METHOD FOR PREDICTING THE RESPONSE OF A CANCER PATIENT TO IMMUNOTHERAPY TREATMENT

Abstract

The invention relates to an in vitro method for predicting the early progressor character of a subject suffering from cancer during an immunotherapy treatment. This method comprises determining, in a blood sample isolated from the subject, the total concentration of circulating free DNA and a concentration of large circulating free DNA fragments, combining these data in a mathematical function so as to obtain a score, and comparing this score to a predetermined reference value. It concludes that the subject has an early progressor character during the immunotherapy treatment when the score obtained is less than or equal to the reference value.

Description

Title of the invention: METHOD FOR PREDICTING THE RESPONSE OF A CANCER PATIENT TO AN IMMUNOTHERAPY TREATMENT

[0001] The present invention falls within the general field of oncology.

[0002] More particularly, the present invention relates to an in vitro method for predicting the early progressor character and / or the early non-progressor character of a subject suffering from cancer during the administration of an immunotherapy treatment.

[0003] Cancer treatment has been the focus of much research for decades. In recent years, a particular effort has been made to develop more individualized therapeutic strategies.

[0004] The approach aimed not at directly attacking the tumor, but at stimulating the immune system, known as immunotherapy, is today recognized as one of the most effective therapeutic strategies for the treatment of a large number of types of cancer. For certain types of cancer, immunotherapy is used routinely, with spectacular results, particularly for small cell lung cancer. Thus, a meta-analysis revealed that 25% of patients treated with immunotherapy showed a response to treatment, prolonging survival. In multivariate analysis, it was also shown that durable responses were more frequent in patients treated with immunotherapy, i.e., their overall survival was improved (Pons-Tostivint et al., 2019, JCO Precision Oncology, 3(3): 1-10).

[0005] However, immunotherapy treatments do not show efficacy in all patients. In addition, they can cause adverse effects in some. For these patients, conventional chemotherapy would have been a better option. These are referred to as patients who are non-responders to immunotherapy treatment. This information is often obtained late, several weeks after the start of immunotherapy treatment, weeks which can lead to a deterioration in the patient's health and significantly reduce their life expectancy. Identifying patients in whom immunotherapy will be effective is a necessity now recognized by the entire immunotherapy community.

[0006] No method currently makes it possible, before starting an immunotherapy treatment, to identify / classify cancer patients who will respond or not respond to this treatment, more precisely to identify patients for whom this treatment will, or will not, prevent progression. of the tumor. A reliable, standardized biomarker that can be used in clinical practice to predict treatment response and assess future efficacy has not yet been identified. However, the existence of such a predictive marker would improve the efficiency of patient care and its underlying ethics, by preventing patients from losing their chance of recovery and unnecessary adverse effects. In addition, new immunotherapy treatments are sold at very high prices. The cost of CAR-T cell treatment is approximately €350,000 per patient, and that of immunomodulator treatment is approximately €75,000 per year. These costs obviously pose problems of access and management, especially since these treatments are only effective in a fraction of patients and do not always provide a major benefit.

[0007] To date, two markers are used to predict the responsiveness of patients to immunotherapy treatments, for information purposes only because the predictive nature of these markers is limited: - the Combined Positive Score (CPS), which assesses the expression of PD-L1 (programmed death ligand-1) in tumor cells and immune cells - this score is equal to the total number of cells marking PD-L1 x 100, divided by the number of viable tumor cells; - and the Tumor Proportion Score (TPS), which only takes into account the expression of PD-L1 by tumor cells - this score is equal to the number of tumor cells marking PD-L1) x 100, divided by the number of viable tumor cells.

[0008] These CPS and TPS markers nevertheless require the implementation of protocols that are often specific to each care center, and no standardization has been carried out to date, which leads to differences in the values of the scores and thresholds applied in the different care centers.

[0009] The present invention aims to provide a method for predicting, with high precision, how a patient suffering from cancer will respond, more precisely whether his tumor will progress early or not, when he is subjected to immunotherapy treatment, before the start of such treatment.

[0010] Additional objectives of the invention are that this method is easy to implement, using equipment commonly available in analysis laboratories, and that it can be applied to the greatest possible number, on the one hand, of types of cancer, and on the other hand, of types of immunotherapy treatments.

[0011] Aiming to develop such a method, the present inventors were interested in the circulating free deoxyribonucleic acid (DNA) present in the blood of patients. This free DNA circulating in the blood plasma is currently the subject of intense clinical research in oncology, because mutations of genomic DNA can be found there present in tumor cells, which guide the therapy to be administered to the patient. For convenience, this circulating free deoxyribonucleic acid will be referred to in this description as “circulating free DNA” or cfDNA.

[0012] It is clearly established that blood carries a small amount of circulating free DNA originating from the release of genetic material from tissues. This cell-free circulating free DNA is in the form of double-stranded DNA with an average size of 150-180 base pairs (bp), corresponding to the coiling of DNA into a nucleosome. Its lifespan is less than two hours, before it is filtered and eliminated from the bloodstream by the spleen, liver and kidneys. All studies agree on a lower quantitative detection of circulating free DNA in healthy individuals and its increase linked to different clinical situations such as stroke and myocardial infarction, intensive muscular exercise, acute renal failure, hepatic cytolysis, trauma, surgery, cancer, the presence of a fetus during gestation...

[0013] In the case of cancer, recent advances in molecular biology and DNA sequencing have made it possible to identify numerous mutations in genes involved in oncogenesis from a particular type of circulating DNA, circulating tumor DNA (ctDNA). In particular, it has been shown that the level of ctDNA would be correlated with the tumor burden and the stage of the cancer. Generally speaking, the studies carried out focus on the use of ctDNA as a tumor biomarker making it possible to monitor the positive or negative progress of the patient during their therapeutic treatment (immunotherapy or others).

[0014] Studies correlating circulating DNA with patient outcome during immunotherapy treatment have been described in the literature. Most of these studies focus on post-treatment monitoring, tracking the level of tumor burden either by measuring ctDNA using standard techniques such as polymerase chain reaction (PCR) or sequencing, or by direct examination of the tumor using imaging.

[0015] From a more general point of view, studies carried out on circulating DNA focus on small DNA fragments, less than 250 base pairs, which are considered to carry the main information, and more precisely on sizes between 70 and 150 base pairs, which are enriched in ctDNA. ctDNA is thus often proposed as an indicator of the progression of the disease.

[0016] It has now been discovered by the present inventors that the analysis of the size profile of the total circulating free DNA of subjects suffering from cancer makes it possible to predict their future response to immunotherapy treatments, more precisely to determine whether or not the subject will be an early progressor when such treatment is administered to him, this upstream of this administration.

