liquid biopsy analysis of nsclc pa ent plasma using ddpcr...
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Liquid Biopsy Analysis of NSCLC Pa�ent Plasma using ddPCR and NGSWeihua Liu, Peng Fang, Chad Galderisi, Cindy Spi�le and Jin Li. MolecularMD, Cambridge, MA and Portland, OR
Introduc�onSince the first liquid biopsy test (Cobas EGFR Muta�on Test v2) was approved by the US Foodand Drug Administra�on (FDA) as a companion diagnos�c assay, the use of liquid biopsy inclinical prac�ce has become a reality. The currently approved liquid biopsy test u�lizes real-�mePCR technology and allows for posi�ve/nega�ve detec�on of EGFR muta�ons (L858R, ex19del,T790M) in cfDNA when NSCLC �ssue is not available or is inadequate. The use of liquid biopsycon�nues to be explored for a wide range of addi�onal applica�ons and indica�ons includingpa�ent screening for combina�on therapies and allele burden monitoring. Therefore, the futureuse of liquid biopsy tes�ng in clinical prac�ce may require technology pla�orms that enable theiden�fica�on of a higher number of targets and/or the absolute quan�fica�on of mutant cfDNAmolecules. Here we use both EGFR droplet digital PCR (ddPCR) and the Oncomine Lung cfDNAAssay (NGS panel) to evaluate allele burden and assess a broader muta�on profile, respec�vely,in a set of plasma samples collected from NSCLC pa�ents.
MethodsPlasma samples (1-2.7ml) were collected at Dana-Farber Cancer Ins�tute. cfDNA was extractedusing the QIAamp DSP circula�ng NA kit. EGFR ddPCR assays were designed by DFCI (PMID:19351754). One third of cfDNA extracted from 1 ml plasma was used for each EGFR ddPCR assay(L858R, T790M and ex19del). The L858R and T790M ddPCR assays detect muta�ons usingmutant specific probes. The ex19del assay allows for the detec�on of a broad range of exon 19dele�ons via the loss of wild-type exon 19 signal. Sixteen plasma samples from pa�ents withEGFR WT tumors were analyzed to assess the background noise (Limit of Blank). The other 37plasma samples were analyzed in a blinded fashion. ddPCR analysis was performed on theQX200 system and data was analyzed using QuantaSo� so�ware.A subset of 12 plasma samples had sufficient cfDNA for addi�onal analysis using the OncomineLung cfDNA Assay on the Ion S5. This NGS panel is designed to detect 150 hotspot muta�ons in11 lung cancer-related genes (ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA,ROS1, and TP53). Torrent Suite So�ware 5.2 was used for NGS data analysis. The HorizonDxcfDNA Mul�plex Reference Standard (MAF 5%, 1%, 0.1% and WT) was also included in theddPCR and NGS studies.
ResultsPosi�ve/Nega�ve Assay Cut-off for EGFR ddPCR: The plasma samples from pa�ents with WTtumors showed large varia�on in the amount of cfDNA extracted ranging from 96 copies to 2032total copies of wild-type EGFR in triplicate wells. No posi�ve droplet was observed in all samplesfor L858R and del19 assays. For the T790M assay, sample TRL_28 showed one double posi�vedroplet and TRL_30 and TRL_32 showed one single posi�ve droplet. Based on the LoBassessment from 16 EGFR nega�ve plasma samples, the cut-off for calling posi�ve muta�on isset at: Poisson es�mated mutant copies ≥ 3 (the actual number of posi�ve droplets ≥ 2) if wildtype copy is ≤ 3000; Poisson es�mated mutant copies ≥ 4.5 (the actual number of posi�vedroplets ≥ 3) if wild type copy is > 3000. These cut-off values were applied to the data generatedin other 32 samples. When the number of copies was below the established posi�ve/nega�vecut off, it was reported as “ND” (Not Detected) (Table 1).
