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NGS is a Natural Evolution from Early Methods
Perhaps the timely analysis of the vast amounts of data from NGS instruments is the biggest and most significant hurdle we have to face to make an impact on cancer care.

With rapid advances in next generation sequencing (NGS) technology and its increased use in clinical settings, the dawn of widespread personalised medicine appears to be upon us. Dr Elaine Mardis, institute Co-director and Director of Technology Development at The Genome Institute, Washington University in St Louis, Missouri, USA, shares her views on the current uses of NGS, the challenges that NGS technologies face, and what can be expected in the future.

What is the significance of NGS to cancer care?
Next Generation Sequencing (NGS) is just a natural evolution from those early methods of fluorescent slab gel and fluorescent capillary electrophoresis that were used to sequence the human and mouse genomes, among others. The significance is huge and growing. NGS gives us this great advantage of generating data that is very comprehensive, yet available to us in a very short time frame. This combination is key for relevance to cancer patients and their oncologists, providing information in a timely manner to allow them to make decisions on how best to treat the disease.

Perhaps the timely analysis of the vast amounts of data from NGS instruments is the biggest and most significant hurdle we have to face to make an impact on cancer care. NGS has these advantages, but also brings with it incredible demands on the computing that is required to tease out the analysis and medical interpretation of the data.

There is also a role for NGS in monitoring patients. Therefore, its use in cancer care is not a one-time application — patients will require monitoring as they are treated with the therapies indicated by the genomics of their cancer. They will require secondary and tertiary sampling of new, recurrent cancers to understand how the genome has changed, and what new therapeutics might be applicable to treating their disease. If we are successful in our aims, NGS is going to become a focus of disease characterisation and disease monitoring in modern molecular pathology.

How do you see disease monitoring influencing patient care?
Monitoring allows clinicians to look at markers in circulation in the blood, often referred to as liquid biopsy. Many, but not all, cancer types actively shed cells into the circulation. A lot of these circulating cells are in the process of apoptosis, so they are actually releasing DNA from the tumor cells into the circulation. The idea of monitoring is to determine if the circulating blood can be used to observe the impact of therapy. So, for example, you sequence a tumor that has been removed, you identify some key markers that are highly unique only to the tumor and not to the individual’s germ line, and then as the patient goes through therapy following surgery, you periodically sample the blood and evaluate it with NGS. Because NGS is digital, if your therapy is effective you should be able to see a decrease over time in the tumor DNA content in the blood.

These have to be very sensitive approaches because there are not huge amounts of tumor DNA in the blood, but it is clearly present for most cancer types.

The other aspect of monitoring is that if treatment is unsuccessful, an increase, or at least a stasis, of the level of tumor DNA in the blood will be observed. This gives clinicians a much more accurate way of monitoring the response of the patient to the identified therapy.

What new NGS technologies should we expect and what hurdles do they need to overcome to be adopted in clinical applications?
A critical path that has not yet been answered by NGS for the human genome is very long read lengths. Most of the technologies we are currently using to sequence whole human genomes are short-read technologies. What I am hoping is next on the horizon—and this could come from a number of different devices— is longer read technologies. These would give us the ability to assemble human chromosomes at the first pass, rather than just aligning the reads to the reference genome; we know the latter is a limited approach at best.

The other hurdle will continue to be accuracy and coverage of the genome, because the biggest worry, especially in the clinical realm, is the notion of false negatives; ie., “What have you missed?” and “Could that missing information also be important?” Certainly, false positives are a worry, but we always have the ability to validate or take a second look using a different technology or a different approach to confirm that the same sites are being altered, which gives us confidence that our NGS analyses are actually correct. But you cannot do that with false negatives because you can’t validate what you missed in the first place. So I think that is going to continue to be an issue. As the methods improve, the read lengths get longer and accuracy increases, we should be able to better address some of these concerns about false negatives.

How soon do you see NGS being routinely used in clinical settings, or is it already in routine use?
I would not call its current use routine. In the specific realm of cancer care, I think within the next two years we will see NGS use becoming widespread because its application to cancer care is very clear. There have been some successes when looking at families, especially those with children who have abnormalities. Again, NGS use is not routine, but increasing, especially for those with abnormalities that do not render to a traditional diagnostic approach.

Probably within the next ten years, as we understand the genome better and the methods and the sequencers improve, genome sequencing will become part of the routine workup of the child, much like the Apgar score and the heel stick for blood are now, and with the exception of children whose parents do not consent, every child will have a genome sequence. This would provide a baseline for identifying their disease susceptibilities and predicting their health as adults.