The discovery is yet another factor that must be considered when developing a liquid biopsy test clinical laboratories can use to detect cancer
How often do disruptive elements present in Liquid biopsies result in misdiagnoses and unhelpful drug therapies for cancer? Researchers at the University of Washington School of Medicine (UW Medicine) in Seattle wanted to know. And the results of their study provide another useful insight for pathologists about the elements that circulate in human blood which must be understood so that liquid biopsy tests can be developed that are not affected by that factor.
Based on their case series study of 69 men with advanced prostate cancer, the UW Medicine researchers determined that 10% of men have a clonal hematopoiesis of indeterminate potential (CHIP) that can “interfere” with liquid biopsies and cause incorrect reports and unneeded prostate cancer treatment, according to their paper published in the journal JAMA Oncology.
The UW Medicine researchers advised testing for “variants in the cell-free DNA (cfDNA)” shed in blood plasma to enable appropriate treatment for people with already diagnosed prostate cancer, noted to a UW Medicine news release.
According to pathologist Colin Pritchard, MD, PhD, Associate Professor of Laboratory Medicine and Pathology at the UW Medicine, who led the research team, “clonal hematopoiesis can interfere with liquid biopsies. For example, mutations in the genes BRCA1, BRCA2, and ATM have been closely linked to cancer development.
“The good news is that, by looking at the blood cellular compartment, you can tell with pretty good certainty whether something is cancer, or something is hematopoiesis,” he said in the news release.
What Does CHIP Interference Mean to a Clinical Laboratory Blood Test?
In their published study, the UW Medicine researchers stressed the “urgent need to understand cfDNA testing performance and sources of test interferences” in light of recent US Food and Drug Administration (FDA) clearance of two PARP inhibitors (PARPi) for prostate cancer:
“We found that a strikingly high proportion of DNA repair gene variants in the plasma of patients with advanced prostate cancer are attributable to CHIP,” the researchers wrote. “The CHIP variants were strongly correlated with increased age, and even higher than expected by age group.
“The high rate of CHIP may also be influenced by prior exposure to chemotherapy,” they added. “We are concerned that CHIP interference is causing false-positive cfDNA biomarker assessments that may result in patient harm from inappropriate treatment, and delays in delivering alternative effective treatment options.
“Without performing a whole-blood control, seven of 69 patients (10%) would have been misdiagnosed and incorrectly deemed eligible for PARP-inhibitor therapy based on CHIP interference in plasma. In fact, one patient in this series had a BRCA2 CHIP clone that had been previously reported by a commercial laboratory testing company with the recommendation to use a PARPi. To mitigate these risks, cfDNA results should be compared to results from whole-blood control or tumor tissue,” the researchers concluded.
To find the clinically relevant CHIP interference in prostate cancer cfDNA testing, researchers used the UW-OncoPlex assay (developed and clinically available at UW Medicine). The assay is a multiplexed next-generation sequencing panel aimed at detecting mutations in tumor tissues in more than 350 genes, according to the UW Medicine Laboratory and Pathology website.
“To improve cfDNA assay performance, we developed an approach that simultaneously analyzes plasma and paired whole-blood control samples. Using this paired testing approach, we sought to determine to what degree CHIP interferes with the results of prostate cancer cfDNA testing,” the researchers wrote in JAMA Oncology.
Men May Receive Unhelpful Prostate Cancer Drug Therapies
The research team studied test results from 69 men with advanced prostate cancer. They analyzed patients’ plasma cfDNA and whole-blood control samples.
Tumor sequencing enabled detection of germline (cells relating to preceding cells) variants from CHIP clones.
The UW Medicine study suggested CHIP variants “accounted for almost half of the somatic (non-germline) DNA repair mutations” detected by liquid biopsy, according to the news release.
Other detailed findings of the UW Medicine Study:
CHIP variants of 2% or more were detected in cfDNA from 13 of 69 men.
Seven men, or 10%, having advanced prostate cancer “had CHIP variants in DNA repair genes used to determine PARPi candidacy.
CHIP variants rose with age: 0% in those 40 to 50; 12.5% in men 51 to 60; 6.3% in those 61 to 70; 20.8% in men 71 to 80; and 71% in men 81 to 90.
Whole-blood control made it possible to distinguish prostate cancer variants from CHIP interference variants.
“Men with prostate cancer are at high risk of being misdiagnosed as being eligible for PARPi therapy using current cfDNA tests; assays should use a whole-blood control sample to distinguish CHIP variants from prostate cancer,” the researchers wrote in JAMA Oncology.
Liquid Biopsies Are ‘Here to Stay’
Surgical oncologist William Cance, MD, Chief Medical and Scientific Officer, American Cancer Society (ACS) in Atlanta, recognizes the challenge of tumor biology to liquid biopsies.
“Genetic abnormalities are only one piece of the puzzle. We need to look comprehensively at tumors for the best therapy, from their metabolic changes and protein signatures in the blood to the epigenetic modifications that may occur, as cancers take hold,” he told Oncology Times. “It’s not just shed DNA in the blood.”
The UW Medicine study demonstrates the importance of understanding how all elements in liquid biopsies interact to affect clinical laboratory test results.
“I think liquid biopsies are here to stay,” Cance told Oncology Times. “They’re all part of precision medicine, tailored to the individual.”
The new method employs a pH sensitive dye and AI algorithms to ‘distinguish between cells originating from normal and cancerous tissue, as well as among different types of cancer’ the researchers said
Might a pH-sensitive dye in tandem with an image analysis solution soon be used to identify cancerous cells within blood samples as well within tissue? Recent research indicates that could be a possibility. If further studies and clinical trials confirm this capability, then anatomic pathologists could gain another valuable tool to use in diagnosing cancers and other types of disease.
Currently, surgical pathologists use a variety of hematoxylin and eosin stains (H/E) to bring out useful features in cells and cell structures. So, staining tissue on glass slides is a common practice. Now, thanks to machine learning and artificial intelligence, anatomic pathologists may soon have a similar tool for spotting cancer cells within both tissue and blood samples.
Researchers at the National University of Singapore (NUS) have developed a method for identifying cancer that uses a pH sensitive dye called bromothymol blue. The dye reacts to various levels of acidity in cancer cells by turning colors. “The pH inside cancer cells tends to be higher than that of healthy cells. This phenomenon occurs at the very early phases of cancer development and becomes amplified as it progresses,” Labroots reported.
In “Machine Learning Based Approach to pH Imaging and Classification of Single Cancer Cells,” published in the journal APL Bioengineering, the NUS researchers wrote, “Here, we leverage a recently developed pH imaging modality and machine learning-based single-cell segmentation and classification to identify different cancer cell lines based on their characteristic intracellular pH. This simple method opens up the potential to perform rapid noninvasive identification of living cancer cells for early cancer diagnosis and further downstream analyses.”
According to an NUS news release, the bromothymol blue dye is “applied onto patients’ cells” being held ex vivo in cell culture dishes. The dye’s color changes depending on the acidity level of the cancer cells it encounters. Microscopic images of the now-visible cancers cells are taken, and a machine-learning algorithm analyzes the images before generating a report for the anatomic pathologist.
The NUS researchers claim the test can provide answers in about half an hour with 95% accuracy, Labroots reported.
“The ability to analyze single cells is one of the holy grails of health innovation for precision medicine or personalized therapy. Our proof-of-concept study demonstrates the potential of our technique to be used as a fast, inexpensive and accurate tool for cancer diagnosis,” said Lim Chwee Teck, PhD, NUS Society Professor and Director of NUS’ Institute for Health Innovation and Technology, in the NUS news release.
AI Cell Analysis versus Laborious Medical Laboratory Steps
By developing an AI-driven method, Professor Lim and the NUS team sought to improve upon time-consuming techniques for identifying cells that traditionally involve using florescent probes, nanoparticles, and labeling steps, or for cells to be fixed or terminated.
“Unlike other cell analysis techniques, our approach uses simple, inexpensive equipment, and does not require lengthy preparation and sophisticated devices. Using AI, we are able to screen cells faster and accurately,” Professor Lim told Labroots. “Furthermore, we can monitor and analyze living cells without causing any toxicity to the cells or the need to kill them.”
The new technique may have implications for cancer detection in tumor tissue as well as in liquid biopsies.
“We are also exploring the possibility of performing the real-time analysis on circulating cancer cells suspended in blood,” Professor Lim said in the NUS news release. “One potential application for this would be in liquid biopsy where tumor cells that escaped from a primary tumor can be isolated in a minimally-invasive fashion from bodily fluids such as blood.”
Diagnosing Cancer in Real Time
The NUS’ method requires more research and clinical studies before it could become an actual tool for anatomic pathologists and other cancer diagnosticians. Additionally, the NUS researchers acknowledged that the focus on only four cell lines (normal cells, benign breast tumor cells, breast cancer cells, and pancreatic cancer cells) limited their study, as did lack of comparison with conventional florescent pH indicators.
Still, the NUS scientists are already planning more studies to advance their concept to different stages of cell malignancy. They envision a “real-time” version of the technique to enable recognition of cells and fast separation of those that need to be referred to clinical laboratories for molecular testing and/or genetic sequencing.
Medical laboratory leaders may want to follow the NUS study. An inexpensive AI-driven method that can accurately detect and classify cancer cells based on pH within the cells is provocative and may be eventually become integrated with other cancer diagnostics.
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.”
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.
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.
“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.
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.