Liquid Assets

Liquid biopsies are becoming increasingly commonplace and, in some cases, are now in clinical use. In cancer, they allow tumor DNA to be sequenced and examined without the need for invasive and potentially risky tissue biopsies. We spoke to two researchers pioneering myriad potential applications of liquid biopsies and circulating tumor DNA (ctDNA). Lao Saal is working to detect occult metastases by identifying and tracking chromosomal rearrangements. Isaac Garcia-Murillas has developed a novel approach to identify the risk of relapse among patients with early-stage breast cancer.

What drew you to cancer research?

Isaac Garcia-Murillas: I’m a molecular biologist by training, and I followed a standard career path – a PhD followed by a postdoctoral fellowship. Much of my work had been on intracellular signaling, but when I joined the lab of oncologist Nick Turner, we started to focus on ctDNA projects. My work took on a whole new meaning when it came to cancer – and to translating our research to the clinic.

Lao Saal: It goes back almost 20 years, to my days at the National Human Genome Institute at the US National Institutes of Health. I’d been studying pediatric cancer genomics and started thinking about ways to apply innovative methods to better understand tumor biology, improve diagnostics, and ultimately deliver precision therapies. After finishing my MD and PhD degrees in New York and gaining some postdoctoral experience, I came to Sweden in 2009 and started my own research group at Lund University.

What are you currently working on?

IGM: My work mostly involves the use of plasma or serum separated from blood, and I am particularly interested in ctDNA and its use as a biomarker for the detection of Minimal Residual Disease (MRD) following neoadjuvant chemotherapy and/or surgery. I’m also studying the use of ctDNA as a biomarker for detection of resistance to targeted therapies. My work is mostly in breast cancer, but I have interest in other solid tumors as well.

LS: Liquid biopsy for cancer is an extremely promising and exciting field, and I believe it will revolutionize how we diagnose, monitor, and treat cancer patients. Over the last couple of years, we have continued working on new technologies for ultrasensitive quantification of ctDNA, and now have several innovative approaches that we, together with partners and collaborators, are applying to measure ctDNA in patients with non-small cell lung cancer, melanoma, breast cancer, acute myeloid leukemia, and ovarian cancer, among others. These technologies are now part of SAGA Diagnostics AB, our cancer genomics company that focuses on molecular diagnostics and cancer liquid biopsies. One of our approaches marries NGS with droplet digital PCR (ddPCR) to measure chromosomal rearrangements as exquisitely specific truncal clonal markers in ctDNA and is particularly suited for patient monitoring and detection of MRD or relapse. We have another method that largely mutes base misincorporation errors – an issue that plagues all methods employing polymerases, such as NGS, qPCR, and ddPCR. With this proprietary method we can achieve an unparalleled limit of detection of one mutant molecule in a background of 100,000 wild-type molecules.

Liquid biopsy hasn’t just seen success in cancer…

LS: Absolutely – I would even go as far as to say that it’s probably gone further into the clinic in the field of noninvasive prenatal diagnostics. However, cancer is very close behind, and there’s a lot of research and work going on. I think liquid biopsies will start to enter the clinical arena much more in the next five years.

There is also some really interesting work in infectious disease and other fields, such as transplant medicine – for example, to monitor whether a transplanted organ is showing signs of rejection.

The benefits are clear – but what are the drawbacks?

IGM: In my opinion, the major pitfalls come from improper sample acquisition, preparation and analysis. The level of sensitivity required by these non-invasive techniques might be hampered by any of the above, rendering results invalid. I also think that liquid biopsies will remain a companion to tissue biopsies, at least for the time being, as there are many questions – including histological and pathological ones – that we cannot currently answer with these techniques.

To fully incorporate liquid biopsies into the management of patients, we need to further investigate their validity through more clinical trials. This will allow a better understanding of the advantages and limitations of these new approaches. I am unable to speculate on timelines, but I have no doubt that through our work and that of our colleagues in the field we will be in a position to start fully incorporating these techniques relatively soon.

LS: For ctDNA analysis to be more useful in earlier stages of cancer or to detect resistance mutations at the earliest moment, we need a method with exceedingly high sensitivity and specificity. That’s one reason we are pushing hard to innovate and improve liquid biopsy technologies. There are also a number of areas where additional knowledge will help us make better use of liquid biopsy. For example, for each cancer type it’s not entirely clear what the optimal schedule for sample collection is, especially in relation to the timing of therapies. We know that ctDNA quantity is closely correlated to tumor burden and that it is a powerful prognostic marker, but how the slope of the curve between ctDNA and tumor volume varies between cancer types is still being figured out. Given the wide successes of immunotherapies, it will be interesting to see how ctDNA analysis may be used to predict response. Depending on the cancer type, different liquid biopsy sources may be more informative, whether it be blood plasma, urine, cerebrospinal fluid, sputum, or other secretions. As a field, we also need to establish clear standards for reporting, so that studies can be compared more easily.

