1. Application of Single-Molecule Amplification and Resequencing Technology for Broad Surveillance of Plasma Mutations in Patients with Advanced Lung Adenocarcinoma 
Zheng Wang, Gang Cheng, Xiaohong Han, Xinlin Mu, Yuhui Zhang, Di Cui, Chang Liu, Li Zhang, Zaiwen Fan, Lingyun Ma, Li Yang, Jing Di, David S. Cram, Yuankai Shi, Dongge Liu 
The Journal of Molecular Diagnostics 
Volume 19, Issue 1, Pages (January 2017)
DOI: /j.jmoldx
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Strategy for plasma detection of oncogenic mutations associated with non–small-cell lung carcinoma. The exonic positions and nature of the known EGFR, KRAS, BRAF, and ERBB2 hot spot mutations are shown. Two sets of bidirectional reverse primers (arrows) were positioned to robust sequences on either side of each mutation site to target and amplify each allele by inverse PCR. For detection of ALK fusions, targeting primers were progressively spaced across exon 19, intron 19, and exon 20, spanning the known ALK breakpoint region. Chr, chromosome.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Analytical sensitivity and specificity of the cSMART assay. Three 30-ng replicates of the Horizon Multiplex I cfDNA Reference Standard with mutation ratios of 5% (A), 1% (B), 0.1% (C), and 0% (D) for the L858R, exon 19 del (E746-A750), and T790M EGFR mutations were analyzed by the cSMART assay (gray bars) and the droplet digital PCR assay (black bars). The horizontal dotted lines on the graphs represent the expected (theoretical) mutation ratios in the reference standard. More than 2000 single-molecule events were measured and used to calculate the observed mutation ratios.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Validation of the cSMART assay for plasma mutation detection. Plasma mutation levels for six common mutation variants are plotted as log2 (mutant templates/2 mL) (y axis) versus 50 healthy control (HC) individuals and 103 non–small-cell lung carcinoma patients (x axis). The vertical dotted line separates the control and patient groups. Red and black dots indicate positive and negative mutation samples, respectively. The positive mutation threshold was arbitrarily defined as one or more mutation templates per 2 mL of plasma for EGFR mutations L858R and exon 19 deletions, the BRAF variant V600X, and ALK fusions. For the KRAS G12X variant and the EGFR mutation T790M, the positive mutation threshold was arbitrarily defined as two or more mutation templates per 2 mL of plasma.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Molecular structure of ALK fusions detected in plasma. EML4-ALK fusion variants 1 and 2 (Patients 9 and 30) and one KIF5B-ALK variant (Patient 102) were detected in plasma. The DNA sequence of the gene fusion determined from reconstruction of the paired end cSMART sequencing reads is shown, together with the hg19 reference coordinates of the breakpoint for each gene partner.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Figure 5
Relative levels of plasma mutations in patients double positive for an activating and resistance EGFR variant. Plasma mutation levels are plotted as log2 (mutant templates/2 mL) (y axis) versus patient number (x axis). All six patients previously identified with an activating mutation (L858R or exon 19 deletion) were subsequently treated with EGFR-TKIs and developed low-level T790M resistance. del, deletion.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7. Figure 6
Plasma versus tumor genotypes for 103 matching samples. Four trends were observed, involving 57 positively concordant, 18 negatively concordant, 9 partially concordant, and 19 discordant samples. Mutations, represented by boxes, are color coded for EGFR, KRAS, BRAF, ERBB2, or ALK fusion mutations, and horizontal bars dividing some boxes indicate the presence of multiple mutations.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions