WO2008119858A1 - Non-invasive in vitro method for detecting transitional carcinoma of the bladder - Google Patents

Non-invasive in vitro method for detecting transitional carcinoma of the bladder Download PDF

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Publication number
WO2008119858A1
WO2008119858A1 PCT/ES2008/000196 ES2008000196W WO2008119858A1 WO 2008119858 A1 WO2008119858 A1 WO 2008119858A1 ES 2008000196 W ES2008000196 W ES 2008000196W WO 2008119858 A1 WO2008119858 A1 WO 2008119858A1
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biomarkers
urine
cancer
combination
protein
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PCT/ES2008/000196
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Spanish (es)
French (fr)
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WO2008119858A8 (en
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Ana Soloaga Villoch
Antonio MARTÍNEZ MARTÍNEZ
Inma RAMOS RODRÍGUEZ
Laureano SIMÓN BUELA
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Laboratorios Salvat, S.A.
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Publication of WO2008119858A1 publication Critical patent/WO2008119858A1/en
Publication of WO2008119858A8 publication Critical patent/WO2008119858A8/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
    • G01N33/57407—Specifically defined cancers
    • 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
    • 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
    • G01N33/57484—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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N2800/34—Genitourinary disorders
    • 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 a non-invasive in vitro method for detecting the presence of transitional bladder carcinoma in an individual by means of urine analysis, as well as to the use of peptide sequences derived from selected proteins and to a kit for carrying out said method.
  • Bladder cancer is the most common cancer of the urinary tract; It is also the fourth most common cancer in men and the eighth most common in women. It includes a broad spectrum of tumors of various histological types such as transitional bladder carcinoma (BTCC, 90%), squamous cell carcinoma (7%), adenocarcinoma (2%) and undifferentiated carcinoma (1%).
  • BTCC transitional bladder carcinoma
  • squamous cell carcinoma 7
  • adenocarcinoma adenocarcinoma
  • undifferentiated carcinoma 1%
  • BTCC prognosis The best indicators of BTCC prognosis are the grade and stage of the tumor. Bladder tumors are cytomorphologically classified from G1 (low grade) to G3 (high grade) in a state of decreasing differentiation and increased aggressiveness of the disease according to the World Health Organization (WHO). With respect to the stage or degree of invasiveness, BTCCs are classified as superficial papillary (Ta and T1), invasive muscle (T2 to T4), and uncommon carcinoma in situ or tumor in situ (TIS).
  • cystoscopy and transurethral biopsy or by resection, all of which are invasive methods.
  • transurethral biopsy all of which are invasive methods.
  • the use of flexible cystoscopes makes the technique less aggressive, although it remains invasive and very uncomfortable and requires some form of anesthesia.
  • cytologies are currently used to control diagnosed and treated bladder cancer patients.
  • the manual cytology of the urine can detect tumors in situ that are not detectable by cystoscopy, as well as tumors located in the upper part of the bladder or in the upper part of the urinary tract, for example ureter, pelvis and kidney;
  • cytology has very low sensitivity for the diagnosis of bladder cancer, not detecting up to 50% of tumors.
  • the results of the cytologies are subtle and can be confused with reactive or degenerative processes.
  • Rasmussen H. H. et al. J Urol 1996, 155: 2113-2119
  • Celis J. E. et al Electrophoretic et al
  • Biopsies from patients with bladder cancer.
  • no markers are identified in any of these references, since the authors do not show a quantification of the differential protein profile in healthy samples versus tumor samples.
  • the present invention provides a non-invasive, highly sensitive, efficient and rapid in vitro method, related to proteins associated with BTCC. It also provides a method for the diagnosis, prognosis and monitoring of BTCC through urine sample analysis.
  • the present invention surprisingly provides a combination of bladder cancer biomarkers for the detection of BTCC by urine analysis, with significant value for the diagnosis, prognosis and / or monitoring of BTCC.
  • Apolipoprotein AI belongs to the family of apolipoproteins A1 / A4 / E and participates in the inverse transport of cholesterol from the tissues to the liver for excretion by promoting cholesterol reflux from the tissues and acting as a cofactor of the lecithin cholesterol acyltransferase (LCAT).
  • Cathepsin D is an acid protease that belongs to the family of peptidases A1 and that activates intracellular protein degradation. It is involved in the pathogenesis of various diseases such as bladder cancer (Ozer E., et al., Urology 1999, 54: 50-5 and loachim E., et al., Anticancer Res 2002, 22: 3383-8), breast cancer and possibly Alzheimer's disease.
  • Cathepsin D is synthesized as an inactive 52 kDa precursor, consisting of two 14 kDa (CATD H) and 34 kDa (CATD K) polypeptides. The inactive peptide is activated by proteolysis of the N-terminal end resulting in the enzymatically active protein of 48 kDa.
  • Peroxiredoxin 2 belongs to the family of ahpc / tsa and is involved in the redox regulation of the cell. Reduces peroxides with reducing equivalents obtained through the thioredoxin system. They have no capacity to receive electrons from glutaredoxin. It can play an important role in the elimination of peroxides generated during metabolism. It could participate in the cascade of signals of growth factors and tumor necrosis factor-alpha by regulating intracellular concentrations of HbO 2 . Increases the activity of natural killer cells (NK).
  • NK natural killer cells
  • Plasma retinol-bound protein belongs to the lipocalin family and is the specific transporter of retinol (alcohol of vitamin A) in blood. It releases retinol from the liver stores to peripheral tissues. In plasma, the RBP-retinol complex interacts with transthyretin, which prevents its loss by filtration through the renal glomeruli. A deficiency of vitamin A induces a post-translational modification in the transporter protein, which blocks its secretion and results in a defective release and supply to the epidermal cells.
  • RETBP Plasma retinol-bound protein
  • Stress induced phosphoprotein 1 is an adapter protein that mediates the association of the molecular chaperones HSC70 and HSP90 (HSPCA and HSPCB).
  • Transthyretin (TTHY, previously called prealbumin) is one of the three proteins that bind to thyroid hormones found in the blood of vertebrates. It is produced in the liver and circulates in the bloodstream, where it binds to retinol and thyroxine. Cancer can be detected by analyzing the expression pattern of at least 4 biomarkers of bladder cancer in a urine sample. Thus, the detection of at least 4 differentially expressed proteins in a urine test sample compared to normal urine is indicative of BTCC.
  • Urine samples are very diverse in terms of their composition, so it is essential to normalize to take into account the differences in total protein concentration and to eliminate trends between samples.
  • the expression levels of a control protein whose urine content is always constant, can be used.
  • transferrin for example, proved to be a protein of constant concentration.
  • biomarkers or bladder cancer proteins identified in the present invention include, but are not limited to CATD, GSTP1, RETBP, STIP1, AMYS, APOA1, PRDX2 and TTHY.
  • One aspect of the present invention relates to a combination of biomarkers for the detection of BTCC comprising the biomarkers CATD 1 GSTP1, RETBP and STIP1 or their transcriptional or post-translational variants.
  • Another aspect of the invention relates to a non-invasive in vitro method comprising: a) detecting and quantifying the combination of CATD, GSTP1, RETBP and STIP1 biomarkers or their transcriptional or post-translational variants, in a urine test sample of a individual; and b) compare the expression value obtained in a) in the urine test sample with the corresponding standard value in normal urine, where variations in the value obtained in a) with respect to the standard value in normal urine are indicative of BTCC. Expression is determined by spectrometry or immunoassays.
  • This method can be adapted to population screening to detect the presence of BTCC and determine the stage or severity of this cancer. In addition, it can be used to evaluate the absence of disease after surgical resection, establish a diagnosis and / or prognosis of this cancer and / or monitor the effect of the treatment administered to an individual suffering from said cancer.
  • Another aspect of the invention relates to the joint use of the peptide sequences derived from the CATD, GSTP1, RETBP and STIP1 biomarkers or their transcriptional or post-translational variants, to detect the presence of BTCC by urine analysis, determine the stage or severity of this cancer, evaluate the absence of disease after surgical resection, establish a diagnosis and / or prognosis of this cancer and / or monitor the effect of the treatment administered to an individual suffering from said cancer, where biomarkers are present in urine.
  • Another aspect of the invention relates to the joint use of nucleotides or peptide sequences derived from the CATD, GSTP1, RETBP and STIP1 biomarkers or their transcriptional or post-translational variants, in screening methods to identify, develop and evaluate the efficacy of agents Therapeutics to treat BTCC.
  • the invention further relates to antibodies against bladder cancer biomarkers mentioned.
  • These antibodies can be presented in a variety of forms appropriate for each use, including, for example, soluble form, immobilized on a substrate, or in combination with a pharmaceutically acceptable carrier.
  • Another aspect of the invention relates to a kit for carrying out the previously described method comprising 1) antibodies that specifically recognize the biomarkers CATD, GSTP1, RETBP and STIP1 in urine and 2) a support in a suitable packaging.
  • a kit for carrying out the previously described method comprising 1) antibodies that specifically recognize the biomarkers CATD, GSTP1, RETBP and STIP1 in urine and 2) a support in a suitable packaging.
  • Cancer refers to the disease that is typically characterized by abnormal or unregulated cell growth, capable of invading adjacent tissues and spreading to other organs.
  • Carcinoma refers to tissue resulting from abnormal or unregulated cell growth.
  • Transportal bladder carcinoma or its abbreviation “BTCC” refers to any proliferative malignant disorder in epithelial cells of the urinary bladder.
  • Tumor refers to any mass of abnormal tissue generated by a neoplastic process, either benign (non-carcinogenic) or malignant (carcinogenic).
  • Bindder cancer proteins or biomarkers are proteins differentially expressed in BTCC, that is, proteins that are expressed differently in the urine of a healthy individual with respect to the urine of a BTCC patient.
  • the group of differentially expressed proteins includes the biomarkers CATD 1 GSTP1, RETBP, STIP1, AMYS, APOA1, PRDX2 and TTHY, as well as any protein in a urine sample, the proportion of which varies at least 2 times when compared 2 different urine samples: one from a healthy individual and one from a BTCC 1 patient where this quantification is carried out using an image analyzer, for example, Progenesis PG220 software.
  • Bladder cancer biomarkers as described may have any length and may comprise additional sequences derived from the native protein and / or heterologous sequences; they include any transcriptional or post-translational variant, as well as any sequence with at least 95% identity with the described sequences, where identity is defined as the percentage of identical residues between two sequences. Those skilled in the art will appreciate that fragments or variants of the sequences can be equally useful in the treatment and detection of cancer.
  • detect biomarkers means determining the existence of biomarkers, while the phrase “quantify biomarkers” means expressing the presence of said biomarkers with a value (for example, an intensity value).
  • a “differentially expressed protein” refers to a protein whose expression pattern varies (increasing or decreasing) in the urine of a BTCC patient compared to the urine of a healthy individual.
  • the “protein extract” corresponds to the supernatant obtained after centrifugation of the urine sample.
  • the term "individual” refers to all species of animals classified as mammals and includes, but is not limited to, domestic and farm animals, primates and humans, and preferably refers to a human, male or female of any age or race.
  • a "healthy individual” is an individual who does not suffer from transitional bladder carcinoma and may include patients with other urological diseases.
  • the term "previously diagnosed” refers to an individual who has received a first positive diagnosis of BTCC.
  • the term “not previously diagnosed” refers to an individual who has never received a positive diagnosis of BTCC (novó diagnosis).
  • the “standard value in normal urine” refers to the quantification of the mean level of expression of the biomarkers detected in individual urine samples of individuals not suffering from BTCC.
  • “Diagnosis” of BTCC refers to the process of identification or determination of the nature and cause of BTCC through the evaluation of one or more biomarkers.
  • the term “prognosis” refers to the probable evolution or course of the disease, that is, the probability of recovery or recurrence.
  • Monitoring means evaluating the presence or absence of BTCC in an individual at different times.
  • Treatment refers to any process, action, application or similar, where an individual submits to medical assistance in order to improve his condition, directly or indirectly.
  • the term "specificity” refers to the ability of a test to exclude the presence of a disease when it is not really present. Specificity is expressed as the number of healthy individuals for whom there is a correct negative test (called true negatives) divided by the sum of the true negatives and the number of healthy individuals for whom there is an incorrect positive test (called false positives) .
  • the term "sensitivity” refers to the ability of a test to detect the presence of a disease when it is truly present. Sensitivity is expressed as the number of sick patients for whom there is a positive test (called true positives), divided by the sum of the true positives and the number of patients for whom there is an incorrect negative test (called false negatives). "Robustness” defines the ability of a numerical method to give the same result despite the variability of the initial samples.
  • the term “gene” refers to a region of double-stranded deoxyribonucleotides that encodes a protein. It can represent a part of a coding sequence or a complete coding sequence.
  • protein indicates at least one molecular chain of amino acids bound intra-molecularly through covalent or non-covalent bonds. The term includes all forms of post-translational modifications, for example glycosylation, phosphorylation or acetylation.
  • the terms “peptide” and “polypeptide” refer to molecular chains of amino acids that They represent a protein fragment.
  • protein and “peptide” are used interchangeably.
  • antibody refers to a Y-shaped protein (known as immunoglobulin) on the surface of B cells that is secreted to the blood or lymph in response to an antigenic stimulus, such as an exogenous protein, bacteria, viruses, parasite or transplanted organ, which has a specific binding for a target molecule called "antigen.”
  • an antigenic stimulus such as an exogenous protein, bacteria, viruses, parasite or transplanted organ.
  • the region of the immunoglobulins that binds to the antigen can be divided into both F (ab ') 2 and Fab fragments.
  • antibody includes monoclonal or polyclonal antibodies, either intact or fragments derived therefrom; and includes human antibodies, humanized antibodies and antibodies of non-human origin.
  • a "non-human antibody” is an antibody generated by an animal species other than Homo sapiens.
  • a “humanized antibody” is a genetically designed antibody in which the minimum part of a mouse antibody is transplanted into a human antibody. Generally, humanized antibodies have 5-10% mouse and 90-95% human.
  • a “human antibody” is an antibody derived from transgenic mice that have human antibody or human cell genes.
  • the “monoclonal antibodies” are homogeneous populations of highly specific antibodies directed to a single antigenic site or “determinant” of the target molecule.
  • Polyclonal antibodies include heterogeneous populations of antibodies that target different antigenic determinants of the target molecule.
  • the term “specific antibody” refers to an antibody specifically generated against a protein (in this case, against a particular marker of bladder cancer).
  • antibody-protein complex refers to a complex formed by an antigen and its specific antibody.
  • combibody combininatorial antibody
  • recombinant Fab antibody refers to a recombinant antibody that only contains the Fab fragment that is univalent and useful when the antibody has a high affinity for its antigen. They can be obtained recombinantly if the protein sequence is known.
  • ScFv antibody fragment refers to a single variable chain fragment (scFv) that can be expressed in bacterial cultures.
  • epitope is an antigenic determinant of a protein; is the amino acid sequence of the protein that recognizes a specific antibody. Said epitopes can comprise a contiguous extension of amino acids (linear epitope) or of non-contiguous amino acids that are close in space thanks to the three-dimensional folding of the polypeptide chain (discontinuous epitopes).
  • solid phase refers to a non-aqueous matrix to which the antibody can bind. Examples of materials for the solid phase include without limitation glass, polysaccharides (for example agarose), polyacrylamide, polystyrene, polyvinyl alcohol and silicones. Examples of solid phase forms are a well or a purification column.
  • dipstick refers to a device that partially submerges (usually at one end) into a liquid to carry out a test that can determine and / or quantify some property of the liquid (chemical, physical, etc.). This type of dipstick is usually made of paper or cardboard and is impregnated with reagents, whose color changes indicate some characteristic of the liquid.
  • support refers to the mechanism or device by which something is driven or transported.
  • packing refers to the container and container prior to sale with the primary objective of facilitating the purchase and use of the product.
  • biochip refers to a multitude of miniaturized devices used to perform biological tests on a solid or fluid support with a high processing capacity.
  • a particular embodiment of the invention relates to a combination of biomarkers for the detection of BTCC comprising the biomarkers CATD 1 GSTP1, RETBP, STIP1, AMYS and APOA1 or their transcriptional or post-translational variants.
  • Another particular embodiment refers to a combination of biomarkers for the detection of BTCC comprising the biomarkers CATD, GSTP1, RETBP, STIP1, AMYS, APOA1 and PRDX2 or their transcriptional or post-translational variants.
  • Another particular embodiment refers to a combination of biomarkers for the detection of BTCC comprising the biomarkers CATD, GSTP1, RETBP, STIP1, AMYS, APOA1 and TTHY or their transcriptional or post-translational variants.
  • Another particular embodiment refers to a combination of biomarkers for the detection of BTCC comprising the biomarkers CATD, GSTP1, RETBP, STIP1, AMYS, APOA1, PRDX2 and TTHY or their transcriptional or post-translational variants.
