November 15, 2015 (Vol. 35, No. 20)

Lisa Heiden Ph.D. Director of Business Development MyBioSource

Eager To Snatch Rare But Valuable Bits of DNA from Blood And Urine, Researchers and Developers Are Staking Claims All Over the Map

With all the buzz surrounding liquid biopsies, people might think circulating cell-free DNA (cfDNA) is a new discovery. Not so, says Iwijn De Vlaminck, Ph.D., assistant professor of biomedical engineering, Cornell University: “cfDNA was actually discovered in the 1940’s, even before the structure of double-stranded DNA.

With the advent of genomics and molecular technologies, it’s all of a sudden becoming very relevant for a wide range of applications.”
 
Cancer cfDNA applications are among those on the verge of entering the clinical mainstream. Significant amounts of tumor-specific cfDNA are found in patients’ bloodstreams and other biological fluids such as urine.
 
The popular term “liquid biopsy” contrasts cfDNA, obtained from biological fluids, with classic tissue biopsy approaches. Both blood- and urine-based liquid biopsies are possible with Trovagene’s Precision Cancer Monitoring approach, which is designed to track specific oncogene mutations over time. According to company CSO Mark Erlander, Ph.D., urine can be “a more viable liquid biopsy than even plasma for some clinical utilities.”
 
Drs. De Vlaminck and Erlander were among the presenters at CHI’s Clinical Applications of Cell-Free DNA, a conference recently held in Washington, DC. Some of the most promising technologies presented there are discussed herein.

Be it from natural apoptotic turnover or aberrant cell death processes, “the components of dead cells don’t disappear magically,” maintained Dr. De Vlaminck. “Bits and pieces can end up in the bloodstream, and so can their genomes. cfDNA is basically DNA that floats around in blood. It’s not contained within cells. It’s essentially remnants of dead cells.”

“It’s a bit of a gold rush,” declared Dr. De Vlaminck. “The gold comes into play because there’s so many useful cfDNA applications, including prenatal, cancer, transplants, infection, and disease, where new approaches can have an impact. Lots of people are attracted to the promise of that.”


Trovagene’s technology can noninvasively detect and quantitate circulating tumor DNA (ctDNA) in urine and plasma. Urine is a par-ticularly convenient source of ctDNA, which can be used to reveal intratumor heterogeneity. Sampling can occur at patients’ homes, and the frequency of testing can be patient-driven.

Barcodes Sift Paydirt from Tailings

At the CHI event, Nickolas E. Papadopoulous, Ph.D., a professor of oncology at Johns Hopkins University and the president of PapGene, focused on cancer applications. “With the same method, the same technology, and the same biological fluid,” he said, “you can monitor cancers, look for therapies, look for diseases, and do early detection.”

Cancer cfDNA technologies rely on detecting mutations that are “by definition present only in tumor cells and not in normal cells,” Dr. Papadopoulos pointed out. “I think the Holy Grail in oncology diagnostics is early detection. Our goal is to revolutionize the field of early detection.”

The caveat is that the smaller the tumor is in the patient, the harder it is to detect in a liquid biopsy, which means that early detection is equivalent to finding a needle in a haystack. “It’s quite a technical challenge when there are only 1–5 mutant tumor DNA fragments in a sea of 10,000 DNA fragments,” exclaimed Dr. Papadopoulos.

To meet this challenge, Johns Hopkins developed the Safe Sequencing System (Safe-SeqS), informed Dr. Papadopoulos. He added that Safe-SeqS has been licensed by PapGene to advance its work on early detection of ovarian and endometrial cancer. “The most important aspect of this technology,” pointed out Dr. Papadopoulos, “is that each molecule is associated with a specific molecular barcode, a unique identifier (UID), at the very first step of the process.”

Each primer has its own molecular barcode. If there are 1,000 different KRAS molecules in the liquid biopsy, there will be 1,000 different UIDs for KRAS. Each molecule goes through all the amplifications with its UID, followed by next-generation sequencing (NGS), mutation calls, and finally a search for any association of the UID with a piece of DNA that has a mutation.

“If a given UID is sometimes associated with a mutation and sometimes not, it means an artifact happened somewhere in the process,” explained Dr. Papadopoulos. “But that’s the whole idea, that’s how we separate real changes in the DNA from errors that take place during the process.”

Barcodes render Safe-SeqS extremely sensitive. One mutant molecule can be detected among 10,000 molecules of wild type. NGS enables scalability: hundreds of different mutations in hundreds of patients can be analyzed at once.


Outfitters Offer Sample and Platform Independence

“It’s like PCR on steroids, you’re actually blocking out the wild type and allowing the mutant to exponentially amplify,” asserted Ben Legendre Jr., Ph.D., referring to Transgenomic’s ICE COLD-PCR enabling technology. “ICE COLD” stands for Improved and Complete Enrichment CO-amplification at Lower Denaturation temperature. According to Dr. Legendre, technical director of laboratory operations, ICE COLD-PCR is a specialized method for PCR enrichment technology that has high-sensitivity mutation detection capabilities.

Analysis of the PCR-based end product can be run on almost any downstream sequencing platform such as digital droplet PCR, NGS, or Sanger sequencing. “Platform independent—can’t stress that enough,” Dr. Legendre interjected.

ICE COLD-PCR offers sample independence, too. Suitable sample types include noninvasive cfDNA liquid biopsies as well as formalin-fixed, paraffin-embedded (FFPE) tissues from surgical biopsies.

The ICE COLD-PCR technology exploits differences in mutant and wild-type binding properties to oligos. “Key to our technology is adding an RS (reference sequence)-oligo that is fully complementary to the wild type,” explained Dr. Legendre. The wild-type and RS-oligo remain preferentially bound together when thermocycler conditions are altered. This is in comparison to the tenuous binding between the mutant and RS-oligo, which becomes denatured at colder temperatures.

When the primers amplify the target segment, the mutant will be exponentially amplified whereas the wild type will only be linearly amplified. “That’s where you get the enabling technology,” Dr. Legendre emphasized. “We have a 100–500-fold improvement of the mutation detection sensitivity across all sequencing platforms.”

“We look at the literature to see what’s clinically actionable,” Dr. Legendre continued. “That’s where we gear our kits.” Kits covering the main activating and resistance mutations in BRAF, EGFR, KRAS, KRAS, and PIK3CA are among those available.


Transgenomic’s ICE COLD-PCR enriches variant alleles from a mixture of wild-type DNA (WT) and mutant DNA (MT). The RS (reference sequence)-oligo binds one strand of the wild-type DNA and mutant DNA. At the critical temperature (Tc), the RS-oligo:mutant DNA heteroduplex is denatured, and the mutant DNA is selectively amplified.

Claims along Red and Yellow Streams

Precision Cancer Monitoring (PCM) is Trovagene’s method of measuring tumor-related gene mutations from tumor cfDNA, which enters the bloodstream and passes through the kidney barrier into the urine. After DNA is extracted, a PCR-NGS enrichment method is employed for mutation detection within a urine sample.

“First it was about plasma versus tissue biopsies,” said Dr. Erlander. “Nobody thought urine was possible, and now yellow is the new red.” Urine is noninvasive versus blood and tissue tests, and larger volumes with greater amounts of DNA can be obtained. Also, emerging data supports the idea of using urine as a method for both detecting and monitoring actionable mutations in metastatic cancer patients throughout therapy.

There are several key clinical utilities for cfDNA. According to Dr. Erlander, these include minimal residual disease surveillance, which amounts to “looking for recurrence in a patient who appears to be disease free.” Other clinical utilities, Dr. Erlander continued, are diagnostic: “Does the patient have a specific mutation that is clinically actionable? And, with respect to drug response monitoring, is the patient responding to the molecularly targeted therapy or chemotherapy?”

More specifically, once the driver or activating mutation has been identified, then testing is really about monitoring the responses to therapy and determining as quickly as possible whether or not the patient is responding. If a patient is responding and the drug is actually killing the cells, “we can see the actual huge spikes of tumor DNA mutations in the urine within one or two days after therapy,” explained Dr. Erlander. This is important because it enables near-real-time assessment of whether a drug is working.


Direct-to-Analyte Nuggets

“We don’t actually do traditional liquid biopsy,” informed Raj Krishnan, Ph.D., CEO, Biological Dynamics. “When it comes to tumor response, we are introducing a ‘direct-to-analyte’ twist on molecular imaging. Our AC Electrokinetics (ACE) platform measures tumor activity by quantifying large molecular weight particles directly from whole blood, serum, and plasma.” The particles consist of cellular debris such as cfDNA, cell membrane fragments, exosomes, oligonucleosomes, and protein aggregates.

“Our TRACE (Treatment Response using ACE) test isolates and tracks a subset of cfDNA that exists if you have cancer,” Dr. Krishnan added. This subset involves cfDNA larger than 300 base pairs that has been shown to correlate with tumor-associated cell death. “Healthy people have little or none of this,” he noted.

The ACE system integrates electronic, microfluidic, and optical functions. Particles are attracted to specific locations where cfDNA is captured on a microelectrode array and quantified using fluorescence microscopy. The levels of large molecular weight cfDNA are correlated with tumor burden.

“TRACE,” declares Dr. Krishnan, “has the potential for monitoring treatment response in multiple cancers.” He likens the TRACE test to existing protein biomarkers such as CEA, CA125, and CA19-9 for monitoring colorectal, ovarian, and pancreatic cancers, respectively. “TRACE, however, can work for multiple cancers, not just a specific one or small subsets of patients within those cancers.”

TRACE is a rapid, repeatable, and inexpensive test that needs only five drops of blood and 20 minutes processing time. Moreover, said Dr. Krishnan, this test involves a straightforward procedure: “Insert the sample into a single-use fluidics cartridge, push a button to pull down the particles, wash them, and then just take a picture.”

Dr. Krishnan summarized TRACE’s mission as follows: “Is the current treatment making the patient better or worse? Our goal is to help doctors get to the right answer.”


Fluorescent images showing high-molecular-weight cell-free DNA (green dye) captured on Biological Dynamics’ ACE platform, which creates an electronic size filter capable of inducing a dipole on particles of interest and attracting the particles to specific locations on a microelectrode array: (A) trace amounts of cell-free DNA (typically found in healthy individuals); (B) significantly higher levels of cfDNA (typically seen in the blood of advanced cancer patients). The company uses this technology to track treatment response in cancer patients.

Getting a Jump on Graft Rejection, Pathogen Intrusion

“Our work is in organ transplantation where the big issue after transplant procedures is transplant rejection,” said Dr. De Vlaminck. “It’s a team effort at Stanford in a study directed by Drs. Kiran Khush, Hannah Valantine, and Stephen Quake.” (Dr. De Vlaminck was a postdoc in the Quake lab.)

The current standard of care is an invasive biopsy of the transplanted organ to monitor rejection. In heart transplants, “a piece of the tissue from the beating organ is taken out and examined under the microscope to look for immune infiltrates and damage,” Dr. De Vlaminck noted. “The organ transplant is also a genome transplant. When there’s injury to the graft during rejection, cells of the transplanted organ enter the bloodstream and have the fingerprint of the foreign donor genome and not the recipient genome.”

cfDNA for organ transplant diagnostics is emerging as a sensitive way to monitor graft health and identify early signs of graft rejection. For example, more donor DNA might mean higher dosages of immunosuppressants are needed.

cfDNA approaches can reduce subjectivity because “if you ask two different pathologists to grade the same biopsy slide they only agree in about 70% of the cases,” Dr. De Vlaminck explained.

“We found that a small number of sequences in our datasets were not human derived, they were actually microbial or fungal derived,” he continued. “With shotgun sequencing, we can test for a multitude of pathogens at once. As long as that agent has a DNA genome and a reference genome, we can identify it.”

The numbers of sequences of particular viruses in the plasma of transplant sequences have been found to strongly correlate with pharmacological immunosuppression. cfDNA approaches for infection definitely represent a promising advance over standard cultures for microbes or PCR applications, methods specific to particular pathogens that must be done one test at a time. 


A researcher at Stanford University holding a tube of blood after centri-fugation. The pale yellow liquid on top is blood plasma containing cell-free DNA. Cell-free DNA in easy-to-obtain samples such as blood or urine is revolutionizing precision medicine approaches in many fields of medicine including oncology, transplantation, and infectious disease. [Quake Lab, Stanford University]

























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