DNA甲基化检测助力肿瘤早期筛查和诊断

doi:10.16150/j.1671-2870.2023.04.011

摘要/Abstract

摘要：

DNA甲基化是国内外癌症早筛采用的常用检测指标。在致癌因素的作用下，抑癌基因启动子区的高甲基化可导致基因表达下调或关闭，从而激活原癌基因的表达，促使肿瘤发生。用于DNA甲基化检测的样本主要采用脱落细胞、血液样本和石蜡包埋组织。常用的检测方法包括特异性聚合酶链反应（methylation-specific polymerase chain reaction, MSP）、核酸质谱、甲基化芯片、亚硫酸氢盐测序、第二代测序技术等。与突变检测相比，DNA甲基化的检测优势在于其肿瘤特异性高、可检测位点多、信号丰度高，且可实现组织溯源。目前，DNA甲基化检测被主要运用在临床脑胶质瘤用药指导、肺癌辅助诊断和高危分流、膀胱癌高危分流和复发监测、宫颈癌高危分流、结直肠癌和胃癌早期筛查及复发监测。在实现大规模临床常规应用前，需注意精准定位DNA甲基化检测的应用场景，相对于耐受性良好的内镜检查，其高危分流的刚需性可能会下降。在辅助诊断病理诊断灵敏度较低的肿瘤、肿瘤监测和预后评估方面，DNA甲基化检测有着重要的作用。随着检测流程和质量管理的规范，DNA甲基化检测会被更广泛地运用于临床，提高肿瘤的早筛和诊断灵敏度。

关键词: 甲基化, 恶性肿瘤, 肿瘤筛查, 早期诊断

Abstract:

DNA methylation is one of the most widely used indicator for early cancer screening. Under the influence of carcinogens, hypermethylation of the promoter region of tumor suppressor genes may lead to downregulation or silencing of gene expression, thereby activating the expression of proto-oncogenes and promoting tumorigenesis. The samples used for DNA methylation detection are mainly exfoliated cells, blood samples and paraffin-embedded tissues. Commonly used detection methods include: methylation-specific polymerase chain reaction (MSP), nucleic acid mass spectrometry, methylation chip, bisulfite sequencing, next-generation sequencing, etc. Compared with mutation detection, the advantages of DNA methylation detection are that it has higher tumor specificity, more detectable sites, higher signal quality, and can achieve tissue traceability. Currently, it is mainly used clinically for medication guidance for brain glioma, aid in diagnosis and high-risk triage for lung cancer, high-risk triage and recurrence monitoring for bladder cancer, high-risk triage for cervical cancer, and early screening and recurrence monitoring for colorectal cancer and gastric cancer. Before large-scale clinical routine application, accurately positioning of the application scenarios for DNA methylation detection must be considered. For well-tolerated endoscopy, the rigid need of DNA methylation detection for high-risk triage may be reduced. DNA methylation detection plays an important role in assisting in the diagnosis of tumors with low pathological diagnosis sensitivity, tumor monitoring and prognosis assessment. With the standardization of testing procedures and quality management, DNA methylation detection will be more widely used to improve the early screening and diagnostic sensitivity of tumors.

Key words: Methylation, Malignant tumors, Cancer screening, Early diagnosis

中图分类号:

R730.4
R56

刘益飞. DNA甲基化检测助力肿瘤早期筛查和诊断[J]. 诊断学理论与实践, 2023, 22(04): 393-401.

LIU Yifei. DNA methylation detection assists early screening and diagnosis of tumors[J]. Journal of Diagnostics Concepts & Practice, 2023, 22(04): 393-401.