  • the first stage of the method for evaluating bladder cancer comprises detecting and quantifying the combination of CATD, GSTP1, RETBP 1 STIP1, AMYS and APOA1 biomarkers or their transcriptional or post-translational variants.
  • the first stage of the method for evaluating bladder cancer comprises detecting and quantifying the combination of CATD 1 GSTP1, RETBP, STIP1, AMYS, APOA1 and PRDX2 biomarkers or their transcriptional or post-translational variants.
  • the first stage of the method for evaluating bladder cancer comprises detecting and quantifying the combination of biomarkers CATD, GSTP1, RETBP 1 STIP1, AMYS 1 APOA1 and TTHY or their transcriptional or post-translational variants.
  • the first stage of the method for evaluating bladder cancer comprises detecting and quantifying the combination of CATD 1 GSTP1, RETBP 1 STIP1, AMYS 1 APOA1, PRDX2 and TTHY biomarkers or their transcriptional or post-translational variants.
  • the method allows to determine the progression of the disease when the same protein or proteins is compared with different samples obtained from the same patient at different times during the evolution of BTCC.
  • the combination of biomarkers can be used to monitor the efficacy of the pharmacological or surgical treatment.
  • the sample to be analyzed is obtained from an individual who has not previously been diagnosed with BTCC. In another particular embodiment, the sample to be analyzed is obtained from an individual who has been previously diagnosed with BTCC. In another particular embodiment, the sample to be analyzed is obtained from an individual who is currently receiving treatment against BTCC. In another particular embodiment, the method comprises obtaining the protein extract of the sample.
  • the method of detecting cancer comprises contacting a urine sample with a molecule that specifically binds to a biomarker of bladder cancer and quantifying said binding.
  • the detection and quantification of proteins comprises a first stage, in which the protein extract of Ia Sample is contacted with a composition of antibodies specific for one or more epitopes of the protein or proteins, and a second stage, in which the complexes formed by antibodies and proteins are quantified.
  • the specific antibodies used for the detection of proteins are of human origin, humanized or of non-human origin and selected from monoclonal or polyclonal antibodies, fragments of intact or recombinant antibodies, combibodies and Fab or scFv antibody fragments.
  • Another embodiment of the invention refers to the joint use of the peptide sequences derived from the CATD, GSTP1, RETBP, STIP1, AMYS and APOA1 biomarkers or their transcriptional or post-translational variants, where the biomarkers are present in urine.
  • Another embodiment of the invention relates to the joint use of the peptide sequences derived from the CATD, GSTP1, RETBP, STIP1, AMYS, APOA1 and PRDX2 biomarkers or their transcriptional or post-translational variants, where the biomarkers are present in urine.
  • Another embodiment of the invention relates to the joint use of the peptide sequences derived from the CATD, GSTP1, RETBP, STIP1, AMYS, APOA1 and TTHY biomarkers or their transcriptional or post-translational variants, where the biomarkers are present in urine.
  • Another embodiment of the invention relates to the joint use of the peptide sequences derived from the CATD, GSTP1, RETBP, STIP1, AMYS, APOA1, PRDX2 and TTHY biomarkers or their transcriptional or post-translational variants, where the biomarkers are present in urine.
  • at least 4 biomarkers present in urine or their transcriptional or post-translational variants are used together.
  • at least 6 biomarkers present in urine or their transcriptional or post-translational variants are used together.
  • at least 7 biomarkers present in urine or their transcriptional or post-translational variants are used together.
  • at least 8 biomarkers present in urine or their transcriptional or post-translational variants are used together.
  • kits for carrying out the previously described method said kit being used to detect the presence of BTCC, determine the stage or severity of this cancer, evaluate the lack of disease after surgical resection, establish a diagnosis and / or prognosis of this cancer and / or monitor the effect of the treatment administered to an individual suffering from said cancer.
  • the kit can comprise a container where the agents that bind to the biomarkers present in urine are located and instructions for its use to determine the BTCC stage.
  • the kit may comprise a compartmentalized support for locating one or more containers, for example vials, tubes or the like.
  • a container may contain a probe that is or may be marked for later detection.
  • the probe may be an antibody or polynucleotide specific for a bladder cancer protein or a bladder cancer gene, respectively.
  • the kit may comprise a mass spectrometry (MS) probe.
  • MS mass spectrometry
  • the kit may also include containers containing nucleotide (s) for amplification or silencing of a target nucleic acid sequence, and / or a container containing reporter media, such as a biotin binding protein, such as avidin or streptavidin, attached to a detectable label, for example, an enzymatic, fluorescent or radioisotopic label.
  • the kit can include the complete amino acid sequence of bladder cancer biomarkers or part of it, or a nucleic acid molecule that encodes such an amino acid sequence.
  • the kit of the invention typically comprises the container described above and one or more containers comprising desirable materials from a commercial and user point of view, including buffers, diluents, filters, needles, syringes and instructions for use. In addition, it may contain a label on the container to indicate that the composition is used for a therapeutic or non-therapeutic application.
  • Another embodiment of the invention relates to a kit for carrying out the previously described method comprising a biochip.
  • kits comprising a biochip, wherein the biochip comprises antibodies for the detection of biomarkers or their transcriptional or post-translational variants.
  • the method of the invention comprises monitoring the stage of bladder carcinoma by quantifying soluble proteins differentially expressed in a urine sample through specific antibodies.
  • any means to identify and quantify these proteins is contemplated.
  • the detection and quantification comprises spectrometry or immunoassay.
  • the spectrometry is generally desorption / ionization mass spectrometry by surface laser (SELDI) or matrix assisted laser desorption / ionization mass spectrometry (MALDI).
  • Immunoassays use unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies).
  • These techniques include western blot, ELISA (enzyme-linked immunoabsorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical or immunohistochemical techniques, techniques based on use of biochips or protein microarrays that include specific antibodies, tests based on the precipitation of colloidal gold in formats such as dipsticks; or chromatographic affinity techniques, ligand binding assays and lectin binding assays.
  • Preferred embodiments of this aspect of the invention are protein microarrays and double ELISA sandwich antibody (DAS-ELISA).
  • Proteins can be quantified with antibodies such as, for example: monoclonal antibodies, polyclonal antibodies, either intact or recombinant fragments thereof, combibodies and Fab or scFv antibody fragments, specific for proteins.
  • These antibodies can be of human origin, humanized or of animal origin.
  • they can be labeled or unlabeled and can be used in a wide variety of assays.
  • Marker molecules that can be used to label antibodies include radionuclides, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes and derivatives. The higher the specificity of the antibody binding, the lower the concentration required for the antigen to be detected.
  • a monoclonal or polyclonal antibody, a fragment thereof, or a combination thereof is anchored to the surface of a solid phase support;
  • the sample to be analyzed is contacted and incubated for a specific time under appropriate conditions for the formation of antigen-antibody complexes.
  • an indicator reagent which consists of a monoclonal or polyclonal antibody, a fragment thereof, or a combination thereof, is linked to a signal generating molecule with the antigen complexes.
  • -antibody under appropriate conditions of time and temperature.
  • the presence of a protein selected from the proteins of the invention is detected in the sample to be analyzed and, if present, the generated signal is quantified and measured. To avoid signal variation Due to the differences in the total protein concentration between samples, all measurements are normalized.
  • the method includes the processing of the samples, the use of 2D electrophoresis to separate proteins from the sample, the selection of differentially expressed proteins by image and statistical analysis of different samples and the use of one or more differentially expressed proteins to generate specific antibodies. to be used as markers of bladder cancer.
  • Comparative proteomic analysis was carried out between samples obtained from healthy individuals (controls) and patients diagnosed with BTCC (Ta, T1-low grade, T1-high grade and T2) in an attempt to identify differentially expressed proteins in the various stages of the cancer and during its progression. Proteins whose differential expression changed more than twice reproducibly were chosen. These were identified by fingerprint of the peptide mass using mass spectrometry and database search.
  • Urine samples (150 in total) were obtained from healthy individuals and patients with transitional bladder carcinoma from the Urology units of hospitals belonging to the Spanish Public Health Network. These samples are classified as follows: a) No Carcinoma (63 samples) including:
  • BTCC patients patients diagnosed with the disease at different stages of development including:
  • the BTCC sample group was accompanied by a biopsy that is the key to its classification in the stages of development of bladder cancer.
  • Urine samples were frozen at -8O 0 C and transferred to the laboratory on dry ice without breaking the cold chain. Samples were kept at -8O 0 C until processed. The samples were very heterogeneous, from yellow urine with little color to red urine with lumps of blood, transparent or containing suspended tissues. The total volume was also very variable, from 5 to 100 mL.
  • the protein concentration of the samples ranged from 20 ⁇ g / mL to 2 mg / mL (the average value being 150 ⁇ g / mL).
  • the urine samples were thawed on ice and centrifuged at 2000xg for 5 minutes at 4 0 C. The supernatant was used to determine the protein concentration.
  • the volume necessary to precipitate 100 ⁇ g of protein was calculated, taking into account that the yield of the precipitation with trichloroacetic acid (TCA) was 75%.
  • TCA trichloroacetic acid
  • the rest of the urine sample was frozen again at -8O 0 C and stored for subsequent 2D electrophoresis (if necessary). After mixing the TCA and the urine for 1 hour on ice, it was centrifuged at 16000xg for 20 minutes at 4 0 C to obtain the pellet of the precipitated proteins This pellet was washed with acetone stored at -2O 0 C, and dried by evaporation of the solvent.
  • the first dimension was IEF (isoelectric focusing) where the proteins were separated according to their charge (pl);
  • the second dimension consisted of SDS-PAGE, where the proteins were separated according to their molecular weight.
  • the dried protein pellets were resuspended in 450 ⁇ l of rehydration buffer (Urea 7M, Tiourea 2M, CHAPS 2%, IPG buffer 2%, bromophenol blue 0.002%) for 1 hour at room temperature .
  • the IPG buffer (Amersham, ref # 17-600-88) was used so that the IEF was carried out in a range of 3-10.
  • IEF For the IEF, Amersham's Ettan TM IPGfor TM Isoelectric Focusing System followed all the manufacturer's recommendations. The IEF was carried out on immobilized pH gradient gels, known as IPG strips, marketed by Amersham (ref # 17-6002-45). The solubilized proteins focused on the gel of the first dimension after a period of 16 hours of active rehydration of the gel at 30 volts. Then the voltage began to rise to 8000 volts, never exceeding a current intensity of 50 ⁇ A for each gel. The IEF stopped at approximately 90000 volt-hours. For the second dimension, 12.5% acrylamide gels of 26 x 20 cm were polymerized in the laboratory using Ettan DALT twelve GeI Caster from Amersham.
  • the gels were run by Large Format Vertical System following all the manufacturer's protocols until the electrophoresis front left the gel.
  • the gels were stained with silver nitrate, using the Amersham staining kit (17-1150-01), following the manufacturer's protocol.
  • the gels were dried and stored for subsequent image analysis of protein spots (FIGURE 1A). For some urine samples, more than one 2D gel was run.
  • the Nonlinear Dynamics Progenesis PG220 software (UK) was used to analyze image files in 300 dpi format (dots per inch) and 8 bits / channel. To increase the resolution the analysis was carried out in discrete areas of the gels. From each of the 4 areas, called A (FIGURE 1 B), K (FIGURE 1C), R (FIGURE 1 D) and S (FIGURE 1E), the best gels were selected and scanned for image analysis.
  • Progenesis PG220 software transforms the information of the flat image into a three-dimensional image, where the intensity of each spot correlates with its volume and with the relative amount of corresponding protein in the urine of the individual. Through this software we obtained some intensity tables of each spot in each gel. These raw data were the basis of subsequent statistical analyzes.
  • FIGURE 2 shows the spot R211 in the area R of a two-dimensional electrophoresis gel (2D) obtained from a urine sample of a healthy individual (FIGURE 2A), of a cancer patient in stage Ta (FIGURE 2B ), of a cancer patient in stage T1 - low grade (FIGURE 2C), of a cancer patient in stage T1 - high degree (FIGURE 2D) and of a cancer patient in stage T2 (FIGURE 2E).
  • 2D two-dimensional electrophoresis gel
  • FIGURE 3 shows the intensity of the R211 spot in CV, Ta, T1-low grade, T1-high grade and T2 samples.
  • the number of samples for each group is indicated in parentheses.
  • FIGURE 4 shows the robustness of the measurement of the intensity for spot R211 in different urine samples. This is expressed as the percentage of gels that maintain the presence or absence of the R211 spot in each gel analyzed. The number of samples for each group is indicated in brackets.
  • Table 2 shows the value of p and the fold change of the statistical analyzes for the R211 spot in samples from different stages of cancer compared to a urine from an individual without cancer (CV).
  • the fold change of the R211 spot was normalized by the total volume of spots (TSV) for each individual gel.
  • the reactivity and sensitivity of polyclonal and monoclonal antibodies were tested (either commercially obtained or generated by immunization protocols).
  • the method generally comprised the preparation of a standard curve ("dose response") for the protein to be monitored.
  • dose response a standard curve for the protein to be monitored.
  • the development of the cancer stage can also be evaluated by monitoring the concentrations of different stage characteristic proteins in the sample.
  • Polyclonal antiserum can be obtained against a given protein using standard methodology, such as that described in numerous available texts and known to those skilled in the art.
  • male New Zealand White rabbits are immunized with protein preparations first in the presence of Freud's complete adjuvant (Gibco, Grand Island, NY) and then every month with the protein in the presence of Incomplete adjuvant for three months.
  • Freud's complete adjuvant Gibco, Grand Island, NY
  • rabbit and mouse sera are used prior to fusion and they are shown to be reactive with protein preparations by means of the standard western blot technique.
  • the proteins found in the gels are cloned and expressed in amounts too small for immunization in E.coli using the expression vector pET28b (+).
  • the crude extracts are obtained under conditions already described (Boronat A., et al., J Bacteriol 1981, 147: 181-85) and run on an SDS-PAGE gel. Recombinant proteins are trimmed from the gel and released from acrylamide. For the generation of antibodies to recombinant proteins, the released proteins are used directly to immunize rabbits as described above.
  • Protein samples (20 ug total protein) were mixed with a loading buffer SDS-PAGE supplemented with 5% ⁇ -mercaptoethanol and incubated at 100 0 C for 5 min, before being loaded on a 6% gel polyacrylamide . By electrophoresis, the proteins were transferred to nitrocellulose membranes. Gels were run and transferred in duplicate. A membrane was incubated with antibodies against the proteins of the invention (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA.) While the second membrane was incubated with an antibody against actin (Amersham, Little Chalfont, UK) as protein load control.
  • the ROC Receiveiver Operating Characteristic
  • FIGURE 1 Two-dimensional (2D) electrophoresis GeI obtained from a urine sample and showing the 4 studied areas A, K, R and S (FIGURE 1A), area A (FIGURE 1 B), area K (FIGURE 1C) , area R (FIGURE 1D) and area S (FIGURE 1 E).
  • FIGURE 2 Spot R211 in the area R of a two-dimensional (2D) electrophoresis gel obtained from urine samples from a healthy individual (FIGURE 2A), from a cancer patient in stage Ta (FIGURE 2B), from a patient of T1-low grade cancer (T1-LG, FIGURE 2C), of a T1-high grade cancer patient (T1-HG, 2D FIGURE) and a T2 stage cancer patient (FIGURE 2E ).
  • 2D two-dimensional
  • FIGURE 3 Intensity of the R211 spot in CV, Ta, T1-low grade (T1-LG), T1-high grade (T1-HG) and T2 samples. The number of samples for each group is indicated in brackets.
  • FIGURE 4 Robustness of the intensity measurement for the R211 spot in different urine samples. This is expressed as the percentage of gels that maintain the presence or absence of the R211 spot in each gel analyzed. The number of samples for each group is indicated in brackets.

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Abstract

The present invention concerns a non-invasive in vitro method for detecting the presence of transitional carcinoma of the bladder in an individual by means of urine analysis, in order to determine the stage or severity of said cancer and monitoring the effect of the treatment administered to an individual suffering from said cancer.

Description

MÉTODO IN VITRO NO INVASIVO PARA DETECTAR CARCINOMA TRANSICIONAL DE VEJIGA IN VITRO NON-INVASIVE METHOD FOR DETECTING TRANSITIONAL CARCINOMA OF BLADDER
SECTOR DE LA TÉCNICA AL CUAL SE REFIERE LA INVENCIÓNSECTOR OF THE TECHNIQUE TO WHICH THE INVENTION IS REFERRED
La presente invención se refiere a un método in vitro no invasivo para detectar Ia presencia de carcinoma transicional de vejiga en un individuo mediante análisis de orina, así como al uso de secuencias peptídicas derivadas de proteínas seleccionadas y a un kit para llevar a cabo dicho método.The present invention relates to a non-invasive in vitro method for detecting the presence of transitional bladder carcinoma in an individual by means of urine analysis, as well as to the use of peptide sequences derived from selected proteins and to a kit for carrying out said method.
ESTADO DE LA TÉCNICA RELATIVO A LA INVENCIÓNSTATE OF THE INVENTION TECHNIQUE
El cáncer de vejiga es el cáncer más común del tracto urinario; es también el cuarto cáncer más común en hombres y el octavo más común en mujeres. Incluye un amplio espectro de tumores de varios tipos histológicos como son carcinoma transicional de vejiga (BTCC, 90%), carcinoma de células escamosas (7%), adenocarcinoma (2%) y carcinoma no diferenciado (1%).Bladder cancer is the most common cancer of the urinary tract; It is also the fourth most common cancer in men and the eighth most common in women. It includes a broad spectrum of tumors of various histological types such as transitional bladder carcinoma (BTCC, 90%), squamous cell carcinoma (7%), adenocarcinoma (2%) and undifferentiated carcinoma (1%).
Los mejores indicadores del pronóstico de BTCC son el grado y estadio del tumor. Los tumores de vejiga se clasifican citomorfológicamente de G1 (grado bajo) a G3 (grado alto) en estado de diferenciación decreciente e incremento de Ia agresividad de Ia enfermedad según Ia Organización Mundial de Ia Salud (OMS). Con respecto al estadio o grado de invasividad, los BTCCs se clasifican como papilar superficial (Ta y T1), invasivo del músculo (T2 a T4), y carcinoma no común in situ o tumor in situ (TIS).The best indicators of BTCC prognosis are the grade and stage of the tumor. Bladder tumors are cytomorphologically classified from G1 (low grade) to G3 (high grade) in a state of decreasing differentiation and increased aggressiveness of the disease according to the World Health Organization (WHO). With respect to the stage or degree of invasiveness, BTCCs are classified as superficial papillary (Ta and T1), invasive muscle (T2 to T4), and uncommon carcinoma in situ or tumor in situ (TIS).
Los tumores de grado bajo normalmente están confinados a Ia mucosa o infiltran las capas superficiales (estadios Ta y T1). La mayoría de tumores de grado alto se detectan como mínimo en el estadio T1 (invadiendo Ia lamina propria). Aproximadamente un 75% de los casos diagnosticados de cáncer de vejiga son superficiales. El 25% restante son invasivos del músculo en el momento del diagnóstico. Los pacientes con BTCC superficial presentan un buen pronóstico aunque el riesgo de recidiva es del 70%. A pesar de este riesgo, los tumores Ta tienden a ser de grado bajo y solamente un 10-15% progresa con invasión del músculo en 2 años. Sin embargo, el porcentaje de cánceres Tϊ que progresa al estadio T2 es más elevado (30-50%).Low-grade tumors are normally confined to the mucosa or infiltrate the superficial layers (stages Ta and T1). The majority of high-grade tumors are detected at least in stage T1 (invading the lamina propria). Approximately 75% of cases diagnosed with bladder cancer are superficial. The remaining 25% are invasive of the muscle at the time of diagnosis. Patients with superficial BTCC have a good prognosis although the risk of recurrence is 70%. In spite of this At risk, Ta tumors tend to be low grade and only 10-15% progress with muscle invasion in 2 years. However, the percentage of Tϊ cancers that progresses to stage T2 is higher (30-50%).
Actualmente, el mejor diagnóstico para el cáncer de vejiga se establece o bien por cistoscopia y biopsia transuretral o por resección, siendo todos ellos métodos invasivos. El uso de cistoscopios flexibles convierte Ia técnica en menos agresiva, aunque sigue siendo invasiva y muy incómoda y requiere alguna forma de anestesia.Currently, the best diagnosis for bladder cancer is established either by cystoscopy and transurethral biopsy or by resection, all of which are invasive methods. The use of flexible cystoscopes makes the technique less aggressive, although it remains invasive and very uncomfortable and requires some form of anesthesia.
La técnica no invasiva predominante para el diagnóstico de BTCC es Ia identificación de células neoplásicas mediante examen morfológico de las células de Ia orina. Así, actualmente se utilizan citologías para controlar pacientes de cáncer de vejiga diagnosticados y tratados. Aunque Ia citología manual de Ia orina puede detectar tumores in situ que no son detectables mediante cistoscopia, así como también tumores localizados en Ia parte superior de Ia vejiga o en Ia parte superior del tracto urinario, por ejemplo uréter, pelvis y riñon; varios estudios han demostrado que Ia citología presenta muy baja sensibilidad para el diagnóstico de cáncer de vejiga, no detectando hasta el 50% de los tumores. Los resultados de las citologías son sutiles y pueden confundirse con procesos reactivos o degenerativos. Además, los estudios citológicos requieren Ia evaluación individual de un experto, Io cual retrasa Ia disponibilidad de los resultados y además introduce subjetividad y variación en los resultados finales. En realidad, no se dispone de ningún método no invasivo altamente sensible y específico para el diagnóstico del cáncer de vejiga (Boman, H., et al., J Urol 2002, 167:80-83).The predominant non-invasive technique for the diagnosis of BTCC is the identification of neoplastic cells by morphological examination of the urine cells. Thus, cytologies are currently used to control diagnosed and treated bladder cancer patients. Although the manual cytology of the urine can detect tumors in situ that are not detectable by cystoscopy, as well as tumors located in the upper part of the bladder or in the upper part of the urinary tract, for example ureter, pelvis and kidney; Several studies have shown that cytology has very low sensitivity for the diagnosis of bladder cancer, not detecting up to 50% of tumors. The results of the cytologies are subtle and can be confused with reactive or degenerative processes. In addition, cytological studies require the individual evaluation of an expert, which delays the availability of the results and also introduces subjectivity and variation in the final results. In reality, there is no highly sensitive and specific non-invasive method for the diagnosis of bladder cancer (Boman, H., et al., J Urol 2002, 167: 80-83).
Se han realizado muchos esfuerzos para mejorar Ia detección no invasiva del cáncer. Avances recientes en el perfil de expresión de células cancerosas mediante técnicas de proteómica, electroforesis bidimensional de alta resolución y espectrometría de masas han hecho posible Ia identificación de proteínas como marcadores de cáncer de vejiga, tales como Ia proteína matricial nuclear NMP22 (Soloway, M.S., et al., J Urol 1996, 156:363-367), el ácido hialurónico y Ia hialuronidasa (Pham HT, et al., Cáncer Res 1997, 57:778-783), los complejos de membrana basal (BTA, Pode, D., et al., J Urol 1999, 161 :443-446), el antígeno carcinoembrionario (CEA, Halim A.B., et al., lnt J Biol Marcadores, 1992; 7:234-239), Ia uroplakina Il (Wu X.R., et al., Cáncer Res 1998; 58:1291-1297), el factor de dispersión/de crecimiento hepatocitario (SF/HGF, Gohji K, et al., J Clin Oncol 2000; 18:2963-2971), las proteínas de Ia familia queratina/citoqueratina tales como citoqueratina 20 (Buchumensky V, et al., J Urol 1998, 160:1971-1974), Ia citoqueratina 18 (Sánchez-Carbayo M, et al., Clin Cáncer Res 2000, 6:3585-3594), Ia proteína del tumor mamario 8-Ka (MAT-8, Morrison BW, et al., J Biol Chem, 1995, 270:2176-2182) y Ia telomerasa (Lee DH, et al., Clinical Cáncer Research, 1998, 4: 535-538).Many efforts have been made to improve non-invasive cancer detection. Recent advances in the profile of cancer cell expression through proteomic techniques, high resolution two-dimensional electrophoresis and mass spectrometry have made it possible to identify proteins as markers of bladder cancer, such as the nuclear matrix protein NMP22 (Soloway, MS, et al., J Urol 1996, 156: 363-367), hyaluronic acid and hyaluronidase (Pham HT, et al., Cancer Res 1997, 57: 778-783), the basement membrane complexes (BTA, Pode, D., et al., J Urol 1999, 161: 443-446), the carcinoembryonic antigen (CEA, Halim AB, et al., Lnt J Biol Markers, 1992; 7: 234-239), Ia uroplakina Il (Wu XR, et al., Cancer Res 1998; 58: 1291-1297), hepatocyte dispersion / growth factor (SF / HGF, Gohji K, et al., J Clin Oncol 2000; 18: 2963-2971), proteins of the keratin / cytokeratin family such as cytokeratin 20 (Buchumensky V, et al., J Urol 1998, 160: 1971-1974), Ia cytokeratin 18 (Sánchez-Carbayo M, et al., Clin Cancer Res 2000, 6: 3585-3594), the 8-Ka breast tumor protein (MAT-8, Morrison BW, et al., J Biol Chem, 1995, 270: 2176-2182) and telomerase (Lee DH, et al., Clinical Cancer Research, 1998, 4: 535-538).
Memon A. A. et al. (Cáncer Detect Prev 2005, 29:249-255) identifican siete proteínas diferencialmente expresadas en líneas celulares y biopsias de cáncer de vejiga. Sin embargo, no utilizan ningún control para comparar.Memon A. A. et al. (Cancer Detect Prev 2005, 29: 249-255) identify seven differentially expressed proteins in cell lines and bladder cancer biopsies. However, they do not use any controls to compare.
Rasmussen H. H. et al. (J Urol 1996, 155 :2113-2119) describen una base de datos de las proteínas más abundantes presentes en Ia orina de pacientes de cáncer de vejiga. Celis J. E. et al (Electrophoresis 1999, 300-309) describen una base de datos de proteínas presentes en biopsias provinientes de pacientes con cáncer de vejiga. Sin embargo, no se identifican marcadores en ninguna de estas referencias, ya que los autores no muestran una cuantificaión del perfil diferencial de proteínas en muestras sanas versus muestras tumorales.Rasmussen H. H. et al. (J Urol 1996, 155: 2113-2119) describe a database of the most abundant proteins present in the urine of bladder cancer patients. Celis J. E. et al (Electrophoresis 1999, 300-309) describe a database of proteins present in biopsies from patients with bladder cancer. However, no markers are identified in any of these references, since the authors do not show a quantification of the differential protein profile in healthy samples versus tumor samples.
Por ahora, no se ha encontrado ningún marcador útil para para predecir Ia prognosis y Ia extensión del cáncer de vejiga en ensayos clínicos (Miyake, H., et al., J Urol 2002; 167:1282-1287).For now, no useful marker has been found to predict the prognosis and extent of bladder cancer in clinical trials (Miyake, H., et al., J Urol 2002; 167: 1282-1287).
DESCRIPCIÓN DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
La presente invención proporciona un método in vitro no invasivo, altamente sensible, eficiente y rápido, relativo a proteínas asociadas con BTCC. También proporciona un método para el diagnóstico, pronóstico y monitorización de BTCC mediante análisis de muestras de orina. La presente invención proporciona sorprendentemente una combinación de biomarcadores de cáncer de vejiga para Ia detección de BTCC mediante análisis de orina, con valor significativo para el diagnóstico, pronóstico y/o monitorización de BTCC.The present invention provides a non-invasive, highly sensitive, efficient and rapid in vitro method, related to proteins associated with BTCC. It also provides a method for the diagnosis, prognosis and monitoring of BTCC through urine sample analysis. The present The invention surprisingly provides a combination of bladder cancer biomarkers for the detection of BTCC by urine analysis, with significant value for the diagnosis, prognosis and / or monitoring of BTCC.
La alfa-amilasa (AMYS) carcinoide, Ia alfa-amilasa pancreática y Ia alfa amilasa 1A pertenecen a Ia familia de las glicosil hidrolasas 13 e hidrolizan enlaces 1 ,4-alfa-glucosídicos en oligosacáridos y polisacáridos, y catalizan Ia primera etapa en Ia digestión del glicógeno y el almidón de Ia dieta. El genoma humano contiene un conjunto de genes que codifican amilasas y que se expresan en niveles elevados o bien en Ia glándula salival o en el páncreas.Carcinoid alpha-amylase (AMYS), pancreatic alpha-amylase and alpha 1-amylase belong to the family of glycosyl hydrolases 13 and hydrolyze 1, 4-alpha-glucosidic bonds in oligosaccharides and polysaccharides, and catalyze the first stage in Ia digestion of glycogen and starch in the diet. The human genome contains a set of genes that encode amylases and that are expressed at elevated levels either in the salivary gland or in the pancreas.
La apolipoproteína A-I (APOA1) pertenece a Ia familia de las apolipoproteínas A1/A4/E y participa en el transporte inverso de colesterol desde los tejidos hasta el hígado para su excreción mediante Ia promoción del reflujo de colesterol desde los tejidos y Ia actuación como cofactor de Ia lecitina colesterol aciltransferasa (LCAT).Apolipoprotein AI (APOA1) belongs to the family of apolipoproteins A1 / A4 / E and participates in the inverse transport of cholesterol from the tissues to the liver for excretion by promoting cholesterol reflux from the tissues and acting as a cofactor of the lecithin cholesterol acyltransferase (LCAT).
La catepsina D (CATD) es una proteasa acida que pertenece a Ia familia de las peptidasas A1 y que activa Ia degradación proteica ¡ntracelular. Está implicada en Ia patogénesis de diversas enfermedades tales como cáncer de vejiga (Ozer E., et al., Urology 1999, 54:50-5 y loachim E., et al., Anticancer Res 2002, 22:3383-8), cáncer de mama y posiblemente enfermedad de Alzheimer. La catepsina D se sintetiza como un precursor inactivo de 52 kDa, formado por dos polipéptidos de 14 kDa (CATD H) y 34 kDa (CATD K). El péptido inactivo se activa por proteólisis del extremo N-terminal resultando en Ia proteína enzimáticamente activa de 48 kDa.Cathepsin D (CATD) is an acid protease that belongs to the family of peptidases A1 and that activates intracellular protein degradation. It is involved in the pathogenesis of various diseases such as bladder cancer (Ozer E., et al., Urology 1999, 54: 50-5 and loachim E., et al., Anticancer Res 2002, 22: 3383-8), breast cancer and possibly Alzheimer's disease. Cathepsin D is synthesized as an inactive 52 kDa precursor, consisting of two 14 kDa (CATD H) and 34 kDa (CATD K) polypeptides. The inactive peptide is activated by proteolysis of the N-terminal end resulting in the enzymatically active protein of 48 kDa.
La glutatión S-transferasa (GSTP1) pertenece a una familia de enzimas que juegan un papel importante en Ia detoxificación mediante catálisis de Ia conjugación de algunos compuestos hidrofóbicos y electrofílicos con glutatión reducido.Glutathione S-transferase (GSTP1) belongs to a family of enzymes that play an important role in detoxification by catalysis of Ia conjugation of some hydrophobic and electrophilic compounds with reduced glutathione.
La peroxiredoxina 2 (PRDX2) pertenece a Ia familia de las ahpc/tsa y está implicada en Ia regulación redox de Ia célula. Reduce los peróxidos con equivalentes reductores obtenidos a través del sistema de tioredoxina. No tienen capacidad para recibir electrones de Ia glutaredoxina. Puede desempeñar un papel importante en Ia eliminación de los peróxidos generados durante el metabolismo. Podría participar en Ia cascada de señales de los factores de crecimiento y factor de necrosis tumoral-alfa regulando concentraciones intracelulares de HbO2. Aumenta Ia actividad de células natural killer (NK).Peroxiredoxin 2 (PRDX2) belongs to the family of ahpc / tsa and is involved in the redox regulation of the cell. Reduces peroxides with reducing equivalents obtained through the thioredoxin system. They have no capacity to receive electrons from glutaredoxin. It can play an important role in the elimination of peroxides generated during metabolism. It could participate in the cascade of signals of growth factors and tumor necrosis factor-alpha by regulating intracellular concentrations of HbO 2 . Increases the activity of natural killer cells (NK).
La proteína unida a retinol plasmático (RETBP) pertenece a Ia familia de las lipocalinas y es el transportador específico de retinol (alcohol de Ia vitamina A) en sangre. Libera retinol de las reservas del hígado hacia los tejidos periféricos. En plasma, el complejo RBP-retinol interacciona con Ia transtiretina, Io que impide su pérdida por filtración a través de los glomérulos renales. Una deficiencia de vitamina A induce una modificación post- traduccional en Ia proteína transportadora, Io cual bloquea su secreción y resulta en una liberación y un suministro defectuosos a las células epidérmicas.Plasma retinol-bound protein (RETBP) belongs to the lipocalin family and is the specific transporter of retinol (alcohol of vitamin A) in blood. It releases retinol from the liver stores to peripheral tissues. In plasma, the RBP-retinol complex interacts with transthyretin, which prevents its loss by filtration through the renal glomeruli. A deficiency of vitamin A induces a post-translational modification in the transporter protein, which blocks its secretion and results in a defective release and supply to the epidermal cells.
