Nanorate sequencing allows researchers to identify changes to individual genetic sequencing letters among millions of DNA letters contained in a single cell
Detecting genetic mutations in cells requires genomic sequencing that, until now, has not been accurate enough to spot minute changes in DNA sequences. Many clinical laboratory scientists know this restricted the ability of genetic scientists to identify cancerous mutations early in individual cells.
Now, researchers at the Wellcome Sanger Institute in the United Kingdom have developed a new method of genetic sequencing that “makes it possible to more accurately investigate how genetic changes occur in human tissues,” according to Genetic Engineering and Biotechnology News (GEN).
This development suggests a new, more sensitive tool may soon be available for anatomic pathologists to speed evaluation of pre-cancerous and cancerous tissues, thereby achieving earlier detection of disease and clinical intervention.
Called Nanorate Sequencing (NanoSeq for short), the new technology enables researchers to detect genetic changes in any human tissues “with unprecedented accuracy,” according to a news release.
How Somatic Mutations Drive Cancer, Aging, and Other Diseases
NanoSeq enables the detection of new mutations in most human cells—the non-dividing cells—GEN explained, calling Wellcome Sangar Institute’s new technology a “breakthrough” in the use of duplex sequencing.
Until now, genomic sequencing has not been “accurate enough” for this level of detection, Sanger stated in the news release. Thus, there was little opportunity to enhance exploration of new mutations in the majority of human cells.
Further, the findings of the Sanger study suggest that cell division may not be the primary cause of somatic mutations (changes in the DNA sequence of a biological cell).
In their paper, the researchers discussed the importance of somatic mutations. “Somatic mutations drive the development of cancer and may contribute to aging and other diseases. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues.
“Here, to overcome these limitations, we developed Nanorate Sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality,” the researchers wrote in Nature.
Refining Duplex Sequencing and Improving PCR Testing
In their study, Sanger researchers assessed duplex sequencing and found errors concentrated at DNA fragment ends. To them, this suggested “flaws” in preparation for DNA sequencing.
Duplex sequencing is an established technique “which sequences both strands of a DNA molecule to remove sequencing and polymerase chain reaction (PCR) errors,” explained a Science Advisory Board article.
Re-evaluating Mutagenesis and Cell Division with NanoSeq
It took the Sanger researchers four years to create NanoSeq. They “carefully refined” duplex sequencing methods using more specific enzymes to aid DNA cutting and bioinformatics analysis, Clinical OMICS noted.
Then, they put NanoSeq’s sensitivity to the test. They wanted to know if its low error rate meant that NanoSeq could enable study of somatic mutations in any tissue. This would be important, they noted, because genetic mutations naturally occur in cells in a range of 15 to 40 mutations per year with some changes leading to cancer.
The scientists compared the rate and pattern of mutation in both stem cells (renewing cells supplying non-dividing cells) and non-dividing cells (the majority of cells) in blood, colon, brain, and muscle tissues.
The Sanger study found:
Mutations in slowly dividing stem cells are on track with progenitor cells, which are more rapidly dividing cells.
Cell division may not be the “dominant process causing mutations in blood cells.”
Analysis of non-dividing neurons and rarely-dividing muscle cells found “mutations accumulate throughout life in cells without cell division and at a similar pace” to blood cells.
“It is often assumed that cell division is the main factor in the occurrence of somatic mutations, with a greater number of divisions creating a greater number of mutations. But our analysis found that blood cells that had divided many times more than others featured the same rates and patterns of mutation. This changes how we think about mutagenesis and suggests that other biological mechanisms besides cell divisions are key,” said Federico Abascal, PhD, First Author and Sanger Postdoctoral Fellow, in the news release.
Using NanoSeq to Scale Up Somatic Mutation Analyses
“NanoSeq will also make it easier, cheaper, and less invasive to study somatic mutation on a much larger scale. Rather than analyzing biopsies from small numbers of patients and only being able to look at stem cells or tumor tissue, now we can study samples from hundreds of patients and observe somatic mutations in any tissue,” said Inigo Martincorena, PhD, Senior Author and Sanger Group Leader, in the news release.
More research is needed before NanoSeq finds its way to diagnosing cancer by anatomic pathology groups. Still, for diagnostics professionals and clinical laboratory leaders, NanoSeq is an interesting development. It appears to be a way for scientists to see genetic changes in single cells and mutations in a handful of cells that evolve into cancerous tumors, as compared to those that do not.
The Sanger scientists plan to pursue larger follow-up NanoSeq studies.