Conclusions Both ddPCR and NGS were able to detect muta�ons at 0.1% MAF with sufficient cfDNAinput. The Oncomine panel confirmed EGFR ddPCR results and detected co-exis�ng PIK3CAmuta�ons. EGFR and PIK3CA muta�ons were present at a similar AF, sugges�ng that EGFRand PIK3CA muta�ons may develop at the same stage of tumorigenesis in these pa�ents. Our study results demonstrate that comprehensive profiling of cfDNA using NGS couldbe cri�cal for the selec�on of appropriate therapies for NSCLC pa�ents while ddPCR is an
ideal pla�orm for monitoring disease burden during the course of therapy.
For Further Informa�onPlease contact [email protected] or visit www.molecularmd.com
Table 4. LoD using 12 ng DNA input in triplicate wells.
Table 8. Results from Oncomine Lung cfDNA Assayfor plasma samples (n=12).
Comparison to ddPCR results from external lab:Plasma samples (n=37) from NSCLC pa�ents that had been previously genotyped forEGFR L858R, Ex19del and T790M muta�ons using ddPCR assays were blinded andtested. MolecularMD analysis determined 12 samples (32%) to be L858R posi�ve, 19samples (51%) to be T790M posi�ve and 20 samples (54%) to be ex19del posi�ve. TheMAF ranged from 0.08-6.68% for L858R, 0.2-20.58% for T790M and 0.62-87.72% forex19del. The ddPCR results between the 2 labs were 100% concordant for L858R and94% concordant for both T790M and ex19del. In all discordant cases, theMolecularMD ddPCR result was posi�ve and the external lab result was nega�ve.When sufficient cfDNA was available, either NGS or an alterna�ve ddPCR assay wasused to resolve discordant results (Table 7). The discordance for these samples couldbe caused by sampling varia�on during cfDNA extrac�on or assay tes�ng to capturethe few copies of mutant molecules present in the plasma samples.Table 6. MolecularMD ddPCR results (n=37).
Results
Precision of EGFR ddPCR : cfDNA was extracted from the HorizonDxD Mul�plex cfDNAReference Standard Set in Synthe�c Plasma and a healthy donor plasma sample. Theextracted cfDNA (10ng input) was analyzed in triplicate wells on 3 different days (Table10). The Reference Standards with 5% and 1% AF were reproducibly detected in 3/3 runs. .Nega�ve samples (healthy donor plasma and HorizonDx WT synthe�c plasma) were callednega�ve in 3/3 runs.
ResultsResults
Table 1. LoB assessment results for EGFR L858R, T790M and del19 ddPCR (n=16).
Limit of Detec�on (LoD) for EGFR ddPCR: The HorizonDx cfDNA Mul�plex Reference StandardSet was serially diluted in wild type cfDNA standard to create 5%, 1%, 0.5%, 0.25% and 0.1%MAF. DNA input for each well was 1 (low), 2 (medium) and 4 ng (high). Triplicate wells wereanalyzed for each dilu�on with each assay on 3 different days by 2 operators. The results weresummarized in Table 2, 3 and 4 for 3, 6, and 12 ng total DNA input in triplicate wells, respec�vely.The rows in grey highlight the lowest MAF detected by each assay. The LoD was defined as theaverage lowest dilu�on that generates a posi�ve muta�on call in 3/3 runs and it increases withcfDNA input (Table 5). In clinical plasma samples, the assay was able to detect 0.08% L858Rmuta�on with high cfDNA input (Table 6: 4.5 L858R mutant copies/5827 wild type copies).Table 2. LoD using 3 ng DNA input in triplicate wells.
Table 3. LoD using 6 ng DNA input in triplicate wells.
Lowest detected average MAF (%)cfDNA input L858R T790M Ex19del3ng 3.51% 1.5% 1.82%6ng 0.33% 0.59% 1.22%12ng 0.31% 0.45% 0.73%
Table 5. Summary of LoD for each EGFR ddPCR assay using 3, 6 and 12 ng DNA input .
