WO2014173860A1

WO2014173860A1 - Methods for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of iron uptake inhibitor and vitamin d receptor agonist

Info

Publication number: WO2014173860A1
Application number: PCT/EP2014/058053
Authority: WO
Inventors: Ivan CRUZ-MOURA; Olivier Hermine; Florence ZYLBERSZTEJN; Etienne PAUBELLE
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Centre National De La Recherche Scientifique (Cnrs); Université Paris Descartes; Fondation Imagine; Université Paris-Sud; Assistance Publique-Hôpitaux De Paris (Aphp)
Priority date: 2013-04-23
Filing date: 2014-04-22
Publication date: 2014-10-30

Abstract

The present invention relates to a method for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist. More specifically, the method of the invention comprises a step of determining the expression level of one marker consisting of the vitamin D in a blood sample obtained from said patient.

Description

METHODS FOR PREDICTING THE RESPONSIVENESS OF A PATIENT AFFECTED WITH A TUMOR TO A TREATMENT WITH A COMBINATION OF IRON UPTAKE INHIBITOR AND VITAMIN D RECEPTOR AGONIST

Related Application

The present application claims priority to European Patent Application No. EP13305531.9, which was filed on April 23, 2013. The European patent application is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION:

The present invention relates to a method for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least vitamin D receptor agonist.

BACKGROUND OF THE INVENTION:

Acute myeloid leukemia (AML) is a group of heterogeneous malignant diseases characterized by uncontrolled cell growth and differentiation arrest \ Prognosis of older patients diagnosed with AML is particularly poor and almost did not change in the last decades ². Although, 40% to 60% of them achieve a complete remission (CR) following with intensive chemotherapy ³, the overall survival for this group of patients (cut off ranging from 60 to 70 years) is between 4 to 7 months ^{4 6} compared to approximately 20 months for the entire population of patients with AML \ Recently it has been shown that the use of demethylating agents (such as 5-azacytidine or decitabine) may induce hematological responses and increase life expectancy in elderly patients ¹. However, management of patients following epigenetic therapy failure is not consensual and best supportive care (BSC) is still proposed for most of these patients. Thus, new therapeutic strategies are strongly needed in this population of AML patients unfit or relapsing/refractory to high dose chemotherapy.

Following the success of differentiating therapies in acute promyelocytic leukemia (APL), great hopes were placed in Vitamin D (VD) and its ability to promote monocyte differentiation of non-APL AML cells ⁸. However, results of clinical studies were disappointing and trials were interrupted due to the occurrence of life-threatening hypercalcemia ⁹. Most elderly patients diagnosed with AML exhibit secondary iron overload because of iterative red blood cell transfusions and, in some cases, an increased of iron absorption due to ineffective erythropoiesis ¹⁰. Interestingly, in myelodysplastic syndromes (MDS), retrospective studies have suggested that iron chelators may increase life expectancy and may decrease the risk of transformation into AML although the molecular mechanism involved remains unknown ⁿ'¹². It has been shown that deferasirox, DFX is able to induce AML cells apoptosis in vitro through inhibition of NF-κΒ pathway ¹³. In addition, our group has shown that in vitro iron chelators are able to promote monocyte differentiation in both normal hematopoietic progenitors and primary AML cells and that iron privation agents acted synergistically with VD to promote cell differentiation ¹⁴. Because of cumulated evidences from experimental data and that both iron chelators and VD analogues are safe drugs and currently used in AML patients, the inventors proposed deferasirox (DFX) and 25- hydroxycholecalciferol combination therapy to a 69-yr-old AML patient, who was refractory to high dose chemotherapy. DFX/VD treatment was associated with reduced blast counts and partial reversal of pancytopenia, accompanied by an increase in monocyte numbers derived from the blast pool ¹⁴. This case-study suggested that the combined DFX/VD therapy could act as a differentiating agent in human AML. Taken together, these observations provided a strong rationale for the use of iron chelators/VD therapy in the setting of high risk or relapsing AML elderly patients after failure or not fit enough for chemotherapy.

Since VD deficiency and iron overload prevalence is high in the elderly AML patients, the association of VD and DFX was given to a subgroup of patients following demethylating agents failure. Here the inventors report a case-control retrospective study (performed in three independent medical centers) aiming to investigate the therapeutic potential of the association of VD and DFX in elderly AML patients following demethylating agents failure.

Thus, prediction of early antitumoral drug response during therapy or even before starting therapy is needed. However, there are no established biomarkers, which may improve the selection of patients who may benefit from the treatment.

The purpose of the present invention is therefore to address this need by providing a new reliable method for predicting whether a patient affected with a tumor is responder or no responder to a treatment with iron chelator and vitamin D analog.

The inventors show that patients who received this combination therapy presented an increased overall survival and that serum VD level prior to the treatment was the stronger factor that positively correlated with overall survival. SUMMARY OF THE INVENTION:

In a first embodiment, the invention relates to a method for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, comprising a step of measuring the level of vitamin D and/or of analog thereof in a blood sample from said patient and a step of comparing the level of vitamin D and/or of analog thereof with a control reference value.

In a second embodiment, the invention also relates to the use of vitamin D and/or of analog thereof as a surrogate marker for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of a) iron uptake inhibitor, and b) at least one vitamin D receptor agonist.

In a third embodiment, the invention also relates to a product of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, as a combined preparation for simultaneous, separate or sequential use in the prevention or treatment of a patient affected with a tumor, which patient being classified as responder by the method according to the invention.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors have shown that the responsiveness of patients affected with a tumor receiving a treatment with a combination of a) iron uptake inhibitor, and b) at least one vitamin D receptor agonist, be accurately predicted by determining the blood level of a marker consisting of vitamin D.

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

The term "surrogate marker" is, in the sense of the invention, a marker which is differentially expressed in responder patients to treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, comparatively to non responder patients to the same treatment. Specifically, a surrogate marker may be any serum lipid steroid derivate which level is different in responder patients when compared to non responder patients. The surrogate markers described herein is namely useful for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist. Thus, the term "surrogate marker" as used herein refers to those biological molecules which are differentially expressed in response to a treatment with a combination of a) iron uptake inhibitor, and b) at least one vitamin D receptor agonist.

According to the invention, the term "patient", is intended for a human or non-human mammal affected or likely to be affected with a tumor. In preferred embodiment the patient is a human affected or likely to be affected with a tumor.

The term "responder" patient, or group of patients, refers to a patient, or group of patients, who show a clinically significant relief in the disease when treated with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, with an increased survival rate. Conversely, a "non responder patient" or group of patients, refers to a patient or group of patients, who do not show a clinically significant relief in the disease when treated with with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist. When the disease is Acute myeloid leukemia (AML), a preferred responder group of patients that is a group that shows normal VD levels (>50 nmol/L)), after treatment with iron uptake inhibitor, such as deferasirox (DFX), and vitamin D analog such as 25-hydroxycholecalciferol (calcifediol).

After being tested for responsiveness to a treatment with a combination of a) at least one iron uptake inhibitor and, b) at least one vitamin D receptor agonist, the patients may thus be prescribed with said with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, with reasonable expectations of success. As show in the example (figure 1C), AML patient with normal vitamin D levels (>50 ng/ml), after treatment deferasirox (DFX), and 25-hydroxycholecalciferol had a significant increase of medial overall survival (OS) compared to the group of vitamin D deficiency (<50 ng/ml).

As used herein the term "overall survival" also called "survival rate" means the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as cancer. The overall survival rate is often stated as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after their diagnosis or the start of treatment.

The treatment of tumor with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist is described in WO2011007208. As defined herein the expression "iron uptake inhibitor" refers to a compound which is active to reduce or to prevent iron uptake by cells.

Preferably, the iron uptake inhibitor is selected from the group consisting of an iron chelator and a transferrin receptor inhibitor.

As used herein, the expressions "iron chelator" or "iron chelating compound" are used indifferently and relate to a compound that binds iron. An iron chelating compound bound or complexed with iron is referred to herein as an iron chelate. Preferably, the iron chelating compound may be a bidentate, a tridentate, a tetradentate or a higher multidentate compound. A bidentate, tridentate, tetradentate or higher multidentate iron chelating compound refers to compounds which bind iron using two, three, four or more separate binding sites, respectively. Iron chelating compounds of the invention include chelation compounds that can bind to all oxidation states of iron including, for example, iron (-II) state, iron (-1) state, iron (0) state, iron (I) state, iron (II) state (ferrous), iron (III) state (ferric), iron (IV) state (ferry 1) and/or iron (V).

Examples of specific bidentate iron chelators comprise l,2-dimethyl-3-hydroxypyridin-

4-one (Deferiprone, DFP or Ferriprox) and 2-deoxy-2-(N-carbamoylmethyl-[N'-2'-methyl-3'- hydroxypyridin-4'- one])-D-glucopyranose (Feralex-G). Examples of specific tridentate iron chelators comprise pyridoxal isonicotinyl hydrazone (PIH), 4,5- dihydro-2-(2,4- dihydroxyphenyl)-4-methylthiazole-4-carboxylic acid (GT56-252), 4,5- dihydro-2-(3'- hydroxypyridin-2'-yl)-4-methylthiazole-4-carboxylic acid (desferrithiocin or DFT) and 4-[3,5- bis(2-hydroxyphenyl)-[l,2,4]triazol-l-yl]benzoic acid (ICL-670, Deferasirox). The iron chelator can also be substituted 3,5-diphenyl-l,2,4-triazoles in the free acid form, salts thereof and its crystalline forms (as described in WO 97/49395). Examples of specific hexadentate iron chelators comprise N,N'-bis(o-hydroxybenzyl)ethylenediamine-N,N'-diaceticacid (HBED), N-(5-C3-L(5 aminopentyl) hydroxycarbamoyl)-propionamido)pentyl)-3(5-(N- hydroxyacetoamido)-pentyl)carbamoyl)-proprionhydroxamic acid (deferoxamine, desferoxamine or DFO or desferal), and hydroxymethyl- starch-bound deferoxamine (S- DFO). Further derivatives of DFO include aliphatic, aromatic, succinic, and methylsulphonic analogs of DFO and specifically, sulfonamide-deferoxamine, acetamide- deferoxamine, propylamide deferoxamine, butylamide-deferoxamine, benzoylamide-deferoxamine, succinamide-derferoxamine, and methylsulfonamide-deferoxamine.

An iron chelator according to the invention can also be a siderophore or a xenosiderophore. Examples of siderophore or xenosiderophore comprise hydroxamates and polycarboxylates. The hydroxamates contain an N- [delta] -hydroxyornithine moiety and are generally categorized into four exemplary families. One category includes rhodotorulic acid, which is the diketopiperazine of N- [delta] -acetyl-L-N [deltaj-hydroxyornithine. Included within this category are derivatives such as dihydroxamate named dimerum acid. A second category includes the coprogens, which contain an N- [delta] -acyl-N- [delta] -hydroxy-L- ornithine moiety. Coprogens also can be considered trihydroxamate derivatives of rhodotorulic acid with a linear structure. A third category includes the ferrichromes, which consist of cyclic peptides containing a tripeptide of N- [delta] -acyl-N- [delta] -hydroxyornithine and combinations of glycine, serine or alanine. The fourth exemplary category includes the fusarinines, also called fusigens, which can be either linear or cyclic hydroxamates. Fusarinine is a compound characterized by N acylation of N-hydroxyornithine by anhydromevalonic acid. The polycarboxylates consist of a citric acid-containing polycarboxylate called rhizoferrin. The molecule contains two citric acid units linked to diaminobutane. Other categories of siderophores useful as iron chelators in the compositions of the invention include, for example, the phenolate-catecholate class of siderophores, hemin, and [beta] -ketoaldehyde phytotoxins .

The iron chelator according to the invention can also be a thiosemicarbazone, Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3AP) which is a synthetic heterocyclic carboxaldehyde thiosemicarbazone with potential antineoplastic activity, pyridoxal isonicotinoyl hydrazone analogs like di-2-pyridylketone thiosemicarbazone (DpT), di-2-pyridylketone isonicotinoyl hydrazone (PKIH) analogs and di-2-pyridylketone-4,4,- dimethyl-3 -thiosemicarbazone (Dp44mT) .

Preferably the iron chelator of the invention is selected from the group consisting of deferoxamine, deferasirox, deferiprone, Triaprine and Dp44mT.

