Tumor Biomarkers and Liquid Biopsy PDF

Pathology and Diagnostics Garlanda - Clinical Pathology - Lecture 4 Tumor Biomarkers and Liquid biopsy 26/11/2024 - Group 2 (Amadio - Grossi) The term “liquid biopsy” defines the collection of tumour-derived material in the bl...

Pathology and Diagnostics Garlanda - Clinical Pathology - Lecture 4 Tumor Biomarkers and Liquid biopsy 26/11/2024 - Group 2 (Amadio - Grossi) The term “liquid biopsy” defines the collection of tumour-derived material in the blood or other body fluids. The most common tumour-related biomarkers assessed on liquid biopsy are circulating tumour DNA (ctDNA), circulating tumour cells (CTCs) and exosomes. This technique has several advantages over canonical tumour biopsy: 1. Being minimally invasive and being accessible for every type of tumor, as the specimen is collected from the blood (in >25% of patients, tissue sample at diagnosis is not available) 2. Feasibility of acquiring samples longitudinally for disease monitoring 3. Might better reflect primary tumor heterogeneity than small tumor biopsies 4. CTCs analysis is likely to reflect the most dangerous cancer clones that seed metastasis, which must have features allowing them to extravasate and intravasate in a vessel 5. Monitoring of CTCs and ctDNA during post-surgical follow-up assessments can enable the detection of disease relapse many months earlier than is possible with current radiological imaging procedures 6. Allows the detection of minimal residual disease (MRD), what is remaining after a surgical intervention 7. Captures the molecular/genetic heterogeneity of metastatic cancers Circulating Tumour Cells (CTCs) CTCs detach from tumour tissue and then migrate usually towards blood vessels, then colonize another tissue thus originating metastases. They may be found as single cells or clusters (CTC clusters) and represent an extraordinary tool to identify patients at risk for metastasis development. Molecular analysis of CTCs allows elucidation on potential mechanisms of tumour dissemination and invasion. Once a blood specimen is available, one can perform: - CTC enumeration - CTC profiling - CTC expansion (to perform functional assays) In this way, the role of CTCs in early dissemination can be understood. 1 CTCs are rare cells, 1 CTC per 1-10 millions of white blood cells, so enrichment procedures are necessary to efficiently identify them. Isolation techniques 1. Negative selection Using mAbs specific for blood cells. For example, when looking for a tumour cell of epithelial origin, antibody against CD45 (leukocyte marker) can be used to eliminate all the leukocytes, so that what is remaining is enriched of epithelial cells 2. Positive selection When some features of the cancer cells are already known: - Biological properties (several kits promote the selection of immunomagnetic systems, they are antibodies recognizing some specific marker of the cancer, which is already known) - Physical properties (normally, cancer cells are larger than normal cells) 3. No selection Everything is stored and then sequenced or stained in an unbiased approach Molecular characterization Once CTCs have been isolated, they can be molecularly analysed to define their origin. This characterization can be of 3 types: 1. Image-based - Immunohistochemistry - Immunocytochemistry - Immunofluorescence 2. Molecular (most important) - PCR based → selected genes - Sequencing → entire genome 3. Functional (once purified) - In vitro - In vivo Clinical applications 2  All of these approaches have many clinical applications, they may provide early detection or diagnosis of the cancer, prognosis, monitoring and treatment. They can also help in detecting metastases earlier, as CTCs are precursors of metastatic cells. In this case, since the genetic profile of the tumour cells is known, the correct drug can be identified based on the genomic modifications of these cells. Therefore, the patient can be stratified and the correct therapeutic pathway can be identified. Screening isn’t really an opportunity, at the moment it is really difficult, it will probably be used in 10 years. 1) Prognostic role One of the clinical applications of CTCs is prognosis. It is known that, depending on the type of cancer, the percentage of patients having a detectable number of CTCs is associated with prognosis. On average, this percentage is around 25-30% of patients, with some exceptions, such as small cell lung cancer (70-95%) and pancreatic cancer (5%). The latter is characterized by a very big capsule of connective tissue, which limits the number of leukocytes able to enter and tumour cells to exit the tumour tissue. Once CTCs are found in a patient, depending on their number, there is a threshold associated with the patient's outcome. 2) Monitoring response to therapy Another possibility involves the monitoring of the patient's response to therapy. In the study shown to the right, the number of CTCs has been investigated after the first cycle of chemotherapy and then after the fourth. Notice that some patients do not respond to chemotherapy, others do even after the first cycle, thus proving the efficacy of the treatment. 3) Identification of alterations associated with resistance to therapy 3  Finally, molecular features of cells resistant to therapy can be assessed. In this case, cells in different patients have been sequenced and then divided into chemosensory and chemorefractory, so that one can identify the genetic profile associated with resistance or sensitivity to a certain chemotherapeutic drug. Limitations - Their presence is rare, even in patients with metastatic disease - The identification and isolation of CTCs generally require complex and expensive methods. For this reason, they will probably be used in the following years - Intrinsic difficulties of single cell analysis - Lack of universal markers able to identify and capture every CTC subsets In this study on non-small cell lung cancer (NSCLC), it was shown that only 28% of patients had at least 5 pulmonary venous CTCs in 7.5 ml of blood. In total, 220 CTCs were isolated, of which only 47% were successfully sequenced. However, even with these limitations, if CTCs are collected at the time of surgery, then sequenced and analysed, the genetic modifications that later on will appear in the metastases can be found. In fact, by analyzing the mutations of primary and circulating tumour cells, it was observed that in CTCs, compared to primary tumour cells, there is a higher mutation overlap with metastases detected 10 months later. This is the demonstration that the most aggressive cells are found in the blood. Therefore, CTCs enumeration and genetic profiling are early predictors of recurrence after surgery. Circulating tumour DNA (ctDNA) 4 Circulating cell-free tumour DNA is released in the circulation by tumour cells undergoing cell death processes. Since in normal conditions, every cell type undergoing apoptosis releases DNA in the bloodstream, ctDNA is contaminated with DNA released by dead circulating blood cells and by ongoing inflammatory processes (circulating free DNA). ctDNA accounts only for 0.1-10% of the total cell free DNA, whose normal plasma levels range from 10 to 100 ng/ml. It is highly fragmented (nearly 143 base pairs) and the dimension of the fragments is associated with cancer. When looking at the DNA found in the blood, the smallest one is mitochondrial DNA of 140 bp, then there is a peak around 140 bp which contains the cancer DNA (in red). Finally, the control DNA is longer and even longer is the extrachromosomal circular DNA. Based on the size, one can purify and select the circulating tumour DNA. ctDNA molecular profile After centrifugation, ctDNA is found in the plasma. At the bottom of the tube will deposit leukocytes and other blood cells, then there is the plasma with ctDNA. With its analysis, molecular information can be found regarding: the genetic and epigenetic profile. 1. Genetic profile: - Mutations - Chromosomal rearrangements - Copy number aberrations 2. Epigenetic profile (associated with cell transcriptional activity and tumor type) : - DNA methylation - DNA fragment lengths → depend on the regions accessible for transcription - Read depth coverage There can also be viral sequences detection, as several tumours are of viral origin. Clinical applications 5  A group of patients may have gene fusion (= aberrant chromosomes), copy number variations, polymorphisms or single nucleotide variations. After undergoing surgery, the liquid biopsy with ctDNA analysis can be performed: the patient may be disease free or still have the persistence of genetic markers associated with the neoplasia (Minimal Residual Disease). According to their markers or genomic features, personalized therapy can be applied. Then, the appearance of new clones with new mutations (clonal evolution of the disease) can be monitored with sequencing. Thanks to this, the treatment can be changed according to the new characteristics of the tumour. Preanalytical considerations in liquid biopsies It is necessary to standardize the workflow, otherwise the material cannot be analysed in any other institute. ctDNA: For cfDNA analysis, avoid genomic DNA release from lysing leukocytes: this DNA would dilute the already potentially small fraction of tumor-derived DNA and can lead to false-negative results Blood collected in EDTA-K3 tubes (or tubes for serum) must be processed within 2 h after venipuncture. cfDNA half life: less than 1 hour To allow greater flexibility for clinical implementation, blood collection tubes containing fixative agents have been developed. These aim to prevent cell membrane lysis and can be processed 7 days or longer after blood draw Achieving the highest extraction efficiency for fragments of cfDNA below 200 bp as this range contains most ctDNA fragments Store extracted ctDNA at -80°C CTCs: For CTCs analysis, different preanalytical steps should be considered. Firstly, for blood collection, preservatives are needed to avoid cell lysis Nucleic acid isolation and amplification from CTCs remains a challenge with different methods prone to errors to identify genetic variations Extracellular vesicle (EV) collection represents a particular challenge as vesicle release from blood cells (especially platelets) after venipuncture needs to be minimised to avoid reduction in purity. To date, no specific EV preservative is commercially available. It is research-only Methods like ultracentrifugation do not distinguish between different subsets of EVs (exosomes, microvesicles) and immunoaffinity methods (Miltenyi®), which target different proteins (CD81, CD63), are capable to separate EVs but are expensive 6  Currently, no consensus has been agreed on which method results in a higher-quality yield After these steps, the following must be carried out. Genotyping cfDNA for somatic genomic alterations found in tumours Genetic variations in ctDNA reflect the mutational landscape of the corresponding tumor tissue Although specificity among detection methods can approach 100%, sensitivity is generally lower and depends on the DNA alteration type. Therefore, with this approach, the capacity to detect the specific marker of the genetic mutation is very high A high concordance (often about 80% or more) has been reported between the mutational profile of driver oncogenes (KRAS, NRAS, BRAF and EGFR) in ctDNA and tumor tissue of colorectal, lung and breast cancer patients. Clonal hematopoiesis (CH) is a natural process in which, with age, in the bone marrow there is the accumulation of somatic mutations of hematopoietic stem cells. It leads to clonal expansion of mutations in blood cells. The most prevalent CH-related mutations are DNMT3A, ASXL1, and TET2, which are genes involved in epigenetic regulation. If these mutations are found, one must verify that they aren’t due to clonal hematopoiesis, in order to avoid false positive results. Sequencing of peripheral blood cells (PBC) and plasma samples from the corresponding patients is essential for accurate determination of tumor-derived mutations from liquid biopsies. Standardization issues There are aspects that should be considered in order to standardize the use of liquid biopsies: 1. Preanalytical variability - Use specialized collecting tubes (such as Streck tubes) to stabilize blood samples at room temperature and prevent lysis of white blood cells - Define the optimal time period between blood draws and plasma processing, as well as centrifugation conditions to reach the maximum final ctDNA yield - Define quantification methods (for example, using fluorescent dyes, spectrophotometry, or qPCR) - Define ctDNA isolation protocols to reach the maximum final ctDNA yield 2. Analytical variability - Intrinsic PCR errors - Nonuniform genomic coverage - Technological errors (for example, related to next-generation sequencing platforms) 3. Biological variability - Take into account both spatial and temporal tumour heterogeneity. The tumour changes over time and, within the same tumour, there are areas with different mutations 7  - Currently, the clinical sensitivities of ctDNA assays are challenging to determine, owing to variability in the tumour stages and types assessed, the sample-processing techniques used, and the targeted molecular alterations across different studies Applications of liquid biopsy The sample is collected from the tumour-affected patient and is analysed. Then, the presence of the driver mutation is followed up. This may be used only for detection of the tumour or for screening with PCR of one or more mutations. In normal conditions, the tumour diagnosis is obtained when the tumour mass is already causing problems to the patient, this is then followed by surgery. However, if the presence of the mutation in the circulation could be assessed at the moment of the diagnosis with a liquid biopsy, the number of mutated DNAs would increase until surgery and then drop. Afterwards, the patient can be further followed to identify a Minimal Residual Disease. A relapse of the primary tumour may occur, but also new clones may develop. The biomarker increases much earlier than imaging-detected lesions, which is why it is important to use this approach for patient follow-up. If a new clone is found, a new therapy is started and, if it doesn’t work, the biomarker continues to increase. Therefore, liquid biopsy is a tool to predict the relapse of the disease. This patient with colorectal cancer has a driver mutation in APC, which is always present (clone 1). It may be followed over time in response to therapy and, if it rises again, new therapies should be applied. The growth after the second therapy suggests that the driver mutation isn’t being targeted. In addition, other mutations may appear thus impacting on the response to the therapy. Imagine that this colorectal cancer is being treated with anti-EGFR antibodies, at first responding but then not anymore because of another mutation downstream of EGFR or a peculiar mutation in the EGFR receptor itself. 8 DNA methylation and cancer Another important point is the epigenetic modifications present in cancer. The term “epigenome” indicates all epigenetic modifications that control gene expression of all cells, including cancer cells. These include: 1. DNA methylation = addition of a methyl group on cytosines, generally followed by a guanine (CpG) 2. Non-coding RNAs = molecules not associated with transcription or encoding proteins, but are used to regulate the expression of other genes 3. Histone modifications = acetylation and methylation of histones associated with DNA accessibility to transcription of different regions of the genome In cancer, DNA methylation is the most investigated epigenetic modification, as it is associated with regulation of transcription of oncogenes. The methylation close to the promoter of these genes is the main mechanism of regulation of oncogene expression. When there is an abnormal silencing of oncosuppressor genes, there may be the activation of oncogenes. Normally, in cancer the methylation of cfDNA is studied because of its possible involvement with the formation of metastases. The most well-studied epigenetic feature of cfDNA is 5-methylcytosine (5mC) at cytosine–guanine (CpG) sites that tend to cluster in CpG islands in the promoter region of genes and are widely known to be involved in gene silencing It is known that the circulating cell-free DNA, found in liquid biopsy, contains molecular features which are typical of the tissue of origin of this DNA. For instance, the methylation features are strictly associated with the properties of a cell to activate or not the transcription of specific genes. Even the fragmentation is a feature of the tissue, fragmentation occurs in the regions between nucleosomes, in the portion of DNA which is not bound to the histones, this is strongly associated with transcriptional features of that tissue that means that the type of fragments will inform us about the source of this nucleic acid. This approach is very important to determine the origin of cancer of the unknown primary tumour, because the DNA that we may find in the circulation is reflecting the tissue of origin. The methylation status, the length of the fragment, the sequence of the fragment is strongly associated with the type of tumour. The analysis of the circulating tumour DNA methylation is very complex. The patterns of DNA methylation are tissue specific but to detect these you need specific approaches, the mixture of modifications implies the need of differential type of analysis and sensitive 9 techniques. The analysis of circulating tumour DNA methylation is not used at the moment in the clinic while we already use the analysis of the mutations of the tumour. The possibility to detect these methylations depends on the abundance of the DNA and also the sequencing depth. DNA methylation analysis will provide information on thousands of tumor-specific and tissue-specific events that are very important to define which tumour is that and which features this tumour has. Study of Nature (2018): proposes the use of the DNA methylome to detect and classify tumours. What is available in the market concerning the biomarkers that we can analyse by liquid biopsy? There are different types of tumours and different genetic markers associated with neoplastic transformations. Companies have generated tests to identify the driver mutations in the most important mutated genes in different types of cancers. In general these markers are for research use only (RUO) and have not been approved in vitro medical devices (IVD). We cannot ask to perform these tests and ask the National health system to reimburse these types of analysis. ctDNA as tumor biomarker There are genetic alterations which are tumour specific: - breast cancer’s typical mutations: PIK3CA mutations, HER2 and ESR1 (estrogen receptor) higher amplification - colorectal cancers: tumor-specific gene alterations of EGFR, BRAF, ALK, KIT, PDGFR, HER2 and KRAS - lung cancers: EGFR mutations and ALK rearrangements (used in the clinic) Markers of residual disease are used to follow and predict the recurrence of the disease and to monitor therapy efficacy - KRAS in metastatic colorectal cancers Increased levels of ctDNA are in general associated with poor survival, only the amount of ctDNA may be used to investigate the prognosis of the patient Therapy efficacy: the level of ctDNA is associated with the tumour burden and may be used as a readout of the therapy efficacy. If the patient responds to the specific therapy, the total amount of ctDNA will decrease. The presence of a particular mutation can guide to a specific treatment that can be provided to the patient. Monitoring emerging resistant mutations is important to understand if we may or may not have a new therapeutic target therapy to propose to patients There are some markers that have been approved by the FDA. - left part: tests that have been approved by the FDA in the US - right part: tests that have been approved by the FD in Japan These are the diagnostic biomarkers that have been approved for some diagnoses depending on the type of tumour. For example, there is the EGF receptor in non-small cell lung cancer. 10 There are target therapies that can be used for this type of mutation, these have been approved as drugs affecting this type of mutation. Approved clinical applications of liquid biopsy - CTC have been approved as prognostic factors by the FDA in the case of breast, colon and prostate; the technology is based on a tool which allows the immunomagnetic purification of CD45-cells. - ctDNA is used as a predictive factor for response to first line treatment with EGFR- tyrosine kinase inhibitors TKI approved by the FDA and EMA (also used in Humanitas). The test will test the presence of mutations in the EGF receptor which are associated with responsiveness to specific TKI. Once these specific mutations are found, target therapy can be applied. Diagnostic applications These tests define the gene of interest and the hotspots, the portions of the gene where these mutations mostly occur. In the case of EGFR it is known that the mutation occurs in specific exons (18,18,20,21), so it is sufficient to define a PCR probe investigating these regions of the gene to have the response on the positivity or negativity to a specific mutation. Tests: - real-time PCR in which the sequences are amplified and then sequenced - digital droplet PCR which has a higher sensitivity, being able to identify very small amounts of mutated DNA ranging from 0.01% - next generation sequencing is not used to look for specific mutations that have already been identified and detected but is an unbiased sequence, so the DNA is extracted, a library is prepared and then it is sequenced. After the analysis, the specific mutations generated in the genome are provided. 11 Clinical condition with indications for liquid biopsy in advanced NSCLC Non-small cell lung cancer is considered since it has been approved by the FDA and EMA and is also used in Humanitas. Let’s imagine we have naive patients that cannot have a biopsy from the tumor tissue. We may test the ctDNA for the EGFR mutations: - EGFR-positive: EGFR- tyrosine kinase inhibitors (TKI) - EGFR-negative: no target therapy to propose so chemotherapy is used The second possibility is if there is a patient with a progressive disease after the administration of the TKI. There is the necessity to understand the mechanism of resistance to this type of target therapy. There is the need to investigate the presence of primary mutations of EGFR which may explain why it is not responding to TKI anymore. There could be a primary mutation which activates the cells that proliferate massively and then eventually there could be a secondary EGF receptor mutation which causes the overgrowth of the cancer cell. By applying the TKI there might be the death of cells which do not have this type of mutation and the survival and enrichment of cells that carry this mutation. There are key driver mutations of the EGF receptor that can be investigated through this approach like the T790M specific mutation. If we don’t detect a mutation then it might be in the 30% of the cases that it is false negative (70% sensitivity). With the false negatives, a tissue biopsy is needed to identify the mutation. If the mutation is found after the biopsy, again a new generation TKI (osimertinib) can be administered. There are several tyrosine kinase inhibitors and we have to apply them in a logical order depending on the mutation which is driving the tumour growth. Another scenario is the performance of a NGS on ctDNA that will help to identify any druggable alterations present in cancer cells that may provide 12  informations about other target therapies to use on the patients in other genes downstream in the signaling cascade, such as ALK and RET rearrangements and BRAF V600E mutations. Chart on past, present, future applications of liquid biopsy in advanced NSCLC In the past the main goal was to identify and quantify the mutations of EGF receptors, now there are standardised techniques that assess the presence of new mutations that are associated with resistance to TKI. At the moment, the NGS with an unbiased approach looking for any type of mutation in the signaling cascade is being validated. In the future it will need to enter the clinic, so there will be the need to provide standardised tests for all the mutations of the downstream cascade, to provide tools to perform NGS as a root in tests to detect a mutation. Then it will be necessary to identify the reasons for resistance of other therapies, like the immunotherapy based on immune checkpoints. There are patients that are resistant to this type of therapy so we have to identify the genetic markers in the tumor, which explain why the patient is not responsive anymore to immune checkpoint inhibitors, looking for the genetic associations with the lack of response to immunotherapy. There is also the study of acquired resistance mechanisms of molecules involved in the signalling cascade in cancer affecting MET, RET, ROS1, NTRK, BRAF, HER2 targeted agents. Microsatellite instability 12-20% of colorectal cancers have a high microsatellite instability. The presence of this type of instability has some implication in prognosis and response to immunotherapy. The genome is particularly unstable and new sequences are generated in the genome of the cancer cell because of the genetic instability. The higher is the mutational load of a cancer cell, the higher is the antigenic load of the cancer cell because any new sequence which is transcribed and translated into a protein will become a new antigen for the new system. When a cancer cell develops this will have an unstable genome, the portion of the genome will be translated in proteins which are abnormal with very important alterations like deletion or insertion of sequence so there might be a complete modification of the frame of reading and the generation of new short sequencing of AA which have nothing to do with the normal proteins. These aminoacidic sequences will behave as antigens for our immune system that has never encountered them. So when there is a tumour that is highly unstable in terms of 13 genome, this will also be a tumor highly responsive to immunotherapy, to immune checkpoint inhibitors. In general T cells will be massively activated by these cancer cells. Essentially the microsatellite instability is associated with poor prognosis in terms of aggressiveness to cancer and poor response to classical chemotherapy but a strong response to immunotherapy. Instability may be detected by immunohistochemistry or PCR. In a 2020 study it was demonstrated that MSI detected by droplet-digital PCR (ddPCR) in blood samples (ctDNA) from colorectal cancer patients showed a clinical specificity and accuracy of 100%. There is a commercially available test (PrecisionPlexTM MSI detection system) that is able to detect, by a PCR-based approach, sequences that differ in microsatellite loci compared to a normal individual. So by PCR there is the amplification of regions of microsatellite instability (peaks) in a patient with cancer (red arrows) but not in a normal patient. A tissue biopsy is performed on a tumour, the DNA of the biopsy is collected and a PCR is performed, obtaining an amplification of the region collected by the PCR. If a different form of widespread analysis is performed, we may have an increase in the number of loci that we may investigate and we may analyse only the region of the tumour we have collected. Since tumours are heterogeneous, when we collect a liquid biopsy from the circulation we may capture the complexity of the tumour cells; so by PCR we may have the same few loci but we catch the heterogeneity because we are capturing the cells of the entire tumour. If a massive sequence of the region of the microsatellite instability is performed, we may detect both the heterogeneity of the tumor and the several loci involved in this microsatellite instability. If we perform a massive sequencing instead of PCR in a liquid biopsy, instead of a tissue biopsy, we will have much more information about the genetic alteration of the cancer cells. 14 miRNA There are several families of miRNA. They are involved in the regulation of transcription of genes, regulation of cell cycle and proliferation or apoptosis, some of them regulate the growth, others work as suppressors, others are involved in epithelial to mesenchymal transition. We can look at miRNA as markers of specific alterations associated with the behaviour of a tumor. The application of circulating miRNA in clinics is quite far. They may be used for the diagnosis of specific tumours, to classify them, to have prognostic and predictive values for therapy response. Example: synthetic miRNA can be used inside lipid nanoparticles and then suppress the miRNA downstream of p53. We may target them and since miRNA are controlling the activity downstream of p53, we may use them as a therapeutic drug, charging them in lipid nanoparticles and then providing them as drugs. Exosomes are particles released by cells, which contain DNA, mRNA, noncoding-RNA, microRNA, proteins, lipids and we may collect exosomes from the circulation. They are a cargo of information including circulating tumor DNA and markers coming from the tumors. They could be used in the future as tumour biomarkers more than the circulating tumor cells. Liquid biopsy overview Collection from the blood of ctDNA or CTC that we find in the plasma and among the cells. - In case of ctDNA we may investigate the mutations, the translocations, the chromosomal aberrations, deletions, insertions, amplifications and the epigenetic marks that are important for detecting the origin of the tumor and classifying the cell types that originate in the tumour. Then we can detect the secreting tumor DNA and we can quantify it, because the quantification is associated with the tumor burden and prognosis. - Secondly, there is the analysis of the cancer’s circulating tumour cells, these are needed to be enriched in a positive or negative manner using antibodies or physical properties. Then, once we have enriched them, we may investigate them through immunocytology using specific biomarkers to define the origin of these cells, their features in terms of protein expression and in terms of sequence, identifying by PCR the different genes or mutations associated with these cells. Functional studies may then be performed in vitro, followed by comprehensive characterization of the CTCs. 15  Then investigation of the genome with different approaches of sequencing to detect the genome, the proteome, the transcriptome is done and finally the function of the cell generating a xenograft is performed. ctDNA: clinical applications - determination of the minimal residual disease MRD - investigation of the heterogeneity of the cells, so the appearance of different clones - identification of the recurrence - identification of mechanisms of residence to specific therapies - Motitoration of the tissue of origin of the mutation and of the primary tumor mutation. With a particular target therapy, cells sensitive to therapy will be eliminated while the resistant ones will overgrow so that the heterogeneity will appear during the course of the disease There are two tables where there are listed advantages and disadvantages in using these types of approaches. 16  CtDNa- driven clinical trials We may use ctDNA to follow a clinical trial, we may need to investigate the mutation detected in the ctDNA to decide which target therapy we can use for the patient. There are different schemes which are possible to be applied in clinical trials and we may decide, based on the presence or not of a mutation in the ctDNa so depending on the presence or absence of a biomarker, to apply the new target therapy if is known for the particular ctDNA analysis or to apply the standard of care therapy also. After the comparison of the results there is the decision, analyzing the number of patients, whether the Health system should invest in providing these approaches and biomarkers that are really changing the life of a lot of patients. When there is the identification of a new biomarker someone has 17 to provide its quality, clinical grade and the Health system will have to pay for it for its entry into the clinical practice. Development of new biomarkers Different biomarkers are collected from the liquid biopsy or the tissue biopsy or through the other possible techniques like genomics, transcriptomics, proteomics and metabolics. This data are analysed in a complex and then are integrated. At the end, the final application will be the use of biomarkers or the information about the pathogenesis (of cancer) in clinics. 18