Genetic Engineering & Biotechnology News

NOV15 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Genetic Engineering & Biotechnology News | | NOVEMBER 15, 2017 | 29 20,000 nanoliter-size droplets that contain randomly dis- tributed nucleic acid molecules. "As a result, the target mol- ecules of interest from the original sample are subdivided into 20,000 individual microreactions," says Dr. Karlin- Neumann. The reactions are performed in a 96-well PCR plate, and after thermocycling, they are transferred to a droplet reader. Positive droplets are identified based on their increased fluorescence. Poisson statistics corrects for multiple occupancies, and software is used to back-calculate the tar- get concentration in the initial volume. Unlike real-time PCR, ddPCR does not require the use of standards and measures absolute concentrations of the tar- gets of interest. "Inherently end-users can get more precision from the measurements," notes Dr. Karlin-Neumann. While in real-time PCR, the presence of inhibitors or unknown con- stituents may compromise cycling efficiency and distort data interpretation, this is not a concern with digital PCR. "An- other gratifying thing about digital PCR in general is that it is much more tolerant of assays that do not have 100% amplification efficiency," he points out. The ddPCR platform provides the first results within 3–4 hours, and an entire 96-well plate can be read within 5 hours. It excels particularly for biomarkers that are known, or for cancers where relatively few recurring mutations drive the malignant process, and it is much more affordable that other complex technologies. "For liquid biopsies, a challenge that needs to be faced and implemented in the clinic is the establishment of clini- cal utility," stresses Dr. Karlin-Neumann. Historically, the response to therapy has been assessed using investigations such as CT scans or other imaging approaches, as well as the levels of imperfect protein biomarkers in the blood. However, as it has been shown for several cancer types, monitoring tumor DNA in the blood also appears to be informative of the relative volume or size of the tumor of origin. The critical question is whether information obtained from changes in tumor DNA from the blood, months before a CT scan, could improve clinical outcomes. "Intuitively, we tend to hope and imagine that this is like- ly to be the case, and there are reasons to believe that clinical utility will be shown both for therapeutic and for reimburse- ment endpoints," says Dr. Karlin-Neumann. Another critical area is to identify biomarkers that are best positioned to represent different cancers. "Some tumors ap- pear not to shed as much as other tumors or when compared to the same tumor in a different location," according to Dr. Karlin-Neumann. For example, intrathoracic lung tumors ap- pear to shed less and, in these instances, even though a muta- tion can be detected in tissue samples, tumor DNA measure- ments in the plasma may not be as informative. "While we need to learn more about the biology of different cancers, we anticipate that circulating tumor DNA levels are likely to be a very good surrogate for clinical outcomes and may be less costly and provide more immediate real-time changes than current clinical measures such as CT scans," he says. Focusing on the First Step Chen-Hsiung Yeh, Ph.D., CSO, Circulogene Theranos- tics, believes that what makes his company different is that the scientists try to make sure that they capture and target every single cfDNA as much as possible at the very first step. Circulogene launched cfDNA liquid biopsy clinical testing services in 2015. "For everybody in the liquid biopsy in- dustry, isolating the DNA or RNA from the specimen is the universal common first step and the most overlooked step," points out Dr. Yeh. While extracting nucleic acids from bacterial or mam- malian cells has become routine in every molecular biology research lab, extracting circulating DNA for clinical applica- tion presents multiple challenges, including the fact that it is highly fragmented, its concentration is low, and a large percentage of the starting material is lost during isolation. "In our approach, we don't need to isolate, purify, or ex- tract the DNA, but we do the genetic manipulation directly on the specimen to enrich DNA," says Dr. Yeh. Because the current industry standard for DNA extraction/concentration leads to substantial sample loss, a large amount of sample would be needed for liquid biopsies to obtain sufficient cfDNA for analyses. "If a large amount of the starting mate- rial is lost in the first step, no matter how good the down- stream detection technology is, the testing result and accu- racy will be greatly jeopardized," notes Dr. Yeh. Using a proprietary enrichment technology, scientists at Circulogene have been able to use low-input volumes, such as 50 µL of unprocessed blood, to recover and yield over 1,000 ng/mL of cfDNA (Figure 2). A key challenge with re- spect to liquid biopsies has been its relatively slow adoption in the medical community. "We have to continuously com- municate with the medical community to point out that the liquid biopsy testing is never intended to replace the current gold standard of tissue biopsy, but that it only tries to fill a gap and complement the limitations of tissue biopsy," says Dr. Yeh. Translational Medicine Insights Molecular Diagnostics The FDA has granted supplementary premarket approval (sPMA) for Myriad Genetics' BRACAnalysis CDx®—a companion diagnostic for AstraZeneca's PARP inhibitor Lynparza® (olaparib)—in patients with HER2-negative metastatic breast cancer. If the test receives full approval, it would be the first and only FDA-approved CDx for use with a PARP inhibitor to identify HER2-negative metastatic breast cancer patients with a BRCA mutation who would benefit from a PARP inhibitor. "The acceptance of the sPMA for BRACAnalysis CDx is a significant step towards enabling personalized medicine for patients with metastatic breast cancer," said Mark C. Capone, president and CEO, Myriad Genetics. "As the pioneer in companion diagnostics for PARP inhibitors, we are excited to once again partner with AstraZeneca and broaden access to Lynparza for even more patients." Myriad said in a statement that it expects the FDA's priority review to conclude in Q3 of 2018. Myriad's sPMA filing comes after positive results from a Phase III OlympiAD trial, which demonstrated that Lynparza significantly reduced the risk of disease progression or death in patients with BRCA-mutated, HER2-negative metastatic breast cancer by 42% compared with stan- dard of care therapy. The results of the OlympiAD trial were published in the New England Journal of Medicine in June. Myriad estimates there are approximately 125,000 patients with metastatic breast cancer who would immediately qualify for the BRACAnalysis CDx test, followed by 60,000 new patients per year on an ongoing basis. This test is the result of an ongoing collaboration with AstraZeneca to develop a novel companion diagnostic test to identify candidates for treatment with olaparib. n Breast Cancer CDx Recieves sPMA Approval Figure 2. Unlike many existing techniques, the in situ enrichment technology from Circulogene Theranostics is capable of near-full DNA recovery of circulating free DNA. Circulogene says that its approach is less labor intensive and is easily integrated into cost-effective, high-throughput analysis platforms.

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