WO2008119858A1 - Méthode in vitro non invasive permettant de détecter un carcinome transitionnel de la vessie - Google Patents

Méthode in vitro non invasive permettant de détecter un carcinome transitionnel de la vessie Download PDF

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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)
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WO2008119858A8 (fr
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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/fr
Publication of WO2008119858A8 publication Critical patent/WO2008119858A8/fr

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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
    • 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
    • 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

La présente invention concerne une méthode in vitro non invasive permettant de détecter la présence d'un carcinome transitionnel de la vessie chez un individu, par analyse d'urine, de déterminer le stade ou la gravité dudit cancer et de surveiller l'effet du traitement administré à l'individu atteint dudit cancer.
PCT/ES2008/000196 2007-03-30 2008-03-28 Méthode in vitro non invasive permettant de détecter un carcinome transitionnel de la vessie WO2008119858A1 (fr)

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KR101345497B1 (ko) 2009-01-12 2013-12-31 이카겐, 인코포레이티드 설폰아미드 유도체
EP2762574A1 (fr) 2013-01-31 2014-08-06 Fina Biotech, S.L. Procédé de diagnostic non invasif pour diagnostiquer un cancer de la vessie

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WO2003003906A2 (fr) * 2001-07-03 2003-01-16 Eos Biotechnology, Inc. Procede de diagnostic du cancer de la vessie, compositions et procedes de criblage de modulateurs du cancer de la vessie
US20040038207A1 (en) * 1999-02-22 2004-02-26 Orntoft Torben F. Gene expression in bladder tumors
WO2004085676A1 (fr) * 2003-03-26 2004-10-07 Progenika Biopharma, S.A. Methode de detection in vitro du carcinome a cellules transitionnelles de la vessie
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US20040038207A1 (en) * 1999-02-22 2004-02-26 Orntoft Torben F. Gene expression in bladder tumors
WO2003003906A2 (fr) * 2001-07-03 2003-01-16 Eos Biotechnology, Inc. Procede de diagnostic du cancer de la vessie, compositions et procedes de criblage de modulateurs du cancer de la vessie
WO2004085676A1 (fr) * 2003-03-26 2004-10-07 Progenika Biopharma, S.A. Methode de detection in vitro du carcinome a cellules transitionnelles de la vessie
WO2007042256A1 (fr) * 2005-10-11 2007-04-19 Laboratorios Salvat, S.A. Methode in vitro non invasive pour detecter un carcinome a cellules transitionnelles de la vessie

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101345497B1 (ko) 2009-01-12 2013-12-31 이카겐, 인코포레이티드 설폰아미드 유도체
EP2762574A1 (fr) 2013-01-31 2014-08-06 Fina Biotech, S.L. Procédé de diagnostic non invasif pour diagnostiquer un cancer de la vessie
WO2014118334A1 (fr) 2013-01-31 2014-08-07 Fina Biotech, S.L Procédé de diagnostic non invasif pour diagnostiquer le cancer de la vessie
US9902998B2 (en) 2013-01-31 2018-02-27 Fina Biotech, S.L. Non-invasive diagnostic method for diagnosing bladder cancer
EP4060048A2 (fr) 2013-01-31 2022-09-21 Fina Biotech, S.L. Procédé de diagnostic non invasif pour diagnostiquer un cancer de la vessie

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