Marion Laig, Brian Ho, Nivedita Majumdar, Le Lac, Frances Chan, Ramesh Sathiyaa, Iain Russel, Paco Cifuentes, Ted Straub, Kamini Varma, David Keys

RESULTS

CONCLUSIONS We developed a panel of rare mutation assays for use in digital PCR with the QuantStudio® 3D Digital PCR System. The assays target mutations in the following genes: AKT1, BRAF, EGFR, ERBB2, IDH1, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PIK3CA, TP53. All assays were optimized for performance, showing excellent signal strength and cluster separation. A subset of assays has been used to test tumor tissue samples and matching cell free DNA (cfDNA).

REFERENCES

1. The Hallmarks of Cancer. Hanahan D and Weinberg RA, 2000, Cell, Vol 100:57-70. 2. Mutations in the p53 Tumor Suppressor Gene: Important Milestones at the Various Steps of Tumorigenesis. Rivlin N et al., Genes & Cancer / vol 2 no 4 (2011), pp.466-474 3. Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing. Borras E et al., BMC Cancer 2011, 11:406 4. Novel mutant-enriched Sequencing Identified High Frequency of PIK3CA Mutations in Pharyngeal Cancer. Qiu W et al., Int J Cancer. 2008 March 1; 122(5): 1189–1194. 5. PIK3CA Mutations Frequently Coexist with RAS and BRAF Mutations in Patients with Advanced Cancers. Janku F et al., PLoS ONE Volume 6 (7): 1-8 6. 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 Linh Vo for testing our assays and Rachel Formosa for reviewing this document.

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For Research Use Only. Not for use in diagnostic procedures.

TaqMan® Rare Mutation Assays for QuantStudio® 3D Digital PCR System

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

ABSTRACT Detection and quantification of mutant alleles in tumor tissue allow for research disease monitoring and the research of drug efficacy. Detection of emerging secondary mutations in the same tumor tissue causing resistance to potential treatment will help guide decisions on future treatment plans. Testing for the presence of mutations in cell free DNA (cfDNA) is a less invasive research method than using tumor tissue. We created a research tool for mutation detection at a sensitivity level of 1% and below. This allows researchers to find correlation between types of mutations and types of tumors and determination of potential secondary mutations. The tool combines TaqMan® SNP Genotyping Assays with digital PCR. A set of assays was optimized for use in digital PCR with the QuantStudio® 3D Digital PCR System. In digital PCR, partitioning the sample into many individual reaction wells facilitates detection and quantification of rare mutant alleles. TaqMan® SNP Genotyping Assays ensure reliable discrimination of mutant and wild-type allele. Our current set of 60 assays covers mutations commonly found in tumor tissues, such as: BRAF V600E, mutations in EGFR exons 19, 20 and 21, KRAS codons 12 and 13, PIK3CA exons 9 and 20, and the JAK2 V617F mutations. All assays were wet-lab tested at a 10% mutation rate and a 1% mutation rate using mutant plasmid spiked into wild-type genomic DNA. Additionally, selected assays were tested at the 0.1% mutation rate using mutant cell lines spiked into wild-type genomic DNA. Wet-lab results confirm that all assays showed superior performance discriminating mutant and wild-type alleles. Mutant alleles were successfully detected as low as 0.1%.

INTRODUCTION The genetic alterations leading to tumorigenesis affect cell processes like cell cycle, programmed cell death (apoptosis), angiogenesis, and cell motility (1). The genes involved in tumorigenesis may show gain of function (oncogenes) or loss of function (tumor suppressor genes) when mutated. The transcription factor p53 is an example of a key tumor suppressor gene involved in multiple pathways in the process of tumorigenesis (2). Mutations in this gene are present in most cancer types. Epidermal growth factor receptor (EGFR), KRAS and BRAF mutations are other examples of mutations present in several cancer types and affect response to treatment (3). PIK3CA mutations have been associated with pharyngeal cancer (4) as well as in some advanced cancers, in combination with KRAS, NRAS and BRAF mutations (5). The discovery of cell-free DNA (cfDNA) now provides a non-invasive method to test for the presence of mutations and monitor tumor progression (6). We are providing a research tool to test for the presence of these mutations in a tumor tissue or cfDNA samples. TaqMan® SNP Genotyping assays with dual VIC® and FAM™ labeled probes simultaneously detect both wild-type and mutant alleles. Detection of both alleles combined with digital PCR allows for easy determination of the percentage of mutant alleles. It enables following tumor response to drug treatment where decreasing levels of mutation are expected.

MATERIALS AND METHODS TaqMan® SNP Genotyping Assays were designed for rare mutation targets using a proprietary bioinformatics design pipeline and then manually optimized, if needed, for use in digital PCR with the QuantStudio® 3D Digital PCR System. Initial designs were wet-lab tested with wild-type genomic DNA with mutant plasmid spiked in at 1% and 10%. If cluster separation and signal intensity were sub-optimal, redesigns were based on initial assay performance. Assay optimization was achieved by increasing primer and/or probe Tm, then retesting the new designs until satisfactory performance was achieved. Selected assays were tested at a low 0.1% mutation rate.

Rare mutation assays for use in digital PCR with the QuantStudio® 3D Digital PCR System offer an easy workflow and quick result.

Figure 3. Rare Mutation Detection Solution

Guaranteed Performance The wet lab–validated Custom TaqMan® SNP Genotyping Assays for rare mutation analysis are covered by the TaqMan® Assays QPCR Guarantee. If the assay doesn’t meet the terms of the guarantee, we’ll replace the assay or credit your account. lifetechnologies.com/taqmanguarantee

Table 1. List of Available Rare Mutation Assays

The list of available assays has been expanded to 60 assays. The assays cover the most common cancer-related mutations in the following genes: AKT1, BRAF, EGFR, ERBB2, IDH1, JAK2, KIT, KRAS, MPL, NRAS, PIK3CA, TP53.

Figure 3. Application of Rare Mutation Assays to “Real-World” Samples

A tumor sample (left) and matching cfDNA (right) were tested with the Rare Mutation Assay for the PIK3CA H1047R mutation. Shown is the scatter plot from QuantStudio® 3D AnalysisSuite™ Software.

Figure 1. Assay Optimization

Initial assay designs were generated with the Applied Biosystems® proprietary bioinformatics assay design pipeline. Adaptation to digital PCR with QuantStudio® 3D and optimization of signal intensity and cluster separation were achieved by increasing primer and probe Tm if needed, based on performance of the initial assay design.

Figure 2. Wet-lab Testing

All assays were wet-lab tested using mutant plasmid spiked into wild-type genomic DNA: A. 10% mutant plasmid B. 1% mutant plasmid. C. Additionally, selected assays were also tested at the 0.1% mutant plasmid to demonstrate high sensitivity.