The hottest track at the 2015 Molecular Med Tri-Con was circulating nucleic acids. Circulating tumor cells were also sizzling, leading to the question: “Which is most important?” The answer? Both, since they provide complementary information. For example, some CTCs are viable and can be expanded to provide a platform for exploring therapeutic options. Circulating nucleic acids provide a quick and sensitive window into tumor status and function. Liquid biopsies to characterize circulating nucleic acids are an attractive alternative to needle biopsies of solids, which all too often miss their target because of tissue heterogeneity.
The emphasis of the circulating nucleic acid track of the liquid biopsy symposium was on cancer diagnostics, but biomarkers for infectious diseases were also discussed. UCLA’s recent problems with Carbapenem-resistant Enterobacteriaceae during endoscopic procedures provided immediate reinforcement that proper diagnosis and remediation of hospital-acquired infections need reliable, sensitive analytics.
Drawing blood for sampling circulating biomarkers, including DNA and RNA, has been a growing research topic for about a decade. Both RNA and DNA need protective structures to inhibit degradation in the bloodstream by nucleases. DNA wrapped around histone structures seems to be protected. Exosomes appear to protect RNA. Circulating RNA can be traced to the host, for example, messenger RNA, or to bacteria in the microbiome, but about 15‒30% is exogenous, meaning that, when sequenced, it maps to outside sources such as food. Bioscientists are still elucidating the roles of these different biomarkers.
Clinical significance of circulating nucleic acids
Chetan Bettegowda, M.D., of Johns Hopkins School of Medicine (Baltimore, Md.), opened the symposium track by discussing the promise and problems with nucleic acid circulating in blood, especially DNA. Free DNA in blood is rapidly degraded by nucleases, so its origin is not always discernable. However, there is clear evidence that high concentrations of DNA in the blood correlate with cancerous cell growth; hence the abbreviation ctDNA—circulating tumor DNA. Probes can be designed to assay for particular mutations associated with cancer, and the measurement of ctDNA provides a near real-time measure of tumor activity. It is particularly useful as an early warning that a tumor is developing resistance to therapy.
Exosomes, endocytic vesicles with a diameter of 30‒120 nm, participate in intercellular communication by delivering proteins and RNA to cells. (“Exosome” should not be confused with “exome.” Exosomes are subcellular particles; exomes are segments of DNA used as probes in next-generation sequencing.) Solid tumors produce exosomes to shape their microenvironment. Professor Pamela Cassidy (Oregon Health and Science University, Portland) described the isolation of microRNAs, which control many biological processes, from exosomes. She is developing sample prep protocols that work with citrate plasma samples, which are expected to be more stable than serum.
Hypermethylation of DNA is often associated with cancer. Kyongchol Kim (CHA University, Seoul, ROK) reported that critical genes showed hypermethylation in 15 out of 32 cancers. Specifically, a methylation panel of the PYCARD, APAF1, MINT3 and BRAC1 genes showed 97.3% sensitivity and 66.4% specificity for the presence of gastric cancer. Further, the circulating cell-free DNA (ccfDNA) concentration returned to normal range after surgical removal of the tumor. In support, Kim noted that 21 of the methylated genes were unmethylated following surgery for a p<0.05.
Liquid biopsies of other fluids
While the exact origin of circulating DNA and RNA is not known, it can be found in body fluids such as cerebrospinal fluid (CSF), blood and urine. Proposed mechanisms for its distribution include necrosis, metastasis, apoptosis and response to chemotherapy. Glioblastomas are an exception, where the blood/brain barrier appears to keep cancer cells from entering the bloodstream.
Dr. Vlada Melnikova, vice president, research and development at Trovagene, Inc. (San Diego, Calif.), described the analysis of nucleic acid biomarkers in urine, which are typically smaller than 300 bp with amplicons in the 35‒45 bp range. Concentration is usually quantified in μg/mL (about 1000 times higher than blood), which extends the dynamic range and improves minimum allele frequency to 0.005% (compared to 5% in blood). Sampling is less invasive and large volumes are available.
