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WO2018162404A1 - Biomarqueur pour l'issue chez des patients atteints de lam - Google Patents

Biomarqueur pour l'issue chez des patients atteints de lam Download PDF

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Publication number
WO2018162404A1
WO2018162404A1 PCT/EP2018/055330 EP2018055330W WO2018162404A1 WO 2018162404 A1 WO2018162404 A1 WO 2018162404A1 EP 2018055330 W EP2018055330 W EP 2018055330W WO 2018162404 A1 WO2018162404 A1 WO 2018162404A1
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dim
kir
cells
clusters
kir7cd57
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PCT/EP2018/055330
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English (en)
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Anne-Sophie CHRETIEN
Cyril FAURIAT
Daniel Olive
Christine ARNOULET
Norbert Vey
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université D'aix Marseille
Institut Jean Paoli & Irene Calmettes
Centre National De La Recherche Scientifique (Cnrs)
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Publication of WO2018162404A1 publication Critical patent/WO2018162404A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to a method for predicting the survival time of a patient suffering from acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • AML Acute Myeloid Leukemia
  • immune escape partly takes the form of NK cell subversion, which includes downregulation of NK triggering receptors such as NKp30, NKp46, DNAM-1 and NKG2D, and upregulation of NK inhibitory receptors such as KIR and NKG2A (Fauriat, Just-Landi et al. 2007; Sanchez-Correa, Gayoso et al. 2012; Khaznadar, Boissel et al. 2015; Sanchez-Correa, Campos et al. 2016). Therefore, and because NK cells are promising tools for therapeutic strategies, an exhaustive knowledge of NK cell dysfunctions in AML is mandatory.
  • NK cell maturation is a multistep process marked by differential expression of several markers, among which CD56, CD 16, NKG2A, KIR and CD57 are of particular importance (Bjorkstrom, Riese et al. (2010)).
  • CD56bright NK cells expressing low levels of CD 16 correspond to a transition between early immature CD56bright CD 16- NK cells and CD56dim CD 16+ NK cells (Beziat, Duffy et al. 2011 ; Frey, Packianathan et al. (1998); Cooper, Fehniger et al. (2001); Hayakawa and Smyth (2006)).
  • NK cells lose expression of NKG2A, and sequentially express KIR.
  • NK cells display different functions during the maturation process, such as migration capacities, cytotoxic functions, cytokine/chemokine production and response to cytokines (Cooley, Weisdorf et al. 2010; Khaznadar, Boissel et al. 2015; Hayakawa and Smyth (2006)). Given these functions are absolutely required for recognition and elimination of leukemic blasts, the clinical outcome may be affected by variations of sub-populations of NK cells with respect to maturation. For instance, increased NK maturation based on the percentage of CD57+ NK cells has been correlated with improved survival in both solid and hematologic malignancies (Nielsen, White et al.
  • NK maturation has been linked to the generation of CD56dim/CD57+/NKG2C+ NK cells defined as memory-like NK cells, and recent studies evidenced the anti-leukemic effect of this NK subpopulation (Muccio, Bertaina et al. 2016; Romee, Rosario et al. 2016; Wagner, Berrien-Elliott et al. 2016).
  • NK cells in AML patients display marked differences in NK maturation compared to healthy subjects, defining three distinct groups of patients according to NK maturation profiles (Chretien, Granjeaud et al. 2015). In the present study, they extended the maturation profile of NK cells in AML to more mature NK cells such as memory-like NK cells in addition to the previously described stages of maturation in a large multicenter cohort allowing them to statistically examine the impact of maturation defects on the clinical outcome of patients.
  • the invention relates to a method for predicting the survival time of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56bright, CD56dim, CD57 and KIR ii) dividing the NK cells is 5 different clusters: CD56bright, CD56dim/KIR-/CD57-, CD56dim/KIR+/CD57-, CD56dim/KIR-/CD57+ and CD56dim/KIR+/CD57+ clusters and iii) providing a good prognosis when the clusters CD56dim/KIR+/CD57-, CD56dim/KIR-/CD57+ and/or CD56dim/KIR+/CD57+ are in a majority compare to the clusters CD56bright, CD56dim/KIR-/CD57- and providing a bad prognosis when the clusters CD56bright and/or CD56dim/
  • a first aspect of the invention relates to a method for predicting the survival time of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bnght , CD56 dim , CD57 and KIR ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 “ , CD56 dim /KIR7CD57 “ , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the clusters CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and/or CD56 dim /KIR + /CD57 + are in a majority compare to the clusters CD56 bright , CD56 dil 7KIR " /CD57 " and providing a bad prognosis when the clusters CD
  • the invention relates to a method for predicting the survival time of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bright , CD56 dim , CD57 and KIR ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 “ , CD56 dim /KIR + /CD57 " , CD56 dil 7KIR7CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the clusters CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + are in a majority compare to the clusters CD56 bright , CD56 dil 7KIR " /CD5T and providing a bad prognosis when the clusters CD56 bright and CD56
  • the invention in another embodiment relates to a method for predicting the survival time of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bnght , CD56 dim , CD57 and KIR ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 " , CD56 dim /KIR + /CD57 " , CD56 dil 7KIR7CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the clusters CD56 dim /KIR + /CD57 " or CD56 dil 7KIR7CD57 + or CD56 dim /KIR + /CD57 + are in a majority compare to the clusters CD56 bright or CD56 dim /KIR " /CD5T and providing a bad prognosis when the clusters CD56 bright
  • the invention relates to a method for predicting the overall survival (OS) of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bnght , CD56 dim , CD57 and KIR ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 “ , CD56 dim /KIR + /CD57 " , CD56 dim /KIR /CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the clusters CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and/or CD56 dim /KIR + /CD57 + are in a majority compare to the clusters CD56 bright , CD56 dim /KIR " /CD5T and providing a bad prognosis when the clusters
  • the invention relates to a method for predicting the overall survival (OS) of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bright , CD56 dim , CD57 and KIR ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 " , CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the clusters CD56 dm VKIR + /CD57 ⁇ , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + are in a majority compare to the clusters CD56 bright , CD56 dim /KIR7CD57 " and providing a bad prognosis when the clusters CD56 bright and CD
  • the invention in another embodiment relates to a method for predicting the overall survival (OS) of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bright , CD56 dim , CD57 and KIR ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 " , CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the clusters CD56 dm VKIR + /CD57 ⁇ or CD56 dim /KIR7CD57 + or CD56 dim /KIR + /CD57 + are in a majority compare to the clusters CD56 bright or CD56 dim /KIR7CD57 " and providing a bad prognosis when the clusters CD56 bright or CD56
  • the invention relates to a method for predicting the relapse-free survival (RFS) of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bright , CD56 dim , CD57 and KIR ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 " , CD56 dim /KIR + /CD57 " , CD56 dim /KIR /CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the clusters CD56 dm VKIR + /CD57 ⁇ , CD56 dim /KIR7CD57 + and/or CD56 dim /KIR + /CD57 + are in a majority compare to the clusters CD56 bright , CD56 dim /KIR7CD57 " and providing a bad prognosis when
  • the invention relates to a method for predicting the relapse-free survival (RFS) of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bright , CD56 dim , CD57 and KIR ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 “ , CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the clusters CD56 dm VKIR + /CD57 " , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + are in a majority compare to the clusters CD56 bright , CD56 dim /KIR7CD57 " and providing a bad prognosis when the clusters CD56
  • the invention relates to a method for predicting the relapse-free survival (RFS) of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bright , CD56 dim , CD57 and KIR ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 " , CD56 dim /KIR + /CD57 " , CD56 dim /KIR /CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the clusters CD56 dm VKIR + /CD57 " or CD56 dim /KIR7CD57 + or CD56 dim /KIR + /CD57 + are in a majority compare to the clusters CD56 bright or CD56 dim /KIR7CD57 " and providing a bad prognosis when the clusters CD56
  • CD57 + and KIR + NK cells denotes NK cells with CD57 and KIR markers at their surface and CD57 " and KIR " NK cells denotes NK cells without CD57 and KIR markers at their surface or with CD57 and KIR at their surface but not detectable by conventional method like FACS.
  • CD56 bright or CD56 dim NK cells denotes NK cells with CD56 at their surface. These cells are thus CD56 + .
  • CD56 bright NK cells denotes NK cells with a high expression of CD56 and CD56 NK cells denotes NK cells with a low expression of CD56 (still detectable by conventional method like FACS).
  • OS Overall survival
  • AML AML
  • the overall survival rate is often stated as a two-year survival rate (notably for AML), which is the percentage of people in a study or treatment group who are alive two years after their diagnosis or the start of treatment.
  • relapse-free survival denotes the time between induction and relapse or death, whatever occurred first.
  • survival time regroups the terms OS and RFS.
  • KIR for "Killer- cell immunoglobulin-like receptors" (also known as CD15a,h and/or CD158bl,b2,j) denotes a family of type I transmembrane glycoproteins expressed on the plasma membrane of natural killer (NK) cells. They regulate the killing function of these cells by interacting with major histocompatibility (MHC) class I molecules, which are expressed on all nucleated cell types.
  • MHC major histocompatibility
  • CD56 also called Neural cell adhesion molecule (NCAM) denotes a homophilic binding glycoprotein expressed on the surface of neurons, glia, skeletal muscle and natural killer cells. NCAM has been implicated as having a role in cell-cell adhesion, neurite outgrowth, synaptic plasticity, and learning and memory.
  • NCAM Neural cell adhesion molecule
  • CD57 also called B3GAT1
  • B3GAT1 denotes a glycoprotein present on lymphocytes as well as cells of neural crest origin.
  • CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset (Lopez verges Blood 2010), and is a marker of replicative senescence on T cells (Sze et al 2001).
  • cluster denotes a population of specifics cells characterized by specifics markers. The inventors highlighted 5 different clusters:
  • the NK cells CD56 bnght cluster denotes a population of NK cells expressing in a high level (high expression) the cell surface marker CD56.
  • the NK cells CD56 /KIR7CD57 " cluster denotes a population of NK cells expressing in a low level (low expression) the cell surface marker CD56 (CD56 dim ), in a low level (low expression) the cell surface marker KIR (KIR ) and in a low level (low expression) the cell surface marker CD57 (CD57 ).
  • the NK cells CD56 dim /KIR7CD57 + cluster denotes a population of NK cells expressing in a low level (low expression) the cell surface marker CD56 (CD56 dim ), in a low level (low expression) the cell surface marker KIR (KIR ) and in a high level (high expression) the cell surface marker CD57 (CD57 + ).
  • the NK cells CD56 dim /KIR + /CD57 + cluster denotes a population of NK cells expressing in a low level (low expression) the cell surface marker CD56 (CD56 dim ), in a high level (high expression) the cell surface marker KIR (KIR + ) and in a high level (high expression) the cell surface marker CD57 (CD57 + ).
  • the inventors showed that when the clusters CD56 bright and CD56 dil 7CD577KIR " represent more than 60% of all the 5 clusters identified, the patient will have a bad prognosis and have an hypo -maturation profile and when the clusters CD56 dm VKIR + /CD57 ⁇ , CD56 dim /KIR7CD57 + and/or CD56 dim /KIR + /CD57 + represent more than 40% of all the 5 clusters, the patient will have a good prognosis and have intermediate maturation or hyper- maturation profile.
  • the invention relates to a method for predicting the survival time of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bnght , CD56 dim , CD57 and KIR ii) divide the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 “ , CD56 dim /KIR7CD57 " , CD56 dim /KIR /CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the frequency of clusters CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and/or CD56 dim /KIR + /CD57 + represent more than 40% of all the clusters and providing a bad prognosis when the frequency of clusters CD56 bnght and/or CD56 dm V
  • the invention relates to a method for predicting the overall survival (OS) of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bnght , CD56 dim , CD57 and KIR ii) divide the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 “ , CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + clusters and iii) providing a good prognosis when the frequency of clusters CD56 dim /KIR + /CD57 ⁇ , CD56 dim /KIR7CD57 + and/or CD56 dim /KIR + /CD57 + represent more than 40% of all the clusters and providing a bad prognosis when the frequency of clusters CD56 bnght and/or CD
  • the invention relates to a method for predicting relapse-free survival
  • RFS acute myeloid leukemia
  • AML acute myeloid leukemia
  • an optional step of statistical classifier or any machine learning algorithm can be added at the end of the method of the invention.
  • a step of classification of the patients with the k-neighbours algorithm see for example Fix, E. et al 1951
  • random forests see for example Leo Breiman, “Random Forests", Machine Learning, vol. 45, 2001, p. 5-32.
  • principal component analysis see for example Pearson K. 1901 "On Lines and Planes of Closest Fit to Systems of Points in Space”. Philosophical Magazine. 2 (11): 559-572
  • support vector machine see for example Cortes C. et al 1995. "Support- vector networks”. Machine Learning. 20 (3): 273-297
  • neural networks see for example Rosenblatt, F. 1958. "The Perceptron: A Probabilistic Model For Information Storage And Organization In The Brain”.
  • Psychological Review. 65 (6): 386-408 or any classifier
  • patient refers to an individual with symptoms of AML.
  • sample denotes, blood, peripheral-blood, serum, plasma, spinal cord, peripheral blood mononuclear cell (PBMC) or purified NK cells.
  • PBMC peripheral blood mononuclear cell
  • the sample is fresh or frozen.
  • measure of the expression level of the markers CD56, CD56 and KIR must be done. Measuring the expression level of CD56, CD57 and KIR can be done by measuring the gene expression level of CD56, CD57 and KIR or by measuring the level of the protein CD56, CD57 and KIR and can be performed by a variety of techniques well known in the art.
  • the expression level of a gene may be determined by determining the quantity of mRNA.
  • Methods for determining the quantity of mRNA are well known in the art.
  • the nucleic acid contained in the samples e.g., cell or tissue prepared from the patient
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
  • LCR ligase chain reaction
  • TMA transcription- mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
  • the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable label).
  • a "detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected either directly or indirectly.
  • a label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • detectable labels include fluorescent molecules (or fiuorochromes).
  • fluorescent molecules or fiuorochromes.
  • Numerous fiuorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies).
  • Examples of particular fiuorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule are provided in U.S. Pat. No.
  • fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphtho fluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron dif uoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties.
  • a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence.
  • Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671.
  • semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics.
  • semiconductor nanocrystals can he produced that emit light of different colors hased on their composition, size or size and composition.
  • quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif).
  • Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • radioisotopes such as 3 H
  • metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+
  • liposomes include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • enzymes for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • an enzyme can he used in a metallographic detection scheme.
  • SISH silver in situ hyhridization
  • Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate.
  • Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate.
  • an oxido-reductase enzyme such as horseradish peroxidase
  • Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
  • ISH in situ hybridization
  • a sample containing target nucleic acid sequence e.g., genomic target nucleic acid sequence
  • a metaphase or interphase chromosome preparation such as a cell or tissue sample mounted on a slide
  • a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence).
  • the slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization.
  • the sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids.
  • the probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium).
  • the chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
  • a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase.
  • fluorescein-labeled avidin or avidin-alkaline phosphatase For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin.
  • FITC fluorescein isothiocyanate
  • samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer).
  • AP alkaline phosphatase
  • Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties.
  • probes labeled with fluorophores including fluorescent dyes and QUANTUM DOTS®
  • fluorophores including fluorescent dyes and QUANTUM DOTS®
  • the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety.
  • a hapten such as the following non- limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin,
  • Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • the detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
  • the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH).
  • the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
  • multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample).
  • a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP.
  • the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn).
  • a first specific binding agent in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn
  • a second specific binding agent in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®,
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the methods of the invention comprise the steps of providing total R As extracted from cumulus cells and subjecting the R As to amplification and hybridization to specific probes, more particularly by means of a quantitative or semiquantitative RT-PCR.
  • the expression level is determined by DNA chip analysis.
  • DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
  • Expression level of a gene may be expressed as absolute expression level or normalized expression level.
  • expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGKl, TFRC, GAPDH, GUSB, TBP and ABL1. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
  • the level of the protein CD56, CD57 and KIR may also be measured and can be performed by a variety of techniques well known in the art.
  • protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.
  • CE-MS capillary electrophoresis-mass spectroscopy technique
  • ELISA ELISA
  • Detection of protein concentration in the sample may also be performed by measuring the level of the protein CD56, CD57 and KIR.
  • the "level of protein” or the “protein level expression” means the quantity or concentration of said protein.
  • the "level of protein” means the level of CD56, CD57 and KIR protein fragments.
  • the "level of protein” means the quantitative measurement of the protein CD56, CD57 and KIR expression relative to a negative control.
  • Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • the presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays such as competition, direct reaction, or sandwich type assays.
  • assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, capillary electrophoresis- mass spectroscopy technique (CE-MS). etc.
  • the reactions generally include revealing labels such as fluorescent, chemio luminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
  • Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value.
  • concentration of protein refers to an amount or a concentration of a transcription product, for instance the protein CD56, CD57 and KIR.
  • a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example.
  • relative units can be employed to describe a concentration.
  • concentration of proteins may refer to fragments of the protein CD56, CD57 and KIR.
  • fragment of CD56, CD57 and KIR protein may also be measured.
  • the detection of the level of CD56, CD57 and KIR can be performed by flow cytometry.
  • the method consists of determining the percentages of NK cells represented in the CD56 bnght cluster, and among the CD56 dim cells, the percentages of NK cells present in the KIR7CD5T, KIR + /CD57 " , KIR7CD57 + , KIR + /CD57 + clusters.
  • the extracellular part of the CD56, CD57 and KIR protein is detected.
  • Predetermined reference values used for comparison may comprise "cut-off or "threshold" values that may be determined as described herein.
  • Each reference (“cut-off) value for CD56, CD57 and KIR expression may be predetermined by carrying out a method comprising the steps of
  • e) optionally, classifying additional samples with any statistical classifier or machine learning algorithm such as hierarchical clustering or the k-neighbours algorithm.
  • the frequency of NK cells in the CD56 bnght cluster, and among the CD56 dim cells, the percentages of NK cells present in the KIR7CD57 " , KIR7CD57 “ , KIR7CD57 + , KIR + /CD57 + clusters has been assessed in 100 AML samples from 100 patients.
  • the 100 samples are classified into several groups according to the proportion of NK cells into the 5 different NK clusters described above, by hierarchical clustering, defining patients with NK hyper-maturation, NK intermediate maturation, and NK hypo-maturation.
  • Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art.
  • Such predetermined reference values of expression level may be determined for any gene defined above.
  • methods of the invention comprise measuring the expression level of at least one further biomarker or prognostic score.
  • biomarker refers generally to a cytogenetic marker, a molecule, the expression of which in a sample from a patient can be detected by standard methods in the art (as well as those disclosed herein), and is predictive or denotes a condition of the subject from which it was obtained.
  • cytogenetic marker a cytogenetic marker
  • prognostic scores may be combined to the biomarkers of the invention in order to improve some parameters such as the specificity (see for example Cornelissen et al. 2012).
  • the other biomarkers may be selected from the group of AML biomarkers consisting of cytogenetics markers (like t(8;21), t(15;17), inv(16) see for example Grimwade et al, 2010or Byrd et al, 2002), lactate dehydrogenase (see for example Haferlach et al 2003), FLT3, NPMl, CEBPa (see for example Thomasger et al, 2002, Dohner et al, 2010).
  • the prognostic scores that may be combined to NKp30 may be for example the disease risk index (DRI) (Armand et al 2012).
  • DRI disease risk index
  • biomarker NKp30 may add to the biomarkers of the invention.
  • biomarkers of the invention denotes the biomarker CD56, CD57, KIR.
  • NKp30 denotes a receptor of the natural cytotoxicity receptors (NCRs) family. NKp30 is a triggering receptor expressed on the plasmatic membrane of NK cells, also known as CD337 or NCR3.
  • another aspect of the invention relates to a method for predicting the survival time of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bnght , CD56 dim , CD57 and KIR and the expression level of NKp30 ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 “ , CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + clusters and comparing the expression level of NKp30 determined at step i) with its predetermined reference value and iii) providing a good prognosis when the clusters CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and/or CD56 dim /KIR + /CD57 + are in a majority compare to the cluster
  • another aspect of the invention relates to a method for predicting the survival time of a patient suffering from acute myeloid leukemia (AML) comprising i) determining in a sample obtained from the patient the proportion of NK cells expressing CD56 bright , CD56 dim , CD57 and KIR and the expression level of NKp30 ii) dividing the NK cells is 5 different clusters: CD56 bright , CD56 dim /KIR7CD57 " , CD56 dim /KIR + /CD57 " , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + clusters and comparing the expression level of NKp30 determined at step i) with its predetermined reference value and iii) providing a good prognosis when the clusters CD56 dim /KIR7CD57 " , CD56 dim /KIR7CD57 + and CD56 dim /KIR + /CD57 + are in a majority compare to the clusters CD56 bright , CD56
  • kits for performing the methods of the invention comprise means for measuring the expression level of the biomarkers of the invention in the sample obtained from the patient.
  • kits may include probes, primers macroarrays or microarrays as above described.
  • the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled.
  • probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers.
  • the kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
  • the kit of the invention may comprise amplification primers that may be pre- labelled or may contain an affinity purification or attachment moiety.
  • the kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.
  • a second aspect of the invention relates to a chemotherapeutic compound and/or an allogeneic stem cell transplantation (allo-SCT) for use in the treatment of AML in a patient with a bad prognosis as described above.
  • allo-SCT allogeneic stem cell transplantation
  • the invention relates to i) a chemotherapeutic compound, and ii) an allo-SCT, as a combined preparation for simultaneous, separate or sequential use in the treatment of AML in patient with a bad prognosis as described above.
  • the invention relates to a method for treating AML in a patient with a bad prognosis as described above comprising administering to said subject in need thereof a chemotherapeutic compound or a allogeneic stem cell.
  • the hematopoietic stem cells come from a donor related or not to the recipient but of the same species.
  • chemotherapeutic compounds may be selected in the group consisting in: fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, i
  • additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, anthracyclines, MDR inhibitors and Ca2+ ATPase inhibitors.
  • further therapeutic active agent can be administred to the patient.
  • This active agent can be a hematopoietic colony-stimulating factor.
  • Suitable hematopoietic colony stimulating factors include, but are not limited to filgrastim, sargramostim, molgramostim and epoietin alpha.
  • the chemotherapeutic compound is the cytarabine or the anthracycline.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • a third aspect of the invention relates to a therapeutic composition
  • a therapeutic composition comprising a chemotherapeutic compound and/or an allogenic stem cell for allo-SCT according to the invention for use in the treatment of AML in patient with a bad prognosis as described above.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Maturation profiles in the peripheral blood are representative of NK maturation profiles in the bone marrow.
  • A) Exemples of maturation profiles on appariated peripheral blood and bone marrow samples by flow cytometry.
  • FIG. 2 Defective NK maturation impacts clinical outcome.
  • OS overall survival
  • RFS Relapse-free survival
  • C Kaplan Meier curves of overall survival
  • RFS Relapse-free survival
  • C Kaplan Meier curves of NKp30 status refined by NK maturation status: patients were classified in two groups according to NKp30 expression. Among patients with high NKp30 expression, patients were divided into two groups according to NK maturation.
  • Figure 3 NK hypomaturation profile is associated low frequency of memory-like NK cells. NK alterations associated with the different maturation phenotypes were explored by mass cytometry.
  • PBMC from 17 additional patients with newly-diagnosed AML and 7 healthy volunteers were analyzed by mass cytometry.
  • Memory-like NK cells were defined as CD56dim/CD57+/NKG2C+ NK cells.
  • A Analysis of the percentage of memory-like NK cells by NK maturation group.
  • B Gating strategy of memory-like NK cells on a Spade tree
  • Allo-SCT allogeneic stem cell transplantation
  • Auto-SCT autologous stem cell transplantation
  • BM bone marrow
  • CR complete remission
  • FAB French- American-British classification
  • M male
  • F female
  • ITD internal tandem duplication
  • MDS myelodysplasia syndrome
  • NA not available
  • Nb number
  • t-AML therapy-related AML
  • OS overall survival
  • RFS Relapse-free survival.
  • Cox regression Multivariate Cox regression models were used to assess the predictive value of NK maturation profile while adjusting for the prognostic factors in the population (age at diagnosis, ELN, leukocytosis, and allogeneic stem cell transplantation as a time-dependent covariate).
  • allo-SCT allogeneic stem cell transplantation
  • CI confidence interval
  • RFS relapse-free survival
  • ELN European Leukemia Net genetic classification
  • HR hazard ratio
  • MDS myelodysplastic syndrome
  • OS overall survival
  • t-AML therapy-related AML.
  • a FACS LSR-Fortessa (BD Biosciences, San Jose, CA) was used for flow cytometry.
  • NK cells were immunostained with Krome Orange-conjugated anti-CD45, (ECD)-conjugated anti-CD3, allophycocyanin-alexafluor 700 (APC AF700)-conjugated anti-CD56, Phycoerythrin cyanin 7 (PC7)-conjugated anti-CD 158b l,b2j, Phycoerythrin cyanin 7 (PC7)- conjugated anti-CD158a,h (further referred to as KIR), Pacific Blue-conjugated anti-CD57, Phycoerythrin (PE)-conjugated NKp30 and Live/dead® Near-IR (Thermo Fisher Scientific, Waltham, MA). All the antibodies used in the study were a kind gift of Beckman-Coulter, Marseille, France.
  • PBMCs were thawed and washed with RPMI with 10% FCS, and incubated RPMI 2%FCS 1/10000 Pierce® Universal Nuclease 5kU (Thermo Fisher Scientific, Waltham, USA) at 37°C with 5% C02 for 30 minutes. Cells were washed and stained with Cisplatin 0.1M for dead cells exclusion. Cells were blocked with 0.5mg/mL Human Fc Block (BD Bioscience). Two million PBMCs were stained 45min at 4° with the extracellular antibodies. Cells were washed and barcoded with the Cell-IDTM 20-Plex Pd Barcoding Kit (Fluidigm) according to the manufacturer's recommendations.
  • NK cells were classified according to maturation markers expression as previously described (Chretien, Granjeaud et al. 2015). Briefly, patients and the mean of HV were clustered according to the percentages of NK cells represented in the CD56bright, KIR-/CD57-, KIR+/CD57-, KIR-/CD57+, KIR+/CD57+ clusters with MeV software using unsupervised hierarchical clustering (HClust, Euclidian distance).
  • OS overall survival
  • RFS relapse-free survival
  • a multivariate Cox regression model was used to assess the predictive value of NKp30 expression while adjusting for other prognostic factors (age at diagnosis, ELN, leukocytosis, and allogeneic stem cell transplantation as a time-dependent covariate).
  • the limit of significance was set at P ⁇ 0.05.
  • AML patients present distinct maturation profiles
  • NK cell subsets In Humans, four parameters define NK cell subsets according to the expression of NKG2A, KIR, CD57 and CD56 (Bjorkstrom, Riese et al. (2010); Lopez- Verges, Milush et al. (2010); Yu, Freud et al. (2013)).
  • CD56 bnght phenotype defines the most immature subset of circulating NK cells.
  • loss of NKG2A and acquisition of KIR and CD57 define several maturation stages. Baseline expression of the maturation parameters CD56, KIR and CD57 on Natural Killer (NK) cells was assessed by flow cytometry.
  • NKG2A was not informative in NK cell cluster definition and was therefore omitted in the clustering process (Chretien, Granjeaud et al. 2015).
  • patients and HV were classified according to the percentages of NK cells represented in the CD56 bright , CD56 dim /KIR7CD57 " , CD56 dim /KIR7CD57 " , CD56 dim /KIR7CD57 + , CD56 dim /KIR + /CD57 + clusters (data not shown). This representation enables to define three distinct groups of patients.
  • NK cells in AML patients display marked differences with regards to maturation, defining three distinct groups of patients, thus confirming previous results obtained in our pilot explorative cohort (Chretien, Granjeaud et al. 2015).
  • Maturation profiles in peripheral blood are representative of NK maturation in the bone marrow
  • NK cell defects in the bone marrow must be at least as pronounced as compared to NK cells from PB. Therefore, we tested whether the measurement of NK maturation parameters in PB accurately reflects NK maturation in BM.
  • cryopreserved BM samples were available.
  • BM NK cells were analyzed by flow cytometry according to the protocol described for peripheral PBMC.
  • NK maturation profiles were similar to the maturation profiles in PB (figure 1 A).
  • NKp30 low phenotype is associated with adverse clinical outcome in AML ((Fauriat, Just- Landi et al. 2007) and submitted manuscript).
  • the following groups were assembled: NKp30 low patients irrespective of maturation profile, NKp30 hlgh /hypomaturation profile patients, and NKp30 hlgh /intermediate or hypermaturation profile patients ( Figures 2C and 2D).
  • the NKp30 low and NKp30 hlgh /hypomaturation groups of patients displayed the worst clinical outcome with lower OS (P ⁇ 0.0001) and RFS (PO.0001).
  • NK hypomaturation profile is associated with a lack of generation of memory-like NK cells in AML
  • NK cells Maturation of NK cells is not terminated with the expression of CD57.
  • recent studies have identified a subset of NK cells expressing CD57 and NKG2C receptors and associated with memory- like properties (Cerwenka and Lanier 2016).
  • We next appended the maturation status of patients with the analysis of CD56 dim /CD57 + /NKG2C + NK cells.
  • PBMC from 17 additional patients with newly-diagnosed AML and 7 healthy volunteers were analyzed by mass cytometry. Classification of patients according to NK maturation profiles was performed as described above and allowed retrieving subgroups of patients based on maturation profile.
  • CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education.” Blood 116(19): 3853-3864. doi: 3810.1182 ood-2010-3804-281675.
  • CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset.

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Abstract

La présente invention porte sur un procédé de prédiction du temps de survie d'un patient souffrant de leucémie myéloïde aiguë (LAM). Les inventeurs ont déclaré que des lymphocytes NK des patients atteints de LAM présentent des différences marquées de maturation NK par rapport aux sujets sains, définissant trois groupes distincts de patients en fonction de profils de maturation NK. Dans cette étude, ils ont analysé le profil de maturation de lymphocytes NK dans une grande cohorte multicentrique (87 patients) leur permettant d'examiner statistiquement l'impact des défauts de maturation sur l'issue clinique des patients. Ainsi, l'invention porte sur un procédé de prédiction du temps de survie d'un patient souffrant de LAM consistant i) à déterminer dans un échantillon obtenu du patient la proportion de lymphocytes NK exprimant CD56bright, CD56dim, CD57 et KIR ii) à diviser les lymphocytes NK en 5 différents agrégats et iii) à émettre un pronostic favorable ou défavorable en fonction de la proportion des différents agrégats.
PCT/EP2018/055330 2017-03-06 2018-03-05 Biomarqueur pour l'issue chez des patients atteints de lam WO2018162404A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN112708675A (zh) * 2020-12-25 2021-04-27 中山大学肿瘤防治中心 骨髓nk细胞联合mcl1抑制剂在抗白血病中的应用
WO2022013256A1 (fr) * 2020-07-15 2022-01-20 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation de cd160 en tant que biomarqueur dans la leucémie aiguë myéloïde
CN114578055A (zh) * 2022-03-01 2022-06-03 暨南大学 γδT细胞的NKG2D/VSTM3作为检测急性髓性白血病标志物中的应用
WO2022236181A1 (fr) * 2021-05-07 2022-11-10 City Of Hope Procédés de détection de cellules nk dysfonctionnelles chez des patients leucémiques

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Publication number Priority date Publication date Assignee Title
WO2022013256A1 (fr) * 2020-07-15 2022-01-20 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation de cd160 en tant que biomarqueur dans la leucémie aiguë myéloïde
CN112708675A (zh) * 2020-12-25 2021-04-27 中山大学肿瘤防治中心 骨髓nk细胞联合mcl1抑制剂在抗白血病中的应用
WO2022236181A1 (fr) * 2021-05-07 2022-11-10 City Of Hope Procédés de détection de cellules nk dysfonctionnelles chez des patients leucémiques
CN114578055A (zh) * 2022-03-01 2022-06-03 暨南大学 γδT细胞的NKG2D/VSTM3作为检测急性髓性白血病标志物中的应用

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