CN113466468A - Fh-缺陷型肾细胞癌的二联诊断标志物及其应用 - Google Patents
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Abstract
本发明涉及疾病的分子诊断技术领域，具体是FH‑缺陷型肾细胞癌的二联诊断标志物及其应用。本发明通过利用非靶向代谢组学，基因检测结合免疫组化和多维影像(CT/MRI/FDG‑PET)，初步发现了小分子化合物succinic‑cys和sado在FH‑缺陷型肾细胞癌患者中显著升高，其浓度与肿瘤负荷呈强正相关。另外，肿瘤细胞小鼠肾脏原位移植模型(CDX)实验显示，血浆标志物浓度随肿瘤生长而同步升高，肿瘤大小决定着标志物浓度水平，验证了临床数据准确性和特异性。这提示小分子化合物succinic‑cys和sado可作为FH‑缺陷型肾细胞癌的诊断标志物，为FH‑缺陷型肾细胞癌的新型诊断与治疗提供新策略。
Description
技术领域
本发明涉及疾病的分子诊断技术领域，具体地说，是FH-缺陷型肾细胞癌的二联诊断标志物及其应用。
背景技术
肿瘤是以细胞异常增殖为特点的综合征。癌基因与抑癌基因的相互制约，协调表达是调节肿瘤生长的重要机制。全球每年约有超过40万患者确诊为肾细胞癌，造成的死亡超过17万人。遗传性平滑肌瘤和肾细胞癌(hereditary leiomyomatosis and renal cellcancer carcinoma，HLRCC)主要由延胡索酸酶基因(fumarate hydratase，FH)的突变、缺失及甲基化失活(Loss ofFunction)造成的，其中约80％的患者为胚系突变，20％为体系突变，HLRCC也被称为FH-缺陷型肾细胞癌。在临床上，罹患疾病的个体主要表现为多处皮肤肉色结节，伴发有II型乳头状肾癌及女性特有的多发子宫肌瘤。在多个已报道的HLRCC队列研究中，该疾病具有广泛的多器官转移，即使很小的肿瘤也会发生转移。与透明细胞癌、集合管癌相比，HLRCC的五年生存率更低，预后更差。在组织病理学上，FH-缺陷型肾细胞癌除了具有典型的乳头状外，还发现有小管状、管状乳头状、实体状等形态，以一种或几种方式混合生长。因此，将FH-缺陷型肾细胞癌与其他肾癌如Xp11转位肾细胞癌、透明细胞乳头状肾癌等鉴别开来具有较大难度。
有文献报道显示免疫组化检测琥珀酰-S-半胱氨酸(2-succinocysteine，2-SC)可提高诊断的准确性，抗体对病灶的细胞核和胞浆呈弥漫性阳性反应。已经有多个队列使用2-SC联合FH免疫组化诊断该疾病，并用二代测序予以验证。虽然该方法的准确性相较于传统方法显著提升，但是2-SC对有一定FH表达的区域仍有强阳反应，表明其特异性还显不足。2-SC联合FH免疫组化法仍需要更大样本量的验证。目前，约有1/2到2/3的病人在确诊时已处于中晚期。医学影像、穿刺活检、免疫组化结合FH基因检测等是确诊的主要手段，但其创伤性侵入、取样困难、低灵敏度和程序复杂严重影响疾病的早筛早诊。
血液肿瘤标志物相对于上述的困难与缺点，具有无创性、高通量、取样方便、检测简单及高特异性和敏感度等优势。血液肿瘤标志物由肿瘤细胞直接产生或由宿主被肿瘤刺激后生成，通过膜转运或细胞崩解进入血液循环。它能准确反映肿瘤发生和发展的过程，已用于肿瘤筛查、早期诊断、临床分期、监测复发/转移、评估预后等临床实践中。
本专利发明人长期从事FH/SDH-缺陷型肾细胞癌的代谢重塑机制研究，对代谢与细胞增殖、周期和侵袭在肿瘤发生中的机制有深入理解。在已取得的成果中，我们率先在血红素加氧酶-1(haem oxygenase-1，HMOX-1)途径(C.Frezza，L.Zheng，Nature，2011)，尿素循环/氨基酸途径(L.Zheng，Cancer&Metabolism，2013)，谷胱甘肽氧化应激途径(L.Zheng，Nat Commun，2015)(J.Reest，Nat Commun，2018，共同通讯作者)等研究中有全新的发现。目前FH-缺陷型肾细胞癌在临床血液标志物领域的研究尚属空白。这些代谢重编程途径是否与血液循环发生物质交换，是以何种方式交换，是否在血液、肝脏、肾脏发生次级代谢而产生新的转化，这些物质是否是肿瘤特异性分子，这些转化与肿瘤的复发与转移存在何种关系，目前并不清楚。本专利对这些科学问题的回答与探究，将对FH-缺陷型肾细胞癌的诊断与治疗提供全新的认识和方案。
