Experimental workflow for picoliter droplet-based digital PCR.

<p>An aqueous phase containing PCR reagents, probes, primers and genomic DNA (fragmented if using DNA extracted from cell culture or frozen tissues) is partitioned into droplets using the RainDrop® Source machine (RainDance Technologies, Billerica, US). After thermal-cycling, droplets are re-injected into RainDrop® Sense instrument, permitting the fluorescence detection of each individual droplet. Analysis is finally performed using the RainDance Technologies Analyst software. Empty droplets correspond to droplets containing no targeted DNA. <i>WT</i>, <i>droplets containing wild-type DNA; MUT</i>, <i>droplets containing mutant DNA</i>.</p

Examples of EGFR L858R castPCR™ and TaqMan® asssays.

<p>Plots obtained from dPCR analysis using EGFR L858R castPCR™ assay (panels A and B) and TaqMan® assay (panels C and D). As negative and positive controls, fragmented human wild-type genomic DNA (A and C) and H1975 cell line genomic DNA (B and D) have been used, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159094#pone.0159094.s006" target="_blank">S6 Fig</a> for probes/primers concentrations). In the lower tables, droplets counts from these experiments are listed. Input ng represents the amount of DNA used in dPCR, previously estimated by Qubit® 2.0 Fluorometer. <i>A</i>.<i>U</i>, <i>arbitrary units; WT</i>, <i>wild-type; Reference</i>, <i>wild-type + mutant DNA; gDNA</i>, <i>genomic DNA</i>.</p

EGFR Del19 screening on lung cancer patients plasma using two-plex assay.

<p>These four plots were obtained from dPCR analysis on DNA extracted from plasma of lung cancer patients. The use of Del19 castPCR™ probe permitted to screen samples containing different deletions on exon 19 (of three, four, five and six amino acids, in panel A, B, C, D, respectively). In the table, event counts from the single experiments are listed. Input ng represents the amount of DNA used in dPCR, previously estimated by Qubit® 2.0 Fluorometer (three μL were used for each sample). Measured allelic frequencies are given for dPCR and NGS analysis. <i>Reference</i>, <i>wild-type + mutant DNA; NA</i>, <i>not analyzed; A</i>.<i>U</i>, <i>arbitrary units; AA</i>, <i>aminoacids</i>.</p

EGFR L858R, L861Q, Del19 and T790M mutation screening on lung cancer patients plasma using dPCR two-plex assay.

<p>Two-plots analysis on plasma DNA samples, whose initial tumor specific mutation has been previously determined by NGS on the tumor tissue. In the table, event counts from each experiment are listed. Input ng represents the amount of DNA used in dPCR, previously estimated by Qubit® 2.0 Fluorometer (three μL were used for each sample). Measured allelic frequencies are given for dPCR and NGS analysis. <i>Reference</i>, <i>wild-type + mutant DNA; NA</i>, <i>not analyzed; A</i>.<i>U</i>, <i>arbitrary units</i>.</p

Mapping of most frequent <i>EGFR</i>, <i>KRAS</i> and <i>TP53</i> mutations.

<p>In <i>EGFR</i> gene (A), most of the mutations occur within Tyrosine Kinase (TK) domain (in light green, those associated with drug resistance). In <i>KRAS</i> (B), the most frequent mutations are located in exon 2 (corresponding to its GTP binding domain), while in <i>TP53</i> (C) they are mainly located in its DNA binding domain. In bold, mutations targeted in the study.</p

Multiplex assays for the most frequent <i>EGFR</i> mutations.

<p>In panel A, B and C, 2D-plots of the three-plex for follow up of the three sensitivity mutations with the T790M resistance mutation. The four-plex is shown in panel D. A pool of fragmented DNA extracted from two cell lines (H1975 harboring L858R and T790M mutations, H1650 harboring Del19 mutation), DNA from FFPE sample (for L861Q mutation) and fragmented wild-type only genomic DNA was used as input. A mix of mutation-specific VIC and/or 6-carboxyfluorescein cast™ and ZEN™ probes was optimized. In the table, event counts from the single experiments are listed (input ng represents the amount of DNA used in dPCR, previously estimated by Qubit® 2.0 Fluorometer). <i>A</i>.<i>U</i>, <i>arbitrary units; WT</i>, <i>wild-type; S</i>, <i>sensitivity mutation; R</i>, <i>resistance mutation</i>.</p

Lysimachia tanakae Maxim.

原著和名: ミヤマコナスビ科名: サクラソウ科 = Primulaceae採集地: 奈良県 玉置山 (大和 玉置山)採集日: 1983/6/26採集者: 萩庭丈壽整理番号: JH036314国立科学博物館整理番号: TNS-VS-98631

Examples of titration series with EGFR castPCR™, ZEN™ and TaqMan® probes.

<p>Serial dilutions of L858R, T790M, Del19, or L861Q mutated DNA (extracted from H1975 cell line, H1650 cell line or FFPE tissue, respectively) in human wild-type genomic DNA. Individual data points are displayed for independent replicates. The expected mutant to wild-type ratio (black line) is shown. Green continuous and dashed lines represent LOB and LOD values, respectively, evaluated from droplets falling into the mutated-DNA cluster and analyzed in a WT gDNA sample for each replicate. For the lowest titration point (0.01%), we used a higher amount of input DNA. Thus, corresponding LOB and LOD values are represented by red lines. Since number of FP was increasing with quantity of input DNA for EGFR p.T790M castPCR™ test, LOB and LOD calculation could not be performed (refer to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159094#pone.0159094.ref008" target="_blank">8</a>]).</p

Study design.

<p>(A) Flowchart of the study population. (B) Time point of ctDNA follow-up. *Includes ALK fusion (<i>n</i> = 3) and ROS1 fusion (<i>n</i> = 1). **PIK3CA p.H1047L. ^†TP53 p.Gly244Cys. ^$KRAS p.G12V, TP53 p.C135Y, and TP53 p.R248W. No mutations were found for <i>AKT1</i>, <i>ERBB2</i>, <i>FBXW7</i>, <i>FGFR2</i>, <i>MET</i>, or <i>NOTCH1</i>.</p

Prognostic impact of positive baseline ctDNA.

<p>(A) PFS and (B) OS in patients with positive and negative ctDNA (<i>n</i> = 105).</p