CN110564851B - 一组用于非超突变型直肠癌分子分型的基因及其应用 - Google Patents
一组用于非超突变型直肠癌分子分型的基因及其应用 Download PDFInfo
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Abstract
本发明公开了一组用于非超突变型直肠癌分子分型的基因,包含SMAD4基因、MUC16基因、FAT4基因、FLG基因、ROBO2基因、NEB基因;根据该基因突变特征组合可对直肠癌进行分子分型,从而筛选在术后复发和死亡风险较低的低危组患者,可按照目前的术后辅助治疗方案进行治疗,避免过度治疗;而另一组术后复发和死亡风险较高的高危组患者,可在目前的术后辅助治疗方案基础上为患者寻找更佳的其他方案。本发明还公开了用于捕获上述基因的试剂盒。
Description
技术领域
本发明属于生物技术领域,涉及一组用于非超突变型直肠癌分子分型的基因,及该组基因在预判直肠癌术后复发转移上的应用。
背景技术
手术、化疗和放疗是传统的癌症治疗手段,目前多数早期患者通过综合治疗后可以获得较好的预后,但这些治疗无法降低所有肿瘤患者的死亡率。主要原因是手术治疗后发生的肿瘤复发或转移,最终导致患者死亡。当前的临床实践中所有的III期肠癌都推荐规范的辅助化疗,一般为含奥沙利铂的联合方案,临床医生根据患者具体情况再做调整。但同一个分期的患者预后可以存在较大差别,不是所有的直肠癌患者都能从术后辅助化疗中受益。因此建立一个区分患者复发和死亡风险度的方法,为个体化辅助治疗提供依据,迫在眉睫。目前,CN108342483A提供了一组五基因组合的用于非超突变型结直肠癌分子分型,但该基因组在非超突变型直肠癌上应用效果不佳,无法区分预后较好和预后较差的患者,也无法区分非超突变型直肠癌患者复发和死亡率风险度。因此,针对非超突变型直肠癌建立一个区分患者复发和死亡风险度的方法,为个体化辅助治疗提供依据,也是迫切需要的。
发明内容
本发明针对直肠癌患者在术后可能发生肿瘤复发或转移,最终导致患者死亡的问题,提供了一组用于预判非超突变型直肠癌术后复发转移的基因及其检测试剂盒。
本发明提供一组用于非超突变型直肠癌分子分型的基因,包括如下基因:SMAD4基因、MUC16基因、FAT4基因、FLG基因、ROBO2基因、NEB基因。
本发明提供一种用于非超突变型直肠癌分子分型的检测试剂盒,所述检测试剂盒包括捕获本发明所述用于直肠癌分子分型的基因的探针。
其中,所述的检测试剂盒较佳地还包括:基因组DNA提取试剂、文库构建试剂、二代测序试剂,选自末端修复酶、末端修复反应缓冲液、DNA连接酶、连接反应缓冲液、含分子标签的接头、文库扩增引物、PCR预混液、接头封闭剂、DNA封闭剂、杂交缓冲液、杂交增强剂、磁珠洗液、杂交洗液、捕获文库PCR引物、质控品、核酸纯化磁珠和链霉亲合素磁珠的一种或多种试剂。
其中,所述基因组DNA提取试剂为本领域常规的基因组DNA提取试剂。
其中,所述文库构建试剂和二代测序试剂为本领域常规使用的试剂,只要能满足对所得序列构建文库并进行二代测序的要求即可。所述的二代测序为本领域常规的二代测序。
本发明所述的检测试剂盒较佳地还包括从检测对象提取检测样本的器械;更佳地,还包括从检测对象或肿瘤患者体内提取组织或血液的器械,所述器械较佳地为任何能用于取血的采血针、注射器等。
本发明所述的检测样本较佳地为来自检测对象的组织,只要能从检测样本中抽提检测对象的基因组DNA即可。所述检测样本较佳地为组织样本、血液、血浆和体液中的一种或几种,更佳地为组织样本,更佳地为石蜡组织样本,优选地为肿瘤细胞含量高的组织。
本发明所述检测试剂盒适用于对确定为非超突变型直肠癌进行进一步检测,使用方法包括以下步骤:
(1)提取血液和组织样本中的基因组DNA双链核酸;
(2)将步骤(1)的所述DNA双链核酸变性处理得到DNA单链,使用捕获探针捕获SMAD4基因、MUC16基因、FAT4基因、FLG基因、ROBO2基因、NEB基因;捕获区域分别如下(参考基因组版本为GRCh37/hg19):
