WO2018162404A1 - Biomarker for outcome in aml patients - Google Patents

Abstract

The present invention relates to a method for predicting the survival time of a patient suffering from acute myeloid leukemia (AML). The inventors have reported that 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. In this study, they analyzed the maturation profile of NK cells in in a large multicenter cohort (87 patients) allowing them to statistically examine the impact of maturation defects on the clinical outcome of patients. Thus, the invention relates to a method for predicting the survival time of a patient suffering from 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 and iii) providing a good or bad prognosis in function of the proportion of the different clusters.

Description

BIOMARKER FOR OUTCOME IN AML PATIENTS

FIELD OF THE INVENTION:

The invention relates to a method for predicting the survival time of a patient suffering from acute myeloid leukemia (AML).

BACKGROUND OF THE INVENTION:

Acute Myeloid Leukemia (AML) is a hematologic malignancy with poor clinical outcome, in particular in patients over 60 years. To date, therapeutic options remain limited. Among hallmarks of cancer, escape to the immune system is generally involved in cancer progression. Besides, the three "E" theory (Mittal, Gubin et al. 2014) clearly defines the immune system as a corner stone of tumor progression and aggressiveness, and therefore of the response to treatment and prolonged remission. In AML, it has been particularly described, for NK cells, with the success of allogeneic stem cell transplantation with KIR/HLA mismatch (Norell, Moretta et al. ; Horowitz, Gale et al. 1990; Ruggeri, Mancusi et al. 2007; Cooley, Weisdorf et al. 2010). In this pathology, 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. More recently, we and others (Mundy-Bosse, Scoville et al. 2016) have evidenced a drastic reduction of immature NK cells in AML patients. However, a more comprehensive view of the NK cell maturation status of NK cells in AML is lacking.

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)). First of all, 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)). Subsequently, NK cells lose expression of NKG2A, and sequentially express KIR. Expression of CD57 marks the acquisition of high cytotoxic potential and decrease of proliferation capacities. Accordingly, 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. 2015). In addition, CMV-induced 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).

SUMMARY OF THE INVENTION:

The inventors have recently reported that 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.

Thus, 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/KIR-/CD57- are in a majority compare to the clusters CD56dim/KIR+/CD57-, CD56dim/KIR-/CD57+ and/or CD56dim/KIR+/CD57+. Particularly, the invention is described by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

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^dil7KIR^" /CD57^" and providing a bad prognosis when the clusters CD56^bright and/or CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD57^", CD56^dim/KIR7CD57⁺ and/or CD56^dim/KIR7CD57⁺.

In one embodiment 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^dil7KIR7CD57⁺ 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^dil7KIR^" /CD5T and providing a bad prognosis when the clusters CD56^bright and CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD57^", CD56^dil7KIR7CD57⁺ and CD56^dim/KIR⁺/CD57⁺.

In another embodiment 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/KIR⁺/CD57^", CD56^dil7KIR7CD57⁺ and CD56^dim/KIR⁺/CD57⁺ clusters and iii) providing a good prognosis when the clusters CD56^dim/KIR⁺/CD57^" or CD56^dil7KIR7CD57⁺ 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 or CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD57^" or CD56^dim/KIR /CD57⁺ or CD56^dim/KIR⁺/CD57⁺.

In one embodiment 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 CD56^bright and/or CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD57^", CD56^dim/KIR7CD57⁺ and/or CD56^dim/KIR⁺/CD57⁺.

In another embodiment 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^dmVKIR⁺/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 CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD57^", CD56^dim/KIR 7CD57⁺ and CD56^dim/KIR⁺/CD57⁺.

In another embodiment 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^dmVKIR⁺/CD57^~ or CD56^dim/KIR7CD57⁺ or CD56^dim/KIR⁺/CD57⁺ are in a majority compare to the clusters _CD56bright _or CD56^dim/KIR7CD57^" and providing a bad prognosis when the clusters CD56^bright or CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD57^" or CD56^dim/KIR 7CD57⁺ or CD56^dim/KIR⁺/CD57⁺.

In one embodiment 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^dmVKIR⁺/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 clusters CD56^bright and/or CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD57^", CD56^dim/KIR 7CD57⁺ and/or CD56^dim/KIR⁺/CD57⁺. In another embodiment 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^dmVKIR⁺/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 CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD57^", CD56^dim/KIR7CD57⁺ and CD56^dim/KIR⁺/CD57⁺.

In another embodiment 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^dmVKIR⁺/CD57^" or CD56^dim/KIR7CD57⁺ or CD56^dim/KIR⁺/CD57⁺ are in a majority compare to the clusters _CD56bright _or CD56^dim/KIR7CD57^" and providing a bad prognosis when the clusters CD56^bright or CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD57^" or CD56^dim/KIR 7CD57⁺ or CD56^dim/KIR⁺/CD57⁺.

As used herein, when the clusters CD56^dmVKIR⁺/CD57^", CD56^dim/KIR7CD57⁺ and/or CD56^dim/KIR⁺/CD57⁺ are in a majority compare to the clusters CD56^bright, CD56^dim/KIR^" /CD57^" the patients are classified as intermediate maturation or hyper-maturation profile.

As used herein, when the clusters CD56^bright and/or CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD5T, CD56^dim/KIR7CD57⁺ and/or CD56^dim/KIR⁺/CD57⁺ the patients are classified as hypomaturation profile.

As used herein and accordingly to the scientifically nomenclature, 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.

As used herein, 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).

As used herein, the term "Overall survival (OS)" denotes the time from diagnosis of a disease such as AML (according to the invention) until death from any cause. 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.

As used herein, the term "relapse-free survival" (RFS) denotes the time between induction and relapse or death, whatever occurred first.

As used herein and according to the invention, the term "survival time" regroups the terms OS and RFS.

As used herein, the term "Good Prognosis" denotes a patient with significantly enhanced probability of survival after treatment.

As used herein and according to all aspects of the invention, the term "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.

As used herein and according to all aspects of the invention, the term "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.

As used herein and according to all aspects of the invention, the term "CD57" also called 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).

As used herein the term "cluster" denotes a population of specifics cells characterized by specifics markers. The inventors highlighted 5 different clusters:

Thus, 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/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 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⁺).

More, the inventors showed that when the clusters CD56^bright and CD56^dil7CD577KIR^" 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^dmVKIR⁺/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.

Thus, in other word, 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^dmVKIR^~ /CD57^" represent more than 60% of all the clusters. Thus, in other word, 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 CD56^dmVKIR^~ /CD57^" represent more than 60% of all the clusters.

Thus, in other word, the invention relates to a method for predicting 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 ofNK 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/orCD56^dmVKIR^~ /CD57^" represent more than 60% of all the clusters.

According to the invention, an optional step of statistical classifier or any machine learning algorithm can be added at the end of the method of the invention. For example 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, can be added.

As used herein, the term "patient" refers to an individual with symptoms of AML.

As used herein and according to all aspects of the invention, the term "sample" denotes, blood, peripheral-blood, serum, plasma, spinal cord, peripheral blood mononuclear cell (PBMC) or purified NK cells. In one embodiment, the sample is fresh or frozen.

For obtaining the proportion of the 5 different clusters highlighted by the inventors, 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.

Typically, 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. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).

Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

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.

Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, 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. Thus, 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 (such as a probe generated by the disclosed methods) 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.

Particular examples of detectable labels include 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 (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene-1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifiuoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 - (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- 1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDarnino fluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difiuorofiuorescein (OREGON GREEN®); fiuorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitro tyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamme B, sulforhodamme 101 and sulfonyl chloride derivative of sulforhodamme 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable 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. Other 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. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the f uorochromes described above, 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. When semiconductor nanocrystals are illuminated with a primary energy source, 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 that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al, Science 281 :20132016, 1998; Chan et al, Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Puhlication No. 2003/0165951 as well as PCT Puhlication No. 99/26299 (puhlished May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors hased on their composition, size or size and composition. For example, 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.

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.

Alternatively, an enzyme can he used in a metallographic detection scheme. For example, silver in situ hyhridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. 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. (See, for example, U.S. Patent Application Puhlication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). 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. (See, for example, U.S. Pat. No. 6,670,113).

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). In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with 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.

For example, a biotinylated probe can be detected using 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. For detection by enzyme activity, 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). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al, Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al, Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al, Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al, Am. .1. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.

Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, 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. 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. 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.

In other examples, 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). As indicated above, 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.

It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, 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). For example, 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. Following exposure of the sample to the probes, 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). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays. 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. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. 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. Such 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.

In a particular embodiment, 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.

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such 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. To determine the expression level, 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. Typically, 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.

According to the invention, 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.

Typically protein concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.

Detection of protein concentration in the sample may also be performed by measuring the level of the protein CD56, CD57 and KIR. In the present application, the "level of protein" or the "protein level expression" means the quantity or concentration of said protein. In another embodiment, the "level of protein" means the level of CD56, CD57 and KIR protein fragments. In still another embodiment, 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. Such 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.

More particularly, 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. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the protein CD56, CD57 and KIR. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the protein CD56, CD57 and KIR. Thus, in a particular embodiment, fragment of CD56, CD57 and KIR protein may also be measured.

In a particular embodiment, the detection of the level of CD56, CD57 and KIR can be performed by flow cytometry. When this method is used, 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.

In another embodiment, 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

a) providing a collection of samples from patients suffering of AML (after diagnosis of AML or after complete remission); b) determining the proposition of each of the 5 clusters (by measuring the expression level of CD56, CD57 and KIR) of the invention for each sample contained in the collection provided at step a);

c) grouping the tissue samples (NK cells) according the proportion of cells in the clusters defined at step b)

d) classifying said samples into several groups according to the proportions of NK cells clusters into the different NK subsets described above; using any statistical classifier or machine learning algorithm, such as hierarchical clustering, defining patients with NK hyper- maturation, NK intermediate maturation, and NK hypo -maturation;

e) optionally, classifying additional samples with any statistical classifier or machine learning algorithm such as hierarchical clustering or the k-neighbours algorithm.

For example 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.

The man skilled in the art also understands that the same technique of assessment of the expression level of a gene should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a gene of a patient subjected to the method of the invention.

Such predetermined reference values of expression level may be determined for any gene defined above.

In a further embodiment of the invention, methods of the invention comprise measuring the expression level of at least one further biomarker or prognostic score.

The term "biomarker", as used herein, 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. Various validated prognostic biomarkers or 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).

For example, 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 Schnittger 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).

Particularly, the biomarker NKp30 may add to the biomarkers of the invention.

As used herein, the term "biomarkers of the invention" denotes the biomarker CD56, CD57, KIR.

As used herein and according to all aspects of the invention, the term "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.

Thus, 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 clusters CD56^bright, CD56^dim/KIR7CD57^~ and when the expression level determined at step i) for NKp30 is higher than its predetermined reference value, providing an intermediate prognosis when the clusters CD56^bright and/or CD56^dmVKIR7CD57^~ are in a majority compare to the clusters CD56^dim/KIR⁺/CD5T, CD56^dim/KIR7CD57⁺ and/or CD56^dim/KIR⁺/CD57⁺ and when the expression level determined at step i) for NKp30 is higher than its predetermined reference value and providing a bad prognosis when the expression level determined at step i) for NKp30 is lower than its predetermined reference value.

Thus, 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^dil7KIR7CD57^~ and when the expression level determined at step i) for NKp30 is higher than its predetermined reference value, providing an intermediate prognosis when the clusters CD56^bright and CD56^dmVKIR7CD57^~ are in a majority compare to the clusters CD56^dim/KIR7CD57^", CD56^dim/KIR 7CD57⁺ and CD56^dim/KIR⁺/CD57⁺ and when the expression level determined at step i) for NKp30 is higher than its predetermined reference value and providing a bad prognosis when the expression level determined at step i) for NKp30 is lower than its predetermined reference value.

A further object of the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of the biomarkers of the invention in the sample obtained from the patient.

The kits may include probes, primers macroarrays or microarrays as above described. For example, the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, 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. Alternatively 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.

In another aspect, 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. In another embodiment, 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.

According to invention, when the patient receives allo-SCT, the hematopoietic stem cells come from a donor related or not to the recipient but of the same species.

According to the invention, 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, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, temolozomide and lavendustin A. In one embodiment, 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.

In another embodiment, 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.

In a particular embodiment, the chemotherapeutic compound is the cytarabine or the anthracycline.

As used herein, the term "treatment" or "treat" refer 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. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at 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 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.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

Particularly, the pharmaceutical compositions contain 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.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

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

FIGURES:

Figure 1: 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. B) Correlation between the frequency of NK cells in the different NK maturation clusters (Pearson correlation). Hyper: hypermaturation; Hypo: hypomaturation; Interm: intermediate maturation.

Figure 2: Defective NK maturation impacts clinical outcome. Kaplan Meier curves of overall survival (OS) (A) and Relapse-free survival (RFS) (B) by NK maturation profile at diagnosis. Kaplan Meier curves (C) and (D) display OS and RFS by 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

Table 1 : Baseline patients characteristics (1/2)

d agnos s .

Table 1 : Baseline patients characteristics (2/2)

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.

Table 2: 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).

Abbreviations: 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.

EXAMPLE:

Material & Methods

Patients and study design

Baseline maturation profile on NK cells at diagnosis was assessed in a total of 87 patients from the LAM2006IR prospective multicenter randomized trial (NCT00860639) of the Groupe Quest Est d'Etude des Leucemies Aigues et autres Maladies du Sang (GOELAMS). Patient samples were collected between November 2007 and April 2012. All patients had previously untreated AML with intermediate cytogenetics. Patients received conventional 3+7 induction chemotherapy with or without the addition of Gemtuzumab Ozogamicin (Hills, Castaigne et al. 2014). Patients with acute promyelocytic leukemia AML and patients above 66 years were excluded. The CMV status was not available. All participants gave written informed consent in accordance with the Declaration of Helsinki. The entire research procedure was approved by the ethical review boards from the IPC and the GOELAMS.

Clinical samples

Peripheral blood mononuclear cells (PBMC) cryopreserved in 90%FCS / 10%DMSO were obtained from randomly selected patients before induction chemotherapy and from healthy volunteers (N=19). Handling, conditioning and storing of samples were performed by the FILOtheque AML (N° BB-0033-00073), tumor bank of the FILO group, Cochin hospital, Paris.

Flow cytometry

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.

Mass cytometry analysis

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-ID™ 20-Plex Pd Barcoding Kit (Fluidigm) according to the manufacturer's recommendations. Cells were washed and samples were combined and stained with metal-labeled anti-PE secondary antibodies 30 min at 4°. Cells were washed and permeabilized with Foxp3 Staining Buffer Set (eBioscience, San Diego, CA, USA) 40 minutes a 4°. Cells were incubated with 0.5mg/mL Human Fc Block 40min at 4°, and stained 40min at 4° in Foxp3 Staining Buffer with the intracellular antibodies. Then cells were washed and labeled overnight with 125nM iridium intercalator (Fluidigm) in Cytofix (BD Biosciences). Finally, cells were diluted in EQTM Four Element Calibration Beads (Fluidigm) before acquisition on a CyTOF2® instrument (Fluidigm).

Patient classification

Patients 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).

Statistical analysis

Statistical analyses were carried out using Graph Pad Prism (Graph Pad Software, San Diego, CA) and SPSS (SPSS software, Chicago, IL). For multiple comparisons, a Kruskal- Wallis test was used followed by a Dunn's post-test. Association between variables was assessed using the Spearman correlation coefficient. For survival analyses, overall survival (OS) was defined as the time from diagnosis until death from any cause, and relapse-free survival (RFS) as the time between induction and relapse or death, whatever occurred first. Patients without an event were censored at the time of their last follow-up. Survival times were estimated by Kaplan-Meier method and compared using the log-rank test. 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.

Results

Baseline patient characteristics

The patient characteristics, stratified by NK maturation profile, are summarized in Table

1. All patients had intermediate cytogenetics. The mean age (±SD) at induction was 46.9 years (±11.3). Median follow-up after diagnosis was 24.9 months. Cytogenetic classification and European Leukemia Net (ELN) genetic classification (Dohner, Estey et al. 2010) (FLT3/CEBPa/NPMl mutational status) were routinely determined in the Biopathology departments of the centers involved in this study.

AML patients present distinct maturation profiles

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. In CD56^dim 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. As previously described, NKG2A was not informative in NK cell cluster definition and was therefore omitted in the clustering process (Chretien, Granjeaud et al. 2015). Using unsupervised hierarchical clustering, 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. Among the 87 patients, 8 (9.2%) had low NK maturation profile, with most NK cells in the CD56^bright and CD56^dim/KIR7CD57^" immature clusters (hypomaturation group); 21 patients (24.1%) had hyper maturation profile, with most NK cells in the CD56dim/KIR+/CD57+ cluster; 58 patients (66.7%) had intermediate maturation profile, with NK cells distributed into the CD56^dil7KIR ^/+/CD57^+/- clusters (Table 1).

In conclusion, we observed that 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

As AML develops in the bone marrow before disseminating in the periphery, we hypothesized that NK cell defects in the bone marrow (BM) 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. For 28 patients of the cohort, cryopreserved BM samples were available. BM NK cells were analyzed by flow cytometry according to the protocol described for peripheral PBMC. In BM, NK maturation profiles were similar to the maturation profiles in PB (figure 1 A). We compared the frequency of NK cells in the CD56^bright, CD56^dim/KIR /CD57^", CD56^dim/KIR⁺/CD57^", CD56^dim/KIR^" /CD57⁺ and CD56^dim/KIR⁺/CD57⁺ clusters in the PB and in the BM with the Pearson correlation coefficient. As expected, BM contained more CD56bright NK cells compared to PB (10.1 vs 3.9%), respectively; P<0.001) and a moderate correlation was observed between the frequencies of CD56^bright NK cells in PB and in BM (r²=0.32, P=0.05, Figure 2B). For all the other clusters of NK cells, correlations coefficients were high (CD56^dim/KIR /CD57^": r²=0.69, PO.0001; CD56^dim/KIR7CD57^": r²=0.96, PO.0001; CD56dim/KIR-/CD57+: r²=0.87, PO.0001; CD56dim/KIR+/CD57+: r²=0.79, P .0001), with no significant difference between PB and BM (Figure 1 B). Thus, the maturation profile of NK cells in PB is representative of the maturation status of NK cells in BM.

Defective NK maturation impacts clinical outcome

Patients were divided into three groups as defined above (NK hypomaturation, intermediate maturation and hypermaturation). In univariate analysis, significant differences in overall survival (OS) (P=0.0006) and relapse-free survival (RFS) (P<0.0001) were observed among the different groups (Figure 2A and B). Patients with hypomaturation profile had reduced OS, with 3-year OS rates of 12.5% vs 57.1% and 57.4% for patients with intermediate and hypermaturation, respectively. Consistently, patients with hypomaturation profile had reduced RFS, with 3-year RFS rates of 0%> vs 52.6% and 73.3% for patients with intermediate and hypermaturation, respectively. In multivariate Cox regression models, NK hypomaturation remained significantly associated with reduced OS and RFS, independent of other factors (HR (hazard ratio)=4.15, P=0.004 and HR=8.23, P=0.003, respectively) (Table 2).

Interestingly, the group of patients with NK hypomaturation did not correspond to the group of patients with NKp30^low phenotype, a NK triggering receptor down-regulated in AML. Since NKp30^low phenotype is associated with adverse clinical outcome in AML ((Fauriat, Just- Landi et al. 2007) and submitted manuscript), we combined maturation-based classification with NKp30 status (data not shown). 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

Maturation of NK cells is not terminated with the expression of CD57. Hence, 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. We then assessed, in the CD56^dim clusters, the percentage of memory-like NK cells, defined as CD56^dim/CD57⁺/NKG2C⁺ NK cells (Lopez- Verges, Milush et al. 2011; Muccio, Bertaina et al. 2016). The frequency of memory- like NK cells was higher, but non-significant, in patients with intermediate or hypermaturation than in healthy subjects (9.0% vs 4.5%, respectively). However, there was a huge difference in frequency of memory-like NK cells between patients with NK hypomaturation profile compared to patients with intermediate or hypermaturation profile (1.5% vs 9.0%, respectively, P<0.01) (Figure 3A and B).

REFERENCES:

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

Bendall, S. C, E. F. Simonds, et al. (2011). "Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum." Science 332(6030): 687-696.

Beziat, V., D. Duffy, et al. (2011). "CD56brightCD16+ NK cells: a functional intermediate stage of NK cell differentiation." J Immunol 186(12): 6753-6761.

Bjorkstrom, N. K., P. Riese, et al. ((2010)). "Expression patterns of NKG2A, KIR, and

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.

Cerwenka, A. and L. L. Lanier (2016). "Natural killer cell memory in infection, inflammation and cancer." Nature Reviews Immunology 16(2): 112-123.

Chretien, A.-S., S. Granjeaud, et al. (2015). "Increased NK cell maturation in patients with Acute Myeloid Leukemia." Frontiers in Immunology 6.

Cooley, S., D. J. Weisdorf, et al. (2010). "Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia." Blood 116(14): 2411-2419. doi: blood-2010-2405-283051.

Cooper, M. A., T. A. Fehniger, et al. (2001). "The biology of human natural killer-cell subsets." Trends Immunol 22(11): 633-640.

Cooper, M. A., T. A. Fehniger, et al. ((2001)). "The biology of human natural killer-cell subsets." Trends Immunol 22(11): 633-640. doi: S1471-4906(1401)02060-02069.

Dohner, FL, E. Estey, et al. (2016). "Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel." Blood: blood-2016-2008-733196.

Dohner, FL, E. H. Estey, et al. (2010). "Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet." Blood 115(3): 453-474. Fauriat, C, S. Just-Landi, et al. (2007). "Deficient expression of NCR in NK cells from acute myeloid leukemia: Evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction." Blood 109(1): 323-330.

Fix, E., Hodges, J.L. (1951); Discriminatory analysis, nonparametric discrimination: Consistency properties. Technical Report 4, USAF School of Aviation Medicine, Randolph Field, Texas.

Frey, M., N. B. Packianathan, et al. ((1998)). "Differential expression and function of L-selectin on CD56bright and CD56dim natural killer cell subsets." J Immunol 161(1): 400- 408.

Hayakawa, Y. and M. J. Smyth ((2006)). "CD27 dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity." J Immunol 176(3): 1517-1524. doi: 1 176/1513/1517.

Hills, R. K., S. Castaigne, et al. (2014). "Addition of gemtuzumab ozogamicin to induction chemotherapy in adult patients with acute myeloid leukaemia: a meta-analysis of individual patient data from randomised controlled trials." The Lancet Oncology 15(9): 986- 996.

Horowitz, M. M., R. P. Gale, et al. (1990). "Graft- versus- leukemia reactions after bone marrow transplantation." Blood 75(3): 555-562.

Khaznadar, Z., N. Boissel, et al. (2015). "Defective NK Cells in Acute Myeloid Leukemia Patients at Diagnosis Are Associated with Blast Transcriptional Signatures of Immune Evasion." J Immunol.

Kong, Y., J. Zhang, et al. (2015). "PD-lhiTIM-3&plus; T cells associate with and predict leukemia relapse in AML patients post allogeneic stem cell transplantation." Blood cancer journal 5(7): e330.

Le Dieu, R., D. C. Taussig, et al. (2009). "Peripheral blood T cells in acute myeloid leukemia (AML) patients at diagnosis have abnormal phenotype and genotype and form defective immune synapses with AML blasts." Blood 114(18): 3909-3916.

Lopez- Verges, S., J. M. Milush, et al. ((2010)). "CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset." Blood 116(19): 3865-3874. doi:3810.1182/blood-2010-3804-282301.

Lopez- Verges, S., J. M. Milush, et al. (2011). "Expansion of a unique CD57+ NKG2Chi natural killer cell subset during acute human cytomegalovirus infection." Proceedings of the National Academy of Sciences 108(36): 14725-14732. Mittal, D., M. M. Gubin, et al. (2014). "New insights into cancer immunoediting and its three component phases—elimination, equilibrium and escape." Curr Opin Immunol 27: 16-25.

Muccio, L., A. Bertaina, et al. (2016). "Analysis of memory- like natural killer cells in human cytomegalovirus-infected children undergoing alphabeta+T and B cell-depleted hematopoietic stem cell transplantation for hematological malignancies." Haematologica 101(3): 371-381.

Mundy-Bosse, B. L., S. D. Scoville, et al. (2016). "MicroRNA-29b mediates altered innate immune development in acute leukemia." The Journal of clinical investigation 126(12).

Nielsen, C. M., M. J. White, et al. (2015). "Functional significance of CD57 expression on human NK cells and relevance to disease." Molecular mechanisms regulating cytotoxic lymphocyte development and function, and their associations to human diseases: 137.

Norell, H., A. Moretta, et al. "At the Bench: Preclinical rationale for exploiting NK cells and gammadelta T lymphocytes for the treatment of high-risk leukemias." J Leukoc Biol 94(6): 1123-1139.

Richards, J. O., X. Chang, et al. (2006). "Tumor growth impedes natural-killer-cell maturation in the bone marrow." Blood 108(1): 246-252.

Richards, J. O., X. Chang, et al. (2006). "Tumor growth impedes natural-killer-cell maturation in the bone marrow." Blood 108(1): 246-252.

Romee, R., M. Rosario, et al. (2016). "Cytokine-induced memory- like natural killer cells exhibit enhanced responses against myeloid leukemia." Sci Transl Med 8(357): 357ral23.

Ruggeri, L., A. Mancusi, et al. (2007). "Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value." Blood 110(1): 433-440.

Sanchez-Correa, B., C. Campos, et al. (2016). "Natural killer cell immunosenescence in acute myeloid leukaemia patients: new targets for immunotherapeutic strategies?" Cancer immunology, immunotherapy: CII 65(4): 453.

Sanchez-Correa, B., I. Gayoso, et al. (2012). "Decreased expression of DNAM-1 on NK cells from acute myeloid leukemia patients." Immunology and Cell Biology 90(1): 109-115.

Sivori, S., M. Falco, et al. (2002). "Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation." Proc Natl Acad Sci U S A 99(7): 4526-4531.

Sun, J. C, J. N. Beilke, et al. (2009). "Adaptive immune features of natural killer cells." Nature 457(7229): 557-561. Sze DM, Giesajtis G, Brown RD, Raitakari M, Gibson J, Ho J, et al. Clonal cytotoxic T cells are expanded in myeloma and reside in the CD8(+)CD57(+)CD28(-) compartment. Blood (2001) 98(9):2817-2710.1182/blood.V98.9.2817.

Wagner, J. A., M. M. Berrien-Elliott, et al. (2016). "Cytokine-Induced Memory-Like Differentiation Enhances Unlicensed Natural Killer Cell Antileukemia and FcgammaRIIIa- Triggered Responses." Biol Blood Marrow Transplant.

Yu, J., A. G. Freud, et al. ((2013)). "Location and cellular stages of natural killer cell development." Trends Immunol 34(12): 573-582. doi:510.1016/j.it.2013.1007.1005.

Claims

CLAIMS:

1. 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^dim/KIR7CD57⁺ and CD56^dim/KIR⁺/CD57⁺ clusters and iii) providing a good prognosis when the clusters CD56^dim/KIR⁺/CD5T, 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 clusters CD56^bright and/or CD56^dim/KIR7CD57^" are in a majority compare to the clusters CD56^dim/KIR⁺/CD57^", CD56^dim/KIR 7CD57⁺ and/or CD56^dim/KIR⁺/CD57⁺.

2. A method according to the claim 1 wherein the sample is blood, peripheral-blood, serum, plasma, spinal cord, peripheral blood mononuclear cell (PBMC) or purified NK cells.

3. A method according to the claims 1 or 2 wherein the expression level of the biomarker NKp30 is also measured.

4. 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 in the claim 1.

5. 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 in claim 1.

6. A method for treating AML in a patient with a bad prognosis as described in claim 1 comprising administering to said subject in need thereof a chemotherapeutic compound or a allogeneic stem cell.