Tools for measuring ctDNA
Solid tumors are usually a collection of heterogeneous cells. Many are not viable. Detecting the few active mutations in a matrix rich in normal cells is a significant problem, and was addressed by Professor Seth Crosby, M.D., of Washington University (St. Louis, Mo.). He described how allele-specific PCR amplification of the mutants can improve detection.
Douglas Horejsh of Promega Corp. (Madison Wis.) was one of several presenters who recognized that circulating DNA could reduce the need for biopsies of solid tumors. These biomarkers are present in very low concentration, so they need to be selectively enriched. Promega has developed a protocol that concentrates the ccfDNA in 4 mL of blood to a volume of 1‒10 μL. With a cohort of seven patients, Promega compared its method with surgical specimens in formalin-fixed, paraffin-embedded (FFPE) tissue samples. The results showed a good correlation of mutations from both sampling procedures. Promega cites these results when asked about the correlation of sampling between FFPE tissue samples and blood. Others reported similar studies in which liquid biopsies provided signals of change earlier than fine needle biopsies or radiography.
Novel isolation technology
RainDance Technologies (San Diego, Calif.) presented “Detecting Circulating Tumor Cells with Fluid Biopsy and Profiling Somatic Tumors with Next-Gen DNA Sequencing.” RainDrop starts with droplet-based digital PCR wherein diluted sample matrix is injected with PCR reagent and then the mixture is injected into a stream of flowing oil. The resulting drops contain at most only one DNA fragment. Most drops are empty. All of the drops are deposited into individual wells on a plate. Thermal cycling is successful only for the drops containing DNA. This first readout provides a measure of the DNA content with a range of several logs. If the primer focused on a particular rare sequence, RainDance can calculate the concentration of the rare allele. Selected active drops can be harvested and further amplified to feed an Illumina (San Diego, Calif.) sequencer.
Necrotic DNA comes from dead cells, and its concentration increases as a result of response to various anticancer treatments. Raj Krishnan, Ph.D., of Biological Dynamics Inc. (San Diego, Calif.), described the TR(ACE) platform for identifying remission, stable or progressive disease states. TR(ACE) traps individual segments of circulating DNA in individual wells created by a combination of dielectrophoresis and an AC-induced electrothermal and electroosmotic flow. The dynamic electric field facilitates frequency-dependent trapping of nanoparticles, even in high-ionic-strength solutions. DNA is captured and counted as individual spots, giving a dynamic range of 3‒1000 pg/μL.
Necrotic DNA is generally larger than 300 bp. Tracking concentration changes is useful in monitoring tumor dynamics and is a measure of the total tumor burden endured by an organism. In one example, in which a patient was diagnosed with colorectal cancer, fluorescence intensity increased from about 5000 during the first three weeks of therapy with cetuximab to 12,000 in the fifth week, indicating that cancer had developed resistance to cetuximab. By way of contrast with necrotic DNA, the traditional biomarker for colorectal cancer, carcinoembryonic antigen (CEA), is not elevated in about 40% of patients.
TR(ACE) provides results within about 20 minutes of sample introduction, which can be as small as 250 μL of serum or plasma. Currently, the company is working on expanding the list of supported assays.
Professor Michael Heller (University of California, San Diego) closed the symposium with a lecture extending Dr. Krishnan’s lecture with more examples demonstrating clinical utility. Heller noted that, before TR(ACE), the smallest detectable tumors had about a million cells. With TR(ACE), it is possible to detect much smaller amounts. Normal background for apoptotic DNA is about 10‒30 ng/mL, but with cancer, the concentration increases quickly to 100‒1000 ng/mL and the size of the DNA also increases from about 180 bp to 100‒10,000 bp. Noncancerous apoptotic DNA is tightly centered at 180 bp.
The entire process from sample to result needs to be quick and seamless. One study showed correlation of cancer stage with what Biological Dynamics calls ACE readout.
Dielectrophoretic (DEP) chips (see below) can be interrogated with a variety of highly specific fluorescent stains to improve the specificity of the image. Dr. Krishnan is also working on extending the platform by adding on-chip PCR amplification to improve detection.
