CN112626216A - 一种检测肿瘤微卫星不稳定性状态的组合物及其应用 - Google Patents
一种检测肿瘤微卫星不稳定性状态的组合物及其应用 Download PDFInfo
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Abstract
本发明涉及分子诊断技术领域，具体是一种检测肿瘤微卫星不稳定性状态的组合物及其应用，所述组合物由PTMAP4、H3F3AP6、RPL22L1、RP11‑267J23.4、MSH4、LYG1、RP11‑372B4.3、TLE6、RP11‑819M15.2、CIRBP‑AS1、FAM151B、EIF5A、SUMO2P17、FECH、MNX1‑AS1组成。本发明通过运用GSVA算法进行差异分析，获取得到各肿瘤相应的截断值，可用于多种肿瘤的微卫星不稳定性(MSI)状态的检测。通过实验验证，发现该方法具有准确性高、特异性好的优点，有很好的应用前景。
Description
技术领域
本发明涉及分子诊断技术领域，具体地说，是一种检测肿瘤微卫星不稳定性状态的组合物及其应用。
背景技术
微卫星不稳定(MSI)，是指由于在DNA复制时插入或缺失突变引起的MS序列长度改变的现象，常由错配修复(MMR)功能缺陷引起。MSI现象于1993年被Jacobs等人在结直肠癌中首次发现，与癌症发生有关，可用于癌症检测。据文献报道，大约15％的结直肠癌中存在MSI-H现象，与MSS特征的结直肠癌相比，其发病机制、预后和对药物的敏感性均不同。在非结直肠癌的实体瘤中，也存在着不同比例的MSI-H现象，MSI状态不同的实体瘤在对Keytruda响应率方面存在显著性差异。因此，准确的区分肿瘤患者的MSI状态，对于提高患者的治疗效果、减轻社会的负担有着极为重要的意义。
目前多重荧光PCR-毛细管电泳法检测MSI状态是国际公认的金标准，提取同一患者的正常组织和肿瘤组织样本DNA，采用多重荧光PCR的方法对检测位点进行扩增，再通过毛细管电泳对扩增产物进行检测，并利用专业软件对两种组织来源检测结果进行对比分析，能准确的给患者MSI状态进行分型。这种方法固然准确，但操作较为繁琐。此外，也有一些研究报道了利用基因数据来计算MSI状态，但都是基于DNA序列且较为复杂，目前仍缺乏在mRNA水平方面的MSI检测方法。因此，一个便于操作，准确性高的MSI检测方法对于肿瘤分型显得尤为重要。
目前的研究显示，错配修复基因在遗传性非息肉性大肠癌(HNPPC)中与MSI密切相关，已在结肠癌中广泛应用。但由于目前缺乏对多种癌症基因组全面系统的分析，仍可能有着具备更高敏感性、特异性和应用价值的基因可以应用于肿瘤微卫星状态的预测。同时，微卫星不稳定性是个连续动态的过程，或许存在一个可以量化的指标。本发明通过对TCGA数据库中1441例涵盖6种肿瘤具有特定MSI状态信息的样本进行基因表达数据分析，筛选出最具有差异的15个上调基因或lncRNA：PTMAP4,H3F3AP6,RPL22L1,RP11-267J23.4,MSH4,LYG1,RP11-372B4.3,TLE6,RP11-819M15.2,CIRBP-AS1,FAM151B,EIF5A,SUMO2P17,FECH,MNX1-AS1。随后，我们将这15个因子设定为一个因子集，运用GSVA算法计算出一个MSI分数，从而实现了MSI状态的量化及预测。该方法操作简便，可定量分析，在敏感性和特异性上具有一定优势。关于本发明一种检测肿瘤微卫星不稳定性状态的组合物及其应用目前还未见报道。
发明内容
本发明的目的是针对现有技术的不足，提供一种检测肿瘤微卫星不稳定性的组合物及其应用。
为实现上述目的，本发明采取的技术方案是：
首先，本发明提供了检测试剂在制备评估肿瘤微卫星不稳定性状态的试剂盒中的应用，所述检测试剂由检测以下基因/lncRNA表达量的试剂组成：PTMAP4、H3F3AP6、RPL22L1、RP11-267J23.4、MSH4、LYG1、RP11-372B4.3、TLE6、RP11-819M15.2、CIRBP-AS1、FAM151B、EIF5A、SUMO2P17、FECH、MNX1-AS1；所述检测试剂作为试剂盒实现评估肿瘤微卫星不稳定性状态的唯一关键成分。
优选地，所述试剂盒内还包括说明书，所述说明书记载如下公式：
GSVA评分＝-2.093101+(0.012068×PTMAP4)-(0.008544×H3F3AP6)+(0.062508×RPL22L1)+(0.073353×RP11-267J23.4)+(0.111126×MSH4)+(0.019458×LYG1)+(0.119968×RP11-372B4.3)+(0.099555×TLE6)+(0.218096×RP11-819M15.2)+(0.15873×FAM151B)+(0.142415×EIF5A)+(0.151092×SUMO2P17)+(0.081567×FECH)+(0.046266×MNX1-AS1)
优选地，所述肿瘤对应疾病包括乳腺癌、结直肠腺癌、食管癌、直肠腺癌、胃腺癌、子宫内膜癌、肝细胞肝癌、肺腺癌、肺鳞癌、肾上腺皮质癌、膀胱尿路上皮癌、宫颈鳞癌和腺癌、弥漫性大B细胞淋巴癌、多行成性胶质细胞瘤、头颈鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾乳头状细胞癌、急性髓细胞样白血病、脑低级别胶质瘤、卵巢浆液性囊腺癌、子宫肉瘤、葡萄膜黑色素瘤、胰腺癌、嗜铬细胞瘤和副神经节瘤、前列腺癌、皮肤黑色素瘤、肉瘤、胸腺癌。
进一步地，各疾病肿瘤的截断值分别为：乳腺癌：0.494、食管癌:0.359、肝细胞肝癌:0.283、结肠腺癌:0.364、肺腺癌:0.494、肺鳞癌:0.926、直肠腺癌:0.431、胃腺癌:0.297、子宫内膜瘤:0.053、肾上腺皮质癌:0.154、膀胱尿路上皮癌:0.157、宫颈鳞癌和腺癌:0.434、弥漫性大B细胞淋巴瘤:0.484、多形成性胶质细胞瘤:0.365、头颈鳞状细胞癌:0.424、肾嫌色细胞癌:0.424、肾透明细胞癌:0.318、肾乳头状细胞癌:0.333、急性髓细胞样白血病:0.401、脑低级别胶质瘤:0.544、卵巢浆液性囊腺癌:0.369、子宫肉瘤:0.243、葡萄膜黑色素瘤:0.151、胰腺癌:0.594、嗜铬细胞瘤和副神经节瘤:0.512、前列腺癌:0.522、皮肤黑色素瘤:0.392、肉瘤:0.243、胸腺癌:0.540。
进一步地，当检测样本GSVA评分高于该肿瘤的截断值时，代表检测样本为MSI-H状态；当检测样本GSVA评分低于该肿瘤的截断值时，代表检测样本为MSI-L状态。
优选地，所述检测样本为新鲜组织肿瘤样本。
其次，本发明提供了如上所述的试剂在制备评估疾病预后生存的试剂盒中的应用。
进一步地，所述疾病包括乳腺癌、结直肠腺癌、食管癌、直肠腺癌、胃腺癌、子宫内膜癌、肝细胞肝癌、肺腺癌、肺鳞癌、肾上腺皮质癌、膀胱尿路上皮癌、宫颈鳞癌和腺癌、弥漫性大B细胞淋巴癌、多行成性胶质细胞瘤、头颈鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾乳头状细胞癌、急性髓细胞样白血病、脑低级别胶质瘤、卵巢浆液性囊腺癌、子宫肉瘤、葡萄膜黑色素瘤、胰腺癌、嗜铬细胞瘤和副神经节瘤、前列腺癌、皮肤黑色素瘤、肉瘤、胸腺癌。
最后，本发明提供了一种评估肿瘤微卫星不稳定性状态的方法，包括微卫星检测位点选择和微卫星不稳定性检测两个过程，所述微卫星不稳定性检测是先检测样本基因/lncRNA表达量(所述基因/lncRNA见上)，再根据GSVA评分计算公式(见上)得到的数值和该肿瘤(见上)的截断值(见上)比较，当得到的数值高于该肿瘤的截断值时，代表检测样本为MSI-H状态；当检测样本GSVA评分低于该肿瘤的截断值时，代表检测样本为MSI-L状态。
本发明优点在于：
本发明首先全面系统性地分析了1400余例样本的全基因组表达数据，涵盖多种肿瘤，并从中发现PTMAP4,H3F3AP6,RPL22L1,RP11-267J23.4,MSH4,LYG1,RP11-372B4.3,TLE6,RP11-819M15.2,CIRBP-AS1,FAM151B,EIF5A,SUMO2P17,FECH,MNX1-AS1在多种肿瘤中具有显著性的差异，适合应用于MSI状态的判断。随后的研究进一步验证了该基因集在MSI状态计算及预测中的应用价值。
本发明首次提出了该基因组合物可用于评估肿瘤微卫星不稳定性状态，较目前常用的方法，具有操作便捷，可定量分析、准确性高、灵敏度和特异性好的优点。
附图说明
附图1是MSI基因集在6种具有明确MSI状态信息的肿瘤中的检验效能。
附图2是MSI基因集在其余23种肿瘤中的预测效能。
附图3是不同MSI预测方法的特异度和灵敏度比较。
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基因集的筛选及效果验证
(一)六种肿瘤的全基因组表达数据分析
1方法
我们首先从TCGA数据库中获得1441例样本的mRNA测序数据，共包含6种肿瘤样本的数据。我们计算每种基因的表达量，以RSEM值表示。随后我们基于Mean≥1000的标准初步筛选那些可能应用于MSI-H和MSI-L鉴别诊断的基因。此处，Mean表示某种基因在肿瘤组织中的平均表达量。之后我们依据调整后P value<0.05从小到大筛选出差异基因，一共筛选出基因673个，其中MSI-H中表达显著高于MSI-L的基因395个，MSI-L中表达远高于MSI-H的基因278个。随后我们选取差异基因构成基因集，分别计算GSVA评分，再进行ROC曲线的验证。
2结果
表1、在MSI-H中上调及下调的前25个差异基因
因为GSVA算法要求基因变化趋势的一致性，我们分别比较上调和下调的基因集。基于先前的一系列研究，我们选取了top10至top25的基因集分别验证每一种肿瘤。最终，比较下来，最佳的预测基因集是MSI-H种top15上调基因构成的因子集：PTMAP4,H3F3AP6,RPL22L1,RP11-267J23.4,MSH4,LYG1,RP11-372B4.3,TLE6,RP11-819M15.2,CIRBP-AS1,FAM151B,EIF5A,SUMO2P17,FECH,MNX1-AS1。其在六种具有明确MSI状态的肿瘤中具有较高的预测效能。除了直肠腺癌(READ)的准确性为88.5％，其余5种准确性均超过90％(见图1)。
(二)效果验证
之后我们利用机器学习随机森林算法，将6种明确MSI状态的肿瘤作为训练集，其余23种未有明确MSI状态信息的肿瘤作为验证集，从而分为MSI-H和MSI-L两组。接着运用MSI相关基因集进行外部验证，其ROC曲线见图2A-2B。由结果可知，除了膀胱尿路上皮癌(BLCA，71.3％)，宫颈鳞癌和腺癌(CESC，86.8％)和葡萄膜黑色素瘤(UVM，66.7％)三种肿瘤的准确性低于90％以为，其余肿瘤MSI预测准确性均高于90％，且普遍在95％以上。
(三)对比实验
综上我们比较了GSVA计算MSI基因集在不同肿瘤中的预测效能，发现在绝大多数肿瘤，该基因集都能较为准确的预测MSI状态。与此同时，我们比较了不同方法预测MSI状态。包括基于R语言的PreMSIm函数、预测MSI状态和Danaher等人提出的基于RNA-seq数据集在NanoString nCounter平台计算MSI评分方法。前者尽管其特异度与本发明的方法几乎相近，但其灵敏度普遍不高。且该方法是定性方法，不能很好的定量评估MSI状态。而后者的灵敏度和特异度普遍不高，且较为复杂。综上，我们的方法是一种较为简便的，可以定量计算的MSI预测方法，具有一定的优势。
以上实验中凡涉及到本发明所述方法其公式为：
GSVA评分＝-2.093101+(0.012068×PTMAP4)-(0.008544×H3F3AP6)+(0.062508×RPL22L1)+(0.073353×RP11-267J23.4)+(0.111126×MSH4)+(0.019458×LYG1)+(0.119968×RP11-372B4.3)+(0.099555×TLE6)+(0.218096×RP11-819M15.2)+(0.15873×FAM151B)+(0.142415×EIF5A)+(0.151092×SUMO2P17)+(0.081567×FECH)+(0.046266×MNX1-AS1)；
