WO2010007464A1

WO2010007464A1 - USE OF DEFENSIN α1 AND/OR DEFENSIN α4, AS A MARKER FOR PREDICTING A RELAPSE IN A PATIENT SUFFERING FROM CHRONIC MYELOID LEUKAEMIA

Info

Publication number: WO2010007464A1
Application number: PCT/IB2008/002795
Authority: WO
Inventors: Béatrice TURCQ; Gabriel Etienne; Maryse Dupouy; François-Xavier MAHON; Bertrand Garbay; Patricia Costaglioli
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS
Priority date: 2008-07-18
Filing date: 2008-07-18
Publication date: 2010-01-21
Anticipated expiration: 2011-01-18
Also published as: EP2313779A2; WO2010007531A2; WO2010007531A3; US20110190220A1

Abstract

The present invention pertains to the use of defensin α1 and/or defensin α4, as a marker for predicting and following the response of a patient suffering from chronic myeloid leukaemia to a treatment with imatinib.

Description

USE OF DEFENSIN αl AND/OR DEFENSIN α4, AS A MARKER FOR PREDICTING A RELAPSE IN A PATIENT SUFFERING FROM CHRONIC MYELOID LEUKAEMIA.

Chronic Myeloid Leukemia (CML) is a clonal hematologic malignant disease of the hematopoietic stem cell in which a t(9;22) (q34;ql l) reciprocal translocation leads to a 22 abnormal chromosome designated Philadelphia chromosome

(Ph). Translocation fuses BCR and ABL genetic sequences resulting in the BCR-ABL hybrid gene coding the fusion protein BCR-ABL (Faderl et al., 1999). This constitutively activated tyrosine kinase protein is responsible of the transformation of BCR-ABL cells.

Recently, novel therapeutic molecules have been used to treat CML patients. Imatinib mesylate (Glivec, or Gleevec in the US; Novartis, Switzerland), hereafter referred to as "imatinib", is a tyrosine kinase inhibitor that blocks the BCR- ABL kinase activity. Imatinib treatment inhibits specifically leukemia cells of CML patients. This molecule inhibits deregulated BCR-ABL protein tyrosine kinase activity and selectively eradicates CML cells. Imatinib is the major therapeutic agent for the treatment of patients with CML.

Imatinib can induce a complete or nearly complete cytogenetic remission in up to 80 percent of patients when they are treated in chronic phase (Deininger et al., 2005).

However, in 20 percent of CML patients, this treatment is inefficient. Indeed, about 20% of CML patients treated with imatinib are or become resistant to treatment. Two kinds of resistances can be distinguished: in primary resistance, patients never respond to treatment; - in secondary resistance, patients first respond to treatment, but after a few months or years, they escape.

In some cases, mutation in the BCR-ABL tyrosine kinase domain can explain a newly occurring resistance. However, in other cases, no mutation allows to explain the resistance phenomenon. In this context, there is a strong need for molecular markers which could help physicians to predict the response of a given patient to imatinib, and/or to detect a secondary resistance at a very early stage, so that the treatment of said patient can be adapted.

To this aim, and as described in the experimental part below, the inventors have used a pangenomic microarray to analyse a great number of genes in

CML patients in different situations (chronic disease before imatinib treatment, during a successful treatment, after relapse due to a secondary resistance, etc.). By doing so, they have identified two prognosis markers for the response to imatinib and for secondary resistance to this treatment. These markers are defensin αl and defensin cc4.

Defensins are small antimicrobial peptides involved in innate and adaptive defense systems. By their antibacterial activities, they permeabilize bacterial membrane. Defensins αl and α4 are synthesized as preprodefensins in the bone marrow precursors of blood granulocytes. The mature defensins are then stored in the granules of neutrophils.

Interestingly, in a transcriptomic study for identifying markers associated with the progression of chronic myeloid leukaemia and resistance to imatinib, Radich et al. failed to identify these defensins as markers for determining a patient's response to imatinib (US 2007/0154931).

In another transcriptomic study, Nakamura and Katagiri demonstrated that defensin αl is over-expressed in CML patients (US 2007/0092519). However, these authors did not try to establish a correlation of the expression level of this defensin with patients' response to imatinib treatment.

The present invention hence pertains to the use of defensin αl and/or defensin α4, as a marker for predicting the response of a patient suffering from chronic myeloid leukaemia to a treatment with a tyrosine kinase inhibitor, and in particular, to a treatment with imatinib. The present invention also relates to a method for predicting relapse in a patient who is treated or who has been treated for a chronic myeloid leukaemia, wherein said method comprises the following steps: a) in vitro measuring the expression level of defensin αl and/or defensin α4; b) comparing the measured expression level of defensin αl and/or defensin α4 to a predetermined threshold; wherein a measured level above said predetermined threshold is indicative of relapse.

In the above method, the in vitro measure can be performed on various kinds of biological samples from said patient, such as blood, serum, saliva or urine samples.

The physician can determine the appropriate threshold to perform the above method by different calculation methods. The physician will chose the calculation method depending on the context. In a preferred embodiment, the physician will use the patient as his/her own reference, by calculating the predetermined threshold as 1 OxP, wherein P is the level of the same marker (defensin αl and/or defensin α4) previously measured in the same patient, when said patient's condition was improving or stabilized. Of course, this can be done only when a "reference biological sample" from this patient is available, i.e., when a biological sample was obtained at a time when the patient's condition was improving or stabilized, which means, in the present context, that the level of BCR-ABL in said patient was decreasing or not detectable.

When no "reference biological sample" from said patient is available, the threshold must be determined by reference to values generally observed in a cohort of patients. As appears from the experimental part below, when the defensin αl expression level is calculated as a ratio between defensin αl mRNA copy number and β-actin mRNA copy number (measured by quantitative PCR), the value of 10⁴ can be taken as predetermined threshold. An appropriate predetermined threshold for defensin α4 is 10, if the expression level is calculated as a ratio between defensin α4 mRNA copy number and β-actin mRNA copy number (measured by quantitative PCR). Of course, the skilled artisan can easily determine the equivalent value when a different technique is used to measure the expression level of defensin αl and/or defensin α4, for example if a gene different from β-actin is used as a reference, of if the expression level is measured at the protein level instead of the mRNA level. By reproducing the inventors' experiments on a larger cohort of patients, the skilled artisan can also determine more precisely a relevant threshold, and calculate the significant statistical parameters (such as sensibility and specificity) corresponding to this threshold.

Another aspect of the present invention is a method for following the evolution of a CML patient, still under treatment and/or under full remission, by regularly measuring the expression level of defensin αl and/or defensin α4 in said patient. According to this embodiment, the method comprises the following steps: a) in vitro measuring the expression level of defensin αl and/or defensin α4 in biological samples from said patient, wherein said biological samples have been obtained at various dates; b) comparing the measured expression levels of defensin αl and/or defensin α4 in said biological samples; wherein an increase in the level of expression of defensin αl and/or defensin α4 in said patient is indicative of relapse.

In this method, biological samples from said patient will be regularly obtained. Advantageously, the expression level of defensin αl and/or defensin α4 will be measured every one to three month(s) during the treatment of the disease. This rhythm can be slowed once the patient is in complete remission and still under treatment. After the arrest of imatinib treatment in case of complete remission, the physician can chose to measure the level of defensin αl and/or defensin α4 every month during at least 6 months, in order to quickly detect a relapse. Of course, the physician will adapt the frequency of the measures according to the evolution of each patient.

As mentioned, the above methods can be performed for anticipating a relapse in a patient still under treatment, for example for a patient treated with imatinib. In this case, an increase in the expression level of defensin αl and/or defensin α4 indicates that the patient's treatment must be changed in order to avoid relapse. Indeed, this can correspond to a secondary resistance to imatinib necessitating an increase of the dose of imatinib given to said patient, or a switch to another tyrosine kinase inhibitor, or a bone marrow graft. The above methods can also be used for detecting relapse in a person who has been treated for a chronic myeloid leukaemia but who is considered in complete remission. Indeed, in case of relapse, the defensin αl and/or defensin α4 expression level will increase a few weeks, possibly a few months before BCR-ABL becomes detectable in said patient. The use of a very early marker of relapse such as those according to the present invention can hence prevent a degradation of the subject's condition, by treating the person again before the appearance of the clinical symptoms.

As disclosed in Example 5 below, the inventors have also shown, in a retrospective study involving 28 CML patients responding to imatinib and 1 1 CML patients who were not good responders to this treatment, that the level of defensins at the beginning of the treatment was statistically superior in patients who are good responders to imatinib.

Hence, according to yet another aspect, the present invention concerns a method for determining if a patient suffering from chronic myeloid leukaemia is likely to be a good responder to a treatment with imatinib, comprising the following steps: a) in vitro measuring the expression level of defensin αl and/or defensin α4; b) comparing the measured expression level of defensin αl and/or defensin α4 to a predetermined threshold; wherein a measured level above said predetermined threshold indicates that said patient is likely to be a good responder to the treatment.

Of course, the predetermined threshold will be calculated, depending on the technology used to measure the level of defensin αl and/or defensin α4, by performing a statistical study on a representative cohort. A "representative cohort" is a cohort of patients who have undergone a chronic myeloid leukaemia and have been treated with imatinib, and for whom the response to imabtinib is known. A representative cohort must comprise at least 35 patients, with at least 10 in each group (responders vs/ non responders). Of course, the skilled artisan can chose to use a bigger cohort for implementing the present invention, and the members of the representative cohort must have been chosen without any selection bias. For example, when defensin αl expression is calculated as the log of the ratio [defensin αl mRNA level / β-actin mRNA level], it can be considered that a patient having a defensin αl expression level above 5 is likely to be a good responder to imatinib.

It is important to early identify patients likely to be primary or secondary resistant to imatinib, since an adapted treatment can be proposed. In particular, these patients can be considered as having priority for a bone marrow graft. When performing the above methods, the skilled artisan can chose to measure either the expression level of defensin α 1 , or the expression level of defensin α.4, or both of them. Of course, the physician can combine these markers to other biological markers of the disease, for example the level of BCR-ABL.

A particular technique to measure the expression level of defensin αl and/or defensin α4, illustrated in the examples below, is quantitative polymerase chain reaction after a reverse transcription step. Examples of primers which can be used for this reaction are disclosed in the experimental part below. As also disclosed below, the skilled artisan can chose to calculate the expression level of defensin αl and/or defensin α4 is as a ratio between the copy number of mRNA encoding said defensin and the copy number of mRNA encoding a reference gene. By "reference gene" is herein meant a gene which has a stable expression. Examples of such a gene are β actin, HuPO, Gus, ABL, ...

Of course, any other technique can be used to measure the expression level of defensin αl and/or defensin α4 in biological samples. For example, this expression level can be measured at the protein level instead of the mRNA level as mentioned above, for example by using an immunoassay. Indeed, antibodies targeting defensin αl and/or defensin α4 are already available, such as the D21 monoclonal antibody sold by HyCuIt biotechnology b. v. (The Netherlands) under the references HM2058 and HM2059. HyCuIt biotechnology b. v. also provides an ELISA kit for detecting human neutrophil defensins, under the references HK314, HK315, HK317, HK321 , HK324 and HK325. Such a kit can be used to perform the present invention.

Since the inventors have demonstrated that CML patients having a higher level of defensin αl and/or defensin α4 at the beginning of imatinib treatment respond to this treatment better than those who have a lower level of defensins, they hypothesized that defensin αl and/or defensin α4 are involved in the immune response against cancer, at least in chronic myeloid leukaemia, and that administration of said defensins to the patients, alone or combined to imatinib, can improve their status. Alternatively, agents causing an increase of defensins expression in patients can be administered instead of defensins themselves. Such agents can be, for example, bacteria or fungi, since defensins are secreted in the primary response to such micro-organisms. Accordingly, the present invention also pertains to the use of defensin αl and/or defensin α4, and/or agents inducing an increase of defensins in humans, for the preparation of a drug for : (i) treating cancer and preventing relapse, in particular in the case of hematologic cancers such as chronic myeloid leukaemia, and/or (ii) increasing the efficiency of imatinib and other tyrosine kinase inhibitors in the treatment of CML.

When used in combination with a tyrosine kinase inhibitor such as imatinib, defensin αl and/or defensin α4, and/or the above-described agent can be administered either at the same time as imatinib, or separately (before of after).

Accordingly, the present invention pertains to a medicinal product comprising (i) a tyrosine kinase inhibitor such as imatinib, and (ii) defensin αl and/or defensin α4, and/or an agent triggering an increase of defensins in humans. The present invention also concerns a kit of parts comprising the same elements, in separate vials or packages.