In some cases, ctDNA liquid biopsies are already entering the clinic. Today, this is most notable in advanced non-small cell lung cancer, and detecting the “druggable” mutations in the EGFR gene, such as L868R, the T790M resistance mutation, and the exon 19 deletions. Gradually, as the evidence accumulates, liquid biopsies will be increasingly used in the clinic and will become rather commonplace. I think of ctDNA as a special kind of cancer biomarker – a biomarker that carries certain biological properties that are inherent and independent of the technology used to detect and quantify it.

How do you think the roles of pathologists and laboratory medicine professionals will change with the rise of liquid biopsy and similar techniques?

LS: I think pathologists will transition to molecular pathology more and more, and be increasingly involved in liquid biopsy diagnostics. As with any new diagnostic approach, there will be a learning curve and a great deal of educational effort will be needed. Many of the technologies could be deployed in local hospitals, given that NGS, qPCR, and ddPCR instruments are increasingly found in many clinical labs. However, the greatest cost-effectiveness and efficiencies of scale will only be possible at very large institutions or at centralized regional service laboratories.

What are the challenges when using ctDNA?

IGM: There are always challenges with any project – most of them technical. There are also biological challenges, and sometimes recruiting patients to provide samples can be difficult too. In our research into detecting aberrations in ctDNA, the main technical challenge is identifying the ctDNA, as the fraction of tumor DNA present in the total circulating DNA is very small. In the case of MRD detection, the loss of materials when extracting ctDNA from plasma was also an issue, as this obviously affects the amount of material available for testing.

Do you have any tips for those looking to implement liquid biopsy?

IGM: Researchers should always take extra care when obtaining and processing samples for molecular analysis – and that’s particularly true for liquid biopsies, which are prone to contamination from other more abundant material coming from the patient. But I personally think that anybody trained in molecular biology techniques and handling human samples should be more than capable of working with liquid biopsies.

LS: People interested in implementing liquid biopsy in their own laboratories should carefully review the various options available, from the resource-intensive NGS approaches to the faster, more sensitive, but less broad qPCR and digital PCR approaches. The dynamic range of ctDNA is extremely wide: anywhere from just one mutant molecule to many tens of thousands may be floating in a 1 mL plasma sample, therefore assay characteristics like sensitivity and specificity are critical, as is the assay’s lower limit of detection. As repeat testing is one of the benefits of liquid biopsies, consideration should be paid to the intended use and cost-effectiveness of the test. In some instances, a wide panel-based approach may be appropriate, whereas in other situations a focused test for just the clinically actionable mutations may be all that is required.

What are your predictions for the future of ctDNA analysis?

IGM: The real revolution that is still to come is the so-called 100-dollar genome. I think that’s going to revolutionize not only cancer research, but genomics research in general. New techniques arrive every few years, but my feeling is we will know more about the genomics of the individual at a larger level, for a much lower price. And this will also raise a lot of ethical and data protection questions…

LS: The fact that a ctDNA sample is so easy to collect, can be used to interrogate all tumor deposits simultaneously, and even carries quantitative prognostic and treatment-predictive molecular information, makes it almost the perfect cancer biomarker. Next, we have to think in terms of large populations, and think about how our research could benefit everyone, regardless of social status or race. At the moment, I think studies have been slightly biased towards certain populations. But as health professionals and researchers we have the duty to make our work available to everybody.

I believe that in the more distant future, ctDNA will be as commonplace and simple as diabetes blood sugar monitoring is today. I think it could also have a role in screening for cancer in apparently healthy people – or perhaps within groups that are considered high risk for certain cancers. The future of this area is really exciting, and it’s great to be driving things forward, and getting closer to delivering precision medicine to cancer patients. Liquid biopsies and ctDNA give us the potential to ensure that people aren’t burdened by therapies they don’t need and instead get access to the therapies they do need – with the ultimate end goal of extending both life and quality of life.

Lao Saal is Associate Professor and Head of the Translational Oncogenomics Unit at Lund University, Sweden. He is also founder and CEO of SAGA Diagnostics.

Isaac Garcia-Murillas is a Senior Scientific Officer at the Institute of Cancer Research in London, UK.