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WO2012113965A1 - Procédé d'obtention de données utiles pour évaluer, prédire et/ou pronostiquer la réponse à un traitement avec des analogues de pyrimidine - Google Patents

Procédé d'obtention de données utiles pour évaluer, prédire et/ou pronostiquer la réponse à un traitement avec des analogues de pyrimidine Download PDF

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Publication number
WO2012113965A1
WO2012113965A1 PCT/ES2012/070115 ES2012070115W WO2012113965A1 WO 2012113965 A1 WO2012113965 A1 WO 2012113965A1 ES 2012070115 W ES2012070115 W ES 2012070115W WO 2012113965 A1 WO2012113965 A1 WO 2012113965A1
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Prior art keywords
pkr
protein
treatment
gene
kit
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Spanish (es)
Inventor
María Ángel GARCÍA CHAVES
Margarita AGUILERA GÓMEZ
Miguel Ángel CALLEJA HERNÁNDEZ
Juan Antonio Marchal Corrales
Mariano ESTEBAN RODRÍGUEZ
Esther CARRASCO PARDO
Gema JIMÉNEZ GONZÁLEZ
Antonia ARÁNEGA JIMÉNEZ
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad de Granada
Servicio Andaluz de Salud
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad de Granada
Servicio Andaluz de Salud
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Publication of WO2012113965A1 publication Critical patent/WO2012113965A1/fr
Anticipated expiration legal-status Critical
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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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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

Definitions

  • the present invention is within medicine and molecular biology, and refers to an in vitro method of obtaining useful data to predict and predict the response to treatment with pyrimidine analogs, and more preferably with 5-fluorouracil (5 -FU), particularly in patients with colon and breast cancer, which allows the establishment of a specific individual (qualitative and quantitative) recognition pattern, establishing different groups of patients
  • Interferon PKR-induced cellular protein is a kinase that plays a fundamental role in the antitumor and antiviral activity that Interferon type I plays.
  • PKR activator is the double stranded RNA that occurs during the infection of various viruses, but is also activated by different types of cellular stress such as polyols such as heparin, and other compounds such as ethanol, arsenic, ceramide, ect .., Its fundamental mechanism of action is to cause the cell protein synthesis to stop and activate the NFkB transcription factor that allows the cell to continue living if the problem that caused its activation is resolved. If cell stress continues, PKR eventually causes cell death in a controlled manner by apoptosis. PKR, therefore, can act as a tumor suppressor and has recently been shown to be critical for tumor suppressor activity of the known p53 protein.
  • 5-fluorouracil 5-fluorouracil
  • the protein involved in this phenomenon is the p53 tumor suppressor. But it is known that more than 50% of tumors have mutations in p53, and that tumor cells with p53 Inactive can die by apoptosis in response to 5-FU chemotherapeutic. Therefore it is unknown how 5FU induces the death of tumor cells in the absence of p53. We have discovered that it is precisely through the activation of the PKR protein that this process is carried out. PKR is activated in response to 5-FU even in the absence of p53, inducing the stop in the synthesis of cellular proteins and finally inducing cell death by apoptosis.
  • p53 together with PKR are key cell markers in the effect of tumor cell death due to apoptosis caused by therapy based on the use of 5-FU or other pyrimidine analogues.
  • PKR and p53 are mutated in tumors can be very useful for the prognosis of response to therapies based on the use of pyrimidine analogues, preferably in 5-FU, as well as their combination with other drugs or cytokines like the IFN. It is known that there is a very considerable percentage of patients who do not respond to chemotherapy based on the use of pyrimidine analogues, and preferably on 5-FU. Given the role that p53 plays in death induced by 5-FU, numerous studies have attempted to demonstrate that the lack of response is due to mutations in said tumor suppressor. Unfortunately, most of the works have failed to demonstrate that relationship, concluding that there must be more cell markers involved in the response to chemotherapy.
  • PKR as a new evaluation marker for cancer treatment with pyrimidine analogs, and preferably with 5-FU.
  • the results of the studies in cell lines and samples of cancer patients show mutations in the genetic sequence of the PKR protein, and alterations in the activity of this protein, which can be used as a marker of the response the pyrimidine analogues, and in particular to 5-FU.
  • a first aspect of the invention relates to the use of the PKR protein (or the pkr gene that encodes it) for obtaining useful data to predict the response to treatment with pyrimidine analogs.
  • the pyrimidine analogs are selected from the list consisting of cytarabine, fluoracil (5-fluoracil or 5- FU), tegafur, carmofur, gemcitabine, capecitabine, azacitidine, decitabine, or any of its salts, isomers, prodrugs, derivatives or corresponding analogs, and combinations thereof.
  • the pyrimidine analog is 5-fluorouracil (5-FU), or any of its salts, isomers, prodrugs, derivatives or the like.
  • Treatment with pyrimidine analogues, and in particular with 5-FU, may be alone, or in combination with other drugs, preferably with IFN alpha.
  • Another aspect of the invention relates to a method of obtaining useful data to predict the response to treatment with pyrimidine analogs, or any of its salts, isomers, prodrugs, derivatives or the like, hereinafter method of the invention, comprising:
  • b) determine the activity of the PKR protein, in the biological sample isolated from (a), or the sequence of the PKR protein or the p / r gene that encodes it.
  • the pyrimidine analogs are selected from the list consisting of cytarabine, fluoracil (5- fluoracil or 5-FU), tegafur, carmofur, gemcitabine, capecitabine, azacitidine, decitabine, or any of its corresponding salts, isomers, prodrugs, derivatives or analogs, and combinations thereof.
  • Treatment with the pyrimidine analogs, and in particular with 5-FU, may be alone, or in combination with other drugs, preferably with IFN alpha.
  • the activity of the PKR protein is determined by detecting the amount of expression of the pkr gene.
  • the activity of the PKR protein is determined by the sequence of the PKR gene.
  • mutations of the sequence encoding PKR are detected. Mutations in said sequence may be indicative of a different activity.
  • the activity of the PKR protein is determined by detecting the amount of PKR protein, and more preferably, by the amount of phosphorylated PKR protein. More preferably, the biological sample of the individual from step (a) are tumor cells.
  • the method of the invention further comprises: c) comparing the amounts obtained in step (b) with a reference amount, or with a reference sequence.
  • the amount or activity of the PKR protein, or the expression of the pkr gene is not sufficient, or the gene has certain polymorphisms, then it can be determined that the patient is not responding to the 5-FU treatment.
  • Steps (b) and / or (c) of the method described above can be fully or partially automated, for example, by means of a robotic sensor device for the determination of PKR activity or the detection of pkr expression levels. , or the identification of several polymorphisms in step (b) or the computerized comparison in step (c).
  • the determination of the activity of the PKR protein allows discriminating between responding individuals and non-responding individuals, thus facilitating the choice and establishment of appropriate therapeutic regimens. This discrimination as understood by an expert in the field is not intended to be correct. in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly.
  • the amount that is significantly statistical can be established by one skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the p-value, Student's test or discriminant functions of Fisher
  • the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%.
  • the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001.
  • the present invention makes it possible to correctly detect the responding individuals differentially by at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a certain group or population analyzed.
  • an "isolated biological sample” includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art.
  • the isolated biological sample are cells, and more preferably tumor cells.
  • the term “individual” is not intended to be limiting in any aspect, and may be of any age, sex and physical condition.
  • the individual has previously been diagnosed with cancer. More preferably, the individual has been diagnosed with colon cancer and / or breast cancer.
  • RNA-dependent protein kinase also referred to as "eukaryotic translation initiation factor 2-alpha kinase 2" (EIF2AK2). Also called EIF2AK1, MGC126524, PKR, PRKR, OTTHUMP00000201320; P1 / elF-2A protein kinase; double stranded RNA activated protein kinase; elF-2A protein kinase 2; interferon-induced, double-stranded RNA-activated protein kinase; interferon-inducible RNA-dependent protein kinase; interferon-inducible elF2alpha kinase; p68 kinase; RNA-activated protein kinase; protein kinase, interferon-inducible double stranded RNA dependent.
  • PKR is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 1, and which would comprise various variants from:
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,
  • nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a) are nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),
  • nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 1, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the PKR protein.
  • the PKR protein activity can be measured by any means known in the state of the art.
  • the presence of polymorphisms or mutations can be determined by DNA sequencing.
  • the amount or concentration of the pkr gene expression product can be brought to out directly or indirectly.
  • Direct measurement refers to the measure of the quantity or concentration of the gene expression product, based on a signal that is obtained directly from the transcript of said gene, or from the protein (PKR), and that is directly correlated with the number of RNA molecules or proteins produced by the gene.
  • Said signal - which we can also refer to as an intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of said products.
  • the indirect measurement includes the measurement obtained from a secondary component or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products).
  • Quantity refers to, but is not limited to, the absolute or relative quantity of the gene expression product, as well as any other value or parameter related to it or that may be derived. of this one.
  • Said values or parameters comprise values of signal strength obtained from any of the physical or chemical properties of said expression product obtained by direct measurement. Additionally, said values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.
  • comparison refers to, but is not limited to, the comparison of the quantity of the expression product of the pkr gene of the biological sample to be analyzed, also called the biological problem sample, with a amount of expression product of one or more desirable reference samples described elsewhere in the present description.
  • the reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample.
  • the comparison described in section (c) of the method of the present invention can be performed manually or assisted by a computer.
  • reference amount refers to the absolute or relative amount of expression product of the pkr gene that allows discriminating individuals responding to 5-FU treatment of non-responders.
  • Suitable reference amounts can be determined by the method of the present invention from a reference sample that can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. Particularly, the reference amount.
  • the reference sequences are SEQ ID NO: 1 for protein, and SEQ ID NO; 2 for the nuecleotidic sequence.
  • variant refers to a protein substantially homologous to the PKR protein.
  • a variant includes additions, deletions or substitutions of amino acids.
  • variant also includes the proteins resulting from posttranslational modifications, such as, but not limited to, glycosylation phosphorylation, sumoylation, methylation or acylation.
  • a protein is "substantially homologous" to the PKR protein when its amino acid sequence has a good alignment with the amino acid sequence SEQ ID NO: 1, that is, when its amino acid sequence has a degree of identity with respect to the amino acid sequence SEQ ID NO: 1, of at least 50%, typically of at least 80%, advantageously of at least 85%, preferably of at least 90%, more preferably at least 95%, and even more preferably at least 99%.
  • the homologous sequences to the PKR protein can be easily identified by one skilled in the art, for example, with the help of an appropriate computer program to compare sequences.
  • the term “functionally equivalent”, as used herein, means that the proteins or fragment / s of the protein / s in question essentially maintain the biological or immunological properties described herein. document. Said capacity can be determined by conventional methods.
  • the "pyrimidine analogues” constitute a well defined group in the ATC code or System of Anatomical, Therapeutic, Chemical Classification (instituted by the World Health Organization, and adopted in Europe). They belong to section ATC L01, section of the ATC classification system within group L, corresponding to antineoplastic and immunomodulating agents. Specifically they are classified as L01 BC Pyrimidine analogues, and in the latest update of 201 1 includes:
  • fluorouracil 5-fluorouracil
  • 5-FU 5-fluorouracil
  • 5- Fluoropyrimidine-2,4-dione a compound with CAS number 51-21-8, of formula (I)
  • capecitabine or pentyl [1 - (3,4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1 H- pyrimidin-4-yl] carbamate, is understood as a compound with CAS number 154361 -50-9, of formula (VI)
  • Azacitidine or 4-amino-1 - -D-ribofuranosyl- 1, 3,5-triazin-2 (1 7) -one, a compound with CAS number 320-67-2, of formula
  • decitabine is understood as, or 4-amino-1- (2-deoxy-bD-erythropentofuranosyl) -1, 3,5-triazin-2 (1 H) -one, a compound with CAS number 2353- 33-5, of formula (VIII)
  • derivative includes both pharmaceutically acceptable compounds, including derivatives of the compound of formula (I), (II), (III), (IV), (V), (VI), (VII), and / or (VIII), which may be used in the manufacture of a medicament, as pharmaceutically acceptable derivatives, since these may be useful in the preparation of pharmaceutically acceptable derivatives.
  • prodrugs of the compound of formula (I), (II), (III), (IV), (V), (VI), (VII), and / or (VIII) are the prodrugs of the compound of formula (I), (II), (III), (IV), (V), (VI), (VII), and / or (VIII) .
  • the term "prodrug” as used herein includes any compound derived from the compound of formula (I), (II), (III), (IV), (V), (VI), (VII), and / or (VIII), for example, esters, including esters of carboxylic acids, amino acid esters, phosphate esters, sulphonate esters of metal salts, carbamates, amides, etc., which, when administered to an individual, are capable of providing, directly or indirectly, the effect of the compound of formula (I) on said individual.
  • said derivative is a compound that increases the bioavailability of the compound of formula (I), (II), (III), (IV), (V), (VI), (VII), and / or (VIII), when administered to an individual or that enhances the release of the compound of formula (I) (II), (III), (IV), (V), (VI), (VII), and / or (VIII), in a biological compartment
  • the nature of said derivative is not critical as long as it can be administered to an individual and provides the compound of formula (I) (II), (III), (IV), (V), (VI), (VII), and / or (VIII), in a biological compartment of an individual.
  • the preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.
  • the detection of the amount of PKR protein is performed by an immunoassay.
  • immunoassay refers to any analytical technique that is based on the reaction of conjugation of an antibody with an antigen.
  • Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot, enzyme-linked immunosorbent assay (ELISA), linear immunoassay (LIA), radioimmunoassay (RIA), immunofluorescence, x-map or protein chips .
  • the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay).
  • ELISA Enzyme-Linked ImmunoSorbent Assay
  • the ELISA is based on the premise that an immunoreactive (antigen or antibody) can be immobilized on a solid support, then bringing that system into contact with a fluid phase containing the complementary reagent that can bind to a marker compound.
  • ELISA Enzyme-Linked ImmunoSorbent Assay.
  • the ELISA is based on the premise that an immunoreactive (antigen or antibody) can be immobilized on a solid support, then bringing that system into contact with a fluid phase containing the complementary reagent that can bind to a marker compound.
  • ELISA enzyme-linked immunosorbent assay
  • ELISA Enzyme-Linked ImmunoSorbent Assay
  • marker compound refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and quantification of the amount of antibodies against to the PKR protein.
  • the marker compound is selected from the list comprising radioisotopes, enzymes, fluorophors or any molecule capable of being conjugated with another molecule or detected and / or quantified directly. This marker compound can bind to the protein directly, or through another compound.
  • directly binding marker compounds are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as 32 P or 35 S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, autoradiography, fluorimetry, or metallography respectively.
  • enzymes such as alkaline phosphatase or peroxidase
  • radioactive isotopes such as 32 P or 35 S
  • fluorochromes such as fluorescein or metal particles
  • Gene expression profile means the gene profile obtained after quantification of mRNA and / or protein produced by the genes of interest or biomarkers, that is, by the pkr gene, in an isolated biological sample.
  • the expression profile of the genes is preferably performed by determining the level of mRNA derived from its transcription, after extracting the total RNA present in the isolated biological sample, which can be performed by protocols known in the state of the art.
  • the mRNA level derived from pkr gene transcription can be determined, for example, but not limited to, by amplification by polymerase chain reaction (PCR), back transcription in combination with polymerase chain reaction (RT).
  • RNA chips made with oligonucleotides deposited by any mechanism; DNA microarrays made with oligonucleotides synthesized in situ by photolithography or by any other mechanism; in situ hybridization using specific probes labeled with any method of marking; by electrophoresis gels; by membrane transfer and hybridization with a specific probe; by nuclear magnetic resonance or any other diagnostic imaging technique using paramagnetic nanoparticles or any other type of detectable nanoparticles functionalized with antibodies or by any other means.
  • the gene expression profile could also be obtained by the detection and / or quantification of the proteins resulting from the translation of the mRNA derived from the transcription of the pkr gene, for example, but not limited to, western blot immunodetection.
  • Quantitative detection of pkr gene expression can be performed more preferably by real-time PCR (RT-PCR or RTqPCR). Real-time detection of amplified products can be carried out through the use of molecules fluorescents that are embedded in the double stranded DNA or by hybridization with different types of probes.
  • RT-PCR real-time PCR
  • Real-time detection of amplified products can be carried out through the use of molecules fluorescents that are embedded in the double stranded DNA or by hybridization with different types of probes.
  • the expression of the pkr gene is detected by RTqPCR using primers SEQ ID NO: 3 - SEQ ID NO: 10, indicated in Table 2.
  • kits or devices comprising the elements necessary to analyze PKR activity, or sequence the pkr gene.
  • step (b) More preferably it comprises the means necessary to compare the amount (of expression of the pkr gene and / or amount of PKR protein) detected in step (b) with a reference amount, or the sequence of the PKR protein or gene, with the reference sequences (SEQ ID NO: 1 and / or SEQ ID NO: 2).
  • the kit of the present invention comprises the means necessary to obtain the sequence encoding the PKR protein, and preferably any of the primers collected in the sequences SEQ ID NO: 3 to S EQ ID NO: 1 0 to carry out the method of the present invention, or any combination thereof.
  • Said kit may comprise all those reagents necessary to analyze the activity of the PKR by means of any of the methods described above in this document. It can also comprise any means to obtain the sequence encoding the PKR protein. Thus, it may include without limitation, any of the primers collected in the sequences SEQ D NO: 3 to SEQ ID NO: 10.
  • it comprises the pair of primers selected from the list comprising: SEQ ID NO: 3 and SEQ ID NO: 4, the pair of primers SEQ ID NO: 5 and EQ ID NO: 6, the pair of primers SEQ ID NO: 7 and EQ ID NO: 8 and the pair of primers SEQ ID NO: 9 and EQ ID NO : 10, or any of its combinations.
  • Another aspect of the invention relates to the sequence primer SEQ ID NO: 3. Another aspect of the invention relates to the sequence primer SEQ ID NO: 4. Another aspect of the invention relates to the sequence primer SEQ ID NO: 5 Another aspect of the invention relates to the sequence primer S EQ IDNO: 6. Another aspect of the invention relates to the sequence primer SEQ ID NO: 7. Another aspect of the invention relates to the sequence primer SEQ ID NO: 8. Another aspect of the invention relates to the sequence primer SEQ ID NO: 9. Another aspect of the invention relates to the sequence primer SEQ ID NO: 10.
  • the kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc.
  • the kit can include all the supports and containers necessary for commissioning and optimization.
  • the kit further comprises instructions for carrying out any of the methods of the invention.
  • Another aspect of the invention relates to the use of the kit or device of the invention for obtaining useful data in the evaluation of the response to treatment with 5-FU, and preferably in individuals diagnosed with cancer.
  • the kit of the invention comprises any of the primers collected in the sequences SEQ D NO: 3 to SEQ ID NO: 10.
  • it comprises the pair of primers selected from the list comprising: SEQ ID NO: 3 and EQ ID NO: 4, the primer pair SEQ ID NO: 5 and EQ ID NO: 6, the primer pair SEQ ID NO: 7 and EQ ID NO: 8 and the primer pair SEQ ID NO: 9 and EQ ID NO: 10, or any of its combinations.
  • polynucleotide and “nucleic acid” are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).
  • amino acid sequence refers to a polymeric form of amino acids of any length, which may be coding or non-coding, Chemically or biochemically modified.
  • (A) SW480, T84, MCF-7 and T47D cells were treated with 1 0 ⁇ of 5-FU at 4, 8, 16 and 24 hours.
  • (C) HCT-1 16 colon cancer cells expressing shRNA versus PKR (shRNA-PKR) or expressing the shRNA control (shRNAc) were treated with 500 Ul / ml of IFNa for 16 hours or treated with 10 ⁇ of 5-FU for 4, 8, 16 and 24 hours. Proteins were extracted and analyzed using the following antibodies: anti-phospho PKR, anti-whole PKR, anti-phospho elF2aanti-whole elF2a and anti- -actin.
  • PKR + + and PKR " ' " MEFs were treated with 5 ⁇ of 5- FU for 48 hours. The cell cycle was analyzed by flow cytometry after staining the cells with Pl.
  • Pl. PKR + + and PKR " ' " MEFs were treated with 5 ⁇ , 1 0 ⁇ and 1 00 ⁇ were 5-FU or 1 00 ⁇ g nnl of Irinotecan for 48 hours. Subsequently the cells were analyzed with Annexin V detection kit.
  • HCT-1 16 p53 + + cells, and HCT-1 16 p53 " ' " were treated with 500 IU / ml of IFNa for 1 6 hours or treated with 1 0 ⁇ of 5-FU for 4, 8, 1 6 and 24 hours .
  • Proteins were extracted and analyzed using the following antibodies: whole p53, PKR, anti phospho PKR, anti whole PKR, anti phospho elF2a, anti whole elF2a and anti b-actin.
  • C HCT-1 16 p53 + + cells, HCT-1 16 p53 " ' " which expressing shRNA against PKR or the shRNA control system were treated with 10 ⁇ of 5-FU for 48 hours. Subsequently the cells were analyzed with Annexin V detection kit.
  • PKR “ ' " and PKR + + MEFs were treated with increasing amounts of 5-FU alone (triangles) or in combination with 50 U l / ml of I FNa (squares) for 6 days.
  • the cell survival curve is represented as a percentage referred to untreated cells.
  • PKR plays an important role in translation inhibition, cell cycle arrest and 5-FU induced apoptosis.
  • PKR protein activation during 5-FU treatment was demonstrated in several tumor cell lines of breast and colon cancer through the detection of phosphorylated PKR protein and phosphorylation of its main substrate elF2a (Figl A) .
  • Figl A MEF cells derived from wild-type (PKR + + ) and knockout (PKR " ' " ) mice were analyzed for various biological functions. As shown in Fig.
  • PKR + + and PKR " ' " MEFs cells were treated with increasing amounts of 5-FU. As shown in Fig 2B, in PKR + + cells, 10 ⁇ of 5-FU were able to significantly induce apoptosis levels that were slightly increased with the high dose of 100 ⁇ of 5-FU. However, apoptosis did not occur in PKR cells " ' " treated. Irinotecam treatment was used as a positive control of the induction of apoptosis in both cell lines. Cell lines of colon cancer HCT-1 16 and breast cancer MCF-7 that expressed shRNA for PKR also showed reduced levels of apoptosis after 5-FU treatment compared to cells expressing control shRNA (Fig. 2C and D).
  • PKR activation by 5-FU occurs independently of p53 It has been previously described that induction of apoptosis in response to 5- FU was mediated by p53. On the other hand, it has been recently suggested that one of the functions of p53 is modular PKR.
  • HCT1 16 p53 + + and HCT1 16 p53 cell lines were analyzed As shown in Figure 3A, PKR and elF2a were phosphorylated during treatment with 5-FU in both lines. In addition, the total PKR level was also increased in the absence of the p53 protein.
  • mRNA PKR PCR was measured in real time in HCT1 cells 16 p53 + + and p53 _. "( Figure 3B). As previously shown, p53 it is involved in apoptosis induced by 5-FU as shown in HCT1 16 cells in p53 + p53 + and _ ⁇ . However, by decreasing evels n PKR interference occurred by reduction significant in 5-FU induced apoptosis in the two cell lines ( Figure 3C). These results suggest the importance of PKR activation in the induction of apoptosis by 5-FU, even in the absence of p53. PKR is involved in the cytotoxic effect of 5-FU as well as in the efficacy of the combined 5-FU / IFNa therapy.
  • PKR is a 5- FU molecular target, which plays an important role in its cytotoxic effect at least, in part, at through the inhibition of translation and through the induction of death by apoptosis of tumor cells in an independent form of p53.
  • Mouse embryonic fibroblasts PKR + / + and PKR - / -, and human colon cells HCT1 16 p53 + / + and p53 - / - were supplied by M Esteban (National Center for Biotechnology, Spain) and B. Vogelstein (Johns Hopkins Oncology Center , USA), respectively.
  • Human cell lines from colon cancer SW-480, T84, RKO, and human cell lines from breast cancer MCF-7, MDA-MB-231, T47D were obtained from the American Type Culture Collection (ATCC) .
  • Interferred cells for PKR (shRNA-PKR) were prepared as indicated below.
  • the MDA-MB-231 cell line was cultured in DMEM / F1 2- medium. All cells were used in exponentially growing experiments. 5-Fluorouracil (Sigma-Aldrich, F6627-1 G) was dissolved in DMSO and stored at -20 ° C. IFNalpha 2b (Intron A) was supplied by Schering-Plow.
  • Apoptosis analysis Cells were incubated for 48 hours with 5-FU alone or in combination with IFN (500IU / ml). IFIa was added 16 hours before 5-FU. After 72 hours, the cells were analyzed using the TACSTM Annexin V-FITC kits (R&D System, TA4638), and Annexin V-APC (ebioscence, 88-8007-74) for the interfered cells. Samples were processed using the FACScan cytometer (Becton Dickson, San Jose, CA, USA) of the instrumentation service of the University of Granada.
  • the cells treated during the established times are lysed in Laemmli buffer and after having undergone SDS-PGE electrophoresis, they are transferred to nitrocellulose membranes (Biorad, 162-01 15), blocked with PBS -5% skimmed milk for 1 hour at room temperature .
  • the primary antibodies used are: total anti-PKR supplied by M Esteban (National Biotechnology Center, Spain), anti-phospho-PKR (Thr 451) (Calbiochem, 527460), anti-phosphoelF2D (Ser 51) (Invitrogen, 44- 728, G), anti-phospho-p53 (Ser15) (Cell signaling, 92845), anti-actin (Sigma, A2228).
  • shRNAmir pGIPZ-lentiviral vectors containing non-silencing sequences (NS) or three specific "hairpin" sequences against PKR (Open Biosystems, RHS4430) were used.
  • the pGIPZ vectors express GFP that allows to identify the transfected cells and puromycin resistance gene that allows to create stable lines.
  • the MCF-7, and HCT1 16 p53 + / + and p53 - / - cell lines were transfected with said vectors using the Amaxa Cell line nucleofector kit V (Lonza Cologne AG, VCA-1003) following the commercial house protocol.
  • the cells were selected with puromycin (Invivogen, 58-58-2) at two days post-transfection at a concentration of i Dg / ml for HCT1 16 and 3 ⁇ / ⁇ for MCF- cells 7. RNA extraction and real-time qRT-PCR analysis.
  • the quantitative PCR reaction was carried out in 96-well plates using TaqMan Gene Expression Assays (Applied Biosystems) and Applied Biosystems 7500 Real Time PCR System that quantifies the amplicons.
  • the taqman probes used were: PKR (Hs00169345_ml) and GAPDH (Hs 99999905_ml) and the reaction was carried out according to the specific Applied Biosystems protocol.

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Abstract

L'invention concerne un procédé in vitro d'obtention de données utiles pour évaluer, prédire et/ou pronostiquer la réponse à un traitement avec des analogues de pyrimidine, et de préférence avec du 5-fluorouracyle (5-FU), ainsi qu'un kit et des utilisations associées.
PCT/ES2012/070115 2011-02-24 2012-02-24 Procédé d'obtention de données utiles pour évaluer, prédire et/ou pronostiquer la réponse à un traitement avec des analogues de pyrimidine Ceased WO2012113965A1 (fr)

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ES201130247A ES2393984B1 (es) 2011-02-24 2011-02-24 Método de obtención de datos útiles para evaluar la respuesta al tratamiento con 5-fluorouracilo (5-FU)
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ES2279266T3 (es) * 2003-08-28 2007-08-16 F. Hoffmann-La Roche Ag Correlacion del cociente de los niveles de expresion del mrna de la timidina fosforilasa y la dihidropirimidina dehidrogenasa en el cancer colorrectal con supervivencia libre de enfermedad en pacientes tratados con 5-fu.
WO2010060999A1 (fr) * 2008-11-27 2010-06-03 Vereniging Vu-Windesheim (Short Name) Prédiction de la réponse clinique à un traitement par un antagoniste du tnf soluble, ou le tnf, ou un agoniste de récepteur du tnf

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ES2279266T3 (es) * 2003-08-28 2007-08-16 F. Hoffmann-La Roche Ag Correlacion del cociente de los niveles de expresion del mrna de la timidina fosforilasa y la dihidropirimidina dehidrogenasa en el cancer colorrectal con supervivencia libre de enfermedad en pacientes tratados con 5-fu.
WO2010060999A1 (fr) * 2008-11-27 2010-06-03 Vereniging Vu-Windesheim (Short Name) Prédiction de la réponse clinique à un traitement par un antagoniste du tnf soluble, ou le tnf, ou un agoniste de récepteur du tnf

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ZHOU Y. ET AL.: "Expression of p68 protein kinase and its prognostic significance during IFN-a therapy in patients with carcinoid tumours.", EUROPEAN JOURNAL OF CANCER., vol. 34, no. 13, 1998, pages 2046 - 2052 *

Cited By (1)

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Publication number Priority date Publication date Assignee Title
US9984982B2 (en) 2013-09-06 2018-05-29 Ictk Co., Ltd. Device and method for generating identification key

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