Fig 1.
Experimental workflow for picoliter droplet-based digital PCR.
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. WT, droplets containing wild-type DNA; MUT, droplets containing mutant DNA.
Fig 2.
Mapping of most frequent EGFR, KRAS and TP53 mutations.
In EGFR gene (A), most of the mutations occur within Tyrosine Kinase (TK) domain (in light green, those associated with drug resistance). In KRAS (B), the most frequent mutations are located in exon 2 (corresponding to its GTP binding domain), while in TP53 (C) they are mainly located in its DNA binding domain. In bold, mutations targeted in the study.
Fig 3.
Examples of EGFR L858R castPCR™ and TaqMan® asssays.
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 S6 Fig 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. A.U, arbitrary units; WT, wild-type; Reference, wild-type + mutant DNA; gDNA, genomic DNA.
Fig 4.
False-positive evaluation in negative controls (human wild-type genomic DNA).
In order to assess the false-positive (FP) events detected in negative control samples, we analysed by dPCR a collection of human wild-type only samples (genomic DNA, refer to S1 Fig for details) with the EGFR, KRAS and TP53 assays described. We used two different amounts of DNA input (20 and 60 ng, depicted by circles and squares respectively). The scatter plot displays the low dynamic range detection of three castPCR™ probes (EGFR p.T790M, TP53 p.R273H and p.R213*), where the number of FP events increased when using 60 ng of starting DNA material (lines represent the mean for each assay). At right, the table shows the LOB and LOD estimation of all assays (refer to [33] for precise formula), calculated from the λFP of each test (where λFP is given by the mean number of false-positives obtained in all experiments realized with 20 ng input DNA). Mean value and standard deviation for each FP measurement for the different assays are shown, both for WT and MUT-DNA containing droplets. (C), castPCR™ probes; (T), TaqMan® probes; (Z), ZEN™ probes; N°, number; FP, false-positive; LOB, Limit of Blank; LOD, Limit of Detection; N/A, not applicable.
Fig 5.
Examples of titration series with EGFR castPCR™, ZEN™ and TaqMan® probes.
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 [8]).
Fig 6.
Examples of titration series with KRAS castPCR™ and TaqMan® probes.
Serial dilutions of G12D and G12S mutated DNA (extracted from A427 and LS123 cell lines, 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.
Fig 7.
Examples of titration series with TP53 castPCR™ and TaqMan® probes.
Serial dilutions of TP53 p.R273H and p.R213* mutated DNA (extracted from HT-29 and SW-684 cell lines, 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 TP53 p.R273H and p.R213*castPCR™ test, LOB and LOD calculation could not be performed (refer to [8]).
Fig 8.
EGFR L858R, L861Q, Del19 and T790M mutation screening on lung cancer patients plasma using dPCR two-plex assay.
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. Reference, wild-type + mutant DNA; NA, not analyzed; A.U, arbitrary units.
Fig 9.
EGFR Del19 screening on lung cancer patients plasma using two-plex assay.
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. Reference, wild-type + mutant DNA; NA, not analyzed; A.U, arbitrary units; AA, aminoacids.
Fig 10.
Multiplex assays for the most frequent EGFR mutations.
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). A.U, arbitrary units; WT, wild-type; S, sensitivity mutation; R, resistance mutation.
Fig 11.
Multiplex panel for the most common EGFR mutations using HdX™ Reference Standards.
A three-plex panel for follow-up of the 3 EGFR sensitivity mutations with the T790M resistance mutation. As input, DNA from FFPE Reference Standards (R.S.) (panels A, B, C), Multiplex I cfDNA (panels D and F) and Multiplex genomic DNA (panel E) from Horizon Diagnostics. The FFPE R.S. contains 50% of genomic DNA and 5% of each mutation, while while the Multiplex DNA is engineered from mutant cell lines for generation of 12.5% EGFR allelic frequency for the four mutations. Multiplex I cfDNA provides a set containing fragmented DNA in a range of low allelic frequencies (from 0.1% to 5%): we showed here only the 5% allelic frequency DNA. 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). A.U, arbitrary units; WT, wild-type; gDNA, genomic DNA.