Markers in the blood
The new test system will help detect lung cancer even before the appearance of a tumor
Elena Morgunova, "Search" No. 18-19 (2016)
A drop of nicotine, alas, still kills. And on a massive scale – more than 1.4 million cases of lung cancer are registered annually in the world. Unfortunately, for a million patients, the diagnosis turns out to be fatal. So, in Russia, more than 88 patients per 100 thousand people are detected, and more than 52 thousand people get sick every year. At the same time, every second person dies from lung cancer in the first year after diagnosis: the insidiousness of the disease is in the absence of pronounced symptoms, because more than 60% of cancer patients have lung cancer detected already at the third or fourth stage, when the chance of cure is only 5-15% (as opposed to 70% at the first stage).
Over the past 20 years, the number of detected cases of lung cancer has doubled, due to both improved screening methods and the spread of smoking. About 87% of lung, bronchial and tracheal cancers are associated with tobacco smoke inhalation. In recent years, an active fight against smoking has been launched, but, nevertheless, there are about 1.25 billion smokers in the world today. And a billion of them are men. It is not surprising that lung cancer is the cause of every third death of men with cancer, in women – 10 times less often.
All these statistics clearly substantiate the importance of the work on the project "Development of a noninvasive diagnostic test system for planning lung cancer therapy based on high-performance sequencing of circulating tumor DNA" within the Federal Target Program "Research and Development in priority areas of development of the scientific and technological complex of Russia for 2014-2020".
The project is carried out by employees of the Laboratory of Molecular Medicine of the Institute of Chemical Biology and Fundamental Medicine (IHBFM) SB RAS. And enthusiastically, because they know that in Russia the level of laboratory diagnostics is far from perfect: in the practice of most polyclinics and medical hospitals, there are a lot of routine, uninformative, outdated research methods.
– Today, oncologists have an urgent need for modern technologies that allow for early diagnosis and effective therapy, - says the project manager, Doctor of Biological Sciences Elena Rykova. – This is shown by the experience of working with partners – Tomsk Research Institute of Oncology and Novosibirsk Research Institute of Pathology of Blood Circulation. Academician E.N. Meshalkin.
Firstly, they believe, it is necessary to make a highly sensitive system for early diagnosis in order to "catch" cancer at the operable stage – surgeons work wonders here, but the patient must get to them on time.
Secondly, it's time to develop personalized treatment methods. For example, if a patient is found to have activating mutations of the EGFR gene that contribute to uncontrolled proliferation of tumor cells, targeted therapy with tyrosine kinase inhibitors, such as commercially available drugs gefitinib, erlotinib, which selectively suppress this receptor, helps him well. However, if there is a concomitant mutation of the KRAS gene in the tumor (in 30% of cases), then the disease becomes resistant to tyrosine kinase inhibitors and expensive targeted therapy does not bring the desired effect. Therefore, when choosing a treatment strategy, an important task is solved – to predict the response of the tumor to therapy.
The third problem is monitoring the condition of patients who have already been treated. Our test system is applicable at all stages. By and large, the goal of the project is to replace the previous model of the therapeutic and diagnostic cycle "identification of biomarkers – treatment" with a more complex and more effective "identification of biomarkers - treatment – assessment of response – correction of therapy – monitoring of resistance (tumor resistance to drugs) – correction of therapy – screening of relapses". By "minimally invasive" is meant a diagnosis based on the detection of tumor DNA circulating in the blood. That is, according to the blood test. The high–tech NGS (next generation sequencing) method - large-scale parallel sequencing of circulating DNA – can take cancer diagnosis and therapy to a whole new level.
Oncology is not only a social problem, but also an economic one. In Europe alone, there are more than 270 laboratories and commercial companies conducting tests for sensitivity and resistance to tyrosine kinase therapy. The European market for related diagnostics has already reached $ 457 million, and by 2018 it will exceed well over a billion. It is clear that large pharmaceutical companies that produce drugs for targeted therapy have been actively investing in firms specializing in the development of diagnostic tools, methods and instrumentation since 2011.
Before launching the project, IHBFM employees conducted a large–scale patent search - analogues of the test system should have the following features: analyze EGFR gene mutations associated with sensitivity and resistance to tyrosine kinase inhibitors, use circulating tumor DNA as a sample, perform non-invasive monitoring of acquired tumor resistance, use high-performance sequencing as a detection method. It is established that there are no complete analogues of such technology.
– As a rule, biopsy material is used in existing patent developments, – says Elena Yurievna. – But in some cases, it is simply impossible to do a biopsy: in the early stages, when a person is unaware of the disease, when the tumor is inaccessible and after surgery, when the tumor is removed and the patient's condition needs to be monitored.
Why are we developing DNA analysis in the blood? First of all, because it makes it possible to diagnose early – fluorography has not yet revealed a tumor, and mutant DNA from tumor cells has already appeared in the blood, and the test system will allow you to find the disease even before clinical manifestations. We have learned to isolate fragments of circulating tumor DNA from the blood plasma, which can be used to detect both a primary, even a small, tumor, and a relapse, wherever it occurs in the body.
The test system being developed will allow for regular examination and compile an up-to-date molecular genetic "portrait" of the tumor, which will help choose the right treatment. In addition to EGFR, we analyze damage to other genes that may be involved in improper cell management, which leads to its malignant transformation. In the course of our work, it was necessary to establish which concomitant mutations can either contribute to the "breakdown" of EGFR, or cancel this undesirable effect.
Therefore, based on data from other studies, we selected 200 target genes for targeted search. The analysis of mutations in the cascade of proteins that are involved in carrying out external signals into the cell will make it possible to predict whether tyrosine kinase inhibitors will help the patient, or the tumor will remain resistant to treatment and it is better to apply a different therapy based on modern radiological, chemical, surgical methods and their combinations.
The first stage of the study was done on tumors to look at the variety of mutations and the frequency of their detection in the tissue of neoplasms. It was necessary to test the possibility of using a high-tech sequencing method in relation to tumor DNA circulating in the blood, which is "cut" into small fragments before entering the blood. It was necessary to take into account the small amount of tumor DNA in the blood, the small size of fragments when creating DNA libraries intended for sequencing, and to solve a number of other technological problems.
In 2015 (the grant was received at the end of 2014), we examined blood samples from more than 40 patients to identify circulating tumor DNA. There are still unresolved problems: the percentage of tumor material in circulating DNA is very low – as a rule, less than 10%. So, of the patients we examined, three had tumor DNA in the biopsy material, but we could not detect it in the blood, which reduces the sensitivity of the test system. But a very good result is 100% specificity of the test (it did not give false positive results).
The result of three years of work on the project (the total amount of funding is 24 million rubles, of which 15 million are budgetary funds) should be a ready–made test system that will allow detecting lung cancer in time, planning effective treatment, and competently monitoring its results.
Among the partners of IHBFM SB RAS are Atlas LLC (CEO Sergey Musienko), co–executor is Ridsens LLC (director is Vladislav Mileyko, at one time an employee of IHBFM who received an award for developing a method for early diagnosis of breast cancer), another co–executor is the Genomics Collective Use Center (curator of the project – Doctor of Biological Sciences Anna Baranova). The research team is distinguished by its youth – half of the participants are under 35. Students of Novosibirsk State University and postgraduates of IHBFM SB RAS are involved in the work. And of course, such a topic could not appear "from scratch".
– In our laboratory of molecular medicine, under the leadership of Candidate of Biological Sciences Pavel Laktionov, we have created our own technological base that allows us to effectively isolate and analyze extracellular nucleic acids from blood and other biological fluids. Kits for DNA and RNA isolation from biological media produced by the company "Biosilica", affiliated with the IHBFM SB RAS, are in demand outside the laboratory and institute, because they are not inferior in quality to imported analogues. The inspirer of all these works is the director of our institute, Academician Valentin Vlasov.
When early studies showed that extracellular nucleic acids may contain "diseased" DNA, this became the motive for launching several projects aimed at solving the problem of diagnosis and personalized therapy, Elena Rykova reveals the secrets. – We are developing methods of DNA and RNA diagnostics of various oncological diseases – prostate, breast, lung cancer. Our research is at the most advanced level and therefore very relevant. According to this project, the main performer is a senior researcher of our laboratory, Candidate of Chemical Sciences Evgeny Morozkin, who since 2016 has been leading research under the RNF grant aimed at developing microRNA markers of prostate cancer. We are also actively investigating epigenetic changes in DNA – in my opinion, a very promising direction for identifying markers of early screening.
We are very keen to bring as close as possible the time when it will be possible to launch technological, accurate and affordable molecular genetic tests into mass production, which will allow detecting cancer at the stage of preventive examination even before clinical manifestations and choosing the optimal therapy based on a simple blood test.
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16.05.2016