ES2299281B1 - METHODS FOR IN VITRO AND FORECAST IN VITRO DIAGNOSIS OF THE CANCER OF PANCREAS, AND FOR THE DEVELOPMENT OF PHARMACOS AGAINST CANCER OF PANCREAS. - Google Patents

Abstract

Methods for diagnosing in vitro and in vitro prognosis of pancreatic cancer, and for drug development in pancreatic cancer.

The present invention relates to an in vitro method to detect the presence of a pancreatic cancer, especially a ductal adenocarcinoma of the pancreas, in an individual, to determine the stage or severity of said cancer in the individual, or to monitor the effect of therapy administered to an individual presenting said cancer; to the search, identification, development and evaluation of the efficacy of compounds for said cancer therapy, in order to develop new medications; as well as agents that inhibit the expression and / or activity of the human AGR2 protein and / or the effects of this expression.

Description

Methods for diagnosing in vitro and in vitro prognosis of pancreatic cancer, and for drug development in pancreatic cancer.

Field of the Invention

The present invention relates to an in vitro method to detect the presence of pancreatic cancer in an individual, to determine the stage or severity of said cancer in the individual, or to monitor the effect of therapy administered to an individual who present said cancer; to the search, identification, development and evaluation of the efficacy of compounds for said cancer therapy, in order to develop new medications; as well as agents that inhibit the expression and / or activity of human AGR2 proteins and / or the effects of this expression.

Background of the invention

Pancreatic cancer was the cause of more than 220,000 deaths in the world, and more than 3,600 in Spain, during the year 2,000 (GLOBOCAN). The clinical behavior of cancer pancreas is homogeneous and always infamous, with no notable differences in survival according to the stage. The number of patients with Pancreatic cancer of good prognosis is insignificant. A possible explanation is that even in patients with tumors small, framed in stage I, the disease is disseminated Your diagnosis in an initial phase, except in cases fortuitous, it is difficult; 75% of patients diagnosed they have advanced disease (Stages III and IV). It is a very aggressive neoplasm, resistant to cytostatic treatments and only between 1 and 4% of cases remain alive at five years of diagnosis, as long as the tumor is located and there are could be completely removed (Warshaw Al., and Fernándes del Castillo C., N. Eng. J. Med., 1992, 326: 455-465; Ahlgren J.D., Semin. Oncol., 1996, 23: 241-250). Is for all that in the case of pancreatic cancer, both the development of early diagnosis systems such as therapies effective, they are crucial for disease control (Byungwoo R., et al., Cancer Res., 2002, 62: 819-826).

Many of the genes and proteins involved in the Pancreatic cancer progression are still unknown; the identification of genes and proteins differentially expressed in association with the tumor invasiveness process, could lead to the identification of biological markers and therapeutic targets, that could have a great value for the diagnosis, the prognosis and the treatment of this disease.

Unexpectedly, the authors of the present invention have discovered, after laborious research and using different techniques (DNA-chips and quantitative RT-PCR), that the expression of the gene encoding the AGR 2 protein, human anterior gradient 2 ( Xenopus laevis ) Homolog, GeneBank code AF088867 is greatly increased in carcinomas of the pancreas, especially in ductal adenocarcinomas of the pancreas, when compared with non-tumor tissue expression from the same individuals, or from individuals affected by pancreatitis. This evidence makes the human AGR2 protein a useful target for the development of new in vitro diagnostic and prognostic methods, and for the identification and development of compounds for pancreatic cancer therapy, especially ductal adenocarcinoma of the pancreas.

In vitro detection of high levels of expression of the human AGR2 gene or of the human AGR2 encoded protein or any combination thereof, in pancreas tissue samples or in other samples, preferably serum, of individuals, will allow early detection of cancer of pancreas, especially ductal adenocarcinoma of the pancreas.

The development of new targeted drugs specifically against the human AGR2 gene or against the protein encoded human AGR2 or any combination of them, is a new approach to treat pancreatic cancer, especially the  ductal adenocarcinoma of the pancreas.

The present invention thus provides a high sensitivity in vitro method for detecting the presence of pancreatic cancer, especially ductal adenocarcinoma of the pancreas, for determining the stage or severity of said cancer in the individual, or for monitoring the effect. of the therapy administered to an individual presenting said cancer, based on the detection and / or quantification of the human AGR2 protein, of the mRNA of the human AGR2 gene or of the corresponding cDNA of the gene in a sample of said individual. Also, the present invention provides targets or tools for the search, identification, development and evaluation of the efficacy of compounds for pancreatic cancer therapy, especially ductal pancreatic adenocarcinoma.

Finally, the invention provides agents characterized in that they inhibit the expression and / or activity of the human protein AGR2, for the treatment of pancreatic cancer, in special ductal adenocarcinoma of the pancreas.

Object of the invention

The present invention has as its main object the development of an in vitro method to detect the presence of pancreatic cancer, especially ductal adenocarcinoma of the pancreas, to determine the stage or severity of said cancer in the individual, or to monitor the effect of the therapy administered to an individual presenting said cancer.

A second object of the present invention is an in vitro method to search, identify, develop and evaluate the efficacy of compounds for pancreatic cancer therapy, especially ductal pancreatic adenocarcinoma.

A further object of the invention lies in the use of sequences derived from the human AGR2 gene, for the in vitro diagnosis and prognosis of pancreatic cancer, especially ductal adenocarcinoma of the pancreas, as well as for the search, identification, development and evaluation of the efficacy of compounds for said therapy
Cancer.

Another object of the present invention consists in provide agents characterized in that they inhibit expression and / or the activity of the human AGR2 protein for the treatment of pancreatic cancer, especially ductal adenocarcinoma of pancreas.

Finally, it is also the subject of the invention. a pharmaceutical composition comprising one or more agents therapeutic together with a pharmaceutically acceptable excipient, for the treatment of pancreatic cancer, especially ductal adenocarcinoma of the pancreas.

Description of the figures

Figure 1: Denaturation curve of PCR product of the target gene, human AGR2 (Tm = 77 ° C) and the gene reference, ribI10 (Tm = 84 ° C), in experiments measuring the gene expression by quantitative RT-PCR in time real, in pancreas samples.

Figure 2: Calculation of the efficiency of amplification of the human AGR2 PCR reactions, in experiments measuring gene expression by Real-time quantitative RT-PCR, in samples of pancreas.

Figure 3: Calculation of the efficiency of amplification of ribI10 PCR reactions, in experiments of gene expression measurement by RT-PCR quantitative in real time, in pancreas samples.

Detailed description of the invention

To facilitate the understanding of this patent application, we explain below the meaning of Some terms and expressions in the context of the invention:

The terms "subject" or "individual" are refer to members of mammalian animal species, and includes, but it is not limited to pets, primates and humans; he subject is preferably a human being, male or female, of Any age or race.

The term "cancer" refers to the disease that is characterized by abnormal growth or uncontrolled cells, capable of invading adjacent tissues and spread to distant organs.

The term "carcinoma" refers to tissue that results from abnormal or uncontrolled cell growth.

The terms "pancreatic cancer", "cancer in the pancreas" or "adenocarcinoma of the pancreas" is refer to any malignant proliferative disorder of cells of the pancreas.

The term "tumor" refers to any abnormal tissue mass resulting from a benign neoplastic process (not cancerous) or malignant (cancerous).

The term "gene" refers to a region of a molecular chain of deoxyribonucleotides that encodes a protein and that may represent a portion of the sequence coding or the entire coding sequence.

The term "DNA" refers to acid deoxyribonucleic. A DNA sequence is a sequence of deoxyribonucleotides.

The term "RNA" refers to acid ribonucleic. An RNA sequence is a sequence of ribonucleotides

The term "mRNA" refers to acid messenger ribonucleic, which is the fraction of total RNA that is Translates to proteins.

The term "cDNA" refers to a nucleotide sequence, complementary to a sequence of mRNA

The phrase "mRNA transcribed from" refers to gene transcription (DNA) in mRNA, as a first step for The gene is expressed and translated into protein.

The term "nucleotide sequence" or "nucleotide sequence" refers interchangeably to a ribonucleotide (RNA) or deoxyribonucleotide sequence (DNA).

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The term "protein" refers to a amino acid molecular chain, linked by covalent bonds or not covalent The term includes all forms of modifications post-translational, for example glycosylation, phosphorylation or acetylation.

The terms "peptide" and "polypeptide" they refer to molecular chains of amino acids that represent a protein fragment The terms "protein" and "peptide", They are used interchangeably.

The term "antibody" refers to a glycoprotein that exhibits a specific binding activity by a target molecule, which is called "antigen." The term "antibody" comprises monoclonal antibodies, or antibodies polyclonal, intact, or fragments thereof; and includes human, humanized and non-human origin antibodies. The "monoclonal antibodies" are homogeneous populations of highly specific antibodies that are directed against a single site or antigenic "determinant". "Antibodies polyclonal "include heterogeneous antibody populations, that are directed against different determinants antigenic

The term "epitope", as used In the present invention, it refers to an antigenic determinant of a protein, which is the amino acid sequence of the protein that a specific antibody recognizes.

The term "solid phase," as used in the present invention, refers to a non-aqueous matrix to which the antibody can bind. Examples of materials for solid phase include glass, polysaccharides, for example agarose, polyacrylamide, polystyrene, polyvinyl alcohol and silicones. Examples of solid phase forms are the well of a plate of test or a purification column.

The terms "oligonucleotide" and "oligonucleotide primer" are used interchangeably and are refer, as used in the present invention, to sequences nucleotides, which are complementary to a sequence nucleotide of the human AGR2 gene. Each primer hybridizes with its target nucleotide sequence and acts as a starting site for the nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase or reverse transcriptase.

The term "probe", as used in The present invention relates to a nucleotide sequence complementary to a nucleotide sequence derived from the human gene AGR2 that can be used to detect that nucleotide sequence derived from said gene.

The term "therapeutic target" refers to nucleotide or peptide sequences, against which you can Clinically design and apply a drug or compound therapeutic.

The term "antagonist" refers to any molecule that inhibits the biological activity of the molecule antagonized Examples of antagonistic molecules include, among others, proteins, peptides, peptide sequence variations natural and small organic molecules (molecular weight less than 500 daltons).

The present invention is based on the discovery that both the expression of the human AGR2 gene, and the concentration of the human AGR2 protein are increased by Ductal adenocarcinoma of an individual's pancreas.

In this regard, the present invention provides, first of all, an in vitro method for detecting the presence of a pancreatic cancer, especially of a ductal adenocarcinoma of the pancreas, in an individual, to determine the stage or severity of said cancer. in the individual, or to monitor the effect of the therapy administered to an individual presenting said cancer, which comprises:

a) the detection and / or quantification of the human AGR2 protein, from the human AGR2 gene mRNA or from the corresponding cDNA of said gene in a sample of said individual, Y

b) comparison of the amount of protein human AGR2, of the amount of mRNA of the human AGR2 gene, or of the amount of the corresponding cDNA of the gene detected in a sample of an individual, with the amount of human protein AGR2, with the amount of the mRNA of the human AGR2 gene or with the amount of corresponding cDNA of the gene detected in the samples of control individuals or in previous samples of the same individual or with normal reference values.

The method provided herein invention is of high sensitivity and specificity, and is based on that subjects or individuals diagnosed with pancreatic cancer, in special pancreatic ductal adenocarcinoma, have levels elevated mRNA transcribed from the human AGR2 gene, or concentrations elevated protein encoded by the human AGR2 gene (protein human AGR2), compared to the corresponding levels in samples from subjects with no clinical history of this carcinoma.

The present method comprises a step of Obtaining the individual's sample. You can work with different fluid samples such as: urine, blood, plasma,  serum, pleural fluid, ascites fluid, synovial fluid, bile, semen, exudate of gastric flow or cerebrospinal fluid. The Sample can also be pancreas tissue that can be obtained by any conventional method, preferably resection surgical

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Samples can be obtained from subjects previously diagnosed, or undiagnosed, of a given type of cancer; or also of a subject under treatment, or who has previously treated against cancer, particularly against pancreatic cancer.

The present method further comprises a step of sample extraction, either to obtain the extract of proteins of this one, or to obtain the total RNA extract. One of these two extracts represents the work material for the next phase Total protein extraction protocols or of the total RNA are well known to the person skilled in the art (Chomczynski P. et al., Anal. Biochern., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532).

Any conventional assay can be used in the context of the invention to detect a cancer, provided that it measures in vitro the level of transcribed mRNA of the human AGR2 gene, or its complementary cDNA, or the concentration of the human AGR2 protein, in samples collected from the individuals to analyze and control individuals.

Thus, this invention provides a method. to detect the presence of pancreatic cancer, especially a ductal adenocarcinoma of the pancreas, in an individual, to determine the stage or severity of said cancer in the individual, or to monitor the effect of the therapy administered to an individual who presents said cancer, based well on the extent of the concentration of the human protein AGR2, or to the extent of the expression level of the human AGR2 gene.

In the event that what is intended to detect be the human AGR2 protein, the method of the invention comprises a first stage of contacting the protein extract of the sample with a composition of one or more specific antibodies against one or more epitopes of the human AGR2 protein and a second quantification stage of the complexes formed by the antibodies and the human AGR2 protein.

There is a wide variety of trials immunological available to detect and quantify the formation of specific antigen-antibody complexes; numerous protein binding assays, competitive and not competitive, have been previously described and a large number of These trials are commercially available.

