Establishment of a digital PCR mutation detection platform for ctDNA in patients with intrahepatic cholangiocarcinoma and its clinical application

doi:10.3969/j.issn.1673-8640.2020.04.016

Abstract

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

CLC Number:

R446.1

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.

Figures/Tables 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