Dielectrophoresis (DEP) is the name given to the phenomenon of the force exerted on a particle such as DNA or cell when subjected to a nonuniform electric field. The field induces electrical polarization in the target creating a dipole. The dipoles experience a force (attractive or repulsive) along the field lines. If the electric field is oscillating, it is possible to trap materials in a pseudo-well with great selectivity, since things that move too fast or slow are ejected from the trap
Prof. Heller feels that the ACE technology will lead to the use of circulating cell-free DNA/RNA in blood as a generic marker for cancer. Once detected, the ccfRNA will be useful in locating the primary tumor. This will be essential in guiding therapy.
Solid tumors are heterogeneous, so solid biopsies often miss important clonality, warned Amir Ali Talasaz, president and founder of Guardant Health, Inc. (Redwood City, Calif.). He advocated that cell-free DNA sequencing provides a more diagnostically useful profile, and noted that Guardant’s 360 digital sequencing platform provides single-molecule DNA sequencing and detects copy number variants, single-nucleotide polymorphisms (SNPs), fusions and indels. Detection limits of the platform have been verified with serial dilutions to a frequency of 0.1%, at which point one is dealing only one or two molecules of DNA. Currently, the test panel contains 68 clinically actionable oncogenes. Analytical specificity is 99.9999%. Comparison of the Guardant 360 with exome sequencing shows that the Guardant made one more call out 1.6 million bases. Dr. Talasaz went on to show examples of remission enabled by accurate identification of the clones. He compared turnaround time for biopsies at ~27 days with assays of blood, which took only one day, but report preparation still took an additional 10 days.
This theme was also picked up by Geraldine Perkins, M.D., Ph.D., of Paris Descartes University, who noted that a minority of subclones are responsible for subsequent therapy resistance. Circulating tumor DNA is only 10‒0.01% of total circulating DNA. The concentration of ctDNA is often below the limit of detection by classic PCR methods. She described emulsion PCR, in which the multitude of cells is diluted and then emulsified, to package one DNA strand in the emulsion volume. Many emulsion drops do not contain any DNA and give no signal. Those that contain a strand of DNA are amplified and produce a signal after several PCR cycles. Classifying and counting the population of drops provides quantitation, even at very low concentration.
Perkins went on to present a case history involving the KRAS oncogene, which is mutated in more than 30% of all adenocarcinomas. Mutated KRAS is a predictive factor of resistance for anti-EGFR (epidermal growth factor receptor) therapy in colorectal cancer, but KRAS mutations are also found in leukemia, lung and pancreatic cancers. Emulsion PCR facilitates detection of 1 mutant in 200,000 wild-type DNA strands. For comparison, bulk experiments have difficulty detecting 1 mutant in 10. With multiplexing and appropriate assays designs that use reporters with different colors, one can detect specific mutants such as G12A, G12V, G12S and G12C. The challenge now is integrating this information into therapy protocols.
Christian Jurinke of Stratec Molecular, GmbH (Berlin, Germany) described a method for extracting cfDNA from up to 4 mL of plasma using magnetic beads with the InviGenius Plus platform. The InviGenius accepts up to 12 primary sample tubes. A variety of kits are available in addition to the cfDNA. The InviGenius packages have CE registration in Europe but are not FDA approved in the U.S.
Personal Genome Diagnostics (Baltimore, Md.) presented a poster describing two assays for ctDNA that detect SNPs and rearrangements. The latter are more common and dangerous. The Plasma Select kit detects mutations across a range of loci. METDetect quantitates MET in blood. MET is deregulated in many types of human malignancies, including cancers of the kidney, liver, stomach, breast and brain. High MET concentration correlates with poor prognosis.
Summary and credits
Cambridge Healthech’s 22nd Molecular Medicine Tri-Conference was held February 15‒20, 2015, at the Moscone Center and Intercontinental Hotel in San Francisco. The CHI program team deserves thanks for ferreting out great topics presented by excellent speakers.
Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected].