WO2023083308A1 - 一种基于dna甲基化的结直肠癌预后评估方法 - Google Patents

一种基于dna甲基化的结直肠癌预后评估方法 Download PDF

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WO2023083308A1
WO2023083308A1 PCT/CN2022/131413 CN2022131413W WO2023083308A1 WO 2023083308 A1 WO2023083308 A1 WO 2023083308A1 CN 2022131413 W CN2022131413 W CN 2022131413W WO 2023083308 A1 WO2023083308 A1 WO 2023083308A1
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methylation
colorectal cancer
prognosis
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陈奇涵
王强
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梅傲科技(广州)有限公司
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  • This article relates to a method for judging the prognosis of colorectal cancer in a subject, especially using the degree of methylation of a methylation site to evaluate and/or predict the prognosis of a colorectal cancer subject.
  • the present invention also relates to a kit for determining the prognosis of a colorectal cancer subject.
  • Colorectal cancer (Colon-Rectal Cancer, CRC), also known as colorectal cancer, is a common malignant tumor of the digestive tract, and its incidence rate is second only to gastric cancer and esophageal cancer.
  • CRC Colorectal Cancer
  • colorectal cancer patients account for the fifth in males and the sixth in females.
  • the incidence of colorectal cancer has gradually increased in the past two decades, and at the same time, its age of onset tends to be aging.
  • colorectal cancer is the second malignant tumor after lung cancer.
  • Early colorectal cancer is often asymptomatic, and symptoms appear only with the enlargement of the cancer and the occurrence of complications.
  • colorectal cancer mainly depends on the size, location, extent of the tumor and the health status of the patient.
  • the principle of treatment for colorectal cancer is surgery-based comprehensive treatment. For patients in the middle and advanced stages, postoperative chemotherapy, radiotherapy, traditional Chinese medicine, and immunotherapy can be selected. Recent studies have found that it is difficult to accurately predict the prognosis of patients with colorectal cancer based on clinical indicators alone. Even at the same stage, some patients have a good prognosis, while others have a poor prognosis. There is an urgent need for markers that can indicate the prognosis of patients Drugs and feasible detection methods, so as to provide a more accurate basis for clinical treatment decisions.
  • CpG islands with concentrated distribution of CpG in the promoter region, ranging in length from 0.5 to 2kb. This region is closely related to the transcriptional regulation of genes. relation.
  • the methylation of CpG islands in certain gene regulatory regions in the human body frequently occurs in related cancer cell tissues, and it has been shown to be related to the onset, progression and prognosis of certain tumors, and drug sensitivity.
  • a method for judging the prognosis of colorectal cancer in a subject based on the degree of DNA methylation comprising:
  • said at least one methylation site is selected from cg08875503, cg21200129, cg01141339, cg02319354, cg04064054, cg10251217, cg17534999, cg21642103, cg17599241, cg07470512, cg2 7124774, cg26700320, cg08339172, cg12258785, cg25583503, cg11390151, cg21789085, cg05695951, cg03982609, cg16478719, cg17088007, cg01939477, cg18885346, cg08650890, cg10984625, cg02450613, cg27388911, cg13039082, cg26146027, cg18131851, c g21587469, cg02
  • the methylation degree of any of the following sites is positively correlated with the risk of poor prognosis of colorectal cancer in said subject: cg08875503, cg21200129, cg01141339, cg02319354, cg04064054, cg10251217, cg17534999, cg21642103 , cg17599241, cg07470512, cg27124774, cg26700320, cg08339172, cg12258785, cg25583503, cg11390151, cg21789085, cg05695951, cg03982609 , cg16478719, cg17088007, cg01939477, cg18885346, cg08650890, cg10984625, cg02450613, cg27388911, cg13039082,
  • the degree of methylation of any of the following sites is inversely correlated with said risk of poor prognosis of colorectal cancer in said subject: cg18776463, cg21789870, cg10313005, cg19660618, cg04546747, cg17560006, cg08481119, cg02405609 , cg04350358, cg14737332, cg12895304, cg27365978, cg08716976, cg17155859, cg12785251, cg12193929, cg03633004, cg16626405, cg24518441 .
  • the at least one methylation site is two or more of the methylation sites listed above.
  • the at least one methylation site is three or more of the methylation sites listed above.
  • the at least one methylation site is five or more of the methylation sites listed above.
  • the methylation sites include cg02162897, cg02857557, cg07020176, cg19708201, and cg26985203.
  • the method further comprises calculating the colorectal cancer prognosis for the subject based on the weight of each methylation site.
  • the weights corresponding to each methylation site are as shown in Table 1 and Table 2.
  • the prognosis of colorectal cancer includes risk of death, and/or risk of recurrence, and/or risk of disease progression.
  • the degree of methylation at the methylation site is detected by methylation chip, pyrosequencing, methylation qPCR, or WGBS.
  • this article provides a method to guide the treatment and follow-up strategy of colorectal cancer, which includes:
  • the present invention provides a kit for judging the prognosis of colorectal cancer, which includes reagents for detecting the degree of methylation of at least one methylation site listed above.
  • the kit includes reagents for detecting the degree of methylation of at least two, at least three, or at least five of the methylation sites listed above.
  • the methylation sites include cg02162897, cg02857557, cg07020176, cg19708201, and cg26985203.
  • the kit is in the form of a methylation chip.
  • the methylation sites and kits provided herein can be used to judge or evaluate the prognosis of colorectal cancer in subjects.
  • Figure 1 shows the survival curves and receiver operating characteristic curves based on the degree of methylation at a single methylation site.
  • Figure 2 shows the survival curves and receiver operating characteristic curves of a simple summation model based on the degree of methylation at 5 methylation sites.
  • Figure 3 shows the survival curve and receiver operating characteristic curve of the methylation degree weight model based on the 5 methylation sites.
  • a "methylation site” herein refers to a position of a nucleotide whose base has a methylation modification.
  • DNA methylation in vertebrates generally occurs at the CpG site (that is, the site where cytosine is immediately followed by guanine in the DNA sequence), and cytosine is converted into 5-methylcytosine by DNA methyltransferase catalysis.
  • CpG sites in the human genome are methylated, but some specific regions, such as CpG islands rich in cytosine and guanine, are usually not methylated.
  • CpG methylation can affect the transcriptional activity of related genes. For example, methylation can inhibit tumor suppressor genes, while demethylation can stimulate the expression of some oncogenes.
  • the specific methylation site such as the methylation site ID represented by "cg+number”
  • Illumina's Infinium MethylationEPIC product file https://webdata.illumina.com/downloads/productfiles/ methylationEPIC/infinium-methylationepic-v-1-0-b5-manifest-file-csv.zip
  • GRCh37 version number: GRCh37
  • degree of methylation also referred to as “level of methylation” refers to the proportion (or frequency) of a specific methylation site in a DNA sample being modified by methylation. Whether a site is methylated can be detected by various methods, including but not limited to, methylation chip, pyrosequencing, methylation qPCR, wgbs, etc.
  • bisulfite treatment can be used to cause the deamination of unmethylated cytosine residues in DNA double-stranded molecules to uracil; while the methylated cytosine residues remain unchanged, which can then be The presence or absence of such changes is detected by, for example, sequencing or PCR amplification reactions (eg, using primers that are complementary only to the target sequence that contains uracil).
  • prognosis refers to the prediction of the possible outcome or consequences of a disease (such as recovery, the appearance or disappearance of certain symptoms, signs and complications, and the death of patients), as well as the possibility of these outcomes or consequences occurring. sexual size. Prognosis is related to various factors such as the type of disease, the physical condition of the patient, whether there is appropriate treatment and whether the measures are taken in time. For example, benign tumors have a complete capsule, do not metastasize, grow slowly, and are easier to treat, so the prognosis is better; malignant tumors often have no clear boundaries, proliferate quickly, easily metastasize, and are difficult to treat thoroughly, so the prognosis is poor. Judgment of prognosis may also include providing temporal cues, such as predicting the likelihood of a certain outcome occurring within a certain period of time.