各疾病肿瘤的截断值分别为：乳腺癌：0.494、食管癌:0.359、肝细胞肝癌:0.283、结肠腺癌:0.364、肺腺癌:0.494、肺鳞癌:0.926、直肠腺癌:0.431、胃腺癌:0.297、子宫内膜瘤:0.053、肾上腺皮质癌:0.154、膀胱尿路上皮癌:0.157、宫颈鳞癌和腺癌:0.434、弥漫性大B细胞淋巴瘤:0.484、多形成性胶质细胞瘤:0.365、头颈鳞状细胞癌:0.424、肾嫌色细胞癌:0.424、肾透明细胞癌:0.318、肾乳头状细胞癌:0.333、急性髓细胞样白血病:0.401、脑低级别胶质瘤:0.544、卵巢浆液性囊腺癌:0.369、子宫肉瘤:0.243、葡萄膜黑色素瘤:0.151、胰腺癌:0.594、嗜铬细胞瘤和副神经节瘤:0.512、前列腺癌:0.522、皮肤黑色素瘤:0.392、肉瘤:0.243、胸腺癌:0.540；
当检测样本GSVA评分高于该肿瘤的截断值时，代表检测样本为MSI-H状态；当检测样本GSVA评分低于该肿瘤的截断值时，代表检测样本为MSI-L状态。
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 (9)
1.检测试剂在制备评估肿瘤微卫星不稳定性状态的试剂盒中的应用，其特征在于，所述检测试剂由检测以下基因/lncRNA表达量的试剂组成：PTMAP4、H3F3AP6、RPL22L1、RP11-267J23.4、MSH4、LYG1、RP11-372B4.3、TLE6、RP11-819M15.2、CIRBP-AS1、FAM151B、EIF5A、SUMO2P17、FECH、MNX1-AS1；所述检测试剂作为试剂盒实现评估肿瘤微卫星不稳定性状态的唯一关键成分。
2.根据权利要求1所述的应用，其特征在于，所述试剂盒内还包括说明书，所述说明书记载如下公式：
GSVA评分＝-2.093101+(0.012068×PTMAP4)-(0.008544×H3F3AP6)+(0.062508×RPL22L1)+(0.073353×RP11-267J23.4)+(0.111126×MSH4)+(0.019458×LYG1)+(0.119968×RP11-372B4.3)+(0.099555×TLE6)+(0.218096×RP11-819M15.2)+(0.15873×FAM151B)+(0.142415×EIF5A)+(0.151092×SUMO2P17)+(0.081567×FECH)+(0.046266×MNX1-AS1) 。
3.根据权利要求1所述的应用，其特征在于，所述肿瘤对应疾病包括乳腺癌、结直肠腺癌、食管癌、直肠腺癌、胃腺癌、子宫内膜癌、肝细胞肝癌、肺腺癌、肺鳞癌、肾上腺皮质癌、膀胱尿路上皮癌、宫颈鳞癌和腺癌、弥漫性大B细胞淋巴癌、多行成性胶质细胞瘤、头颈鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾乳头状细胞癌、急性髓细胞样白血病、脑低级别胶质瘤、卵巢浆液性囊腺癌、子宫肉瘤、葡萄膜黑色素瘤、胰腺癌、嗜铬细胞瘤和副神经节瘤、前列腺癌、皮肤黑色素瘤、肉瘤、胸腺癌。
4.根据权利要求3所述的应用，其特征在于，各疾病肿瘤的截断值分别为：乳腺癌：0.494、食管癌:0.359、肝细胞肝癌:0.283、结肠腺癌:0.364、肺腺癌:0.494、肺鳞癌:0.926、直肠腺癌:0.431、胃腺癌:0.297、子宫内膜瘤:0.053、肾上腺皮质癌:0.154、膀胱尿路上皮癌:0.157、宫颈鳞癌和腺癌:0.434、弥漫性大B细胞淋巴瘤:0.484、多形成性胶质细胞瘤:0.365、头颈鳞状细胞癌:0.424、肾嫌色细胞癌:0.424、肾透明细胞癌:0.318、肾乳头状细胞癌:0.333、急性髓细胞样白血病:0.401、脑低级别胶质瘤:0.544、卵巢浆液性囊腺癌:0.369、子宫肉瘤:0.243、葡萄膜黑色素瘤:0.151、胰腺癌:0.594、嗜铬细胞瘤和副神经节瘤:0.512、前列腺癌:0.522、皮肤黑色素瘤:0.392、肉瘤:0.243、胸腺癌:0.540。
