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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 罗宇文等 / 基于微滴式数字 PCR 检测肠癌病人游离环状 DNA KRAS 突变的新方法 Mar. 25, 2018, 34(3): Chin J Biotech, All rights reserved 生物技术与方法 407 PCR DNA KRAS 罗宇文, 李瑶 ,. PCR DNA KRAS., 2018, 34(3): Luo YW, Li Y. Detection of KRAS mutation in colorectal cancer patients cfdna with droplet digital PCR. Chin J Biotech, 2018, 34(3): 摘要 : 基于微滴式数字聚合酶链式反应 (Droplet digital polymerase chain reaction, ddpcr) 设计一种检测肠癌游离循环 DNA (Circulating cell free DNA, cfdna) 中 KRAS (V-Ki-ras2 Kirsten ratsarcoma viral oncogene homolog) 基因突变的新方法并评估其灵敏度和准确性 根据肠癌病人 KRAS 基因的突变类型设计并合成, 采用 ddpcr 扩增并评估其灵敏度和准确性 ; 根据 AMRS-PCR 引物设计原理设计 KRAS 基因的实时定量 PCR 扩增引物并评估其准确性, 进而比较 ddpcr 和 qpcr 二者之间的优缺点 ; 最后针对 52 例肠癌病人的 cfdna 采用 ddpcr 进行检测, 研究 ddpcr 在 cfdna KRAS 基因突变检测的应用 成功使用 ddpcr 和 qpcr 两种方法对 KRAS 野生型及 7 种突变型建立检测方法, 使用质粒标准品及实际样品验证该两种方法可行并对其假阳性率 线性范围及检测下限等性能进行了评价, 最后成功对 52 例临床患者和 20 例正常人的血浆 cfdna 样本进行检测, 临床灵敏度为 97.64%, 临床特异性为 81.43% ddpcr 的检测性能优于 qpcr,lod 达到个位数 DNA 拷贝, 最低可确认突变浓度达到 0.01% 0.04% 样本提取效率在方法学建立中也十分重要, 直接影响到灵敏度和 Cut Off 值的判定 临床患者检测结果显示其 KRAS 突变率接近报道水平 : 微滴式数字聚合酶链式反应, 肠癌游离循环 DNA,KRAS, 液态活检 Received: September 8, 2017; Accepted: December 11, 2017 Corresponding author: Yao Li. Tel: ; yaoli@fudan.edu.cn

2 408 ISSN CN /Q Chin J Biotech Detection of KRAS mutation in colorectal cancer patients cfdna with droplet digital PCR Yuwen Luo, and Yao Li Institute of Genetics, School of Life Sciences, Fudan University, Shanghai , China Abstract: This study aims to develop a new method for the detection of KRAS mutations related to colorectal cancer in cfdna, and to evaluate the sensitivity and accuracy of the detection. We designed a method of cfdna based KRAS detection by droplets digital PCR (ddpcr). The theoretical performance of the method is evaluated by reference standard and compared to the ARMS PCR method. Two methods, ddpcr and qpcr, were successfully established to detect KRAS wild type and 7 mutants. Both methods were validated using plasmid standards and actual samples. The results were evaluated by false positive rate, linearity, and limit of detection. Finally, 52 plasma cfdna samples from patients and 20 samples from healthy people were tested, the clinical sensitivity is 97.64%, clinical specificity is 81.43%. ddpcr method shows higher performance than qpcr. The LOD of ddpcr method reached single digits of cfdna copies, it can detect as low as 0.01% to 0.04% mutation abundance. Keywords: droplet digital PCR colorectal cancer, cell free DNA, KRAS, liquid biopsy DNA (Circulating cell free DNA cfdna) DNA 180 bp cfdna DNA (Circulating cell free tumor DNA ctdna) [1] cfdna [2] [3] cfdna PCR 30 pg 1% PCR (Droplet digital pcr ddpcr) PCR 0.001% [4] PCR PCR DNA PCR [5] [6] PCR (Limits of detection LOD) [7] ddpcr PCR [8-9] PCR PCR ddpcr cfdna KRAS ( PCR) LOD PCR

3 罗宇文等 / 基于微滴式数字 PCR 检测肠癌病人游离环状 DNA KRAS 突变的新方法 409 PCR PCR 1 材料与方法 1.1 标准品处理 KRAS G12C G12V G12D G12R G12S G12A G13D ( ( ) ) XbaⅠ( ( ) ) 1.2 血浆 cfdna 的提取 Cell-Free DNA BCT ( Streck Inc. USA) 5 ml g 20 min g 10 min QIAamp Circulating Nucleic Acid Kit (Qiagen) cfdna QIAamp Mini PCR 50 μl K 60 1 h 30 μl 微滴式数字 PCR 引物设计及扩增 Beacon Designer 8.10 (Primer Biosoft) ( Beacon-Designer.shtml) Blast 7 KRAS KRAS KRAS cfdna KRAS (5 -GACTGAATATAAACT TGTGGTA-3 ; 5 -GTCCACAAAATGATTCTGA-3 ) FAM Taqman KRAS HEX Taqman 1 ( ) PCR (Bio-Rad) 8 Sample 8 PCR 8 20 μl 1 buffer control (Bio-Rad) (Bio-Rad) 8 Oil 70 μl (Bio-Rad) 8 (Bio-Rad) QX200 Droplet Generator(Bio-Rad) (Bio-Rad) 8 Sample 表 1 微滴式数字 PCR 探针序列 Table 1 Sequence of digital PCR probes Type Sequence G12C FAM-5 -ACTCTTGCCTACGCCACAAG-3 -BHQ1 G12S FAM- 5 -ACTCTTGCCTACGCCACTAG-3 -BHQ1 G12R FAM-5 -ACTCTTGCCTACGCCACGAG-3 -BHQ1 G12V FAM-5 -ACTCTTGCCTACGCCAACAG-3 -BHQ1 G12D FAM-5 -ACTCTTGCCTACGCCAT CAG-3 -BHQ1 G12A FAM-5 -ACTCTTGCCTAC GCCAGCAG-3 -BHQ1 G13D FAM-5 -ACTCTT GCCTACGTCACCAG-3 -BHQ1 W T HEX-5 -ACTCTTGCCTACGCCACCAG-3 -BHQ cjb@im.ac.cn

4 410 ISSN CN /Q Chin J Biotech 96 PCR PCR 2.5 /s PCR 96 QX200 Droplet Reader (Bio-Rad) QuantaSoft (Bio-Rad) RED FAM HEX QuantaSoft PCR ( copies/μl) 1.4 聚合酶链式扩增阻碍突变系统引物设计及检测 ARMS- PCR PCR 使用通用引物, PNA PNA 2 PNA ( ) 5 -TGGAGCTGGT GGCGTAGGC-P04-3 HEX Taq-man 5 -HEX-TCTGAATTAGCTGTATCGTCAAG GCACTCT-BHQ 方法学论证 cfdna cfdna 表 2 qpcr 引物序列 Table 1 Sequence of qpcr primers Type Sequence (5 3 ) G12C AACTTGT GGTAGTTGGAGCGT G12S ATAAACTTG TGGTAGTTGGAGCTA G12R ATAAACTT GTGGTAGTTGGAGCCC G12V AAACTT GTGGTAGTTGGAGCGGT G12D CTTGTG GTAGTTGGAGCTTA G12A AACTTGTGG TAGTTGGAGCTGC G13D GTGGTAGTTG GAGCTGGTAA Universal ACCTCTATTGTTGGATCATATTCGT C OD 260 (OD 260 >1) OD 260 /OD 280 (1.6<OD 260 /OD 280 <1.9) cfdna cfdna ddpcr qpcr LOD (Limit of quantification LOQ) (CV%) ddpcr ddpcr KRAS Cut Off (Limit of blank LOB) LOD LOQ 1 LOB LOB LOD LOD LOQ LOD LOQ LOQ LOD LOD 95% LOD LOD

5 罗宇文等 / 基于微滴式数字 PCR 检测肠癌病人游离环状 DNA KRAS 突变的新方法 411 图 1 Fig. 1 检测方法动态范围关系图 Dynamic range schematic of diagnostic method. 1.6 统计学分析 SPSS v21.0 ( en.softonic.com/) qpcr ± ddpcr Ct / 95% 2 结果 2.1 ddpcr 理论性能检测 ddpcr 7 KRAS ( ) 3 100% 2A 2B ddpcr PCR [10] 2 G12C G12D 0.01% 0.04% 0.1% CV 25% 0% R 健康人样本的 ddpcr 检测及理论 LOD 值 20 ddpcr ( 4) 2 copies/μl 1 2 droplets ddpcr LOD LOD 95% (LOD 1) ddpcr FPR=false droplets number/wells (False positive rate FPR) FPR 95% ( ) [11] LOD 95% LOD (LOD 2) 3 LOD cjb@im.ac.cn

6 412 ISSN CN /Q Chin J Biotech 图 2 ddpcr 扩增微滴图 ( 以 G12D 标准品梯度稀释检测结果为例 ). (A) ddpcr 扩增微滴一维图,Ch1 为 FAM 通道 ( 突变型 ),Ch2 为 HEX 通道 ( 野生型 ). 黑灰色微滴为无扩增阴性微滴, 蓝色和绿色微滴分别为 FAM 和 HEX 通道中 PCR 阳性扩增微滴, 突变比例从左至右依次降低. (B) ddpcr 扩增微滴二维图, 纵轴为 FAM 荧光增量, 横轴为 HEX 荧光增量, 微滴在坐标系内聚类为 4 簇 : 左下为无模板微滴, 左上为仅有 FAM 信号的微滴, 右下为仅有 HEX 信号的微滴, 右上为同时发出 FAM 与 HEX 信号的双阳性微滴. Fig. 2 ddpcr amplification chart (G12D as an example). (A) ddpcr 1D amplification charts, Ch1 is FAM Channel (mutant type) and Ch2 is HEX channel (wild type). In these charts, black points signify PCR negative droplets, blue and green points respectively signify PCR positive droplets in FAM channel and HEX channel, the mutation abundances descend from left to right. (B) ddpcr 2D amplification chart, Y axis shows amplitude in FAM channel and X axis shows amplitude in HEX channel, the droplets are separated in 4 clusters: lower left points signify no template droplets, upper left points signify the FAM signal positive droplets, lower right points signify HEX signal positive droplets, upper right points signify double positive droplets in both FAM and HEX channel.

7 罗宇文等 / 基于微滴式数字 PCR 检测肠癌病人游离环状 DNA KRAS 突变的新方法 413 图 3 KRAS 基因不同突变型标准品梯度稀释线性方程图. (A) KRAS 基因 G12C 突变型标准品梯度稀释线性方程图. (B) KRAS 基因 G12D 突变型标准品梯度稀释线性方程图. (C) KRAS 基因 G12A 突变型标准品梯度稀释线性方程图. (D) KRAS 基因 G12V 突变型标准品梯度稀释线性方程图. (E) KRAS 基因 G12R 突变型标准品梯度稀释线性方程图. (F) KRAS 基因 G12S 突变型标准品梯度稀释线性方程图. (G) KRAS 基因 G13D 突变型标准品梯度稀释线性方程图. G12D 及 G12C 的浓度梯度为 100% 0.01%,10 倍稀释 ; 其余突变型的稀释梯度为 1% 0.2% 0.1% 0.04% Fig. 3 ddpcr linear curves of KRAS mutant abundances. (A) shows linear curve of KRAS G12C type. (B) shows linear curve of KRAS G12D type. (C) shows linear curve of KRAS G12A type. (D) shows linear curve of KRAS G12V type. (E) shows linear curve of KRAS G12R type. (F) shows linear curve of KRAS G12S type. (G) shows linear curve of KRAS G13D type. The samples of G12D and G12C are tenfold diluted from 100% to 0.01%, the dilution gradients of other mutant types are 1%, 0.2%, 0.1% and 0.04% cjb@im.ac.cn

8 414 ISSN CN /Q Chin J Biotech 图 4 Fig. 4 阴性对照检测的微滴一维图 1D droplets chart of negative control tests. 表 3 两种不同方法判定的 ddpcr KRAS 突变检测法的 LOD Table 3 LODs of ddpcr KRAS detection evaluated by 2 different algorithms LOD G12C G12S G12R G12V G12D G12A G13D LOD algorithm 1 (copies/well) LOD algorithm 2 (positive droplets/well) Note: LOD algorithm 1 is according to normal distribution, LOD algorithm 2 is according to Poisson distribution, There are significant differences between LODs and negative controls in all mutant type, P < QX200 ddpcr nl 20 μl copies/μl=20 ( ln(1 PD/20 000))/0.89 PD LOD=3 positive droplets/well 3.49 QX200 ddpcr ( ) % 0.01% 0.04% CV<25% PCR KRAS LOD 0.01% 0.04%

9 罗宇文等 / 基于微滴式数字 PCR 检测肠癌病人游离环状 DNA KRAS 突变的新方法 实时定量 PCR 理论性能检测 5 Ct>35 0.1% Ct 12% Ct 2 50% qpcr ( ) Ct 2.4 健康人样本的实时定量 PCR 检测及理论 LOD 值 16 PCR Ct 50 Ct 40 Ct PCR LOD 4 LOD 0.1% Ct G12S 0.1% 表 4 qpcr 法在各突变检测位点的理论 LOD (Ct 值 ) Table 4 The oretical LODs of qpcr methods in all mutant type (Ct value) Mutant G12S G12R G12C G12D G12A G12V G13D type LOD Note: There are significant differences between LODs and negative controls in all mutant type, P< 误差与重复性 6 ddpcr qpcr LOQ CV ddpcr CV qpcr 2.6 ddpcr 实际样本检测结果 ddpcr 7 KRAS LOD 1 LOD PCR cfdna ( 5) LOD 1 图 5 qpcr 浓度梯度 Ct 值曲线 Fig. 5 Ct/concentration gradient curve of qpcr. 图 6 ddpcr 与 qpcr 变异系数对照图 Fig. 6 Comparison of variable coefficients between ddpcr and qpcr. X axis shows cjb@im.ac.cn

10 416 ISSN CN /Q Chin J Biotech 表 5 基于 ddpcr 测得临床样本 KRAS 野生型 突变型拷贝数换算出的突变含量 Table 5 Clinical sample mutation abundances calculated from ddpcr detection results of KRAS wild and mutant type copy numbers Sample G12R G12C G12A G12S G12D G12V G13D % 10.13% 0.00% 14.89% 0.00% 1.52% 10.52% % 0.00% 0.00% 9.79% 2.12% 0.00% 1.86% % 0.00% 2.39% 4.99% 0.00% 0.00% 5.54% % 4.76% 4.41% 15.00% 2.78% 3.88% 12.18% % 19.60% 17.65% 38.14% 47.10% 26.83% 45.51% % 4.35% 0.00% 12.89% 0.00% 1.50% 16.29% % 0.10% 0.25% 0.80% 0.26% 0.30% 0.00% % 0.00% 1.96% 0.00% 8.09% 4.44% 0.00% % 0.00% 0.00% 22.48% 0.00% 0.00% 7.69% % 8.23% 5.24% 8.11% 0.00% 3.06% 0.00% % 0.00% 54.55% 75.12% 55.10% 71.05% 0.00% % 0.00% 38.14% 10.42% 43.50% 4.59% 0.00% % 0.00% 42.86% 55.45% 20.62% 1.93% 13.73% % 0.00% 8.97% 0.00% 17.53% 3.61% 0.00% % 50.00% 0.00% 44.13% 4.11% 0.00% 0.00% % 0.37% 4.63% 1.39% 0.28% 0.40% 0.00% % 0.00% 5.19% 18.75% 7.53% 3.19% 0.00% % 0.00% 21.11% 2.95% 5.98% 0.00% 0.00% % 0.00% 5.14% 6.86% 0.00% 3.41% 0.00% % 0.06% 0.00% 0.13% 0.06% 0.03% 0.00% % 1.25% 0.00% 4.97% 0.00% 1.35% 0.00% % 2.27% 1.26% 17.43% 1.23% 8.11% 0.00% % 0.00% 15.79% 0.00% 0.00% 1.26% 0.00% % 0.00% 3.23% 56.00% 2.74% 23.08% 0.00% % 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% % 28.57% 0.00% 0.00% 0.00% 0.00% 13.73% % 6.04% 0.00% 0.00% 5.41% 0.00% 0.00% % 0.00% 0.00% 0.00% 0.00% 1.57% 0.00% % 0.00% 0.00% 2.32% 0.00% 0.00% 0.00% % 0.00% 0.00% 0.00% 0.00% 1.19% 0.00% % 1.45% 0.00% 3.38% 62.79% 0.00% 0.00% % 0.00% 0.00% 0.54% 0.00% 1.71% 0.00% % 0.00% 0.00% 9.88% 0.00% 6.54% 0.00% % 0.00% 0.00% 0.00% 56.90% 0.00% 0.00% % 0.00% 10.83% 0.00% 0.00% 0.00% 0.00% % 0.00% 0.00% 0.00% 45.00% 0.00% 0.00% % 0.00% 0.00% 35.38% 0.81% 0.00% 0.00% % 33.33% 25.00% 1.99% 48.28% 1.01% 13.95% % 10.45% 4.19% 0.00% 5.51% 6.35% 0.00% % 0.98% 1.43% 1.13% 0.46% 0.69% 0.00% % 6.18% 3.34% 0.00% 1.15% 0.00% 1.39% % 0.00% 4.19% 0.00% 5.23% 8.31% 0.00% % 0.00% 0.00% 0.00% 0.00% 3.60% 0.00% % 1.57% 0.00% 0.00% 1.10% 0.00% 0.00%

11 罗宇文等 / 基于微滴式数字 PCR 检测肠癌病人游离环状 DNA KRAS 突变的新方法 % 0.00% 0.00% 1.16% 11.50% 0.00% 0.00% % 1.58% 0.00% 0.00% 0.52% 1.08% 0.89% % 0.00% 0.00% 0.00% 0.00% 0.00% 3.33% % 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% % 6.25% 12.00% 0.00% 0.00% 11.29% 3.41% % 0.40% 0.00% 0.00% 2.24% 0.32% 0.00% % 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% % 2.57% 0.49% 0.00% 0.00% 0.59% 0.00% Note: the bold numbers show the positive repeated verification results of samples under LOD (false negative in single test). The numbers with underline show the negative repeated verification results of samples under LOD (false positive in single test) LOD = /( + ) = /( + ) 97.64% 81.43% 2.7 cfdna 提取得率 cfdna ( 7) 5.19 ng/ml OD 260 ddpcr 95% DNA 0.1% DNA 3 DNA LOD LOD 6 图 7 Fig. 7 本研究中 52 个结直肠癌病人血液 cfdna 提取效率 Blood cfdna extraction efficiency of 52 colorectal cancer patients sample in this study cjb@im.ac.cn

12 418 ISSN CN /Q Chin J Biotech 表 6 以本次研究结果建立的采血量与样本极限 LOD 关系表 Table 6 Relationship between total blood capacity and sample LOD according to the result of this study Sample LOD Total copies Mass Extraction efficiency (ng/ml) Total blood requirement (ml) 1/ ng ~2 1/ ng 5.19 ~20 1/ μg ~200 3 讨论 KRAS RAS EGFR 12p12.1 p21 [12] EGFR 38% KRAS >96% [13] KRAS EGFR EGFR [14] KRAS 30% 40% KRAS [15] KRAS DNA [16] 1974 Sorrells RB [17] (DNA RNA) [18] cfdna [19] (FISH) [20] TKI ddpcr [21-22] ddpcr LOD 3.49 copies/well LOD 0.01% 0.04% R 2 25% qpcr Ct ddpcr LOD LOQ

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