qBiomarker™ Somatic Mutation PCR Arrays: A Novel Tool
for Pathway-Focused Cancer Mutation Profiling
Samuel Long, Min You, ...
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Som aacr2011poster

  1. 1. qBiomarker™ Somatic Mutation PCR Arrays: A Novel Tool for Pathway-Focused Cancer Mutation Profiling Samuel Long, Min You, Liang Wang, Xiaofang Jiang and Yexun Wang QIAGEN, 6951 Executive Way, Frederick, MD 21703 Assay Differentiation Window (ADW), Assay Efficiency and Assay Detection Limit for 4 Pathway Arrays Recent advances in sequencing technology have led to a significant increase in the number of known cancer somatic mutations. Translating such information into therapeutic benefits requires the molecular characterization of all major human tumor types and the advent of research tools that enable interrogation of multiple mutations in multiple cancer genes simultaneously, with high accuracy and scalability. A new system of real-time PCR based somatic mutation arrays has been developed as a pathway-focused cancer mutation profiling tool. By combining allele specific amplification (ARMS®) and 5’ hydrolysis probe detection, the PCR assays on these arrays are designed to detect as little as 1% somatic mutation in a background of wild type genomic DNA. Each array contains assays for detecting the most frequent and functionally verified mutations for multiple genes within a specific pathway that has been implicated in a variety of cancers. For example, the PCR panels cover pathways such as the EGFR, PDGFR and c-Met pathways, each on separate, ready-for-sample PCR plates. Each targeted mutation on the array is carefully chosen from curated, comprehensive somatic mutation databases (including the COSMIC database) and peer-reviewed scientific literature. The qBiomarker Somatic Mutation PCR Arrays, with their comprehensive content coverage, are an ideal tool for studying mutations in the context of whole pathway and have the potential for discovering and verifying biomarkers for a variety of human cancers. KRAS c.34G>T p.G12C BRAF c.1391G>T p.G464V EGFR c.2573T>G p.L858R KRAS c.38G>A p.G13D Signal generated on mutant template Signal generated on WT gDNA template All assays on the arrays have close to 100% PCR efficiency. The following is a summary of average ADW and assay efficiencies on the 4 qBiomarker™ Somatic Mutation PCR arrays. Figure #1 EGFR pathway In this study, data is presented to demonstrate the consistency and reliability of array performance using a variety of sample types (fresh, frozen or FFPE samples from cell lines and tissues) and under a large range of sample quality conditions (such as FFPE tumor samples from different sources). In addition, some of the results were further verified by DNA sequencing using Pyrosequencing ®. Somatic Mutation Array Performance in FFPE Samples The assay differentiation window was defined as the Ct difference between the signals generated on wildtype genomic DNA background and on 100% mutant template by a mutation assay. Assays were verified to have a minimum differentiation window of 8 but are usually higher. The BRAF V600E assay ADW is shown as follows: Fluorescence (dR) Abstract ERBB2 pathway PDGFR pathway Average ADW 11.15 12.33 11.41 FLT3 pathway 11.73 Average efficiency 104.7% 107.1% 102.3% In an off-site beta-testing project, the human EGFR pathway somatic mutation array was used to profile somatic mutations in 4 FFPE cell line samples, a wildtype FFPE placenta sample (control), and 9 lung adenocarcinoma FFPE tissue samples (Asterand) of mainly (8/9) Caucasian origin with mixed gender and smoking status. The array detected BRAF and KRAS mutations in the FFPE MDA-MB-231 cell line, which were independently confirmed by DxS KRAS mutation kit and Pyrosequencing. The array also detected one KRAS somatic mutation and one EGFR somatic mutation in two FFPE lung adenocarcinoma samples. The EGFR mutation rate is in agreement with expected EGFR mutation rate (~10%) in lung cancer adenocarcinoma samples of such origin. 102.5% The majority of assays have mutation detection limit of 1% or lower. Shown below is the detection limit performance data for the BRAF V600E mutation assay. A series of 10 ng genomic DNA samples, which contain genomic DNA from A375 cell line (containing BRAF V600E mutation) mixed with WT genomic DNA at different ratios, were tested on Somatic Mutation Assay for BRAF V600E with or without whole genome amplification (with QIAGEN Repli-g UltraFast kit). Mutation detection limit for this assay is determined to be ~2%. Somatic Mutation PCR Array Data Confirmed by Pyrosequencing EGFR BRAF CHART or PICTURE 39 No WGA WGA KRAS 37 35 Ct qBiomarker Mutation PCR Array Technology Principles • Allele-specific PCR amplification with ARMS (amplification refractory mutation system) primers 33 31 29 • Mutant amplicon detection by hydrolysis probe 27 Additional mismatch Mutation of interest 25 1 2 4 8 10 20 50 Human EGFR pathway somatic mutation PCR array detected BRAF, EGFR and KRAS mutations in FFPE lung adenocarcinoma samples (Cybrdi) of Asian, female, non-smoker origin. EGFR somatic mutations occurred in 30% of the samples, as expected. Sample quality varies by 8 Ct as measured by gene copy assays on the array. 100 % mutant DNA Mutant template 296-1 KRAS c.35G>T 25% Somatic Mutation Assay Cross-reactivity Performance Cross-reactivity tests were performed for all assay clusters containing assays that potentially recognize another assay’s template due to imperfect base-pairing and read-through. Shown below are the cross-reactivity test results (represented by Ct between the Ct obtained for a mutation assay with a template and the Ct obtained for the mutation assay with its own template) for the KRAS Codon 12 and 13 cluster mutation assays. A Ct=0 (cells highlighted in yellow) is set for a mutation assay with its own template. Cells containing Ct<6 values are highlighted in blue. The results indicate that the G12V assay shows significant cross-reactivity with the G12R mutant template, and the G13V assay shows moderate cross-reactivity with the G13A mutant template. Wildtype template Sample requirement • 5-10 ng genomic DNA isolated from fresh tissues/cells for each array OR • 200-500 ng DNA from FFPE sections for each array Template => p.G12S p.G12R p.G12C p.G12D p.G12A p.G12V p.G13S p.G13R p.G13C p.G13D p.G13A p.G13V Extraction (opt.)Amplification DNA mini or FFPE kit Detection 30 min Analysis qPCR Array and Assay REPLI-g for Fresh sample 1.5hr KRAS KRAS KRAS KRAS KRAS KRAS KRAS KRAS KRAS KRAS KRAS KRAS Template 2hr 10-20 min => Assay Somatic Mutation PCR Array Workflow p.G12S p.G12R p.G12C p.G12D p.G12A p.G12V p.G13S p.G13R p.G13C p.G13D p.G13A p.G13V 0.00 7.20 9.06 7.60 10.10 7.19 10.55 10.88 8.54 7.72 9.04 7.96 7.42 0.00 6.46 7.60 10.10 3.87 10.00 9.81 7.97 7.47 8.82 7.90 7.42 6.48 0.00 7.60 9.48 7.17 10.55 10.80 8.56 7.63 8.80 7.96 7.42 8.31 8.26 0.00 10.10 7.01 9.05 10.21 7.87 7.62 8.35 7.96 7.42 9.18 7.49 7.60 0.00 6.99 10.11 10.93 8.14 7.59 8.21 7.96 7.42 9.56 7.88 7.60 9.31 0.00 7.54 10.77 9.16 7.58 8.99 7.96 7.42 9.82 9.20 7.60 10.10 7.19 0.00 8.62 7.50 8.02 9.04 7.96 7.42 9.26 9.20 7.59 9.02 7.19 9.46 0.00 6.99 7.78 9.04 7.96 7.42 9.51 9.20 7.60 10.10 6.98 10.13 9.59 0.00 7.68 7.43 7.96 7.42 9.67 9.20 7.46 10.10 7.19 9.80 10.31 8.06 0.00 6.88 7.96 7.42 9.40 9.20 7.60 10.10 7.05 10.55 9.99 8.97 7.21 0.00 5.24 7.42 9.43 9.20 7.60 10.10 7.19 9.96 10.94 8.83 7.97 8.46 0.00 Human EGFR Pathway Somatic Mutation PCR Array Layout Methods 1 2 3 4 5 6 7 8 9 10 11 12 AKT1 B BRAF BRAF BRAF BRAF BRAF BRAF BRAF BRAF EGFR EGFR EGFR p.E17K p.G464V p.G466V p.G469A p.L597V p.V600M p.V600E p.V600A p.V600G p.G719S p.G719C p.G719A EGFR A EGFR EGFR EGFR EGFR EGFR EGFR EGFR EGFR EGFR EGFR EGFR p.E746_A750 del p.E746_A750 del p.E746_T751 del p.E746_T751 >A p.L747_A750 >P p.E746_S752 >D p.L747_E749 del p.L747_S752d el p.L747_T751 del p.S768I p.V769_D770 insASV p.D770_N771 insG EGFR E EGFR EGFR EGFR KRAS KRAS KRAS KRAS KRAS KRAS KRAS p.T790M p.L858M p.L858R p.L861Q p.Q61R p.Q61L p.Q61H p.G12S p.G12R p.G12C p.G12D KRAS KRAS KRAS KRAS KRAS KRAS KRAS KRAS HRAS HRAS p.G12A p.G12V p.G13S p.G13R p.G13C p.G13D p.G13A p.G13V p.Q22K p.Q61K p.Q61R HRAS HRAS HRAS HRAS HRAS HRAS NRAS NRAS NRAS p.G13R p.G13C p.Q61K p.Q61P p.Q61R NRAS NRAS NRAS NRAS NRAS NRAS MEK1 MEK1 MEK1 p.G12A p.G13R p.G13D p.G13A p.G13V p.A18T Q56P K57N D67N PIK3CA PIK3CA PIK3CA PIK3CA PIK3CA PTEN PTEN PTEN PTEN NRAS c.182A>T p.Q61L PIK3CA c.3140A>G p.H1047R p.P539R p.E542K p.E545K p.E545G p.E545D p.H1047R p.H1047L p.R130* 276-2 KRAS c.35G>T 35% 3237-1 EGFR c.2236_2250del15 20% 266-1 EGFR c.2236_2250 deletion 12% 317-2 KRAS c.35G>C 10% KRAS G12 somatic mutations and EGFR deletion mutations in these samples were confirmed by Pyrosequencing. Representative pyrograms are shown. Conclusions • Utilizing allele-specific PCR amplification with ARMS primers combined with probe detection, assays on qBiomarker™ Somatic Mutation PCR arrays achieve sufficient specificity and sensitivity for detecting as low as 1% mutant alleles in wild type genomic DNA background. • Simple workflow and sample requirement allows fresh, frozen or FFPE samples from a variety of sources and with a wide range of quality to be routinely profiled in any laboratory with access to real-time PCR instruments; sample input requirement is small (as little as 200 ng per sample/array). • Comprehensive content coverage on each array allows for studying mutations in the context of a pathway and enables discovering and verifying clinical biomarkers for a variety of human cancers involving the selected signaling pathway. Bibliography PIK3CA c.1633G>A p.E545K • Mutation profiling results obtained on qBiomarker™ Somatic Mutation PCR Array have been confirmed by Pyrosequencing. PTEN p.Q61H p.G12S p.G12R p.G12C p.G12D p.G12V p.R233* p.R173C p.R173H p.R130Q p.R130G AKT1 PTEN H KRAS c.38G>A p.G13D P124L PIK3CA KRAS c.35G>T p.G12V MEK1 p.G12D BRaf c.1799T>A p.V600E p.Q61L NRAS p.G12S PIK3CA G KRAS c.38G>A p.G13D NRAS NRAS F KRAS c.34G>A p.G12S p.Q61L HRAS BRAF c.1391G>T p.G464V HRAS HRAS D EGFR p.H773_V774 insH KRAS C Somatic Mutation Array Performance in Cancer Cell Lines 256-1 KRAS c.35G>A 17% BRAF EGFR KRAS HRAS NRAS MEK1 PIK3CA PTEN SMPC SMPC Copy Number Assay Copy Number Assay Copy Number Assay Copy Number Assay Copy Number Assay Copy Number Assay Copy Number Assay Copy Number Assay Copy Number Assay Positive PCR Control Positive PCR Control Mutations were chosen from curated, comprehensive somatic mutation databases and peer-reviewed scientific literature. Assays were designed to detect the most frequent, functionally verified, and biologically significant mutations in the particular pathway. The content of the human EGFR pathway array is presented above. References • Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF. Nucleic Acids Res. 1989 Apr 11;17(7):2503-16 200 ng of gDNA from WT or well-characterized cancer cell lines was profiled on the human EGFR somatic mutation PCR array. All previously identified mutations in the EGFR pathway in these cell lines were called correctly using the Ct method. A01-H01: mutation assays; H02-H10: gene copy assays; SMPC: PCR control assay • Targeting the EGFR and the PKB pathway in cancer. Klein S, Levitzki A. Curr Opin Cell Biol. 2009 Apr; 21(2):185-93 • Update on HER-2 as a target for cancer therapy: intracellular signaling pathways of ERBB2/HER-2 and family members. Olayioye MA. Breast Cancer Res. 2001; 3(6):385-9. • Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations in acute promyelocytic leukemia: a systematic review. Beitinjaneh A, Jang S, Roukoz H, Majhail NS. Leuk Res. 2010 Jul; 34(7):831-6. • Clinical significance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromal tumours. Lasota J, Miettinen M. Histopathology. 2008 Sep; 53(3):245-66. AACR Abstract # 1157 The qBiomarker Somatic Mutation PCR Arrays are intended for molecular biology applications. They are neither intended for the diagnosis, prevention, or treatment of a disease, nor have been verified for such use either alone or in combination with other products. Corresponding author email: Samuel.Long@qiagen.com Sample & Assay Technologies