Thus, the human AGR2 protein can be quantified with antibodies such as: monoclonal antibodies, polyclonal, intact or recombinant fragments of them, combibodies and Fab or scFv fragments of antibodies, specific against the human AGR2 protein; being these human antibodies, Humanized or of non-human origin. The antibodies that are used in these tests they may be marked or not; no antibodies markings can be used in agglutination tests; the labeled antibodies can be used in a wide variety of essays. Marker molecules that can be used to Marking antibodies include radionuclides, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes and derivatives.

There is a wide variety of well known assays, which can be used in the present invention, which use unlabeled antibodies (primary antibody) and labeled antibodies (secondary antibody); These techniques include Western-blot or Western blotting, ELISA (Enzyme-Linked immunosorbent assay or enzyme-linked immunosorbent assay), RIA (Radioimmunoassay or Radioimmunoassay), Competitive EIA (Competitive enzyme immunoassay or Competitive enzyme immunoassay), DAS-ELISA (Double antibody sandwich-ELISA or ELISA sandwich test with double antibody), petrochemical and immunohistochemical techniques, techniques based on the use of biochips or microarrays of proteins that include specific antibodies or tests based on colloidal precipitation in formats such as dipsticks . Other ways to detect and quantify the human AGR2 protein include affinity chromatography techniques, ligand binding assays or lectin binding assays.

The preferred immunoassay in the method of the invention is a double antibody sandwich ELISA assay ( DAS-ELISA ). In this immunoassay any antibody or combination of antibodies, specific against one or more epitopes of the human AGR2 protein can be used. As an example of one of the many possible formats of this assay, an antibody, monoclonal or polyclonal, or a fragment of this antibody, or a combination of antibodies, which cover a solid phase, are contacted with the sample to be analyzed, and they are incubated for a time and under appropriate conditions to form the antigen-antibody complexes. After washing under appropriate conditions to remove non-specific complexes, an indicator reagent, comprising a monoclonal or polyclonal antibody, or a fragment of this antibody, or a fragment of this antibody, is incubated with the antigen-antibody complexes. combination of these antibodies, bound to a compound generating a signal. The presence of the human AGR2 protein in the sample to be analyzed is detected and quantified, if it exists, by measuring the generated signal. The amount of human AGR2 protein present in the test sample is proportional to that signal.

In the case that it is intended to detect the mRNA or cDNA corresponding to the human AGR2 gene and not the proteins, the method of the invention for detecting carcinoma in vitro has different stages. Thus, once the sample is obtained and the total RNA is extracted, according to the method of the invention, the detection of the mRNA or the corresponding cDNA of the human AGR2 gene is performed, which comprises a first stage of amplification of the total RNA extract or the corresponding cDNA synthesized by reverse transcription from mRNA, and a second stage of quantification of the amplification product of mRNA or human AGR2 cDNA.

An example of mRNA amplification is to re-transcribe the mRNA into cDNA (RT), followed by the Polymerase Chain Reaction (PCR), using oligonucleotide primers, the sequences of the primers used being 5'-AGAAATTGGCAGAGCAGTTTGTC-3 '( SEQ ID NO 1) and 5'-GCCATCAGGAGAAAGGTGTTTG-3 '(SEQ ID NO 2) for human AGR2; PCR is an amplification technique of a certain nucleotide sequence (target) contained in a mixture of nucleotide sequences. In the PCR, an excess of a pair of oligonucleotide primers is used, which hybridize with the complementary strands of the target nucleotide sequence. Next, an enzyme with polymerase activity (DNA Taq Polymerase) extends each primer, using the target nucleotide sequence as a template. The products of the extension are then converted into target sequences, after dissociation of the original target strand. New primer molecules hybridize and polymerase extends them; The cycle is repeated to exponentially increase the number of target sequences. This technique is described in US patents 4683195 and US 4683202. Many methods have been previously described for detecting and quantifying the products of PCR amplification, of which anyone can be used in this invention. In a preferred method of the invention, the amplified product is detected by agarose gel electrophoresis, as follows: five microliters of the amplification product is subjected to electrophoresis separation in an agarose gel at a concentration of 2% , in a 0.5x TBE buffer at 100 vdc, for one hour. After electrophoresis, the gel is stained with ethidium bromide and the amplification product is visualized by illuminating the gel with ultraviolet (uv) light; As an alternative to staining, and preferred embodiment, the amplified product can be transferred to a nylon membrane by Southern blotting or Southern blotting techniques, to be detected with a properly labeled human AGR2 gene cDNA probe.

In another example the mRNA detection is performed transferring the mRNA to a nylon membrane, using techniques transfer such as Northern blot or Northern transfer, and detecting it with specific probes of the mRNA or the corresponding cDNA of the human AGR2 gene.