  • the "positive correlation” between the degree of methylation and the poor prognosis of colorectal cancer means that the higher the degree of methylation at this site, the higher the risk of poor prognosis of the patient;
  • the “negative correlation” of poor prognosis means that the lower the degree of methylation of the site, the higher the risk of poor prognosis of the patient.
  • the prognosis of colorectal cancer may include events such as colorectal cancer recurrence, progression, metastasis, or patient death. High risk of poor prognosis refers to disease recurrence or progression, and/or high risk of patient death.
  • the degree of methylation at a single methylation site is used to assess the prognosis of colorectal cancer in a subject.
  • multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 1, 50, 100 or more) methylation sites to evaluate the prognosis of colorectal cancer in subjects.
  • the degree of methylation at 5 methylation sites is detected and used to assess the prognosis of colorectal cancer in the subject.
  • the prognosis of colorectal cancer is assessed by simple summation of methylation levels at multiple (two or more) methylation sites.
  • the plurality of methylated sites includes at least one methylated site positively associated with poor prognosis of colorectal cancer and at least one methylated site negatively associated with poor prognosis of colorectal cancer .
  • the index coefficient of each methylation site is also considered (or weight), that is, a weighted model of these methylation sites is used to evaluate the prognosis of colorectal cancer.
  • a training set can be constructed based on known or available methylation degree and prognosis data of subject samples (such as tumor biopsies or surgical samples) to determine specific methylation sites.
  • subject samples such as tumor biopsies or surgical samples
  • the weight of the chemicalization site can be determined by a multiple linear regression model.
  • a "subject” as used herein refers to a diagnosis, treatment, or clinical trial object, which can be either a disease patient or a healthy individual.
  • the methylation sites provided herein can be used to judge the prognosis of colorectal cancer patients.
  • these methylation sites can be used to prepare a kit for judging the prognosis of colorectal cancer patients.
  • the kit may include, for example, bisulfite (such as sodium bisulfite) for processing the sample to be tested, amplification primers or hybridization probes for specific methylation sites, sequencing reagents, and the like.
  • the kit can also exist in the form of a methylation chip that can simultaneously detect the methylation degree of multiple methylation sites.
  • Example 1 A single methylation site is used to predict the prognosis of colorectal cancer
  • sample TCGA-NH-A50U the methylation degree of five sites is 0.936554, 0.939474, 0.567004, 0.881240 and 0.816545, and the sum score is 1.133701; sample TCGA-F4-6461, five The degree of methylation of each site was 0.940247, 0.935072, 0.582139, 0.879909 and 0.730888 respectively, and the sum score was 1.023483. Both are in the prognostic high risk group based on the score.
  • Figure 2 is the result of predicting the prognosis of 294 patients through this model.
  • the judgment result, HR is 4.2
  • AUC is 0.798, far better than the judgment of a single site.
  • Example 3 Build a prognostic model by combining the above sites and assigning different weight coefficients
  • Example 2 Also take the three samples mentioned in Example 2 as an example for calculation: sample TCGA-NH-A50U, the methylation degrees of the five sites are 0.936554, 0.939474, 0.567004, 0.881240 and 0.816545, and the sum score is 13.94448174; For sample TCGA-F4-6461, the methylation degrees of the five sites were 0.940247, 0.935072, 0.582139, 0.879909 and 0.730888, and the sum score was 13.50687649. Both are in the prognostic high risk group based on the score.
  • Figure 3 shows the results of predicting the prognosis of 294 patients through this model.
  • the judgment result, HR is 11, AUC is 0.826, the result is better than the judgment of simple sum of five sites.

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Abstract

提供了基于DNA甲基化程度判断受试者中结直肠癌预后的方法，包括1)检测来自所述受试者的肿瘤样本的DNA上至少一个甲基化位点的甲基化程度；以及2)将所检测的甲基化程度与已知结直肠癌预后的患者的相应甲基化位点的甲基化程度进行比较，以评估和/或预测所述受试者结直肠癌的预后。还提供了用于判断受试者中结直肠癌预后的试剂盒。

Description

一种基于DNA甲基化的结直肠癌预后评估方法
本申请要求于2021年11月12日提交至中国专利局、申请号为202111340420.0、发明名称为“一种基于DNA甲基化的结直肠癌预后评估方法”的中国专利申请的优先权，在此通过引用将其全文并入本文。
技术领域
本文涉及判断受试者中结直肠癌预后的方法，尤其是利用甲基化位点的甲基化程度来评估和/或预测结直肠癌受试者的预后。本文还涉及用于判断结直肠癌受试者预后的试剂盒。
背景技术
结直肠癌(Colon-Rectal Cancer，CRC)，又称大肠癌，是常见的消化道恶性肿瘤，发生率仅次于胃癌和食道癌。在我国常见恶性肿瘤死亡中，结直肠癌患者在男性占第五位，女性占第六位。近二十年来结直肠癌的发病率在逐渐增加，同时，其发病年龄趋向老龄化。在西方发达国家，结直肠癌是仅次于肺癌的第二位恶性肿瘤。早期大肠癌常无症状，随着癌肿的增大与并发症的发生才出现症状。结直肠癌的治疗方法主要取决于肿瘤的大小、位置、病变程度及病人的健康状况。结直肠癌的治疗原则是以手术为主的综合治疗方式，对中晚期的患者，术后可以选择化疗、放疗、中医中药、免疫治疗等。最近研究发现，结直肠癌患者仅按临床指标难以准确预测其预后特征，即使在同一分期，部分患者预后良好，而另一些结直肠癌患者的预后仍然很差，迫切需要能提示患者预后的标志物和可行的检测方法，从而提供更精准的临床治疗决策依据。
基因组的DNA甲基化异常与肿瘤的发生一直是医学界研究的热点之一，细胞周期、DNA修复、血管生成和细胞凋亡等都涉及到相关基因的甲基化。DNA高甲基化最可能的调控作用是通过抑制关键基因的表达，从而决定该细胞的命运，如对于肿瘤细胞中DNA甲基化异常现象的研究已经在多种肿瘤中取得了许多重大的进展。哺乳动物中，甲基化通常仅影响DNA链上鸟嘌呤前的胞嘧啶(CpG)。正常细胞中CpG双核苷酸的甲基化分布并不均一，大约50％的基因在启动子区域有CpG集中分布的CpG岛存在，长度0.5～2kb不等，该区域与基因的转录调控有着密切关系。人体某些基因调控区域的CpG岛甲基化在相关癌细胞组织中频繁出现，显示出与某些肿瘤的发病、病程进展和预后、用药敏感性等相关。