图/表 2

表1

表2

基因甲基化检测靶标基因

序号	基因缩写	基因名称	生物学作用	异常甲基化的主要肿瘤类型
1	MGMT	O6-甲基鸟嘌呤-DNA-甲基转移酶	该基因编码的蛋白质是一种DNA修复蛋白，参与细胞抵御烷基化剂的诱变和毒性。	脑胶质瘤、胶质母细胞瘤、结直肠癌、肺癌、淋巴瘤
2	SHOX2	矮小同源盒基因	在胚胎形成期对骨骼、心脏和神经系统的发育作用重大。	肺癌、乳腺癌、肾癌、前列腺癌、结直肠癌、胶质瘤、头颈部癌
3	RASSF1A	Ras相关区域家族1A	抑癌基因，其调控的靶基因涉及基因转录、信号转导、细胞骨架、细胞周期、细胞黏附及凋亡等多方面。	肺癌、乳腺癌、头颈部鳞状细胞癌、肾细胞癌、神经母细胞瘤、子宫内膜癌
4	PTGER4	前列腺素E受体	该基因编码的蛋白质可以激活T细胞因子信号转导，介导PGE2诱导的早期生长反应1（EGR1）的表达，调节环氧化酶2 mRNA的水平和稳定性，并导致糖原合成酶激酶3的磷酸化。	肺癌、结直肠癌、恶性间皮瘤、乳腺癌、头颈部肿瘤
5	Twist1	twist家族bHLH转录因子1	该基因在多种人类癌症中高度甲基化和过表达，编码的蛋白质促进肿瘤细胞的侵袭和转移，以及转移复发。	膀胱癌、乳腺癌、胶质瘤、胃癌、胰腺癌、结直肠癌
6	ASTN1	星形肌动蛋白1	该基因是一种神经元粘附分子，在发育中的大脑皮质区域（包括大脑、海马体、小脑和嗅球），神经胶质引导年轻的有丝分裂后神经母细胞迁移所需。	宫颈癌、肝癌、胶质瘤、子宫内膜癌
7	DLX1	无远端同源框1	该基因可能作为来自多个TGF-β超家族成员信号的转录调节因子发挥作用。编码的蛋白质可能在颅面模式的控制以及前脑抑制性神经元的分化和存活中起作用。	宫颈癌、乳腺癌、前列腺癌、肾癌、卵巢癌、胃癌
8	ITGA4	整合素亚单位α4	在细胞表面粘附和信号转导中起作用，是治疗多发性硬化、克罗恩病和炎症性肠病的治疗靶点。	宫颈癌、胃癌、结直肠癌、胰腺癌、肺癌、口腔癌
9	RXFP3	松弛素家族肽受体3	该蛋白正向调节胞质分裂。	宫颈癌、子宫内膜癌、胶质瘤、乳腺癌
10	SOX17	SRY-box转录因子17	该基因编码SOX转录因子家族的一个成员，参与胚胎发育的调节和细胞命运的决定。	宫颈癌、肺癌、胃癌、食管癌、乳腺癌
11	ZNF671	锌指蛋白671	使DNA结合转录因子活性和RNA聚合酶Ⅱ顺式调节区序列特异性DNA结合活性。	宫颈癌、卵巢癌、喉癌、鼻咽癌、肾癌、食管癌
12	PAX1	配对盒1	该基因可能是肿瘤抑制基因，在胚胎发生期间的模式形成中起作用，并可能对脊柱的发育至关重要。	宫颈癌、卵巢癌、食管癌、头颈部癌、子宫内膜癌、口腔癌
13	JAM3	连接粘附分子3	该基因内含子的突变与脑出血性破坏、室管膜下钙化和先天性白内障相关。	宫颈癌、食管癌、结直肠癌、前列腺癌
