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WO2022101414A1 - Méthode de détermination de pronostic de ldgcb - Google Patents

Méthode de détermination de pronostic de ldgcb Download PDF

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Publication number
WO2022101414A1
WO2022101414A1 PCT/EP2021/081532 EP2021081532W WO2022101414A1 WO 2022101414 A1 WO2022101414 A1 WO 2022101414A1 EP 2021081532 W EP2021081532 W EP 2021081532W WO 2022101414 A1 WO2022101414 A1 WO 2022101414A1
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WIPO (PCT)
Prior art keywords
patient
sample
proteins
level
protein
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PCT/EP2021/081532
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English (en)
Inventor
Karl-Johan MALMBERG
Eivind Heggernes ASK
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Universitetet i Oslo
Oslo Universitetssykehus hf
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Universitetet i Oslo
Oslo Universitetssykehus hf
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Publication of WO2022101414A1 publication Critical patent/WO2022101414A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • G01N2333/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4 or KC
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5428—IL-10
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • a method of determining the prognosis of a patient diagnosed with diffuse large B cell lymphoma The determined prognosis may be used to guide therapy for the patient.
  • a method of treating a patient diagnosed with DLBCL the method comprising first determining the patient’s prognosis, and then selecting an appropriate therapy for the patient depending on their prognosis.
  • a kit suitable for carrying out the prognostic method is also provided.
  • Diffuse large B-cell lymphoma is the most common non-Hodgkin lymphoma (NHL), accounting for 30-40 % of cases. The annual incidence is about 5-8 per 100 000, and the median age at presentation is 70 years.
  • the most common treatment for DLBCL is currently “R-CHOP”, a chemotherapy/immunotherapy regime comprising rituximab (an anti- CD20 antibody), cyclophosphamide, doxorubicin hydrochloride, vincristine and prednisolone.
  • R-CHOP a chemotherapy/immunotherapy regime comprising rituximab (an anti- CD20 antibody), cyclophosphamide, doxorubicin hydrochloride, vincristine and prednisolone.
  • WO 2005/038057 discloses a method of determining DLBCL prognosis based on 6 biomarkers: specifically a weighted expression of the genes LM02, BCL6, FN1, CCND2, SCYA3 and BCL2.
  • prognosis can be considered indicative of the response to standard treatments, in that a poor prognosis is indicative of a likely poor response to standard treatments. It is important to be able to identify patients unlikely to respond to standard treatments for DLBCL so that alternative therapies can rapidly be deployed.
  • the present invention is advantageous because it is based on protein expression levels, rather than gene expression. It is known that protein expression is more closely linked to phenotypes than gene expression, because mRNA translation may be regulated meaning that mRNA levels do not correlate well with protein levels.
  • the present invention may be implemented straightforwardly using a simple blood test.
  • the present inventors have identified that the plasma levels of the proteins CD137, IL-10, CXCL9 and CD134 in DLBCL patients correlate with the patient response to current therapies and thus survival of the disease.
  • the inventors have further found that the prognosis of DLBCL patients can be determined with a high level of accuracy based on an analysis of the levels of these proteins in plasma. The method described herein can thus be used to determine the prognosis of DLBCL patients, and to guide their treatment.
  • a method for determining the prognosis of a patient diagnosed with diffuse large B cell lymphoma comprising the steps: a) providing a cell-free blood-derived sample from the patient; b) determining the level in the sample of the proteins CD137, IL-10, CXCL9 and CD134; and c) comparing the level to a reference value, wherein a level above the reference value indicates poor survival prognosis, and a level below the reference value indicates good survival prognosis.
  • a method of treating a patient diagnosed with diffuse large B cell lymphoma comprising the steps: a) providing a cell-free blood-derived sample from the patient; b) determining the level in the sample of the proteins CD137, IL-10, CXCL9 and CD134; and c) comparing the level to a threshold, wherein the therapy for the patient is selected based on whether the level is above the threshold or below the threshold.
  • DLBCL diffuse large B cell lymphoma
  • binding agents which specifically bind CD134, and from said binding determining the levels of the proteins in the sample.
  • kits for use in determining the concentration of proteins in a sample comprising up to 10 different specific binding agents, including:
  • the first aspect provided herein is a method of determining the prognosis of a patient diagnosed with diffuse large B cell lymphoma (DLBCL).
  • patient is meant a human subject who has been diagnosed with DLBCL.
  • the patient may be any human, i.e. a male or female of any age or ethnicity.
  • the patient may have been diagnosed with any DLBCL subtype of any stage.
  • Diagnosis of DLBCL may have been made by any method known in the art, e.g. biopsy.
  • the prognosis will be performed shortly after the patient has been diagnosed, e.g. prior to the beginning of treatment, so that the prognosis can be used to inform selection of the treatment.
  • the method may comprise a final step of selecting a therapy for the patient based on whether the patient has a good prognosis or a poor prognosis. More generally, provided herein is a method for detecting the proteins CD137, IL-10, CXCL9 and CD134 in cell-free sample from a DLBLC patient, that is a patient diagnosed with DLBLC. More particularly, the levels of the proteins in the sample are determined.
  • the prognosis of the patient as referred to herein may mean the likelihood of the patient recovering from the cancer, i.e., being cured.
  • prognosis may mean the likelihood of the patient surviving the cancer for a certain number of years, e.g., the likelihood of the patient surviving 10, 5, 4, 3, 2 or 1 years, or at least e.g. 6, 9 or 12 months.
  • the prognosis refers to the likelihood of the patient having progression- free survival, for instance for a defined period of time, e.g., progression-free survival for a period of at least 6 or 9 months, or 1 , 2, 3, 4 or 5 years.
  • progression-free survival is meant, as is standard in the art, survival without the cancer progressing, or getting worse.
  • the method provided herein can be used to identify those patients with a good prognosis and those with a poor prognosis.
  • “Prognosis” may alternatively be referred to herein as “survival prognosis”. Both terms mean the likelihood of the patient being cured, surviving for a certain period of time or enjoying progression-free survival for a defined period of time.
  • the prognosis is based on the likelihood that the patient will respond to current therapies, and thus can be understood as meaning the likelihood of the patient being cured by/surviving for a defined period of time/enjoying progression-free survival for a defined period of time, when treated with current first line therapies, e.g. R-CHOP.
  • the method may be for predicting the response of a patient diagnosed with diffuse large B cell lymphoma (DLBCL) to therapy.
  • the therapy may in a further embodiment be R-CHOP.
  • a “good” prognosis means that the patient has a better likelihood of surviving the cancer if treated with current therapies than if the patient is left untreated.
  • a good prognosis means that the patient has a likelihood of at least 30, 40, 50, 60, 70, 75, 80, 85 or 90 % of achieving the assessed level of survival, e.g. of being cured of DLBCL, surviving the cancer for a defined period of time or enjoying progression-free survival, as discussed above, when treated with current therapies.
  • a “poor” prognosis means that the patient has a likelihood of less than 50, 40, 30, 25, 20, 15 or 10 % of achieving the assessed level of survival.
  • a patient’s prognosis is determined by comparison of protein levels in plasma or serum (or other appropriate clinical sample) to a reference value. This is discussed further below, but the reference value may be selected based on the analysis to be performed. If the analysis is intended to determine the likelihood of a person being cured, it may be set to a particular level, if to determine the likelihood of a person surviving for e.g. 3 years, it may be set to a different level, or if it is to determine the likelihood of progression- free survival for e.g. 1 year, it may be set to a different level again. The appropriate level can be selected by the skilled person.
  • the method for determining the prognosis of the patient comprises first providing a cell-free blood-derived sample from the patient.
  • blood-derived sample is meant any sample derived from the blood of the patient.
  • the sample is cell-free, meaning that it does not contain any, or contains essentially or substantially no blood cells (or other cells) from the patient (or indeed any other person).
  • the sample is thus a processed blood sample.
  • the blood-derived sample may be a fraction of a whole blood sample.
  • a “cell-free sample” is a sample which has been processed to remove cells.
  • the “cell- free” sample is thus depleted of cells (e.g. blood cells) using any technique known in the art (e.g. centrifugation, as discussed further below).
  • the cell depletion (or cell removal) step removes substantially all cells from the blood sample, but if it is not successful in removing all cells from the blood sample, the sample analysed according to the method provided herein may contain a small remnant of cells.
  • a “cell-free” sample as defined herein generally comprises at most 5 %, preferably 4, 3, 2, 1 or 0.1 % of the concentration of cells as a comparable unprocessed blood sample (e.g. an unprocessed blood sample taken from the same patient at the same time). Processing of blood may also include other steps in addition to cell removal.
  • the blood sample, or fraction thereof may or may not be subjected to, for example, contacting with various agents, such as anticoagulants or any other component which may be present in a blood collection vessel.
  • Blood may be obtained from the patient using standard clinical methods, e.g. venipuncture (phlebotomy), fingerprick or heelprick.
  • the blood sample is likely to be a venous blood sample, but arterial blood is equally suitable. Any suitable method may be used to obtain a sample of blood from the subject, as known to skilled practitioners in the art such as clinicians, nurses and phlebotomists.
  • the methods provided herein further comprise taking a blood sample from the subject.
  • the blood sample may be taken from the subject using the techniques listed above.
  • the step of taking a blood sample from the subject may be, and indeed is likely to be, performed by a different individual to the individual who performs the prognostic method upon the blood-derived sample.
  • the blood sample may be taken by e.g. a physician, nurse or phlebotomist, while the prognostic method may be performed by e.g. a pathologist or clinical biochemist.
  • the blood-derived sample is obtained by processing of a blood sample obtained from the patient.
  • the methods provided herein may further comprise processing a blood sample from the subject to yield a blood-derived sample.
  • processing may include fractionation, specifically obtaining a fraction of blood from which blood cells have been removed (or essentially removed, as discussed above).
  • the blood-derived sample is a plasma sample.
  • Plasma or blood plasma
  • Plasma is the liquid component of blood, as is known to the skilled person. Plasma may be obtained from whole blood by removal of blood cells from the blood. Appropriate methods for separation of blood cells and plasma (i.e. blood fractionation) are well known in the art. For instance, plasma may be obtained by centrifugation of whole blood, which is the current standard procedure in the art.
  • an anticoagulant is added to the blood sample upon collection.
  • Suitable anticoagulants are well known in the art and include sodium EDTA and potassium EDTA, sodium citrate, heparin and potassium oxalate.
  • Appropriate centrifugation protocols are known in the art, for instance an exemplary centrifugation protocol would be to spin whole blood containing an anticoagulant at 1,300 x g for 20 mins at 4°C.
  • the upper phase of centrifuged whole blood is the plasma, which may be removed from the other phases using e.g., a pipette.
  • the methods provided herein may thus comprise steps of adding an anticoagulant to a blood sample from the subject, and subsequently centrifuging the blood sample in order to isolate a plasma sample from the subject.
  • the blood-derived sample is a serum sample.
  • Serum or blood serum
  • Serum is blood plasma lacking clotting factors. Serum can also be considered the liquid fraction of blood remaining following clotting.
  • Methods to isolate serum from blood are well known in the art. Generally, serum is obtained by collecting a whole blood sample, and allowing the sample to clot by leaving it undisturbed at room temperature, for e.g. 15-30 mins or as long as is required for a clot to form. Thus, unlike the process of plasma isolation, no anticoagulant is added to a blood sample if it is desired to isolate the serum.
  • the clotted blood is then centrifuged to separate the serum and the blood clot.
  • a suitable centrifugation protocol is to spin the sample at about 1 ,500 x g for 10 mins at 4°C.
  • serum which may be removed from the other phases using e.g. a pipette.
  • the methods provided herein may thus comprise steps of allowing a blood sample from the subject to clot, and subsequently centrifuging the blood sample in order to obtain a serum sample from the subject.
  • the next step in the method is to determine the level in the sample (i.e. the cell-free blood-derived sample) of the proteins CD137, IL-10, CXCL9 and CD134.
  • CD137 is also referred to as TNFRSF9 or 4-1 BB, and has the UniProt accession number Q07011 (and the sequence set forth in SEQ ID NO: 1); IL-10 has the UniProt accession number P22301 (and the sequence set forth in SEQ ID NO: 2); CXCL9 has the UniProt accession number Q07325 (and the sequence set forth in SEQ ID NO: 3); and CD134 is also referred to as TNFRSF4, and has the UniProt accession number P43489 (and the sequence set forth in SEQ ID NO: 4).
  • CD137 is defined herein as encompassing any protein which has an amino acid sequence with at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and which is recognised by an antibody which specifically binds a protein of SEQ ID NO: 1.
  • CD137 may alternatively be defined as encompassing any protein which has an amino acid sequence with at least 95 %, 98 % or 99 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and which is recognised by an antibody which specifically binds a protein of SEQ ID NO: 1.
  • CD137 is defined as the protein of SEQ ID NO: 1.
  • IL-10 is defined herein as encompassing any protein which has an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and which is recognised by an antibody which specifically binds a protein of SEQ ID NO: 2.
  • IL-10 is defined as the protein of SEQ ID NO: 2.
  • CXCL9 is defined herein as encompassing any protein which has an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 and which is recognised by an antibody which specifically binds a protein of SEQ ID NO: 3.
  • CXCL-9 is defined as the protein of SEQ ID NO: 3.
  • CD134 is defined herein as encompassing any protein which has an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and which is recognised by an antibody which specifically binds a protein of SEQ ID NO: 4.
  • IL-10 is defined as the protein of SEQ ID NO: 4.
  • Sequence identity may be assessed by any convenient method. However, for determining the degree of sequence identity between sequences, computer programmes that make pairwise or multiple alignments of sequences are useful, for instance EMBOSS Needle or EMBOSS stretcher (both Rice, P.
  • Sequence alignments and % identity calculations may be determined using for instance standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1.
  • the standard EMBOSS Needle parameters may be used: matrix BLOSUM62, gap opening penalty 10, gap extension penalty 0.5. Any other suitable parameters may alternatively be used.
  • the “level” of each protein in the sample may mean the absolute level of the protein, e.g., its concentration.
  • the level of each protein may mean a relative level or normalised level.
  • a relative level is an amount which is determined in relation to the level of another protein. This may be for example a control protein, or a different protein from the group of four.
  • a normalised level may be a level of the protein adjusted to take into account other factors or variables, or may be normalised to a healthy level (e.g., the median level of the protein in the healthy population). Normalisation of determined levels or amounts is a routine matter in the art.
  • a relative or normalised level of each protein may be a level determined by e.g. comparing the amount of each protein in the sample to a control.
  • the “amount” may be a concentration of the protein or an amount expressed in alternative (e.g. arbitrary) units, depending on the method used to determine the level.
  • the control may be e.g. a healthy control, e.g. the average level of the protein in healthy people. Comparison to a healthy control will provide a relative/normalised level of the protein in the sample, showing the degree of up- or down-regulation of the protein in the sample compared to a healthy control. Alternatively, the level of each protein in the sample may be normalised based on the level of a chosen comparator protein in the same sample.
  • the levels (e.g. concentrations) of the proteins of interest are determined using an immunoassay.
  • immunoassay is meant any assay which utilises an antibody in detection/quantification of the protein of interest.
  • a specific binding agent is an agent (i.e. molecule) which binds specifically to a particular binding partner. More particularly, a specific binding agent is capable of binding to its target in a manner which may be distinguished from binding to a non-target molecule. Thus, binding to a non-target molecule may be negligible or substantially reduced as compared to binding to a target molecule.
  • An antibody is an example of a specific binding agent.
  • a protein is “recognised” by an antibody or other binding molecule if it is specifically bound by that antibody or other binding molecule.
  • a specific binding agent may alternatively be defined as a specific binding partner for a given protein. Whether an antibody specifically binds a particular molecule, e.g. protein, can be determined by standard techniques in the art, e.g. using ELISA, Western-blot or SPR, etc.
  • antibody is used herein to refer broadly to any and all types of antibody molecule or antibody fragment.
  • the term thus includes any molecule which is a full-length immunoglobulin molecule, or a fragment or derivative thereof. Accordingly, subsumed under this term is any antibody-type or antibody-derived molecule or fragment, or more generally any molecule which comprises an antigen-binding domain derived or obtained from an antibody (e.g. an immunoglobulin molecule, such as a native antibody), or based on the antigen-binding domain of an antibody.
  • An antibody may alternatively be defined as immunological binding agent, or an immunointeractive agent.
  • An antibody may accordingly be of any desired or convenient species, class or subtype, it may be natural, derivatised or synthetic.
  • Poly- or monoclonal antibodies are included, and any fragment thereof, as described further below.
  • Antibody derivatives such as single chain antibodies, chimeric antibodies and other synthetically made or altered antibody-like molecules are all included.
  • Antibody fragments include, for example, Fab, F(ab’)2, Fab’ and Fv fragments, all of which are well-known and understood in the art.
  • Antibody derivatives include scFv molecules, which are also well known in the art.
  • a preferred type of immunoassay for determining the levels of the proteins in the sample is ELISA, particularly quantitative ELISA.
  • Methods for performing quantitative ELISA are well known in the art.
  • kits for the performance of quantitative ELISA are commercially available and include instructions/protocols.
  • Any form of ELISA assay may be used in the methods of the invention, including direct ELISA, indirect ELISA and sandwich ELISA.
  • Sandwich ELISA is preferred for use in the invention, since only the antigen of interest is captured onto the ELISA plate during the assay. Protocols for performing ELISA sandwich assays are well known in the art.
  • DELFIA dissociation-enhanced lanthanide fluorescence immunoassay
  • Any type of DELFIA may be used in the methods provided herein, including for instance a sandwich DELFIA (which is equivalent to a sandwich ELISA).
  • DELFIA is a similar technology to ELISA, except in a DELFIA assay, as is known in the art, rather than conjugating the detection antibody to an enzyme as in ELISA, the detection enzyme is conjugated to a lanthanide ion, generally Europium.
  • the lanthanide ion is released from the antibody to which it was conjugated, and detected by fluorescence.
  • fluorescence For instance, Europium ions have an excitation wavelength of 320 or 340 nm and a fluorescence wavelength at 615 nm.
  • Reagents for performance of DELFIA reactions are available from PerkinElmer (USA), including a Europium labelling kit, for labelling of the detection antibody. DELFIAs can be performed using kits from PerkinElmer in accordance with the manufacturer’s instructions.
  • Luminex assay Another preferred type of immunoassay for determining the levels of the proteins in the sample is a Luminex assay.
  • Any type of Luminex assay may be used in the methods provided herein.
  • microbeads having defined fluorescent properties are conjugated to specific binding molecules (such as antibodies) specific for a target.
  • the assay can be multiplexed by conjugating microbeads with different fluorescent properties to each different species of binding molecule (such that a particular and unique fluorescent signature is associated with the binding molecules for a particular target).
  • the bead-binding molecule conjugates are incubated with a sample of interest, washed and then incubated with a mixture of (generally) biotinylated antibodies against all targets.
  • the beads are incubated with a streptavidin-phycoerythrin (PE) reporter.
  • PE streptavidin-phycoerythrin
  • the resulting conjugates are read using a dual laser detection instrument: one laser classifies the beads based on their fluorescent signature (thus enabling identification of the targets); a second laser determines the magnitude of the PE-based signal, which is in direct proportion to the amount of each analyte of interest bound to the beads, enabling quantification of the amount of each target identified as present in the sample.
  • Kits for performing Luminex assays can be obtained from several sources, e.g. R&D Systems or Thermo Fisher Scientific (both USA).
  • An alternative type of immunoassay is a proximity assay.
  • Proximity assays rely on the principle of “proximity probing”. In these methods an analyte is detected by the binding of multiple (i.e. two or more, generally two or three) probes, which when brought into proximity by binding to the analyte (hence “proximity probes”) allow a signal to be generated.
  • the proximity probes comprises a nucleic acid domain (or moiety) linked to the analyte-binding domain (or moiety) of the probe, and generation of the signal involves an interaction between the nucleic acid moieties and/or a further functional moiety which is carried by the other probe(s).
  • signal generation is dependent on an interaction between the probes (more particularly between the nucleic acid or other functional moieties/domains carried by them) and hence only occurs when the necessary probes have bound to the analyte, thereby lending improved specificity to the detection system.
  • Exemplary proximity assays include the proximity ligation assay (PLA) and proximity extension assay (PEA).
  • the immunoassay is a PEA.
  • PEA and PLA are described in WO 01/61037; PEA is further described in WO 03/044231, WO 2004/094456, WO 2005/123963, WO 2006/137932 and WO 2013/113699.
  • nucleic acid moieties linked to the analyte-binding domains of a probe pair hybridise to one another when the probes are in close proximity (i.e. when bound to a target), and are then extended using a nucleic acid polymerase.
  • the extension product forms a reporter nucleic acid, detection of which demonstrates the presence in a sample of interest of a particular analyte (the analyte bound by the relevant probe pair).
  • nucleic acid moieties linked to the analyte-binding domains of a probe pair come into proximity when the probes of the probe pair bind their target, and may be ligated together, or alternatively they may together template the ligation of separately added oligonucleotides which are able to hybridise to the nucleic acid domains when they are in proximity.
  • the ligation product is then amplified, acting as a reporter nucleic acid.
  • Multiplex analyte detection using PEA or PLA may be achieved by including a unique barcode sequence in the nucleic acid moiety of each probe.
  • a reporter nucleic acid molecule corresponding to a particular analyte may be identified by the barcode sequences it contains.
  • Kits for performing proximity assays, particularly PEAs can be obtained from Olink Proteomics AB (Sweden), and used in accordance with the manufacturer’s instructions.
  • the reference value is a dichotomising reference value, wherein a level of the proteins above the reference value indicates a poor prognosis for the patient, and a level of the proteins below the reference value indicates a good prognosis for the patient.
  • the reference value may thus be a value which discriminates, or differentiates, between patients with a poor and a good prognosis.
  • the skilled person is able to select a suitable reference value to separate patients with good and poor prognoses, depending on the type of prognosis being analysed (e.g. likelihood of cure, likelihood of survival for a defined period, likelihood of progression-free survival for a defined period). This may also depend on the technique used to determine the protein level, and whether for example an absolute protein concentration is determined, or a relative value for the level of the protein.
  • the level of each protein may individually be compared to separate reference value, or the levels of each protein may be combined (e.g. averaged or to generate a score) and the single resulting value compared to the reference value. If the level of each protein is compared to a reference value, the overall level of the proteins may be deemed to be above the reference value (and thus to indicate a poor prognosis) when a defined number of proteins have a level above their reference value. For instance, in particular embodiments if any one, two or three of the proteins have a level above their reference value, this is indicative of a poor prognosis for the patient. In another embodiment, the level of the proteins is only deemed to be above the reference value (and thus to indicate a poor prognosis) if all proteins are present in the sample at a level above their respective reference values.
  • the level of each protein may be weighted equally, or alternatively the levels of the four different proteins may be weighted differently to generate the single value.
  • the reference value is the level (e.g. plasma concentration, etc., as discussed above) of the proteins in a healthy control sample.
  • the level in a healthy control sample may be an average level obtained from analysis of multiple healthy control samples, i.e. samples taken from multiple healthy volunteers.
  • a healthy volunteer is meant an individual who does not have DLBCL, and preferably does not have cancer or any chronic health condition, and who is medically well when the sample is taken from them.
  • the level of the proteins in a healthy control sample may be a defined quantile in the protein level distribution in the healthy population. For instance, it may be determined that patients with a level of the protein equal to or above e.g. the 95 th percentile of the distribution in healthy patients have a poor prognosis. In this example, 5 % of healthy individuals would have a level of the protein falling within the range that indicates a poor prognosis in patients suffering from DLBCL.
  • the reference value is a threshold value that has been identified as dichotomising DLBCL patients with good and poor prognoses.
  • the skilled person is capable of determining an appropriate threshold value depending on the specific prognosis being analysed, as discussed above.
  • the threshold value may be a concentration (e.g. plasma concentration) which has been identified as prognostic.
  • a separate threshold value may be used for each protein, as described above, and the level of each protein individually compared to a protein-specific threshold (the threshold may be e.g. an absolute or relative concentration of the protein of interest).
  • the threshold may be e.g. an absolute or relative concentration of the protein of interest.
  • the patient is determined to have a poor prognosis.
  • any two or three of the four may be at a level above their threshold and any one or two of the proteins at a level below their threshold.
  • the levels of at least three of the four proteins are above the respective protein-specific thresholds, the patient is determined to have a poor prognosis.
  • any three of the four may be at a level above their threshold and any one of the proteins at a level below its threshold.
  • the patient is determined to have a poor prognosis when the levels of each of the proteins CD137, IL-10, CXCL9 and CD134 (i.e. when the levels of all four of the proteins) are above their thresholds.
  • the threshold for CD137 is a plasma concentration in the range 100 to 120 pg/ml, for instance about 111 pg/ml
  • the threshold for IL-10 is a plasma concentration in the range 0.9 to 1.1 pg/ml, for instance about 0.96 pg/ml
  • the threshold for CXCL9 is a plasma concentration in the range 100 to 120 pg/ml, for instance about 110 pg/ml
  • the CD134 (TNFRSF4) threshold is a plasma concentration in the range 15 to 20 pg/ml, for instance about 17 pg/ml.
  • a patient is defined as having a poor prognosis for progression-free survival if all proteins have plasma concentrations above the thresholds.
  • the thresholds for each protein are set at a level higher than the protein levels in approximately 90 % of DLBCL patients with a good prognosis.
  • the threshold for CD137 is a plasma concentration of about 1500 pg/ml; the threshold for IL-10 is a plasma concentration of about 10 pg/ml; the threshold for CXCL9 is a plasma concentration of about 650 pg/ml; and the threshold for CD134 (TNFRSF4) is a plasma concentration of about 80 pg/ml.
  • a patient is defined as having a poor prognosis for progression-free survival if at least two proteins have plasma concentrations above the thresholds.
  • the cut-off, or threshold value differing levels of specificity may be selected as the cut-off, or threshold value.
  • a value of 80, 85 or 95 % or higher, e.g. 98, 99, or 100 % specificity may be selected (e.g. the threshold value may be set such that 80, 85, 90, 95, 98, 99, or 100 % of DLBCL patients with a good prognosis in a cohort tested for protein levels display a protein level at or below the threshold, good prognosis being determined for example by progression-free or disease event-free survival for a given time period, e.g. 2. 3, 4 or 5 years or more).
  • a skilled person may select an appropriate cutoff or threshold level for a protein based on e.g. the test used for the protein, the cohort size etc.
  • a threshold value may be set at a value where the determined protein level is not within the range of experimental variation, and this may be applicable to any means of determining the protein level.
  • the threshold may be set a level where the most robust statistical results are obtained, or at a less stringent or lower level, for example, the lowest value in the noise distribution (variation) may be used as the threshold.
  • experimental variation e.g. the level of false positives
  • experimental background noise or more particularly the distribution thereof, may be used to set the threshold for a protein level.
  • the method may comprise determining the concentrations of the proteins of interest (generally an absolute concentration, though a relative or normalised concentration may also be suitable) and calculating a score based on the concentrations. The score is then compared to the threshold.
  • concentrations of the proteins of interest generally an absolute concentration, though a relative or normalised concentration may also be suitable
  • the calculation method will determine whether a high or low score is indicative of good prognosis.
  • the inventors have found that higher plasma concentrations of CD137, IL-10, CXCL9 and CD134 are indicative of worse prognosis in DLBCL patients.
  • the present method refers to a level above the reference value indicating poor prognosis and a level below the reference value indicating good prognosis.
  • “above” the reference value does not mean that the level (e.g. score) is necessarily numerically higher than the reference value (though commonly that is the case).
  • a level “above” the reference value is a level that indicates higher protein concentrations, and thus a worse prognosis
  • a level “below” the reference value is a level that indicates lower protein concentrations, and thus better prognosis.
  • a level “above” the reference value, that indicates a worse prognosis may be numerically lower than the reference value
  • a level “below” the reference value, that indicates good prognosis may be numerically higher than the reference value, if the level is a score designed such that high protein concentrations yield a low score, and vice versa.
  • the score may be calculated by any suitable means known in the art.
  • One suitable means is to determine the distribution of plasma concentrations of each protein in DLBCL patients. Once the distribution is determined, each new patient is assigned a score for each protein based on where within the distribution they fall, e.g. in which centile (for instance each protein could be given a score between 0.1 and 1, in which case a patient in the 9 th centile for a given protein could be awarded a score of 0.9). In this example, across all proteins each patient would receive a score between 0.4 and 4, with a higher total score indicating a worse prognosis. A cut-off for good or poor prognosis would be applied, depending on the particular prognosis being calculated (as detailed above), with scores above the cut-off (i.e. threshold) indicating a poor prognosis.
  • Another suitable means to calculate a score is to determine a threshold level for each protein which is indicative of poor prognosis. A score is then calculated based on the number of the four proteins present in the patient at a plasma concentration above the respective thresholds.
  • the threshold level for each protein may be determined by any suitable means known in the art. For instance, an ROC (receiver operating characteristic) analysis may be performed for each protein using a cohort of DLBCL patients, to determine a suitable threshold for the concentration of each protein indicative of poor prognosis. Alternatively an arbitrary quantile may be applied to the distribution across a patient cohort to set a threshold. For instance the median plasma concentration may be used for each protein, such that half of patients will fall either side of the threshold.
  • the scores assigned for each protein may be equally weighted, or differently weighted (e.g. a different score may be assigned for each different protein falling above the threshold).
  • a threshold is applied to determine whether the patient has good or poor prognosis. As mentioned above, the threshold is selected based on the nature of the prognostic analysis performed.
  • Another suitable means to calculate a score is to perform a regression analysis of the relationship between survival and plasma concentrations of the proteins in a cohort of DLBCL patients. This allows weightings to be applied to the concentrations of the four proteins, by which the concentrations of the proteins are multiplied (or divided), to be determined. By multiplying (or dividing) each protein concentration by a weighting factor, account can be made for the natural differences between plasma concentrations of the four proteins, which may differ by several orders of magnitude, and also for the fact that the concentrations of certain of the four proteins may correlate more closely with prognosis than others, and so need to be weighted more heavily when calculating the score.
