Method for predicting the response of a patient with cancer to an immunotherapy treatment

Abstract

The invention relates to an in vitro method for predicting the early-progressor character of a subject with cancer during an immunotherapy treatment. This method comprises determining, in a blood sample taken from the subject, the total concentration of circulating-free DNA and a concentration of fragments of circulating-free DNA of large size, combining these data into 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 obtained score is less than or equal to the reference value.

Description

[0001] METHOD FOR PREDICTING THE RESPONSE OF A CANCER PATIENT TO IMMUNOTHERAPY TREATMENT

[0002] The present invention falls within the general field of oncology.

[0003] 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.

[0004] 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.

[0005] 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. In certain types of cancer, immunotherapy is used routinely, with spectacular results, particularly in 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).

[0006] However, immunotherapy treatments are not effective in all patients. Furthermore, they can cause side effects in some. For these patients, conventional chemotherapy would have been a better option. These patients are referred to as non-responders to immunotherapy treatment. This information is often obtained late, several weeks after the start of immunotherapy treatment, weeks that 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 recognized today by the entire immunotherapy community.No method currently allows, before starting an immunotherapy treatment, to identify / classify cancer patients who will respond or not to this treatment, more precisely to identify patients for whom this treatment will, or will not, prevent tumor progression. A reliable, standardized biomarker that can be used in clinical practice to predict response to treatment and assess its 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 coverage, 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 response of patients to immunotherapy treatments, for information purposes only because the predictive nature of these markers is limited:

[0008] - 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;

[0009] - 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.

[0010] These CPS and TPS markers nevertheless require the implementation of protocols that are often specific to each care center, and no standardization has been achieved to date, which leads to differences in the values ​​of the scores and thresholds applied in the different care centers. The present invention aims to propose a method for predicting, with high precision, how a cancer patient 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. 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 circulating free deoxyribonucleic acid (DNA) present in the blood of patients. This free DNA circulating in blood plasma is currently the subject of intense clinical research in oncology, because mutations of the genomic DNA present in tumor cells can be found there, which guide the therapy to be administered to the patient. For convenience, this circulating free deoxyribonucleic acid will be referred to in the present description as "circulating free DNA" or cfDNA.

[0012] It is well established that blood carries a small amount of circulating cell-free DNA resulting from the release of genetic material from tissues. This cell-free circulating cell-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 cell-free DNA in healthy individuals and its increase related 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, etc.

[0013] In the case of cancer, recent advances in molecular biology and DNA sequencing have made it possible to identify numerous gene mutations 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 tumor burden and the stage of the cancer. Generally speaking, the studies carried out focus on the use of ctDNA as a tumor biomarker to monitor the positive or negative evolution 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 follow-up, monitoring 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 by 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 specifically 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, 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 cancer management protocol 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 early progressor characteristics, also conventionally in itself in the medical field, is understood to mean a subject suffering from cancer for whom, on the first image of the tumor acquired by medical imaging, in particular by X-ray scanner, after the start of immunotherapy treatment, compared to the image acquired by the same medical imaging technique before the start of this treatment, an increase of more than 20% of the tumor lesions identified on the imaging, that is to say that 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 immunotherapy treatment.

[0020] As indicated above, circulating cell-free DNA also means extracellular DNA present in the patient's blood plasma. This circulating cell-free DNA includes circulating tumor cell-free DNA and circulating non-tumor cell-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 for determining whether this patient will be an early progressor or an early non-progressor during the administration of an immunotherapy treatment.Nothing in the prior art, which only deals with small circulating free DNA fragments, well below 500 base pairs, suggested such a result. Thus, according to the present invention, an in vitro method is proposed 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 / determining, in an isolated blood sample which has been obtained from said subject, preferably before said immunotherapy treatment:.

[0022] - the total concentration of free deoxyribonucleic acid circulating in said blood sample, called total cfDNA concentration,

[0023] - 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, result of the mathematical function, this score reflecting the probability that the subject presents or not an early progressor characteristic and / or an early non-progressor characteristic during the immunotherapy treatment,c / comparison of this score with a first predetermined reference value and / or a second predetermined reference value, and d / determination of the early progressor character and / or the early non-progressor character of the subject during the immunotherapy treatment on the basis of the result of this comparison.,

[0024] In particular, depending on the mathematical function, steps c / and d / may consist either of steps d / and d1 / and / or c2 / and d2 / : c1 / comparison of this score with a first predetermined reference value, and d1 / conclusion that the subject has an early progressor character during the 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 has an early non-progressor character during the immunotherapy treatment when the score is greater than this second reference value, or of steps c3 / and d3 / and / or c4 / and d4 / : c3 / comparison of this score with a first predetermined reference value,and d3 / conclusion that the subject presents an early non-progressor character during the immunotherapy treatment when this score is less than or equal to this first reference value, and / or c4 / comparison of this score with a second predetermined reference value, and d4 / conclusion that the subject presents an early progressor character during the immunotherapy treatment when the score is greater than this second reference value.,

[0025] 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 / determining, in an isolated blood sample which has been obtained from said subject, preferably before said immunotherapy treatment:

[0026] - the total concentration of free deoxyribonucleic acid circulating in said blood sample, called total cfDNA concentration,

[0027] - 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, c1 / comparison of this score with a first predetermined reference value,and d1 / conclusion 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, or c4 / comparison of this score with a second predetermined reference value, and d4 / conclusion that the subject presents an early progressor character during the immunotherapy treatment when the score is greater than this second reference value.,

[0028] 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 / determining, in an isolated blood sample which has been obtained from said subject, preferably before said immunotherapy treatment:

[0029] - the total concentration of free deoxyribonucleic acid circulating in said blood sample, called total cfDNA concentration,

[0030] - 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 with a second predetermined reference value,and d2 / conclusion that the subject presents an early non-progressor character during the immunotherapy treatment when the score is higher than this second reference value, or c3 / comparison of this score with a first predetermined reference value, and d3 / conclusion that the subject presents an early non-progressor character during the immunotherapy treatment when this score is lower than or equal to this first reference value.,

[0031] 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 immunotherapy treatment. It then comprises, after the step of obtaining the score:

[0032] - a step d / of comparing this score to the first predetermined reference value, and a step d1 / 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, or a step c4 / of comparing this score to a second predetermined reference value, and a step d4 / of concluding that the subject presents an early progressor character during the immunotherapy treatment when the score is greater than this second reference value, and

[0033] - 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 or a step c3 / of comparing this score to a first predetermined reference value, and a step d3 / of concluding that the subject presents an early non-progressor character during the immunotherapy treatment when this score is lower than or equal to this first reference value.

[0034] 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 character of early non-progressor and / or the character of early progressor with respect to an immunotherapy treatment of a patient suffering from cancer, even before the start of such treatment. The result of the method according to the invention thus constitutes a particularly advantageous aid for decision-making, by the clinician, who will take it into account within the overall clinical picture, whether or not to administer an immunotherapy treatment for each given patient.Such a method proves to be particularly advantageous in that it makes it possible, for example, to avoid administering immunotherapy treatment to patients who have been predicted as early progressors, for whom this treatment will not have the desired efficacy, and / or, on the contrary, to administer immunotherapy treatment with confidence to patients who have been predicted as early non-progressors.

[0035] The precision (or accuracy) of prediction of the method according to the invention has in particular been verified in prospective observational studies carried out on cohorts of, respectively, 51 patients and 155 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).

[0036] In a conventional manner, the term precision here designates the proportion of patients who have been correctly classified by the method according to the invention.

[0037] The performance of statistical prediction methods is generally evaluated by plotting a receiver operating characteristic curve (ROC curve) and measuring the area 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 51 patients, a method according to an embodiment of the invention made it possible to predict an early progressor character of patients with an AUC as high as 0.833, which clearly demonstrates its good performance.

[0038] "Sensitivity" here means, in a conventional manner, the probability of the method identifying early progressors (or, as the case may be, early non-progressors) as positive, i.e., identifying true positives. "Specificity" means the probability of the method not identifying early non-progressors (or, as the case may be, early progressors) as positive, i.e., not identifying true negatives.

[0039] Furthermore, in this 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.

[0040] 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 the level of specificity required for each prediction. Such a determination can be carried out either empirically or theoretically.

[0041] 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:

[0042] - if the score is less than or equal to the common reference value, the subject presents an early progressor character or, according to the chosen mathematical formula, an early non-progressor character, during immunotherapy treatment, and / or

[0043] - if the score is higher than the common reference value, the subject presents an early non-progressor character or, depending on the mathematical formula chosen, an early progressor character, during immunotherapy treatment.

[0044] In alternative embodiments of the invention, the first reference value and the second reference value are different, the first reference value being lower than the second reference value. The method may then comprise a step of concluding that: - if the score is lower than or equal to the first reference value, the subject has an early progressor character, or, according to the chosen mathematical formula, an early non-progressor character, during the immunotherapy treatment,

[0045] - if the score is higher than the second reference value, the subject presents an early non-progressor character or, according to the chosen mathematical formula, an early non-progressor character, during immunotherapy treatment,

[0046] - 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.

[0047] The prediction method according to the invention, the results of which are based on a statistical analysis, advantageously allows in particular:

[0048] - a prediction-stratification before therapy of early non-progressor patients and / or early progressor patients during immunotherapy treatment, thus allowing personalized care;

[0049] - patient stratification allowing for more effective clinical studies,

[0050] - for the patient, a saving of time increasing the chances of success of his therapy, by avoiding side effects or induced effects;

[0051] - simplicity and speed of implementation by analysis carried out from a blood sample already taken from the patient for other purposes;

[0052] - 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.

[0053] 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 administration of this treatment, or after, preferably just after, within a few hours or a few days following the start of administration of the treatment.

[0054] The method according to the invention does not in itself require any step applied to the patient's body. The analysis steps it involves are carried out using whole blood samples that have been taken from the patient beforehand, in a conventional manner.

[0055] The subject to which the method according to the invention is applied is preferably a mammal. It is preferably a human.

[0056] The method according to the invention may also meet one or more of the characteristics described below, implemented in isolation or in each of their technically effective combinations.

[0057] The mathematical function implemented in the method according to the invention can be any type of multivariate function.

[0058] In addition to the variables of large cfDNA concentration and total cfDNA concentration, it may use other variables. In particular, it may use, as variables, several different large cfDNA concentrations, each corresponding to a predetermined analytical size range different from the others. It is recalled that for the purposes of the present invention, the expression "analytical size range" designates any size range included in sizes greater than 500 base pairs. It may also or otherwise use, as variables, other clinical or biological information relating to the patient.

[0059] 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.

[0060] In particular embodiments of the invention, the mathematical function comprises, as a variable, the ratio between at least said large cfDNA concentration and said total cfDNA concentration. For example, it consists of the ratio between at least said large cfDNA concentration and said total cfDNA concentration.

[0061] 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.

[0062] The mathematical function implemented according to the invention may alternatively be equal to the sum of different concentrations of large cfDNA, each associated with a predetermined analytical size range, this sum being divided by the total cfDNA concentration.

[0063] When the mathematical function is the ratio of a large cfDNA concentration to the total cfDNA concentration, or a sum of such ratios, the method comprises steps d / and d1 / , and / or c2 / and d2 / described above, i.e. it concludes that the subject exhibits early progressor character during immunotherapy treatment when the score is less than or equal to the first reference value, and / or the subject exhibits early non-progressor character during immunotherapy treatment when the score is greater than the second reference value.

[0064] The mathematical function implemented according to the invention can otherwise combine the above variables in any other way, and in particular be part of the framework of linear regression.

[0065] In particular, it may have been obtained by the following steps:

[0066] - assessment of the character of early progressor or early non-progressor, during immunotherapy treatment, in a cohort of cancer patients, for whom the values ​​of the markers described above and below are known,

[0067] - and carrying out a logistic regression analysis to evaluate and weight the independent discriminative value of each of these markers for the prediction of early progressor or early non-progressor status during immunotherapy treatment,

[0068] - so as to obtain the targeted mathematical function.

[0069] 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.

[0070] 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.

[0071] In particular alternative embodiments of the invention, each predetermined size range, or analytical size range, is defined by its minimum value and its maximum value, i.e., 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 larger and / or larger size ranges are more particularly preferred within the scope of the invention, compared to the smaller and / or smaller size ranges.

[0072] 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.

[0073] In particular embodiments of the invention, at least one predetermined size range / analytical size range is the size range between 1650 and 4000 base pairs.

[0074] The mathematical function used according to the invention can combine all combinations of the variables each associated with a range of analytical size specific to it, which are mentioned above.

[0075] 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 may be cited: the size range greater than or equal to 1650 base pairs, in particular between 1650 and 4000 base pairs, and the size range from 580 to 1649 base pairs.

[0076] In particular embodiments of the invention, the method comprises establishing a curve representing the concentration, as a function of size, of the free deoxyribonucleic acid fragments circulating in said blood sample, and determining the size, expressed in base pairs, corresponding to the second peak on said curve, this size being designated herein by the expression "Position peak2". The mathematical function then preferably comprises, as a variable, said Position peak2.