14	BMPR1A	骨形态发生蛋白受体类型1A	通过与2种不同类型的丝氨酸(苏氨酸)激酶受体形成异聚复合物来转导信号。	肝癌、头颈癌、唾液腺肿瘤、乳腺癌、卵巢癌、结直肠癌
15	PLAC8	胎盘相关8	该基因促进胰腺神经内分泌肿瘤和肺癌细胞生长，通过阻断自噬促进乳腺癌耐药，诱导鼻咽癌上皮间质转化等。	肝癌、宫颈癌、肺癌、乳腺癌、胰腺癌
16	Septin9	隔膜蛋白9	该基因是卵巢肿瘤抑制基因的候选基因。该基因的突变可引起遗传性神经痛性肌萎缩，该基因的染色体易位导致急性粒-单核细胞白血病。	结直肠癌、胃癌、宫颈癌、肝癌、头颈部癌、乳腺癌、膀胱癌、肝癌、鼻咽癌
17	RNF180	环指蛋白180	预测参与去甲肾上腺素代谢过程、蛋白酶体泛素依赖性蛋白分解代谢过程的正向调节、以及5 -羟色胺的代谢过程。	胃癌、肺癌、肝癌、结直肠癌、乳腺癌
18	BCAT1	支链氨基酸转氨酶1	该基因缺陷可引起高脂血症和高亮氨酸异亮氨酸血症。	结直肠癌、胰腺癌、胶质母细胞瘤、头颈部癌、肺癌
19	SDC2	多配体蛋白聚糖2	介导细胞结合、细胞信号传导和细胞骨架组织，通过其细胞外基质蛋白受体参与细胞增殖、细胞迁移和细胞-基质相互作用。	结直肠癌、胃癌、卵巢癌、宫颈癌、食管癌
20	SFRP2	分泌型卷曲相关蛋白2	可作为Wnt信号通路的可溶性调节剂，该基因甲基化是结直肠癌存在的潜在标志物。	胃癌、结直肠癌、肺癌、前列腺癌、黑素瘤、骨肉瘤、口腔鳞状细胞癌
21	TFPI2	组织因子途径抑制剂2	抑癌基因，该基因编码的蛋白可抑制多种丝氨酸蛋白酶，包括因子Ⅶa/组织因子、因子Ⅹa、纤溶酶、胰蛋白酶、糜蛋白酶和血浆激肽释放酶。	胃癌、结直肠癌、肝癌、宫颈癌、食管癌、肺癌
22	NPY	神经肽Y	该基因编码的神经肽影响许多生理过程，包括皮质兴奋性、应激反应、食物摄入、昼夜节律和心血管功能，可能是各种代谢性疾病和心血管疾病的危险因素。	胃癌、结直肠癌、胰腺癌、乳腺癌、头颈癌、口腔鳞状细胞癌
23	FGF5	成纤维细胞生长因子5	癌基因，参与多种生物学过程，包括胚胎发育、细胞生长、形态发生、组织修复、肿瘤生长和侵袭等。	胃癌、结直肠癌、食管癌、乳腺癌、肺癌、胶质瘤
24	PDX1	胰十二指肠同源盒1	该基因编码转录激活因子，激活包括胰岛素、生长抑素、葡萄糖激酶、胰岛淀粉样多肽和葡萄糖转运蛋白2型，并参与胰腺的早期发育。	胃癌、结直肠癌、胰腺癌、卵巢癌
25	BMP3	骨形成蛋白3	结合各种TGF-β受体，导致调节基因表达的SMAD家族转录因子的募集和激活。通过调节其他配体对TGF-β受体的利用，抑制成骨细胞分化，并负性调节骨密度。	胃癌、结直肠癌、胆道癌、胰腺癌、乳腺癌
26	NDRG4	N-myc下调基因家族成员4	该基因编码的蛋白对原代星形胶质细胞的细胞周期进展和生存是必需的，并可能参与血管平滑肌细胞有丝分裂信号的调节。	胃癌、结直肠癌、胰腺癌、食管癌、甲状腺癌

表2

参考文献 30

[1]	SUNG H, FERLAY J, SIEGEL R L, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J]. CA Cancer J Clin, 2021, 71(3):209-249. doi: 10.3322/caac.v71.3 URL
[2]	LI S, TOLLEFSBOL T O. DNA methylation methods: Global DNA methylation and methylomic analyses[J]. Methods, 2021, 187:28-43. doi: 10.1016/j.ymeth.2020.10.002 pmid: 33039572
[3]	LIU MC, OXNARD GR, KLEIN EA, et al. Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA[J]. Ann Oncol, 2020, 31(6):745-759. doi: 10.1016/j.annonc.2020.02.011 pmid: 33506766
[4]	YIZHAK K, AGUET F, KIM J, et al. RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues[J]. Science, 2019, 364(6444):eaaw0726. doi: 10.1126/science.aaw0726 URL
[5]	杨林林, 蒋涛, 隋亚鑫, 等. DNA甲基化检测技术[J]. 标记免疫分析与临床, 2020, 27(5):898-904.
	YANG L L, JIANG T, SUI Y X, et al. DNA Methylation Detection Technology[J]. Lab Immunoassay & Clin Med, 2020, 27(5):898-904.
[6]	杨晋生, 古选民, 方军超. 胶质瘤MGMT启动子甲基化及其临床意义[J]. 临床肿瘤学杂志, 2019, 24(2):153-157.
	YANG J S, GU X M, FANG J C. Methylation of MGMT promoter in glioma and its clinical significance[J]. Chin Clin Oncol, 2019, 24(2):153-157.
[7]	樊星, 刘幸, 柴睿超, 等. 2020版美国国立综合癌症网络脑胶质瘤临床实践指南解读[J]. 中华神经外科杂志, 2021, 37(6): 541-545.
	FAN X, LIU X, CHAI R C, et al. Interpretation of the 2020 National Comprehensive Cancer Network Clinical Practice Guidelines for Gliomas in the United States[J]. Chin J Neurosurg, 2021, 37(6): 541-545.
[8]	ZHANG C, YU W, WANG L, et al. DNA methylation analysis of the SHOX2 and RASSF1A panel in bronchoalveolar lavage fluid for lung cancer diagnosis[J]. J Cancer, 2017, 8(17):3585-3591. doi: 10.7150/jca.21368 pmid: 29151944
[9]	陈瑞英, 刘雅, 孙婷, 等. 支气管肺泡灌洗液中SHOX2和RASSF1A基因甲基化检测对肺癌的诊断价值[J]. 郑州大学学报(医学版), 2019, 54(5):732-736.
	CHEN R Y, LIU Y, SUN T, et al. Value of DNA methylation analysis of SHOX2 and RASSF1A in bron-choalveolar lavage fluid in diagnosis of lung cancer[J]. J Zhengzhou Univ( Med Sci), 2019, 54(5):732-736.
[10]	张彤, 王晓伟, 吴俊秀, 等. SHOX2和RASSF1A基因甲基化在非小细胞肺癌小活检样本临床病理诊断中的应用价值[J]. 诊断病理学杂志, 2022, 29(5):389-394.
	ZHANG T, WANG X W, WU J X, et al. Application value of SHOX2 and RASSF1A gene methylation in clinicopathological diagnosis of small biopsy samples of non-small cell lung cancer[J]. J Diag Pathol, 2022, 29(5):389-394.
[11]	SHI J, CHEN X, ZHANG L, et al. Performance evaluation of SHOX2 and RASSF1A methylation for the aid in diagnosis of lung cancer based on the analysis of FFPE specimen[J]. Front Oncol, 2020, 10:565780. doi: 10.3389/fonc.2020.565780 URL
[12]	ZHANG N, LIU Z, LI K, et al. DNA methylation analysis of the SHOX2 and RASSF1A panel using cell-free DNA in the diagnosis of malignant pleural effusion[J]. J Oncol, 2023, 2023:5888844.
[13]	LIANG C, LIU N, ZHANG Q, et al. A detection panel of novel methylated DNA markers for malignant pleural effusion[J]. Front Oncol, 2022, 12:967079. doi: 10.3389/fonc.2022.967079 URL
[14]	XING W, SUN H, YAN C, et al. A prediction model based on DNA methylation biomarkers and radiological characteristics for identifying malignant from benign pulmonary nodules[J]. BMC Cancer, 2021, 21(1):263. doi: 10.1186/s12885-021-08002-4 pmid: 33691657
[15]	TAN W S, FEBER A, SARPONG R, et al. Who should be investigated for haematuria? Results of a contemporary prospective observational study of 3556 patients[J]. Eur Urol, 2018, 74(1):10-14. doi: S0302-2838(18)30184-2 pmid: 29653885
[16]	张晗, 林琳, 杞朝梅, 等. DNA甲基化在宫颈病变和宫颈癌中的临床研究进展[J]. 中国妇幼保健, 2021, 36(20):4873-4875.
