WO2022219128A1 - In vitro tests allowing to identify the potential for mdm2 inhibitors to induce the selection of mutations in patients suffering from a myeloproliferative neoplasm - Google Patents

Abstract

TP53 mutations are the most frequent mutations in all cancer subtypes and their acquisition are key events leading to the transformation of chronic myeloproliferative neoplasms (MPN) to leukemia. The MDM2 ubiquitin ligase is responsible for proteasome-dependent p53 degradation. MDM2 inhibitors (nutlins) may rescue p53 from degradation and have been evaluated in a variety of cancers. The inventors report herein evidence of a direct effect of MDM2 inhibition on the selection of MPN patients' cells harboring TP53 mutations. To decipher whether these mutations can arise in a specific molecular context they used a DNA single cell approach to determine the clonal architecture of TP53 mutations. They observed that: (i) clonal evolution in MPN frequently consists of sequential branching instead of linear consecutive acquisition of mutations in the same clone; (ii) TP53 mutations are late events mainly occurring in the driver clone; (iii) additional mutations don't appear to influence the selection by MDM2 inhibitor treatment. This is the first demonstration that a treatment can directly favor the emergence of potentially aggressive MPN subclones. As the molecular environment doesn't affect the potential selection of TP53 mutated cells by MDM2 inhibitors, such events could occur irrespective of the subtype of cancer.

Description

IN VITRO TESTS ALLOWING TO IDENTIFY THE POTENTIAL FOR MDM2 INHIBITORS TO INDUCE THE SELECTION OF MUTATIONS IN PATIENTS SUFFERING FROM A MYELOPROLIFERATIVE NEOPLASM

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular oncology.

BACKGROUND OF THE INVENTION:

BCR-ABL1 negative myeloproliferative neoplasms (MPNs) form a group of disorders characterized by the acquisition of phenotype-driving genetic lesions in hematopoietic stem cells, including mutations in th eJAK2 (JAK2^V617F and exon 12), CALR and MPL genes. MPNs are heterogeneous with a chronic phase inconstantly followed by evolution to more aggressive diseases like myelofibrosis or acute myeloid leukemia (AML). This heterogeneity is likely due to the acquisition of numerous additional mutations during the course of the disease that have been reported to impact the clinical outcome (1-4). Among these, the acquisition of TP53 mutations is identified in 16% of cases of MPN (5) and is associated with a poorer prognosis (6). The p53 protein is considered to be a main barrier to cancer development because of its multiple anti -oncogenic functions (7-9). Acquired mutations in TP53 leading to inactive forms of the protein are among the most frequent mutations in all types of cancer and are considered key events in the transformation of chronic MPN to acute leukemia.

Recently, anti-cancer therapies were developed to either restore the mutant p53 protein conformation (10, 11) or reinforce the wild-type p53 functions by inhibiting its degradation using MDM2 inhibitors. MDM2, an E3 ubiquitin ligase, targets p53 for degradation by the proteasome (12-14). Several MDM2 inhibitors have been developed, the best described being nutlins, a family of small molecules blocking the p53-MDM2 interactions leading to stabilization of p53 (15). These drugs have been evaluated in patients with solid cancers and hematological malignancies with promising results. Although the impact of nutlins on wild- type p53 is well documented, the differential impact of these drugs on mutant vs wild-type proteins is not known. This question is highly relevant in myeloid disorders such as MPNs where ZP53-mutated cells can emerge secondarily during the course of the disease. Based on the selective activity of nutlins on JAK2^V617F mutated PV progenitor cells (16) a phase 1 trial was conducted using idasanutlin in PV patients (NCT02407080) (17). Importantly, the Inventors recently showed the expansion of 7/^J53-mutated clones in some of the patients included in this trial (18). In five patients, variant allelic frequencies (VAFs) of pre-existing ZP53-mutant sub-clones increased under treatment and subsequently decreased after therapy discontinuation suggesting a drug-dependent selection of clones with leukemic potential in MPNs. Several questions arose from this observation: Does MDM2 inhibition exert a direct selective pressure allowing the emergence of ZP53-mutated clones? Does the molecular context (additional mutations) of ZP53-mutated cells impact on the selection potential of these drugs? Is it possible to better predict the risk for an individual patient to develop ZP53-mutated clones receiving idasanutlin therapy?

SUMMARY OF THE INVENTION:

As defined by the claims, the present invention relates to methods of determining whether a patient suffering from a myeloproliferative neoplasm is at risk of developing at least one ZP53-mutated tumoral clone under therapy with a MDM2 inhibitor.

DETAILED DESCRIPTION OF THE INVENTION:

In this study, the Inventors developed an in vitro assay using patient’ cells which confirmed a direct effect of this MDM2 inhibitor on clonal selection which has the potential to predict the clonal evolution in patients prior to treatment. They also used an innovative single cell DNA next generation sequencing (NGS) approach to decipher the molecular context of ZP53-mutated cells emergence.

The present invention relates to a method of determining whether a patient suffering from a myeloproliferative neoplasm is at risk of developing at least one ZP53-mutated tumoral clone under therapy with a MDM2 inhibitor comprising the steps of i) culturing a sample of hematopoietic stem and progenitors cells (HSPC) obtained from the patient in presence of an amount of the MDM2 inhibitor for at least 5 days, and ii) detecting the presence of TP53- mutated tumoral clones wherein detection of said clones indicates that the patient is at risk of developing ZP53-mutated tumoral clones.

As used herein, the term “myeloproliferative neoplasm” or “MPN” has its general meaning in the art and include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) that are a diverse but inter-related group of clonal disorders of pluripotent hematopoietic stem and progenitor cells that share a range of biological, pathological and clinical features including the relative overproduction of one or more cell types from myeloid origin with growth factor independency/hypersensitivity, marrow hypercellularity, extramedullary hematopoiesis, spleno- and hepatomegaly, and thrombotic and/or hemorrhagic diathesis. An international working group for myeloproliferative neoplasms research and treatment (IWG-MRT) has been established to delineate and define these conditions (see for instance Vannucchi et al, CA Cancer J. Clin., 2009, 59:171-191), and those disease definitions are to be applied for purposes of this specification. MPN has traditionally been considered to be synonymous with ‘preleukemia’ because of the increased risk of transformation into acute myelogenous leukemia (AML).

In some embodiments, the patients harbour at least one mutation in JAK2 , MPL, or CALR. Mutations in the JAK2 (e.g. JAK2^V617F and exon 12), CALR and MPL genes are characteristic of an acquisition of phenotype-driving genetic lesions in hematopoietic stem cells in BCR-ABL1 negative myeloproliferative neoplasms (MPNs) disorders.

As used herein the term “JAK2” has its general meaning in the art and refers to the Janus Kinase 2 protein. The amino acid sequence of human JAK2 is well known in the art. Human JAK2 sequences are, for example, represented in the NCBI database (www.ncbi.org; www.ncbi.nlm.nih.gov), for example, under accession number NP 004963. Typical myeloproliferative neoplasm associated mutation is the JAK2^V617F mutation which refers to the point mutation (1849 G for T) in exon 14, which causes the substitution of phenylalanine for valine at codon 617 in the JAK homology JH2 domain. Other examples of JAK2 mutations include exon 12 mutations which can be substitutions, deletions, insertions and duplications, and all occur within a 44 nucleotides region in the JAK2 gene which encompasses amino acids 533-547 at the protein level.

As used herein, the term “MPL” has its general meaning in the art and refers to the MPL proto-oncogene, thrombopoietin receptor. The amino acid sequence of human MPL is well known in the art. Human MPL sequences are, for example, represented in the NCBI database (www.ncbi.org; www.ncbi.nlm.nih.gov), for example, under accession number NP_005364.1. Typical MPL mutation include MPLS505N; MPLW515L, MPLW515K and the rare MPLW515A, MPLW515R and MPLW515S mutations. The tryptophan residue (W) in position 515 at the intracellular juxtamembrane boundary normally inhibits dimerization of the Mpl transmembrane helix and thereby prevents receptor self-activation. Replacing W515 with another amino acid, for example, leucine, lysine, or arginine, leads to loss of this inhibition and results in a constitutively active Mpl. Alternative mutations have also been reported in rare cases including V501A, S505C, A506T, V507I, G509C, L510P, R514K and R519T.

As used herein, the term “CALR” has its general meaning in the art and refers to the calreticulin which is a multifunctional protein that acts as a major Ca⁽²⁺⁾ -binding (storage) protein in the lumen of the endoplasmic reticulum. The amino acid sequence of human CALR is well known in the art. Human CALR sequences are, for example, represented in the NCBI database (www.ncbi.org; www.ncbi.nlm.nih.gov), for example, under accession number NP 004334.1. Typical, myeloproliferative neoplasm-associated mutations are insertions, deletions or insertions and deletions (indels) and occur in exon 9 resulting in a +1 base pair frame-shift of the coding sequence and generation of a translated protein with a novel C- terminus that lacks a retrieval sequence (KDEL) typical of normal CALR and other endoplasmic reticulum resident proteins. Specific examples include CALRdel52/type I; c.l092_1143del; L367 fs*46 or a 5-bp insertion CALRins5/type II; cl 154 1155insTTGTC; K385 fs*47.

As used herein, the term “P53” has its general meaning in the art and refers the tumor human suppressor protein containing transcriptional activation, DNA binding, and oligomerization domains. The P53 protein responds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. Mutations in this gene are associated with a variety of human cancers, including hereditary cancers such as Li-Fraumeni syndrome, since P53 is an anti oncogene protein which has an essential role in the protection of cells against cancer.

As used herein, the term “LP5J” refers to the gene encoding P53 protein. The NCBI reference gene ID is 7151 and is accessible under Ensembl:ENSG00000141510 MIM:191170.

As used the term “TP53 mutation” has its general meaning in the art and refers to any mutation which results into a dysfunction of the protein leading to the loss of its transcriptional activity associated with a negative effect on the wild type protein in heterozygous status. P53 loss of function mutations have fully been exemplified in the prior art and thus the skilled man in the art can easily identify TP53 mutations (Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M. Impact of mutant P53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database.Hum Mutat. 2007 Jun;28(6):622-9) (http://P53.iarc.fr/). TP53 mutations are mainly missense mutations. Examples of TP53 mutations include but are not limited to R175H, Y163C, C242Y, Y243H, G245S, M246V, R248W, R248Q, R273H, C277Y and C277F. TP 53 mutations are major oncogenic events, strongly associated with the development of many cancers.

As used herein, term “tumoral clone” has its general meaning in the art and refers to a population of tumor cells formed by repeated division from a common cell. The expression “ TP53 mutated tumoral clone” refers to a tumoral clone that harbors at least one TP 53 mutation.

As used herein, the term “MDM2” has its general meaning in the art and refers to E3 ubiquitin-protein ligase Mdm2 P53 binding protein that is a P53-associated protein (Oliner, J. D., et al., Nature 358 (1992) 80-83; Momand, J., et al., Cell 69 (1992) 1237-1245; Chen, J., et al., Mol. Cell. Biol. 13 (1993) 4107-4114; and Bueso-Ramos C. E., et al., Blood 82 (1993) 2617-2623). It is a nuclear phosphoprotein that binds and inhibits transactivation by tumor protein P53, as part of an autoregulatory negative feedback loop. MDM2 also regulates the wild-type P53 protein function by inducing its degradation.

As used herein, the term “MDM2 inhibitor” has its general meaning in the art and refers to therapeutic agents that inhibit the MDM2-P53 interaction. MDM2 inhibitors are usually used as drugs in the field of cancer to stabilize wild-type P53 protein and promote an anti -oncogenic activity. Besides peptides and antibodies, several classes of small-molecule inhibitors with distinct chemical structures have now been reported including, but not limited to, a benzodiazepinedione, a sulphonamide, a chromenotriazolopyrimidine, a morpholinone, a piperidinone, a terphenyl, a chalcone, a pyrazole, an imidazole, an imidazole-indole, an isoindolinone, a pyrrolidinone, a piperidine, a naturally derived prenylated xanthone, a stapled peptide, a benzothi azole, and stictic acid. (Shangary et al., (2008) Ann. Rev. Pharmacol. Toxicol. 49: 223-241). These are derivatives of cis-imidazoline (see e.g. Vassilev et al., (2004) Science 303: 844-848), spiro-oxindole (Ding et al., (2005) J. Am. Chem. Soc. 127: 10130- 10131; Shangary et al., (2008) Proc. Natl. Acad. Sci. USA 105: 3933-3938; Ding et al., (2006) J. Med. Chem. 49: 3432-3435; Shangary et al., (2008) Mol. Cancer Ther. 7: 1533-1542), benzodiazepinedione (Grasberger et al., (2005) J. Med. Chem. 48: 909-912; Parks et al., (2005) Bioorg. Med. Chem. Lett. 15: 765-770; Koblish et al., (2006) Mol. Cancer Ther. 5: 160-169), terphenyl (Yin et al., (2005) Angew. Chem. Int. Ed. Engl. 44: 2704-2707; Chen et al., (2005) Mol. Cancer Ther. 4: 1019-1025), quilinol (Lu, Y., (2006) J. Med. Chem. 49: 3759-3762), chalcone (Stoll et al, (2001) Biochemistry 40: 336-44) and sulfonamide (Galatin et al., (2004) J. Med. Chem. 47: 4163-4165). Examples of MDM2 inhibitors and their mechanism of action and effect on P53 levels are also discussed in Hoe et al., (2014) Nature Revs. 13: 217-236, incorporated herein by reference in its entirety. Methods for identifying MDM2 inhibitors are well known in the art (e.g. Han AR, Durgannavar T, Ahn D, Chung SJ. A FRET-Based Fluorescent Probe to Screen Anticancer Drugs, Inhibiting p73 Binding to MDM2. Chembiochem. 2021 Mar 2;22(5):830-833. doi: 10.1002/cbic.202000660. Epub 2020 Nov 20. PMID: 33103305). For instance, a screening assay using a biosensor involving the physical principle of surface plasm on resonance (SPR) may be performed. For this, P53 may be immobilized on a surface and MDM2 may be put in contact concomitantly with the potential inhibitor. If the potential inhibitor is a MDM2 inhibitor, the percentage of binding between p53 and MDM2 decrease in dose-dependent manner. To investigate the mode of binding of the MDM2 inhibitor, a crystal structure can also be determined.

In some embodiments, the MDM2 inhibitor is a nutlin. As used herein, the term “nutlin” refers to cis-imidazoline analogs that inhibit the interaction between MDM2 and tumor suppressor P53, and which were discovered by screening a chemical library by Vassilev et al., (2004) Science 303: 844-848. Nutlin- 1, Nutlin-2 and Nutlin-3 were all identified in the same screen. However, Nutlin-3, (((±)-4-[4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy- phenyl)-4,5-dihydro-imidazole-l-carbonyl]-piperazin-2-one) is the compound most commonly used in anti-cancer studies. Inhibiting the interaction between MDM2 and P53 stabilizes P53 and is thought to selectively induce a growth-inhibiting state called senescence in cancer cells. The term “nutlin” as used herein further refers to enantiomers and stereoisomers. The more potent of the two enantiomers, (-)-Nutlin-3 (Nutlin-3 A) ((-)-4-(4,5-Bis(4-chlorophenyl)-2-(2- isopropoxy-4-methoxyphenyl)-4,5-dihydro-lH-imidazole-l-carbonyl)piperazin-2-one can now be synthesized in a highly enantioselective fashion and is arbitrarily referred to as enantiomer because it appears as the first peak from chiral purification of racemic nutlin-3 although its absolute stereocenter assignment is not known. The term “nutlin” may further refer to “second-generation” nutlin” derivatives such as, but not limited to, RG7388 (ChemieTek, Indianapolis, IN) (described by Ding et al. (2013) JMed Chem. 56: 5979-5983 and incorporated herein by reference in its entirety) and to derivatives described in, for example, US Patent Applications 20150211073 and 20170008904.

In some embodiments, the MDM2 inhibitor is Idasanutlin. As used herein, the term “Idasanutlin” has its general meaning in the art and refers to 4-[(2R,3S,4R,5S)-3-(3-chloro-2- fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-(2,2-dimethylpropyl)pyrrolidine-2- amido]-3-methoxybenzoic acid.

As used herein, the term "treatment" or "therapy" 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.]). As used herein, the term "risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of conversion. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk.

The method of the present invention is thus particularly suitable for determining whether the patient is at risk of developing a secondary acute leukemia.

As used herein, the term "acute leukemia" means a disease that is characterized by a rapid increase in the numbers of immature blood cells that transform into malignant cells, rapid progression and accumulation of the malignant cells, which spill into the bloodstream and spread to other organs of the body.

Thus the method of the present invention is particularly suitable for determining whether a patient will be eligible for the therapy with the MDM2 inhibitor. The method of the present invention is particularly suitable also for the physician to decide that the patient shall be carefully monitored during and after the treatment so as to select the most accurate clinical decisions. As used herein, the term “hematopoietic stem and progenitor cells” or “HSPCs” refers to blood cells that have the capacity to self-renew and to differentiate into precursors of blood cells. These precursor cells are immature blood cells that cannot self-renew and must differentiate into mature blood cells. HSPCs are typically CD34⁺ but display a number of phenotypes, such as Lin-CD34⁺CD38XD90⁺CD45RA , Lin-CD34⁺CD38XD90XD45RA Lin-CD34⁺CD38⁺IL-3aloCD45RA , and Lin-CD34⁺CD38⁺CD10⁺ (Daley et al., Focus 18:62- 67, 1996; Pimentel, E., Ed., Handbook of Growth Factors Vol. IP: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994). In some embodiments, the hematopoietic stem and progenitor cells are isolated form peripheral blood cells.

Any culture medium suitable for growth, survival and differentiation of hematopoietic stem and progenitor cells may be used. Typically, it consists of a base medium containing nutrients (a source of carbon, amino acids), a pH buffer and salts, which can be supplemented with serum of human or other origin and/or growth factors and/or antibiotics to which cytokines are added. Typically, the base medium can be RPMI 1640, DMEM, IMDM, X-VIVO or AIM- V medium, all of which are commercially available standard media.

In some embodiments, the culture medium comprises an amount of IL-3 (i.e. interleukin-3), an amount of SCF (i.e. stem cell factor), an amount of Flt-3 ligand (i.e. FMS- like tyrosine kinase 3), an amount of IL-6 (i.e. interleukin-6) and an amount of TPO (i.e. thrombopoietin). The cytokines can be obtained from a variety of sources. They may be purified or recombinant protein and are typically commercially available from different companies, for example R&D Systems or PeproTech.

In some embodiment, the present invention relates to a method of determining whether a patient suffering from a myeloproliferative neoplasm (MPN) is at risk of developing at least one ZP53-mutated tumoral clone under therapy with a MDM2 inhibitor comprising the steps of i) culturing in a culture medium comprising an amount of IL-3 (i.e. interleukin-3), an amount of SCF (i.e. stem cell factor), an amount of Flt-3 ligand (i.e. FMS-like tyrosine kinase 3), an amount of IL-6 (i.e. interleukin-6) and an amount of TPO (i.e. thrombopoietin) a sample of hematopoietic stem and progenitors cells (HSPC) obtained from the patient in presence of an amount of the MDM2 inhibitor for at least 10 days, and ii) detecting the presence and quantifying of ZP53-mutated tumoral clones wherein detection of said clones indicates that the patient is at risk of developing ZP53-mutated tumoral clones and the quantification in comparison with non-treated condition allows to predict the expansion of a pre-existing TP53- mutated clone.

Typically, IL-3 is used in an amount comprised between 40ng/mL and 60ng/mL and is preferably about 50 ng/mL.

Typically, SCF is used in an amount comprised between 80ng/mL and 120ng/mL and is preferably about 100 ng/mL.

Typically, IL-6 is used in an amount comprised between 40ng/mL and 60ng/mL and is preferably about 50 ng/mL.

Typically, Flt-3 ligand is used in an amount comprised between 80ng/mL and 120ng/mL and is preferably about 100 ng/mL.

Typically, TPO is used in an amount comprised between lOng/mL and 30ng/mL and is preferably about 20 ng/mL.

Typically, the MDM2 inhibitor is used in an amount comprised between lOnM and 30nM and is preferably about 20nM.

Typically, in some embodiments, the culture of the hematopoietic stem and progenitor cells is carried out for about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the culture of the hematopoietic stem and progenitor cells is carried out for at least 10 days.

In some embodiments, detection of the TP53 mutated tumoral clones is performed by any method well known in the art. In some embodiments, the detection of the TP53 mutated tumoral clones is performed by sequencing. Typically, nucleic acids are extracted and purified from the hematopoietic stem and progenitor cells at the end of the culture and sequencing is performed. According to the present invention, a plurality of reads is thus obtained. As used herein, the term “read” refers to a sequence read from a portion of a nucleic acid sample. Typically, a read represents a short sequence of contiguous base pairs in the sample. The read may be represented symbolically by the base pair sequence in A, T, C, and G of the sample portion, together with a probabilistic estimate of the correctness of the base (quality score). As used herein, the term “sequencing” generally means a process for determining the order of nucleotides in a nucleic acid. A variety of methods for sequencing nucleic acids is well known in the art and can be used. In some embodiments, next generation sequencing is carried out. As used herein, the term “next generation sequencing” has its general meaning in the art and refers to sequencing technologies having increased throughput as compared to traditional Sanger- and capillary electrophoresis-based approaches, for example with the ability to generate hundreds of thousands or millions of relatively short sequence reads at a time. Next-generation sequencers are well known in the art and can include a number of different sequencers based on different technologies, such as Illumina (Solexa) sequencing, Roche 454 sequencing, Ion torrent sequencing, SOLiD sequencing, and the like. An example of a sequencing technology that can be used in the present methods is the Illumina platform. The Illumina platform is based on amplification of DNA on a solid surface (e.g., flow cell) using fold-back PCR and anchored primers (e.g., capture oligonucleotides). For sequencing with the Illumina platform, DNA is thus fragmented, and adapters are added to both terminal ends of the fragments (see the preceding step). DNA fragments are attached to the surface of flow cell channels by capturing oligonucleotides which are capable of hybridizing to the adapter ends of the fragments. The DNA fragments are then extended and bridge amplified. After multiple cycles of solid-phase amplification followed by denaturation, an array of millions of spatially immobilized nucleic acid clusters or colonies of single-stranded nucleic acids are generated. Each cluster may include approximately hundreds to a thousand copies of single-stranded DNA molecules of the same template. The Illumina platform uses a sequencing-by-synthesis method where sequencing nucleotides comprising detectable labels (e.g., fluorophores) are added successively to a free 3 'hydroxyl group. After nucleotide incorporation, a laser light of a wavelength specific for the labeled nucleotides can be used to excite the labels. An image is captured and the identity of the nucleotide base is recorded. These steps can be repeated to sequence the rest of the bases. Sequencing according to this technology is described in, for example, U.S. Patent Publication Application Nos. 2011/0009278, 2007/0014362, 2006/0024681, 2006/0292611, and U.S. Pat. Nos. 7,960,120, 7,835,871, 7,232,656, and 7,115,200, each of which is incorporated herein by reference in its entirety.

In some embodiments, the method of the present invention further comprises the steps of i) implementing an algorithm on data that results from the sequencing as to obtain an algorithm output; and ii) determining the probability that the patient will develop at least one TP53 mutated tumoral clone under therapy with the MDM2 inhibitor.