La fosfoproteína inducida por estrés 1 (STIP1) es una proteína adaptadora que media Ia asociación de las chaperonas moleculares HSC70 y HSP90 (HSPCA y HSPCB).Stress induced phosphoprotein 1 (STIP1) is an adapter protein that mediates the association of the molecular chaperones HSC70 and HSP90 (HSPCA and HSPCB).
La transtiretina (TTHY, antes llamada prealbúmina) es una de las tres proteínas que se unen a hormonas tiroideas encontradas en Ia sangre de los vertebrados. Se produce en el hígado y circula al torrente sanguíneo, donde se une a retinol y tiroxina. El cáncer puede ser detectado analizando el patrón de expresión de al menos 4 biomarcadores de cáncer de vejiga en una muestra de orina. Así, Ia detección de al menos 4 proteínas diferencialmente expresadas en una muestra test de orina en comparación con orina normal es indicativa de BTCC.Transthyretin (TTHY, previously called prealbumin) is one of the three proteins that bind to thyroid hormones found in the blood of vertebrates. It is produced in the liver and circulates in the bloodstream, where it binds to retinol and thyroxine. Cancer can be detected by analyzing the expression pattern of at least 4 biomarkers of bladder cancer in a urine sample. Thus, the detection of at least 4 differentially expressed proteins in a urine test sample compared to normal urine is indicative of BTCC.
Las muestras de orina son muy diversas en cuanto a su composición, por Io que es esencial normalizar para tener en cuenta las diferencias en Ia concentración total de proteína y para eliminar tendencias entre muestras. Para normalizar niveles de señales se pueden usar los niveles de expresión de una proteína control, cuyo contenido en orina es siempre constante. En Ia bibliografía existen numerosos ejemplos de estas proteínas invariables. En Ia presente invención, Ia transferrina, por ejemplo, demostró ser una proteína de concentración constante.Urine samples are very diverse in terms of their composition, so it is essential to normalize to take into account the differences in total protein concentration and to eliminate trends between samples. To normalize signal levels, the expression levels of a control protein, whose urine content is always constant, can be used. In the literature there are numerous examples of these invariable proteins. In the present invention, transferrin, for example, proved to be a protein of constant concentration.
Biomarcadores o proteínas de cáncer de vejiga representativas identificadas en Ia presente invención incluyen, aunque no se limitan a CATD, GSTP1 , RETBP, STIP1 , AMYS, APOA1 , PRDX2 y TTHY.Representative biomarkers or bladder cancer proteins identified in the present invention include, but are not limited to CATD, GSTP1, RETBP, STIP1, AMYS, APOA1, PRDX2 and TTHY.
Un aspecto de Ia presente invención se refiere a una combinación de biomarcadores para Ia detección de BTCC que comprende los biomarcadores CATD1 GSTP1 , RETBP y STIP1 o sus variantes transcripcionales o post- traduccionales.One aspect of the present invention relates to a combination of biomarkers for the detection of BTCC comprising the biomarkers CATD 1 GSTP1, RETBP and STIP1 or their transcriptional or post-translational variants.
Otro aspecto de Ia invención se refiere a un método in vitro no invasivo que comprende: a) detectar y cuantificar Ia combinación de biomarcadores CATD, GSTP1 , RETBP y STIP1 o sus variantes transcripcionales o post- traduccionales, en una muestra test de orina de un individuo; y b) comparar el valor de expresión obtenido en a) en Ia muestra test de orina con el correspondiente valor estándar en orina normal, donde variaciones en el valor obtenido en a) respecto al valor estándar en orina normal son indicativas de BTCC. La expresión se determina por espectrometría o inmunoensayos. Este método se puede adaptar a cribado poblacional para detectar Ia presencia de BTCC y determinar el estadio o Ia severidad de este cáncer. Además, puede usarse para evaluar Ia ausencia de enfermedad después de resección quirúrgica, establecer un diagnóstico y/o pronóstico de este cáncer y/o monitorizar el efecto del tratamiento administrado a un individuo que padece dicho cáncer.Another aspect of the invention relates to a non-invasive in vitro method comprising: a) detecting and quantifying the combination of CATD, GSTP1, RETBP and STIP1 biomarkers or their transcriptional or post-translational variants, in a urine test sample of a individual; and b) compare the expression value obtained in a) in the urine test sample with the corresponding standard value in normal urine, where variations in the value obtained in a) with respect to the standard value in normal urine are indicative of BTCC. Expression is determined by spectrometry or immunoassays. This method can be adapted to population screening to detect the presence of BTCC and determine the stage or severity of this cancer. In addition, it can be used to evaluate the absence of disease after surgical resection, establish a diagnosis and / or prognosis of this cancer and / or monitor the effect of the treatment administered to an individual suffering from said cancer.
Otro aspecto de Ia invención se refiere al uso conjunto de las secuencias peptídicas derivadas los biomarcadores CATD, GSTP1 , RETBP y STIP1 o sus variantes transcripcionales o post-traduccionales, para detectar Ia presencia de BTCC mediante análisis de orina, determinar el estadio o severidad de este cáncer, evaluar Ia ausencia de enfermedad después de resección quirúrgica, establecer un diagnóstico y/o pronóstico de este cáncer y/o monitorizar el efecto del tratamiento administrado a un individuo que padece dicho cáncer, donde los biomarcadores están presentes en orina.Another aspect of the invention relates to the joint use of the peptide sequences derived from the CATD, GSTP1, RETBP and STIP1 biomarkers or their transcriptional or post-translational variants, to detect the presence of BTCC by urine analysis, determine the stage or severity of this cancer, evaluate the absence of disease after surgical resection, establish a diagnosis and / or prognosis of this cancer and / or monitor the effect of the treatment administered to an individual suffering from said cancer, where biomarkers are present in urine.
Otro aspecto de Ia invención se refiere al uso conjunto de los nucleótidos o secuencias peptídicas derivados de los biomarcadores CATD, GSTP1 , RETBP y STIP1 o sus variantes transcripcionales o post-traduccionales, en métodos de cribaje para identificar, desarrollar y evaluar Ia eficacia de agentes terapéuticos para tratar BTCC.Another aspect of the invention relates to the joint use of nucleotides or peptide sequences derived from the CATD, GSTP1, RETBP and STIP1 biomarkers or their transcriptional or post-translational variants, in screening methods to identify, develop and evaluate the efficacy of agents Therapeutics to treat BTCC.
La invención se refiere además a anticuerpos contra los biomarcadores de cáncer de vejiga mencionados. Estos anticuerpos pueden presentarse en una gran variedad de formas apropiadas para cada uso, incluyendo, por ejemplo, forma soluble, inmovilizados sobre un sustrato, o en combinación con un soporte farmacéuticamente aceptable.The invention further relates to antibodies against bladder cancer biomarkers mentioned. These antibodies can be presented in a variety of forms appropriate for each use, including, for example, soluble form, immobilized on a substrate, or in combination with a pharmaceutically acceptable carrier.
Otro aspecto de Ia invención se refiere a un kit para llevar a cabo el método previamente descrito que comprende 1) anticuerpos que reconocen específicamente los biomarcadores CATD, GSTP1 , RETBP y STIP1 en orina y 2) un soporte en un embalaje adecuado. A efectos de Ia invención se han utilizado las siguientes definiciones:Another aspect of the invention relates to a kit for carrying out the previously described method comprising 1) antibodies that specifically recognize the biomarkers CATD, GSTP1, RETBP and STIP1 in urine and 2) a support in a suitable packaging. For the purposes of the invention the following definitions have been used:
"Cáncer" se refiere a Ia enfermedad que se caracteriza típicamente por un crecimiento celular anormal o no regulado, capaz de invadir tejidos adyacentes y extenderse a otros órganos. "Carcinoma" se refiere al tejido resultante de un crecimiento celular anormal o no regulado. "Carcinoma transicional de vejiga" o su abreviación "BTCC" se refiere a cualquier desorden proliferativo maligno en células epiteliales de Ia vejiga urinaria. "Tumor" se refiere a cualquier masa de tejido anormal generada por un proceso neoplástico, ya sea benigno (no cancerígeno) o maligno (cancerígeno)."Cancer" refers to the disease that is typically characterized by abnormal or unregulated cell growth, capable of invading adjacent tissues and spreading to other organs. "Carcinoma" refers to tissue resulting from abnormal or unregulated cell growth. "Transitional bladder carcinoma" or its abbreviation "BTCC" refers to any proliferative malignant disorder in epithelial cells of the urinary bladder. "Tumor" refers to any mass of abnormal tissue generated by a neoplastic process, either benign (non-carcinogenic) or malignant (carcinogenic).
Las "proteínas de cáncer de vejiga o biomarcadores" son proteínas expresadas diferencialmente en BTCC, es decir, proteínas que se expresan de forma diferente en Ia orina de un individuo sano respecto a Ia orina de un paciente de BTCC. En Ia presente invención el grupo de proteínas diferencialmente expresadas incluye los biomarcadores CATD1 GSTP1 , RETBP, STIP1 , AMYS, APOA1 , PRDX2 y TTHY, así como también cualquier proteína de una muestra de orina, cuya proporción varía al menos 2 veces cuando se comparan 2 muestras de orina diferentes: una de un individuo sano y otra de un paciente de BTCC1 donde esta cuantificación se lleva a cabo mediante un analizador de imágenes, por ejemplo, el software Progenesis PG220. Los biomarcadores de cáncer de vejiga tal como se han descrito pueden tener cualquier longitud y pueden comprender secuencias adicionales derivadas de Ia proteína nativa y/o secuencias heterólogas; incluyen cualquier variante transcripcional o post-traduccional, así como también cualquier secuencia con al menos un 95% de identidad con las secuencias descritas, donde identidad se define como el porcentaje de residuos idénticos entre dos secuencias. Los expertos en Ia materia podrán apreciar que fragmentos o variantes de las secuencias pueden ser igualmente útiles en el tratamiento y detección del cáncer. La frase "detectar biomarcadores" significa determinar Ia existencia de biomarcadores, mientras que Ia frase "cuantificar biomarcadores" significa expresar Ia presencia de dichos biomarcadores con un valor (por ejemplo un valor de intensidad). La frase "indicativo de BTCC" significa que variaciones encontradas en el valor de expresión de los biomarcadores en una muestra test de orina respecto al correspondiente valor estándar en orina normal prueban Ia presencia de BTCC. "Variación" significa un cambio en el nivel de expresión de una proteína."Bladder cancer proteins or biomarkers" are proteins differentially expressed in BTCC, that is, proteins that are expressed differently in the urine of a healthy individual with respect to the urine of a BTCC patient. In the present invention the group of differentially expressed proteins includes the biomarkers CATD 1 GSTP1, RETBP, STIP1, AMYS, APOA1, PRDX2 and TTHY, as well as any protein in a urine sample, the proportion of which varies at least 2 times when compared 2 different urine samples: one from a healthy individual and one from a BTCC 1 patient where this quantification is carried out using an image analyzer, for example, Progenesis PG220 software. Bladder cancer biomarkers as described may have any length and may comprise additional sequences derived from the native protein and / or heterologous sequences; they include any transcriptional or post-translational variant, as well as any sequence with at least 95% identity with the described sequences, where identity is defined as the percentage of identical residues between two sequences. Those skilled in the art will appreciate that fragments or variants of the sequences can be equally useful in the treatment and detection of cancer. The phrase "detect biomarkers" means determining the existence of biomarkers, while the phrase "quantify biomarkers" means expressing the presence of said biomarkers with a value (for example, an intensity value). The phrase "indicative of BTCC" means that variations found in the expression value of the biomarkers in a urine test sample with respect to the corresponding standard value in normal urine prove the presence of BTCC. "Variation" means a change in the level of expression of a protein.
Una "proteína expresada diferencialmente" se refiere a una proteína cuyo patrón de expresión varía (aumentando o disminuyendo) en Ia orina de un paciente de BTCC en comparación con Ia orina de un individuo sano.A "differentially expressed protein" refers to a protein whose expression pattern varies (increasing or decreasing) in the urine of a BTCC patient compared to the urine of a healthy individual.
El "extracto de proteínas" corresponde al sobrenadante obtenido después de Ia centrifugación de Ia muestra de orina.The "protein extract" corresponds to the supernatant obtained after centrifugation of the urine sample.
El término "individuo" se refiere a todas las especies de animales clasificados como mamíferos e incluye, aunque sin limitación, animales domésticos y de granja, primates y humanos, y preferiblemente se refiere a un humano, macho o hembra de cualquier edad o raza. Un "individuo sano" es un individuo que no padece carcinoma transicional de vejiga y puede incluir pacientes de otras enfermedades urológicas. El término "previamente diagnosticado" se refiere a un individuo que ha recibido un primer diagnóstico positivo de BTCC. El término "no previamente diagnosticado" se refiere a un individuo que nunca ha recibido un diagnóstico positivo de BTCC (diagnóstico de novó).The term "individual" refers to all species of animals classified as mammals and includes, but is not limited to, domestic and farm animals, primates and humans, and preferably refers to a human, male or female of any age or race. A "healthy individual" is an individual who does not suffer from transitional bladder carcinoma and may include patients with other urological diseases. The term "previously diagnosed" refers to an individual who has received a first positive diagnosis of BTCC. The term "not previously diagnosed" refers to an individual who has never received a positive diagnosis of BTCC (novó diagnosis).
El "valor estándar en orina normal" se refiere a Ia cuantificación de Ia media del nivel de expresión de los biomarcadores detectados en muestras individuales de orina de individuos que no padecen BTCC. "Diagnosis" de BTCC se refiere al proceso de identificación o determinación de Ia naturaleza y causa de BTCC mediante Ia evaluación de uno o más biomarcadores. El término "prognosis" se refiere a Ia probable evolución o curso de Ia enfermedad, es decir, Ia probabilidad de recuperación o recurrencia. "Monitorizar" significa evaluar Ia presencia o ausencia de BTCC en un individuo a diferentes tiempos. "Tratamiento" se refiere a cualquier proceso, acción, aplicación o similar, donde un individuo se somete a ayuda médica con el objeto de mejorar su condición, directa o indirectamente.The "standard value in normal urine" refers to the quantification of the mean level of expression of the biomarkers detected in individual urine samples of individuals not suffering from BTCC. "Diagnosis" of BTCC refers to the process of identification or determination of the nature and cause of BTCC through the evaluation of one or more biomarkers. The term "prognosis" refers to the probable evolution or course of the disease, that is, the probability of recovery or recurrence. "Monitoring" means evaluating the presence or absence of BTCC in an individual at different times. "Treatment" refers to any process, action, application or similar, where an individual submits to medical assistance in order to improve his condition, directly or indirectly.
El término "especificidad" se refiere a Ia capacidad de un test de excluir Ia presencia de una enfermedad cuando verdaderamente no está presente. La especificidad se expresa como el número de individuos sanos para los cuales existe un test negativo correcto (llamados verdaderos negativos) dividido por Ia suma de los verdaderos negativos y el número de individuos sanos para los cuales existe un test positivo incorrecto (llamados falsos positivos). El término "sensibilidad" se refiere a Ia capacidad de un test de detectar Ia presencia de una enfermedad cuando está verdaderamente presente. La sensibilidad se expresa como el número de pacientes enfermos para los cuales existe un test positivo (llamados verdaderos positivos), dividido por Ia suma de los verdaderos positivos y el número de pacientes para los cuales existe un test negativo incorrecto (llamados falsos negativos). "Robustez" define Ia capacidad de un método numérico para dar el mismo resultado a pesar de Ia variabilidad de las muestras iniciales.The term "specificity" refers to the ability of a test to exclude the presence of a disease when it is not really present. Specificity is expressed as the number of healthy individuals for whom there is a correct negative test (called true negatives) divided by the sum of the true negatives and the number of healthy individuals for whom there is an incorrect positive test (called false positives) . The term "sensitivity" refers to the ability of a test to detect the presence of a disease when it is truly present. Sensitivity is expressed as the number of sick patients for whom there is a positive test (called true positives), divided by the sum of the true positives and the number of patients for whom there is an incorrect negative test (called false negatives). "Robustness" defines the ability of a numerical method to give the same result despite the variability of the initial samples.
El término "gen" se refiere a una región de los desoxiribonucleótidos de doble hebra que codifica una proteína. Puede representar una parte de una secuencia codificante o una secuencia codificante completa. El término "proteína" indica al menos una cadena molecular de aminoácidos unidos ¡ntermolecularmente a través de enlaces covalentes o no covalentes. El término incluye todas las formas de modificaciones post-traduccionales, por ejemplo glicosilación, fosforilación o acetilación. Los términos "péptido" y "polipéptido" se refieren a cadenas moleculares de aminoácidos que representan un fragmento de proteína. Los términos "proteína" y "péptido" se usan indistintamente.The term "gene" refers to a region of double-stranded deoxyribonucleotides that encodes a protein. It can represent a part of a coding sequence or a complete coding sequence. The term "protein" indicates at least one molecular chain of amino acids bound intra-molecularly through covalent or non-covalent bonds. The term includes all forms of post-translational modifications, for example glycosylation, phosphorylation or acetylation. The terms "peptide" and "polypeptide" refer to molecular chains of amino acids that They represent a protein fragment. The terms "protein" and "peptide" are used interchangeably.
El término "anticuerpo" se refiere a una proteína en forma de Y (conocida como inmunoglobulina) en Ia superficie de células B que se secreta a Ia sangre o linfa en respuesta a un estímulo antigénico, tal como una proteína exógena, bacteria, virus, parásito u órgano transplantado, que presenta una unión específica para una molécula diana llamada "antígeno". La región de las ¡nmunoglobulinas que se une al antígeno se puede dividir tanto en fragmentos F(ab')2 como Fab. El término "anticuerpo" incluye anticuerpos monoclonales o policlonales, ya sean intactos o fragmentos derivados de los mismos; e incluye anticuerpos humanos, anticuerpos humanizados y anticuerpos de origen no humano. Un "anticuerpo no-humano" es un anticuerpo generado por una especie animal diferente de Homo sapiens. Un "anticuerpo humanizado" es un anticuerpo diseñado genéticamente en el cual Ia mínima parte de un anticuerpo de ratón se transplanta a un anticuerpo humano. Generalmente, los anticuerpos humanizados tienen un 5-10% de ratón y un 90-95% de humano. Un "anticuerpo humano" es un anticuerpo derivado de ratones transgénicos que tienen genes de anticuerpos humanos o de células humanas. Los "anticuerpos monoclonales" son poblaciones homogéneas de anticuerpos altamente específicos dirigidos a un único sitio antigénico o "determinante" de Ia molécula diana. "Anticuerpos policlonales" incluyen poblaciones heterogéneas de anticuerpos que se dirigen a diferentes determinantes antigénicos de Ia molécula diana. El término "anticuerpo específico" se refiere a un anticuerpo generado específicamente contra una proteína (en este caso, contra un marcador particular de cáncer de vejiga). El término "complejo anticuerpo-proteína" se refiere a un complejo formado por un antígeno y su anticuerpo específico. El término "combibody" (anticuerpo combinatorial) se refiere a un anticuerpo dispuesto en Ia superficie de fagos filamentosos, Io cual permine el cribado directo de librerías de ADNc para Ia expresión de anticuerpos reactivos de superficie celular, sin Ia necesidad de producción y purificación usando bacterias o sistemas de células eucariotas. El término "anticuerpo Fab recombinante" se refiere a un anticuerpo recombinante que sólo contiene el fragmento Fab que es univalente y útil cuando el anticuerpo tiene una alta afinidad por su antígeno. Pueden obtenerse recombinantemente si Ia secuencia proteica es conocida. El término "fragmento de anticuerpo ScFv " se refiere a un fragmento de una sola cadena variable (scFv) que se puede expresar en cultivos de bacterias.The term "antibody" refers to a Y-shaped protein (known as immunoglobulin) on the surface of B cells that is secreted to the blood or lymph in response to an antigenic stimulus, such as an exogenous protein, bacteria, viruses, parasite or transplanted organ, which has a specific binding for a target molecule called "antigen." The region of the immunoglobulins that binds to the antigen can be divided into both F (ab ') 2 and Fab fragments. The term "antibody" includes monoclonal or polyclonal antibodies, either intact or fragments derived therefrom; and includes human antibodies, humanized antibodies and antibodies of non-human origin. A "non-human antibody" is an antibody generated by an animal species other than Homo sapiens. A "humanized antibody" is a genetically designed antibody in which the minimum part of a mouse antibody is transplanted into a human antibody. Generally, humanized antibodies have 5-10% mouse and 90-95% human. A "human antibody" is an antibody derived from transgenic mice that have human antibody or human cell genes. The "monoclonal antibodies" are homogeneous populations of highly specific antibodies directed to a single antigenic site or "determinant" of the target molecule. "Polyclonal antibodies" include heterogeneous populations of antibodies that target different antigenic determinants of the target molecule. The term "specific antibody" refers to an antibody specifically generated against a protein (in this case, against a particular marker of bladder cancer). The term "antibody-protein complex" refers to a complex formed by an antigen and its specific antibody. The term "combibody" (combinatorial antibody) refers to an antibody disposed on the surface of filamentous phages, which allows direct screening of cDNA libraries for the expression of reactive antibodies on cell surface, without the need for production and purification using bacteria or eukaryotic cell systems. The term "recombinant Fab antibody" refers to a recombinant antibody that only contains the Fab fragment that is univalent and useful when the antibody has a high affinity for its antigen. They can be obtained recombinantly if the protein sequence is known. The term "ScFv antibody fragment" refers to a single variable chain fragment (scFv) that can be expressed in bacterial cultures.
Un "epítopo" es un determinante antigénico de una proteína; es Ia secuencia de aminoácidos de Ia proteína que reconoce un anticuerpo específico. Dichos epítopos pueden comprender una extensión contigua de aminoácidos (epítopo lineal) o de aminoácidos no contiguos que se encuentran próximos en el espacio gracias al plegamiento tridimensional de Ia cadena polipetídica (epítopos discontinuos). El término "fase sólida" se refiere a una matriz no acuosa a Ia cual puede unirse el anticuerpo. Ejemplos de materiales para Ia fase sólida incluyen sin limitación vidrio, polisacáridos (por ejemplo agarosa), poliacrilamida, poliestireno, alcohol polivinílico y siliconas. Ejemplos de formas en fase sólida son un pocilio o una columna de purificación.An "epitope" is an antigenic determinant of a protein; is the amino acid sequence of the protein that recognizes a specific antibody. Said epitopes can comprise a contiguous extension of amino acids (linear epitope) or of non-contiguous amino acids that are close in space thanks to the three-dimensional folding of the polypeptide chain (discontinuous epitopes). The term "solid phase" refers to a non-aqueous matrix to which the antibody can bind. Examples of materials for the solid phase include without limitation glass, polysaccharides (for example agarose), polyacrylamide, polystyrene, polyvinyl alcohol and silicones. Examples of solid phase forms are a well or a purification column.
El término "dipstick" se refiere a un dispositivo que se sumerge parcialmente (normalmente por un extremo) dentro de un líquido para llevar a cabo un test que puede determinar y/o cuantificar alguna propiedad del líquido (química, física, etc). Este tipo de dipstick está usualmente fabricado en papel o cartón y está impregnado con reactivos, cuyos cambios de color indican alguna característica del líquido. El término "soporte" se refiere al mecanismo o dispositivo por el cual se conduce o se transporta algo. El término "embalaje" se refiere al contenedor y envase previo a Ia venta con el objetivo primario de facilitar Ia compra y el uso del producto.The term "dipstick" refers to a device that partially submerges (usually at one end) into a liquid to carry out a test that can determine and / or quantify some property of the liquid (chemical, physical, etc.). This type of dipstick is usually made of paper or cardboard and is impregnated with reagents, whose color changes indicate some characteristic of the liquid. The term "support" refers to the mechanism or device by which something is driven or transported. The term "packaging" refers to the container and container prior to sale with the primary objective of facilitating the purchase and use of the product.
El término "biochip" se refiere a una multitud de dispositivos miniaturizados empleados para realizar pruebas biológicas sobre un soporte sólido o fluido con una alta capacidad de procesamiento. Una realización particular de Ia invención se refiere a una combinación de biomarcadores para Ia detección de BTCC que comprende los biomarcadores CATD1 GSTP1 , RETBP, STIP1 , AMYS y APOA1 o sus variantes transcripcionales o post-traduccionales.The term "biochip" refers to a multitude of miniaturized devices used to perform biological tests on a solid or fluid support with a high processing capacity. A particular embodiment of the invention relates to a combination of biomarkers for the detection of BTCC comprising the biomarkers CATD 1 GSTP1, RETBP, STIP1, AMYS and APOA1 or their transcriptional or post-translational variants.
Otra realización particular se refiere a una combinación de biomarcadores para Ia detección de BTCC que comprende los biomarcadores CATD, GSTP1 , RETBP, STIP1 , AMYS, APOA1 y PRDX2 o sus variantes transcripcionales o post-traduccionales.Another particular embodiment refers to a combination of biomarkers for the detection of BTCC comprising the biomarkers CATD, GSTP1, RETBP, STIP1, AMYS, APOA1 and PRDX2 or their transcriptional or post-translational variants.
Otra realización particular se refiere a una combinación de biomarcadores para Ia detección de BTCC que comprende los biomarcadores CATD, GSTP1 , RETBP, STIP1, AMYS, APOA1 y TTHY o sus variantes transcripcionales o post-traduccionales.Another particular embodiment refers to a combination of biomarkers for the detection of BTCC comprising the biomarkers CATD, GSTP1, RETBP, STIP1, AMYS, APOA1 and TTHY or their transcriptional or post-translational variants.
Otra realización particular se refiere a una combinación de biomarcadores para Ia detección de BTCC que comprende los biomarcadores CATD, GSTP1 , RETBP, STIP1 , AMYS, APOA1 , PRDX2 y TTHY o sus variantes transcripcionales o post-traduccionales.Another particular embodiment refers to a combination of biomarkers for the detection of BTCC comprising the biomarkers CATD, GSTP1, RETBP, STIP1, AMYS, APOA1, PRDX2 and TTHY or their transcriptional or post-translational variants.
En otra realización particular, Ia primera etapa del método para evaluar cáncer de vejiga comprende detectar y cuantificar Ia combinación de biomarcadores CATD, GSTP1 , RETBP1 STIP1 , AMYS y APOA1 o sus variantes transcripcionales o post-traduccionales.In another particular embodiment, the first stage of the method for evaluating bladder cancer comprises detecting and quantifying the combination of CATD, GSTP1, RETBP 1 STIP1, AMYS and APOA1 biomarkers or their transcriptional or post-translational variants.
En otra realización particular, Ia primera etapa del método para evaluar cáncer de vejiga comprende detectar y cuantificar Ia combinación de biomarcadores CATD1 GSTP1 , RETBP, STIP1 , AMYS, APOA1 y PRDX2 o sus variantes transcripcionales o post-traduccionales.In another particular embodiment, the first stage of the method for evaluating bladder cancer comprises detecting and quantifying the combination of CATD 1 GSTP1, RETBP, STIP1, AMYS, APOA1 and PRDX2 biomarkers or their transcriptional or post-translational variants.
En otra realización particular, Ia primera etapa del método para evaluar cáncer de vejiga comprende detectar y cuantificar Ia combinación de biomarcadores CATD, GSTP1 , RETBP1 STIP1 , AMYS1 APOA1 y TTHY o sus variantes transcripcionales o post-traduccionales.In another particular embodiment, the first stage of the method for evaluating bladder cancer comprises detecting and quantifying the combination of biomarkers CATD, GSTP1, RETBP 1 STIP1, AMYS 1 APOA1 and TTHY or their transcriptional or post-translational variants.
En otra realización particular, Ia primera etapa del método para evaluar cáncer de vejiga comprende detectar y cuantificar Ia combinación de biomarcadores CATD1 GSTP1 , RETBP1 STIP1 , AMYS1 APOA1 , PRDX2 y TTHY o sus variantes transcripcionales o post-traduccionales.In another particular embodiment, the first stage of the method for evaluating bladder cancer comprises detecting and quantifying the combination of CATD 1 GSTP1, RETBP 1 STIP1, AMYS 1 APOA1, PRDX2 and TTHY biomarkers or their transcriptional or post-translational variants.
En otra realización particular, el método permite determinar Ia progresión de Ia enfermedad cuando Ia misma proteína o proteínas se compara con diferentes muestras obtenidas del mismo paciente a diferentes tiempos durante Ia evolución de BTCC.In another particular embodiment, the method allows to determine the progression of the disease when the same protein or proteins is compared with different samples obtained from the same patient at different times during the evolution of BTCC.
En otra realización particular, Ia combinación de biomarcadores puede ser utilizada para monitorizar Ia eficacia del tratamiento farmacológico o quirúrgico.In another particular embodiment, the combination of biomarkers can be used to monitor the efficacy of the pharmacological or surgical treatment.
En otra realización particular, Ia muestra a analizar se obtiene de un individuo a quien no se Ie ha diagnosticado previamente BTCC. En otra realización particular, Ia muestra a analizar se obtiene de un individuo a quien se Ie ha diagnosticado previamente BTCC. En otra realización particular, Ia muestra a analizar se obtiene de un individuo que en Ia actualidad está recibiendo tratamiento contra BTCC. En otra realización particular, el método comprende Ia obtención del extracto de proteínas de Ia muestra.In another particular embodiment, the sample to be analyzed is obtained from an individual who has not previously been diagnosed with BTCC. In another particular embodiment, the sample to be analyzed is obtained from an individual who has been previously diagnosed with BTCC. In another particular embodiment, the sample to be analyzed is obtained from an individual who is currently receiving treatment against BTCC. In another particular embodiment, the method comprises obtaining the protein extract of the sample.
En otra realización particular, el método para detectar cáncer comprende poner en contacto una muestra de orina con una molécula que se une específicamente a un biomarcador de cáncer de vejiga y cuantificar dicha unión.In another particular embodiment, the method of detecting cancer comprises contacting a urine sample with a molecule that specifically binds to a biomarker of bladder cancer and quantifying said binding.
En otra realización particular, Ia detección y cuantificación de proteínas comprende una primera etapa, en Ia cual el extracto de proteína de Ia muestra se pone en contacto con una composición de anticuerpos específicos para uno o más epítopos de Ia proteína o proteínas, y una segunda etapa, en Ia cual se cuantifican los complejos formados por anticuerpos y proteínas.In another particular embodiment, the detection and quantification of proteins comprises a first stage, in which the protein extract of Ia Sample is contacted with a composition of antibodies specific for one or more epitopes of the protein or proteins, and a second stage, in which the complexes formed by antibodies and proteins are quantified.
En otra realización particular, los anticuerpos específicos usados para Ia detección de proteínas son de origen humano, humanizados o de origen no humano y seleccionados entre anticuerpos monoclonales o policlonales, fragmentos de anticuerpos intactos o recombinantes, combibodies y fragmentos de anticuerpo Fab o scFv.In another particular embodiment, the specific antibodies used for the detection of proteins are of human origin, humanized or of non-human origin and selected from monoclonal or polyclonal antibodies, fragments of intact or recombinant antibodies, combibodies and Fab or scFv antibody fragments.
Otra realización de Ia invención se refiere al uso conjunto de las secuencias peptídicas derivadas de los biomarcadores CATD, GSTP1 , RETBP, STIP1 , AMYS y APOA1 o sus variantes transcripcionales o post-traduccionales, donde los biomarcadores están presentes en orina.Another embodiment of the invention refers to the joint use of the peptide sequences derived from the CATD, GSTP1, RETBP, STIP1, AMYS and APOA1 biomarkers or their transcriptional or post-translational variants, where the biomarkers are present in urine.
Otra realización de Ia invención se refiere al uso conjunto de las secuencias peptídicas derivadas de los biomarcadores CATD, GSTP1 , RETBP, STIP1 , AMYS, APOA1 y PRDX2 o sus variantes transcripcionales o post- traduccionales, donde los biomarcadores están presentes en orina.Another embodiment of the invention relates to the joint use of the peptide sequences derived from the CATD, GSTP1, RETBP, STIP1, AMYS, APOA1 and PRDX2 biomarkers or their transcriptional or post-translational variants, where the biomarkers are present in urine.
Otra realización de Ia invención se refiere al uso conjunto de las secuencias peptídicas derivadas de los biomarcadores CATD, GSTP1 , RETBP, STIP1 , AMYS, APOA1 y TTHY o sus variantes transcripcionales o post- traduccionales, donde los biomarcadores están presentes en orina.Another embodiment of the invention relates to the joint use of the peptide sequences derived from the CATD, GSTP1, RETBP, STIP1, AMYS, APOA1 and TTHY biomarkers or their transcriptional or post-translational variants, where the biomarkers are present in urine.
Otra realización de Ia invención se refiere al uso conjunto de las secuencias peptídicas derivadas de los biomarcadores CATD, GSTP1 , RETBP, STIP1 , AMYS, APOA1 , PRDX2 y TTHY o sus variantes transcripcionales o post- traduccionales, donde los biomarcadores están presentes en orina. En otra realización, se usan conjuntamente al menos 4 biomarcadores presentes en orina o sus variantes transcripcionales o post-traduccionales. En otra realización, se usan conjuntamente al menos 6 biomarcadores presentes en orina o sus variantes transcripcionales o post-traduccionales. En otra realización, se usan conjuntamente al menos 7 biomarcadores presentes en orina o sus variantes transcripcionales o post-traduccionales. En otra realización, se usan conjuntamente al menos 8 biomarcadores presentes en orina o sus variantes transcripcionales o post-traduccionales.Another embodiment of the invention relates to the joint use of the peptide sequences derived from the CATD, GSTP1, RETBP, STIP1, AMYS, APOA1, PRDX2 and TTHY biomarkers or their transcriptional or post-translational variants, where the biomarkers are present in urine. In another embodiment, at least 4 biomarkers present in urine or their transcriptional or post-translational variants are used together. In another embodiment, at least 6 biomarkers present in urine or their transcriptional or post-translational variants are used together. In another embodiment, at least 7 biomarkers present in urine or their transcriptional or post-translational variants are used together. In another embodiment, at least 8 biomarkers present in urine or their transcriptional or post-translational variants are used together.
Otra realización particular de Ia invención se refiere a un kit para llevar a cabo el método previamente descrito, siendo empleado dicho kit para detectar Ia presencia de BTCC, determinar el estadio o severidad de este cáncer, evaluar Ia falta de enfermedad después de resección quirúrgica, establecer un diagnóstico y/o pronóstico de este cáncer y/o monitorizar el efecto del tratamiento administrado a un individuo que padece dicho cáncer.Another particular embodiment of the invention relates to a kit for carrying out the previously described method, said kit being used to detect the presence of BTCC, determine the stage or severity of this cancer, evaluate the lack of disease after surgical resection, establish a diagnosis and / or prognosis of this cancer and / or monitor the effect of the treatment administered to an individual suffering from said cancer.
El kit puede comprender un contenedor donde se ubican los agentes que se unen a los biomarcadores presentes en orina e instrucciones de su uso para determinar el estadio de BTCC. Además, el kit puede comprender un soporte compartimentalizado para ubicar uno o más contenedores, por ejemplo viales, tubos o similares. Por ejemplo, un contenedor puede contener una sonda que es o puede estar marcada para ser posteriormente detectada. La sonda puede ser un anticuerpo o polinucleótido específico para una proteína de cáncer de vejiga o un gen de cáncer de vejiga, respectivamente. Alternativamente, el kit puede comprender una sonda de espectrometría de masas (MS). El kit puede incluir también contenedores que contengan nucleótido(s) para amplificación o silenciamiento de una secuencia de ácido nucleico diana, y/o un contenedor que contenga medios reportadores, tal como una proteína ligante de biotina, como por ejemplo, avidina o estreptavidina, unida a una marca detectable, por ejemplo, una marca enzimática, fluorescente o radioisotópica. El kit puede incluir Ia secuencia de aminoácidos completa de los biomarcadores de cáncer de vejiga o parte de ella, o una molécula de ácido nucleico que codifica tal secuencia aminoacídica. El kit de Ia invención comprende típicamente el contenedor descrito anteriomente y uno o más contenedores que comprenden materiales deseables desde un punto de vista comercial y de usuario, incluyendo tampones, diluyentes, filtros, agujas, jeringas e instrucciones para el uso. Además, puede contener una etiqueta en el contenedor para indicar que Ia composición se usa para una aplicación terapéutica o no terapéutica.The kit can comprise a container where the agents that bind to the biomarkers present in urine are located and instructions for its use to determine the BTCC stage. In addition, the kit may comprise a compartmentalized support for locating one or more containers, for example vials, tubes or the like. For example, a container may contain a probe that is or may be marked for later detection. The probe may be an antibody or polynucleotide specific for a bladder cancer protein or a bladder cancer gene, respectively. Alternatively, the kit may comprise a mass spectrometry (MS) probe. The kit may also include containers containing nucleotide (s) for amplification or silencing of a target nucleic acid sequence, and / or a container containing reporter media, such as a biotin binding protein, such as avidin or streptavidin, attached to a detectable label, for example, an enzymatic, fluorescent or radioisotopic label. The kit can include the complete amino acid sequence of bladder cancer biomarkers or part of it, or a nucleic acid molecule that encodes such an amino acid sequence. The kit of the invention typically comprises the container described above and one or more containers comprising desirable materials from a commercial and user point of view, including buffers, diluents, filters, needles, syringes and instructions for use. In addition, it may contain a label on the container to indicate that the composition is used for a therapeutic or non-therapeutic application.
Otra realización de Ia invención se refiere a un kit para llevar a cabo el método previamente descrito que comprende un biochip.Another embodiment of the invention relates to a kit for carrying out the previously described method comprising a biochip.
Otra realización de Ia invención se refiere a un kit que comprende un biochip, donde el biochip comprende anticuerpos para Ia detección de biomarcadores o sus variantes transcripcionales o post-traduccionales.Another embodiment of the invention relates to a kit comprising a biochip, wherein the biochip comprises antibodies for the detection of biomarkers or their transcriptional or post-translational variants.
Como se ha indicado anteriormente, el método de Ia invención comprende Ia monitorización del estadio del carcinoma de vejiga mediante Ia cuantificación de proteínas solubles expresadas diferencialmente en una muestra de orina a través de anticuerpos específicos. Como será apreciado por un experto en Ia materia, se contempla cualquier medio para identificar y cuantificar estas proteínas.As indicated above, the method of the invention comprises monitoring the stage of bladder carcinoma by quantifying soluble proteins differentially expressed in a urine sample through specific antibodies. As will be appreciated by an expert in the field, any means to identify and quantify these proteins is contemplated.
Típicamente, Ia detección y cuantificación comprende espectrometría o inmunoensayo. La espectrometría es generalmente espectrometría de masas de desorción/ionización mediante láser de superficie (SELDI) o espectrometría de masas de desorción/ionización mediante láser asistida por matriz (MALDI). Los inmunoensayos utilizan anticuerpos no marcados (anticuerpos primarios) y anticuerpos marcados (anticuerpos secundarios). Estas técnicas incluyen western blot, ELISA (ensayo inmunoabsorbente unido a enzima), RIA (radioinmunoensayo), EIA competitivo (inmunoensayo enzimático competitivo), DAS-ELISA (ELISA sandwich de doble anticuerpo), técnicas inmunocitoquímicas o inmunohistoquímicas, técnicas basadas en el uso de biochips o microarrays de proteína que incluyen anticuerpos específicos, ensayos basados en Ia precipitación de oro coloidal en formatos tales como dipsticks; o técnicas de afinidad cromatográfica, ensayos de unión a ligando y ensayos de unión a lectina. Las realizaciones preferidas de este aspecto de Ia invención son microarrays de proteínas y doble anticuerpo sandwich ELISA (DAS-ELISA).Typically, the detection and quantification comprises spectrometry or immunoassay. The spectrometry is generally desorption / ionization mass spectrometry by surface laser (SELDI) or matrix assisted laser desorption / ionization mass spectrometry (MALDI). Immunoassays use unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies). These techniques include western blot, ELISA (enzyme-linked immunoabsorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical or immunohistochemical techniques, techniques based on use of biochips or protein microarrays that include specific antibodies, tests based on the precipitation of colloidal gold in formats such as dipsticks; or chromatographic affinity techniques, ligand binding assays and lectin binding assays. Preferred embodiments of this aspect of the invention are protein microarrays and double ELISA sandwich antibody (DAS-ELISA).
Las proteínas pueden cuantificarse con anticuerpos tales como, por ejemplo: anticuerpos monoclonales, anticuerpos policlonales, ya sean fragmentos intactos o recombinantes de estos, combibodies y fragmentos de anticuerpos Fab o scFv, específicos para proteínas. Estos anticuerpos pueden ser de origen humano, humanizados o de origen animal. Por otro lado, pueden ser marcados o no marcados y pueden usarse en un amplia variedad de ensayos. Las moléculas marcadoras que pueden usarse para marcar anticuerpos incluyen radionúclidos, enzimas, fluoróforos, reactivos quimioluminiscentes, sustratos enzimáticos o cofactores, inhibidores enzimáticos, partículas, colorantes y derivados. Cuanto más alta es Ia especificidad de Ia unión del anticuerpo, menor es Ia concentración requerida para que el antígeno pueda detectarse.Proteins can be quantified with antibodies such as, for example: monoclonal antibodies, polyclonal antibodies, either intact or recombinant fragments thereof, combibodies and Fab or scFv antibody fragments, specific for proteins. These antibodies can be of human origin, humanized or of animal origin. On the other hand, they can be labeled or unlabeled and can be used in a wide variety of assays. Marker molecules that can be used to label antibodies include radionuclides, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes and derivatives. The higher the specificity of the antibody binding, the lower the concentration required for the antigen to be detected.
Como ejemplo de uno de los posibles formatos de este ensayo, un anticuerpo monoclonal o policlonal, un fragmento del mismo, o una combinación de estos ellos se ancla a Ia superficie de un soporte de fase sólida; se contacta con Ia muestra a analizar y se incuba durante un tiempo específico en condiciones apropiadas para Ia formación de complejos antígeno-anticuerpo. Posteriormente al lavado en condiciones apropiadas para eliminar complejos no específicos, se incuba un reactivo indicador, que consiste en un anticuerpo monoclonal o policlonal, un fragmento del mismo, o una combinación de ellos, ligado a una molécula generadora de una señal con los complejos antígeno-anticuerpo en condiciones apropiadas de tiempo y temperatura. Se detecta Ia presencia de una proteína seleccionadas entre las proteínas de Ia invención en Ia muestra a analizar y, si está presente, se cuantifica y se mide Ia señal generada. Para evitar variación de señales debida a las diferencias en Ia concentración total de proteína entre muestras, todas las medidas son normalizadas.As an example of one of the possible formats of this assay, a monoclonal or polyclonal antibody, a fragment thereof, or a combination thereof is anchored to the surface of a solid phase support; The sample to be analyzed is contacted and incubated for a specific time under appropriate conditions for the formation of antigen-antibody complexes. After washing under appropriate conditions to remove non-specific complexes, an indicator reagent, which consists of a monoclonal or polyclonal antibody, a fragment thereof, or a combination thereof, is linked to a signal generating molecule with the antigen complexes. -antibody under appropriate conditions of time and temperature. The presence of a protein selected from the proteins of the invention is detected in the sample to be analyzed and, if present, the generated signal is quantified and measured. To avoid signal variation Due to the differences in the total protein concentration between samples, all measurements are normalized.
A continuación, se describe el método generalizado para obtener y analizar el contenido total de proteína de muestras de orina humana (en adelante referidas como muestras). El método comprende el procesado de las muestras, el uso de electroforesis 2D para separar proteínas de Ia muestra, Ia selección de proteínas expresadas diferencialmente mediante análisis de imagen y estadística de diferentes muestras y el uso de una o más proteínas expresadas diferencialmente para generar anticuerpos específicos para usarse como marcadores de cáncer de vejiga.Next, the generalized method for obtaining and analyzing the total protein content of human urine samples (hereinafter referred to as samples) is described. The method includes the processing of the samples, the use of 2D electrophoresis to separate proteins from the sample, the selection of differentially expressed proteins by image and statistical analysis of different samples and the use of one or more differentially expressed proteins to generate specific antibodies. to be used as markers of bladder cancer.
El análisis proteómico comparativo se llevó a cabo entre muestras obtenidas de individuos sanos (controles) y pacientes diagnosticados con BTCC (Ta, T1 -grado bajo, T1 -grado alto y T2) en un intento para identificar proteínas expresadas diferencialmente en los diversos estadios del cáncer y durante Ia progresión del mismo. Se eligieron las proteínas cuya expresión diferencial cambió más de dos veces de manera reproducible. Estas fueron identificadas mediante huella de Ia masa peptídica usando espectrometría de masas y búsqueda en bases de datos.Comparative proteomic analysis was carried out between samples obtained from healthy individuals (controls) and patients diagnosed with BTCC (Ta, T1-low grade, T1-high grade and T2) in an attempt to identify differentially expressed proteins in the various stages of the cancer and during its progression. Proteins whose differential expression changed more than twice reproducibly were chosen. These were identified by fingerprint of the peptide mass using mass spectrometry and database search.
La presente invención se ilustra mediante los siguientes ejemplos. Dichos ejemplos no deben considerarse limitativos de Ia invención.The present invention is illustrated by the following examples. Said examples should not be considered as limiting the invention.
I. Identificación de proteínas expresadas diferencialmente Con el fin de identificar proteínas expresadas diferencialmente durante Ia progresión de cáncer de vejiga, se compararon los perfiles de proteínas de orinas sanas con aquellas de pacientes con tumor de vejiga en estadio temprano y avanzado utilizando una aproximación proteómica.I. Identification of differentially expressed proteins In order to identify differentially expressed proteins during bladder cancer progression, healthy urine protein profiles were compared with those of patients with early and advanced bladder tumor using a proteomic approach.
Se obtuvieron muestras de orina (150 en total) de individuos sanos y pacientes de carcinoma transicional de vejiga de las unidades de Urología de hospitales pertenecientes a Ia Red de Ia Salud Pública Española. Estas muestras se clasifican como sigue: a) No Carcinoma (63 muestras) incluyendo:Urine samples (150 in total) were obtained from healthy individuals and patients with transitional bladder carcinoma from the Urology units of hospitals belonging to the Spanish Public Health Network. These samples are classified as follows: a) No Carcinoma (63 samples) including:
- Controles (CV1 32 muestras): pacientes que habían tenido cáncer de vejiga a los que se aplicó una resección del tumor vesical y estaban siendo controlados (donde Ia cistoscopia negativa no mostró otras alteraciones).- Controls (CV 1 32 samples): patients who had had bladder cancer to whom a bladder tumor resection was applied and were being controlled (where the negative cystoscopy showed no other alterations).
- Pacientes de otras enfermedades del tracto urológico (31 muestras, incluyendo entre otras hiperplasia benigna de próstata, cáncer de próstata). b) Pacientes de BTCC (87 muestras): pacientes diagnosticados con Ia enfermedad en diferentes estadios de desarrollo incluyendo:- Patients of other urological tract diseases (31 samples, including among other benign prostatic hyperplasia, prostate cancer). b) BTCC patients (87 samples): patients diagnosed with the disease at different stages of development including:
-Ta (21 muestras)-Ta (21 samples)
-T1 -grado bajo (29 muestras)-T1 -Low grade (29 samples)
-T1 -grado alto (19 muestras)-T1 - high grade (19 samples)
-T2 (18 muestras)-T2 (18 samples)
El grupo de muestras de BTCC iba acompañado de una biopsia que es Ia clave para su clasificación en los estadios de desarrollo de cáncer de vejiga.The BTCC sample group was accompanied by a biopsy that is the key to its classification in the stages of development of bladder cancer.
A. Procesado de las muestras de orina y separación de las proteínas. Se congelaron muestras de orina a -8O0C y se transfirieron al laboratorio en hielo seco sin romper Ia cadena de frío. Se mantuvieron las muestras a - 8O0C hasta que se procesaron. Las muestras fueron muy heterogéneas, desde orinas amarillas con poco color hasta orinas rojas con grumos de sangre, transparentes o conteniendo tejidos en suspensión. El volumen total también fue muy variable, desde 5 hasta 100 mL La concentración de proteína de las muestras oscilaba entre 20 μg/mL y 2 mg/mL (siendo el valor medio 150 μg/mL). Para obtener el contenido de proteína, las muestras de orina se descongelaron en hielo y se centrifugaron a 2000xg durante 5 minutos a 40C. El sobrenadante se utilizó para determinar Ia concentración de proteína. Se calculó el volumen necesario para precipitar 100 μg de proteína, teniendo en cuenta que el rendimiento de Ia precipitación con ácido tricloroacético (TCA) era del 75%. El resto de Ia muestra de orina se congeló otra vez a -8O0C y se almacenó para posteriores electroforesis 2D (en caso necesario). Tras mezclar el TCA y Ia orina durante 1 hora en hielo, se centrifugó a 16000xg durante 20 minutos a 4 0C para obtener el pellet de las proteínas precipitadas. Este pellet se lavó con acetona almacenada a -2O0C, y se secó por evaporación del disolvente. Para llevar a cabo los experimentos de electroforesis bidimensionales (2D), Ia primera dimensión fue IEF (isoelectroenfoque) donde las proteínas se separaron en función de su carga (pl); Ia segunda dimensión consistió en SDS-PAGE, donde las proteínas se separaron en función de su peso molecular. Para llevar a cabo Ia primera dimensión, los pellets secados de proteínas se resuspendieron en 450 μl de tampón de rehidratación (Urea 7M, Tiourea 2M, CHAPS 2%, tampón IPG 2%, azul de bromofenol 0.002%) durante 1 hora a temperatura ambiente. El tampón IPG (Amersham, ref# 17-600-88) se usó de manera que el IEF se llevó a cabo en un rango de 3-10. Para el IEF, Ettan™ IPGfor™ Isoelectric Focusing System de Amersham se siguieron todas las recomendaciones del fabricante. El IEF se llevó a cabo en geles de gradiente de pH inmovilizado, conocidos como IPG strips, comercializados por Amersham (ref# 17-6002- 45). Las proteínas solubilizadas se enfocaron en el ,gel de Ia primera dimensión tras un periodo de 16 horas de rehidratación activa del gel a 30 voltios. Entonces se comenzó a subir el voltaje hasta alcanzar los 8000 voltios, sin rebasar nunca una intensidad de corriente de 50 μA por cada gel. El IEF se paró aproximadamente a los 90000 voltios-hora. Para Ia segunda dimensión, se polimerizaron geles de acrilamida al 12.5% de 26 x 20 cm en el laboratorio mediante Ettan DALT twelve GeI Caster de Amersham. Los geles se corrieron mediante Large Format Vertical System siguiendo todos los protocolos del fabricante hasta que el frente de electroforesis salió del gel. Los geles se tiñeron con nitrato de plata, utilizando el kit de tinción de Amersham (17-1150-01), siguiendo el protocolo del fabricante. Los geles se secaron y se almacenaron para el análisis de imagen posterior de spots proteicos (FIGURA 1A). Para algunas muestras de orina se corrió más de un 2D-gel.A. Processing of urine samples and protein separation. Urine samples were frozen at -8O 0 C and transferred to the laboratory on dry ice without breaking the cold chain. Samples were kept at -8O 0 C until processed. The samples were very heterogeneous, from yellow urine with little color to red urine with lumps of blood, transparent or containing suspended tissues. The total volume was also very variable, from 5 to 100 mL. The protein concentration of the samples ranged from 20 μg / mL to 2 mg / mL (the average value being 150 μg / mL). To obtain the protein content, the urine samples were thawed on ice and centrifuged at 2000xg for 5 minutes at 4 0 C. The supernatant was used to determine the protein concentration. The volume necessary to precipitate 100 μg of protein was calculated, taking into account that the yield of the precipitation with trichloroacetic acid (TCA) was 75%. The rest of the urine sample was frozen again at -8O 0 C and stored for subsequent 2D electrophoresis (if necessary). After mixing the TCA and the urine for 1 hour on ice, it was centrifuged at 16000xg for 20 minutes at 4 0 C to obtain the pellet of the precipitated proteins This pellet was washed with acetone stored at -2O 0 C, and dried by evaporation of the solvent. To carry out the two-dimensional electrophoresis (2D) experiments, the first dimension was IEF (isoelectric focusing) where the proteins were separated according to their charge (pl); The second dimension consisted of SDS-PAGE, where the proteins were separated according to their molecular weight. To carry out the first dimension, the dried protein pellets were resuspended in 450 μl of rehydration buffer (Urea 7M, Tiourea 2M, CHAPS 2%, IPG buffer 2%, bromophenol blue 0.002%) for 1 hour at room temperature . The IPG buffer (Amersham, ref # 17-600-88) was used so that the IEF was carried out in a range of 3-10. For the IEF, Amersham's Ettan ™ IPGfor ™ Isoelectric Focusing System followed all the manufacturer's recommendations. The IEF was carried out on immobilized pH gradient gels, known as IPG strips, marketed by Amersham (ref # 17-6002-45). The solubilized proteins focused on the gel of the first dimension after a period of 16 hours of active rehydration of the gel at 30 volts. Then the voltage began to rise to 8000 volts, never exceeding a current intensity of 50 μA for each gel. The IEF stopped at approximately 90000 volt-hours. For the second dimension, 12.5% acrylamide gels of 26 x 20 cm were polymerized in the laboratory using Ettan DALT twelve GeI Caster from Amersham. The gels were run by Large Format Vertical System following all the manufacturer's protocols until the electrophoresis front left the gel. The gels were stained with silver nitrate, using the Amersham staining kit (17-1150-01), following the manufacturer's protocol. The gels were dried and stored for subsequent image analysis of protein spots (FIGURE 1A). For some urine samples, more than one 2D gel was run.
B. Análisis de los spots en los qeles.B. Analysis of the spots in the qeles.
Cada gel se escaneó para obtener una mapa de spots proteicos para el análisis de imagen. El software Progenesis PG220 de Nonlinear Dynamics (UK) se utilizó para analizar archivos de imágenes en formato 300 dpi (puntos por pulgada) y 8 bits/canal. Para incrementar Ia resolución el análisis se llevó a cabo en áreas discretas de los geles. De cada una de las 4 áreas, llamadas A (FIGURA 1 B), K (FIGURA 1C), R (FIGURA 1 D) y S (FIGURA 1E), los mejores geles fueron seleccionados y escaneados para análisis de imagen. El software Progenesis PG220 transforma Ia información de Ia imagen plana en una imagen tridimensional, donde Ia intensidad de cada spot correlaciona con su volumen y con Ia cantidad relativa de proteína correspondiente en Ia orina del individuo. Mediante este software se obtuvieron unas tablas de intensidades de cada spot en cada gel. Estos datos crudos fueron Ia base de posteriores análisis estadísticos.Each gel was scanned to obtain a map of protein spots for image analysis. The Nonlinear Dynamics Progenesis PG220 software (UK) was used to analyze image files in 300 dpi format (dots per inch) and 8 bits / channel. To increase the resolution the analysis was carried out in discrete areas of the gels. From each of the 4 areas, called A (FIGURE 1 B), K (FIGURE 1C), R (FIGURE 1 D) and S (FIGURE 1E), the best gels were selected and scanned for image analysis. Progenesis PG220 software transforms the information of the flat image into a three-dimensional image, where the intensity of each spot correlates with its volume and with the relative amount of corresponding protein in the urine of the individual. Through this software we obtained some intensity tables of each spot in each gel. These raw data were the basis of subsequent statistical analyzes.
Para llevar a cabo el análisis estadístico de cada spot individual y comparar su intensidad en dos grupos diferentes (por ejemplo CV y Ta) hubo que comprobar que estos conjuntos de datos seguía una distribución normal. Para comparar los spots pertenecientes a dos grupos se aplicó un test t de Student, y se obtuvo el valor p: este era el margen de error teórico de Ia asignación de un valor de intensidad de este spot a un subgrupo o a otro. Se seleccionaron solamente aquellos spots con valores de p <0.05. Se calculó el fold change, ratio media de dichos spots referida a 1) el volumen total de spots de Ia muestra, esto es el contenido total de proteína, o a 2) una proteína expresada de manera constante, cuya cantidad es constante en todas las muestras; o a 3) una segunda proteína expresada diferencialmente de Ia misma muestra.To carry out the statistical analysis of each individual spot and compare its intensity in two different groups (for example CV and Ta) it was necessary to verify that these data sets followed a normal distribution. To compare the spots belonging to two groups, a Student's t-test was applied, and the p-value was obtained: this was the theoretical margin of error of the assignment of an intensity value of this spot to a subgroup or another. Only those spots with values of p <0.05 were selected. The fold change was calculated, the average ratio of said spots referring to 1) the total volume of spots in the sample, that is, the total protein content, or 2) a protein expressed constantly, the quantity of which is constant in all samples ; or a 3) a second differentially expressed protein of the same sample.
También se tuvo en cuenta el valor n o número de muestras. La identificación de dichos spots se llevó a cabo mediante espectroscopia MALDI-TOF (espectrometría de masas de desorción/ionización mediante láser asistida por matriz). Aquellas muestras de orina cuyas electroforesis 2D habían mostrado spots estadísticamente significativos fueron sometidas de nuevo a electroforesis 2D y los spots fueron recortados del gel. Las proteínas se sometieron a digestión y se analizaron mediante un espectrómetro MALDI- TOF. La huella de Ia masa peptídica permitió Ia identificación de CATD, GSTP1 , RETBP, STIP1 , AMYS, APOA1 , PRDX2 y TTHY (ver tabla 1). Así, estas 8 proteínas, para las cuales se había demostrado que se expresaban díferencialmente en pacientes diagnosticados con BTCC, se identificaron como marcadores biológicos con valor significativo para el diagnóstico, pronóstico monitorización y tratamiento de Ia enfermedad.The value not number of samples was also taken into account. The identification of these spots was carried out by MALDI-TOF spectroscopy (desorption / ionization mass spectrometry by matrix-assisted laser). Those urine samples whose 2D electrophoresis had shown statistically significant spots were again subjected to 2D electrophoresis and the spots were trimmed from the gel. The proteins were digested and analyzed by a MALDI-TOF spectrometer. The footprint of the peptide mass allowed the identification of CATD, GSTP1, RETBP, STIP1, AMYS, APOA1, PRDX2 and TTHY (see table 1). So, These 8 proteins, for which it had been shown that they were expressed diferentially in patients diagnosed with BTCC, were identified as biological markers with significant value for the diagnosis, prognosis, monitoring and treatment of the disease.
Tabla 1Table 1
Figure imgf000024_0001
Figure imgf000024_0001
Por ejemplo, Ia FIGURA 2 muestra el spot R211 en el área R de un gel de electroforesis bidimensional (2D) obtenido de una muestra de orina de un individuo sano (FIGURA 2A), de un paciente de cáncer en el estadio Ta (FIGURA 2B), de un paciente de cáncer en el estadio T1 -grado bajo (FIGURA 2C), de un paciente de cáncer en el estadio T1 -grado alto (FIGURA 2D) y de un paciente de cáncer en el estadio T2 (FIGURA 2E). Puede observarse que Ia intensidad del spot R211 está claramente disminuida en las muestras de pacientes con cáncer en distintos estadios, cuando se compara con Ia muestra de orina sana. Se demostró que estas diferencias eran significativas después de llevar a cabo un análisis estadístico con Progenesis PG220 software. La FIGURA 3 muestra Ia intensidad del spot R211 en muestras CV, Ta, T1 -grado bajo, T1 -grado alto y T2. El número de muestras para cada grupo está indicado entre paréntesis. La FIGURA 4 muestra Ia robustez de Ia medición de Ia intensidad para el spot R211 en diferentes muestras de orina. Ésta se expresa como el porcentaje de geles que mantienen Ia presencia o ausencia del spot R211 en cada gel analizado. El número de muestras para cada grupo se indica entre paréntesis.For example, FIGURE 2 shows the spot R211 in the area R of a two-dimensional electrophoresis gel (2D) obtained from a urine sample of a healthy individual (FIGURE 2A), of a cancer patient in stage Ta (FIGURE 2B ), of a cancer patient in stage T1 - low grade (FIGURE 2C), of a cancer patient in stage T1 - high degree (FIGURE 2D) and of a cancer patient in stage T2 (FIGURE 2E). It can be seen that the intensity of the R211 spot is clearly diminished in the samples of cancer patients at different stages, when compared with the healthy urine sample. It was shown that these differences were significant after carrying out a statistical analysis with Progenesis PG220 software. FIGURE 3 shows the intensity of the R211 spot in CV, Ta, T1-low grade, T1-high grade and T2 samples. The number of samples for each group is indicated in parentheses. FIGURE 4 shows the robustness of the measurement of the intensity for spot R211 in different urine samples. This is expressed as the percentage of gels that maintain the presence or absence of the R211 spot in each gel analyzed. The number of samples for each group is indicated in brackets.
En Ia tabla 2 se muestra el valor de p y el fold change de los análisis estadísticos para el spot R211 en muestras de diferentes estadios de cáncer en comparación con una orina de un individuo sin cáncer (CV). El fold change del spot R211 fue normalizado por el volumen total de spots (TSV) para cada gel individual.Table 2 shows the value of p and the fold change of the statistical analyzes for the R211 spot in samples from different stages of cancer compared to a urine from an individual without cancer (CV). The fold change of the R211 spot was normalized by the total volume of spots (TSV) for each individual gel.
Tabla 2Table 2
Figure imgf000025_0001
Figure imgf000025_0001
II. Desarrollo de anticuerpos para las proteínas encontradas en Ia muestra de orinaII. Development of antibodies to the proteins found in the urine sample
Se ensayó Ia reactividad y sensibilidad de los anticuerpos policlonales y monoclonales (ya fueran obtenidos comercialmente o generados mediante protocolos de inmunización). El método generalmente comprendió Ia preparación de una curva estándar ("dosis respuesta") para Ia proteína a monitorizar. Como se ha indicado anteriormente, el desarrollo del estadio del cáncer puede también evaluarse monitorizando las concentraciones de distintas proteínas características de estadio en Ia muestra.The reactivity and sensitivity of polyclonal and monoclonal antibodies were tested (either commercially obtained or generated by immunization protocols). The method generally comprised the preparation of a standard curve ("dose response") for the protein to be monitored. As indicated above, the development of the cancer stage can also be evaluated by monitoring the concentrations of different stage characteristic proteins in the sample.
A. Protocolos de inmunización 1. Anticuerpos policlonalesA. Immunization protocols 1. Polyclonal antibodies
Se puede obtener antisuero policlonal contra una proteína dada usando metodología estándar, tal como Ia descrita en numerosos textos disponibles y conocidos por los expertos en Ia materia. En este ejemplo se inmunizan conejos machos de raza New Zealand White con preparaciones de proteína primero en presencia de coadyuvante completo de Freud (Gibco, Grand Island, N.Y.) y después cada mes con Ia proteína en presencia del coadyuvante incompleto durante tres meses. Como antisuero policlonal se utilizan sueros de conejo y de ratón previamente a Ia fusión y demuestran ser reactivos con las preparaciones de proteína mediante Ia técnica estándar de western blot.Polyclonal antiserum can be obtained against a given protein using standard methodology, such as that described in numerous available texts and known to those skilled in the art. In this example, male New Zealand White rabbits are immunized with protein preparations first in the presence of Freud's complete adjuvant (Gibco, Grand Island, NY) and then every month with the protein in the presence of Incomplete adjuvant for three months. As polyclonal antiserum, rabbit and mouse sera are used prior to fusion and they are shown to be reactive with protein preparations by means of the standard western blot technique.
2. Generación de proteínas recombinantes2. Generation of recombinant proteins
Se clonan y se expresan las proteínas encontradas en los geles en cantidades demasiado pequeñas para Ia inmunización en E.coli usando el vector de expresión pET28b(+). Los extractos crudos se obtienen en condiciones ya descritas (Boronat A., et al., J Bacteriol 1981 , 147:181-85) y se corren en un gel SDS-PAGE. Se recortan del gel las proteínas recombinantes y se liberan de Ia acrilamida. Para Ia generación de anticuerpos para las proteínas recombinantes, las proteínas liberadas se usan directamente para inmunizar conejos como se ha descrito anteriormente.The proteins found in the gels are cloned and expressed in amounts too small for immunization in E.coli using the expression vector pET28b (+). The crude extracts are obtained under conditions already described (Boronat A., et al., J Bacteriol 1981, 147: 181-85) and run on an SDS-PAGE gel. Recombinant proteins are trimmed from the gel and released from acrylamide. For the generation of antibodies to recombinant proteins, the released proteins are used directly to immunize rabbits as described above.
B. Experimentos de western blotB. Western blot experiments
Muestras de proteína (20 μg de proteína total) se mezclaron con un tampón de carga SDS-PAGE suplementado con 5% β-mercaptoetanol y se incubaron a 1000C durante 5 min, antes de ser cargados en un gel del 6% de poliacrilamida. Mediante electroforesis, las proteínas se transfirieron a membranas de nitrocelulosa. Se corrieron geles y se transfirieron por duplicado. Una membrana se incubó con anticuerpos contra las 40 proteínas de Ia invención (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA.) mientras que Ia segunda membrana se incubó con un anticuerpo contra actina (Amersham, Little Chalfont, UK) como control de carga de proteína. Finalmente, las membranas se hibridaron con un segundo anticuerpo conjugado con peroxidasa (Amersham) y se detectó Ia señal quimioluminiscente usando el sistema ECL (Amersham) con una película quimioluminiscente de alta sensibilidad (Hyperfilm ECL, Amersham). III. Validación de muestras test de orina usando experimentos de western blot. Diversos biomarcadores fueron probados en 40 muestras ciegas de orina, que comprendían: a) No Carcinomas (12 muestras) b) BTCCs incluyendo: Ta (8 muestras), T1 -grado bajo (6 muestras), T1 -grado alto (6 muestras) y T2 (8 muestras).Protein samples (20 ug total protein) were mixed with a loading buffer SDS-PAGE supplemented with 5% β-mercaptoethanol and incubated at 100 0 C for 5 min, before being loaded on a 6% gel polyacrylamide . By electrophoresis, the proteins were transferred to nitrocellulose membranes. Gels were run and transferred in duplicate. A membrane was incubated with antibodies against the proteins of the invention (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA.) While the second membrane was incubated with an antibody against actin (Amersham, Little Chalfont, UK) as protein load control. Finally, the membranes were hybridized with a second peroxidase-conjugated antibody (Amersham) and the chemiluminescent signal was detected using the ECL system (Amersham) with a high sensitivity chemiluminescent film (Hyperfilm ECL, Amersham). III. Validation of urine test samples using western blot experiments. Several biomarkers were tested in 40 blind urine samples, which included: a) No Carcinomas (12 samples) b) BTCCs including: Ta (8 samples), T1-low grade (6 samples), T1-high grade (6 samples) and T2 (8 samples).
Teniendo en cuenta que el resultado de Ia validación es continuo entre los dos estados clínicos posibles (sano, enfermo), es decir, que puede producirse un solapamiento entre Ia partición real en sano/enfermo, y Ia obtenida a través de un punto de corte elegido teórico, se utilizaron las curvas ROC (Receiver Operating Characterístic) para evaluar Ia bondad de Ia prueba. Las curvas ROC proporcionan una representación global de Ia exactitud diagnóstica mediante una representación de los pares (1 -especificidad, sensibilidad) obtenidos al considerar todos los posibles valores de corte de Ia prueba. Para cada valor de corte, los valores de sensibilidad y especificidad se obtienen mediante las siguientes fórmulas:Taking into account that the result of the validation is continuous between the two possible clinical states (healthy, sick), that is, that an overlap can occur between the real partition in healthy / sick, and that obtained through a cut-off point chosen theoretically, the ROC (Receiver Operating Characteristic) curves were used to evaluate the goodness of the test. The ROC curves provide a global representation of the diagnostic accuracy by means of a representation of the pairs (1 -specificity, sensitivity) obtained when considering all possible cut-off values of the test. For each cut-off value, the sensitivity and specificity values are obtained by the following formulas:
Sensibilidad = Verdaderos positivosSensitivity = True Positive
(Verdaderos positivos + Falsos negativos)(True positive + False negative)
Especificidad = Verdaderos negativosSpecificity = True Negatives
(Verdaderos negativos + Falsos positivos)(True negative + False positive)
Los valores de las curvas ROC de las distintas combinaciones se muestran en Ia tabla 3.The values of the ROC curves of the different combinations are shown in Table 3.
Tabla 3Table 3
Figure imgf000027_0001
DESCRIPCIÓN DE LAS FIGURAS
Figure imgf000027_0001
DESCRIPTION OF THE FIGURES
FIGURA 1 : GeI de electroforesis bidimensional (2D) obtenido de una muestra de orina y mostrando las 4 áreas estudiadas A, K, R y S (FIGURA 1A), el área A (FIGURA 1 B), el área K (FIGURA 1C), el área R (FIGURA 1D) y el área S (FIGURA 1 E).FIGURE 1: Two-dimensional (2D) electrophoresis GeI obtained from a urine sample and showing the 4 studied areas A, K, R and S (FIGURE 1A), area A (FIGURE 1 B), area K (FIGURE 1C) , area R (FIGURE 1D) and area S (FIGURE 1 E).
FIGURA 2: Spot R211 en el área R de un gel de electroforesis bidimensional (2D) obtenido de muestras de orina de un individuo sano (FIGURA 2A), de un paciente de cáncer en el estadio Ta (FIGURA 2B), de un paciente de cáncer en el estadio T1 -grado bajo (T1-LG, FIGURA 2C), de un paciente de cáncer en el estadio T1 -grado alto (T1-HG, FIGURA 2D) y de un paciente de cáncer en el estadio T2 (FIGURA 2E).FIGURE 2: Spot R211 in the area R of a two-dimensional (2D) electrophoresis gel obtained from urine samples from a healthy individual (FIGURE 2A), from a cancer patient in stage Ta (FIGURE 2B), from a patient of T1-low grade cancer (T1-LG, FIGURE 2C), of a T1-high grade cancer patient (T1-HG, 2D FIGURE) and a T2 stage cancer patient (FIGURE 2E ).
FIGURA 3: Intensidad del spot R211 en muestras CV, Ta, T1-grado bajo (T1- LG), T1 -grado alto (T1-HG) y T2. El número de muestras para cada grupo se indica entre paréntesis.FIGURE 3: Intensity of the R211 spot in CV, Ta, T1-low grade (T1-LG), T1-high grade (T1-HG) and T2 samples. The number of samples for each group is indicated in brackets.
FIGURA 4: Robustez de Ia medición de intensidad para el spot R211 en diferentes muestras de orina. Ésta se expresa como el porcentaje de geles que mantinenen Ia presencia o ausencia del spot R211 en cada gel analizado. El número de muestras para cada grupo se indica entre paréntesis. FIGURE 4: Robustness of the intensity measurement for the R211 spot in different urine samples. This is expressed as the percentage of gels that maintain the presence or absence of the R211 spot in each gel analyzed. The number of samples for each group is indicated in brackets.

Claims

REIVINDICACIONES
1. Una combinación de biomarcadores para Ia detección de BTCC que comprende los biomarcadores CATD (ID SEC 1 , ID SEC 2), GSTP1 (ID SEC 3), RETBP (ID SEC 4) y STIP1 (ID SEC 5) o sus variantes transcripcionales o post-traduccionales.1. A combination of biomarkers for the detection of BTCC comprising the biomarkers CATD (SEQ ID 1, SEQ ID 2), GSTP1 (SEQ ID 3), RETBP (ID SEQ 4) and STIP1 (SEQ ID 5) or its transcriptional variants or post-translational.
2. Combinación según Ia reivindicación 1 que comprende además los biomarcadores AMYS (ID SEC 6), APOA1 (ID SEC 7) o sus variantes transcripcionales o post-traduccionales.2. A combination according to claim 1, further comprising the AMYS biomarkers (SEQ ID 6), APOA1 (SEQ ID 7) or their transcriptional or post-translational variants.
3. Combinación según Ia reivindicación 2 que comprende además el biomarcador PRDX2 (ID SEC 8) o sus variantes transcripcionales o post- traduccionales.3. Combination according to claim 2, further comprising the PRDX2 biomarker (SEQ ID 8) or its transcriptional or post-translational variants.
4. Combinación según Ia reivindicación 2 ó 3 que comprende además el biomarcador TTHY (ID SEC 9) o sus variantes transcripcionales o post- traduccionales.4. A combination according to claim 2 or 3, further comprising the TTHY biomarker (SEQ ID 9) or its transcriptional or post-translational variants.
5. Un método in vitro no invasivo que comprende: a) detectar y cuantificar Ia combinación de biomarcadores definida en Ia reivindicación 1 en una muestra test de orina de un individuo; y b) comparar el valor de expresión obtenido en a) en Ia muestra test de orina con el correspondiente valor estándar en orina normal, donde variaciones en el valor obtenido en a) respecto al valor estándar en orina normal son indicativas de carcinoma transicional de vejiga.5. A non-invasive in vitro method comprising: a) detecting and quantifying the combination of biomarkers defined in claim 1 in a urine test sample of an individual; and b) compare the expression value obtained in a) in the urine test sample with the corresponding standard value in normal urine, where variations in the value obtained in a) with respect to the standard value in normal urine are indicative of transitional bladder carcinoma.
6. Método según Ia reivindicación 5, donde Ia etapa a) comprende detectar y cuantificar Ia combinación de biomarcadores definida en Ia reivindicación 2.6. Method according to claim 5, wherein step a) comprises detecting and quantifying the combination of biomarkers defined in claim 2.
7. Método según Ia reivindicación 5, donde Ia etapa a) comprende detectar y cuantificar Ia combinación de biomarcadores definida en Ia reivindicación 3. 7. Method according to claim 5, wherein step a) comprises detecting and quantifying the combination of biomarkers defined in claim 3.
8. Método según Ia reivindicación 5, donde Ia etapa a) comprende detectar y cuantificar Ia combinación de biomarcadores definida en Ia reivindicación 4.8. Method according to claim 5, wherein step a) comprises detecting and quantifying the combination of biomarkers defined in claim 4.
9. Método según cualquiera de las reivindicaciones 5 a 8, donde el método se emplea para detectar Ia presencia de BTCC, determinar el estadio o severidad de este cáncer, evaluar Ia ausencia de enfermedad después de resección quirúrgica, establecer un diagnóstico y/o pronóstico de este cáncer y/o monitorizar el efecto de Ia tratamiento administrado a un individuo que padece dicho cáncer.9. Method according to any of claims 5 to 8, wherein the method is used to detect the presence of BTCC, determine the stage or severity of this cancer, assess the absence of disease after surgical resection, establish a diagnosis and / or prognosis of this cancer and / or monitor the effect of the treatment administered to an individual suffering from said cancer.
10. Método según cualquiera de las reivindicaciones 5 a 9, donde Ia muestra de orina a analizar se obtiene de un individuo a quien no se Ie ha diagnosticado previamente carcinoma transicional de vejiga.10. Method according to any of claims 5 to 9, wherein the urine sample to be analyzed is obtained from an individual who has not previously been diagnosed with transitional bladder carcinoma.
11. Método según cualquiera de las reivindicaciones 5 a 9, donde Ia muestra de orina a analizar se obtiene de un individuo a quien se Ie ha diagnosticado previamente carcinoma transicional de vejiga.11. Method according to any of claims 5 to 9, wherein the urine sample to be analyzed is obtained from an individual who has previously been diagnosed with transitional bladder carcinoma.
12. Método según cualquiera de las reivindicaciones 5 a 9, donde Ia muestra de orina a analizar se obtiene de un individuo que está recibiendo tratamiento en Ia actualidad contra carcinoma transicional de vejiga.12. Method according to any of claims 5 to 9, wherein the urine sample to be analyzed is obtained from an individual currently receiving treatment against transitional bladder carcinoma.
13. Método según cualquiera de las reivindicaciones 5 a 12, donde Ia detección y cuantificación de proteínas comprende una primera etapa, en Ia cual el extracto de proteína de Ia muestra se pone en contacto con una composición de anticuerpos específicos para uno o más epítopos de las proteínas de Ia reivindicación 1 , y una segunda etapa, en Ia cual se cuantifican los complejos anticuerpo-proteína.13. Method according to any of claims 5 to 12, wherein the detection and quantification of proteins comprises a first stage, in which the protein extract of the sample is contacted with a specific antibody composition for one or more epitopes of the proteins of claim 1, and a second stage, in which the antibody-protein complexes are quantified.
14. Método según Ia reivindicación 13, donde los anticuerpos son de origen humano, humanizados o de origen no humano y seleccionados entre anticuerpos monoclonales o policlonales, fragmentos de anticuerpos intactos o recombinantes, combibodies y fragmentos de anticuerpo Fab o scFv.14. Method according to claim 13, wherein the antibodies are of human origin, humanized or of non-human origin and selected from monoclonal or polyclonal antibodies, intact or recombinant antibody fragments, combibodies and Fab or scFv antibody fragments.
15. Método según Ia reivindicación 13 ó 14, donde Ia detección y cuantificación de los complejos anticuerpo-proteína, las técnicas utilizadas se seleccionan del grupo que comprende: western blot, ELISA (ensayo inmunoabsorbente unido a enzima), RIA (radioinmunoensayo), EIA competitivo (inmunoensayo enzimático competitivo), DAS-ELISA (ELISA sandwich de doble anticuerpo), técnicas inmunocitoquímicas o inmunohistoquímicas, técnicas basadas en el uso de biochips o microarrays de proteína que incluyen anticuerpos específicos, ensayos basados en Ia precipitación de oro coloidal en formatos tales como dipsticks; o por técnicas de afinidad cromatográfica, ensayos de unión a ligando y ensayos de unión a lectina.15. Method according to claim 13 or 14, wherein the detection and quantification of antibody-protein complexes, the techniques used are selected from the group comprising: western blot, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), EIA competitive (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical or immunohistochemical techniques, techniques based on the use of biochips or protein microarrays that include specific antibodies, tests based on colloidal gold precipitation in such formats as dipsticks; or by chromatographic affinity techniques, ligand binding assays and lectin binding assays.
16. Método según cualquiera de las reivindicaciones 5 a 15, donde el método se utiliza para monitorizar Ia eficacia del tratamiento farmacológico o quirúrgico.16. Method according to any of claims 5 to 15, wherein the method is used to monitor the efficacy of the pharmacological or surgical treatment.
17. Método según cualquiera de las reivindicaciones 5 a 16, donde el método se utiliza para determinar Ia progresión de Ia enfermedad cuando Ia misma proteína o proteínas se comparan en diferentes muestras obtenidas de un mismo paciente a diferentes tiempos durante Ia evolución del carcinoma transicional de vejiga.17. Method according to any of claims 5 to 16, wherein the method is used to determine the progression of the disease when the same protein or proteins are compared in different samples obtained from the same patient at different times during the evolution of transitional carcinoma of bladder.
18. Uso in vitro de las secuencias peptídicas derivadas de los biomarcadores de Ia combinación definida en Ia reivindicación 1 para detectar Ia presencia de BTCC mediante análisis de orina, determinar el estadio o severidad de este cáncer, evaluar Ia ausencia de enfermedad después de resección quirúrgica, establecer un diagnóstico y/o pronóstico de este cáncer y/o monitorizar el efecto de Ia tratamiento administrado a un individuo que padece dicho cáncer, donde los biomarcadores están presentes en orina. 18. In vitro use of the peptide sequences derived from the biomarkers of the combination defined in claim 1 to detect the presence of BTCC by urine analysis, determine the stage or severity of this cancer, evaluate the absence of disease after surgical resection , establish a diagnosis and / or prognosis of this cancer and / or monitor the effect of the treatment administered to an individual suffering from said cancer, where biomarkers are present in urine.
19. Uso según Ia reivindicación 18 de las secuencias peptídicas derivadas de los biomarcadores de Ia combinación definida en Ia reivindicación 2.19. Use according to claim 18 of the peptide sequences derived from the biomarkers of the combination defined in claim 2.
20. Uso según Ia reivindicación 18 de las secuencias peptídicas derivadas de los biomarcadores de Ia combinación definida en Ia reivindicación 3.20. Use according to claim 18 of the peptide sequences derived from the biomarkers of the combination defined in claim 3.
21. Uso según Ia reivindicación 18 de las secuencias peptídicas derivadas de los biomarcadores de Ia combinación definida en Ia reivindicación 4.21. Use according to claim 18 of the peptide sequences derived from the biomarkers of the combination defined in claim 4.
22. Uso según cualquiera de las reivindicaciones 18 a 21 , donde variaciones en el nivel de expresión de uno o más biomarcadores en una muestra test de orina respecto al correspondiente valor estándar en orina normal son indicativas de carcinoma transicional de vejiga.22. Use according to any of claims 18 to 21, wherein variations in the level of expression of one or more biomarkers in a urine test sample with respect to the corresponding standard value in normal urine are indicative of transitional bladder carcinoma.
23. Uso conjunto de los nucleótidos o secuencias peptídicas derivados de los biomarcadores de Ia combinación definida en Ia reivindicación 1 , en métodos para cribar, identificar, desarrollar y evaluar Ia eficiencia de compuestos en el carcinoma transicional de vejiga.23. Joint use of nucleotides or peptide sequences derived from biomarkers of the combination defined in claim 1, in methods to screen, identify, develop and evaluate the efficiency of compounds in bladder transitional carcinoma.
24. Un kit para llevar a cabo el método previamente descrito en cualquiera de las reivindicaciones 5 a 17 que comprende 1) anticuerpos que reconocen específicamente Ia combinación de biomarcadores definida en Ia reivindicación 1 y 2) un soporte en un embalaje adecuado.24. A kit for carrying out the method previously described in any one of claims 5 to 17 comprising 1) antibodies that specifically recognize the combination of biomarkers defined in claim 1 and 2) a support in a suitable package.
25. Un kit según Ia reivindicación 24 que se emplea para detectar Ia presencia de carcinoma transicional de vejiga, determinar el estadio o severidad de este cáncer, evaluar Ia falta de enfermedad después de resección quirúrgica, establecer un diagnóstico y/o pronóstico de este cáncer y/o monitorizar el efecto del tratamiento administrado a un individuo que padece dicho cáncer. 25. A kit according to claim 24 which is used to detect the presence of transitional bladder carcinoma, determine the stage or severity of this cancer, evaluate the lack of disease after surgical resection, establish a diagnosis and / or prognosis of this cancer and / or monitor the effect of the treatment administered to an individual suffering from said cancer.
26. Un kit según la reivindicación 24 ó 25 que comprende un biochip.26. A kit according to claim 24 or 25 comprising a biochip.
27. Un kit según Ia reivindicación 26, donde el biochip comprende anticuerpos para Ia detección de proteínas de Ia combinación de biomarcadores definida en Ia reivindicación 1. 27. A kit according to claim 26, wherein the biochip comprises antibodies for the detection of proteins of the combination of biomarkers defined in claim 1.
PCT/ES2008/000196 2007-03-30 2008-03-28 Non-invasive in vitro method for detecting transitional carcinoma of the bladder WO2008119858A1 (en)

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