  • the score for a new patient is calculated by multiplying (or dividing) the plasma concentration of each protein by its weighting factor, and then adding together the four weighted concentrations.
  • An appropriate threshold is applied to the patient’s score to determine the patient’s prognosis (as mentioned above, the threshold can be selected by the skilled person based on the specific prognosis being analysed, e.g. likelihood of cure, likelihood of survival for a defined time period, etc.).
  • the prognosis for a DLBCL patient can be used to determine the most appropriate therapy for the patient.
  • a poor prognosis determined by the method above is indicative of a likelihood of a poor response to current standard treatments for DLBCL (in particular treatment based on rituximab).
  • a good prognosis determined by the method above is indicative of a likelihood of a good response to current standard treatments for DLBCL.
  • a method of treating a patient diagnosed with DLBCL comprising performing a prognostic analysis as described above, and selecting the therapy by which to treat the patient based on the prognosis. That is to say, the therapy is selected based on whether the level of the proteins is above the threshold, such that a first therapy option is selected if the patient has a good prognosis, and a second (alternative) therapy option is selected if the patient has a poor prognosis.
  • the method may further comprise administering the therapy selected.
  • this aspect of the invention may be seen as providing an anti-DLBCL agent for use in the treatment of DLBCL in a patient, wherein the patient has been stratified according to the prognostic method described herein and the agent selected accordingly.
  • the anti-DLBCL agent may be any therapeutic agent, or therapy, which is effective against DLBCL, and may include notably a standard therapy for the condition.
  • the anti-DLBCL agent may be a chemotherapeutic agent and/or immunotherapeutic agent.
  • a patient with a good prognosis has a relatively high likelihood of responding to current treatments.
  • a patient with a good prognosis i.e. having a level of the proteins of interest below the reference value
  • a patient with a good prognosis may be treated with current standard treatments.
  • a patient with a good prognosis may be treated with rituximab.
  • Rituximab may be used as part of a standard combination therapy regimen, e.g. R-CHOP or R-CHOEP.
  • a patient with a good prognosis may be treated with rituximab in combination with cyclophosphamide, doxorubicin hydrochloride, vincristine and prednisolone (i.e.
  • the patient may be treated with an R-CHOP regimen).
  • a patient with a good prognosis may alternatively be treated with rituximab in combination with cyclophosphamide, doxorubicin hydrochloride, vincristine, etoposide and prednisolone (i.e. the patient may be treated with an R-CHOEP regimen).
  • Such regimens are well known to the skilled person.
  • a patient with a good prognosis may be treated with a therapy designed to have reduced toxicity (reduced, that is, relative to existing standard therapies, such as R-CHOP).
  • a therapy designed to have reduced toxicity reduced, that is, relative to existing standard therapies, such as R-CHOP.
  • the patient may be treated with fewer or lower doses of the therapy, or drugs associated with high toxicity may be omitted from the treatment regime.
  • a patient with a poor prognosis has a relatively low likelihood of responding to current treatments (i.e. treatments centred on rituximab). Therefore a patient with a poor prognosis (i.e. having a level of the proteins of interest above the reference value) may be treated with an alternative treatment regimen.
  • a patient with a poor prognosis may be treated by immunotherapy, in particular utilising a chimeric antigen receptor (CAR) or a T cell receptor (TCR) which targets DLBCL cancer cells, or B cells more generally.
  • a T cell or NK cell may be modified to express a suitable CAR or TCR, and infused into the patient. Such methods are well known in the art. Methods by which NK cells can be modified to express TCRs are disclosed in WO 2016/116601.
  • a patient may be treated with a TCR or CAR that recognises CD20 or CD19.
  • TCRs or CARs may be generated by the skilled person using methods known in the art.
  • An example of a suitable therapeutic for this purpose is brexucabtagene autoleucel (Kite Pharma), a CAR-T cell therapy targeting CD19.
  • Brexucabtagene autoleucel has been licensed for treatment of mantle cell lymphoma, a nonHodgkin B cell lymphoma, and according to the methods provided herein may also be used to treat DLBCL.
  • the immunotherapy used to treat a DLBCL patient with poor prognosis may alternatively (or additionally) comprise administration of a checkpoint inhibitor to the patient.
  • Checkpoint inhibitors act by blocking the activity of immune checkpoints. Immune checkpoints keep the immune system in check by preventing the killing of healthy cells and autoimmunity. They act as a “brake” on the immune system by preventing T cell activation. Checkpoint proteins are expressed on the surface of immune cells and bind to checkpoint ligands on the surface of target cells or antigen-presenting cells, resulting in inhibition of immune cell activity. Suitable checkpoint inhibitors include PD-1 inhibitors (in particular anti-PD-1 and anti-PD-L1 antibodies) and CTLA-4 inhibitors (in particular anti-CTLA-4 antibodies).
  • Suitable checkpoint inhibitors that may be used to treat patients with a poor prognosis include Nivolumab (Bristol-Myers Squibb), a human monoclonal anti-PD1 lgG4 antibody; Pembrolizumab, a humanized lgG4 anti-PD-1 antibody (Merck); Atezolizumab, a fully humanised anti-PD-L1 antibody (Genentech); and Durvalumab, a human anti-PD-L1 antibody (Medimmune/Astrazeneca).
  • Nivolumab Black-Myers Squibb
  • Pembrolizumab a humanized lgG4 anti-PD-1 antibody
  • Atezolizumab a fully humanised anti-PD-L1 antibody (Genentech)
  • Durvalumab a human anti-PD-L1 antibody (Medimmune/Astrazeneca).
  • binding agents which specifically bind CD134, and from said binding determining the levels of the proteins in the sample.
  • the binding agents (or specific binding agents) and sample used in the method of this aspect may be as described above. Determination of the levels of the proteins in the sample may similarly be performed as described above.
  • the binding agents are antibodies and the method comprises performing an immunoassay to determine the levels (e.g. concentrations) of the proteins in the sample. Suitable immunoassays include ELISA, DELFIA, Luminex assays and proximity assays such as PEA, as detailed above.
  • the sample is generally a plasma or serum sample.
  • the binding agent may be capable of disclosing, or indicating, the presence of the protein in question.
  • the binding agent may be detectable, or capable of being detected to determine the presence, or more particularly the level, of the protein.
  • the agent may be provided in a format capable of being detected, i.e. configured such that from the binding of the binding agent to the protein the level of the protein may be determined.
  • the agent may be labelled, or provided with a tag or reporter moiety which allows the binding agent, and hence the protein to be detected, and the amount of protein present to be assessed (i.e. determined).
  • the binding agent is typically detectable in a quantitative manner.
  • the label, tag, or reporter etc. may be directly or indirectly detectable.
  • the binding agent may be provided with a directly signal-giving label etc. (e.g. a fluorescent or coloured label etc.), or it may be provided with a tag or reporter which may be detected in detection reaction, for example as in a PEA as described above, or in an enzymatic reaction.
  • a directly signal-giving label etc. e.g. a fluorescent or coloured label etc.
  • a tag or reporter which may be detected in detection reaction, for example as in a PEA as described above, or in an enzymatic reaction.
  • the sample may be a sample taken at or after diagnosis of the patient with DLBCL. In a particular embodiment it is sample taken at, about or around the time of diagnosis.
  • kits for performing the prognostic method described above are for use in determining the amount or concentration of proteins in a sample (that is to say, the kit is suitable for use in determining the amount or concentration of proteins in a sample).
  • the kit comprises up to 10 different specific binding agents.
  • “different” specific binding agents is meant different species of specific binding agents (each having a different structure, in particular a different target binding domain). Each different specific binding agent may specifically bind a different target protein, but this is not required and in some cases the kit comprises multiple (i.e. at least 2) different specific binding agents which bind the same target protein.
  • the different specific binding agents which bind the same target protein bind the protein at different epitopes, such that both/all the different specific binding agents which bind the same target protein can bind the protein at the same time.
  • the kit comprises one or more binding agents which specifically bind CD137, one or more binding agents which specifically bind IL-10, one or more binding agents which specifically bind CXCL9, and one or more binding agents which specifically bind CD134.
  • the kit comprises the same number of specific binding agents against each target protein.
  • the kit comprises one specific binding agent which specifically binds CD137, one specific binding agent which specifically binds IL-10, one specific binding agent which specifically binds CXCL9, and one specific binding agent which specifically binds CD134.
  • the kit comprises two specific binding agents which specifically bind CD137, two specific binding agents which specifically bind IL-10, two specific binding agents which specifically bind CXCL9, and two specific binding agents which specifically bind CD134.
  • the kit comprises up to 10 different specific binding agents.
  • the kit must comprise a minimum of 4 different specific binding agents (one to bind each of the target proteins) and so the kit comprises in the range of 4 to 10 different specific binding agents.
  • the kit may comprise only specific binding agents that recognise CD137, IL-10, CXCL9 and CD134.
  • the kit may comprise specific binding agents that recognise from 4 to 10 different targets).
  • the kit comprises two specific binding agents against each target protein, clearly it may comprise specific binding agents that recognise 4 or 5 different targets (for instance the 4 target proteins CD137, IL-10, CXCL9 and CD134, plus optionally a control target).
  • Each species of specific binding agent may be provided in a separate container.
  • all species of specific binding agent against the same target may be provided in the same container, and species of binding agent against different targets provided in separate containers.
  • the kit may thus be used to detect the proteins CD137, IL-10, CXCL9 and CD134 in a sample of interest, e.g. in the context of a prognostic analysis as described above.
  • the specific binding agents may thus be detection agents, designed for use in a detection assay, for instance in an ELISA, DELFIA, Luminex assay or proximity assay (in particular a PEA). That is to say, the specific binding agents may be provided in a format suitable for performing a detection assay, in particular in a format suitable for performing an ELISA, DELFIA, Luminex assay or proximity assay (in particular a PEA).
  • the specific binding agents may be provided conjugated to an enzyme suitable for use in ELISA, e.g. horseradish peroxidase (HRP) or alkaline phosphatase (AP).
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • the specific binding agents may be provided conjugated to a lanthanide ion, particularly a Europium ion.
  • a first set of specific binding agents may be provided conjugated to fluorescent microbeads, and a second set of specific binding agents may be provided conjugated to biotin.
  • the specific binding agents may be provided conjugated to nucleic acid domains capable of interacting to form a reporter nucleic acid molecule.
  • the kit comprises two specific binding agents that recognise each target protein.
  • One of the two specific binding agents that recognise each target may be provided immobilised on an assay plate (e.g. an ELISA plate or a DELFIA plate) and the other specific binding agent may be provided in solution conjugated to an appropriate detection moiety (i.e. an enzyme if the kit is for an ELISA, or a lanthanide ion if the kit is for DELFIA).
  • a Luminex assay also uses a “sandwich” technique and thus when the kit is for performing a Luminex assay, it preferably comprises two specific binding agents that recognise each target protein, one conjugated to a fluorescent bead and the other to a first member of a binding pair (commonly biotin, as mentioned above). Similarly, if the kit is for a proximity assay it will comprise two specific binding agents against each target protein, each conjugated to a nucleic acid domain. Where the kits are for use in sandwich assays and thus comprise two different specific binding agents against each target, as mentioned above the two binding agents recognise different epitopes on the target.
  • the specific binding agents are antibodies, as defined above.
  • the specific binding agents are monoclonal antibodies (i.e. monoclonal full-length antibodies).
  • the kit may further comprise reagents and/or equipment for performing an ELISA, a DELFIA, a Luminex assay or a proximity assay (as appropriate depending on the format of the specific binding agents provided).
  • an appropriate plate may be provided, preferably to which a specific binding agent against each protein of interest has already been conjugated, for a sandwich assay.
  • Appropriate buffers may also be provided.
  • a suitable substrate particularly a chromogenic substrate, may be provided.
  • a streptavidinphycoerythrin conjugate may be provided, along with a plate to hold the beads during the assay and appropriate buffers.
  • buffers and reagents for DNA amplication e.g. for PCR, such as a DNA polymerase, primers and dNTPs may also be provided
  • a ligase enzyme may be provided.
  • a kit as provided herein may further comprise instructions (e.g. an instruction manual) for performing an immunoassay to determine the levels of the proteins of interest in a sample.
  • the instructions may further detail how to perform the prognostic analysis method described above.
  • Figure 1 shows the overall survival probability of patients in the Oslo cohort stratified on clusters as detailed in Example 1. Patients in cluster 2 had significantly worse overall survival compared with the other patients in the Oslo cohort.
  • Figure 3 shows the concordance for Cox overall survival models for scores with different numbers of proteins included. Proteins are included by order of absolute difference between score groups. The red vertical line highlights the lowest number of proteins needed to achieve optimal concordance. These are specifically indicated as 4-1 BB, IL-10, CXCL9 and TNFRSF4.
  • Figure 4 shows the probability of overall survival in the Oslo cohort, stratified on the SPD scores created from the four proteins selected in Figure 3.
  • Figure 5 shows the probability of overall survival in the St. Louis cohort, stratified on the SPD scores created from the four proteins selected in Figure 3.
  • Figure 6 shows a comparison of concordances in both cohorts for Cox overall survival models with scores based on all possible combinations of the four proteins selected in Figure 3.
  • Figure 7 shows the probability of progression-free survival in the (A) Oslo and (B) St. Louis cohorts, stratified on the SPD scores created from the four proteins selected in Figure 3.
  • Figure 8 shows box plots comparing the plasma concentrations of 4-1 BB, IL-10, CXCL9 and TNFRSF4 in healthy donors (HD) and DLBCL patients stratified as in Fig. 2 (high or mid/low).
  • “high” stratified patients are represented by the left-hand box, “mid/low” stratified patients by the central box and healthy donors by the right-hand box.
  • Figure 9 shows overall survival (A) and progression-free survival (B) for patients scored as described in Example 2, below. As shown by the p values, stratification based on this scoring system separates patients into populations with a significantly different risk of death or disease progression.
  • Cluster 1 comprised all healthy controls and approximately two thirds of the patients. The majority of healthy controls clustered closely together and displayed low relative concentrations of most of the proteins.
  • Cluster 2 comprising the remaining one third of the patients, was characterized by high levels of a majority of the assessed proteins. Notably, patients in this unique cluster had significantly worse overall survival, compared with all other patients in the Oslo cohort (Fig. 1).
  • SPD systemic protein deviation scoring algorithm
  • the St Louis cohort was used for validation, the 4-protein based SPD score was strongly correlated with survival also in the St. Louis cohort (Fig. 5). In both cohorts, the 4-protein SPD score outperformed all SPD scores based on any combination of 1-3 of the 4 proteins (Fig. 6). The 4-protein based SPD score was also strongly correlated with progression-free survival in both cohorts (Fig. 7). This showed that analysis using these proteins provides an accurate method for determining the prognosis of DLBCL patients.
  • Plasma samples were collected from patients, and healthy donor peripheral blood samples were obtained from the blood bank at Oslo University Hospital. Patient plasma samples were collected using EDTA as anticoagulant and cryopreserved. Serum was collected by centrifugation, aliquoted and cryopreserved. Healthy blood samples were processed in the same way as patient samples. Serum and Plasma Protein Quantification
  • NPX Normalized Protein expression
  • Luminex assay Alternatively, donor samples were analysed by Luminex assay in order to calculate absolute protein concentrations.
  • the Luminex assays were performed using standard techniques in the art.
  • hinges correspond to the 25th and 75th percentile, while whiskers range to the most extreme values, but no longer than 1.5 times the inter-quartile range, and data points outside that range were plotted individually.
  • patients are ranked from lowest to highest protein value.
  • the ranks are subsequently divided by the number of patients, so that the patient with the lowest protein value is given a value of 1 divided by the number of patients, and the patient with the highest value is given the value 1.
  • These values are then summed for each patient, giving the resulting scores a theoretical maximum equal to the number of patients.
  • the selected proteins were first ordered based on the absolute difference between the previously created score groups. Then, multiple sets of scores were created as described above, including increasing numbers of the top proteins from the sorted list. For each set of scores, a Cox proportional hazards regression model was created with “high” (above 2/3-quantile) score as a binary variable. The concordances of these models were then compared, and the lowest number of proteins yielding optimal concordance was considered the optimal protein count.
  • Serum concentrations for the four proteins (4-1BB, IL-10, CXCL9 and TNFRSF4) were measured by Luminex assay for 50 of the 51 patients in the Oslo cohort. None of the St. Louis patients were included.
  • Each patient was assigned a score relating to their plasma concentration of each protein.
  • a concentration above the threshold was assigned a score of 1 ; a concentration equal to or below the threshold was assigned a score of 0.
  • the patients’ scores were then summed, giving a total score between 0 and 4. The results were as follows:
  • a score of 0- 3 indicates a 5 % risk of death/relapse/progression within 2 years, while the same risk for patients with a score of 4 is 46 %.
  • Fig. 9 compares overall survival (A) and progression-free survival (B) in patients with a score of 0-3 and patients with a score of 4, similarly showing the difference in prognosis between these two patient groups.
  • Serum protein concentrations were measured by Luminex assay. The fifth highest serum concentrations of each protein in the 42 progression-free patients were calculated, and the threshold set just above that level. The following thresholds were selected:
  • Shaded cells indicate values above the threshold for the protein in question; triple-bordered cells indicate the fifth highest concentration in the progression-free group.
  • 7/8 patients who did not experience at least 2 years progression-free survival were correctly called by the analysis of having cancer poor prognosis, a sensitivity of 87.5 %; 3 patients who experienced at least 2 years progression-free survival were incorrectly called as having cancer poor prognosis, a specificity of 92.9 %.

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Abstract

L'invention concerne une méthode pour déterminer le pronostic d'un patient diagnostiqué avec un lymphome diffus à grandes cellules B (LDGCB). La méthode est basée sur l'évaluation des taux des protéines CD137, IL-10, CXCL9 et CD134 dans un échantillon dérivé du sang (p. ex., le plasma) du patient et la comparaison de ces taux à une valeur de référence, qui définit si le pronostic du patient est bon ou mauvais.
PCT/EP2021/081532 2020-11-12 2021-11-12 Méthode de détermination de pronostic de ldgcb Ceased WO2022101414A1 (fr)

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