In a particular embodiment the amplification and quantification of the mRNA corresponding to the human AGR2 gene is performs at the same time by quantitative RT-PCR to real time (Q-PCR).

The final step of the method of the invention to detect in vitro the presence of cancer, in a sample from an individual, comprises comparing the amount of human AGR2 protein, the amount of mRNA of the human AGR2 gene or the amount of the corresponding cDNA of the gene. detected in a sample of an individual, with the amount of human AGR2 protein, the amount of mRNA of the human AGR2 gene, or the amount of the corresponding cDNA of the gene detected in the samples of control subjects or in previous non-tumor samples of the same individual, or with normal reference values.

In its second object, the invention also provides an in vitro method for identifying and evaluating the efficacy of agents for pancreatic cancer therapy, especially ductal pancreatic adenocarcinoma, comprising:

a) contact a cell culture tumor, with the candidate compound, under the conditions and during the appropriate time to allow them to interact,

b) detect and quantify the levels of expression of the human AGR2 gene or at least one of the proteins human AGR2, and

c) compare these expression levels with the of control cultures of untreated tumor cells with the candidate compound.

The quantification of the expression levels of the human AGR2 gene, or the human AGR2 protein is performed in a similar manner as indicated in the method of the invention to detect in vitro the presence of a pancreatic cancer, especially a ductal adenocarcinoma of pancreas, in an individual.

When an agent lowers the levels of gene and / or protein expression of the human AGR2 protein, or reverse the effects of said high expression, preferably By decreasing the levels of cell proliferation, this agent is becomes a candidate for cancer therapy.

Therefore, another object of the invention is refers to the use of nucleotide or peptide sequences derived of the human AGR2 gene, in search methods, identification, development and evaluation of the efficacy of compounds for therapy of pancreatic cancer, especially ductal adenocarcinoma of pancreas. Highlight the importance acquired lately by binding drug screening methods, competitive or not, from the potential drug molecule to the therapeutic target.

Another additional object of the invention relates to to the use of nucleotide or peptide sequences derived from the gene human AGR2 to detect the presence of pancreatic cancer, in special ductal adenocarcinoma of the pancreas, to determine the stage or severity of said cancer in the individual, or to monitor the effect of therapy administered to an individual who present said cancer.

Another object of the invention consists in provide agents characterized in that they inhibit expression and / or the activity of the human protein AGR2. These agents, which they can identify and evaluate according to the present invention, they can be selected from the group formed by:

a) an antibody, or combination of antibodies, specific against one or more epitopes present in the protein human AGR2, preferably a human monoclonal antibody or humanized; it may also be a fragment of the antibody, a single chain antibody or an antibody anti-idiotype,

b) cytotoxic agents, such as toxins, molecules with radioactive atoms, or agents chemo-therapeutic, including, without limitation, small organic and inorganic molecules, peptides, phosphopeptides, antisense molecules, ribozymes, siRNAs, molecules triple helix, etc., which inhibit the expression and / or activity of the human AGR2 protein and

c) human protein antagonist compounds AGR2 that inhibit one or more of the functions of the human protein AGR2.

Finally, it is also an object of the present invention a pharmaceutical composition comprising a therapeutically effective amount of one or more agents of the mentioned above together with one or more excipients and / or transport substances. In addition said composition may contain any other active ingredient that inhibits the function of the human protein AGR2.

Excipients, transport substances and auxiliary substances have to be pharmaceutically and pharmacologically tolerable, so that they can be combined with other components of the formulation or preparation and do not exert adverse effects on the treated organism. The compositions Pharmaceuticals or formulations include those that are suitable for oral or parenteral administration (including subcutaneous, intradermal, intramuscular and intravenous), although the best route of Administration depends on the patient's condition. Formulations They can be in the form of single doses. The formulations are prepared according to known methods in the field of pharmacology. The quantities of active substances for administered may vary depending on the particularities of the therapy.

The following examples illustrate the invention.

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Example 1 Differential analysis of human AGR2s gene expression in pancreas tissue samples, using the Human Genome U133 DNA arrays microarrays 1.1. Materials and methods

Microarrays The GeneChip Test 3 microarrays (Affymetrix, Santa Clara) were used, which allow to test the quality of the RNA, prior to the expression analysis with the GeneChip Human Genome U133A array (Affymetrix, Santa Clara), which represents 13,220 complete sequences of annotated genes; the human AGR2 gene is represented in the microarray by the probe set 209173_at; each set of probes is composed of 11 sense oligonucleotides of 25 nucleotides in length, designed based on the sequence Hs. 12802 of Unigene, or AB007860 of
Genebank.

Samples The samples studied came from clinically classified biopsies, obtained by surgical resection: Biopsies of non-tumor pancreatic tissue, from individuals presenting with ductal adenocarcinoma of the pancreas ("non-tumor") (n = 1); biopsies of non-tumor but inflamed pancreatic tissue, from individuals without pancreatic ductal adenocarcinoma, but with chronic pancreatitis ("chronic pancreatitis") (n = 1); biopsies of non-tumor, but inflamed, pancreatic tissue, from individuals presenting with ductal adenocarcinoma of the pancreas and pancreatitis ("pancreatitis") (n = 1); Tumor pancreatic tissue biopsies from patients presenting with ductal pancreatic adenocarcinoma (n = 10) in one of the following stages: Stage I, tumor limited to the pancreas, Stage III, tumor extended to regional lymph nodes and Stage IVB, there is metastasis in distant tissues or organs. All samples were classified clinically and histologically (grade and stage) at the Central Hospital of Asturias, the same hospital where the samples had been collected, following the precepts of the Helsinki declaration. The samples were frozen in liquid nitrogen immediately after extraction and kept at -80 until the time of analysis.

For each type of tumor the following cases:

- Stadium tumor I:

7 samples

- Stadium tumor III:

1 sample

- Stadium tumor IV:

2 samples

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GeneChip gene expression analysis

The analysis was carried out with total RNA from individual subjects. The 13 samples that were analyzed are described in Table 1.

TABLE 1 Sample Description analyzed

one

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CDNA synthesis

The total RNA of each of the biopsies was obtained by homogenizing the tissue in TRizol® Reagent (Life Technologies), following the recommendations of the provider. The resulting total RNA was cleaned with the Rneasy kit (QIAGEN) (Chomczynski P. et al ., Anal. Biochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532). From each total RNA preparation, 10 µg was used as a starting material for the synthesis of the first strand of cDNA with the enzyme reverse transcriptase SuperScript ™ II RNase (Life Technologies), using as an primer an oligo-dT oligonucleotide which It contained the T7 phage RNA polymerase promoter sequence. The second strand of cDNA was synthesized using E. coli DNA polymerase I enzymes (Invitrogen Life Technologies), E. coli DNA ligase (Invitrogen Life Technologies), E. coli Rnasa H (Invitrogen Life Technologies), and DNA polymerase of phage T4 (Invitrogen Life Technologies). The biotin-labeled cRNA was synthesized using the ENZO BioArray? HighYield? Transcript Labeling Kit (Enzo Diagnostics Inc). After in vitro transcription, unincorporated nucleotides were removed using the Rneasy columns (QIAGEN).

Array hybridization and scanning

15 µg of each biotinylated cRNA was fragmented at 94 ° C for 35 minutes in a buffer solution containing 40 mM Tris-Acetate (pH 8.1), 100 mM KOAc and 30 mM MgOAc. The fragmented cRNA was mixed with hybridization buffer (100 mM MONTH, 1M NaCl, 20 mM EDTA, 0.01% Tween 20) and heated at 99 ° for 5 minutes and then at 45 ° for 5 minutes, to then be loaded into the array from Affymetrix. The first array in which hybridization was performed was Affymetrix Test 3. This array allows testing RNA quality prior to expression analysis in the Affymetrix® GeneChip® Human Genome 133 A (HG-U133A).

For hybridization, arrays were incubated in a 45º rotary kiln for 16 hours and with a rotation 60 rpm constant.

The washing and staining of each array was carried out at the Affymetrix® Fluid Station. A program was used washing and staining that included:

-

10x2 wash cycles with SSPE-T 6x (0.9 m NaCl, 60 mM NaH 2 PO4, 6 mM EDTA, 0.01% Tween 20) at 25 °,

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4x15 cycles with 0.1 mM MONTH, 0.1M NaCl, 0.01% Tween 20 at 50º,

-

Staining of biotinylated cRNA with a streptavidin-phycoerythrin conjugate (10 \ mug / mlMolecular Probes)

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10x4 wash cycles with SSPE-T at 25º

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Staining with an antibody anti-streptavidin for 10 minutes

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Staining a conjugate Streptavidin-phycoerythrin (1 mg / ml, Molecular Probes) for 10 minutes

-

15x4 wash cycles with SSPE-T at 30º

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The arrays were scanned at 560 nm using a confocal microscope that uses laser emission (Agilent GeneArray Scanner) The analysis of the intensity readings was performed with Microarray Suite 5.0 software. For the comparison of arrays, these were scaled to a total intensity of 100.

