1. Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples 
Laetitia Borsu, Julie Intrieri, Linta Thampi, Helena Yu, Gregory Riely, Khedoudja Nafa, Raghu Chandramohan, Marc Ladanyi, Maria E. Arcila 
The Journal of Molecular Diagnostics 
Volume 18, Issue 6, Pages (November 2016)
DOI: /j.jmoldx
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Assay design. A: Genomic sequence of the EGFR exon 20 with primer and probe localization. Intron sequence in lower case; exons in capital letters with corresponding amino acid under each codon; the beginning and end of the exon are marked by downward arrows. B: The probes are designed with standard 5′ fluorophores TET or 6FAM and a 3′ Q. The probe sequence contains LNA nucleotides. All probes also contain a second internal Z Q. The incorporation of an internal Z Q decreases the distance between the 5′ dye and Q and, in concert with the 3′ Q, provides greater overall dye quenching, lowering background, and increasing signal detection. C: The 5′ to 3′ exonuclease activity of the polymerase degrades the probe, physically separating the Qs from the fluorophore, which results in measurable fluorescence. LNA, locked nucleic acid; Q, quencher; R, reporter; Z, zen.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
EGFR T790M assessment by dPCR. A total of 71 cases were tested. Initial assessment was based on a high-sensitivity LNA-PCR Sanger Sequencing assay to establish the T790M status. Cases were divided into two sets. Set 1 encompassed cases submitted for comprehensive testing by NGS. Set 2 encompassed cases in which MSK-IMPACT testing was not ordered. dPCR was performed on both sets either based on MSK-IMPACT libraries or directly from DNA. For a subset of cases in set 1 (28 cases) testing was also performed on direct DNA based on availability for paired direct comparisons. Among cases tested on the libraries, there was 100% success. For cases tested directly from DNA, one case was nonevaluable and two (from the paired set 1) were borderline. dPCR, digital PCR; LNA, locked nucleic acid; MSK-IMPACT, Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets; NGS, next-generation sequencing.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Representative dPCR plots. A–C: Negative (A), positive (B), and equivocal (C) representative plots from three FFPE tumor samples using 40-ng library inputs. Ovals designate the EGFR T790 wild-type (upper left), T790M mutant gate (lower right), and empty droplet gate (lower left). Negative case shows no droplets in the T790M gate. In positive samples, clusters are typically tight and exhibit little variability. Depending on the amount of background noise, droplets from outside the gate may extend into the positive T790M gate, but these are dispersed and do not form a tight cluster. These cases are interpreted as equivocal because very low levels of a mutation may be hidden by the background. These cases may be resolved by repeating the assay with higher DNA input. dPCR, digital PCR; FFPE, formalin-fixed, paraffin-embedded.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Correlation between dPCR and MSK-IMPACT variant frequencies. Scatterplot showing variant frequencies for the same samples analyzed with the dPCR platform as a function of the variant frequencies obtained with the MSK-IMPACT platform shows excellent linearity with Pearson's correlation coefficient of r =  Inset shows the expansion of the area of lowest region for details. dPCR, digital PCR; MSK-IMPACT, Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Figure 5
Comparison of dPCR performed on NGS libraries versus direct DNA. Twenty-eight FFPE samples were tested concurrently using dPCR on NGS libraries and direct DNA to assess assay performance. Results demonstrate that 12 cycles of random oligonucleotide-primed PCR amplification used to prepare the NGS libraries do not introduce major genome representation artifacts in our samples. A: Scatterplot shows direct comparison of T790M variant frequencies obtained on libraries and DNA and shows good correlation at all frequencies (correlation coefficient R2 of 0.98). B: Comparison of droplet occupancy for testing on DNA and libraries. Results are normalized and expressed as the percentage of expected occupancy based on the DNA input (total occupied droplets generated/expected occupied droplet count for input, ×100). Red box shows the distribution based on the NGS libraries; green displays the distribution based on testing directly from genomic DNA. One hundred percent expected occupancy corresponds to theoretical maximal yield of 308 occupied droplets/ng DNA, assuming two EGFR copies per cell. In most samples analyzed directly from genomic DNA, yield is less than expected. Preparations from libraries have higher occupancy, reflecting higher quality and more consistent amount of amplifiable DNA after barcoding and clean-up steps. Higher-than-expected occupancy is related to copy number gains. The bar at 200% expected occupancy level corresponds to a twofold increase in the number of expected EGFR droplets. Samples yielding droplets above this level (with fold increases of 2.1, 2.6, 3.2, and 9.8) are associated with EGFR amplification. Corresponding MSK-IMPACT copy number analysis for four outlier samples above the red box are associated with equivalent EGFR amplification with fold changes of 2.4, 2.8, 2.8, and 11.2, respectively. dPCR, digital PCR; FFPE, formalin-fixed, paraffin-embedded; MSK-IMPACT, Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets; NGS, next-generation sequencing; VF, variant frequency.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7. Figure 6
Sensitivity study results. A: dPCR plots of sequential dilutions from 50% to 0.01%. B: Linearity at all dilution levels. Y axis was calculated T790M variant frequencies are log10 transformed. X axis is dilution levels on the x axis: 1 is undiluted, 2 (50%), 3 (25%), 4 (12.5%), 5 (6.25%), 6 (3.13%), 7 (1.56%), 8 (1%), 9 (0.1%) to 10 (0.01% dilution). dPCR, digital PCR; VF, variant frequency. Ovals designate the EGFR T790 wild-type (upper left), T790M mutant gate (lower right), and empty droplet gate (lower left).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions