MicroRNAs in urine could prove to be promising biomarkers in clinical laboratory tests designed to diagnose brain tumors regardless of the tumor’s size or malignancy, paving the way for early detection and treatment
Researchers at Nagoya University in Japan have developed a liquid biopsy test for brain cancer screening that, they claim, can identify brain tumors in patients with 100% sensitivity and 97% specificity, regardless of the tumor’s size or malignancy. Pathologists will be interested to learn that the research team developing this technology says it is simple and inexpensive enough to make it feasible for use in mass screening for brain tumors.
Neurologists, anatomic pathologists, and histopathologists know that brain tumors are one of the most challenging cancers to diagnose. This is partly due to the invasive nature of biopsying tissue in the brain. It’s also because—until recently—clinical laboratory tests based on liquid blood or urine biopsies were in the earliest stages of study and research and are still in development.
Thus, a non-invasive urine test with this level of accuracy that achieves clinical status would be a boon for the diagnosis of brain cancer.
Researchers at Japan’s Nagoya University believe they have developed just such a liquid biopsy test. In a recent study, they showed that microRNAs (tiny molecules of nucleic acid) in urine could be a promising biomarker for diagnosing brain tumors. Their novel microRNA-based liquid biopsy correctly identified 100% of patients with brain tumors.
Well-fitted for Mass Screenings of Brain Cancer Patients
According to the National Cancer Institute (NCI), brain and other central nervous system (CNS) cancers represent 1.3% of all new cancer cases and have a five-year survival rate of only 32.6%.
In their published study, the Nagoya University scientists wrote, “There are no accurate mass screening methods for early detection of central nervous system (CNS) tumors. Recently, liquid biopsy has received a lot of attention for less-invasive cancer screening. Unlike other cancers, CNS tumors require efforts to find biomarkers due to the blood–brain barrier, which restricts molecular exchange between the parenchyma and blood.
“Additionally, because a satisfactory way to collect urinary biomarkers is lacking, urine-based liquid biopsy has not been fully investigated despite the fact that it has some advantages compared to blood or cerebrospinal fluid-based biopsy.
“Here, we have developed a mass-producible and sterilizable nanowire-based device that can extract urinary microRNAs efficiently. … Our findings demonstrate that urinary microRNAs extracted with the nanowire device offer a well-fitted strategy for mass screening of CNS tumors.”
The Nagoya University researchers focused on microRNA in urine as a biomarker for brain tumors because “urine can be collected easily without putting a burden on the human body,” said Atsushi Natsume, MD, PhD, Associate Professor in the Department of Neurosurgery at Nagoya University and a corresponding author of the study, in a news release.
A total of 119 urine and tumor samples were collected from patients admitted to 14 hospitals in Japan with CNS cancers between March 2017 and July 2020. The researchers used 100 urine samples from people without cancer to serve as a control for their test.
To extract the microRNA from the urine and acquire gene expression profiles, the research team designed an assembly-type microfluidic nanowire device using nanowire scaffolds containing 100 million zinc oxide nanowires. According to the scientists, the device can be sterilized and mass-produced, making it suitable for medical use. The instrument can extract a significantly greater variety and quantity of microRNAs from only a milliliter of urine compared to traditional methods, such as ultracentrifugation, the news release explained.
Simple Liquid-biopsy Test Could Save Thousands of Lives Each Year
While further studies and clinical trials will be necessary to affirm the noninvasive test’s accuracy, the Nagoya University researchers believe that, with the inclusion of additional technologies, a urine-based microRNA test could become a reliable biomarker for detecting brain tumors.
“In the future, by a combination of artificial intelligence and telemedicine, people will be able to know the presence of cancer, whereas doctors will be able to know the status of cancer patients just with a small amount of their daily urine,” Natsume said in the news release.
Biomarkers found in urine or blood samples that provide clinical laboratories with a simple, non-invasive procedure for early diagnosis of brain tumors could greatly increase the five-year survival rate for thousands of patients diagnosed with brain cancer each year. Such diagnostic technologies are also appropriate for hospitals and physicians interested in advancing patient-centered care.
Decision is part of UK effort to diagnose 75% of all cancers at stage I or stage II by 2028 and demonstrates to pathologists that the technology used in liquid biopsy tests is improving at a fast pace
Pathologists and medical laboratory scientists know that when it comes to liquid biopsy tests to detect cancer, there is plenty of both hope and hype. Nevertheless, following a successful pilot study at the Christie NHS Foundation Trust in Manchester, England, which ran from 2015-2021, the UK’s National Health Service (NHS) is pushing forward with the use of liquid biopsy tests for certain cancer patients, The Guardian reported.
NHS’ decision to roll out the widespread use of liquid biopsies—a screening tool used to search for cancer cells or pieces of DNA from tumor cells in a blood sample—across the UK is a hopeful sign that ongoing improvements in this diagnostic technology are reaching a point where it may be consistently reliable when used in clinical settings.
The national program provides personalized drug therapies based on the genetic markers found in the blood tests of cancer patients who have solid tumors and are otherwise out of treatment options. The liquid biopsy creates, in essence, a match-making service for patients and clinical trials.
Liquid Biopsy Genetic Testing for Cancer Patients
“The learnings from our original ‘Target’ study in Manchester were that genetic testing needs to be done on a large scale to identify rare genetic mutations and that broader access to medicines through clinical trials being undertaken across the country rather than just one site are required,” Matthew Krebs, PhD, Clinical Senior Lecturer in Experimental Cancer Medicine at the University of Manchester, told The Guardian.
Krebs, an honorary consultant in medical oncology at the Christie NHS Foundation Trust, led the Target National pilot study.
“This study will allow thousands of cancer patients in the UK to access genetic testing via a liquid biopsy. This will enable us to identify rare genetic mutations that in some patients could mean access to life-changing experimental medicines that can provide great treatment responses, where there are otherwise limited or no other treatment options available.”
Detecting cancers at earlier stages of disease—when treatment is more likely to result in improved survival—has become a strategic cancer planning priority in the UK, theBMJ noted.
“The NHS is committed to diagnosing 75% of all cancers at stage I or II by 2028, from around 50% currently,” the BMJ wrote. “Achieving such progress in less than a decade would be highly ambitious, even without disruption caused by the COVID-19 pandemic. In this context, considerable hope has been expressed that blood tests for circulating free DNA—sometimes known as liquid biopsy—could help achieve earlier detection of cancers.”
The Guardian noted that the UK’s initiative will use a liquid biopsy test made by Swiss-healthcare giant Roche.
In her article “The Promise of Liquid Biopsies for Cancer Diagnosis,” published in the American Journal of Managed Care (AJMC) Evidence-based Oncology, serial healthcare entrepreneur and faculty lecturer at Harvard Medical School Liz Kwo, MD, detailed the optimism surrounding the “revolutionary screening tool,” including its potential for:
identifying mechanisms of resistance to therapies,
measuring remaining disease after treatment,
assessing cancer relapse or resistance to treatment, and
eliminating risk surrounding traditional biopsies.
The AJMC article estimated the liquid biopsy market will be valued at $6 billion by 2030. However, Kwo also noted that clinical adoption of liquid biopsies in the US continues to face challenges.
Welch compared the investor hype surrounding liquid biopsies to that of the now-defunct blood testing company Theranos, which lured high-profile investors to pour millions into its unproven diagnostic technology.
“Effective cancer screening requires more than early detection. It also requires that starting therapy earlier helps people live to older ages than they would if they started treatment later,” he wrote. “If that doesn’t happen, liquid biopsies will only lead to people living longer with the knowledge they have a potentially incurable disease without extending their lives. These people would be subjected to cancer therapies and their toxicities earlier, but at a time when they would otherwise be experiencing no cancer-related signs or symptoms.”
And so, while there’s much excitement about the possibility of a minimally invasive way to detect cancer, anatomic pathology groups and clinical laboratories will have to wait and see if the hype and hope surrounding liquid biopsies is substantiated by further research.
Smaller cities and rural towns are finding the NWSS a useful early warning tool for tracking COVID-19 in their communities
In a move that mirrors similar programs around the world, the federal Centers for Disease Control and Prevention (CDC) now monitors sewage nationwide and records levels of SARS-CoV-2 in an effort to prevent new outbreaks of COVID-19 and spot any new variants of the coronavirus.
Advances in gene sequencing technologies are enabling the CDC’s National Wastewater Surveillance System (NWSS), and in many communities, clinical laboratories and health system laboratories have worked with local health authorities to test wastewater since onset of the pandemic.
“What started as a grassroots effort by academic researchers and wastewater utilities has quickly become a nationwide surveillance system with more than 34,000 samples collected representing approximately 53 million Americans,” noted epidemiologist Amy Kirby, PhD (above) during the telebriefing.
Kirby is a Senior Service Fellow in the Waterborne Disease Prevention Branch at the CDC.
“Currently, CDC is supporting 37 states, four cities, and two territories to help develop wastewater surveillance systems in their communities. More than 400 testing sites around the country have already begun their wastewater surveillance efforts,” she added.
Genetic Sequencing Enables Tracking of Virus and Bacteria
At the time of the telebriefing, the federal agency anticipated having an additional 250 sites online within a few weeks and even more sites added within the coming months. Many of the participating sites are sequencing the genes of their biological samples and reporting that data to the CDC.
“So, we’ve seen from very early days in the pandemic that rates of detection in wastewater correlate very well with other clinical indicators, like pace rates and hospitalization and test positivity,” Kirby stated. “That data continues to come in and it continues to be a very solid indicator of what’s going on in the community.”
Wastewater, also referred to as sewage, includes water from toilets, showers, and sinks that may contain human fecal matter and water from rain and industrial sources. To use the CDC’s wastewater surveillance system:
Wastewater is collected from a community area served by the surveillance system as it flows into a local water treatment plant.
Collected samples are sent to an environmental or public health laboratory where they are tested for SARS-CoV-2.
Health departments submit the testing data to the CDC through the online NWSS Data Collection and Integration for Public Health Event Response (DCIPHER) portal.
The DCIPHER system then analyzes the data and reports the results back to the health department for use in their COVID-19 response.
Beginning in February 2022, members of the public can view the results of collected data online through the CDC’s COVID Data Tracker.
Wastewater Sampling Is a ‘Critical Early Warning System’
According to the CDC NWSS website, there are many advantages to using wastewater surveillance in the fight against COVID-19, including:
Wastewater can capture the presence of the virus shed by people both with and without symptoms.
Health officials can determine if infections are increasing or decreasing within a certain monitoring site.
Wastewater surveillance does not depend on people having access to healthcare or the availability of COVID-19 testing.
It is possible to implement wastewater surveillance in many communities as nearly 80% of the US population are served by municipal wastewater collection systems.
“These built-in advantages can inform important public health decisions, such as where to allocate mobile testing and vaccination sites,” Kirby said. “Public health agencies have also used wastewater data to forecast changes in hospital utilization, providing additional time to mobilize resources and preparation for increasing cases.”
The wastewater sampling represents a critical early warning system for COVID-19 surges and variants, and the CDC hopes this type of sampling and research can be utilized in the future for other infectious diseases.
“Wastewater surveillance can be applicable to a wide variety of health concerns. And so, we are working to expand the National Wastewater Surveillance platform to use it for gathering data on other pathogens, and we expect that work to commence by the end of this year,” Kirby said. “Our targets include antibiotic resistance, foodborne infections like E. Coli, salmonella, norovirus, influenza, and the emerging fungal pathogen Candida Auris.”
Critical Surveillance Tool for Microbiology Laboratories
Independent of the nation’s network of public health laboratories, expansion of this program may give microbiology and clinical laboratories in smaller cities and rural towns an opportunity to test wastewater specimens in support of local wastewater monitoring programs.
As the CDC develops this surveillance network into a more formal program, microbiology labs may find it useful to learn which infectious diseases are showing up in their localities, often days or weeks before any patients test positive for the same infectious agents.
That would give pathologists and clinical laboratory leaders an early warning to be on the alert for positive test results of infectious diseases that wastewater monitoring has confirmed exist in the community.
At that reduced cost, clinical laboratories in developing countries with no access to WGS could have it as a critical tool in their fight against the spread of deadly bacteria and viruses
New research into a low-cost way to sequence bacterial genomes—for as little as $10—is predicted to give public health authorities in low- and middle-income countries (LMICs) a new tool with which to more quickly identify and control disease outbreaks.
This new approach offers an alternative to more expensive Whole genome sequencing (WGS) methodologies, which clinical laboratories in developed countries typically use to identify and track outbreaks of infectious diseases. And with SARS-CoV-2 variants resulting in increased COVID-19 infections, the ability to perform low-cost, rapid, and accurate WGS is becoming increasingly important.
But for many developing countries that need it the most, the cost of WGS has kept this critical technology out of reach.
Now, a global consortium of scientists has successfully established an efficient and inexpensive pipeline for the worldwide collection and sequencing of bacterial genomes. The large-scale sequencing method could potentially provide researchers in LIMCs with tools to sequence large numbers of bacterial and viral pathogens. This discovery also could strengthen research collaborations and help tackle future pandemics.
The team of scientists, led by researchers at the Earlham Institute and the University of Liverpool, both located in the UK, are confident their technology can be made accessible to clinical laboratories in LMICs around the globe.
The international team of scientists aimed their innovative WGS approach at streamlining the collection and sequencing of bacterial isolates (variants). The researchers collected more than 10,400 clinical and environmental bacterial isolates from several LMICs in less than a year. They optimized their sample logistics pipeline by transporting the bacterial isolates as thermolysates from other countries to the UK. Those isolates were sequenced using a low cost, low input automated method for rapid WGS. They then performed the gene library construction and DNA sequencing analysis for a total reagent cost of less than $10 per genome.
The scientists focused their research on Salmonella enterica, a pathogen that causes infections and deadly diseases in human populations. Non-typhoidal Salmonella (NTS) have been associated with enterocolitis, a zoonotic disease in humans linked to industrial food production.
Because the disease is common in humans, there have been more genome sequences generated for Salmonella than any other type of germ.
“In recent years, new lineages of NTS serovars Typhimurium and Enteritidis have been recognized as common causes of invasive bloodstream infections (iNTS disease), responsible for about 77,000 deaths per year worldwide,” the researchers wrote in their Gen Biology paper. “Approximately 80% of deaths due to iNTS disease occurs in sub-Saharan Africa, where iNTS disease has become endemic.”
Increasing Access to Genomics Technologies in Developing Countries
The research consortium 10,000 Salmonella Genomes Project (10KSG) led the large-scale WGS initiative. The alliance involves contributors from 25 institutions in 16 countries and was designed to generate information relevant to the epidemiology, drug resistance, and virulence factors of Salmonella using WGS techniques.
“One of the most significant challenges facing public health researchers in LMICs is access to state-of-the-art technology, Jay Hinton, PhD, Professor of Microbial Pathogenesis at the University of Liverpool and one of the paper’s authors, told Technology Networks. “For a combination of logistical and economic reasons, the regions associated with the greatest burden of severe bacterial disease have not benefited from widespread availability of WGS. The 10,000 Salmonella genomes project was designed to begin to address this inequality.”
The authors noted in their study that the costs associated with sequencing have remained high mostly due to sample transportation and library construction and the fact that there are only a few centers in the world that have the ability to handle large-scale bacterial genome projects.
“Limited funding resources led us to design a genomic approach that ensured accurate sample tracking and captured comprehensive metadata for individual bacterial isolates, while keeping costs to a minimum for the Consortium,” Hall told Genetic Engineering and Biotechnology News(GEN). “The pipeline streamlined the large-scale collection and sequencing of samples from LMICs.”
“The number of publicly available sequenced Salmonella genomes reached 350,000 in 2021 and are available from several online repositories,” he added. “However, limited genome-based surveillance of Salmonella infections has been done in LMICs, and the existing dataset did not accurately represent the Salmonella pathogens that are currently causing disease across the world.”
The $10 cost is designed to help healthcare systems in developing countries identify the specific genetic composition of infectious diseases. That’s the necessary first step for developing a diagnostic test that enables physicians to make an accurate diagnosis and initiate appropriate therapy.
“The adoption of large-scale genome sequencing and analysis of bacterial pathogens will be an enormous asset to public health and surveillance in LMI countries,” molecular microbiologist Blanca Perez Sepulveda, PhD, told GEN. Sepulveda is a postdoctoral Researcher at the University of Liverpool and one of the authors of the study.
Improvement in next-generation sequencing technology has reduced costs, shortened turnaround time (TAT), and improved accuracy of whole genome sequencing. Once this low-cost method for collecting and transporting bacterial sequences becomes widely available, clinical laboratories in developing countries may be able to adopt it for genome analysis of different strains and variants of bacteria and viruses.
CRISPR-Act 3.0 could significantly increase crop yields and plant diversity worldwide and help fight against global hunger and climate change
Clinical laboratory professionals and pathologists who read Dark Daily are highly aware of CRISPR gene editing technology. We’ve covered the topic in multiple ebriefings over many years. But how many know there’s a version of CRISPR specifically designed for editing and activating plant genes?
Scientists at the University of Maryland (UMD) developed a new version of CRISPRa (CRISPR Activation) for plants which they claim has four to six times the activation capacity of currently available CRISPRa systems and can activate up to seven genes at once. They call their new and improved CRISPRa technology “CRISPR-Act 3.0.”
According to a paper published in the journal Nature Plants, titled, “CRISPR-Act3.0 for Highly Efficient Multiplexed Gene Activation in Plants,” the UMD researchers developed “a highly robust CRISPRa system working in rice, Arabidopsis (rockcress), and tomato, CRISPR-Act 3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenes Cas9 (dSpCas9).
CRISPR-Act 3.0 Increases Function of Multiple Genes Simultaneously
The UMD researchers successfully applied CRISPR-Act 3.0 technology to activate many types of genes in plants, including the ability to expedite the breeding process via faster flowering. They hope that activating genes in plants to improve functionality will result in better plants and crops.
“Through activation, you can really uplift pathways or enhance existing capacity, even achieve a novel function. Instead of shutting things down, you can take advantage of the functionality already there in the genome and enhance what you know is useful,” said Yiping Qi, PhD, associate professor, Department of Plant Science and Landscape Architecture at the University of Maryland, in a UMD new release.
The scientists also noted that there may be other advantages to this type of multiplexed activation of genes.
“Having a much more streamlined process for multiplexed activation can provide significant breakthroughs. For example, we look forward to using this technology to screen the genome more effectively and efficiently for genes that can help in the fight against climate change and global hunger,” Qi added. “We can design, tailor, and track gene activation with this new system on a larger scale to screen for genes of importance, and that will be very enabling for discovery and translational science in plants.”
The researchers hope this technology can have a major impact on the efficiency of crop and food production.
“This type of technology helps increase crop yield and sustainably feed a growing population in a changing world,” Qi said. “I am very pleased to continue to expand the impacts of CRISPR technologies.”
Feeding the World’s Hungry with CRISPR
CRISPR is a robust tool used for editing genomes that typically operates as “molecular scissors” to cut DNA. CRISPR-Act 3.0, however, uses deactivated CRISPR-Cas9 which can only bind and not cut. This allows the system to work on the activation of proteins for designated genes of interest by binding to certain segments of DNA. The UMD researchers believe there is significant potential for expanding the multiplexed activation further, which could alter and improve genome engineering.
“People always talk about how individuals have potential if you can nurture and promote their natural talents,” Qi said in the UMD news release. “This technology is exciting to me because we are promoting the same thing in plants—how can you promote their potential to help plants do more with their natural capabilities? That is what multiplexed gene activation can do, and it gives us so many new opportunities for crop breeding and enhancement.”
CRISPR is being developed and enhanced in many research settings, and knowledge of how to best use the gene editing technology is rapidly advancing. Though more research on CRISPR-Act 3.0 is needed to ensure its reliability, it’s exciting to consider the potential of gene activation for massively increasing crop yield worldwide.
Not to mention how new CRISPR technologies continue to drive innovations in clinical laboratory diagnostics and precision medicine treatments.
This “Virus Trap” might eventually be manufactured by clinical laboratories for the diagnostic process
Clinical laboratory managers and pathologists will be fascinated by this new treatment coming out of Germany for viral infections. It’s an entirely different technology approach to locating and neutralizing live viruses that may eventually be able to control anti-viral-resistant strains of specific viruses as well.
As virologists and microbiologists are aware, even in our present era of technological and medical advances, viral infections are extremely difficult to treat. There are currently no effective antidotes against most viral infections and antibiotics are only successful in fighting bacterial infections.
Thus, this new technology developed by a research team at the Technical University of Munich (TUM) in Munich, Germany, that uses DNA origami to neutralize and trap viruses and render them harmless is sure to gain swift attention, especially given the world’s battle with the SARS-CoV-2 Omicron variant.
DNA origami is the nanoscale folding of DNA to create two- and three-dimensional complex shapes that can be manufactured with a high degree of precision at the nanoscale. Researchers have been working with and enhancing this technique for about 15 years.
However, scientists at TUM wondered if they could create such hollow structures based on the capsules that encompass viruses to entrap those viruses. They developed a method that made it possible to create artificial hollow bodies the size of a virus and explored using those hollow bodies as a type of “virus trap.”
The researchers theorized that if those hollow bodies could be lined on the inside with virus-binding molecules, they could tightly bind the viruses and remove them from circulation. For this method to be successful, however, those hollow bodies had to have large enough openings to ensure the viruses could get into the shells.
“None of the objects that we had built using DNA origami technology at that time would have been able to engulf a whole virus—they were simply too small,” said Hendrik Dietz, PhD, Professor of Physics at TUM and an author of the study in a press release. “Building stable hollow bodies of this size was a huge challenge,” he added.
So, the team of researchers used the icosahedron geometric shape, which is an object comprised of 20 sides. They engineered the hollow bodies for their virus trap from three-dimensional, triangular plates which had to have slightly beveled edges to ensure the binding points would assemble properly to the desired objects.
“In this way, we can now program the shape and size of the desired objects using the exact shape of the triangular plates,” Dietz explained. “We can now produce objects with up to 180 subunits and achieve yields of up to 95%. The route there was, however, quite rocky, with many iterations.”
By varying the binding points on the edges of the triangles, the scientists were able to create closed hollow spheres and spheres with openings or half-shells that could be utilized as virus traps. They successfully tested their virus traps on adeno-associated viruses (AAV) and hepatitis B viruses in cell cultures.
“Even a simple half-shell of the right size shows a measurable reduction in virus activity,” Dietz stated in the press release. “If we put five binding sites for the virus on the inside—for example suitable antibodies—we can already block the virus by 80%. If we incorporate more, we achieve complete blocking.”
The team irradiated their finished building blocks with ultraviolet (UV) light and then treated the outside with polyethylene glycol and oligolysine. This process prevented the DNA particles from being immediately degraded in body fluids. Those particles were stable in mouse serum for 24 hours. The TUM scientists plan to test their building blocks on living mice soon.
“We are very confident that this material will also be well tolerated by the human body,” Dietz said.
Could Clinical Laboratories Manufacture Components of the Virus Traps?
The researchers noted that the starting materials for their virus traps can be mass produced at a very reasonable cost and may have other uses.
“In addition to the proposed application as a virus trap, our programmable system also creates other opportunities,” Dietz said. “It would also be conceivable to use it as a multivalentantigen carrier for vaccinations, as a DNA or RNA carrier for gene therapy, or as a transport vehicle for drugs.”
There is much research yet to be done on this cutting-edge technology. However, for this therapy to be appropriate for a patient, a specimen of the virus will need to be identified and studied. Then, the DNA origami would be tailored to capture that specific virus. Thus, it’s conceivable that clinical laboratories, if used for the diagnostic step, might also be able to then manufacture the virus trap that is customized to locate, surround, and neutralize that specific virus.