	ZHANG H, LIN L, QI C M, et al. Clinical research progress of DNA methylation in cervical lesions and cervical cancer[J]. Matern Child Health Care China, 2021, 36(20):4873-4875.
[17]	SCHMITZ M, EICHELKRAUT K, SCHMIDT D, et al. Performance of a DNA methylation marker panel using liquid-based cervical scrapes to detect cervical cancer and its precancerous stages[J]. BMC Cancer, 2018, 18(1):1197. doi: 10.1186/s12885-018-5125-8 pmid: 30509219
[18]	SHI L, YANG X, HE L, et al. Promoter hypermethylation analysis of host genes in cervical intraepithelial neoplasia and cervical cancers on histological cervical specimens[J]. BMC Cancer, 2023, 23(1):168. doi: 10.1186/s12885-023-10628-5 pmid: 36803573
[19]	LI B, GUO R, LAI T, et al. The application of PAX1 methylation detection and HPV E6/E7 mRNA detection in cervical cancer screening[J]. J Obstet Gynaecol Res, 2021, 47(8):2720-2728. doi: 10.1111/jog.v47.8 URL
[20]	SONG L, YU H, JIA J, et al. A systematic review of the performance of the SEPT9 gene methylation assay in colorectal cancer screening, monitoring, diagnosis and prognosis[J]. Cancer Biomark, 2017, 18(4):425-432. doi: 10.3233/CBM-160321 pmid: 28128742
[21]	XU F, YU S, HAN J, et al. Detection of circulating tumor DNA methylation in diagnosis of colorectal cancer[J]. Clin Transl Gastroenterol, 2021, 12(8):e00386. doi: 10.14309/ctg.0000000000000386 URL
[22]	BURKE C A, LIEBERMAN D, FEUERSTEIN J D. AGA clinical practice update on approach to the use of noninvasive colorectal cancer screening options: commentary[J]. Gastroenterology, 2022, 162(3):952-956. doi: 10.1053/j.gastro.2021.09.075 URL
[23]	XIE L, JIANG X, LI Q, et al. Diagnostic value of methy-lated Septin9 for colorectal cancer detection[J]. Front Oncol, 2018, 8:247. doi: 10.3389/fonc.2018.00247 URL
[24]	LI S, CHEN M, LI Y, et al. Prenatal epigenetics diets play protective roles against environmental pollution[J]. Clin Epigenetics, 2019, 11(1):82. doi: 10.1186/s13148-019-0659-4 pmid: 31097039
[25]	LU P, ZHU X, SONG Y, et al. Methylated Septin 9 as a promising biomarker in the diagnosis and recurrence monitoring of colorectal cancer[J]. Dis Markers, 2022, 2022:7087885.
[26]	LIU W, HU P, LIU J, et al. mSEPT9 can monitor the response and predict the prognosis of stage Ⅳ colorectal cancer patients with liver metastasis undergoing potentially curative surgery[J]. J Surg Res, 2021, 267:485-494. doi: 10.1016/j.jss.2021.06.008 URL
[27]	XU H, CHEN H, HU J, et al. Feasibility of quantification based on novel evaluation with stool DNA and fecal immunochemical test for colorectal cancer detection[J]. BMC Gastroenterol, 2022, 22(1):384. doi: 10.1186/s12876-022-02470-z pmid: 35963995
[28]	陈旻, 黄一鸣, 赵晖, 等. 胰腺癌血浆循环游离DNA甲基化预测模型的构建及应用[J]. 肿瘤, 2021, 41(08): 541-549.
	CHEN Min, HUANG Yiming, ZHAO Hui, et al. Construction and application of plasma cell-free DNA methylation prediction model for pancreatic cancer[J]. Tumor, 2021, 41(08): 541-549. doi: 10.3781/j.issn.1000-7431.2021.2105-0331
[29]	刘玉玲, 梁顺顺, 徐慧莉, 等. miR-103/MTHFR轴抑制肝细胞癌中LINE-1基因的甲基化水平[J]. 肿瘤, 2022, 42(02): 93-107. doi: 10.3781/j.issn.1000-7431.2022.2012-1124
	LIU Yuling, LIANG Shunshun, XU Huili, et al. MiR-103/MTHFR axis inhibits the DNA methylation of LINE-1 gene in hepatocellular carcinoma[J]. Tumor, 2022, 42(2): 93-107.
[30]	郑荣寿, 孙可欣, 张思维, 等. 2015年中国恶性肿瘤流行情况分析[J]. 中华肿瘤杂志, 2019, 41(1):19-28.
	ZHENG R S, SUNK X, ZHANG S W, et al. Report of cancer epidemiology in China, 2015[J]. Chin J Oncol, 2019, 41(1):19-28.

