CN113151479A - 一种检测肺腺癌细胞周期进展通路相关基因突变的试剂盒 - Google Patents

一种检测肺腺癌细胞周期进展通路相关基因突变的试剂盒 Download PDF

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CN113151479A
CN113151479A CN202110511263.9A CN202110511263A CN113151479A CN 113151479 A CN113151479 A CN 113151479A CN 202110511263 A CN202110511263 A CN 202110511263A CN 113151479 A CN113151479 A CN 113151479A
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单光耀
毕国澍
卞赟艺
姚光宇
张毅
薛亮
詹成
范虹
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Abstract

本发明涉及分子诊断技术领域，具体是一种检测肺腺癌细胞周期进展通路相关基因(CDKN2A、CCND1、CCNE1、CDK4、RB1)突变的组合物及其应用，所述组合物由检测相关基因表达量的试剂组成。本发明通过运用RNA测序、LASSO和二分类Logistic回归筛选与细胞周期进展通路相关的基因，并构建评分Score，通过ROC分析得到肺腺癌中Score相应的截断值，可用于肺腺癌细胞周期进展通路相关基因突变的预测。该发明通过利用基因组合物表达量来预测肺腺癌特定基因突变，经验证发现该预测方法具有准确性高，特异性好的优点，有很好的应用前景。

Description

一种检测肺腺癌细胞周期进展通路相关基因突变的试剂盒
技术领域
本发明涉及分子诊断技术领域，具体地说，是一种检测肺腺癌细胞周期进展通路相关基因突变的试剂盒及其应用。
背景技术
肺癌作为最常见的恶性肿瘤，其死亡率在世界范围内高居所有肿瘤榜首。肺腺癌是肺癌中最常见的亚型，占据肺癌病例的40％以上。针对肺腺癌的各种靶向药物的出现明显提高了肺腺癌患者的预后，使肺腺癌的治疗已经进入分子时代。如何能够准确评估肺腺癌的分子特征，对于患者的精准治疗和减轻社会负担意义重大。
维持持续性增殖信号是肿瘤标志之一，对于促进肿瘤生长及播散有重要意义。在肺腺癌中，CDKN2A、CCND1、CCNE1、CDK4和RB1基因具有较高的突变率且与细胞周期进展高度相关。上述基因的突变可使肿瘤细胞摆脱正常细胞***信号的调控进入持续增殖模式，进而促进肿瘤生长和转移，影响患者预后。第三代CDK(cyclin-dependent kinase)抑制剂已被美国FDA(Food and DrugAdministration)批准用于特定类型乳腺癌的治疗，可使患者从中获益，展现出细胞周期疗法在肿瘤治疗中的巨大前景。目前肺腺癌细胞周期进展通路相关基因突变的检测并非临床常规项目，单独检测价格昂贵，仍可能有兼具准确性和应用价值的检测手段可以应用于肺腺癌患者个体化治疗方案的制定。
关于本发明一种检测肺腺癌细胞周期进展通路相关基因突变的试剂盒目前还未见报道。
发明内容
本发明的目的是针对现有技术的空白，提供一种检测肺腺癌细胞周期进展相关基因的组合物及其应用。
第一方面，本发明提供了检测试剂在制备评估肺腺癌细胞周期进展通路相关基因CDKN2A、CCND1、CCNE1、CDK4、RB1中任一基因突变的试剂盒中的应用，所述检测试剂由检测以下基因表达量的试剂组成：GFRA3、IGF2、CST2、PADI2、PLA2G12B、LRP4、DLX3、TRIM17、MYOZ1、FFAR2、CNIH2、ZNF713、GBP6、FGF9、CAMK2N2、EYA1、CRISP3、FBXO43、LIPK、PAGE5、TAF7L、SV2B、FER1L5、KIR2DL1、ASB11、C10orf82、ASTN1、NXPH1、GCG、PLA2G2F、MAGEB16、TMEM151B、CHRM2、CALML5、ZACN；所述检测试剂作为试剂盒实现评估肺腺癌细胞周期进展通路相关基因突变的唯一关键成分。