发明内容
本发明的目的是针对现有技术的不足，提供FH-缺陷型肾细胞癌的诊断标志物。
为实现上述目的，本发明采取的技术方案是：
第一方面，本发明提供了小分子化合物succinic-cys和sado中的任一种或其组合作为生物标志物在制备FH-缺陷型肾细胞癌的诊断试剂或试剂盒中的应用。
优选地，所述诊断试剂或试剂盒用于判断受试样品属于或不属于FH-缺陷型肾细胞癌。
第二方面，本发明提供了小分子化合物succinic-cys和sado中的任一种或其组合作为生物标志物在制备鉴别FH-缺陷型肾细胞癌与其他类型的肾细胞癌例如：Xp11转位肾细胞癌、透明细胞乳头状肾癌的诊断试剂或试剂盒中的应用。
第三方面，本发明提供了小分子化合物succinic-cys和sado中的任一种或其组合作为生物标志物在制备FH-缺陷型肾细胞癌的生存预后试剂或试剂盒中的应用。
第四方面，本发明提供了小分子化合物succinic-cys和sado中的任一种或其组合作为生物标志物在制备FH-缺陷型肾细胞癌的治疗效果的试剂或试剂盒中的应用。
本发明在起始阶段的研究积累中，利用非靶向代谢组学，基因检测结合免疫组化和多维影像(CT/MRI/FDG-PET)，初步发现了小分子化合物succinic-cys和sado在FH-缺陷型肾细胞癌患者中显著升高，其浓度与肿瘤负荷呈强正相关。FH-/-肿瘤细胞小鼠肾脏原位移植模型(CDX)实验显示，血浆标志物浓度随肿瘤生长而同步升高，肿瘤大小决定着标志物浓度水平，验证了临床数据准确性和特异性。
HLRCC相关肾细胞癌具有发病早及侵袭性高等特点，青少年时期可出现转移性肾癌。因此，对于有明确FH突变的的儿童群体，NCCN指南建议8或10岁时开始每年一次MRI或CT检查进行监测。除了HLRCC外，FH的双等位基因突变(纯合或复合杂合突变)能够在儿童中引起延胡索酸水合酶缺乏症(Fumarase deficiency，FD)。延胡索酸水合酶缺乏症是一种罕见的常染色体隐性遗传疾病，发病率尚未确切，约1/300000，在儿童早期引起严重的神经系统损害，其特征是脑病伴有癫痫发作和肌张力减退。由于FD患儿有发生FH缺陷型肾细胞癌的风险，因此，对于有FH突变的儿童应重点排查且高度重视，而本专利中小分子化合物succinic-cys和sado作为诊断标志物，适用于儿童和青少年群体中的FH缺陷型的早筛早诊。
本发明优点在于：
1、目前关于FH-缺陷型肾细胞癌的鉴定主要依赖于医学影像、穿刺活检、免疫组化结合FH基因检测等主要手段，缺乏有效的分子诊断方法及治疗指导标志物。本发明从临床诊断治疗热点和难点出发，以代谢重塑分子机制研究为坚实基础，突破血液标志物对肾细胞癌的诊断空白。本发明通过研究发现，琥珀酸型代谢物succinic-cys和sado在患者血浆中显著上升；小鼠CDX模型实验显示其血浆浓度随肿瘤的生长而升高，这提示小分子化合物succinic-cys和sado可用来诊断FH-缺陷型肾细胞癌，且具有潜在的预后和FH-缺陷型肾细胞癌治疗价值。
2、本发明的各标志物具备很高的诊断效用。
3、本发明的两种标志物联合具备很高的诊断效用。
附图说明
附图1是FH-缺陷型肾细胞癌患者血浆标志物的ROC诊断AUC筛选；其中
A、在发现组中，FH-野生型、FH缺陷型肾细胞癌患者的主成分分析图；
B、在发现组中，FH-野生型、FH缺陷型肾细胞癌患者和正常对照人群的ROC曲线的AUC值排序；
C、在发现组中，FH-野生型、FH-缺陷型肾细胞癌患者血浆代谢标志物的维恩图；
D、在发现组中，AUC排序图中代谢物的热图聚类分析；
E、在发现组中，FH-野生型、FH-缺陷型肾细胞癌患者血浆代谢标志物的相关性分析；
F、在发现组中，代谢物通路聚类分析。
附图2是SCID-NOD鼠肾脏原位分别注射Fh1fl/fl；HRASG12V和Fh1-/-；HRASG12V细胞建立CDX模型。
附图3是SCID-NOD鼠CDX模型中血浆代谢物浓度随着肿瘤生长的情况；其中
A、在SCID-NOD鼠CDX模型中，肿瘤的生长情况及尾静脉采血时间图(每周)；
B、在SCID-NOD鼠CDX模型中，代谢物suc-cys,sado,argsuc,saicar-riboside等代谢物随时间在血浆中的浓度变化；
C、在SCID-NOD鼠中，13C-化合物在体内的降解情况和稳定性。
附图4是肿瘤标志物suc-cys和sado的产生机制；其中
A、sGSH在体外通过GGT1酶促反应生成suc-cys-gly,suc-cys-gly在体外通过DPEP1生成suc-cys；
B、sGSH在SCID-NOD鼠体内生成生成suc-cys-gly和suc-cys；
C、sGSH在老鼠的肾脏组织lysate中生成suc-cys-gly和suc-cys；GGT1和DPEP1在肾脏组织中高表达；
D、sGSH生成suc-cys-gly和suc-cys的代谢机制图；
E、在老鼠MEF细胞中sg-ADSL敲除；
F、MEF细胞中，sado在sg-ADSL敲除中的代谢机制。
附图5是嘌呤合成途径ADSL酶反应反转机制和sado生成机制图。
附图6是血浆标志物suc-cys和sado在肾癌病人和正常人群中的AUC诊断曲线。
具体实施方式
下面结合具体实施方式，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1血浆标志物在FH-缺陷型肾癌患者(discovery cohort/发现队列)中的发现
1实验样本及分组
通过大样本群基因组测序和免疫组化筛查，收集和建立了2006年至今上海儿童医学中心和上海仁济医院FH-缺陷型肾细胞癌的患者队列，血浆标志物在FH-缺陷型肾癌患者(discovery cohort/发现队列)中的发现通过对35例FH-缺陷型肾细胞癌患者的血浆质量检控发现10例发生溶血，由此我们的FH-缺陷型队列(发现队列)组成如下：FH-缺陷型肾细胞癌患者(n＝25)；FH-野生型肾细胞癌患者(n＝55)；正常对照组(n＝65)。
2实验方法
2.1对145个血浆样本进行非靶向代谢组学分析，一共检测到1065个化合物。通过PCA分析，FH-野生型和FH-缺陷型肾细胞癌患者可以区分开来(附图1A)。通过对FH-缺陷型肾细胞癌ROC诊断曲线(FDR<0.01)进行分析，得到20个最佳诊断化合物(附图1A)。排名前五位的分别是：succinic-cys、sado、saicar-riboside、succinic-cys-gly和creatine-riboside(附图1B)，进一步对这20个化合物进行聚类分析，发现在FH-缺陷型患者中显著升高(附图1D)。
2.2为了确证标志物筛选的正确性与重复性，我们建立了第二种方法进行分析，即三重筛选分析：FH-缺陷型组vs正常对照组差异分析(q<0.01，2<FC<0.5，火山图)；FH-缺陷型组vs FH-野生型组差异分析(q<0.01，2<FC<0.5，火山图)；FH-缺陷型组肿瘤体积vs代谢物浓度关联度分析法(correlation coefficient)。将该三重筛选条件所得数据互相取交集，通过维恩图分析，我们发现succinic-cys、sado、creatine-riboside和succinic-cys-gly是唯一同时符合这三个条件的代谢物(附图1C)。
第二种方法的三个化合物与第一种方法的ROC诊断曲线筛选所得化合物一致，强烈预示了这三个代谢物是FH-缺陷型肾细胞癌诊断的血浆标志物。最后我们还对肿瘤组织进行代谢组学相关性分析和通路富集发现上述血浆代谢物是来源于嘌呤通路和谷胱甘肽通路(附图1E,附图1F)。
实施例2小鼠体内肾脏原位肿瘤模型的建立与标志物的发现
1实验方法
1.1小鼠体内肾脏原位肿瘤模型的建立
为了模拟肿瘤的生长和血液代谢物水平的关系，我们建立了肿瘤细胞来源的小鼠肾脏原位移植模型。通过分别过表达Fh1fl/fl；HRASG12V和Fh1-/-；HRASG12V，我们得到两株肿瘤细胞，并将其分别注射入小鼠肾脏(附图2)(Fh1fl/fl；HRASG12V，n＝5；Fh1-/-；HRASG12V，n＝5)。
1.2标志物的发现
每周通过超声影像对肿瘤生长进行监测，每周采集尾静脉血一次，直到小鼠由于肿瘤负荷过大而死亡(附图3A)，然后对小鼠血浆代谢物分析。
2实验结果
