结直肠癌基因DNA甲基化标志物筛查的价值

摘要

结直肠癌是全球最常见的恶性肿瘤之一, 通过人群早期筛查和诊断结直肠癌是疾病防治的关键. 近年来发现了一系列启动子高甲基化的相关基因, 在结直肠癌的发生发展中发挥着重要的作用. 多数在肿瘤组织中发生高甲基化基因的启动子片段在血液、粪便标本中同样被检测到. 这些高甲基化基因对结直肠癌诊断的敏感性和特异性均明显优于传统筛查指标如血癌胚抗原和粪便潜血等. 本文将重点介绍目前结直肠癌基因DNA甲基化标志物筛查的研究进展以及将来可能的研究方向.

关键词: 结直肠癌; DNA甲基化; 筛查

核心提示: 结直肠癌患者血液、粪便标本中包括细胞质分裂基因9(Septin9)、分泌型卷曲相关蛋白2(secreted frizzled related protein 2)在内多个基因甲基化的检出率明显高于健康对照者. 甚至早在TNM 1期或Duke A期结直肠癌患者中也能检测到高甲基化的DNA片段. 将不同基因的甲基化检测和突变、血红蛋白等指标结合起来综合分析可进一步提高检测的敏感性.

Value of DNA methylation markers in colorectal cancer screening

Meng Xue, Liang-Jing Wang

Meng Xue, Liang-Jing Wang, Department of Gastroenterology, the Second Affiliated Hospital, School of Medicine and Institute of Gastroenterology, Zhejiang University, Hangzhou 310009, Zhejiang Province, China

Supported by: National Natural Science Foundation of China, Nos. 81472214, 8130207 and 81272678; Science and Technology Innovation Team of Zhejiang Province, No. 2013TD13.

Correspondence to: Liang-Jing Wang, Chief Physician, Researcher, Department of Gastroenterology, the Second Affiliated Hospital, School of Medicine and Institute of Gastroenterology, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, Zhejiang Province, China. wanglj76@hotmail.com

Received: April 28, 2015
Revised: May 21, 2015
Accepted: June 1, 2015
Published online: October 18, 2015

Abstract

Colorectal cancer (CRC) is one of the most common malignant tumors in the world. Recognizing CRC at an early stage by population-based screening is crucial in the prevention and treatment of CRC. Numerous candidate genes, which play important roles in the development and progression of CRC, have been found to be hyper-methylated in the promoter regions in recent studies. The promoter fragments of those hyper-methylated genes in tumor tissues have also been detected in the blood and fecal specimens, with higher sensitivity and specificity than traditional markers in the screening of CRC, including carcino-embryonic antigen (CEA) and fecal occult blood test. Here, we will discuss what we have already known about the DNA methylation markers for CRC screening and the potential research direction in the future.

Key Words: Colorectal cancer; DNA methylation; Screening

0 引言

结直肠癌是消化系最常见的恶性肿瘤之一, 50岁以下年轻人群出现结直肠癌的比例也有明显的增加[1]. 我国结直肠癌的发病率低于西方国家, 但在一些结直肠癌高发区域, 其发病率已接近胃癌和肝癌[2]. 然而多数患者在诊断时已进展到晚期, 部分患者丧失了手术根治的机会. 因此, 寻找和建立结直肠癌筛查和早期诊断的有效手段是研究热点.

粪便潜血、血清肿瘤标志物[如癌胚抗原(carcino embryonie antigen, CEA)]是目前结直肠癌非侵入性筛查的主要手段. 然而, 其诊断效率和准确性仍不甚令人满意[3]. 结直肠镜侵入性检查的准确性相对较高, 却因其检查前肠道准备和检查过程的不适感而难以在人群中大规模开展[4]. 基因DNA高甲基化是结直肠癌发生发展中的一个早期事件, 既往在结直肠癌肿瘤组织的研究已筛选到大量异常启动子区高甲基化的基因[5]. 然而, 由于肿瘤组织取材的不便而不适合用于人群中筛查. 近十年来, 随着DNA甲基化检测技术的不断改进, 血液、粪便、尿液等标本中均可检测到不同基因DNA高频率甲基化, 使其应用于结直肠癌筛查成为可能[6]. 本文将对近年来结直肠癌DNA甲基化标志物的筛查方法和临床价值做一综述.

1 结直肠癌单基因甲基化检测

错配修复基因1(mutl homolog l, MLH1)是结直肠癌中最早报道的异常血液基因DNA甲基化指标. 2001年的一项研究[7]对18例结直肠癌患者血清中提取的DNA进行甲基化特异性PCR检测, 有3例检测到高甲基化的MLH1. 此后, 在结直肠癌患者的血液样本中, 检测到不同基因启动子的异常甲基化[7-35](表1), 如细胞周期蛋白依赖激酶抑制剂2A(cyclin-dependent kinase inhibitor 2A, CDKN2A)[10]、无芒相关同源盒基因4(aristaless-like homeobox 4, ALX4)[13]、Runt相关转录因子3(Runt-related transcription factor 3, RUNX3)[11]等, 其甲基化的频率均明显高于健康对照者. Epi proColon是最早商业化并进入临床试验, 以细胞质分裂基因9(Septin9)基因为基础开发的甲基化检测试剂盒. Tóth等[22]采用Epi proColon对结直肠癌和健康对照患者的血浆进行甲基化检测, 92例患者血浆中甲基化Septin9的阳性率高达95.7%(88/92), 而无肿瘤的对照者的阳性率仅为15.2%(14/92). 在随后两项类似的研究[24,25]中, 虽然结直肠癌患者血浆Septin9甲基化的比例低于这项研究, 但仍明显高于健康对照者.

表1 结直肠癌筛查中血液为基础的甲基化指标.

基因	作者, 年份	标本类型	检测方法	健康对照		结直肠癌		AUC面积	P值
				总例数	甲基化n(%)	总例数	甲基化n(%)
单基因
MLH1	Grady, 2001	血清	MSP	N/A	N/A	18	3(16.7)	N/A	N/A
	Leung, 2005	血清	MethyLight	N/A	N/A(2.4)	N/A	N/A(42.9)	0.71	0.001
	Wallner, 2006	血清	MethyLight	20	0(0.0)	103	13(12.6)	N/A	0.090
CDKN2A	Zou, 2002	血清	MSP	10	0(0.0)	20	14(70.0)	N/A	0.001
	Tan, 2007	血浆	MSP	10	0(0.0)	17	12(70.6)	N/A	0.001
CDH4	Miotto, 2004	全血	MSP	17	0(0.0)	46	32(69.6)	N/A	0.001
HLTF	Leung, 2005	血清	MethyLight	N/A	N/A(7.3)	N/A	N/A(32.7)	0.63	0.015
	Wallner, 2006	血清	MethyLight	20	0(0.0)	103	31(30.1)	N/A	0.005
APC	Leung, 2005	血清	MethyLight	N/A	N/A(0.0)	N/A	N/A(6.1)	0.53	0.210
ALX4	Ebert, 2006	血清	qMSP	30	9(30.0)	20	25(83.3)	0.84	0.001
	Tänzer, 2010	血浆	MethyLight	22	13(59.1)	5	4(80.0)	N/A	0.289
	He, 2010	血浆	MethyLight	170	11(6.5)	182	87(47.8)	N/A	0.001
HPP1	Wallner, 2006	血清	MethyLight	20	0(0.0)	103	24(23.3)	N/A	0.016
RUNX3	Tan, 2007	血浆	MSP	10	0(0.0)	17	11(64.7)	N/A	0.001
RASSF1a	Tan, 2007	血浆	MSP	10	0(0.0)	17	4(23.5)	N/A	0.097
	Cassinotti, 2012	血浆	Methyl-microarray	30	17(56.7)	30	26(86.7)	N/A	0.010
CDH1	Tan, 2007	血浆	MSP	10	0(0.0)	17	3(17.6)	N/A	0.159
Septin9	Lofton-Day, 2008	血浆	qMSP	179	25(14.0)	133	92(69.2)	0.8	0.001
	Grützmann, 2008	血浆	qMSP	285	25(8.8)	378	193(51.1)	N/A	0.001
	deVos, 2009	血浆	qMSP	327	45(13.8)	187	138(73.8)	N/A	0.001
	Tänzer, 2010	血浆	MethyLight	34	4(11.8)	33	27(81.8)	N/A	0.001
	He, 2010	血浆	MethyLight	170	6(3.5)	182	136(74.7)	N/A	0.001
	Warren, 2011	血浆	qMSP	94	11(11.7)	50	45(90.0)	N/A	0.001
	Ahlquist, 2012	血浆	qMSP	48	13(27.1)	30	18(60.0)	N/A	0.004
	Tóth, 2012	血浆	Epi proColon	92	14(15.2)	92	88(95.7)	N/A	0.001
	Su, 2014	全血	MSP	62	4(6.5)	172	152(88.4)	N/A	0.001
	Church, 2014	血浆	Epi proColon	1457	126(8.6)	53	27(50.9)	N/A	0.001
	Potter, 2014	血浆	Epi proColon	444	97(21.8)	44	30(68.2)	N/A	0.001
TMEFF2	Lofton-Day, 2008	血浆	qMSP	179	55(30.7)	133	86(64.7)	0.72	0.001
	He, 2010	血浆	MethyLight	170	8(4.7)	182	129(70.9)	N/A	0.001
Vimentin	Li, 2009	血浆	Methyl-BEAMing	110	8(5.3)	81	48(40.9)	0.81	0.001
Wif-1	Lee, 2009	血浆	MSP	276	26(9.4)	243	89(36.6)	0.64	0.001
AKAP12	Liu, 2010	血清	MS-HRM	50	4(8.0)	100	48(48.0)	N/A	0.001
SFRP2	Tang, 2011	血浆	MSP	30	0(0.0)	169	113(66.9)	N/A	0.001
NeuroG1	Herbst, 2011	血清	MethyLight	45	9(20.0)	97	61(62.9)	0.73	0.001
TFPI2	Hibi, 2011	血清	qMSP	20	0(0.0)	215	39(18.1)	N/A	0.037
DLC1	Wu, 2011	血清	MSP	45	4(8.9)	85	36(42.4)	N/A	0.001
CYCD2	Cassinotti, 2012	血浆	Methyl-microarray	30	14(46.7)	30	28(93.3)	N/A	0.001
HIC1	Cassinotti, 2012	血浆	Methyl-microarray	30	2(6.7)	30	19(63.3)	N/A	0.001
PAX5	Cassinotti, 2012	血浆	Methyl-microarray	30	19(63.3)	30	29(96.7)	N/A	0.001
RB1	Cassinotti, 2012	血浆	Methyl-microarray	30	14(46.7)	30	27(90.0)	N/A	0.001
SRBC	Cassinotti, 2012	血浆	Methyl-microarray	30	2(6.7)	30	10(33.3)	N/A	0.01
SDC2	Oh, 2013	血清	qMSP	125	6(4.8)	131	114(87.0)	N/A	0.001
Rassf2	Lyu, 2014	血清	MSP	59	0(0.0)	59	16(27.1)	N/A	0.001
SFRP1	Lyu, 2014	血清	MSP	59	0(0.0)	59	18(30.5)	N/A	0.001
CHAM	Pedersen, 2014	血浆	qMSP	74	5(6.8)	73	40(54.8)	N/A	0.001

MSP: 甲基化特异性PCR; MethyLight: 荧光定量甲基化; qMSP: 定量MSP; Methyl-microarray: 甲基化微阵列; Epi proColon: 商业化的Septin9基因甲基化检测试剂盒; MS-HRM: 甲基化敏感的高分辨率溶解曲线; N/A: 无记录; AUC: 曲线下面积.

全身各部位器官的恶性肿瘤均会引起血液中甲基化指标的异常. 因此, 血液中一些甲基化的指标并不能特异性地反映来源于结直肠癌. 此外, 在结直肠癌的早期阶段, 释放到血液循环中的异常甲基化片段含量过低而难以检测出来. 而在早期或晚期的结直肠癌中, 均有一定量的异常甲基化片段从癌肿病灶脱落到粪便中[21]. 因此, 粪便DNA甲基化的指标可能在结直肠癌的早期诊断中更有意义. Müller等[36]在2004年发表的研究结果最早对结直肠癌患者的粪便DNA甲基化指标进行了报道. 此研究采用了一种高通量的甲基化定量MethyLight检测方法, 对粪便中提取的DNA进行10个基因的甲基化检测, 发现结直肠癌患者中分泌型卷曲相关蛋白2(secreted frizzled related protein 2, SFRP2)、孕激素受体(progesterone receptor, PGR)和SFRP5基因的出现甲基化的比例明显高于正常对照人群. 此后, 其他研究在结直肠癌患者的粪便中鉴定了更多DNA甲基化的指标(表2)[26,29,37-66].

表2 结直肠癌筛查中粪便为基础的甲基化指标.

基因	作者, 年份	检测方法	健康对照		结直肠癌		AUC面积	P值
			总例数	甲基化n(%)	总例数	甲基化n(%)
单基因
SFRP2	Müller, 2004	MethyLight	13	3(23.1)	23	19(82.6)	N/A	0.001
	Huang, 2007	MSP	24	1(4.2)	52	49(94.2)	N/A	0.001
	Wang, 2008	MSP	30	2(6.7)	69	60(87.0)	N/A	0.001
	Nagasaka, 2009	COBRA	113	9(8.0)	84	53(63.1)	N/A	0.001
	Chang, 2010	MSP	31	0(0.0)	30	18(60.0)	N/A	0.001
	Tang, 2011	MSP	30	2(6.7)	169	142(84.0)	N/A	0.001
	Zhang, 2014	MSP	30	1(3.3)	48	29(60.4)	N/A	0.001
	Lu, 2014	MSP	40	4(10.0)	56	32(57.1)	N/A	0.001
PGR	Müller, 2004	MethyLight	26	8(30.8)	23	18(78.3)	N/A	0.001
SFRP5	Müller, 2004	MethyLight	26	9(34.6)	23	18(78.3)	N/A	0.002
HIC1	Lenhard, 2005	MSP	31	0(0.0)	26	11(42.3)	N/A	0.001
Vimentin	Chen, 2005	MSP	107	8(7.5)	94	43(45.7)	N/A	0.001
	Itzkowitz, 2007	MSP	122	16(13.1)	40	29(72.5)	N/A	0.001
	Itzkowitz, 2008	MSP	363	62(17.1)	82	63(76.8)	N/A	0.001
	Baek, 2009	MSP	37	0(0.0)	60	23(38.3)	N/A	0.001
	Li, 2009	Methyl-BEAMing	38	2(5.3)	22	9(40.9)	N/A	0.001
	Zhang, 2011	MSP	30	0(0.0)	60	32(53.3)	N/A	0.001
	Amiot, 2014	qMSP	157	0(0.0)	90	29(32.2)	N/A	0.001
	Kisiel, 2013	QuARTS	N/A	N/A(11.0)	N/A	N/A(89.0)	0.97	N/A
HLTF	Itzkowitz, 2007	MSP	122	9(7.4)	40	15(37.5)	N/A	0.001
HPP1	Huang, 2007	MSP	24	0(0.0)	52	37(71.2)	N/A	0.001
MGMT	Huang, 2007	MSP	24	0(0.0)	52	25(48.1)	N/A	0.001
	Baek, 2009	MSP	37	5(13.5)	60	31(51.7)	N/A	0.001
	Azuara, 2010	MSP & Melting curve	15	0(0.0)	28	9(32.1)	N/A	0.014
	Kang, 2011	MSP	26	1(3.8)	69	38(55.1)	N/A	0.001
CDKN2A	Abbaszadegan, 2007	MSP	20	0(0.0)	25	5(20.0)	N/A	0.034
	Chang, 2010	MSP	31	1(3.2)	30	12(40.0)	N/A	0.001
	Azuara, 2010	MSP & Melting curve	13	0(0.0)	30	9(30.0)	N/A	0.026
	Kang, 2011	MSP	26	0(0.0)	69	36(52.2)	N/A	0.001
SFRP1	Zhang, 2007	MSP	15	2(13.3)	29	18(62.1)	N/A	0.002
	Kim, 2009	qMSP	15	0(0.0)	20	11(55.0)	N/A	0.001
	Salehi, 2012	MSP	25	2(8.0)	25	13(52.0)	N/A	0.006
EN1	Mayor, 2009	Melting curve	30	1(3.3)	30	8(26.7)	N/A	0.011
TFPI2	Glöckner, 2009	qMSP	87	11(12.6)	84	67(79.8)	N/A	0.001
	Zhang, 2011	MSP	30	4(13.3)	60	45(75.0)	N/A	0.001
GATA4	Li, 2009	Methyl-BEAMing	110	8(5.3)	81	48(40.9)	0.81	0.001
NDRG4	Melotte, 2009	qMSP	75	3(4.0)	75	42(56.0)	N/A	0.001
	Lu, 2014	MSP	40	1(2.5)	56	16(28.6)	N/A	0.001
	Kisiel, 2013	QuARTS	N/A	N/A(11.0)	N/A	N/A(100.0)	0.85	N/A
ITGA4	Ausch, 2009	qMSP	28	6(21.4)	75	69(92.0)	N/A	0.001
	Chang, 2010	MSP	31	0(0.0)	30	11(36.7)	N/A	0.001
B4GALT1	Kim, 2009	qMSP	10	2(20.0)	16	9(56.3)	N/A	0.069
OSMR	Kim, 2009	qMSP	96	4(4.2)	89	35(39.3)	N/A	0.001
	Zhang, 2011	MSP	30	0(0.0)	60	41(68.3)	N/A	0.001
	Bosch, 2012	qMSP	101	7(6.9)	65	25(38.5)	N/A	0.001
MLH1	Baek, 2009	MSP	37	0(0.0)	60	18(30.0)	N/A	0.001
RASSF2	Nagasaka, 2009	COBRA	113	6(5.3)	84	38(45.2)	N/A	0.001
ESR1	Kato, 2009	nMSP	N/A	N/A	49	32(65.3)	N/A	N/A
RARB2	Azuara, 2010	MSP & Melting curve	13	0(0.0)	34	11(32.4)	N/A	0.019
APC	Azuara, 2010	MSP & Melting curve	15	0(0.0)	28	9(32.1)	N/A	0.014
MAL	Kang, 2011	MSP	26	1(3.8)	69	54(78.3)	N/A	0.001
PHACTR3	Bosch, 2012	qMSP	101	4(4.0)	65	40(61.5)	0.78- 0.87	0.001
SPG20	Zhang, 2013	MSP	30	0(0.0)	96	77(80.2)	N/A	0.001
SLIT2	Azuara, 2013	Melting Curve	44	0(0.0)	16	4(25.0)	N/A	0.001
Septin9	Carmona, 2013	Pyrosequence	N/A	N/A	35	7(20.0)	N/A	N/A
FBN1	Guo, 2013	MSP	30	2(6.7)	75	54(72.0)	N/A	0.001
p33ING1b	He, 2014	nMSP	20	1(5.0)	61	45(73.8)	N/A	0.001
Wif1	Zhang, 2014	MSP	30	0(0.0)	48	27(56.3)	N/A	0.001
	Amiot, 2014	qMSP	157	1(0.6)	90	17(18.9)	N/A	0.001
ALX4	Amiot, 2014	qMSP	157	2(1.3)	90	9(10.0)	N/A	0.001
GATA5	Lu, 2014	MSP	40	7(17.5)	56	47(83.9)	N/A	0.001
VIM	Lu, 2014	MSP	40	6(15.0)	56	23(41.1)	N/A	0.006
BMP3	Kisiel, 2013	QuARTS	N/A	N/A(11.0)	N/A	N/A(100.0)	0.97	N/A
EYA4	Kisiel, 2013	QuARTS	N/A	N/A(11.0)	N/A	N/A(100.0)	0.95	N/A

MethyLight: 荧光定量甲基化; MSP: 甲基化特异性PCR; COBRA: 重亚硫酸钠处理限制性酶切图谱分析; Methy-BEAMing: 磁珠乳液扩增法检测甲基化; qMSP: 定量MSP; QuARTS: 等位基因特异性实时定量目标和信号放大; Melting curve: 溶解曲线; nMSP: 巢式MSP; Pyrosequence: 焦磷酸测序法; N/A: 无记录; AUC: 曲线下面积.

和在血液中的研究不同, 粪便中很少有Septin9甲基化检测的研究, 唯一的一项报道中, 结直肠癌患者粪便中出现Septin9甲基化的比例仅为20%[66]. ColoSure是以粪便标本提取DNA样本, 通过定量检测粪便中波形蛋白(Vimentin)基因高甲基化而开发的第一个商业化结直肠癌筛选试剂盒. Li等[26]采用ColoSure进行的研究, 发现结直肠癌患者粪便中Vimentin出现甲基化的比例为40.9%(9/22), 明显高于健康对照者.

除了血液和粪便的标本, 一些研究检测了其他非组织样本中DNA的异常甲基化. Song等[67]对从尿液中提取的DNA进行甲基化检测, 发现20例结直肠癌患者中有75%均呈现出Vimentin基因启动子的高甲基化, 而在健康对照者中, 这一比例仅为10%(2/20). 另一项研究[68]检测了肠道灌洗液中15个基因的异常甲基化片段, 发现其中miR-124-3、LOC386758和SFRP1基因甲基化诊断结直肠癌的敏感性分别为71.8%、79.5%和74.4%. 直肠毛刷是可以直接取到黏膜标本的微创检测方法, Reddy等[69]从结直肠癌患者的黏膜毛刷中提取DNA, 以此为模板成功检测到了结肠腺瘤样息肉(adenomatosis polyposis coli, APC)和进行性色素沉着基因1(hyperpigmentation progressive 1, HPP1)基因的甲基化条带. 但上述标本来源的基因DNA甲基化检测, 对结直肠癌的敏感性和特异性有待大样本的前瞻性研究证实.

2 结直肠癌联合多基因甲基化指标

不同结直肠癌患者存在不同的异常基因甲基化谱, 以单一基因的甲基化检测结果来筛查结直肠癌可能存在敏感性和特异性不足的问题. 一些研究开始尝试联合检测不同基因的甲基化水平来综合筛查结直肠癌, 取得了良好的效果.

Leung等[8]同时检测了结直肠癌患者血清中MLH1、解旋酶样转录因子(helicase-like transcription factor, HLTF)和APC的甲基化水平, 发现单独标志物诊断结直肠癌的敏感性均不足50%, 而三者联合检测敏感性提高到57%. Cassinotti等[16]采用甲基化小芯片检测结直肠癌患者血浆中一系列基因的甲基化, 发现联合D型细胞周期蛋白2(cyclin type D2, CYCD2)、癌中高甲基化基因1(hypermethylated in cancer 1, HIC1)、配对盒子基因5(paired box 5, PAX5)、Ras相关结构域家族基因1[Ras association(RalGDS/AF-6) domain family member 1, Rassf1a]、视网膜神经胶质瘤基因1(retinoblastoma, RB1)和CD2(CD2 molecule, SRBC)基因诊断结直肠癌的敏感性高达83.3%.

对于粪便标本, 不仅可以将不同基因的甲基化检测结果进行组合, 还可以和K-ras突变、血红蛋白等的检测结果进行综合分析. Ahlquist等[70]综合分析粪便中的Vimentin甲基化和K-ras、APC突变, 在19例结直肠癌患者中, 有11例(57.9%)结果为阳性. 同一个研究中心在2012年的一项研究[21]中, 采用甲基化定量检测方法QuARTS同时检测200余例结直肠癌患者和健康对照者粪便中Vimentin、N-myc下游调节基因4(N-myc downstream regulated gene 4, NDRG4)、骨形态形成蛋白3(bone morphogenetic protein3, BMP3)和组织因子通路抑制剂2(tissue factor pathway inhibitor 2, TFPI2)的甲基化, 结合突变型K-ras、血红蛋白的检测结果, 敏感性达84.9%, 特异性为90.1%.

3 早期结直肠癌的基因异常甲基化检测

Duke分期和肿瘤-淋巴结-转移(tumor-node-metastasis, TNM)分期均是临床上常用的结直肠癌分期方法, 早期Duke A期或TNM 1期的结直肠癌确诊无疑将大大提高患者根治的机会, 其中部分患者还可通过创伤较小的内镜下黏膜切除术(endoscopic mucosal resection, ESD)完整切除肿瘤. 已有研究结果显示早期结直肠癌甲基化阳性检出率亦明显高于健康对照者. Grützmann等[18]研究发现85例TNM 1期结直肠癌患者血浆甲基化Septin9的阳性率为41.2%(35例), 明显高于健康对照者的8.8%(25/285). Li等[26]报道了22例Duke A期结直肠癌和110例健康对照者中Vimentin出现甲基化的比例, 前者亦明显高于后者(50.0% vs 5.3%). 在2013年的一项报道中, 26例TNM 1期结直肠癌患者血清多配体蛋白聚糖(syndecan, SDC)甲基化出现的频率高达92.3%(24例), 而对照组中这一比例仅为4.8%(6/125)[33].

4 结直肠癌甲基化筛选指标和其他方法的比较

在Tóth等[22]的研究中, 作者对结直肠癌患者血浆Septin9的甲基化和其他筛查指标如血清CEA、粪便潜血进行了比较, 发现前者的敏感性(95.7%)明显高于血清CEA(51.8%)和粪便潜血(68.2%). He等[65]比较了61例结肠癌患者的粪便p33^ING1b甲基化和粪便潜血的阳性率, 发现45例(73.8%)患者p33^ING1b出现了甲基化, 明显高于粪便潜血的阳性率(49.2%). 粪便免疫化学检测(FIT)以血红蛋白的特异性抗原抗体反应为基础, 对结直肠癌患者的检测敏感性优于粪便潜血试验[71]. 2014年新英格兰医学杂志发表的一篇临床研究[72]纳入了65例结直肠癌患者, 同时进行了以甲基化指标为主的粪便多靶点检测(NDRG4和BMP3的甲基化+K-ras突变+血红蛋白检测)和FIT检测, 发现前者的敏感性为92.3%, 明显高于后者(73.8%). 此外, 本研究还纳入了2896例早期腺瘤和758例进展期癌前病变(进展期腺瘤和直径≥1 cm的锯齿状息肉), 粪便多靶点检测的阳性率分别为17.2%和42.3%, 明显高于FIT检测的7.6%和23.7%.

5 结论

目前大便潜血和结直肠镜仍是临床上结直肠癌筛查指南推荐的主要手段, 然而其中任何一种手段都难以在准确性、经济性和人群接受性上同时令人满意. 一些非侵入性检查方法不断涌现, 如血循环miRNA、CEA、N-甲基转移酶等, 其中DNA高甲基是结直肠癌发生的早期事件, 为结直肠癌的筛查提供了新的选择.

既往研究在结直肠癌患者的血液和粪便中发现大量基因的启动子呈高甲基化状态, 且多数甲基化指标的敏感性和特异性要优于CEA和粪便潜血. 目前已有多个商业化的试剂盒用于临床实践, 为血液、粪便的甲基化规范检测提供了参考. DNA甲基化、血红蛋白、突变型K-ras等的联合检测可以明显提高对于结肠癌和早期腺瘤的检出率, 反映了其在临床应用中的巨大潜力.

由于结直肠癌的发生发展是一个多阶段的过程, 其中涉及一系列抑癌基因的高甲基化, 单一基因DNA甲基化检测的敏感性和特异性在不同人群中存在着较大的差别. 在未来的临床实践中, 开展有金标准对照的大样本、多中心研究, 综合分析这些甲基化指标在结直肠癌筛查中的价值, 将会有力推动结直肠癌的早诊早治工作.

评论

背景资料

结直肠癌是消化系最常见的恶性肿瘤之一, 给我国人民生活带来巨大的家庭和经济负担. 甲基化是肿瘤发生发展中的早期事件, 近年来的临床研究在结直肠癌患者的血液、粪便等非侵入性检测可取得的标本中检测到一系列启动子高甲基化的基因. 这些指标对结直肠癌诊断的敏感性和特异性均明显优于传统筛查指标如血癌胚抗原和粪便潜血等, 提示在结直肠癌筛查领域应用的诱人前景.

同行评议者

邓安梅, 教授, 主任医师, 长海医院; 白雪, 副主任医师, 中国人民解放军北京军区总医院普通外科

研发前沿

当前在结直肠癌患者血液、粪便中有大量启动子高甲基化的基因被鉴定出来, 系统分析这些指标来综合预测评判结直肠癌风险的大小是未来可能的研究方向.

相关报道

Zitt等详细总结了结直肠癌筛查工作中, 粪隐血、结肠镜、甲基化等方法的优缺点、推荐筛查频率等, Ganepola等则对各种方法的花费情况进行了总结.

创新盘点

本文主要是在前人研究的基础上, 对结直肠癌筛查中非侵入性甲基化指标进行总结提炼. 按单基因、组合基因、早期指标及和其他方法的比较4个方面对此领域的既往研究进行了全面系统的评述, 并详细列举了每项研究的甲基化检测方法.

应用要点

本文在总结既往研究的基础上, 提出了该领域未来研究的可能方向, 可能对未来相关研究方案的设计具有一定的指导意义.

名词解释

甲基化: 是指从活性甲基化合物上将甲基催化转移到其他化合物的过程, 可发生在DNA的核酸和蛋白质的氨基酸上.

同行评价

DNA甲基化作为结直肠癌早期筛选的分子标志物, 是目前重要的研究方向之一. 本文综述了该领域的最新研究进展, 提供了充足的有意义的信息, 具有一定的指导意义.

备注

编辑:韦元涛 电编:都珍珍

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