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1.2. Results

Differential analysis of the expression of the human AGR2 gene in biopsies of tumor pancreatic tissue, with respect to biopsies of non-tumor pancreatic tissue, with or without pancreatitis, was performed from the comparison data of arrays obtained using the software Affymetrix The parameters that were taken into account (in the order in which they appear in the list) were: i) Detection . Indicates whether the transcript is Present (P), Absent (A) or Marginal (M), ii) Change : Indicates whether the expression of a given transcript Increases (I), Decreases (D), Does not Change (NC), Increases Marginally (MI), or Marginally Decrease (MD), iii) Signal Loq Ratio (SLR) : Indicates the level of expression change between the baseline (control) and a problem sample. This change is expressed as the log_ {2} of the ratio ( fold change or number of times the gene expression is elevated or repressed in the problem-tumor sample versus the control-non-tumor sample). An SLR value of 1 (equivalent to a fold change of 2) is considered significant, for transcripts whose expression increases against the control and -1, for transcripts whose expression decreases against the control.

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TABLE 2 Results obtained for the human gene AGR2 Acc. AF088867. Result of the comparisons versus the sample PA36 non-tumor control

2

TABLE 3 Results obtained for the gene human gene AGR2 Acc. AF088867. Result of the comparisons versus the sample PA46 control with nancreatitis

3

Differential analysis of gene expression human AGR2 in tumor stages with respect to control no PA36 tumor, or to control with pancreatitis (PA46), showed that the expression levels of the human AGR2 gene in the stages Tumors were increased in all tumors analyzed, except in 2 of the 7 biopsies of ductal pancreatic adenocarcinoma of stage I. The increases were consistently greater than compare tumors versus control with pancreatitis, with increments of more than 100 times (SLR = 6.7 and SLR = 11.5), which at compare them against the non-tumor control PA36, with increases that they ranged between 3 and 60 times (SLR = 1.4 and SLR = 5.7) (Tables 16 and 17).

1.3. Discussion

These results showed that the expression of the human AGR2 gene was increased in the vast majority of ductal adenocarcinomas of the pancreas analyzed, both against PA36 non-tumor tissue biopsies, as opposed to biopsies of chronic pancreatitis.

Example 2 Differential analysis of human AGR2 gene expression in samples of pancreas tissue, using the technique of Real-time quantitative RT-PCR 2.1. Materials and methods

The method used consists of the reverse transcription of the mRNA to cDNA and its subsequent amplification in a LightCycler device (Roche ), using SYBR Green for the detection of the amplified product. The quantification is performed in real time and allows the relative expression of the sequence to be calculated in different samples in the linear amplification phase of the reaction.

Samples The samples studied in the expression analysis of the human AGR2s genes came from clinically classified biopsies, obtained by surgical resection: Biopsies of non-tumor pancreatic tissue, from individuals presenting with pancreatic ductal adenocarcinoma ("non-tumor") (n = 1 ); biopsies of non-tumor, but inflamed pancreatic tissue, from individuals without pancreatic ductal adenocarcinoma, but with chronic pancreatitis (n = 1) ("chronic pancreatitis"); Tumor pancreatic tissue biopsies, from patients presenting with ductal adenocarcinoma of the pancreas (n = 5) in one of the following stages: Stage I, tumor limited to the pancreas and Stage IV, there is metastasis in distant tissues or organs.

All samples were classified histologically (grade and stage) at the Central Hospital of Asturias, the same hospital where the samples had been collected  following the precepts of the Declaration of Helsinki. The samples they were frozen in liquid nitrogen immediately after extraction and kept at -80ºC until the moment of its analysis.

Quantitative RT-PCR in Real Time . The analysis was carried out with total RNA from individual subjects. The samples that were analyzed are described in Tables 4.

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TABLE 4 Description of the samples analyzed for the gene human AGR2

4

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CDNA synthesis

The total RNA of each of the biopsies was obtained by homogenizing the tissue in TRizol® Reagent (Life Technologies), following the recommendations of the provider. The resulting total RNA was cleaned with the Rneasy kit (QIAGEN) (Chomczynski P. et al ., Anal. Biochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532). RNA was quantified spectrophotometrically and 5 µg of total RNA was digested with DNase I. 1 pg of DNAse treated RNA was used as a starting material for the synthesis of the first strand of cDNA with the enzyme SuperScript ™ reverse transcriptase. II RNase (Life Technologies), using as an primer an oligo-dT oligonucleotide containing the promoter sequence of the phage RNA polymerase T7. Aliquots of the diluted cDNA were prepared at the working concentration.