[0017] In the present description, the term “early non-progressor subject”, or subject with early non-progressor characteristics, conventionally in itself in the medical field, is understood to mean a subject suffering from cancer for whom there is observed, on the first image of the tumor acquired by medical imaging, in particular by X-ray scanner, after the start of the immunotherapy treatment, compared to the image acquired by the same medical imaging technique before the start of this treatment, an increase of less than 20% of the tumor lesions identified on the imaging, that is to say that the tumor lesions have decreased, have remained stable or have increased by less than 20% (in surface area). We then speak more commonly of progression of less than 20% of the cancerous tumor on the first imaging evaluation after the start of the immunotherapy treatment.

[0018] The time of acquisition of the first medical imaging image after the start of immunotherapy treatment is determined according to the conventional protocol for the management of cancer patients in force in the field, and depends on the particular type of cancer concerned. Typically, this time is 6 weeks after the start of immunotherapy treatment for lung cancers, and 12 weeks for other cancers.

[0019] In contrast, an early progressor, or a subject with an early progressor character, also conventionally in itself in the medical field, is understood to mean a subject suffering from cancer for whom an increase of more than 20% of the tumor lesions identified on the imaging is observed on the first image of the tumor acquired by medical imaging, in particular by X-ray scanner, after the start of the immunotherapy treatment, compared to the image acquired by the same medical imaging technique before the start of this treatment, i.e. the tumor lesions have increased by 20% or more (in surface area). We then more commonly speak of progression of more than 20% of the cancerous tumor on the first imaging assessment after the start of the immunotherapy treatment.

[0020] As indicated above, circulating free DNA also means the extracellular DNA present in the patient's blood plasma. This circulating free DNA includes circulating tumor free DNA and circulating non-tumor free DNA.

[0021] More particularly, it has been discovered by the present inventors that patients with early non-progressor characteristics, for whom the tumor does not progress or progresses little, or even regresses, in the initial period of an immunotherapy treatment, in the first weeks of this treatment, have a higher level of large circulating free DNA fragments than patients with early progressor characteristics. Thus, the concentration, in particular the relative concentration, of circulating free DNA fragments of a patient suffering from cancer, the size of which is greater than 500 base pairs, constitutes a parameter which can be used in a method making it possible to determine to determine whether this patient will be an early progressor or an early non-progressor when receiving immunotherapy treatment. Nothing in the prior art, which only looks at small circulating free DNA fragments, well below 500 base pairs, suggested such a result.

[0022] Thus, according to the present invention, there is provided an in vitro method for predicting the early progressor character and / or the early non-progressor character of a subject suffering from cancer during an immunotherapy treatment, i.e. when an immunotherapy treatment will be administered to him. This method falls under statistical analysis methods. It comprises steps of: a / determination, in an isolated blood sample which has been obtained from said subject, preferably before said immunotherapy treatment: - the total concentration of free deoxyribonucleic acid circulating in said blood sample, called total cfDNA concentration, - at least one concentration, in said blood sample, of circulating free deoxyribonucleic acid fragments whose size is included in a predetermined size range, said predetermined size range, which will be designated in the present description by the expression "analytical size range", being included in sizes greater than 500 base pairs, the concentration thus determined being called in the present description "large cfDNA concentration", b / combination in a mathematical function of at least said large cfDNA concentration, and said total cfDNA concentration, so as to obtain a score, this score reflecting the probability that the subject will or will not have an early progressor characteristic and / or an early non-progressor characteristic during the immunotherapy treatment, And : cl / comparison of this score with a first predetermined reference value, and dl / conclusion that the subject presents an early progressor character during immunotherapy treatment when this score is less than or equal to this first reference value, and / or c2 / comparison of this score with a second predetermined reference value, and d2 / conclusion that the subject presents an early non-progressor character during immunotherapy treatment when the score is higher than this second reference value.

[0023] In particular embodiments of the invention, the method is an in vitro method for predicting the early progressor character of a subject suffering from cancer during an immunotherapy treatment, which comprises steps of: a / determination, in an isolated blood sample which has been obtained from said subject, preferably before said immunotherapy treatment: - the total concentration of free deoxyribonucleic acid circulating in said blood sample, called total cfDNA concentration, - at least one concentration, in said blood sample, of circulating free deoxyribonucleic acid fragments whose size is included in a predetermined size range, said predetermined size range, which will be designated in the present description by the expression "analytical size range", being included in sizes greater than 500 base pairs, the concentration thus determined being called in the present description "large cfDNA concentration", b / combination in a mathematical function of at least said large cfDNA concentration, and said total cfDNA concentration, so as to obtain a score, this score reflecting the probability that the subject will or will not have an early progressor characteristic during the immunotherapy treatment, cl / comparison of this score with a first predetermined reference value,and dl / conclusion that the subject presents an early progressor character during immunotherapy treatment when this score is less than or equal to this first reference value.,

[0024] In particular embodiments of the invention, the method is an in vitro method for predicting the early non-progressor character of a subject suffering from cancer during an immunotherapy treatment, which comprises steps of: a / determination, in an isolated blood sample which has been obtained from said subject, preferably before said immunotherapy treatment: - the total concentration of free deoxyribonucleic acid circulating in said blood sample, called total cfDNA concentration, - at least one concentration, in said blood sample, of circulating free deoxyribonucleic acid fragments whose size is included in a predetermined size range, said predetermined size range, which will be designated in the present description by the expression "analytical size range", being included in sizes greater than 500 base pairs, the concentration thus determined being called in the present description "large cfDNA concentration", b / combination in a mathematical function of at least said large cfDNA concentration, and said total cfDNA concentration, so as to obtain a score, this score reflecting the probability that the subject presents or does not present an early non-progressor character during the immunotherapy treatment, c2 / comparison of this score to a second predetermined reference value, and d2 / conclusion that the subject presents an early non-progressor character during immunotherapy treatment when the score is higher than this second value of reference.

[0025] The method according to the invention may otherwise be an in vitro method for predicting the early progressor character and the early non-progressor character of a subject suffering from cancer during an immunotherapy treatment. It then comprises, after the step of obtaining the score: - a step cl / of comparing this score to the first predetermined reference value, and a step dl / of concluding that the subject presents an early progressor character during the immunotherapy treatment when this score is less than or equal to this first reference value, And - a step c2 / of comparing this score to the second predetermined reference value, and a step d2 / of concluding that the subject presents an early non-progressor character during the immunotherapy treatment when the score is higher than this second reference value.

[0026] As indicated above, the method according to the invention, which takes into consideration the content of large cfDNA contained in the blood sample from the subject, makes it possible to predict, with high precision, the early non-progressor character and / or the early progressor character with respect to an immunotherapy treatment of a patient suffering from cancer, even before the start of such treatment. Such a method proves to be particularly advantageous in that it makes it possible, for example, to avoid administering the immunotherapy treatment to patients who have been predicted as early progressors and / or who have not been predicted as early non-progressors, for whom this treatment will not have the desired efficacy.

[0027] The precision (or accuracy) of prediction of the method according to the invention was notably verified in a prospective observational study carried out on a cohort of 51 patients suffering from different types of cancer, and having been subjected to different types of immunotherapy treatments (the method according to the invention having been implemented on blood samples taken from the patients before the start of these treatments).

[0028] Conventionally in itself, the term precision here designates the proportion of patients having been correctly classified by the method according to the invention.

[0029] The performance of statistical prediction methods is generally evaluated by plotting a receiver operating characteristic curve (ROC curve) and measuring the amount under the curve (AUC). The ROC curve is established by plotting the sensitivity versus (1-specificity) after classification of patients, based on the results obtained by the prediction method. The closer the AUC value is to 1, the higher the sensitivity and specificity of the method, and the more efficient the method is. In the study on the aforementioned cohort of patients, a method according to an implementation mode The implementation of the invention allowed to predict an early progressor character of patients with an AUC as high as 0.833, which clearly demonstrates its good performance.

[0030] “Sensitivity” here means, in a conventional manner, the probability of the process of identifying as positive early progressors (or, as the case may be, early non-progressors), i.e., to identify true positives. "Specificity" refers to the probability that the procedure will not identify early non-progressors (or, as the case may be, early progressors) as positive, i.e., not to identify true negatives.

[0031] Furthermore, in the present description, the term "positive predictive value" (PPV) means the probability for a subject to be an early progressor (or, as the case may be, an early non-progressor) when the result of the method predicts it as such. The term "negative predictive value" (NPV) means the probability for a subject to be an early non-progressor (or, as the case may be, an early progressor) when the result of the method predicts it as such.

[0032] Each of the reference values used in the method according to the invention is preferably a threshold value, also commonly referred to as a “cut-off value”. It is within the skill of a person skilled in the art to know how to establish such reference values, in particular such threshold values, for the prediction method according to the invention, depending on the level of sensitivity and specificity required for each prediction. Such a determination can be carried out either empirically or theoretically.

[0033] In particular embodiments of the invention, said first reference value and said second reference value are identical, and designated by the expression "common reference value". Then, the method comprises a step of concluding that: - if the score is less than or equal to the common reference value, the subject presents an early progressor character during immunotherapy treatment, and / or - if the score is higher than the common reference value, the subject presents an early non-progressor character during immunotherapy treatment.

[0034] In alternative embodiments of the invention, the first reference value and the second reference value are different, the first reference value being less than the second reference value. The method may then comprise a step of concluding that: - if the score is less than or equal to the first reference value, the subject presents an early progressor character during immunotherapy treatment, - if the score is higher than the second reference value, the subject presents a early non-progressor character during immunotherapy treatment, - if the score is higher than the first reference value and lower than or equal to the second reference value, the method does not allow the subject's response to immunotherapy treatment to be satisfactorily predicted.

[0035] The prediction method according to the invention, the results of which are based on a statistical analysis, advantageously allows in particular: - a prediction-stratification before therapy of early non-progressor patients and / or early progressor patients during immunotherapy treatment, thus allowing personalized care; - for the patient, a saving of time increasing the chances of success of his therapy, by avoiding side effects or induced effects; - simplicity and speed of implementation by analysis carried out from a blood sample already taken from the patient for other purposes; - a reduction in the very significant costs of immunotherapy treatments, which can be targeted only at patients predicted as early non-progressors by the method according to the invention.

[0036] The blood sample used in the method according to the invention has preferably been isolated from the subject by a blood sample taken before the start of the immunotherapy treatment. The invention does not, however, exclude that this blood sample has been taken at the time of the administration of this treatment, or after, preferably just after, within a few hours or a few days following the start of the administration of the treatment.

[0037] The method according to the invention is in itself devoid of any step applied to the patient's body. The analysis steps it involves are carried out using whole blood samples which have been taken from the patient beforehand, in a conventional manner in itself.

[0038] The subject to which the method according to the invention is applied is preferably a mammal. It is preferably a human.

[0039] The method according to the invention may furthermore meet one or more of the characteristics described below, implemented in isolation or in each of their technically effective combinations.

[0040] The mathematical function implemented in the method according to the invention can be any type of multivariate function.

[0041] In addition to the variables that are the large cfDNA concentration and the total cfDNA concentration, it can use other variables. In particular, it can use, as variables, several different large cfDNA concentrations, each corresponding to a predetermined analytical size range and different from the others. It is recalled that for the purposes of the present invention, the expression "analytical size range" means any size range within the sizes greater than 500 base pairs. It may also or otherwise use, as variables, other clinical or biological information relating to the patient.

[0042] Each of the variables contained in the mathematical function according to the invention can be weighted by a coefficient which is specific to it and which can be equal to 1, or different from 1.

[0043] In particular embodiments of the invention, the mathematical function is the ratio between at least said concentration of large cfDNA, and said concentration of total cfDNA.

[0044] This is in particular the ratio between a concentration of large cfDNA and the concentration of total cfDNA. This function can then be described as the relative concentration of cfDNA fragments whose size is included in the predetermined analytical size range, present in the blood sample of the subject studied. As indicated above, it has been discovered by the present inventors that such a relative concentration constitutes an indicator / biomarker making it possible to effectively predict the response (in terms of early progression or not of the tumor) of a patient suffering from cancer when subjected to immunotherapy treatment.

[0045] The mathematical function implemented according to the invention may otherwise be equal to the sum of different concentrations of large cfDNA, each being associated with a predetermined analytical size range, this sum being divided by the total cfDNA concentration.

[0046] It can otherwise combine the above variables in any other way, and in particular be part of the framework of linear regression.

[0047] In particular embodiments of the invention, each predetermined size range, or analytical size range, is defined solely by its minimum value, i.e., it is a range defined as including sizes greater than, or greater than or equal to, a given minimum value.

[0048] Thus, in particular embodiments of the invention, at least one predetermined size range / analytical size range is the size range greater than 500 base pairs, preferably the size range greater than or equal to 580 base pairs, more preferably the size range greater than or equal to 600 base pairs, and even more preferably the size range greater than or equal to 1500 base pairs. Preferably, it is the size range greater than or equal to 1600 base pairs, or even greater than or equal to 1650 base pairs, or even greater than or equal to 1700 base pairs.

[0049] In particular alternative embodiments of the invention, each predetermined size range, or analytical size range, is defined by its value minimum and its maximum value, that is, it is a range defined as including sizes greater than, or greater than or equal to, a given minimum value, and less than, or less than or equal to, a given maximum value. In such a configuration, the higher and / or wider size ranges are more particularly preferred within the scope of the invention, compared to the lower and / or less wide size ranges.

[0050] In particular embodiments of the invention, at least one predetermined size range / analytical size range is the size range between 580 and 1649 base pairs.

[0051] The mathematical function used according to the invention can combine all combinations of the variables each associated with a range of analytical size which is specific to it, which are mentioned above.

[0052] Among the predetermined size ranges particularly preferred in the context of the invention, in the context in which the mathematical function implemented is the ratio between a concentration of large cfDNA (i.e. cfDNA fragments in the predetermined size range) and the total cfDNA concentration, examples of analytical size ranges include the size range greater than or equal to 1650 base pairs, and the size range from 580 to 1649 base pairs.

[0053] The score obtained by the method according to the invention advantageously constitutes a generic indicator of the early progressor character and / or the early non-progressor character of a subject studied, independent of both the type of cancer from which the subject is suffering and the type of immunotherapy treatment which could be administered to him.

[0054] In particular, the method according to the invention advantageously makes it possible to predict the early progressor character and / or the early non-progressor character of a subject suffering from any type of cancer, and in particular, but not limited to, a metastatic tumor pathology, a melanoma, a kidney cancer, in particular clear cell cancer, a urothelial carcinoma of the bladder, a squamous cell carcinoma of the head and neck, or even a small cell or non-small cell bronchial cancer, or a plurality of such cancers.

[0055] The immunotherapy treatment for which the aim is to know the early progressor character and / or the early non-progressor character of the subject can be of any type. It can in particular use nivolumab, ripilimumab, pembrolizumab and / or one or more anti-PD-Ll antibodies such as atezolizumab, avelumab and / or durvalumab.

[0056] The prediction by the method according to the invention is furthermore also effective with regard to configurations in which the immunotherapy treatment is carried out jointly with other therapeutic treatment methods, such as a chemotherapy treatment and / or targeted therapy when possible.

[0057] Obtaining the blood sample from the subject, from which the method according to the invention is applied, may have been carried out in any conventional manner. It may, for example, have been carried out by taking a blood sample from the subject in tubes containing a buffer based on ethylenediaminetetraacetic acid (EDTA) or in specific tubes for obtaining circulating DNA, such as the tubes marketed by Streck under the name Cell-Free DNA BCT® (Streck) or by Roche Diagnostic under the name “Cell-free DNA collection tube”, according to the supplier's recommendations.

[0058] In particular embodiments of the invention, the determination of said total cfDNA concentration and the determination of said at least one large cfDNA concentration are carried out by direct analysis of the blood sample isolated from the subject, i.e. a whole blood sample.

[0059] In preferred embodiments of the invention, the method comprises a prior step of obtaining a plasma sample from this whole blood sample.

[0060] The step of selectively obtaining the plasma sample from the whole blood sample isolated from the patient by blood collection is preferably carried out within a few days following the blood collection, for example within 6 hours after the blood collection if EDTA buffer tubes are used, and within 7 days after the blood collection if specific tubes for obtaining circulating free DNA are used, such as the tubes mentioned above.

[0061] This step can be carried out in any conventional manner, for example by centrifugation.

[0062] In order to limit the release of cellular DNA contained in the circulating cells present in the whole blood sample, which would cause the dilution of the circulating free DNA, a protocol including two centrifugations is preferably favored according to the invention. Preferably, the step of obtaining the plasma sample, from a whole blood sample obtained from the patient, of the method according to the invention thus comprises: - a first gentle centrifugation, for example at a speed between 1200 and 1600 g, preferably at room temperature, i.e. at 20°C + / - 5°C; - recovery of the plasma (supernatant), without removing the cell wafer separating the plasma and the red blood cells; - a second centrifugation at a faster speed, for example between 3000 and 16000 g, preferably also at room temperature; - and the recovery of the plasma (supernatant), for example by aspiration, without removing the pellet formed.

[0063] Regardless of the aforementioned tubes, the plasma sample thus obtained can typically be stored at -20°C for a period of less than or equal to 1 month, or at -80°C for periods of more than 1 month, before its analysis for the determination of the different cfDNA concentrations necessary for the implementation of the method according to the invention.

[0064] The determination of the total cfDNA concentration and the determination of the large cfDNA concentration(s) in the blood sample can be carried out by any method conventional in itself for those skilled in the art. Depending on the particular technique implemented, they can be carried out directly on the whole blood sample or on the plasma sample obtained from this blood sample, or after a step of extracting the cfDNA from one or other of these samples.

[0065] Thus, in particular embodiments of the invention, which are particularly advantageous in terms of the speed and simplicity of implementation of the method according to the invention, the determination of said total cfDNA concentration and the determination of said at least one large cfDNA concentration are carried out by direct analysis of the plasma sample obtained from the whole blood sample isolated from the subject, i.e. they are carried out directly on this sample itself, without a prior DNA extraction step.

[0066] In alternative particular embodiments of the invention, the method comprises a step of extracting the circulating free deoxyribonucleic acid from the blood sample, or where appropriate from the plasma sample obtained from this blood sample, so as to obtain an extract containing the circulating free deoxyribonucleic acid contained therein. The determination of the total cfDNA concentration and the determination of said at least one large cfDNA concentration are then carried out by analyzing the circulating free deoxyribonucleic acid extract thus obtained.

[0067] The step of extracting the cfDNA from the blood sample, and where appropriate from the plasma sample, can be carried out in any conventional manner, for example by a technology using magnetic beads, or by means of a kit marketed for this purpose, by the companies IDSolutions (IDXtract Kit), Promega (Maxwell® RSC ccfDNA plasma Kit) or Qiagen (QIAamp Circulating Nucleic Acid Kit) for example.

[0068] The analysis of the blood sample, the plasma sample, or the cfDNA extract obtained according to the invention, to determine the total cfDNA concentration and one or more large cfDNA concentrations (each associated with a different analytical size range) can be carried out by any technique known to those skilled in the art for this purpose.

[0069] It can, for example, be carried out by polymerase chain reaction (PCR), or by capillary electrophoresis technology, using a commercially available device, such as the Fragment Analyzer and 7100 CE system from Agilent Technologies, the QIAxcel offered by the company Qiagen, or the GenomeLab system from the company Sciex. These techniques require prior extraction of the circulating free DNA from the blood sample or the plasma sample.

[0070] Preferably, the determination of the different concentrations of cfDNA is carried out by a technique based on the so-called pLas technology, as described in particular in documents WO 2016 / 016470, WO 2014 / 020271, or the publication by Ranchon et al., Lab Chip, 2016, 16(7): 1243-53. Schematically, the pLas technology operates in two stages respectively of concentration and separation carried out online. First, the DNA is concentrated via a capillary system formed by the junction of a small capillary and another capillary of larger section. The solution containing the DNA is made to flow in a laminar manner in the large capillary, and an electric field is used to slow down the migration. Due to the shear provided by the laminar flow, this counter-electrophoresis causes a transverse force, dependent on the size of the DNA, which pushes the DNA towards the walls.The change in flow speed and electric field at the constriction stops the DNA and concentrates it like a pancake. Indeed, upstream of the constriction, the flow and counter-electrophoresis are slow, causing a weak transverse force. The DNA is therefore in the mass of the flow, and advances towards the constriction. On the other hand, downstream of the constriction, the flow and counter-electrophoresis are fast, strongly pressing the DNA to the wall where the laminar flow is very weak, and where counter-electrophoresis dominates. The DNA then moves back towards the constriction, by counter-electrophoresis, along the wall. This pancake is then released by the progressive decrease in the electric field, which also allows the separation operation to be carried out according to the size of the fragments.

[0071] The analysis of the blood sample, the plasma sample, or the cfDNA extract obtained according to the invention, to determine the total cfDNA concentration and one or more large cfDNA concentrations (each associated with a different analytical size range) can in particular be carried out by the method, based on the pLas technology above, as described in document FR 3128231 or the publication by Boutonnet et al., Analytical Chemistry, 2023, 95(24): 9263-70. This method has the advantage of being able to be implemented directly on a plasma sample, without prior extraction of the circulating DNA contained therein, and it advantageously makes it possible to easily and economically determine the characterization of the size profile of the cfDNA with a sen increased reliability compared to other analysis techniques. This method can in particular be implemented using the device described in documents WO 2017 / 009566 and Andriamanampisoa et al., Analytical Chemistry, 2018, 90(6): 3766-74, and marketed under the name Biabooster by Adelis Technologies.

[0072] In essence, this method comprises at least one iteration of an alternation: - a step of laminar flow of the sample to be analyzed in a capillary in a first direction of flow, the capillary being provided with at least one local restriction of its section and comprising an analysis buffer, during which the sample is subjected to a first electrical potential difference whose action on the nucleic acid molecules is opposite to the first direction of flow and causes the retention of nucleic acid molecules in the capillary, - a laminar flow step of the sample, in a second flow direction opposite to the first flow direction, and, after the last iteration of the alternation, a step of separation by laminar flow of the sample in the capillary in the first direction of flow, during which the sample is subjected to an electrical potential difference less than or equal to the first potential difference, the action of which on the nucleic acid molecules is opposite to the first direction of flow and causes partial retention of nucleic acid molecules in the capillary. This method further comprises, during or after the separation step, a step of measuring a fluorescence time profile of the fluorescent nucleic acid molecules and a step of converting the fluorescence time profile into a concentration profile of nucleic acid molecules of different lengths, by implementing a fluorescence profile of a standard sample, the concentration of which is known for each length of fluorescent nucleic acid molecule present in the sample. The nucleic acid molecules were made fluorescent by one of the techniques known to those skilled in the art, for example by adding to the analysis buffer an intercalating fluorophore, that is to say a molecule which fluoresces little in the free state, and which fluoresces a lot when it is intercalated between the bases of the DNA. The guaranteed technical uncertainty of such a method, based on pLas technology, is advantageously 5 to 10%. The size repeatability is better than 1%. In particular, this method advantageously allows the determination of the total cfDNA concentration and the concentration(s) of large cfDNA in the plasma sample with a very good degree of approximation.

[0073] It has further been discovered by the present inventors that the score obtained within the framework of the prediction method according to the invention also constitutes a good statistical indicator of the prognosis of overall survival without progression of the subject suffering from cancer. treated with immunotherapy, as well as its 3-year survival.

[0074] The characteristics and advantages of the invention will appear more clearly in the light of the following examples of implementation, provided for purely illustrative purposes and in no way limiting the invention, with the support of figures 1 to 8, in which:

[0075] [Fig-1] [Fig.l] shows a curve representing the fluo intensity profile time-dependent growth, obtained by analyzing, using a method based on pLas technology, a plasma sample from a cancer patient containing cfDNA.

[0076] [Fig.2] [Fig.2] shows a curve representing the intensity profile of fluo rescence as a function of time, obtained by analysis, by a method based on pLas technology, of a standard sample containing DNA fragments of known size and concentration.

[0077] [Fig.3] [Fig.3] shows a curve representing the DNA concentration profile circulating free, expressed in picograms per pl per base pair (bp), as a function of size, obtained from the curve in [Fig.l].

[0078] [Fig.4] [Fig.4] shows a histogram representing the distribution of the circulating free DNA concentration for different size ranges, obtained from the curve in [Fig.3].

[0079] [Fig.5] [Fig.5] represents ROC curves relating to the prediction of the character of early progressor of a patient suffering from cancer during immunotherapy treatment, obtained for an indicator according to the invention, the relative concentration of cfDNA fragments of size greater than or equal to 1650 bp in a plasma sample of said patient, in a / for a training batch, in b / for a test batch, and in c / for the total cohort; for each curve, the area under the curve (“AUC”) is indicated in the figure.

[0080] [Fig.6] [Fig.6] represents ROC curves relating to the prediction of the character of early progressor of a patient suffering from cancer during immunotherapy treatment, obtained for an indicator according to the invention, the relative concentration of cfDNA fragments of size between 580 and 1649 bp in a plasma sample of said patient, in a / for a training batch, in b / for a test batch, and in c / for the total cohort; for each curve, the area under the curve (“AUC”) is indicated in the figure.

[0081] [Fig.7] [Fig.7] represents the box plots obtained for a cohort of 51 patients for two indicators according to the invention, in a / the relative concentration of cfDNA fragments of size greater than or equal to 1650 bp in a plasma sample of said patient, in b / the relative concentration of cfDNA fragments of size between 580 and 1649 bp in this plasma sample.

[0082] [Fig.8] [Fig.8] shows a graph representing, as a function of time, the curve of progression-free survival of cancer patients who have undergone immunotherapy treatment, determined by the Kaplan-Meier method, according to whether they have a score higher (light curve) or lower (dark curve) than the threshold value of 0.034, this score being predicted by a method according to the invention taking into account the relative concentration of cfDNA fragments of size greater than or equal to 1650 bp in the plasma samples of the patients; the number of patients (“individuals at risk”) from the initial cohort included in the study at each evaluation time, respectively with a score lower than 0.034 (top line) and a score higher than 0.034 (bottom line) is indicated in the table below the graph.

[0083] This study is based on a cohort of 51 patients with 5 types of cancer pathologies: melanoma (20%), ENT (squamous cell carcinoma of the head and neck) (20%), lung (non-small cell) (21%), kidney (18%), bladder (21%).

[0084] The response to immunotherapy treatment of patients refers to the response to the treatments used which are: nivolumab, ipilimumab and / or pembrolizumab, these treatments being able to be combined with targeted therapy, the administration of anti-PD-Ll antibodies or chemotherapy treatment.

[0085] The characteristics of the patients in the cohort at the start of the study are summarized in Table 1.

[0086] [Tables 1] Characteristics All Bladder ENT Kidney Melanoma Lung Number of patients 51 11 10 9 10 11 Average age (years) 65 68 60 64 66 66 Male 39 8 8 7 7 9 Female 12 3 2 2 3 2 Treatment performed Nivolumab 13 0 9 0 1 3 Pembrolizumab 13 9 0 2 0 2 Nivolumab + ipilimumab 10 0 0 1 9 0 Nivolumab + targeted therapy 2 1 1 0 0 0 Pembrolizumab + targeted therapy 6 1 0 5 0 0 Pembrolizumab + anti-PD-L1 1 0 0 1 0 0 Pembrolizumab + chemotherapy 5 0 0 0 0 5 Pembrolizumab + chemotherapy + targeted therapy 1 0 0 0 0 1 Number of metastases 0 12 1 6 0 2 3 1 12 4 2 4 1 1 2 16 4 1 4 5 2 3 6 1 1 1 1 2 4 3 1 0 0 0 2 6 1 0 0 0 0 1 7 1 0 0 0 1 0

[0087] Table 1 - Characteristics of patients in the cohort

[0088] The assessment of whether or not patients progress during immunotherapy treatment is carried out by analyzing tumor images acquired by scanner using the standard iRECIST methodology (“iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics”, Seymour et al., LANCET Oncology, vol 18, issue 3, el43-el52, 2017), 6 to 12 weeks after the start of treatment, depending on the type of cancer (6 weeks for lung cancer, 12 weeks for other cancers). The clinical data monitored are: serum lactate dehydrogenase (LDH) level, level of metastases, age, sex, smoker or non-smoker, tumor proportion score (TPS).

[0089] 1 / Preparation of plasma samples

[0090] The samples used according to the invention are prepared from whole blood taken from the individual. For this purpose, the whole blood is collected on Cell-Free DNA Collection Tubes according to the recommendations of the supplier Roche Diagnostics.

[0091] Each sample thus collected is subjected to two successive centrifugations: - a first gentle centrifugation at 1600 g, at room temperature (20°C + / - 5°C), for 10 min, this first centrifugation being carried out within a period of 7 days maximum after collection; the plasma (supernatant) is recovered without removing the cell pellet, - and a 2nd centrifugation at a faster speed, at 4500 g, also at room temperature (20°C + / - 5°C) and for 10 minutes; the plasma is aspirated without removing the formed pellet.

[0092] The plasma thus recovered can be used immediately, or it can be stored at -20°C for a period of less than or equal to 1 month, or -80°C for periods of more than 1 month, before its analysis.

[0093] 2 / Analysis of plasma samples for circulating free DNA concentrations in different size ranges

[0094] The total concentration of circulating free DNA in the plasma samples, and the concentration profiles as a function of size, are determined by a method based on pLas microfluidic technology. More particularly, the protocol implemented is as described in the publication by Boutonnet et al., Analytical Chemistry, 2023, 95(24): 9263-70.

[0095] The material used is as follows: - Agilent G7100A CE electrophoresis system equipped with a capillary incorporating pLAS technology, the diameter of which is provided with a local restriction, - Picometrics fluorescence detector, Zetalif® LED 480.

[0096] The protocol implemented is schematically as follows.

[0097] The plasma samples are first subjected to pretreatment with proteinase K in the presence of detergent, so as to release the nucleic acids from the vesicles and nucleoprotein complexes in which they are most often trapped. To this end, they are placed in the presence of an aqueous solution of proteinase K (2 mg / ml) and non-ionic surfactant NP-40 (1%) at 56°C for 2 h with stirring. vigorous (900 rpm), then recovered by centrifugation.

[0098] Each plasma sample is injected into the electrophoresis system, the temperature of which is maintained at 25°C. The different ingredients and solutions used for the analysis are as follows: - RNASE: RNase Away (Biolab), - BSA water: 50 mg / ml BSA solution in purified water, - HCl 0.1 M: 0.1 M hydrochloric acid solution in water, - PVA 2%: polyvinyl alcohol solution in water (800 mg in 40 ml of water), - TAE buffer: Tris-Acetate-EDTA (TAE) 0.5X + bovine serum albumin (BSA) 0.5 mg / mL, pH 8, polyvinylpyrrolidone (PVP) 360kDa 5% (w / v), water, - TAE buffer + dye: TAE buffer + SybrGreen 2X (Sigma), - PIPES buffer: 30 mM Bis-tris, 10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), pH 6.5, 1 mM EDTA, 5% (w / v) PVP 360KDa, water, - PIPES stamp + dye: PIPES stamp + SYBR Green 2X.

[0099] The exact program implemented in the electrophoresis system is detailed in Tables 2 to 4.

[0100] [Tables2] Input Output Pressure (bar) Voltage (kV) Time (min) Preconditioning RNASE Bin 10 0 4 RNASE Bin -9.9 0 4 WATER BSA Bin 10 0 4 HCl 0.1 M Bin 10 0 2 PVA 2% Bin 10 0 3 TAE Buffer Bin 10 0 6 Sample Injection Bin 5 0 1.33 (80s) Transfer Buffer TAE Buffer 5 0 0.17 (10s)

[0101] Table 2 - Electrophoresis system program - preconditioning, injection and transfer

[0102] [Tables3] Input Output Pressure (bar) Voltage (kV) Time (min) Concentration with returns TAE buffer + dye (pH 8) TAE buffer (pH 8) 11 25 0 11 25 1 -11 0 1.01 -11 0 1.75 11 25 1.76 11 25 2.76 -11 0 2.77 -11 0 3.52 11 25 3.53 11 25 4.53 -11 0 4.54 -11 0 5.29 11 25 5.3 11 25 7.3 -11 0 7.31 -11 0 8.06 11 25 8.07 11 25 11.07 -11 0 11.08 -11 0 11.58 11 25 11.59 11 25 16 -11 0 16.01 -11 0 16.51 11 25 16.52 11 25 20.52 -11 0 20.53 -11 0 21.03

[0103] Table 3 - Electrophoresis system program - Concentration with returns

[0104] [Tables4] Input Output Pressure (bar) Voltage (kV) Time (min) Buffer change and end of concentration at 7 bars PIPES buffer + dye PIPES buffer 7 30 0 7 30 9 Separation PIPES buffer + dye PIPES buffer 7 30 9 7 10 9.40 7 5 9.80 7 0.6 13 7 0.1 24 7 0 24.1

[0105] Table 4 - electrophoresis system program - buffer change and end of concentration, separation

[0106] At the end of the analysis, a fluorescence intensity profile as a function of time is obtained for each plasma sample analyzed. An example of such a profile is shown in [Fig.l].

[0107] The fluorescence profile is then converted into a concentration profile using the fluorescence intensity profile of a standard sample comprising DNA fragments whose respective sizes, ranging from 100 base pairs ("bp") to 1500 base pairs, are known and for which, for each size of DNA fragment, the migration time, the fluorescence intensity and the concentration are known (making it possible to know the fluorescence to concentration conversion factor for each migration time of the peaks of the standard sample).

[0108] This standard sample has the composition indicated in Table 5:

[0109] [Tables5] DNA size (bp) 100 150 200 300 400 500 600 700 800 1000 1500 Conc. (Pg / pl) 9 3 5 4 3 1.7 1.4 0.7 1.4 1.4 1.4

[0110] Table 5 - Composition of the standard sample (Conc. denotes the concentration)

[0111] The migration profile of this standard sample in the device used is shown on [Fig.2].

[0112] These data are used to carry out, on the one hand, a calibration of the fluorescence, and, on the other hand, a calibration of the migration times.

[0113] For fluorescence calibration, the area of each peak of the standard sample is measured (in RFU.min), then divided by the concentration of the corresponding fragment. To obtain an instantaneous conversion factor, the result is divided by the time unit (1 min), and we therefore obtain, for each of the peaks in [Fig.2], a fluorescence value per pg.pL for each migration time of the standard sample. Virtual points can be added outside the curve expressing the conversion factor (RFU / pg / pL.min) as a function of the migration time thus obtained, for short and long migration times. A linear interpolation is carried out between the points of this curve, in a conventional manner in itself, to determine the fluorescence conversion factor into concentration for all migration times located between the first and last peak of the standard sample.A slight linear extrapolation is made on both sides to slightly expand the range of migration times in which the conversion factor is known. Here, the extrapolation is made between 75 and 100 bp and between 1500 and 1650 bp. This gives a curve composed of pieces of straight lines, which is smoothed using splines of degree 2, in a classical manner. This smoothed curve is used to convert the fluorescence value into a concentration value throughout the analysis. Thus, the fluorescence versus time curve for each given sample is converted into a concentration versus time curve.

[0114] For the calibration of migration times, the fragments of the standard sample give “migration time / DNA size” pairs. A linear interpolation is made, in a conventional manner in itself, between each point to obtain a conversion of migration times into DNA size. Additional points can be added to the curve representing the DNA size as a function of the migration time thus obtained, the migration times of these additional points being calculated relative to the closest experimental DNA fragments in the standard sample, in order to gain precision in the calibration of the DNA size, without having to increase the number of DNA fragments in the standard sample. After a new linear interpolation, a curve representing the DNA size as a function of the migration time is obtained.

[0115] Using these two calibrations, that of fluorescence versus concentration, and that of migration time versus DNA size, we convert, for each plasma sample analyzed, the fluorescence curve as a function of time into a concentration curve as a function of size.

[0116] For each plasma sample analyzed, a concentration intensity profile of cfDNA as a function of size is thus obtained. An example of such a profile is shown on [Fig.3].

[0117] By this method, concentrations are only quantitatively determined between 75 and 1650 base pairs. Thus, for fragments smaller than 75 base pairs and fragments larger than 1650 base pairs, a relative concentration value, in arbitrary units, is calculated. This relative value is, however, highly representative of the actual value.

[0118] To determine the concentration of cfDNA in each desired size range, the minimum and maximum sizes of the interval are converted to minimum and maximum times according to the time / size calibration, and then the integral of the time / concentration curve is calculated between these minimum and maximum times. This gives the absolute concentration of cfDNA in each desired size range, expressed in pg / pL.

[0119] For each plasma sample analyzed, from the individual concentrations of each size, a histogram of the percentages of the following different size ranges is constructed: size less than 75 bp, from 75 to 239 bp, from 240 to 369 bp, from 370 to 579 bp, from 580 to 1649 bp, greater than or equal to 1650 bp. An example of such a histogram is shown in [Fig.4].

[0120] For each plasma sample analyzed, the total concentration of cfDNA is further calculated. In this example, this total concentration is approximated to the concentration of DNA between the sizes 75 bp and 1650 bp, the value thus approximated being very close to the actual value.

[0121] Finally, for each plasma sample analyzed, the relative concentration of DNA in each of the above size ranges is calculated, this relative concentration being equal to the absolute concentration of DNA in this size range, determined as indicated above, divided by the total concentration of DNA.

[0122] 3 / Statistical analysis

[0123] Preliminary statistical tests including classification by univariate logistic regression, univariate and multivariate COX regression and Kaplan-Meier estimation to classify early progressors and early non-progressors on a first set of 25 patients having a distribution of pathologies equivalent to that of the entire cohort of 51 patients were first carried out, for the following panels of potential indicators: relative concentrations of cfDNA fragments in plasma, for the following respective size ranges: size less than 75 bp, from 75 to 239 bp, from 240 to 369 bp, from 370 to 579 bp, from 580 to 1649 bp, greater than or equal to 1650 bp.

[0124] The two indicators that characterize the patient's response (early progressors or early non-progressors under immunotherapy treatment) are the relative concentrations of cDNA fragments in the plasma for the following size ranges: size greater than or equal to 1650 base pairs, and size between 580 and 1649 base pairs.

[0125] The mathematical functions (indicators) used for statistical analysis, named PI and P2, are therefore as follows:

[0126] [Math.l] Concentration of cfDNA fragments of size greater than or equal to 1650 bp Total concentration of cfDNA

[0127] [Math.2] Concentration of cfDNA fragments between 580 and 1649 bp Total concentration of cfDNA

[0128] The cohort of 51 patients is divided into two data sets: a training set (70% - 35 patients) and a test set (30% - 16 patients). This distribution between training set and test set is classic and corresponds to the standard methodology of this type of statistical analysis. The two sets (training and test) present a balanced distribution between patients in progression, pathology and sex.

[0129] The statistical data are established in relation to the clinical data obtained by the iRECIST methodology 6 to 12 weeks after the start of immunotherapy treatment, depending on the type of cancer.

[0130] For each indicator, a ROC curve is performed on the training batch to determine the threshold that minimizes the distance from the optimal point (0,1). The method used to set the optimal threshold value is the BOOTSTRAP method. For 100 experiments, patients are randomly sampled with replacement between the training batch (“in bag”) and the test batch (“out of bag”).

[0131] For each indicator, the threshold is optimized and defined as the median of the 100 thresholds obtained by the BOOTSTRAP method.

[0132] These thresholds are then used on the training batch and then the test batch, and the following metrics are determined: - area under the curve (AUC), - classification accuracy, - sensitivity, - specificity, - positive predictive value (PPV): probability that the patient predicted as an early progressor is an early progressor, - negative predictive value (NPV): probability that the patient predicted as an early non-progressor is an early non-progressor.

[0133] The thresholds determined by the BOOTSTRAP method are further used for calculate a 95% confidence interval of the threshold values by extracting the 2.5% and 97.5% quantiles.

[0134] The ROC curves obtained for each indicator, respectively for the training batch of 35 patients, the test batch of 16 patients and the total cohort, are shown in [Fig.5] for the PI indicator and in [Fig.6] for the P2 indicator. The values of the area under the curve (AUC) are indicated in the figures for each of the curves. It is observed that this value is high for both indicators, and is higher for the PI indicator ([Fig.5]) than for the P2 indicator ([Fig.6]). For comparison, for example, for the indicator that is the relative concentration of cfDNA fragments in the size range greater than or equal to 240 bp, an AUC of only 0.65 is obtained for the total cohort.

[0135] [Fig.7] shows the box plots obtained for each of the PI indicators and P2, for the entire cohort. We observe that the results are statistically different between early progressors and early non-progressors.

[0136] All the results obtained, for the training batch (“A”) and for the test batch (“T”) are summarized in table 6, for each of the indicators PI and P2.

[0137] [Tableauxô] PI Indicator P2 Threshold 0.034 0.072 Quantile 2.5% 0.028 0.062 Quantile 97.5% 0.043 0.122 AUC A 0.830 0.693 AUC T 0.833 0.817 Accuracy A 0.800 0.771 Accuracy T 0.875 0.812 Sensitivity A 0.733 0.533 Sensitivity T 0.833 0.500 Specificity A 0.850 0.950 Specificity T 0.900 1.000 PPV A 0.786 0.889 PPVT 0.833 1.000 NPV A 0.810 0.731 NPVT 0.900 0.769

[0138] Table 6 - Characteristic statistical values for the PI and P2 indicators

[0139] For the PI indicator, we obtain a sensitivity of 0.833 and a specificity of 0.900, for a threshold (score below which the patient is considered an early progressor) of 0.034 (confidence interval of 0.028 to 0.043).

[0140] The P2 indicator has an excellent specificity of 1 considering a threshold of 0.072 (confidence interval of 0.062 to 0.122). This indicator thus has a good predictive power of progression or not of the tumor at the first examination: if it is low, then there will very probably be (better than 90%) progression of the disease at the first examination. On the other hand, if it is high, its predictive power of an absence of early progression is moderate, lower than that of the PI marker, which presents the best compromise between sensitivity and specificity.

[0141] For the test batch, the progression-free survival curves and survival probabilities were estimated by the Kaplan-Meier method, and compared to the prediction results by the PI indicator according to the invention, with the threshold value of 0.034. The results are shown in [Fig.8]. It is observed that the curves are well correlated and characteristic of a long response, and therefore of a good prognosis of progression-free survival. Thus, the PI indicator constitutes a marker allowing the prognosis of progression-free survival.

Claims

Claims

1. An in vitro method for predicting early progressor character and / or early non-progressor character during immunotherapy treatment of a subject suffering from cancer, comprising steps of: a / determining, in an isolated blood sample obtained from said subject before said immunotherapy treatment: - the total concentration of circulating free deoxyribonucleic acid in said blood sample, called total cfDNA concentration, - at least one concentration, in said blood sample, of circulating free deoxyribonucleic acid fragments whose size is included in a predetermined size range, said predetermined size range being included in sizes greater than 500 base pairs, called large cfDNA concentration, b / combining in a mathematical function at least said large cfDNA concentration, and said total cfDNA concentration,so as to obtain a score, and: cl / comparison of said score with a first predetermined reference value, and dl / conclusion that said subject presents an early progressor character during said immunotherapy treatment when said score is less than or equal to said first reference value, and / or c2 / comparison of said score with a second predetermined reference value, and d2 / conclusion that said subject presents an early non-progressor character during said immunotherapy treatment when said score is greater than said second reference value.,

2. The method of claim 1, wherein said first reference value and said second reference value are identical.

3. The method of claim 1, wherein said first reference value is less than said second reference value.

4. A method according to any one of claims 1 to 3, wherein said mathematical function is the ratio of at least said large cfDNA concentration to said total cfDNA concentration.

5. A method according to any one of claims 1 to 4, wherein said predetermined size range is the size range greater than 500 base pairs, preferably greater than or equal to 600 base pairs, and more preferably greater than or equal to 1500 base pairs.

6. A method according to one of claims 1 to 4, wherein said predetermined size range is the size range greater than or equal to 1650 base pairs.

7. A method according to any one of claims 1 to 4, wherein said predetermined size range is the size range between 580 and 1649 base pairs.

8. A method according to any one of claims 1 to 7, comprising a step of obtaining a plasma sample from said blood sample.

9. The method of claim 8, wherein the determination of said total cfDNA concentration and the determination of said at least one large cfDNA concentration are performed by direct analysis of said plasma sample.

10. Method according to any one of claims 1 to 9, comprising a step of extracting circulating free deoxyribonucleic acid from said blood sample, or where appropriate from said plasma sample, and according to which the determination of said total cfDNA concentration and the determination of said at least one large cfDNA concentration are carried out by analyzing the extract of circulating free deoxyribonucleic acid thus obtained.

11. A method according to any one of claims 1 to 10, wherein said subject is a human.

12. The method of any one of claims 1 to 11, wherein said cancer is a metastatic tumor pathology, melanoma, kidney cancer, urothelial carcinoma of the bladder, squamous cell carcinoma of the head and neck, or non-small cell lung cancer.

13. A method according to any one of claims 1 to 12, wherein said immunotherapy treatment uses nivolumab, ripilimumab, pembrolizumab, atezolizumab, avelumab and / or durvalumab.

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