Comparison to Oncomine Lung cfDNA Assay: A subset of samples (n=12) that hadsufficient remaining cfDNA was also analyzed by NGS using the Oncomine Lung cfDNAAssay. There was 100% concordance between the NGS and ddPCR results generatedby MolecularMD. EGFR T790M muta�on was detected as low as 0.18% MAF with8.5ng plasma cfDNA input in the NGS assay (Table 8 in green). In addi�on, PIK3CAmuta�ons were found to co-exist with EGFR ex19 dele�ons in 3/12 plasma samples byNGS (Table 8 in yellow). The MAF of PIK3CA muta�ons and EGFR muta�ons wascomparable in the same samples (TRL23: 24.3% for EGFR del19 and 10.4% T790M vs20.2% for PIK3CA H1047R; TRL48: 7.8% for EGFR del19 vs 3.4% for PIK3CA E545K;TRL51: 0.85% for EGFR del19 vs 1.02% for PIK3CA E545K).HorizonDx cfDNA Mul�plex Reference Standard set with 5%, 1%, 0.1% MAF and wildtype was analyzed using NGS along with plasma samples. Muta�ons were detected aslow as 0.1% MAF using 20ng input (Table 9). No muta�on was detected for cfDNA wildtype standard.
Table 9. Results from Oncomine Lung cfDNA Assayfor HorizonDx Mul�plex Standards.Sample Name Gene Muta�on MAF (%) Read Depth
HorizonDx Std 5%
NRAS p.Q61K 5.58 19439NRAS p.A59T 5.48 19471PIK3CA p.E545K 6.83 21514EGFR p.E746_A750delELREA 5.6 21819EGFR p.V769_D770insASV 4.67 17838EGFR p.T790M 7.26 20260EGFR p.L858R 3.79 16875KRAS p.G12D 6.73 22394
HorizonDx Std 1%
NRAS p.Q61K 1.4 22483NRAS p.A59T 0.61 22474PIK3CA p.E545K 1.43 24444EGFR p.E746_A750delELREA 1.18 24477EGFR p.V769_D770insASV 1.04 19300EGFR p.T790M 0.9 22226EGFR p.L858R 0.79 19783KRAS p.G12D 1.4 26832
HorizonDx Std 0.1%
NRAS p.Q61K 0.12 30463NRAS p.A59T 0.09 30450PIK3CA p.E545K 0.09 28803EGFR p.E746_A750delELREA 0.09 31376EGFR p.V769_D770insASV 0.06 24824EGFR p.T790M 0.08 30445EGFR p.L858R 0.16 23471KRAS p.G12D 0.14 32305
Table 10. Results of Precision Study for ddPCR assays.
Sample Name Gene Muta�on MAF (%) Read DepthTRL_23 PIK3CA p.H1047R 20.22 49657TRL_23 EGFR p.E746_A750delELREA 24.29 63630TRL_23 EGFR p.T790M 10.36 35757TRL_43 EGFR p.L747_E749delLRE 86.34 54733TRL_43 EGFR p.T790M 11.8 26533TRL_45 EGFR p.E746_A750delELREA 37.38 52341TRL_45 EGFR p.T790M 17.94 38125TRL_46 EGFR p.L747_E749delLRE 39.49 55918TRL_46 EGFR p.T790M 0.6 30066TRL_47 EGFR p.L747_E749delLRE 4.46 18503TRL_47 EGFR p.T790M 0.18 14658TRL_48 PIK3CA p.E545K 3.42 31061TRL_48 EGFR p.E746_A750delELREA 7.79 27856TRL_49 EGFR p.E746_A750delELREA 3.07 33238TRL_50 EGFR p.E746_A750delELREA 2.59 37631TRL_51 PIK3CA p.E545K 1.02 95255TRL_51 EGFR p.E746_A750delELREA 0.85 77810TRL_52 EGFR p.T790M 0.27 39003TRL_52 EGFR p.L858R 0.52 57698TRL_53 EGFR p.T790M 0.38 31483TRL_53 EGFR p.L858R 0.85 59324TRL_54 EGFR p.T790M 15.06 45911TRL_54 EGFR p.L858R 1.37 95345
L858R T790M Ex19delSample Mut Copies Wt Copies MAF (%) Mut Copies Wt Copies MAF (%) Mut Copies Wt Copies MAF (%)TRL_26 0 397.5 0 0 322.5 0 0 285 0TRL_27 0 412.5 0 0 285 0 0 315 0TRL_28 0 585 0 1.58 375 ND 0 427.5 0TRL_29 0 742.5 0 0 502.5 0 0 630 0TRL_30 0 382.5 0 1.5 307.5 ND 0 300 0TRL_32 0 915 0 1.58 630 ND 0 772.5 0TRL_33 0 472.5 0 0 345 0 0 397.5 0TRL_34 0 465 0 0 420 0 0 420 0TRL_35 0 120.75 0 0 97.5 0 0 96 0TRL_36 0 900 0 0 660 0 0 772.5 0TRL_37 0 690 0 0 495 0 0 585 0TRL_38 0 1050 0 0 810 0 0 900 0TRL_39 0 2032.5 0 0 1477.5 0 0 1755 0TRL_40 0 1687.5 0 0 1207.5 0 0 1402.5 0TRL_41 3 847.5 0 0 585 0 0 667.5 0TRL_42 0 645 0 0 412.5 0 0 570 0
Run 1 Run 2 Run 3
Sample Target Mutant Copies WT Copies MAF (%) Mutant Copies WT Copies MAF (%) Mutant Copies WT Copies MAF (%)Average MAF (%)
StDev of MAF (%)
WT EGFR_L858R 0 990 0.00 0 952.5 0.00 0 667.5 0.00 0.00 0.005.00% EGFR_L858R 37.5 1065 3.40 27.75 975 2.77 38.25 840 4.36 3.51 0.801.00% EGFR_L858R 9.75 915 1.05 1.5 900 0.17 16.5 870 1.86 1.03 0.850.50% EGFR_L858R 5.25 870 0.60 2.25 832.5 0.27 1.575 772.5 0.20 0.36 0.210.25% EGFR_L858R 0 825 0.00 0 922.5 0.00 1.5 765 0.20 0.07 0.110.10% EGFR_L858R 0 1117.5 0.00 0 1012.5 0.00 0 1005 0.00 0.00 0.00
WT EGFR_T790M 0 802.5 0.00 0 735 0.00 0 525 0.00 0.00 0.005.00% EGFR_T790M 25.5 817.5 3.02 35.25 765 4.40 46.5 667.5 6.51 4.65 1.761.00% EGFR_T790M 4.5 772.5 0.58 16.5 720 2.24 10.5 615 1.68 1.50 0.840.50% EGFR_T790M 2.25 577.5 0.39 5.25 682.5 0.76 0 502.5 0.00 0.38 0.380.25% EGFR_T790M 2.25 660 0.34 0 705 0.00 0 540 0.00 0.11 0.200.10% EGFR_T790M 0 825 0.00 0 810 0.00 0 697.5 0.00 0.00 0.00
WT EGFR_Ex19del 0 450 0.00 0 465 0.00 0 337.5 0.00 0.00 0.005.00% EGFR_Ex19del 22.5 480 4.48 37.5 562.5 6.25 37.5 345 9.80 6.84 2.711.00% EGFR_Ex19del 7.5 397.5 1.85 7.5 457.5 1.61 7.5 367.5 2.00 1.82 0.200.50% EGFR_Ex19del 0 412.5 0.00 0 487.5 0.00 0 330 0.00 0.00 0.000.25% EGFR_Ex19del 0 510 0.00 0 510 0.00 0 352.5 0.00 0.00 0.000.10% EGFR_Ex19del 0 502.5 0.00 0 457.5 0.00 0 442.5 0.00 0.00 0.00
Run 1 Run 2 Run 3
Sample Target Mutant Copies WT Copies MAF (%) Mutant Copies WT Copies MAF (%) Mutant Copies WT Copies MAF (%)Average MAF (%)
StDev of MAF (%)
WT EGFR_L858R 0 1665 0.00 0 1957.5 0.00 0 1282.5 0.00 0.00 0.005.00% EGFR_L858R 87 1702.5 4.86 102.75 1897.5 5.14 76.5 1605 4.55 4.85 0.291.00% EGFR_L858R 14.25 1417.5 1.00 8.25 1770 0.46 15 1717.5 0.87 0.78 0.280.50% EGFR_L858R 3.75 1492.5 0.25 6 2085 0.29 6.75 1462.5 0.46 0.33 0.110.25% EGFR_L858R 2.25 1275 0.18 0 1777.5 0.00 1.5 1545 0.10 0.09 0.090.10% EGFR_L858R 3.75 1515 0.25 2.25 1875 0.12 0 1740 0.00 0.12 0.12
WT EGFR_T790M 0 1215 0.00 0 1470 0.00 0 952.5 0.00 0.00 0.005.00% EGFR_T790M 54.75 1147.5 4.55 63.75 1312.5 4.63 39 990 3.79 4.33 0.471.00% EGFR_T790M 6.75 1035 0.65 9.75 1215 0.80 3.75 1140 0.33 0.59 0.240.50% EGFR_T790M 3.75 1207.5 0.31 2.25 1305 0.17 10.5 907.5 1.14 0.54 0.530.25% EGFR_T790M 1.5 1087.5 0.14 1.5 1342.5 0.11 0 1080 0.00 0.08 0.070.10% EGFR_T790M 0 1207.5 0.00 6 1470 0.41 1.5 1290 0.12 0.17 0.21
WT EGFR_Ex19del 0 900 0.00 0 855 0.00 0.00 712.50 0.00 0.00 0.005.00% EGFR_Ex19del 52.5 1012.5 4.93 67.5 937.5 6.72 52.50 900.00 5.51 5.72 0.911.00% EGFR_Ex19del 7.5 780 0.95 15 780 1.89 7.50 892.50 0.83 1.22 0.580.50% EGFR_Ex19del 0 802.5 0.00 7.5 915 0.81 7.50 735.00 1.01 0.61 0.540.25% EGFR_Ex19del 0 855 0.00 0 877.5 0.00 7.50 847.50 0.88 0.29 0.510.10% EGFR_Ex19del 0 847.5 0.00 0 862.5 0.00 0.00 1080.00 0.00 0.00 0.00
Run 1 Run 2 Run 3
Sample Target Mutant Copies WT Copies MAF (%) Mutant Copies WT Copies MAF (%) Mutant Copies WT Copies MAF (%)Average MAF (%)
StDev of MAF (%)
WT EGFR_L858R 0 3315 0.00 0 3090 0.00 0 2647.5 0.00 0.00 0.005.00% EGFR_L858R 183 3787.5 4.61 150.75 2977.5 4.82 170.25 3105 5.20 4.88 0.301.00% EGFR_L858R 30 3382.5 0.88 33 2940 1.11 21 3315 0.63 0.87 0.240.50% EGFR_L858R 18.75 3390 0.55 18.75 3030 0.62 16.5 2677.5 0.61 0.59 0.040.25% EGFR_L858R 15 3480 0.43 6 2902.5 0.21 9 2925 0.31 0.31 0.110.10% EGFR_L858R 7.5 3855 0.19 1.5 3090 0.05 4.5 3495 0.13 0.12 0.07
