Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell.

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Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell Lung Cancer Guanshan Zhu, Xin Ye, Zhengwei Dong, Ya Chao Lu, Yun Sun, Yi Liu, Rose McCormack, Yi Gu, Xiaoqing Liu The Journal of Molecular Diagnostics Volume 17, Issue 3, Pages 265-272 (May 2015) DOI: 10.1016/j.jmoldx.2015.01.004 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Design of the two assays for detection of EGFR E19-Dels and L858R. For E19-Del assay, a PNA blocker was introduced to block the amplification of wild-type EGFR allele, and a TaqMan assay located on EGFR exon2 (E2) was included as internal control. For L858R assay, FAM- and VIC-labeled probes were designed to target the mutant and wild-type EGFR alleles, respectively. E19-Del, exon 19 deletion; PNA, peptide nucleic acid; Mt, mutant allele; Wt, wild-type allele. The Journal of Molecular Diagnostics 2015 17, 265-272DOI: (10.1016/j.jmoldx.2015.01.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Selective sensitivity of the two assays for E19-Del and L858R mutations. Up to 1:2500 dilution of mutant to wild-type EGFR alleles, at least two positive droplets are stably detected by both ddPCR assays. The numbers shown in the positive area are the amounts of EGFR mutant allele-positive droplets by detection. ddPCR, droplet digital PCR; EGFR, epidermal growth factor receptor; E19-Del, exon 19 deletion; Mt, mutant allele; Wt, wild-type allele. The Journal of Molecular Diagnostics 2015 17, 265-272DOI: (10.1016/j.jmoldx.2015.01.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Summary of EGFR mutant fraction and total DNA input of 33 EGFR mutation-positive plasma samples (one sample is shown twice for E19-Del and L858R mutation, respectively). The fractions of either EGFR-E19-Del– or EGFR-L858R–mutant DNA of all 33 samples are shown in the order of increased values (black columns). Total DNA inputs (GEs/reaction) of all these 33 samples were displayed with gray columns. Samples 3, 4, 7, and 11 are the ones negative for the same EGFR mutations in the paired tumor tissues (arrows). EGFR, epidermal growth factor receptor; E19-Del, exon 19 deletion; GE, genome equivalent. The Journal of Molecular Diagnostics 2015 17, 265-272DOI: (10.1016/j.jmoldx.2015.01.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions