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14 | AUGUST 2014 | GENengnews.com | Genetic Engineering & Biotechnology News trials and access to experimental therapies should be considered instead of another line of chemotherapy. Negative selection of CTCs has particu- lar advantages when analyzing the mRNA content of tumor cells, as "binding of any- thing to the surface of a membrane initiates signal transduction and starts changing the expression of certain molecules in a cell," ex- plained Dr. McCormack. Negative-selection methods also allow for single-cell sorting for NGS applications. In addition, Janssen Di- agnostics has had success culturing CTCs, which can then be used to test for drug sus- ceptibility or resistance. David Miyamoto, M.D., Ph.D., instruc- tor in radiation oncology, Massachusetts General Hospital, described the successive generations of microfuidic devices a team of bioengineers, biologists, and clinicians at MGH have developed to isolate CTCs from blood samples. Through improvements and modifcations in design and materials, the chip-based device has evolved to be able to capture single CTCs as well as clusters of CTCs and to provide enhanced adherence across the chip surface. MGH has tested its second-generation "herringbone CTC chip" in several pilot studies in various cancers including prostate, breast, and melanoma. Prototypes of a third- generation chip, the CTC iChip (the "i" stands for "inertial focusing device"), is in develop- ment in collaboration with Johnson & John- son. These prototypes have been engineered to facilitate downstream assays. Instead of the CTCs remaining trapped on the device, as with previous versions of the chip, the CTC iChip releases the CTCs into solution. "This allows for a range of applications and enables single-cell analysis," noted Dr. Miyamoto. "You can isolate single cells" for RNA expression analysis, DNA analysis, or mutation analysis, for example. A key advantage of the third-generation device is its use of both positive and negative selection for CTC isolation, according to Dr. Miyamoto. Negative selection is achieved by coating all of the non-CTCs in a sample with magnetic beads, a procedure that targets the non-CTCs for removal, leaving the CTCs unperturbed. Dr. Miyamoto is studying the use of the device as a prognostic tool in pros- tate cancer, and in particular to isolate CTCs in blood samples from patients with meta- static castration-resistant prostate cancer to assay for androgen receptor signaling prior to treatment with second-generation andro- gen receptor targeting agents. "I think CTC technology is going beyond the enumeration and examination of molec- ular pathways that are activated or turned off as a result of therapies," projected Dr. Miyamoto. "[It is becoming] a tool to guide targeted therapies and advance personalized medicine." ExoCap™, a new kit available from JSR Life Sciences for research applications, uses a magnetic bead-based isolation method to extract and enrich exosomes for use with cell culture supernatants without the need for ultracentrifugation. The magnetic beads are coupled to antibodies that recognize an- tigens on the surface of exosomes. The four ExoCap kits include an antibody targeted to capture exosomes with the EpCAM, CD9, CD63, or CD81 surface antigen, and the Composite ExoCap Kit, which combines all four antibodies. "Binding [of the antibody-bead com- plexes] should not alter the exosomes," as- serted Kenneth Henry, Ph.D., senior research scientist. The company has shown that the vesicles isolated are characteristic of exo- somes. They have a lipid bilayer membrane and a particle size distribution of about 100 nm, and they contain cargo of the sort found in exosomes. Studies are under way to demonstrate whether these exosomes are biologically ac- tive once they are released from the beads. Nucleic acid analysis can be done directly from isolated exosomes that remain bound to the magnetic beads, Dr. Henry explained, or users can add a reagent included in the kit to release the exosomes from the beads for subsequent analysis. Genotyping ctDNA Mark Sausen, Ph.D., director of R&D; at Personal Genome Diagnostics (PGDx), identifes three main areas of technology and applications development in the commercial- ization of noninvasive diagnostic approaches for using NGS in cancer. These include the capture and characterization of CTCs; the isolation and analysis of ctDNA; and exo- some-based approaches. PGDx is focusing on ctDNA, and ear- lier this year the company introduced the METDetect™ assay for detecting amplif- cations and structural changes of the MET cancer gene in blood samples from cancer patients. These changes are associated with the response to specifc targeted therapies in clinical trials as well as resistance to other targeted therapies. Furthermore, these structural alterations can serve as markers for monitoring treat- ment response and of overall prognosis and cancer recurrence. Whereas the mere pres- ence of ctDNA has been used as a prognos- tic marker—for example, to detect minimal residual disease following surgical resection and as an indication of a greater chance for relapse—more recently, molecular tools such as NGS have been applied to ctDNA to look for a particular genotype. Detection and NGS of ctDNA "is a very powerful tool to evaluate the genotype of a tumor without a biopsy," said Dr. Sausen. Both CTCs and ctDNA have advantages and challenges, and these may depend on the application you are using or clinical question you are asking. Overall, "it seems the levels of ctDNA are higher than the levels of CTCs in a particular blood sample," Dr. Sausen ob- served. A challenge in isolating CTCs is not to miss them, as their numbers can be quite low. With ctDNA, continued Dr. Sausen, "you are looking for somatic mutations that oc- cur at low levels (with a large range)—from <0.1 to >50%—in the presence of a lot of wild-type DNA." Detecting very low-level mutations in ctDNA is particularly problem- atic given that the error rates of NGS instru- ments can be approximately 1%, although experimental and bioinformatics approaches can overcome this limitation. Norgen Biotek specializes in technology for collecting RNA or DNA from CTCs, exosomes, and other types of samples. Its silicon carbide resin "is much more sensi- tive than traditional silica-based columns," according to Bernard Lam, Ph.D., a senior scientist at the company. It can be used to isolate DNA or RNA from single cells and can capture rare copies of transcripts with- out the need for carrier RNA. "By adjusting the chemistry, you can choose to elute either DNA or RNA, or both," explained Dr. Lam. The samples are eluted in small volumes "and can go directly into next-generation ap- plications," he added. The silicon carbide resin is also applicable for collecting microRNAs. "The technology has no bias," Dr. Lam asserted, and can cap- ture larger mRNA transcripts and smaller miRNAs without having to skew the chem- istry to account for the different GC content in various RNA species. Biostabilization at Ambient Conditions CTC technologies are among the many advanced molecular diagnostic tools be- ing developed that require a high degree of precision, and "if something goes wrong from [sample] collection to analysis" it can introduce a lot of variability into the sam- ple "and mask the results," cautioned Rolf Müller, Ph.D., president, CSO, and founder of Biomatrica. Sample destabilization and degradation will affect the outcomes of an- alytical methods including detection of cell surface markers and cell-free DNA as well as genomic, transcriptomic, and proteomic analyses. At present, "99% of biostability is done using cold chain management," noted Dr. Müller. Whether current approaches to cold chain management will remain adequate, however, is unclear. Biostability demands are expected to grow with the rise in omics- based tests that require intact DNA, RNA, proteins, and metabolites. Another relevant trend is the commercial-scale development of diagnostics that rely on isolated CTCs or other live cell samples. Scaling up the cold chain is costly, com- plex, and unreliable, according to Dr. Mül- ler, and interruptions in the cold chain can result in freeze-thaw cycles, causing "tre- mendous cell stress—especially for CTCs— and cell lysis and degradation of nucleated cells." Furthermore, cell surface markers "are extremely sensitive to freezing and thawing," which can result in changes in biomarker profles. Biomatrica has developed a technology platform for the preservation of biomateri- als at ambient temperatures that includes the stabilization of DNA, RNA, and pro- teins; blood and other patient samples; diag- nostic assays; and live mammalian cells. It provides an alternative to cold chain storage and transport and cryopreservation. Different types of biological samples and different applications have varying require- ments for biostability. Accordingly, Biomat- rica not only offers off-the-shelf biostabili- zation reagents, it also uses its proprietary screening technology and library of biostabi- lizers to optimize the collection of a particu- lar sample type and develop a custom stabili- zation approach for a specifc assay. Earlier this year, Biomatrica and Ameri- can Type Culture Collection (ATCC) signed a licensing agreement. As per the agree- ment, Biomatrica will supply ATCC with its DNAstable ® and RNAstable ® reagents for the stabilization of DNA and RNA stan- dards at room temperature. Tumor Markers Continued from page 12 DRUG DISCOVERY Side-by-side comparison of miRNA recovery eciency using a standardized sample of ultra- centrifuged exosomes from HT-29 cell culture supernatant spiked into buer. JSR Life Sciences Todd Skrinar CORRECTION In the July issue of GEN, pictures of the co-authors of the POV article entitled "Drug R&D;: Big Data for Big Returns" were incorrectly labeled. Todd Skrinar and Thaddeus Wolfram are shown Thaddeus Wolfram below with their correct headshots.