Keys to Better Liquid Biopsy Assay Sensitivity

“So as everyone here is aware, I’m sure, detection of circulating tumor DNA is challenging. There’s very little of it, to start with.” Hardly a revolutionary statement by Tony Godfrey, PhD, (Associate Chair, Surgical Research and Associate Professor of Surgery, Boston University School of Medicine) but an important acknowledgement from a leading expert of the difficulty faced by laboratorians in unlocking the full promise of liquid biopsy assays. Both he and Bob Daber, PhD, (President and CTO of Genosity) detailed how they overcame common ctDNA challenges in their labs during SeraCare’s AMP Corporate Workshop in San Antonio, Texas. Watch the video to see the practical advice they gave in their presentations.

Bob Daber, with whom we collaborated on our NGS-based assay validation eBook, discussed the challenges that are unique or more pronounced in ctDNA assay validation. Chief among them being limited access to samples for the variety of studies needed to have true confidence in your assay. Drawing on his years of clinical genomics and bioinformatics expertise, including building the tumor sequencing lab at BioReference, Dr Daber talked about how ground-truth data from known-negative and known-positive materials are critical to determining your assay’s sensitivity and specificity; two attributes that take on even greater importance in liquid biopsy.

Tony Godfrey’s presentation highlighted how access to patient-like ctDNA reference standards allowed his team to refine their SiMSen-Seq technology by reducing background error rates, evaluating absolute copy numbers, and improving sensitivity. Dr Godfrey talked about how his team was able to confidently detect  0.1% mutant allele frequency, and how ground-truth reference materials help them improve performance. Something that wouldn’t have been possible with the inherent variability and scarcity of remnant specimens.

Both presentations are full of actionable information and instructive data from the speakers’ own labs. Watch the video for free today to learn how you can have more confidence in your liquid biopsy assay.

 

This blog post was originally published on the SeraCare blog.

Every DNA counts – and we would know

The National Measurement Laboratory at LGC turned 30 years old this year, and to celebrate we’ve been looking back at notable accomplishments, and looking at where we are now. Clinical measurement is one field where our scientists have excelled and innovated throughout our time.

biology-clinic-doctor-4154Clinical measurement “is the development, use, on-going support, and maintenance of technology for diagnosing, aiding or treating patients.” Modern medicine wouldn’t be possible if we couldn’t rely on the accuracy of clinical tests and diagnosis. Poor measurement can lead to misdiagnosis, incorrect prescription and dosage of medicine, or false interpretation of data. Therefore, reliable certified reference materials are absolutely necessary to ensure the quality and accuracy of clinical measurement.

Throughout the last 30 years, the National Measurement Laboratory (NML) at LGC has worked in this area to ensure that testing methods and reference materials are of the highest quality.

In one case study from 2006¹, scientists in the NML developed isotope dilution liquid chromatography-mass spectrometry (IDMS) methodologies that were then used to generate reference values for clinical reference materials (CRM), some of which led to the analysis of creatine in frozen serum and testosterone in frozen serum CRMs.

In another blog post, we outlined the work we’ve done to improve Alzheimer’s diagnosis, which could lead to techniques for earlier diagnosis of the disease, and in another, we illustrate the importance of harmonising newborn blood sport screening tests to ensure infants are diagnosed and treated early so that they can live as normal lives as possible.

An important part of working in the field of clinical medicine and measurement is communicating our knowledge with other scientists and medical professionals to ensure that good measurement is being performed consistently across the board. We have worked with the NHS and England’s Chief Scientific Officer Sue Hill on doing just that as part of the Knowledge Transfer Partnership Programme, which aims to improve patient care through new approaches to measurement.

And now, our scientists can even count DNA and measure changes to that DNA over time. Identification and targeting of specific genetic sequences forms the basis of many promising advanced healthcare solutions such as: precision (personalised) medicine in cancer, gene therapies to end genetic disorders in children and the detection of pathogenic and non-pathogenic bacteria in a wide spectrum of infectious and autoimmune diseases.

However, the new methods and technologies currently being developed will only achieve their full potential if we can ensure they are safe and can be reproduced. High accuracy reference methods are one of the key factors in supporting their development into routine application.

Using tests for guiding treatment of colorectal cancer as a model, our scienists outlined in a paper published in Clinical Chemistry how a range of dPCR assays and platforms compared and how precisely they measured the cancer mutation. An inter-laboratory study of clinical and National Measurement Institute laboratories demonstrated reproducibility of the selected method. Together these results reveal the unprecedented accuracy of dPCR for copy number concentration of a frequently occurring gene mutation used to decide on drug treatment.

This study has shown that using high-accuracy dPCR measurements can support the traceable standardisation, translation and implementation of molecular diagnostic procedures that will advance precision medicine.

All of this just goes to show you how far we’ve come in 30 years!

¹VAM Bulletin, Issue 35, Autumn 2006, pp 13. ‘Case Study 3: IDMS certification of clinical reference materials using LC-MS/MS”

Delivering impact to support AIDS research

LGC is helping to ensure that research into a cure for HIV is based on sound fundamental measurements.

Over 36 million people currently live with HIV, with approximately 2 million becoming infected each year (WHO 2015). Although HIV can be successfully managed with combination antiretroviral therapy (cART), the therapy must be continued indefinitely as no cure presently exists. This can be challenging in regions with high HIV prevalence and long-term use can potentially have toxic side effects.

One barrier to curing HIV is the presence of infected host cells that are not targeted by current therapies but lay dormant (so-called ‘viral reservoir’). These cells have the potential to become re-activated so novel strategies to cure HIV aim to target this reservoir. To determine whether these new approaches are successful, accurate and robust, methods for measuring HIV DNA are required.

The Molecular and Cell Biology team at LGC perform research to support accurate and reliable measurement as part of our National Measurement Laboratory (NML) role. Recent work by NML scientists comparing different molecular methods (qPCR, digital PCR) for quantification of HIV DNA has raised some concerns around the current popular choice of calibrator used to compare results between HIV clinical studies (8E5, ATCC® CRL-8993). It appears to lose HIV DNA copies during cell growth, potentially producing misleading estimates of how much HIV DNA is present and affecting whether novel strategies towards curing HIV are deemed successful or not.

Based in part on our work, the NIH AIDS Reagent Program, which provides critical reagents and resources to support research in the areas of AIDS therapeutics and vaccine development, has recently highlighted the potential instability of the standard on its reagent database to support the research community and enable the best chances of success.

 

 

Citation:

Busby E et al. Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR (2017) Sci Rep 7(1):1209. doi:10.1038/s41598-017-01221-5

Rediscovering the lost soldiers of Fromelles

The Battle of Fromelles took place on 19-20 July 1916, and is still known as “the worst 24 hours in Australian history,” as 5,533 Australian soldiers lost their lives in the battle. Most of the men were reinterred after the war, but in 2006, the remains of two hundred and fifty soldiers were discovered in unmarked graves near Fromelles.

In 2009, our specialist DNA team began to work with the Australian and British governments, as well as the Commonwealth War Graves Commission to help identify these soldiers using the most current DNA methods. Using DNA extracted from remains, like teeth, we can compare samples from the remains to living relatives of World War I soldiers with a combination of mitochondrial and Y-chromosome DNA.

Earlier this week it was announced that nine more Australian soldiers who had fallen at Fromelles have been identified using advanced DNA analysis. This is a huge moment for the families of the men and all those involved in the work for the last nine years.

Vic Moore, one of our DNA experts, has worked on the project since the remains were excavated. She said, “This year we have been able to provide closure for nine different families, bringing the total number of soldiers identified at Fromelles to 159 from the 250 soldiers originally recovered.”

The battle, discovery and research is a fascinating story which also caught the attention of playwright and author Lynn Brittney, who wrote ‘Dig for the Diggers’ in 2010, just after the work had begun. The play recounts the story of the fictional Mick Feeney while two forensic scientists examine his bones.

“I got the idea for ‘Dig for the Diggers’ after reading a piece in the paper about the War Graves Commission finding more bodies and the DNA testing started on the Australian relatives. I thought it was so fascinating and then I read up about the Battle of Fromelles and was so appalled by the disaster that I felt I had to write something about it,” said Lynn. “I am deeply impressed that the scientists in your organisation were involved in the forensic work on the soldiers from the battle.”

After researching the battle and the present day research, Lynn was moved by the story of the brave men who were getting their names and identities back. She explained, “I chose to write about the ‘ghost’ of the first body to be disinterred and how he viewed what was happening to his remains. It told the story of Australia’s involvement through his eyes and how ‘the worst 24 hours in Australia’s history’ panned out.”

The play has been performed extensively in Australia and was even performed last week as part of this year’s One Act Play Festival as drama groups commemorate the First World War. Director Christine Mace, whose group The Athelstan Players performed the play last week, called it “very emotional and moving”.

Captain Kenneth Mortimer, one of the nine soldiers identified. Image via Australian War Memorial

It’s easy to imagine that the nine men who have been identified had much in common with the play’s fictional protagonist. One of the men, Alexander McCulloch was 35 years old at the time of his death, while Captain Kenneth Mortimer was only 20 years old.

The men will be honoured at a commemoration ceremony this July on the 102nd anniversary of the battle, and new headstones will replace the old anonymous ones, marking their identities for the first time in over a century.

“Even after 102 years, being able to provide a name to an unknown grave can have a massive impact to the families, as it allows them to finally know what happened to their loved ones, and know where their final resting place lies,” reflected Vic.

Work continues to identify the remaining 91 soldiers with the aim that one day, each of the recovered diggers will be laid to rest. Read more about the identities of the soldiers here.

Our top 5 favourite scientific breakthroughs in history

British Science Week kicked off on the 9th March and this year’s theme is ‘Exploration & Discovery’, which encompasses the spirit of scientific enquiry. The week is a ten day celebration of science, technology, engineering and maths.

As humans, we love to celebrate big moments in history and retell stories that help us understand our own story. Famous thinkers often become legends who attain ‘larger than life’ status. But it’s important to remember that our heroes of science pursued science every day and dedicated themselves to their work. Innovations are often accomplished over the course of lifetimes with the help of many scientists.

