Methods for predicting active disease or progressive disease under therapy in a subject suffering from chronic lymphocytic leukemia

Abstract

Monitoring active disease or progressive disease under therapy in chronic lymphocytic leukemia (CLL) represents a challenge to earlier and better adapt therapeutic strategy, notably in the era of targeted therapies in which minimal residual detection or mutations are sometimes not associated to poor clinical outcome. By following CLL patients before treatment (Binet stages A and B / C) or during targeted therapy, the Inventors developed a new flow cytometric method, based on CD69, CD49d, CD20 and CD279 expression at the surface of CD19+ / CD5+ B leukemic cells. Analyses of these markers alone or in combination show that CD69 / CD49d / CD20 / CD279 co-expression (quadruple population, QP) > 0.5% is the best criterion predicting CLL active disease or progression under therapy. This new flow cytometry immunophenotyping could help clinicians to monitor CLL evolution and quickly adapt their therapeutic strategy. Accordingly, the present invention relates to an ex vivo method for predicting active Chronic Lymphocytic Leukemia (CLL) or progressive CLL under therapy in a subject suffering from CLL, comprising the step of quantifying a population of CD69+ / CD49d+ / CD20+ / CD279+ cells in a sample obtained from the subject.

Description

[0001] METHODS FOR PREDICTING ACTIVE DISEASE OR PROGRESSIVE DISEASE UNDER THERAPY IN A SUBJECT SUFFERING FROM CHRONIC LYMPHOCYTIC LEUKEMIA FIELD OF THE INVENTION: The present invention is in the field of medicine and relates in particular to oncology. BACKGROUND OF THE INVENTION: iwCLL guidelines1well defined chronic lymphocytic leukemia (CLL) diagnosis, staging, and indication of treatments. At diagnosis, Binet or RAI staging systems defining sub- types of patients1, is now completed by CLL-IPI prognostic index2leading to wait and watch strategy or initiation of therapy. In case of progression or symptomatic / active disease, additional criteria are defined and lead to therapeutic indication.1Recently International Prognostic Score for early stage CLL (IPS-E) has been proposed to predict time to first treatment for asymptomatic patients (Binet stage A).3This predictive score based on IGHV mutational status (UM-IGHV)4, high absolute lymphocyte count (>15 x 109 G / L) and presence of palpable lymph nodes could give an advice to clinicians. For patients needing treatments, a wide range of therapies are available, ranging from cytostatic agents combined or not with monoclonal antibodies, to targeted therapies.5Follow- up of clinical response is mainly based on the use of multi-color flow cytometry, PCR, or NGS to detect measurable residual disease (MRD).6–11These methods are also useful for BCL-2 inhibitor therapy such as venetoclax12, but unfortunately not really applicable for BTK inhibitors treatment such as ibrutinib in monotherapy.13,14Indeed prolonged ALC was often observed in ibrutinib-treated patients15and the decrease of ALC was not correlated to a better outcome.16Under ibrutinib treatment, the well-reported acquired resistance mechanism was driven by BTK and / or PLCγ2 mutations,17induced by ibrutinib selective pressure, but these mutations were not detected in 30% of ibrutinib-relapsed patients.18,19Some independent phenotypic markers, such as CD49d, CD69 or CD279 on B leukemic cells have also been reported as associated to CLL evolution under ibrutinib therapy and linked to ibrutinib outcome.20–24Finally, single cell analyses revealed an increased expression of CD49d, CD69, CD279 and CD20 under ibrutinib progression.25 Nevertheless, no clear biological markers are available to better monitor rapid disease progression in asymptomatic patients (Binet stage A) to B / C stage or under targeted therapy such as ibrutinib or venetoclax. SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to an ex vivo method for predicting active Chronic Lymphocytic Leukemia (CLL) or progressive CLL under therapy in a subject suffering from CLL, comprising the step of quantifying a population of CD69+ / CD49d+ / CD20+ / CD279+ cells in a sample obtained from the subject. DETAILED DESCRIPTION OF THE INVENTION: In this study, we developed a new multicolor flow cytometric method, based on CD69, CD49d, CD20 and CD279 co-expression at the surface of CD19+ / CD5+ B leukemic cells. This quadruple population (QP) monitoring allows to better follow CLL evolution and shortly predicts CLL progression in both untreated and targeted-therapies treated patients. In a first aspect, the present invention relates to an ex vivo method for predicting active disease or progressive disease under therapy in a subject suffering from Chronic Lymphocytic Leukemia (CLL), comprising the step of determining the expression of at least one biomarker selected in the group consisting of CD69, CD49d, CD20 and CD279 in population of cells in a sample obtained from the subject. As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine or a primate. In a preferred embodiment, the subject is a human. In some embodiments, the subject is asymptomatic. In some embodiments, the subject suffering from CLL is classified as A stage, B stage or C stage according to Binet staging system1. In some embodiments, the subject suffering from CLL is classified as low risk (Stage 0), intermediate risk (Stage I or II) or high risk (Stage III or IV) according to Rai staging