Other characteristics of the invention will also become apparent in the course of the description which follows of the biological assays which have been performed in the framework of the invention and which provide it with the required experimental support, without limiting its scope. Figures legends

Figure 1 : BCR-ABL/ABL and defensin αl/β actin evolution in a secondary resistant patient.

Figure 2: evolution of the expression ratios BCR-ABL/ABL and defensin αl/β actin in a patient who stopped imatinib therapy. Figure 3: comparison of defensin αl expression at JO imatinib in both responders and non-responders populations. Dashes indicate mean values

Examples

The experimental data described below have been obtained by using the materials and methods which follow.

Patient characteristics

A total of 44 patients were enrolled in this study. The mean age at diagnosis time of all patients was 52 years (range: 19-79), with 16 female and 28 male patients. The mean time between diagnosis and imatinib treatment was 21 months (range 0-87). Twenty-six patients received another treatment prior to imatinib therapy.

Peripheral blood samples used for this study were collected after informed consent was obtained in accordance with the Declaration of Helsinki. Plasmids

Defαl , Defα4, βACT and HuPO PCR fragments were obtained using DEFαl forward primer 5'-AGGCTCAAGGAAAAACATGG-B ' (SEQ ID NO: 1) and reverse primer 5'-GCAGAATGCCCAGAGTCTTC-S' (SEQ ID NO: 2), DEFα4 forward primer 5'-GCAGCTGAGCTTGCAGAATA-S' (SEQ ID NO: 3) and reverse primer 5 '-GGACAAAGTATAGGAGAAACAACCA-S ' (SEQ ID NO: 4), ACT forward primer 5' AGCATCGGGTGATGTTCATT-S' (SEQ ID No: 5) and reverse primer 5- ATTAC AAGC ATGCGTC ACC A-3' (SEQ ID No: 6) HuPO forward primer 5TGGAGGGTGTCCGCAATGTT-S ' (SEQ ID NO: 7) and reverse primer 5'- GA AGGCCTTGACCTTTTC AG-3' (SEQ ID No: 8) respectively. These fragments were inserted into the pCR®2.1-Topo vector using the TOPO TA Cloning® kit (Invitrogen). The resulting plasmids were used as template DNA at concentrations ranging from 10 to 10 copies/μl to produce standard curves. RQ-PCR Total RNA from leucocytes was extracted using Trizol reagent method (Invitrogen). DNA contaminants were removed by DNase I treatment (Ambion). RNA concentration was determined by OD260. cDNA was synthesized from 1 μg of total RNA with random hexamers in a final volume of 20 μl (Roche). Quantitative polymerase chain reaction (RQ-PCR) was carried out in 96-weIl ABgene plates using the Mx3OO5P system (Stratagene) with the Sybr green Master mix reaction (Stratagene). All reactions were performed in a total volume of 25 μl and contained 2 μl of cDNA and 6.25 μM of each primer set. Each sample was analyzed in triplicate. Negative controls without added reverse transcriptase were performed. The primers used for amplification of DEFαl , DEFα4, ACT and HuPO were the same as described above. Thermal cycling was performed at 95°C for 10 min followed by 40 cycles comprising each a denaturation step at 95⁰C for 30 s, and an annealing/extension step at 58°C for 45 s. Amplification of the appropriate product was verified by continuous monitoring of the fluorescence; the temperature was ramped from 58°C to 95°C by steps of 0.1 °C to generate a melting curve. BCR-ABL/ABL transcript levels were quantified as previously described (Colombat et al., 2006).

Microarray experiments

Total RNA was purified on Rneasy Mini Kit column for removal of small fragments that affect RT-reaction and hybridization quality (Qiagen). Microarray used were the 22K Human Agilent Microarray. The targets for Agilent DNA microarray analysis were prepared according to the manufacter's instructions. The amount of starting total RNA for each reaction was 500 ng. Briefly, first strand cDNA synthesis was generated using a T7-linked oligo-dT primer, followed by second strand synthesis. An in vitro transcription reaction was performed to generate cRNA containing Cy 3 -CTP or Cy5-CTP. Labeling and hybridization of RNA for microarray analysis were performed using the Agilent low RNA input linear amplification kit. Microarray hybridizations were carried out on Agilent Human oligonucleotide microarrays using 0.75 μg Cy3-labeled "A" sample and 0.75 μg Cy5-labeled "B" sample. Hybridizations were carried out overnight using the Agilent hybridization kit and a "22K" chamber hybridization oven. The arrays were washed once in 6 x SSC and 0.005% Triton X- 102 10 min at room temperature and once in 0.1 x SSC and 0.005% Triton X- 102 5 min at 4°C. Microarrays were scanned using Dual Laser Microarray Scanner (Agilent Technologies).

Statistical analysis

Statistical analysis was carried out using Mann- Whitney test.

Example 1 : identification of defensin αl and defensin α4 as potential markers for relapse of CML patients treated by imatinib

The inventors have performed a transcriptomic study of the imatinib resistance in three patients exhibiting a secondary resistance. Differential RNA expression profiles were analysed using microarray. For each patient taken separately, gene expression of said patient when responding to treatment was compared to gene expression of the same patient during relapse. It was noted that the BCR-ABL levels were about the same in both conditions (respond and relapse). By doing so, the inventors could get rid of genetic variability, since each patient was his own control; moreover, the results were not due to the variability of BCR-ABL level. The median time between CML diagnosis and the imatinib treatment was 38 months. Agilent 22K human 60-mer oligonucleotide pan genomic microarrays (Agilent technologies) were used for this study. Four experiments (a dye-swap and a duplicate) were done for each patient. The selective criteria were drastic. A gene was selected only if the expression difference was positive in each experiment; moreover, the inventors selected only genes presenting a log ratio above 0.5 or below -0.5, with a p-value below 0.001. This corresponds at least to a three times variation expression.

With these criteria, genes coding for the defensin αl and defensin α4 were highly over-expressed, about 50 times when patients relapsed.

Microarray results were then confirmed by quantitative reverse transcriptase polymerase chain reaction assay (RT-PCRq), which is more sensitive. With this technique, the over-expression was about 300 times when patients relapsed. Example 2: defensin αl and defensin α4 levels in primary resistant patients

The inventors then investigated defensin αl and defensin α4 expressions in responder patients to the imatinib treatment and in patients primary resistant to the treatment at different periods of their disease. The reference gene used in the RT-PCR quantitative experiment disclosed in Examples 1 to 5 was the gene encoding the β actin. Other reference genes can be used alternatively. In particular, the gene coding the acidic ribosomal PO protein (HuPO) could advantageously be used, since it is expressed at a higher level than the gene encoding β actin, and hence, its use would allow to perform the experiments on smaller samples. In what follows, the defensin level in hence calculated as: defensin αl copy number / β-actin copy number.

In primary resistant patients, defensin levels before imatinib were about 10 and 3 to 6 months later, defensin levels were about 10⁴. In responder patients, defensin levels were also about 10⁷- 10⁶ and 3 to 9 months after imatinib therapy, defensin levels ranged between 0.9 and 129. In this later case, there was a strong decrease of the imatinib rate.

Example 3: defensin αl level in secondary resistant patients From the first results disclosed in Examples 1 and 2, the inventors assumed that in secondary resistant patients the relapse was accompanied by an increase of defensin αl and defensin α4 mRNA, whereas in primary resistant patient the defensin mRNA remained high and in responder patient there was a strong and persistent reduction of the defensin transcription level.

The inventors thus looked more specifically at secondary resistant patients. For these patients, BCR-ABL/ABL gene expression ratio and defensin αl/β actin gene expression ratio were measured at different times. Total RNA was extracted from leucocytes from a single blood tube and then treated with Dnase I to remove residual contamination of genomic DNA. RNA was transcribed in cDNA and a PCRq assay was realized to determine defensin αl/β actin and BCR-ABL/ABL copy numbers ratios. These results allow to quantify the expression level of defensin αl and BCR- ABL/ABL. Example of BCR-ABL/ABL and defensin αl/β actin genes expression curves in a secondary resistant patient is shown in Figure 1.

As appears from Figure 1, defensin αl/β actin ratio increases 6 months before the BCR-ABL/ABL ratio.

To validate that the defensin αl could be used as an early predictive marker of imatinib resistance, 8 secondary resistant patients were then studied. In all these cases, defensin αl expression increased in average 6 months before BCR-ABL. Example 4: defensin αl and defensin α4 levels in patients in remission

Patients in complete molecular remission are patients who have had repeated negative results by BCR-AB L/ABL real-time polymerase chain reaction during at least 2 years. The inventors investigated the defensin levels in some of these patients, who have stopped imatinib therapy.

An example of BCR-ABL/ABL and defensin αl/β actin gene expression evolution over time in a patient who stopped imatinib therapy is shown in Figure 2. For this patient, the defensin αl/β actin gene expression ratio increased one month after the imatinib therapy stop, whereas the BCR-ABL/ABL gene expression ratio increased two months after the imatinib therapy stop.

Three other patients in complete molecular remission have been analyzed. One patient who stopped imatinib therapy two years ago did not relapse. For this patient, BCR-ABL is still undetectable and the defensin αl expression is stable. The two other patients relapsed after they stopped imatinib therapy and the increase of defensin αl expression occurred at the same time and 3 months before the increase of BCR-ABL/ABL gene expression.

These experiments reinforce the role of defensin αl as an early predictive marker of CML relapse.

Similar results were obtained with defensin α4.

Defensin αl and defensin α4 expression levels can hence be used as early predictive genetic markers of CML patient relapse.

Even with low BCR-ABL/ABL level, undetectable Philadelphia chromosome and normal blood formula, it is now possible to predict relapse in CML patients. In this way, treatment can be changed rapidly, for example by increasing imatinib doses, using other tyrosine kinase inhibitors or performing a bone marrow transplantation.

Example 5: defensin αl and defensin α4 levels as markers for predictinfi a patient's response to imatinib treatment

Defensin αl expression levels were measured at day 0 of imatinib therapy, in 28 responder patients and 1 1 non-responder patients (3 primary resistant and 8 secondary resistant patients). The results are given in Figure 3.

In responder and non-responder populations, defensin αl expression medians are 5.8 and 4.7 respectively and defensin αl expression means are 5.8 and 4.2 respectively (wherein defensin αl expression is calculated as the log of the ratio [defensin αl copy number / β-actin copy number]). Mann- Whitney test was used to test I l

for significant difference between defensin αl expression and response of patients. Level of significance was set at 3%. The statistical power of the test is 84%. Statistical analysis showed significantly defensin αl expression difference between responder versus non-responder population. Similar results are obtained with defensin α4.

Defensin αl and defensin α4 can hence be used also as predictive markers of imatinib treatment response.

The identification of defensin αl or defensin α4 as early predictive genetic markers is essential for the treatment of CML. Example 6: defensin α2 and defensin α3 can also be used for predicting the evolution of a patient suffering or having suffered from chronic myeloid leukemia

The same experiments as described above are performed for investigating the levels of defensins α2 and α3 during chronic myeloid leukemia treatment. Similar results as described above are obtained, which demonstrates that all the aspects of the invention disclosed in detail above can be extended to the use of defensin α2 and defensin α3.

Example 7: Use of defensins as therapeutic agents

Higher levels of defensin αl found in imatinib good responder patients before the treatment highlights the possibility of a putative role of defensins in tumor suppression. Such a role was suggested by Bullard et al. for the defensin βl in prostate cancer (Bullard et al., 2008). This tumor suppressor effect of defensin αl (and of defensins α4, α2 and α3) could be necessary for the success of imatinib treatment. A further study is performed to determine if these defensins can be used as therapeutic or prophylactic agents (to prevent a relapse of CML) either alone or in combination with imatinib. In such a case, defensins can be administered either as such, or an agent inducing the secretion of defensins can be used instead, such as bacteria or fungi.

REFERENCES

Bullard, R. S., Gibson, W., Bose, S. K., Belgrave, J. K., Eaddy, A. C, Wright, C. J., Hazen-Martin, D. J., Lage, J. M., Keane, T. E., Ganz, T. A., and Donald, C. D. (2008). Functional analysis of the host defense peptide Human Beta Defensin-1 : new insight into its potential role in cancer. MoI Immunol 45, 839-848.