5.根据权利要求4所述的应用，其特征在于，当检测样本GSVA评分高于该肿瘤的截断值时，代表检测样本为MSI-H状态；当检测样本GSVA评分低于该肿瘤的截断值时，代表检测样本为MSI-L状态。
6.根据权利要求5所述应用，其特征在于，所述检测样本为新鲜组织肿瘤样本。
7.权利要求1所述的试剂在制备评估疾病预后生存的试剂盒中的应用。
8.根据权利要求7所述的应用，其特征在于，所述疾病包括乳腺癌、结直肠腺癌、食管癌、直肠腺癌、胃腺癌、子宫内膜癌、肝细胞肝癌、肺腺癌、肺鳞癌、肾上腺皮质癌、膀胱尿路上皮癌、宫颈鳞癌和腺癌、弥漫性大B细胞淋巴癌、多行成性胶质细胞瘤、头颈鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾乳头状细胞癌、急性髓细胞样白血病、脑低级别胶质瘤、卵巢浆液性囊腺癌、子宫肉瘤、葡萄膜黑色素瘤、胰腺癌、嗜铬细胞瘤和副神经节瘤、前列腺癌、皮肤黑色素瘤、肉瘤、胸腺癌。
9.一种评估肿瘤微卫星不稳定性状态的方法，其特征在于，包括微卫星检测位点选择和微卫星不稳定性检测两个过程，所述微卫星不稳定性检测是先检测样本基因/lncRNA表达量，再根据GSVA评分计算公式得到的数值和该肿瘤的截断值比较，当得到的数值高于该肿瘤的截断值时，代表检测样本为MSI-H状态；当检测样本GSVA评分低于该肿瘤的截断值时，代表检测样本为MSI-L状态；
所述基因/lncRNA由：PTMAP4、H3F3AP6、RPL22L1、RP11-267J23.4、MSH4、LYG1、RP11-372B4.3、TLE6、RP11-819M15.2、CIRBP-AS1、FAM151B、EIF5A、SUMO2P17、FECH、MNX1-AS1组成；
所述GSVA评分＝-2.093101+(0.012068×PTMAP4)-(0.008544×H3F3AP6)+(0.062508×RPL22L1)+(0.073353×RP11-267J23.4)+(0.111126×MSH4)+(0.019458×LYG1)+(0.119968×RP11-372B4.3)+(0.099555×TLE6)+(0.218096×RP11-819M15.2)+(0.15873×FAM151B)+(0.142415×EIF5A)+(0.151092×SUMO2P17)+(0.081567×FECH)+(0.046266×MNX1-AS1)
所述肿瘤对应疾病包括乳腺癌、结直肠腺癌、食管癌、直肠腺癌、胃腺癌、子宫内膜癌、肝细胞肝癌、肺腺癌、肺鳞癌、肾上腺皮质癌、膀胱尿路上皮癌、宫颈鳞癌和腺癌、弥漫性大B细胞淋巴癌、多行成性胶质细胞瘤、头颈鳞状细胞癌、肾嫌色细胞癌、肾透明细胞癌、肾乳头状细胞癌、急性髓细胞样白血病、脑低级别胶质瘤、卵巢浆液性囊腺癌、子宫肉瘤、葡萄膜黑色素瘤、胰腺癌、嗜铬细胞瘤和副神经节瘤、前列腺癌、皮肤黑色素瘤、肉瘤、胸腺癌；
各疾病肿瘤的截断值分别为：乳腺癌：0.494、食管癌:0.359、肝细胞肝癌:0.283、结肠腺癌:0.364、肺腺癌:0.494、肺鳞癌:0.926、直肠腺癌:0.431、胃腺癌:0.297、子宫内膜瘤:0.053、肾上腺皮质癌:0.154、膀胱尿路上皮癌:0.157、宫颈鳞癌和腺癌:0.434、弥漫性大B细胞淋巴瘤:0.484、多形成性胶质细胞瘤:0.365、头颈鳞状细胞癌:0.424、肾嫌色细胞癌:0.424、肾透明细胞癌:0.318、肾乳头状细胞癌:0.333、急性髓细胞样白血病:0.401、脑低级别胶质瘤:0.544、卵巢浆液性囊腺癌:0.369、子宫肉瘤:0.243、葡萄膜黑色素瘤:0.151、胰腺癌:0.594、嗜铬细胞瘤和副神经节瘤:0.512、前列腺癌:0.522、皮肤黑色素瘤:0.392、肉瘤:0.243、胸腺癌:0.540。
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