺癌中的MT1G和CRABP1的低表达有关;Int J Cancer104:735-744,2003)。该文章提供了正常的甲状腺组织、乳头状癌及乳头状癌滤泡变异体(FVPTC)的研究结果。但是并未涉及滤泡状腺瘤以及滤泡状腺瘤相对滤泡状癌的诊断。Liu等人揭示了在乳头状甲状腺癌细胞中,金属硫蛋白的8个功能亚型的表达均可能是由于受到镉元素的刺激和ERK1/2激酶的激活。这些亚型在其中表达的细胞的特征是具有较短的GI期,并在细胞周期中迅速转换至S期和M期(作者:ZM·Liu ZM、CA·Hasselt、FZ·Song FZ、AC·Vlantis AC、MG·Cherian、J·Koropatnick和GG·Chen GG;名称:金属硫蛋白功能亚型在乳头状甲状腺癌中的表达;Mol Cell Endocrinol302:92-98,2009)。Liu等人所做的关于金属硫蛋白在甲状腺肿瘤中的表达研究只基于一例病人的一种乳头状癌(KAT5)。该报告无法提供来自不同病人临床材料的研究结果,这样的研究结果才能确保各种情况并说明实际状况。该文献也未涉及有关滤泡状癌的滤泡状腺瘤的诊断问题,而专注于分析一种乳头状癌中金属硫蛋白表达水平的调控机制。
受钙离子影响的甲状腺退行发育性癌细胞中也会发生金属硫蛋白亚型诱导,其表达增加同样导致细胞周期中GI期缩短(作者:ZM·Liu、GG·Chen、CK·Shum、AC·Vlantis、MG·Cherian、J·Koropatnick和CA·van Hasselt;名称:甲状腺退行发育性癌细胞中功能亚型MT1和MT2的钙诱导;FEBSLett.581:2465-2472,2007)。该项研究依然未涉及通过测定金属硫蛋白的表达来区分滤泡状癌和滤泡状腺瘤。
因此,亟需一种有效的甲状腺滤泡状腺瘤和甲状腺滤泡状癌癌变的标记物,便于正确诊断并选择正确的治疗性干预。尽管对金属硫蛋白在各种恶性肿瘤中的作用已研究了多年,但它在上述几种甲状腺肿瘤中预测值的大小尚未得到证实。
发明内容
本发明的主题是一种在体外区分包含滤泡状腺瘤和滤泡状癌的甲状腺癌变的方法,其特征在于采用抗金属硫蛋白(MT)的单克隆抗体,利用免疫组织化学方法检测患者的癌组织样本中MT的表达。根据Remmele的半定量免疫反应积分(IRS)方法在光学显微镜下评估金属硫蛋白表达的增加度。其中金属硫蛋白表达的增加度的半定量IRS值等于或大于8,则确定具有滤泡状癌。
优选地,评估金属硫蛋白亚型MT I和MT II的表达。
优选地,检测源自经胸腺切除术且肿瘤已摘除的患者的物料中金属硫蛋白的表达。
本发明的另一个较优的实施例中,利用同步的病理组织检查上述病变确定滤泡状癌或滤泡状腺瘤的存在。
本发明的下一主题是一种区分包含滤泡状腺瘤和滤泡状癌的甲状腺癌变的试剂盒,该试剂盒包含抗MT的单克隆抗体。
本发明的一个优选实施例中,试剂盒包含抗金属硫蛋白亚型MT I和MT II的单克隆抗体。
本发明的另一主题是金属硫蛋白在区分滤泡状癌和滤泡状腺瘤型甲状腺肿瘤中的用途。
优选地,金属硫蛋白亚型为MT I和MT II。