尽管如此，目前仍缺乏用于判断结直肠癌预后的特异性甲基化位点和方法。
发明内容
在一方面，本文提供了一种基于DNA甲基化程度判断受试者中结直肠癌预后的方法，其包括：
1)检测来自所述患者的肿瘤病理样本的DNA上至少一个甲基化位点的甲基化程度；以及
2)将所检测的甲基化程度与已知结直肠癌预后情况的患者的相应甲基化位点的甲基化程度进行比较，以评估和预测所述结直肠癌的预后，
其中所述至少一个甲基化位点选自cg08875503、cg21200129、cg01141339、cg02319354、cg04064054、cg10251217、cg17534999、cg21642103、cg17599241、cg07470512、cg27124774、cg26700320、cg08339172、cg12258785、cg25583503、cg11390151、cg21789085、cg05695951、cg03982609、cg16478719、cg17088007、cg01939477、cg18885346、cg08650890、cg10984625、cg02450613、cg27388911、cg13039082、cg26146027、cg18131851、cg21587469、cg02837456、cg02414499、cg06756436、cg00513735、cg26985203、cg10497345、cg01582438、cg05275995、cg22282038、cg08455031、cg15618108、cg07679571、cg18885210、cg19489885、cg19708201、cg12135573、cg14944647、cg07652786、cg27097923、cg19272720、cg11697474、cg04155793、cg12268453、cg20966148、cg21748751、cg04609801、cg04790887、cg08751994、cg20991819、cg17782848、cg05129477、cg13656701、cg06794355、cg00937510、cg20861822、cg05939926、cg02379079、cg24568905、cg12806497、cg03635685、cg01674874、cg14654643、cg12608727、cg19692782、cg08021727、cg00886954、cg14562086、cg11781854、cg06782350、cg09102447、cg14102634、cg14191024、cg13609861、cg17429457、cg04842472、cg15436354、cg13143743、cg14344955、cg14708905、cg17924243、cg21125143、cg11691189、cg14124917、cg11819637、cg22121495、cg15438951、cg22707438、cg11454936、cg06528823、cg25028639、cg09274091、cg25352592、cg10248231、cg20705390、cg09447675、cg18954541、cg23024326、cg08341924、cg23235497、cg09944899、cg23754382、cg25889918、cg02524010、cg00107630、cg18343556、cg09121415、cg17475304、cg20173984、cg14703482、cg07372500、cg14311811、cg00333583、cg01796478、cg00569982、cg10272968、cg17980283、cg27298551、cg10496030、cg15109744、cg14851345、ch.1.590396F、cg19588570、cg04173182、cg25449585、cg04297264、cg08098127、cg12062572、cg19931206、cg27329737、cg10588471、cg14727452、cg15001633、cg19311431、cg21922841、cg15829826、cg01020079、cg15323936、cg20455931、cg00595039、cg19754694、cg11356589、cg11599526、cg12497187、cg16932672、cg13535231、cg14968844、cg05597838、cg08923060、cg18059321、cg10662787、cg06211272、cg02857557、ch.17.53379803R、cg01061553、cg00088042、cg26547124、cg02691091、cg01103443、cg15830441、cg12660383、cg24276151、cg22722731、cg11183445、cg05679613、cg22281363、cg15114744、cg25325446、cg19067392、cg12604858、cg27133684、cg17948297、cg27298830、cg03671660、cg18349937、cg25706474、cg15149876、cg10171557、cg12184854、cg22463793、cg10601476、cg22616506、cg09790523、cg04138846、cg03124769、cg19827167、cg18776463、cg21789870、cg10313005、 cg19660618、cg04546747、cg17560006、cg08481119、cg02405609、cg04350358、cg14737332、cg12895304、cg27365978、cg08716976、cg17155859、cg12785251、cg12193929、cg03633004、cg16626405、cg24518441、cg19368383、cg20695611、cg02162897、cg05612346、cg10313043、cg26661922、cg26060817、cg08921986、cg21548029、cg17178218、cg02219116、cg01341076、cg15276922、cg22021076、cg11659391、cg14506686、cg23552821、cg01034813、cg00256074、cg22971029、cg00599163、cg18394567、cg09386653、cg15225534、cg10385101、cg04560668、cg09353563、cg01963906、cg00690901、cg19742623、cg05737153、cg02630888、cg04742384、cg14893161、cg16523463、cg13606421、cg12970081、cg22699052、cg22291365、cg26354017、cg16581476、cg10002066、cg07020176、cg06599483、cg19995539、cg14159672、cg08198187、cg16970604、cg02416336、cg27016549、cg02850689、cg04091325、cg13417891、cg12034943、cg24605338、cg03449040、cg16673477、cg26384993、cg05755354、cg26864950、cg13123165、cg13218573、cg26235748、cg05906166、cg08169949、cg07157834、cg19486756、cg25361663、cg04482110、cg15989318、cg06963182、cg01233744、cg10805721、cg15757853、cg26922007、cg20845639、cg07614018、cg11965913、cg00349573、cg07167872、cg07916654、cg19548479、cg08377924、cg18222083和cg00506886。
在一些实施方案中，如下任一位点的甲基化程度与所述受试者的所述结直肠癌预后不良的风险正相关：cg08875503、cg21200129、cg01141339、cg02319354、cg04064054、cg10251217、cg17534999、cg21642103、cg17599241、cg07470512、cg27124774、cg26700320、cg08339172、cg12258785、cg25583503、cg11390151、cg21789085、cg05695951、cg03982609、cg16478719、cg17088007、cg01939477、cg18885346、cg08650890、cg10984625、cg02450613、cg27388911、cg13039082、cg26146027、cg18131851、cg21587469、cg02837456、cg02414499、cg06756436、cg00513735、cg26985203、cg10497345、cg01582438、cg05275995、cg22282038、cg08455031、cg15618108、cg07679571、cg18885210、cg19489885、cg19708201、cg12135573、cg14944647、cg07652786、cg27097923、cg19272720、cg11697474、cg04155793、cg12268453、cg20966148、cg21748751、cg04609801、cg04790887、cg08751994、cg20991819、cg17782848、cg05129477、cg13656701、cg06794355、cg00937510、cg20861822、cg05939926、cg02379079、cg24568905、cg12806497、cg03635685、cg01674874、cg14654643、cg12608727、cg19692782、cg08021727、cg00886954、cg14562086、cg11781854、cg06782350、cg09102447、cg14102634、cg14191024、cg13609861、cg17429457、cg04842472、cg15436354、cg13143743、cg14344955、cg14708905、cg17924243、cg21125143、cg11691189、cg14124917、cg11819637、cg22121495、cg15438951、cg22707438、cg11454936、cg06528823、cg25028639、cg09274091、cg25352592、cg10248231、cg20705390、cg09447675、cg18954541、cg23024326、cg08341924、cg23235497、cg09944899、cg23754382、cg25889918、cg02524010、cg00107630、cg18343556、cg09121415、cg17475304、cg20173984、cg14703482、cg07372500、cg14311811、cg00333583、cg01796478、cg00569982、cg10272968、cg17980283、cg27298551、cg10496030、cg15109744、cg14851345、 ch.1.590396F、cg19588570、cg04173182、cg25449585、cg04297264、cg08098127、cg12062572、cg19931206、cg27329737、cg10588471、cg14727452、cg15001633、cg19311431、cg21922841、cg15829826、cg01020079、cg15323936、cg20455931、cg00595039、cg19754694、cg11356589、cg11599526、cg12497187、cg16932672、cg13535231、cg14968844、cg05597838、cg08923060、cg18059321、cg10662787、cg06211272、cg02857557、ch.17.53379803R、cg01061553、cg00088042、cg26547124、cg02691091、cg01103443、cg15830441、cg12660383、cg24276151、cg22722731、cg11183445、cg05679613、cg22281363、cg15114744、cg25325446、cg19067392、cg12604858、cg27133684、cg17948297、cg27298830、cg03671660、cg18349937、cg25706474、cg15149876、cg10171557、cg12184854、cg22463793、cg10601476、cg22616506、cg09790523、cg04138846、cg03124769和cg19827167。
