Who is warned is armed

According to the tumor DNA circulating in the blood, it is possible to diagnose lung cancer relapses very early

Vyacheslav Kalinin, "Elements"

Fig.1. Monitoring the evolution of lung cancer for early detection of relapses after tumor removal. In clinical studies of the TRACERx consortium (article by Jamal-Hanjani et al., 2017), complete sequencing of coding regions of the genome from various sites (1-4) of a surgically removed non-small cell lung cancer tumor was performed for one hundred patients. DNA mutations present in the tumor were identified (indicated by colored crosses). Among them, clonal mutations present in all tumor cells and subclonal mutations that arose later and were present only in some areas were identified. A phylogenetic tree was constructed for each patient, showing the evolution of the tumor genome, the accumulation of mutations during its growth. Based on these data, Abbosh and co-authors (article by C. Abbosh et al., 2017) developed a test in which tumor DNA (ctDNA) circulating in blood plasma was analyzed. This test detected cancer recurrences (in this case metastases localized in another lung) long before they could be found using computed tomography. A drawing from the synopsis to the articles discussed in Nature.

Scientists from the British TRACERx consortium sequenced the DNA of lung cancer cells from various tumor sites surgically removed from one hundred patients. They were able to trace the evolution of the genome of each tumor: clonal mutations were identified, with which the development of cancer began, and subclonal ones that arose later. The set of mutations in each patient turned out to be individual. Knowing which mutations to look for, another group of scientists from the same consortium monitored the DNA circulating in the blood plasma of patients after the removal of tumors. This made it possible to detect recurrences and metastases of primary tumors long before they became visible on tomograms. The works discussed are an important step towards the introduction of effective personalized methods of diagnosis and treatment into clinical practice.

According to the World Health Organization, lung cancer is the most common cause of death from cancer. Smoking is considered to be the most significant factor provoking it, but people who have never smoked and have not had obvious contacts with other known risk factors (asbestos, radon, etc.) can also get sick. About 1.8 million new cases of lung cancer are registered annually in the world and about 1.6 million patients die from it.

According to the histological picture of tumor cells, lung cancer is divided into several types. The most frequent of them (about 80% of cases) – poorly treatable non-small cell lung cancer, NSCLC (see Non-small-cell lung carcinoma, NSCLC). Among NSCLC, squamous cell and large cell carcinoma, adenocarcinoma and some other rare subtypes are distinguished by histology. NSCLC very often metastases to various organs, and every year more than a million patients die from metastases, not from the primary tumor. Preventive postoperative chemotherapy increases the survival rate of patients by only 5%. This may be due to the high genetic heterogeneity of tumor cells: differently mutated cancer cells may be sensitive or, conversely, resistant to completely different drugs.

The British TRACERx Consortium (TRAcking non-small cell lung Cancer Evolution through therapy [Rx]) is conducting a large-scale study on NSCLC tumors, involving several clinics and several hundred patients. His results for one hundred patients are published in M. Jamal-Hanjani et al. in The New England Journal of Medicine. Complete sequencing of DNA coding sequences from various sites of remote tumors revealed mutations present in the tumor of each patient. Some of them were found in almost all sites (clonal mutations) – obviously, because they appeared in the early stages of tumor development. Other mutations, along with clonal ones, were found only in some areas and, therefore, arose later (subclonal mutations). The sets of mutations in different patients were individual.

As expected, mutations of EGFR, MET, BRAF and TP53 genes, which are known as oncogenesis drivers (that is, they are associated with the initiation and development of cancerous tumors), were often detected as clonal. Among the subclonal ones, there could be both mutations that aggravate the malignancy of cells (that is, their ability to uncontrolled growth), and those that are not directly related to cancer, but arose as a result of a general violation of the processes of repair of damaged DNA. Based on the distribution of mutations, phylogenetic trees were constructed showing the history of tumor growth and progression at the molecular level (Fig. 1, 2).

Fig. 2. A phylogenetic tree showing the evolutionary relationships of various tumor sites and metastasis by mutations found in the primary tumor and metastasis of one of the patients. Colored circles – clones of mutations found in tumor DNA circulating in the bloodstream (ctDNA); gray circles – undetected; large circle – clonal mutations. The numbers 135, 167 are the number of mutations; the length of the uncrossed branches of the tree is proportional to the number of mutations. The red dotted line is a branch to relapse (metastasis). Colored or white areas in rectangles denote pairs of primers, with the help of which it was possible to detect respectively colored mutations. amp – amplification, del – deletion, P1 – primary tumor, R1–R5 – primary tumor sites, M1 – metastasis. Figure from the article under discussion by Abbosh et al., 2017

A logical continuation and development of these studies is the work of Abbosh et al., also carried out within the TRACERx project and published in the journal Nature. To trace the evolution of NSCLC tumors in the patient's body during clinical observations and treatment allowed the previously used, but relatively new approach – liquid biopsy. With a conventional biopsy, the doctor takes a fragment of a cancerous tumor for examination, but at the same time it is possible to obtain information only about a limited area of the tumor, and most importantly, the tumor must first be detected (for example, using computer or magnetic resonance imaging). The principle of liquid biopsy is based on the analysis of DNA, cDNA circulating in blood plasma (see Circulating tumor DNA, ctDNA). It appears as a result of the decay and renewal of tissues constantly occurring in the body. In cancer, not only fragments of DNA of normal cells can be found in the blood, but also DNA of dying cells from various parts of the tumor. cDNA can theoretically be sequenced (to determine the nucleotide sequence) and thus obtain comprehensive information about neoplasms that develop in the body, including those that are not yet visible on tomograms.

The difficulty was that cDNA can only make up less than 1% of the DNA found in blood plasma. To find this "needle in a haystack", British scientists purposefully searched for those mutations that were discovered as a result of DNA sequencing from various sites of a previously removed tumor of the same patient. The authors of the work have significantly improved the procedure for detecting mutations. They used the so-called multiplex polymerase chain reaction to amplify (increase the number, accumulation) of target DNA fragments with subsequent sequencing. At the same time, in one reaction mixture, the authors amplified and then sequenced not one, as usual, but several DNA fragments at once. This greatly accelerated the work. In the study under consideration, up to 28 different fragments were analyzed simultaneously. And the methods of highly efficient sequencing (new generation sequencing) made it possible to capture and determine the nucleotide sequence of a mutant DNA fragment against the background of thousands of normal fragments.

For each of the 96 patients participating in the study, 10 to 22 anomalies (point mutations, deletions, gene amplifications, chromosome rearrangements) were selected from among those found in the DNA of the removed tumor. To begin with, scientists tried to detect these anomalies in the stored blood plasma taken from patients before the removal of the tumor. This was partially achieved: at least two anomalies were detected in 48% of cases (46 out of 96) and one anomaly in another 12 cases. The success rate of detection was different for different subtypes of NSCLC: in squamous cell carcinoma, in which the tumor is rapidly renewed and its extensive necrosis forms, mutations were detected in 97% of cases (30 out of 31), and in adenocarcinomas, in which necrosis is not so extensive, only in 19% (11 out of 58).These anomalies could then be detected by subsequent liquid biopsies.

For 24 patients (of those whose cDNA was detected in the blood taken before surgery), relapses and metastases were monitored after surgery by regular testing of DNA from blood plasma. The result was very impressive. Soon after the operation, the cDNA almost completely disappeared. And relapses or metastases could be detected by a reliable amount of newly appearing cDNA in an average of 70 days, and in the case shown in Fig. 3, even 347 days before they were detected by computed tomography. Thus, a liquid biopsy turns out to be much more sensitive than a computed tomography. This is important from at least two points of view. Firstly, treatment of relapses should be started as early as possible. Secondly, the results may allow us to determine which therapy should be applied. So, for one of the patients, it was shown that cells carrying clonal oncogenic amplification of the ERRB2 gene recur. In this case, it is advisable to use chemotherapeutic agents that effectively suppress the product of this gene.

Fig. 3. Detection of mutant cDNA allows early recognition of cancer recurrence after surgery. Two mutations were determined – clonal (blue circles) and subclonal (red triangles); VAF – variant allele frequency. After reliable detection of recurrence by clonal mutation (mutant cDNA accounted for 0.1% of the total plasma DNA), computed tomography (CT – computed tomography) was performed, which initially gave a negative result (Normal CT) and only after 347 days positive (R – relapse, "relapse"). Figure from the article under discussion by C. Abbosh et al., 2017.

Another important finding of the authors was the ability to use a liquid biopsy to estimate the size of the tumor by the frequency of occurrence of the mutant variant relative to the total volume of blood plasma DNA. According to the estimates made, the proportion of mutant DNA 0.1% corresponds to about 300 million tumor cells (Fig. 4). Thus, a liquid biopsy can allow evaluating the effectiveness of therapy: if the tumor size decreases, this will affect the amount of cDNA.

Fig. 4. The frequency of occurrence of mutant cDNA in relation to total DNA in blood plasma correlates with the size of the tumor. a. The size of tumors determined by computed tomography correlates with the frequency of occurrence of clonal mutations in blood plasma DNA (VAF).
LUCD – squamous cell lung cancer, LUAD – lung adenocarcinoma, Other – other types of cancer. The red-brown area shows 95 percent confidence intervals. b. Predicted frequency of occurrence of clonal mutations in blood plasma DNA at hypothetical tumor sizes.
Figure from an article by Abbosh et al., 2017

Despite the fact that liquid biopsy with cDNA analysis has shown high efficiency for monitoring cancer recurrence, it remains technically quite complex (DNA amplification and sequencing are required), time-consuming and simply expensive. Therefore, it is too early to talk about its introduction into broad medical practice. But the cost of biotechnologies, especially sequencing, is constantly decreasing, and the application of this precision individual approach in clinical practice may become real in the near future.

Sources:
1) Jamal-Hanjani et al. (TRACERx Consortium). Tracking the Evolution of Non-Small-Cell Lung Cancer // The New England Journal of Medicine. 2017. V. 376. P. 2109–2121.
2) Abbosh et al. (TRACERx Consortium, PEACE consortium). Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution // Nature. 2017. V. 545. P. 446–451.
3) Alberto Bardelli. Medical research: Personalized test tracks cancer relapse // Nature.2017. V. 545. P. 417-418 (synopsis to the articles discussed).

Portal "Eternal youth" http://vechnayamolodost.ru  30.08.2017