2.1对小鼠血浆代谢物分析结果如附图3B所示，succinic-cys、sado、succinic-cys-gly、mal和fum水平在Fh1-/-；HRASG12V小鼠中随时间显著升高，而在Fh1fl/fl；HRASG12V小鼠中浓度水平较低甚至无法检测。在5只Fh1-/-；HRASG12V不同小鼠中，同一时间采样点的血浆标志物水平取决于该时间点的肿瘤负荷的大小，即正比例于肿瘤尺寸，提示上述代谢物来源于肿瘤刺激，且浓度由肿瘤负荷决定。
2.2为了验证这些代谢物是否可以在小鼠血浆中较稳定的存在，我们使用了13C-15N尾静脉注射体内示踪联合液质分析法检测上述代谢物，结果表明13C2-fum在血液中大量转化成13C2-mal，进一步有一小部分转化成天冬氨酸(附图3C)，这解释了mal在FH-缺陷型肾癌患者体内异常升高的原因以及fum不稳定的现象。此外，15N1 13C3-succinic-cys和15N1 13C2-sado的示踪结果表明，它们可能会以尿液排泄，但其在血浆中无任何生物/化学转化，以原型状态存在，提示两者稳定性较好，适合作为血液标志物(附图3C)。
实施例3小分子化合物sado和succinic-cys的代谢机制研究
1实验方法
Suc-cys可能是由sGSH降解生成的。我们使用重组蛋白体外实验进行验证。Sado是DNA从头合成途径中ADSL的生成产物。我们使用CRISPR-Cas9技术，构建了稳定敲除ADSL的细胞株。
2实验结果
我们前期阐述了sGSH的合成是由谷胱甘肽与fum通过非酶催化反应生成的(L.zheng，Nat Commun，2015)。此次，我们发现sGSH可降解成suc-cys-gly，且该反应需要酶参与。通过γ-谷氨酰转肽酶体外酶活反应，发现suc-cys-gly是由sGSH经由GGT-1生成的。同时suc-cys-gly经由DPEP1生成suc-cys(附图4A)。我们将sGSH通过鼠尾静脉注射，发现sGSH在体内可降解生成suc-cys-gly和suc-cys。说明sGSH体外降解和体内降解都是酶参与的(附图4B)。在小鼠器官新鲜组织裂解液中，sGSH可快速降解生成suc-cys-gly和suc-cys。负责酶促生成反应的GGT1和DPEP1在小鼠的各器官组织中，肾脏最高，这个和肾脏肿瘤的发生密切相关(附图4C)。在ADSL敲除细胞实验中，我们发现sado、adenyl-succinate、SAICAribose、IMP、Inosine等显著上升(附图4E，F)。由于adenyl-succinate的磷酸酯键不稳定且极易水解，生成sado。因此sado的异常升高是由fum在FH-缺陷型细胞中的大量累积，造成ADSL的逆向反应，从而造成该通路的代谢紊乱(附图5)。我们将suc-cys和sado应用在临床诊断中，其AUC曲线的sado,suc-cys及其联合诊断均在0.88以上(附图6)。
综上所述，小分子化合物sado和succinic-cys在FH-缺陷型肾细胞癌中表达增高，且与患者生存预后相关，小分子化合物sado和succinic-cys可作为检测FH-缺陷型肾细胞癌的标志物和治疗的潜在靶点。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (5)
1.小分子化合物succinic-cys和sado中的任一种或其组合作为生物标志物在制备FH-缺陷型肾细胞癌的诊断试剂或试剂盒中的应用。
2.根据权利要求1所述的应用，其特征在于，所述诊断试剂或试剂盒用于判断受试样品属于或不属于FH-缺陷型肾细胞癌。
3.小分子化合物succinic-cys和sado中的任一种或其组合作为生物标志物在制备鉴别FH-缺陷型肾细胞癌与Xp11转位肾细胞癌或透明细胞乳头状肾癌的诊断试剂或试剂盒中的应用。
4.小分子化合物succinic-cys和sado中的任一种或其组合作为生物标志物在制备FH-缺陷型肾细胞癌的生存预后试剂或试剂盒中的应用。
5.小分子化合物succinic-cys和sado中的任一种或其组合作为生物标志物在制备FH-缺陷型肾细胞癌的治疗效果的试剂或试剂盒中的应用。
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