在一些实施方案中，如下任一位点的甲基化程度与所述受试者的所述结直肠癌预后不良的风险负相关：cg18776463、cg21789870、cg10313005、cg19660618、cg04546747、cg17560006、cg08481119、cg02405609、cg04350358、cg14737332、cg12895304、cg27365978、cg08716976、cg17155859、cg12785251、cg12193929、cg03633004、cg16626405、cg24518441、cg19368383、cg20695611、cg02162897、cg05612346、cg10313043、cg26661922、cg26060817、cg08921986、cg21548029、cg17178218、cg02219116、cg01341076、cg15276922、cg22021076、cg11659391、cg14506686、cg23552821、cg01034813、cg00256074、cg22971029、cg00599163、cg18394567、cg09386653、cg15225534、cg10385101、cg04560668、cg09353563、cg01963906、cg00690901、cg19742623、cg05737153、cg02630888、cg04742384、cg14893161、cg16523463、cg13606421、cg12970081、cg22699052、cg22291365、cg26354017、cg16581476、cg10002066、cg07020176、cg06599483、cg19995539、cg14159672、cg08198187、cg16970604、cg02416336、cg27016549、cg02850689、cg04091325、cg13417891、cg12034943、cg24605338、cg03449040、cg16673477、cg26384993、cg05755354、cg26864950、cg13123165、cg13218573、cg26235748、cg05906166、cg08169949、cg07157834、cg19486756、cg25361663、cg04482110、cg15989318、cg06963182、cg01233744、cg10805721、cg15757853、cg26922007、cg20845639、cg07614018、cg11965913、cg00349573、cg07167872、cg07916654、cg19548479、cg08377924、cg18222083和cg00506886。
在一些实施方案中，所述至少一个甲基化位点为上文所列甲基化位点中的两个或更多个。
在一些实施方案中，所述至少一个甲基化位点为上文所列甲基化位点中的三个或更多个。
在一些实施方案中，所述至少一个甲基化位点为上文所列甲基化位点中的五个或更多个。
在一些实施方案中，所述甲基化位点包括cg02162897、cg02857557、cg07020176、cg19708201和cg26985203。
在一些实施方案中，所述方法还包括根据每个甲基化位点的权重来计算所述受试者的所述结直肠癌的预后。
在一些实施方案中，对应于各个甲基化位点的权重如表1和表2中所示。
在一些实施方案中，所述结直肠癌的预后包括死亡风险、和/或复发风险、和/或疾病进展风险。
在一些实施方案中，通过甲基化芯片、焦磷酸测序、甲基化qPCR、或WGBS检测所述甲基化位点的甲基化程度。
另一方面，本文提供了一种指导结直肠癌治疗和随访策略的方法，其包括：
1)以所述药物对患有结直肠癌的受试者给药；或/和以其他治疗手段对患有结直肠癌的受试者进行治疗；
2)以上述方法判断所述结直肠癌预后，
其中根据所述结直肠癌预后来指导用所述药物等治疗手段进行结直肠癌治疗和相应的随访的策略。
另一方面，本文提供了一种判断结直肠癌预后的试剂盒，其包括用于检测至少一个上文所列甲基化位点的甲基化程度的试剂。
在一些实施方案中，所述试剂盒包括用于检测至少两个、至少三个、或至少五个上文所列甲基化位点的甲基化程度的试剂。
在一些实施方案中，所述甲基化位点包括cg02162897、cg02857557、cg07020176、cg19708201和cg26985203。
在一些实施方案中，所述试剂盒为甲基化芯片的形式。
本文提供的甲基化位点和试剂盒可用于判断或评估受试者结直肠癌的预后。
附图说明
图1显示了基于单一甲基化位点的甲基化程度的生存曲线和受试者工作特征曲线。
图2显示了基于5个甲基化位点的甲基化程度简单加和模型的生存曲线和受试者工作特征曲线。
图3显示了基于5个甲基化位点的甲基化程度权重模型的生存曲线和受试者工作特征曲线。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
本文中“甲基化位点”指其碱基具有甲基化修饰的核苷酸的位置。脊椎动物的DNA甲基化一般发生在CpG位点(即DNA序列中胞嘧啶后紧连鸟嘌呤的位点)，经DNA甲基转移酶催化将胞嘧啶转化为5-甲基胞嘧啶。人类基因组中大多CpG位点已被甲基化，但是在某 些特定区域，如富含胞嘧啶和鸟嘌呤的CpG岛则通常未被甲基化。CpG甲基化可影响相关基因的转录活性，例如，甲基化可抑制抑癌基因，而去甲基化则刺激某些癌基因的表达，这些情况下都有可能导致癌症发生。在本文中，具体的甲基化位点，例如通过“cg+数字”方式表示的甲基化位点ID，由Illumina公司Infinium MethylationEPIC产品文件(https://webdata.illumina.com/downloads/productfiles/methylationEPIC/infinium-methylationepic-v-1-0-b5-manifest-file-csv.zip)提供，在人类(Homo sapiens)基因组(版本号：GRCh37)中具有一一对应的位置关系。另外，在下文表1和表2中也分别通过“染色体编号+位置编号”方式进行了进一步的说明。
本文中“甲基化程度”，也可称为“甲基化水平”，指DNA样本中某一特定甲基化位点被甲基化修饰的比例(或频率)。可通过多种方式来检测一个位点是否被甲基化，包括但不限于，甲基化芯片、焦磷酸测序、甲基化qPCR、wgbs等。在一个具体实例中，可利用亚硫酸氢盐处理造成DNA双链分子中未甲基化的胞嘧啶残基脱氨基，成为尿嘧啶；而甲基化的胞嘧啶残基保持不变，随后可通过例如测序或PCR扩增反应(例如，所采用的引物仅与包含尿嘧啶的靶序列互补)检测是否存在这种变化。
本文中“预后(prognosis)”指对某种疾病的可能结局或后果(如康复，某种症状、体征和并发症等的出现或消失及患者死亡)的预测，以及这些结局或后果发生的可能性大小。预后与疾病的种类、患者的身体状况、有无适当的治疗措施及措施采取是否及时等多种因素有关。例如，良性肿瘤多有完整的包膜、不转移、生长慢，较易治疗，因此预后较佳；恶性肿瘤则多无清楚的界限、增殖快、容易转移、治疗不易彻底，因此预后不良。对预后的判断也可以包括提供时间线索，如预测某段时间内发生某种结局的可能性。
本文中，甲基化程度与结直肠癌的预后不良“正相关”指该位点甲基化程度越高，则患者预后不良的风险越高；相应地，甲基化程度与结直肠癌的预后不良“负相关”指该位点甲基化程度越低，则患者预后不良的风险越高。结直肠癌的的预后可包括结直肠癌的复发、进展、转移或患者死亡等事件。预后不良的风险高指疾病复发或进展，和/或患者死亡风险高。
在本文提供的判断结直肠癌的预后的方法的一些实施方案中，采用单一甲基化位点的甲基化程度来评估受试者的结直肠癌的预后。在优选的实施方案中，采用多个(例如，2个、3个、4个、5个、6个、7个、8个、9个、10个、15个、20个、30个、40个、50个、100个或更多个)甲基化位点的甲基化程度的组合来评估受试者的结直肠癌的预后。例如，在一些实例中，检测5个甲基化位点的甲基化程度并用来评估受试者的结直肠癌的预后。在一些实施方案中，采用多个(两个或两个以上)甲基化位点的甲基化程度的简单加和来评估结直肠癌的预后。在一些实施方案中，该多个甲基化位点包括至少一个与结直肠癌的预后不良正相关的甲基化位点和至少一个与结直肠癌的预后不良负相关的甲基化位点。在更优选的实施方案中，采用多个(两个或两个以上)甲基化位点的甲基化程度来评估结直肠癌的预后时，还考虑每个甲基化位点的指标系数(或权重)，即采用这些甲基化位 点的加权模型来评估结直肠癌的预后。在已知甲基化位点组合的情况下，可基于已知或可获得的受试者样本(如肿瘤的活检或手术样本)的甲基化程度和预后数据构建训练集来确定具体甲基化位点的权重。在一个具体实例中，可通过多元线性回归模型来确定甲基化位点的权重。
本文所用的“受试者”，指诊断、治疗、或临床试验对象，既可以是疾病患者，也可以为健康个体。
本文提供的甲基化位点可用于判断结直肠癌患者的预后。例如，可利用这些甲基化位点来制备用于判断结直肠癌患者预后的试剂盒。该试剂盒例如可以包括，用于对待测样本进行处理的亚硫酸氢盐(例如亚硫酸氢钠)、针对特定甲基化位点的扩增引物或杂交探针、测序试剂等。该试剂盒还可以以可同时检测多这个甲基化位点的甲基化程度的甲基化芯片的形式的存在。
以下通过具体实施例来进一步说明本发明。
实施例1 单一甲基化位点用于结直肠癌预后预测
通过TCGA数据库中公开的结直肠癌病人肿瘤组织样本450k甲基化芯片测序结果以及我们自己用850k甲基化芯片测序的192例结直肠癌患者肿瘤组织样本，我们挑选出了多个位点作为判断结直肠癌患者预后预测的标志物。这些位点分别列在表1和表2中。所选出的可用于结直肠癌预后预测的甲基化位点共分为两类，即甲基化程度与预后不良呈现正相关(194个，表1)与负相关(106个，表2)。对于每一个位点，我们在TCGA的294例病人中进行预测，并与实际的预后情况进行比较。通过对判断出的高风险和低风险人群(147 vs 147)的预后情况进行综合比较，我们对每个位点判断结果的风险系数(HR)和AUC值进行计算，HR值越大则判断效果越好，AUC约接近1则判断效果越好。统计结果见表1和表2。另外，参见图1，我们针对每个位点还绘出了生存曲线和受试者工作特征曲线(AOC)。
表1 与预后不良正相关的甲基化位点和相应统计数据
标志物编号 染色体 位置 hr rocauc
cg19827167 chr1 93846650 1.61297 0.62686
cg03124769 chr7 139109085 1.76162 0.66494
cg04138846 chr5 179848032 3.36386 0.66657
cg09790523 chr17 80921264 1.98789 0.65519
cg22616506 chr2 99162468 2.23496 0.56578
cg10601476 chr17 1533990 2.86477 0.69976
cg22463793 chr17 1899038 2.04945 0.67561
cg12184854 chr3 189808454 1.64503 0.63894
cg10171557 chr20 35668899 2.20204 0.65401
