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TUTORIAL Translational Medicine Because of this unique fusion technology, Phusion DNA Polymerase generates PCR products with a level of accuracy and speed unattainable with a single enzyme, even for GC-rich templates. It also has extremely high fdelity, with an error rate approximately 50fold lower than that of Taq DNA polymerase and 6-fold lower than that of Pfu DNA polymerase, as determined by a modifed lacIbased method (Figure 2) and later confrmed using Roche/454 and Illumina sequencing. By using a DNA polymerase with such a low error-rate, one can be highly confdent that mutations detected by BEAMing are refective of the original ctDNA template, rather than introduced by an error-prone DNA polymerase. BEAMing in Action: Detecting PIK3CA Mutations in ctDNA Recently, a research team led by Ben H. Park, M.D., Ph.D., of the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center and including scientists from Inostics worked to assess the suitability of BEAMing for detecting PIK3CA mutations in ctDNA, using plasma samples from patients with metastatic breast cancer. PIK3CA is the most commonly mutated oncogene in breast cancer, and aberrant PI3K pathway signaling is being actively studied as a prognostic marker for disease outcome and a predictive marker for developing and administering highly targeted therapies. Part of the study involved using BEAMing to perform a retrospective screen of 49 patients with metastatic breast cancer. The aim was to use the new technique to detect PIK3CA mutations in matched ctDNA and archival tumor samples, to assess whether blood samples might replace biopsies as a reliable method for assessing tumor genotype. Of the 49 patients tested, 14 exhibited a PIK3CA mutation (Table). In every case, the result obtained using DNA from the biopsy matched that using the corresponding ctDNA sample, indicating that BEAMing of ctDNA is a reliable method for assessing tumor genotype without the need for a biopsy. With multiple inhibitors of phosphoinositide 3-kinases currently in development, BEAMing has clear applications for clinical trial design and execution, as well as future clinical treatments designed to target subsets of patients exhibiting distinct tumor genotypes. Conclusions BEAMing is a powerful method for detecting known genetic mutations in a blood sample, even when at very low copy numbers. It is much more sensitive than most competing techniques, while also enabling copy-number quantifcation. The accuracy of the method relies upon the utilization of high-fdelity DNA polymerases, which minimize error rate and ensure that the detection of false positives/negatives does not signifcantly lower the diagnostic value of the techFrank Diehl is CSO and managing director at Inostics, and Edita Smergeliene (edita. smergeliene@thermofsher.com) is product portfolio manager for gene detection at Thermo Fisher Scientifc. Website: www.thermofsher.com. nique. By enabling the analysis of ctDNA via noninvasive biopsies such as systemic blood samples, BEAMing has many applications including optimizing patient recruitment during clinical trials. In the future, BEAMing may also prove valuable as a diagnostic technique for making informed treatment decisions, as well as for tracking the occurrence of resistance mutations during and after treatment, so that medication can be adjusted accordingly to maximize therapy effectiveness. PIK3CA Mutations Mutation Amino acid Observed frequency / expected frequency change (N = 49) (%)/(%) 1633G>A 545E>K 3 (6.1)/(4.4) 3140A>G 1047H>R 10 (20.4)/(13.3) 3140A>T 1047H>L 1 (2)/(1.1) Total number of mutations 14 (28.6)/(18.7) PIK3CA mutations identified retrospectively in peripheral blood of patients with breast cancer. Note: the BEAMing of ctDNA and breast tumor tissue yielded results with 100% concordance (full dataset originally published by Higgins et al., 2012). One Automated Analyzer for Fast Comprehensive Cell Culture Analysis The Power of One Flexible Modular Design BioProfle FLEX can reduce cell culture analysis time, labor, and operating costs by consolidating multiple analyzers into a single, easy to use workstation. BioProfle FLEX can be customized with 1–4 modules to consolidate up to 15 vital cell culture tests: Module 1: Glucose, Lactate, Glutamine, Glutamate, Ammonium, pH, PCO2, PO2, Sodium, Potassium, Calcium Module 2: Cell Density/Cell Viability Module 3: Osmolality Module 4: IgG or Phosphate One compact workstation, up to 15 Cell Culture assays, including IgG or Phosphate One small, 1 mL sample conserves cell culture mass and end product One fast, 6 minute analysis saves at least one hour over multiple instrument analyses One integrated data source simplifes data collection, analysis, and archiving One validation saves time vs validation of multiple instruments New IgG and Phosphate Modules The IgG or Phosphate modules offer: Results in less that 6 minutes with no sample preparation required 0.1 to 10.0 g/L IgG measurement range 0.2 to 10 mmol/L Phosphate measurement range Other BioProfle FLEX Options On-Line Autosampler with automated sampling from up to 10 bioreactors OPC Connectivity Test Menu Gluc Lac Gln Glu NH4+ pH PO2 PCO2 Na+ K+ Ca++ CD CV Osm IgG PO4 www.BioProfleFLEX.com Genetic Engineering & Biotechnology News | GENengnews.com | September 1, 2013 | 49