The expression "transferrin receptor inhibitor" refers to any compound which binds to the transferrin receptor and inhibits or prevents fixation of transferrin or inhibits or prevents the internalisation of the transferrin receptor/transferrin complex into cells.

Transferrin is the main blood plasma protein involved in the transport of iron. Iron loaded transferrin (Fe-Tf) binds to its cellular receptor (TfR) and the complex Fe-Tf/TfR is internalized in a vesicle. The pH of the vesicle is acidified wich induces the release of iron by transferrin. The receptor is then recycled to the cell surface (Irie et al. (1987) Am J Med Sci 293: 103-11).

The transferrin receptor inhibitor according to the invention can for example be selected from the group consisting of an anti-transferrin receptor antibody, synthetic or native sequence peptides and small molecule antagonists and aptamers which bind to the transferrin receptor.

Preferably, the transferrin receptor inhibitor according to the invention is an anti- transferrin receptor antibody, more preferably an monoclonal antibody. More preferably, this antibody is A24 (deposited at the CNCM (Collection nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, Paris) on May 10, 2001, under number 1-2665) notably described in WO 2005/111082 (Moura et al. (2001) J Exp Med 194(4):417-25), Moura et al. (2004) Blood 103(5): 1838-45, Lepelletier et al. (2007) Cancer Res 67(3): 1145- 54).

As used herein the term "antibody" is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, chimeric, humanized or human antibodies, diabodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and also antibody fragments. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2.

As defined herein the expression "vitamin D receptor agonist" refers to a compound which is able to activate vitamin D receptor (VDR), and preferably is able to induce cell differentiation upon binding.

Preferably, the "vitamin D receptor agonist" is selected from the group consisting of vitamin D and/or vitamin D analog and/or vitamin D receptor modulator.

As used herein the term "vitamin D" comprises all the forms of vitamin D or its precursor, as for example vitamin Dl, vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), vitamin D4 (22-dihydroergocalciferol) and vitamin D5 (sitocalciferol). Preferably, the "vitamin D" according to the invention is vitamin D3, more preferably vitamin D3 in its active form l ,25-dihydroxy cholecalciferol D3 (l,25-(OH)2D3 or calcitriol).

Preferably, the "vitamin D precursor" according to the invention is vitamin D3 precursor (i.e calcifediol (vitamin D3 precursor also known as calcidiol, 25- hy droxy cho lecalcifero 1)) .

As intended herein a "vitamin D analog" or a "vitamin D receptor modulator" are able to bind to the vitamin D receptor (VDR) and preferably are able to induce cell differentiation upon binding.

Tests for determining the capacity of a vitamin D analog or of a vitamin D receptor modulator to bind to the vitamin D receptor are well known to the person skilled in the art. Preferably, this capacity can be evaluated by estimating the specific binding of the analog or of the vitamin D receptor modulator on a cell extract. For example, in a typical binding experiment, soluble cell extract obtained by sonication is incubated with increasing concentration of vitamin D analog or of vitamin D receptor modulator. Bounds and free analogs can be separated by the hydroxylapatite method. Specific binding can be calculated by subtracting non-specific binding obtained in the presence of an excess l,25-(OH)2D3 from the total binding measured in absence of l,25-(OH)2D3 (Skowronski et al. (1995) Endocrynology 136(1): 20-26).

The capacity of the analog or of vitamin D receptor modulator to induce cell differentiation can be measured by various methods well known to the person skilled in the art. For example, this capacity can be estimated by the measure of the induction of monocyte differentiation or CDl lb/CD14 expression (a marker of cellular differentiation) in a LNCaP cell line after incubation with the analog or with the vitamin D receptor modulator as described in Skowronski et al. (op. cit.).

Many vitamin D analogs are well known in the state of the art. The expression "vitamin D analog" notably encompasses vitamin D metabolites, vitamin D derivatives and vitamin D precursors, preferably it encompasses vitamin D3 metabolites, vitamin D3 derivatives and vitamin D3 precursors.

Vitamin D analogues according to the invention can retain the secosteroid structure with modified side chain structures around the C-24 position. For example, vitamin D can be paricalcitol (19-nor-la(OH)2D2), ILX23-7553 (16-ene-23-yne-l ,25(OH)2D3), OCT (Maxacalcitol, 22-oxa-la,25(OH)2D3) and EB1089 (Seocalcitol, l -dihydroxy-22,24-diene- 24,26,27-trihomo-vitamin D3).

More preferably, the vitamin D analog according to the invention is selected from the group consisting paricalcitol, OCT, EB1089, 14-epi-analog of 1,25D₃, inecalcitol [19-nor-14- epi-23-yne-l,25-(OH)₂D₃; TX522].

Vitamin D receptor modulators (VDRMs) according to the invention are preferably non-secosteroidal compounds (mostly chemical entities) that have been shown to be less hypercalcemic than the VD analogues, as for example the compounds mentioned in US 2008/0200552, WO2005051936, WO2005051938, WO2005051893, and WO2006069154. More preferably, the at least one vitamin D receptor modulator (VDRMs) is selected from the group consisting of LY2108491, LY2109866 and LG190119 (Ma et al. (2006) J Clin Invest 116(4):892-904, Polek et al (2001) Prostate 49(3):224-33).

In the context of the invention, the term "treatment or prevention" means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. In particular, the treatment of the disorder may consist in reducing the number of malignant cells. Most preferably, such treatment leads to the complete depletion of the malignant cells.

Preferably, the individual to be treated is a human or non-human mammal (such as a rodent (mouse, rat), a feline, a canine, or a primate) affected or likely to be affected with cancer. Preferably, the individual is a human.

The terms "cancer" "malignancy" and "tumors" refer to or describe the pathological condition in mammals that is typically characterized by unregulated cell growth. More precisely, in the use of the invention, diseases, namely tumors that express vitamin D receptor are most likely to respond to vitamin D modulators. Furthermore, inventors make hypothesis that iron deprivation by using iron uptake inhibitor restores sensibility in the vitamin D by surexpression of its receptor in the cell surface. In particular, the cancer may be associated with a solid tumor or unregulated growth of undifferentiated hematopoietic bone marrow cells (hematopoietic stem cell). Examples of cancers that are associated with solid tumor formation include breast cancer, bladder cancer, uterine/cervical cancer, oesophageal cancer, pancreatic cancer, colon cancer, colorectal cancer, kidney cancer, ovarian cancer, prostate cancer, head and neck cancer, non-small cell lung cancer and stomach cancer. Preferably the solid tumor is hormone sensitive breast cancer.

Preferably, the cancer or malignancy or tumor according to the invention is due to an unregulated growth of undifferentiated hematopoietic bone marrow cells (hematopoietic stem cell).

As intended herein the expression "hematopoietic stem cell (HSC)" refers to adult multipotent stem cells that give rise to all the blood cell types including for example myeloid lineages (monocytes and macrophages, neutrophils, basophils, eosinophils), erythrocytes, megakaryocytes/platelets,, and lymphoid lineages (T-cells, B-cells, NK-cells).

The expression "hematopoietic stem cell malignancy" or "hematopoietic malignancy" according to the invention comprises acute myeloid leukemia (AML), acute lymphoblastic leukemia, Chronic myeloid, lymphoid leukemia, lymphoma and myelodysplasia syndrome (as defined in 2001 WHO classification). Preferably, the cancer according to the invention is selected from the group consisting of myelodysplastic syndrome and acute myeloid leukaemia.

Methods for predicting the responsiveness of a patient according to the invention:

A first aspect of the invention consists of a method for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, comprising a step of measuring the level of vitamin D and/or at analog thereof in a blood sample from said patient and a step of comparing the expression level of vitamin D with control reference value.

In a particular embodiment, methods of the invention are suitable for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, wherein said iron uptake inhibitor is selected in the group consisting of an iron chelator and a transferrin receptor inhibitor.

In preferred embodiments, the iron chelator is selected from the group consisting of deferoxamine, deferasirox, deferiprone, Triapine and Dp44mT.

In preferred embodiments, the transferrin receptor inhibitor is an anti-transferrin receptor antibody, and in most preferred embodiment the transferrin receptor inhibitor is monoclonal antibody A24.

In preferred embodiments, the vitamin D is vitamin D3 (cholecalciferol) or its precursor (calcifedio 1) .

In preferred embodiments, the analog of vitamin D is selected from the group consisting of calcifedio 1, paricalcitriol, OCT and EB1089.

In preferred embodiments, vitamin D receptor modulator is selected from the group consisting of LY2108491, LY2109866 and LG190119.

Patients who have been clinically diagnosed as being affected with any tumor sensitive to a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, are of particular interest in the invention. However in a preferred embodiment the patient is affected with a hematopoietic malignancy. According to this embodiment, the hematopoietic malignancy is selected in the group consisting of myelodysplasia syndrome multiple myeloma, non-Hodgkin's lymphoma, acute and chronic leukemia (e.g. acute myeloid leukemia (AML) and any hematopoietic malignancy in which deferasirox (DFX), and 25- hydroxycholecalciferol showed efficacy.

In preferred embodiment the haemathematological malignancy is selected in the group consisting of myelodysplasia syndrome and acute myeloid leukemia (AML).

Samples that may be used for performing the methods according to the invention is blood sample obtained from a patient before the treatment. Determination of the level vitamin D (such as vitamin D3) can be performed by a variety of techniques and method any well known method in the art: RIA kits (DiaSorin; IDS, Diasource) Elisa kits (IDS (manual) IDS (adapted on open analyzers) Immunochemiluminescent automated methods (DiaSorin Liaison, Roche Elecsys family, IDS iS YS) ( Janssen MJ, Steroids, nov 2012)

Control reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of vitamin D in biological samples previously collected from the patient under testing.

A "control reference value" can be a "threshold value" or a "cut-off value". Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data (see figure 1 C). Preferably, the person skilled in the art may compare the vitamin D levels (obtained according to the method of the invention) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the vitamin D level (or ratio, or score) determined in a blood sample derived from one or more subjects who are responders to tumor treatment. In one embodiment of the present invention, the threshold value may also be derived from vitamin D level (or ratio, or score) determined in a blood sample derived from one or more subjects who are non-responders to tumor treatment. Furthermore, retrospective measurement of the vitamind levels (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.

As indicated above, at the end of the method according to the invention, level value found for the vitamin D (or analog thereof) in the patient tested superior to the control reference value above indicates (>50 nmol/L) that the patient tested consists of a responder to the treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist.

Use of surrogate marker for predicting the responsiveness of a patient: Another aspect of the invention is the use of vitamin D or analog thereof as a surrogate marker for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist.

According to this aspect, this use of vitamin D or analog thereof as a surrogate marker comprise a step of measuring the level of the vitamin D or analog thereof in a blood sample from said patient and a step of comparing the level of vitamin D or analog thereof with control reference value.

In this embodiment, control reference value is determined as for the method of prediction of the invention.

The level of vitamin D or analog thereof may be determined by quantifying lipid according to the methods as described above.

In a particular embodiment, the use of vitamin D or analog thereof as surrogate biomarkers for predicting the responsiveness of a patient affected with an acute myeloid leukemia (AML) to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist.

In another particular embodiment, the use of vitamin D as surrogate biomarker for predicting the responsiveness of a patient affected with a tumor to a treatment with deferasirox (DFX), and 25-hydroxycholecalciferol (calcifediol).

In a preferred embodiment, the use of vitamin D as surrogate biomarker for predicting the responsiveness of a patient affected with an acute myeloid leukemia (AML) to a treatment with deferasirox (DFX), and 25-hydroxycholecalciferol (calcifediol).

Therapeutic methods:

A further aspect of the invention relates to a method for the treatment of a tumor in a patient, such as hematologic malignancy or solid tumor.

Said method comprises the following steps: a) determining whether a patient affected with a tumor is a responder or a non responder to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, by performing the method as above described; and

b) administering a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist to said patient, if said patient has been determined as consisting of a responder, at step a) above.

In a particular embodiment, said iron uptake inhibitor is selected in the group consisting of an iron chelator and a transferrin receptor inhibitor

In preferred embodiments, the vitamin D is vitamin D3 cholecalciferol, or its precursor calcifediol.

In preferred embodiments, the analog of vitamin D is selected from the group consisting of paricalcitriol, OCT and EB1089.

In another preferred embodiment, the tumor affecting the patient is an acute myeloid leukemia (AML).

A further aspect of the invention is a product of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, as a combined preparation for simultaneous, separate or sequential use in the prevention or treatment of a patient affected with a tumor, which patient being classified as responder by the method as above described.

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

Figure 1: DFX/VD association induces myeloid differentiation and increases overall survival in elderly AML patients.

(A) Kaplan-Meier estimated OS in DFX/VD and BSC treated patients (B) Multivariate analysis. Forest plot of the odds ratio. (C) OS within subgroups presenting normal VD levels (>50 nmol/L) or VD deficiency (<50 nmol/L). (D) Monocytes numbers in VD/DFX treated patients (E) Creatinine levels in treated patients.

EXAMPLE:

Material & Methods

Patients

A retrospective chart review of 17 elderly AML patients following demethylating agents failure was performed in three French centers. The combination of DFX VD was proposed to patients who were not eligible to any other treatment. Matched controls (13 individuals) were patients receiving best supportive care (BSC) during the same period. There were no differences in blood tests panels or AML prognostic factors between combination therapy and BSC (data not shown). The choice between combination therapy and BSC was made at the discretion of physicians based on his practice but not on clinical or blood tests panels. In each case, the dose of DFX was adapted based on ferritin and creatinine levels. DFX initial dose was of 20-30 mg/kg per day and VD was used at 100,000 units orally weekly.

Ethics statement

This study received ethics approval from the Human Ethics Committee of the Necker hospital (IRB registration #00001072). According to French legislation no written consents are necessary for observational retrospective studies. All data were analyzed anonymously. Each patient was identified with a personal number. Patients were aware that their data were stored in a specific database, but were not informed that these data were used for research purposes. This procedure has been disclosed to the Ethics Committee that, in accordance with national legislation, approved it.

Statistical analyses

Statistical analyses were performed with Graphpad Prism® and SAS JMP8®. X² statistic and Fisher exact test for discrete variables were used to compare proportions, and Student t test was used for continuous variables. The onset of treatment was considered for both groups as the time from which patients had stopped to receive 5-azacytidine treatment. Overall survival (OS) was defined as the time from the initiation of therapy to death from any cause and was censored at the date of last information. Survival was estimated by the method of Kaplan-Meier and was compared by the use of the log-rank test. Multivariate analysis was performed using Cox proportional hazard model. Statistical significance was defined as p- value <0.05 (*), <0.01 (**), or <0.001 (***).

Results

In our retrospective case-control study the median age was not statistically different between the DFX/VD (76 years; range, 63-84) and BSC patients (71 years; range 58-85) groups (Table 1). Most patients were diagnosed with AML with multilineage dysplasia (DFX/VD cases 70%, BSC controls 76%>) and cytogenetic prognosis groups were distributed homogeneously between the patients and controls (Table 1). In addition, there were no significant differences in blast infiltration, leukocytosis, neutropenia, systemic iron and phospho-calcium parameters (Table 1). All patients received 5-azacytidine (median of 8 and 7 courses for the cases and the controls, respectively).

When evaluating patients' outcome between the two groups we observed that median survival of treated patients was significantly increased relative to BSC group (10.4 months versus 4 months, p=0.002) (figure 1A). Multivariate analysis realized in the DFX VD group showed that among the several blood parameters evaluated only serum levels of VD prior to treatment was able to predict patients' outcome (figure IB). Patients with normal VD levels (>50 nmol/L)) had a significant increase of median OS compared to the group of VD deficiency (<50 nmol/L)) (21.2 versus 7.1 months respectively; p=0.04) (figure 1C). Serum VD levels were not correlated to nutritional status (data not shown)

By evaluating hematological parameters at 6 months, we observed that 4 treated patients (out of 17 still alive at this time point; 23.5%) had significant increased monocyte numbers (figure ID). The treatment did not decrease the need of transfusion in both VD/DFX and BSC groups. Serum creatinine levels did not incresead following DFX/VD therapy (figure IE) and hypercalcemia or hepatotoxicity (data not shown) were not observed suggesting the safety of the combined therapy in this population of patients..

Kantarjian et al have shown that in elderly patients AML, OS was of 4 months ⁶. Furthermore, in a recent study that reviewed the outcome of patients with newly-diagnosed AML aged 65 or older treated with demethylating agents CR rate was of 28% and median survival of only 6.5 months. Moreover, no difference in the outcome was observed according to prognosis subgroups (cytogenetics, FLT3 mutational status, age...)¹⁵. Here, we show that the DFX/VD differentiating therapy has a beneficial effect in elderly AML patients following demethylating agents failure. Although no patient has achieved CR, we observed that the DFX/VD association was able to improve drastically OS without toxicity. BSC patients were similar with respect to clinical and biological parameters and exhibited poor OS, which was comparable to the reported in the literature ⁶. The elevation of monocyte levels suggest that this increase in overall survival might be due to a differentiating effect promoted by the use of the combined therapy. In a recent study, Nasr et al have shown that arsenic trioxide and retinoids, in addition of their differentiating effect, were able to affect leukemic initiating cells (LIC)¹⁶. Therefore, further studies should evaluate the impact of DFX/VD on LIC. These encouraging results from this retrospective study should, however, be verified in a large randomized prospective multicenter study.

Table 1: Patient baseline characteristics.

Cases Control

Characteristics P

rnedim fnngi) Median (range)

Number of 5-azacyti dine courses 8(3-24) 7(2-18) ns

LAM 1 or LAM with fflultineage dysplasia {¾) 70 76 OS leukocytes (G.L^"1) 0,3(0-55) 3,8 (0.8 -.34) ns granulocytes (Gi^"1} 0.1 (0-5,6) 0-8 {0.3 -8) ns monoctes (G.L¹) 0 (0-1,8) 0,1 (0-2,4) ns blood blast Iwel { } 15(1-87) 23(0-83) ns medullary blast count ( ) 26(4-71) 22(16-54) ns favorable karpt e (%f 0 0

intermediate karyotype (%) 82 84 ns adverse risk karyotype {¾) 18 15 ns ferritin (μ&ί^'1) 1412(1310-1850) 1500(1100-1910) ns creatinine {pnwli^*1} 67 (50-124) 71(60-139) ns calcium (mmol.L^"1) 2,21(2,12-236) 2.10(1,95-2.24) ns phosphates (mm#I.L⁴| 1.11 (0.91-1.43J 1.1SJ0J6-1J7J ns vitamin 0 {25-OH; nmoll^"1) 33.5 (5.5 - 117) 37 nd

proteins fgl^'1) 68 (€5-75) 70 (SO -74) ns 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.

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Claims

CLAIMS:

1. A method for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, comprising a step of measuring the level of vitamin D and/or analog thereof in a blood sample from said patient and a step of comparing the level of vitamin D with a control reference value.

2. The method according to claim 1, wherein the iron uptake inhibitor is selected from the group consisting of an iron chelator and a transferrin receptor inhibitor.

3. The method according to claim 2, wherein the iron chelator is selected from the group consisting of deferoxamine, deferasirox, deferiprone, Triapine and Dp44mT.

4. The method according to claims 1 to 3, wherein the vitamin D receptor agonist is selected from the group consisting of vitamin D, vitamin D analog and vitamin D receptor modulator.

5. The method according to anyone claims 4 wherein the vitamin D is vitamin D3 or its precursor selected from the group consisting of cholecalciferol and calcifediol.

6. The method according to anyone claims 4 wherein the vitamin D analog is selected from the group consisting of paricalcitol, OCT, EB1089.

7. The method according to anyone claims 1 to 6 wherein the iron uptake inhibitor is deferasirox (DFX), and vitamin D is 25-hydroxycholecalciferol.

8. The method according to anyone claims 1 to 7, wherein the tumor is a hematopoietic malignancy.

9. The method according to claim 8, wherein the tumor is selected in the group consisting of myelodysplastic syndrome and acute myeloid leukemia.

10. The method according to claims 1 to 7, wherein the tumor is a solid tumor like breast cancer.

11. A product of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist, as a combined preparation for simultaneous, separate or sequential use in the prevention or treatment of a patient affected with a tumor, which patient being classified as responder by the method according to any one claims 1 to 10.

12. Use of vitamin D and/or analog thereof as surrogate markers for predicting the responsiveness of a patient affected with a tumor to a treatment with a combination of a) at least one iron uptake inhibitor and b) at least one vitamin D receptor agonist.