在某些实施例中，所述试剂盒内还包括说明书，所述说明书记载如下公式：Score＝(-0.3591×GFRA3)+(-0.2567×IGF2)+(-0.4489×CST2)+(0.3886×PADI2)+(-0.6545×PLA2G12B)+(0.5672×LRP4)+(0.6257×DLX3)+(-0.8741×TRIM17)+(-0.7005×MYOZ1)+(0.9237×FFAR2)+(1.5149×CNIH2)+(2.6282×ZNF713)+(1.2654×GBP6)+(-2.6386×FGF9)+(-1.4028×CAMK2N2)+(-1.3872×EYA1)+(0.6539×CRISP3)+(-1.8037×FBXO43)+(-3.4313×LIPK)+(0.9605×PAGE5)+(1.0385×TAF7L)+(1.6826×SV2B)+(-3.6871×FER1L5)+(0.6506×KIR2DL1)+(1.3617×ASB11)+(-2.9494×C10orf82)+(-5.6024×ASTN1)+(2.2708×NXPH1)+(1.6415×GCG)+(-5.7800×PLA2G2F)+(1.1841×MAGEB16)+(1.7702×TMEM151B)+(3.0550×CHRM2)+(-0.1535×CALML5)+(0.4328×ZACN)。
在某些实施例中，所述肺腺癌的Score截断值为1.946。
在某些实施例中，所述检测样本为新鲜组织肿瘤样本。
在某些实施例中，所述评估为，当检测样本评分Score≥1.946时，代表该样本存在细胞周期进展通路相关基因突变的可能性较大；当检测样本评分Score<1.946时，代表该样本存在细胞周期进展通路相关基因突变的可能性较小。
第二方面，本发明提供了一种评估肺腺癌细胞周期进展通路相关基因CDKN2A、CCND1、CCNE1、CDK4、RB1中任一基因突变的试剂盒，至少包括特异性检测以下基因表达量的试剂：GFRA3、IGF2、CST2、PADI2、PLA2G12B、LRP4、DLX3、TRIM17、MYOZ1、FFAR2、CNIH2、ZNF713、GBP6、FGF9、CAMK2N2、EYA1、CRISP3、FBXO43、LIPK、PAGE5、TAF7L、SV2B、FER1L5、KIR2DL1、ASB11、C10orf82、ASTN1、NXPH1、GCG、PLA2G2F、MAGEB16、TMEM151B、CHRM2、CALML5、ZACN。
第三方面，本发明提供了一种评估肺腺癌细胞周期进展通路相关基因CDKN2A、CCND1、CCNE1、CDK4、RB1中任一突变的方法，该方法用于非疾病诊断和治疗目的，包括如下步骤：
(1)检测样本以下基因表达量：GFRA3、IGF2、CST2、PADI2、PLA2G12B、LRP4、DLX3、TRIM17、MYOZ1、FFAR2、CNIH2、ZNF713、GBP6、FGF9、CAMK2N2、EYA1、CRISP3、FBXO43、LIPK、PAGE5、TAF7L、SV2B、FER1L5、KIR2DL1、ASB11、C10orf82、ASTN1、NXPH1、GCG、PLA2G2F、MAGEB16、TMEM151B、CHRM2、CALML5、ZACN；
(2)计算：Score＝(-0.3591×GFRA3)+(-0.2567×IGF2)+(-0.4489×CST2)+(0.3886×PADI2)+(-0.6545×PLA2G12B)+(0.5672×LRP4)+(0.6257×DLX3)+(-0.8741×TRIM17)+(-0.7005×MYOZ1)+(0.9237×FFAR2)+(1.5149×CNIH2)+(2.6282×ZNF713)+(1.2654×GBP6)+(-2.6386×FGF9)+(-1.4028×CAMK2N2)+(-1.3872×EYA1)+(0.6539×CRISP3)+(-1.8037×FBXO43)+(-3.4313×LIPK)+(0.9605×PAGE5)+(1.0385×TAF7L)+(1.6826×SV2B)+(-3.6871×FER1L5)+(0.6506×KIR2DL1)+(1.3617×ASB11)+(-2.9494×C10orf82)+(-5.6024×ASTN1)+(2.2708×NXPH1)+(1.6415×GCG)+(-5.7800×PLA2G2F)+(1.1841×MAGEB16)+(1.7702×TMEM151B)+(3.0550×CHRM2)+(-0.1535×CALML5)+(0.4328×ZACN)；
(3)判断：当Score评分≥1.946时，代表该样本存在细胞周期进展通路相关基因突变的可能性较大；当Score评分<1.946时，代表该样本存在细胞周期进展通路相关基因突变的可能性较小。
与现有技术相比，本发明具有以下技术效果：
1.本发明首次提出了该基因组合物可用于评估肺腺癌是否存在细胞周期进展通路相关基因(CDKN2A、CCND1、CCNE1、CDK4、RB1)的突变，可以有效指导肺腺癌患者的个体化治疗，提高临床获益，同时避免不必要的医疗资源浪费。
2.肺腺癌细胞周期进展通路相关基因突变目前在临床上属于非常规检测项目，单独检测上述基因突变费用较高。该发明可通过临床上已广泛应用的RNA测序技术判定肺腺癌患者是否存在上述基因突变，具有经济实惠，准确性高、灵敏度和特异性好的优点。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式描述中所需要使用的附图作简单地介绍。