Colombat, M., Fort, M. P., Chollet, C, Marit, G., Roche, C, Preudhomme, C, Reiffers, J., Praloran, V., and Mahon, F. X. (2006). Molecular remission in chronic myeloid leukemia patients with sustained complete cytogenetic remission after imatinib mesylate treatment. Haematologica 91, 162-168.

Deininger, M., Buchdunger, E., and Druker, B. J. (2005). The development of imatinib as a therapeutic agent for chronic myeloid leukemia. Blood 105, 2640-2653.

Faderl, S., Talpaz, M., Estrov, Z., O'Brien, S., Kurzrock, R., and Kantarjian, H. M. (1999). The biology of chronic myeloid leukemia. N Engl J Med 341, 164-172.

Claims

1. Use of defensin αl and/or defensin α4, as a marker for predicting the response of a patient suffering from chronic myeloid leukaemia to a treatment with a tyrosine kinase inhibitor.

2. The use of claim 1 , wherein said inhibitor is imatinib.

3. A method for predicting relapse in a patient who is treated or who has been treated for a chronic myeloid leukaemia, wherein said method comprises the following steps: a) in vitro measuring the expression level of defensin αl and/or defensin α4; b) comparing the measured expression level of defensin αl and/or defensin α4 to a predetermined threshold; wherein a measured level above said predetermined threshold is indicative of relapse.

4. The method of claim 3, wherein said predetermined threshold is calculated as 1OxP, wherein P is the level of the same marker previously measured in the same patient, when said patient's condition was improving or stabilized.

5. The method of claim 3, wherein said predetermined threshold is 10⁴- fold the expression level of β-actin.

6. A method for following the evolution of a patient who is treated or who has been treated for a chronic myeloid leukaemia, wherein said method comprises the following steps: a) in vitro measuring the expression level of defensin αl and/or defensin α4 in biological samples from said patient, wherein said biological samples have been obtained at various dates; b) comparing the measured expression levels of defensin αl and/or defensin α4 in said biological samples; wherein an increase in the level of expression of defensin αl and/or defensin α4 in said patient is indicative of relapse.

7. A method for determining if a patient suffering from chronic myeloid leukaemia is likely to be a good responder to a treatment with imatinib, comprising the following steps: a) in vitro measuring the expression level of defensin αl and/or defensin α4; b) comparing the measured expression level of defensin αl and/or defensin α4 to a predetermined threshold; wherein a measured level above said predetermined threshold indicates that said patient is likely to be a good responder to the treatment.

8. The method according to any of claims 3 to 7, wherein the expression level of defensin αl is measured.

9. The method according to any of claims 3 to 7, wherein the expression level of defensin α4 is measured.

10. The method according to any of claims 3 to 7, wherein the expression levels of both defensin αl and defensin α4 are measured.

1 1. The method according to any of claims 3 to 10, wherein the expression level of defensin αl and/or defensin α4 is measured by quantitative polymerase chain reaction.

12. The method according to any of claims 3 to 1 1, wherein the expression level of defensin αl and/or defensin α4 is calculated as a ratio between the copy number of mRNA encoding said defensin and the copy number of mRNA encoding a reference gene.

13. A medicinal product comprising at least a tyrosine kinase inhibitor and an agent selected in the group consisting of defensin αl , defensin α4 and agents inducing an increase of defensin αl and/or defensin α4 when administered to a human.

14. A kit of parts comprising at least a tyrosine kinase inhibitor and an agent selected in the group consisting of defensin αl, defensin α4 and agents inducing an increase of defensin αl and/or defensin α4 when administered to a human.

15. Use of at least an agent selected in the group consisting of defensin αl , defensin α4 and agents inducing an increase of defensin αl and/or defensin α4 when administered to a human, for the preparation of a therapeutic composition for treating chronic myeloid leukaemia and preventing relapse.

16. The use of claim 15, wherein said composition is used in combination with a tyrosine kinase inhibitor.

17. Use of at least an composition selected in the group consisting of defensin αl, defensin α4 and agents inducing an increase of defensin αl and/or defensin α4 when administered to a human, for the preparation of a therapeutic composition for increasing the efficiency of a tyrosine kinase inhibitor in the treatment of chronic myeloid leukemia.

18. The medicinal product of claim 13, the kit of claim 14, or the use of claim 16 or claim 17, wherein said tyrosine kinase inhibitor is imatinib.