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Clinical Laboratories and Pathology Groups

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Researchers at Wellcome Sanger Institute Develop New Tool to Analyze Genetic Changes and Role of Cell Division in Human Tissue

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.

The Wellcome Sanger Institute researchers published their findings in the journal Nature, titled, “Somatic Mutation Landscapes at Single-Molecule Resolution.”

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.

Robert Osborne, PhD

“Detecting somatic mutations that are only present in one or a few cells is incredibly technically challenging. You have to find a single letter change among tens of millions of DNA letters and previous sequencing methods were simply not accurate enough,” said Robert Osborne, PhD (above), former Principal Staff Scientist at Sanger who led development of NanoSeq, in the news release. Osborne is now COO of Biofidelity, a cancer diagnostics developer in Cambridge, United Kingdom. This research may eventually give clinical laboratories and surgical pathologists useful new tools that enable earlier, more accurate diagnosis of cancer. (Photo copyright: Cambridge Independent.)

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.

—Donna Marie Pocius

Related Information:

NANOSEQ: Nanorate Sequencing, Ultra-Accurate Detection of Somatic Mutations

Major Advance Enables Study of Genetic Mutations in Any Tissue

NanoSeq Technique Improved to Detect New Non-Dividing Cell Mutations

Somatic Mutation Landscape at Single-Molecule Resolution

New Method Allows for Study of Genetic Changes in Individual DNA Molecules

Sanger Institute Improves NanoSeq Method to Detect New Mutations in Non-Dividing Cells

Wellcome Sanger Institute’s NanoSeq Sequencing Breakthrough Enables Study of DNA Mutations from Any Human Tissue

University of Queensland Researches May Have Found a Universal Biomarker That Identifies Cancer in Various Human Cells in Just 10 Minutes!

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.

Such a diagnostic test would be a boon to clinical laboratories and anatomic pathology groups involved in cancer diagnosis and the development of precision medicine treatments.

One Test, 90% Accuracy, Many Cancers

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.

Methylation are marks that indicate whether pieces of DNA should be read,” Dino DiCarlo, PhD, Professor in the Department of Bioengineering and Biomedical Engineering, University of California Los Angeles (UCLA) and Director of Cancer Nanotechnology at UCLA’s Jonsson Comprehensive Cancer Center, told USA Today.


“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.  

—Donna Marie Pocius

Related Information:

Nano-Signature Discovery Could Revolutionize Cancer Diagnosis

Epigentically Reprogrammed Methylation Landscape Drives the DNA Self-Assembly and Serves as a Universal Cancer Biomarker

One Test to Diagnose Them All: Researchers Exploit Cancers’ Unique DNA Signature

Cancer Researchers in Australia Develop Universal Blood Test

Universal 10-Minute Cancer Test in Sight

A 10-Minute, Universal Blood Test for Cancer

Sound Wave Acoustic Tweezers Locate and Isolate Circulating Tumor Cells in Liquid Biopsies; Could Lead to Less Invasive Cancer Diagnostics and Treatments

Pathologists will be interested to learn that this latest version of the acoustic tweezer device requires about five hours to identify the CTCs in a sample of blood

Medical laboratory leaders and pathologists are well aware that circulating tumor cells (CTCs) released by primary tumors into the bloodstream are fragile and easily damaged. Many studies have sought to find ways to separate CTCs from surrounding cells. Such a process could then be used as an early-detection biomarker to detect cancer from a sample of blood.

One team of researchers believe it has a way to accomplish this. These researchers are using sound waves to gently detect and isolate CTCs in blood samples. In turn, this could make it possible to diagnose cancer using “liquid biopsies” as opposed to invasive conventional biopsies.

Researchers from Carnegie Mellon University (CMU) in collaboration with researchers from the Massachusetts Institute of Technology (MIT) and Pennsylvania State University (Penn State) have developed a method for using acoustic tweezers and sound waves to separate blood-borne cancer cells from white blood cells. The research team believes this new device could one day replace invasive biopsies, according to a CMU article. (more…)

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