附图1是该预测模型的受试者工作特性曲线(ROC)
附图2是TCGA数据库分析得到的656个差异基因
具体实施方式
下面结合附图和实施方式对本发明进行详细说明。
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式作简单地介绍，显而易见地，下面描述中的实施例是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些实施例获得其他的实施方式。
实施例1肺腺癌细胞周期进展通路相关基因突变的评分模型构建
1.方法
我们首先从TCGA数据库中获得510个肺腺癌样本数据，根据是否存在细胞周期进展相关基因(CDKN2A、CCND1、CCNE1、CDK4、RB1)突变将样本分为突变组(n＝57)和野生组(n＝453)，进而计算并筛选出656个差异基因。通过对510个样本的上述656个差异基因的表达量数据[log2(FPKM+1)形式]进行LASSO回归分析，我们得到了与细胞周期进展通路相关基因突变状态显著相关的基因，最终挑选出35个基因为：GFRA3、IGF2、CST2、PADI2、PLA2G12B、LRP4、DLX3、TRIM17、MYOZ1、FFAR2、CNIH2、ZNF713、GBP6、FGF9、CAMK2N2、EYA1、CRISP3、FBXO43、LIPK、PAGE5、TAF7L、SV2B、FER1L5、KIR2DL1、ASB11、C10orf82、ASTN1、NXPH1、GCG、PLA2G2F、MAGEB16、TMEM151B、CHRM2、CALML5、ZACN。
通过二分类Logistic回归构建评分模型Score＝(-0.3591×GFRA3)+(-0.2567×IGF2)+(-0.4489×CST2)+(0.3886×PADI2)+(-0.6545×PLA2G12B)+(0.5672×LRP4)+(0.6257×DLX3)+(-0.8741×TRIM17)+(-0.7005×MYOZ1)+(0.9237×FFAR2)+(1.5149×CNIH2)+(2.6282×ZNF713)+(1.2654×GBP6)+(-2.6386×FGF9)+(-1.4028×CAMK2N2)+(-1.3872×EYA1)+(0.6539×CRISP3)+(-1.8037×FBXO43)+(-3.4313×LIPK)+(0.9605×PAGE5)+(1.0385×TAF7L)+(1.6826×SV2B)+(-3.6871×FER1L5)+(0.6506×KIR2DL1)+(1.3617×ASB11)+(-2.9494×C10orf82)+(-5.6024×ASTN1)+(2.2708×NXPH1)+(1.6415×GCG)+(-5.7800×PLA2G2F)+(1.1841×MAGEB16)+(1.7702×TMEM151B)+(3.0550×CHRM2)+(-0.1535×CALML5)+(0.4328×ZACN)。
最后，我们基于该评分模型计算每个样本的得分，通过ROC曲线获取最佳阈值点，即截断值，截断值为1.946。根据截断值将TCGA样本分为两组，评分高于该截断值归于突变组，评分低于该截断值归于野生组。
实验结果表明：该预测模型灵敏度为0.877，特异度为0.918。
实施例2效果验证
将复旦大学附属中山医院胸外科收集的34例肺腺癌样本进行RNA测序并检测了细胞周期进展通路相关基因(CDKN2A、CCND1、CCNE1、CDK4、RB1)突变，将RNA表达数据带入该预测模型得到每个样本的Score评分，评分高于截断值1.946的为突变组，低于截断值1.946的为野生组。
实验结果表明：该预测模型灵敏度为0.833，特异度为0.893。
以上所述仅为本发明的实施方式，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (7)

1.检测试剂在制备评估肺腺癌细胞周期进展通路相关基因CDKN2A、CCND1、CCNE1、CDK4、RB1中任一基因突变的试剂盒中的应用，其特征在于，所述检测试剂由检测以下基因表达量的试剂组成：GFRA3、IGF2、CST2、PADI2、PLA2G12B、LRP4、DLX3、TRIM17、MYOZ1、FFAR2、CNIH2、ZNF713、GBP6、FGF9、CAMK2N2、EYA1、CRISP3、FBXO43、LIPK、PAGE5、TAF7L、SV2B、FER1L5、KIR2DL1、ASB11、C10orf82、ASTN1、NXPH1、GCG、PLA2G2F、MAGEB16、TMEM151B、CHRM2、CALML5、ZACN；所述检测试剂作为试剂盒实现评估肺腺癌细胞周期进展通路相关基因突变的唯一关键成分。
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：Score＝(-0.3591×GFRA3)+(-0.2567×IGF2)+(-0.4489×CST2)+(0.3886×PADI2)+(-0.6545×PLA2G12B)+(0.5672×LRP4)+(0.6257×DLX3)+(-0.8741×TRIM17)+(-0.7005×MYOZ1)+(0.9237×FFAR2)+(1.5149×CNIH2)+(2.6282×ZNF713)+(1.2654×GBP6)+(-2.6386×FGF9)+(-1.4028×CAMK2N2)+(-1.3872×EYA1)+(0.6539×CRISP3)+(-1.8037×FBXO43)+(-3.4313×LIPK)+(0.9605×PAGE5)+(1.0385×TAF7L)+(1.6826×SV2B)+(-3.6871×FER1L5)+(0.6506×KIR2DL1)+(1.3617×ASB11)+(-2.9494×C10orf82)+(-5.6024×ASTN1)+(2.2708×NXPH1)+(1.6415×GCG)+(-5.7800×PLA2G2F)+(1.1841×MAGEB16)+(1.7702×TMEM151B)+(3.0550×CHRM2)+(-0.1535×CALML5)+(0.4328×ZACN)。
3.根据权利要求1所述的应用，其特征在于，所述肺腺癌的Score截断值为1.946。
4.根据权利要求1所述应用，其特征在于，所述检测样本为新鲜组织肿瘤样本。
5.根据权利要求1所述应用，其特征在于，所述评估为，当检测样本评分Score≥1.946时，代表该样本存在细胞周期进展通路相关基因突变的可能性较大；当检测样本评分Score<1.946时，代表该样本存在细胞周期进展通路相关基因突变的可能性较小。
6.一种评估肺腺癌细胞周期进展通路相关基因CDKN2A、CCND1、CCNE1、CDK4、RB1中任一基因突变的试剂盒，其特征在于，至少包括特异性检测以下基因表达量的试剂：GFRA3、IGF2、CST2、PADI2、PLA2G12B、LRP4、DLX3、TRIM17、MYOZ1、FFAR2、CNIH2、ZNF713、GBP6、FGF9、CAMK2N2、EYA1、CRISP3、FBXO43、LIPK、PAGE5、TAF7L、SV2B、FER1L5、KIR2DL1、ASB11、C10orf82、ASTN1、NXPH1、GCG、PLA2G2F、MAGEB16、TMEM151B、CHRM2、CALML5、ZACN。
7.一种评估肺腺癌细胞周期进展通路相关基因CDKN2A、CCND1、CCNE1、CDK4、RB1中任一突变的方法，其特征在于，该方法用于非疾病诊断和治疗目的，包括如下步骤：
(1)检测样本以下基因表达量：GFRA3、IGF2、CST2、PADI2、PLA2G12B、LRP4、DLX3、TRIM17、MYOZ1、FFAR2、CNIH2、ZNF713、GBP6、FGF9、CAMK2N2、EYA1、CRISP3、FBXO43、LIPK、PAGE5、TAF7L、SV2B、FER1L5、KIR2DL1、ASB11、C10orf82、ASTN1、NXPH1、GCG、PLA2G2F、MAGEB16、TMEM151B、CHRM2、CALML5、ZACN；
(2)计算：Score＝(-0.3591×GFRA3)+(-0.2567×IGF2)+(-0.4489×CST2)+(0.3886×PADI2)+(-0.6545×PLA2G12B)+(0.5672×LRP4)+(0.6257×DLX3)+(-0.8741×TRIM17)+(-0.7005×MYOZ1)+(0.9237×FFAR2)+(1.5149×CNIH2)+(2.6282×ZNF713)+(1.2654×GBP6)+(-2.6386×FGF9)+(-1.4028×CAMK2N2)+(-1.3872×EYA1)+(0.6539×CRISP3)+(-1.8037×FBXO43)+(-3.4313×LIPK)+(0.9605×PAGE5)+(1.0385×TAF7L)+(1.6826×SV2B)+(-3.6871×FER1L5)+(0.6506×KIR2DL1)+(1.3617×ASB11)+(-2.9494×C10orf82)+(-5.6024×ASTN1)+(2.2708×NXPH1)+(1.6415×GCG)+(-5.7800×PLA2G2F)+(1.1841×MAGEB16)+(1.7702×TMEM151B)+(3.0550×CHRM2)+(-0.1535×CALML5)+(0.4328×ZACN)；
(3)判断：当Score评分≥1.946时，代表该样本存在细胞周期进展通路相关基因突变的可能性较大；当Score评分<1.946时，代表该样本存在细胞周期进展通路相关基因突变的可能性较小。
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