Marion Laig1, Ana Justino2, Rosella Petraroli1, Le Lac1, Dieu Linh Vo1, Kamini Varma1, José Carlos Machado2, José Luis Costa2

1Thermo Fisher Scientific, 180 Oyster Point Blvd., South San Francisco, CA 2IPATIMUP, Porto, Portugal

RESULTS Rare mutation analysis by digital PCR on QuantStudio 3D confirmed the low mutation frequency detected by NGS. Both technologies showed matching results for all eleven samples tested at mutation rates below 1%. Both technologies also detected expected mutation rates based on the time points. CONCLUSIONS We confirmed the sensitivity and accuracy of NGS for low frequency mutation detection. Rare mutation analysis by digital PCR on QuantStudio 3D is an excellent application to verify NGS results in samples for cancer research.

REFERENCES

Mills GB. 2012. An emerging toolkit for targeted cancer therapies. Genome Res. 22(2):177-182

Kinde I. et al. 2011. Detection and quantification of rare mutations with massively parallel sequencing. PNAS 108(23):9530-9535

Yates LR and Campbell PJ. 2012. Evolution of the cancer genome. Nat Rev Gen 13:795-806.

Ma C et al. 2011. T790M and acquired resistance of EGFR TKI: a literature review of clinical reports. J Thorac Dis. 3(1): 10–18

Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies. Bettegowda C et al., Sci Transl Med. 2014 February 19; 6(224)

ACKNOWLEDGEMENTS We thank Alicia Burt and Rachel Formosa for reviewing this document.

TRADEMARKS/LICENSING © 2015 Thermo Fisher Scientific Inc. All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. For Research Use Only. Not for use in diagnostic procedures.

Rare Mutation Analysis Using Digital PCR on QuantStudio™ 3D to Verify Ion AmpliSeq™ Next Generation Sequencing for Cancer Research

Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com

ABSTRACT We identified mutations in eleven cell free (cf) DNA samples by next generation sequencing (NGS) using the Ion AmpliSeq™ Colon & Lung Cancer Research Panel and the Ion PGM™ System. Since detection of low frequency mutant alleles may not always be called confidently in NGS, we verified results by rare mutation analysis using digital PCR on the QuantStudio™ 3D Digital PCR System as an independent method. We show that frequencies detected are consistent for both methods for low frequency mutant alleles at and below 1%.

INTRODUCTION Next generation sequencing (NGS) is becoming an increasingly important analysis tool for the discovery of biomarkers in cancer research and to study tumor heterogeneity in samples. Although NGS is an excellent discovery tool, detection of low frequency mutant alleles may not always be called confidently due to an inherent low error rate of the DNA polymerase used (fidelity). It is therefore beneficial to verify detection of rare alleles with an independent method. In this study, we set out to confirm NGS sensitivity and accuracy detecting low frequency mutations using rare mutation analysis by digital PCR as an independent technology. We present data showing consistency in low level mutation detection in cf DNA samples between two methods: NGS and Rare Mutation Analysis using digital PCR.

MATERIALS AND METHODS Eleven cell free DNA samples isolated from plasma were typed using the Ion AmpliSeq panel and the Ion Proton PGM. The same samples were then analyzed by digital PCR to confirm the presence of the mutations detected. Samples represented a mixture of individual samples as well as the same sample taken at different time points. Mutations were identified and quantified by digital PCR using TaqMan™ Rare Mutation Assays (Applied Biosystems) for the following mutations: EGFR T790M (COSMIC ID 6240, Assay ID AHRSROS), L858R (COSMIC ID 6224, Assay ID AHRSRSV), and exon 19 deletions E746_A750delELREA (COSMIC ID 6223, Assay ID AHLJ0XO) and L747_P753>S (COSMIC ID 12370, Assay ID AHZAGZG), and KRAS mutations G12V (COSMIC ID 520, Assay ID AHX1IHY) and G12D (COSMIC ID 521, Assay ID AH6R5PI). Experiments were run according to the TaqMan Rare Mutation Assays protocol.

Figure 3. Ion PGM System and QuantStudio 3D Rare Mutation Detection Solution

Table 1. Determination of Allelic Fractions in the EGFR and KRAS Genes in cell free DNA Samples

Allelic fractions of low frequency mutations in the EGFR and KRAS genes were determined in cell free (cf)DNA by sequencing using the Ion AmpliSeq panel and verified by Rare Mutation Analysis using digital PCR. For digital PCR, two chips were run for each mutation per sample. Allelic frequencies were consistent between the two technologies at low percentages of 1% and below. * One of two chips showed no mutation detection, the fraction represents the average of two chips ? Based on one positive read

Figure 2. Detection of EGFR T790M using the Ion AmpliSeq Colon & Lung Cancer Research Panel

Ion AmpliSeq results displayed in the Integrative Genomics Viewer (IGV, Broad Institute) Software detecting the T790M mutation in the EGFR gene in the same sample as above (left), detection of wild-type only in the same sample as above (right).

Mutant p.T790M Wild-type

Figure 1. Detection of the EGFR T790M Mutation by Digital PCR

Digital PCR results displayed in QuantStudio 3D AnalysisSuite™ Cloud Software showing the detection of the T790M mutation in the EGFR gene in one sample (left, mutation in the outlined FAM cluster), detection of wild-type only (VIC) in another sample (right).

Mutant p.T790M Wild-type

All samples were originally sequenced using the Ion AmpliSeq panel and the Ion PGM System (left) and verified with Rare Mutation Assays by digital PCR using the QuantStudio 3D (right). https://www.thermofisher.com/us/en/home/life-science/pcr/digital-pcr/rare-mutation-analysis.html