WO2022180236A1 - N-acetyl-d-glucosamine as a diagnostic biomarker of a disease induced by mast cell activation - Google Patents
N-acetyl-d-glucosamine as a diagnostic biomarker of a disease induced by mast cell activation Download PDFInfo
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- WO2022180236A1 WO2022180236A1 PCT/EP2022/054855 EP2022054855W WO2022180236A1 WO 2022180236 A1 WO2022180236 A1 WO 2022180236A1 EP 2022054855 W EP2022054855 W EP 2022054855W WO 2022180236 A1 WO2022180236 A1 WO 2022180236A1
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- mast cell
- cell activation
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Classifications
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Definitions
- N-ACETYL-D-GLUCOSAMINE AS A DIAGNOSTIC BIOMARKER OF A DISEASE INDUCED BY MAST CELL ACTIVATION
- the field of the present invention is that of medicine and diagnostic, in particular diagnostic of a disease induced by mast cell activation.
- the invention more particularly relates to N-acetyl-D-glucosamine (GlcNAc) and uses thereof as a biomarker in the diagnosis of a disease induced by mast cell activation.
- the invention also relates to methods of assessing the severity of a disease induced by mast cell activation, of predicting or monitoring the evolution of a disease induced by mast cell activation, of determining the efficacy of a therapy of a disease induced by mast cell activation in a subject, as well as kits and their uses for implementing these methods.
- the mast cell is a tissue resident granulocyte, active in the allergic response but also playing a vital role in the innate immune response, immune tolerance, wound healing and angiogenesis.
- Mast cell may be activated by both IgE-dependent and IgE-independent mechanisms. Once activated, it releases preformed mediators stored in granules, including histamine, heparin, proteases (e.g., tryptase), de novo synthetized arachidonic acid metabolites (e.g., prostaglandin D2 and leukotriene C4); as well as cytokines (e.g., TNFa) and chemokines.
- mediators stored in granules including histamine, heparin, proteases (e.g., tryptase), de novo synthetized arachidonic acid metabolites (e.g., prostaglandin D2 and leukotriene C4); as well as cytokines (e.g
- mast cell activation can be highly varied in their presentation, depending on the affected organ.
- Common manifestations of mast cell activation are “allergic type” phenomena, such as for example asthma, hives or rhinitis.
- More heterogeneous disorders are also induced by abnormal or excessive mast cell activation.
- the measurement of the mast cell rate in tissues allows the discrimination of disorders into two distinct main groups.
- the first group is characterized by inappropriate activation of mast cells without proliferation, and the second by an accumulation of mast cells.
- the mast cell activation syndrome belongs to the first category identified above. It is a condition in which the patient experiences repeated episodes of the symptoms of anaphylaxis and allergic symptoms such as tachycardia, hypotension, syncope, itching hives, swelling, flushing, wheezing, difficult breathing, nausea with abdominal pain and severe diarrhea. High levels of mast cell mediators are released during those episodes. Increases in serum mast cell tryptase and in urine levels of N-methylhistamine, 1 IB -Prostaglandin F2a (1 lB-PGF2a) and/or Leukotriene E4 (LTE4) are the only tests useful for the diagnosis of MCAS. However, serum mast cell tryptase should be drawn between 30 minutes and two hours after the start of an episode, and the urine tests are to be performed on a 24-hour collection of urine that is started immediately (i.e., at the beginning of an episode).
- mastocytosis results from a clonal, neoplastic proliferation of morphologically and immunophenotypically abnormal mast cells (MCs) that accumulate in one or more organs.
- Mastocytosis includes cutaneous mastocytosis, mast cell sarcoma (MCS) and systemic mastocytosis (SM).
- SM Systemic mastocytosis
- ISM indolent SM
- SSM smoldering SM
- ASM aggressive SM
- MCL mast cell leukemia
- ISM and SSM are grouped together as advanced SM (AdvSM).
- Systemic mastocytosis is a KIT-driven hematopoietic neoplasm characterized by the accumulation of abnormal neoplastic mast cells (MCs) in various organs and, mainly, the bone marrow (BM) (Metcalfe DD, 2008; Metz M et al., 2007; Siraganian RP, 2003).
- MCs abnormal neoplastic mast cells
- BM bone marrow
- SM pathology ranges from benign chronic manifestations to very aggressive tumors with limited therapeutic options available to date (Gilreath JA et al. 2019; Hartmann K et al., 2020).
- ISM indolent SM
- MC mast cells
- AdvSM advanced SM
- KIT D816V mutation is identified as a major cause of the disease and is associated with most clinical forms of SM (Bibi S et al, 2014; Garcia- Montero AC, 2006; Garcia-Montero AC et al., 2016; Kitamura Y and Hirotab S, 2004).
- Oncometabolites are conventional metabolites that, when aberrantly accumulated, have pro-oncogenic functions mainly via epigenetic dysregulation (Yang M et al., 2013; Morin A et al., 2014; Yong C et al., 2020).
- SM The major diagnostic criterion for SM is the presence of multifocal clusters of morphologically abnormal mast cells in the bone marrow. Minor diagnostic criteria include elevated serum tryptase level, abnormal mast cell expression of CD25 and/or CD2, and presence of KITD816V mutation.
- AdvSM advanced systemic mastocytosis
- Inventors now herein advantageously provide simple to implement methods of diagnosing, assessing the severity, predicting and monitoring the evolution, of a disease induced by mast cell activation in a subject.
- N-acetyl-D-glucosamine GIcNAc
- GIcNAc N-acetyl-D-glucosamine
- the present invention is useful for classifying a sample issued from a subject as being associated to mastocytosis (in particular systemic mastocytosis), mast cell activation syndrome (MCAS), asthma or allergy.
- the present invention thus relates to the use of N-acetyl-D-glucosamine (GIcNAc) as a biomarker of a disease induced by mast cell activation, the disease being preferably selected from mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis (CM); mast cell activation syndrome (MCAS); asthma; and allergy.
- SM systemic mastocytosis
- MCS mast cell sarcoma
- CM mast cell activation syndrome
- asthma asthma
- allergy also described herein is an in vitro or ex vivo method for diagnosing a disease induced by mast cell activation in a subject. This method typically comprises the steps of:
- step (ii) diagnosing a disease induced by mast cell activation in the subject if the concentration of GlcNAc as measured in step (i) is above a reference GlcNAc ’s concentration.
- the method of the invention enables classifying a diagnosed systemic mastocytosis (SM) in a subject, in particular into a non-advanced SM (non-AdvSM) or advanced SM (AdvSM), more particularly into indolent SM (ISM), smoldering SM (SSM), SM with an associated hematological neoplasm (SM-AHN), aggressive SM (ASM), mast cell leukemia (MCL), or even more particularly into indolent SM (ISM) or aggressive SM (ASM).
- SM systemic mastocytosis
- Inventors in particular herein provide an in vitro or ex vivo method for assessing the severity of a disease induced by mast cell activation in a subject, preferably systemic mastocytosis (SM), in particular for differentially diagnosing between non-advanced SM (non-AdvSM) and advanced SM (AdvSM) in a subject, more particularly between indolent SM (ISM) and i) aggressive SM (ASM), ii) SM with an associated hematological neoplasm (SM-AHN) or iii) mast cell leukemia (MCL), and wherein the method typically comprises:
- SM systemic mastocytosis
- Inventors in particular herein provide an in vitro or ex vivo method for assessing the severity of SM, wherein the reference control sample is a sample obtained from a healthy subject, and wherein a concentration of GlcNAc in the biological sample of the subject that is 6 to 8 times greater than the concentration of GlcNAc in the reference control sample is indicative of non-AdvSM, in particular indolent SM (ISM); and a concentration of GlcNAc in the biological sample of the subject that is more than 8-fold greater than the concentration of GlcNAc in the reference control sample is indicative of advanced SM (AdvSM), in particular aggressive SM (ASM) or SM with an associated hematological neoplasm (SM-AHN).
- AdvSM advanced SM
- ASM aggressive SM
- SM-AHN hematological neoplasm
- Inventors in particular herein provide an in vitro or ex vivo method for assessing the severity of SM, wherein the biological sample is preferably a fluid sample as herein described below, wherein a concentration of GlcNAc in the biological sample of the subject that is equal to or above the reference concentration of GlcNAc is indicative of AdvSM, in particular ASM or SM-AHN, and a concentration of GlcNAc in the biological sample of the subject that is below the reference concentration of GlcNAc is indicative of non-AdvSM, in particular ISM.
- an in vitro or ex vivo method for assessing/predicting the risk for a subject affected or diagnosed with non-AdvSM, in particular ISM, to develop AdvSM, in particular ASM or SM-AHN comprising a step of determining the concentration of GlcNAc in a biological sample obtained from a subject affected or diagnosed with non-advanced SM, in particular ISM, wherein a concentration of GlcNAc above a reference concentration of GlcNAc indicates that the subject is at risk to develop AdvSM, in particular ASM or SM-AHN.
- mast cell activation preferably selected from mastocytosis, mast cell sarcoma (MCS), mast cell activation syndrome (MCAS), asthma and allergy, and even more preferably systemic mastocytosis (SM).
- MCS mast cell sarcoma
- MCAS mast cell activation syndrome
- asthma and allergy preferably systemic mastocytosis
- SM systemic mastocytosis
- step (c) comparing the concentrations of GlcNAc as measured in steps (a) and (b) to monitor the evolution of the disease in the subject, wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative of a favorable evolution of the disease in the subject, a concentration of GlcNAc as measured in step (b) identical to the concentration of GlcNAc as measured in step (a) is indicative of a stabilization of the disease in the subject, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative of an unfavorable evolution of the disease in the subject.
- This in vitro or ex vivo method for monitoring the evolution of a disease induced by mast cell activation in a subject can in particular be implemented for monitoring SM evolution, for example in a subject affected with non-AdvSM, in particular indolent SM (ISM) or smoldering SM (SSM).
- ISM indolent SM
- SSM smoldering SM
- Inventors further herein describe an in vitro or ex vivo method for determining the efficacy of a therapy of a disease induced by mast cell activation which is preferably selected from mastocytosis, mast cell sarcoma, mast cell activation syndrome (MCAS), asthma and allergy, and even more preferably systemic mastocytosis (SM), in a subject affected with said disease.
- the method comprises the steps of:
- step (b) measuring the concentration of GlcNAc in a sample obtained from a subject once started the administration of a therapy of a disease induced by mast cell activation; and (c) comparing the concentrations of GlcNAc as measured in steps (a) and (b), wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative that the therapy is effective in the treatment of the disease induced by mast cell activation, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative that the therapy is not effective in the treatment of the disease induced by mast cell activation; or, alternatively, wherein the method comprises the steps of:
- step (ii) comparing the concentration of GlcNAc as measured in step (i) with a reference concentration of GlcNAc, wherein, a concentration of GlcNAc as measured in step (i) below the GlcNAc reference concentration is indicative that the therapy is effective in the treatment of the disease induced by mast cell activation, and a concentration of GlcNAc as measured in step (i) above the GlcNAc reference concentration is indicative that the therapy is not effective in the treatment of the disease induced by mast cell activation.
- This in vitro or ex vivo method for determining the efficacy of therapy may further comprises a step of determining, in a biological sample from the subject, the transcript level of one or more enzymes involved in the hexosamine biosynthesis pathway (HBP), such as GFAT1, PGM3, NAGK, OGT and OGA.
- HBP hexosamine biosynthesis pathway
- the methods herein described are performed on a biological sample obtained from a living subject, typically from a mammal for example from a human subject or a domestic animal, in particular a pet.
- a particular subject is a subject having a mutated kit gene or expressing a mutated KIT receptor.
- the biological sample is preferably a biological fluid sample, preferably selected from a blood, plasma, serum, urine and bone marrow aspirate sample; or a solid sample, preferably a bone marrow biopsy sample.
- kits for measuring the concentration of GlcNAc in a biological sample of a subject.
- the kit comprises an anti-O-linked N-acetylglucosamine (O-GlcNAc) binding agent, for example RL2 monoclonal antibody, a molecule allowing the binding agent detection; or a N-Acetyl-D- Glucosamine Kinase (NAGK).
- O-GlcNAc anti-O-linked N-acetylglucosamine
- NAGK N-Acetyl-D- Glucosamine Kinase
- the kit also comprises a leaflet providing GlcNAc reference concentration(s).
- the present disclosure also relates to the use of a kit as herein described on a biological sample obtained from a subject, for diagnosing a disease induced by mast cell activation, preferably selected from mastocytosis, mast cell activation syndrome (MCAS), asthma and allergy, assessing the severity of systemic mastocytosis; assessing/predicting the risk for a subject affected or diagnosed with non- AdvSM to develop AdvSM, preferably prior to the onset of AdvSM’s symptoms; monitoring or predicting the evolution of a disease induced by mast cell activation; and/or monitoring the efficacy of a therapy of a disease induced by mast cell activation, in the subject.
- a disease induced by mast cell activation preferably selected from mastocytosis, mast cell activation syndrome (MCAS), asthma and allergy
- MCAS mast cell activation syndrome
- FIG. 1 Metabolomic profiling of systemic mastocytosis (SM).
- A Clinical spectrum of patients with SM.
- B Schematic representation of the untargeted metabolomic approach used to identify metabolite signatures in SM plasma samples.
- C Z-score scatterplots of plasma metabolites from indolent and advanced SM patients. Each data point is expressed as the number of standard deviations (SDs) from the mean of the controls (healthy donors). The y-axis shows all values for each metabolite on the same horizontal line. The z-scores are the SDs from the control mean, revealing the changes relative to the healthy donor group.
- D Z-score scatterplots present results similar to those in (C) but relative to the indolent SM group.
- E Principal component analysis (PCA) of metabolites measured in indolent (white dots and advanced (black dots) SM plasma samples.
- PCA Principal component analysis
- FIG. 1 Metabolomic alterations associated with systemic mastocytosis (SM) severity.
- N-acetyl-D-glucosamine (GlcNAc) is a biomarker of advanced systemic mastocytosis (AdvSM).
- C Receiver operating characteristic (ROC) curve of GlcNAc concentrations between indolent SM (ISM) and advanced SM (ASM) patient samples.
- E Schematic representation of the hexosamine biosynthetic pathway (HBP) and the use of GlcNAc in the salvage pathway indicating relevant enzymes [O-linked-GlcNAc transferase (OGT), the O-GlcNAcase OGA and GlcNAc kinase (NAGK)].
- G Lysates from three bone marrow biopsies from patients with mast cell (MC) activation syndrome (MCAS), ISM and ASM were immunoblotted with RL2 antibodies that detect O-GlcNAcylation
- G Quantification of the immunoblot shown in (F) by ImageJ software.
- the values in the graphs are presented as the means ⁇ SDs (non-significant, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001; unpaired t test, two tailed).
- FIG. 4 N-acetyl-D-glucosamine (GlcNAc) exacerbates the severity of an advanced systemic mastocytosis (ASM) phenotype.
- GlcNAc N-acetyl-D-glucosamine
- (B) Schema of in vivo administration of GlcNAc to ROSA D816-Gluc transplanted mice after irradiation with 1.5 Gy (n 10, 5 per group).
- FIG. 5 N-acetyl-D-glucosamine (GlcNAc) increases mast cell (MC) susceptibility to IgE- mediated stimulation.
- GlcNAc N-acetyl-D-glucosamine
- BMMC bone marrow derived MC
- GlcNAc GlcNAc
- Inventors herein advantageously describe efficient methods allowing the diagnosis of a disease induced by mast cell activation as well as kits for implementing said methods.
- the present invention is based on the discovery that N-acetyl-D-glucosamine (GlcNAc) is a powerful tool when used as a biomarker for the diagnosis of a disease induced by mast cell in a subject.
- GlcNAc N-acetyl-D-glucosamine
- GlcNAc can discri inate between subjects suffering from a disease induced by mast cell activation and subjects suffering from other myeloproliferative disorders not related to mast cell such as acute myeloid leukemias, chronic myeloid leukemia, myelodysplastic and myeloproliferative syndrome, and essential thrombocythemia.
- Inventors also discovered that by determining the concentration of GlcNAc in a biological sample of a subject, it is possible to identify the particular disease induced by mast cell activation the subject is suffering of or is prone to develop.
- GlcNAc N-acetyl-D-glucosamine
- the disease is preferably selected from mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis; mast cell activation syndrome (MCAS); asthma; and allergy.
- SM systemic mastocytosis
- MCS mast cell sarcoma
- MCAS mast cell activation syndrome
- N-acetyl-D-glucosamine also identified in the art as 2-acetamino-2-deoxy- -D-glucose or 2- (acetylamino)-2-deoxy-D-glucose, is a monosaccharide that usually polymerizes linearly through (1,4)- b-linkages.
- the molecular formula of this amino monosaccharide is CsHisNOe, and its molecular weight is 221.21. In general, it is a white and slightly sweet powder that melts at 221 °C.
- the solubility of GlcNAc is 25% in water, and 1% aqueous solutions are colorless and clear.
- GlcNAc is the monomeric unit of the polymer chitin, the second most abundant carbohydrate after cellulose. In addition to being a structural component of homogeneous polysaccharides like chitin, GlcNAc is also a constituent of heterogeneous polysaccharides. In mammals, GlcNAc is a component of glycoproteins, proteoglycans, glycosaminoglycans (GAGs) and other connective tissue building blocks.
- biomarker refers to a biological molecule whose presence and/or concentration can be detected and correlated typically with a known condition.
- the biomarker can be used to classify a sample from a subject as a “disease induced by mast cell activation” sample, to associate [i.e.
- sub-classify such a sample for example with one of SM various forms or clinical subtypes, to assess the severity of the disease or disorder associated with mast cell activation, and/or to assess the efficiency of a treatment in a sick subject or in a subject diagnosed with a disease induced by mast cell activation, or in the context of preventive treatment in a subject suspected of being, or identified as, at risk of developing such a disease or disorder.
- Appropriate (secondary) diagnostic markers suitable for use in the present invention, in addition to GlcNAc, are identified below.
- Mast cell are of hematopoietic lineage. Mast cells reside close to blood vessels, nerves and lymphatic ducts in tissues that interface with the external environment (e.g., airways, gastrointestinal tract, uterus, skin). Mast cells enter the circulation as progenitors rather than mature cells. At this stage, progenitor cells in human subjects can express both FceRI and KIT (CD117). In human subjects, immediate mast cell precursors from blood were recently defined as Lin CD34 hl CDl 17 mt/hl FcsRI + cells.
- mast cells initiate inflammatory site formation through three coupled sets of events: (1) release of pre-formed cytoplasmic granules containing vasoactive amines such as histamine and serotonin, proteases such as chymase and tryptase, proteoglycans such as herapin (2) de novo biosynthesis of bioactive lipid mediators such as LTB4, LTC4, PAF, PGD2 and PGE2 and (3) de novo transcriptional activation of genes coding for a range of cytokines and chemokines such as II- 1 b, 11-4, IL-6, CXCL8, IL-10, CCL2, TNF-oc, and growth factors such as FGF-2, PDGF, TGF-bI and VEGF.
- Mediators can be secreted from mast cells through the release of granules or the release of secretory vesicles.
- disease induced by mast cell activation is meant any condition or disorder that is caused by inappropriate, in particular excessive, release of mediators by mast cells (that occurs during mast cell degranulation).
- the expression “disease induced by mast cell activation” encompasses mastocytosis, mast cell activation syndrome (MCAS), asthma, and allergy.
- mastocytosis is meant any condition that is caused by clonal proliferation of mast cells, with an abnormal accumulation of these cells in tissues including the skin, bone marrow and gastrointestinal tract. Although mastocytosis is associated with an increased number of mast cells, its presentation is not necessarily accompanied by symptoms of mast cell activation. As apparent in the all description, the expression “mastocytosis” encompasses systemic mastocytosis (SM), mast cell sarcoma (MCS) and cutaneous mastocytosis (CM).
- SM systemic mastocytosis
- MCS mast cell sarcoma
- CM cutaneous mastocytosis
- CM cutaneous mastocytosis
- cytokines and histamines which lead to itching and skin lesions.
- Cutaneous mastocytosis in the skin has several different patterns: urticaria pigmentosa, diffuse cutaneous mastocytosis, or isolated mastocytoma.
- CM is usually benign and self-limiting in time.
- Mast cell sarcoma is a rare, neoplastic disease characterized by locally destructive sarcoma-like growth of a solitary mass, composed of atypical mast cells, and without systemic involvement. It can affect any organ and the symptoms depend on the location. Cells are medium to large, pleomorphic or epithelioid, with oval, bilobed or multilobulated nuclei, sometimes prominent multinucleated giant cells. The disease closely resembles other neoplasms and may share associated markers, however the tumor is positive for mast cell tryptase.
- SM Systemic mastocytosis
- MCAS Mest cell activation syndrome
- asthma refers to airway hyperactivity and reversible airways obstruction.
- Pathological derangements at the tissue level include constriction of airway smooth muscle, increased vascular permeability resulting in edema of airways, outpouring of mucus from goblet cells and mucus glands, parasympathetic nervous system activation, denudation of airway epithelial lining cells, and influx of inflammatory cells. Underlying these tissue effects are direct effects of potent mediators secreted following physical, inflammatory, or immunological activation and degranulation.
- the early phase of the asthmatic reaction is mediated by histamine and other mast cell mediators that induce rapid effects on target organs and tissues, particularly smooth muscle.
- the pathophysiologic sequence of asthma is initiated by mast cell activation in response to allergen binding to IgE.
- the term “allergy” designates any condition or disorder characterized by the undesirable, in particular excessive, release of histamine by mast cells.
- Several biomarkers have been identified as markers of mast cells activation. Some of these markers such as tryptase, urinary N-methylhistamine, leukotriene E4 (LTE4), prostaglandin F2 alpha (PGF2oc), prostaglandin D2, markers of oxidative stress and cytokines (IL-6, IL-31) are currently used as minor markers for diagnosis of a disease induced by mast cell activation, in particular of asthma, allergy, MCAS and non-AdvSM. Tryptase diagnostic specificity for example is limited by the presence of high tryptase levels in severe allergic reaction.
- Inventors now advantageously identify biomarkers that are not dependent of an episode of mast cell activation leading to mast cell degranulation. Moreover, inventors develop tools and methods capable of discriminating between diseases induced by mast cell activation, in particular between non-advanced SM (non-AdvSM) and advanced-SM (AdvSM).
- non-AdvSM non-advanced SM
- AdvSM advanced-SM
- Inventors herein describe an in vitro or ex vivo method for diagnosing a disease induced by mast cell activation, preferably selected from mastocytosis, mast cell activation syndrome (MCAS), asthma and allergy, in a subject.
- This method preferably comprises the steps of:
- step (ii) diagnosing a disease induced by mast cell activation, preferably selected from mastocytosis, mast cell activation syndrome, asthma and allergy, in the subject if the concentration of GlcNAc as measured in step (i) is above a reference GlcNAc’ s concentration.
- diagnosis refers to the detection or identification of a pathology, disease, disorder or dysfunctional state as herein defined, typically a disease induced by mast cell activation, or to the evaluation/assessment (dosing, comparison) of the severity or of the stage of such a pathology, disease, disorder or dysfunctional state in a subject.
- the term “subject” refers to any testable subject and typically designates a patient.
- the subject is any member of the animal kingdom, preferably a mammal.
- the subject is a domestic animal, in particular a pet, such as for example a dog or a cat.
- a particular domestic animal is an animal suspected of suffering of, or diagnosed as suffering of, mast cell sarcoma (MCS).
- MCS mast cell sarcoma
- the subject is a human being.
- the subject, in the human subject has a mutated kit gene or expresses a mutated KIT receptor.
- the invention may involve either an individual or an entire population. The subject may be tested whatever his/her age or sex.
- biological sample includes any biological sample from a subject.
- the biological sample may be a solid sample or a fluid sample.
- Typical examples of biological fluid samples usable in the context of the present invention may be selected from a blood, plasma, serum, urine and bone marrow aspirate sample.
- the biological sample is a plasma, a blood or a serum sample, even more preferably a plasma sample.
- sample may be used pure or diluted.
- Typical example of biological solid sample usable in the context of the present invention is a bone marrow biopsy sample.
- Another particular sample is a sample comprising mast cells bearing a mutation in the KIT receptor.
- the KIT mutation is preferably located within the activation loop of the receptor at residue 816, and is in particular D816X, where X is V, H, I, F or Y.
- Mast cells can be for example KITD816X mast cells where X is V, H, I, F or Y.
- chromatography may comprise Gas Chromatography (GC), Liquid Chromatography (LC), High Pressure Liquid Chromatography (HPLC) and Ultra High Pressure Liquid Chromatography (UHPLC).
- Spectroscopy may comprise Ultraviolet- Visible (UV/Vis), Infrared (IR), Near Infrared (NIR) and Nuclear Magnetic Resonance (NMR).
- Mass analyzers/spectrometry may comprise ESI-QqQ, ESI-QqTOF, Matrix-assisted laser desorption/ionization (MALDI)-QqQ, MALDI-QqTOF and MALDI-TOF-TOF.
- MALDI Matrix-assisted laser desorption/ionization
- mass analyzers/spectrometry comprises Quadrupole Mass Analyzers, Ion Trap Mass Analyzers, TOF (Time of Flight) Mass Analyzer, Orbitrap mass analyzer, Magnetic Sector Mass Analyzer, Electrostatic Sector Mass Analyzer, Ion Cyclotron Resonance (ICR) and combinations of mass analyzers, including single quadrupole (Q) and triple quadrupole (QqQ), QqTOF, TOF-TOF and Q-Orbitrap.
- Quadrupole Mass Analyzers Ion Trap Mass Analyzers
- TOF Time of Flight Mass Analyzer
- Orbitrap mass analyzer Magnetic Sector Mass Analyzer
- Electrostatic Sector Mass Analyzer Electrostatic Sector Mass Analyzer
- ICR Ion Cyclotron Resonance
- Q single quadrupole
- QqQ triple quadrupole
- QqTOF quadrupole
- TOF-TOF-TOF TOF-TOF
- Q-Orbitrap Q-Orbitrap
- GlcNAc N-acetyl-D-glucosamine
- the concentration of GlcNAc can also be measured based on the same samples (i.e., blood, plasma, serum, urine, bone marrow aspirate preferably blood, plasma or serum), on a phosphorylated product of GlcNAc, using a kinase assay (EIA).
- EIA kinase assay
- N-acetylglucosamine kinase GlcNAc kinase or NAGK; E.C. 2.7.1.59
- an enzyme of the sugar-kinase/Hsp70/actin super family is a prominent enzyme in amino sugar metabolism that catalyzes the conversion of GlcNAc to GlcNAc-6-phosphate (GlcNAc-6P).
- the specific activity of NAGK enzyme is directly correlated to the amount of GlcNAc present in the sample of the subject.
- the concentration of GlcNAc can be determined indirectly by measuring the O-GlcNAcylation of proteins, using for example a binding agent that specifically binds to O-linked N-acetylglucosamine (O-GlcNAc), such as for example an anti-O-linked N-acetylglucosamine (O- GlcNAc) binding agent, preferably a monoclonal antibody, such as for example RL2, HGAC85, 18B10.C7 or CTD110.6 monoclonal antibody. A second molecule allowing the binding agent detection can be required.
- Concentration of GlcNAc can be determined using variety of known methods such as for example immunoassay, immunosorbent assay (ELISA), radioimmunoassay (RIA), and the like.
- reference GlcNAc reference GlcNAc
- reference GlcNAc reference GlcNAc
- concentration control GlcNAc
- cut-off GlcNAc cut-off GlcNAc
- the reference value can also be a statistic or discriminating value/threshold, i.e., a value which has been determined by measuring the parameter in both a healthy control population and a population with a known pathology, disease, disorder or dysfunctional state as herein described.
- the discriminating value identifies the diseased population with a specificity and/or a sensitivity, both predetermined by the skilled person, based on an analysis of the relation between the parameter values and the known clinical data of the healthy control subject/population and of the diseased patient subject/population. The discriminating value determined in this manner is valid for the same experimental setup in future individual tests.
- Reference GlcNAc can, in principle, be calculated for a cohort of subjects as specified above based on the average or mean values for a given biomarker by applying standard methods of statistics.
- accuracy of a test such as a method aiming to diagnose an event, or not, is best described by its receiver-operating characteristics (ROC).
- ROC receiver-operating characteristics
- Each point on the ROC plot represents a sensitivity/specificity pair corresponding to a particular decision threshold.
- a threshold can be derived from the ROC curve allowing for the diagnosis or prediction for a given event with a proper balance of sensitivity and specificity, respectively. Accordingly, the reference to be used for the method of the present invention, i.e.
- a threshold which allows to discriminate between subjects who have a disease induced by mast cell activation and those who don’t have a disease induced by mast cell activation can be generated, preferably, by establishing a ROC analysis for said cohort as described above and deriving one or more threshold amounts or concentrations therefrom.
- the ROC plot allows deriving adjustable suitable thresholds. It will be understood that an optimal sensitivity is desired for excluding a disease induced by mast cell activation (i.e., a rule out) whereas an optimal specificity is envisaged for a subject to be identified as having a disease induced by mast cell activation (i.e. a rule in).
- the concentration of GlcNAc determined in step (i) of the method of the present invention is compared to more than one reference GlcNAc’s concentration, e.g. a reference GlcNAc’s concentration for discriminating between the diseases induced by mast cell activation, and a reference GlcNAc’s concentration for ruling out a particular disease induced by mast cell activation among said diseases, typically for discriminating between healthy subjects and subjects suffering of a disease induced by mast cell activation.
- a reference GlcNAc concentration for discriminating between the diseases induced by mast cell activation
- a reference GlcNAc concentration for ruling out a particular disease induced by mast cell activation among said diseases, typically for discriminating between healthy subjects and subjects suffering of a disease induced by mast cell activation.
- the reference GlcNAc’s value in plasma sample of a healthy control population is below 0.46 mM.
- a concentration of GlcNAc in plasma above the reference GlcNAc’s value of 0.46 pM is indicative of a disease induced by mast cell activation and allows the classification of a tested subject as a “diseased” subject, i.e., a subject suffering of a disease induced by mast cell activation.
- the in vitro or ex vivo method of the invention allows differentially diagnosing between diseases induced by mast cell activation and non-mast cells-related myeloproliferative disorders such as acute myeloid leukemias, chronic myeloid leukemia, myelodysplastic and myeloproliferative syndrome, and essential thrombocythemia in a subject.
- the in vitro or ex vivo method for differentially diagnosing between disease induced by mast cell activation and non-mast cells-related myeloproliferative disorders preferably acute myeloid leukemias, chronic myeloid leukemia, myelodysplastic and myeloproliferative syndrome, and essential thrombocythemia in a subject comprises the steps of:
- step (ii) diagnosing a disease induced by mast cell activation in the subject if the concentration of GlcNAc as measured in step (i) is above a reference GlcNAc’s concentration.
- a concentration of GlcNAc in plasma above the reference GlcNAc’s value of 0.46 pM, for example above 0.8 pM is indicative of a disease induced by mast cell activation and allows the classification of a tested subject as a subject suffering of a disease induced by mast cell activation and not from a non-mast cells-related myeloproliferative disorder such as acute myeloid leukemias, chronic myeloid leukemia, myelodysplastic and myeloproliferative syndrome, and essential thrombocythemia.
- the reference GlcNAc s concentrations to be used for the method of the present invention are also helpful to discriminate between the particular diseases induced by mast cell activation.
- the in vitro or ex vivo method of the invention allows the diagnosis of asthma or allergy in a subject, for example vs mastocytosis, in particular SM, or vs MCAS.
- the in vitro or ex vivo method for diagnosing asthma or allergy comprises the steps of: (i) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject; and
- step (ii) diagnosing asthma or allergy in the subject if the concentration of GlcNAc as measured in step (i) is above a first reference GlcNAc’ s concentration and, preferably below a second reference GlcNAc’ s concentration.
- the first and the second reference GlcNAc ’s concentrations are different.
- a concentration of GlcNAc in a plasma sample of a subject above 0.46 mM and below 1.7 mM indicates that the subject is suffering of asthma or allergy.
- ROC receiver operating characteristic
- the in vitro or ex vivo method of the invention allows the diagnosis of mast cell activation syndrome (MCAS) or non-advanced SM (non-AdvSM), in particular indolent systemic mastocytosis (ISM) or smoldering SM (SSM), in a subject.
- MCAS mast cell activation syndrome
- non-AdvSM non-advanced SM
- ISM indolent systemic mastocytosis
- SSM smoldering SM
- the in vitro or ex vivo method for diagnosing mast cell activation syndrome (MCAS) or non-advanced SM (non-AdvSM), in particular indolent systemic mastocytosis (ISM) or smoldering SM (SSM), in a subject comprises the steps of:
- step (ii) diagnosing mast cell activation syndrome (MCAS) or non-advanced SM (non-AdvSM), in particular indolent systemic mastocytosis (ISM) or smoldering SM (SSM), in the subject if the concentration of GlcNAc as measured in step (i) is above a first reference GlcNAc’ s concentration and below a second GlcNAc’ s concentration.
- MCAS mast cell activation syndrome
- non-AdvSM non-advanced SM
- ISM indolent systemic mastocytosis
- SSM smoldering SM
- the first and the second reference GlcNAc ’s concentrations are different.
- a concentration of GlcNAc in a plasma sample of a subject above 1.7 pM and below 2.8 pM indicates that the subject is suffering of mast cell activation syndrome (MCAS) or non-advanced SM (non-AdvSM), in particular of indolent systemic mastocytosis (ISM) or smoldering SM (SSM).
- MCAS mast cell activation syndrome
- non-AdvSM non-advanced SM
- ISM indolent systemic mastocytosis
- SSM smoldering SM
- the cut-off value that allow to discriminate between asthmatic or allergic patient population and a MCAS or non-AdvSM, in particular ISM or SSM, patient populations is 2.2 pM for a sensitivity of 0.9 and specificity of 0.8.
- the in vitro or ex vivo method of the invention allows the diagnosis of advanced SM (AdvSM), in particular aggressive SM (ASM), SM with an associated hematological neoplasm (SM-AHN) or mast cell leukemia (MCL), in a subject.
- AdvSM advanced SM
- ASM aggressive SM
- SM-AHN SM with an associated hematological neoplasm
- MCL mast cell leukemia
- the in vitro or ex vivo method for diagnosing advanced SM comprises the steps of:
- step (ii) diagnosing advanced SM (AdvSM), in particular ASM, SM-AHN or MCL, in the subject if the concentration of GlcNAc as measured in step (i) is above a reference GlcNAc’ s concentration.
- AdvSM advanced SM
- a concentration of GlcNAc in a plasma sample of a subject above 2.8 mM indicates that the subject is suffering of advanced SM (AdvSM), in particular ASM, SM-AHN or MCL.
- AdvSM advanced SM
- ASM advanced SM
- SM-AHN SM-AHN
- MCL advanced SM
- a concentration of GlcNAc in a biological sample (as herein defined) of a subject above 2.8 pM and, preferably, below 7, 8, 9 or 10 pM for example, indicates that the subject is suffering of advanced SM (AdvSM), in particular ASM, SM-AHN or MCL.
- AdvSM advanced SM
- ROC receiver operating characteristic
- the level of concentration of GlcNAc in a biological sample of the subject preferably in a plasma or serum sample, enables to discriminate between the different forms of AdvSM. For example, using ROC analysis, a concentration of GlcNAc in a plasma or serum sample of a subject below 4.5 pM indicates that the subject has ASM. A concentration of GlcNAc in a plasma or serum sample of a subject above 4.5 pM indicates that the subject has advanced SM-AHN.
- the in vitro or ex vivo method of the invention can be implemented for diagnosing a systemic mastocytosis (SM), in particular non-advanced SM (non-AdvSM) or advanced SM (AdvSM), more particularly indolent SM (ISM), smoldering SM (SSM), SM with an associated hematological neoplasm (SM-AHN), aggressive SM (ASM), mast cell leukemia (MCL), even more particularly indolent SM (ISM) or aggressive SM (ASM).
- SM systemic mastocytosis
- ISM in particular non-advanced SM
- AdvSM advanced SM
- ISM indolent SM
- SSM smoldering SM
- SM-AHN hematological neoplasm
- ASM aggressive SM
- MCL mast cell leukemia
- ISM indolent SM
- ASM aggressive SM
- ASM aggressive SM
- ASM aggressive
- SM systemic mastocytosis
- the level of concentration of GlcNAc in a biological sample of the subject enables to discriminate between the different forms of SM.
- a concentration of GlcNAc in a plasma or serum sample of a subject below 3.8 mM indicates that the subject has non-AdvSM, in particular indolent SM (ISM) or smoldering SM (SSM).
- ISM indolent SM
- SSM smoldering SM
- a concentration of GlcNAc in a plasma or serum sample of a subject above 3.8 mM indicates that the subject has advanced SM (AdvSM), in particular ASM, SM-AHN or MCL.
- biomarkers suitable for the diagnosis of SM in combination with GlcNAc and more particularly for discriminating between non-AdvSM, in particular ISM, and AdvSM, in particular ASM, SM-AHN or MCL.
- ISM non-AdvSM
- AdvSM in particular ASM, SM-AHN or MCL.
- the in vitro or ex vivo method of the invention for diagnosing a disease induced by mast cell activation further comprises a step of determining, in a biological sample from the subject which may be the same sample used for measuring the concentration of GlcNAc, or may be a different sample, the concentration of one or more secondary (/additional) biomarkers involved in the nicotinate/nicotinamide metabolism, pyrimidine metabolism, purine metabolism, glycerophospholipid metabolism, amino sugar and nucleotide sugar metabolism; arginine biosynthesis, and/or one carbon pool by folate metabolism, preferably in the nicotinate/nicotinamide metabolism and/or in the amino sugar metabolism (i.e. protein glycosylation).
- Samples used to measure the concentration of GlcNAc and one or more biomarkers belonging to the seven metabolic pathways as disclosed herein may be of the same or different nature.
- the sample used for measuring the concentration of one or more biomarkers is of the same nature as that used for measuring the concentration of GlcNAc.
- a fluid sample such as a serum or plasma sample can be used for measuring the concentration of GlcNAc and also for measuring the concentration of one or more distinct biomarkers.
- the sample used for measuring the concentration of the one or more biomarkers is of a nature different from that used for measuring the concentration of GlcNAc.
- one sample is a serum or plasma sample and the other sample is a (liquid or solid) bone marrow sample.
- 58 can be used as selective biomarkers of AdvSM, in particular ASM, and non-AdvSM, in particular ISM, in combination with GlcNAc, based on a relative fold change of anyone of said 58 biomarkers above 1.5 (as herein listed below).
- the in vitro or ex vivo method of the invention for diagnosing a disease induced by mast cell activation further comprises a step of determining from the biological sample from the subject a concentration of one or more (secondary) biomarkers, for example biomarkers involved in nicotinate/nicotinamide metabolism such as L- kynurenine and/or quinolinate, and/or biomarkers involved in amino sugar metabolism such as dihydroxyacetone phosphate, wherein a variation of the concentration of said one or more biomarkers, such as an increase if the biomarker(s) is/are L-kynurenine and/or quinolinate, and/or or a decrease if the biomarker is dihydroxyacetone phosphate, when compared to a reference concentration of said one or more biomarkers, is indicative of AdvSM.
- secondary biomarkers for example biomarkers involved in nicotinate/nicotinamide metabolism such as L- kynurenine and/or quinolinate, and/
- Each determined concentration is compared to a reference concentration.
- the comparison can be absolute or relative depending of the nature of the reference concentration which can be obtained from a healthy subject/population or from a subject/population having a disease induced by mast cell activation, such as ISM.
- concentrations in the sample of the tested subject of L-kynurenine and/or quinolinate above their respective reference concentrations, and/or a concentration of dihydroxyacetone phosphate below its reference concentration is/are indicative of AdvSM.
- a decrease in the concentration of dihydroxyacetone phosphate in the sample of the tested subject compared to the reference concentration of dihydroxyacetone phosphate in the ISM reference sample is indicative of AdvSM and/or an increase in the concentrations of L-kynurenine and/or quinolinate in the sample of the tested subject compared to the reference concentration of L- kynurenine and/or quinolinate in the ISM reference sample, is indicative of AdvSM.
- Mastocytosis is a disorder characterized by the abnormal proliferation and accumulation of mast cells.
- mast cells derive from a clonal progenitor carrying a gain-of-function mutation in KIT.
- KIT (CD117) is a Type III receptor tyrosine kinase that is expressed by MC notably.
- One example of the KIT transcription product of the kit gene has the sequence of NCBI reference sequence NM_000222.2.
- SCF stem cell factor
- Gain-of-function somatic mutations in the KIT tyrosine kinase domain have been found to occur in a majority of cases of SM, at least in adult subjects.
- Other mutations associated to AdvSM have been observed at position 816, in particular D816V, D816H, D816I, D816F and D816Y.
- Other less common ( ⁇ 5%) somatic KIT mutations identified in adults suffering of SM include K509I, F522C, V560G, D815K, insVI815-816 and D820G (Cem Akin, J Allergy Clin Immunol 2017;140:349-55).
- KIT mutation associated to AdvSM are observed in mast cells of the bone marrow, but also in other myeloid/lymphoid lineages, such as CD34 + hematopoietic stem and precursor cells, eosinophils, monocytes, and maturing neutrophils, and, to a lesser extent, also in T lymphocytes.
- Treatment protocol for diseases induced by mast cell activation generally depends on the severity of the disease. It is thus essential to determine this aspect with certainty and as early as possible.
- the in vitro or ex vivo diagnostic method of the invention offers a valuable means for the practitioner to categorize patients suffering from a disease induced by mast cell activation.
- the practitioner can subsequently categorize the subject as having mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis (CM); mast cell activation syndrome (MCAS); asthma; or allergy depending on the GlcNAc concentration in the sample.
- SM systemic mastocytosis
- MCS mast cell sarcoma
- CM mast cell activation syndrome
- asthma or allergy depending on the GlcNAc concentration in the sample.
- the in vitro or ex vivo method of the invention for diagnosing a disease induced by mast cell activation further comprises a step of administering a treatment for a disease induced by mast cell activation to a subject diagnosed as having mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis (CM); mast cell activation syndrome (MCAS); asthma; and allergy.
- SM systemic mastocytosis
- MCS mast cell sarcoma
- CM mast cell sarcoma
- MCAS mast cell activation syndrome
- asthma asthma
- allergy allergy
- This subsequent treatment step follows the diagnosis made on the basis of the GlcNAc alone, or in combination with all or part of the other (secondary) biomarkers mentioned above.
- Such treatment can be a treatment as defined below depending on the type of disease induced by mast cell activation which is concerned.
- the subject diagnosed as having mastocytosis in particular anyone of the herein described particular mastocytosis, is a subject who has not been diagnosed with a GlcNac biomarker, typically a subject who has been diagnosed has suffering of such mastocytosis by using a biomarker selected from tryptase, urinary histamine metabolites, urinary prostaglandin D2, urinary leukotriene E4 and metabolites thereof, and thus who is likely not to have been correctly diagnosed.
- a biomarker selected from tryptase, urinary histamine metabolites, urinary prostaglandin D2, urinary leukotriene E4 and metabolites thereof, and thus who is likely not to have been correctly diagnosed.
- ISM patients have a normal life expectancy and treatment is generally limited to anaphylaxis/prevention of symptoms, and/or to the prevention or treatment of osteoporosis.
- the main objective is to reduce the symptoms of MC activation, such as pruritus, flushes and gastrointestinal cramps.
- antihistamines HI anti-Hl
- anti- H2 are used for gastrointestinal tract manifestations.
- anti- H2 are effective and can be combined with anti-Hl, with di-sodium cromoglycate or with leukotriene inhibitors.
- Corticoids can suppress antihistamines recalcitrant symptoms.
- biphosphonates are recommended with adequate supplementation of calcium and vitamin D.
- symptomatic treatment may be the only therapy. Avoidance of known triggers, prophylactic prescription of an epi-pen, and medications such as antihistamines, antileukotrienes, cromolyn sodium, omalizumab and aspirin may all have a role in the prevention or treatment of MC- mediated symptoms.
- GlcNAc concentration of patients can be assessed regularly to monitor disease evolution and appropriately adjust therapy.
- introduction of targeted or non-targeted cytoreductive therapy is now possible.
- Non-targeted therapies include interferon-(IFNalpha), which may be effective in a subset of patients, and cladribine (2CdA) which provides high and, in some patients, long lasting, response rates.
- Targeted therapy is with KIT tyrosine kinase inhibitors (KIT TKIs).
- KIT TKIs KIT tyrosine kinase inhibitors
- small molecule inhibitors that target mutant-KIT may be used, including FDA approved midostaurin (RYDAPT) or avapritinib (AYVAKIT).
- Other TKIs included imatinib mesylate or masitinib may be indicated for the patients who do not have the KITD816V mutation.
- Symptomatic treatment is mainly with antihistamines (anti-Hl and anti-H2).
- ASM-AHN Treatment of ASM-AHN primarily targets the AHN component, if an aggressive disease such as acute myeloid leukemia is present. Allogeneic stem cell transplant can be considered in such patients, or in those with relapsed/refractory advanced SM. Imatinib has a limited therapeutic role in SM; effective cytoreduction is limited to subjects having imatinib-sensitive KIT mutations.
- Management of MCF treatment includes chemotherapy, with or without interferon alpha or cladribine.
- poly- chemotherapy protocols otherwise used for high-risk AML
- SCT stem cell transplantation
- the outcome after allogeneic SCT is better for those prepared with ablative conditioning compared with less-intensive (nonmyeloablative) conditioning.
- splenectomy is indicated. Hydroxyurea is a palliative treatment.
- mast cell patients take baseline medications, including a second generation HI and H2 antihistamine, a mast cell stabilizer and a leukotriene inhibitor.
- SM systemic mastocytosis
- ISM indolent SM
- ASM aggressive SM
- SM-AHN hematological neoplasm
- MCL mast cell leukemia
- assessing includes determining if an element is present or not, and if present, optionally determining the amount or level, in particular the concentration, of said element.
- determining evaluates the amount or level
- assessing includes determining if an element is present or not, and if present, optionally determining the amount or level, in particular the concentration, of said element.
- determining evaluates the amount or level, in particular the concentration, of said element.
- assessing includes determining if an element is present or not, and if present, optionally determining the amount or level, in particular the concentration, of said element.
- determining evaluating
- assessing assaying
- the correlation takes into account the amount or concentration of GlcNAc in the sample of a subject affected with SM compared to a control/reference amount or concentration of GlcNAc (e.g., in healthy subjects in whom a disease induced by mast cell activation is absent).
- the in vitro or ex vivo method is for assessing the severity of SM, in particular for differentially diagnosing between non-AdvSM, in particular indolent SM (ISM), and advanced SM (AdvSM), in particular aggressive SM (ASM) or SM with an associated hematological neoplasm (SM- AHN), in a subject.
- a particular method of this kind comprises a step of comparing the concentration of GlcNAc in the biological sample of the subject with a reference concentration of GlcNAc, which is the concentration of GlcNAc in a biological sample obtained from a healthy subject.
- a concentration of GlcNAc in the biological sample of the subject that is 6 to 8 times greater than the concentration of GlcNAc in the reference control sample is indicative of non-AdvSM, in particular indolent SM (ISM) or smoldering SM (SSM); and a concentration of GlcNAc in the biological sample of the subject that is more than 8 fold greater than the concentration of GlcNAc in the reference control sample is indicative of advanced SM (AdvSM), in particular aggressive SM (ASM) or SM with an associated hematological neoplasm (SM- AHN).
- AdvSM advanced SM
- ASM aggressive SM
- SM- AHN hematological neoplasm
- Inventors herein disclose the plasma metabolome profiling of SM. Inventors in particular herein identify for the first time N-acetyl-D-glucosamine (GlcNAc) as the most predictive biomarker of SM severity. As apparent from data disclosed in the experimental section of the present description, high plasma levels of GlcNAc in patients with advanced SM correlate with the activation of the GlcNAc-fed hexosamine biosynthesis pathway (HBP) in patients’ BM aspirates.
- HBP hexosamine biosynthesis pathway
- the biological sample of the subject is a fluid sample, preferably a plasma or serum sample, and the reference concentration of GlcNAc is 2.8 mM.
- a concentration of GlcNAc in the biological (plasma or serum) sample of the subject that is equal to or above the reference (plasma or serum) concentration of GlcNAc is indicative of AdvSM, in particular ASM or SM-AHN
- a concentration of GlcNAc in the biological (plasma or serum) sample of the subject that is below the reference (plasma or serum) concentration of GlcNAc is indicative of non-AdvSM, in particular ISM or SSM.
- ISM patients with multilineage KIT D816V mutations have been shown to have a higher risk of progression to AdvSM than patients with mast cells-restricted KIT D816V.
- the diagnostic standard is mutation analysis of KIT in bone marrow (BM) cells. If a BM aspirate is not available, the analysis can be performed on the marrow smear or from a paraffin-embedded biopsy sample.
- BM bone marrow
- these methods are invasive and not easy to implement, especially since the number of neoplastic MCs in the BM of ISM patients is very low. This sets the requirement for highly sensitive methods of mutation analysis.
- GlcNAc can be used as a biomarker for assessing/predicting the risk for a subject affected or diagnosed with non-AdvSM, in particular ISM or SSM to develop AdvSM, in particular ASM or SM-AHN and herein describe an in vitro or ex vivo method for assessing/predicting the risk for a subject affected or diagnosed with non-AdvSM, in particular ISM or SSM, to develop AdvSM, in particular ASM or SM-AHN.
- the method comprises a step of determining the concentration of GlcNAc in a biological sample obtained from a subject affected or diagnosed with non-AdvSM, in particular ISM or SSM, wherein a concentration of GlcNAc above a reference concentration of GlcNAc indicates that the subject is at risk to develop AdvSM, in particular ASM or SM-AHN.
- a concentration of GlcNAc above a reference concentration of GlcNAc indicates that the subject is at risk to develop AdvSM, in particular ASM or SM-AHN.
- the reference concentration of GlcNAc useful to predict the risk for a subject affected or diagnosed with ISM to develop ASM is 3.8 mM, as determined by ROC analysis.
- inventors herein provide an in vitro or ex vivo method for assessing/predicting the risk for a subject affected or diagnosed with non-AdvSM, in particular ISM, to develop AdvSM, in particular ASM or SM-AHN, wherein the method comprises the steps of:
- a concentration of GlcNAc as measured in step (b) below or identical to the concentration of GlcNAc as measured in step (a) is indicative that the subject is not at risk to develop AdvSM, in particular ASM or SM-AHN, and
- a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative that the subject is at risk to develop AdvSM, in particular ASM or SM-AHN.
- a follow-up of patient affected with a disease induced by mast cell activation being essential in a clinical point of view so as to optimally adapt the treatment to the course of the disease inventors herein provide an in vitro or ex vivo method for monitoring the evolution of a disease induced by mast cell activation, preferably selected from mastocytosis, mast cell activation syndrome, asthma and allergy, in particular SM, in a subject affected with said disease.
- This method preferably comprises the steps of:
- step (c) comparing the concentrations of GlcNAc as measured in steps (a) and (b) to monitor the evolution of the disease in the subject, wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative of a favorable evolution of the disease in the subject, a concentration of GlcNAc as measured in step (b) identical to the concentration of GlcNAc as measured in step (a) is indicative of a stabilization of the disease in the subject, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative of an unfavorable evolution of the disease in the subject, which preferably requires an adaptation of the treatment.
- monitoring is meant detecting and following any change in the parameters of a disease. These parameters can be physiologic or symptomatic parameters. Monitoring involves periodic measurements that guide the management of a chronic or recurrent condition such as, in the context of the present invention, a disease induced by mast cell activation.
- “favorable evolution of the disease” is meant a regression, at least in part, of the symptoms or clinical signs associated with a disease induced by mast cell activation.
- “unfavorable evolution of the disease” relates to a worsening of clinical symptoms or signs associated with a disease induced by mast cell activation. This worsening can include for example higher release of mast cell mediators, more organs affected with mast cell infiltrate, or any other neurologic, gastrointestinal, cardiovascular, constitutional signs classically associated with a disease induced by mast cell activation.
- the “stabilization of the disease” means that there is no modulation, i.e., neither improvement nor worsening, of the clinical symptoms or signs associated with a disease induced by mast cell activation.
- monitoring before treatment should establish the need or not for treatment and then a baseline/threshold above which it becomes possible to judge the response to a treatment or changes in the patient's condition.
- the objective of monitoring is to ensure that measures stay within reasonable limits, called control limits.
- the control limits ensure that relevant/significative changes in the level of the target biomarker(s) are detected while minimizing misdiagnosis or errors.
- the monitoring interval can be several days, weeks or months. The interval depends on evolution/course (progression, stabilization or regression) of the disease.
- the in vitro or ex vivo method is for monitoring the evolution of non-AdvSM, in particular ISM or SSM, in a subject wrongly or rightly diagnosed as affected with non-AdvSM, in particular ISM or SSM.
- a particular method for monitoring the evolution of non-AdvSM, in particular ISM or SSM comprises the steps of:
- step (c) comparing the concentrations of GlcNAc as measured in steps (a) and (b) to monitor the evolution of non-AdvSM, in particular ISM or SSM, in the subject, wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative of a favorable evolution of non-AdvSM, in particular ISM or SSM, in the subject, a concentration of GlcNAc as measured in step (b) identical to the concentration of GlcNAc as measured in step (a) is indicative of a stabilization of non-AdvSM, in particular ISM or SSM, in the subject, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative of an unfavorable evolution of non-AdvSM, in particular ISM or SSM, in the subject.
- step (b) may be repeated as many times as necessary to monitor the evolution of the disease over a period considered as relevant by the practitioner. Monitoring can also be implemented once a treatment for a disease induced by mast cell activation has started.
- a therapy of a disease induced by mast cell activation selected preferably from mastocytosis, mast cell activation syndrome, asthma and allergy, in particular SM, in a subject affected with said disease.
- This method preferably comprises the steps of:
- step (c) comparing the concentrations of GlcNAc as measured in steps (a) and (b), wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative that the therapy is effective in the treatment of the disease induced by mast cell activation, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative that the therapy is not effective, or not effective enough, in the treatment of the disease induced by mast cell activation; or, alternatively, wherein the method comprises the steps of:
- step (ii) comparing the concentration of GlcNAc as measured in step (i) with a reference concentration of GlcNAc, wherein, a concentration of GlcNAc as measured in step (i) below the GlcNAc reference concentration is indicative that the therapy is effective in the treatment of the disease induced by mast cell activation, and a concentration of GlcNAc as measured in step (i) above the GlcNAc reference concentration is indicative that the therapy is not effective, or not effective enough, in the treatment of the disease induced by mast cell activation.
- step (b) may be repeated as many times as deemed necessary by the practitioner during the course of the disease and until the subject is cured or stabilization at an acceptable level (i.e. compatible with an acceptable life for the diseased subject).
- any of the herein described in vitro or ex vivo method for determining the efficacy of a therapy of a disease induced by mast cell activation further comprises a step of determining, in a biological sample from the subject, the transcript level of one or more enzymes involved in the hexosamine biosynthesis pathway (HBP), such as GFAT1, PGM3, NAGK, OGT and OGA.
- HBP hexosamine biosynthesis pathway
- Bone marrow aspirate which contains mast cells, is a particularly suitable biological sample to perform transcription study.
- the reference sample may in particular be a sample previously collected from said subject, in particular before monitoring or evaluation has started.
- kits for measuring the concentration of GlcNAc in a biological sample of a subject comprising an anti-O-linked N-acetylglucosamine (O-GlcNAc) binding agent, for example RL2, HGAC85, 18B10.C7 or CTD110.6 monoclonal antibody, a molecule allowing the binding agent detection; or a N-Acetyl-D-Glucosamine Kinase (NAGK), and optionally a leaflet providing GlcNAc reference concentration(s).
- O-GlcNAc anti-O-linked N-acetylglucosamine
- NAGK N-Acetyl-D-Glucosamine Kinase
- the kit for measuring the concentration of GlcNAc in a biological sample of a subject comprises an anti-N-acetylglucosamine (GlcNAc) binding agent that binds GlcNAc, preferably that selectively binds GlcNAc and does not bind O-GlcNAc; a molecule allowing the binding agent detection; or a N-Acetyl-D-Glucosamine Kinase (NAGK), and optionally a leaflet providing GlcNAc reference concentration(s).
- GlcNAc anti-N-acetylglucosamine
- the kit may further comprise primers and probes for assessing the transcript level of one or more enzymes involved in the hexosamine biosynthesis pathway (HBP), such as GFAT1, PGM3, NAGK, OGT and OGA in a biological sample of a subject.
- HBP hexosamine biosynthesis pathway
- Another particular kit also comprises for example:
- KIT mutation in particular KITD816X where X is V, H, I, F or Y, in a biological sample of a subject;
- kits on a biological sample obtained from a subject, for diagnosing a disease induced by mast cell activation typically selected from mastocytosis, mast cell activation syndrome, asthma and allergy, preferably SM; for assessing the severity of systemic mastocytosis; for assessing/predicting the risk for a subject affected with non-AdvSM to develop AdvSM, preferably prior to the onset of AdvSM’s symptoms; monitoring the evolution of a disease induced by mast cell activation and/or for determining the efficacy of a therapy of a disease induced by mast cell activation typically selected from mastocytosis, mast cell activation syndrome, asthma and allergy, preferably SM, in the subject.
- Plasma was isolated from whole blood of patient collected in heparinized tube and stored at -80°C until analysis. Blood and bone marrow aspirate material were obtained at diagnosis, after written informed consent was given. Data and samples collection were approved by the ethics committee of the Necker Hospital (Paris, France) and conducted in compliance with the precepts of the Helsinki protocol. Healthy subjects without mastocytosis were used as controls.
- ROSA K " WT and ROSA K " D816v were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% heat inactivated fetal calf serum FCS, 100 pg/mL streptomycin, 100 IU/mL penicillin, 2 mM L-glutamine and ImM sodium pyruvate.
- ROSA KITWT cells were grown with additional 80ng/mL murine stem cell factor.
- Bibi et al. (Bibi S, 2016) generated the ROSA HT D816v Gluc mast cells (kindly provided by M. Arock, Paris, France).
- ROSA KIT D816v were obtained by the transduction of ROSA KIT D816v with lentiviral particles LV-Gluc-CFP.
- ROSA KIT D816v Gluc cells expressed both GFP and CFP (cyan fluorescent protein).
- Murine bone marrow-derived mast cells (BMMCs) were cultured in Opti-MEM Reduced Serum medium supplemented with 10% heat inactivated fetal calf serum FCS, 100 mg/mL streptomycin, 100 IU/mL penicillin, 2-mercaptoethanol, 2 mM L- glutamine and 1 mM sodium pyruvate in presence of IL-3.
- Kit D814V F1 mice Gerbaulet et al. generated the Kit D814V F1 mice.
- the Kit D814V transgene harboring the murine equivalent of the human kit D816V mutation, is expressed upon Cre-mediated excision of the loxP- flanked transcriptional stop element in adult mice. These mice were crossed to Mxl-Cre mice to generate Kit D814V F1 ; Mxl-Cre mice and their progeny were used for subsequent experiments.
- Cre expression was induced by 3 intra-peritoneal (i.p.) injections of 250 pg polyinosinic:polycytidylic (pI:C) every second day to activate the expression of the transgenic Kit D814V allele.
- Bone marrow derived mast cells were differentiated by culturing bone marrow from Kit D814V F1 ; Mxl-Cre and Kit WT; Mxl-Cre mice for 4 to 6 weeks in IL-3 supplemented media. Mast cell phenotype was confirmed by flow cytometry analysis with antibodies specific for c-Kit (2B8; eBiosciences) and the multi-chain activating receptor FceRI (MAR-1; eBiosciences). All animal studies were performed in the animal facility at Cancer Research Center of Marseilles (CRCM) in compliance with the laws and approved by the French animal ethics committees (Agreement APAFIS N°6743- 2016091513513606 v4). Protocols were carried out in accordance with guidelines for animal care and protection.
- Polar metabolites were extracted from the plasma of patient in ice-cold methanol following the protocol described in a previous study (Yuan M et al., 2012). All supernatants were evaporated on a SpeedVac concentrator to a pellet using no heat and sent on dry ice to the mass spectrometry core of Beth Israel Deaconess Medical Center (BIDMC) of Boston led by JM Asara. Endogenous metabolite profiles were obtained using a positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform using an AB/SCIEX 5500 QTRAP hybrid triple quadrupole mass spectrometer. Metabolites were identified by their m/z retention time, and through comparison to library entities of purified known standards.
- Bioinformatic analysis of the metabolomic profiling was performed using R software and MetaboAnalyst 4.0. (Chong J et al., 2019).
- To normalize the LC-MS/MS peak areas inventors selected metabolites that were detected in at least 50% of the samples. The remaining missing values were replaced by half of the minimum peak area value measured in the data set. Then, the samples’ peak area values were normalized by probabilistic quotient normalization, using the “Healthy” or ISM samples as reference. The final normalized values were log2 transformed. For computation of the z-scores, each metabolite was centered by the reference samples’ mean and scaled by the reference samples’ standard deviation.
- RMA Robust Multichip Average
- ROSA K " WT and ROSA KIT D816v mast cells were seeded in low glucose (1 g/L) IMDM supplemented with 10% heat inactivated fetal calf serum FCS, 100 pg/mL streptomycin, 100 IU/mL penicillin, 2 mM L-glutamine and ImM sodium pyruvate at day 0 with or without 20 mM GlcNAc.
- Cells numbers were counted by automated cell counting on a Cellometer Auto T4 (Nexcelom Bioscience) at day 2, day 4 and day 8.
- Mast cell proliferation in response to GlcNAc in the cell culture medium was also measured by BrdU incorporation using an APC BrdU Flow Kit from BD Bioscience following manufacturer instructions. Flow cytometry analysis was performed with BD FSRFortessaTM (Becton Dickinson, Franklin Fakes, NJ, USA). Propidium iodide staining was used to gate out the dead population. Degranulation assays
- PE Annexin V Kit BD Pharmingen
- mast cells in low glucose (1 g/L) IMDM complete medium in the presence or absence of GlcNAc 100 mM for 24 hours were stimulated for 30 minutes with IgE 0.5 pg/mL and DNP- OVA 0.2 pg/mL, before staining using a PE Annexin V Kit (BD Pharmingen) following the manufacturer’s instruction exactly.
- Cells were seeded in low glucose medium with or without GlcNAc 100 mM overnight at 37°C. Cells were then stimulated with IgE-anti-DNP 0.5 pg/mL and DNP-OVA 0.2 pg/mL for 3 hours. Brefeldin A 10 pg/mL was added in the last 2 hours of stimulation for inhibiting protein exocytosis. Cells were collected, washed in PBS, re-suspended in 100 pL of Cytofix/Cytoperm (BD PharmingenTM) and incubated 20 minutes on ice.
- Cytofix/Cytoperm BD PharmingenTM
- TNF-a release was carried out using the Mouse TNF-a High Sensitivity ELISA (eBioscience). Briefly, BMMC cells were incubated with or without GlcNAc 100 mM for 24 hours, then stimulated with IgE 0.5 pg/mL and DNP-OVA 0.2 pg/mL for 9 hours. 50 pL of each supernatant sample were added into the anti-mouse TNF-a coated micro-wells, followed by incubation with a biotin- conjugated and streptavidin-horseradish peroxidase (HRP) at RT for 2 hours and 1 hour, respectively.
- HRP biotin- conjugated and streptavidin-horseradish peroxidase
- Amplification reagent including biotinyl-tyramide and streptavidin-HRP was used to multiply the available HRP molecules for substrate reaction. 100 m ⁇ of substrate solution containing tetramethyl- benzidine was added at RT for 10 minutes. Reaction was terminated by adding 1M phosphoric acid. The absorbance values were measured at 450 nm using the FLUOstar OPTIMA microplate reader.
- Immunodetection was performed with an anti-OGlcNAc (RL2) (1:2000) (Abeam), an anti-OGT (1:1000) (Abeam), an anti-OGA (1:20000), or an anti-NAGK (1:10000) antibody overnight at 4°C.
- An anti-ERK2 antibody (1:20000) was used for loading controls.
- Incubation with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) was performed at RT 1 hour. Enhanced chemiluminescence and X-ray films were used to detect protein expression signals.
- PCA passive cutaneous anaphylaxis
- mice Twenty- four hours later, mice were injected intravenously with 0.5 mg DNP-OVA in 200 pi 0.5% Evans Blue dye (Sigma-Aldrich) in PBS. lh later, the mice were euthanized and ear sections were excised. The extravasated blue dye was then extracted from the tissues by incubation in form amide 63 °C overnight, and its intensity (correlated to the extent of extravasation) measured spectrophotometrically (OD 650). PCA response was quantified by the amount of dye leaked at IgE-injected sites. Data are expressed as Evans Blue dye in ng/mg tissue.
- mice (005557) were obtained from the Jackson Laboratory and bred in a pathogen-free environment at the animal facility of Cancer Research Center of Marseille (CRCM). Animal experiments were performed in accordance with guidelines established by the Institutional Animal Committee. lOxlO 6 ROSA KIT D816V-Gluc cells were injected to mice (2 groups, 5 mice per group) 24h after sublethally irradiation with 1.5 Gy of total body irradiation. Intraperitoneal (IP) injection of 300 mg/kg GlcNAc was administered 5 consecutive days per week for 7 weeks. In addition, treated animals had ad libitum access to drinking water supplemented with 50 mg/mL GlcNAc.
- IP Intraperitoneal
- GlcNAc has been used safely in multiple clinical trials; no adverse effects have been observed in previous chronic toxicity and carcinogenicity studies after administration of 300 mg/kg GlcNAc in dogs or even after administration of up to 2000 mg/kg/day GlcNAc. Engraftment was assessed after 4 weeks using quantitative measurement of Glue activity in plasma, and quantitative measurement of GFP+ or CFP+ cells in peripheral blood using flow cytometry (Bibi S et al., 2016). 24 Weight loss >20%, ruffled coat, hunched back, weakness, reduced motility, were monitored daily and considered endpoints. All experiments were performed in compliance with the laws and protocols approved by animal ethics committees (Agreement APAFIS N°6743-2016091513513606 v4).
- Glue activity was determined on 5 m ⁇ of plasma mixed with 100 m ⁇ of water soluble CTZ (50 gig/iTiL) followed by immediate acquisition of photon counts using a luminometer (Perkin Elmer) as previously described.
- Metabolomic alterations associated with SM severity are associated with Metabolomic alterations associated with SM severity.
- BMMCs BM-derived MCs
- ASM one type of AdvSM
- inventors filtered the entire human genome to select only metabolic transcripts, which constitute 12% of the human genome (3638 transcripts) and encode for known metabolic enzymes or transporters (Figure 2D).
- GlcNAc is a circulating predictive metabolite of SM aggressiveness.
- AdvSM patient samples showed significantly decreased dihydroxyacetone phosphate level (white dot), one of the major metabolites involved in glycolysis and markedly increased levels of GlcNAc, kynurenine, and quinolinate (black dots).
- GlcNAc is part of the hexosamine biosynthesis pathway that is responsible for protein glycosylation, and kynurenine/quinolinate are part of oxidative processes leading to the production of nicotinamide adenine dinucleotide (NAD).
- NAD nicotinamide adenine dinucleotide
- inventors measured absolute concentrations of GlcNAc by LC-MS/MS in an independent cohort of randomly selected KIT D816V samples from the first cohort mixed with new randomly selected KIT D816V patient samples from the CEREMAST database.
- the overall receiver-operating characteristic (ROC) curves for GlcNAc indicated that its AdvSM predictive value was high, with an area under the curve (AUC) of 0.97 for serum samples (Figure 3C). Notably, an AUC of 1.0 indicates perfect prediction, and an AUC of 0.5 indicates prediction equivalent to random selection.
- the concentration threshold to discriminate ISM from AdvSM was 3.8 mM, as calculated by the pROC package.
- GlcNAc concentration was significantly discriminant between patients with mast cell activation syndrome (MCAS) (2.7 mM ⁇ 0.4) and allergic and asthmatic patients combined (1.8 mM+0.2). Definitive criteria for MCAS diagnosis do not exist yet and GlcNAc could be a useful tool for distinguishing different forms of mast-cell related pathologies.
- SM ISM or AdvSM
- MCAS myelodysplastic and myeloproliferative syndrome
- E essential thrombocythemia
- Serum tryptase level is used as a minor criterion for SM as it also rises during anaphylaxis.
- GlcNAc levels were not correlated with any other chem ical parameters available for the samples in the cohort.
- inventors observed that high levels of GlcNAc were associated with AdvSM patients presenting gastrointestinal (GI) symptoms such as diarrhea, nausea, and abdominal pain due to MCs mediator release leading to a noticeable weight loss. Altogether these data indicate that GlcNAc is a circulating predictive metabolite of SM aggressiveness.
- GI gastrointestinal
- HBP hexosamine biosynthesis pathway
- GlcNAc fuels the hexosamine biosynthesis pathway (HBP) by the GlcNAc-salvage pathway, which is activated during glucose deficiency and has pro-survival effects (Figure 3E).