序号	肿瘤类型	样本类型	注册企业	检测指标	注册证
个性化用药
1	脑胶质瘤	石蜡组织	基因科技（上海）	MGMT	20193400101
辅助诊断
2	肺癌	肺泡灌洗液脱落细胞（补充临床实验样本痰液、胸水、穿刺样本）	上海甲预生命（上海透景生命控股子公司）	SHOX2/RASSF1A	20173403354
高危分流
3	膀胱癌	尿液脱落细胞	安徽达健医学	Twist1	20223401301
4	宫颈癌	宫颈脱落细胞	上海捷诺生物	ASTN1/DLX1/ITGA4/RXFP3/SOX17/ZNF671	20223401036
5	宫颈癌	宫颈脱落细胞	北京起源聚禾	PAX1/JAM3	20233400253
6	宫颈癌	宫颈脱落细胞	湖南宏雅基因	PAX1	20233400944
7	肺癌	血液	北京艾克伦	SHOX2/RASSF1A/PTGER4	20223400203
早期筛查
8	胃癌	血液	博尔成（北京）	Septin9/RNF180	20203400447
9	结直肠癌	血液	博尔成（北京）	Septin9	20153401481
10	结直肠癌	血液	苏州为真生物	Septin9	20183400103
11	结直肠癌	血液	上海甲预生命（上海透景生命控股子公司）	Septin9	20193400316
12	结直肠癌	血液	深圳优圣康	Septin9	20233400151
13	结直肠癌	血液	北京艾克伦	Septin9/SDC2/BCAT1	20223401371
14	结直肠癌	粪便	杭州诺辉健康	粪便潜血+Kras突变+BMP3/NDRG4甲基化	20203400845
15	结直肠癌	粪便	广州康立明	SDC2	20183400506
16	结直肠癌	粪便	厦门艾德生物	SDC2	20213400007
17	结直肠癌	粪便	上海锐翌生物	SDC2/SFRP2	20223400637
18	结直肠癌	粪便	武汉艾米森	SDC2/TFPI2	20223400373
19	结直肠癌	粪便	安徽达健医学	SDC2/NPY/FGF5/PDX1	20233400970