金属硫蛋白(MT)是一族低分子量蛋白,该蛋白主要存在于动物中。这种蛋白保守性强,在正常组织和癌组织的各不同位置呈现出四种主要亚型。金属硫蛋白多肽链由61至68个氨基酸组成(依据不同的亚型),其中30%的氨基酸残基是半胱氨酸残基。一个金属硫蛋白分子由两个结构域通过赖氨酸二聚体连接而成。金属硫蛋白的生理学作用在于结合并输送锌离子和铜离子,通过结合给有机体消解有毒重金属,如镉,汞和铅。在密集分裂的细胞(包括癌细胞)中,观察到金属硫蛋白的表达增加,将锌离子(Zn)运送至参与DNA复制的酶,同时金属硫蛋白的表达增加还防止细胞凋亡。金属硫蛋白还可能对一些化疗的活性产生影响(它们使在如蒽环类药物等细胞抑制剂的新陈代谢中形成的自由基失活,或者结合如顺铂等其它物质)。金属硫蛋白的表达在滤泡状腺瘤和滤泡状癌之间的差异表示可将金属硫蛋白用作区分上述病变的辅助标记物。此外,金属硫蛋白是比Ki-67更好的用于区分滤泡状腺瘤型病变和滤泡状癌型病变的诊断标记物,其原因在于上述不同肿瘤类型的金属硫蛋白的表达在统计上有显著差异。而抗原Ki-67的表达则缺乏这种显著性差异。所述诊断方法的应用构成了这两种甲状腺肥厚性病变的组织病理诊断的辅助方法。
附图说明
图1表示金属硫蛋白在滤泡状腺瘤(1A)及滤泡状癌(1B)中的细胞核-细胞质表达以及细胞核抗原Ki-67在滤泡状腺瘤(1C)及滤泡状癌(1D)中的表达。
图2表示金属硫蛋白表达在甲状腺不同病变下的增强。统计上在滤泡状癌和乳头状癌(p<0.001)以及滤泡状癌和滤泡状腺瘤(p<0.05)之间均存在显著差异。在滤泡状癌的表达在统计上显著高于在髓状癌的表达(图2;P<0.0001)。统计上在乳头状癌的表达(p<0.0001)和在髓状癌(p<0.005)的表达均显著低于在肿瘤状甲状腺肿的表达。
图3显示了抗原Ki-67表达在甲状腺不同病变下的增强。髓状癌和滤泡状腺瘤(p<0.05)以及髓状癌和肿瘤状甲状腺肿(p<0.05)之间在统计上均具显著差异。
具体实施方式
本研究所采用的甲状腺各种病变的186块石蜡块均来自弗罗茨瓦夫医学院病理形态学系。所检测的石蜡块包括92例癌症,其中48例乳头状癌、35例滤泡状癌、9例髓状癌、31例腺瘤以及63例肿瘤状甲状腺肿。
A.免疫组织化学法
将肿瘤活组织切片固定于10%缓冲福尔马林中,进行脱水,然后将其包埋在石蜡块中。所有免疫组织化学(IHC)反应都在4μm厚的石蜡切片上进行,在二甲苯去除石蜡,再水化。对于免疫组织化学抗Ki-67反应,将切片置入柠檬酸盐缓冲液(pH6.10mM)在95-98℃的温度下温育20分钟,从而使表位暴露。将其置入3%H2O2中温育5分钟来封闭内源性过氧化物酶活性。MT I/II(克隆E9)和Ki-67(克隆MIB-1)的表达用小鼠单克隆抗体(产自丹麦格洛斯楚普的DakoCytomation公司)测定。检测的抗原可通过生物素化的抗体和辣根过氧化物酶共轭标记的链霉亲和素(DakoCytomation LSAB+System-HRP)显现。酶作用物为二氨基联苯胺(DAB,DakoCytomation)。利用阴性对照进行各反应。利用苏木精对各样本进行对比染色。
B.免疫组织化学反应强度评估
我们使用根据Remmele的半定量免疫反应积分法来评估金属硫蛋白表达的强度,并考虑了显色反应的强度和阳性癌细胞占样本的百分比。该数值范围从0至12:0表示无反应,1-2表示弱反应,3-4表示中等反应,6-12表示强反应。癌细胞中抗原Ki-67的表达强度通过表示阳性细胞占所有癌细胞百分比的数值来评估。该数值对应下述比例:0表示无反应,1代表1-10%,2代表11-25%,3代表26-50%,4代表>50%。
C.统计分析
利用软件Prism5.0package(美国加州GraphPad公司出品)统计分析结果。我们还进行了斯皮尔曼相关性检验(Spearman correlation tests),克鲁斯卡尔-沃利斯检验(Kruskal-Wallis test)和曼-惠特尼U检验(Mann-Whitney U test)。当p<0.05时,视为具有统计上的显著差异。
在检测的186例甲状腺病变中,在180例(96,7%)中发现有金属硫蛋白在细胞核-细胞质的表达(图1A、1B)。金属硫蛋白在73%肿瘤状甲状腺肿例、70.9%滤泡状腺瘤例(图1A)、45.8%乳头状癌例和85.7%滤泡状癌例(图1B)中表现出高表达(6-12pkt)。而金属硫蛋白在33%髓状癌例中有高表达。
金属硫蛋白在滤泡状癌中呈现最高表达(9.14±3.32)稍低但依然保持极高水平的表达存在于肿瘤状甲状腺肿(7.25±3.41)和滤泡状腺瘤(6.54±3.40),在乳头状癌(4.47±3.14)和髓状癌(3.22±2.54)中的表达最低。金属硫蛋白的表达在各甲状腺病变之间存在统计上的显著差异性(图2)。
金属硫蛋白表达的强度在滤泡状癌(图1B)和滤泡状腺瘤(图1A)之间存在统计上的显著差异,这一点很难通过组织结构区分(图2,P<0.05)。在乳头状癌中的表达在统计上显著高于在滤泡状癌(图2,P<0.0001)和髓状癌(图2,P<0.0001)中的表达。在乳头状癌(图2,P<0.0001)和髓状癌(图2,P<0.005)中的表达在统计上显著低于在肿瘤状甲状腺肿中的表达。
在131例检查的病变中,我们在118例中发现抗原Ki-67的细胞核表达。在滤泡状癌中的表达(1.13±0.92)最高(图3),依次降低为滤泡状腺瘤(1.08±0.4)、乳头状癌(1.05±0.52)和肿瘤状甲状腺肿(1.05±0.22),最低的是髓状癌(0.56±0.53)。
抗原Ki-67表达的强度在滤泡状腺瘤(图1C)和滤泡状癌(图1D)之间没有统计上的显著差异。而该抗原表达在髓状癌和肿瘤状甲状腺肿(p<0.05)之间以及髓状癌和滤泡状腺瘤(p<0.05)之间具有统计上的显著差异(图3)。
通过斯皮尔曼检验所评估出的金属硫蛋白和抗原Ki-67的表达在如滤泡状腺瘤和滤泡状癌等病变中的依赖性表示上述抗原间缺乏相关性(r=0.12,p>0.05)。
Claims (8)
1.一种区分包含滤泡状腺瘤和滤泡状癌的甲状腺癌变的方法,其特征在于采用抗金属硫蛋白单克隆抗体,利用免疫组织化学方法评估一患者的一癌组织样本中的金属硫蛋白(MT)的表达,根据Remmele的半定量免疫反应积分法在一光学显微镜下评估所述金属硫蛋白表达的强度,其中若所述金属硫蛋白的表达强度的半定量免疫反应积分值等于或大于8,确定具有滤泡状癌。
2.根据权利要求1所述的方法,其特征在于检测金属硫蛋白亚型MT I和MT II的表达。
3.根据权利要求1或2所述的方法,其特征在于检测源自经胸腺切除术且肿瘤已摘除的患者的物料中金属硫蛋白的表达。
4.根据权利要求1-3中任一权利要求所述的方法,其特征在于对具有滤泡状癌或滤泡状腺瘤的确定伴有对所述病变进行一充分的组织评估。
5.一种区分包含滤泡状腺瘤和滤泡状癌的甲状腺癌变的试剂盒其特征在于包含抗金属硫蛋白的单克隆抗体。
6.根据权利要求5所述的试剂盒,其特征在于包含抗金属硫蛋白亚型MT I和MT II的单克隆抗体。
7.金属硫蛋白在区分滤泡状癌和滤泡状腺瘤型甲状腺肿瘤中的用途。
8.根据权利要求7所述的用途,其特征在于所述金属硫蛋白亚型是MTI和MT II。
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