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TUTORIAL Translational Medicine BEAMing for Cancer Detecting Tumor Mutations in Peripheral Blood Using Digital PCR Frank Diehl and Edita Smergeliene Highly specifc biomarkers that provide information about cancer initiation, development, metastasis, and response to therapy have the potential to offer prognostic insight, while also facilitating the design and implementation of targeted treatments. Because of the genetic complexity and diversity of cancer, these biomarkers are most useful when they identify a specifc group of patients that would beneft from a highly targeted treatment, specifcally optimized for their disease subtype. However, this is further complicated by the continued accrual of somatic mutations over time, for example, those driven by selective pressures exerted by the host immune system and external drug treatments. Therefore, it is necessary to regularly evaluate the biomarker signature of a cancer for changes over the course of treatment and during remission. Cataloguing the genetic status of a given tumor traditionally involves carrying out sequencing analysis of fresh and/or archival biopsy specimens. However, there are several limitations to this approach. For example, tumor biopsies are often contaminated with the DNA of normal somatic cells, while the cancer cell population within a tumor tends to be genetically heterogeneous in nature. The use of archival tissue samples, particularly if fxed and embedded in paraffn, BETTER DETECTION UPSTREAM. MAXIMUM PRODUCTION DOWNSTREAM. Whether you work in drug discovery, upstream or downstream bioprocessing, Enzo offers a range of innovative products to help you maintain cell line viability, optimize and monitor product integrity, and maximize yield. Quantify Visible and Sub-visible Protein Aggregates Detect LMW and HMW PEG Molecules ProteoStat® Detection Reagent y = 291.2x R2 = 0.988 Tiofavin T y = 4.56x R2 = 0.517 % of Aggregate ® PROTEOSTAT AGGREGATION DETECTION KIT PEGYLATED PROTEIN ELISA KIT Discover these tools and more at: www.enzolifesciences.com/bioprocess © 2013 Enzo Life Sciences, Inc. All rights reserved. 48 | September 1, 2013 | GENengnews.com | Genetic Engineering & Biotechnology News Figure 1. The key steps of BEAMing digital PCR analysis Figure 2. The Phusion DNA polymerase is a hybrid enzyme that delivers high PCR fidelity (relative fidelity of different DNA polymerases shown, where fidelity = 1/error rate). can introduce further sources of error due to DNA degradation, which can interfere with analysis. Lastly, tumors can often develop in areas of the body diffcult or impossible to access surgically, and can therefore not be assessed using this approach. Noninvasive Biomarker Analysis Using Peripheral Blood Fortunately, rapid developments in the technology and protocols required for reliably isolating and analyzing circulating tumor DNA (ctDNA) in peripheral blood are making it easier to obtain the necessary samples without the need for physical biopsies. One such method, developed by the team at Inostics, utilizes digital PCR to detect somatic tumor mutations in ctDNA with high sensitivity. The technique combines emulsion digital PCR and fow cytometry. Known as BEAMing, it employs a combination of beads, emulsifcation, amplifcation, and magnetics to achieve the necessary level of sensitivity. The BEAMing process (Figure 1) starts with isolating and purifying the DNA present in blood plasma, which then goes through a pre-amplifcation step using conventional PCR to amplify the genetic section of interest using primers incorporating known tag sequences. These DNA templates are then amplifed again via emulsion PCR, employing primers directed at these sequence tags and covalently bound to magnetic microbeads via streptavidin–biotin interactions. By designing the system this way, the PCR products generated in each emulsion droplet will remain physically affxed to the microbeads at the end of the reaction, allowing them to be easily separated and purifed using a magnet. The method also provides a digital readout of copy number, as it has been experimentally optimized to ensure that each emulsion droplet will contain a maximum of one microbead and one DNA template molecule. This makes it possible to detect even very rare mutant templates at copy ratios greater than 1:10,000. Following the emulsion PCR reaction and subsequent purifcation, the DNA attached to the beads is denatured and probed to determine the presence and number of known mutant variations. Any number of a range of fuorescence-based methods for distinguishing two alleles can be utilized for this step, as long as beads housing the wild-type or mutant DNA can be easily differentiated using fow cytometry. Importance of Accurate Amplifcation The number of ctDNA copies circulating in the peripheral blood stream is very low, especially in comparison to wild-type DNA. For this reason, DNA amplifcation using PCR is an important part of the BEAMing process, necessary to ensure reliable detection. However, PCR is not an error-free reaction, and any experimental variability introduced by the process increases the risk of false-positive detection, thereby reducing the effectiveness of the diagnostic test. This risk can be minimized by using a high-fdelity DNA polymerase such as Thermo Scientifc™ Phusion™ High-Fidelity DNA Polymerase, an engineered enzyme containing a DNA-binding domain fused to a Pyrococcus-like proofreading polymerase.