As used herein, the term “algorithm” is any mathematical equation, algorithmic, analytical or programmed process, or statistical technique that takes one or more continuous parameters and calculates an output value, sometimes referred to as an “index” or “index value.” Non-limiting examples of algorithms include sums, ratios, and regression operators, such as coefficients or exponents, biomarker value transformations and normalizations (including, without limitation, those normalization schemes based on clinical parameters, such as gender, age, or ethnicity), rules and guidelines, statistical classification models, and neural networks trained on historical populations. Of particular interest are structural and syntactic statistical classification algorithms, and methods of risk index construction, utilizing pattern recognition features, including established techniques such as cross-correlation, Principal Components Analysis (PCA), factor rotation, Logistic Regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), Support Vector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), as well as other related decision tree classification techniques, Shrunken Centroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, Support Vector Machines, and Hidden Markov Models, among others. Other techniques may be used in survival and time to event hazard analysis, including Cox, Weibull, Kaplan-Meier and Greenwood models well known to those of skill in the art.

In some embodiments, the method of the present invention comprises the use of a machine learning algorithm. The machine learning algorithm may comprise a supervised learning algorithm. Examples of supervised learning algorithms may include Average One- Dependence Estimators (AODE), Artificial neural network (e.g., Backpropagation), Bayesian statistics (e.g., Naive Bayes classifier, Bayesian network, Bayesian knowledge base), Case- based reasoning, Decision trees, Inductive logic programming, Gaussian process regression, Group method of data handling (GMDH), Learning Automata, Learning Vector Quantization, Minimum message length (decision trees, decision graphs, etc.), Lazy learning, Instance-based learning Nearest Neighbor Algorithm, Analogical modeling, Probably approximately correct learning (PAC) learning, Ripple down rules, a knowledge acquisition methodology, Symbolic machine learning algorithms, Subsymbolic machine learning algorithms, Support vector machines, Random Forests, Ensembles of classifiers, Bootstrap aggregating (bagging), and Boosting. Supervised learning may comprise ordinal classification such as regression analysis and Information fuzzy networks (IFN). Alternatively, supervised learning methods may comprise statistical classification, such as AODE, Linear classifiers (e.g., Fisher's linear discriminant, Logistic regression, Naive Bayes classifier, Perceptron, and Support vector machine), quadratic classifiers, k-nearest neighbor, Boosting, Decision trees (e.g., C4.5, Random forests), Bayesian networks, and Hidden Markov models. The machine learning algorithms may also comprise an unsupervised learning algorithm. Examples of unsupervised learning algorithms may include artificial neural network, Data clustering, Expectation- maximization algorithm, Self-organizing map, Radial basis function network, Vector Quantization, Generative topographic map, Information bottleneck method, and IBSEAD. Unsupervised learning may also comprise association rule learning algorithms such as Apriori algorithm, Eclat algorithm and FP-growth algorithm. Hierarchical clustering, such as Single linkage clustering and Conceptual clustering, may also be used. Alternatively, unsupervised learning may comprise partitional clustering such as K-means algorithm and Fuzzy clustering. In some instances, the machine learning algorithms comprise a reinforcement learning algorithm Examples of reinforcement learning algorithms include, but are not limited to, temporal difference learning, Q-learning and Learning Automata. Alternatively, the machine learning algorithm may comprise Data Pre-processing.

In some embodiments, the output obtained by the algorithm is a score. As used herein, the term “score” refers to a piece of information, usually a number that conveys the result of the subject on a test. A risk scoring system separates a patient population into different risk groups; herein the process of risk stratification classifies the patients into very high-risk, high- risk, intermediate-risk and low-risk groups.

A further object of the present invention relates to a kit or device for performing the method of the present invention, comprising means for carrying out the culture and detecting the TP53 mutated tumoral clones as described above. In some embodiments, the kits or devices of the present invention comprise at least one sample collection container for sample collection. Collection devices and container include but are not limited to syringes, lancets, collection tubes. In some embodiments, the container contains the predetermined amount of the different cytokines (e.g. IL-3, SCF, IL-6, FLT-3 Ligand and TPO) and the MDM2 inhibitor. In some embodiments, the kits or devices described herein further comprise instructions for using the kit or device and interpretation of results. In some embodiments, the kit or device of the present invention further comprises a microprocessor to implement an algorithm so as to determine the probability that the patient will develop TP53 mutated tumoral clones. In some embodiments, the kit or device of the present invention further comprises a visual display and/or audible signal that indicates the probability determined by the microprocessor.

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: Increase of mutant TP53 VAFs in HSPC of 7P5.?-mutated MPN patients upon MDM2 inhibitor treatment. (A) Comparison of various TP53 mutations VAFs in CD34+ cells of MPN1 patient after 10 days with or without 20 nM of idasanutlin. (B) Comparison of all TP 53 mutations VAFs in CD34+ cells of 8 different MPN patients after 10 days in presence or absence of idasanutlin 20 nM. (C) Mean +/- SD of VAFs for all TP 53 mutants found in 8 MPN patients after 10 days in presence or absence of idasanutlin 20 nM or Ruxolitinib 70nM in 4 other MPN patients. A paired t-test was used, ** p < 0.01, ns: non significant Figure 2: Absence of variation of non TP53 mutations in HSPC of 7P5J-mutated MPN patients upon MDM2 inhibitor treatment. Comparison of VAFs for mutations found in other genes in CD34 positive cells of the same MPN patients. NT: Not treated. **: p < 0.01 using a Student test for statistical comparison.

EXAMPLE:

Material and methods

Patients. MPN patients were selected on the basis of having one driver mutation and at least one TP53 mutation detectable by NGS. Most of these patients also harbored mutations in other genes. Clinical and molecular characteristics are given in Table 1.

In vitro assay CD34⁺ cells from MPN patients after NGS analysis were prospectively cryopreserved. Peripheral mononuclear cells were isolated from whole blood using Ficoll (Eurobio) and CD34⁺ cells sorted using a column-free immunomagnetic approach (EasySep®, StemCell). 10⁵ CD34⁺ cells were cultured in CTS StemPro™ HSC expansion medium (Thermo Fisher) in which the following cytokines were added: IL-3 (50 ng/mL), SCF (100 ng/mL), TPO (20 ng/mL), IL-6 (50 ng/mL), FLT-3 Ligand (100 ng/mL). Idasanutlin (provided by Roche) was added at 20 nM. After 10 days of culture, cells were harvested, DNA extracted and submitted to NGS analysis.

NGS analysis. We used a capture-based custom NGS panel (Sophia Genetics) targeting 36 myeloid genes (ABU; ASXL1; BRAF; CALR; CBL; CCND2; CEBPA; CSF3R; CUX1; DNMT3A; ETNK1; ETV6; EZH2; FLT3; HRAS; IDH1; IDH2; IKZF1; JAK2; KIT; KRAS; MPL; NFE2; NPM1; NRAS; PTPN11; RUNX1; SETBP1; SF3B1; SH2B3; SRSF2; TET2; TP53; U2AF1; WT1; ZRSR2). Libraries were prepared on 200ng extracted from whole blood DNA (Qiagen) and sequencing was performed on a MiSeq® instrument (Illumina). Bioinformatics were performed at Sophia Genetics (Switzerland) using the SOPHIA DDM software and significant variants were retained with a sensitivity of 1%.

Single cell analysis. The same sample cultured in vitro was used for single cell analysis. CD34⁺ were resuspended in Tapestri® cell buffer (Mission Bio) and quantified using an automatic cell counter (Biorad). Viable single cells (3,000-4,000 cells/mΐ) were encapsulated using a Tapestri® microfluidics cartridge (Mission Bio), lysed, and barcoded. Barcoded samples were then subjected to targeted PCR amplification of a custom 96 amplicons covering 17 genes known to be mutated in MPN (. ASXL1 ; CALR; CBL; DNMT3A; EZH2; IDHF, IDH2, JAK2; KRAS; MPL; NFE2; NRAS; SF3B1; SRSF2; TET2; TP53; U2AF1). PCR products were removed from individual droplets, purified with Ampure XP beads (Beckman Coulter), and used as a template for PCR to incorporate Illumina i5/i7 indices. PCR products were purified a second time, quantified via an Agilent Bioanalyzer and pooled to be sequenced. Library pools were sequenced on aNextSeq instrument (Illumina). The Tapestri Insights® pipeline was used to filter variants (data not shown) and samples were included if they harbored three or more protein-encoding, non-synonymous/insertion/deletion variants and more than 1000 cells with definitive genotype for all protein-coding variants within the sample. We next sought to define genetic clones, which we identified as cells that possessed identical genotype calls for the protein-encoding variants of interest. Importantly, almost 95% of the panel amplicons in each sample had sufficient coverage to annotate variants. The estimated median allele dropout (ADO) rate was 8.74% (IQR: 6.5% - 10.6%, data not shown). From the variants annotated for each sample, we first removed those with low quality (<20% in the Tapestri Insights® software) and low frequency (< 1% cells) (data not shown). As a result, we detected a total of 37 different variants passing the pre-filtering step across the 8 patients (data not shown) on the NGS analysis reference, with a median of 4 variants per patient (IQR: 3.25 - 4.75). All variants detected in NGS analysis below the 1% threshold were not taken into account in the single cell analysis. Study approval. The study was accepted by the local IRB (Comite d’Evaluation de TEthique des projets de Recherche Biomedicale (CEERB) Paris Nord) and patients have given informed consent for the study.

Results

Direct and rapid selection of 7P5.?-mutated clones under MDM2 treatment

After the observation of increased allelic frequencies of TP53 mutations in 5 patients treated in vivo with idasanutlin, an MDM2 inhibitor, we addressed the question of a direct effect of this drug on the selection of mutated cells. We aimed at reproducing the clinical observation with a short-term in vitro culture of patient samples using a controlled medium to support the hypothesis of a direct targeted cellular effect of MDM2 inhibition. Eight patients harboring at least the presence of one driver mutation and one TP53 mutation were examined for the impact of idasanutlin on the selection of ZP53-mutated subclones. Patient’ clinical and molecular characteristics are depicted in Table 1. We first determined the optimal culture conditions and idasanutlin concentrations that limited cellular proliferation of CD34⁺ cells from 3 MPN patients (data not shown). A dose of 20nM idasanutlin was identified as optimal and used in the subsequent experiments.

CD34⁺ cells from ZP53-mutated patients were cultured with or without idasanutlin. After 10 days the cells were retrieved and NGS sequenced as previously described (19). To validate the culture conditions, we compared the allelic frequencies (VAF) of every mutation found after 10 days without treatment to those found in whole blood DNA. For a total of 37 mutations, we observed an excellent correlation between VAFs measured by NGS after culture of CD34+ cells and VAFs measured in bulk whole blood DNA (R=0.979, p< 0.0001) (data not shown) demonstrating that the culture conditions reproduced the in vivo situation without altering the molecular architecture of MPN cells. To estimate the accuracy of this assay for reproducing the in vivo selection of ZP53-mutated clones, we analyzed cells taken from a patient (#MPN1) who had experienced an expansion of TP 53 mutant clones in vivo during idasanutlin therapy (18) and found a similar increase of TP53 mutation VAFs in CD34⁺ cells in vitro (Figure 1A). We concluded the in vitro culture conditions we defined closely replicated the in vivo process of a clonal selection of TP53 mutated cells. We then tested 7 additional patient samples using this assay and observed an increase of each TP53 mutant VAF after idasanutlin treatment compared to the untreated (NT) conditions (Figure IB). In patients with several TP53 mutations, the VAF of each of these mutations increased independently. Altogether, the mean VAF of TP53 mutations was significantly higher in presence of the MDM2 inhibitor treatment compared to the untreated condition (7.38%, us 3.34% respectively, p < 0.01) (Figure 1C). Importantly, such increase was not observed under in vitro exposure to ruxolitinib (Figure 1C). To evaluate the specificity of the selection for TP53 mutations, we considered in each sample the variants found in all genes. Contrary to TP53 mutations, the VAF of mutations found in other genes showed no significant variation after culture with idasanutlin (driver mutations, NT: 38,29% vs Idasanutlin: 39,93%, p=0,25, additional non-TP53 mutations, NT : 11,59% vs idasanutlin: 11,98%, p=0,30, strongly indicating a specific selection of the TP53 mutation-bearing cells by idasanutlin (Figure 2).