[0077] An example of a method for establishing the curve representing the concentration, as a function of the size, of the free deoxyribonucleic acid fragments circulating in the blood sample, is described in detail below in the present description. The second peak observed on this curve, that is to say the second starting from the value 0 and going towards the highest sizes, corresponds to the dinucleosome. It has been discovered by the present inventors that the integration of this value into the mathematical function implemented improves the prediction performance of the method according to the invention.

[0078] In preferred embodiments of the invention, the method comprises determining the concentration of circulating free deoxyribonucleic acid in said blood sample for each size between 160 and 220 base pairs, and calculating the sum of each of said concentrations to the power of 0.1, said sum being called Sumi6o-22o. The mathematical function implemented then preferably comprises, as a variable, said Sumi6o-22o. This parameter Sumi6o-22o can be defined by the following equation:

[0079] In preferred embodiments of the invention, the mathematical function comprises, as variables:

[0080] - at least one ratio between a concentration of large cfDNA and the concentration of total cfDNA, called relative concentration of large cfDNA, for example the relative concentration of cfDNA of size greater than or equal to 1650 base pairs, in particular between 1650 and 4000 base pairs, and / or the relative concentration of cfDNA of size between 580 and 1649 base pairs,

[0081] - and at least Position pic2 and / or Sommei6o-22o.

[0082] For example, the mathematical function can be expressed by equation (1 a) or by equation (1 b):

[0083] Score = 1 / (1 + Exp(-(a1 + b1 x P1 + c1 x Sumi6o-22o + d1 x Position pic2))) Equation (1 a)

[0084] Score = 1 - 1 / (1 + Exp(-(a1 + b1 x P1 + c1 x Sommei6o-22o + d1 x Position pic2))) Equation (1 b) in which:

[0085] - a1, b1, c1 and d1 are constant coefficients,

[0086] - variable P1 is the relative concentration of cfDNA of size greater than or equal to 1650 base pairs in the blood sample,

[0087] - the variable Sommei6o-22o is as defined above, and expressed in pg / pl to the power of 0.1,

[0088] - the variable Position pic2 is as defined above, and expressed in base pairs.

[0089] The coefficients a1, b1, c1 and d1 are preferably such that:

[0090] - -40.0 < a1 < -30.0, preferably -38.0 < a1 < -37.0, for example -37.2 < a1 < -37.1,

[0091] - and / or 30.0 < b1 < 40.0, preferably 32.0 < b1 < 33.0, for example 32.0 < b1 < 32.1,

[0092] - and / or -1.0 < c1 < 0.0, preferably -0.1 < c1 < 0.0,

[0093] - and / or 0.0 < d1 < 1.0, preferably 0.0 < d1 < 0.2, for example 0.1 < d1 < 0.2. In such embodiments, particularly suitable for patients suffering from lung cancer, the first reference value and the second reference value are identical, and equal to 0.5.

[0094] With regard to equation (1 a), it is concluded according to the invention that the subject has an early progressor character during the immunotherapy treatment when the score is less than or equal to this reference value, and that the subject has an early non-progressor character during the immunotherapy treatment when the score is greater than this reference value. Steps c / and d / of the method according to the invention thus consist of steps d / and d1 / , and c2 / and d2 / described above. With regard to equation (1 b), it is concluded according to the invention that the subject has an early non-progressor character during the immunotherapy treatment when the score is less than or equal to this reference value, and that the subject has an early progressor character during the immunotherapy treatment when the score is greater than this reference value.Steps c / and d / of the method according to the invention thus consist of steps c3 / and d3 / , and c4 / and d4 / described above.

[0095] In particular embodiments of the invention, the method comprises determining the number of neutrophils and the number of lymphocytes in said blood sample. The mathematical function then preferably comprises, as a variable, the ratio between said number of neutrophils and said number of lymphocytes.

[0096] The number of neutrophils and the number of lymphocytes in the blood sample can be determined by any method known to those skilled in the art, for example by blood count.

[0097] It has been discovered by the present inventors that the integration of this clinical marker into the mathematical function according to the invention can improve the performance of the prediction.

[0098] In preferred embodiments of the invention, the mathematical function comprises, as variables:

[0099] - at least one ratio between a concentration of large cfDNA and the concentration of total cfDNA, called relative concentration of large cfDNA, for example the relative concentration of cfDNA of size greater than or equal to 1650 base pairs, in particular between 1650 and 4000 base pairs, and / or the relative concentration of cfDNA of size between 580 and 1649 base pairs,

[0100] - at least Position pic2 and / or Sommei6o-22o.

[0101] - and at least the ratio between the number of neutrophils and the number of lymphocytes in the blood sample, this ratio being called Rn / L

[0102] The mathematical function can then be expressed in particular by equation (2a) or by equation (2b):

[0103] Score = 1 / (1 + Exp(-(a2 + b2 x P1 + c2 x Sumi6o-22o + d2 x Position pic2 + e2 x Rn / I)))

[0104] Equation (2a) Score = 1 - 1 1 (1 + Exp(-(a2 + b2 x P1 + c2 x Sumi6o-22o + d2 x Position pic2 + e2 x

[0105] Rn / I))) Equation (2b) in which:

[0106] - a2, b2, c2, d2 and e2 are constant coefficients,

[0107] - variable P1 is the relative concentration of cfDNA of size greater than or equal to 1650 base pairs in the blood sample,

[0108] - the variable Sommei6o-22o is as defined above, and expressed in pg / pl to the power of 0.1,

[0109] - the variable Position pic2 is as defined above, and expressed in base pairs,

[0110] - Rn / I the ratio between the number of neutrophils and the number of lymphocytes in the blood sample.

[0111] As a first example, particularly suitable for patients with head and neck squamous cell carcinoma (HNSCC), the coefficients a2, b2, c2, d2 and e2 in equations (2a) and (2b) are preferably such that:

[0112] - 2.0 < a2 < 3.0, preferably 2.2 < a2 < 2.3,

[0113] - and / or 10.0 < b2 < 20.0, preferably 19.0 < b2 < 20.0, for example 19.4 < b2 < 19.5,

[0114] - and / or -1.0 < c2 < 0.0, preferably -0.1 < c2 < 0.0,

[0115] - and / or 0.0 < d2 < 1.0, preferably 0.0 < d2 < 0.1, for example 0.04 < d2 < 0.05,

[0116] - and / or -3.0 < e2 < -2.0, preferably -2.5 < e2 < -2.0, for example -2.3 < e2 < -2.2.

[0117] As a second example, particularly suitable for patients with lung cancer, the coefficients a2, b2, c2, d2 and e2 in equations (2a) and (2b) are preferably such that:

[0118] - 30.0 < a2 < -20.0, preferably -28.0 < a2 < -27.0, for example -27.4 < a2 < -27.3,

[0119] - and / or -7.0 < b2 < -6.0, preferably -6.4 < b2 < -6.2, for example -6.4 < b2 < - 6.3,

[0120] - and / or -1.0 < c2 < 0.0, preferably -0.2 < c2 < 0.0, for example -0.2 < c2 < -01,

[0121] - and / or 0.0 < d2 < 1.0, preferably 0.0 < d2 < 0.2, for example 0.1 < d2 < 0.2, - and / or -1.0 < e2 < 0.0, preferably -0.1 < e2 < 0.0.

[0122] As a third example, particularly suitable for patients with all types of cancer except melanomas, the coefficients a2, b2, c2, d2 and e2 in equations (2a) and (2b) are preferably such that:

[0123] - -20.0 < a2 < -10.0, preferably -17.0 < a2 < -16.0, for example -16.2 < a2 < -16.1,

[0124] - and / or 20.0 < b2 < 30.0, preferably 27.0 < b2 < 28.0, for example 27.2 < b2 < 27.3,

[0125] - and / or -1.0 < c2 < 0.0, preferably -0.1 < c2 < 0.0,

[0126] - and / or 0.0 < d2 < 1.0, preferably 0.0 < d2 < 0.2, for example 0.0 < d2 < 0.1,

[0127] - and / or -1.0 < e2 < 0.0, preferably -0.2 < e2 < 0.0, for example -0.2 < e2 < -0.1.

[0128] In such embodiments, the first reference value and the second reference value are identical, and equal to 0.5. With regard to equation (2a), it is concluded according to the invention that the subject has an early progressor character during the immunotherapy treatment when the score is less than or equal to this reference value, and that the subject has an early non-progressor character during the immunotherapy treatment when the score is greater than this reference value. Steps c / and d / of the method according to the invention thus consist of steps d / and d1 / , and c2 / and d2 / described above.

[0129] With regard to equation (2b), it is concluded according to the invention that the subject has an early non-progressor character during the immunotherapy treatment when the score is less than or equal to this reference value, and that the subject has an early progressor character during the immunotherapy treatment when the score is greater than this reference value. Steps c / and d / of the method according to the invention thus consist of steps c3 / and d3 / , and c4 / and d4 / described above.

[0130] The score obtained by the method according to the invention, which is the result of the mathematical function, 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.

[0131] 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, 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 lung cancer), or a plurality of such cancers.

[0132] The immunotherapy treatment for which the aim is to determine the early progressor and / or early non-progressor status of the subject can be of any type. It can notably involve nivolumab, lipilimumab, pembrolizumab and / or one or more anti-PD-L1 antibodies such as atezolizumab, avelumab and / or durvalumab.

[0133] 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 in conjunction with other therapeutic treatment methods, such as chemotherapy treatment and / or targeted therapy when possible.

[0134] 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 an ethylenediaminetetraacetic acid (EDTA) buffer 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.

[0135] In particular embodiments of the invention, the determination of said total cfDNA concentration and the determination of said at least one large cfDNA concentration, and where appropriate the establishment of the curve representing the concentration, as a function of the size, of the circulating free deoxyribonucleic acid fragments and / or the determination of the circulating free deoxyribonucleic acid concentration for each size between 160 and 220 base pairs, are carried out by direct analysis of the blood sample isolated from the subject, i.e. a whole blood sample. In preferred embodiments of the invention, the method comprises a prior step of obtaining a plasma sample from this whole blood sample.

[0136] 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 of the blood sample being taken, for example within 6 hours of the blood sample being taken if EDTA buffer tubes are used, and within 7 days of the blood sample being taken if specific tubes for obtaining circulating free DNA are used, such as the tubes mentioned above.

[0137] This step can be carried out in any conventional way, for example by centrifugation.

[0138] 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 preferred 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:

[0139] - 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;

[0140] - recovery of the plasma (supernatant), without removing the cell wafer separating the plasma and the red blood cells;

[0141] - a second centrifugation at a faster speed, for example between 3000 and 16000 g, preferably also at room temperature;

[0142] - and the recovery of the plasma (supernatant), for example by aspiration, without removing the pellet formed.

[0143] 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 concentrations of cfDNA necessary for the implementation of the method according to the invention.

[0144] The determination of the total cfDNA concentration and the determination of the concentration(s) of large cfDNA in the blood sample, and where appropriate the establishment of the curve representing the concentration, as a function of the size, of the circulating free deoxyribonucleic acid fragments and / or the determination of the circulating free deoxyribonucleic acid concentration for each size between 160 and 220 base pairs, can be carried out by any method conventional in itself for those skilled in the art. Depending on the particular technique used, 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.

[0145] 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, and where appropriate the establishment of the curve representing the concentration, as a function of the size, of the circulating free deoxyribonucleic acid fragments and / or the determination of the circulating free deoxyribonucleic acid concentration for each size between 160 and 220 base pairs, 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.

[0146] 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, and where appropriate the establishment of the curve representing the concentration, as a function of the size, of the circulating free deoxyribonucleic acid fragments and / or the determination of the circulating free deoxyribonucleic acid concentration for each size between 160 and 220 base pairs, are then carried out by analyzing the circulating free deoxyribonucleic acid extract thus obtained.

[0147] 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.

[0148] 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), and where appropriate the curve representing the concentration, as a function of the size, of the circulating free deoxyribonucleic acid fragments and / or the circulating free deoxyribonucleic acid concentration for each size between 160 and 220 base pairs, can be carried out by any technique known to those skilled in the art for this purpose.

[0149] For example, it can be performed by polymerase chain reaction (PCR), gel electrophoresis, or capillary electrophoresis technology, using a commercially available device, such as the Agilent Technologies 7100 CE Fragment Analyzer and System, the QIAxcel offered by Qiagen, or the GenomeLab system from Sciex. These techniques require prior extraction of circulating free DNA from the blood sample or plasma sample.

[0150] 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 fashion in the large capillary, and an electric field is used to slow down the migration. Because of the shear provided by the laminar flow, this counter-electrophoresis reveals 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 into a ring. 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 ring 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.

[0151] 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), and where appropriate the curve representing the concentration, as a function of the size, of the circulating free deoxyribonucleic acid fragments and / or the circulating free deoxyribonucleic acid concentration for each size between 160 and 220 base pairs, may 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 cfDNA with increased sensitivity 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.

[0152] In essence, this method involves at least one iteration of an alternation:

[0153] - 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,

[0154] - a step of laminar flow of the sample, in a second direction of flow opposite to the first direction of flow, 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.

[0155] 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.

[0156] 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.

[0157] 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 by immunotherapy, as well as of his survival at 3 years.

[0158] It has further been observed by the present inventors that the parameter Position pic2, as defined above, alone makes it possible to predict with good performance the character of early progressor and / or the character of early non-progressor during an immunotherapy treatment of a subject suffering from cancer, and in particular lung cancer.

[0159] Thus, another aspect of the invention relates to an in vitro method for predicting the early progressor character and / or the early non-progressor character during an immunotherapy treatment of a subject suffering from cancer, in particular lung cancer, this method comprising steps of: 1 / establishing a curve representing the concentration, as a function of the size, of the free deoxyribonucleic acid fragments circulating in an isolated blood sample obtained from said subject before said immunotherapy treatment,

[0160] 2 / determination of the size, expressed in base pairs, corresponding to the second peak on said curve, this size being designated herein by the expression “Position peak2”, and

[0161] 3a / comparison of said size “Position peak2” to a first predetermined reference value, and 4a / 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

[0162] 3b / comparison of said size “Position peak2” to a second predetermined reference value, and 4b / conclusion that said subject presents an early non-progressor character during said immunotherapy treatment when said score is higher than said second reference value.

[0163] This method may meet one or more of the characteristics described above in this description, not relating to one or more variables of the mathematical function with the exception of the variable Position pic2.

[0164] The characteristics and advantages of the invention will appear more clearly in the light of the examples of implementation below, provided for purely illustrative purposes and in no way limiting the invention, with the support of figures 1 to 16, in which:

[0165] Figure 1 shows a curve representing the fluorescence intensity profile as a function of time, obtained by analysis, by a method based on pLas technology, of a plasma sample from a cancer patient containing cfDNA.

[0166] Figure 2 shows a curve representing the fluorescence intensity profile 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.

[0167] Figure 3 shows a curve representing the circulating free DNA concentration profile, expressed in picograms per pl per base pair (bp), as a function of size, obtained from the curve in Figure 1.

[0168] Figure 4 shows a histogram representing the distribution of circulating free DNA concentration for different size ranges, obtained from the curve in Figure 3.

[0169] Figure 5 represents ROC curves relating to the prediction of the early progressor character of a patient suffering from cancer during an 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.

[0170] Figure 6 represents ROC curves relating to the prediction of the early progressor character of a patient suffering from cancer during an 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.

[0171] Figure 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.

[0172] Figure 8 shows a graph representing, as a function of time, the progression-free survival curve of patients with cancer who have been subjected to 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.

[0173] Figure 9 represents, in a / the box plots obtained for a cohort of 68 patients suffering from lung cancer (54 patients with the status of non-early progressor NEP and 14 patients with the status of early progressor EP), for an indicator according to the invention (relative concentration of cfDNA fragments of size greater than or equal to 1650 bp in a plasma sample of said patient), and in b / a ROC curve relating to the prediction of the early progressor character of a patient suffering from lung cancer during an immunotherapy treatment, obtained for the same indicator.

[0174] Figure 10 shows a graph representing the negative predictive value (NPV) as a function of specificity for a cohort of 68 patients with lung cancer and the indicator according to the invention “relative concentration of cfDNA fragments of size greater than or equal to 1650 bp in a plasma sample of said patient”.

[0175] Figure 1 1 shows graphs representing, as a function of time, the progression-free survival curve of patients with cancer (in a / , all cancers, in b / , lung cancer) and having been subjected to immunotherapy treatment, determined by the Kaplan-Meier method, according to whether they have a score higher (dark curve) or lower (light curve) than the threshold value (in a / 0.039, in b / 0.037), 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 numbers of patients (“individuals at risk”) of the initial cohort included in the study at each evaluation time, respectively with a score lower than the threshold value (top line) and a score lower than the threshold value (bottom line) are indicated in the tables below the graphs.

[0176] Figure 12 shows graphs representing, as a function of time, the progression-free survival curve of patients with cancer (in a / , all cancers, in b / , lung cancer) and having been subjected to immunotherapy treatment, determined by the Kaplan-Meier method, according to whether they have a score higher (dark curve) or lower (light curve) than the threshold value (in a / 307.2, in b / 304.8), this score being predicted by a method according to the invention taking into account the position of peak 2 on the curve representing the concentration, as a function of the size, of the cfDNA fragments in the plasma samples of the patients; the numbers of patients (“individuals at risk”) of the initial cohort included in the study at each evaluation time, respectively with a score lower than the threshold value (top line) and a score lower than the threshold value (bottom line) are indicated in the tables below the graphs.

[0177] Figure 13 shows a graph representing, as a function of time, the progression-free survival curve of patients with cancer who have been subjected to immunotherapy treatment, determined by the Kaplan-Meier method, according to whether they have a score higher (dark curve) or lower (light curve) than the threshold value (0.086), this score being predicted by a method according to the invention taking into account the relative concentration of cfDNA fragments of size between 580 and 1649 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 the threshold value (top line) and a score lower than the threshold value (bottom line) is indicated in the table below the graph.Figure 14 shows a graph representing, as a function of time, the progression-free survival curve of patients with lung cancer who have been subjected to immunotherapy treatment, determined by the Kaplan-Meier method, according to whether they have a score higher (dark curve) or lower (light curve) than the threshold value (0.5), this score being predicted by a method according to the invention taking into account several parameters of cfDNA size in the plasma samples of the patients; the number of patients (“individuals at risk”) of the initial cohort included in the study at each evaluation time, respectively with a score lower than the threshold value (top line) and a score lower than the threshold value (bottom line) is indicated in the table below the graph.

[0178] Figure 15 shows a graph representing, as a function of time, the progression-free survival curve of patients with lung cancer who have been subjected to immunotherapy treatment, determined by the Kaplan-Meier method, according to whether they have a score higher (dark curve) or lower (light curve) than the threshold value (0.5), this score being predicted by a method according to the invention taking into account several parameters of cfDNA size in the patients' plasma samples and a clinical marker; 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 the threshold value (top line) and a score lower than the threshold value (bottom line) is indicated in the table below the graph.

[0179] Figure 16 shows a graph representing, as a function of time, the progression-free survival curve of patients with HNSCC cancer who have been subjected to immunotherapy treatment, determined by the Kaplan-Meier method, according to whether they have a score higher (dark curve) or lower (light curve) than the threshold value (0.5), this score being predicted by a method according to the invention taking into account several parameters of cfDNA size in the patients' plasma samples and a clinical marker; the number of patients ("at-risk individuals") from the initial cohort included in the study at each evaluation time, respectively with a score lower than the threshold value (top line) and a score lower than the threshold value (bottom line) is indicated in the table below the graph. A / Study 1

[0180] 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%).

[0181] Response to immunotherapy treatment of patients refers to the response to the treatments used, which are: nivolumab, lipilimumab and / or pembrolizumab, these treatments can be combined with targeted therapy, administration of anti-PD-L1 antibodies or chemotherapy treatment. The characteristics of the patients in the cohort at the start of the study are summarized in Table 1.

[0182] Table 1 - Characteristics of patients in the cohort

[0183] The assessment of whether patients progress during immunotherapy treatment is performed by analyzing tumor images acquired by CT scan 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, e143-e152, 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).

[0184] A.1 / Preparation of plasma samples

[0185] The samples used according to the invention are prepared from whole blood taken from the individual. For this purpose, whole blood is collected on Cell-Free DNA Collection Tubes according to the recommendations of the supplier Roche Diagnostics.

[0186] Each sample thus collected is subjected to two successive centrifugations:

[0187] - a 1 ère gentle centrifugation at 1600 g, at room temperature (20°C + / - 5°C), for 10 min, this 1 ère centrifugation being carried out within a maximum period of 7 days after collection; the plasma (supernatant) is recovered without removing the cell pellet,

[0188] - and a 2 èmecentrifugation 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 pellet formed. 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 longer than 1 month, before analysis.

[0189] A.2 / Analysis of plasma samples for circulating free DNA concentrations in different size ranges

[0190] The total circulating free DNA concentration in plasma samples, and the concentration profiles as a function of size, are determined by a method based on pLas microfluidic technology. More specifically, the protocol implemented is as described in the publication by Boutonnet et al., Analytical Chemistry, 2023, 95(24): 9263-70.

[0191] The material used is as follows:

[0192] - Agilent G7100A CE electrophoresis system equipped with a capillary incorporating pLAS technology, the diameter of which is provided with a local restriction,

[0193] - Picometrics fluorescence detector, Zetalif® LED 480.

[0194] The protocol implemented is schematically as follows.

[0195] Plasma samples are first pretreated with proteinase K in the presence of detergent to release 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 vigorous stirring (900 rpm), then recovered by centrifugation.

[0196] 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:

[0197] - RNASE: RNase Away (Biolab),

[0198] - BSA water: 50 mg / ml BSA solution in purified water,

[0199] - HCl 0.1 M: 0.1 M hydrochloric acid solution in water,

[0200] - PVA 2%: polyvinyl alcohol solution in water (800 mg in 40 ml of water),

[0201] - 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,

[0202] - TAE buffer + dye: TAE buffer + SybrGreen 2X (Sigma),

[0203] - 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,

[0204] - PIPES stamp + dye: PIPES stamp + SYBR Green 2X.

[0205] The exact program implemented in the electrophoresis system is detailed in Tables 2 to 4. Table 2 - Electrophoresis system program - preconditioning, injection and transfer

[0206]

[0207] Table 3 - Electrophoresis system program - Concentration with returns

[0208] Table 4 - Electrophoresis system program - Buffer change and end of concentration, separation A Tissue 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 Figure 1.

[0209] 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).

[0210] This standard sample has the composition indicated in Table 5:

[0211] Table 5 - Composition of the standard sample (Conc. denotes the concentration) The migration profile of this standard sample in the device used is shown in Figure 2.

[0212] 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.

[0213] 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 unit of time (1 min), and thus, for each of the peaks in Figure 2, a fluorescence value per pg.pL is obtained 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, 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 produces a curve composed of pieces of straight lines, which is smoothed using splines of degree 2, in a conventional 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.

[0214] For migration time calibration, the fragments in the standard sample provide "migration time / DNA size" pairs. A linear interpolation is performed, in a conventional manner, between each point to convert migration times to DNA size. Additional points can be added to the resulting DNA size versus migration time curve, with the migration times of these additional points calculated relative to the closest experimental DNA fragments in the standard sample, to improve DNA size calibration accuracy without having to increase the number of DNA fragments in the standard sample. After further linear interpolation, a DNA size versus migration time curve is obtained.

[0215] 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.

[0216] 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 in Figure 3.

[0217] 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, however, is highly representative of the actual value. To determine the cfDNA concentration in each desired size range, the minimum and maximum sizes of the interval are converted into 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.

[0218] 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 Figure 4. For each plasma sample analyzed, the total concentration of cfDNA is also 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 real value.

[0219] 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.

[0220] A.3 / Statistical analysis

[0221] 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 with 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, 75 to 239 bp, 240 to 369 bp, 370 to 579 bp, 580 to 1649 bp, greater than or equal to 1650 bp.

[0222] The two indicators that characterize patient response (early progressors or early non-progressors under immunotherapy treatment) are the relative concentrations of cDNA fragments in plasma for the following size ranges: size greater than or equal to 1650 base pairs, and size between 580 and 1649 base pairs.

[0223] The mathematical functions (indicators) used for the statistical analysis, named P1 and P2, are therefore the following: Concentration of cfDNA fragments of size greater than or equal to 1650 bp Total concentration of cfDNA

[0224] Concentration of cfDNA fragments between 580 and 1649 bp Total concentration of cfDNA

[0225] The cohort of 51 patients is divided into two datasets: a training batch (70% - 35 patients) and a test batch (30% - 16 patients). This distribution between training batch and test batch is classic and corresponds to the standard methodology for this type of statistical analysis. Both batches (training and test) present a balanced distribution between patients in progression, pathology and sex.

[0226] Statistical data are established against clinical data obtained by iRECIST methodology 6 to 12 weeks after the start of immunotherapy treatment, depending on the type of cancer.

[0227] 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”).

[0228] For each indicator, the threshold is optimized and defined as the median of the 100 thresholds obtained by the BOOTSTRAP method.

[0229] These thresholds are then used on the training batch and then the test batch, and the following metrics are determined:

[0230] - area under the curve (AUC),

[0231] - classification accuracy,

[0232] - sensitivity,

[0233] - specificity,

[0234] - positive predictive value (PPV): probability that the patient predicted as an early progressor is an early progressor,

[0235] - negative predictive value (NPV): probability that the patient predicted as an early non-progressor is an early non-progressor.

[0236] The thresholds determined by the BOOTSTRAP method are further used to calculate a 95% confidence interval of the threshold values ​​by extracting the 2.5% and 97.5% quantiles.

[0237] 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 Figure 5 for indicator P1 and in Figure 6 for indicator P2. 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 indicator P1 (Figure 5) than for indicator P2 (Figure 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.

[0238] Figure 7 shows the box plots obtained for each of the P1 and P2 indicators for the entire cohort. We observe that the results are statistically different between early progressors and early non-progressors.

[0239] 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 P1 and P2.

[0240] Table 6 - Characteristic statistical values ​​for indicators P1 and P2 For indicator P1, 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).

[0241] The P2 indicator has an excellent specificity of 1 considering a threshold (below which the patient is considered an early progressor) 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 P1 marker, which presents the best compromise between sensitivity and specificity.

[0242] 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 P1 indicator according to the invention, with the threshold value of 0.034. The results are shown in Figure 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 P1 indicator constitutes a marker allowing the prognosis of progression-free survival.

[0243] B / Study 2

[0244] This study is based on a cohort of 155 patients (including the 51 patients from Study 1) with 5 types of cancer pathologies: melanoma, ENT (head and neck squamous cell carcinoma, hereinafter referred to as HNSCC), lung (non-small cell), kidney and bladder.

[0245] The response to immunotherapy treatment of patients refers to the response to the treatments used which are: nivolumab, lipilimumab and / or pembrolizumab, these treatments can be combined with targeted therapy, the administration of anti-PD-L1 antibodies or chemotherapy treatment.

[0246] The characteristics of the cohort patients at the start of the study are summarized in Table 7 and Table 8. early, NEP = early non-progressor

[0247] Table 8 - Complete characteristics of patients in the cohort

[0248] For each patient, the curve representing the concentration of cfDNA as a function of size is established as described in the description of Study 1, and as illustrated in Figure 3. The position of peak 2 (expressed in base pairs), for example 308 base pairs for the example in Figure 3, is determined, as are the different concentrations of cfDNA in the desired ranges of values.

[0249] Statistical analysis was performed as described with reference to Study 1.

[0250] B.1 / Preliminary statistical analyses The mathematical functions (indicators) used for the statistical analysis are P1 and P2 described with reference to Study 1, as well as the parameter Position peak2.

[0251] For each mathematical function, ROC curves are established, and the area under the curve (AUC) values ​​and p-values, established respectively by the t-test and Wilcoxon test methods in a classical manner, with respect to known EP or NEP statuses, are determined.

[0252] The results obtained are shown in Table 9, for lung and bladder cancers, as well as for all cancers affecting patients in the cohort.

[0253] Table 9 - Characteristic statistical results for indicators P1, P2 and Position pic2 - n = number of patients, t = t test, Wile. = Wilcoxon

[0254] It is observed that from the point of view of the p-value, that is to say the certainty of having a different distribution between the early progressor (EP) and early non-progressor (NEP) groups, the P1 and P2 indicators, as well as the Position peak2 indicator, are discriminating for lung cancer and bladder cancer.

[0255] The lowest p-values ​​are obtained when considering all pathologies. This shows that these indicators have a different distribution between EP and NEP in the other pathologies as well. When the p-value is combined with the AUC, we observe that the P1 indicator performs best.

[0256] For each indicator and each type of cancer, the threshold that minimizes the distance from the optimal point (0.1) (reference value), established from the ROC curve, is indicated in Table 10. Scores above these threshold values ​​indicate early non-progressor status.

[0257] NEP

[0258] Depending on the information desired for the patient, the threshold values ​​may be chosen differently, in particular higher values ​​if the objective is to prioritize the specificity of prediction of an early progressor status rather than the sensitivity of prediction.

[0259] B.2 / Performance of indicator P1 on lung cancer

[0260] For the lung cancer patient group, the box plots and ROC curve for the P1 indicator are shown in Figure 9, respectively in a / and b / . The AUC obtained is equal to 0.79. The graph representing the NPV as a function of the specificity is shown in Figure 10. It is observed that the P1 indicator allows to predict approximately 3 / 4 of the early non-progressors with an NPV of 90%.

[0261] The “Position peak2” indicator shows similar performance to the P1 indicator in lung cancer.

[0262] B.3 / Multivariate analysis - cfDNA size parameters

[0263] This analysis takes into account the following size parameters:

[0264] - P1,

[0265] - Position peak2, expressed in base pairs,

[0266] - Sommei6o-22o, expressed in pg / pl to the power of 0.1, which is defined by the following formula:

[0267] A logistic regression was performed, specifically to predict early progressor (EP) patients, by dividing the data into a training set (75% of the data) and a test set (25% of the data, 16 patients), for a total of 64 lung cancer patients. The logistic regression was adjusted on the training set by cross-validation and then tested on the test set.

[0268] The results on the test set are shown in Table 1 1 .

[0269] Table 1 1 - Performance of a logistic regression on the P1 markers,

[0270] Position peak2 and Sommei6o-22o for the test set not participating in the training, for patients suffering from lung cancer (n=16) The performance is good; according to these data, it would be possible to avoid giving immunotherapy to 2 / 3 of EP patients, while omitting to give immunotherapy to 8% of NEP patients.

[0271] The complete mathematical formula, named S1 , is:

[0272] 51 = 1 - 1 / (1 + Exp(-(-37.13 + 32.04xP1 - 0.08x(Sum i6o-22o) + 0.13x(Position pic2))))

[0273] The threshold value (above which the patient is considered an early progressor) is equal to 0.5.

[0274] B.4 / Multivariate analysis - cfDNA size parameters and clinical marker

[0275] This analysis takes into account the following size parameters:

[0276] - P1,

[0277] - Position peak2, expressed in base pairs,

[0278] - Sommei6o-22o, expressed in pg / pl to the power of 0.1,

[0279] - ratio between the number of neutrophils and the number of lymphocytes (“Rn / I”), these numbers being determined by blood count. a / HNSCC cancer

[0280] A logistic regression was performed, specifically to predict early progressor (EP) patients, by dividing the data into a training set (25 patients) and a test set (7 patients), for a total of 32 patients with HNSCC. The logistic regression was adjusted on the training set by cross-validation and then tested on the test set.

[0281] On the whole data set, we obtain very good performances:

[0282] - Sensitivity = 83%

[0283] - Specificity = 86%

[0284] - PPV: 88%

[0285] - NPV: 80%.

[0286] The complete mathematical formula, named S2, is:

[0287] 52 = 1 - 1 / (1 + Exp(-(2.29 + 19.46xP1 - 0.07x(Sommei6o-22o) + 0.047x(Position pic2) - 2.27xRn / l)))

[0288] The threshold value (above which the patient is considered an early progressor) is equal to 0.5. b / Lung cancer

[0289] A logistic regression was performed, specifically to predict early progressor (EP) patients, by dividing the data into a training set (42 patients) and a test set (13 patients), for a total of 55 lung cancer patients. The logistic regression was adjusted on the training set by cross-validation and then tested on the test set.

[0290] On the whole data set, we obtain very good performances:

[0291] - Sensitivity = 44%

[0292] - Specificity = 96%

[0293] - PPV: 67%

[0294] - NPV: 90%.

[0295] The complete mathematical formula, named S3, is:

[0296] 53 = 1 - 1 / (1 + Exp(-(-27.35 - 6.30xP1 - 0.2x(Sommei60-22o) +0.12x(Position peak2) - 0.04xRn / l))) The threshold value (above which the patient is considered an early progressor) is equal to 0.5. c / All cancers excluding melanoma

[0297] A logistic regression was performed, specifically to predict early progressor (EP) patients, by dividing the data into a training set (83 patients) and a test set (27 patients), for a total of 110 patients with non-melanoma cancer. The logistic regression was adjusted on the training set by cross-validation and then tested on the test set.

[0298] On the whole data set, we again obtain very good performances:

[0299] - Sensitivity = 81%

[0300] - Specificity = 81%

[0301] - PPV: 68%

[0302] - NPV: 89%.

[0303] In particular, it is observed that the NPV is high: the conclusion of the prediction process allows immunotherapy treatment to be administered to the patient with good confidence that he will not be an early progressor.

[0304] The complete mathematical formula, named S4, is:

[0305] 54 = 1 - 1 / (1 + Exp(-(-16.18 + 27.26xP1 - 0.075x(Sommei6o-22o) + 0.056x(Position pic2) - 0.19xRn / l)))

[0306] The threshold value (above which the patient is considered an early progressor) is equal to 0.5. It emerges from the above results that the combination of the size markers chosen in accordance with the invention and the ratio “number of neutrophils / number of lymphocytes” further increases the performance of predicting an early progressor characteristic, compared to what can be predicted by the combination of these size markers alone.

[0307] B.5 / Progression-free survival curves

[0308] Progression-free survival curves and survival probabilities were estimated by the Kaplan-Meier method, and compared with the prediction results by different of the above indicators, with the corresponding cut-off value, for different types of cancer. Confidence intervals were obtained by the BOOTSTRAP method.

[0309] The results are shown:

[0310] - in figure 1 1 for indicator P1, in a / for all cancers and in b / for lung cancer alone;

[0311] - in figure 12 for the Position peak2 indicator, in a / for all cancers and in b / for lung cancer alone;

[0312] - in figure 13 for indicator P2, for all cancers;

[0313] - in figure 14 for indicator S1, for lung cancer;

[0314] - in figure 15 for indicator S3, for lung cancer;

[0315] - and in Figure 16 for indicator S2, for HNSCC cancer.

[0316] 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, with the exception of that of Figure 16, for which the indicator nevertheless correctly identifies patients with a low life expectancy, less than or equal to 4 months. Thus, the indicators chosen in accordance with the invention constitute a tool allowing the prognosis of progression-free survival.

[0317] This study conducted on 155 patients confirms the predictive power associated with the presence of large fragments in circulating free DNA.

Claims

CLAIMS 1. In vitro method for predicting the early progressor character and / or the early non-progressor character during an 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 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 being included in sizes greater than 500 base pairs, called 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, c / comparison of said score with a first predetermined reference value and / or a second predetermined reference value, and d / determination of the early progressor character and / or the early non-progressor character of said subject during said immunotherapy treatment on the basis of the result of said comparison.

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 comprises, as a variable, the ratio between at least said large cfDNA concentration, and said total cfDNA concentration.

5. The method of claim 4, wherein said mathematical function is the ratio of at least said large cfDNA concentration to said total cfDNA concentration.

6. Method according to any one of claims 1 to 5, according to which 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.

7. Method according to one of claims 1 to 5, according to which said predetermined size range is the size range greater than or equal to 1650 base pairs.

8. Method according to one of claims 1 to 5, according to which said predetermined size range is the size range between 1650 and 4000 base pairs.

9. A method according to any one of claims 1 to 5, wherein said predetermined size range is the size range between 580 and 1649 base pairs.

10. A method according to any one of claims 1 to 9, comprising establishing a curve representing the concentration, as a function of size, of the free deoxyribonucleic acid fragments circulating in said blood sample, and determining the size, expressed in base pairs, corresponding to the second peak on said curve, called Position peak2, and wherein said mathematical function includes, as a variable, said Position peak2.

11. A method according to any one of claims 1 to 10, comprising determining the concentration of circulating free deoxyribonucleic acid in said blood sample for each size between 160 and 220 base pairs, and calculating the sum of each of said concentrations to the power of 0.1, said sum being called Sumi6o-22o, and wherein said mathematical function comprises, as a variable, said Sumi6o-22o.

12. A method according to any one of claims 1 to 11, comprising determining the number of neutrophils and the number of lymphocytes in said blood sample, and wherein said mathematical function comprises, as a variable, the ratio between said number of neutrophils and said number of lymphocytes.

13. A method according to any one of claims 1 to 12, comprising a step of obtaining a plasma sample from said sample of blood.

14. Method according to claim 13, according to which the determination of said total cfDNA concentration and the determination of said at least one large cfDNA concentration, and where appropriate the establishment of the curve representing the concentration, as a function of the size, of the circulating free deoxyribonucleic acid fragments and / or the determination of the circulating free deoxyribonucleic acid concentration for each size between 160 and 220 base pairs, are carried out by direct analysis of said plasma sample.

15. Method according to any one of claims 1 to 14, 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, and where appropriate reestablishment of the curve representing the concentration, as a function of the size, of the circulating free deoxyribonucleic acid fragments and / or the determination of the circulating free deoxyribonucleic acid concentration for each size between 160 and 220 base pairs, are carried out by analyzing the circulating free deoxyribonucleic acid extract thus obtained.

16. A method according to any one of claims 1 to 15, wherein said subject is a human.

17. The method of any one of claims 1 to 16, 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.

18. Method according to any one of claims 1 to 17, according to which said immunotherapy treatment uses nivolumab, lipilimumab, pembrolizumab, atezolizumab, avelumab and / or durvalumab.

Images