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• • G—PHYSICS
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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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  • C12Q2600/00—Oligonucleotides characterized by their use
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
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  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/54—Determining the risk of relapse

Definitions

• the present invention provides an assay method for the in vitro diagnosis of resistance to azacitidine treatment in a patient.
• the invention also relates to an in vitro assay kit for predicting a patient's resistance to azacitidine treatment and the use of such a kit.
• Azacitidine is a hypomethylating agent producing 40% to 60% response in both diseases.
• MDS Myelodysplastic syndromes
• AML acute myeloid leukemia
• MDS myeloid hemopathies that develop from bone marrow stem cells, with precursors of the granulosa lineage, corresponding to white blood cells, of the corresponding erythroblastic lineage. red blood cells, lineages megakaryocyte corresponding to platelets and histio-monocytic line.
• MDS are characterized by major maturation disorders of one or three medullary, granular, erythroblastic and megakaryocytic lineages responsible for cytopenia. MDS can also progress to acute leukemia (LA).
• LA acute leukemia
• the classical diagnosis is based on the cytological study of blood and marrow, cytogenetics and molecular biology. MDS include various refractory anemias or cytopenias as well as 5q- syndrome.
• LMAs are characterized by the rapid proliferation of medullary precursors of the three lineages, granular, erythroblastic and megakaryocytic leading to accumulation in the blood and marrow of immature cells, destroying normal hematopoiesis. Their diagnosis is based on the same techniques as for MDS. They include undifferentiated, poorly differentiated, myeloblastic, monoblastic, and yelomonoblastic LMAs, as well as acute erythroblastic leukemias and acute megakaryoblastic leukemias.
• Primary or secondary AML may be responsible for tumors in various organs or tissues (skin, lymph nodes, breast, gastrointestinal tract, spleen, etc.) producing myeloid sarcoma, formerly known as chloroma or granulocytic sarcoma. They can reveal acute leukemia and pose difficult diagnostic problems with malignant lymphoma.
• prognostic scoring systems are available to predict and predict the overall survival of patients treated with hypomethylating agents. These systems are based on a prognostic score evaluated in subgroups of patients. These are risk groups defined by the karyotype study and certain clinical characteristics of the patients. However, the results associated with these scoring systems are unreliable predictors of response.
• the only known method for determining whether a patient is resistant to azacitidine treatment is to administer this treatment for at least 6 months and determine whether this treatment has an effect or not.
• azacitidine treatment When recommended for patients with MDS and / or AML, azacitidine treatment is injected subcutaneously into the upper arm, thigh or abdomen daily for 7 days. and is followed by a rest period of 21 days. It can lead to many more or less serious side effects such as intracranial bleeding, sepsis, changes in blood pressure, lethargy, feeling of general malaise, hair loss. In addition, the cost of azacitidine treatment is considerable. It is estimated at around 80,000 euros per year of treatment. nowadays, no measurement makes it possible to diagnose reliably and inexpensively whether a patient is sensitive or resistant to azacitidine treatment.
• the Applicant has been able to demonstrate a link between the level of expression of the BCL2L10 protein in a biological fluid sample taken from a patient and the sensitivity of this patient to azacitidine treatment.
• BCL2L10 is defined as being able to correspond to the gene, to the RNA transcript or to the BCL2L10 protein.
• the BCL2L10 gene is a member of the Bcl-2 family, which exhibits anti-apoptotic activity in vitro.
• the BCL2L10 protein shares with the Bcl-2 family of proteins the BH1, BH4 and BH2 domains.
• the BH3 domain which is characteristic of proapoptotics of the Bcl-2 family is, for its part, absent from the BCL2L10 protein.
• BCL2L10 may interact with members of the Bcl-2 family including Bcl-2, Bcl-xL and Bax to regulate apoptosis in different settings.
• Certain publications such as, for example, the article "Loss of BCL2L10 protein expression as prognostic predictor for poor clinical outcome in gastric carcinoma, Histopathology 2010, 57, 814-82" present BCL2L10 as an anti-apoptotic gene.
• BCL2L10 Overexpression of BCL2L10 has been described as suppressing apoptosis by inhibition of cytochrome C release by mitochondria.
• the publication suggests that it is a low expression level of BCL2L10 that is correlated with a low chance of response to azacitidine. Moreover, even if a high methylation level of the BCL2L10 gene was actually related to resistance to azacitidine treatment, these data could not have been correlated with the level of expression of the BCL2L10 protein. Indeed, the level of methylation of a gene is not necessarily related to the expression of the protein derived from this gene. This is particularly the case for BCL2L10.
• the solution to the problem posed relates to an in vitro analysis method making it possible to diagnose resistance to azacitidine treatment in a patient, by using the BCL2L10 protein contained in a sample of biological fluid taken from said patient as well as Biological molecules specifically binding the BCL2L10 protein, characterized in that:
• this threshold value being between 20 and 60%
• resistance to azacitidine treatment is diagnosed in a patient having a percentage of cells expressing the BCL2L10 protein in said biological fluid greater than said reference threshold value.
• the Applicant has demonstrated the existence of a link between the percentage of cells of a biological fluid of a patient that express the BCL2L10 protein and the phenomenon of azacitidine resistance.
• This method makes it possible to diagnose the phenomenon of azacitidine resistance in a patient even before administering said molecule of azacitidine to the patient. This method also allows, so to predict the relapse of a patient who has previously been sensitive to azacitidine treatment.
• a second subject of the invention relates to an in vitro analysis kit for carrying out the in vitro analysis method according to the invention, said kit comprising biological molecules specifically fixing the BCL2L10 protein in cells derived from collected biological fluids. on patients.
• a third subject of the invention concerns the use of an in vitro analysis kit according to the invention, for the implementation of a method for monitoring an azacitidine treatment in order to predict relapse.
• AZA-R azacitidine resistant SKM1 myeloid cells
• SKM1-R azacitidine resistant SKM1 myeloid cells
• AZA-S or SKM1-S cells are azacitidine sensitive cells.
• Figure 1 shows the results of a screening, commonly called “screening” of cells from SKM1 cell lines, expressing the Bcl-2 protein.
• the SKM1-S and SKM1-R cells are treated with ⁇ of azacitidine for 24 hours.
• Western blot experiments ("western blot" in English) are then performed in order to evaluate the amounts of Bcl-2, Mcl-1, Bcl-xl and BCL2L10 proteins.
• An anti-HSP60 antibody was used as a charge control.
• Figures 2 to 6 show the expression of the BCL2L10 protein in the SKM1-S and SKM1-R cell lines with LMA.
• Figure 5 depicts a Reverse Transcript Polymerase Chain Reaction RT-PCR reverse transcriptase polymerase chain reaction analysis of the SKM1-S and SKM1-R cells mRNA.
• Figure 6 shows the results of a western blot for visualizing the protein level of BCL2L10 in SKM1-S and SKM1-R cells.
• Figures 7 to 10 show the resensitization of SKM1-R cells to azacitidine followed by the extinction of the expression of the BCL2L10 gene. Extinguishing the expression of the BCL2L10 gene is commonly referred to as “knockdown", in this case BCL2L10 knockdown.
• the SKM1-S and SKM1-R cells are transfected with siRNA Luc interfering RNA, siRNA BCL2L10 or siRNA Bcl-2. After 72 hours of transfection, the cells are stimulated with azacitidine at ⁇ .
• Figure 8 shows the results of caspase 3 labeling visualized by flow cytometry 24 hours after the addition of azacitidine to ⁇ .
• FIG. 9 shows the results of a marking of Propidium Iodide (PI) by flow cytometry, 24 hours after the addition of azacitidine to ⁇ .
• PI Propidium Iodide
• FIG. 10 represents the results of Western blots carried out 24 hours after the addition of azacitidine at ⁇ in order to determine the inhibition of the expression of BCL2L10 and of Bcl-2.
• Figure 11 shows the expression of western blot BCL2L10, Bcl-2 and ERK proteins on fresh bone marrow samples from 7 healthy patients, 7 azacitidine-sensitive patients and 5 azacitidine-resistant patients. . Western blot results are shown for two patients in each subgroup.
• FIG. 12 represents the expression of the BCL2L10 and ERK proteins analyzed using the Image J software (Image J is a free software for the processing of images written in Java by the National Institute of Health (NIH)) and the quantification of the ratio of BCL2L10 expression relative to ERK expression.
• Image J is a free software for the processing of images written in Java by the National Institute of Health (NIH)
• NASH National Institute of Health
• FIG. 13 represents the quantification of the expression of the BCL2L10 and ERK proteins analyzed using the Image J software and the quantification of the ratio of the expression of BCL2L10 with respect to the expression of ERK.
• Figures 14 to 17 illustrate that in azacitidine-resistant patients with MDS or AML the percentage of cells expressing BCL2L10 protein in the bone marrow is increased.
• the percentage of cells expressing the BCL2L10 protein is quantified by flow cytometry in 32 patients with MDS or of AML and under azacitidine treatment and in 8 healthy patients, all from cohort 1.
• Figure 15 the percentage of cells expressing the BCL2L10 protein is quantified by flow cytometry in frozen samples in DMSO from 14 patients with low-risk MDS, 31 patients with high-risk MDS or AML and treated with azacitidine, all from cohort 2.
• BCL2L10 is quantified by flow cytometry in frozen samples in DMSO from 16 patients with high-risk MDS, or patients at diagnosis, as shown in Figure 16.
• BCL2L10 is quantified by flow cytometry in frozen samples in DMSO from 15 patients with high-risk MDS or AML, all on azacitidine treatment, as shown in Figure 17.
• Figures 18a and 18b show the correlation between the percentage of cells expressing the BCL2L10 protein and the overall survival of treated patients with MDS or AML.
• Figures 19 to 22 make it possible to validate the protein quantification technique of BCL2L10 by flow cytometry.
• FIG. 19 cells from an HEK293 line have been transfected with either pcDNA3 expression plasmids incorporating the N- end of the Myc epitope tag of BCL2L10, or with expression plasmids pcDNA3 integrating the N-terminal portion of the Myc epitope tag alone.
• the level of protein expression of BCL2L10 was quantified by flow cytometry. The results of this experiment are shown in Figure 19.
• Figures 21 and 22 show the protein level of BCL2L10 visualized by western blot.
• An anti-HSP60 antibody is used as a charge control.
• the present invention relates to an analytical method for the in vitro diagnosis of resistance to azacitidine treatment in patients, in particular by performing a quantification of the expression of the BCL2L10 protein by the total cells of a fluid. organic.
• the patients are human beings.
• the method of analysis is particularly suitable for patients with hematological malignancies such as myeloid hemopathies. More particularly, patients have AML or MDS.
• the biological fluid is a fluid from the human body.
• biological fluid include bone marrow, blood, cerebrospinal fluid, urine.
• the biological fluid according to the invention is bone marrow.
• total cells includes all the cells present in the biological fluid removed.
• the total cells include hematopoietic stem cells (HSCs) and medullary stroma cells which are hematopoietic cells.
• the biological molecules specifically binding the BCL2L10 protein are molecules capable of binding specifically to the BCL2L10 protein.
• they are monoclonal or polyclonal antibodies, soluble receptors or aptamers, preferentially monoclonal or polyclonal antibodies.
• the biological molecules specifically binding the BCL2L10 protein are monoclonal antibodies.
• biological molecules specifically fixing the BCL2L10 protein mention may be made of the monoclonal antibody anti-BCL2L10 referenced "# 3869" by the company "Cell Signaling Technologies".
• the reference threshold value also called “cut-off” value, corresponds to a percentage of BCL2L10 positive cells, that is to say a percentage of cells expressing the BCL2L10 protein, in a biological fluid.
• the reference threshold value is between 20 and 60%, preferably between 30 and 55%, more preferably, this reference threshold value is equal to 50%.
• the method of analysis according to the invention makes it possible to diagnose resistance to azacitidine treatment in a patient.
• These patients treated with azacitidine should generally undergo bone marrow punctures every 1, 3, and 6 months as part of their treatment, then every 3 months thereafter.
• this bone marrow sample taken as part of a conventional monitoring of azacitidine treatment can also be used for the analysis method according to the invention. It is therefore not necessarily necessary to perform specific bone marrow punctures in patients for this diagnosis of resistance to treatment with azacitidine.
• the measurement of the percentage of cells of the biological fluid expressing the BCL2L10 protein is carried out by flow cytometry (immunophenotyping), by hydrophobic interaction chromatography (HIC), or by reaction of quantitative chain polymerization (in English qPCR for "quantitative Polymerase Chain Reaction”).
• this measurement is performed by flow cytometry (immunophenotyping).
• the invention also provides an in vitro assay method for diagnosing resistance to azacitidine treatment in a patient by detecting overexpression of the gene.
• BCL2L10 contained in a biological fluid sample taken from said patient, characterized in that:
• the percentage of total cells of said biological fluid expressing BCL2L10 is calculated by detecting overexpression of the BCL2L10 gene; comparing said calculated percentage with a reference threshold value, this threshold value being between 20 and 60%; and
• resistance to azacitidine treatment is diagnosed in a patient having a percentage of cells expressing BCL2L10 in said biological fluid greater than said reference threshold value.
• the detection of the overexpression of the BCL2L10 gene is carried out by the CGH comparative genomic hybridization method, the flow cytometry method, the ELISA method, the DNA microarray method, or by quantitative chain polymerization reaction (qPCR ).
• the detection of the overexpression of the BCL2L10 gene is carried out by the CGH comparative genomic hybridization method, by the DNA chip method or by a quantitative chain polymerization reaction (qPCR). More preferably still, the detection of the overexpression of the BCL2L10 gene is carried out by the DNA chip method or quantitative chain polymerization reaction (qPCR).
• the invention also relates to an in vitro assay kit comprising biological molecules specifically binding the BCL2L10 protein in cells from a biological fluid sample taken from a patient, said kit for predicting the resistance of said patient to a treatment with azacitidine in a patient having a percentage of cells, in said biological fluid expressing the BCL2L10 protein, greater than a reference threshold value of between 20 and 60%.
• an in vitro assay kit comprising at least one reagent selected from the group consisting of:
• Another subject of the invention relates to the use of a kit or the method according to the invention, for the implementation of a method of monitoring an azacitidine treatment in order to predict the relapse. .
• the use of the kit or the method according to the invention also makes it possible to adapt the treatment as a function of the response of the patient.
• said patient when it is diagnosed the resistance to azacitidine treatment in a patient, in particular suffering from myelodysplastic syndrome and / or acute myeloid leukemia, said patient is furthermore administered a treatment alternative comprising at least one anti-tumor agent and / or an anti-inflammatory agent.
• said patient is administered an antitumour compound chosen from alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors, and antitumor antibiotics.
• an antitumour compound chosen from alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors, and antitumor antibiotics.
• antitumour agent that may be used according to the invention, mention may be made in particular of acadesine, also called AICAR for 5-Aminoimidazole-4-carboxamide-1-D-ribofuranoside, and acadesine derivatives.
• actinomycin D amsacrine
• anthracyclines such as doxorubicin or daunorubicin
• aracytin aracytin
• ATRA All-trans retinoic acid
• bleomycin bleomycin
• bortezomib busulfan
• camptothecin derivatives camptothecin derivatives
• cisplatin carboplatin, chlorambucil, decitabine, depakine, docetaxel
• TKIs tyrosine kinase inhibitors
• - Ri is chosen from
• a naphthyl group optionally substituted by one or more substituted alkyl or amine groups having from 1 to 4 carbon atoms,
• benzyl group optionally substituted by one or more substituted alkyl or amine groups having from 1 to 4 carbon atoms,
• a phenyl group optionally substituted by a halogen chosen from Cl, Br, I and F, a thiophene group,
• a phenyl or -C ⁇ C-phenyl group optionally substituted by a halogen
• a -C ⁇ C-2-methoxynaphthalene group their racemates, enantiomers, diastereoisomers and mixtures thereof, tautomers and pharmaceutically acceptable salts thereof.
• the derivatives of acadesine are compounds of general formula:
• R3 -C ⁇ C-2-methoxynaphthalene
• Example 1 Validation of the cytometry technique for the detection of BCL2L10.
• Azacitidine resistant SKM1 (AZA) cells labeled SKM1-R, defective for both apoptosis and autophagy processes, were generated.
• SKM1-R cells show increased expression of BCL2L10 protein (Bcl-B), an antigenic apoptotic of the Bcl-2 family, but the SKM1-R and SKM1-S cells show equivalent levels of Bcl-2, Bcl-xL and Mcl-1 proteins, as shown in Figure 1.
• BCL2L10 proteins An increase in the expression of BCL2L10 proteins was also found in the mass of SKM1-R cells before limited dilution, indicating that the overexpression of BCL2L10 is related to azacitidine resistance (AZA) and is not due to a clonal effect.
• AZA azacitidine resistance
• HEK293 cells were first transfected with Myc-labeled BCL2L10 Myc-BCL2L10 construct and the transfection efficiency was evaluated using an anti-Myc antibody, as shown in FIG. BCL2L10 proteins were confirmed by Western Blot using an anti-BCL2L10 monoclonal antibody as shown in Figure 21.
• siRNA was used, in order to extinguish the expression of the BCL2L10 gene in HE 293 cells.
• the expression of BCL2L10 was also analyzed by western blot on samples from patients when the amount of material to be analyzed was sufficient.
• the results presented in FIGS. 11 to 13 show that the level of BCL2L10 relative to the level of BCL-2 is variable according to the patients.
• the ERK protein was used as an internal control for each patient sample. This made it possible to show that the protein expression of BCL2L10 versus ERK is very weak in healthy patients, as shown in FIG. 12. In contrast, the expression of the Bcl-2 protein is not significantly different in the 3 groups of patients as shown in Figure 13. The results suggest that the expression of BCL2L10 predicts azacitidine resistance in patients with MDS.
• Example 4 Expression of BCL2L10 protein is a biomarker of azacitidine resistance in patients with MDS.
• the mean value for freshly isolated bone marrow samples from healthy and azacitidine-sensitive patients is 0%, with values ranging from 0 to 18%, and 8%, respectively. with values ranging from 0 to 40% of cells expressing the BCL2L10 protein, while the average value for bone marrow cells from patients resistant to azacitidine is 85%, with values ranging from 57 to 99 %, of cells expressing the BCL2L10 protein with a p-value less than 0.0001, as illustrated by Figure 11.
• Figure 11 When comparing retrospectively samples from 14 patients with low-grade MDS.
• Example 5 The percentage of cells expressing the BCL2L10 protein predicts the overall survival of patients with MDS and AML.
• threshold value also called “cut-off”
• 50% of cells expressing the BCL2L10 protein on the total cells of the biological fluid
• FIG. 18b shows Kaplanier overall survival curves of the two groups of AZA-treated SMD or LAM patients with more or less than 50% of cells expressing BCL2L10 in their bone marrow.

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• 2012
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• 2013
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  • 2013-02-28 BR BR112014021173A patent/BR112014021173A2/pt not_active IP Right Cessation
  • 2013-02-28 CA CA2865684A patent/CA2865684A1/fr not_active Abandoned
  • 2013-02-28 US US14/381,406 patent/US20150094217A1/en not_active Abandoned
  • 2013-02-28 WO PCT/FR2013/000055 patent/WO2013128089A1/fr active Application Filing
  • 2013-02-28 CN CN201380013105.4A patent/CN104321649A/zh active Pending
  • 2013-02-28 IN IN1795MUN2014 patent/IN2014MN01795A/en unknown
• 2014
  • 2014-08-27 IL IL234332A patent/IL234332A0/en unknown

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