Use of soluble CD95l as a prognostic marker in posttransplant lymphoproliferative disorders

Abstract

PTLDs arise after solid organ and hematopoietic stem cell transplants, with occurrences linked to immunosuppressive therapy and EBV status. Initial treatment typically involves reducing immunosuppressive therapy and using rituximab, with further options including immunochemotherapy or adoptive cellular therapy for refractory cases. Now, the inventors hypothesized that the concentration of sCD95L could exert a biological function and affect clinical outcomes by modulating the immune response. Using the K-VIROGREF cohort, they dosed by ELISA sCD95L in 175 transplant patients with PTLD and 16 transplant controls. Plasma levels of sCD95L were elevated in transplant recipients. Interestingly, in patients with PTLD who expressed higher concentrations of sCD95L, clinical outcomes were better than those with lower concentrations, especially in patients with central nervous system (CNS) involvement where sCD95L was a marker of good prognostic. Thus, sCD95L is a novel prognostic marker that might guide treatment strategies in patients with PTLD, especially in those with CNS involvement. Therefore, the present invention relates to the use of soluble CD95L as a prognostic marker in post-transplant lymphoproliferative disorders.

Description

[0001] USE OF SOLUBLE CD95L AS A PROGNOSTIC MARKER IN POST-TRANSPLANT LYMPHOPROLIFERATIVE DISORDERS FIELD OF THE INVENTION:

[0002] The present invention is in the field of medicine, in particular hemato-oncology.

[0003] BACKGROUND OF THE INVENTION:

[0004] Post-Transplant Lymphoproliferative Disorders (PTLDs) are heterogeneous tumors arising after solid organ transplantation (SOT) and allogeneic hematopoietic stem cells transplantation (HSCT). PTLDs occur in 1-2% of SOT with an incidence for Non-Hodgkin Lymphoma of 194 per 100000 person-years and an increased risk according to the immunosuppressive regimen and the Epstein-Barr virus (EBV) serological status of the recipient / donor couple(l). Reduction of the immunosuppressive therapy to release the antitumor response by immune cells, usually followed by rituximab treatment, an anti-CD20 monoclonal antibody, is the first approach to treat patients with PTLD and this regimen deeply modifies the clinical outcomes (2-4). Rituximab-refractory patients will receive either immunochemotherapy (R-CHOP)(5) or adoptive cellular therapy (tabelecleucel) for EBV-positive PTLDs (6).

[0005] CD95 (Fas or APO1) is a ubiquitously expressed transmembrane protein that belongs to the Tumor Necrosis Factor (TNF) receptor superfamily (7). Its membrane-bound ligand, CD95L (mCD95L), also known as FasL, belongs to the TNF superfamily (8) and can be cleaved by metalloproteases to release a soluble ligand (sCD95L) (9). Recent studies have demonstrated that mCD95L expression by memory T cells can halt the immune response by inducing the accelerated differentiation of T memory stem cells (TSCM) through cell-cell contact(10,11). TSCM are a rare lymphocyte subpopulation with self-renewal and multipotency properties, that allow to reconstitute the entire memory and effector T-cell subsets (12). We have recently established that membrane-bound CD95L expressed by T cells can also impair BCR signaling through a caspase-dependent degradation of CD19 (13). On the other hand, after cleavage by metalloproteases, the soluble CD95L can stimulate immune cells including Thl7 lymphocytes (14, 15) and neutrophils (16) contributing to the elimination of tumor cells (17) and the severity of autoimmune disorders (14,15,18). Unlike its transmembrane counterpart, sCD95L fails to trigger apoptosis and exerts inflammatory signaling pathways (14,15,19). Because mCD95L can extinguish the T-cell-mediated antitumor response (10,11), and mitigate BCR activation inB cells (13), while its soluble counterpart stimulates the immune response, we postulate that the sCD95L concentration might reflect an efficient antitumor response.

[0006] SUMMARY OF THE INVENTION:

[0007] The present invention is defined by the claims. In particular, the present invention relates to the use of soluble CD95L as a prognostic marker in post-transplant lymphoproliferative disorders.

[0008] DETAILED DESCRIPTION OF THE INVENTION:

[0009] CD95L is a transmembrane cytokine mainly expressed by activated T and NK cells to contract the immune response through cell-cell contact. Conversely, after cleavage by metalloproteases, this ligand releases a sCD95L that stimulates the immune response and its antitumor activity. In Post-Transplant Lymphoproliferative Disorders (PTLDs), the inventors hypothesized that the concentration of sCD95L could exert a biological function and affect clinical outcomes by modulating the immune response. Using the K-VIROGREF cohort, they dosed sCD95L by ELISA in 175 transplant patients with PTLD and 16 transplant controls. Plasma levels of sCD95L were elevated in transplant recipients. Interestingly, in patients with PTLD who expressed higher concentrations of sCD95L, clinical outcomes were better than those with lower concentrations, especially in patients with central nervous system (CNS) involvement where sCD95L was a good prognostic marker. On a cellular aspect, the high concentration of sCD95L was associated with a reduced percentage of peripheral blood NK and NKT-like cells suggesting that either sCD95L could promote the trafficking of these cells within the tumor or modulate their differentiation and / or survival. In conclusion, sCD95L is a novel and good prognostic marker that might guide treatment strategies in patients with PTLD, especially in those with CNS involvement.

[0010] Main definitions:

[0011] As used herein, the term “patient” refers to a transplant patient who has undergone either solid organ transplantation (SOT) or allogeneic hematopoietic stem cells transplantation (HSCT).

[0012] As used herein, the term “post-transplant lymphoproliferative disorder” or “PTLD” refers to various lymphoid and / or plasmacytic proliferations that occur due to immunosuppression in patients who have undergone solid organ transplantation (SOT) or allogeneic hematopoietic stem cell transplantation (HSCT). PTLD includes a range of conditions from benign lymphoidhyperplasia to aggressive lymphomas. The Epstein-Barr virus (EBV) is a significant factor in the development of PTLD. EBV can remain dormant in the body and reactivate under immunosuppressed conditions. In transplant patients, EBV infects B cells, leading to their unchecked proliferation due to weakened immune surveillance, thus heightening the risk of PTLD. Nearly 50% of PTLD cases are EBV positive.

[0013] As used herein, the term “Central Nervous System Post-Transplant Lymphoproliferative Disorder” refers to a subset of PTLDs that specifically involve the central nervous system, including the brain and spinal cord. These disorders are characterized by abnormal lymphoid proliferation within the CNS, occurring in the context of immunosuppression following transplantation. CNS-PTLDs can present with a variety of neurological symptoms, such as headaches, seizures, cognitive impairment, and focal neurological deficits, depending on the location and extent of the lesions. Diagnosing CNS-PTLD typically involves imaging studies, such as MRI, and may require biopsy for histopathological confirmation. Treatment strategies often include reducing immunosuppressive therapy, followed by targeted therapies such as rituximab, chemotherapy, or radiotherapy, tailored to the specific characteristics and extent of the CNS involvement.

[0014] As used herein, the expression “EBV positive tumor” refers to the presence of Epstein-Barr virus (EBV) within the tumor cells. This status is determined through diagnostic tests that identify EBV DNA, RNA, or proteins within the tumor. The presence of EBV in tumor cells is significant as it can influence the development, progression, and treatment response of PostTransplant Lymphoproliferative Disorders (PTLD), particularly in the context of immunosuppression following transplantation.

[0015] As used herein, the term “survival time” indicates the duration of the patient’s life from the diagnosis of PTLD. As used herein, the expression “short survival time” indicates that the patient will have a survival time that will be lower than the median observed in the general population of patients suffering from PTLD. When the patient will have a short survival time, it is meant that the patient will have a “poor prognosis”. Inversely, the expression “long survival time” indicates that the patient will have a survival time that will be higher than the median (or mean) observed in the general population of patients suffering from PTLD. When the patient will have a long survival time, it is meant that the patient will have a “good prognosis”.As used herein, the term “predicting” refers to the process of using measurable indicators or biomarkers to forecast clinical outcomes or disease progression. In the context of the present invention, predicting involves assessing the levels of sCD95L in a patient's sample to estimate their survival time.

[0016] As used herein, the term “CD95L,” also known as “FasL,” has its general meaning in the art and refers to the transmembrane cytokine that is mainly expressed by activated T and NK cells. It plays a crucial role in contracting the immune response through cell-cell contact. The gene that encodes for CD95L is known as the FASLG gene. An exemplary amino acid sequence of CD95L is shown as SEQ ID NO:1 CD95L can be cleaved by metalloproteases to release its soluble form, known as soluble CD95L, which stimulates the immune response and exhibits antitumor activity. Thus, the term “soluble CD95L” or “sCD95L” has its general meaning in the art and refers to the soluble ligand produced by the cleavage of the transmembrane CD95L.

[0017] SEQ ID NO: 1 >sp | P48023 | TNFL6_HUMAN Tumor necrosis factor ligand

[0018]

[0019] superfamily member 6 0S=Homo sapiens OX=9606 GN=FASLG PE=1 SV=1 MQQPFNYPYPQIYWVDSSASSPWAPPGTVLPCPTSVPRRPGQRRPPPPPPPPPLPPPPPP PPLPPLPLPPLKKRGNHSTGLCLLVMFFMVLVALVGLGLGMFQLFHLQKELAELRESTSQ MHTASSLEKQIGHPSPPPEKKELRKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVKYKKGG LVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMWA RSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL

[0020] As used herein, the term “sample” encompasses any biological material obtained from a patient, including but not limited to blood, serum, plasma, tissue, or other bodily fluids. In particular, a blood sample is a commonly utilized form for measuring sCD95L levels due to its accessibility and the comprehensive information it can provide regarding a patient's immunological status. As used herein, the term “blood sample” means any blood sample derived from the subject. Collections of blood samples can be performed by methods well known to those skilled in the art. In some embodiments, the blood sample is a serum or plasma sample.

[0021] As used herein, the term “level” refers to the measurable concentration or amount of a specific substance within a given sample. In the context of the present invention, the level of sCD95L in a patient's sample is used as a predictive marker for the patient's survival time. This level can provide critical insights into the patient's prognosis and guide therapeutic decisions.As used herein, the term “correlates” refers to the statistical relationship between two variables. In the context of this invention, it specifically means the association between the level of sCD95L in a patient's sample and their survival time. A higher or lower level of sCD95L can be indicative of the patient's prognosis, providing valuable information for predicting overall survival (OS), progression-free survival (PFS), and disease-free survival (DFS). This correlation helps in understanding the progression of post-transplant lymphoproliferative disorder (PTLD) and aids in making informed therapeutic decisions.

[0022] As used herein, the term “high” refers to a measure that is significantly greater than normal, greater than a standard, such as a predetermined reference value or a subgroup measure, or that is relatively greater than another subgroup measure. For example, high levels of sCD95L refers to a level of sCD95L that is greater than a normal sCD95L level. A normal sCD95L level may be determined according to any method available to one skilled in the art. A high level of sCD95L may also refer to a level equal to or greater than a predetermined reference value, such as a predetermined cutoff. A high level of sCD95L may also refer to a level of sCD95L wherein a high sCD95L subgroup has relatively greater levels of sCD95L than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low. In some cases, a “high” level may comprise a range of levels that is very high and a range of levels that is “moderately high”, where moderately high is a level that is greater than normal but less than “very high”.

[0023] As used herein, the term “low” refers to a level that is less than normal, or less than a standard, such as a predetermined reference value or a subgroup measure that is relatively less than another subgroup level. For example, a low level of sCD95L means a level of sCD95L that is less than a normal level in a particular set of samples of patients. A normal level of sCD95L measure may be determined according to any method available to one skilled in the art. A low level of sCD95L may also mean a level that is less than a predetermined reference value, such as a predetermined cutoff. A low level of sCD95L may also mean a level wherein a low level sCD95L subgroup is relatively lower than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thuscreating a group whose measure is low (i.e., less than the median) with respect to another group whose measurement is high (i.e., greater than the median).

[0024] Methods of the present invention:

[0025] The first object of the present invention relates to a method of predicting the survival time of a patient having a post-transplant lymphoproliferative disorder comprising determining the level of sCD95L in a sample obtained from the patient wherein said level correlates with the patient’s survival time.

[0026] In some embodiments, the method of the present invention is particularly suitable for predicting the survival time of patients having central nervous system post-transplant lymphoproliferative disorder.

[0027] In some embodiments, the patient has a EBV positive status.

[0028] The method of the present invention is particularly suitable for predicting the overall survival (OS) or the progression-free survival (PFS). As used herein, the term “overall survival” or “OS” refers to the length of time from either the date of diagnosis or the initiation of therapy for a disease, such as PTLD, that patients are alive. As used herein, the term “progression-free survival” or “PFS” refers to the length of time during and after the treatment of a disease that patients are alive in remission or with a stable disease. It measures the number of people who are disease-free or still have PTLD, but without any progression, including people who may have had responded to treatment, but where the cancer has not disappeared completely.

[0029] According to the present invention, low levels of sCD95L correlate with a poor prognosis, suggesting a shorter survival time for the patient. Conversely, high levels of sCD95L correlate with a good prognosis, indicating a longer survival time.

[0030] Thus, in some embodiments the method of the present invention comprises the steps of i) determining the level of sCD95L in a sample obtained from the patient ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient has a poor prognosis when the level determined at step i) is lower than the predeterminedreference value or concluding that the patient has a good prognosis when the level determined at step i) is higher than the predetermined reference value.

[0031] Typically, the predetermined reference value is a threshold or cutoff value. Typically, a "threshold value" or "cutoff value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based on the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skill in the art. For example, retrospective measurement in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of sCD95L in a group of reference, one can use algorithmic analysis to statistically treat the levels determined in samples to be tested and thus obtain a classification standard having significance for sample classification. The full name of the ROC curve is the receiver operator characteristic curve, which is also known as the receiver operation characteristic curve. It is mainly used for clinical and biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cutoff values (thresholds or critical values, boundary values between normal and abnormal diagnostic test results) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate, and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point with high sensitivity and specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result improves as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used to draw the ROC curve, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER. SAS, DESIGNROC. FOR, MULTIREADER POWER. SAS,CREATE-ROC. SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0032] In some embodiments, the predetermined reference value is the level of sCD95L determined in a population of healthy individuals. Typically, it is concluded that the patient has a poor prognosis when the level of sCD95L is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 fold lower than the level determined in a population of healthy individuals. Conversely, it is concluded that the patient has a good prognosis when the level of sCD95L is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 fold higher than the level determined in a population of healthy individuals.

[0033] In some embodiments, the predetermined reference value may be typically determined by carrying out a method comprising the steps of

[0034] a) providing a collection of samples from PTLD patients;

[0035] b) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding PTLD patient (e.g. the duration of PFS and / or OS);

[0036] c) providing a serial of arbitrary quantification values;

[0037] d) determining the level of sCD95L for each sample contained in the collection provided at step a);

[0038] e) classifying said samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising samples that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising samples that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of samples are obtained for the said specific quantification value, wherein the samples of each group are separately enumerated;

[0039] f) calculating the statistical significance between (i) the quantification value obtained at step e) and (ii) the actual clinical outcome of the patients from which samples contained in the first and second groups defined at step f) derive;

[0040] g) reiterating steps f) and g) until every arbitrary quantification value provided at step d) is tested;h) setting the said predetermined reference value as consisting of the arbitrary quantification value for which the highest statistical significance (most significant) has been calculated at step g). For example, the level of sCD95L has been assessed for 100 samples of 100 patients. The 100 samples are ranked according to the level of sCD95L. Sample 1 has the highest level and sample 100 has the lowest level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding cancer patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated. The predetermined reference value is then selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the level of sCD95L corresponding to the boundary between both subsets for which the p value is minimum is considered as the predetermined reference value. It should be noted that the predetermined reference value is not necessarily the median value of levels of sCD95L.

[0041] Practically, high statistical significance values (e.g. low P values) are generally obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in some embodiments, instead of using a definite predetermined reference value, a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off value as described above. For example, according to this specific embodiment of a "cut-off value, the outcome can be determined by comparing the level of sCD95L with the range of values which are identified. In some embodiments, a cut-off value thus consists of a range of quantification values, e.g. centred on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum p-value which is found). For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. Therefore, a patient may be assessed by comparing values obtained by measuring the level of sCD95L, where values greater than 5 reveal a good prognosis and values less than 5 reveal a poor prognosis. In someembodiments, a patient may be assessed by comparing values obtained by measuring the level of sCD95L and comparing the values on a scale, where values above the range of 4-6 indicate a good prognosis and values below the range of 4-6 indicate a poor prognosis, with values falling within the range of 4-6 indicating an intermediate prognosis.

[0042] In some embodiments, the predetermined value is about 305 pg / ml as described in the EXAMPLE.

[0043] According to the invention, the measure of the level of sCD95L can be performed by a variety of techniques. Typically, the methods may comprise contacting the sample with a binding partner capable of selectively interacting with sCD95L in the sample. In some aspects, the binding partners are antibodies, such as, for example, monoclonal antibodies or even aptamers. The aforementioned assays generally involve the binding of the partner (i.e. antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

[0044] The level of sCD95L may be measured by using standard immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin / avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation.

[0045] An exemplary biochemical test for identifying specific proteins employs a standardized test format, such as ELISA test, although the information provided herein may apply to the development of other biochemical or diagnostic tests and is not limited to the development of an ELISA test (see, e.g., Molecular Immunology: A Textbook, edited by Atassi et al. Marcel Dekker Inc., New York and Basel 1984, for a description of ELISA tests). It is understood that commercial assay enzyme-linked immunosorbant assay (ELISA) kits for various plasma constituents are available. Therefore, ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize sCD95L. A sample containing or suspected of containing sCD95L is then added to the coated wells. After a periodof incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

[0046] Typically, levels of immunoreactive sCD95L in a sample may be measured by an immunometric assay on the basis of a double-antibody "sandwich" technique, with a monoclonal antibody specific for sCD95L (Cayman Chemical Company, Ann Arbor, Michigan). According to said embodiment, said means for measuring sCD95L level are for example i) a sCD95L buffer, ii) a monoclonal antibody that interacts specifically with sCD95L, iii) an enzyme-conjugated antibody specific for sCD95L and a predetermined reference value of sCD95L.

[0047] Measuring the level of sCD95L (with or without immunoassay-based methods) may also include separation of the compounds: centrifugation based on the compound’s molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the compound's affinity for the particular solid-phase that is used. Once separated, said one or two biomarkers proteins may be identified based on the known "separation profile" e.g., retention time, for that compound and measured using standard techniques.

[0048] Alternatively, the separated compounds may be detected and measured by, for example, a mass spectrometer.

[0049] The method as disclosed herein is particularly suitable for selecting a therapeutic regimen or determining if a certain therapeutic regimen is more appropriate for a patient identified as having a poor prognosis. Typically, when it is concluded that the patient has a poor prognosis, reducing the level of immunosuppressive therapy can be decided (i.e, reducing of the the quantity of the regimen or the frequency of the immunosuppressive therapy). As used herein, the term "immunosuppressive therapy" refers to treatments that inhibit or prevent activity of the immune system to reduce inflammation and prevent rejection of transplanted organs or tissues. This can include the use of immunosuppressive drugs, such as corticosteroids, calcineurin inhibitors, antiproliferative agents, and monoclonal antibodies. In someembodiments, in addition to the reduction of the immunosuppressive therapy, the patient can be eligible to a therapeutic intervention such as the administration of B cell depleting antibodies and / or chemotherapy. Typical B cell depleting antibodies include but are not limited to anti-CD20 monoclonal antibodies [e.g., Rituximab (Roche), Ibritumomab tiuxetan (Bayer Schering), Tositumomab (GlaxoSmithKline), AME-133v (Applied Molecular Evolution), Ocrelizumab (Roche), Ofatumumab (HuMax-CD20, Gemnab), TRU-015 (Trubion), and IMMU-106 (Immunomedics)]. The patient can also be eligible for immunochemotherapy, such as the widely recognized regimen R-CHOP. R-CHOP stands for Rituximab, Cyclophosphamide, Doxorubicin Hydrochloride (Hydroxydaunomycin), Vincristine (Oncovin), and Prednisone. Patients having a CNS-PTLD are directly eligible to a therapeutic intervention that consists in administering them with a combination of Rituximab, methotrexate and aracytine. More particularly, patients having a poor prognosis and especially those who are refractory immunochemotherapy can be eligible for adoptive cellular therapy, such as tabelecleucel. Tabelecleucel is a therapy designed to treat patients with Epstein-Barr Virus-associated diseases, including those who have undergone hematopoietic cell transplant. This therapy harnesses the power of the patient's immune system by using specifically engineered T-cells to target and eliminate diseased cells.

[0050] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0051] FIGURES:

[0052] Figure 1. sCD95L is up-regulated in transplant patients and is a prognostic marker in PTLD patients. A. Quantification of soluble CD95L in plasma of healthy subjects (n=29) and indicated transplant patients (PTLD, n=175; Transplant Controls, n=16). Statistical analyses were performed with Mann-Whitney test, ****p<0.0001. B. Overall Survival (OS) and Progression-Free survival (PFS) according to the concentration of sCD95L in the entire PTLD cohort (threshold 305 pg / ml) (n=175). Differences in survival between groups were assessed using the Log-rank test. C. Quantification of soluble CD95L in plasma of PTLD patients according to the EBV status of the tumor and the Overall Survival in EBV positive (n=88; upper panel) and EBV negative patients (n=86; lower panel). Statistical analyses were performed with t-test assay; differences in survival between groups were assessed using the Log-rank test. D.

[0053] Quantification of soluble CD95L in plasma of PTLD patients according to serological EBVstatus (at time of transplantation) and their corresponding Overall Survival in EBV seronegative (n=19; upper panel) and EBV seropositive patients (n=82; lower panel). Statistical analyses were performed with Mann-Whitney test, ***p=0.0006; differences in survival between groups were assessed using the Log-rank test.

[0054] Figure 2. sCD95L is a prognostic factor for PTLD patients with CNS involvement. A.

[0055] Overall Survival (OS) according to CNS involvement in PTLD patients (n= 175: yes, n=39; no, n=136). Differences in survival between groups were assessed using the Log-rank test. B.

[0056] Quantification of soluble CD95L in plasma of PTLD patients with (n=39) or without CNS involvement (n=136). Statistical analyses were performed with t-test assay, *p=0.0123. C.

[0057] Overall Survival (OS, upper panel) and Progression-Free survival (PFS, lower panel) according to the concentration of sCD95L in the CNS-PTLD patients (n=39). Differences in survival between groups were assessed using the Log-rank test. D. Overall Survival (OS, upper panel) and Progression-Free survival (PFS, lower panel) according to the concentration of sCD95L in non-CNS PTLD patients (threshold 305 pg / ml) (n=136). Differences in survival between groups were assessed using the Log-rank test.

[0058] Figure 4. sCD95L is a predictive factor for survival. (A) Forest plot for univariate Cox proportional hazard regression analyses of predicting factors for progression-free survival. (B) Forest plot for univariate Cox proportional hazard regression analyses of predicting factors for overall survival. CNS, central nervous system; EBV, Epstein-Barr virus; mTOR, mammalian target of rapamycin; NK, natural killer; NKT, natural killer T; sCD95L, soluble CD95 ligand; PTLD, posttransplant lymphoproliferative disorder.

[0059] EXAMPLE:

[0060] Methods:

[0061] Patients and samples

[0062] We conducted a national multicenter, retrospective study of 175 adult transplant recipients diagnosed with PTLD from July 2014 to October 2023. Patients were consecutively recruited at 20 French medical centers through the K-VIROGREF Study Group, at diagnosis or relapse of PTLD, before chemotherapy. Diagnosis of PTLD, including assessment of tumor EBV status (by in situ hybridization of EBV-encoded small RNAs [EBER] and / or latent membrane protein 1 [LMP1] histochemical stains), was performed on formalin-fixed paraffin embedded tissues by hematopathologists at their respective institutions. Patients were classified as EBV-positive PTLD (n=88) or EBV-negative PTLD (n=86) according to EBV status of the tumor, with one missing status (Table 1). PTLD-free transplant controls (TCs, n=16) were recruited from thekidney (n=9) and liver (n=7) transplant units of the Pitie-Salpetriere Hospital (Paris, France). Blood samples from patients with PTLD (at time of inclusion in the K-VIROGREF study) and TCs (during their follow-up) were collected in their treating centers, then centralized and stored at the Department of Immunology of the Pitie-Salpetriere Hospital. This study was approved by institutional research ethics committee (Comite de Protection des Personnes Ile-de-France VII, No. PP13-022) and conducted in accordance to the human-experimentation guidelines of the declaration of Helsinki. All patients provided written informed consent. Healthy donors were collected at the University Hospital of Limoges and stored at the CRIBL Lab, University of Limoges (Limoges, France).

[0063] CD95L ELISA

[0064] Soluble CD95L was measured retrospectively by ELISA according to the manufacturer’s instructions (Abeam®) in frozen plasma of patients with PTLD, transplant controls without PTLD and healthy donors.

[0065] Flow cytometry

[0066] Flow cytometry analyses were performed on fresh blood cells, at time of inclusion, in the Department of Immunology and CIMI-INSERM 1135, Paris, France. Percentages and absolute numbers of T and B lymphocytes and NK cells were determined on EDTA-collected whole blood samples with an automated AQUIOS CL flow cytometry system (Beckman Coulter) using commercial kits AQUIOS Tetra-1 Panel (#B23533) and Tetra-2 + Panel (#B23534) according to manufacturer’s instructions (Beckman Coulter). AQUIOS Tetra-1 Panel Monoclonal Antibody Reagents is a four-color monoclonal antibody cocktail consisting of CD45-FITC / CD4-RD1 / CD8-ECD / CD3-PC5 and AQUIOS Tetra-2+ Panel Monoclonal Antibody Reagents is a four-color monoclonal antibody cocktail consisting of CD45-FITC / (CD56 + CD16)-RD1 / CD19-ECD / CD3-PC5.

[0067] Statistical analysis

[0068] Quantitative data were described using mean, median, standard deviation (SD), interquartile range (IQR), and compared between 2 groups using t-Test or Mann-Whitney assays. Qualitative data were described using numbers, percentages, and compared between 2 groups using Chi2 or Fisher exact tests. All tests were two-tailed, and p<0.05 was considered significant. Correlations were determined with the Spearman rank correlation coefficient. Follow-up was estimated with the reverse Kaplan Meier method. Overall Survival (OS) was measured fromthe date of PTLD diagnosis to the date of death. Progression Free Survival (PFS) was calculated from the time of diagnosis of PTLD to the time of relapse / progression or death from any cause, whichever occurred first. Survival outcomes were estimated using the Kaplan Meier method, and differences in survival between groups were assessed using the Log-rank test. Hazard ratios (HR) were reported with their 95% Confidence Intervals (Cis). All statistical analyses were performed on GraphPad Prism v10.0 (GraphPad Software) and Medistica.pvalue.io (an online biostatistics software by Medistica®).

[0069] Results:

[0070] Using the K-VIROGREF cohort (20) (not shown), we analyzed 175 patients with PTLD and compared them to 16 TCs patients. Clinical data were comparable between the two groups (data not shown). Among the 175 patients with PTLD, the median age at transplantation was 47 years (IQR, 34-57), and median age at PTLD diagnosis was 58 years (IQR, 45-67) (Table 1). The most common transplanted organ was kidney (57%), followed by liver (22%), heart (12%), lung (5%) and hematopoietic stem cells (5%). EBV serological status of the recipients was obtained at time of transplantation for 101 patients, of whom 19 were seronegative. At time of PTLD diagnosis, most of patients were receiving at least a two-drug immunosuppressive therapy (median 2; range 0-3); the immunosuppressive regimens comprised anti-calcineurin (cyclosporine, tacrolimus) or mammalian target of rapamycin (mTOR) inhibitors (everolimus, sirolimus) in 90%, antiproliferative drugs (azathioprine, mycophenolic acid derivatives) in 70% and / or glucocorticosteroids in 56% of the patients, respectively. Regarding histology, 51% of tumors were positive for EBV, and 55.5% had a monomorphic B-cell type (21). Overall, 19.5% (n=34) of patients had a primary CNS-PTLD and 3% (n=5) a systemic PTLD with CNS involvement. Of note, among all the patients with a CNS-PTLD (n=39), 87% (n=34) had an EBV-positive tumor. Patients’ characteristics are summarized in Table 1.

[0071] The dosage of sCD95L revealed a significant increased concentration of sCD95L in plasma of transplant patients compared to healthy donors (n=29), and regardless of PTLD diagnosis (Figure 1A, p<0.0001). We did not observe any organ-specific variations in the concentration of sCD95L, except for heart-transplanted patients in whom a lower concentration of sCD95L was detected (data not shown, p=0.007) when compared to kidney transplant recipients.

[0072] We next investigated whether sCD95L concentration could be correlated with the clinical outcomes in patients with PTLD. Using Receiver-Operating Characteristic (ROC) plots forsurvivals, a sCD95L concentration threshold for OS at 5 years was fixed at 305 pg / mL (data not shown, AUC=0.7056). Interestingly, at a median follow-up of 47.7 months for the entire cohort, patients with PTLD and high concentrations of plasma sCD95L (above 305 pg / mL) had longer PFS and OS compared to patients with low plasma sCD95L levels (Figure 1B). Indeed, median PFS and OS were not reached in patients with sCD95L > 305 pg / ml, whereas in patients with sCD95L < 305 pg / ml, median survival estimates were at 22.1 and 41.5 months, respectively (HR for PFS: 0.606, 95% CI: 0.389-0.944, p=0.023 and HR for OS: 0.565, 95%CI: 0.354-0.901, p=0.014). To assess whether sCD95L concentrations could discriminate PTLD patients, we further categorized patients according to their levels of sCD95L with a 305 pg / ml threshold (Table 2). The only differences in univariate analysis between the 2 groups were fewer recipients with a negative Epstein-Barr virus (EBV) serological status at transplantation (p<0.01) and higher rates of CNS localizations (p<0.01) in the sCD95L < 305 pg / ml group.

[0073] Since EBV is involved in the pathogenesis of PTLD in 50 to 60% of patients (22), we next evaluated whether the concentration of sCD95L was related to the EBV status of the tumor and / or the recipient serological status (at time of transplantation). Although no difference in sCD95L concentrations was observed between patients with EBV-positive and EBV-negative tumors (Figure 1C), high sCD95L concentrations in patients with EBV-positive tumor were associated with longer OS as compared to sCD95L-low patients (Figure 1C, HR for OS: 0.520, 95%CI: 0.268-0.939, p=0.030). On the other hand, survival outcomes in patients with EBV-negative tumor were similar regardless of their sCD95L concentrations. Regarding the EBV serological status of the recipients (before transplant, n=101), the concentration of sCD95L at PTLD diagnosis was increased in seronegative recipients (n=19) as compared to the seropositive counterparts (n=82) (Figure 1D, p=0.0006). As primo-infected EBV subjects are known to have higher concentrations of sCD95L than uninfected ones (23), this observation could be related to the post-transplant primo-infection of EBV- seronegative recipients undergoing an acute immune response and the upregulation of sCD95L. Nonetheless, the sCD95L concentration was associated neither with EBV-DNAemia (data not shown) nor with the clinical outcomes in seropositive or seronegative recipients (FigurelD).

[0074] Because CNS-PTLD patients are known to have a poor survival (24) (Figure 2A, median OS at 19.5 months vs not reached, p=0.0001), we wondered whether sCD95L could discriminate clinical outcomes in these patients (Table 3). Thirty-four patients had a primary, localized, CNS-PTLD, and 5 additional patients had a systemic CNS-PTLD with a similar poorer OS ascompared to patients with no CNS involvement (data not shown). Consistent with a better clinical outcome associated with elevated concentrations of sCD95L > 305 pg / ml, we observed that patients with CNS-PTLD (n=39) exhibited lower sCD95L concentrations compared to patients without CNS involvement (n=136) (Figure 2B, p=0.0123). Of note, 35% of heart-transplanted patients had a CNS involvement compared to 20% in other transplant recipients that could explain the lower concentrations of sCD95L measured in the heart-transplanted patients with PTLD (data not shown). Regarding outcomes, the median PFS and OS were 11.6 months and 14.8 months, respectively, in patients with CNS-PTLD and sCD95L < 305 pg / ml, whereas they were both at 47.8 months in patients with CNS-PTLD and sCD95L > 305 pg / ml (HR for PFS: 0.3645, 95%CI: 0.1678-0.7916, p=0.03 and HR for OS: 0.3705, 95%CI: 0.1704-0.8059, p=0.03) (Figure 2C). Interestingly, when patients with CNS involvement were excluded from the PTLD cohort, the sCD95L concentration threshold lost its prognostic value for both OS and PFS (Figure 2D).

[0075] In an attempt to explain the association between high concentration of sCD95L and the treatment efficacy in patients with CNS-PTLD, we postulated that this link could be due to the immunostimulatory role of sCD95L, as observed in auto-immune disorders (14,15). We therefore assessed whether CD4+and CD8+T lymphocytes (CD45+CD3+CD4+and CD45+CD3+CD8+, respectively), B lymphocytes (CD45+CD3-CD19+), NK (CD45+CD3‘ CD16+and / or CD56+) and NKT-like (CD45+CD3+CD16+and / or CD56+) cells were associated with the sCD95L concentrations in patients with PTLD (n=159) (Table 2) or CNS-PTLD (n=36) (Table 3). The percentage of NKT-like cells was significantly reduced both in blood of patients with PTLD (data not shown) and CNS-PTLD (data not shown), with elevated concentrations of sCD95L, while NK cells were only found down-regulated in sCD95L-high patients with CNS-PTLD (data not shown). Although no correlation was observed between the percentage of NK cells nor NKT-like cells and sCD95L concentrations in patients with CNS-PTLD (data not shown), a trend suggested an inverted correlation. An inverted correlation with sCD95L and NKT-like cells was measured in the entire cohort of patients with PTLD (data not shown).

[0076] Finally, we performed univariate Cox proportional hazards regression analyses to identify clinical and biological features associated with PFS and OS at time of diagnosis in PTLD patients (Fig. 3). Interestingly, only sCD95L >305 pg / mL and higher CD45+ and CD3+ cell counts were associated with a better prognosis. A poorer clinical outcome was associated with age >60 years, CNS involvement, and higher percentage of NK and NKT like cells. Multivariateanalyses revealed that for OS, CNS involvement, CD3+ cell counts, and sCD95L were independent prognostic markers.

[0077] Discussion:

[0078] Because the sCD95L concentration is similar in transplant patients with or without PTLD, we conclude that this cytokine is probably not involved in the disease occurrence. However, we were able to show that sCD95L represents a robust biomarker in patients with EBV-positive PTLD, particularly in patients with CNS-PTLD, and that high sCD95L concentrations are associated with prolonged survivals. The beneficial impact of sCD95L might be related to its pro-inflammatory effect, that might promote an antitumor response by modulating the localization and / or survival / differentiation of peripheral blood “innate” cells, such as NKT-like and NK cells.

[0079] In conclusion, in this pioneer work, we were able to show that the expression of the metalloprotease-cleaved CD95L is increased in all transplant recipients compared to healthy donors, regardless of the immunosuppressive environment. Additionally, high concentrations of this cytokine is a good prognostic marker in patients with PTLD, particularly in CNS-PTLDs.

[0080] TABLES:

[0081] Table 1: Clinical characteristics of the patients with PTLD and according to CNS involvement.

[0082] All PTLDs non-CNS CNS n p test (n = 175) (n = 136) (n = 39) TRANSPLANTATI ON

[0083] Sex, n Male 114 (65.0%) 91 (67.0%) 23 (59.0%) 0.36 Chi2

[0084] Female 61 (35.0%) 45 (33%) 16 (41.0%)

[0085] Age at 46.8 (33.6-57.0) 44.5 54.0 17 0.014 Welc transplantation, (32.5; (45.0; 5 h median [IQR] 44.5) 58.0)

[0086] EBV serological Positive 82 (47.0%) 67 (49.0%) 15 (38.0%) 0.43 Fishe status (recipient), n r Negativ 19 (11.0%) 15 (11.0%) 4 (10.0%) e

[0087] NA 74 (42.0%) 54 (40.0%) 20 (51.0%)Transplant organ, n Kidney 100 (57.0%) 79 (58%) 21 (54.0%) 0.76 Fishe r Liver 35 (20.0%) 28 (21.0%) 7 (18.0%)

[0088] Heart 21 (12.0%) 14 (10.0%) 7 (18.0%)

[0089] Lung 10 (6.0%) 8 (6.0%) 2 (5.0%)

[0090] HSC 9 (5.0%) 7 (5.0%) 2 (5.0%)

[0091] PTLD

[0092] Age at PTLD 58.4 [44.6; 67.0] 58.0 61.0 17 <0.01 Welc diagnosis, (42.5;66.5 (54.5;68.0 5 h median [IQR] ) )

[0093] Time from 112.8 [48.8; 176 115.5 107.5 17 0.96 Welc transplantation.6] (49.0;176. (54.5;168. 5 h (mos), 0) 5)

[0094] median [IQR]

[0095] Early PTLD (<12 No 155 (89.0%) 118 (87.0 37 (95.0%) 0.25 Fishe mos), n %) r Yes 19 (11.0%) 17 (13.0%) 2 (5.0%)

[0096] Current IS drugs, n 0 3 (1.5%) 3 (2.0%) 0 (0%) 0.068 Fishe r 1 30 (17.5%) 22 (16.5%) 8 (20.5%)

[0097] 2 76 (44.0%) 65 (48.5%) 11 (28.0%)

[0098] 3 64 (37.0%) 44 (33.0%) 20 (51.5%)

[0099] CsA, tacrolimus, No 18 (10.5%) 9 (6.5%) 9 (23.0%) <0.01 Fishe everolimus, r sirolimus, n

[0100] Yes 155 (89.5%) 125 (93.5 30 (77.0%)

[0101] %)

[0102] MMF, No 52 (30.0%) 45 (33.5%) 7 (18.0%) 0.061 Chi2 azathioprine..., n

[0103] Yes 121 (70.0%) 89 (66.5%) 32 (82.0%) Corticosteroids, n No 76 (43.5%) 65 (48.0%) 11 (28.0%) 0.027 Chi2

[0104] Yes 98 (56.5%) 70 (52.0%) 28 (72.0%)EBV status Negativ 86 (49.5%) 81 (60.0%) 5 (13.0%) <0.00 Chi2 (tumor), n e 1

[0105] Positiv 88 (50.5%) 54 (40.0%) 34 (87.0%) e

[0106] EBV-DNAemia ns 112 (64.0%) 91 (67.0%) 21 (54.0%) 0.095 Fishe log, n r + 17 (10.0%) 15 (11.0%) 2 (5.0%)

[0107] ++ 26 (15.0%) 18 (13.0%) 8 (21.0%)

[0108] +++ 20 (11.0%) 12 (9.0%) 8 (21.0%)

[0109] ORR, n no 35 (21.0%) 28 (22.0%) 7 (18.0%) 0.65 Chi2 respons

[0110] e

[0111] PR or 131 (79.0%) 100 (78.0 31 (82.0%) better %)

[0112] Alive or Dead, n Alive 104 (59%) 92 (68.0%) 12 (31.0%) <0.00 Chi2

[0113] 1 Dead 71 (41%) 44 (32.0%) 27 (69.0%)

[0114] Relapse or No 96 (55.0%) 84 (62.0%) 12 (31.0%) <0.00 Chi2 Death, n 1

[0115] Yes 79 (45.0%) 52 (38.0%) 27 (69.0%)

[0116] SCD95L, median 290 (195;480) 316 (198; 248 (165; 17 <0.01 Welc (IQR) 504) 348) 5 h SCD95L>305, n No 93 (53.0%) 65 (48.0%) 28 (72.0%) <0.01 Chi2

[0117] Yes 82 (47.0%) 71 (52.0%) 11 (28.0%)

[0118] Table 2: Clinical and biological characteristics of patients with PTLD according to sCD95L levels (threshold 305 pg / ml).

[0119] All PTLDs sCD95L<305 sCD95L>305 n p test (n = 175) (n = 93) (n = 82) TRANSPLANTATI ON

[0120] Sex, n Male 114 (65.0 67 (72.0%) 47 (57.0%) 0.04 Chi2

[0121] %) 1Female 61 (35.0% 26 (28.0%) 35 (43.0%)

[0122] )

[0123] Age at 46.8 48.1 [33.7; 57. 44.6 [33.1; 56. 17 0.3 Welc transplantation, (33.6- 5] 2] 5 h median [IQR] 57.0)

[0124] EBV serological Positive 82 (47.0% 48 (52.0%) 34 (41.0%) <0.0 Chi2 status ) 1 (recipient), n

[0125] Negativ 19 (11.0% 3 (3.0%) 16 (20.0%) e )

[0126] NA 74 (42.0% 42 (45.0%) 32 (39.0%)

[0127] )

[0128] Transplant Kidney 100 (57.0 50 (54.0%) 50 (61.0%) 0.44 Fishe organ, n %) r Liver 35 (20.0% 18 (19.0%) 17 (21.0%)

[0129] )

[0130] Heart 21 (12.0% 15 (16.0%) 6 (7.0%)

[0131] )

[0132] Lung 10 (6.0%) 6 (6.5%) 4 (5.0%)

[0133] HSC 9 (5.0%) 4 (4.5%) 5 (6.0%)

[0134] PTLD

[0135] Age at PTLD, 58.4 58.7 [47.3; 68. 58.1 [43.2; 64. 17 0.12 Welc diagnosis [44.6; 67.0 4] 1] 5 h median [IQR] ]

[0136] Time from 112.8 112.8 (61.5- 110.5 (34.5- 17 0.54 Welc transplantation [48.8; 176. 168.5) 181.1) 5 h (mos), 6]

[0137] median [IQR]

[0138] Early PTLD (<12 No 155 (89.0 86 (92.0%) 69 (85.0%) 0.12 Chi2 mos), n %)

[0139] Yes 19 (11.0% 7 (8.0%) 12 (15.0%)

[0140] )

[0141] Current IS drugs, n 0 3 (2.0%) 1 (1.0%) 2 (2.5%) 0.6 Fishe r 1 30 (17.0% 15 (16.0%) 15 (19.0%)

[0142] )

[0143] 2 76 (40.0% 38 (41.0%) 38 (47.0%)

[0144] )3 64 (40.0% 38 (41.0%) 26 (32.0%)

[0145] )

[0146] CsA, tacrolimus, No 18 (10.0% 8 (9.0%) 10 (12.0%) 0.43 Chi2 everolimus, )

[0147] sirolimus, n

[0148] Yes 155 (90.0 84 (91.0%) 71 (88.0%)

[0149] %)

[0150] MMF, No 52 (30.0% 26 (28.0%) 26 (32.0%) 0.58 Chi2 azathioprine..., n )

[0151] Yes 121 (70.0 66 (72.0%) 55 (68.0%)

[0152] %)

[0153] Corticosteroids, n No 76 (44.0% 37 (40.0%) 39 (48.0%) 0.33 Chi2

[0154] )

[0155] Yes 98 (56.0% 55 (60.0%) 43 (52.0%)

[0156] )

[0157] EBV status negativ 86 (49.0% 48 (52.0%) 38 (47.0%) 0.54 Chi2 (tumor), n e )

[0158] positive 88 (51.0% 45 (48.0%) 43 (53.0%)

[0159] )

[0160] Systemic Yes 141 (81.0 69 (74.0%) 72 (88.0%) 0.02 Chi2 involvement, n %) 3

[0161] No 34 (19.0% 24 (26.0%) 10 (12.0%)

[0162] )

[0163] CNS Yes 39 28 (30.0%) 11 (13.0%) <0.0 Chi2 involvement, n (22.0%) 1

[0164] No 136 65 (70.0%) 71 (87.0%)

[0165] (78.0%)

[0166] Stade Ann Arbor, n allograft 3 (2.0%) 2 (2.0%) 1 (1.5%) 0.1 Fishe r 1 4 (2.5%) 3 (3.5%) 1 (1.5%)

[0167] 1 CNS 34 (20.0% 24 (26.5%) 10 (13.0%)

[0168] )

[0169] 2 10 (6.0%) 4 (4.5%) 6 (7.5%)

[0170] 3 10 (6.0%) 3 (3.5%) 7 (9.0%)

[0171] 4 102 (60.5 50 (55.5%) 52 (66.5%)

[0172] %)

[0173] 4CNS 5 (3.0%) 4 (4.5%) 1 (1.5%)

[0174] EBV-DNAemia ns 112 (64.0 59 (63.0%) 53 (65.0%) 1 Chi2 log, n %)+ 17 (10.0% 9 (10.0%) 8 (10.0%)

[0175] )

[0176] ++ 26 (15.0% 14 (15.0%) 12 (15.0%)

[0177] )

[0178] +++ 20 (11.0% 11 (12.0%) 9 (11.0%)

[0179] )

[0180] ORR, n no 35 (21.0% 20 (23.0%) 15 (19.0%) 0.53 Chi2 respons )

[0181] e

[0182] PR or 131 (79.0 67 (77.0%) 64 (81.0%) better %)

[0183] Alive or Dead, n Alive 104 (59.0 51 (55.0%) 53 (65.0%) 0.19 Chi2

[0184] %)

[0185] Dead 71 (41.0% 42 (45.0%) 29 (35.0%)

[0186] )

[0187] Relapse or no 96 (55.0% 47 (51.0%) 49 (60.0%) 0.22 Chi2 Death, n )

[0188] yes 79 (45.0% 46 (49.0%) 33 (40.0%)

[0189] )

[0190] SCD95L<305 SCD95L>305 n p test (n = 93) 1 (n = 82)

[0191] CD45+ 868 883 [466;1220] 828 [432; 1378 15 0.09 Welc Lymphocytes (438; 1326 ] 9 h counts / mm3, medi )

[0192] an

[0193] [IQR]

[0194] CD45+CD3+ %, 76.0 75.0 77.0 (64.0; 15 0.28 Welc median [IQR] [64.0; 83.8 (64.5;83.5) 84.0) 9 h ]

[0195] CD45+CD3+ 644 618 [302; 947] 650 [318; 1072 15 0.19 Welc counts / mm3, [310; 1012] ] 9 h median [IQR]

[0196] CD45+CD3+CD4+ 35.0 37.0 [29.0; 44. 33.5 [22.8; 47. 15 0.42 Welc %, [26.1; 46.0 8] 0] 9 h median [IQR] ]

[0197] CD45+CD3+CD4+ 333 333 [152; 510] 332 [131; 566] 15 0.9 Welc counts / mm3, [136; 534] 9 h median [IQR]CD45+CD3+CD8+ 31.0 31.0 [21.6; 42. 30.5 [19.8; 44. 15 0.92 Welc %, [21.3; 43.5 0] 0] 9 h median [IQR] ]

[0198] CD45+CD3+CD8+ 253 266 [146; 424] 241 [146; 434] 15 0.26 Welc counts / mm3, [146; 434] 9 h median [IQR]

[0199] Ratio CD4 / CD8, 1.20 1.20 [0.790; 1. 1.20 [0.600; 1. 15 0.55 Welc median [IQR] [0.700; 1.8 82] 83] 9 h 2]

[0200] CD45+CD3 CD16+10.0 10.0 [5.14; 19. 10.1 [5.97; 19. 15 0.7 Welc and / or CD56+[5.40; 19.0 0] 5] 9 h NK cells %, ]

[0201] median [IQR]

[0202] CD45+CD3 CD16+89.0 87.0 [38.0; 157 93.0 [59.0; 162 15 0.21 Welc and / or CD56+[40.0; 158] ] ] 9 h NK cells

[0203] counts / mm3,

[0204] median [IQR]

[0205] CD45+CD3+CD16+6.21 8.60 [4.38; 14. 5.00 [3.47; 9.0 15 <0.0 Welc and / or CD56+[4.00; 13. 8] 0] 8 1 h NKT-like cells %, 0]

[0206] median [IQR]

[0207] CD45+CD3+CD16+53.0 53.5 [37.0; 107 52.5 [29.0; 92. 15 0.1 Welc and / or CD56+[32.0; 99.8 ] 2] 8 h NKT-like cells ]

[0208] counts / mm3,

[0209] median [IQR]

[0210] CD45+CD3-CD19+ 8.00 7.77 [2.61; 16. 8.00 [4.34; 14. 15 0.48 Welc %, [3.00; 15.0 0] 0] 5 h median [IQR] ]

[0211] CD45+CD3- 63.0 57.0 [20.2; 124 66.0 [28.0; 141 15 0.21 Welc CD19+ [21.0; 136] ] ] 5 h counts / mm3,

[0212] median [IQR]

[0213] Table 3: Clinical and Biological characteristics of patients with CNS-PTLD according to sCD95L levels (threshold 305 pg / ml).

[0214] CNS SCD95L<305 SCD95L>305 n p test (n = 28) (n = 11)TRANSPLANTATION

[0215] Sex, n Male 18 (64.0%) 5 (45.0%) 2 0.31 Fisher 3

[0216] Female 10 (36.0%) 6 (55.0%) 1

[0217] 6

[0218] Age at 53.4 [43.7; 59.3] 54.0 [45.7; 55.5] 3 0.7 Mann- transplantation, 9 Whitney median [IQR]

[0219] EBV serological status Positive 12 (43.0%) 3 (27.0%) 1 0.09 Fisher (recipient), n 5 9

[0220] Negative 1 (4.0%) 3 (27.0%) 4

[0221] NA 15 (54.0%) 5 (45.0%) 2

[0222] 0

[0223] Transplant organ, n Kidney 14 (50.0%) 7 (64.0%) 2 0.84 Fisher 1

[0224] Liver 5 (18.0%) 2 (18.0%) 7

[0225] Heart 6 (21.0%) 1 (9.0.0%) 7

[0226] Lung 1 (4.0%) 1 (9.0%) 2

[0227] HSC 2 (7.0%) 0 (0%) 2

[0228] PTLD

[0229] Age at PTLD 61.0 [55.5; 67.6] 61.6 [52.0; 66.9] 3 0.96 Mann- diagnosis, 9 Whitney median [IQR]

[0230] Time from 107 [66.0; 155] 121 [38.1; 186] 3 0.96 Mann-transplantation (mos), 9 Whitney median [IQR]

[0231] Early PTLD (<12 No 26 (93.0%) 11 (100%) 3 1 Fisher mos), n 7

[0232] Yes 2 (7.0%) 0 (0%) 2

[0233] Current IS drugs, n 1 6 (21.0%) 2 (18.0%) 8 0.06 Fisher 1

[0234] 2 5 (18.0%) 6 (55.0%) 1

[0235] 1

[0236] 3 17 (61.0%) 3 (27.0%) 2

[0237] 0

[0238] CsA, tacrolimus, 0 6 (21.0%) 3 (27.0%) 9 0.69 Fisher everolimus,

[0239] sirolimus, n1 22 (79.0%) 8 (73.0%) 3

[0240] 0

[0241] MMF, 0 5 (18.0%) 2 (18.0%) 7 1 Fisher azathioprine..., n

[0242] 1 23 (82.0%) 9 (82.0%) 3

[0243] 2 Corticosteroids, n 0 6 (21.0%) 5 (45.0%) 1 0.23 Fisher 1

[0244] 1 22 (79.0%) 6 (55.0%) 2

[0245] 8

[0246] EBV status (tumor), n 0 3 (11.0%) 2 (18.0%) 5 0.61 Fisher 1 25 (89.0%) 9 (82.0%) 3

[0247] 4

[0248] EBV-DNAemia log, n ns 13 (46.0%) 8 (73.0%) 2 0.5 Fisher 1

[0249] + 2 (7.0%) 0 (0%) 2

[0250] ++ 6 (21.0%) 2 (18.0%) 8

[0251] +++ 7 (25.0%) 1 (9.0%) 8

[0252] ORR, n no 5 (19.0%) 2 (18.0%) 7 1 Fisher respons

[0253] e

[0254] PR or 22 (81.0%) 9 (82.0%) 3

[0255] better 1

[0256] Alive or Dead, n Alive 6 (21.0%) 6 (55.0%) 1 0.06 Fisher 2 1

[0257] Dead 22 (79.0%) 5 (45.0%) 2

[0258] 7

[0259] SCD95L<305 SCD95L>305 n p test (n = 28) (n = 11)

[0260] SCD95L, median [IQR 200 [146; 254] 422 [366; 525] 3

[0261] ] 9

[0262] CD45+ Lymphocytes 524 [171; 938] 634 [580; 1126] 3 0.26 Mann- counts / mm3, 6 Whitney median [IQR]

[0263] CD45+CD3+ %, 74.5 [63.6; 82.5] 78.7 [72.5; 82.0] 3 0.46 Mann- median [IQR] 6 Whitney CD45+CD3+ 382 [146; 672] 481 [414; 816] 3 0.23 Mann- counts / mm3, 6 Whitneymedian [IQR]

[0264] CD45+CD3+CD4+ %, 36.7 [30.0; 42.5] 29.5 [23.8; 44.8] 3 0.49 Mann- median [IQR] 6 Whitney CD45+CD3+CD4+ 206 [78.5; 382] 258 [150; 433] 3 0.56 Mann- counts / mm3, 6 Whitney median [IQR]

[0265] CD45+CD3+CD8+ %, 33.4 [22.2; 42.0] 36.0 [28.2; 49.5] 3 0.2 Mann- median [IQR] 6 Whitney CD45+CD3+CD8+ 163 [66.5; 278] 266 [183; 348] 3 0.12 Mann- counts / mm3, 6 Whitney median [IQR]

[0266] Ratio CD4 / CD8, 1.15 [0.755; 1.89 0.905 [0.450; 1.60 3 0.31 Mann- median [IQR] ] ] 6 Whitney CD45+CD3 CD16+15.5 [7.94; 23.4] 5.69 [5.00; 8.59] 3 0.02 Mann- and / orCD56+6 1 Whitne NK cells %, y median [IQR]

[0267] CD45+CD3 CD16+81.5 [28.8; 130] 57.5 [36.2; 74.8] 3 0.48 Mann- and / or CD56+6 Whitney NK cells counts / mm3,

[0268] median [IQR]

[0269] CD45+CD3+CD16+13.5 [7.19; 17.0] 6.50 [4.75; 8.00] 3 0.02 Mann- and / orCD56+6 1 Whitne NKT-like cells %, y median [IQR]

[0270] CD45+CD3+CD16+59.5 [40.2; 98.0] 60.0 [29.0; 75.0] 3 0.63 Mann- and / or CD56+6 Whitney NKT-like cells

[0271] counts / mm3,

[0272] median [IQR]

[0273] CD45+CD3-CD19+ 7.27 [2.78; 15.4] 12.0 [8.40; 14.0] 3 0.1 Mann- %, 6 Whitney median [IQR]

[0274] CD45+CD3-CD19+ 60.5 [11.0; 102] 94.0 [53.2; 152] 3 0.08 Mann- counts / mm3, 6 3 Whitney median [IQR]

[0275] REFERENCES:Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of predicting the survival time of a patient having a post-transplant lymphoproliferative disorder comprising determining the level of sCD95L in a sample obtained from the patient wherein said level correlates with the patient’s survival time.

2. The method according to claim 1 wherein the patient has a central nervous system posttransplant lymphoproliferative disorder.

3. The method according to claim 1 or 2 wherein the patient has a EBV positive tumor.

4. The method according to any one of claims 1 to 3 wherein low levels of sCD95L correlate with a poor prognosis, suggesting a shorter survival time for the patient whereas, high levels of sCD95L correlate with a good prognosis, indicating a longer survival time.

5. The method according to any one of claims 1 to 4 that comprises the steps of i) determining the level of sCD95L in a sample obtained from the patient ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient has a poor prognosis when the level determined at step i) is lower than the predetermined reference value or concluding that the patient has a good prognosis when the level determined at step i) is higher than the predetermined reference value.

6. The method according to any one of claims 1 to 5 wherein when it is concluded that the patient has poor prognosis, then the patient is administered with a reduction of the level of immunosuppressive therapy along with immunochemotherapy.

7. The method according to claim 6 wherein the patient is also eligible for adoptive cellular therapy, such tabelecleucel.

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