cg15149876 chr8 124976673 2.7834 0.69702
cg25706474 chr2 238252516 1.87564 0.61705
cg18349937 chr12 132817495 1.45082 0.61002
cg03671660 chr21 39182981 1.64862 0.61709
cg27298830 chr3 31935817 1.73562 0.61764
cg17948297 chr8 22226929 2.19999 0.64118
cg27133684 chr17 17801332 1.67249 0.62492
cg12604858 chr6 38094126 2.28545 0.69411
cg19067392 chr12 130807085 2.02419 0.60946
cg25325446 chr5 140558616 2.03499 0.61992
cg15114744 chr1 44027114 1.89459 0.60118
cg22281363 chr8 53986480 1.91408 0.66398
cg05679613 chr4 87832891 1.77443 0.61136
cg11183445 chr8 65633567 1.42314 0.63796
cg22722731 chr4 139860047 1.4501 0.58103
cg24276151 chr17 82010251 1.96917 0.60823
cg12660383 chr4 123250468 1.85214 0.58401
cg15830441 chr13 20774005 1.35786 0.66031
cg01103443 chr16 260592 2.43533 0.64324
cg02691091 chr17 47915445 2.16617 0.6351
cg26547124 chr12 104216766 2.40535 0.64777
cg00088042 chr10 102132552 1.92812 0.57378
cg01061553 chr12 110453662 1.75148 0.64933
ch.17.53379803R chr17 57947443 1.84145 0.60989
cg02857557 chr10 119819049 1.68865 0.56551
cg06211272 chr1 58547003 1.63887 0.59263
cg10662787 chr1 206612062 1.55355 0.57914
cg18059321 chr3 51895026 2.35966 0.53785
cg08923060 chr12 120446899 1.50495 0.55597
cg05597838 chr16 19180474 2.27009 0.62362
cg14968844 chr8 1882602 1.72521 0.60406
cg13535231 chr9 122241498 2.20358 0.65698
cg16932672 chr18 2571334 2.2248 0.6661
cg12497187 chr15 59445530 2.04527 0.62833
cg11599526 chr5 151069948 2.27045 0.59523
cg11356589 chr2 144237505 1.93688 0.64463
cg19754694 chr7 131041508 2.80891 0.697
cg00595039 chr8 142378848 2.74755 0.66498
cg20455931 chr22 42615024 1.55739 0.5668
cg15323936 chr3 195275566 2.30862 0.67011
cg01020079 chr12 50842921 1.51446 0.60196
cg15829826 chr11 65386389 2.24323 0.59448
cg21922841 chr17 74747992 1.65703 0.6133
cg19311431 chr2 161235420 2.53422 0.66639
cg15001633 chr8 102253753 2.26142 0.68707
cg14727452 chr11 117413051 1.87937 0.62544
cg10588471 chr13 112978330 1.68713 0.64718
cg27329737 chr3 151212284 1.69112 0.59574
cg19931206 chr6 11138417 2.44587 0.68505
cg12062572 chr15 34979814 2.43551 0.62451
cg08098127 chrX 13663687 2.50771 0.6862
cg04297264 chr14 77475727 1.7991 0.62456
cg25449585 chr8 28824712 2.45475 0.62543
cg04173182 chr4 53579868 1.93305 0.66664
cg19588570 chr6 100136277 1.88653 0.64959
ch.1.590396F chr1 17019481 2.22922 0.65833
cg14851345 chr6 63713901 1.91395 0.65739
cg15109744 chr1 180948737 1.53288 0.60341
cg10496030 chr11 76799422 1.63723 0.68005
cg27298551 chr12 53075071 2.15406 0.63232
cg17980283 chr2 44929503 1.91101 0.62017
cg10272968 chr19 2611107 1.42625 0.55355
cg00569982 chr6 75237856 1.96721 0.64081
cg01796478 chr17 58661581 2.23897 0.59952
cg00333583 chr15 85046043 2.44744 0.66928
cg14311811 chr18 31497900 2.00115 0.7104
cg07372500 chr5 177940012 1.72785 0.62994
cg14703482 chr4 122837604 2.06596 0.61325
cg20173984 chr6 84226264 2.04095 0.59808
cg17475304 chr5 177085895 1.88083 0.61607
cg09121415 chr12 49064754 2.45509 0.66754
cg18343556 chr3 50278953 1.47242 0.59817
cg00107630 chr9 117412130 2.58263 0.64105
cg02524010 chr16 69083776 2.21459 0.65779
cg25889918 chr2 179051457 1.92651 0.66829
cg23754382 chr22 29316483 2.42204 0.65468
cg09944899 chr4 22516853 2.07543 0.65138
cg23235497 chr1 110150278 2.47084 0.68123
cg08341924 chr14 24262336 1.55083 0.6091
cg23024326 chr6 36794066 2.3445 0.62512
cg18954541 chr20 46651346 1.45275 0.64504
cg09447675 chr12 46483683 1.85549 0.66723
cg20705390 chr1 1330798 2.29337 0.64995
cg10248231 chr1 3423306 2.84523 0.61911
cg25352592 chr1 6421091 1.93416 0.64798
cg09274091 chr14 24312731 1.86903 0.6721
cg25028639 chr11 9863350 2.23157 0.61807
cg06528823 chr10 32014179 1.65151 0.68818
cg11454936 chr6 166771507 1.97682 0.65162
cg22707438 chr11 46342577 1.62872 0.63143
cg15438951 chr12 95995916 1.93533 0.61456
cg22121495 chr17 73423922 2.17858 0.64536
cg11819637 chr3 184377717 2.23875 0.69623
cg14124917 chr2 9950845 1.79199 0.63086
cg11691189 chr2 47516602 2.23223 0.69671
cg21125143 chr11 43880562 1.70789 0.6118
cg17924243 chr5 174383890 2.60155 0.63128
cg14708905 chr6 30145297 2.12719 0.63585
cg14344955 chr17 73748586 2.92034 0.67503
cg13143743 chr3 177852906 2.12223 0.61943
cg15436354 chr19 8052991 2.13674 0.60642
cg04842472 chr7 39833252 1.71843 0.6345
cg17429457 chr12 109096665 2.19811 0.692
cg13609861 chr9 130905461 2.37167 0.62748
cg14191024 chr11 110199784 1.6166 0.57415
cg14102634 chr6 30828980 1.64505 0.63463
cg09102447 chr8 56994579 1.31075 0.56444
cg06782350 chr15 40768196 2.22711 0.59922
cg11781854 chr19 39246050 2.15196 0.55901
cg14562086 chr17 50201904 1.69862 0.68738
cg00886954 chr8 143973545 1.72479 0.61626
cg08021727 chr16 4006878 2.1851 0.65251
cg19692782 chr1 112359593 1.78179 0.63823
cg12608727 chr1 64245350 2.48063 0.71691
cg14654643 chr2 222322800 1.90165 0.64958
cg01674874 chrX 49158677 2.52302 0.6849
cg03635685 chr22 24154674 2.06108 0.65148
cg12806497 chr2 218811198 1.84855 0.67185
cg24568905 chr8 21756905 1.60846 0.65583
cg02379079 chr20 38346440 2.12831 0.65206
cg05939926 chr13 111227680 1.90627 0.63619
cg20861822 chr20 31818904 1.60093 0.60568
cg00937510 chr5 88269652 1.65114 0.64416
cg06794355 chr1 16688304 2.04198 0.69519
cg13656701 chr9 130915105 2.3346 0.68679
cg05129477 chr1 20718452 2.16368 0.64646
cg17782848 chr5 170579828 1.42589 0.6393
cg20991819 chr1 218444427 1.46324 0.55611
cg08751994 chr7 96086082 1.96565 0.60956
cg04790887 chr15 58223345 1.54902 0.61912
cg04609801 chr16 28597855 2.59997 0.54613
cg21748751 chr6 35342090 1.63102 0.69856
cg20966148 chr22 21126229 1.84927 0.65535
cg12268453 chr11 120549438 2.4578 0.66496
cg04155793 chr16 89686439 2.83854 0.62693
cg11697474 chr6 170273290 1.70912 0.62536
cg19272720 chr1 204320316 2.81377 0.67672
cg27097923 chr6 82361716 1.96535 0.65605
cg07652786 chr2 240786636 1.74039 0.62283
cg14944647 chr11 43581307 1.92889 0.63995
cg12135573 chr7 101166716 2.41041 0.65238
cg19708201 chr1 237833593 2.73382 0.70519
cg19489885 chr5 179257415 1.9455 0.64125
cg18885210 chr2 120758957 2.74408 0.60226
cg07679571 chrX 70492373 2.17198 0.66852
cg15618108 chr1 203075549 2.48942 0.62503
cg08455031 chr6 37652329 2.1292 0.59142
cg22282038 chr8 41647976 2.06694 0.56693
cg05275995 chr22 17580231 1.79689 0.63931
cg01582438 chr20 63829806 1.56798 0.64308
cg10497345 chr4 89250887 1.59823 0.63469
cg26985203 chr11 43881492 1.69529 0.61632
cg00513735 chr5 176880765 2.13712 0.63156
cg06756436 chr18 615936 2.12655 0.63223
cg02414499 chr17 77560962 1.78149 0.69317
cg02837456 chr11 94767446 2.58751 0.64315
cg21587469 chr12 55930032 2.31359 0.70811
cg18131851 chr5 141192309 1.84098 0.58879
cg26146027 chr3 24495398 1.40595 0.60163
cg13039082 chr11 77412286 1.49275 0.62055
cg27388911 chr3 24495786 1.79605 0.68157
cg02450613 chr5 37839987 2.46346 0.67296
cg10984625 chr2 237691215 1.73987 0.64047
cg08650890 chr6 32096836 1.6598 0.63711
cg18885346 chr6 52088316 1.83567 0.67293
cg01939477 chr11 43581329 1.78666 0.63453
cg17088007 chr22 37062658 1.78895 0.5913
cg16478719 chr14 36513966 1.80787 0.6221
cg03982609 chr22 43344792 1.58225 0.65016
cg05695951 chrX 46577883 1.48027 0.63333
cg21789085 chr8 2076514 1.48055 0.58476
cg11390151 chr1 2456371 1.6724 0.59677
cg25583503 chr4 69096958 2.1976 0.61534
cg12258785 chr3 24495916 1.65136 0.67474
cg08339172 chr17 65696869 1.73225 0.57999
cg26700320 chr22 48550254 1.74131 0.64404
cg27124774 chr5 141192315 1.58878 0.5585
cg07470512 chr20 33667246 1.62675 0.63206
cg17599241 chr5 83562857 1.62126 0.62044
cg21642103 chr6 32096879 1.62215 0.61772
cg17534999 chr11 127002720 1.93748 0.59056
cg10251217 chr16 71577616 1.26657 0.58739
cg04064054 chr16 569455 1.45156 0.59884
cg02319354 chr5 73234359 1.813 0.65071
cg01141339 chr5 73234371 1.864 0.64624
cg21200129 chr9 23821568 1.65045 0.59656
cg08875503 chr21 17607990 1.73185 0.55318
表2 与预后不良负相关的甲基化位点和相应统计数据
标志物编号 染色体 位置 hr rocauc
cg00506886 chr19 38483710 1.47725 0.57856
cg18222083 chr17 43211988 2.11454 0.53637
cg08377924 chr17 43211609 2.33099 0.54599
cg19548479 chr17 43211718 2.21597 0.56713
cg07916654 chr10 133127725 1.36975 0.62207
cg07167872 chr1 205850335 1.90427 0.63178
cg00349573 chr1 242448524 1.76468 0.63837
cg11965913 chr1 205850278 1.48274 0.60605
cg07614018 chr19 18700765 2.04218 0.5815
cg20845639 chr8 23573373 1.2965 0.61108
cg26922007 chr11 5059222 2.10866 0.6091
cg15757853 chr18 23138337 1.45445 0.54649
cg10805721 chr6 82365470 1.81566 0.60422
cg01233744 chr11 125778347 1.64698 0.57564
cg06963182 chr2 84894962 1.5132 0.60209
cg15989318 chr10 104666452 1.55533 0.54865
cg04482110 chr17 43212102 2.11294 0.58109
cg25361663 chr7 635933 1.36653 0.60756
cg19486756 chr5 150901629 1.31118 0.55411
cg07157834 chr1 205850481 1.54989 0.60697
cg08169949 chr1 27915021 2.0293 0.66236
cg05906166 chr19 16326246 1.36443 0.60255
cg26235748 chr14 85621167 1.66876 0.62492
cg13218573 chr1 9951174 2.03691 0.58959
cg13123165 chr11 122235528 1.75653 0.60723
cg26864950 chr19 1395366 1.34617 0.58893
cg05755354 chr10 14330597 1.47085 0.63545
cg26384993 chr14 72812750 1.7718 0.68039
cg16673477 chr19 18701207 2.20011 0.60008
cg03449040 chrX 10126323 1.95352 0.66654
cg24605338 chrX 17377733 1.86423 0.64466
cg12034943 chr19 18700980 2.12358 0.60134
cg13417891 chr17 37476891 1.24303 0.56645
cg04091325 chr15 83446681 1.85432 0.71007
cg02850689 chr1 2459908 1.48292 0.59584
cg27016549 chr11 131909965 1.65009 0.65053
cg02416336 chr21 30598706 1.55044 0.62091
cg16970604 chr8 60413244 1.45835 0.65476
cg08198187 chr12 31729662 1.59067 0.60258
cg14159672 chr1 205850051 1.72503 0.61867
cg19995539 chr12 120101333 1.48043 0.60434
cg06599483 chr12 113161553 2.13304 0.59316
cg07020176 chr15 25228251 1.71376 0.59975
cg10002066 chr13 114130717 1.77965 0.63623
cg16581476 chr15 25180671 1.25433 0.62521
cg26354017 chr1 205849960 1.70558 0.62798
cg22291365 chr15 74599098 1.87308 0.57297
cg22699052 chr17 805357 1.64682 0.62032
cg12970081 chr19 50770667 1.73226 0.66548
cg13606421 chr3 16174673 2.02907 0.57974
cg16523463 chr12 110920575 1.5039 0.59241
cg14893161 chr1 205850123 1.77394 0.63829
cg04742384 chr20 56001359 1.79445 0.56797
cg02630888 chr4 15656197 1.76673 0.68373
cg05737153 chr4 5839903 1.64652 0.62793
cg19742623 chr13 109157880 1.86321 0.60977
cg00690901 chr10 59283390 1.46828 0.58
cg01963906 chr1 27350749 1.76194 0.64219
cg09353563 chr15 70702096 1.56531 0.56721
cg04560668 chr19 35136256 1.57749 0.62922
cg10385101 chr7 668091 1.67951 0.61887
cg15225534 chr6 33899170 1.94797 0.64623
cg09386653 chr7 5121900 1.66004 0.65034
cg18394567 chr17 2855081 1.90208 0.63178
cg00599163 chr2 161243984 1.46202 0.58473
cg22971029 chr14 91352153 1.47406 0.61742
cg00256074 chr16 30374829 1.65177 0.64353
cg01034813 chr16 27475726 1.52351 0.56009
cg23552821 chr1 998656 1.91053 0.64231
cg14506686 chr7 48455736 1.62477 0.65631
cg11659391 chr2 207681837 1.75516 0.63114
cg22021076 chr11 1161469 1.74277 0.69891
cg15276922 chr8 98658616 1.25001 0.60249
cg01341076 chr7 36084795 1.54926 0.63369
cg02219116 chr9 137279504 1.42964 0.58404
cg17178218 chr1 7292189 1.32869 0.65105
cg21548029 chr5 141136093 2.00024 0.67443
cg08921986 chr3 77317350 1.35177 0.61833
cg26060817 chr5 14406567 1.79129 0.63965
cg26661922 chr16 69917917 1.50056 0.62574
cg10313043 chr10 98260938 1.64767 0.64405
cg05612346 chr11 17884332 1.92184 0.62572
cg02162897 chr2 38073394 2.04387 0.67308
cg20695611 chr17 37493542 1.61393 0.63773
cg19368383 chr10 98434621 1.32394 0.57561
cg24518441 chr1 16148621 1.60403 0.60912
cg16626405 chr20 34709620 2.39304 0.64573
cg03633004 chr7 2647900 1.67991 0.67072
cg12193929 chr5 140857900 1.49157 0.59275
cg12785251 chr6 33320164 1.35304 0.61842
cg17155859 chr11 64259069 1.70637 0.61458
cg08716976 chr2 3242023 1.80621 0.61977
cg27365978 chr10 104333901 1.47633 0.65113
cg12895304 chr13 100155241 1.52461 0.58814
cg14737332 chr10 112950121 1.64932 0.62242
cg04350358 chr6 117675178 1.80102 0.6315
cg02405609 chr4 182890529 1.8049 0.60008
cg08481119 chr2 112054347 3.44397 0.67648
cg17560006 chr3 134250749 1.70929 0.64782
cg04546747 chr11 1764322 2.11095 0.62614
cg19660618 chr12 49828376 1.63855 0.61512
cg10313005 chr7 128739512 2.05923 0.59757
cg21789870 chr18 74148452 2.23069 0.62279
cg18776463 chr12 14771098 1.56648 0.6098
实施例2 通过组合上述位点建立预后模型
尽管单一位点已经能够初步预测结直肠癌患者的预后，但通过多个位点的组合能够更好的预测。这里我们以一个包括5个位点的模型为例cg02162897+cg02857557+cg07020176+cg19708201+cg26985203，其中第一、三位点甲基化程度与预后不良呈现负相关，其余三个位点正相关。
以3个样本为例进行计算：样本TCGA-NH-A50U，五个位点甲基化程度分别为0.936554、0.939474、0.567004、0.881240和0.816545，加和得分为1.133701；样本TCGA-F4-6461，五个位点甲基化程度分别为0.940247、0.935072、0.582139、0.879909和0.730888，加和得分为1.023483。此二者基于得分均处于预后高风险组。样本TCGA-F4-6463，五个位点甲基化程度分别为0.945746、0.821139、0.675280、0.531395和0.304300，加和得分为0.035808。此样本基于得分均处于预后低风险组。
图2为通过该模型对294例病人进行预后预测的结果。其判断结果，HR为4.2，AUC为0.798，远好于单一位点的判断情况。
实施例3 通过组合上述位点并赋以不同的权重系数建立预后模型
尽管简单的组合多个位点能够很好的预测结直肠癌患者的预后，在模型的训练中如果考虑各个位点在预测中的重要性，能够进一步提高预测的准确度。这里我们同样以实施例2中的5个位点为例，使用公式-9.38113*cg02162897+20.43697*cg02857557-3.02883*cg07020176+3.10983*cg19708201+3.07058*cg26985203进行判断。
同样以实施例2中提到的3个样本为例进行计算：样本TCGA-NH-A50U，五个位点甲基化程度分别为0.936554、0.939474、0.567004、0.881240和0.816545，加和得分为13.94448174；样本TCGA-F4-6461，五个位点甲基化程度分别为0.940247、0.935072、0.582139、0.879909和0.730888，加和得分为13.50687649。此二者基于得分均处于预后高风险组。样本TCGA-F4-6463，五个位点甲基化程度分别为0.945746、0.821139、0.675280、0.531395和0.304300，加和得分为8.45104422。此样本基于得分均处于预后低风险组。
图3显示了为通过该模型对294例病人进行预后预测的结果。其判断结果，HR为11，AUC为0.826，结果好于五个位点简单的加和的判断情况。

Claims (17)

  1. 基于DNA甲基化程度判断受试者中结直肠癌预后的方法，包括：
    1)检测来自所述受试者的肿瘤样本的DNA上至少一个甲基化位点的甲基化程度；以及
    2)将所检测的甲基化程度与已知结直肠癌预后的患者的相应甲基化位点的甲基化程度进行比较，以评估和/或预测所述结直肠癌的预后，
    其中所述至少一个甲基化位点选自cg08875503、cg21200129、cg01141339、cg02319354、cg04064054、cg10251217、cg17534999、cg21642103、cg17599241、cg07470512、cg27124774、cg26700320、cg08339172、cg12258785、cg25583503、cg11390151、cg21789085、cg05695951、cg03982609、cg16478719、cg17088007、cg01939477、cg18885346、cg08650890、cg10984625、cg02450613、cg27388911、cg13039082、cg26146027、cg18131851、cg21587469、cg02837456、cg02414499、cg06756436、cg00513735、cg26985203、cg10497345、cg01582438、cg05275995、cg22282038、cg08455031、cg15618108、cg07679571、cg18885210、cg19489885、cg19708201、cg12135573、cg14944647、cg07652786、cg27097923、cg19272720、cg11697474、cg04155793、cg12268453、cg20966148、cg21748751、cg04609801、cg04790887、cg08751994、cg20991819、cg17782848、cg05129477、cg13656701、cg06794355、cg00937510、cg20861822、cg05939926、cg02379079、cg24568905、cg12806497、cg03635685、cg01674874、cg14654643、cg12608727、cg19692782、cg08021727、cg00886954、cg14562086、cg11781854、cg06782350、cg09102447、cg14102634、cg14191024、cg13609861、cg17429457、cg04842472、cg15436354、cg13143743、cg14344955、cg14708905、cg17924243、cg21125143、cg11691189、cg14124917、cg11819637、cg22121495、cg15438951、cg22707438、cg11454936、cg06528823、cg25028639、cg09274091、cg25352592、cg10248231、cg20705390、cg09447675、cg18954541、cg23024326、cg08341924、cg23235497、cg09944899、cg23754382、cg25889918、cg02524010、cg00107630、cg18343556、cg09121415、cg17475304、cg20173984、cg14703482、cg07372500、cg14311811、cg00333583、cg01796478、cg00569982、cg10272968、cg17980283、cg27298551、cg10496030、cg15109744、cg14851345、ch.1.590396F、cg19588570、cg04173182、cg25449585、cg04297264、cg08098127、cg12062572、cg19931206、cg27329737、cg10588471、cg14727452、cg15001633、cg19311431、cg21922841、cg15829826、cg01020079、cg15323936、cg20455931、cg00595039、cg19754694、cg11356589、cg11599526、cg12497187、cg16932672、cg13535231、cg14968844、cg05597838、cg08923060、cg18059321、cg10662787、cg06211272、cg02857557、ch.17.53379803R、cg01061553、cg00088042、cg26547124、cg02691091、cg01103443、cg15830441、cg12660383、cg24276151、cg22722731、cg11183445、cg05679613、cg22281363、cg15114744、cg25325446、cg19067392、cg12604858、cg27133684、cg17948297、cg27298830、cg03671660、cg18349937、cg25706474、cg15149876、cg10171557、cg12184854、cg22463793、cg10601476、cg22616506、 cg09790523、cg04138846、cg03124769、cg19827167、cg18776463、cg21789870、cg10313005、cg19660618、cg04546747、cg17560006、cg08481119、cg02405609、cg04350358、cg14737332、cg12895304、cg27365978、cg08716976、cg17155859、cg12785251、cg12193929、cg03633004、cg16626405、cg24518441、cg19368383、cg20695611、cg02162897、cg05612346、cg10313043、cg26661922、cg26060817、cg08921986、cg21548029、cg17178218、cg02219116、cg01341076、cg15276922、cg22021076、cg11659391、cg14506686、cg23552821、cg01034813、cg00256074、cg22971029、cg00599163、cg18394567、cg09386653、cg15225534、cg10385101、cg04560668、cg09353563、cg01963906、cg00690901、cg19742623、cg05737153、cg02630888、cg04742384、cg14893161、cg16523463、cg13606421、cg12970081、cg22699052、cg22291365、cg26354017、cg16581476、cg10002066、cg07020176、cg06599483、cg19995539、cg14159672、cg08198187、cg16970604、cg02416336、cg27016549、cg02850689、cg04091325、cg13417891、cg12034943、cg24605338、cg03449040、cg16673477、cg26384993、cg05755354、cg26864950、cg13123165、cg13218573、cg26235748、cg05906166、cg08169949、cg07157834、cg19486756、cg25361663、cg04482110、cg15989318、cg06963182、cg01233744、cg10805721、cg15757853、cg26922007、cg20845639、cg07614018、cg11965913、cg00349573、cg07167872、cg07916654、cg19548479、cg08377924、cg18222083和cg00506886。
  2. 如权利要求1所述的方法，其中如下任一位点的甲基化程度与所述受试者的所述结直肠癌预后不良的风险正相关：
    cg08875503、cg21200129、cg01141339、cg02319354、cg04064054、cg10251217、cg17534999、cg21642103、cg17599241、cg07470512、cg27124774、cg26700320、cg08339172、cg12258785、cg25583503、cg11390151、cg21789085、cg05695951、cg03982609、cg16478719、cg17088007、cg01939477、cg18885346、cg08650890、cg10984625、cg02450613、cg27388911、cg13039082、cg26146027、cg18131851、cg21587469、cg02837456、cg02414499、cg06756436、cg00513735、cg26985203、cg10497345、cg01582438、cg05275995、cg22282038、cg08455031、cg15618108、cg07679571、cg18885210、cg19489885、cg19708201、cg12135573、cg14944647、cg07652786、cg27097923、cg19272720、cg11697474、cg04155793、cg12268453、cg20966148、cg21748751、cg04609801、cg04790887、cg08751994、cg20991819、cg17782848、cg05129477、cg13656701、cg06794355、cg00937510、cg20861822、cg05939926、cg02379079、cg24568905、cg12806497、cg03635685、cg01674874、cg14654643、cg12608727、cg19692782、cg08021727、cg00886954、cg14562086、cg11781854、cg06782350、cg09102447、cg14102634、cg14191024、cg13609861、cg17429457、cg04842472、cg15436354、cg13143743、cg14344955、cg14708905、cg17924243、cg21125143、cg11691189、cg14124917、cg11819637、cg22121495、cg15438951、cg22707438、cg11454936、cg06528823、cg25028639、cg09274091、cg25352592、cg10248231、cg20705390、cg09447675、cg18954541、cg23024326、cg08341924、cg23235497、cg09944899、cg23754382、cg25889918、cg02524010、cg00107630、cg18343556、cg09121415、cg17475304、 cg20173984、cg14703482、cg07372500、cg14311811、cg00333583、cg01796478、cg00569982、cg10272968、cg17980283、cg27298551、cg10496030、cg15109744、cg14851345、ch.1.590396F、cg19588570、cg04173182、cg25449585、cg04297264、cg08098127、cg12062572、cg19931206、cg27329737、cg10588471、cg14727452、cg15001633、cg19311431、cg21922841、cg15829826、cg01020079、cg15323936、cg20455931、cg00595039、cg19754694、cg11356589、cg11599526、cg12497187、cg16932672、cg13535231、cg14968844、cg05597838、cg08923060、cg18059321、cg10662787、cg06211272、cg02857557、ch.17.53379803R、cg01061553、cg00088042、cg26547124、cg02691091、cg01103443、cg15830441、cg12660383、cg24276151、cg22722731、cg11183445、cg05679613、cg22281363、cg15114744、cg25325446、cg19067392、cg12604858、cg27133684、cg17948297、cg27298830、cg03671660、cg18349937、cg25706474、cg15149876、cg10171557、cg12184854、cg22463793、cg10601476、cg22616506、cg09790523、cg04138846、cg03124769和cg19827167。
  3. 如权利要求1或2所述的方法，其中如下任一位点的甲基化程度与所述受试者的所述结直肠癌预后不良的风险负相关：
    cg18776463、cg21789870、cg10313005、cg19660618、cg04546747、cg17560006、cg08481119、cg02405609、cg04350358、cg14737332、cg12895304、cg27365978、cg08716976、cg17155859、cg12785251、cg12193929、cg03633004、cg16626405、cg24518441、cg19368383、cg20695611、cg02162897、cg05612346、cg10313043、cg26661922、cg26060817、cg08921986、cg21548029、cg17178218、cg02219116、cg01341076、cg15276922、cg22021076、cg11659391、cg14506686、cg23552821、cg01034813、cg00256074、cg22971029、cg00599163、cg18394567、cg09386653、cg15225534、cg10385101、cg04560668、cg09353563、cg01963906、cg00690901、cg19742623、cg05737153、cg02630888、cg04742384、cg14893161、cg16523463、cg13606421、cg12970081、cg22699052、cg22291365、cg26354017、cg16581476、cg10002066、cg07020176、cg06599483、cg19995539、cg14159672、cg08198187、cg16970604、cg02416336、cg27016549、cg02850689、cg04091325、cg13417891、cg12034943、cg24605338、cg03449040、cg16673477、cg26384993、cg05755354、cg26864950、cg13123165、cg13218573、cg26235748、cg05906166、cg08169949、cg07157834、cg19486756、cg25361663、cg04482110、cg15989318、cg06963182、cg01233744、cg10805721、cg15757853、cg26922007、cg20845639、cg07614018、cg11965913、cg00349573、cg07167872、cg07916654、cg19548479、cg08377924、cg18222083和cg00506886。
  4. 如权利要求1-3任一项所述的方法，其中所述至少一个甲基化位点为权利要求1中所列甲基化位点中的两个或更多个。
  5. 如权利要求1-4任一项所述的方法，其中所述至少一个甲基化位点为权利要求1中所列甲基化位点中的三个或更多个。
  6. 如权利要求1-5任一项所述的方法，其中所述至少一个甲基化位点为权利要求1中所列甲基化位点中的五个或更多个。
  7. 如权利要求1-6任一项所述的方法，其中所述甲基化位点包括cg02162897、cg02857557、cg07020176、cg19708201和cg26985203。
  8. 如权利要求1-7任一项所述的方法，还包括根据每个甲基化位点的权重来评估和/或预测所述受试者的所述结直肠癌的预后。
  9. 如权利要求1-8中任一项所述的方法，其中所述结直肠癌的预后包括死亡风险，和/或复发风险，和/或疾病进展风险。
  10. 如权利要求1-9中任一项所述的方法，其中通过甲基化芯片、焦磷酸测序、甲基化qPCR、或WGBS检测所述甲基化位点的甲基化程度。
  11. 评估结直肠癌治疗方法的治疗效果的方法，包括
    1)以结直肠癌治疗方法对患有结直肠癌的受试者进行治疗；以及
    2)以权利要求1-10任一项所述的方法判断所述结直肠癌的预后，
    其中根据所述结直肠癌的预后来评估所述结直肠癌治疗方法的治疗效果。
  12. 如权利要求11所述的方法，其中所述治疗方法包括药物治疗、手术治疗和/或放射治疗。
  13. 确定结直肠癌受试者的随访策略的方法，包括以权利要求1-10任一项所述的方法判断所述结直肠癌预后不良的风险，并根据所述结直肠癌预后不良的风险来确定所述随访策略。
  14. 判断结直肠癌预后的试剂盒，包括用于检测至少一个权利要求1中所列甲基化位点的甲基化程度的试剂。
  15. 如权利要求14所述的试剂盒，包括用于检测至少两个、至少三个、或至少五个权利要求1中所列甲基化位点的甲基化程度的试剂。
  16. 如权利要求14或15所述的试剂盒，其中所述甲基化位点包括cg02162897、cg02857557、cg07020176、cg19708201和cg26985203。
  17. 如权利要求14-16任一项所述的试剂盒，其为甲基化芯片形式。
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