- HBP hexosamine biosynthesis pathway
- GlcNAc enters the HBP pathway by the GlcNAc kinase (NAGK) that is activated under metabolic stress conditions.
- GAGK GlcNAc kinase
- the HBP pathway activation leads to posttranslational modification of proteins by glycosylation, particularly O-GlcNAcylation of protein serine and/or threonine residues by O-linked GlcNAc transferase (OGT).
- O-linked GlcNAc transferase O-linked GlcNAc transferase
- b-N-acetylglucosaminidase is responsible for the reverse reaction.
- protein extracts from BM aspirate were immunoblotted with an RL2 antibody that recognizes O-GlcNAc moieties on proteins.
- Three patient samples per group were assessed: ISM, AdvSM and MC activation syndrome (MCAS), a syndrome characterized by MC activation without clonal expansion of MCs. Variations in band intensities were noted, but all SM samples (ISM and AdvSM) exhibited evidence of increased O- GlcN Acylation compared to samples from patients with MCAS.
- Inventors’ data showed that increased levels of GlcNAc in the plasma of patients with AdvSM were associated with the activation of the enzymes of the HBP pathway at the transcriptional level in sorted patient KIT D816V MCs and to high protein O-GlcNAcylation levels in patient’s BM aspirate enriched in MCs.
- GlcNAc exacerbates the severity of a KIT D816V+ SM phenotype.
- GlcNAc As high levels of GlcNAc are correlated to AdvSM, inventors tested the hypothesis that GlcNAc promotes proliferation of MCs carrying KIT D816V mutation. Human ROSA KIT D816v MCs incubated with GlcNAc showed significantly higher proliferation rates than their non-treated counterparts. GlcNAc had no effect on ROS A KIT WT cell proliferation during the course of the experiment. Assessment of BrdU incorporation confirmed these observations. The absence of an effect of GlcNAc on KIT WT MCs proliferation was not due to an inability of GlcNAc to enter the cells. Indeed, inventors found that GlcNAc was able to activate the HBP, as assessed by western blot analysis of O-GlcNAcylation levels.
- ROSA KIT D816v MCs have been reported to efficiently engraft in NOD/SCID IL-2Ry ; (NSG) mice, giving rise to an ASM/MCL-like disease in vivo.
- NSG NOD/SCID IL-2Ry ;
- Glue Gaussia princeps luciferase
- GlcNAc increases MC susceptibility to IgE-mediated stimulation.
- BMMC murine bone marrow-derived MCs
- FcsRI FcsRI
- MCs treated with GlcNAc showed a significant increased ability to degranulate, as indicated both by the release of b- hexosaminidase (an enzyme normally stored in cytoplasmic granules) into the culture supernatant and an increase of Annexin V staining as Annexin V levels increase at sites of secretory granule fusion with the plasma membrane during degranulation.
- b- hexosaminidase an enzyme normally stored in cytoplasmic granules
- Annexin V staining as Annexin V levels increase at sites of secretory granule fusion with the plasma membrane during degranulation.
- the production of TNF-a, IL6 and IL-13 was also increased in GlcNAc- treated KIT D814V BMMC, as assessed both by intracellular staining and ELISA for TNF-a.
- Ear MCs of Kit D814V/flox -Cre mice pretreated with GlcNAc (or PBS) by intradermal injection were sensitized with anti-DNP IgE antibodies before systemic injection of antigen together with Evans blue dye.
- inventors quantified the cutaneous extravasation of the dye after extraction from the tissue. In agreement with the increased responses observed in GlcNAc-treated MCs in vitro, they observed significantly enhanced responses of GlcNAc-treated MCs during acute response in vivo. Overall, inventors’ data indicate that GlcNAc exacerbates MC responses.
- GlcNAc as a biomarker for diagnosing a disease induced by mast cell activation.
- the level of concentration of GlcNAc in plasma sample of a subject allows to classify patients according to the disease they are affected with, in particular, it is possible to discriminate between mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis (CM); mast cell activation syndrome (MCAS); asth a and allergy.
- SM systemic mastocytosis
- MCS mast cell sarcoma
- CM mast cell activation syndrome
- asth a and allergy SM
- SM is a heterogeneous disease
- inventors have been able to demonstrate that plasma metabolic profiling was a sufficiently sensitive means to discriminate among SM for s differing according to levels of aggressiveness.
- the correlation between the concentration of circulating GlcNAc and SM severity indicates that plasma GlcNAc detection is an efficient noninvasive approach by which to identify patients at greater risk of disease progression, thereby alerting physicians for the need for closer follow-up or the pursuit of early interventional therapy in these “high-risk” indolent cases.
- the absence of correlation the inventors observed between GlcNAc and tryptase concentrations in plasma suggests that GlcNAc levels are not dependent upon mast cell activation.
- GlcNAc is helpful to identify patients with modestly increased concentration of tryptase that are likely to have an advanced SM phenotype.
- biomarkers have been identified as being secondary indicators for diagnosing a disease induced by mast cell activation. These biomarkers belong to pathways involved in nicotinate/nicotinamide metabolism, pyrimidine metabolism, purine metabolism, glycerophospholipid metabolism, amino sugar and nucleotide sugar metabolism; arginine biosynthesis, and one carbon pool by folate metabolism.
- Inventors demonstrate that a variation in the transcript level of one or more enzymes involved in the hexosamine biosynthesis pathway (HBP) in mast cells, such as GFAT1, PGM3, NAGK, OGT and OGA can be considered as secondary indicators for diagnosing a disease induced by mast cell activation.
- HBP hexosamine biosynthesis pathway
- Inventors developed noninvasive methods using GlcNAc for diagnosing a disease induced by mast cell activation or for assessing the severity of a disease induced by mast cell activation; for monitoring the evolution of a disease induced by mast cell activation; or for determining the efficacy of a therapy of a disease induced by mast cell activation.
- Greiner G, Witzeneder N, Berger A, et al. CCL2 is a KIT D816V-dependent modulator of the bone marrow microenvironment in systemic mastocytosis. Blood. 2017;129:371-382. Hanssens K, Brenet F, Agopian J, et al. SRSF2-p95 hotspot mutation is highly associated with advanced for s of mastocytosis and mutations in epigenetic regulator genes. Haematologica. 2014;99:830-5.
- Metcalfe DD Mast cells and mastocytosis. Blood. 2008;112(4):946-956. Metz M, Maurer M. Mast cells— key effector cells in immune responses. Trends Immunol. 2007;28:234-41.
- Pardanani A Systemic mastocytosis in adults: 2019 update on diagnosis, risk stratification and management. Am J Hematol. 2019;94:363-7.
- Tefferi A Fevine RE, Fim K-H, et al. Frequent TET2 mutations in systemic mastocytosis: clinical, KITD816V and FIP1F1-PDGFRA correlates. Feukemia. 2009;23(5):900-904.
Abstract
The invention relates to the field of medicine and diagnostic. Inventors herein describe the use of GlcNAc as a biomarker of a disease induced by mast cell activation. Methods are also described for diagnosing a disease induced by mast cell activation; for assessing the severity a disease induced by mast cell activation; for monitoring the evolution a disease induced by mast cell activation; or for determining the efficacy of a therapy of a disease induced by mast cell activation. Kits are also described as well as uses thereof.
Description
N-ACETYL-D-GLUCOSAMINE AS A DIAGNOSTIC BIOMARKER OF A DISEASE INDUCED BY MAST CELL ACTIVATION
FIELD OF THE INVENTION
The field of the present invention is that of medicine and diagnostic, in particular diagnostic of a disease induced by mast cell activation. The invention more particularly relates to N-acetyl-D-glucosamine (GlcNAc) and uses thereof as a biomarker in the diagnosis of a disease induced by mast cell activation. The invention also relates to methods of assessing the severity of a disease induced by mast cell activation, of predicting or monitoring the evolution of a disease induced by mast cell activation, of determining the efficacy of a therapy of a disease induced by mast cell activation in a subject, as well as kits and their uses for implementing these methods.
PRIOR ART
The mast cell is a tissue resident granulocyte, active in the allergic response but also playing a vital role in the innate immune response, immune tolerance, wound healing and angiogenesis. Mast cell may be activated by both IgE-dependent and IgE-independent mechanisms. Once activated, it releases preformed mediators stored in granules, including histamine, heparin, proteases (e.g., tryptase), de novo synthetized arachidonic acid metabolites (e.g., prostaglandin D2 and leukotriene C4); as well as cytokines (e.g., TNFa) and chemokines.
The disorders induced by the activation of mast cell can be highly varied in their presentation, depending on the affected organ. Common manifestations of mast cell activation are “allergic type” phenomena, such as for example asthma, hives or rhinitis. More heterogeneous disorders are also induced by abnormal or excessive mast cell activation.
The measurement of the mast cell rate in tissues allows the discrimination of disorders into two distinct main groups. The first group is characterized by inappropriate activation of mast cells without proliferation, and the second by an accumulation of mast cells.
The mast cell activation syndrome (MCAS) belongs to the first category identified above. It is a condition in which the patient experiences repeated episodes of the symptoms of anaphylaxis and allergic symptoms such as tachycardia, hypotension, syncope, itching hives, swelling, flushing, wheezing, difficult breathing, nausea with abdominal pain and severe diarrhea. High levels of mast cell mediators are released during those episodes. Increases in serum mast cell tryptase and in urine levels of N-methylhistamine, 1 IB -Prostaglandin F2a (1 lB-PGF2a) and/or Leukotriene E4 (LTE4) are the only tests useful for the diagnosis of MCAS. However, serum mast cell tryptase should be drawn between 30
minutes and two hours after the start of an episode, and the urine tests are to be performed on a 24-hour collection of urine that is started immediately (i.e., at the beginning of an episode).
Unlike MCAS, mastocytosis results from a clonal, neoplastic proliferation of morphologically and immunophenotypically abnormal mast cells (MCs) that accumulate in one or more organs. Mastocytosis includes cutaneous mastocytosis, mast cell sarcoma (MCS) and systemic mastocytosis (SM).
Systemic mastocytosis (SM) encompasses the 5 categories of SM defined in 2016 by the World Health Organization according to their location and aggressiveness: indolent SM (ISM), smoldering SM (SSM), aggressive SM (ASM), SM with an associated hematological neoplasm (SM-AHN), and mast cell leukemia (MCL). The latter three sub-classifications are associated with reduced overall survival and are grouped together as advanced SM (AdvSM). ISM and SSM are grouped together as non-advanced SM (non-Adv SM).
Systemic mastocytosis is a KIT-driven hematopoietic neoplasm characterized by the accumulation of abnormal neoplastic mast cells (MCs) in various organs and, mainly, the bone marrow (BM) (Metcalfe DD, 2008; Metz M et al., 2007; Siraganian RP, 2003). SM pathology ranges from benign chronic manifestations to very aggressive tumors with limited therapeutic options available to date (Gilreath JA et al. 2019; Hartmann K et al., 2020). Indeed, patients with indolent SM (ISM) have symptoms related primarily on mast cells (MC) mediators release and exhibit a normal life expectancy (Escribano L et al, 1998); in contrast, in patient with forms of advanced SM (AdvSM), the clinical course is aggressive, with organ impairment and failure related to excessive MCs infiltration and mediators release, leading to a shortened life expectancy (Pardanani A, 2019; Valent P et al, 2017; Georgin-Lavialle S et al, 2013).
To date, little is known regarding the mechanisms contributing to the malignant expansion of neoplastic MCs and their progenitors in patients with AdvSM. The KIT D816V mutation is identified as a major cause of the disease and is associated with most clinical forms of SM (Bibi S et al, 2014; Garcia- Montero AC, 2006; Garcia-Montero AC et al., 2016; Kitamura Y and Hirotab S, 2004). Possible explanations for the diverse clinical presentations include the co-occurrence of KIT D816V together with secondary genetic lesions (Schwaab J et al., 2013; Jawhar M et al., 2016; Tefferi A et al., 2009; Hanssens K et al., 2014), epigenetic (De Vita S et al., 2014; Leoni C et al., 2015; Leoni C et al., 2017; Lyberg K et al., 2017; Martinelli G et al., 2018; Soucie E et al., 2012; Wedeh G et al., 2015) and/or microenvironment alterations (Gangemi S et al. , 2015 ; Greiner G et al., 2017; Hoermann G et al., 201 1 ; Mayado A et al., 2016; Tobio A et al., 2020; Mueller N et al., 2018) that modify the disease phenotype in a given SM case.
Reprogramming of cellular metabolism occurs as a direct consequence of not only oncogenic mutations but also environmental cues that affect the abundance of oncometabolites by eliciting oncogenic cascades (Collins RRJ et al., 2017; Khatami F et al., 2019; Sciacovelli M and Frezza C, 2016; Yang M
et al., 2013). Oncometabolites are conventional metabolites that, when aberrantly accumulated, have pro-oncogenic functions mainly via epigenetic dysregulation (Yang M et al., 2013; Morin A et al., 2014; Yong C et al., 2020).
Thus, multiple genetic and epigenetic mechanisms contribute to the onset and severity of SM; however, little is known to date about the metabolic underpinnings underlying SM aggressiveness. This has impeded the development of strategies to leverage metabolic dependencies when existing KIT-targeted treatments fail.
The major diagnostic criterion for SM is the presence of multifocal clusters of morphologically abnormal mast cells in the bone marrow. Minor diagnostic criteria include elevated serum tryptase level, abnormal mast cell expression of CD25 and/or CD2, and presence of KITD816V mutation. Established advanced systemic mastocytosis (AdvSM) is characterized by organ damage due to infiltration of mast cells.
Given the extraordinary array of MC mediators and of their effects which may be direct or indirect, local or remote, acute, or delayed and chronic in cells/tissues/organs/systems throughout the body, diseases induced by mast cell activation belong to an area especially prone to misdiagnosis. Underdiagnosis and overdiagnosis are both problematic for patients, typically delaying or preventing patients from accessing effective treatment. Thus, there is a need for new diagnostic tools and procedure options that are fast, efficient, and able to distinguish SM from other diseases induced by mast cell activation.
Inventors now herein advantageously provide simple to implement methods of diagnosing, assessing the severity, predicting and monitoring the evolution, of a disease induced by mast cell activation in a subject.
SUMMARY OF THE INVENTION
Inventors herein identify for the first time N-acetyl-D-glucosamine (GIcNAc) as a particularly interesting biomarker of diseases induced by mast cell activation. They have developed a simple tool for discriminating between several diseases associated with, typically induced by, mast cell activation. As a non-limiting example, the present invention is useful for classifying a sample issued from a subject as being associated to mastocytosis (in particular systemic mastocytosis), mast cell activation syndrome (MCAS), asthma or allergy.
The present invention thus relates to the use of N-acetyl-D-glucosamine (GIcNAc) as a biomarker of a disease induced by mast cell activation, the disease being preferably selected from mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis (CM); mast cell activation syndrome (MCAS); asthma; and allergy.
Also described herein is an in vitro or ex vivo method for diagnosing a disease induced by mast cell activation in a subject. This method typically comprises the steps of:
(i) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject; and
(ii) diagnosing a disease induced by mast cell activation in the subject if the concentration of GlcNAc as measured in step (i) is above a reference GlcNAc ’s concentration.
The method of the invention enables classifying a diagnosed systemic mastocytosis (SM) in a subject, in particular into a non-advanced SM (non-AdvSM) or advanced SM (AdvSM), more particularly into indolent SM (ISM), smoldering SM (SSM), SM with an associated hematological neoplasm (SM-AHN), aggressive SM (ASM), mast cell leukemia (MCL), or even more particularly into indolent SM (ISM) or aggressive SM (ASM).
Inventors in particular herein provide an in vitro or ex vivo method for assessing the severity of a disease induced by mast cell activation in a subject, preferably systemic mastocytosis (SM), in particular for differentially diagnosing between non-advanced SM (non-AdvSM) and advanced SM (AdvSM) in a subject, more particularly between indolent SM (ISM) and i) aggressive SM (ASM), ii) SM with an associated hematological neoplasm (SM-AHN) or iii) mast cell leukemia (MCL), and wherein the method typically comprises:
(a) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject;
(b) comparing the concentration of GlcNAc in the biological sample of the subject with a reference concentration of GlcNAc and correlating the concentration of GlcNAc in the biological sample with the severity of the SM in the subject.
Inventors in particular herein provide an in vitro or ex vivo method for assessing the severity of SM, wherein the reference control sample is a sample obtained from a healthy subject, and wherein a concentration of GlcNAc in the biological sample of the subject that is 6 to 8 times greater than the concentration of GlcNAc in the reference control sample is indicative of non-AdvSM, in particular indolent SM (ISM); and a concentration of GlcNAc in the biological sample of the subject that is more than 8-fold greater than the concentration of GlcNAc in the reference control sample is indicative of advanced SM (AdvSM), in particular aggressive SM (ASM) or SM with an associated hematological neoplasm (SM-AHN).
Inventors in particular herein provide an in vitro or ex vivo method for assessing the severity of SM, wherein the biological sample is preferably a fluid sample as herein described below, wherein a concentration of GlcNAc in the biological sample of the subject that is equal to or above the reference concentration of GlcNAc is indicative of AdvSM, in particular ASM or SM-AHN, and a concentration
of GlcNAc in the biological sample of the subject that is below the reference concentration of GlcNAc is indicative of non-AdvSM, in particular ISM.
Further herein described is an in vitro or ex vivo method for assessing/predicting the risk for a subject affected or diagnosed with non-AdvSM, in particular ISM, to develop AdvSM, in particular ASM or SM-AHN, wherein the method comprises a step of determining the concentration of GlcNAc in a biological sample obtained from a subject affected or diagnosed with non-advanced SM, in particular ISM, wherein a concentration of GlcNAc above a reference concentration of GlcNAc indicates that the subject is at risk to develop AdvSM, in particular ASM or SM-AHN.
Also herein described is an in vitro or ex vivo method for monitoring the evolution of a disease induced by mast cell activation which is preferably selected from mastocytosis, mast cell sarcoma (MCS), mast cell activation syndrome (MCAS), asthma and allergy, and even more preferably systemic mastocytosis (SM). Typically, the method comprises the steps of:
(a) measuring the concentration of GlcNAc in a sample obtained from a subject at To;
(b) measuring the concentration of GlcNAc in a distinct sample obtained from said subject at Ti, Ti being posterior to To; and
(c) comparing the concentrations of GlcNAc as measured in steps (a) and (b) to monitor the evolution of the disease in the subject, wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative of a favorable evolution of the disease in the subject, a concentration of GlcNAc as measured in step (b) identical to the concentration of GlcNAc as measured in step (a) is indicative of a stabilization of the disease in the subject, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative of an unfavorable evolution of the disease in the subject.
This in vitro or ex vivo method for monitoring the evolution of a disease induced by mast cell activation in a subject can in particular be implemented for monitoring SM evolution, for example in a subject affected with non-AdvSM, in particular indolent SM (ISM) or smoldering SM (SSM).
Inventors further herein describe an in vitro or ex vivo method for determining the efficacy of a therapy of a disease induced by mast cell activation which is preferably selected from mastocytosis, mast cell sarcoma, mast cell activation syndrome (MCAS), asthma and allergy, and even more preferably systemic mastocytosis (SM), in a subject affected with said disease. Typically, the method comprises the steps of:
(a) measuring the concentration of GlcNAc in a sample obtained from a subject prior to the administration of a therapy of a disease induced by mast cell activation;
(b) measuring the concentration of GlcNAc in a sample obtained from a subject once started the administration of a therapy of a disease induced by mast cell activation; and
(c) comparing the concentrations of GlcNAc as measured in steps (a) and (b), wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative that the therapy is effective in the treatment of the disease induced by mast cell activation, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative that the therapy is not effective in the treatment of the disease induced by mast cell activation; or, alternatively, wherein the method comprises the steps of:
(i) measuring the concentration of GlcNAc in a sample obtained from a subject once started the administration of a therapy of a disease induced by mast cell activation; and
(ii) comparing the concentration of GlcNAc as measured in step (i) with a reference concentration of GlcNAc, wherein, a concentration of GlcNAc as measured in step (i) below the GlcNAc reference concentration is indicative that the therapy is effective in the treatment of the disease induced by mast cell activation, and a concentration of GlcNAc as measured in step (i) above the GlcNAc reference concentration is indicative that the therapy is not effective in the treatment of the disease induced by mast cell activation.
This in vitro or ex vivo method for determining the efficacy of therapy may further comprises a step of determining, in a biological sample from the subject, the transcript level of one or more enzymes involved in the hexosamine biosynthesis pathway (HBP), such as GFAT1, PGM3, NAGK, OGT and OGA.
The methods herein described are performed on a biological sample obtained from a living subject, typically from a mammal for example from a human subject or a domestic animal, in particular a pet. A particular subject is a subject having a mutated kit gene or expressing a mutated KIT receptor.
The biological sample is preferably a biological fluid sample, preferably selected from a blood, plasma, serum, urine and bone marrow aspirate sample; or a solid sample, preferably a bone marrow biopsy sample.
Inventors also herein describe a kit for measuring the concentration of GlcNAc in a biological sample of a subject. The kit comprises an anti-O-linked N-acetylglucosamine (O-GlcNAc) binding agent, for example RL2 monoclonal antibody, a molecule allowing the binding agent detection; or a N-Acetyl-D- Glucosamine Kinase (NAGK). Optionally, the kit also comprises a leaflet providing GlcNAc reference concentration(s).
The present disclosure also relates to the use of a kit as herein described on a biological sample obtained from a subject, for diagnosing a disease induced by mast cell activation, preferably selected from mastocytosis, mast cell activation syndrome (MCAS), asthma and allergy, assessing the severity of systemic mastocytosis; assessing/predicting the risk for a subject affected or diagnosed with non- AdvSM to develop AdvSM, preferably prior to the onset of AdvSM’s symptoms; monitoring or
predicting the evolution of a disease induced by mast cell activation; and/or monitoring the efficacy of a therapy of a disease induced by mast cell activation, in the subject.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Metabolomic profiling of systemic mastocytosis (SM).
(A) Clinical spectrum of patients with SM. (B) Schematic representation of the untargeted metabolomic approach used to identify metabolite signatures in SM plasma samples. (C) Z-score scatterplots of plasma metabolites from indolent and advanced SM patients. Each data point is expressed as the number of standard deviations (SDs) from the mean of the controls (healthy donors). The y-axis shows all values for each metabolite on the same horizontal line. The z-scores are the SDs from the control mean, revealing the changes relative to the healthy donor group. (D) Z-score scatterplots present results similar to those in (C) but relative to the indolent SM group. (E) Principal component analysis (PCA) of metabolites measured in indolent (white dots and advanced (black dots) SM plasma samples.
Figure 2: Metabolomic alterations associated with systemic mastocytosis (SM) severity.
(A) Indolent SM-based z-score plot of 59 metabolites significantly deregulated between samples from patients with indolent and AdvSM. Each dot represents one metabolite in one SM sample, and the color indicates the disease form (gray, indolent; black, advanced) (B) Metabolome view from pathway analysis performed using MetaboAnalyst 4.0. Each circle represents a different pathway, and the circle size and the circle marked with numbers indicate the pathway impact and p-value respectively. Selected pathways with high impact and/or high p-values are labeled. (C) Pathway analysis performed using the HumanCyc database showing the smallest set of pathways that includes the differential metabolites between ISM and AdvSM. (D) Outline of the transcriptomic approach used to highlight significant dysregulated metabolic transcripts in sorted bone marrow mast cells (MCs) from SM patients. (E) Enrichment of over-activated pathways between different groups of SM patients, as identified by Enrich. The adjusted p-values are shown as -log (p) values on the x-axis.
Figure 3: N-acetyl-D-glucosamine (GlcNAc) is a biomarker of advanced systemic mastocytosis (AdvSM).
(A) Volcano plot of statistical significance against fold change between the indolent and advanced forms of SM showing the most significantly differentially regulated metabolites linked to disease severity. (B) GlcNAc levels in SM-related plasma (n=20, 10 Indolent SM and 10 Advanced SM), MCAS (n=5), in allergic (n=5) and asthmatic patients (n=5) and in healthy donor (HD) plasma (n=5) measured by Liquid Chromatography-Mass Spectrometry (LC-MS/MS). (C) Receiver operating characteristic (ROC) curve of GlcNAc concentrations between indolent SM (ISM) and advanced SM (ASM) patient samples. (D) GlcNAc and tryptase levels in SM patient samples. (E) Schematic representation of the hexosamine
biosynthetic pathway (HBP) and the use of GlcNAc in the salvage pathway indicating relevant enzymes [O-linked-GlcNAc transferase (OGT), the O-GlcNAcase OGA and GlcNAc kinase (NAGK)]. (F) Lysates from three bone marrow biopsies from patients with mast cell (MC) activation syndrome (MCAS), ISM and ASM were immunoblotted with RL2 antibodies that detect O-GlcNAcylation (G) Quantification of the immunoblot shown in (F) by ImageJ software. (H) Normalized transcript levels of HBP enzymes from microarray analysis performed in MCs sorted from bone marrow samples from healthy donors (HD) (n=5) and patients presenting ISM (n=14) or ASM (n=12) disorders. The values in the graphs are presented as the means ± SDs (non-significant, *p<0.05, **p<0.01, ***p<0.001; unpaired t test, two tailed).
Figure 4: N-acetyl-D-glucosamine (GlcNAc) exacerbates the severity of an advanced systemic mastocytosis (ASM) phenotype.
(A) The proliferation of human MCs, ROSA KIT WT and ROSA KIT D816V cells, was assessed after treatment with or without GlcNAc, and the cells were counted using a Cellometer Auto T4 at days 0, 2, 4 and 8. (B) Schema of in vivo administration of GlcNAc to ROSA D816-Gluc transplanted mice after irradiation with 1.5 Gy (n=10, 5 per group). (C) Measure of GlcNAc activity (in relative luciferase units, RLU) in PBS- or GlcNAc-treated mice from week 3 (W3) to week 7 (W7). (D) Number of Gluc-i- cells circulating in the plasma of mice from W3 to W7, as measured by FACS. (E) Number of GFP+ and CFP+ cells in the bone marrow of PBS- or GlcNAc-treated mice, as measured by FACS. (F) Number of GFP+ and CFP+ cells in the spleens of PBS- or GlcNAc-treated mice, as measured by FACS. (G) Weights of spleens at W7 in PBS or GlcNAc-treated mice. The values in the graphs are presented as the means ± SDs (non-significant, *p<0.05, **p<0.01, ***p<0.001; unpaired t test, two tailed).
Figure 5: N-acetyl-D-glucosamine (GlcNAc) increases mast cell (MC) susceptibility to IgE- mediated stimulation.
(A) Degranulation of bone marrow derived MC (BMMC) KITD814V after treatment with or without GlcNAc was measured by assessment of beta-hexosaminidase release (n=3). Cells not stimulated with DNP-OVA or ionomycin were considered negative controls, whereas ionomycin stimulation was used as a positive control for MC degranulation ability. (B) Degranulation of MCs was measured by annexin V staining upon stimulation with IgE and antigen complexes for 30 min. (C) Intracellular staining (left histogram) or release (right histogram) of TNF-a in MCs stimulated with IgE and antigen incubated with PBS or GlcNAc. (D) Same as C for intracellular IL-13. (E) Same as C for intracellular IL-6. (F) Schematic representation of the experimental design for passive cutaneous anaphylaxis. (G) Acute dye extravasation was measured by assessment of the optical density (OD) at 650 nm (n=20 in each group). The values in the graphs are presented as the means ± SDs (non-significant, *p<0.05, **p<0.01, ***p<0.001; unpaired t test, two tailed).
Figure 6: N -acetyl-D-glucosamine (G!cNAc) is a biomarker of diseases induced by mast cells but not of other non-mast cells-related myeloproliferative disorders.
Plasmatic GlcNAc levels measured by LC-MS/MS in patients suffering from a disease induced by mast cells activation [n=20 (10 Indolent SM and 10 Advanced SM), MCAS (n=5), allergic (n=5), asth atic (n=5)]; in healthy donor (HD) (n=5); and in patients suffering from non-mast cells-related myeloproliferative disorders [acute leukemias (n=9), chronic myeloid leukemia (CML; n=10), myelodysplastic and myeloproliferative syndrome (MDS/MNP; n=7), and essential thrombocythemia (ET) (n=10)].
DETAILED DESCRIPTION OF THE INVENTION
Knowing the diversity of mediators, metabolites, cytokines and chemokines released by activated mast cells, identifying and selecting relevant biomarkers allowing the diagnosis, in a patient, of a disease induced by mast cell activation is a challenge. Identifying sub-groups of diseases induced by mast cells is even more complex.
Inventors herein advantageously describe efficient methods allowing the diagnosis of a disease induced by mast cell activation as well as kits for implementing said methods. The present invention is based on the discovery that N-acetyl-D-glucosamine (GlcNAc) is a powerful tool when used as a biomarker for the diagnosis of a disease induced by mast cell in a subject. In particular, inventors have shown that GlcNAc makes it possible to discriminate between healthy subjects and subjects suffering from a disease induced by the activation of mast cells. Interestingly, they were also able to demonstrate that GlcNAc can discri inate between subjects suffering from a disease induced by mast cell activation and subjects suffering from other myeloproliferative disorders not related to mast cell such as acute myeloid leukemias, chronic myeloid leukemia, myelodysplastic and myeloproliferative syndrome, and essential thrombocythemia. Inventors also discovered that by determining the concentration of GlcNAc in a biological sample of a subject, it is possible to identify the particular disease induced by mast cell activation the subject is suffering of or is prone to develop.
Herein described for the first time is the use of N-acetyl-D-glucosamine (GlcNAc) as a biomarker of a disease induced by mast cell activation. The disease is preferably selected from mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis; mast cell activation syndrome (MCAS); asthma; and allergy.
N-acetyl-D-glucosamine (GlcNAc), also identified in the art as 2-acetamino-2-deoxy- -D-glucose or 2- (acetylamino)-2-deoxy-D-glucose, is a monosaccharide that usually polymerizes linearly through (1,4)- b-linkages. The molecular formula of this amino monosaccharide is CsHisNOe, and its molecular weight is 221.21. In general, it is a white and slightly sweet powder that melts at 221 °C. The solubility of GlcNAc is 25% in water, and 1% aqueous solutions are colorless and clear. GlcNAc is the monomeric
unit of the polymer chitin, the second most abundant carbohydrate after cellulose. In addition to being a structural component of homogeneous polysaccharides like chitin, GlcNAc is also a constituent of heterogeneous polysaccharides. In mammals, GlcNAc is a component of glycoproteins, proteoglycans, glycosaminoglycans (GAGs) and other connective tissue building blocks.
As used herein, the term “biomarker” refers to a biological molecule whose presence and/or concentration can be detected and correlated typically with a known condition. In the context of the present invention, the biomarker can be used to classify a sample from a subject as a “disease induced by mast cell activation” sample, to associate [i.e. (sub-)classify] such a sample for example with one of SM various forms or clinical subtypes, to assess the severity of the disease or disorder associated with mast cell activation, and/or to assess the efficiency of a treatment in a sick subject or in a subject diagnosed with a disease induced by mast cell activation, or in the context of preventive treatment in a subject suspected of being, or identified as, at risk of developing such a disease or disorder. Appropriate (secondary) diagnostic markers suitable for use in the present invention, in addition to GlcNAc, are identified below.
Mast cell are of hematopoietic lineage. Mast cells reside close to blood vessels, nerves and lymphatic ducts in tissues that interface with the external environment (e.g., airways, gastrointestinal tract, uterus, skin). Mast cells enter the circulation as progenitors rather than mature cells. At this stage, progenitor cells in human subjects can express both FceRI and KIT (CD117). In human subjects, immediate mast cell precursors from blood were recently defined as Lin CD34hlCDl 17mt/hlFcsRI+ cells. In response to a variety of stimuli including CNS derived agents and physical stressors, mast cells initiate inflammatory site formation through three coupled sets of events: (1) release of pre-formed cytoplasmic granules containing vasoactive amines such as histamine and serotonin, proteases such as chymase and tryptase, proteoglycans such as herapin (2) de novo biosynthesis of bioactive lipid mediators such as LTB4, LTC4, PAF, PGD2 and PGE2 and (3) de novo transcriptional activation of genes coding for a range of cytokines and chemokines such as II- 1 b, 11-4, IL-6, CXCL8, IL-10, CCL2, TNF-oc, and growth factors such as FGF-2, PDGF, TGF-bI and VEGF. Mediators can be secreted from mast cells through the release of granules or the release of secretory vesicles.
By “disease induced by mast cell activation” is meant any condition or disorder that is caused by inappropriate, in particular excessive, release of mediators by mast cells (that occurs during mast cell degranulation). As apparent in the all description, the expression “disease induced by mast cell activation” encompasses mastocytosis, mast cell activation syndrome (MCAS), asthma, and allergy.
By “mastocytosis” is meant any condition that is caused by clonal proliferation of mast cells, with an abnormal accumulation of these cells in tissues including the skin, bone marrow and gastrointestinal tract. Although mastocytosis is associated with an increased number of mast cells, its presentation is not necessarily accompanied by symptoms of mast cell activation. As apparent in the all description, the
expression “mastocytosis” encompasses systemic mastocytosis (SM), mast cell sarcoma (MCS) and cutaneous mastocytosis (CM).
In “cutaneous mastocytosis” (CM), the excessive production of mast cells in the skin leads to an increased release of cytokines and histamines, which lead to itching and skin lesions. Cutaneous mastocytosis in the skin has several different patterns: urticaria pigmentosa, diffuse cutaneous mastocytosis, or isolated mastocytoma. CM is usually benign and self-limiting in time.
Mast cell sarcoma (MCS) is a rare, neoplastic disease characterized by locally destructive sarcoma-like growth of a solitary mass, composed of atypical mast cells, and without systemic involvement. It can affect any organ and the symptoms depend on the location. Cells are medium to large, pleomorphic or epithelioid, with oval, bilobed or multilobulated nuclei, sometimes prominent multinucleated giant cells. The disease closely resembles other neoplasms and may share associated markers, however the tumor is positive for mast cell tryptase.
Systemic mastocytosis (SM) is a more aggressive variant of mastocytosis with extra-cutaneous involvement that may be associated with multiorgan dysfunction/failure and shortened survival. Unlike CM, which is mainly present during childhood, SM is generally seen in adult patients.
“Mast cell activation syndrome” (MCAS) designates a severe constellation of symptoms within the broader group of disorders induced by mast cell activation. The following cumulative criteria are associated to MCAS: i) episodic multisystem symptoms consistent with mast cell activation, ii) appropriate response to medications targeting mast cell activation and iii) documented increase in validated systemic markers of mast cell activation (which may be detected in serum or urine) during a symptomatic period compared with the patient’s baseline values. Clinically available and validated markers of MCAS are tryptase, urinary histamine metabolites, urinary prostaglandin D2, urinary leukotriene E4 and metabolites thereof (Cem Akin, J Allergy Clin Immunol 2017;140:349-55).
The term “asthma” refers to airway hyperactivity and reversible airways obstruction. Pathological derangements at the tissue level include constriction of airway smooth muscle, increased vascular permeability resulting in edema of airways, outpouring of mucus from goblet cells and mucus glands, parasympathetic nervous system activation, denudation of airway epithelial lining cells, and influx of inflammatory cells. Underlying these tissue effects are direct effects of potent mediators secreted following physical, inflammatory, or immunological activation and degranulation. The early phase of the asthmatic reaction is mediated by histamine and other mast cell mediators that induce rapid effects on target organs and tissues, particularly smooth muscle. The pathophysiologic sequence of asthma is initiated by mast cell activation in response to allergen binding to IgE.
The term “allergy” designates any condition or disorder characterized by the undesirable, in particular excessive, release of histamine by mast cells.
Several biomarkers have been identified as markers of mast cells activation. Some of these markers such as tryptase, urinary N-methylhistamine, leukotriene E4 (LTE4), prostaglandin F2 alpha (PGF2oc), prostaglandin D2, markers of oxidative stress and cytokines (IL-6, IL-31) are currently used as minor markers for diagnosis of a disease induced by mast cell activation, in particular of asthma, allergy, MCAS and non-AdvSM. Tryptase diagnostic specificity for example is limited by the presence of high tryptase levels in severe allergic reaction. In this regard, plasmatic or serum tryptase levels are dependent on mast cell degranulation resulting from mast cell activation. Therefore, high tryptase levels can only be detected when patient blood sampling occurs close to an episode of mast cell degranulation. However, an erroneous measure of tryptase level can lead to misdiagnosis or underdiagnosis of a disease induced by mast cell activation. Moreover, the measurement of three of the above-mentioned minor markers is required to diagnose a systemic mastocytosis.
Inventors now advantageously identify biomarkers that are not dependent of an episode of mast cell activation leading to mast cell degranulation. Moreover, inventors develop tools and methods capable of discriminating between diseases induced by mast cell activation, in particular between non-advanced SM (non-AdvSM) and advanced-SM (AdvSM).
Inventors herein describe an in vitro or ex vivo method for diagnosing a disease induced by mast cell activation, preferably selected from mastocytosis, mast cell activation syndrome (MCAS), asthma and allergy, in a subject. This method preferably comprises the steps of:
(i) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject; and
(ii) diagnosing a disease induced by mast cell activation, preferably selected from mastocytosis, mast cell activation syndrome, asthma and allergy, in the subject if the concentration of GlcNAc as measured in step (i) is above a reference GlcNAc’ s concentration.
The terms “diagnostic” and “diagnosing” refer to the detection or identification of a pathology, disease, disorder or dysfunctional state as herein defined, typically a disease induced by mast cell activation, or to the evaluation/assessment (dosing, comparison) of the severity or of the stage of such a pathology, disease, disorder or dysfunctional state in a subject.
The term “subject” refers to any testable subject and typically designates a patient. The subject is any member of the animal kingdom, preferably a mammal. In a particular aspect, the subject is a domestic animal, in particular a pet, such as for example a dog or a cat. A particular domestic animal is an animal suspected of suffering of, or diagnosed as suffering of, mast cell sarcoma (MCS). In another particular aspect, the subject is a human being. In again another particular aspect, the subject, in the human subject, has a mutated kit gene or expresses a mutated KIT receptor. The invention may involve either an individual or an entire population. The subject may be tested whatever his/her age or sex.
The term “biological sample” includes any biological sample from a subject. The biological sample may be a solid sample or a fluid sample. Typical examples of biological fluid samples usable in the context of the present invention may be selected from a blood, plasma, serum, urine and bone marrow aspirate sample. Preferably, the biological sample is a plasma, a blood or a serum sample, even more preferably a plasma sample. In the methods of the invention, sample may be used pure or diluted. Typical example of biological solid sample usable in the context of the present invention is a bone marrow biopsy sample.
Another particular sample is a sample comprising mast cells bearing a mutation in the KIT receptor. The KIT mutation is preferably located within the activation loop of the receptor at residue 816, and is in particular D816X, where X is V, H, I, F or Y. Mast cells can be for example KITD816X mast cells where X is V, H, I, F or Y.
For the measurement of the GlcNAc concentration in the biological sample a quantitative or semi- quantitative analytical method such as chromatography, spectroscopy or mass spectrometry is employed, while mass spectrometry is particularly preferred. The chromatography may comprise Gas Chromatography (GC), Liquid Chromatography (LC), High Pressure Liquid Chromatography (HPLC) and Ultra High Pressure Liquid Chromatography (UHPLC). Spectroscopy may comprise Ultraviolet- Visible (UV/Vis), Infrared (IR), Near Infrared (NIR) and Nuclear Magnetic Resonance (NMR). Mass analyzers/spectrometry may comprise ESI-QqQ, ESI-QqTOF, Matrix-assisted laser desorption/ionization (MALDI)-QqQ, MALDI-QqTOF and MALDI-TOF-TOF. More preferably, mass analyzers/spectrometry comprises Quadrupole Mass Analyzers, Ion Trap Mass Analyzers, TOF (Time of Flight) Mass Analyzer, Orbitrap mass analyzer, Magnetic Sector Mass Analyzer, Electrostatic Sector Mass Analyzer, Ion Cyclotron Resonance (ICR) and combinations of mass analyzers, including single quadrupole (Q) and triple quadrupole (QqQ), QqTOF, TOF-TOF and Q-Orbitrap. Preferred is the use of LC-tandem mass spectrometry.
On blood, plasma, serum, urine, bone marrow aspirate sample, preferably blood, plasma or serum sample, the absolute concentration of N-acetyl-D-glucosamine (GlcNAc) can be determined directly on free cellular GlcNAc using liquid chromatography coupled to mass spectrometry (LC-MS/MS).
The concentration of GlcNAc can also be measured based on the same samples (i.e., blood, plasma, serum, urine, bone marrow aspirate preferably blood, plasma or serum), on a phosphorylated product of GlcNAc, using a kinase assay (EIA). N-acetylglucosamine kinase (GlcNAc kinase or NAGK; E.C. 2.7.1.59), an enzyme of the sugar-kinase/Hsp70/actin super family is a prominent enzyme in amino sugar metabolism that catalyzes the conversion of GlcNAc to GlcNAc-6-phosphate (GlcNAc-6P). The specific activity of NAGK enzyme is directly correlated to the amount of GlcNAc present in the sample of the subject.
In bone marrow biopsy sample, the concentration of GlcNAc can be determined indirectly by measuring the O-GlcNAcylation of proteins, using for example a binding agent that specifically binds to O-linked
N-acetylglucosamine (O-GlcNAc), such as for example an anti-O-linked N-acetylglucosamine (O- GlcNAc) binding agent, preferably a monoclonal antibody, such as for example RL2, HGAC85, 18B10.C7 or CTD110.6 monoclonal antibody. A second molecule allowing the binding agent detection can be required. Concentration of GlcNAc can be determined using variety of known methods such as for example immunoassay, immunosorbent assay (ELISA), radioimmunoassay (RIA), and the like.
The terms “reference GlcNAc’s value”, “reference GlcNAc’s concentration”, “control GlcNAc’s value” and “cut-off GlcNAc’s value” can refer to a basal value corresponding to the mean of values (measured levels, quantities or concentrations) obtained with the biological sample(s) of a reference subject or population, typically of a healthy subject or of a population or cohort of healthy subjects, i.e., of subjects who do not suffer from a pathology, disease, disorder or dysfunctional state as herein described, the mean of values depending typically on the number of tested samples for a given reference subject or on the size of the reference population. The reference value can also be a statistic or discriminating value/threshold, i.e., a value which has been determined by measuring the parameter in both a healthy control population and a population with a known pathology, disease, disorder or dysfunctional state as herein described. The discriminating value identifies the diseased population with a specificity and/or a sensitivity, both predetermined by the skilled person, based on an analysis of the relation between the parameter values and the known clinical data of the healthy control subject/population and of the diseased patient subject/population. The discriminating value determined in this manner is valid for the same experimental setup in future individual tests.
Reference GlcNAc’s value can, in principle, be calculated for a cohort of subjects as specified above based on the average or mean values for a given biomarker by applying standard methods of statistics. In particular, accuracy of a test such as a method aiming to diagnose an event, or not, is best described by its receiver-operating characteristics (ROC). Each point on the ROC plot represents a sensitivity/specificity pair corresponding to a particular decision threshold. Dependent on a desired confidence interval, a threshold can be derived from the ROC curve allowing for the diagnosis or prediction for a given event with a proper balance of sensitivity and specificity, respectively. Accordingly, the reference to be used for the method of the present invention, i.e. a threshold which allows to discriminate between subjects who have a disease induced by mast cell activation and those who don’t have a disease induced by mast cell activation can be generated, preferably, by establishing a ROC analysis for said cohort as described above and deriving one or more threshold amounts or concentrations therefrom. Dependent on a desired sensitivity and specificity for a diagnostic method, the ROC plot allows deriving adjustable suitable thresholds. It will be understood that an optimal sensitivity is desired for excluding a disease induced by mast cell activation (i.e., a rule out) whereas an optimal specificity is envisaged for a subject to be identified as having a disease induced by mast cell activation (i.e. a rule in). In a particular aspect, the concentration of GlcNAc determined in step (i) of the method of the present invention is compared to more than one reference GlcNAc’s concentration,
e.g. a reference GlcNAc’s concentration for discriminating between the diseases induced by mast cell activation, and a reference GlcNAc’s concentration for ruling out a particular disease induced by mast cell activation among said diseases, typically for discriminating between healthy subjects and subjects suffering of a disease induced by mast cell activation.
For example, the reference GlcNAc’s value in plasma sample of a healthy control population is below 0.46 mM. In this example, a concentration of GlcNAc in plasma above the reference GlcNAc’s value of 0.46 pM is indicative of a disease induced by mast cell activation and allows the classification of a tested subject as a “diseased” subject, i.e., a subject suffering of a disease induced by mast cell activation.
Interestingly, inventors also showed that GlcNAc is a biomarker of diseases induced by mast cell activation but not of non-mast cells-related myeloproliferative disorders. Therefore, in one embodiment, the in vitro or ex vivo method of the invention allows differentially diagnosing between diseases induced by mast cell activation and non-mast cells-related myeloproliferative disorders such as acute myeloid leukemias, chronic myeloid leukemia, myelodysplastic and myeloproliferative syndrome, and essential thrombocythemia in a subject.
In a particular aspect, the in vitro or ex vivo method for differentially diagnosing between disease induced by mast cell activation and non-mast cells-related myeloproliferative disorders preferably acute myeloid leukemias, chronic myeloid leukemia, myelodysplastic and myeloproliferative syndrome, and essential thrombocythemia in a subject, comprises the steps of:
(i) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject; and
(ii) diagnosing a disease induced by mast cell activation in the subject if the concentration of GlcNAc as measured in step (i) is above a reference GlcNAc’s concentration.
For example, a concentration of GlcNAc in plasma above the reference GlcNAc’s value of 0.46 pM, for example above 0.8 pM, is indicative of a disease induced by mast cell activation and allows the classification of a tested subject as a subject suffering of a disease induced by mast cell activation and not from a non-mast cells-related myeloproliferative disorder such as acute myeloid leukemias, chronic myeloid leukemia, myelodysplastic and myeloproliferative syndrome, and essential thrombocythemia.
The reference GlcNAc’s concentrations to be used for the method of the present invention are also helpful to discriminate between the particular diseases induced by mast cell activation.
The in vitro or ex vivo method of the invention allows the diagnosis of asthma or allergy in a subject, for example vs mastocytosis, in particular SM, or vs MCAS.
In a particular aspect, the in vitro or ex vivo method for diagnosing asthma or allergy, in a subject, comprises the steps of:
(i) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject; and
(ii) diagnosing asthma or allergy in the subject if the concentration of GlcNAc as measured in step (i) is above a first reference GlcNAc’ s concentration and, preferably below a second reference GlcNAc’ s concentration.
The first and the second reference GlcNAc ’s concentrations are different.
For example, a concentration of GlcNAc in a plasma sample of a subject above 0.46 mM and below 1.7 mM indicates that the subject is suffering of asthma or allergy.
Using receiver operating characteristic (ROC) curve analysis, the cut-off value that allows to discriminate between healthy control population and a asthmatic or allergic patient population is 1 pM for a sensitivity of 1 and specificity of 1.
In another particular aspect, the in vitro or ex vivo method of the invention allows the diagnosis of mast cell activation syndrome (MCAS) or non-advanced SM (non-AdvSM), in particular indolent systemic mastocytosis (ISM) or smoldering SM (SSM), in a subject.
In this particular aspect, the in vitro or ex vivo method for diagnosing mast cell activation syndrome (MCAS) or non-advanced SM (non-AdvSM), in particular indolent systemic mastocytosis (ISM) or smoldering SM (SSM), in a subject, comprises the steps of:
(i) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject; and
(ii) diagnosing mast cell activation syndrome (MCAS) or non-advanced SM (non-AdvSM), in particular indolent systemic mastocytosis (ISM) or smoldering SM (SSM), in the subject if the concentration of GlcNAc as measured in step (i) is above a first reference GlcNAc’ s concentration and below a second GlcNAc’ s concentration.
The first and the second reference GlcNAc ’s concentrations are different.
For example, a concentration of GlcNAc in a plasma sample of a subject above 1.7 pM and below 2.8 pM indicates that the subject is suffering of mast cell activation syndrome (MCAS) or non-advanced SM (non-AdvSM), in particular of indolent systemic mastocytosis (ISM) or smoldering SM (SSM).
Using receiver operating characteristic (ROC) curve analysis, the cut-off value that allow to discriminate between asthmatic or allergic patient population and a MCAS or non-AdvSM, in particular ISM or SSM, patient populations, is 2.2 pM for a sensitivity of 0.9 and specificity of 0.8.
In a further particular aspect, the in vitro or ex vivo method of the invention allows the diagnosis of advanced SM (AdvSM), in particular aggressive SM (ASM), SM with an associated hematological neoplasm (SM-AHN) or mast cell leukemia (MCL), in a subject.
In this particular aspect, the in vitro or ex vivo method for diagnosing advanced SM (AdvSM), in particular ASM, SM-AHN or MCL, in a subject, comprises the steps of:
(i) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject; and
(ii) diagnosing advanced SM (AdvSM), in particular ASM, SM-AHN or MCL, in the subject if the concentration of GlcNAc as measured in step (i) is above a reference GlcNAc’ s concentration.
For example, a concentration of GlcNAc in a plasma sample of a subject above 2.8 mM indicates that the subject is suffering of advanced SM (AdvSM), in particular ASM, SM-AHN or MCL.
In another example, a concentration of GlcNAc in a biological sample (as herein defined) of a subject above 2.8 pM and, preferably, below 7, 8, 9 or 10 pM for example, indicates that the subject is suffering of advanced SM (AdvSM), in particular ASM, SM-AHN or MCL.
Using receiver operating characteristic (ROC) curve analysis, the cut-off value that allows to discriminate between a MCAS or non-AdvSM, in particular ISM or SSM, patient populations and an AdvSM, in particular ASM, SM-AHN or MCL, patient population is 3.8 pM for a sensitivity of 1 and specificity of 0.9.
When a diagnosis of AdvSM is established, the level of concentration of GlcNAc in a biological sample of the subject, preferably in a plasma or serum sample, enables to discriminate between the different forms of AdvSM. For example, using ROC analysis, a concentration of GlcNAc in a plasma or serum sample of a subject below 4.5 pM indicates that the subject has ASM. A concentration of GlcNAc in a plasma or serum sample of a subject above 4.5 pM indicates that the subject has advanced SM-AHN.
The in vitro or ex vivo method of the invention can be implemented for diagnosing a systemic mastocytosis (SM), in particular non-advanced SM (non-AdvSM) or advanced SM (AdvSM), more particularly indolent SM (ISM), smoldering SM (SSM), SM with an associated hematological neoplasm (SM-AHN), aggressive SM (ASM), mast cell leukemia (MCL), even more particularly indolent SM (ISM) or aggressive SM (ASM).
As used herein, the term "diagnosing" with reference to systemic mastocytosis (SM) refers to a method or process of determining if a subject has or does not have SM.
When a diagnosis of systemic mastocytosis is established, the level of concentration of GlcNAc in a biological sample of the subject, preferably in a plasma or serum sample, enables to discriminate between the different forms of SM. For example, using ROC analysis, a concentration of GlcNAc in a
plasma or serum sample of a subject below 3.8 mM indicates that the subject has non-AdvSM, in particular indolent SM (ISM) or smoldering SM (SSM). A concentration of GlcNAc in a plasma or serum sample of a subject above 3.8 mM indicates that the subject has advanced SM (AdvSM), in particular ASM, SM-AHN or MCL.
By studying plasma metabolomic profiles of patients with varying degrees of SM severity and validated diagnosis, inventors identified other (secondary) biomarkers suitable for the diagnosis of SM in combination with GlcNAc, and more particularly for discriminating between non-AdvSM, in particular ISM, and AdvSM, in particular ASM, SM-AHN or MCL. These biomarkers are illustrative of highly deregulated pathways in subject affected with SM.
Thus, in a particular aspect, the in vitro or ex vivo method of the invention for diagnosing a disease induced by mast cell activation, preferably systemic mastocytosis, further comprises a step of determining, in a biological sample from the subject which may be the same sample used for measuring the concentration of GlcNAc, or may be a different sample, the concentration of one or more secondary (/additional) biomarkers involved in the nicotinate/nicotinamide metabolism, pyrimidine metabolism, purine metabolism, glycerophospholipid metabolism, amino sugar and nucleotide sugar metabolism; arginine biosynthesis, and/or one carbon pool by folate metabolism, preferably in the nicotinate/nicotinamide metabolism and/or in the amino sugar metabolism (i.e. protein glycosylation).
Samples used to measure the concentration of GlcNAc and one or more biomarkers belonging to the seven metabolic pathways as disclosed herein may be of the same or different nature. In a particular aspect, the sample used for measuring the concentration of one or more biomarkers is of the same nature as that used for measuring the concentration of GlcNAc. For example, a fluid sample, such as a serum or plasma sample can be used for measuring the concentration of GlcNAc and also for measuring the concentration of one or more distinct biomarkers. In another particular aspect, the sample used for measuring the concentration of the one or more biomarkers is of a nature different from that used for measuring the concentration of GlcNAc. For example, one sample is a serum or plasma sample and the other sample is a (liquid or solid) bone marrow sample.
Among more than 306 metabolites identified, 58 can be used as selective biomarkers of AdvSM, in particular ASM, and non-AdvSM, in particular ISM, in combination with GlcNAc, based on a relative fold change of anyone of said 58 biomarkers above 1.5 (as herein listed below).
In a more particular aspect, the in vitro or ex vivo method of the invention for diagnosing a disease induced by mast cell activation, preferably systemic mastocytosis, further comprises a step of determining from the biological sample from the subject a concentration of one or more (secondary) biomarkers, for example biomarkers involved in nicotinate/nicotinamide metabolism such as L- kynurenine and/or quinolinate, and/or biomarkers involved in amino sugar metabolism such as dihydroxyacetone phosphate, wherein a variation of the concentration of said one or more biomarkers, such as an increase if the biomarker(s) is/are L-kynurenine and/or quinolinate, and/or or a decrease if the biomarker is dihydroxyacetone phosphate, when compared to a reference concentration of said one or more biomarkers, is indicative of AdvSM. Each determined concentration is compared to a reference concentration. The comparison can be absolute or relative depending of the nature of the reference concentration which can be obtained from a healthy subject/population or from a subject/population having a disease induced by mast cell activation, such as ISM. In a particular example of such a method, when the reference concentration is obtained from a ISM subject population, concentrations in the sample of the tested subject of L-kynurenine and/or quinolinate above their respective reference concentrations, and/or a concentration of dihydroxyacetone phosphate below its reference concentration, is/are indicative of AdvSM. In another particular example of such a method, if the reference concentration is obtained from a population having a disease induced by mast cell activation, for example ISM, a decrease in the concentration of dihydroxyacetone phosphate in the sample of the tested subject compared to the reference concentration of dihydroxyacetone phosphate in the ISM reference sample, is indicative of AdvSM and/or an increase in the concentrations of L-kynurenine
and/or quinolinate in the sample of the tested subject compared to the reference concentration of L- kynurenine and/or quinolinate in the ISM reference sample, is indicative of AdvSM.
Mastocytosis is a disorder characterized by the abnormal proliferation and accumulation of mast cells. In some AdvSM, mast cells derive from a clonal progenitor carrying a gain-of-function mutation in KIT. KIT (CD117) is a Type III receptor tyrosine kinase that is expressed by MC notably. One example of the KIT transcription product of the kit gene has the sequence of NCBI reference sequence NM_000222.2. The interaction between KIT and its ligand, the stem cell factor (SCF), plays a key role in regulating MC proliferation, MC maturation, MC adhesion, MC chemotaxis, as well as in regulating the tyrosine kinase activity of KIT. Gain-of-function somatic mutations in the KIT tyrosine kinase domain, particularly the D816V mutation, have been found to occur in a majority of cases of SM, at least in adult subjects. Other mutations associated to AdvSM have been observed at position 816, in particular D816V, D816H, D816I, D816F and D816Y. Other less common (<5%) somatic KIT mutations identified in adults suffering of SM include K509I, F522C, V560G, D815K, insVI815-816 and D820G (Cem Akin, J Allergy Clin Immunol 2017;140:349-55). KIT mutation associated to AdvSM are observed in mast cells of the bone marrow, but also in other myeloid/lymphoid lineages, such as CD34+ hematopoietic stem and precursor cells, eosinophils, monocytes, and maturing neutrophils, and, to a lesser extent, also in T lymphocytes.
Treatment protocol for diseases induced by mast cell activation generally depends on the severity of the disease. It is thus essential to determine this aspect with certainty and as early as possible. The in vitro or ex vivo diagnostic method of the invention offers a valuable means for the practitioner to categorize patients suffering from a disease induced by mast cell activation. In particular, the practitioner can subsequently categorize the subject as having mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis (CM); mast cell activation syndrome (MCAS); asthma; or allergy depending on the GlcNAc concentration in the sample.
Thus, in a particular aspect, the in vitro or ex vivo method of the invention for diagnosing a disease induced by mast cell activation, preferably systemic mastocytosis, further comprises a step of administering a treatment for a disease induced by mast cell activation to a subject diagnosed as having mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis (CM); mast cell activation syndrome (MCAS); asthma; and allergy. This subsequent treatment step follows the diagnosis made on the basis of the GlcNAc alone, or in combination with all or part of the other (secondary) biomarkers mentioned above. Such treatment can be a treatment as defined below depending on the type of disease induced by mast cell activation which is concerned.
In another particular aspect, the subject diagnosed as having mastocytosis, in particular anyone of the herein described particular mastocytosis, is a subject who has not been diagnosed with a GlcNac biomarker, typically a subject who has been diagnosed has suffering of such mastocytosis by using a
biomarker selected from tryptase, urinary histamine metabolites, urinary prostaglandin D2, urinary leukotriene E4 and metabolites thereof, and thus who is likely not to have been correctly diagnosed.
Therapy for systemic mastocytosis is primarily symptomatic; no available therapy is curative.
ISM patients have a normal life expectancy and treatment is generally limited to anaphylaxis/prevention of symptoms, and/or to the prevention or treatment of osteoporosis. The main objective is to reduce the symptoms of MC activation, such as pruritus, flushes and gastrointestinal cramps. In case of pruritus or skin manifestations, antihistamines HI (anti-Hl) are used. For gastrointestinal tract manifestations, anti- H2 are effective and can be combined with anti-Hl, with di-sodium cromoglycate or with leukotriene inhibitors. Corticoids can suppress antihistamines recalcitrant symptoms. For patients with SM and osteoporosis, biphosphonates are recommended with adequate supplementation of calcium and vitamin D.
In SSM patients, symptomatic treatment may be the only therapy. Avoidance of known triggers, prophylactic prescription of an epi-pen, and medications such as antihistamines, antileukotrienes, cromolyn sodium, omalizumab and aspirin may all have a role in the prevention or treatment of MC- mediated symptoms.
Regular follow-up and assessment for transformation to more aggressive systemic mastocytosis is desirable. Thanks to the present invention, GlcNAc concentration of patients can be assessed regularly to monitor disease evolution and appropriately adjust therapy. In patients who progress to more advanced variants of the disease, introduction of targeted or non-targeted cytoreductive therapy is now possible.
Patients with advanced SM, in particular ASM, frequently need MC cytoreductive therapy to prevent, slow or treat disease-related organ dysfunction. Non-targeted therapies include interferon-(IFNalpha), which may be effective in a subset of patients, and cladribine (2CdA) which provides high and, in some patients, long lasting, response rates. Targeted therapy is with KIT tyrosine kinase inhibitors (KIT TKIs). In particular, small molecule inhibitors that target mutant-KIT may be used, including FDA approved midostaurin (RYDAPT) or avapritinib (AYVAKIT). Other TKIs included imatinib mesylate or masitinib may be indicated for the patients who do not have the KITD816V mutation. Symptomatic treatment is mainly with antihistamines (anti-Hl and anti-H2).
Treatment of ASM-AHN primarily targets the AHN component, if an aggressive disease such as acute myeloid leukemia is present. Allogeneic stem cell transplant can be considered in such patients, or in those with relapsed/refractory advanced SM. Imatinib has a limited therapeutic role in SM; effective cytoreduction is limited to subjects having imatinib-sensitive KIT mutations.
Management of MCF treatment includes chemotherapy, with or without interferon alpha or cladribine. For MCF patients with rapid progression and those who are resistant against 2CdA or midostaurin, poly-
chemotherapy (protocols otherwise used for high-risk AML) is usually recommended. In patients who are young and fit and have a suitable donor, stem cell transplantation (SCT) should be considered after successful debulking. The outcome after allogeneic SCT is better for those prepared with ablative conditioning compared with less-intensive (nonmyeloablative) conditioning. In subject having splenomegaly with hypersplenism, splenectomy is indicated. Hydroxyurea is a palliative treatment.
In the context of MCAS, management of symptoms of mast cell activation is complex and individualized. Typically, mast cell patients take baseline medications, including a second generation HI and H2 antihistamine, a mast cell stabilizer and a leukotriene inhibitor.
Inventors herein described an in vitro or ex vivo method for assessing the severity of a systemic mastocytosis (SM) in a subject, in particular for differentially diagnosing between non-advanced SM (non-AdvSM) and advanced SM (AdvSM) in a subject, more particularly between indolent SM (ISM) and i) aggressive SM (ASM), ii) SM with an associated hematological neoplasm (SM-AHN) or iii) mast cell leukemia (MCL). The method preferably comprises:
(a) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject; and
(b) comparing the concentration of GlcNAc in the biological sample of the subject with a reference concentration of GlcNAc and correlating the concentration of GlcNAc in the biological sample with the severity of the SM in the subject.
As used herein, the term "assessing" includes determining if an element is present or not, and if present, optionally determining the amount or level, in particular the concentration, of said element. The terms "determining", "evaluating", "assessing" and "assaying" can be used interchangeably and can include quantitative and/or qualitative determinations. The term "measuring" involves a quantitative determination.
The correlation takes into account the amount or concentration of GlcNAc in the sample of a subject affected with SM compared to a control/reference amount or concentration of GlcNAc (e.g., in healthy subjects in whom a disease induced by mast cell activation is absent).
In a particular aspect, the in vitro or ex vivo method is for assessing the severity of SM, in particular for differentially diagnosing between non-AdvSM, in particular indolent SM (ISM), and advanced SM (AdvSM), in particular aggressive SM (ASM) or SM with an associated hematological neoplasm (SM- AHN), in a subject. A particular method of this kind comprises a step of comparing the concentration of GlcNAc in the biological sample of the subject with a reference concentration of GlcNAc, which is the concentration of GlcNAc in a biological sample obtained from a healthy subject. In the context of this method, a concentration of GlcNAc in the biological sample of the subject that is 6 to 8 times greater than the concentration of GlcNAc in the reference control sample (of the same nature than the biological sample of the subject, i.e. for example a bone marrow sample if the biological sample of the subject is
a bone marrow sample) is indicative of non-AdvSM, in particular indolent SM (ISM) or smoldering SM (SSM); and a concentration of GlcNAc in the biological sample of the subject that is more than 8 fold greater than the concentration of GlcNAc in the reference control sample is indicative of advanced SM (AdvSM), in particular aggressive SM (ASM) or SM with an associated hematological neoplasm (SM- AHN).
Inventors herein disclose the plasma metabolome profiling of SM. Inventors in particular herein identify for the first time N-acetyl-D-glucosamine (GlcNAc) as the most predictive biomarker of SM severity. As apparent from data disclosed in the experimental section of the present description, high plasma levels of GlcNAc in patients with advanced SM correlate with the activation of the GlcNAc-fed hexosamine biosynthesis pathway (HBP) in patients’ BM aspirates.
In another particular in vitro or ex vivo method for assessing the severity of SM, in particular for differentially diagnosing between non-AdvSM, in particular indolent SM (ISM) or SSM, and advanced SM (AdvSM), in particular ASM or SM-AHN, in a subject, the biological sample of the subject is a fluid sample, preferably a plasma or serum sample, and the reference concentration of GlcNAc is 2.8 mM. In the context of this method, a concentration of GlcNAc in the biological (plasma or serum) sample of the subject that is equal to or above the reference (plasma or serum) concentration of GlcNAc is indicative of AdvSM, in particular ASM or SM-AHN, and a concentration of GlcNAc in the biological (plasma or serum) sample of the subject that is below the reference (plasma or serum) concentration of GlcNAc is indicative of non-AdvSM, in particular ISM or SSM.
ISM patients with multilineage KIT D816V mutations have been shown to have a higher risk of progression to AdvSM than patients with mast cells-restricted KIT D816V. The diagnostic standard is mutation analysis of KIT in bone marrow (BM) cells. If a BM aspirate is not available, the analysis can be performed on the marrow smear or from a paraffin-embedded biopsy sample. However, these methods are invasive and not easy to implement, especially since the number of neoplastic MCs in the BM of ISM patients is very low. This sets the requirement for highly sensitive methods of mutation analysis.
Inventors now advantageously reveal that GlcNAc can be used as a biomarker for assessing/predicting the risk for a subject affected or diagnosed with non-AdvSM, in particular ISM or SSM to develop AdvSM, in particular ASM or SM-AHN and herein describe an in vitro or ex vivo method for assessing/predicting the risk for a subject affected or diagnosed with non-AdvSM, in particular ISM or SSM, to develop AdvSM, in particular ASM or SM-AHN. Typically, the method comprises a step of determining the concentration of GlcNAc in a biological sample obtained from a subject affected or diagnosed with non-AdvSM, in particular ISM or SSM, wherein a concentration of GlcNAc above a reference concentration of GlcNAc indicates that the subject is at risk to develop AdvSM, in particular ASM or SM-AHN. For example, in a serum sample, the reference concentration of GlcNAc useful to
predict the risk for a subject affected or diagnosed with ISM to develop ASM is 3.8 mM, as determined by ROC analysis.
In a particular aspect, inventors herein provide an in vitro or ex vivo method for assessing/predicting the risk for a subject affected or diagnosed with non-AdvSM, in particular ISM, to develop AdvSM, in particular ASM or SM-AHN, wherein the method comprises the steps of:
(a) measuring the concentration of GlcNAc in a sample obtained from a subject at To;
(b) measuring the concentration of GlcNAc in a distinct sample obtained from said subject at Ti, Ti being posterior to To; and
(c) comparing the concentrations of GlcNAc as measured in steps (a) and (b) to assess/predict said risk, wherein:
- a concentration of GlcNAc as measured in step (b) below or identical to the concentration of GlcNAc as measured in step (a) is indicative that the subject is not at risk to develop AdvSM, in particular ASM or SM-AHN, and
- a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative that the subject is at risk to develop AdvSM, in particular ASM or SM-AHN.
A follow-up of patient affected with a disease induced by mast cell activation being essential in a clinical point of view so as to optimally adapt the treatment to the course of the disease, inventors herein provide an in vitro or ex vivo method for monitoring the evolution of a disease induced by mast cell activation, preferably selected from mastocytosis, mast cell activation syndrome, asthma and allergy, in particular SM, in a subject affected with said disease. This method preferably comprises the steps of:
(a) measuring the concentration of GlcNAc in a sample obtained from a subject at To;
(b) measuring the concentration of GlcNAc in a distinct sample obtained from said subject at Ti, Ti being posterior to To; and
(c) comparing the concentrations of GlcNAc as measured in steps (a) and (b) to monitor the evolution of the disease in the subject, wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative of a favorable evolution of the disease in the subject, a concentration of GlcNAc as measured in step (b) identical to the concentration of GlcNAc as measured in step (a) is indicative of a stabilization of the disease in the subject, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative of an unfavorable evolution of the disease in the subject, which preferably requires an adaptation of the treatment.
By “monitoring” is meant detecting and following any change in the parameters of a disease. These parameters can be physiologic or symptomatic parameters. Monitoring involves periodic measurements
that guide the management of a chronic or recurrent condition such as, in the context of the present invention, a disease induced by mast cell activation.
By “favorable evolution of the disease” is meant a regression, at least in part, of the symptoms or clinical signs associated with a disease induced by mast cell activation. On the contrary, “unfavorable evolution of the disease” relates to a worsening of clinical symptoms or signs associated with a disease induced by mast cell activation. This worsening can include for example higher release of mast cell mediators, more organs affected with mast cell infiltrate, or any other neurologic, gastrointestinal, cardiovascular, constitutional signs classically associated with a disease induced by mast cell activation. The “stabilization of the disease” means that there is no modulation, i.e., neither improvement nor worsening, of the clinical symptoms or signs associated with a disease induced by mast cell activation.
Monitoring before treatment should establish the need or not for treatment and then a baseline/threshold above which it becomes possible to judge the response to a treatment or changes in the patient's condition. The objective of monitoring is to ensure that measures stay within reasonable limits, called control limits. The control limits ensure that relevant/significative changes in the level of the target biomarker(s) are detected while minimizing misdiagnosis or errors. The monitoring interval can be several days, weeks or months. The interval depends on evolution/course (progression, stabilization or regression) of the disease.
In a particular aspect, the in vitro or ex vivo method is for monitoring the evolution of non-AdvSM, in particular ISM or SSM, in a subject wrongly or rightly diagnosed as affected with non-AdvSM, in particular ISM or SSM. A particular method for monitoring the evolution of non-AdvSM, in particular ISM or SSM comprises the steps of:
(a) measuring the concentration of GlcNAc in a sample obtained from a subject at To;
(b) measuring the concentration of GlcNAc in a distinct sample obtained from said subject at Ti, Ti being posterior to To; and
(c) comparing the concentrations of GlcNAc as measured in steps (a) and (b) to monitor the evolution of non-AdvSM, in particular ISM or SSM, in the subject, wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative of a favorable evolution of non-AdvSM, in particular ISM or SSM, in the subject, a concentration of GlcNAc as measured in step (b) identical to the concentration of GlcNAc as measured in step (a) is indicative of a stabilization of non-AdvSM, in particular ISM or SSM, in the subject, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative of an unfavorable evolution of non-AdvSM, in particular ISM or SSM, in the subject.
In the in vitro or ex vivo method for monitoring the evolution of a disease induced by mast cell activation, preferably SM, step (b) may be repeated as many times as necessary to monitor the evolution of the disease over a period considered as relevant by the practitioner.
Monitoring can also be implemented once a treatment for a disease induced by mast cell activation has started.
Further described herein is an in vitro or ex vivo method for determining the efficacy of a therapy of a disease induced by mast cell activation selected preferably from mastocytosis, mast cell activation syndrome, asthma and allergy, in particular SM, in a subject affected with said disease. This method preferably comprises the steps of:
(a) measuring the concentration of GlcNAc in a sample obtained from a subject prior to the administration of a therapy of a disease induced by mast cell activation, for example a mastocytosis therapy;
(b) measuring the concentration of GlcNAc in a sample obtained from a subject once started the administration of a therapy of a disease induced by mast cell activation; and
(c) comparing the concentrations of GlcNAc as measured in steps (a) and (b), wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative that the therapy is effective in the treatment of the disease induced by mast cell activation, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative that the therapy is not effective, or not effective enough, in the treatment of the disease induced by mast cell activation; or, alternatively, wherein the method comprises the steps of:
(i) measuring the concentration of GlcNAc in a sample obtained from a subject once started the administration of a therapy of a disease induced by mast cell activation, for example a mastocytosis therapy; and
(ii) comparing the concentration of GlcNAc as measured in step (i) with a reference concentration of GlcNAc, wherein, a concentration of GlcNAc as measured in step (i) below the GlcNAc reference concentration is indicative that the therapy is effective in the treatment of the disease induced by mast cell activation, and a concentration of GlcNAc as measured in step (i) above the GlcNAc reference concentration is indicative that the therapy is not effective, or not effective enough, in the treatment of the disease induced by mast cell activation.
In the herein described in vitro or ex vivo method for determining the efficacy of a therapy of a disease induced by mast cell activation, preferably SM, step (b) may be repeated as many times as deemed necessary by the practitioner during the course of the disease and until the subject is cured or stabilization at an acceptable level (i.e. compatible with an acceptable life for the diseased subject).
Inventors have also identified disturbances in hexosamine biosynthesis pathway (HBP) in samples of subjects affected with AdvSM. In particular, a modification of the transcript level occurs for enzymes involved in the HBP pathway in mast cells.
In a particular aspect, any of the herein described in vitro or ex vivo method for determining the efficacy of a therapy of a disease induced by mast cell activation further comprises a step of determining, in a biological sample from the subject, the transcript level of one or more enzymes involved in the hexosamine biosynthesis pathway (HBP), such as GFAT1, PGM3, NAGK, OGT and OGA.
Bone marrow aspirate, which contains mast cells, is a particularly suitable biological sample to perform transcription study.
In the context of a method for monitoring the evolution of the disease in a subject or for evaluating the efficacy of a treatment of a disease induced by mast cell activation in a subject, the reference sample may in particular be a sample previously collected from said subject, in particular before monitoring or evaluation has started.
Also herein described is a kit for measuring the concentration of GlcNAc in a biological sample of a subject, wherein the kit comprises an anti-O-linked N-acetylglucosamine (O-GlcNAc) binding agent, for example RL2, HGAC85, 18B10.C7 or CTD110.6 monoclonal antibody, a molecule allowing the binding agent detection; or a N-Acetyl-D-Glucosamine Kinase (NAGK), and optionally a leaflet providing GlcNAc reference concentration(s).
In another particular aspect, the kit for measuring the concentration of GlcNAc in a biological sample of a subject comprises an anti-N-acetylglucosamine (GlcNAc) binding agent that binds GlcNAc, preferably that selectively binds GlcNAc and does not bind O-GlcNAc; a molecule allowing the binding agent detection; or a N-Acetyl-D-Glucosamine Kinase (NAGK), and optionally a leaflet providing GlcNAc reference concentration(s).
The kit may further comprise primers and probes for assessing the transcript level of one or more enzymes involved in the hexosamine biosynthesis pathway (HBP), such as GFAT1, PGM3, NAGK, OGT and OGA in a biological sample of a subject.
Another particular kit also comprises for example:
- means for assessing the concentration of dihydroxyacetone phosphate, kynurenine and quinolinate biomarkers in a biological sample of a subject;
- primers, probes or any other suitable means for detecting a KIT mutation, in particular KITD816X where X is V, H, I, F or Y, in a biological sample of a subject; and/or
- means for measuring the concentration of tryptase, urinary histamine metabolites, urinary prostaglandin D2, urinary leukotriene E4 and/or any metabolite thereof, in a biological sample of a subject.
Further herein described is the use of a herein above described kit on a biological sample obtained from a subject, for diagnosing a disease induced by mast cell activation typically selected from mastocytosis,
mast cell activation syndrome, asthma and allergy, preferably SM; for assessing the severity of systemic mastocytosis; for assessing/predicting the risk for a subject affected with non-AdvSM to develop AdvSM, preferably prior to the onset of AdvSM’s symptoms; monitoring the evolution of a disease induced by mast cell activation and/or for determining the efficacy of a therapy of a disease induced by mast cell activation typically selected from mastocytosis, mast cell activation syndrome, asthma and allergy, preferably SM, in the subject.
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
EXEMPLE
MATERIALS & METHODS
Patient data
Diagnoses were established according to criteria provided by the World Health organization. Adults with systemic mastocytosis were enrolled in a prospective national multi-centric study, sponsored by the French association for initiative and research on mast cell and mastocytosis (AFIRMM). The cohort consisted in 25 SM-related plasma samples: specifically, 12 plasma specimens from patients with AdvSM, including patients with ASM (n=4), SM-AHN (n=6), and MCL (n=2), and 13 specimens from individuals with ISM. Plasma was isolated from whole blood of patient collected in heparinized tube and stored at -80°C until analysis. Blood and bone marrow aspirate material were obtained at diagnosis, after written informed consent was given. Data and samples collection were approved by the ethics committee of the Necker Hospital (Paris, France) and conducted in compliance with the precepts of the Helsinki protocol. Healthy subjects without mastocytosis were used as controls.
Cell lines
The human mast cell lines ROSAK" WT and ROSAK" D816v were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% heat inactivated fetal calf serum FCS, 100 pg/mL streptomycin, 100 IU/mL penicillin, 2 mM L-glutamine and ImM sodium pyruvate. ROSAKITWT cells were grown with additional 80ng/mL murine stem cell factor. Bibi et al. (Bibi S, 2016) generated the ROSAHT D816v Glucmast cells (kindly provided by M. Arock, Paris, France). They were obtained by the transduction of ROSAKIT D816v with lentiviral particles LV-Gluc-CFP. ROSAKIT D816v Gluc cells expressed both GFP and CFP (cyan fluorescent protein). Murine bone marrow-derived mast cells (BMMCs) (see below) were cultured in Opti-MEM Reduced Serum medium supplemented with 10% heat inactivated
fetal calf serum FCS, 100 mg/mL streptomycin, 100 IU/mL penicillin, 2-mercaptoethanol, 2 mM L- glutamine and 1 mM sodium pyruvate in presence of IL-3.
Mice and BMMCs
Gerbaulet et al. generated the Kit D814VF1 mice. The Kit D814V transgene, harboring the murine equivalent of the human kit D816V mutation, is expressed upon Cre-mediated excision of the loxP- flanked transcriptional stop element in adult mice. These mice were crossed to Mxl-Cre mice to generate Kit D814VF1; Mxl-Cre mice and their progeny were used for subsequent experiments. In mice carrying the Mxl-Cre allele, Cre expression was induced by 3 intra-peritoneal (i.p.) injections of 250 pg polyinosinic:polycytidylic (pI:C) every second day to activate the expression of the transgenic Kit D814V allele. Bone marrow derived mast cells (BMMCs) were differentiated by culturing bone marrow from Kit D814VF1; Mxl-Cre and Kit WT; Mxl-Cre mice for 4 to 6 weeks in IL-3 supplemented media. Mast cell phenotype was confirmed by flow cytometry analysis with antibodies specific for c-Kit (2B8; eBiosciences) and the multi-chain activating receptor FceRI (MAR-1; eBiosciences). All animal studies were performed in the animal facility at Cancer Research Center of Marseilles (CRCM) in compliance with the laws and approved by the French animal ethics committees (Agreement APAFIS N°6743- 2016091513513606 v4). Protocols were carried out in accordance with guidelines for animal care and protection.
Metabolomic profiling and bioinformatic analysis
Polar metabolites were extracted from the plasma of patient in ice-cold methanol following the protocol described in a previous study (Yuan M et al., 2012). All supernatants were evaporated on a SpeedVac concentrator to a pellet using no heat and sent on dry ice to the mass spectrometry core of Beth Israel Deaconess Medical Center (BIDMC) of Boston led by JM Asara. Endogenous metabolite profiles were obtained using a positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform using an AB/SCIEX 5500 QTRAP hybrid triple quadrupole mass spectrometer. Metabolites were identified by their m/z retention time, and through comparison to library entities of purified known standards. Bioinformatic analysis of the metabolomic profiling was performed using R software and MetaboAnalyst 4.0. (Chong J et al., 2019). To normalize the LC-MS/MS peak areas, inventors selected metabolites that were detected in at least 50% of the samples. The remaining missing values were replaced by half of the minimum peak area value measured in the data set. Then, the samples’ peak area values were normalized by probabilistic quotient normalization, using the “Healthy” or ISM samples as reference. The final normalized values were log2 transformed. For computation of the z-scores, each metabolite was centered by the reference samples’ mean and scaled by the reference samples’ standard deviation. By plotting the resulting z-scores as scatterplot, we obtain a view of metabolite changes in all patient samples relative to the reference group. For the differential analysis of Indolent versus AdvSM,
t-tests were used to compare peaks between ISM vs. AdvSM groups and fold changes were calculated for each metabolite. A cutoff threshold of 0.05 for p-value and fold change was used to select the most significant metabolites. The Benjamini-Hochberg method was used to correct p-value for multiple testing. For the ISM vs. AdvSM analysis, the Benjamini-Hochberg false discovery rate (FDR) was 0.13. Hierarchical clustering was performed on the log transformed normalized data (mean centered and scaled by standard deviation). Clustering was performed using the Ward method and Pearson’s correlation was used for distance calculation.
Metabolic transcript screening
Gene expression profiles from highly purified bone marrow mast cells of SM patient were generated in a previous study (Teodosio C, 2013). The raw data are available from the corresponding author on request. Briefly, RNA was extracted from sorted MCs from 33 subjects, including 5 control subjects and 26 patients with SM (ISM (n=14), ASM (n= 12)). Sequencing was performed on the Affymetrix GeneChip Human Gene 1.0 ST Array with standard protocols. Expression data were extracted and normalized with the Robust Multichip Average (RMA) method (Bioconductor, Package oligo) and filtered from a precompiled Gene Ontology (GO) terms list to restrain analysis to metabolic transcripts. The list of the 3,638 genes annotated with the term “metabolic”, encoding all known human metabolic enzymes and transporters, is available on request. To identify differentially expressed metabolic genes in SM, we used t-tests to compare for each gene the mean expression in two groups samples. The Benjamini-Hochberg (BH) method was used to correct p-value for multiple testing. We calculated fold changes for each gene as well. Genes with BH FDR < 0.05 and fold change > 3 were selected, giving a list of 512 genes (ASM vs HD) and 343 genes (ISM vs HD). The intersection of the two lists is shown as a Venn diagram in the Figure. 2E. Identification of enriched GO functional categories in the set of metabolic genes deregulated only in ASM (180 genes) or ISM (11 genes) signatures respectively was performed with Enrich (adjusted p-value<0.05, Fischer exact test) (Kuleshow MV et al., 2016). The complete lists of functional categories are provided in Table S3.
Cell Proliferation
ROSAK" WT and ROSAKIT D816v mast cells were seeded in low glucose (1 g/L) IMDM supplemented with 10% heat inactivated fetal calf serum FCS, 100 pg/mL streptomycin, 100 IU/mL penicillin, 2 mM L-glutamine and ImM sodium pyruvate at day 0 with or without 20 mM GlcNAc. Cells numbers were counted by automated cell counting on a Cellometer Auto T4 (Nexcelom Bioscience) at day 2, day 4 and day 8. Mast cell proliferation in response to GlcNAc in the cell culture medium was also measured by BrdU incorporation using an APC BrdU Flow Kit from BD Bioscience following manufacturer instructions. Flow cytometry analysis was performed with BD FSRFortessa™ (Becton Dickinson, Franklin Fakes, NJ, USA). Propidium iodide staining was used to gate out the dead population.
Degranulation assays
Degranulation was monitored by the release of b-hexosaminidase into the culture supernatants as described in a previous study (Leoni C, 2017). Briefly, mast cells/mL were preloaded with IgE-anti DNP 0.15 pg/mL, with or without GlcNAc 100 mM in a low glucose (1 g/L) medium. Cells were resuspended in Tyrode’s buffer-0.1% BSA and stimulated with dinitrophenylated oval hum in (DNP-OVA) 0.125 pg/mL (Sigma-Aldrich) for 2h at 37°C. As positive control, cells were stimulated for 2h with 2pM ionomycin (Sigma-Aldrich). After stimulation, 50 pi of supernatant was collected in a flat-bottom 96- wells plate. Cell pellets were then lysed in 0.5% Triton X-100 in Tyrode’s buffer, and 50 pi from the cell lysate was transferred to a second flat-bottom 96-wells plate. Samples of the supernatant and cell lysate were then incubated for lh with 50pl b-hexosaminidase substrate (4 mM, 4-nitrophenyl-N-acetyl- b-D-glucosaminide; Sigma). The reaction was stopped with 0.2M Glycine (pH 10.7), and the absorbance was read at 405 nm on a FLUOstar OPTIMA microplate reader. The percentage of degranulation was calculated as the ratio between the absorbance of the supernatants and the total absorbance of the supernatants and cell lysates. % Degranulation = ((A Sup)/(A Sup+A Lysate)) x 100. Alternatively, degranulation was assessed using PE Annexin V Kit (BD Pharmingen), because during the membrane fusion process of degranulation, annexin V binding occurs at sites of secretory granule exposure to the cell surface. Briefly, mast cells in low glucose (1 g/L) IMDM complete medium in the presence or absence of GlcNAc 100 mM for 24 hours, were stimulated for 30 minutes with IgE 0.5 pg/mL and DNP- OVA 0.2 pg/mL, before staining using a PE Annexin V Kit (BD Pharmingen) following the manufacturer’s instruction exactly.
Intracellular cytokine staining
Cells were seeded in low glucose medium with or without GlcNAc 100 mM overnight at 37°C. Cells were then stimulated with IgE-anti-DNP 0.5 pg/mL and DNP-OVA 0.2 pg/mL for 3 hours. Brefeldin A 10 pg/mL was added in the last 2 hours of stimulation for inhibiting protein exocytosis. Cells were collected, washed in PBS, re-suspended in 100 pL of Cytofix/Cytoperm (BD Pharmingen™) and incubated 20 minutes on ice. Cells were then washed and incubated with the following antibodies: anti- TNF-a-PE/Cy7 or anti-IL-6-PE (BioLegend) on ice for 20 minutes. Flow cytometry analyses were performed on a BD LSRFortessa™.
Enzyme-linked immunosorbent assay (ELISA)
The measurement of TNF-a release was carried out using the Mouse TNF-a High Sensitivity ELISA (eBioscience). Briefly, BMMC cells were incubated with or without GlcNAc 100 mM for 24 hours, then stimulated with IgE 0.5 pg/mL and DNP-OVA 0.2 pg/mL for 9 hours. 50 pL of each supernatant sample were added into the anti-mouse TNF-a coated micro-wells, followed by incubation with a biotin-
conjugated and streptavidin-horseradish peroxidase (HRP) at RT for 2 hours and 1 hour, respectively. Amplification reagent including biotinyl-tyramide and streptavidin-HRP was used to multiply the available HRP molecules for substrate reaction. 100 mΐ of substrate solution containing tetramethyl- benzidine was added at RT for 10 minutes. Reaction was terminated by adding 1M phosphoric acid. The absorbance values were measured at 450 nm using the FLUOstar OPTIMA microplate reader.
Western blots
For protein extracts, patient bone marrow aspirates were lysed in RIPA buffer (50 mM Tris HC1, pH7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X100, 0.5% sodium deoxycholate) supplemented with phosphatase (PhosSTOPEASYPack; Roche) and protease inhibitors (Sigma). Protein concentration was measured with a Bradford protein assay (Life Science Technology). Samples (30 pg) were run on a 10% SDS-polyacrylamide gels and transferred to PVDF membrane. Immunodetection was performed with an anti-OGlcNAc (RL2) (1:2000) (Abeam), an anti-OGT (1:1000) (Abeam), an anti-OGA (1:20000), or an anti-NAGK (1:10000) antibody overnight at 4°C. An anti-ERK2 antibody (1:20000) was used for loading controls. Incubation with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) was performed at RT 1 hour. Enhanced chemiluminescence and X-ray films were used to detect protein expression signals.
Passive cutaneous anaphylaxis
In vivo passive cutaneous anaphylaxis (PCA) experiments were performed as previously described (Leoni C et al., 2017).20 Briefly, 20 mg GlcNAc resuspended in PBS was administered by intraperitoneal injection to half of the mice (n=20) at DO, D1 and D2 and PBS was administered to the other half. Mice were anesthetized with isoflurane at D2, 2 hours after the last injection. In each group, the left ear was sensitized by intradermal injection of 10 mΐ of 1 pg anti-DNP IgE or vehicle in the right ear. Twenty- four hours later, mice were injected intravenously with 0.5 mg DNP-OVA in 200 pi 0.5% Evans Blue dye (Sigma-Aldrich) in PBS. lh later, the mice were euthanized and ear sections were excised. The extravasated blue dye was then extracted from the tissues by incubation in form amide 63 °C overnight, and its intensity (correlated to the extent of extravasation) measured spectrophotometrically (OD 650). PCA response was quantified by the amount of dye leaked at IgE-injected sites. Data are expressed as Evans Blue dye in ng/mg tissue.
Xenogenic transplantation of ROSA KITD816V-Gluc cells in NOD-SCID IL-2R g-/- (NSG) mice
NSG mice (005557) were obtained from the Jackson Laboratory and bred in a pathogen-free environment at the animal facility of Cancer Research Center of Marseille (CRCM). Animal experiments were performed in accordance with guidelines established by the Institutional Animal Committee. lOxlO6 ROSA KIT D816V-Gluc cells were injected to mice (2 groups, 5 mice per group)
24h after sublethally irradiation with 1.5 Gy of total body irradiation. Intraperitoneal (IP) injection of 300 mg/kg GlcNAc was administered 5 consecutive days per week for 7 weeks. In addition, treated animals had ad libitum access to drinking water supplemented with 50 mg/mL GlcNAc. GlcNAc has been used safely in multiple clinical trials; no adverse effects have been observed in previous chronic toxicity and carcinogenicity studies after administration of 300 mg/kg GlcNAc in dogs or even after administration of up to 2000 mg/kg/day GlcNAc. Engraftment was assessed after 4 weeks using quantitative measurement of Glue activity in plasma, and quantitative measurement of GFP+ or CFP+ cells in peripheral blood using flow cytometry (Bibi S et al., 2016).24 Weight loss >20%, ruffled coat, hunched back, weakness, reduced motility, were monitored daily and considered endpoints. All experiments were performed in compliance with the laws and protocols approved by animal ethics committees (Agreement APAFIS N°6743-2016091513513606 v4). For measurement of Glue activity ex vivo, peripheral blood of engrafted mice was collected in heparin pre-coated tubes. Glue activity was determined on 5 mΐ of plasma mixed with 100 mΐ of water soluble CTZ (50 gig/iTiL) followed by immediate acquisition of photon counts using a luminometer (Perkin Elmer) as previously described.
Quantification and Statistical Analysis
Statistics were calculated using Prism 6 software (Graphpad Software, Inc). Data were represented as mean ±SD. The significance of the differences between groups was determined via paired or unpaired t tests or two-way ANOVA unless otherwise indicated, details of which including values and number of replicates can be found in Figures and legends.
RESULTS
Metabolomic profiling of KIT-dependent SM.
To profile the metabolome of SM (Figure 1A), inventors used liquid chromatography coupled with hybrid quadrupole mass spectrometry to investigate the relative levels of metabolites across plasma samples from 25 patients with varying degree of SM severity and validated diagnosis (Figure IB). In total, high-throughput profiling identified 306 polar metabolites involved in all major metabolic pathways. Compared to healthy controls, the metabolic profiles for the patients with SM exhibited robust alterations across all metabolites identified (Figure 1C). As these differences could be representative of a biological system in a generally dysregulated physiological state rather than being specific to an aberrant MCs phenotype, inventors reduced the metabolome heterogeneity to an ISM-based z-score plot. Then, they were able to identify metabolic alterations in SM that are specific to advanced forms of the disease (Figure ID). Next, they investigated whether these differences could be used to discriminate patients according to their diagnosis. Principal component analysis (PGA) was used to investigate the general interrelations between indolent (ISM) and advanced (“AdvSM”) groups through analysis of clustering and outliers among the samples. The PCI versus PC2 score plot obtained from the PGA model
of the data set showed two clusters (Figure IE). To identify metabolite signatures, inventors screened for differentially abundant metabolites between ISM and AdvSM forms. They found a total of 59 out of the 306 metabolites to be differentially abundant between these two classes (p-vaiue<0.05, fold change >1.5). To visualize the relationships among the 59 altered metabolites, hierarchical unsupervised clustering was used to arrange the metabolites based on their relative levels across samples. Among the perturbed metabolites, 25 were up regulated in AdvSM samples, whereas 34 were down regulated. The metabolites mostly clustered according to disease aggressiveness rather than KIT mutation status. Together, these data show that, although SM is a heterogeneous disease, plasma metabolic profiles can be used to discriminate among different levels of SM aggressiveness, confirming the possibility of using metabolomics for patient stratification.
Metabolomic alterations associated with SM severity.
The most relevant metabolites responsible for the observed discrimination of SM samples were identified (false discovery rate-adjusted p-value <0.05, fold change >1.5; Figure 2A) and subjected to pathway analysis. Mapping the differential metabolic profiles to their respective biochemical pathways as outlined in the Kyoto Encyclopedia of Genes and Genomes (KEGG, release 92) showed that seven pathways were significantly altered in AdvSM compared to ISM (p-value <0.05). These metabolic perturbations were involved in nicotinate/nicotinamide, pyrimidine, purine, glycerophospholipid, and amino sugar/ nucleotide sugar metabolism, arginine biosynthesis and one carbon pool by folate metabolism, as shown in Figure 2B. These pathways provide building blocks for nucleotides, proteins, fatty acids, membrane lipids and ATP, which are required for the rapid growth of cells. Inventors found similar results using a new pathway covering approach that uses set theory (MetaCyc database) (Caspi R et al., 2020), rather than a statistical model, to suggest the smallest set of pathways that includes the differential metabolites between ISM and AdvSM (Figure 2C).
To confirm these findings at the RNA level in neoplastic MCs, inventors used DNA microarray data from a published study (Teodosio C et al., 2013) that analyzed the gene expression profiles of highly purified BM-derived MCs (BMMCs) from patients with ISM and ASM (one type of AdvSM) who carried the D816V KIT mutation. To identify transcripts involved in metabolic processes that were differentially expressed between ASM and ISM samples, inventors filtered the entire human genome to select only metabolic transcripts, which constitute 12% of the human genome (3638 transcripts) and encode for known metabolic enzymes or transporters (Figure 2D). Unlike the metabolomic approach, principal component analysis (PGA) of the transcriptomic data revealed a weak separation of ISM and ASM patients. However, using the Significance Analysis of Microarrays method, inventors found that 180 transcripts (34%) were significantly deregulated (upregulated) only in ASM compared to ISM samples (Figure 2E). Subsequent corrected distribution analysis showed that the ASM metabolic signature was highly enriched in pathways associated with oxidation-reduction processes (respiratory and mitochondrial electron transport) and amino acid glycosylation (glycosaminoglycan metabolism,
O-linked glycosylation) (Figure 2E,). These data recapitulated the findings at the metaboiomic levels as metabolites involved in both protein O-GlcNAcylation and, NAD biosynthesis (oxido-reduction) were identified among the most deregulated pathways (Figure 2C). Thus, the abundance and considerable diversity of transcripts encoding key enzymes in these two processes and the detection of intermediate metabolites of these pathways (the nicotinate/nicotinamide and amino sugar/nucleotide sugar metabolism pathways) in the serum of patients strongly indicate that AdvSM mainly presents with oxidative phosphorylation and protein glycosylation alterations.
GlcNAc is a circulating predictive metabolite of SM aggressiveness.
To determine whether any components of the 2 metabolic pathways that are significantly altered in AdvSM compared to ISM, as described above, are potential markers of disease aggressiveness, inventors performed a volcano plot analysis of the ISM versus AdvSM metabolite significance versus fold change (Figure 3A). Notably, compared with ISM samples, AdvSM patient samples showed significantly decreased dihydroxyacetone phosphate level (white dot), one of the major metabolites involved in glycolysis and markedly increased levels of GlcNAc, kynurenine, and quinolinate (black dots). GlcNAc is part of the hexosamine biosynthesis pathway that is responsible for protein glycosylation, and kynurenine/quinolinate are part of oxidative processes leading to the production of nicotinamide adenine dinucleotide (NAD). Inventors found that GlcNAc, kynurenine and quinolinate were significantly positively correlated in the same samples. GlcNAc was the most discriminant of the three metabolites in terms of fold change and significance, indicating that it is the most robust biomarker of AdvSM. To test this, inventors measured absolute concentrations of GlcNAc by LC-MS/MS in an independent cohort of randomly selected KIT D816V samples from the first cohort mixed with new randomly selected KIT D816V patient samples from the CEREMAST database. GlcNAc levels were significantly increased in ISM specimens (n=10) (2.8 mM±0.6) compared to samples from a healthy donor population (n=5) (0.46 mM±0.06). Additionally, there were even greater increases in GlcNAc levels in AdvSM serum samples (n=10) compared to ISM serum samples (n=10) (4.7 mM+1.12) (Figure 3B). The overall receiver-operating characteristic (ROC) curves for GlcNAc indicated that its AdvSM predictive value was high, with an area under the curve (AUC) of 0.97 for serum samples (Figure 3C). Notably, an AUC of 1.0 indicates perfect prediction, and an AUC of 0.5 indicates prediction equivalent to random selection. The concentration threshold to discriminate ISM from AdvSM was 3.8 mM, as calculated by the pROC package. Interestingly, GlcNAc concentration was significantly discriminant between patients with mast cell activation syndrome (MCAS) (2.7 mM±0.4) and allergic and asthmatic patients combined (1.8 mM+0.2). Definitive criteria for MCAS diagnosis do not exist yet and GlcNAc could be a useful tool for distinguishing different forms of mast-cell related pathologies. Inventors also measured plasmatic GlcNAc levels by LC-MS/MS in patients affected with acute myeloid leukemias (n=9), chronic myeloid leukemia (CML; n=10), myelodysplastic and myeloproliferative syndrome (MDS/MNP; n=7), and essential thrombocythemia (ET) only (n=10). Comparison with SM (ISM or
AdvSM), MCAS, allergic or asthmatic specimens shows that GlcNAc is a biomarker of diseases induced by mast cell activation but not of other non-mast cells-related myeloproliferative disorders (Figure 6).
Serum tryptase level is used as a minor criterion for SM as it also rises during anaphylaxis. We found that GlcNAc levels were not correlated with tryptase levels (Pearson r=0.2) in the same sample, suggesting that GlcNAc may be useful for identifying patients with modestly increased tryptase levels that are likely to have an aggressive disease phenotype (Figure 3D). GlcNAc levels were not correlated with any other chem ical parameters available for the samples in the cohort. However, inventors observed that high levels of GlcNAc were associated with AdvSM patients presenting gastrointestinal (GI) symptoms such as diarrhea, nausea, and abdominal pain due to MCs mediator release leading to a noticeable weight loss. Altogether these data indicate that GlcNAc is a circulating predictive metabolite of SM aggressiveness.
The hexosamine biosynthesis pathway (HBP) is activated in AdvSM.
GlcNAc fuels the hexosamine biosynthesis pathway (HBP) by the GlcNAc-salvage pathway, which is activated during glucose deficiency and has pro-survival effects (Figure 3E). GlcNAc enters the HBP pathway by the GlcNAc kinase (NAGK) that is activated under metabolic stress conditions. The HBP pathway activation leads to posttranslational modification of proteins by glycosylation, particularly O-GlcNAcylation of protein serine and/or threonine residues by O-linked GlcNAc transferase (OGT). b-N-acetylglucosaminidase is responsible for the reverse reaction. Inventors hypothesized that high levels of GlcNAc might correlate with increased HBP activity in AdvSM patients. To measure HBP activity in patients with MC-related diseases, protein extracts from BM aspirate were immunoblotted with an RL2 antibody that recognizes O-GlcNAc moieties on proteins. Three patient samples per group were assessed: ISM, AdvSM and MC activation syndrome (MCAS), a syndrome characterized by MC activation without clonal expansion of MCs. Variations in band intensities were noted, but all SM samples (ISM and AdvSM) exhibited evidence of increased O- GlcN Acylation compared to samples from patients with MCAS. The highest levels of O-GlcNAcylated proteins were found in the context of AdvSM (Figure 3F-G), for which bone marrow MCs infiltration is the highest. For comparison, previous studies have shown low levels of O-GlcNAcylated proteins in normal PBMCs and CD34+ human cells (Shi Y et al., 2012).
Because protein O-GlcNAcylation levels reflect the activity of the HBP pathway, the expression of a number of HBP enzymes was determined in human MCs sorted from patient BM with SM (ISM and aggressive SM (ASM), a form of AdvSM) and healthy donors (HD) from microarray analysis (Teodosio C et al., 2013). Consistent with the results obtained with RL2 antibody, most enzymes in this pathway (GFAT1, PGM3, NAGK, OGT and OGA) were up regulated in SM samples compared to HD control samples. The levels of the rate-limiting enzymes of glycolysis (PFKL) and the tricarboxylic acid cycle (TCA) (IDH2, AC02), pathways branched to HBP, were unchanged (Figure 3H). The transcripts levels of the enzymes of the HBP pathway were the same between ISM and ASM, which is in agreement
with PC A analysis showing that the transcriptomic profiles obtained in this microarray analysis were not predictive of disease severity. Inventors’ data showed that increased levels of GlcNAc in the plasma of patients with AdvSM were associated with the activation of the enzymes of the HBP pathway at the transcriptional level in sorted patient KIT D816V MCs and to high protein O-GlcNAcylation levels in patient’s BM aspirate enriched in MCs.
GlcNAc exacerbates the severity of a KIT D816V+ SM phenotype.
As high levels of GlcNAc are correlated to AdvSM, inventors tested the hypothesis that GlcNAc promotes proliferation of MCs carrying KIT D816V mutation. Human ROSAKIT D816v MCs incubated with GlcNAc showed significantly higher proliferation rates than their non-treated counterparts. GlcNAc had no effect on ROS AKIT WT cell proliferation during the course of the experiment. Assessment of BrdU incorporation confirmed these observations. The absence of an effect of GlcNAc on KIT WT MCs proliferation was not due to an inability of GlcNAc to enter the cells. Indeed, inventors found that GlcNAc was able to activate the HBP, as assessed by western blot analysis of O-GlcNAcylation levels. Therefore, inventors believe that differences in specific proteins O-GlcNAcylation that are not detectable by a bulk analysis, could be responsible for the difference in proliferation they observed between these two genotypes. These data suggest an oncogenic cooperation between KIT D816V signaling and GlcNAc leading to increased MCs proliferation.
ROSA KIT D816v MCs have been reported to efficiently engraft in NOD/SCID IL-2Ry ; (NSG) mice, giving rise to an ASM/MCL-like disease in vivo. To evaluate the effect of GlcNAc on the severity of the disease, inventors used a humanized in vivo model of KIT D816V advanced SM using a ROSA subclone, ROSA HT D816V Gluc engineered to naturally secrete Gaussia princeps luciferase (Glue) as a reporter (Bibi S et al, 2016). This model leads to an AdvSM phenotype in 4 weeks with neoplastic MCs invading the BM, blood and different vital organs. ROSAKIT D816V GIuc cells were derived from ROSA KIT D816v cells stably expressing the KITD816V mutant/GFP and transduced with a lentiviral vector expressing Glue and CFP. Disease progression is then monitored using Glue activity as a reporter and GFP+ (green fluorescence protein) and CFP+ (cyan fluorescent protein) as markers of Glue expressing cells. Therefore, inventors intravenously injected ROSA HT D816V Gluc cells (n=10) into irradiated NSG mice, and treated half of the cohort with either GlcNAc or PBS. Upon measuring the activity of Glue in the plasma of the GlcNAc-treated and PBS-treated controls, they found significantly higher luciferase activity in the GlcNAc-treated group than in the control group starting at week 4, and an almost 3 -fold increase was reached by week 7. The higher level of Glue activity was correlated with an increased number of Gluc+ cells, as analyzed by flow cytometry for the presence of GFP+ cells in peripheral blood (PB) samples. To further investigate disease progression, BM and spleen samples were analyzed for the presence of GFP+ ROSA KIT D816V Gluc cells. They observed increased numbers of medullary neoplastic MCs without increased invasion of SM clones into the spleen in the GlcNAc-treated group compared with the control group, despite the finding that 3 out of the 5 mice in the treated group presented
splenomegaly. In addition, they observed rapid health deterioration in the GlcNAc-treated mice associated with splenomegaly, which prompted them to sacrifice all the animals at week 7 instead of week 12, the previously described end point for a ROSA HT D816V Gluc cell-injected cohort. Overall, inventors’ data show that GlcNAc increases human KIT D816V MCs proliferation in vitro and exacerbates the severity of a KIT D816V SM model phenotype in vivo by strongly increasing circulating neoplastic MCs burden.
GlcNAc increases MC susceptibility to IgE-mediated stimulation.
To deter ine whether GlcNAc increases the susceptibility of neoplastic MCs to IgE and antigen-triggered activation, inventors studied the effect of GlcNAc on MC degranulation. For this assay, they used murine bone marrow-derived MCs (BMMC), as human MCs in culture express very low levels of the high-affinity IgE receptor, FcsRI (Saleh R et al., 2014). WT KIT BMMC and D814V KIT BMMC cells (carrying the murine homolog of the human KIT D816V mutation) were differentiated in culture with IL-3 from total BM of KitD814V/flox -Cre mice. Interestingly, MCs treated with GlcNAc showed a significant increased ability to degranulate, as indicated both by the release of b- hexosaminidase (an enzyme normally stored in cytoplasmic granules) into the culture supernatant and an increase of Annexin V staining as Annexin V levels increase at sites of secretory granule fusion with the plasma membrane during degranulation. The production of TNF-a, IL6 and IL-13 (cytokines secreted by MCs at high levels in response to IgE/antigen stimulation) was also increased in GlcNAc- treated KIT D814V BMMC, as assessed both by intracellular staining and ELISA for TNF-a. Interestingly, unlike the effect of GlcNAc on MCs proliferation, the increase in GlcNAc-induced MCs responses were independent of the mutational status of KIT. Because MCs stimulated with GlcNAc respond to IgE/antigen stimulation with enhanced cytokine production and degranulation activity, inventors assessed acute responses (which are primarily dependent on the release of MC granules) in an in vivo model of anaphylaxis-associated vascular permeability (passive cutaneous anaphylaxis). Ear MCs of KitD814V/flox -Cre mice pretreated with GlcNAc (or PBS) by intradermal injection were sensitized with anti-DNP IgE antibodies before systemic injection of antigen together with Evans blue dye. As a measure of MC activation, inventors quantified the cutaneous extravasation of the dye after extraction from the tissue. In agreement with the increased responses observed in GlcNAc-treated MCs in vitro, they observed significantly enhanced responses of GlcNAc-treated MCs during acute response in vivo. Overall, inventors’ data indicate that GlcNAc exacerbates MC responses.
CONCLUSION
Inventors herein describe for the first time the use of GlcNAc as a biomarker for diagnosing a disease induced by mast cell activation. Moreover, the level of concentration of GlcNAc in plasma sample of a subject allows to classify patients according to the disease they are affected with, in
particular, it is possible to discriminate between mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis (CM); mast cell activation syndrome (MCAS); asth a and allergy.
Although SM is a heterogeneous disease, inventors have been able to demonstrate that plasma metabolic profiling was a sufficiently sensitive means to discriminate among SM for s differing according to levels of aggressiveness. The correlation between the concentration of circulating GlcNAc and SM severity indicates that plasma GlcNAc detection is an efficient noninvasive approach by which to identify patients at greater risk of disease progression, thereby alerting physicians for the need for closer follow-up or the pursuit of early interventional therapy in these “high-risk” indolent cases. The absence of correlation the inventors observed between GlcNAc and tryptase concentrations in plasma suggests that GlcNAc levels are not dependent upon mast cell activation. Thus, GlcNAc is helpful to identify patients with modestly increased concentration of tryptase that are likely to have an advanced SM phenotype.
To a lesser extent, other biomarkers have been identified as being secondary indicators for diagnosing a disease induced by mast cell activation. These biomarkers belong to pathways involved in nicotinate/nicotinamide metabolism, pyrimidine metabolism, purine metabolism, glycerophospholipid metabolism, amino sugar and nucleotide sugar metabolism; arginine biosynthesis, and one carbon pool by folate metabolism.
Inventors also demonstrate that a variation in the transcript level of one or more enzymes involved in the hexosamine biosynthesis pathway (HBP) in mast cells, such as GFAT1, PGM3, NAGK, OGT and OGA can be considered as secondary indicators for diagnosing a disease induced by mast cell activation.
Inventors developed noninvasive methods using GlcNAc for diagnosing a disease induced by mast cell activation or for assessing the severity of a disease induced by mast cell activation; for monitoring the evolution of a disease induced by mast cell activation; or for determining the efficacy of a therapy of a disease induced by mast cell activation.
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Claims
1. Use of N-acetyl-D-glucosamine (GlcNAc) as a biomarker of a disease induced by mast cell activation selected from mastocytosis, in particular systemic mastocytosis (SM), mast cell sarcoma (MCS) or cutaneous mastocytosis (CM); mast cell activation syndrome (MCAS); asthma; and allergy.
2. An in vitro or ex vivo method for diagnosing a disease induced by mast cell activation in a subject, the method comprising the steps of:
(i) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject; and
(ii) diagnosing a disease induced by mast cell activation in the subject if the concentration of GlcNAc as measured in step (i) is above a reference GlcNAc ’s concentration.
3. The method according to claim 2, wherein the disease is systemic mastocytosis (SM), in particular non-advanced SM (non-AdvSM) or advanced SM (AdvSM), more particularly indolent SM (ISM), smoldering SM (SSM), SM with an associated hematological neoplasm (SM-AHN), aggressive SM (ASM), mast cell leukemia (MCL), even more particularly indolent SM (ISM) or aggressive SM (ASM).
4. An in vitro or ex vivo method for assessing the severity of systemic mastocytosis (SM) in a subject, in particular for differentially diagnosing between non-advanced SM (non-AdvSM) and advanced SM (AdvSM) in a subject, more particularly between indolent SM (ISM) and i) aggressive SM (ASM), ii) SM with an associated hematological neoplasm (SM-AHN) or iii) mast cell leukemia (MCL), and wherein the method comprises:
(a) measuring the concentration of N-acetyl-D-glucosamine (GlcNAc) in a biological sample obtained from a subject;
(b) comparing the concentration of GlcNAc in the biological sample of the subject with a reference concentration of GlcNAc and correlating the concentration of GlcNAc in the biological sample with the severity of the SM in the subject.
5. The in vitro or ex vivo method for assessing the severity of SM according to claim 4, wherein the reference control sample is a sample obtained from a healthy subject, and wherein a concentration of GlcNAc in the biological sample of the subject that is 6 to 8 times greater than the concentration of GlcNAc in the reference control sample is indicative of non-AdvSM, in particular indolent SM
(ISM); and a concentration of GlcNAc in the biological sample of the subject that is more than 8 fold greater than the concentration of GlcNAc in the reference control sample is indicative of AdvSM, in particular aggressive SM (ASM) or SM with an associated hematological neoplasm (SM-AHN).
6. The in vitro or ex vivo method for assessing the severity of SM according to claim 4, wherein a concentration of GlcNAc in the biological sample of the subject that is equal to or above the reference concentration of GlcNAc is indicative of AdvSM, in particular ASM or SM-AHN, and a concentration of GlcNAc in the biological sample of the subject that is below the reference concentration of GlcNAc is indicative of non- AdvSM, in particular ISM.
7. An in vitro or ex vivo method for assessing/predicting the risk for a subject affected or diagnosed with non- AdvSM, in particular ISM, to develop AdvSM, in particular ASM or SM-AHN, wherein the method comprises a step of determining the concentration of GlcNAc in a biological sample obtained from a subject affected or diagnosed with non-AdvSM, in particular ISM, wherein a concentration of GlcNAc above a reference concentration of GlcNAc indicates that the subject is at risk to develop AdvSM, in particular ASM or SM-AHN.
8. An in vitro or ex vivo method for monitoring the evolution of a disease induced by mast cell activation selected from mastocytosis, mast cell sarcoma, mast cell activation syndrome, asthma and allergy, preferably SM, in a subject affected with said disease, wherein the method comprises the steps of:
(a) measuring the concentration of GlcNAc in a sample obtained from a subject at To;
(b) measuring the concentration of GlcNAc in a distinct sample obtained from said subject at Ti, Ti being posterior to To; and
(c) comparing the concentrations of GlcNAc as measured in steps (a) and (b) to monitor the evolution of the disease in the subject, wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative of a favorable evolution of the disease in the subject, a concentration of GlcNAc as measured in step (b) identical to the concentration of GlcNAc as measured in step (a) is indicative of a stabilization of the disease in the subject, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative of an unfavorable evolution of the disease in the subject.
9. An in vitro or ex vivo method for determining the efficacy of a therapy of a disease induced by mast cell activation selected from mastocytosis, mast cell sarcoma, mast cell activation syndrome,
asthma and allergy, preferably SM, in a subject affected with said disease, wherein the method comprises the steps of:
(a) measuring the concentration of GlcNAc in a sample obtained from a subject prior to the administration of a therapy of a disease induced by mast cell activation;
(b) measuring the concentration of GlcNAc in a sample obtained from a subject once started the administration of a therapy of a disease induced by mast cell activation; and
(c) comparing the concentrations of GlcNAc as measured in steps (a) and (b), wherein, a concentration of GlcNAc as measured in step (b) below the concentration of GlcNAc as measured in step (a) is indicative that the therapy is effective in the treatment of the disease induced by mast cell activation, and a concentration of GlcNAc as measured in step (b) above the concentration of GlcNAc as measured in step (a) is indicative that the therapy is not effective in the treatment of the disease induced by mast cell activation; or, alternatively, wherein the method comprises the steps of:
(i) measuring the concentration of GlcNAc in a sample obtained from a subject once started the administration of a therapy of a disease induced by mast cell activation; and
(ii) comparing the concentration of GlcNAc as measured in step (i) with a reference concentration of GlcNAc, wherein, a concentration of GlcNAc as measured in step (i) below the GlcNAc reference concentration is indicative that the therapy is effective in the treatment of the disease induced by mast cell activation, and a concentration of GlcNAc as measured in step (i) above the GlcNAc reference concentration is indicative that the therapy is not effective in the treatment of the disease induced by mast cell activation.
10. The method according to claim 9, wherein the method further comprises a step of determining, in a biological sample from the subject, the transcript level of one or more enzymes involved in the hexosamine biosynthesis pathway (HBP), such as GFAT1, PGM3, NAGK, OGT and OGA.
11. The method according to anyone of claims 1 to 10, wherein the subject is a mammal, for example a human subject or a domestic animal, in particular a pet, more particularly a subject having a mutated kit gene or expressing a mutated KIT receptor.
12. The method according to anyone of claims 1 to 11, wherein the biological sample is a biological fluid sample, preferably selected from a blood, plasma, serum, urine and bone marrow aspirate sample; or a solid sample, preferably a bone marrow biopsy sample.
13. A kit for measuring the concentration of GlcNAc in a biological sample of a subject, wherein the kit comprises an anti-N-acetylglucosamine (GlcNAc) binding agent that selectively binds to GlcNAc and does not bind to O-GlcNAc; a molecule allowing the binding agent detection; or a N-
Acetyl-D-Glucosamine Kinase (NAGK), and optionally a leaflet providing GlcNAc reference concentration(s).
14. Use of a kit as described in claim 13 on a biological sample obtained from a subject, for diagnosing a disease induced by mast cell activation as described in claims 1 to 3; assessing the severity of systemic mastocytosis as described in claims 4 to 6; assessing/predicting the risk for a subject affected or diagnosed with non-AdvSM to develop AdvSM as described in claim 7, preferably prior to the onset of AdvSM’s symptoms; monitoring the evolution of a disease induced by mast cell activation as described in claim 8; and/or for determining the efficacy of a therapy of a disease induced by mast cell activation in the subject as described in claims 9 and 10.
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