1. Molecular Analysis of Circulating Cell-Free DNA from Lung Cancer Patients in Routine Laboratory Practice 
Stephan Bartels, Sascha Persing, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann 
The Journal of Molecular Diagnostics 
Volume 19, Issue 5, Pages (September 2017)
DOI: /j.jmoldx
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
dPCR result plots for the 5% cfDNA reference standard (HD777) and the EGFR_6240 assay (p.T790M). A wild-type EGFR allele will produce a VIC-positive signal (wild-type probe is VIC dye labeled), and a p.T790M mutated allele will produce a FAM-positive signal (mutated probe is FAM dye labeled). If two EGFR alleles, a wild-type and a mutated allele, are amplified together in a single well, this will produce a VIC and a FAM double-positive signal. If no EGFR allele is present in a reaction well, no FAM or VIC signal will be produced during the amplification process. Blue lines highlight the analysis threshold chosen for the EGFR_6240 assay (Materials and Methods).
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
Mean allele frequencies and SDs of the sensitivity testing for each of the four cfDNA reference standards measured with dPCR EGFR assays and NGS. Four independent replicates were performed for the three different dPCR assays with the four reference standard DNA samples, respectively. NGS mean allele frequencies and SDs originate from four independently synthesized libraries. Dashed lines indicate the expected allele frequencies provided by the manufacturer of the reference standards of 5%, 1%, and 0.1%, respectively. n = 4 in each group.
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
dPCR analyses results from the EGFR_6240 (p.T790M) assay tested with four different cfDNA reference standards. Results are plotted the same as in Figure 1. A: Wild-type cfDNA reference standard (HD776) without any FAM dye positive signal. B: cfDNA reference standards with 0.1% allele frequency of EGFR p.T790M mutation (HD779; B); three FAM-positive signals were detected (red circle). C and D: The 1% allele frequency of the mutation (HD778; C) and 5.0% allele frequency of the mutation (HD777; D) are shown. Thresholds for FAM and VIC dye signals are listed in Materials and Methods.
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
Aligned sequencing reads visualized in the Integrative Genomics Viewer browser version and sorted by base at position c A: cfDNA reference standards with EGFR wild-type (HD776) and three false-positive c.2369C>T variant calls. B: cfDNA reference standards with 0.1% allele frequency of EGFR p.T790M mutation (HD779) and 13 true-positive c.2369C>T variant calls.
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
cfDNA patient samples under study. Twenty-five patient samples were tested with three molecular approaches to compare the results of p.T790M mutation profiling. One sample failed in the NGS analysis. In addition, low amounts of cfDNA exclude three samples from qPCR analysis and one sample from dPCR analysis. Thirty additional cfDNA patient samples were analyzed with this NGS approach (10 selected patient samples were sequenced additionally with the Oncomine lung cfDNA assay to compare the results of EGFR p.T790M mutation analysis).
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. Supplemental Figure S1
Patient cfDNA dPCR results with true-positive (A) and false-positive (B) variant signals. Patient samples 16 (A) and 18 (B) are shown. Red cirlces indicate true-positive signals (A) and the false-positive signals (B). In the repetition, Patient 18 did not show any single- or double-positive signals (Supplemental Table S3).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

8. Supplemental Figure S2
qPCR EGFR_6240 mutation detection assay results of HD200 formalin-fixed, paraffin-embedded reference standard (Horizon Discovery) and HD778 cfDNA reference standard in duplicate. Both reference standards have an EGFR p.T790M mutation with 1.0% allelic frequency. Mutation assay (HD200 mut, HD778 mut) and reference assay (HD200 ref, HD778 ref) show a ΔCT value of 10 for HD200 (reference assay mean CT, 25.5; and mutation assay CT, 35.5) as well as for HD778 (reference assay mean CT, 26.5; and mutation assay CT, 36.5). ref, reference assay; mut, mutation assay.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

9. Supplemental Figure S3
qPCR EGFR_6240 mutation detection assay results of cfDNA reference standards HD778 [1.0% allelic frequency of EGFR p.T790M mutation, HD778 reference assay (ref), and HD778 mutation assay (mut)] and HD779 (0.1% allelic frequency of EGFR p.T790M mutation, HD779 ref, and HD779 mut). Mutation detection assay signal of HD779 (HD779 mut) does not reach the cycle threshold.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

10. Supplemental Figure S4
Distribution of the cfDNA concentration (in ng/μL) of the 55 patient samples under study. The mean value is 2.13 ng/μL.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

11. Supplemental Figure S5
Sorted EGFR exon 19 reads from Patient 7 shown in the Integrative Genomics Viewer browser. Twenty-three p.E746_A750del variant reads in 11,269 total reads are shown (0.2% allelic frequency of the mutation).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

12. Supplemental Figure S6
Aligned sequencing reads from EGFR exon 20 with p.T790M mutation of Patient 3 (left rectangle). On the 3′-end of the amplicon, a second nucleotide variant (c.2411A>G, p.E804G) can be observed (right rectangle).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

13. Supplemental Figure S7
cfDNA concentration values for patients with detectable EGFR primary mutation (mut +) and patients with known EGFR primary mutations that could not be detected in the blood (mut −). Median values are 1.5 and 1.1 ng, respectively. n = 25 (mut +); n = 15 (mut −).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions