ICC患者血浆ctDNA突变检测数字PCR平台的建立及临床应用价值

doi:10.3969/j.issn.1673-8640.2020.04.016

摘要/Abstract

摘要：

目的建立KRAS G12D、TP53 C242S、IDH1 R132C突变数字聚合酶链反应（dPCR）检测平台,并初步评估其检测性能及临床应用价值。方法选取行切除手术的肝内胆管细胞癌（ICC）患者22例,设计KRASG12D、TP53 C242S、IDH1 R132C突变位点的引物及探针,建立dPCR突变检测平台。并采用不同浓度的自配标准品质粒验证该平台的准确性、精密度、空白限、功能灵敏度及线性范围。将外周血dPCR检测结果与外周血Oseq-ctDNA及组织Oseq-T靶向测序结果进行比较。ICC患者行切除术后,每6个月采集1次外周血并跟踪随访,评估该突变检测平台对ICC患者术后疗效监测的作用。结果 dPCR平台的准确性良好[3种突变（KRAS G12D、TP53 C242S和IDH1 R132C）3个丰度的检测结果与理论值的偏差均<±15%],批内和批间精密度[变异系数（CV）]均<20%,空白限为4拷贝,功能灵敏度为0.1%,且在0.1%~10.0%范围内线性良好。ROC曲线分析结果显示,ctDNA突变谱诊断ICC的曲线下面积（AUC）为0.659,敏感性为31.8%,特异性为100%;与糖类抗原19-9（CA19-9）联合检测诊断ICC的AUC为0.841,敏感性为68.2%,特异性为100%。dPCR平台的检测结果与Oseq-ctDNA测序结果有较高的一致性（kappa=0.792,P=0.007）。随访结果显示,有1例ICC患者术后18个月KRAS G12D突变升高,与影像学检查确认的复发时间一致。结论建立了检测KRAS G12D、TP53 C242S、IDH1 R132C突变的dPCR平台,可用于ICC患者的辅助诊断、疗效评估及术后动态监测。

关键词: 循环肿瘤DNA, 糖类抗原19-9, 数字聚合酶链反应, 肝内胆管细胞癌

Abstract:

Objective To establish a digital polymerase chain reaction（dPCR） detection platform for KRAS G12D,TP53 C242S and IDH1 R132C mutations,and to preliminarily evaluate its detection performance and clinical application value. Methodse A total of 22 intrahepatic cholangiocarcinoma（ICC） patients who underwent resection were enrolled. The primers and probes of KRAS G12D,TP53 C242S and IDH1 R132C mutation sites were designed to establish a dPCR mutation detection platform. The accuracy,precision,detection limit of blank,functional sensitivity and linear range of the platform were validated according to self-made standard quality particles with different concentrations. The results of dPCR in Oseq-ctDNA in peripheral blood and Oseq-T targeted sequencing in tissues were compared. Peripheral blood samples were collected and followed up every 6 months after resection to evaluate the role of the platform in monitoring the efficiency and prognosis of ICC patients. Results The accuracy of dPCR platform was good,and the deviation among the 3 abundances of the 3 mutations（KRAS G12D,TP53 C242S and IDH1 R132C） and the theoretical value was less than ±15%. The detection limit of blank was 4 copies,the functional sensitivity was 0.1%,and the linearity was good in the range of 0.1%-10%. In 22 ICC patients,the sensitivity,specificity and area under curve（AUC） of ctDNA were 31.8%,100% and 0.659,those for carbohydrate antigen 19-9（CA19-9） combined with ctDNA were 68.2%,100% and 0.841. The consistency of dPCR and Oseq-ctDNA in peripheral blood was high（kappa=0.792,P=0.007）. The results of follow-up showed that the copy number of KRAS G12D mutation was increased in 1 ICC patient after resection for 18 months,which were the same as the recurrence time confirmed by imaging. Conclusions A dPCR detection platform for detecting KRAS G12D,TP53 C242S and IDH1 R132C mutations has been established,which can be used in the diagnosis,efficiency evaluation and dynamic monitoring of ICC patients after resection.

Key words: Circulating tumor DNA, Carbohydrate antigen 19-9, Digital polymerase chain reaction, Intrahepatic cholangiocarcinoma

中图分类号:

R446.1

戴谦, 黄斐, 王宇鹏, 黄傲, 成剑文, 潘柏申, 郭玮, 周俭, 樊嘉, 杨欣荣, 王蓓丽. ICC患者血浆ctDNA突变检测数字PCR平台的建立及临床应用价值[J]. 检验医学, 2020, 35(4): 354-362.

DAI Qian, HUANG Fei, WANG Yupeng, HUANG Ao, CHENG Jianwen, PAN Baishen, GUO Wei, ZHOU Jian, FAN Jia, YANG Xinrong, WANG Beili. Establishment of a digital PCR mutation detection platform for ctDNA in patients with intrahepatic cholangiocarcinoma and its clinical application[J]. Laboratory Medicine, 2020, 35(4): 354-362.

图/表 10

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基因位点	引物序列（5'~3'）	引物大小/bp
KRAS G12D
F	CCGAGTGGAAGGAAATTTGC	20
R	CACCACCACACTATGTCGAAAAG	23
Probe1	TATTTGGATGACAAAAAC	18
Probe2	TGGATGACAGAAAC	14
TP53 C242S
F	CCTGAGGTTGGCTCTGACTGTA	22
R	CCAGTGTGATGATGGTGAGGAT	22
Probe1	CATGAACGGGAGGC	14
Probe2	CATGAACCGGAGGC	14
IDH1 R132C
F	CTTGTGAGTGGATGGGTAAAACCTA	25
R	CACATTATTGCCAACATGACTTACTTGAT	29
Probe1	AAGCATGACAACCTATG	17
Probe2	ATCATAGGTCATCATGC	17

基因位点	引物序列（5'~3'）	引物大小/bp
KRAS G12D
F	CCGAGTGGAAGGAAATTTGC	20
R	CACCACCACACTATGTCGAAAAG	23
Probe1	TATTTGGATGACAAAAAC	18
Probe2	TGGATGACAGAAAC	14
TP53 C242S
F	CCTGAGGTTGGCTCTGACTGTA	22
R	CCAGTGTGATGATGGTGAGGAT	22
Probe1	CATGAACGGGAGGC	14
Probe2	CATGAACCGGAGGC	14
IDH1 R132C
F	CTTGTGAGTGGATGGGTAAAACCTA	25
R	CACATTATTGCCAACATGACTTACTTGAT	29
Probe1	AAGCATGACAACCTATG	17
Probe2	ATCATAGGTCATCATGC	17

突变位点	靶值	第1次检测	第2次检测	第3次检测	均值	偏差
KRASG12D	5.00	4.60	4.40	4.30	4.43	-11.40
	1.00	1.21	0.90	1.09	1.07	7.00
	0.50	0.45	0.42	0.41	0.43	-14.00
TP53 C242S	5.00	4.78	5.10	4.30	4.73	-5.40
	1.00	0.89	0.80	0.94	0.88	-12.00
	0.50	0.43	0.36	0.49	0.43	-14.00
IDH1 R132C	7.80	9.00	8.00	6.20	7.73	-0.90
	1.56	2.10	1.10	1.30	1.50	-4.00
	0.78	0.82	1.01	0.75	0.86	10.26

突变位点	靶值	第1次检测	第2次检测	第3次检测	均值	偏差
KRASG12D	5.00	4.60	4.40	4.30	4.43	-11.40
	1.00	1.21	0.90	1.09	1.07	7.00
	0.50	0.45	0.42	0.41	0.43	-14.00
TP53 C242S	5.00	4.78	5.10	4.30	4.73	-5.40
	1.00	0.89	0.80	0.94	0.88	-12.00
	0.50	0.43	0.36	0.49	0.43	-14.00
IDH1 R132C	7.80	9.00	8.00	6.20	7.73	-0.90
	1.56	2.10	1.10	1.30	1.50	-4.00
	0.78	0.82	1.01	0.75	0.86	10.26

项目	批内精密度			批间精密度
项目	x	s	CV/%	x	s	CV/%
KRASG12D
高丰度	21.78	1.20	5.50	21.82	1.13	5.19
低丰度	2.47	0.28	11.46	2.47	0.28	11.46
TP53 C242S
高丰度	19.68	0.85	4.32	19.03	0.83	4.36
低丰度	2.21	0.17	7.51	2.37	0.35	14.57
IDH1 R132C
高丰度	16.49	0.92	5.61	16.65	1.28	7.68
低丰度	1.86	0.17	8.99	1.80	0.21	11.49