Breakthrough assay a ‘tenfold improvement over any prior assay for TERT mutations in the blood for brain tumors,’ MGH says in an affirmation of a diagnostic technology clinical labs might soon use
In recent years, investors have poured tens of millions of dollars into companies that promised to create non-invasive cancer tests which use liquid biopsy technology. Medical laboratory scientists even watched some of these companies hype their particular liquid biopsy tests before clinical studies generated data demonstrating that these tests produced accurate, reliable, and reproducible results.
For diagnosing cancer, a liquid biopsy test typically uses a blood sample with the goal of finding and identifying circulating tumor cells. Harvard Medical School researchers at Massachusetts General Hospital (MGH) believe they have developed just such a blood test. Their assay utilizes an enhanced form of liquid biopsy to detect and monitor one of the more prevalent types of brain tumor in adults—a glioma—and, according to a Harvard news release, can detect the presence of glioma at a significantly higher “overall sensitivity” than other similar liquid-biopsy tests.
Gliomas start in glia cells contained in the brain or spine. They account for about 30% of all brain and central nervous system tumors and 80% of all malignant brain tumors.
During their study, MGH researchers compared blood samples and tumor biopsy tissues from patients diagnosed with a glioma. They discovered that an assay they developed—a droplet digital polymerase chain reaction (ddPCR) blood test—could detect and monitor two types of telomerase reverse transcriptase (TERT) promoter gene mutations—C228T and C250T. These two gene mutations promote cancer growth and are present in more than 60% of all gliomas. The mutations are also present in 80% of all high-grade gliomas, which are the most aggressive and life-threatening types of the cancer.
In the press release, instructor in Neurosurgery at MGH and one of the study’s authors, Leonora Balaj, PhD, said, “By ‘supercharging’ our ddPCR assay with novel technical improvements, we showed for the first time that the most prevalent mutation in malignant gliomas can be detected in blood, opening a new landscape for detection and monitoring of the tumors.”
Bob Carter, MD, PhD (above), is neurosurgical oncologist and Chief of Neurosurgery at MGH, a Professor of Neurosurgery at Harvard Medical School, and one of the study’s authors. In the MGH press release he said, “We envision the future integration of tests like this one into the clinical care of our patients with brain tumors. For example, if a patient has a suspected mass on MRI scanning, we can take a blood sample before the surgery and assess the presence of the tumor signature in the blood and then use this signature as a baseline to monitor as the patient later receives treatment, both to gauge response to the treatment and gain early insight into any potential recurrence.” What Carter describes is precision medicine and could open new diagnostic opportunities for anatomic pathology groups and clinical laboratories. (Photo copyright: Massachusetts General Hospital.)
MGH’s Ten-Fold Improvement over Previous ddPCR Assays
A liquid biopsy is the sampling and analysis of non-solid tissue in the body—primarily blood. MGH’s liquid-biopsy method detects cancer by examining fragments of tumor DNA circulating in the bloodstream. Since the technique is mostly non-invasive, tests can be performed more frequently to track tumors and mutations and monitor treatment progression. Prior to this new method, brain tumors had been difficult to detect using liquid biopsies.
“Liquid biopsy is particularly challenging in brain tumors because mutant DNA is shed into the bloodstream at a much lower level than any other types of tumors,” Balaj said in the press release.
However, MGH’s new ddPCR assay has an overall sensitivity rate of 62.5% and a specificity of 90%, which represents a tenfold improvement over prior assays for TERT mutations in the blood.
And when testing the performance of the ddPCR assay in tumor tissue, the MGH researchers discovered their results were the same as results from a previous independently-performed clinical laboratory assessment of TERT mutations within collected tumor specimens. They also found that their assay could detect TERT mutations when looking at blood plasma samples collected at other facilities.
The researchers believe that their test could be performed in most clinical laboratories and can be utilized to follow the course of disease in cancer patients. The MGH researcher’s goal is to expand and adapt the blood test to diagnose, differentiate, and monitor other types of brain tumors in addition to gliomas.
Of course, more research will be needed before MGH’s new assay can become a vital tool in the fight against disease. However, this type of genetic analysis may soon provide pathologists with new techniques to more accurately diagnose and monitor cancers, and to provide healthcare providers with valuable data regarding which therapies would be the most beneficial for individual patients, a key element of precision medicine.
The researchers unveiled a diagnostic device that uses microfluidic technology to identify cell types in blood by their size. The device also “can isolate individual cancer cells from patient blood samples,” according to a news release.
The ability to isolate circulating tumor cells could enable clinical laboratories to perform diagnostic cancer tests on liquid biopsies and blood samples. Dark Daily reported on various studies involving liquid biopsies—an alternative to invasive and costly cancer diagnostic procedures, such as surgery and tissue biopsies—in previous e-briefings.
“This new microfluidics chip lets us separate cancer cells from whole blood or minimally diluted blood. Our device is cheap and doesn’t require much specimen preparation or dilution, making it fast and easy-to-use,” said Ian Papautsky, PhD, Professor of Bioengineering at University of Illinois at Chicago, in the news release. He is shown above with members of the Papautsky Lab, which has been developing “microfluidic systems and point- of-care sensors for public health applications.” (Photo copyright: University of Illinois at Chicago.)
Searching for ‘Purity’
The UIC and QUT researchers were motivated by the
information-rich nature of circulating tumor cells. They also saw opportunity
for escalated “purity” in results, as compared to past studies.
In the paper, they acknowledged the work of other scientists
who deployed microfluidic technology affinity-based methods to differentiate
tumor cells in blood. Past studies (including previous work by the authors)
also explored tumor cells based on size and difference from white blood cells.
“While many emerging systems have been tested using patient samples, they share a common shortcoming: their purity remains to be significantly improved. High purity is in strong demand for circulating tumor cell enumeration, molecular characterization, and functional assays with less background intervention from white blood cells,” the authors wrote in their paper.
How the Device Works
The scientists say their system leverages “size-dependent
inertial migration” of cells. According to the news release:
Blood passes through “microchannels” formed in
plastic in the device;
“Inertial migration and shear-induced diffusion”
separate cancer cells from blood;
Tiny differences in size determine a cell’s
attraction to a location; and
Cells separate to column locations as the liquid
moves.
In other words, the device works as a filter sorting out, in
blood samples, the circulating tumor cells based on their unique size, New
Atlas explained.
93% of Cancer Cells Recovered by Device
When the researchers tested their new device:
Researchers placed 10 small-cell-lung cancer cells into five-milliliter samples of healthy blood;
The blood was then flowed through the device; and
93% of the cancer cells were recovered.
“A 7.5 milliliter tube of blood, which is typical volume for
a blood draw, might have 10 cancer cells and 35- to 40-billion blood cells. So,
we are really looking for a needle in a haystack,” Papautsky stated in the news
release.
The graphic above illustrates how, in the lab, the microfluidic device enabled the researchers to separate out cancer cells in six of the eight lung cancer samples they studied. (Graphic copyright: Ian Papautsky, PhD/University of Illinois at Chicago/New Atlas.)
“We report on a novel multi-flow microfluidic system for the
separation of circulating tumor cells with high purity. The microchannel takes
advantage of inertial migration of cells. The lateral migration of cells
strongly depends on cell size in our microchannel, and label-free separation of
circulating tumor cells from white blood cells is thus achieved without
sophisticated sample predation steps and external controls required by
affinity-based and active approaches,” the researchers wrote in their paper.
The researchers plan wider trials and the addition of
biomarkers to enable cancer DNA detection, New Atlas reported, which described
the UIC/QUT study as part of a “new wave of diagnostics.”
With so much focus on liquid biopsy research, it may be
possible for medical laboratories to one day not only diagnose cancer through
blood tests, but also to find the disease earlier and in a more precise way
than with traditional tissue sample analysis.
This research could lead to a useful liquid biopsy test that would be a powerful new tool for clinical laboratories and anatomic pathologists
Cancer researchers have long sought the Holy Grail of
diagnostics—a single biomarker that can quickly detect cancer from blood or
biopsied tissue. Now, researchers in Australia may have found that treasure. And
the preliminary diagnostic test they have developed reportedly can return
results in just 10 minutes with 90% accuracy.
In a news release, University of Queensland researchers discussed identifying a “simple signature” that was common to all forms of cancer, but which would stand out among healthy cells. This development will be of interest to both surgical pathologists and clinical laboratory managers. Many researchers looking for cancer markers in blood are using the term “liquid biopsies” to describe assays they hope to develop which would be less invasive than a tissue biopsy.
“This unique nano-scaled DNA signature appeared in every type of breast cancer we examined, and in other forms of cancer including prostate, colorectal, and lymphoma,” said Abu Sina, PhD, Postdoctoral Research Fellow at the Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland (UQ), in the news release.
“We designed a simple test using gold nanoparticles that
instantly change color to determine if the three-dimensional nanostructures of cancer
DNA are present,’ said Matt
Trau, PhD, Professor of Chemistry at the University of Queensland, and
Deputy Director and Co-Founder of UQ’s AIBN, in the news release.
The team’s test is preliminary, and more research is needed before
it will be ready for Australia’s histopathology laboratories (anatomic
pathology labs in the US). Still, UQ’s research is the latest example of how
increased knowledge of DNA is making it possible for researchers to identify
new biomarkers for cancer and other diseases.
“We certainly don’t know yet whether it’s the holy grail for
all cancer diagnostics, but it looks really interesting as an incredibly simple
universal marker of cancer, and as an accessible and inexpensive technology
that doesn’t require complicated lab-based equipment like DNA sequencing,” Trau
added.
The UQ researchers published their study in the journal Nature Communications. In it, they noted that “Epigenetic reprogramming in cancer genomes creates a distinct methylation landscape encompassing clustered methylation at regulatory regions separated by large intergenic tracks of hypomethylated regions. This methylation landscape that we referred to as ‘Methylscape’ is displayed by most cancer types, thus may serve as a universal cancer biomarker.”
While methyl patterning is not new, the UQ researchers say they were the first to note the effects of methyl pattern in a particular solution—water. With the aid of transmission electron microscopy, the scientists saw DNA fragments in three-dimensional structures in the water. But they did not observe the signature in normal tissues in water.
“To date, most research has focused on the biological consequences of DNA Methylscape changes, whereas its impact on DNA physicochemical properties remains unexplored,” UQ scientists Matt Trau, PhD (left), Abu Sina, PhD (center), and Laura Carrascosa (right), wrote in their study. “We exploit these Methylscape differences to develop simple, highly sensitive, and selective electrochemical or colorimetric one-step assays for the detection of cancer.” (Photo copyright: University of Queensland.)
Their test averaged 90% accuracy during the testing of 200
human cancer samples. Furthermore, the researchers found the DNA structure to
be the same in breast, prostate, and bowel cancers, as well as lymphomas, noted
The Conversation.
“We find that DNA polymeric
behavior is strongly affected by differential patterning of methylcytosine
leading to fundamental differences in DNA solvation and DNA-gold affinity
between cancerous and normal genomes,” the researchers wrote in NatureCommunications.“We exploit
these methylscape differences to develop simple, highly sensitive, and
selective electrochemical or one-step assays for detection of cancer.”
Next Steps for the
“Gold Test”
“This approach represents an exciting step forward in
detecting tumor DNA in blood samples and opens up the possibility of a generalized
blood-based test to detect cancer, Ged Brady, PhD, Cancer Research UK
Manchester Institute, told The
Oxford Scientist. “Further clinical studies are required to evaluate
the full clinic potential of the method.”
Researchers said the next step is a larger clinical study to
explore just how fast cancer can be detected. They expressed interest in
finding different cancers in body fluids and at various stages. Another opportunity
they envision is to use the cancer assay with a mobile device.
DiCarlo told USA Today
that such a mobile test could be helpful to clinicians needing fast answers for
people in rural areas. However, he’s also concerned about false positives. “You
don’t expect all tumors to have the same methylation pattern because there’s so
many different ways that cancer can develop,” he told USA Today. “There
are some pieces that don’t exactly align logically.”
The UQ researchers have produced an intriguing study that differs
from other liquid biopsy papers covered by Dark Daily. While their test may need to be used in combination with other
diagnostic tests—MRI, mammography, etc.—it has the potential to one day be used
by clinical laboratories to quickly reveal diverse types of cancers.
Using GPIIb/IIIa inhibition, and ion chelation, researchers have developed a “universal” method for preserving blood up to 72 hours while keeping it viable for advanced rare-cell applications
However, preserving sample quality is an essential part of analytical accuracy. This is particularly true in precision oncology and other specialties where isolating rare cells (aka, low abundance cells), such as circulating tumor cells (CTCs), is a key component to obtaining information and running diagnostics.
Should further testing validate their findings and methodology, this change could allow greater use of central laboratories and other remote testing facilities that previously would not be available due to distance and sample travel time.
Keeping Blood Alive Is Not Easy
“At Mass. General, we have the luxury of being so integrated with the clinical team that we can process blood specimens in the lab typically within an hour or two after they are drawn,” stated lead author Keith Wong, PhD, former Research Fellow, MGH-CEM, and now Senior Scientist at Rubius Therapeutics, Boston, in a Mass General press release. “But to make these liquid biopsy technologies routine lab tests for the rest of the world, we need ways to keep blood alive for much longer than several hours, since these assays are best performed in central laboratories for reasons of cost-effectiveness and reproducibility.”
Study authors Wong and co-lead author Shannon Tessier, PhD, Investigator at MGH-CEM, noted that current FDA-approved blood stabilization methods for CTC assays use chemical fixation—a process that can result in degradation of sensitive biomolecules and kill the cells within the sample.
Without stabilization, however, breakdown of red cells, activation of leukocytes (white blood cells), and clot formation can render the results of analyzing a sample useless, or create issues with increasingly sensitive equipment used to run assays and diagnostics.
“We wanted to slow down the biological clock as much as possible by using hypothermia, but that is not as simple as it sounds,” says Tessier. “Low temperature is a powerful means to decrease metabolism, but a host of unwanted side effects occur at the same time.”
Researchers started by using hypothermic treatments to slow degradation and cell death. However, this created another obstacle—aggressive platelet coagulation. By introducing glycoprotein IIb/IIIa inhibitors, they found they could minimize this aggregation.
Keith Wong, PhD (left), a former Research Fellow, MGH-CEM, and now Senior Scientist at Rubius Therapeutics in Boston; and Shannon Tessier, PhD (right), Investigator at MGH-CEM, co-authored a study to develop a whole blood stabilization method that preserves sample integrity for up to 72 hours, making it possible to transport blood specimens further distances to central clinical laboratories for processing. (Photo copyrights: LinkedIn.)
Prior to microfluidic processing of their test samples, researchers applied a brief calcium chelation treatment. The result was efficient sorting of rare CTCs from blood drawn up to 72 hours prior, while keeping RNA intact and retaining cell viability.
“The critical achievement here,” says Tessier, “Is that the isolated tumor cells contain high-quality RNA that is suitable for demanding molecular assays, such as single-cell qPCR, droplet digital PCR, and RNA sequencing.”
Their testing involved 10 patients with metastatic prostate cancer. Sample integrity was verified by comparing CTC analysis results between fresh samples and preserved samples from the same patients using MGH-CEM’s own microfluidic CTC-iChip device.
Results showed a 92% agreement across 12 cancer-specific gene transcripts. For AR-V7, their preservation method achieved 100% agreement. “This is very exciting for clinicians,” declared David Miyamoto, MD, PhD, of Massachusetts General Hospital Cancer Center in the press release. “AR-V7 mRNA can only be detected using CTCs and not with circulating tumor DNA or other cell-free assays.”
Methodology Concerns and Future Confirmations
“Moving forward, an extremely exciting area in precision oncology is the establishment of patient-specific CTC cultures and xenograft models for drug susceptibility,” the study authors noted. “The lack of robust methods to preserve viable CTCs is a major roadblock towards this Holy Grail in liquid biopsy. In our preliminary experiments, we found that spiked tumor cells in blood remain highly viable (>80%) after 72 hours of hypothermic preservation.”
Despite this, they also acknowledge limitations on their current findings. The first is the need for larger-scale validation, as their testing involved a 10-patient sample group.
Second, they note that further studies will be needed to “more completely characterize whole-transcriptome alterations as a result of preservation, and to what extent they can be stabilized through other means, such as further cooling (e.g., non-freezing sub-zero temperatures) or metabolic depression.”
Researchers also note that their approach has multiple advantages for regulatory approval and further testing—GPIIb/IIIa inhibitors are both low-cost and already approved for clinical use, implementation requires no modification of existing isolation assays, and cold chain protocols are already in place allowing for easy adaptation to fit the needs of pathology groups, medical laboratories, and other diagnostics providers handling samples.
While still in its early stages, the methods introduced by the researchers at MGH-CEM show potential to allow both the facilities collecting samples and the clinical laboratories processing them greater flexibility and increased accuracy, as high-sensitivity assays and diagnostics continue to power the push toward personalized medicine and expand laboratory menus across the industry.
Liquid biopsy tests hold much promise. But inconsistencies in their findings provoke scrutiny and calls from researchers for further development before they can be considered reliable enough for diagnostic use
Many commercial developers of liquid biopsy tests tout the accuracy and benefits of their diagnostic technology. However, there are an equal number of medical laboratory experts who believe that this technology is not yet reliable enough for clinical use. Critics also point out that these tests are being offered as Laboratory Developed Tests (LDTs), which are internally developed and validated and have not undergone regulatory review.
Dark Daily has published several e-briefings on researchers who have sent the same patient samples to different genetic testing labs and received back materially different test results. Now, a new study by Johns Hopkins University concludes that liquid biopsy technology “must improve” before it should be relied upon for diagnostic and treatment decision making.
‘Certification for Medical Laboratories Must Improve’
Liquid Biopsy is the term for drawing whole blood and looking for cancer/tumor cells circulating in the blood stream. This is one factor in the imprecision of a liquid biopsy. Did the blood sample drawn actually have tumor cells? After all, only a limited number of tumor cells, if present, are in circulation.
Gonzalo Torga, MD (above left), and Kenneth J. Pienta, MD (above right), are the two Johns Hopkins Medicine doctors who conducted the recent study into the efficacy of liquid biopsy laboratory developed tests (LDTs) offered by different medical laboratory companies. They published their findings in JAMA Oncology. (Photos copyright: Johns Hopkins.)
In reporting the DNA findings and results from the two medical laboratory companies, researchers discovered that the results completely matched in only three of the 40 patients! The Johns Hopkins researchers are concerned that patients could be prescribed certain cancer treatments based on which lab company’s liquid biopsy test their physician orders, instead of an accurate identification of the unique mutations in their tumors.
“Liquid biopsy is a promising technology, with an exceptional potential to impact our ability to treat patients, but it is a new technology that may need more time and experience to improve,” Gonzalo Torga, MD, Postdoctoral Fellow and Instructor at Johns Hopkins, and the lead author of the study, told Forbes. “We can’t tell from these studies which laboratory’s panel is better, but we can say that certification for these laboratories must improve.”
Unlocking New View of Tumors
Two commercial tests were used for the study:
Guardant360 from Guardant Health, Inc., uses digital sequencing to analyze genomic data points at the single molecular level. It examines 73 genes, including all National Comprehensive Cancer Network (NCCN) listed genes. The test searches for DNA fragments among billions of cells and digitally tags each fragment. This process unlocks a view of tumors that is not seen with tissue biopsies, which helps doctors prescribe the best treatment options for a particular patient.
“As a simple blood test, it provides physicians with a streamlined, cost-effective method to identify genomic alterations that can comprehensively influence a patient’s therapy response,” Helmy Eltoukhy, PhD, co-founder and Chief Executive Officer at Guardant Health, told MDBR.
“The only way of keeping ahead of those diseases and tracking those mutations has been through surgery, through doing a tissue biopsy and physically cutting a piece of the tumor out and sequencing it,” Eltoukhy noted in an interview with Xconomy. “What we’re able to do is essentially get the same, or sometimes better performance to tissue biopsy, but through two teaspoons of blood.”
According to the Guardant Health website, it takes just 14 days for a full report from Guardant360 to reach the ordering physician. In addition, the blood test provides samples with an adequate level of cell-free DNA to test 99.8% of the time and reduces errors and false positives found in standard sequencing methods by 1,000 times. It is common for samples used for tissue sequencing to have insufficient DNA for testing 20% to 40% of the time.
“We believe that conquering cancer is at its core a big data problem, and researchers have been data-starved,” explained Eltoukhy in VentureBeat. “Our launch of the world’s first commercial comprehensive liquid biopsy sparked a boom in cancer data acquisition. Every physician who orders one of our tests, and every patient whose tumor DNA we sequence, adds to this larger mission by improving our understanding of this complex disease.”
PlasmaSELECT-R64, manufactured by Personal Genome Diagnostics (PGDx), evaluates a targeted panel of 64 genes that have biological and functional relevance in making treatment decisions. PGDx announced the expanded version of its PlasmaSELECT assay in March of 2017.
“We are proud to launch the revolutionary PlasmaSELECT 64 expanded assay just six months after we introduced the most accurate, clinically actionable liquid biopsy tumor profiling assay to the market,” said Doug Ward, Chief Executive Officer at PGDx, in a press release. “This update is the first liquid biopsy assay that includes MSI (microsatellite instability) testing as a biomarker for high tumor mutational load, thereby providing cancer patients and their oncologists with information on whether they might be candidates for immuno-oncology therapies. The ability to generate DNA tumor profiling non-invasively using blood or plasma offers many advantages and makes genomic testing more accessible and usable.”
Regulations of LDTs Could be Needed to Improve Liquid Biopsy Tests
There are pathologists and clinical laboratory professionals who believe the technology behind liquid biopsies is not yet reliable enough for clinical use. The tests are being offered as LDTs, which are internally developed and validated, and the Food and Drug Administration (FDA) allows LDTs to be sold without regulatory reviews at this time. However, there are discussions regarding if and how to regulate LDTs, the outcome of which could impact how clinical laboratories are allowed to market the LDTs they develop.
Clearly, liquid biopsies are still in their relatively early stages of development. More testing and evaluation is needed to determine their efficacy. However, their potential to revolutionize cancer detection and care is obvious and a strong motivator for LTD developers, which means there will be future developments worth noting.