VCU scientists used the technique to measure mutations associated with acute myeloid leukemia, potentially offering an attractive alternative to DNA sequencing
More accurate but less-costly cancer diagnostics are the Holy Grail of cancer research. Now, research scientists at Virginia Commonwealth University (VCU) say they have developed a clinical laboratory diagnostic technique that could be far cheaper and more capable than standard DNA sequencing in diagnosing some diseases. Their method combines digital polymerase chain reaction (dPCR) technology with high-speed atomic force microscopy (HS-AFM) to generate nanoscale-resolution images of DNA.
The technique allows the researchers to measure polymorphisms—variations in gene lengths—that are associated with many cancers and neurological diseases. The VCU scientists say the new technique costs less than $1 to scan each dPCR reaction.
“We chose to focus on FLT3 mutations because they are difficult to [diagnose], and the standard assay is limited in capability,” said physicist Jason Reed, PhD, Assistant Professor in the Virginia Commonwealth University Department of Physics, in a VCU press release.
Reed is an expert in nanotechnology as it relates to biology and medicine. He led a team that included other researchers in VCU’s physics department as well as physicians from VCU Massey Cancer Center and the Department of Internal Medicine at VCU School of Medicine.
Validating the Clinical Laboratory Test
The physicists worked with two VCU physicians—hematologist/oncologist Amir Toor, MD, and hematopathologist Alden Chesney, MD—to compare the imaging technique to the LeukoStrat CDx FLT3 Mutation Assay, which they described as the “current gold standard test” for diagnosing FLT3 gene mutations.
The researchers said their technique matched the results of the LeukoStrat test in diagnosing the mutations. But unlike that test, the new technique also can measure variant allele frequency (VAL). This “can show whether the mutation is inherited and allows the detection of mutations that could potentially be missed by the current test,” states the VCU press release.
“We plan to continue developing and testing this technology in other diseases involving DNA structural mutations,” Reed said. “We hope it can be a powerful and cost-effective tool for doctors around the world treating cancer and other devastating diseases driven by DNA mutations.”
“In our approach we first used digital PCR, in which a mixed sample is diluted to less than one target molecule per aliquot and the aliquots are amplified to yield homogeneous populations of amplicons,” he said. “Then, we deposited each population onto an atomically-flat partitioned surface.”
The VCU researchers “scanned each partition with high-speed atomic force microscopy, in which an extremely sharp tip is rastered across the surface, returning a 3D map of the surface with nanoscale resolution,” he said. “We wrote code that traced the length of each imaged DNA molecule, and the distribution of lengths was used to determine whether the aliquot was a wild type [unmutated] or variant.”
In Diagnostics World, Reed said the method “doesn’t really have any more complexity than a PCR assay itself. It can easily be done by most lab technicians.”
Earlier Research
A VCU press release from 2017 noted that Reed’s research team had developed technology that uses optical lasers (similar to those in a DVD player) to accelerate the scanning. The researchers previously published a study about the technique in Nature Communications, and a patent is currently pending.
“DNA sequencing is a powerful tool, but it is still quite expensive and has several technological and functional limitations that make it difficult to map large areas of the genome efficiently and accurately,” Reed said in the 2017 VCU press release. “Our approach bridges the gap between DNA sequencing and other physical mapping techniques that lack resolution. It can be used as a stand-alone method or it can complement DNA sequencing by reducing complexity and error when piecing together the small bits of genome analyzed during the sequencing process.”
Using CRISPR technology, the team also developed what they described as a “chemical barcoding solution,” placing markers on DNA molecules to identify genetic mutations.
New DNA Clinical Laboratory Testing?
Cancer diagnostics are constantly evolving and improving. It is not clear how long it will be before VCU’s new technique will reach clinical laboratories that perform DNA testing, if at all. But VCU’s new technique is intriguing, and should it prove viable for clinical diagnostic use it could revolutionize cancer diagnosis. It is a development worth watching.
Pathologists and clinical laboratory scientists know that influenza vaccines typically produce short-lived protection and researchers have new clues as to why this is true
With so much interest in development of a COVID-19 vaccine, findings by researchers at Atlanta’s Emory Vaccine Center into why the vaccine for influenza (Flu) is so short-lived offer a new window on how the body’s immune system responds to invading viruses and what happens to the immunity over time.
Because the autumn influenza season is just weeks away, these insights into the body’s immune response to influenza will be of interest to clinical laboratories that provide testing for influenza, as well as SARS-CoV-2, the coronavirus that causes COVID-19.
Clinical laboratory managers recognize that an influenza vaccine is an annual imperative for people—especially the elderly and those with existing comorbidities—and medical laboratory tests are typically used to diagnose the illness and identify which strains of viruses are present. The flu vaccine is even more important amid the COVID-19 pandemic, infectious disease authorities say.
The scientists at the Emory Vaccine Center published their findings in the journal Science.
Not so with influenza vaccines. The immunity they impart generally only lasts for a single flu season and are “lost within one year,” the Emory study notes.
As Genetic Engineering and Biotechnology News (GEN) explains, the influenza genome has eight RNA segments which can change as the virus enters a cell. This antigenic shift creates new influenza strains that require updated vaccines, GEN noted.
However, the Emory researchers stated that “The fact that a small number did persist over one year raises prospects that the longevity of flu vaccines can be improved and provides key information for the development of universal vaccines against influenza.”
Bone Marrow Has Major Role in Producing New Flu Antibodies
The Emory study focused on the influenza vaccine’s role in how it affects the immune system and what needs to change to create a longer-lasting influenza vaccine. “Our results suggest that most bone marrow plasma cells (BMPC) generated by influenza vaccination in adults are short-lived. Designing strategies to enhance their persistence will be key,” the Emory researchers wrote in Science.
The scientists analyzed bone marrow from 53 healthy volunteers (age 20 to 45). An Emory news release states that bone marrow is the “home base for immune cells producing antibodies.”
Besides the bone marrow, the researchers also examined blood samples from the volunteers, all of which was collected between 2009 and 2018:
before influenza vaccination,
one month after influenza vaccination, and
one year post vaccination.
Through DNA sequencing the samples, the Emory researchers found the number of flu-specific cells increased from 0.8% to 1.9% after one month. They concluded that an annual vaccine does increase antibody-producing cells for influenza in bone marrow.
However, in follow-up visits one year after vaccination, they found that the number of cells present in the volunteers had fallen back to the starting point.
“Specific cells produced by the vaccine … produced unique antibodies that can be identified using sequencing techniques,” Carl Davis, PhD, postdoctoral fellow in the Rafi Ahmed Laboratory at Emory and first author of the paper, said in the news release, adding, “We could see that these new antibodies expanded in the bone marrow one month after vaccination and then contracted after one year.”
He continued, “On the other hand, antibodies against influenza that were in the bone marrow before the vaccine was given stayed at a constant level over one year.”
Vaccine Adjuvants Help Boost Immunity
A vaccine additive called an adjuvant could be the answer to extending the power of influenza vaccines, the Emory scientists noted.
“Just getting to the bone marrow is not enough. A plasma cell has to find a niche within the bone marrow and establish itself there and undergo gene expression and metabolism changes that promote longevity,” Rafi Ahmed, PhD, Director of the Emory Vaccine Center, said in the news release.
“It’s totally crazy (that the most commonly used influenza vaccines don’t include an adjuvant), Ahmed told Science. “I’m hoping that things will change in the influenza vaccine world, and 10 years from now, you will not be getting any nonadjuvanted vaccines.”
Are Adjuvants the Answer for COVID-19 Vaccines?
According to USA Today, about 20-million “essential” workers will likely be the first to receive the new COVID-19 vaccine and participate in check-in text messages with the Centers for Disease Control and Prevention (CDC) by the end of 2020.
In its COVID-19 vaccine testing, Novavax, a late-state biotechnology company, suggests that “an adjuvant is critical to its vaccine working well,” National Public Radio (NPR) reported in “The Special Sauce That Makes Some Vaccines Work.” However, vaccine developers may be reluctant to share their adjuvant research.
“Adjuvants end up being very proprietary. It’s kind of the secret sauce on how to make your protein vaccine work,” Barney Graham, MD, PhD, Deputy Director, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, told NPR.
Still, a study published in Immunopharmacology revealed potential adjuvants for the COVID-19 vaccine based on vaccine studies of other coronaviruses. While there are many adjuvants available, not all have safety track records that can be leveraged to gain clearance from regulatory bodies, the researchers pointed out. But some do.
“CpG 1018, MF59, and AS03 are already approved for human vaccine and their inclusion may expedite the vaccine development process. Further, Protollin has shown promising results in pre-clinical studies,” the authors wrote.
Clinical laboratories that provide influenza testing will want to follow these types of research studies. Findings on immunity will affect development of vaccines that medical labs provide—including for COVID-19.
Many other healthcare systems also are partnering with private genetic testing companies to pursue research that drive precision medicine goals
It is certainly unusual when a major health network announces that it will give away free genetic tests to 10,000 of its patients as a way to lay the foundation to expand clinical services involving precision medicine. However, pathologists and clinical laboratory managers should consider this free genetic testing program to be the latest marketplace sign that acceptance of genetic medicine continues to move ahead.
Notably, it is community hospitals that are launching this
new program linked to clinical laboratory research that uses genetic tests for
specific, treatable conditions. The purpose of such genetic research is to
identify patients who would benefit from test results that identify the best
therapies for their specific conditions, a core goal of precision medicine.
Clinical laboratory leaders will be interested in this
initiative, as well other partnerships between healthcare systems and private
genetic testing companies aimed at identifying and enrolling patients in
research studies for disease treatment protocols and therapies.
The Future of Precision Medicine
Modern Healthcare reported that data from the WholeMe DNA study, which was funded through donations to the AdventHealth Foundation, also will be used by the healthcare network for research beyond FH, as AdventHealth develops its genomics services. The project’s cost is estimated to reach $2 million.
“Genomics is the future of medicine, and the field is rapidly evolving. As we began our internal discussions about genomics and how to best incorporate it at AdventHealth, we knew research would play a strong role,” Wes Walker MD, Director, Genomics and Personalized Health, and Associate CMIO at AdventHealth, told Becker’s Hospital Review.
“We decided to focus on familial hypercholesterolemia
screening initially because it’s a condition that is associated with
life-threatening cardiovascular events,” he continued. “FH is treatable once
identified and finding those who have the condition can lead to identifying
other family members who are subsequently identified who never knew they had
the disease.”
The AdventHealth Orlando website states that participants in the WholeMe study receive information stored in a confidential data repository that meets HIPAA security standards. The data covers ancestry and 22 other genetic traits, such as:
Asparagus Odor Detection
Bitter Taste
Caffeine Metabolism
Cilantro Taste Aversion
Circadian Rhythm
Coffee Consumption
Delayed Sleep
Earwax Type
Endurance vs Power
Exercise Impact on Weight
Eye Color
Freckling
Hair Curl and Texture
Hand Grip Strength
Height
Lactose Tolerance
Sleep Duration
Sleep Movement
Sleeplessness
Sweet Tooth
Tan vs. Sunburn
Waist Size
Those who test positive for a disease-causing FH variant will be referred by AdventHealth for medical laboratory blood testing, genetic counseling, and a cardiologist visit, reported the Ormond Beach Observer.
One in 250 people have FH, and 90% of them are undiagnosed,
according to the FH Foundation,
which also noted that children have a 50% chance of inheriting FH from parents
with the condition.
AdventHealth plans to expand the free testing beyond central
Florida to its 46 other hospitals located in nine states, Modern Healthcare
noted.
Other Genetics Data Company/Healthcare Provider Partnerships
Business Insider noted that Helix has focused on clinical partnerships for about a year and seems to be filling a niche in the genetic testing market.
“Helix is able to sidestep the costs of direct-to-consumer
marketing and clinical test development, while still expanding its customer
base through predefined hospital networks. And the company is in a prime
position to capitalize on providers’ interest in population health management,”
Business Insider reported.
Ochsner’s program is the first “fully digital population
health program” aimed at including clinical genomics data in primary care in an
effort to affect patients’ health, FierceHealthcare
reported.
Hereditary breast and ovarian cancer due to
mutations in BRCA1 and BRCA2 genes;
Lynch
syndrome, associated with colorectal and other cancers; and
FH.
Color also offers genetic testing and whole genome sequencing services to NorthShore’s DNA10K program, which plans to test 10,000 patients for risk for hereditary cancers and heart diseases, according to news release.
And, Jefferson Health offered Color’s genetic testing to the healthcare system’s 33,000 employees, 10,000 of which signed up to learn their health risks as well as ancestry, a Color blog post states.
“Understanding the genome warning signals of every patient will be an essential part of wellness planning and health management,” said Geisinger Chief Executive Officer David Feinberg, MD, when he announced the new initiative at the HLTH (Health) Conference in Las Vegas. “Geisinger patients will be able to work with their family physician to modify their lifestyle and minimize risks that may be revealed,” he explained. “This forecasting will allow us to provide truly anticipatory healthcare instead of the responsive sick care that has long been the industry default across the nation.”
It will be interesting to see how and if genetic tests—free
or otherwise—will advance precision medicine goals and population health
treatments. It’s important for medical laboratory leaders to be involved in health
network agreements with genetic testing companies. And clinical laboratories should
be informed whenever private companies share their test results data with
patients and primary care providers.
By offering DTC preventative gene sequencing, hospital leaders
hope to help physicians better predict cancer risk and provide more accurate
diagnoses
Two Boston health systems, Brigham and Women’s Hospital and Massachusetts General Hospital (MGH), are the latest to open preventative gene sequencing clinics and compete with consumer gene sequencing companies, such as 23andMe and Ancestry, as well as with other hospital systems that already provide similar services.
This may provide opportunities for clinical laboratories. However, some experts are concerned that genetic sequencing may not be equally available to patients of all socioeconomic classes. Nor is it clear how health systems plan to pay for the equipment and services, since health insurance companies continue to deny coverage for “elective” gene sequencing, or when there is not a “clear medical reason for it, such as for people with a long family history of cancer,” notes STAT.
Therefore, not everyone is convinced of the value of gene sequencing to either patients or hospitals, even though advocates tout gene sequencing as a key element of precision medicine.
Is Preventative Genetic Sequencing Ready for the Masses?
Brigham’s Preventive Genomics Clinic offers comprehensive DNA sequencing, interpretation, and risk reporting to both adults and children. And MGH “plans to launch its own clinic for adults that will offer elective sequencing at a similar price range as the Brigham,” STAT reported.
The Brigham and MGH already offer similar gene sequencing services as other large health systems, such as Mayo Clinic and University of California San Francisco (UCSF), which are primarily used for research and cancer diagnoses and range in price depending on the depth of the scan, interpretation of the results, and storage options.
However, some experts question whether offering the
technology to consumers for preventative purposes will benefit anyone other
than a small percentage of patients.
“It’s clearly not been demonstrated to be cost-effective to promote this on a societal basis,” Robert Green, MD, MPH, medical geneticist at Brigham and Women’s Hospital, and professor of genetics at Harvard, told STAT. “The question that’s hard to answer is whether there are long-term benefits that justify those healthcare costs—whether the sequencing itself, the physician visit, and any downstream testing that’s stimulated will be justified by the situations where you can find and prevent disease.”
Additionally, large medical centers typically charge more
for genomic scans than consumer companies such as 23andMe and Ancestry. Hospital-based
sequencing may be out of the reach of many consumers, and this concerns some
experts.
“The idea that genomic sequencing is only going to be
accessible by wealthy, well-educated patrons who can pay out of pocket is
anathema to the goals of the publicly funded Human Genome Project,” Jonathan
Berg, MD, PhD, Genetics Professor, University of North Carolina at Chapel
Hill, told Scientific
American.
And, according to the American Journal of Managed Care, “It’s estimated that by 2021, 100 million people will have used a direct-to-consumer (DTC) genetic test. As these tests continue to gain popularity, there is a need for educating consumers on their DTC testing results and validating these results with confirmatory testing in a medical-grade laboratory.”
This is why it’s critical that clinical laboratories and
anatomic pathology groups have a genetic testing and gene sequencing strategy,
as Dark
Daily reported.
David Bick, MD, Chief Medical Officer at the HudsonAlpha Institute for Biotechnology and Medical Director of the Smith Family Clinic for Genomic Medicine, told Scientific American, “there’s just more and more interest from patients and families not only because of 23andMe and the like, but because there’s just this understanding that if you can find out information about your health before you become sick, then really our opportunity as physicians to do something to help you is much greater.”
Is Preventative Genomics Elitist?
As large medical centers penetrate the consumer genetic
testing market some experts express concerns. In a paper he wrote for Medium,
titled, “Is Preventive Genomics Elitist?” Green asked, “Is a service like this
further widening the inequities in our healthcare system?”
Green reported that while building the Preventive Genomics Clinic at Brigham, “we … struggled with the reality that there is no health insurance coverage for preventive genomic testing, and our patients must therefore pay out of pocket. This is a troubling feature for a clinic at Brigham and Women’s Hospital, which is known for its ties to communities in Boston with diverse ethnic and socioeconomic backgrounds.”
Most of Brigham’s early genetics patients would likely be “well-off,
well-educated, and largely white,” Green wrote. “This represents the profile of
typical early adopters in genetic medicine, and in technology writ large. It
does not, however, represent the Clinic’s ultimate target audience.”
More Data for Clinical Laboratories
Nevertheless, preventive genomics programs offered by large
health systems will likely grow as primary care doctors and others see evidence
of value.
Therefore, medical laboratories that process genetic
sequencing data may soon be working with growing data sets as more people reach
out to healthcare systems for comprehensive DNA sequencing and reporting.