system1. In some embodiments, the subject suffering from CLL is classified as low risk (score 0-1), intermediate risk (score 2-3), high risk (score 4-6) or very high risk (score 7-10) according to the CCL International Prognostic Index2. In some embodiments, the subject suffers from relapsed CLL. As used herein, the term “Chronic Lymphocytic Leukemia” or “CLL” has its general meaning in the art and refers to a type of slow-growing leukemia affecting B-lymphocytes. Methods for diagnosing CLL are well-known in the art1. As used herein, the term “sample” refers to any biological sample obtained from the purpose of evaluation ex vivo, such as whole blood, serum, plasma, amniotic fluid, brain / spinal cord fluid, liquor, cerebrospinal fluid, sputum, throat and pharynx secretions and other mucous membrane secretions, synovial fluids, ascites, tear fluid, lymph fluid and urine. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a peripheral blood sample. In some embodiments, the sample is a sample of (i) purified blood leukocytes, (ii) peripheral blood mononuclear cells or PBMC, (iii) purified lymphocytes, (iv) purified B cells, (v) purified CD19+ / CD5+ lymphocytes, (vi) red blood cells lysis sample or (vii) a sample comprising a population of CD19+ / CD5+ cells. In some embodiments, the sample is a tissue sample, in particular a lymphoid tissue sample, cells isolated from tissue samples or bone marrow aspiration. In the context of the present invention, the term “at least one biomarker” encompasses one, two, three, four or more biomarkers. In some embodiments, the method further comprises detecting the expression of CD19 and / or CD5. In the context of the present invention, the expression “CD69+ / CD49d+ / CD20+ / CD279+ cells” has to be understood as cells CD69+, CD49d+, CD20+ and CD279+ (quadruple positive population). In the context of the present invention, the expression “CD19+ / CD5+ cells” has to be understood as cells CD19+ and CD5+. As used herein, the term “CD69” refers to a transmembrane C-Type lectin protein encoded by the CD69 gene (NCBI Gene: 969; Ensembl : ENSG00000110848). An exemplary amino acid sequence for CD69 is represented in SEQ ID NO:1. sapiens OX=9606 GN=CD69 PE=1 SV=1 MSSENCFVAE NSSLHPESGQ ENDATSPHFS TRHEGSFQVP VLCAVMNVVF ITILIIALIA LSVGQYNCPG QYTFSMPSDS HVSSCSEDWV GYQRKCYFIS TVKRSWTSAQ NACSEHGATL AVIDSEKDMN FLKRYAGREE HWVGLKKEPG HPWKWSNGKE FNNWFNVTGS DKCVFLKNTE VSSMECEKNL YWICNKPYK As used herein, the term “CD49d” refers to a protein member of the integrin alpha chain family of protein encoded by the ITGA4 gene (NCBI Gene: 3676; Ensembl: ENSG00000115232). An exemplary amino acid sequence for CD69 is represented in SEQ ID NO:2. SEQ ID NO:2 >sp|P13612|ITA4_HUMAN Integrin alpha-4 OS=Homo sapiens HGANRWLLVG APTANWLANA SVINPGAIYR CRIGKNPGQT CEQLQLGSPN GEPCGKTCLE ERDNQWLGVT LSRQPGENGS IVTCGHRWKN IFYIKNENKL PTGGCYGVPP DLRTELSKRI APCYQDYVKK FGENFASCQA GISSFYTKDL IVMGAPGSSY WTGSLFVYNI TTNKYKAFLD KQNQVKFGSY LGYSVGAGHF RSQHTTEVVG GAPQHEQIGK AYIFSIDEKE LNILHEMKGK KLGSYFGASV CAVDLNADGF SDLLVGAPMQ STIREEGRVF VYINSGSGAV MNAMETNLVG SDKYAARFGE SIVNLGDIDN DGFEDVAIGA PQEDDLQGAI YIYNGRADGI SSTFSQRIEG LQISKSLSMF GQSISGQIDA DNNGYVDVAV GAFRSDSAVL LRTRPVVIVD ASLSHPESVN RTKFDCVENG WPSVCIDLTL CFSYKGKEVP GYIVLFYNMS LDVNRKAESP PRFYFSSNGT SDVITGSIQV SSREANCRTH QAFMRKDVRD ILTPIQIEAA YHLGPHVISK RSTEEFPPLQ PILQQKKEKD IMKKTINFAR FCAHENCSAD LQVSAKIGFL KPHENKTYLA VGSMKTLMLN VSLFNAGDDA YETTLHVKLP VGLYFIKILE LEEKQINCEV TDNSGVVQLD CSIGYIYVDH LSRIDISFLL DVSSLSRAEE DLSITVHATC ENEEEMDNLK HSRVTVAIPL KYEVKLTVHG FVNPTSFVYG SNDENEPETC MVEKMNLTFH VINTGNSMAP NVSVEIMVPN SFSPQTDKLF NILDVQTTTG ECHFENYQRV CALEQQKSAM QTLKGIVRFL SKTDKRLLYC IKADPHCLNF LCNFGKMESG KEASVHIQLE GRPSILEMDE TSALKFEIRA TGFPEPNPRV IELNKDENVA HVLLEGLHHQ RPKRYFTIVI ISSSLLLGLI VLLLISYVMW KAGFFKRQYK SILQEENRRD SWSYINSKSN DD As used herein, the term “CD20” refers to a protein member of the membrane-spanning 4A gene family and encoded by the MS4A1 gene (NCBI Gene: 931; Ensembl: ENSG00000156738). An exemplary amino acid sequence for CD69 is represented in SEQ ID NO:3. MTTPRNSVNG TFPAEPMKGP IAMQSGPKPL FRRMSSLVGP TQSFFMRESK TLGAVQIMNG LFHIALGGLL MIPAGIYAPI CVTVWYPLWG GIMYIISGSL LAATEKNSRK CLVKGKMIMN SLSLFAAISG MILSIMDILN IKISHFLKME SLNFIRAHTP YINIYNCEPA NPSEKNSPST QYCYSIQSLF LGILSVMLIF AFFQELVIAG IVENEWKRTC SRPKSNIVLL SAEEKKEQTI EIKEEVVGLT ETSSQPKNEE DIEIIPIQEE EEEETETNFP EPPQDQESSP IENDSSP As used herein, the term “CD279” refers to a cell surface receptor encoded by the PDCD1 gene (NCBI Gene: 5133; Ensembl: ENSG00000188389). An exemplary amino acid sequence for CD69 is represented in SEQ ID NO:4. 1 OS=Homo sapiens OX=9606 GN=PDCD1 PE=1 SV=3 MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL In some embodiments, the expression of the biomarker is determined by a flow- cytometric method. As used herein, the term "flow cytometric method" refers to a technique for counting cells of interest, by suspending them in a stream of fluid and passing them through an electronic detection apparatus. Flow cytometric methods allow simultaneous multiparametric analysis of the physical and / or chemical parameters of up to thousands of events per second, such as fluorescent parameters. Modern flow cytometric instruments usually have multiple lasers and fluorescence detectors. Preferentially, the cytometer has at least three lasers (Blue, Red, Violet). In some embodiments, the expression of the biomarker is determined by the flow cytometric method described in material and methods section (see “immunofluorescence staining and analysis”). For cell surface staining, fresh PBMC from blood samples are used after Ficoll purification or red blood cell lysis. Levels of expression of the QP biomarkers are assessed in said samples. Exemplary titrated antibodies that can be used for labeling are : BV605 anti-human CD19 (Cat# 302244; 1 µg / mL), PE / Cy7 anti-human CD5 (Cat# 300622; 1 µg / mL), BV421 anti-human CD49d (Cat# 304322; 1 µg / mL), APC anti-human CD20 (Cat# 302310; 1 µg / mL), Mouse IgG isotype controls BV605 (Cat# 400162; 1 µg / mL), PE / Cy7 (Cat# 400126; 1 µg / mL), BV421 (Cat# 400158; 1 µg / mL), APC (Cat# 400322; 1 µg / mL) from BioLegend; PE anti-human CD69 (Cat# IM1943U; 1 / 100), PE mouse IgG isotype control (Cat# A09141; 1 / 100) from Beckman-Coulter; FITC mouse anti-human CD279 (Cat# 557860; 10µg / mL), FITC mouse IgG isotype control (Cat# 555748; 10µg / mL) from BD Pharmingen. Cells are incubated in PBS 1% SVF for 20 min at 4°C in the dark with: (i) isotype controls; (ii) anti-CD19 / anti-CD5 antibodies + isotype controls for CD69, CD49d, CD20, CD279 (specific control); (iii) anti-CD19 / anti-CD5 antibodies + anti-CD69, anti-CD49d, anti- CD20, anti-CD279 antibodies. After washing, samples can be measured as example on a BDTMLSR II cytometer and analyzed with BD FACS DivaTMsoftware (BD Bioscience). All samples are analyzed compared to isotype controls or specific control (for multiplex labeling). The cytometric systems of the present disclosure may include a number of additional components, such as data output devices, e.g., monitors, printers, and / or speakers, softwares (e.g. (Flowjo, DIVA, CytoBank….), data input devices, e.g., interface ports, a mouse, a keyboard, etc., fluid handling components, power sources, etc. More particularly, the sample is contacted with a panel of antibodies specific for the specific market of the population of cells of the interest. Such antibodies or antigen-binding fragments are available commercially from vendors such as R&D Systems, BD Biosciences, e- Biosciences, Biolegend, Proimmune and Miltenyi, or can be raised against these cell-surface markers by methods known to those skilled in the art. In some embodiments, an agent that specifically bind to a cell-surface marker, such as an antibody or antigen-binding fragment, is labelled with a tag to facilitate the isolation and detection of population of cells of the interest. As used herein, the terms "label" or "tag" refer to a composition capable of producing a detectable signal indicative of the presence of a target, such as, the presence of a specific cell-surface marker in a sample. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods to isolate and detect the cancer cells. Non-limiting examples of fluorescent labels or tags for labeling the agents such as antibodies for use in the methods of invention include Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Succinimidyl ester, Methoxycoumarin, Succinimidyl ester, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, PerCPeFluor 710, PE- CF594, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, BV 785, BV711, BV421, BV605, BV510 or BV650. As used herein, the term “Active Chronic Lymphocytic Leukemia” has its general meaning in the art1. Exemplary criteria characterizing active CLL include : - Evidence of progressive marrow failure as manifested by the development of, or worsening of, anemia and / or thrombocytopenia. Cutoff levels of Hb <10 g / dL or platelet counts <100 × 109 / L are generally regarded as indication for treatment. However, in some patients, platelet counts <100 × 109 / L may remain stable over a long period; this situation does not automatically require therapeutic intervention. - Massive (ie, ≥6 cm below the left costal margin) or progressive or symptomatic splenomegaly. - Massive nodes (ie, ≥10 cm in longest diameter) or progressive or symptomatic lymphadenopathy. - Progressive lymphocytosis with an increase of ≥50% over a 2-month period, or lymphocyte doubling time (LDT) <6 months. LDT can be obtained by linear regression extrapolation of absolute lymphocyte counts obtained at intervals of 2 weeks over an observation period of 2 to 3 months; patients with initial blood lymphocyte counts <30 × 109 / L may require a longer observation period to determine the LDT. Factors contributing to lymphocytosis other than CLL (eg, infections, steroid administration) should be excluded. - Autoimmune complications including anemia or thrombocytopenia poorly responsive to corticosteroids. - Symptomatic or functional extranodal involvement (eg, skin, kidney, lung, spine). - Disease-related symptoms as defined by any of the following: o Unintentional weight loss ≥10% within the previous 6 months. o Significant fatigue (ie, ECOG performance scale 2 or worse; cannot work or unable to perform usual activities). o Fevers ≥100.5°F or 38.0°C for 2 or more weeks without evidence of infection. o Night sweats for ≥1 month without evidence of infection. As used herein, the term “Progressive Chronic Lymphocytic Leukemia under therapy” has its general meaning in the art1. Treatments against CLL includes, but are not limited to, Ibrutinib, Idelalisib, Acalabrutinib, Zanubrutinib, Venetoclax, Alemtuzumab, Obinutuzumab, Rituximab, Ofatumumab, Cladribine, Fludarabine, Epcoritamab, Glofitamab, Pirtobrutinib, Ublituximab, Linperlisib, Mosunetuzumab, Relmacabtagene, Loncastuximab, Lisocabtagene, Orelabrutinib, Tafasitamab, Brexucabtagene, Inebilizumab, Tirabrutinib, Tislelizumab, Zanubrutinib, Selinexor, Polatuzumab, Talazoparib, Duvelisib, Copanlisib, Umbralisib, Pembrolizumab, Nivolumab, Pentostatin, Lenalidomide, Bendamustine, Chlorambucil, Cyclophosphamide, Prednisone, Dexamethasone. In a preferred embodiment, the treatment is a BTK inhibitor (e.g. Ibrutinib, Pirtobrutinib, Zanubrutinib or Acalabrutinib) or a BCL-2 inhibitor (e.g. Venetoclax). In a preferred embodiment, the treatment is Ibrutinib or Venetoclax. Exemplary criteria characterizing progressive CLL under therapy include : - Lymphadenopathy : appearance of any new lesion such as enlarged lymph nodes (≥1.5 cm), splenomegaly, hepatomegaly, or other organ infiltrates or an increase by ≥50% in greatest determined diameter of any previous site (≥1.5 cm) - An increase in the spleen size by ≥50% or the de novo appearance of splenomegaly - An increase in the liver size of ≥50% of the extent enlargement of the liver below the costal margin defined by palpation, or the de novo appearance of hepatomegaly - An increase in the number of blood lymphocytes by 50% or more with at least 5 × 109 / L B lymphocytes - Transformation to a more aggressive histology (Richter syndrome or Richter transformation) - Occurrence of cytopenia (neutropenia, anemia, or thrombocytopenia) directly attributable to CLL and unrelated to autoimmune cytopenias Typically, the term “Predicting Active CLL or Progressive CLL under therapy” includes, but are not limited to, detection before clinical symptoms or before it can be diagnosed by clinical tests. In some embodiments, the present invention relates to an ex vivo method for predicting active CLL or progressive CLL under therapy in a subject suffering from CLL, comprising the step of quantifying a population of CD69+ / CD49d+ / CD20+ / CD279+ cells in a sample obtained from the subject, wherein the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells correlates with the risk of active CLL or progressive CLL under therapy in said subject. In some embodiments, the ex vivo method for predicting active CLL or progressive CLL under therapy in a subject suffering from CLL, comprises the step of quantifying CD69+ / CD49d+ / CD20+ / CD279+ cells in a population of CD19+ / CD5+ cells in a sample obtained from the subject, wherein the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells correlates with the risk of active CLL or progressive CLL under therapy in said subject. As used herein, the term “risk” in the context of the present invention, relates to the probability that an event will occur over a specific time period and can mean a subject's “absolute” risk or “relative” risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p / (l-p) where p is the probability of event and (l-p) is the probability of no event) to no-conversion. “Risk evaluation,” or “evaluation of risk” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of conversion. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk. In some embodiments, the present invention may be used so as to discriminate those at risk from normal. In some embodiments, the ex vivo method for predicting active CLL or progressive CLL under therapy in a subject suffering from CLL comprises the step of: i) Quantifying a population of CD69+ / CD49d+ / CD20+ / CD279+ cells in a sample obtained from the subject; and ii) comparing the level determined at step i) with a predetermined reference value Wherein the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells determined at step i) correlates with the risk of active CLL or progressive CLL under therapy in the subject. As used herein, the term “predetermined reference value” refers to a threshold value or a cut-off value. A "threshold value", “reference value” or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the level of the marker of the invention (e.g. the at least one biomarker) in properly banked historical patient samples may be used in establishing the predetermined corresponding reference value. In some embodiments, the predetermined corresponding reference value is the median measured in the population of the patients for the marker of in the invention. In some embodiments, 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 so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of the marker of the invention in a group of reference, one can use algorithmic analysis for the statistic treatment of the levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. 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 cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) 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 having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better 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 quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of 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. In some embodiments, the ex vivo method for predicting active CLL or progressive CLL under therapy in a subject suffering from CLL comprises the steps of: i) Quantifying CD69+ / CD49d+ / CD20+ / CD279+ cells in a population of CD19+ / CD5+ cells in a sample obtained from the subject; and ii) concluding that the subject is at risk of active CLL or progressive CLL under therapy when the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells determined at step i) represents at least 0.5% of the population of CD19+ / CD5+ cells. Another aspect of the present invention relates to a method of treating CLL in a subject in need thereof, comprising the step of quantifying a population of CD69+ / CD49d+ / CD20+ / CD279+ cells in a sample obtained from the subject, wherein the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells correlates with the risk of active CLL in said subject. In some embodiments, the method of treating CLL in a subject in need thereof, comprises the steps of : i) Quantifying CD69+ / CD49d+ / CD20+ / CD279+ cells in a population of CD19+ / CD5+ cells in a sample obtained from the subject; ii) concluding that the subject is at risk of active CLL when the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells determined at step i) represents at least 0.5% of the population of CD19+ / CD5+ cells; and iii) Administering said subject with a treatment against CLL. Another aspect of the present invention relates to a method of monitoring a treatment in a subject suffering from CLL, comprising the step of quantifying a population of CD69+ / CD49d+ / CD20+ / CD279+ cells in a sample obtained from the subject, wherein the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells correlates with the risk of progressive CLL under therapy in said subject. In some embodiments, the method of monitoring a treatment in a subject suffering from CLL comprises the steps of : i) Quantifying CD69+ / CD49d+ / CD20+ / CD279+ cells in a population of CD19+ / CD5+ cells in a sample obtained from the subject; ii) concluding that the subject is at risk of progressive CLL under therapy when the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells determined at step i) represents at least 0.5% of the population of CD19+ / CD5+ cells; and iii) Discontinuing said treatment. As used herein, the terms “treating”, “treatment” or “therapy” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disorder or suspected to have contracted the disorder as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The term encompasses both drug administration and non-drug treatment. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., treatment against CLL) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. 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. FIGURES: Figure 1: Analysis of CD69, CD49d, CD279 and CD20 expression in CLL patients. (A) Schematic representation of flow cytometry gating strategy for the analysis of markers alone or in combination in CD19+ / CD5+ leukemic cells. CD19+ / CD5+ population was defined compared to isotype control; for multiplex labeling, gates were defined on CD19+ / CD5+ population compared to specific control (see methods) (B) Pearson r analysis of CD69, CD49d, CD279, CD20. (C) Yule coefficient of bio-markers (alone or in combination) in CLL patients according to their clinical status (under or not ibrutinib therapy); PD: progressive disease, no PD: no progressive disease. (D) Youden test efficacy of bio-markers (alone or in combination) in CLL patients according to their clinical status as in (C). Figure 2: CD69 / CD49d / CD20 / CD279 co-expression (QP) as a predictive marker of evolution and progression (A) Quantification of QP according to clinical stages of CLL patients under or not targeted therapies. SLO: second lymphoid organs; *p < 0.05; **p < 0.01 (B) BTK mutational status and QP frequency analysis in ibrutinib-treated patients according to progression and / or response. (C) Time to next treatment (TTNT) analysis according to CD49d and QP criteria in patients under ibrutinib treatment; (D) Overall survival analysis according to CD49d and QP criteria in patients under ibrutinib treatment. ns: not significant. (E) Swimmer plot follow-up of QP in ibrutinib-progressive CLL patients. TTNT: Time to next treatment. EXAMPLE: Material and Methods Patients. Peripheral blood samples from CLL patients (Table 1) were obtained from the Hematology Department with written informed consent and referenced in INSERM cell bank. According to French law, INSERM cell bank has been registered with the Ministry of Higher Education and Research (DC- 2013- 1903) after being approved by an ethic committee. Clinical and Biological annotations of the samples have been reported to the Comité National Informatique et Liberté. Patients under targeted therapy (ibrutinib, venetoclax) were included in clinical trials (clinicaltrials.gov NCT02824159 and NCT02005471). Immunofluorescence staining and analysis. For cell surface staining, fresh PBMC from blood samples were used after Ficoll purification or red blood cell lysis according to manufacturer instructions (RBC Lysis buffer, BioLegend). Levels of expression of the QP markers were assessed in 11 samples and showed no statistical difference between Ficoll purified PBMC and Red blood cells lysis samples (r=0.981), confirming that either type of samples can be used for this analysis. Titrated antibodies used for labeling were: BV605 anti- human CD19 (Cat# 302244; 1 µg / mL), PE / Cy7 anti-human CD5 (Cat# 300622; 1 µg / mL), BV421 anti-human CD49d (Cat# 304322; 1 µg / mL), APC anti-human CD20 (Cat# 302310; 1 µg / mL), Mouse IgG isotype controls BV605 (Cat# 400162; 1 µg / mL), PE / Cy7 (Cat# 400126; 1 µg / mL), BV421 (Cat# 400158; 1 µg / mL), APC (Cat# 400322; 1 µg / mL) from BioLegend; PE anti-human CD69 (Cat# IM1943U; 1 / 100), PE mouse IgG isotype control (Cat# A09141; 1 / 100) from Beckman-Coulter; FITC mouse anti-human CD279 (Cat# 557860; 10µg / mL), FITC mouse IgG isotype control (Cat# 555748; 10µg / mL) from BD Pharmingen. Cells were incubated in PBS 1% SVF for 20 min at 4°C in the dark with: (i) isotype controls; (ii) anti- CD19 / anti-CD5 antibodies + isotype controls for CD69, CD49d, CD20, CD279 (specific control); (iii) anti-CD19 / anti-CD5 antibodies + anti-CD69, anti-CD49d, anti-CD20, anti- CD279 antibodies. After washing, samples were measured on a BDTMLSR II cytometer (30 000 events from CD19+ / CD5+ gated population) and analyzed with BD FACS DivaTMsoftware (BD Bioscience). All samples were analyzed compared to isotype controls or specific control (for multiplex labeling) following the gating strategy described in Figure 1A. Statistical analyses of medical and biological data. Statistical analysis of biological data was done using One way Anova (*p < 0.05; **p < 0.01), or Chi2test when appropriate. Single markers correlation was done using Pearson r correlation coefficient analysis. Yule coefficient was used to measure association between variables, Youden’s index was calculated to determine the performance of our test. Results Surface expression of CD69, CD49d, CD279 and CD20 markers (alone or in combination) was analyzed following gating strategy described in Figure 1A, in a cohort of 74 patients (Table 1), treated or not with targeted therapies. Positivity for each marker was defined as previously described,22,24or by frequency analysis in the whole cohort compared to specific control on CD19+ / CD5+ gated B leukemic cells for single or multiplex labeling. The weak correlations, measured by Pearson r analysis, between CD69, CD49d, CD279 and CD20 supported their independence (Figure 1B). To evaluate the best marker or combination predicting CLL evolution, we compared each group of patients using Yule coefficient measurement combined to Chi2analysis. Results shown in Figure 1C demonstrated that although some markers alone or some combination can discriminate different CLL stages or progressive disease, co-expression of CD69+ / CD49d+ / CD279+ / CD20+ (quadruple positive population, QP+) > 0.5% on B leukemic cells, was the best combination in all comparisons. This was also confirmed using Youden test efficacy (Figure 1D). Altogether, these data showed that QP>0.5% was the best marker linked to CLL progression in both untreated patients (stage B / C vs stage A) and targeted therapy- exposed patients (relapsing vs responding disease). QP quantification was determined in all patients of the cohort (Figure 2A). Statistical analyses revealed an increase of this B leukemic cell sub-population in stage B / C vs stage A untreated patients, as well as in patients presenting a progressive disease under ibrutinib (exposed >18 months) or under venetoclax (all patients having been pre-exposed to ibrutinib). Interestingly, we observed a high percentage of QP+in the secondary lymphoid organs of ibrutinib-treated patients (diagnosed as Richter transformation), suggesting that this sub- population putatively re-circulate from niches where activation signals are delivered. Progressive disease under ibrutinib treatment was originally associated to a selection- induced resistance mechanism, driven by BTK hotspot mutations.16,17We thus compared BTK mutational status and QP frequencies in the ibrutinib-treated patient’s cohort. As shown in Figure 2B, all patients displaying progressive disease exhibit a QP>0.5% (100%), while only 64% of patients carried BTK mutation. In non-progressive patient’s cohort, the only patient (1 / 25, 4%) displaying a QP > 0.5% after 18 months of ibrutinib, later decreased this percentage (< 0.5% after 2 years of therapy). These results strongly demonstrate that QP+was a more reliable marker of “CLL activation status” and therefore progression, than BTK mutational status (preceding the overt relapses for almost one year). Moreover, since none of the patients reached undetectable MRD after 18 months under ibrutinib, due to permanent hyper- lymphocytosis (PR-L, partial response with lymphocytosis), we confirmed that QP > 0.5% criterion was independent of the percentage (r = -0.17; p=0.42) or absolute number (r = 0.012; p=0.44) of circulating B leukemic cells and so doing, stands for a reliable dynamic proxy of long-lasting ibrutinib response. CD49d / VLA-4 (integrin a4 chain) expression emerged as a microenvironmental factor that contributes to BTKi resistance in CLL23. We next compared the time to next treatment (TTNT) and overall survival (OS) according to CD49d and QP+in the ibrutinib-exposed cohort. As shown in Figure 2C, QP > 0.5%, better than CD49d>30%, was significantly associated to a shorter TTNT in our cohort but not with overall survival (Figure 2D). Furthermore, swimmer plot analysis of TTNT were done in relapsing patients. In the sub-group of patients in whom serial assessments were available, detection of QP+cells preceded overt relapse (Figure 2E). Interestingly, for all patients showing a QP>0.5%, a discontinuation of BTKi therapy was done after a median of 47.2 months. Conclusion Altogether our data strongly showed that QP monitoring represent a rapid, easy feasible, new bio-marker to early predict CLL evolution before treatment, and progression under targeted therapies, especially BTK inhibitors for which MRD standard is difficult to apply. This could be a benefit for clinicians for better patient follow-up and therapeutic adjustments. TABLES Table 1: Clinical characteristics of the patients

[0002] 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. 1. Hallek M, Cheson BD, Catovsky D, et al. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. Blood. 2018;131(25):2745–2760. 2. International CLL-IPI working group. An international prognostic index for patients with chronic lymphocytic leukaemia (CLL-IPI): a meta-analysis of individual patient data. The Lancet Oncology.2016;17(6):779–790. 3. Condoluci A, Terzi Di Bergamo L, Langerbeins P, et al. International prognostic score for asymptomatic early-stage chronic lymphocytic leukemia. Blood.2020;135(21):1859– 1869. 4. Suárez-Cabrera A, Fiallo-Suárez DV, Stuckey R, et al. Genetic Testing at Diagnosis Has Prognostic Value in Patients with Chronic Lymphocytic Leukemia including at Early Stages. Diagnostics.2022;12(8):1802. 5. Hallek M, Al‐Sawaf O. Chronic lymphocytic leukemia: 2022 update on diagnostic and therapeutic procedures. American J Hematol.2021;96(12):1679–1705. 6. Michallet A-S, Letestu R, Le Garff-Tavernier M, et al. A fixed-duration, measurable residual disease–guided approach in CLL: follow-up data from the phase 2 ICLL- 07 FILO trial. Blood.2021;137(8):1019–1023. 7. Rawstron AC, Fazi C, Agathangelidis A, et al. A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study. Leukemia.2016;30(4):929–936. 8. Van Der Velden VHJ, Cazzaniga G, Schrauder A, et al. Analysis of minimal residual disease by Ig / TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data. Leukemia.2007;21(4):604–611. 9. Hengeveld PJ, Van Der Klift MY, Kolijn PM, et al. Detecting measurable residual disease beyond 10−4 by an IGHV leader-based NGS approach improves prognostic stratification in CLL. Blood.2023;141(5):519–528. 10. Munir T, Cairns DA, Bloor A, et al. Chronic Lymphocytic Leukemia Therapy Guided by Measurable Residual Disease. N Engl J Med.2024;390(4):326–337. 11. Wierda WG, Allan JN, Siddiqi T, et al. Ibrutinib Plus Venetoclax for First-Line Treatment of Chronic Lymphocytic Leukemia: Primary Analysis Results From the Minimal Residual Disease Cohort of the Randomized Phase II CAPTIVATE Study. JCO. 2021;39(34):3853–3865. 12. Scarfò L, Heltai S, Albi E, et al. Minimal residual disease–driven treatment intensification with sequential addition of ibrutinib to venetoclax in R / R CLL. Blood. 2022;140(22):2348–2357. 13. Heltai S, Ghia P, Scarfò L. Relevance of Minimal Residual Disease in the Era of Targeted Agents. Cancer J.2019;25(6):410–417. 14. Wang XV, Hanson CA, Tschumper RC, et al. Measurable residual disease does not preclude prolonged progression-free survival in CLL treated with ibrutinib. Blood. 2021;138(26):2810–2827. 15. Herman SEM, Niemann CU, Farooqui M, et al. Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study. Leukemia.2014;28(11):2188–2196. 16. Barrientos JC, Burger JA, Byrd JC, et al. Characterizing the kinetics of lymphocytosis in patients with chronic lymphocytic leukemia treated with single-agent ibrutinib. Leukemia & Lymphoma.2019;60(4):1000–1005. 17. Quinquenel A, Fornecker L-M, Letestu R, et al. Prevalence of BTK and PLCG2 mutations in a real-life CLL cohort still on ibrutinib after 3 years: a FILO group study. Blood. 2019;134(7):641–644. 18. Bonfiglio S, Sutton L-A, Ljungström V, et al. BTK and PLCG2 remain unmutated in one-third of patients with CLL relapsing on ibrutinib. Blood Advances. 2023;7(12):2794–2806. 19. Woyach JA, Ghia P, Byrd JC, et al. B-cell Receptor Pathway Mutations Are Infrequent in Patients with Chronic Lymphocytic Leukemia on Continuous Ibrutinib Therapy. Clinical Cancer Research.2023;29(16):3065–3073. 20. Smyth E, Aidan K, David OB, et al. Low CD49d expression in newly diagnosed chronic lymphocytic leukaemia may be associated with high‐risk features and reduced treatment‐free‐intervals. European J of Haematology.2022;109(5):441–446. 21. Grzywnowicz M, Karabon L, Karczmarczyk A, et al. The function of a novel immunophenotype candidate molecule PD-1 in chronic lymphocytic leukemia. Leukemia & Lymphoma.2015;56(10):2908–2913. 22. Tissino E, Pozzo F, Benedetti D, et al. CD49d promotes disease progression in chronic lymphocytic leukemia: new insights from CD49d bimodal expression. Blood. 2020;135(15):1244–1254. 23. Alsadhan A, Chen J, Gaglione EM, et al. CD49d Expression Identifies a Biologically Distinct Subtype of Chronic Lymphocytic Leukemia with Inferior Progression- Free Survival on BTK Inhibitor Therapy. Clinical Cancer Research.2023;29(18):3612–3621. 24. Del Poeta G, Del Principe MI, Zucchetto A, et al. CD69 is independently prognostic in chronic lymphocytic leukemia: a comprehensive clinical and biological profiling study. Haematologica.2012;97(2):279–287. 25. Cadot S, Valle C, Tosolini M, et al. Longitudinal CITE-Seq profiling of chronic lymphocytic leukemia during ibrutinib treatment: evolution of leukemic and immune cells at relapse. Biomark Res.2020;8(1):72.

Claims

CLAIMS:

1. An ex vivo method for predicting active Chronic Lymphocytic Leukemia (CLL) or progressive CLL under therapy in a subject suffering from CLL, comprising the step of quantifying a population of CD69+ / CD49d+ / CD20+ / CD279+ cells in a population of CD19+ / CD5+ cells in a sample obtained from the subject.

2. The ex vivo method according to claim 1, comprising the steps of: i) Quantifying a population of CD69+ / CD49d+ / CD20+ / CD279+ cells in a population of CD19+ / CD5+ cells in a sample obtained from the subject; and ii) comparing the level determined at step i) with a predetermined reference value wherein the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells determined at step i) correlates with the risk of active CLL or progressive CLL under therapy in the subject.

3. The ex vivo method according to claim 1, comprising the steps of: i) Quantifying CD69+ / CD49d+ / CD20+ / CD279+ cells in a population of CD19+ / CD5+ cells in a sample obtained from the subject; and ii) concluding that the subject is at risk of active CLL or progressive CLL under therapy when the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells determined at step i) represents at least 0.5% of the population of CD19+ / CD5+ cells.

4. A method of treating CLL in a subject in need thereof, comprising the steps of : i) Quantifying CD69+ / CD49d+ / CD20+ / CD279+ cells in a population of CD19+ / CD5+ cells in a sample obtained from the subject; ii) concluding that the subject is at risk of active CLL when the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells determined at step i) represents at least 0.5% of the population of CD19+ / CD5+ cells; and iii) Administering said subject with a treatment against CLL.

5. A method of monitoring a treatment in a subject suffering from CLL, comprising the steps of : i) Quantifying CD69+ / CD49d+ / CD20+ / CD279+ cells in a population of CD19+ / CD5+ cells in a sample obtained from the subject;ii) concluding that the subject is at risk of progressive CLL under therapy when the quantity of CD69+ / CD49d+ / CD20+ / CD279+ cells determined at step i) represents at least 0.5% of the population of CD19+ / CD5+ cells; and iii) Discontinuing said treatment.

6. The method according to any of claims 1 to 5, wherein the treatment is a BTK inhibitor.

7. The method according to claim 6, wherein the BTK inhibitor is Ibrutinib.

8. The method according to any of claims 1 to 5, wherein the treatment is a BCL-2 inhibitor.

9. The method according to claim 8, wherein the BCL-2 inhibitor is Venetoclax.

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