We are constantly building on the knowledge of the past to take science into the future, and it’s exciting to think that we could each play a part in something big. After all, there are often just a few steps between ‘business as usual’ and ‘making history’. So keep up the good work!

To celebrate the spirit of exploration and discovery, here’s a look at our top five favourite scientific breakthroughs:

Genomics/DNA: While the term ‘genomics’ was only coined in 1986, by geneticist Tom Roderick, the actual study of the human genome is more extensive than that. A genome is defined as all the genetic information of an organism, and therefore genomics, the study of the complete genetic material of these organisms.

Gregor Mendel

Selective breeding has been practiced for thousands of years, but it wasn’t until the Augustinian friar Gregor Mendel undertook his studies in the mid-19th century that modern genetics as we know it was born. Do you remember practicing Mendel’s laws in school, determining traits in offspring based on dominant and recessive traits? It was the most fun to be had in biology.

Later, British Nobel Prize-winners James D. Watson and Francis Crick published the discovery of the helical structure of DNA, based on work done by Rosalind Franklin and Raymond Gosling, and then molecular biologists began to sequence nucleic acids. By 2001, the Human Genome Project completed a rough draft of the human genome, a feat which is being replicated with the 1000 Genomes Project. Now, scientists are using genomics to forge the way forward in personalised medicine, conservation, synthetic biology and gene editing. This all within the relatively short space of 150 years!

Domestication of plants & fermentation: Perhaps not a ‘discovery’, the domestication of plants definitely changed the course of human history, allowing populations to settle and grow. Plant domestication first occurred about 10,000 years ago in the Middle East. This change from hunter-gatherer societies to agricultural societies is largely seen as the beginning of the rise of civilisation.

Often, crops would go bad before they could be consumed, so in order to make the yields last longer and feed more, humans began to use a chemical process called fermentation in the Neolithic Age. This process converts sugars and carbohydrates to acids, gases or alcohol, and it was used to preserve food and beverages. Some of our favourite food and drinks were invented thanks to fermentation, including beer, wine, yoghurt, kimchi and sauerkraut (not that this is the only reason it made the list).

Alexander Fleming in his St Mary’s lab in London

Penicillin/antibiotics: Discovered in 1928 by Scottish scientist Alexander Fleming, penicillin became the world’s first true antibiotic. By the time Fleming made this discovery, scientists had reported the antibacterial properties of some moulds, including penicillium. But they were unable to successfully harness these properties. For his part, Fleming recounted that his historically famous discovery was a lucky accident. After mistakenly leaving a Petri dish containing Staphylococci exposed in his lab, he returned from holiday and noticed it had grown a blue-green mould. The mould slowed the growth of the bacteria around it, and after studying this effect, Fleming was able to use his ‘mould juice’ (blegh) to kill a range of harmful bacteria.

Ultimately, this discovery has greatly reduced the number of deaths from infection, playing an enormous role in improving the mortality rates around the globe. Today, antimicrobial resistance is a growing concern, and medical professionals warn that if we do not discover new classes of antibiotics, infections could kill as many as ten million people a year by 2050. But scientists are looking for new antibiotics in unexpected places, like toilet seats, dog food bowls, and even laptop keyboards.

Steam engine: Another British invention, the steam engine is not so much a scientific breakthrough as it is a series of breakthroughs over the course of one hundred years, and it certainly changed the course of human history. This invention has roots in Roman times, but it wasn’t until the 17th century when Englishman Thomas Savery developed a model of the steam engine that it became a promising innovation. Soon after, another Englishman, Thomas Newcomen, and Scottish engineer James Watt made the design more efficient and the rest, as they say, was history.

James Watt’s steam engine at the Thinktank museum in Birmingham (© Copyright Ashley Dace)

Connected to a piston and cylinder, a boiler filled with water is heated until the water turns to steam. Once the steam expands, it travels through the cylinder and moves the piston first forward, and then, once the steam is cooled, backward. This back-and-forth process, attached to a larger machine, moves the machine forward, in what must be one of the most rudimentary explanations ever of this amazing process. This engine was adapted for use in boats, cars, and, of course, trains. The idea that people began to cross continents in record time just by turning a liquid into a gas over and over is pretty bonkers when you think about it.

Periodic table: This one may be last on our list, but it’s definitely not last in our hearts. Chemists have spent a lot of time throughout history on the classification of chemical elements, but when Russian chemistry professor Dmitri Mendeleev got hold of it, things changed. He published his version in 1869, much to the chagrin of German chemist Julius Lothar Meyer, who published his version just one year later in 1870 and probably thought we’d all be talking about “Meyer’s Table” right now.

Like others before him, Mendeleev saw when elements were listed in order of atomic weight, elements at certain intervals shared physical and chemical properties. But Mendeleev left gaps in the table, predicting where an element hadn’t yet been discovered and it’s properties. He also took care to classify elements into ‘chemical families’. And just like any good developer, he released an updated version in 1871. Adjustments have been made from time to time, when new elements have been discovered or to make the table more easily readable, but Mendeleev is still considered the Father of the Periodic Table.

What are your favourite breakthroughs?