In four of the eight patients tested, we identified TP53 mutations in the treated sample that were not detected in the untreated sample or in the whole blood DNA. We tracked these emerging mutations by carefully checking the raw sequencing data from whole blood using IGV software and identified in each case a significant amount of mutant reads (data not shown). For patient #MPN1 who received idasanutlin therapy, the presence of these low level mutations was further confirmed by their detection in a subsequent blood sample taken 3 months later. In patient #MPN4 treated only with hydroxyurea for 20 years, the mutation (p.Y176C) detected after in vitro culture using idasanutlin was eventually found in a follow-up whole blood sample taken one year later. This observation confirmed prior experiments suggesting that idasanutlin is not likely to induce TP53 mutations but rather participates in their selection (18).

Altogether, using this new in vitro culture assay with defined and controlled medium combined with NGS analysis after 10 days of culture, we confirmed in 8 different patients and for a total of 16 TP 53 mutations a direct and rapid effect of idasanutlin exposure on the selection of ZP53-mutated MPN CD34⁺ cells. Importantly, this test appears able to uncover TP53- mutated clones that may be selected in vivo before initiating treatment with an MDM2 inhibitor, even when they are undetectable in patients’ blood using NGS.

Single cell-derived clonal architecture and selection of TP53-mutant MPN cells

Both driver and non-driver mutations found in MPN cells target important regulatory pathways such as intracellular signaling, DNA methylation, histone modifications or splicing processes that could modulate the effects of drugs like MDM2 inhibitors. To better decipher the impact of MDM2 inhibition on clonal selection we next investigated the co-occurrence and order of mutation acquisition for each of the 8 patients tested in vitro. Clonal architecture was determined using the single-cell high-throughput microfluidic Tapestri platform® coupled to the Tapestri Insights® bioinformatics software (Mission Bio). As recently reported (25) this approach permitted accurate analysis of thousands of single cells per specimen. From 8 patient samples a total of 45,443 cells were sequenced with a median of 4,923 cells per sample (interquartile range [IQR]: 3820 - 8172). The panel designed for this study covered 17 of the most frequent MPN mutated genes. The median sequencing coverage was 100 reads per amplicon per cell (IQR: 48.07 - 127.2). To validate the accuracy of the variant identification in single cells, we compared the allelic frequency of each mutation found with single cell analysis with the VAFs found with NGS of whole blood DNA. Among 37 variants identified by whole blood NGS and covered by the panel, 100% (33 SNV and 4 indels) were also detected using the single-cell approach. For each variant there was an excellent correlation between the proportions of mutant alleles detected at single-cell level and the VAFs measured with whole blood DNA (data not shown). When considering all the variants together, the allelic fractions quantified by single-cell and whole blood NGS were highly correlated (R=0.991, p< 0.0001, data not shown).

The results of the single cell analysis allowed us to infer the order of acquisition of somatic events and hence reconstitute the clonal architecture for each patient (data not shown).

In five patients we observed a major clone harboring 1-3 variants and comprising 32% to 75% of the cells associated with 2 to 9 minor subclones with less than 10% of the cells (#MPN1 to 5, data not shown). In three patients several clones of comparable size were present without one dominant clone (#MPN6 to 8) (data not shown). In most cases the first molecular event was a driver mutation in JAK2 or CALR. Only in #MPN2 and #MPN3 patients were DNMT3A and U2AF1 mutations, respectively, the initial events (data not shown).

Several striking observations on the clonal architecture of MPN rapidly emerged from this study. First, clonal evolution of MPN was rarely linear with sequential accumulation of mutations in the same clone. To the contrary, we observed branching evolution with several clones evolving simultaneously in the majority of samples (data not shown). Another important finding was that JAK2^V617F homozygous subclones were present in all JAK2^,¾/7/- mutated patients evaluated. In #MPN1 patient, homologous recombination giving rise to a homozygous clone occurred early in disease development and was present in almost all the single cells tested. In other patients, homologous recombination occurred as a late event and was present in a minor subclone (#MPN3), or even in several minor subclones (#MPN8). Finally, we observed that TP53 mutations always appeared late during clonal evolution, and exclusively in clones harboring a driver mutation (JAK2 or CALR in this study). Although the number of patients tested was limited, it is unlikely that these conclusions can be attributed to chance as this association was observed in 8/8 patients. Furthermore, we tested several patients with multiple TP53 mutations which all developed in distinct clones, but always in cells carrying the driver mutation. For example, in #MPN1 a major JAK2IASXL1 mutated clone was present from which emerged three subclones, each with a different TP53 mutation (data not shown). Multiple independent ZP53-mutated subclones within the driver clone were also detected in #MPN3 and #MPN4 patients (data not shown). The absence of JAK2 wild- type/ TP 53 mutated cells was unexpected since JAK2^V617F positive MPN may evolve to JAK2 wild-type / TP53 mutated acute leukemia while genotypic reversion by mitotic recombination or gene deletion has been previously excluded as an explanation for the development of JAK2 wild-type leukemia in JAK2 -mutated patients (22).

Interestingly, several patients had additional and independent clones with mutations affecting epigenetic genes ( TET2 and DNMT3A ) in small numbers of cells (#MPN5, 6 and 7, data not shown). According to the advanced age of these patients (83, 82 and 67 years), these clones could simply reflect clonal hematopoiesis of indeterminate potential (CHIP) without oncogenic potential occurring independently of MPN-related clones.

The concomitant analysis on the same samples of MDM2 inhibitor treatment and single cell DNA clonal architecture allowed us to determine the molecular background in which TP53 mutations are acquired at the single cell level, and whether this background may affect their selection by MDM2 inhibitors. We found that TP53 mutations may be acquired in three distinct scenarios: either in a clone mutated for a driver gene and an epigenetic gene (TET2, DNMT3A , ASXL1 or EZH2), which is the most frequent situation (#MPN1, 2, 6), or in a clone mutated for a driver gene and a splicing factor (U2AF1 in #MPN3) or in a clone with a driver mutation only that has undergone homologous recombination (#MPN7) or not (#MPN4, 5, 8). Thus, TP53 mutations may be acquired in a large array of molecular landscapes without evidence for a specific association with a particular pathway alteration, with the notable exception of the presence of driver mutations. With idasanutlin treatment in vitro , we observed a significant increase in the VAFs of every ZP53-mutated clone. We did not identify any additional mutations that might favor or antagonize idasanutlin-dependent selection of ZP53-mutated clones, but numbers of clones were relatively small and confirmatory studies would be of interest. Nevertheless, our study shows that the selection by idasanutlin at the single cell level was due to the presence of TP53 mutation itself, independently of associated mutations.

Discussion

In the aim of preventing leukemic transformation, understanding which circumstances may favor the selection of cells carrying TP53 mutations is crucial. MPN patients are treated for years with various anti-proliferative drugs, some treatments have been clearly shown to favor the transformation process (23). However, the participation of long-term treatment in MPN patients in the clonal selection of TP53 mutations is still elusive. Particularly, hydroxyurea has been suspected but no definitive correlation has been established (5). Recent anti-cancer therapeutic approaches aim at enhancing the p53 tumor suppressor activity, notably using MDM2 inhibitors such as nutlins (15) to rescue p53 from degradation (24). This novel class of drugs is now tested in a wide variety of cancers including hematological malignancies.

Several parameters may influence the clonal evolution in vivo and to demonstrate a direct effect of idasanutlin in the expansion of ZP53-mutated clones we performed in vitro culture of patients-derived HSPCs in a defined medium. We observed after a short delay (10 days) a selection of ZP53-mutated cells at the expense of wild-type cells. Importantly, this was reproducible in 8 out of 8 patients, with 16 different TP 53 mutations targeting the DNA binding domain between exon 5 and 8 (five mutations in exon 5, four mutations in exon 6, five in exon 7 and two in exon 8). In patients harboring several TP53 mutations, we observed that each individual mutation VAF increased. In the same time, none of the VAF of other mutations present in these patients increased. This demonstrated that MDM2 inhibitor directly favors the expansion of cells harboring TP53 mutations, an effect not observed in this study with JAK inhibition using ruxolitinib, and raises the question of the mechanism by which the inhibition of MDM2 acts on clonal selection. One explanation is that idasanutlin treatment activates the p53 pathway and cell death in wild-type p53 cells, which would not occur in p53 mutant cells with loss of wild-type functions. The other explanation is that mutations confer gain of function mutations to p53 leading to positive selection of mutated cells. The possibility of gain of function mutations in p53 is still debated but it would be interesting to test the drug on TP53- mutated or inactivated cells to explore a possible gain of function of the mutated forms in the survival of these cells under treatment. Thus, we developed an in vitro assay using patient’ cells which has the potential to uncover ZP53-mutated clones that were not detected by NGS. The results presented herein establish for the first time the causality of this treatment in the selection of MPN sub-clones with a high potential for leukemic transformation. We developed an in vitro assay that may help identify patients with ZP53-mutated clones undetectable with standard NGS before starting an MDM2 inhibitor. With the exception of driver mutations, we did not identify any gene or pathway being redundantly mutated and co segregating with the TP53 mutations, suggesting that only TP53 mutations confer a selective advantage under idasanutlin therapy. Therefore, one can suspect that selection of TP53 mutated cells may occur whatever the tissue and the subtype of cancer.

Tables:

Table 1: Clinical and molecular characteristics of patients harboring at least the presence of one driver mutation and one TP53 mutation. REFERENCES:

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Claims

CLAIMS:

1. A method of determining whether a patient suffering from a myeloproliferative neoplasm (MPN) is at risk of developing at least one ZP53-mutated tumoral clone under therapy with a MDM2 inhibitor comprising the steps of i) culturing a sample of hematopoietic stem and progenitors cells (HSPC) obtained from the patient in presence of an amount of the MDM2 inhibitor for at least 5 days, and ii) detecting the presence of ZP53-mutated tumoral clones wherein detection of said clones indicates that the patient is at risk of developing ZP53-mutated tumoral clones.

2. The method of claim 1 wherein the patient harbours at least one mutation in JAK2, MPL, or CALR.

3. The method of clam 1 wherein the MDM2 inhibitor is a nutlin.

4. The method of claim 1 wherein the MDM2 inhibitor is Idasanutlin.

5. The method of claim 1 wherein the culture medium comprises an amount of IL-3 (i.e. interleukin-3), an amount of SCF (i.e. stem cell factor), an amount of Flt-3 ligand (i.e. FMS-like tyrosine kinase 3), an amount of IL-6 (i.e. interleukin-6) and an amount of TPO (i.e. thrombopoietin).

6. The method of claim 1 wherein the culture of the hematopoietic stem and progenitor cells is carried out for about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

7. The method of claim 1 wherein the detection of the ZP53-mutated tumoral clones is performed by sequencing.

8. The method of claim 1 which further comprises the steps of i) implementing an algorithm on data that results from the sequencing as to obtain an algorithm output; and ii) determining the probability that the patient will develop at least one ZP53-mutated tumoral clone under therapy with the MDM2 inhibitor.

9. A kit or device for performing the method according to claim 1 to 9, comprising means for carrying out the culture and detecting the ZP53-mutated tumoral clones.