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Liquid biopsies – beacons of hope for cancer diagnosis?

Researchers around the world, including researchers from the Freiburg University Medical Center, are working on developing simple blood tests, so-called liquid biopsies, that can be used to obtain accurate information about tumour staging and the progress of therapy. An international liquid biopsy symposium was held in Freiburg in October 2015 and has produced valuable insights.

Prof. Dr. Nikolas von Bubnoff has been assistant medical director of the Freiburg University Medical Center’s Department of Tumour Biology since 2012. © Freiburg University Medical Center

Prof. Dr. Nikolas von Bubnoff is an oncologist and assistant medical director at the Freiburg University Medical Center. As a scientist, von Bubnoff is specifically interested in the development of new, molecular cancer therapies and is studying the molecular causes for the failure of generally effective therapies. This has led to the use of liquid biopsies which he and his team have now established for resolving oncological issues. Liquid biopsies are non-invasive diagnostic tests using a patient's blood. In the case of cancer patients, such blood tests can provide different types of information on the disease.

They can be used to analyse circulating tumour cells that have been shed into the blood from the primary tumour. The number of tumour cells in a given blood volume provides information about the metastasisation of the tumour. In addition, molecular methods can be used to investigate circulating tumour cells for the presence of surface molecules that might make suitable drug targets. Such cell analyses are sometimes also used to monitor the course and success of therapy. Liquid biopsies are also used to detect and analyse tiny fragments of tumour DNA that are shed into the blood.

The analysis of tumour cells and tumour DNA fragments is hotly debated by scientists worldwide. Von Bubnoff explains: “Both methods are used to find answers on tumour staging and treatment success. Technological progress in this field is enormous. Some researchers swear by the analysis of circulating cells while others set more store by the analysis of circulating DNA fragments. The recent symposium has clearly shown that DNA analysis has decisive advantages over cell analysis. Personally, I think that a combination of both approaches is an excellent way of determining cancer staging.”

Liquid biopsies – two methods, one objective

Liquid biopsy used to analyse freely circulating (fc) DNA in the blood. The schematic shows the technique as applied to GIST (gastrointestinal stroma tumours). © Freiburg University Medical Center, Annual Report 2014

The liquid biopsy method established by von Bubnoff and his team in Freiburg involves as little as eight to nine millilitres of patient blood. The tumour-specific DNA fragments contained in the blood are amplified using PCR (polymerase chain reaction) and analysed using digital PCR and next-generation sequencing (NGS). The method is so sensitive that it can detect the fragment that contains tumour-specific mutations from among 10,000 fragments. However, the researchers had to specifically modify conventional PCR methods in order to achieve this high sensitivity. “Standard NGS protocols are not designed for such high sensitivity detection,” says von Bubnoff.

This method facilitates the continuous genetic typing of a tumour. “Up until now, it has only been possible to genotype solid tumours with tissue samples. However, the removal of tissue samples carries some risks for patients, biopsies are painful and take time. Tissue samples therefore cannot be obtained as frequently as blood samples. Tissue samples only provide us with a static genetic fingerprint of a tumour at a single time point and before treatment commences. Given the complexities of tumour heterogeneity, a tissue sample may therefore not be a true representation of the tumour’s molecular profile. Thanks to liquid biopsies, we are now able to reproduce the clonal evolution of a tumour. As each treatment is a dynamic process, the reproduction of a tumour’s clonal evolution is interesting for both academic and therapeutic reasons,” says von Bubnoff. The Freiburg University Medical Center team has now been using the technique for monitoring the progress of lung cancer and malignant melanoma therapies both during and after treatment.

Von Bubnoff has already filed two international patents for liquid biopsy methods that detect DNA fragments of gastrointestinal stroma tumours (GIST) in blood. Von Bubnoff is also in contact with companies in the Dresden area with whom he hopes to be able to develop commercial test methods based on the two aforementioned patents. “These are pilot projects in Freiburg for which we have received seed grants. We are currently working on a new funding proposal that we intend to use for implementing the project at different sites and advance the project further,” says von Bubnoff.

The technique initially targets therapeutic diagnostics, but might later be used for tumour screening

Liquid biopsies can be used to analyse tumour-specific mutations in blood plasma. The schematic shows how digital PCR (dPCR) detects any decrease in tumour-specific mutations (in the protein kinase BRAF gene) in the blood of a melanoma patient. © Freiburg University Medical Center

In principle, liquid biopsies are also suitable for cancer screening. However, von Bubnoff wants to curb unrealistic expectations about the technique. “I believe it is too early to use such systems for cancer screening in terms of cancer prevention. The techniques still have to go through two to three development cycles before they are sensitive enough to capture tiny quantities of circulating DNA. Even then, the techniques need to be shown to be capable of reliably detecting tumours that cannot be detected with other techniques and also that they do not lead to false negative findings.”

The sensitivity and the limits of the validity of liquid biopsies were among the most discussed issues at the Freiburg symposium. Von Bubnoff summarises the outcome: “With the new methods, it is possible to determine after 10 to 14 days, and hence much earlier and more accurately than before, whether a patient is responding to therapy or not. This applies in principle to all types of cancer, although the method cannot always be used, for example for glioblastomas and other tumours beyond the blood-brain barrier.” In the case of these tumours, only a tiny number of DNA fragments are shed into the blood. And the method is not yet sensitive enough to detect such a small number. But von Bubnoff believes it is only a matter of time before suitable test systems are available.

Recent progress gives cause for optimism. The scientists believe that liquid biopsies will one day be suitable for monitoring therapeutic progress as well as offer promising options for cancer screening, albeit in the distant future. Moreover, the unexpected and massive interest among visitors and speakers in the first liquid biopsy symposium in Freiburg in October 2015 reflects the method's potential. The 250 top experts who participated in the symposium clearly exceeded the organisers' expectations. The organising team from the CCCF (Tumour Centre of the Freiburg University Medical Center) and their cooperation partners at the Cancéropôle du Grand-Est in Strasbourg, rated the huge success of the symposium as a clear signal to schedule another liquid biopsy meeting in 2017.

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