乳腺癌组织样本中 HER2基因拷贝数的检测分析

doi:10.3969/j.issn.1674-8115.2022.11.011

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (11): 1589-1597.doi: 10.3969/j.issn.1674-8115.2022.11.011

• 论著 · 技术与方法 • 上一篇

乳腺癌组织样本中 HER2基因拷贝数的检测分析

孙亚蒙¹(), 马晔²(), 郭润生²()

^1.上海柏辰生物科技有限公司，上海 200233
^2.上海健康医学院附属嘉定区中心医院普外科，上海 201800

收稿日期:2022-05-07 接受日期:2022-10-18 出版日期:2022-11-28 发布日期:2023-01-04
通讯作者: 马晔,郭润生 E-mail:symbuster_1986@163.com;malele2013@sina.com;grs982600@163.com
作者简介:孙亚蒙（1986—），男，博士，电子信箱： symbuster_1986@163.com。第一联系人：（马晔、郭润生并列第一作者）
基金资助:
上海市嘉定区卫健委课题(2019-KY-13)

Detection and analysis of copy number of HER2 gene in breast cancer tissue samples

SUN Yameng¹(), MA Ye²(), GUO Runsheng²()

^1.Shanghai Bio-Chain Biological Technology Co. , Ltd, Shanghai, 200233, China
^2.Department of General Surgery, Jiading District Central Hospital, Shanghai University of Medicine& Health Sciences, Shanghai, 201800, China

Received:2022-05-07 Accepted:2022-10-18 Online:2022-11-28 Published:2023-01-04
Contact: MA Ye,GUO Runsheng E-mail:symbuster_1986@163.com;malele2013@sina.com;grs982600@163.com
Supported by:
Project of Shanghai Jiading District Health Commission(2019-KY-13)

摘要/Abstract

摘要：

目的·利用微滴式数字PCR系统（droplet digital PCR system，ddPCR）建立乳腺癌组织福尔马林固定石蜡包埋（formalin-fixed paraffin embedded，FFPE）样本和血浆样本中 HER2基因拷贝数检测体系，并对检测体系进行性能评估，为临床辅助诊疗提供科学依据。方法·收集2020年1月—2021年6月就诊于上海健康医学院附属嘉定区中心医院普外科乳腺癌患者组织样本12例及其中4例配对血浆样本用于检测体系评价，同期收集健康志愿者血浆样本24例及就诊的乳腺癌组织切片样本77例，提取核酸，用于建立检测体系空白检测限及性能评价。设计及筛选检测体系，使用ddPCR检测体系的空白检测限、精密度、灵敏度及线性范围，使用高通量测序（next generation sequencing，NGS）验证ddPCR检测一致性，并与荧光原位杂交（fluorescence in situ hybridization，IHC）/免疫组化（immunohistochemistry，FISH）金标准方法比对检测体系准确性。定量资料比较采用 χ ² 检验和配对样本 t检验进行分析，使用Poisson概率函数分析体系空白检测限，变异系数（coefficient of variation，CV）评价灵敏度及精密度，线性回归相关系数（ R ² ）评价线性范围。结果·使用ddPCR平台建立了乳腺癌患者组织切片DNA中 HER2基因拷贝数定量和扩增检测体系，配对样本 t检验评价：与NGS方法检测组织核酸和血浆游离核酸样本结果相比，检测拷贝数结果相关系数0.987，扩增判读一致性100%。评估检测体系的批内精密度为6.8%、批间精密度为9.4%，灵敏度为1 ng，线性良好（ R ² >0.98）。与传统的IHC/FISH方法相比，检测体系的灵敏度和特异度分别为84.6%（95% CI 64.3%~95.0%）和76.5%（95% CI 62.2%~86.8%）， Kappa为0.57，一致率为79.2%。结论·使用ddPCR建立乳腺癌患者 HER2拷贝数检测体系，临床检测一致性良好，可能为乳腺癌患者的诊疗和预后判断的研究提供辅助手段。

关键词: 乳腺癌, 福尔马林固定石蜡包埋, 血浆, 微滴式数字PCR, HER2拷贝

Abstract:

Objective ·A copy number detection system of HER2 genes in FFPE samples and plasma samples of breast cancer patients was established by using droplet digital PCR system (ddPCR), and then the performance of the detection system was evaluated to provide a scientific basis for clinical auxiliary diagnosis and treatment. Methods ·To evaluate the detection system, 12 tissue samples of breast cancer patients and 4 paired plasma samples were collected from the Department of General Surgery, Jiading District Central Hospital, Shanghai University of Medicine & Health Sciences from January 2020 to June 2021. In the same period, 24 plasma samples of healthy volunteers and 77 tissue sections of breast cancer patients were collected to extract nucleic acids, to establish limit of blank (LoB) and performance evaluation of detection system respectively. The detection system was designed and screened, and subsequently LoB, precision, sensitivity and linear range were also evaluated. We used next generation sequencing (NGS) to verify the consistency of ddPCR detection system, and compared the accuracy of the detection system with fluorescence in-situ hybridization (IHC)/immunohistochemistry (FISH) gold standard method. χ ² test and matched samples t-test were used for comparison of quantitative data. Poisson probability function was used to analyze the blank detection limit of the system, the coefficient of variation (CV) was used to evaluate the sensitivity and precision, and linear regression correlation coefficient ( R ² ) was used to evaluate the linear range. Results ·The ddPCR platform was used to establish the quantitative and amplification detection system of HER2 gene copy number in tissue section DNA of breast cancer patients. Compared with the results of tissue nucleic acid and plasma free nucleic acid samples detected by NGS method, the correlation coefficient of copy number detection results was 0.987, and the consistency of amplification was 100%. The intra assay precision and inter assay precision of the evaluation system were 6.8% and 9.4%, respectively. The sensitivity was 1 ng and the linearity was good ( R²>0.98). Compared with the traditional IHC/FISH method, the sensitivity and specificity were 84.6% (95% CI 64.3%~95.0%) and 76.5% (95% CI 62.2%~86.8%), Kappa was 0.57, and the consistency rate was 79.2%. Conclusion ·A copy number detection system for HER2 in breast cancer patients is established by using ddPCR platform, with good consistency in clinical detection, providing auxiliary means for the diagnosis, treatment and prognosis of breast cancer patients.

Key words: breast cancer, formalin-fixed paraffin embedded (FFPE), plasma, droplet digital PCR (ddPCR), HER2 copy number

中图分类号:

R446.1

孙亚蒙, 马晔, 郭润生. 乳腺癌组织样本中 HER2基因拷贝数的检测分析[J]. 上海交通大学学报（医学版）, 2022, 42(11): 1589-1597.

SUN Yameng, MA Ye, GUO Runsheng. Detection and analysis of copy number of HER2 gene in breast cancer tissue samples[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(11): 1589-1597.

图/表 12

参考文献 25

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Gene	Forword primer （5′→3′）	Reverse primer （5′→3′）	Probe sequence （5′→3′）
HER2	TCACTCATATCCTCCTCTTTCTGC	AATTTTCACATTCTCCCCATCAG	FAM-CAGGGCATCTGGATC-MGB
EIF2C1	GCTGCTAGGCTTTCCTGTTC	GCCTATTTTCCTGCATCTTCT	HEX-AGGCCCCAAAACCCTAAAC-MGB

Gene	Forword primer （5′→3′）	Reverse primer （5′→3′）	Probe sequence （5′→3′）
HER2	TCACTCATATCCTCCTCTTTCTGC	AATTTTCACATTCTCCCCATCAG	FAM-CAGGGCATCTGGATC-MGB
EIF2C1	GCTGCTAGGCTTTCCTGTTC	GCCTATTTTCCTGCATCTTCT	HEX-AGGCCCCAAAACCCTAAAC-MGB

Sample	Detection System 1			BC-HER2-2019
Sample	HER2 gene copy number （copies）	Reference gene copy number （copies）	Copy amplification ［（ HER2/Reference）］×2	HER2 gene copy number （copies）	Reference gene copy number （copies）	Copy amplification ［（ HER2/Reference）］×2
HV-1	2 400	3 600	1.33	3 180	3 200	1.99
HV-2	2 150	3 450	1.25	3 050	3 100	1.97
HV-3	2 600	3 700	1.41	3 200	3 280	2.05

Sample	Detection System 1			BC-HER2-2019
Sample	HER2 gene copy number （copies）	Reference gene copy number （copies）	Copy amplification ［（ HER2/Reference）］×2	HER2 gene copy number （copies）	Reference gene copy number （copies）	Copy amplification ［（ HER2/Reference）］×2
HV-1	2 400	3 600	1.33	3 180	3 200	1.99
HV-2	2 150	3 450	1.25	3 050	3 100	1.97
HV-3	2 600	3 700	1.41	3 200	3 280	2.05

