Genetic Engineering & Biotechnology News

NOV15 2017

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28 | NOVEMBER 15, 2017 | | Genetic Engineering & Biotechnology News in a liquid biopsy, and a lot of work needs to be done before we can advance this further into a test that is detecting sub- clinical cancer and impact cure rates." The study of molecular changes in cfDNA is intimately linked to technologies that are best positioned to address specific biological questions. "For focused questions, such as looking for EGFR resistance mutations, digital PCR is a very cost-effective and scalable method that allows us to study many specimens," says Dr. Oxnard. Other questions, par- ticularly if they are exploratory in nature, require broader panels, such as NGS. "As people talk about bigger and bigger assays to study plasma, like whole-exome sequencing, it is important to benchmark our newer assays against existing assays," con- tinues Dr. Oxnard. Often, several analyses need to be con- ducted using the same sample of biological material. This is easier and commonly performed for solid tumors, for which the biological material can be divided for parallel tests. "In comparison, cfDNA is not a resource that can be di- vided into multiple ways, and the inability to line up multiple assays and test them against each other is a huge challenge simply because of the scarcity of specimens," he points out. One of the efforts in his lab focuses on developing and validating standards that are reliable, highly quantitative, and can be used as established standards that can be tested against multiple assays. "This will be a lot more difficult for cfDNA than for solid tumors," explains Dr. Oxnard. New Detection Approaches Researchers at the Johns Hopkins School of Medicine have developed a different approach for early detection using cfDNA, in a way that is unbiased with respect to the position of mutations that exist in an individual tumor, according to Victor E. Velculescu, M.D., Ph.D., professor of oncology and pathology and codirector of cancer biology. Historically, a substantial body of work exploring bio- markers has relied on late-stage tumors or used information obtained about patients' primary tumor to guide subsequent efforts to identify blood-based biomarkers in the same indi- viduals. The scarcity of noninvasive methods for early-stage detection has been an ongoing and acute challenge in cancer management. To address the much-needed development of noninvasive early-stage biomarkers, Dr. Velculescu and colleagues recently developed a panel of 58 cancer-related genes that are frequently mutated in several common tumor types, and used it to interro- gate mutations in the cfDNA from the plasma. "We evaluated this approach using plasma from breast, colorectal, lung, and ovarian cancer patients, and we were encouraged to see that we detected somatic mutations in two-thirds to three-quarters of the patients even with early-stage disease, while no alterations were seen in healthy individuals," says Dr. Velculescu. The challenge with detecting mutations in circulating tu- mor DNA is that mutations are usually infrequent and the DNA is diluted, which makes regular sequencing approaches less informative. "In order to detect alterations at such low levels, we had to develop a methodology that would permit sensitive and specific detection," explains Dr. Velculescu. The new methodology developed by his team, called tar- geted error correction sequencing (TEC-seq), relies on pu- rifying free DNA from the plasma and generating a library that is used for very deep sequencing. "The sequencing of many molecules multiple times allowed the identification of true alterations," says Dr. Velculescu. Algorithms used as part of this methodology can distin- guish DNA in the blood originating from multiple sources, including the tumor, the germline, and blood cells. "The abil- ity to detect these mutations during early-stage disease could permit a substantial change in the way patients are treated," maintains Dr. Velculescu. In the analysis of cfDNA, substantial efforts and hopes are focusing on extracting clinical and prognostic informa- tion from its qualitative and quantitative characterization. "The presence of circulating tumor DNA is very important at the time of diagnosis, but may also be informative about a worse type of disease depending on its levels," according to Dr. Velculescu. For example, cell-free circulating tumor DNA may originate not only from the primary tumor, but also from undetected metastatic lesions. In a subset of patients with resectable colorectal cancer, higher preoperative levels of circulating tumor DNA were associated with disease recurrence and decreased overall survival. "We need to conduct larger trials for patients with each of these tumor types in various populations to show that this is something that has the sensitivity and specificity that is consistent with what we observed and could be useful in a clinical setting," says Dr. Velculescu. Role for Digital PCR Digital PCR in a well-designed assay gives high specificity and sensitivity, particularly for some of the most difficult-to- distinguish mutations, such as single nucleotide polymor- phisms, which are an important source of drivers in tumor oncogenes, says George Karlin-Neumann, Ph.D., director of scientific affairs at Bio-Rad's digital biology center. At the recent Next Generation Summit held in Washing- ton, DC, Dr. Karlin-Neumann talked about Bio-Rad's Drop- let Digital PCR (ddPCR™), which combines microfluidics with water-oil emulsion droplet technology and allows the absolute quantitation of nucleic acids (Figure 1). In ddPCR, the amplification reaction is partitioned into Cell-Free DNA Drives Liquid Biopsy Testing Translational Medicine Continued from page 1 Figure 1. In the Bio-Rad Droplet Digital™ PCR (ddPCR™) platform, the amplification reaction is partitioned into 20,000 individual droplet microreactions, and after amplification of the target DNA, its concentration is calculated from the fraction of positive droplets.

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