WT EGFR_T790M 3 2490 0.12 0 2302.5 0.00 0 1920 0.00 0.04 0.075.00% EGFR_T790M 142.5 2722.5 4.97 130.5 2392.5 5.17 90 2175 3.97 4.71 0.641.00% EGFR_T790M 21 2460 0.85 24.75 2235 1.10 26.25 2317.5 1.12 1.02 0.150.50% EGFR_T790M 12 2557.5 0.47 12.75 2340 0.54 5.25 1927.5 0.27 0.43 0.140.25% EGFR_T790M 11.25 2430 0.46 15.75 2497.5 0.63 5.25 1995 0.26 0.45 0.180.10% EGFR_T790M 1.5 2805 0.05 3 2490 0.12 3.75 2482.5 0.15 0.11 0.05
WT EGFR_Ex19del 0 1755 0.00 0 1837.5 0.00 0.00 1605.00 0.00 0.00 0.005.00% EGFR_Ex19del 90 1890 4.55 142.5 1837.5 7.20 172.50 1860.00 8.49 6.74 2.011.00% EGFR_Ex19del 22.5 1687.5 1.32 15 1605 0.93 22.50 1882.50 1.18 1.14 0.200.50% EGFR_Ex19del 15 1740 0.85 7.5 1830 0.41 15.00 1597.50 0.93 0.73 0.280.25% EGFR_Ex19del 0 1755 0.00 7.5 1725 0.43 0.00 1732.50 0.00 0.14 0.250.10% EGFR_Ex19del 7.5 1627.5 0.46 0 1695 0.00 15.00 1980.00 0.75 0.40 0.38
L858R T790M Ex19del
Sample ID Mut Copies Wt Copies MAF (%) Mut Copies Wt Copies MAF (%) Mut Copies Wt Copies MAF (%)TRL_1 67.5 7612.5 0.88 18.75 4770 0.39 0 5932.5 0.00TRL_2 0 727.5 0.00 0 405 0.00 4 637.5 0.62TRL_3 0 667.5 0.00 0 397.5 0.00 4 577.5 0.69TRL_4 255 9645 2.58 61.5 5512.5 1.10 0 7762.5 0.00TRL_5 0 1042.5 0.00 5.25 652.5 0.80 15 832.5 1.77TRL_6 0 480 0.00 0 345 0.00 0 465 0.00TRL_8 114.75 2197.5 4.96 3 1462.5 0.20 0 1890 0.00TRL_9 0 1155 0.00 58.5 712.5 7.59 210 667.5 23.93TRL_10 0 795 0.00 0 517.5 0.00 52.5 630 7.69TRL_11 660 10432.5 5.95 3.75 6630 ND 0 8662.5 0.00TRL_12 0 3442.5 0.00 0 2175 0.00 255 2452.5 9.42TRL_13 87 1215 6.68 6.75 975 0.69 0 1027.5 0.00TRL_14 0 2197.5 0.00 10.5 1297.5 0.80 825 1297.5 38.87TRL_15 0 2340 0.00 126.75 1260 9.14 480 1575 23.36TRL_16 4.5 5827.5 0.08 1.35 4110 ND 0 5010 0.00TRL_17 0 442.5 0.00 0 375 0.00 0 322.5 0.00TRL_18 57 4725 1.19 11.25 3067.5 0.37 0 3847.5 0.00TRL_19 117.75 2070 5.38 1.425 1200 ND 0 1612.5 0.00TRL_20 22.5 1980 1.12 3 1275 0.23 0 1530 0.00TRL_21 0 2632.5 0.00 96.75 1830 5.02 907.5 1470 38.17TRL_22 0 585 0.00 0 382.5 0.00 187.5 375 33.33TRL_23 0 7050 0.00 465 3667.5 11.25 2062.5 4215 32.86TRL_24 0 615 0.00 0 367.5 0.00 0 502.5 0.00TRL_25 0 825 0.00 0 622.5 0.00 0 750 0.00
TRL_31 0 1237.5 0.00 1.5 862.5 ND 7.5 1020 0.73TRL_43 0 8392.5 0.00 697.5 4965 12.32 6645 930 87.72TRL_44 0 532.5 0.00 0 300 0.00 0 412.5 0.00TRL_45 0 4680 0.00 585 2257.5 20.58 1650 2340 41.35TRL_46 0 1680 0.00 3.75 930 0.40 600 847.5 41.45TRL_47 0 2820 0.00 16.5 1875 0.90 112.5 2122.5 5.03TRL_48 0 795 0.00 0 517.5 0.00 45 555 7.50TRL_49 0 1170 0.00 0 847.5 0.00 22.5 780 2.80TRL_50 0 1012.5 0.00 0 615 0.00 30 742.5 3.88TRL_51 0 1162.5 0.00 0 682.5 0.00 15 945 1.56TRL_52 84.75 10162.5 0.83 18 5797.5 0.31 0 7560 0.00TRL_53 72.75 9592.5 0.75 20.25 5677.5 0.36 0 7290 0.00TRL_54 56.25 2925 1.89 345 1522.5 18.47 0 2265 0.00
Table 7. Discordant ddPCR results between MolecularMD and the external lab.Sample ID MolecularMD ddPCR result MAF Third method resultsTRL_5 T790M posi�ve 0.8% Insufficient cfDNATRL_47 T790M posi�ve 0.9% Confirmed posi�ve by NGSTRL_3 Ex19del posi�ve 0.7% Insufficient cfDNATRL_31 Ex19del posi�ve 0.7% Confirmed posi�ve by third ddPCR run using Biorad assay
Run 1 Run 2 Run 3
Sample Target Mutant Copies WT Copies MAF (%) Mutant Copies WT Copies MAF (%) Mutant Copies WT Copies MAF (%)Average MAF (%)
StDev of MAF (%)
Healthy Donor EGFR_L858R 0 1080 0.00 0 930 0.00 0 1020 0.00 0.00 0.00HDcfDNA WT EGFR_L858R 0 2902.5 0.00 0 3450 0.00 0 2655 0.00 0.00 0.00HDcfDNA 5% EGFR_L858R 119.25 2310 4.90 167.25 3585 4.50 150 2775 5.10 4.83 0.31HDcfDNA 1% EGFR_L858R 39.75 3270 1.19 42 3877.5 1.08 19.5 3195 0.60 0.96 0.31HDcfDNA 0.1% EGFR_L858R 0 3082.5 0.00 12 3727.5 0.31 5.25 2925 0.17 0.16 0.16Healthy Donor EGFR_T790M 2.25 690 ND 0 637.5 0.00 0 645 0.00 0.00 0.00HDcfDNA WT EGFR_T790M 0 2272.5 0.00 1.5 2212.5 ND 2.25 2010 0.10 0.00 NAHDcfDNA 5% EGFR_T790M 90.75 1695 5.10 131.25 2490 5.00 100.5 2032.5 4.70 4.93 0.21HDcfDNA 1% EGFR_T790M 21.75 2317.5 0.92 30.75 2932.5 1.00 20.25 2347.5 0.90 0.94 0.05HDcfDNA 0.1% EGFR_T790M 0 2197.5 0.00 3.75 2737.5 0.13 6 2100 0.29 0.14 0.15Healthy Donor EGFR_Ex19del 0 810 0.00 0 2002.5 0.00 0 900 0.00 0.00 0.00HDcfDNA WT EGFR_Ex19del 0 1575 0.00 0 2265 0.00 0 1605 0.00 0.00 0.00HDcfDNA 5% EGFR_Ex19del 97.5 1485 6.16 120 2227.5 5.11 112.5 1732.5 6.10 5.79 0.59HDcfDNA 1% EGFR_Ex19del 22.5 1710 1.30 22.5 1950 1.14 7.5 1935 0.39 0.94 0.49HDcfDNA 0.1% EGFR_Ex19del 0 1627.5 0.00 0 772.5 0.00 0 1785 0.00 0.00 0.00