Sample No.	Concentration/（ng·μL ^-1）	HER2 Gene copy	Sample No.	Concentration/（ng·μL ^-1）	HER2 Gene copy
1	0.44	1.80	13	0.21	2.24
2	0.19	2.20	14	0.10	2.20
3	0.54	2.08	15	0.16	2.22
4	0.26	2.01	16	0.10	1.20
5	0.95	2.58	17	0.38	2.37
6	0.62	2.00	18	0.13	1.87
7	0.23	2.90	19	0.49	1.99
8	0.29	2.27	20	0.32	1.62
9	0.32	1.99	21	0.10	2.40
10	0.21	2.28	22	0.24	1.75
11	0.23	2.08	23	0.19	2.19
12	0.38	2.17	24	0.74	1.69

乳腺癌组织样本中 HER2基因拷贝数的检测分析

Detection and analysis of copy number of HER2 gene in breast cancer tissue samples

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Metrics

Sample No.	Test date	Copy amplification ［（ HER2/Reference）］×2
		Batch 1		Batch 2		Batch 3
		Repeat 1	Repeat 2	Repeat 1	Repeat 2	Repeat 1	Repeat 2
1	Day 1	1.74	2.14	2.16	2.14	1.82	1.94
2	Day 2	1.84	1.88	2.36	2.18	1.92	1.88
3	Day 3	2.08	1.98	2.38	2.50	2.20	1.82
4	Day 4	2.12	2.26	1.83	2.14	1.80	1.90
5	Day 5	2.10	2.10	2.24	2.04	2.18	1.92
X_mean		2.05
Intra batch standard deviation		0.14
inter batch standard deviation		0.19
Intra batch precision/%		6.80
inter batch precision/%		9.40

Loading quantity	Repeat	HER2 Gene copy number （copies/20 μL）	Reference Gene copy number （copies/20 μL）	Copy amplification ［（ HER2/Reference）］×2	CV/%
10 ng	1	2 780	2 940	1.89	3.30
	2	3 100	3 120	1.99
	3	3 160	3 140	2.01
1 ng	1	226	274	1.64	12.50
	2	234	244	1.91
	3	276	260	2.11
0.1 ng	1	15.20	14.00	2.20	26.00
	2	9.60	14.40	1.30
	3	13.00	15.40	1.70

HER2∶ reference	Theoretical value			Actual value
HER2∶ reference	HER2 gene copy number （copies/20 μL）	Reference gene copy number （copies/20 μL）	Copy amplification ［（ HER2/ reference）］×2	HER2 gene copy number （copies/20 μL）	Reference gene copy number （copies/20 μL）	Copy amplification ［（ HER2/ reference）］×2
1∶1	3 260	3 190	2.04	3 160	3 360	1.89
2∶1	5 984	3 044	3.93	6 460	3 240	2.99
3∶1	8 708	2 899	6.01	8 900	3 000	5.94
4∶1	13 914	3 496	7.96	16 140	3 740	8.62
5∶1	14 482	2927	9.90	16 760	3 360	10.00

No.	NGS copy amplification	ddPCR copy amplification
1 ^a	1.90	1.62
2 ^a	1.90	1.86
3 ^a	1.90	1.62
4 ^a	5.00	4.27
5 ^a	6.00	4.21
6 ^a	13.10	14.10
7 ^a	8.00	6.64
8 ^a	18.00	16.00
9 ^a	2.40	1.73
9 ^b	2.10	1.84
10 ^a	2.20	1.77
10 ^b	1.80	1.41
11 ^a	5.00	3.50
11 ^b	1.80	1.49
12 ^a	5.00	3.85
12 ^b	1.70	1.20

ddPCR	FISH		Specificity ［% （95% CI）］	Sensitivity ［% （95% CI）］	Kappa value
ddPCR	Positive	Negative	Specificity ［% （95% CI）］	Sensitivity ［% （95% CI）］	Kappa value
Positive	22	12	76.5 （62.2—86.8）	84.6 （64.3—95.0）	0.57
Negative	4	39	76.5 （62.2—86.8）	84.6 （64.3—95.0）	0.57

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