针对捕获对象设计探针为本领域的常用技术手段,探针的序列包括但不限于如上表所示区域。
(3)将步骤(2)所捕获的DNA单链进行测序,得到血液和组织样本中的核酸序列;
(4)将步骤(3)所得到的核酸序列进行自动化处理,计算组织样本中的6个基因的变异位点数量。若合计变异位点数量为0,则为6基因野生型;若合计变异位点数量大于0,则为6基因突变型。
其中步骤(1-4)所述的提取方法、文库构建、测序方法和基因变异位点计算方法均为本领域常规方法。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明所取得的有益效果是:本发明首次提出了非超突变型直肠癌的复发和死亡风险标志物,该组标志物可用于区分预后较差和预后较好的患者,提示患者在术后发生复发转移的可能性,筛选在术后复发和死亡风险较低的低危组患者,可按照目前的术后辅助治疗方案进行治疗,避免过度治疗;而另一组术后复发和死亡风险较高的高危组患者,可在目前的术后辅助治疗方案基础上为患者寻找更佳的其他方案,有较好的临床指导意义。
附图说明
图1显示了6基因模型与非超突变型直肠癌患者(ZJU数据集)术后生存时间的分析结果。与6基因野生型组的患者相比,6基因突变型组的患者预后更差,死亡风险更高。
图2显示了6基因模型与非超突变型直肠癌患者(TCGA数据集)术后生存时间的分析结果。与6基因野生型组的患者相比,6基因突变型组的患者预后更差,死亡风险更高。
图3显示了对非超突变型直肠癌患者(TCGA数据集),随机选取6个基因,进行了10000次多重置换检验的结果,并与本发明的6基因模型的Log10(P值)比较。结果说明本发明的6基因预后预测模型显著优于随机选择的同样数量基因的预后预测模型。
图4显示了6基因突变型的非超突变型III期直肠癌(混合数据集)术后生存时间的分析结果。与6基因野生型组的患者相比,6基因突变型组的患者预后更差,死亡风险更高。
图5显示了5基因模型与非超突变型直肠癌患者(ZJU数据集)术后生存时间的分析结果。
图6显示了5基因模型与非超突变型直肠癌患者(TCGA数据集)术后生存时间的分析结果。
具体实施方式
本发明对直肠癌的基因组突变谱进行了深入分析,筛选得到了一组直肠癌基因突变特征和组合,并在独立的直肠癌队列上做了验证,根据该基因突变特征组合可对直肠癌进行分子分型,从而筛选在术后复发和死亡风险较低的低危组患者,可按照目前的术后辅助治疗方案进行治疗,避免过度治疗;而另一组术后复发和死亡风险较高的高危组患者,可在目前的术后辅助治疗方案基础上为患者寻找更佳的其他方案。
下面结合具体实施例,进一步阐述本发明,但并不因此将本发明限制在所述的实施例范围之中。本领域的普通技术人员可以理解:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书。
实施例1 :基因组DNA样品的制备和肿瘤体细胞突变位点的测定
为检测直肠癌的体细胞突变,本发明设计了一个包括524个基因的基因群(表1),定制了针对这个基因群的捕获探针。使用该捕获探针完成了157例直肠癌组织标本的靶向测序工作。所有标本来自患者手术切除的组织标本,经病理诊断后多余的部分用于测序研究。此工作经浙江大学医学院附属第二医院人体研究伦理委员会批准进行。此部分157例肿瘤患者为ZJU数据集。
表1:524基因列表
ABCA13 | ASPM | C20orf54 | COL6A3 | EIF3E | FAT4 | HEMGN | LCORL |
ABCA6 | ATAD5 | C2orf44 | COL6A6 | EIF4A2 | FBN2 | HIST1H2BC | LEKR1 |
ABCA8 | ATM | C9 | CRB1 | ELF3 | FBXW7 | HIST1H2BM | LGR5 |
ABCA9 | ATP10A | CA4 | CRTC1 | ELMO1 | FCRL1 | HIVEP2 | LIFR |
ABCB1 | ATP1A2 | CACNA1B | CSF2RA | ELMOD2 | FETUB | HLA-B | LIG1 |
ACOX2 | ATP1B2 | CASP8 | CSMD1 | EP300 | FLG | HMCN1 | LIG4 |
ACSL6 | ATP8A2 | CBL | CSMD3 | EPCAM | FLG2 | HNRNPL | LMNA |
ACTC1 | ATR | CCDC141 | CTNNB1 | ERBB2 | FLI1 | HRNR | LPHN3 |
ACVR1B | ATXN1 | CCDC168 | CTNND2 | ERBB4 | FOXA1 | HSPA8 | LRIF1 |
ACVR2A | AUTS2 | CCDC62 | CTSA | ERCC1 | FOXE1 | HYDIN | LRP1B |
ADAM21 | AXIN1 | CCDC66 | CUX1 | ERCC5 | FREM2 | IDO2 | LRP2 |
ADAM29 | AXIN2 | CCDC73 | DCAF6 | ERCC6 | FRG1 | IGSF3 | LRRC4C |
ADAM7 | AZGP1 | CCDC88A | DCHS2 | ERICH3 | FRY | IL15 | LRRD1 |
ADAMTS12 | B2M | CCNE1 | DDX6 | EXO1 | FSHR | IRF5 | LRRIQ1 |
ADAMTS20 | B3GNT6 | CD40LG | DEFB108B | EYS | FSIP2 | ITLN2 | LRRK2 |
ADCY2 | B3GNT9 | CD44 | DNAH11 | F10 | FSTL5 | ITPR1 | LRRN3 |
ADM2 | BAGE3 | CD58 | DNAH14 | F13B | GABRG3 | IVL | LRRTM3 |
AGBL2 | BAI3 | CD7 | DNAH3 | F5 | GALNT12 | JAK2 | MAGEB3 |
AKT1 | BAIAP2L2 | CDC42EP1 | DNAH5 | FAM123B | GGT1 | KCNJ5 | MAGEL2 |
ALPK2 | BAP1 | CDH1 | DNAH6 | FAM135B | GLI3 | KCNN3 | MAL2 |
ALS2CR11 | BAX | CDKN2A | DNAH7 | FAM153B | GLT8D2 | KCNS2 | MANSC4 |
ALS2CR12 | BCL9 | CFAP58 | DNAH8 | FAM184A | GLTPD2 | KCNT2 | MAP1B |
ALX4 | BCL9L | CHEK2 | DNAJC24 | FAM194B | GNAT2 | KDM6A | MAP2K1 |
AMPD1 | BIVM-ERCC5 | CLEC18B | DOCK10 | FAM71E2 | GNG12 | KIAA0408 | MAP2K4 |
ANKRD12 | BLM | CLUL1 | DOCK2 | FAN1 | GOLGA4 | KIF4B | MAP3K19 |
ANKRD30A | BMPR1A | CMYA5 | DPP10 | FANCA | GOLGB1 | KIRREL | MAPK1 |
APC | BMPR2 | CNBD1 | DPY19L2 | FANCD2 | GPN1 | KMT2B | MAPK8IP1 |
APOBEC3B | BRAF | CNTN1 | DSEL | FANCE | GPR112 | KRAS | MDM2 |
APOBEC3F | BRCA1 | CNTN6 | DUX4 | FANCI | GPR149 | KRTAP10-6 | MFRP |
ARHGAP15 | BRCA2 | CNTNAP5 | DYNC2H1 | FANCM | GREM1 | KRTAP4-5 | MKI67 |
ARHGAP32 | BRIP1 | COL11A1 | EBLN1 | FAT1 | GRIA2 | KRTAP5-5 | MLH1 |
ARHGEF12 | C17orf70 | COL12A1 | EDNRB | FAT2 | GRIN2A | KTN1 | MLH3 |
ARID1A | C1ORF74 | COL18A1 | EFHB | FAT3 | HDGFRP2 | LCA5 | MNX1 |
MSH2 | NOLC1 | PLD5 | QSER1 | SCN9A | ST8SIA6 | TRAK2 | ZFP36L2 |
MSH3 | NOTCH1 | PLXNC1 | RABGGTB | SEC63 | STAG1 | TRIM49B | ZFPM1 |
MSH6 | NOTCH2 | PMS1 | RAD21 | SEMA3D | STK11 | TRNT1 | ZFPM2 |
MTOR | NPIPB5 | PMS2 | RAD50 | SERPINF1 | STK31 | TSGA10 | ZNF208 |
MTUS1 | NRAS | POLD1 | RAD51AP2 | SHKBP1 | STON1-GTF2A1L | TSHZ3 | ZNF233 |
MTUS2 | NTHL1 | POLD3 | RALGAPA1 | SI | STPG2 | TSPO2 | ZNF267 |
MUC1 | NTSR2 | POLE | RALY | SLC12A1 | SUFU | TUSC3 | ZNF285 |
MUC12 | NUDT11 | POLE2 | RASA1 | SLC13A1 | SYCP1 | UACA | ZNF334 |
MUC16 | NUP107 | POLE4 | RASSF2 | SLC25A51 | SYNE1 | UBE4A | ZNF382 |
MUC17 | NUP50 | POLN | RB1 | SLC30A8 | SYT16 | UBN1 | ZNF417 |
MUC4 | OPRD1 | POLQ | RBM12 | SLC4A10 | TBC1D7 | UBR5 | ZNF469 |
MUC6 | OR11H12 | POSTN | RBMX | SLC5A10 | TBL1XR1 | UGT2A2 | ZNF479 |
MUTYH | PABPC1 | POTEC | RBMXL3 | SLCO1B1 | TBP | UGT3A2 | ZNF521 |
MYCBP2 | PAFAH1B2 | POTEE | REV3L | SLCO6A1 | TCF7L2 | UNC13C | ZNF569 |
MYCN | PARP1 | POTEH | RFC1 | SLITRK1 | TDRD3 | UNC5CL | ZNF600 |
MYO16 | PARP4 | POTEJ | RGS17 | SLITRK4 | TDRD6 | UNC93A | ZNF665 |
MYO3B | PAX1 | PPM1E | RGS22 | SLX4 | TEAD2 | USH2A | ZNF671 |
MYO9A | PBRM1 | PRDM9 | RHCG | SMAD2 | TELO2 | UTP23 | ZNF679 |
MYOCD | PCBP1 | PREX2 | RHOA | SMAD3 | TG | UTS2B | ZNF727 |
MYPOP | PCDH9 | PRKAA1 | RIMS2 | SMAD4 | TGFBR2 | VN1R2 | ZNF728 |
NAALAD2 | PCDHB15 | PRKRIR | RIN3 | SNTG2 | TGIF1 | VPS37B | ZNF729 |
NALCN | PCGF6 | PRSS35 | RMI1 | SORBS2 | TLR4 | VTI1A | ZNF735 |
NBEA | PCLO | PSCA | RNF43 | SOX2 | TLR7 | WBSCR17 | ZNF750 |
NBPF15 | PCSK7 | PSG5 | ROBO2 | SOX9 | TM6SF1 | WDR11 | ZNF761 |
NBPF3 | PHF2 | PTCH1 | RP1 | SPARCL1 | TMEM173 | XIRP2 | ZNF787 |
NEB | PIEZO2 | PTEN | RPA4 | SPATA31D1 | TMEM184A | XKR9 | ZNF804A |
NEFH | PIK3CA | PTPLA | RPL22 | SPDYE6 | TMPRSS13 | XPC | ZNF804B |
NFE2L2 | PIK3R1 | PTPN11 | RPS20 | SPG20 | TNNI3K | XRCC1 | ZNF831 |
NIN | PIK3R3 | PTPRK | RSPO2 | SPIDR | top2B | ZBED9 | ZNF850 |
NKX2-1 | PKD2L2 | PTPRT | RSPO3 | SPP1 | top3B | ZDBF2 | |
NLRP14 | PLCE1 | PTPRZ1 | RYR2 | SPTA1 | topAZ1 | ZFC3H1 | |
NME8 | PLCL1 | PYHIN1 | SACS | SPZ1 | TP53 | ZFHX4 | |
NMI | PLCZ1 | QRFPR | SCN1A | SSBP1 | TPM3 | ZFP28 |
实施例2 :非超突变型直肠癌的基因突变预后预测模型的建立
由于超突变型和非超突变型直肠癌的发生机制、预后和疗效等均存在较大区别,因此本发明首先将直肠癌患者区分为超突变型和非超突变型两组。突变负荷率小于等于10Mut/Mb的肿瘤定义为非超突变型肿瘤。实施例1中的157例直肠癌(ZJU数据集)中,148例被确定为非超突变型。另外为了验证模型的稳定性和普适性,本发明下载了来自TGCA的直肠癌数据共计112例作为独立验证的数据(TCGA数据集),根据同样的标准其中107例被定为非超突变型直肠癌。进一步,本发明要求大于15个月随访时间的患者数据用于预后模型的建立,因此来自ZJU数据集的144例为训练集,来自TCGA数据集的62例为测试集,用于后续分析。后续分析的所有数据均为非超突变型肿瘤。
首先,本发明筛选了ZJU数据集中的直肠癌高频基因(突变频率≥5%)合计36个基因用于后续分析。这些基因的组合形成一个包括n个基因的突变特征,用于构建预后预测模型。在n个基因中携带有1个以上突变位点的患者为突变型,在n个基因中均无突变位点存在的为野生型。其次,本发明使用单因素比例风险回归模型评价了每个突变组合特征和总体生存时间之间的关系,使用ZJU数据集作为训练集,使用TCGA数据集作为测试集。为评价模型预测的生存时间和实际的生存时间之间的符合情况,本发明对测试集计算了C-index指数。为了寻找最小的能区分患者预后的基因组合特征,本发明从一个基因开始逐个增加基因数量,直到C-index值不再增加时,本发明得到了最小且最佳的基因突变组合特征。使用上述策略,本发明建立了一个包括6个基因的突变特征组合,包括如下基因:SMAD4基因、MUC16基因、FAT4基因、FLG基因、ROBO2基因、NEB基因。该模型将直肠癌患者区分为六基因突变型和六基因野生型两类,根据ZJU数据集计算得到风险比为2.11(95%置信区间=1.18-3.74, P<0.01)。将此模型应用于独立的TCGA数据集,计算得到风险比为3.52(95%置信区间=1.38-9.01,P=0.005),见图1和图2,与6基因野生型组的患者相比,6基因突变型组的患者预后更差,死亡风险更高。最后,为进一步验证该模型是否为过拟合模型,本发明还进行了多重置换检验的测试,随机选择同样数量的基因,按照同样的模型训练和验证流程重复进行10000次,逐一记录模型在验证集中的P值,将P值的分布情况列出后与六基因模型比较,见图3,结果说明本发明的6基因预后预测模型显著优于随机选择的同样数量基因的预后预测模型。
实施例3 :应用六基因预后预测模型分析非超突变型III期直肠癌的术后死亡风险
根据临床上常用的AJCC癌症分期系统,直肠癌可分为0期到IV期,不同分期的治疗方案有区别较大。因此本发明对临床上较多见的III期直肠癌分别作了分析。结果表明,六基因模型在III期直肠癌中与术后生存时间显著相关,风险比为3.89(95%置信区间=1.88-8.02,P<0.001),见图4,说明与6基因野生型组的患者相比,6基因突变型组的患者预后更差,死亡风险更高。同时,六基因野生组和六基因突变组中接受术后辅助化疗患者比例无显著差异(P=0.934)。
需要注意的是,本发明所使用的为回顾性收集的研究数据,不同患者所接受的化疗方案有所差别。且由于剔除了随访时间少于15个月的数据,剔除了部分临床信息不全的数据之后,符合要求的数据量较少,本实施例整合了来自ZJU和TCGA的数据。此部分的结果仅为对六基因模型和直肠癌的术后化疗获益之间的关系的初步提示,要确证此关系尚待进一步的多中心前瞻性研究。
实施例4:五基因模型无法预测非超突变型直肠癌的术后死亡风险
本发明人此前公布的专利“一组用于非超突变型结直肠癌分子分型的基因及其应用”中提出的五基因模型可用于非超突变型结直肠癌的分子分型,也可用于非超突变型结肠癌的分子分型,但在非超突变型直肠癌上应用效果不佳。根据ZJU数据集的分析结果(图5)和TCGA数据集(图6)的分析结果,此前公布专利中的五基因模型均无法区分预后较差和预后较好的患者,本发明提出的六基因组合弥补了上述缺陷,可用于区分预后较差和预后较好的非超突变型结直肠癌患者,提示患者在术后发生复发转移的可能性,筛选在术后复发和死亡风险较低的低危组患者,可按照目前的术后辅助治疗方案进行治疗,避免过度治疗;而另一组术后复发和死亡风险较高的高危组患者,可在目前的术后辅助治疗方案基础上为患者寻找更佳的其他方案,有较好的临床指导意义。
Claims (5)
1.一组用于非超突变型直肠癌分子分型的基因探针,其特征在于,由捕获SMAD4基因、MUC16基因、FAT4基因、FLG基因、ROBO2基因、NEB基因的探针组成;所述探针的捕获区域如下所示:
(1)染色体编号chr18,区域起始位置48573407,终止位置48604847,区域为外显子区域及外显子内含子交接区域,用于捕获SMAD4基因;
(2)染色体编号chr19,区域起始位置8959598,区域终止位置9091824,区域为外显子区域及外显子内含子交接区域,用于捕获MUC16基因;
(3)染色体编号chr4,区域起始位置126237554,区域终止位置126414087,区域为外显子区域及外显子内含子交接区域,用于捕获FAT4基因;
(4)染色体编号chr1,区域起始位置152275166,区域终止位置152287942,区域为外显子区域及外显子内含子交接区域,用于捕获FLG基因;
(5)染色体编号chr3,区域起始位置75955845,区域终止位置77699115,区域为外显子区域及外显子内含子交接区域,用于捕获ROBO2基因;
(6)染色体编号chr2,区域起始位置152341850,区域终止位置152591001,区域为外显子区域及外显子内含子交接区域,用于捕获NEB基因。
2.如权利要求1所述的一组基因探针在制备用于非超突变型直肠癌分子分型的试剂盒中的应用。
3.一种用于非超突变型直肠癌分子分型的检测试剂盒,其特征在于,所述检测试剂盒包括:权利要求1中所述的基因探针。
4.如权利要求3所述的检测试剂盒,其特征在于,所述检测试剂盒包括:基因组DNA提取试剂、文库构建试剂、二代测序试剂。
5.如权利要求3所述的检测试剂盒,其特征在于,所述检测试剂盒还包括从检测对象提取检测样本的器械。
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