WO2011018090A1 - Predictive marker for hydroxyurea resistance - Google Patents

Predictive marker for hydroxyurea resistance Download PDF

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Publication number
WO2011018090A1
WO2011018090A1 PCT/DK2010/050207 DK2010050207W WO2011018090A1 WO 2011018090 A1 WO2011018090 A1 WO 2011018090A1 DK 2010050207 W DK2010050207 W DK 2010050207W WO 2011018090 A1 WO2011018090 A1 WO 2011018090A1
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Prior art keywords
catalase
hydroxyurea
neoplastic disorder
inhibitors
tyrosine kinase
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PCT/DK2010/050207
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French (fr)
Inventor
Stig Uggerhøj ANDERSEN
Trine Heidi Juul Andersen
Jan-Elo Bjarne JØRGENSEN
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Andersen Stig Uggerhoej
Trine Heidi Juul Andersen
Joergensen Jan-Elo Bjarne
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Publication of WO2011018090A1 publication Critical patent/WO2011018090A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention relates to the field of sub-typing cancers.
  • the present invention relates to methods for predicting the sensitivity of cancer to hydroxyurea and to methods for predicting the sensitivity of a cancer to combinatorial therapies comprising both the administration of tyrosine kinase inhibitors and catalase inhibitors.
  • Blood cancers are often treated with cytotoxic agents that prevent cell divisions.
  • chemotherapeutic resistance in the cancer cells.
  • Detailed knowledge of mechanisms causing chemo- resistance could allow for development of valuable and accurate methods for predicting therapy efficacy. These prediction methods may also be relevant when deciding whether a patient should have a monotherapy or a combinatorial therapy comprising more than one active component.
  • WO2008/074131 discloses a method for predicting therapeutic efficacy of a treatment of cancer patients with oxidative stress based therapies, wherein an elevated level of an oxidative stress adaption marker is indicative of resistance towards these therapies.
  • Catalase is mentioned as an oxidative stress adaption marker.
  • WO2005/078127 discloses a method for detecting a genetic variation or polymorphism, i.e. a mutation, in a catalase gene, wherein the presence or absence of a variant genotype of the catalase gene is indicative of an increased risk towards cancer, especially colon and rectal cancer. It is further disclosed that the method may be used for determining whether a subject will benefit from treatment with a drug.
  • WO 2006/136391 discloses a combinatorial therapy of cancer diseases comprising tyrosine kinase inhibitors and optionally hydroxyurea.
  • HU Hydroxyurea
  • RNR ribonucleotide reductase
  • hydroxyurea is suggested in combinatorial therapy of cancers together with tyrosine kinase inhibitors.
  • Predictive markers of when such combinatorial therapy may be advantageously for the patient are relatively unknown.
  • an improved method for distinguishing between hydroxyurea resistant and sensitive cancers would be advantageous, and in particular a more efficient and/or reliable method for distinguishing between patients with hydroxyurea resistant and sensitive blood cancers would be advantageous.
  • more reliable methods for evaluating whether a synergistic effect is present when combinational therapy is evaluated against monotherapy would be advantageously.
  • hydroxyurea appears to be a catalase inhibitor.
  • subtypes of neoplastic disorders indeed exist which show a differentiated expression of the catalase enzyme and that the sensitivity to hydroxyurea depends on the catalase status.
  • combinational therapy with hydroxyurea and tyrosine kinase inhibitors are well- known therapies (or suggested therapies) it appears that testing for the expression of catalase in a sample (e.g.
  • cancerous material would assist in determining whether a disease should be treated with a monotherapy with hydroxyurea or a combinational therapy of tyrosine kinase inhibitors and a catalase inhibitors, such as hydroxyurea.
  • the methods of the invention may also be used to predict whether a synergistic effect of tyrosine kinases and catalase inhibitors will be present when a neoplstic disorder is treated.
  • an object of the present invention relates to providing a method for determining hydroxyurea resistance using a sample. Another object relates to assisting in determining whether a disease should be treated with a monotherapy with hydroxyurea or a combinational therapy of tyrosine kinase inhibitors and a catalase inhibitor, such as hydroxyurea.
  • one aspect of the invention relates to a method for sub-typing a neoplastic disorder, said method comprising
  • neoplastic disorder is likely to be a hydroxyurea resistant subtype of said neoplastic disorder or a hydroxyurea sensitive subtype of said neoplastic disorder.
  • the neoplastic disorder may be divided into a hydroxyurea sensitive or hydroxyurea resistant subtype. Thus, it may be possible to decide whether hydroxyurea is a suitable treatment or if another treatment should be selected.
  • the method may be appropriate in assisting in the decision of selecting the concentration of hydroxyurea to be administrated to a patient, since a sample showing low catalase activity may require a higher dose of hydroxyurea to achieve a suppressive effect on the neoplastic disorder.
  • a method for patient specific optimization of a treatment regime is provided.
  • hydroxyurea resistant and hydroxyurea sensitive relate both to monotherapy with hydroxyurea and combinational therapy with hydroxyurea and at least one further active component.
  • the invention relates to a method for sub-typing a neoplastic disorder, said method comprising
  • composition comprising one or more
  • tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
  • the method of the invention may be employed by the clinician to assist the diagnosing of the subtype of a cancer and further in the clinical decision on the treatment.
  • the object is to subject only those patients to hydroxyurea treatment that will likely benefit from this treatment.
  • Hydroxyurea treatment may be in the form of monotherapy or combinatorial therapy e.g. as decribed above. Predicting responsiveness
  • Another aspect of the present invention relates to a method for predicting responsiveness of a neoplastic disorder to hydroxyurea treatment, said method comprising
  • the invention relates to a method for predicting responsiveness of a neoplastic disorder to hydroxyurea treatment or treatment with a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors, said method comprising
  • a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors.
  • Yet another aspect of the present invention is to provide a method of monitoring the status of a neoplastic disorder in a subject, said method comprising
  • neoplastic disorder has progressed to become a hydroxyurea resistant subtype of said neoplastic disorder, regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder, or the sensitivity to hydroxyurea of said neoplastic disorder is maintained.
  • the invention relates to a method of monitoring the status of a neoplastic disorder in a subject, said method comprising
  • neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
  • - has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • the method of monitoring the status of a neoplastic disorder in a subject comprises a) providing at least two biological samples obtained from a subject at different time points,
  • neoplastic disorder has progressed to become a hydroxyurea resistant subtype of said neoplastic disorder, regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder, or the sensitivity to hydroxyurea of said neoplastic disorder is maintained.
  • the method further comprises evaluating whether it is likely that said neoplastic disorder has progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • the catalase status of a neoplastic disorder can be compared over time, allowing the clinician to evaluate the progression of hydroxyurea sensitivity. It is to be understood that the two or more samples may be compared to each other before being compared to said threshold level or said known standard.
  • Still another aspect of the present invention is to provide a kit for performing the method according to the invention, comprising
  • Figure 1 shows detected amino acid mutations in Arabidopsis thaliana giving rise to a lowered catalase activity and hydroxyurea resistance and the corresponding position in the human catalase protein sequence.
  • Figure 2 shows the alignment of the Arabidopsis thaliana catalase 2 protein sequence and Homo sapiens catalase protein sequence. The amino acid positions listed in Figure 1, which when mutated in Arabidopsis thaliana cause hydroxyurea resistance and lowered catalase activity, are highlighted.
  • Figure 3 shows the relative catalase activity in 1) control group, 2) patients diagnosed with chronic myeloid leukemia (CML), 3) a patient diagnosed with CML and treated with high-dose HU for three weeks immediately prior to sampling, and 4) patients diagnosed with chronic lymphoid leukemia (CLL).
  • Diamonds indicate data points. Error bars indicate standard deviations.
  • Figure 4 shows HU uptake by wild type and HU resistant mutants. Eleven days old wild type and mutant seedlings grown on drug-free medium were moved to plates containing 25 mM HU and incubated for 8 or 24 hours before shoots were harvested. After 24 hours, the HU concentration in seedling shoots approached that of the medium for both wild type and HU resistant plants. HU could not be detected in plants grown on drug-free medium. Error bars indicate SEM.
  • Figure 5 shows inhibition of RNR activity by HU in wild type and HU resistant mutants.
  • RNR activity in extracts from wild type and HU resistant mutants is shown.
  • no HU was added to the RNR reaction.
  • 1 mM or 5 mM HU was added to the RNR reaction. Error bars indicate SEM.
  • Figure 6 shows HU response in wild type and HU-resistant mutants.
  • A More than 90% of wild type plants germinated on 2.5 mM HU, whereas less than 1/20,000 germinated on 3 mM HU. Two HU-resistant mutants germinating at significantly higher concentrations of HU than the wild type are shown.
  • B-C Seven days old seedlings grown on 3 mM HU.
  • B Wild type.
  • C cat2-9.
  • Figure 7 shows HU inhibition of catalase-mediated decomposition of H2O2.
  • A Catalase activity in plant and rat protein preparations in the absence (- HU) and presence of 1 mM HU (+ HU) is shown. Triplicate measurements were performed and error bars indicate standard deviations.
  • B Fractional activity, with respect to catalase activity under the same conditions in the absence of HU, is plotted versus log [cHU (mM)]. The black line indicates the best fit to a sigmoidal response curve.
  • C Catalase activity plotted versus concentration in the presence of varying concentrations of HU (mM). Black lines indicate the best fit to a
  • Figure 8 shows binding site of HU determined by crystallography.
  • A Omit 2mFo- DFc electron density of the active site funnel with HU contoured at 1.1 ⁇ . Water molecules are indicated with arrows. The ligand and the two water molecules shown with density were omitted for map calculations.
  • B Water-mediated interactions of HU with the main chain of Argl26 and the side chain of Aspl27. Putative hydrogen bonds are shown with dashed lines. The heme group is shown in the background.
  • C HU acts as a stopper in the substrate tunnel leading to the heme-containing active site. HU is shown as sticks and catalase in a surface presentation. See Table Sl for data collection and refinement statistics.
  • FIG. 9 shows catalase activity in patient blood samples.
  • Catalase activity in blood samples from a control group Normal
  • Values were normalized to the average activity of the Normal group. Closed triangles indicate that patients were undergoing HU-treatment at the time of sampling. SEM for technical replicates and more detailed diagnoses, where available, are shown in Figure 10.
  • Figure 10 shows catalase activity in patient blood samples.
  • CML Chronic myeloid leukemia
  • AML Acute myeloid leukemia
  • MF MF
  • PV Polycytaemia vera
  • TC Trombocytosis
  • CLL Chronic lymphatic leukemia
  • L L
  • Lymphoma Activity: Catalase activity normalized to the average of the Normal group; SEM : Standard error of the mean; HU : NA: information not available.
  • hydroxyurea (HU) is also known under the synonym hydroxycarbamide.
  • Hydroxyurea has the chemical formula CH 4 N 2 O 2 .
  • IUPAC name Hydroxyurea.
  • CAS number 127-07-1. It is to be understood that derivatives of hydroxyurea and hydroxyurea degradation products are included in the present invention.
  • the catalase status of a sample refers to the catalase enzyme activity level, the RNA level of catalase, the protein level of catalase, the catalase gene mutation status in said sample or combinations of these parameters.
  • the (biological) sample of the invention refers to a sample obtained from a subject.
  • the sample may be a biopsy or a body fluid sample such as a blood sample.
  • the sample may be processed before subjecting the sample to any of the methods of the invention. For example a sub-fraction of the sample may be isolated such as a sub-population of cells e.g. by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the term sensitive or sensitivity refers to the susceptibility of e.g. a tissue or cell population to treatment with a drug or drug composition compared to the reference standard, cell, or cell population.
  • the standard may for example be the corresponding healthy tissue, cell, or cell population, for example be the corresponding non-neoplastic counterpart (wild type), or non- malignant non-neoplastic counterpart.
  • the terms a sensitive or a susceptible cell and a sensitive or a susceptible tissue refer to cells or tissues obtained from a patient, wherein said cells or tissue is likely to respond to the administration of said drug or composition. Thus, the clinician would consider using said drug or composition for the treatment of said patient. Resistance
  • resistant or resistance refers to the reduced susceptibility of e.g. a tissue or cell population to the treatment a drug or drug composition compared to the above reference standard, cell, or cell population.
  • Susceptibility to the treatment with a drug or drug composition may typically be monitored as the response to the treatment with a drug or drug composition in terms of rate of survival and/or growth rate in the presence of said drug or composition.
  • increased survival or maintained or increased growth compared to the reference indicates resistance to said drug or drug composition.
  • the drug may be hydroxyurea and the drug composition may be a composition comprising hydroxyurea. It is to be understood that a sample of the invention may have some degree of resistance, without having lost all activity of a certain biomarker e.g. catalase activity. However, the change in susceptibility of the cells in the sample to e.g. hydroxyurea may be of importance in the clinical decision on the treatment of the patient
  • Neoplastic disorder is herein defined as a disorder comprising an abnormal mass of tissue or cells as a result of neoplasia.
  • Neoplasia is the abnormal proliferation of cells.
  • Neoplasms may be benign, pre-malignant or malignant.
  • solid tumour is synonymous with a neoplasm that has formed a lump.
  • Some neoplastic disorders do not cause a lump; they include haematological leukemia and non- solid tumours such as most forms of carcinoma in situ. Cancer is a malignant neoplastic disorder.
  • conservative substitution refers to a gene product wherein a substitution of one amino acid with another with generally similar properties (size, hydrophobicity, etc) likely does not seriously affect the overall function of the protein.
  • Conservative amino acid substitution as used herein relates to the substitution of one amino acid (within a predetermined group of amino acids) for another amino acid (within the same group), wherein the amino acids exhibit similar or substantially similar characteristics.
  • Preferred conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
  • a gene product according to the invention may comprise, within the same gene product, or among different gene products, at least one substitution, such as a plurality of substitutions introduced independently of one another. It is clear from the above outline that the same gene product may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above.
  • Tyrosine kinases play a critical role in the modulation of growth factor signaling. Activated forms of these enzymes can cause increases in tumor cell proliferation and growth, induce antiapoptotic effects, and promote angiogenesis and metastasis. In addition to activation by growth factors, protein kinase activation by somatic mutation is a common mechanism of tumor genesis. Because all of these effects are initiated by receptor tyrosine kinase activation, they are key targets for inhibitors (Arora A, Scholar EM. Role of tyrosine kinase inhibitors in cancer therapy. J Pharmacol Exp Ther. 2005 Dec;315(3) :971-9. Table 1, table 2 and figure 1 in Arora A and Scholar EM List different tyrosine kinase inhibitors which are hereby incorporated by reference.
  • tyrosine kinase inhibitors which are known drugs in the treatment of different neoplastic disorders are given below:
  • Sorafenib (BAY 43-9006) :
  • Leflunomide (SUlOl) Systematic (IUPAC) name: 5-methyl-N-[4-(trifluoromethyl) phenyl]-isoxazole-4-carboxamide
  • Sub-typing a neoplastic disorder such as a cancer in relation to hydroxyurea sensitivity would be a strong tool in cancer diagnosis. This may be done by knowing one or more biomarkers related to hydroxyurea sensitivity.
  • the invention relates to a method for sub-typing a neoplastic disorder, said method comprising
  • neoplastic disorder is likely to be a hydroxyurea resistant subtype of said neoplastic disorder or a hydroxyurea sensitive subtype of said neoplastic disorder.
  • the invention relates to method for sub-typing a neoplastic disorder, said method comprising
  • composition comprising one or more
  • tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
  • the neoplastic disorder may be divided into hydroxyurea sensitive or hydroxyurea resistant subtypes. Thus, it may be possible to evaluate whether it is likely that
  • hydroxyurea is a suitable treatment or if an alternative treatment is preferable.
  • the method may be appropriate in assisting the clinician in the decision of selecting the concentration of hydroxyurea to be administrated to a patient, since a sample showing low catalase activity may require a higher dose of hydroxyurea to achieve a suppressive effect on the neoplastic disorder.
  • the method of the invention may be employed by the clinician to assist the diagnosing of the subtype of a neoplastic disorder such as a cancer and further in the clinical decision on the treatment.
  • a neoplastic disorder such as a cancer
  • the invention relates to a method wherein said at least one parameter being equal to or less than said predetermined threshold level or said known standard present in said neoplastic disorder with known sensitivity to treatment with hydroxyurea is indicative of the neoplastic disorder being a hydroxyurea resistant subtype of said neoplastic disorder.
  • an amount of catalase protein or catalase RNA in a sample being equal to or less than a predetermined threshold level or standard may be indicative of the neoplastic disorder (e.g. a cancer) being hydroxyurea resistant.
  • the catalase activity in the sample being equal to or less than a predetermined threshold level or standard may be indicative of the cancer being hydroxyurea resistant.
  • the mutational status of the catalase gene may also be indicative of the neoplastic disorder (e.g. cancer) being hydroxyurea resistant.
  • the invention relates to a method wherein said at least one parameter being greater than said predetermined threshold level or said known standard present in a neoplastic disorder with known sensitivity to treatment with hydroxyurea is indicative of the neoplastic disorder being a hydroxyurea sensitive subtype of said neoplastic disorder.
  • an amount of catalase protein or catalase RNA in a sample being greater than a predetermined threshold level or standard may be indicative of the neoplastic disorder (e.g. cancer) being hydroxyurea sensitive.
  • a catalase activity in the sample being greater than a predetermined threshold level or standard may be indicative of the cancer being hydroxyurea sensitive.
  • the mutational status of the catalase gene may also be indicative of the cancer being hydroxyurea sensitive.
  • the invention may also be used for assisting in the evaluation of whether a neoplastic disorder is a subtype more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors or a subtype equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • the invention relates to a method, wherein the at least one parameter being greater than said predetermined threshold level indicates a neoplastic disorder being a subtype more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • the invention in another embodiment relates to a method, wherein the at least one parameter being lower or equal to said predetermined threshold level indicates a neoplastic disorder being a subtype equally of less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • composition or “preparation” is also to be understood, as "kit of parts” in the sense that the combination partners e.g. tyrosine kinase inhibitor (a) and catalase inhibitor (b) as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b) i.e., simultaneously or at different time points.
  • the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
  • the ratio of the total amounts of the combination partner (a) and (b) to each other being administered in the combined preparation/composition can be varied, e.g.
  • an advantage of the present invention may be to assist in determining whether the effect on the treated disease in the combined use of the parts is larger than the effect which would be obtained by use of only one of the combination partners.
  • an advantage of the present invention may be to assist in determining whether there is at least one beneficial effect, e.g., a mutual enhancing of the effect of the combination partners (a) and (b), in particular a synergism, e.g.
  • a neoplastic subtype may comprise more than one of characteristics disclosed in the present invention.
  • a subtype may comprise two of the characteristics, such as being 1) both a hydroxyurea resistant subtype and a subtype equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors and 2) being a hydroxyurea sensitive subtype and a subtype being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • catalase inhibitors may have side effects beside the desired effect it may be advantageously to evaluate whether such administration is expected to have the desired effect.
  • a catalase inhibitor such as hydroxyurea
  • the administration of a catalase inhibitor, such as hydroxyurea should therefore preferably not be administered to subject until the catalase status of the relevant sample has been determined.
  • hydroxyurea is an inhibitor of catalase.
  • the catalase inhibitor is
  • the threshold value or standard may be in the form of known catalase values obtained from a number of samples obtained from hydroxyurea resistant samples (e.g the average catalase activity or catalase RNA level of the samples), hydroxyurea sensitive samples or both. These values could then used to form the base level of the amount of catalase (protein level/RNA level/protein activity) which is required to form a threshold or standard for indicating that the cancer is a hydroxyurea resistant subtype or another subtype according to the invention.
  • the threshold or standard may also take further parameters into account such as knowledge of e.g. gender, age, medical history, and blood type.
  • a threshold level which enables the discrimination between two populations using standard statistical methodology (e.g. by receiver operating characteristic (ROC) curve analysis). Accordingly, the skilled person would know how to employ such methods to set threshold levels and standard levels for biomarkers in relation to protein level, RNA level and protein activity.
  • ROC receiver operating characteristic
  • the prediction may be combined with other biomarkers, such as the status of other proteins and/or data obtained from histological or cytological analyses of the sample or another corresponding sample obtained from the same subject. In this way the power of the prediction can be increased.
  • catalase gene mutation status is used as a predictive marker the known DNA sequence of catalase or a consensus sequence can be used as a standard. If the analysis shows that the gene comprises one or more mutations, the one or more mutations can be compared to a list of known mutations resulting in a lowered catalase activity. Such mutations may cause an altered protein sequence, the introduction of stop codons in the RNA leading to the expression of a dysfunctional catalase protein, mutations in non-transcribed regions (both 5'- UTR and 3'-UTR regions) such as promoter and enhancer sequences leading to an altered expression and mutations resulting in an altered splice pattern.
  • the invention relates to a method for predicting responsiveness of a neoplastic disorder to hydroxyurea treatment, said method comprising
  • the invention relates to a method for predicting responsiveness of a neoplastic disorder to hydroxyurea treatment or treatment with a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors, said method comprising
  • the prediction of the responsiveness of a neoplastic disorder to hydroxyurea treatment or treatment with a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors may be used in the clinical evaluation of the disease before a treatment is initiated.
  • a clinical evaluation may include selecting a drug or drugs and assessing the dose of the drug(s) to be administrated. It may also be an advantage to evaluate more than one parameter to improve the method.
  • at least two parameters are measured, such as three parameters or such as four parameters.
  • the invention relates to a method, wherein the neoplastic disorder is predicted to be resistant to hydroxyurea if the at least one parameter is equal to or less than said
  • an amount of catalase protein or catalase RNA in a sample lower or similar to a
  • predetermined threshold level or a known standard may be indicative of the neoplastic disorder being resistant to hydroxyurea treatment.
  • a catalase activity in the sample lower or similar to a predetermined threshold level or a known standard may be indicative of the neoplastic disorder being resistant to hydroxyurea treatment.
  • the mutational status of the catalase gene may also be indicative of the cancer being resistant to hydroxyurea treatment.
  • the invention relates to a method, wherein the neoplastic disorder is predicted to be sensitive to the hydroxyurea if the at least one parameter is greater than said predetermined threshold level or said known standard present in the neoplastic disorder with known sensitivity to treatment with hydroxyurea.
  • an amount of catalase protein or catalase RNA in a sample higher than a predetermined threshold level or a known standard may be indicative of the neoplastic disorder being sensitive to hydroxyurea treatment.
  • a catalase activity in the sample higher than a predetermined threshold level or a known standard may be indicative of the neoplastic disorder being sensitive to hydroxyurea treatment.
  • the mutational status of the catalase gene may also be indicative of the cancer being sensitive to hydroxyurea treatment.
  • catalase As a biomarker to predict the responsiveness of a neoplastic disorder to be more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, it may be an advantage to compare at least one parameter of the catalase status to a predetermined threshold value or a known standard to determine if the neoplastic disorder is indicative of being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • the neoplastic disorder is predicted to be more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors if the at least one parameter is greater than said predetermined threshold level or known standard.
  • the neoplastic disorder is predicted to be equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors if the at least one parameter is equal to or less than said predetermined threshold level or known standard.
  • a neoplastic disorder may be predicted to be 1) both hydroxyurea resistant and equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors and 2) both being a hydroxyurea sensitive and a being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • the administration of catalase inhibitors to a subject may have side effects beside the desired effect it may be advantageously to evaluate whether such administration is expected to have the desired effect.
  • a catalase inhibitor such as hydroxyurea
  • the administration of a catalase inhibitor, such as hydroxyurea should preferably not be administered to a subject until the catalase status of the relevant sample has been determined.
  • WO 2006/136391 discloses a combinatorial therapy of cancer diseases comprising tyrosine kinase inhibitors and optionally hydroxyurea.
  • hydroxyurea is an inhibitor of catalase and it may therefore be relevant to determine the catalase status before a combinatorial treatment as disclosed in e.g. WO 2006/136391 is initiated. By doing this at may be determined whether a specific subject will have a synergistic effect of the combined treatment compared to the monotherapies.
  • the invention relates to a method of monitoring the status of a neoplastic disorder in a subject, said method comprising
  • RNA level of catalase ii. the RNA level of catalase
  • protein level of catalase iii. the protein level of catalase
  • the invention relates to a method of monitoring the status of a neoplastic disorder in a subject, said method comprising
  • neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
  • - has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • the method of monitoring the status of a neoplastic disorder in a subject comprises
  • neoplastic disorder has progressed to become a hydroxyurea resistant subtype of said neoplastic disorder, regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder, or the sensitivity to hydroxyurea of said neoplastic disorder is maintained.
  • the method further relates to indicating whether the neoplastic disorder has progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a
  • composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors. It is to be understood that the two or more samples may be compared to each other before being compared to said threshold level or said known standard,
  • the catalase status of a neoplastic disorder can be compared over time, allowing the clinician to evaluate the progression of the hydroxyurea sensitivity or sensitivity to a combinatorial treatment as described above. It may also be an advantage to evaluate more than one parameter in the at least two samples to improve the method. Thus, in one embodiment of the invention at least two parameters are measured such as three parameters or such as four parameters. When comparing the at least two samples from a subject with respect to one or more parameters, it may be advantageous to evaluate the progression of hydroxyurea sensitivity.
  • the invention relates to a method, wherein it is likely that said neoplastic disorder has progressed to become a hydroxyurea resistant subtype of said neoplastic disorder if the at least one parameter is lower than said predetermined threshold level or said known standard present in the neoplastic disorder with known sensitivity to treatment with hydroxyurea, and if the value is lower in the sample obtained at the latest point in time.
  • Development of resistance is a known problem during treatment of a neoplastic disorder, therefore comparing different samples from a subject over time may be advantageous.
  • a resistant subtype of a neoplastic disorder may progress to a sensitive subtype.
  • one embodiment of the invention relates to a method, wherein it is likely that said neoplastic disorder has progressed to become a hydroxyurea sensitive subtype of said neoplastic disorder if the at least one parameter is greater than said predetermined threshold level or said known standard present in the neoplastic disorder with known sensitivity to treatment with hydroxyurea and if the value is greater in the sample obtained at the latest point in time. Going from a resistant subtype towards a sensitive subtype may occur e.g. in the case where the subject is stimulated with a substance increasing catalase activity or it could be a spontaneous event.
  • the invention relates to a method, wherein it is likely that said sensitivity to hydroxyurea in the neoplastic disorder is maintained if the value is equal in the samples obtained at different points in time.
  • the neoplastic disorder may be maintained in a hydroxyurea sensitive state or in a hydroxyurea resistant state.
  • the at least one parameter in the samples may be compared to said predetermined threshold level or said known standard.
  • Acquiring data indicating that the hydroxyurea sensitivity is likely unchanged may be used to suggest maintaining the current treatment with hydroxyurea if the neoplastic disorder is still sensitive to hydroxyurea treatment or alternatively to keep using an alternative treatment if the data indicates that the neoplastic disorder is still resistant to hydroxyurea treatment. Over time the neoplastic disorder may have changed its responsiveness towards a combinatorial treatment.
  • the invention relates to a method wherein it is likely that said neoplastic disorder has progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors disorder if the at least one parameter is greater than said predetermined threshold level or said known standard, and if the value is greater in the sample obtained at the latest point in time.
  • the invention relates to a method wherein it is likely that said neoplastic disorder has progressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors if the at least one parameter is lower than said predetermined threshold level or said known standard present in the neoplastic disorder and if the value is lower in the sample obtained at the latest point in time.
  • a switch to or from a therapy with hydroxyurea or with a tyrosine kinase inhibitor and a catalase inhibitor may be advantageously if a change in the status of catalase is measured.
  • the one or more tyrosine kinase inhibitor is selected from the group consisting of 4-[(4- methylpiperazin-l-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2- yl)amino]phenyl]benzamide (imatinib), 4-[(4-Methyl-l-piperazinyl)methyl]-N-[4- methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamide
  • the one or more catalase inhibitors are selected from the group consisting of hydroxyurea and 3-Amino-Triazole.
  • the catalase inhibitor is one used in the treatment of a neoplastic disorder.
  • the catalase inhibitor is hydroxyurea as discovered by the inventors.
  • CAS number for hydroxyurea is 127-07-1.
  • the invention relates to a method wherein a sample such as a blood sample obtained from a subject is provided.
  • the cells in the sample are then analyzed for catalase activity by measuring the turnover of hydrogen peroxide using photospectrometry.
  • a sub-population of the cells may be isolated (e.g by FACS sorting).
  • a low activity of catalase is indicative of the subject being resistant to hydroxyurea treatment and/or is indicative of the subject being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition without one or more catalase inhibitors.
  • the status of catalase can be measured in different ways using different parameters.
  • the invention relates to a method, wherein said at least one parameter is catalase enzyme activity in said sample.
  • Catalase possesses at least two enzymatic functions; an enzymatic reaction leading to decomposition of hydrogen peroxide wherein hydrogen peroxide is converted into water and oxygen, and a second peroxidase reaction wherein hydrogen peroxide is decomposed to water with the aid of an additional compound RH 2 .
  • RH 2 may be in the form of phenols, formic acid, formaldehyde and alcohols.
  • the measurement also shows if the activity has been influenced by e.g. post-translational modifications such as phosphorylations and interactions with other proteins. This is not the case when evaluating the DNA or RNA status.
  • Catalase activity may be monitored in different ways.
  • the invention relates to a method, wherein said catalase enzyme activity is
  • the invention relates to a method, wherein said substrate is selected from the group consisting of hydroxyurea, hydrogen peroxide and sodium perborate.
  • Sodium perborate may also be used as a substrate for catalase since it may be more stable than hydrogen peroxide (H 2 O 2 ).
  • Sodium perborate may be measured by titrating KMnO 4 in the presence of sulphuric acid. The person skilled in the art knows how to optimize such assays.
  • Hydrogen peroxide may also be used as a substrate for catalase.
  • the invention relates to a method, wherein said substrate is hydrogen peroxide. Measuring hydrogen peroxide may be done by
  • the invention relates to a method, wherein said turnover is determined as the change in absorbance at 240 nm.
  • the turnover of hydrogen peroxide may also be monitored using enzymatic assays.
  • the invention relates to a method, wherein said turnover of hydrogen peroxide is measured using horseradish peroxidise (HRP) as a reporter enzyme.
  • HRP horseradish peroxidise
  • kits which may also be used for performing the assay include OxiSelectTM supplied by Cell Biolabs, Inc., the Catalase Assay Kit supplied by Sigma-Aldrich, Inc. and The Amplex ® Red Catalase Assay Kit supplied by Molecular Probes. Before analysis the cells may be sorted using FACS.
  • the invention relates to a method wherein a sample such as a blood sample is obtained from subject.
  • the cells in the sample are then analyzed for the amount of catalase RNA using Q-PCR. Before analysis the cells may be sorted using FACS.
  • a low amount of catalase RNA is indicative of the subject being resistant to hydroxyurea treatment and/or indicative of the subject being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition without one or more catalase inhibitors.
  • the catalase status of a sample may also be determined by measuring the RNA level of catalase.
  • the invention relates to a method, wherein said at least one parameter is the RNA level of catalase in said sample. Measuring RNA levels may be advantageous in cases where catalase activity assays are not appropriate. This could be in cases where only small amounts of a sample are available or where an amplification step is necessary. It may also be advantageous where the sample has been fixed in a fixative making activity measurement inappropriate. Such fixatives could be cross-linking fixatives such as formalin.
  • the invention relates to a method, wherein assessment of the RNA level of catalase comprises the use a technique selected from the group consisting of PCR, Q-PCR, RT-PCR, a hybridization technique, and micro-arrays.
  • PCR-techniques have the advantage of a large amplification and allow for real-time monitoring. Design of primers and amplification programs are standard techniques for the person skilled in the art. Hybridization techniques like Northern blotting may be preferred if there is no access to quantitative PCR- equipment.
  • Microarrays have the advantage of multiplexing, allowing several biomarkers to be measured simultaneously. This speeds up the testing phase and saves sample materials. FACS may be applied to the samples before analysis to remove the contribution from e.g. non-malignant cells to the read-out.
  • the invention relates to method wherein a sample such as a blood sample is obtained from subject.
  • the cells in the sample are then analyzed for the presence of the catalase protein using a monoclonal or polyclonal antibody against the catalase protein which is subsequently detected using a secondary antibody.
  • a low amount of catalase is indicative of the subject being resistant to hydroxyurea treatment and/or indicative of the subject being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition without one or more catalase inhibitors.
  • the cells may be sorted using FACS.
  • the invention relates to a method, wherein said at least one parameter is the protein level of catalase in said sample.
  • Protein level measurement may also be used when the sample has been in a fixative making activity measurement inappropriate.
  • fixatives could be cross-linking fixatives such as formalin. In some instances cross-linking fixatives also results in degradation of nucleic acids, making both DNA and RNA analysis difficult.
  • the invention relates to a method, wherein assessment of the protein level of catalase comprises the use a technique selected from the group consisting of immuno-based assays and mass spectrometry.
  • immuno-based assays are western blotting, ELISA- techniques, immunohistochemistry, immunocytochemistry and protein arrays.
  • Mass spectrometry (MS) may also be advantageous in some cases, since it is very sensitive and several proteins may be monitored simultaneously. A further advantage by MS is that antibodies are avoided. FACS may be applied to the samples before analysis.
  • the predetermined threshold of the invention may be determined by any suitable statistical method.
  • Receiver operating characteristic (ROC) curve analysis is a classification model for a mapping of instances into a certain class/group.
  • ROC curve analysis may be used determine the classifier boundary between groups of samples (or patients) for which the classifier boundary between classes must be determined by a threshold value, for instance to determine whether a person is likely to have subtype of a neoplasm or not, or if a patient is likely to respond to a treatment or not.
  • a threshold value for instance to determine whether a person is likely to have subtype of a neoplasm or not, or if a patient is likely to respond to a treatment or not.
  • the predetermined threshold level is selected by receiver operating characteristic (ROC) curve analysis.
  • This methodology may be applied to determining the predetermined threshold for method comprising the assessment of catalase enzyme activity, catalase RNA or protein level.
  • the method comprises:
  • neoplastic disorder is likely to be a hydroxyurea resistant subtype of said neoplastic disorder or a hydroxyurea sensitive subtype of said neoplastic disorder; thereby predicting the sensitivity or resistance of said neoplastic disorder to treatment with hydroxyurea; or thereby indicating whether it is likely that said neoplastic disorder has progressed to become a
  • hydroxyurea resistant subtype of said neoplastic disorder regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder, the sensitivity to hydroxyurea of said neoplastic disorder is maintained, progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • the invention relates to a method wherein a sample such as a blood sample is obtained from a subject. Genomic DNA is extracted from the cells in the sample and the entire or parts of the genomic sequence of the catalase gene is determined using first a DNA amplification method (e.g. a PCR method) and then standard sequencing techniques or re-sequencing by array hybridization or other appropriate means of acquiring sequence information. Both cDNA and genomic DNA can be sequenced. Before analysis the cells may be sorted using FACS.
  • a DNA amplification method e.g. a PCR method
  • a sequence deviating from the standard sequence at either the protein level and/or the DNA level is indicative of the subject being resistant towards hydroxyurea treatment and/or indicative of the subject being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition without one or more catalase inhibitors.
  • the catalase status may also be determined by querying the DNA status of a genome.
  • the invention relates to a method, wherein said at least one parameter is the catalase gene mutation status, and said catalase gene mutation status is assessed by detecting the presence or absence of a mutation in the catalase gene.
  • Determining the catalase gene mutational status provides information which is normally not obtained by looking at protein or RNA levels. Looking at the DNA sequence can be used for determining if the patient has a hereditary character making him or her less suitable for hydroxyurea treatment. Mutations may also have occurred spontaneously in some cells (e.g. cancer cells). If the analysis shows that the gene (or the sequence surrounding the gene) comprises one or more mutations, this or these mutations can be checked against known mutations resulting in a lowered catalase activity.
  • Such mutations may be mutations resulting in an altered protein sequence, the introduction of stop codons in the RNA, mutations in non-transcribed regions (both 5'-UTR and 3'-UTR regions) such as promoter and enhancer sequences resulting in an altered expression and mutations resulting in an altered splice pattern.
  • a standard sequence is required.
  • the invention relates to a method, wherein said known standard is the nucleic acid sequence encoding catalase set forth in at least one of SEQ ID NO:2 and SEQ ID NO:3.
  • the known DNA sequence of catalase can be used as a standard.
  • SEQ ID NO:2 shows the cDNA sequence of catalase
  • SEQ ID NO: 3 shows the genomic sequence of catalase.
  • Checking the genomic sequence gives additional information concerning mutations positioned in e.g. introns relating to e.g. a changed splicing pattern, whereas looking at the cDNA sequence may provide information concerning the actual splicing pattern.
  • mutations may be in the form of deletions of one or more nucleic acids, insertions of one or more nucleic acids.
  • the invention relates to a method, wherein said known standard is a list comprising at least one nucleic acid sequence encoding catalase with no or reduced catalyse enzyme activity.
  • the list of nucleic acid mutations coding for a polypeptide sequence indicative of a lowered catalase activity compared to a reference such a wild-type catalase may be used by the clinician in the methods of the invention to evaluate the genetic background of the neoplastic disorder and indicate whether neoplasms is likely to be resistant or not.
  • Such a list may also be a list of amino acid positions in the catalase protein which is known to have a lower catalase activity than the standard catalase protein sequence.
  • the invention relates to a method, wherein said at least one nucleic acid sequence encodes a catalase polypeptide with no or reduced catalase enzyme activity, said catalase polypeptide is selected from the group consisting of polypeptides deviating at position 71, 83, 88, 114, 121, 124, 147, 192, 195, 261, 275, 277, 303, 342, 354, 463, 503 and 517 of SEQ ID NO: 1.
  • the deviation is a non-conservative amino acid substitution.
  • Mutations which are likely to change the expression level of catalase or the activity of the catalase enzyme may be in form of 1) non-synonymous mutations, relating to mutations in a codon which result in an altered amino acid sequence, 2) nonsense mutations, relating to a point mutation in a sequence of DNA that results in a premature stop codon, 3) deletions or insertions at one or more of the positions which may result in a changed reading frame resulting in a non- functional protein product, and 4) inversions, resulting in a changed coding sequence. 5) Large deletions, 6) Rearrangements of the catalase gene.
  • the invention relates to a method, wherein the presence of at least one amino acid substitution in at least one of the positions selected from the group consisting of position 71, 83, 88, 114, 121, 124, 147, 192, 195, 261, 275, 277, 303, 342, 354, 463, 503 and 517 of SEQ ID NO: 1, is indicative of the neoplastic disorder being a hydroxyurea resistant subtype and/or indicative of the subject being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition without one or more catalase inhibitors.
  • an indication of a subtype of a neoplastic disorder can be determined by analyzing the catalase gene at the DNA level.
  • the DNA status can also be determined by sequencing the transcribed RNA, with the limitations that non- transcribed regions of the gene will not be sequenced. Precise amino acid changes resulting in decreased catalase activity are listed in figure 1.
  • the invention relates to mutations selected from the list in figure 1.
  • the standard is a consensus sequence of a nucleic acid or amino acid sequence encoding a functional catalase enzyme with an activity comparable to the wild type catalase enzyme. In another embodiment, the standard is a consensus sequence of a nucleic acid or amino acid sequence encoding a catalase enzyme with a reduced activity comparable to the wild type catalase enzyme.
  • the human genome normally comprises two alleles of each gene.
  • the invention relates to a method, wherein the presence of the mutation is heterozygous or homozygous. Both heterozygote and homozygote mutations may result in an altered hydroxyurea response, since not all mutations can be fully complemented by the presence of a non-mutated catalase protein.
  • loss-of-heterozygosity LOH may lead to hydroxyurea resistance. LOH may reflect the appearance of another mutation, deletion etc. in the other catalase allele causing the loss-of-function of that allele.
  • the invention relates to a method, wherein said sample is obtained from a subject currently receiving treatment for said neoplastic disorder. In this way unnecessary sampling is avoided. It may furthermore be advantageous if the samples are obtained from a person under treatment with hydroxyurea.
  • said treatment comprises the
  • the invention relates to a method, wherein the neoplastic disorder is a cancer.
  • the cancer may both be of a solid cancer or a leukemia. Therefore, in an embodiment the invention relates to a method, wherein the cancer is selected from the group consisting of leukemia and solid tumor cancers.
  • the method may be especially suited for the evaluation of haematological neoplasms such as leukemia cancers. Therefore, one embodiment of the invention relates to a method, wherein said neoplastic disorder is a haematological neoplasm.
  • the haematological neoplasm is a malignant haematological neoplasm.
  • the invention chronic myeloid leukemia (CML), chronic lymphatic leukemia (CLL), polycytemia vera, thrombocytosis,
  • the invention relates to a method, wherein the cancer is a chronic myeloid leukemia (CML).
  • CML chronic myeloid leukemia
  • leukemia may be interpreted as "hematological malignancies”.
  • solid tumor cancer is selected from the group consisting of glioblastoma and gastrointestinal stromal tumors.
  • the sample to be analysed by the method of the invention may be provided from several sources. Therefore, in an embodiment the invention relates to a method, wherein the sample is a blood sample or tissue sample.
  • the sample is a blood sample it may be an advantage to sort the samples into specific cell populations (preferably healthy and malignant cell populations) e.g. by the use of flow cytometry e.g. FACS) or affinity beads.
  • the sample is cells isolated from a biopsy or body fluid sample such as a blood sample such as by means of FACS sorting or affinity-bead purification (e.g. antibody coated magnetic beads).
  • FACS sorting or affinity-bead purification e.g. antibody coated magnetic beads.
  • the person skilled in the art knows how to optimize a cell isolation method.
  • the different populations can then subsequently be analyzed for catalase status according to the invention. Sorting cells prior to analysis allows healthy cells, with high, normal or low catalase activity, to be removed from the cells to be analyzed for catalase status.
  • Tissue samples may be provided from e.g. solid tumors or skin biopsies.
  • the sample is provided from a human subject.
  • kits making the user capable of performing the method of the invention. Therefore, in an embodiment the invention relates to a kit for performing the method of the invention, comprising
  • kits By having both the instruction manual and at least one reagent comprised in a kit practitioners may be able to perform the invention.
  • One of the reagents present in the kit may be hydrogen peroxide.
  • the invention relates to a kit, wherein said at least one reagent is hydrogen peroxide.
  • Hydrogen peroxide may be used when the assay relates to testing for catalase activity.
  • Other reagents which may be present in such a kit could be selected from the group consisting of sodium perborate, KMnO4 and sulphuric acid.
  • the kit may comprise primers for amplification part of the catalase gene or mRNA.
  • the kit may comprise antibodies against catalase (monoclonal or polyclonal), secondary antibodies and detection reagents.
  • the kit may comprise microarrays that can be used to specifically detect changes in the catalase gene with respect to a reference sequence.
  • a kit may also comprise reagents for performing two or more of the assays described above.
  • Arabidopsis thaliana ecotype Columbia (CoI-O) wild type and mutant plants were grown on Vi Murashige and Skoog medium (Duchefa, M0222) solidified with 0.9% agar in growth chambers at 21°C, 16/8 hours light/dark cycles and 50 ⁇ mol m ' V 1 photosynthetic photon flux.
  • HU US Biological, H9120
  • HU stock solution was added to the medium immediately before pouring plates.
  • RNR activity was quantified as described in (Jong AY, Yu K, Zhou B, Frgala T, Reynolds CP, et al. (1998) A simple and sensitive ribonucleotide reductase assay. J Biomed Sci 5: 62-68). Cytidine 5'-diphosphate, triammonium salt, [2-14C]- (Moravek) was used for labeling. 300 mg of plant tissue per sample was collected and ground in liquid nitrogen.
  • HU was quantified as described in (Bachir D, Hulin A, Huet E, Habibi A, Nzouakou R, et al. (2007) Plasma and urine hydroxyurea levels might be useful in the management of adult sickle cell disease. Hemoglobin 31 : 417-425). Between 8 and 30 mg of leaf tissue per sample was collected and ground in phosphate buffered saline (PBS). The concentration of HU in plant shoots was calculated assuming a water content of 85%. Catalase enzyme assay and materials
  • Catalase activity was quantified by monitoring the rate of H 2 O 2 breakdown at 240 nm.
  • GraphPad Prism 5 was used for statistical analysis and curve fitting.
  • Plant extracts were prepared by grinding ca. 60 mg of tissue in PBS. Rat erythrocytes were isolated by centrifugation and lysed in distilled water. Blood samples were drawn into 10 ml ethylenediaminetetraacetic acid (EDTA) stabilized tubes. Less than one hour after vein puncture samples were processed into mononuclear cells (MNC) by Lymphoprep differential centrifugation at 2100 rpm for 10 minutes. The interface mononuclear cell suspension was harvested and washed twice with PBS.
  • EDTA ethylenediaminetetraacetic acid
  • Isolated MNCs were counted and subsequently frozen in dimethyl sulfoxide (DMSO) in liquid nitrogen.
  • Cell lysates were prepared from 5xlO 6 cells thawed at 37°C, spun at 300 x g for 5 minutes, washed with PBS and finally lysed in distilled water.
  • Catalase activity was normalized to the total protein concentration for each sample.
  • a commercially available aqueous catalase solution was used (Sigma, C3155).
  • Crystals of bovine liver catalase (Sigma, C3155) were obtained through vapor diffusion by mixing a solution of catalase dissolved in 0.5 M sodium phosphate buffer pH 6.6 at 22 mg/ml in a 1 : 1 ratio with reservoir buffer containing 12% PEG4000 (w/v). The crystals were soaked with HU by leaving them briefly in cryo buffer (30% MPD, 8.4% PEG4000, 50 mM HU) before flash freezing in liquid nitrogen. Data were collected at MAX-Lab 911-2 and at ESRF ID14-2 and processed with XDS. The structure was determined by molecular replacement (RCSB entry: 4BLC) using PHASER and iteratively rebuilt in Coot and refined with PHENIX. REFINE.
  • HU was primarily recognized through two water molecules. One of these linked the HU alcohol and the main chain carbonyl group of Argl26 (Fig 8B). The HU amide was less well recognized, and the amino- and carbonyl group could at this resolution be distinguished on the basis of electron density (omit 2mFo-DFc). In one of the four catalase subunits within the asymmetric unit, a water molecule could bridge the amide group to the side chain of Aspl27 through hydrogen bonds, suggesting that here the amino group coordinated this water ( Figure 8B). For this reason, the orientation with the amide amino group directed against side chain of Aspl27 was imposed for all four copies, even though the electron density for this water was not strong in the remaining three catalase subunits. Crystallographic data
  • R-factor ⁇ h
  • R-free is identical to R- factor on a subset of test reflections not used in refinement.
  • HU taken up by Arabidopsis thaliana should inhibit its primary target RNR and kill plants at a concentration physiologically relevant to cancer treatment.
  • HU a concentration physiologically relevant to cancer treatment.
  • the Arabidopsis thaliana catalase 2 protein (seq ID. NO:4) was aligned to the
  • This Ki value is comparable to those previously published for RNR inhibition by HU (0.07-0.13 mM).
  • peak plasma levels can reach concentrations of 1 mM after HU administration to blood cancer patients, HU treatment could have an impact on cellular catalase activity.
  • the HU-catalase interaction may therefore be of relevance to clinicians with regard to both therapeutic and side effects of HU treatment.
  • these data clearly shows that HU inhitbits catalase activity and that this inhibition is most likely due to competitive inhibition.
  • HU is lodged in the catalase substrate tunnel.
  • HU resistance in Arabidopsis is that loss of catalase function prevents catalase-mediated conversion of HU to a cytotoxic.
  • the catalytic cycle of catalase can be divided into two steps: activation by one molecule of hydrogen peroxide to compound I and subsequent reaction of compound I with a second molecule of hydrogen peroxide returning the enzyme to its resting state.
  • Chronic myeloid leukemias are characterized by high catalase activity.
  • HU chronic myeloid leukemia
  • CML chronic myeloid leukemia
  • CLL chronic lymphatic leukemia
  • CML chronic myeloid leukemia
  • catalase is a biomarker in relation to evaluating the potential effect of HU treatment in a malignant disease.
  • the levels of catalase may be used for subtyping a neoplastic disorder.
  • Malaria is a mosquito-borne infectious disease caused by a eukaryotic protist of the genus Plasmodium. It is widespread in tropical and subtropical regions, including parts of the Americas, Asia, and Africa.
  • Plasmodium parasite Five species of the Plasmodium parasite can infect humans; the most serious forms of the disease are caused by Plasmodium falciparum. Malaria caused by Plasmodium vivax, Plasmodium ovale and Plasmodium malariae causes milder disease in humans that are not generally fatal. A fifth species, Plasmodium knowlesi, is a zoonosis that causes malaria in macaques but can also infect humans
  • catalase may play a role in protecting parasites and their host cells from oxidative damage.
  • Glucose-6- phosphate dehydrogenase deficiency affords some protection against malaria and the underlying cause appears to be a higher susceptibility to oxidative damage due to decreased catalase activity.
  • catalase prevents killing of malaria parasites by activated macrophages and reduces the efficiency of the prime anti- malarial drug artemisinine, which depends on generation of reactive oxygen species for activity.
  • HU as a catalase inhibitor in anti-malarial treatment regimens could limit infection by exacerbating the oxidative challenge to parasite-hosting cells.
  • HU as monotherapy has shown a remarkable anti-malarial effect in a cerebral malaria mouse model (Pino P, Taoufiq Z, Brun M, Tefit M, Franetich JF, et al. (2006) Effects of hydroxyurea on malaria, parasite growth and adhesion in experimental models. Parasite Immunol 28: 675-680).
  • an immunosuppressant such as HU to patients suffering from infectious disease
  • the effect of hydroxyurea in malaria may depend on the status of catalase in the patient.
  • the invention relates to a method for sub-typing a subject suffering from malaria, said method comprising
  • the threshold level or said known standard thereby indicating whether the malaria patient is likely to be a hydroxyurea resistant subtype of said malaria or a hydroxyurea sensitive subtype of said malaria.
  • the samples When obtaining a sample from a malaria patient it would preferably be a sample selected from the group consisting of blood samples or tissue samples. More specifically the samples could be purified blood cells such as red blood cells (erythrocytes), which are known to be of particularly importance in malaria infections since the erythrocytes are a site of multiplication of the parasite.
  • erythrocytes red blood cells
  • said at least one parameter being equal to or less than said predetermined threshold level or said known standard present in said sample with known sensitivity to treatment with hydroxyurea is indicative of the subject being a hydroxyurea resistant subtype of said infectious disease.
  • said at least one parameter being greater than said predetermined threshold level or said known standard present in said infectious disease with known sensitivity to treatment with hydroxyurea is indicative of the subject being a hydroxyurea sensitive subtype of said infectious disease.
  • kits according to the invention may also be used for determining the effect of hydroxyurea treatment in a subject suffering from malaria.
  • the present invention is based on the novel discovery that there is a connection between the catalase status in a sample and the sensitivity towards hydroxyurea and that hydroxyurea is an inhibitor of catalase.
  • the invention relates to methods for sub-typing neoplastic disorders thereby assisting in determining optimal treatment regimes for said neoplastic disorders.
  • determining the catalase status in a sample it may be possible to assist in determining whether said neoplastic disorder is indicative of being a hydroxyurea urea resistant subtype or a hydroxyurea resistant subtype.
  • catalase inhibitors such as hydroxyurea
  • the invention furthermore relates to methods for assisting in
  • determining whether a neoplastic disorder is a subtype more sensitive to a composition or treatment protocol comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors (such as hydroxyurea) than to a composition (or treatment protocol) comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
  • a composition or treatment protocol comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors (such as hydroxyurea) than to a composition (or treatment protocol) comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.

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Abstract

The present invention is based on the novel discovery that there is a connection between the catalase status in a sample and the sensitivity towards hydroxyurea and that hydroxyurea is an inhibitor of catalase. Thus, the invention relates to methods for sub-typing neoplastic disorders thereby assisting in determining optimal treatment regimes for said neoplastic disorders. By determining the catalase status in a sample it may be possible to assist in determining whether said neoplastic disorder is indicative of being a hydroxyurea urea resistant subtype or a hydroxyurea resistant subtype. Since catalase inhibitors (such as hydroxyurea) may be used in combination with tyrosine kinase inhibitors, the invention furthermore relates to methods for assisting in determining whether a neoplastic disorder is a subtype more sensitive to a composition or treatment protocol comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors (such as hydroxyurea) than to a composition(or treatment protocol)comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.

Description

PREDICTIVE MARKER FOR HYDROXYUREA RESISTANCE
Technical field of the invention
The present invention relates to the field of sub-typing cancers. In particular the present invention relates to methods for predicting the sensitivity of cancer to hydroxyurea and to methods for predicting the sensitivity of a cancer to combinatorial therapies comprising both the administration of tyrosine kinase inhibitors and catalase inhibitors. Background of the invention
Blood cancers are often treated with cytotoxic agents that prevent cell divisions.
Unfortunately, relapses are frequent due to development of chemotherapeutic resistance in the cancer cells. Detailed knowledge of mechanisms causing chemo- resistance could allow for development of valuable and accurate methods for predicting therapy efficacy. These prediction methods may also be relevant when deciding whether a patient should have a monotherapy or a combinatorial therapy comprising more than one active component.
WO2008/074131 discloses a method for predicting therapeutic efficacy of a treatment of cancer patients with oxidative stress based therapies, wherein an elevated level of an oxidative stress adaption marker is indicative of resistance towards these therapies. Catalase is mentioned as an oxidative stress adaption marker.
WO2005/078127 discloses a method for detecting a genetic variation or polymorphism, i.e. a mutation, in a catalase gene, wherein the presence or absence of a variant genotype of the catalase gene is indicative of an increased risk towards cancer, especially colon and rectal cancer. It is further disclosed that the method may be used for determining whether a subject will benefit from treatment with a drug.
WO 2006/136391 discloses a combinatorial therapy of cancer diseases comprising tyrosine kinase inhibitors and optionally hydroxyurea.
Hydroxyurea (HU) is a known ribonucleotide reductase (RNR) inhibitor still used extensively in blood cancer therapy. Resistance towards HU is also a well-known problem in blood cancer therapies. When treated with HU, most patients suffering from myeloproliferative disorders initially respond well with normalization of blood counts, but ultimately the malignant process progresses due to development of HU-resistance through an unknown mechanism. Early on, it became clear that HU limits the cellular supply of deoxyribonucleotides by acting as an inhibitor of ribonucleotide reductase (RNR). The importance of the HU-RNR interaction was bolstered by a number of cell line studies, where changes in RNR properties were shown to influence HU resistance. In this context, relatively little attention has been given to alternative potential HU targets, perhaps both due to lack of evidence for their in vivo importance and to the dominant position of the HU-RNR interaction in the field.
Similar, hydroxyurea is suggested in combinatorial therapy of cancers together with tyrosine kinase inhibitors. Predictive markers of when such combinatorial therapy may be advantageously for the patient are relatively unknown. This is a clear drawback since many cancer drugs are toxic to the person and it would therefore be an improvement if prediction could be made on when such therapy would be preferred. Hence, an improved method for distinguishing between hydroxyurea resistant and sensitive cancers would be advantageous, and in particular a more efficient and/or reliable method for distinguishing between patients with hydroxyurea resistant and sensitive blood cancers would be advantageous. In addition more reliable methods for evaluating whether a synergistic effect is present when combinational therapy is evaluated against monotherapy would be advantageously.
Summary of the invention
Using a plant screening model, the inventors made the novel finding that hydroxyurea appears to be a catalase inhibitor. In an additional screening of patient material it was found that subtypes of neoplastic disorders indeed exist which show a differentiated expression of the catalase enzyme and that the sensitivity to hydroxyurea depends on the catalase status. Furthermore since combinational therapy with hydroxyurea and tyrosine kinase inhibitors are well- known therapies (or suggested therapies) it appears that testing for the expression of catalase in a sample (e.g. cancerous material) would assist in determining whether a disease should be treated with a monotherapy with hydroxyurea or a combinational therapy of tyrosine kinase inhibitors and a catalase inhibitors, such as hydroxyurea. In other words, the methods of the invention may also be used to predict whether a synergistic effect of tyrosine kinases and catalase inhibitors will be present when a neoplstic disorder is treated.
Thus, an object of the present invention relates to providing a method for determining hydroxyurea resistance using a sample. Another object relates to assisting in determining whether a disease should be treated with a monotherapy with hydroxyurea or a combinational therapy of tyrosine kinase inhibitors and a catalase inhibitor, such as hydroxyurea.
In particular, it is an object of the present invention to provide a method that solves the above-mentioned problems of the prior art by taking advantage of an identified correlation between hydroxyurea sensitivity and catalase activity. Sub-typing a neoplastic disorder
Thus, one aspect of the invention relates to a method for sub-typing a neoplastic disorder, said method comprising
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby indicating whether the neoplastic disorder is likely to be a hydroxyurea resistant subtype of said neoplastic disorder or a hydroxyurea sensitive subtype of said neoplastic disorder.
By using the status of catalase to subtype a neoplastic disorder, the neoplastic disorder may be divided into a hydroxyurea sensitive or hydroxyurea resistant subtype. Thus, it may be possible to decide whether hydroxyurea is a suitable treatment or if another treatment should be selected.
Similarly, the method may be appropriate in assisting in the decision of selecting the concentration of hydroxyurea to be administrated to a patient, since a sample showing low catalase activity may require a higher dose of hydroxyurea to achieve a suppressive effect on the neoplastic disorder. Thus, a method for patient specific optimization of a treatment regime is provided.
It is to be understood that in the present context the wordings "hydroxyurea resistant" and "hydroxyurea sensitive" relate both to monotherapy with hydroxyurea and combinational therapy with hydroxyurea and at least one further active component.
Thus, in an additional aspect the invention relates to a method for sub-typing a neoplastic disorder, said method comprising
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby indicating whether the neoplastic disorder is likely to be
- a hydroxyurea resistant subtype of said neoplastic disorder,
- a hydroxyurea sensitive subtype of said neoplastic disorder,
- a subtype more sensitive to a composition comprising one or more
tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
a subtype equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
In a clinical setting, the method of the invention may be employed by the clinician to assist the diagnosing of the subtype of a cancer and further in the clinical decision on the treatment. The object is to subject only those patients to hydroxyurea treatment that will likely benefit from this treatment. Hydroxyurea treatment may be in the form of monotherapy or combinatorial therapy e.g. as decribed above. Predicting responsiveness
Another aspect of the present invention relates to a method for predicting responsiveness of a neoplastic disorder to hydroxyurea treatment, said method comprising
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one of the parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase, and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby predicting the sensitivity or resistance of said neoplastic disorder to treatment with hydroxyurea.
By predicting the responsiveness of a neoplastic disorder to hydroxyurea treatment, it may be possible to decide whether hydroxyurea is a suitable treatment or if another treatment should be selected. Similarly, the method may be appropriate in assisting in the decision of selecting the concentration of hydroxyurea to be administrated to a patient. In yet an aspect the invention relates to a method for predicting responsiveness of a neoplastic disorder to hydroxyurea treatment or treatment with a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors, said method comprising
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one of the parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase, and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby predicting the sensitivity or resistance of said neoplastic disorder to treatment with hydroxyurea or to treatment with a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors.
Monitoring the status of a neoplastic disorder
Yet another aspect of the present invention is to provide a method of monitoring the status of a neoplastic disorder in a subject, said method comprising
a) providing at least two biological samples obtained from a subject at
different time points,
b) assessing in said samples at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter of said at least two biological
samples,
thereby indicating whether it is likely that said neoplastic disorder has progressed to become a hydroxyurea resistant subtype of said neoplastic disorder, regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder, or the sensitivity to hydroxyurea of said neoplastic disorder is maintained.
In a further aspect the invention relates to a method of monitoring the status of a neoplastic disorder in a subject, said method comprising
a) providing at least two biological samples obtained from a subject at
different time points,
b) assessing in said samples at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter of said at least two biological
samples,
thereby indicating whether it is likely that said neoplastic disorder has
- progressed to become a hydroxyurea resistant subtype of said
neoplastic disorder,
- regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder,
- the sensitivity to hydroxyurea of said neoplastic disorder is maintained,
- progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
- has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
In one embodiment the method of monitoring the status of a neoplastic disorder in a subject, comprises a) providing at least two biological samples obtained from a subject at different time points,
b) assessing in said samples at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard,
e) compare said evaluation in said at least two samples,
thereby indicating whether it is likely that said neoplastic disorder has progressed to become a hydroxyurea resistant subtype of said neoplastic disorder, regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder, or the sensitivity to hydroxyurea of said neoplastic disorder is maintained. In an additional embodiment the method further comprises evaluating whether it is likely that said neoplastic disorder has progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors. By assessing the catalase status from at least two samples from a subject obtained at different time-points, the catalase status of a neoplastic disorder can be compared over time, allowing the clinician to evaluate the progression of hydroxyurea sensitivity. It is to be understood that the two or more samples may be compared to each other before being compared to said threshold level or said known standard.
Kit
Still another aspect of the present invention is to provide a kit for performing the method according to the invention, comprising
a) at least one reagent,
b) an instruction manual explaining how to carry out the methods according to the preceding claims. By having both the instruction manual and at least one reagent in a kit practitioners may be enabled to perform the methods of the invention.
Brief description of the figures
Figure 1
Figure 1 shows detected amino acid mutations in Arabidopsis thaliana giving rise to a lowered catalase activity and hydroxyurea resistance and the corresponding position in the human catalase protein sequence.
Figure 2
Figure 2 shows the alignment of the Arabidopsis thaliana catalase 2 protein sequence and Homo sapiens catalase protein sequence. The amino acid positions listed in Figure 1, which when mutated in Arabidopsis thaliana cause hydroxyurea resistance and lowered catalase activity, are highlighted.
Figure 3
Figure 3 shows the relative catalase activity in 1) control group, 2) patients diagnosed with chronic myeloid leukemia (CML), 3) a patient diagnosed with CML and treated with high-dose HU for three weeks immediately prior to sampling, and 4) patients diagnosed with chronic lymphoid leukemia (CLL). Diamonds indicate data points. Error bars indicate standard deviations.
Figure 4
Figure 4 shows HU uptake by wild type and HU resistant mutants. Eleven days old wild type and mutant seedlings grown on drug-free medium were moved to plates containing 25 mM HU and incubated for 8 or 24 hours before shoots were harvested. After 24 hours, the HU concentration in seedling shoots approached that of the medium for both wild type and HU resistant plants. HU could not be detected in plants grown on drug-free medium. Error bars indicate SEM.
Figure 5
Figure 5 shows inhibition of RNR activity by HU in wild type and HU resistant mutants. RNR activity in extracts from wild type and HU resistant mutants is shown. In the control, no HU was added to the RNR reaction. Where indicated, 1 mM or 5 mM HU was added to the RNR reaction. Error bars indicate SEM.
Figure 6
Figure 6 shows HU response in wild type and HU-resistant mutants. (A) More than 90% of wild type plants germinated on 2.5 mM HU, whereas less than 1/20,000 germinated on 3 mM HU. Two HU-resistant mutants germinating at significantly higher concentrations of HU than the wild type are shown. (B-C) Seven days old seedlings grown on 3 mM HU. (B) Wild type. (C) cat2-9.
Figure 7
Figure 7 shows HU inhibition of catalase-mediated decomposition of H2O2. (A) Catalase activity in plant and rat protein preparations in the absence (- HU) and presence of 1 mM HU (+ HU) is shown. Triplicate measurements were performed and error bars indicate standard deviations. (B) Fractional activity, with respect to catalase activity under the same conditions in the absence of HU, is plotted versus log [cHU (mM)]. The black line indicates the best fit to a sigmoidal response curve. (C) Catalase activity plotted versus concentration in the presence of varying concentrations of HU (mM). Black lines indicate the best fit to a
competitive inhibition model. Figure 8
Figure 8 shows binding site of HU determined by crystallography. (A) Omit 2mFo- DFc electron density of the active site funnel with HU contoured at 1.1 σ. Water molecules are indicated with arrows. The ligand and the two water molecules shown with density were omitted for map calculations. (B) Water-mediated interactions of HU with the main chain of Argl26 and the side chain of Aspl27. Putative hydrogen bonds are shown with dashed lines. The heme group is shown in the background. (C) HU acts as a stopper in the substrate tunnel leading to the heme-containing active site. HU is shown as sticks and catalase in a surface presentation. See Table Sl for data collection and refinement statistics.
Figure 9
Figure 9 shows catalase activity in patient blood samples. Catalase activity in blood samples from a control group (Normal), patients diagnosed with chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelofibrosis (MF), myelomatosis (MM), unspecified myeloproliferative disease (MP), polycytaemia vera (PV), thrombocytosis (TC), chronic lymphatic leukemia (CLL), or lymphoma (L). Values were normalized to the average activity of the Normal group. Closed triangles indicate that patients were undergoing HU-treatment at the time of sampling. SEM for technical replicates and more detailed diagnoses, where available, are shown in Figure 10.
Figure 10
Figure 10 shows catalase activity in patient blood samples.
Legends: CML: Chronic myeloid leukemia; AML: Acute myeloid leukemia; MF:
Myelofibrosis; MM : Myelomatosis; MP: Unspecified myeloproliferative disease;
PV: Polycytaemia vera; TC: Trombocytosis; CLL: Chronic lymphatic leukemia; L:
Lymphoma; Activity: Catalase activity normalized to the average of the Normal group; SEM : Standard error of the mean; HU : NA: information not available.
The present invention will now be described in more detail in the following.
Detailed description of the invention
Definitions
Prior to discussing the present invention in further details, the following terms and conventions will be defined :
Hydroxyurea
Hydroxyurea (HU) is also known under the synonym hydroxycarbamide.
Hydroxyurea has the chemical formula CH4N2O2. IUPAC name: Hydroxyurea. CAS number: 127-07-1. It is to be understood that derivatives of hydroxyurea and hydroxyurea degradation products are included in the present invention.
Catalase status
The catalase status of a sample refers to the catalase enzyme activity level, the RNA level of catalase, the protein level of catalase, the catalase gene mutation status in said sample or combinations of these parameters.
Biological sample
The (biological) sample of the invention refers to a sample obtained from a subject. The sample may be a biopsy or a body fluid sample such as a blood sample. The sample may be processed before subjecting the sample to any of the methods of the invention. For example a sub-fraction of the sample may be isolated such as a sub-population of cells e.g. by fluorescence activated cell sorting (FACS).
Sensitive
As used herein the term sensitive or sensitivity refers to the susceptibility of e.g. a tissue or cell population to treatment with a drug or drug composition compared to the reference standard, cell, or cell population. The standard (reference) may for example be the corresponding healthy tissue, cell, or cell population, for example be the corresponding non-neoplastic counterpart (wild type), or non- malignant non-neoplastic counterpart. The terms a sensitive or a susceptible cell and a sensitive or a susceptible tissue (e.g neoplasm, tumor, or cancer) refer to cells or tissues obtained from a patient, wherein said cells or tissue is likely to respond to the administration of said drug or composition. Thus, the clinician would consider using said drug or composition for the treatment of said patient. Resistance
As used herein the term resistant or resistance refers to the reduced susceptibility of e.g. a tissue or cell population to the treatment a drug or drug composition compared to the above reference standard, cell, or cell population. Susceptibility to the treatment with a drug or drug composition may typically be monitored as the response to the treatment with a drug or drug composition in terms of rate of survival and/or growth rate in the presence of said drug or composition. Thus, increased survival or maintained or increased growth compared to the reference indicates resistance to said drug or drug composition. The drug may be hydroxyurea and the drug composition may be a composition comprising hydroxyurea. It is to be understood that a sample of the invention may have some degree of resistance, without having lost all activity of a certain biomarker e.g. catalase activity. However, the change in susceptibility of the cells in the sample to e.g. hydroxyurea may be of importance in the clinical decision on the treatment of the patient
Neoplastic disorder
Neoplastic disorder is herein defined as a disorder comprising an abnormal mass of tissue or cells as a result of neoplasia. Neoplasia is the abnormal proliferation of cells. Neoplasms may be benign, pre-malignant or malignant. The term solid tumour is synonymous with a neoplasm that has formed a lump. Some neoplastic disorders do not cause a lump; they include haematological leukemia and non- solid tumours such as most forms of carcinoma in situ. Cancer is a malignant neoplastic disorder.
Conservative substitution
As used herein the term "conservative substitution" refers to a gene product wherein a substitution of one amino acid with another with generally similar properties (size, hydrophobicity, etc) likely does not seriously affect the overall function of the protein.
Thus, Conservative amino acid substitution as used herein relates to the substitution of one amino acid (within a predetermined group of amino acids) for another amino acid (within the same group), wherein the amino acids exhibit similar or substantially similar characteristics.
Within the meaning of the term "conservative amino acid substitution" as applied herein, one amino acid may be substituted for another within the groups of amino acids indicated herein below:
• Amino acids having polar side chains (Asp, GIu, Lys, Arg, His, Asn, GIn, Ser, Thr, Tyr, and Cys,)
• Amino acids having non-polar side chains (GIy, Ala, VaI, Leu, He, Phe, Trp, Pro, and Met)
• Amino acids having aliphatic side chains (GIy, Ala VaI, Leu, He)
• Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro)
• Amino acids having aromatic side chains (Phe, Tyr, Trp)
• Amino acids having acidic side chains (Asp, GIu)
• Amino acids having basic side chains (Lys, Arg, His)
• Amino acids having amide side chains (Asn, GIn)
• Amino acids having hydroxy side chains (Ser, Thr)
• Amino acids having sulphor-containing side chains (Cys, Met),
• Neutral, weakly hydrophobic amino acids (Pro, Ala, GIy, Ser, Thr)
• Hydrophilic, acidic amino acids (GIn, Asn, GIu, Asp), and
• Hydrophobic amino acids (Leu, He, VaI)
Preferred conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine.
Accordingly, a gene product according to the invention may comprise, within the same gene product, or among different gene products, at least one substitution, such as a plurality of substitutions introduced independently of one another. It is clear from the above outline that the same gene product may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above.
Tyrosine kinase inhibitors
Tyrosine kinases play a critical role in the modulation of growth factor signaling. Activated forms of these enzymes can cause increases in tumor cell proliferation and growth, induce antiapoptotic effects, and promote angiogenesis and metastasis. In addition to activation by growth factors, protein kinase activation by somatic mutation is a common mechanism of tumor genesis. Because all of these effects are initiated by receptor tyrosine kinase activation, they are key targets for inhibitors (Arora A, Scholar EM. Role of tyrosine kinase inhibitors in cancer therapy. J Pharmacol Exp Ther. 2005 Dec;315(3) :971-9. Table 1, table 2 and figure 1 in Arora A and Scholar EM List different tyrosine kinase inhibitors which are hereby incorporated by reference.
A non-limiting list of tyrosine kinase inhibitors which are known drugs in the treatment of different neoplastic disorders are given below:
Imatinib:
Systematic (IUPAC) name: 4-[(4-methylpiperazin-l-yl)methyl]-N-[4-methyl-3-
[(4-pyridin-3-ylpyrimidin-2-yl)amino] phenyl] benzamide
Identifiers CAS number: 52459-95-5
Imatinib mesylate:
Systematic (IUPAC) name: 4-[(4-Methyl-l-piperazinyl)methyl]-N-[4-methyl-3-
[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamide monomethanesulfonate
CAS Registry Number: 220127-57-1
Dasatinib:
Systematic (IUPAC) name: N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2- hydroxyethyl)-l-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazole carboxamide monohydrate
Identifiers CAS number: 302962-49-8 Nilotinib:
Systematic (IUPAC) name: 4-methyl-N-[3-(4-methyl-lH-imidazol-l-yl)- 5- (trifluoromethyl)phenyl]-3- [(4-pyridin-3-ylpyrimidin-2-yl) amino] benzamide Identifiers CAS number: 641571-10-0(base)
Sunitinib:
Systematic (IUPAC) name: N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-l,2- dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-lH-pyrrole-3-carboxamide Identifiers CAS number: 341031-54-7.
Gefitinib (Iressa) :
CAS number 184475-35-2 Erlotinib (OSI-774; Tarceva) :
CAS number 183321-74-6
Lapatinib (GW-572016) :
CAS number 231277-92-2
Canertinib (CI-1033):
CAS number 267243-28-7
Semaxinib (SU5416) :
CAS number 194413-58-6,
Vatalanib (PTK787/ZK222584) :
Systematic (IUPAC) name :N-(4-chlorophenyl)-4-(pyndin-4-ylmethyl)phthalazin- 1-amine
CAS number 212141-54-3
Sorafenib (BAY 43-9006) :
Systematic (IUPAC) name : 4-[4-[[4-chloro-3- (tπfluoromethyl)phenyl]carbamoylamino]
phenoxy]-N-methyl-pyridine-2-carboxamide CAS number 284461-73-0
Leflunomide (SUlOl) :Systematic (IUPAC) name: 5-methyl-N-[4-(trifluoromethyl) phenyl]-isoxazole-4-carboxamide
CAS number 75706-12-6
It is to be understood that the above list of tyrosine kinase inhibitors forms part of the present invention. The person skilled in the art may be able to find additional tyrosine kinase inhibitors relevant. Sub-typing a neoplastic disorder
Sub-typing a neoplastic disorder such as a cancer in relation to hydroxyurea sensitivity would be a strong tool in cancer diagnosis. This may be done by knowing one or more biomarkers related to hydroxyurea sensitivity. Thus, in a first aspect the invention relates to a method for sub-typing a neoplastic disorder, said method comprising
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby indicating whether the neoplastic disorder is likely to be a hydroxyurea resistant subtype of said neoplastic disorder or a hydroxyurea sensitive subtype of said neoplastic disorder.
In a alternative aspect the invention relates to method for sub-typing a neoplastic disorder, said method comprising
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one parameter selected from the group of: i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby indicating whether the neoplastic disorder is likely to be
- a hydroxyurea resistant subtype of said neoplastic disorder,
- a hydroxyurea sensitive subtype of said neoplastic disorder,
- a subtype more sensitive to a composition comprising one or more
tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
a subtype equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
By using the status of catalase to subtype a neoplastic disorder, the neoplastic disorder may be divided into hydroxyurea sensitive or hydroxyurea resistant subtypes. Thus, it may be possible to evaluate whether it is likely that
hydroxyurea is a suitable treatment or if an alternative treatment is preferable. Similarly, the method may be appropriate in assisting the clinician in the decision of selecting the concentration of hydroxyurea to be administrated to a patient, since a sample showing low catalase activity may require a higher dose of hydroxyurea to achieve a suppressive effect on the neoplastic disorder.
It may also be an advantage to evaluate more than one parameter to increase the statistic power of the method. Thus in an embodiment of the invention at least two parameters are measured, such as three parameters or such as four parameters. In a clinical setting, the method of the invention may be employed by the clinician to assist the diagnosing of the subtype of a neoplastic disorder such as a cancer and further in the clinical decision on the treatment. When using catalase as a biomarker in relation to susceptibility to hydroxyurea, it may be an advantage to compare at least one parameter of the catalase status to a predetermined threshold value or a known standard to determine if the sample is indicative of being catalase resistant. Thus, in an embodiment the invention relates to a method wherein said at least one parameter being equal to or less than said predetermined threshold level or said known standard present in said neoplastic disorder with known sensitivity to treatment with hydroxyurea is indicative of the neoplastic disorder being a hydroxyurea resistant subtype of said neoplastic disorder. Thus, an amount of catalase protein or catalase RNA in a sample being equal to or less than a predetermined threshold level or standard may be indicative of the neoplastic disorder (e.g. a cancer) being hydroxyurea resistant. Similarly, the catalase activity in the sample being equal to or less than a predetermined threshold level or standard may be indicative of the cancer being hydroxyurea resistant. Furthermore, the mutational status of the catalase gene may also be indicative of the neoplastic disorder (e.g. cancer) being hydroxyurea resistant.
When catalase is used as a biomarker in relation to hydroxyurea sensitivity, it may also be an advantage to compare at least one parameter of the catalase status to a predetermined threshold value or a known standard to determine if the sample is indicative of being hydroxyurea sensitive. Thus, in another embodiment the invention relates to a method wherein said at least one parameter being greater than said predetermined threshold level or said known standard present in a neoplastic disorder with known sensitivity to treatment with hydroxyurea is indicative of the neoplastic disorder being a hydroxyurea sensitive subtype of said neoplastic disorder. Thus, an amount of catalase protein or catalase RNA in a sample being greater than a predetermined threshold level or standard may be indicative of the neoplastic disorder (e.g. cancer) being hydroxyurea sensitive. Similarly, a catalase activity in the sample being greater than a predetermined threshold level or standard may be indicative of the cancer being hydroxyurea sensitive. Furthermore, the mutational status of the catalase gene may also be indicative of the cancer being hydroxyurea sensitive.
As described above, the invention may also be used for assisting in the evaluation of whether a neoplastic disorder is a subtype more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors or a subtype equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
Thus, in an embodiment the invention relates to a method, wherein the at least one parameter being greater than said predetermined threshold level indicates a neoplastic disorder being a subtype more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors. In another embodiment the invention relates to a method, wherein the at least one parameter being lower or equal to said predetermined threshold level indicates a neoplastic disorder being a subtype equally of less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
The term "composition" or "preparation" is also to be understood, as "kit of parts" in the sense that the combination partners e.g. tyrosine kinase inhibitor (a) and catalase inhibitor (b) as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b) i.e., simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partner (a) and (b) to each other being administered in the combined preparation/composition can be varied, e.g. in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to the particular disease, age, sex, body weight, etc. of the patients. Thus, an advantage of the present invention may be to assist in determining whether the effect on the treated disease in the combined use of the parts is larger than the effect which would be obtained by use of only one of the combination partners. In addition, an advantage of the present invention may be to assist in determining whether there is at least one beneficial effect, e.g., a mutual enhancing of the effect of the combination partners (a) and (b), in particular a synergism, e.g. a more than additive effect, additional advantageous effects, less side effects, a combined therapeutic effect in a non-effective dosage of one or both of the combination partners (a) and (b), and very preferably a strong synergism of the combination partners ((a) and (b).
In the present context it is to be understood that a neoplastic subtype may comprise more than one of characteristics disclosed in the present invention. Thus, in an embodiment a subtype may comprise two of the characteristics, such as being 1) both a hydroxyurea resistant subtype and a subtype equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors and 2) being a hydroxyurea sensitive subtype and a subtype being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
Since the administration of catalase inhibitors to a subject may have side effects beside the desired effect it may be advantageously to evaluate whether such administration is expected to have the desired effect. As described in the present invention, subtypes of cancers exist which have different expression levels of catalase, the administration of a catalase inhibitor, such as hydroxyurea, should therefore preferably not be administered to subject until the catalase status of the relevant sample has been determined.
As described in present text the inventors have discovered that hydroxyurea is an inhibitor of catalase. Thus, in an embodiment the catalase inhibitor is
hydroxyurea. In these cases where the protein level, the RNA level or protein activity of catalase are used as a parameter, the threshold value or standard may be in the form of known catalase values obtained from a number of samples obtained from hydroxyurea resistant samples (e.g the average catalase activity or catalase RNA level of the samples), hydroxyurea sensitive samples or both. These values could then used to form the base level of the amount of catalase (protein level/RNA level/protein activity) which is required to form a threshold or standard for indicating that the cancer is a hydroxyurea resistant subtype or another subtype according to the invention. The threshold or standard may also take further parameters into account such as knowledge of e.g. gender, age, medical history, and blood type. The person skilled in the art would know to determine a threshold level which enables the discrimination between two populations using standard statistical methodology (e.g. by receiver operating characteristic (ROC) curve analysis). Accordingly, the skilled person would know how to employ such methods to set threshold levels and standard levels for biomarkers in relation to protein level, RNA level and protein activity.
The prediction may be combined with other biomarkers, such as the status of other proteins and/or data obtained from histological or cytological analyses of the sample or another corresponding sample obtained from the same subject. In this way the power of the prediction can be increased.
In the case catalase gene mutation status is used as a predictive marker the known DNA sequence of catalase or a consensus sequence can be used as a standard. If the analysis shows that the gene comprises one or more mutations, the one or more mutations can be compared to a list of known mutations resulting in a lowered catalase activity. Such mutations may cause an altered protein sequence, the introduction of stop codons in the RNA leading to the expression of a dysfunctional catalase protein, mutations in non-transcribed regions (both 5'- UTR and 3'-UTR regions) such as promoter and enhancer sequences leading to an altered expression and mutations resulting in an altered splice pattern. Since mutations in introns may result in an altered splicing pattern or expression, in some instances it may also be an advantage to sequence the genomic sequence of catalase and not only the coding sequence. Responsiveness of a neoplastic disorder to hydroxyurea treatment It may also be advantageous to be able to predict whether or not and to what extent a neoplastic disorder is susceptible to hydroxyurea treatment. Therefore, in another aspect the invention relates to a method for predicting responsiveness of a neoplastic disorder to hydroxyurea treatment, said method comprising
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one of the parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase, and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby predicting the sensitivity or resistance of said neoplastic disorder to treatment with hydroxyurea.
In a further aspect the invention relates to a method for predicting responsiveness of a neoplastic disorder to hydroxyurea treatment or treatment with a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors, said method comprising
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one of the parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase, and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby predicting the sensitivity or resistance of said neoplastic disorder to treatment with hydroxyurea or to treatment with a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors. The prediction of the responsiveness of a neoplastic disorder to hydroxyurea treatment or treatment with a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors may be used in the clinical evaluation of the disease before a treatment is initiated. A clinical evaluation may include selecting a drug or drugs and assessing the dose of the drug(s) to be administrated. It may also be an advantage to evaluate more than one parameter to improve the method. Thus in an embodiment of the invention at least two parameters are measured, such as three parameters or such as four parameters.
When using catalase as a biomarker to predict the responsiveness of a neoplastic disorder to hydroxyurea treatment, it may be an advantage to compare at least one parameter of the catalase status to a predetermined threshold value or a known standard to determine if the neoplastic disorder is indicative of being resistant to hydroxyurea treatment. Thus, in an embodiment the invention relates to a method, wherein the neoplastic disorder is predicted to be resistant to hydroxyurea if the at least one parameter is equal to or less than said
predetermined threshold level or said known standard present in a neoplastic disorder with known sensitivity to treatment with hydroxyurea. Thus, an amount of catalase protein or catalase RNA in a sample lower or similar to a
predetermined threshold level or a known standard may be indicative of the neoplastic disorder being resistant to hydroxyurea treatment. Similarly, a catalase activity in the sample lower or similar to a predetermined threshold level or a known standard may be indicative of the neoplastic disorder being resistant to hydroxyurea treatment. Furthermore, the mutational status of the catalase gene may also be indicative of the cancer being resistant to hydroxyurea treatment.
When using catalase as a biomarker to predict the responsiveness of a neoplastic disorder to hydroxyurea treatment, it may also be an advantage to compare at least one parameter of the catalase status to a predetermined threshold value or a known standard to determine if the neoplastic disorder is indicative of being responsive to hydroxyurea treatment. Thus, in an embodiment the invention relates to a method, wherein the neoplastic disorder is predicted to be sensitive to the hydroxyurea if the at least one parameter is greater than said predetermined threshold level or said known standard present in the neoplastic disorder with known sensitivity to treatment with hydroxyurea. Thus, an amount of catalase protein or catalase RNA in a sample higher than a predetermined threshold level or a known standard may be indicative of the neoplastic disorder being sensitive to hydroxyurea treatment. Similarly, a catalase activity in the sample higher than a predetermined threshold level or a known standard may be indicative of the neoplastic disorder being sensitive to hydroxyurea treatment. Furthermore, the mutational status of the catalase gene may also be indicative of the cancer being sensitive to hydroxyurea treatment.
When using catalase as a biomarker to predict the responsiveness of a neoplastic disorder to be more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, it may be an advantage to compare at least one parameter of the catalase status to a predetermined threshold value or a known standard to determine if the neoplastic disorder is indicative of being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors. Thus, in an embodiment the neoplastic disorder is predicted to be more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors if the at least one parameter is greater than said predetermined threshold level or known standard.
In another embodiment the neoplastic disorder is predicted to be equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors if the at least one parameter is equal to or less than said predetermined threshold level or known standard.
In the present context it is to be understood that the methods of the invention may result in more than one prediction. Thus, in an embodiment a neoplastic disorder may be predicted to be 1) both hydroxyurea resistant and equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors and 2) both being a hydroxyurea sensitive and a being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
Since the administration of catalase inhibitors to a subject may have side effects beside the desired effect it may be advantageously to evaluate whether such administration is expected to have the desired effect. As described in the present invention, subtypes of cancers exist which have different expression levels of catalase, the administration of a catalase inhibitor, such as hydroxyurea, should preferably not be administered to a subject until the catalase status of the relevant sample has been determined.
For example, WO 2006/136391 discloses a combinatorial therapy of cancer diseases comprising tyrosine kinase inhibitors and optionally hydroxyurea. As disclosed in the present invention hydroxyurea is an inhibitor of catalase and it may therefore be relevant to determine the catalase status before a combinatorial treatment as disclosed in e.g. WO 2006/136391 is initiated. By doing this at may be determined whether a specific subject will have a synergistic effect of the combined treatment compared to the monotherapies. Monitoring the status of a neoplastic disorder in a subject
It may also be advantageous to monitor the status of a neoplastic disorder in relation to a biomarker indicative of the hydroxyurea sensitivity of the neoplastic disorder. Thus, in yet an aspect, the invention relates to a method of monitoring the status of a neoplastic disorder in a subject, said method comprising
a) providing at least two biological samples obtained from a subject at
different time points,
b) assessing in said samples at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase, iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter of said at least two biological
samples,
thereby indicating whether it is likely that said neoplastic disorder has progressed to become a hydroxyurea resistant subtype of said neoplastic disorder, regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder, or the sensitivity to hydroxyurea of said neoplastic disorder is maintained. In an additional aspect the invention relates to a method of monitoring the status of a neoplastic disorder in a subject, said method comprising
a) providing at least two biological samples obtained from a subject at
different time points,
b) assessing in said samples at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter of said at least two biological
samples,
thereby indicating whether it is likely that said neoplastic disorder has
- progressed to become a hydroxyurea resistant subtype of said
neoplastic disorder,
- regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder,
- the sensitivity to hydroxyurea of said neoplastic disorder is maintained,
- progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
- has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
In one embodiment, the method of monitoring the status of a neoplastic disorder in a subject, comprises
a) providing at least two biological samples obtained from a subject at
different time points,
b) assessing in said samples at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard,
e) compare said evaluation in said at least two samples,
thereby indicating whether it is likely that said neoplastic disorder has progressed to become a hydroxyurea resistant subtype of said neoplastic disorder, regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder, or the sensitivity to hydroxyurea of said neoplastic disorder is maintained. In yet an embodiment the method further relates to indicating whether the neoplastic disorder has progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a
composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors. It is to be understood that the two or more samples may be compared to each other before being compared to said threshold level or said known standard,
By assessing the catalase status from at least two samples from a subject obtained at different time-points, the catalase status of a neoplastic disorder can be compared over time, allowing the clinician to evaluate the progression of the hydroxyurea sensitivity or sensitivity to a combinatorial treatment as described above. It may also be an advantage to evaluate more than one parameter in the at least two samples to improve the method. Thus, in one embodiment of the invention at least two parameters are measured such as three parameters or such as four parameters. When comparing the at least two samples from a subject with respect to one or more parameters, it may be advantageous to evaluate the progression of hydroxyurea sensitivity. Thus, in an embodiment the invention relates to a method, wherein it is likely that said neoplastic disorder has progressed to become a hydroxyurea resistant subtype of said neoplastic disorder if the at least one parameter is lower than said predetermined threshold level or said known standard present in the neoplastic disorder with known sensitivity to treatment with hydroxyurea, and if the value is lower in the sample obtained at the latest point in time. Development of resistance is a known problem during treatment of a neoplastic disorder, therefore comparing different samples from a subject over time may be advantageous.
In certain instances a resistant subtype of a neoplastic disorder may progress to a sensitive subtype. Thus, one embodiment of the invention relates to a method, wherein it is likely that said neoplastic disorder has progressed to become a hydroxyurea sensitive subtype of said neoplastic disorder if the at least one parameter is greater than said predetermined threshold level or said known standard present in the neoplastic disorder with known sensitivity to treatment with hydroxyurea and if the value is greater in the sample obtained at the latest point in time. Going from a resistant subtype towards a sensitive subtype may occur e.g. in the case where the subject is stimulated with a substance increasing catalase activity or it could be a spontaneous event.
It may also be the case that the sensitivity has not changed over time. Thus, in yet an embodiment, the invention relates to a method, wherein it is likely that said sensitivity to hydroxyurea in the neoplastic disorder is maintained if the value is equal in the samples obtained at different points in time. The neoplastic disorder may be maintained in a hydroxyurea sensitive state or in a hydroxyurea resistant state. To get an indication whether the neoplastic disorder is maintained in a hydroxyurea sensitive state or in a hydroxyurea resistant state the at least one parameter in the samples may be compared to said predetermined threshold level or said known standard.
Acquiring data indicating that the hydroxyurea sensitivity is likely unchanged may be used to suggest maintaining the current treatment with hydroxyurea if the neoplastic disorder is still sensitive to hydroxyurea treatment or alternatively to keep using an alternative treatment if the data indicates that the neoplastic disorder is still resistant to hydroxyurea treatment. Over time the neoplastic disorder may have changed its responsiveness towards a combinatorial treatment. Thus, in yet an embodiment the invention relates to a method wherein it is likely that said neoplastic disorder has progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors disorder if the at least one parameter is greater than said predetermined threshold level or said known standard, and if the value is greater in the sample obtained at the latest point in time. In yet another embodiment the invention relates to a method wherein it is likely that said neoplastic disorder has progressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors if the at least one parameter is lower than said predetermined threshold level or said known standard present in the neoplastic disorder and if the value is lower in the sample obtained at the latest point in time.
If a change in the catalase status has taken place of time, it may indicate that a change in therapy could be advantageously. Thus, a switch to or from a therapy with hydroxyurea or with a tyrosine kinase inhibitor and a catalase inhibitor (such as hydroxyurea) may be advantageously if a change in the status of catalase is measured. Tyrosine kinase inhibitors
Different types of tyrosine kinase inhibitors may form part of the present examples. Thus, in an embodiment of the present invention the one or more tyrosine kinase inhibitor is selected from the group consisting of 4-[(4- methylpiperazin-l-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2- yl)amino]phenyl]benzamide (imatinib), 4-[(4-Methyl-l-piperazinyl)methyl]-N-[4- methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamide
monomethanesulfonate (imatinib mesylate), N-(2-chloro-6-methylphenyl)-2-[[6- [4-(2-hydroxyethyl)-l-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazole carboxamide monohydrate (dasatinib), 4-methyl-N-[3-(4-methyl-lH-imidazol-l- yl)- 5-(trifluoromethyl)phenyl]-3- [(4-pyridin-3-ylpyrimidin-2-yl)
amino]benzamide (nilotinib) and N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-l,2- dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-lH-pyrrole-3-carboxamide
(sunitinib). Catalase inhibitors
Different types of catalase inhibitors may form part of the invention. Thus, in an embodiment the one or more catalase inhibitors are selected from the group consisting of hydroxyurea and 3-Amino-Triazole. Preferably the catalase inhibitor is one used in the treatment of a neoplastic disorder. Thus, in a specific
embodiment the catalase inhibitor is hydroxyurea as discovered by the inventors. CAS number for hydroxyurea is 127-07-1. Catalase enzyme activity
In an embodiment the invention relates to a method wherein a sample such as a blood sample obtained from a subject is provided. The cells in the sample are then analyzed for catalase activity by measuring the turnover of hydrogen peroxide using photospectrometry. Before cells of the sample are analyzed for catalase activity a sub-population of the cells may be isolated (e.g by FACS sorting). A low activity of catalase is indicative of the subject being resistant to hydroxyurea treatment and/or is indicative of the subject being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition without one or more catalase inhibitors.
The status of catalase can be measured in different ways using different parameters. In one embodiment the invention relates to a method, wherein said at least one parameter is catalase enzyme activity in said sample. Catalase possesses at least two enzymatic functions; an enzymatic reaction leading to decomposition of hydrogen peroxide wherein hydrogen peroxide is converted into water and oxygen, and a second peroxidase reaction wherein hydrogen peroxide is decomposed to water with the aid of an additional compound RH2. RH2 may be in the form of phenols, formic acid, formaldehyde and alcohols. It may be an advantage to measure one or both of the enzymatic reactions catalysed by catalase instead of measuring the DNA or RNA status, since the activity is the most direct measurement of the catalytic activity. When measuring the activity of catalase in a sample, the measurement also shows if the activity has been influenced by e.g. post-translational modifications such as phosphorylations and interactions with other proteins. This is not the case when evaluating the DNA or RNA status.
Catalase activity may be monitored in different ways. Thus, in an embodiment the invention relates to a method, wherein said catalase enzyme activity is
determined by measuring the turnover of a catalase substrate. Different types of catalase substrates may be used. Thus, in another embodiment the invention relates to a method, wherein said substrate is selected from the group consisting of hydroxyurea, hydrogen peroxide and sodium perborate.
Sodium perborate may also be used as a substrate for catalase since it may be more stable than hydrogen peroxide (H2O2). Sodium perborate may be measured by titrating KMnO4 in the presence of sulphuric acid. The person skilled in the art knows how to optimize such assays.
Hydrogen peroxide may also be used as a substrate for catalase. Thus, in another embodiment, the invention relates to a method, wherein said substrate is hydrogen peroxide. Measuring hydrogen peroxide may be done by
photospectrometry. Thus in yet an embodiment, the invention relates to a method, wherein said turnover is determined as the change in absorbance at 240 nm. The turnover of hydrogen peroxide may also be monitored using enzymatic assays. Thus, in a further embodiment, the invention relates to a method, wherein said turnover of hydrogen peroxide is measured using horseradish peroxidise (HRP) as a reporter enzyme. Examples of kits which may also be used for performing the assay include OxiSelect™ supplied by Cell Biolabs, Inc., the Catalase Assay Kit supplied by Sigma-Aldrich, Inc. and The Amplex® Red Catalase Assay Kit supplied by Molecular Probes. Before analysis the cells may be sorted using FACS.
RNA level of catalase
In an embodiment the invention relates to a method wherein a sample such as a blood sample is obtained from subject. The cells in the sample are then analyzed for the amount of catalase RNA using Q-PCR. Before analysis the cells may be sorted using FACS. A low amount of catalase RNA is indicative of the subject being resistant to hydroxyurea treatment and/or indicative of the subject being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition without one or more catalase inhibitors.
The catalase status of a sample may also be determined by measuring the RNA level of catalase. Thus, in an embodiment, the invention relates to a method, wherein said at least one parameter is the RNA level of catalase in said sample. Measuring RNA levels may be advantageous in cases where catalase activity assays are not appropriate. This could be in cases where only small amounts of a sample are available or where an amplification step is necessary. It may also be advantageous where the sample has been fixed in a fixative making activity measurement inappropriate. Such fixatives could be cross-linking fixatives such as formalin. Thus, in yet an embodiment, the invention relates to a method, wherein assessment of the RNA level of catalase comprises the use a technique selected from the group consisting of PCR, Q-PCR, RT-PCR, a hybridization technique, and micro-arrays. PCR-techniques have the advantage of a large amplification and allow for real-time monitoring. Design of primers and amplification programs are standard techniques for the person skilled in the art. Hybridization techniques like Northern blotting may be preferred if there is no access to quantitative PCR- equipment. Microarrays have the advantage of multiplexing, allowing several biomarkers to be measured simultaneously. This speeds up the testing phase and saves sample materials. FACS may be applied to the samples before analysis to remove the contribution from e.g. non-malignant cells to the read-out.
Catalase protein level
In an embodiment the invention relates to method wherein a sample such as a blood sample is obtained from subject. The cells in the sample are then analyzed for the presence of the catalase protein using a monoclonal or polyclonal antibody against the catalase protein which is subsequently detected using a secondary antibody. A low amount of catalase is indicative of the subject being resistant to hydroxyurea treatment and/or indicative of the subject being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition without one or more catalase inhibitors. Before analysis the cells may be sorted using FACS.
In certain cases it may be advantageous to measure the protein level of catalase in a sample. Thus, in an embodiment the invention relates to a method, wherein said at least one parameter is the protein level of catalase in said sample. Protein level measurement may also be used when the sample has been in a fixative making activity measurement inappropriate. Such fixatives could be cross-linking fixatives such as formalin. In some instances cross-linking fixatives also results in degradation of nucleic acids, making both DNA and RNA analysis difficult.
Furthermore, in many pathological institutions immuno-based assays are the preferred technique, since the staining patterns can be compared to the visual appearance of the sample. Thus, in another embodiment, the invention relates to a method, wherein assessment of the protein level of catalase comprises the use a technique selected from the group consisting of immuno-based assays and mass spectrometry. Examples of immuno-based assays are western blotting, ELISA- techniques, immunohistochemistry, immunocytochemistry and protein arrays. Mass spectrometry (MS) may also be advantageous in some cases, since it is very sensitive and several proteins may be monitored simultaneously. A further advantage by MS is that antibodies are avoided. FACS may be applied to the samples before analysis.
Threshold level, Standard
The predetermined threshold of the invention may be determined by any suitable statistical method.
Receiver operating characteristic (ROC) curve analysis is a classification model for a mapping of instances into a certain class/group. Receiver operating
characteristic (ROC) curve analysis may be used determine the classifier boundary between groups of samples (or patients) for which the classifier boundary between classes must be determined by a threshold value, for instance to determine whether a person is likely to have subtype of a neoplasm or not, or if a patient is likely to respond to a treatment or not. Thus, in one embodiment of the present invention the predetermined threshold level is selected by receiver operating characteristic (ROC) curve analysis.
This methodology may be applied to determining the predetermined threshold for method comprising the assessment of catalase enzyme activity, catalase RNA or protein level.
Accordingly, in a further embodiment of the present invention, the method comprises:
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase;
b) constructing a percentile plot of said at least one of the parameter; c) constructing a ROC (receiver operating characteristics) curve based on said at least one of the parameter;
d) selecting a desired sensitivity;
e) determining from the ROC curve the specificity corresponding to the desired sensitivity;
f) determining from the percentile plot the level corresponding to the determined specificity; and
g) thereby indicating depending on the method in question whether the neoplastic disorder is likely to be a hydroxyurea resistant subtype of said neoplastic disorder or a hydroxyurea sensitive subtype of said neoplastic disorder; thereby predicting the sensitivity or resistance of said neoplastic disorder to treatment with hydroxyurea; or thereby indicating whether it is likely that said neoplastic disorder has progressed to become a
hydroxyurea resistant subtype of said neoplastic disorder, regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder, the sensitivity to hydroxyurea of said neoplastic disorder is maintained, progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
Catalase DNA status
In an embodiment the invention relates to a method wherein a sample such as a blood sample is obtained from a subject. Genomic DNA is extracted from the cells in the sample and the entire or parts of the genomic sequence of the catalase gene is determined using first a DNA amplification method (e.g. a PCR method) and then standard sequencing techniques or re-sequencing by array hybridization or other appropriate means of acquiring sequence information. Both cDNA and genomic DNA can be sequenced. Before analysis the cells may be sorted using FACS. A sequence deviating from the standard sequence at either the protein level and/or the DNA level is indicative of the subject being resistant towards hydroxyurea treatment and/or indicative of the subject being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition without one or more catalase inhibitors.
The catalase status may also be determined by querying the DNA status of a genome. Thus, in an embodiment the invention relates to a method, wherein said at least one parameter is the catalase gene mutation status, and said catalase gene mutation status is assessed by detecting the presence or absence of a mutation in the catalase gene.
Determining the catalase gene mutational status provides information which is normally not obtained by looking at protein or RNA levels. Looking at the DNA sequence can be used for determining if the patient has a hereditary character making him or her less suitable for hydroxyurea treatment. Mutations may also have occurred spontaneously in some cells (e.g. cancer cells). If the analysis shows that the gene (or the sequence surrounding the gene) comprises one or more mutations, this or these mutations can be checked against known mutations resulting in a lowered catalase activity. Such mutations may be mutations resulting in an altered protein sequence, the introduction of stop codons in the RNA, mutations in non-transcribed regions (both 5'-UTR and 3'-UTR regions) such as promoter and enhancer sequences resulting in an altered expression and mutations resulting in an altered splice pattern. To be able to determine if the sequence comprises one or more mutations, a standard sequence is required. Thus, in another embodiment the invention relates to a method, wherein said known standard is the nucleic acid sequence encoding catalase set forth in at least one of SEQ ID NO:2 and SEQ ID NO:3.
In the case the catalase gene mutation status is used as a predictive marker the known DNA sequence of catalase can be used as a standard. The difference between SEQ ID NO:2 and SEQ ID NO:3 is that SEQ ID NO:2 shows the cDNA sequence of catalase whereas SEQ ID NO: 3 shows the genomic sequence of catalase. Checking the genomic sequence gives additional information concerning mutations positioned in e.g. introns relating to e.g. a changed splicing pattern, whereas looking at the cDNA sequence may provide information concerning the actual splicing pattern. Furthermore, mutations may be in the form of deletions of one or more nucleic acids, insertions of one or more nucleic acids. Both deletions and additions will normally result in a changed reading frame resulting in a nonfunctional protein product. Sequencing techniques relating to sequencing genomic DNA, RNA and cDNA are known to the person skilled in the art. It is to be understood that sequences positioned both upstream and downstream of the coding sequence may be also sequenced for mutations.
Catalase mutations
Since not all mutations resulting in a lowered or no catalase activity can be predicted from the sequence analysis itself, it may be an advantage to know certain mutations which result in a lowered or no catalase activity. Thus in yet an embodiment the invention relates to a method, wherein said known standard is a list comprising at least one nucleic acid sequence encoding catalase with no or reduced catalyse enzyme activity. The list of nucleic acid mutations coding for a polypeptide sequence indicative of a lowered catalase activity compared to a reference such a wild-type catalase may be used by the clinician in the methods of the invention to evaluate the genetic background of the neoplastic disorder and indicate whether neoplasms is likely to be resistant or not. Such a list may also be a list of amino acid positions in the catalase protein which is known to have a lower catalase activity than the standard catalase protein sequence. Thus, in a further embodiment the invention relates to a method, wherein said at least one nucleic acid sequence encodes a catalase polypeptide with no or reduced catalase enzyme activity, said catalase polypeptide is selected from the group consisting of polypeptides deviating at position 71, 83, 88, 114, 121, 124, 147, 192, 195, 261, 275, 277, 303, 342, 354, 463, 503 and 517 of SEQ ID NO: 1. In yet a further embodiment, the deviation is a non-conservative amino acid substitution.
Mutations which are likely to change the expression level of catalase or the activity of the catalase enzyme may be in form of 1) non-synonymous mutations, relating to mutations in a codon which result in an altered amino acid sequence, 2) nonsense mutations, relating to a point mutation in a sequence of DNA that results in a premature stop codon, 3) deletions or insertions at one or more of the positions which may result in a changed reading frame resulting in a non- functional protein product, and 4) inversions, resulting in a changed coding sequence. 5) Large deletions, 6) Rearrangements of the catalase gene. It is to be understood that synonymous mutations relating to mutations in a codon which does not result in an altered amino acid sequence is likely not to change the expression level or activity of the catalase enzyme. Similarly, a conservative substitution is also likely not to cause any serious effect of the enzyme activity. Amino acid substitutions in the Arabidopsis thaliana catalase 2 protein leading to decreased catalase activity are listed in figure 1. The corresponding positions in the human catalase enzyme are also listed in figure 1. Thus, in an embodiment the invention relates mutations selected from the list in figure 1 showing the corresponding human sequence position. An alignment of the amino acid sequences of human catalase protein and Arabidopsis thaliana protein is shown in figure 2.
The presence of a mutation in the catalase gene, may be used to determine if the neoplastic disorder is a hydroxyurea resistant subtype. Thus, in an embodiment the invention relates to a method, wherein the presence of at least one amino acid substitution in at least one of the positions selected from the group consisting of position 71, 83, 88, 114, 121, 124, 147, 192, 195, 261, 275, 277, 303, 342, 354, 463, 503 and 517 of SEQ ID NO: 1, is indicative of the neoplastic disorder being a hydroxyurea resistant subtype and/or indicative of the subject being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition without one or more catalase inhibitors.
In this way an indication of a subtype of a neoplastic disorder can be determined by analyzing the catalase gene at the DNA level. The DNA status can also be determined by sequencing the transcribed RNA, with the limitations that non- transcribed regions of the gene will not be sequenced. Precise amino acid changes resulting in decreased catalase activity are listed in figure 1. Thus, in an embodiment the invention relates to mutations selected from the list in figure 1.
In one embodiment, the standard is a consensus sequence of a nucleic acid or amino acid sequence encoding a functional catalase enzyme with an activity comparable to the wild type catalase enzyme. In another embodiment, the standard is a consensus sequence of a nucleic acid or amino acid sequence encoding a catalase enzyme with a reduced activity comparable to the wild type catalase enzyme.
Heterozygosity and homozygosity
The human genome normally comprises two alleles of each gene. Thus, in another embodiment the invention relates to a method, wherein the presence of the mutation is heterozygous or homozygous. Both heterozygote and homozygote mutations may result in an altered hydroxyurea response, since not all mutations can be fully complemented by the presence of a non-mutated catalase protein. In the case of a heterozygosity resulting in hydroxyurea susceptibility response, loss-of-heterozygosity (LOH) may lead to hydroxyurea resistance. LOH may reflect the appearance of another mutation, deletion etc. in the other catalase allele causing the loss-of-function of that allele. It would be relevant to analyze samples from subjects which are already diagnosed with a neoplastic disorder. Thus, in an embodiment the invention relates to a method, wherein said sample is obtained from a subject currently receiving treatment for said neoplastic disorder. In this way unnecessary sampling is avoided. It may furthermore be advantageous if the samples are obtained from a person under treatment with hydroxyurea. Thus, in another embodiment the invention relates to a method, wherein said treatment comprises the
administration of hydroxyurea to said subject. Hydroxyurea treatment is used in the treatment of certain cancers. Thus, in yet another embodiment the invention relates to a method, wherein the neoplastic disorder is a cancer. The cancer may both be of a solid cancer or a leukemia. Therefore, in an embodiment the invention relates to a method, wherein the cancer is selected from the group consisting of leukemia and solid tumor cancers.
Leukemia
The method may be especially suited for the evaluation of haematological neoplasms such as leukemia cancers. Therefore, one embodiment of the invention relates to a method, wherein said neoplastic disorder is a haematological neoplasm. In a further embodiment, the haematological neoplasm is a malignant haematological neoplasm. In yet a further embodiment the invention chronic myeloid leukemia (CML), chronic lymphatic leukemia (CLL), polycytemia vera, thrombocytosis,
myelofibrosis hypereosinophilic syndrome and acute myeloid leukemia (AML).. Leukemia is often treated with hydroxyurea. Since leukemias have a tendency to develop resistance, the method may be especially suited for leukemia patients receiving a hydroxyurea treatment. Patients with chronic myeloid leukemia (CML), often receives hydroxyurea treatment. Thus, in yet an embodiment the invention relates to a method, wherein the cancer is a chronic myeloid leukemia (CML).
In the present context the term "leukemia" may be interpreted as "hematological malignancies".
Solid tumors
The methods according to the invention may also be relevant for solid tumors. In an embodiment solid tumor cancer is selected from the group consisting of glioblastoma and gastrointestinal stromal tumors.
Sampling
The sample to be analysed by the method of the invention may be provided from several sources. Therefore, in an embodiment the invention relates to a method, wherein the sample is a blood sample or tissue sample.
When the sample is a blood sample it may be an advantage to sort the samples into specific cell populations (preferably healthy and malignant cell populations) e.g. by the use of flow cytometry e.g. FACS) or affinity beads. Thus, in one embodiment of the present invention the sample is cells isolated from a biopsy or body fluid sample such as a blood sample such as by means of FACS sorting or affinity-bead purification (e.g. antibody coated magnetic beads). The person skilled in the art knows how to optimize a cell isolation method. The different populations can then subsequently be analyzed for catalase status according to the invention. Sorting cells prior to analysis allows healthy cells, with high, normal or low catalase activity, to be removed from the cells to be analyzed for catalase status. In a thought example wherein half the cells are "normal" cells having an expression level of 100% and the other half is malignant cells having an expression level of 0%, the final result will give a result saying that all the cells have an expression level of 50%, which would be wrong for all cells in the sample. Therefore, cell sorting can be used to increase the power of the analysis. The same noise may be present in the case of DNA, RNA and protein analysis.
Tissue samples may be provided from e.g. solid tumors or skin biopsies. In an embodiment of the invention the sample is provided from a human subject.
Kit
It may be an advantage to have a kit making the user capable of performing the method of the invention. Therefore, in an embodiment the invention relates to a kit for performing the method of the invention, comprising
a) at least one reagent,
b) an instruction manual explaining how to carry out the methods according to the preceding claims.
By having both the instruction manual and at least one reagent comprised in a kit practitioners may be able to perform the invention. One of the reagents present in the kit may be hydrogen peroxide. Thus in a further embodiment, the invention relates to a kit, wherein said at least one reagent is hydrogen peroxide.
Hydrogen peroxide may be used when the assay relates to testing for catalase activity. Other reagents which may be present in such a kit (or substitute hydrogen peroxide) could be selected from the group consisting of sodium perborate, KMnO4 and sulphuric acid. When the assay is optimized for detecting the mutational status or RNA level the kit may comprise primers for amplification part of the catalase gene or mRNA. When the assay is optimized for detecting the amount of catalase enzyme in a sample the kit may comprise antibodies against catalase (monoclonal or polyclonal), secondary antibodies and detection reagents. When the assay is optimized for detecting mutations in the catalase gene, the kit may comprise microarrays that can be used to specifically detect changes in the catalase gene with respect to a reference sequence. A kit may also comprise reagents for performing two or more of the assays described above. It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.
The invention will now be described in further details in the following non-limiting examples.
Examples
Methods
Plant growth conditions
Arabidopsis thaliana ecotype Columbia (CoI-O) wild type and mutant plants were grown on Vi Murashige and Skoog medium (Duchefa, M0222) solidified with 0.9% agar in growth chambers at 21°C, 16/8 hours light/dark cycles and 50 μmol m'V1 photosynthetic photon flux. HU (US Biological, H9120) was dissolved in water to prepare a 2.5 M stock solution. HU stock solution was added to the medium immediately before pouring plates.
RNR activity assay
RNR activity was quantified as described in (Jong AY, Yu K, Zhou B, Frgala T, Reynolds CP, et al. (1998) A simple and sensitive ribonucleotide reductase assay. J Biomed Sci 5: 62-68). Cytidine 5'-diphosphate, triammonium salt, [2-14C]- (Moravek) was used for labeling. 300 mg of plant tissue per sample was collected and ground in liquid nitrogen.
Quantification of HU content
HU was quantified as described in (Bachir D, Hulin A, Huet E, Habibi A, Nzouakou R, et al. (2007) Plasma and urine hydroxyurea levels might be useful in the management of adult sickle cell disease. Hemoglobin 31 : 417-425). Between 8 and 30 mg of leaf tissue per sample was collected and ground in phosphate buffered saline (PBS). The concentration of HU in plant shoots was calculated assuming a water content of 85%. Catalase enzyme assay and materials
Catalase activity was quantified by monitoring the rate of H2O2 breakdown at 240 nm. GraphPad Prism 5 was used for statistical analysis and curve fitting. Plant extracts were prepared by grinding ca. 60 mg of tissue in PBS. Rat erythrocytes were isolated by centrifugation and lysed in distilled water. Blood samples were drawn into 10 ml ethylenediaminetetraacetic acid (EDTA) stabilized tubes. Less than one hour after vein puncture samples were processed into mononuclear cells (MNC) by Lymphoprep differential centrifugation at 2100 rpm for 10 minutes. The interface mononuclear cell suspension was harvested and washed twice with PBS. Isolated MNCs were counted and subsequently frozen in dimethyl sulfoxide (DMSO) in liquid nitrogen. Cell lysates were prepared from 5xlO6 cells thawed at 37°C, spun at 300 x g for 5 minutes, washed with PBS and finally lysed in distilled water. Catalase activity was normalized to the total protein concentration for each sample. For experiments with purified enzyme, a commercially available aqueous catalase solution was used (Sigma, C3155).
Crystallization and structure determination
Crystals of bovine liver catalase (Sigma, C3155) were obtained through vapor diffusion by mixing a solution of catalase dissolved in 0.5 M sodium phosphate buffer pH 6.6 at 22 mg/ml in a 1 : 1 ratio with reservoir buffer containing 12% PEG4000 (w/v). The crystals were soaked with HU by leaving them briefly in cryo buffer (30% MPD, 8.4% PEG4000, 50 mM HU) before flash freezing in liquid nitrogen. Data were collected at MAX-Lab 911-2 and at ESRF ID14-2 and processed with XDS. The structure was determined by molecular replacement (RCSB entry: 4BLC) using PHASER and iteratively rebuilt in Coot and refined with PHENIX. REFINE. Data collection and refinement statistics are presented below. HU was primarily recognized through two water molecules. One of these linked the HU alcohol and the main chain carbonyl group of Argl26 (Fig 8B). The HU amide was less well recognized, and the amino- and carbonyl group could at this resolution be distinguished on the basis of electron density (omit 2mFo-DFc). In one of the four catalase subunits within the asymmetric unit, a water molecule could bridge the amide group to the side chain of Aspl27 through hydrogen bonds, suggesting that here the amino group coordinated this water (Figure 8B). For this reason, the orientation with the amide amino group directed against side chain of Aspl27 was imposed for all four copies, even though the electron density for this water was not strong in the remaining three catalase subunits. Crystallographic data
Data collection
Beam line ESRF ID14-1
Wavelength 0.93
Spacegroup P2i2121
Unit-cell parameters a=86.38 A, b= 139.88 A,
c=227.81 A
Unique reflections 162671
Resolution (A) 1 45-2.05 (2.2-2.05)
Redundancy 3.25 (3.18)
Completeness (%) 93.9 (94.2)
Mean I/sigma 16.11 (3.30)
R merge (%)2 12.2 (53.2)
Refinement
Resolution (A) 2.05
R-factor3/R-free4 (%) 17.3/20.6
Reflections (work/test) 160671/2000
Number of atoms in the asymmetric
unit
Protein 16064
Ligands (Hem group and hydroxyurea) 196
Water 1731
R.m.s. deviations Bonds (A)/ Angles (°) 0.01/1.09
Ramachandran plot (%)
Most favored 86.3%
Additionally allowed 12.8%
Generously allowed 0.5%
Disallowed 0.5% Values in parentheses are for outer shells. 2Rmerge = (∑h∑j=i,N I Ih-Ih(J) I /∑N x Ih) for the intensity of reflection h measured N times. 3R-factor =∑h| F0| - | Fc| /∑h| F0| , where Fc is the calculated structure factor scaled to F0. 4R-free is identical to R- factor on a subset of test reflections not used in refinement.
Example 1
Arabidopsis as a model for studying HU toxicity
Plant growth depends on the activity of pluripotent stem cells within the root and shoot apical meristems, allowing for easy scoring of plant stem cell viability. This makes plants convenient for studying stem cell resistance to cytotoxic agents such as HU. Ideally, HU taken up by Arabidopsis thaliana (Arabidopsis) should inhibit its primary target RNR and kill plants at a concentration physiologically relevant to cancer treatment. To examine these criteria, we first investigated plant uptake of HU by measuring the HU concentration in plants grown for 8 or 24 hours on 25 mM HU. Roots, not shoots, were in contact with the medium, and we measured HU-content in leaves to ensure quantifying only internalized HU. After 24 hours, the concentration in plant shoots approached that of the medium, showing that Arabidopsis seedlings take up HU (Figure 4). Second, we measured the effect of HU on Arabidopsis RNR activity in protein extracts and found a clear inhibition (Figure 5), supporting previously published results showing that HU can quench the tyrosyl radical of a purified subunit of Arabidopsis RNR. Having established HU uptake and RNR-inhibition, we then quantified HU toxicity in Arabidopsis to establish screening conditions. On medium containing 2.5 mM HU the germination rate was more than 90% but dropped sharply to less than 1/20,000 on 3 mM HU, which demonstrated the feasibility of a tight and sensitive screen (Figure 6A). A 3 mM HU concentration is comparable to the peak plasma levels of 1 mM HU detected after HU administration to blood cancer patients, indicating that screening conditions were physiologically relevant. Example 2
Detection of catalase genotypes leading to hydroxyurea resistance
250,000 EMS mutagenized Arabidopsis thaliana seeds were screened on medium containing 3 mM HU and more than twenty independent HU-resistant mutants were recovered. Resistant mutants were easily distinguished from the wild type by their elongated roots and expanded cotelydons (Figure 6B-C) and they tolerated HU concentrations more than five times higher than that lethal to the wild type.
One of the resistant mutants in A. thaliana Columbia background was crossed to 5 the closely related ecotype A. thaliana Landsberg erecta and the offspring were allowed to self-fertilize to produce a genetic mapping population. Mapping delimited a region containing five genes, and candidate gene sequencing identified a point mutation in catalase 2. Sequencing the catalase open reading frame in all identified HU resistant mutants revealed lesions in the catalase gene in the vast 10 majority of mutants, confirming that the causative mutation had been identified (SEQ ID. NO:5 provides the cDNA sequence of the Arabidopsis thaliana catalase 2 gene, and SEQ ID. NO:6 provides the genomic sequence of the Arabidopsis thaliana catalase 2 gene).
15 18 unique alleles were recovered. All catalase mutants were then assayed for catalase activity with regard to breakdown of hydrogen peroxide. When compared to the wild type, all mutants displayed a significant decrease (95%CL, 1-way ANOVA, Tukey's post-test, Graphpad Prism) in catalase activity (Figure 1).
The Arabidopsis thaliana catalase 2 protein (seq ID. NO:4) was aligned to the
20 human protein ortholog of catalase (seq ID.NO: 1) to identify the positions in the human catalase protein corresponding to the positions in Arabidopsis thaliana catalase 2 protein having a lowered catalase activity. Alignment is shown in figure 2. The discovered mutants in Arabidopsis thaliana corresponded, following alignment, to positions 71, 83, 88, 114, 121, 124, 147, 192, 195, 261, 275, 277,
25 303, 342, 354, 463, 503 and 517 in the homo sapiens amino acid sequence. It is shown that the mutations discovered, corresponding to position 277, 303, 342, 354, 463, 503 and 517 in homo sapiens result in either the generation of stop codons or interferes with splicing. Therefore mutations resulting in an amino acid change, the introduction of a stop codon or a change in the splicing pattern, may
30 result in a decrease in catalase activity. Thus, mutations at these positions are likely to be candidates for mutations causing loss of catalase activity, which results in hydroxyurea resistance.
Two previously described catalase 2 loss-of-function T-DNA insertion mutants also displayed HU resistance, reconfirming correct identification of the causal mutation
35 (Figure 1). This also indicates that loss of catalase activity is sufficient for inducing HU-resistance and that no specific structural changes, modifying enzyme specificity are required.
Example 3
Hydroxyurea (HU) inhibits catalase
To study the HU resistance mechanism, we investigated the effect of HU on catalase-mediated hydrogen peroxide decomposition. In complex mixtures, we found that addition of HU inhibited hydrogen peroxide decomposition in both plant extracts and rat erythrocyte lysates (Figure 7A). To confirm that the HU-catalase interaction was direct and did not require additional components, we then used purified bovine catalase for quantitative characterization. Measuring catalase activity in the presence of 12 mM H2O2 and varying concentrations of inhibitor produced a sigmoidal response-curve, from which we determined an IC50 value of 0.44 +/- 0.02 mM (95% CL) using non-linear regression (Figure 7B). Next, both substrate (H2O2) and inhibitor (HU) concentrations were varied to allow
determination of the mode of inhibition using curve fitting (Figure 7C). Best fits of the collected data to the models "competitive inhibition", "noncompetitive inhibition" and "uncompetitive inhibition" were compared. Competitive inhibition was the preferred model with a probability of >99.99% according to Akaike's method, with AICc scores of 26,80 (noncompetitive) and 78,54 (uncompetitive). Based on the best fit to the competitive inhibition model, we calculated the following kinetic characteristics: Km = 13.52 (11.22 to 15.82 mM), Ki = 0.23 (0.19 to 0.26 mM), and Vmax = 29 (27 to 31 μM/s) (95% CLs indicated). If Ki was instead calculated from the IC50 value obtained from the data shown in Figure 4B using the Cheng-Prusoff equation, a value of Ki = IC50 / (1+ [S]/Km) = 0.23 mM was still obtained. This Ki value is comparable to those previously published for RNR inhibition by HU (0.07-0.13 mM). As peak plasma levels can reach concentrations of 1 mM after HU administration to blood cancer patients, HU treatment could have an impact on cellular catalase activity. The HU-catalase interaction may therefore be of relevance to clinicians with regard to both therapeutic and side effects of HU treatment. In addition these data clearly shows that HU inhitbits catalase activity and that this inhibition is most likely due to competitive inhibition. Thus, when selecting treatments comprising hydroxyurea it appears highly relevant to determine the catalase status for the disease to be treated. This goes for both monotherapies with catalase inhibitors such as hydroxyurea and for combinatorial treatments of e,g. Catalase inhibitors (such as hydroxyurea) and tyrosine kinase inhibitors.
Example 4
HU is lodged in the catalase substrate tunnel.
To characterize the HU-catalase interaction at the molecular level, we soaked preformed crystals of bovine catalase in a cryo-protecting solution containing 50 mM HU and obtained X-ray diffraction data with a maximum resolution of 2.04 A (See method section) enabling us to detect small ligands and water molecules. In the electron density map we observed a strong density in the entrance to the catalytic site compatible with HU, with the density for the alcohol tail in particular discriminating it from a more spherical molecule (Figure 8A). No other differences with respect to the previously solved structure of catalyse were observed. The HU ligand was trapped in the funnel-shaped entrance tunnel 17 A above the iron atom of the heme, with the amide group pointing towards the constriction of the funnel leading to the active site (Figure 8B-C). Without conformational changes relative to the solved structure, the constricted funnel is apparently too narrow to allow HU to enter the active site. Trapping of the inhibitor at the funnel
constriction leaves no space for a hydrogen peroxide to diffuse into the active site, suggesting that HU could function as a competitive inhibitor without directly entering the active site. The simplest explanation for HU resistance in Arabidopsis is that loss of catalase function prevents catalase-mediated conversion of HU to a cytotoxic. The catalytic cycle of catalase can be divided into two steps: activation by one molecule of hydrogen peroxide to compound I and subsequent reaction of compound I with a second molecule of hydrogen peroxide returning the enzyme to its resting state. Our structural data showing HU lodged in the substrate tunnel of resting state catalase suggests that the HU-catalase reaction could be analogous to the reaction of catalase with its inhibitor 3-amino-l,2,4-triazole in that access to the active site is only granted when catalase is converted to compound I. Such a mechanism would also explain why a glucose/glucose oxidase hydrogen-peroxide generating system, which produces a steady stream of hydrogen peroxide increasing the steady-state level of compound I, is required for the HU-catalase reaction in vitro, but not for the reaction between HU and peroxidise.
Example 5
Chronic myeloid leukemias are characterized by high catalase activity.
HU is known to effectively decrease the white blood cell count in chronic myeloid leukemia (CML), whereas it is rarely used in the treatment of chronic lymphatic leukemia (CLL). We measured catalase activity in samples from blood cancer patients (leukocytes isolated from blood samples) and found elevated catalase activity levels in CML samples, whereas CLL samples presented activities within the normal range (Figure 3). One CML sample appeared to be an outlier displaying normal catalase activity (Figure 3). This sample originated from a patient that had been treated with high-dose HU for three weeks immediately prior to sampling. In an additional study we proceeded to study the potential of catalase activity as a biomarker in circulating blood cells from patients with hematological malignancies. Most patient samples displayed catalase activity levels within the normal range with the exception of isolated mononuclear cells from four patients suffering from chronic myeloid leukemia (CML), where the catalase activity was four times increased compared to the normal average and lymphoid malignancies. Four other CML samples had activity levels within the normal range, and for two of these, patients were undergoing HU treatment at the time of sampling (Figure 9-10).
These data indicate that with respect to cancer treatment, CML cells with elevated levels of catalase activity could be HU sensitive due to increased catalase- mediated conversion of HU to cytotoxic compounds. Thus catalase is a biomarker in relation to evaluating the potential effect of HU treatment in a malignant disease. In addition the levels of catalase may be used for subtyping a neoplastic disorder.
Furthermore, the data indicates that when selecting treatments comprising hydroxyurea is appears highly relevant to determine the catalase status for the disease to be treated. This goes for both monotherapies with catalase inhibitors such as hydroxyurea and for combinatorial treatments of e.g. catalase inhibitors (such as hydroxyurea) and tyrosine kinase inhibitors. Example 6
HU in the treatment of malaria Malaria is a mosquito-borne infectious disease caused by a eukaryotic protist of the genus Plasmodium. It is widespread in tropical and subtropical regions, including parts of the Americas, Asia, and Africa.
Five species of the Plasmodium parasite can infect humans; the most serious forms of the disease are caused by Plasmodium falciparum. Malaria caused by Plasmodium vivax, Plasmodium ovale and Plasmodium malariae causes milder disease in humans that are not generally fatal. A fifth species, Plasmodium knowlesi, is a zoonosis that causes malaria in macaques but can also infect humans
The inhibitory effect of HU on catalase-mediated hydrogen peroxide
decomposition documented here effectively makes HU the first clinically available catalase inhibitor. Usually, the antioxidative effect of catalase is desirable but a catalase inhibitor could prove clinically useful under certain circumstances. As an example, we would like to consider malaria, where catalase may play a role in protecting parasites and their host cells from oxidative damage. Glucose-6- phosphate dehydrogenase deficiency affords some protection against malaria and the underlying cause appears to be a higher susceptibility to oxidative damage due to decreased catalase activity. Further, catalase prevents killing of malaria parasites by activated macrophages and reduces the efficiency of the prime anti- malarial drug artemisinine, which depends on generation of reactive oxygen species for activity. Thus, including HU as a catalase inhibitor in anti-malarial treatment regimens could limit infection by exacerbating the oxidative challenge to parasite-hosting cells. Indeed, HU as monotherapy has shown a remarkable anti-malarial effect in a cerebral malaria mouse model (Pino P, Taoufiq Z, Brun M, Tefit M, Franetich JF, et al. (2006) Effects of hydroxyurea on malaria, parasite growth and adhesion in experimental models. Parasite Immunol 28: 675-680). Though it may seem illogical to prescribe an immunosuppressant such as HU to patients suffering from infectious disease, there could be cases, including HIV and possibly malaria, where the challenge to the pathogen outweighs the suppression of the host immune system. The effect of hydroxyurea in malaria may depend on the status of catalase in the patient. Thus, in an aspect the invention relates to a method for sub-typing a subject suffering from malaria, said method comprising
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby indicating whether the malaria patient is likely to be a hydroxyurea resistant subtype of said malaria or a hydroxyurea sensitive subtype of said malaria.
When obtaining a sample from a malaria patient it would preferably be a sample selected from the group consisting of blood samples or tissue samples. More specifically the samples could be purified blood cells such as red blood cells (erythrocytes), which are known to be of particularly importance in malaria infections since the erythrocytes are a site of multiplication of the parasite.
Whether a patient suffering from a malaria infection is likely to be resistant or sensitive towards hydroxyurea treatment, is likely to depend on the level of catalase. Thus, in an embodiment said at least one parameter being equal to or less than said predetermined threshold level or said known standard present in said sample with known sensitivity to treatment with hydroxyurea is indicative of the subject being a hydroxyurea resistant subtype of said infectious disease.
In another embodiment said at least one parameter being greater than said predetermined threshold level or said known standard present in said infectious disease with known sensitivity to treatment with hydroxyurea is indicative of the subject being a hydroxyurea sensitive subtype of said infectious disease. The person skilled in the art would of course be able to transfer the other aspects and embodiments presented in the present application into aspects and
embodiments relating to malaria such as predicting responsiveness of a malaria disorder to hydroxyurea treatment and monitoring the status of a malaria disorder, Similar a kit according to the invention may also be used for determining the effect of hydroxyurea treatment in a subject suffering from malaria.
In summary, the present invention is based on the novel discovery that there is a connection between the catalase status in a sample and the sensitivity towards hydroxyurea and that hydroxyurea is an inhibitor of catalase. Thus, the invention relates to methods for sub-typing neoplastic disorders thereby assisting in determining optimal treatment regimes for said neoplastic disorders. By determining the catalase status in a sample it may be possible to assist in determining whether said neoplastic disorder is indicative of being a hydroxyurea urea resistant subtype or a hydroxyurea resistant subtype. Since catalase inhibitors (such as hydroxyurea) may be used in combination with tyrosine kinase inhibitors, the invention furthermore relates to methods for assisting in
determining whether a neoplastic disorder is a subtype more sensitive to a composition or treatment protocol comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors (such as hydroxyurea) than to a composition (or treatment protocol) comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.

Claims

Claims
1. A method for sub-typing a neoplastic disorder, said method comprising
a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby indicating whether the neoplastic disorder is likely to be
- a hydroxyurea resistant subtype of said neoplastic disorder,
- a hydroxyurea sensitive subtype of said neoplastic disorder,
a subtype more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
a subtype equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
2. The method according to claim 1, wherein said at least one parameter being equal to or less than said predetermined threshold level or said known standard present in said neoplastic disorder with known sensitivity to treatment with hydroxyurea is indicative of the neoplastic disorder being a hydroxyurea resistant subtype of said neoplastic disorder.
3. The method according to claim 1, wherein said at least one parameter being greater than said predetermined threshold level or said known standard present in a neoplastic disorder with known sensitivity to treatment with hydroxyurea is indicative of the neoplastic disorder being a hydroxyurea sensitive subtype of said neoplastic disorder.
4. The method according to claim 1, wherein the at least one parameter being greater than said predetermined threshold level indicates a neoplastic disorder being a subtype more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
5. The method according to claim 1, wherein the at least one parameter being lower or equal to said predetermined threshold level indicates a neoplastic disorder being a subtype equally of less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
6. A method for predicting responsiveness of a neoplastic disorder to hydroxyurea treatment or treatment with a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors, said method comprising a) providing a biological sample obtained from a subject,
b) assessing in said sample at least one of the parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase, and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard, thereby predicting the sensitivity or resistance of said neoplastic disorder to treatment with hydroxyurea or to treatment with a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors.
7. The method of claim 6, wherein the neoplastic disorder is predicted to be resistant to hydroxyurea if the at least one parameter is equal to or less than said predetermined threshold level or said known standard present in a neoplastic disorder with known sensitivity to treatment with hydroxyurea.
8. The method of claim 6, wherein the neoplastic disorder is predicted to be sensitive to the hydroxyurea if the at least one parameter is greater than said predetermined threshold level or said known standard present in the neoplastic disorder with known sensitivity to treatment with hydroxyurea.
9. The method according to claim 6, wherein the neoplatic disorder is predicted to be more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors if the at least one parameter is greater than said predetermined threshold level or known standard.
10. The method according to claim 6, wherein the neoplatic disorder is predicted to be equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors if the at least one parameter is equal to or less than said predetermined threshold level or known standard.
11. A method of monitoring the status of a neoplastic disorder in a subject, said method comprising
a) providing at least two biological samples obtained from a subject at
different time points,
b) assessing in said samples at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status, c) comparing the at least one parameter of said at least two biological samples,
thereby indicating whether it is likely that said neoplastic disorder has
- progressed to become a hydroxyurea resistant subtype of said
neoplastic disorder,
- regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder,
- the sensitivity to hydroxyurea of said neoplastic disorder is maintained,
- progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
- has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
12. A method according to claim 11, said method comprising
a) providing at least two biological samples obtained from a subject at
different time points,
b) assessing in said samples at least one parameter selected from the group of:
i. catalase enzyme activity,
ii. the RNA level of catalase,
iii. the protein level of catalase and
iv. the catalase gene mutation status,
c) comparing the at least one parameter to a corresponding predetermined threshold level or a known standard,
d) evaluating the at least one parameter relative to said predetermined
threshold level or said known standard,
e) compare said evaluation in said at least two samples,
thereby indicating whether it is likely that said neoplastic disorder has - progressed to become a hydroxyurea resistant subtype of said neoplastic disorder,
- regressed to become a hydroxyurea sensitive subtype of said neoplastic disorder,
- the sensitivity to hydroxyurea of said neoplastic disorder is maintained,
- progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors, or
- has regressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
13. The method according to claim 11 or 12, wherein it is likely that said neoplastic disorder has progressed to become a hydroxyurea resistant subtype of said neoplastic disorder if the at least one parameter is lower than said
predetermined threshold level or said known standard present in the neoplastic disorder with known sensitivity to treatment with hydroxyurea, and if the value is lower in the sample obtained at the latest point in time.
14. The method according to claim 11 or 12 or, wherein it is likely that said neoplastic disorder has progressed to become a hydroxyurea sensitive subtype of said neoplastic disorder if the at least one parameter is greater than said predetermined threshold level or said known standard present in the neoplastic disorder with known sensitivity to treatment with hydroxyurea, and if the value is greater in the sample obtained at the latest point in time.
15. The method according to claim 11 or 12, wherein it is likely that said sensitivity to hydroxyurea in the neoplastic disorder is maintained if the value is equal in the samples obtained at different points in time.
16. The method according to claim 11 or 12, wherein it is likely that said neoplastic disorder has progressed to become a subtype of said neoplastic disorder being more sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors disorder if the at least one parameter is greater than said predetermined threshold level or said known standard, and if the value is greater in the sample obtained at the latest point in time.
17. The method according to claim 11 or 12, wherein it is likely that said neoplastic disorder has progressed to become a subtype of said neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a
composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors if the at least one parameter is lower than said
predetermined threshold level or said known standard present in the neoplastic disorder and if the value is lower in the sample obtained at the latest point in time.
18. The method according to any of the preceding claims, wherein the one or more tyrosine kinase inhibitors are selected from the group consisting of 4-[(4- methylpiperazin-l-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2- yl)amino] phenyl] benzamide (imatinib), 4-[(4-Methyl-l-piperazinyl)methyl]-N-[4- methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamide
monomethanesulfonate (imatinib mesylate), N-(2-chloro-6-methylphenyl)-2-[[6- [4-(2-hydroxyethyl)-l-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazole carboxamide monohydrate (dasatinib), 4-methyl-N-[3-(4-methyl-lH-imidazol-l- yl)- 5-(trifluoromethyl)phenyl]-3- [(4-pyridin-3-ylpyrimidin-2-yl)
amino]benzamide (nilotinib) and N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-l,2- dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-lH-pyrrole-3-carboxamide (sunitinib).
19. The method according to any of the preceding claims, wherein the one or more catalase inhibitors are selected from the group consisting of hydroxyurea and 3-Amino-Triazole.
20. The method according to any of the preceding claims , wherein said at least one parameter is catalase enzyme activity in said sample.
5
21. The method according to claim 20, wherein said catalase enzyme activity is determined by measuring the turnover of a catalase substrate.
22. The method according to claim 21, wherein said substrate is selected from the 10 group consisting of hydroxyurea, hydrogen peroxide and sodium perborate.
23. The method according to claim 22, wherein said substrate is hydrogen peroxide.
15 24. The method according to claim 23, wherein said turnover is determined as the change in absorbance at 240 nm.
25. The method according to claim 24, wherein said turnover of hydrogen peroxide is measured using horseradish peroxidise (HRP) as a reporter enzyme.
20
26. The method according to any of claims 1-19, wherein said at least one parameter is the RNA level of catalase in said sample.
27. The method according to claim 26, wherein assessment of the RNA level of 25 catalase comprises the use a technique selected from the group consisting of PCR,
Q-PCR, RT-PCR, a hybridization technique and microarrays.
28. The method according to any of claims 1-19, wherein said at least one parameter is the protein level of catalase in said sample.
30
29. The method according to claim 28, wherein assessment of the protein level of catalase comprises the use of a technique selected from the group consisting of immuno-based assays and mass spectrometry.
30. The method according to claim 1-19, wherein said at least one parameter is the catalase gene mutation status, and said catalase gene mutation status is assessed by detecting the presence or absence of a mutation in the catalase gene.
31. The method according to claim 30, wherein said known standard is the nucleic acid sequence encoding catalase set forth in at least one of SEQ ID NO: 2 and SEQ ID NO:3
32. The method according to claim 30, wherein said known standard is a list comprising at least one nucleic acid sequence encoding catalase with no or reduced catalase enzyme activity.
33. The method according to claim 32, wherein said at least one nucleic acid sequence encodes a catalase polypeptide with no or reduced catalase enzyme activity, said catalase polypeptide is selected from the group consisting of polypeptides deviating at position 71, 83, 88, 114, 121, 124, 147, 192, 195, 261, 275, 277, 303, 342, 354, 463, 503 and 517 of SEQ ID NO: 1
34. The method according to any of claims 30-33, wherein the presence of at least one amino acid substitution in at least one of the positions selected from the group consisting of position 71, 83, 88, 114, 121, 124, 147, 192, 195, 261, 275, 277, 303, 342, 354, 463, 503 and 517 of SEQ ID NO: 1, is indicative of the neoplastic disorder being a hydroxyurea resistant subtype and/or the neoplastic disorder being equally or less sensitive to a composition comprising one or more tyrosine kinase inhibitors and one or more catalase inhibitors than to a
composition comprising one or more tyrosine kinase inhibitors without the one or more catalase inhibitors.
35. A method according to any of claims 30-34, wherein the presence of the mutation is heterozygous or homozygous.
36. The method according to any of the preceding claims, wherein said sample is obtained from a subject currently receiving treatment for said neoplastic disorder.
37. The method according to claim 36, wherein said treatment comprises the administration of at least one of catalase inhibitors and tyrosine kinase inhibitors to said subject.
5 38. The method according to any of the preceding claims, wherein the neoplastic disorder is a cancer.
39. The method according to claim 38, wherein the cancer is selected from the group consisting of leukemia and solid tumor cancers.
10
40. The method according to claim 39, wherein cancer is a leukemia selected from the group consisting of chronic myeloid leukemia (CML), chronic lymphatic leukemia (CLL), polycytemia vera, thrombocytosis, myelofibrosis hypereosinophilic syndrome and acute myeloid leukemia (AML).
15
41. The method according to claim 40, wherein the cancer is a chronic myeloid leukemia (CML).
42. The method according to claim 39, wherein the solid tumor cancer is selected 20 from the group consisting of glioblastoma and gastrointestinal stromal tumors.
43. The method according to any of the preceding claims, wherein the sample is a blood sample or tissue sample.
25 44. A kit for performing the method of any one of preceding claims, comprising a) at least one reagents,
b) an instruction manual explaining how to carry out the methods according to the preceding claims.
30 45. The kit according to claim 36, wherein said at least one reagent is hydrogen peroxide.
PCT/DK2010/050207 2009-08-11 2010-08-11 Predictive marker for hydroxyurea resistance WO2011018090A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005078127A1 (en) 2004-02-13 2005-08-25 Oy Jurilab Ltd Method for detecting the risk of cancer, coronary heart disease, and stroke by analysing a catalase gene
WO2006136391A2 (en) 2005-06-23 2006-12-28 Novartis Ag Treatment of solid tumor disease with combinations comprising imatinib and an efflux pump inhibitor
WO2008074131A1 (en) 2006-12-20 2008-06-26 Mcmaster University Skin and salivary tests for the detection of sensitivity to oxidative stress based therapies

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005078127A1 (en) 2004-02-13 2005-08-25 Oy Jurilab Ltd Method for detecting the risk of cancer, coronary heart disease, and stroke by analysing a catalase gene
WO2006136391A2 (en) 2005-06-23 2006-12-28 Novartis Ag Treatment of solid tumor disease with combinations comprising imatinib and an efflux pump inhibitor
WO2008074131A1 (en) 2006-12-20 2008-06-26 Mcmaster University Skin and salivary tests for the detection of sensitivity to oxidative stress based therapies

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ARORA A; SCHOLAR EM: "Role of tyrosine kinase inhibitors in cancer therapy", J PHARMACOL EXP THER, vol. 315, no. 3, December 2005 (2005-12-01), pages 971 - 9
BACHIR D; HULIN A; HUET E; HABIBI A; NZOUAKOU R ET AL.: "Plasma and urine hydroxyurea levels might be useful in the management of adult sickle cell disease", HEMOGLOBIN, vol. 31, 2007, pages 417 - 425
BRUCHOVA H ET AL: "GENE EXPRESSION PROFILING IN CHRONIC MYELOID LEUKEMIA PATIENTS TREATED WITH HYDROXYUREA", LEUKEMIA AND LYMPHOMA, HARWOOD ACADEMIC PUBLISHERS, CHUR, CH, vol. 43, no. 6, 1 January 2002 (2002-01-01), pages 1289 - 1295, XP008065882, ISSN: 1042-8194 *
JONG AY; YU K; ZHOU B; FRGALA T; REYNOLDS CP ET AL.: "A simple and sensitive ribonucleotide reductase assay", J BIOMED SCI, vol. 5, 1998, pages 62 - 68
JUUL TRINE ET AL: "The in Vivo Toxicity of Hydroxyurea Depends on Its Direct Target Catalase", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 285, no. 28, July 2010 (2010-07-01), pages 21411 - 21415, XP002602139, ISSN: 0021-9258 *
KWEE J K ET AL: "Possible role of catalase in chronic myeloid leukemia (CML) cells resistant to As203", FREE RADICAL BIOLOGY AND MEDICINE, vol. 31, no. 10, November 2001 (2001-11-01), & 8TH ANNUAL MEETING OF THE OXYGEN SOCIETY; RESEARCH TRIANGLE PARK, NORTH CAROLINA, USA; NOVEMBER 15-19, 2001, pages S139, XP002570813, ISSN: 0891-5849 *
LUQUE ET AL.: "Possible role of catalase activity as a biomarker for Gleevec-resistant CML patients", FREE RADICAL BIOLOGY AND MEDICINE, vol. 37, no. Supp. 1, 1 January 2004 (2004-01-01), pages S172 - S172, XP002570812 *
PINO P; TAOUFIQ Z; BRUN M; TEFIT M; FRANETICH JF ET AL.: "Effects of hydroxyurea on malaria, parasite growth and adhesion in experimental models", PARASITE IMMUNOL, vol. 28, 2006, pages 675 - 680

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