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Amplification

The synthesized cDNA was amplified using specific primers of the human AGR2 gene (SEQ ID NO 1 and SEQ ID NO 2) and specific primers of the gene encoding the human L10 ribosomal protein (SEQ ID NO 4 and SEQ ID NO 5). Real-time PCR reactions were prepared using the LightCycler-FastStart DNA master SYBR Green I kit (Roche ), following the manufacturer's instructions. The amplification program consisted of 1 cycle of 95 ° C for 10 min ("hot start") followed by 45 cycles of 95 ° C (denaturation) for 10 sec, 60 ° C (banding) for 5 sec, 72 ° C (amplification and fluorescence acquisition) for 10 sec. The denaturation curve analysis program consisted of a pulse cycle of 95 ° C, 65 ° C for 15 sec, and a pulse of 95 ° C during the amplification and acquisition step.

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Quantification

First, the specificity of PCR products analyzing denaturation curves. Subsequently, as a relative measure of gene expression, calculated the relationship between the abundance of mRNAs transcribed from human AGR2 and the abundance of ribI10 transcribed mRNAs and it normalized the relationship data in each of the samples tumor based on the values of the control sample. To calculate The efficiency of the PCR reactions (human AGR2 and ribI10) is built, for each gene sequence, a standard curve made with serial dilutions of cDNA. At cDNA concentrations template for reactions in the standard curve were given values arbitrary 10, 5, 2.5, 1.25 and 0.625. The efficiency was calculated using the equation:

E = 10 -1 / P

where E is the amplification efficiency and p is the slope of the standard line.

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The relationship of gene expression values It was determined using the following equation, which relates the experimental amplification data and corrects the error caused by the difference in efficiency of the reactions of PCR:

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where E is the amplification efficiency, Cp is the cut-off point , target is human AGR2, reference is ribI10, control is the control sample (not tumor or chronic pancreatitis) and sample is the tumor sample.

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2.2. Results Denaturation Curves Analysis

The analysis of the PCR products demonstrated the specific amplification of two products with denaturation temperatures close to those expected according to the theoretical calculation of the primer design software, PrimerExpress (Applied Biosystems ) (Figures 1-6).

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Calculation of the efficiency of PCR reactions

From each of the 5 dilutions of cDNA, amplified two replicates and the cut-off points (Cp) of each of they were represented in a graph with respect to the logarithm of the cDNA concentration to build the standard line (Tables 5-6; Figures 2-3).

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TABLE 5 Cut-off points ( Cp ) of the amplification of the 5 dilutions of cDNA for each replica amplified with primers of the human AGR2 gene

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TABLE 6 Cut-off points ( Cp ) of the amplification of the 5 cDNA dilutions for each replica amplified with the ribI10 gene primers

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Quantification of the expression change of the human AGR2 gene

Two replicas of each sample (healthy and tumor) are amplified with human AGR2 and ribI10 specific primers. From the cut-off points generated in these amplifications, the expression changes of the human AGR2 gene were calculated, in the tumor samples with respect to non-tumor control PA36 and control of pancreatitis PA46, applying the equation described above. Experimental data and final quantification are described in Tables 7 and 8 for the human AGR2 gene.

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TABLE 7 Experimental cut-off points of ribI10 and human AGR2 in the 5 tumor samples and controls

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TABLE 8 Overexpression values (Fold Change) of human AGR2 in the 5 tumor samples with respect to the non-tumor control PA36 and regarding the control of pancreatitis PA46

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The results confirmed the data obtained measuring the difference in gene expression with DNA-chips (example 1), that is, gene expression human AGR2 was strongly increased in all samples Tumor, except PA33, for the non-tumor sample PA36 and regarding the sample with chronic pancreatitis PA46, with a average increase in expression of 18 times, with respect to non-tumor samples without pancreatitis (PA36) and 19 times, with regarding non-tumor samples with pancreatitis (PA46) (Table 9); no difference can be seen when compared to a healthy sample or sample with pancreatitis, which is different from the result with DNA-chips in which the increase was greater than compare with the control sample with pancreatitis. The sensibility and the reliability of quantification are greater in the technique of Quantitative RT-PCR than in the technique of DNA-chips, which can be considered semiquantitative, so we consider that this quantitative PCR result, in which the expression of the human AGR2 gene is strongly increased in all tumor samples, except PA33, regarding healthy samples and regarding samples with Pancreatitis, is more correct.

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2.3. Discussion

These results confirmed that the increase of expression of the human AGR2 gene, is associated with adenocarcinoma ductal pancreas.

Claims (19)

1. In vitro method to detect the presence of pancreatic cancer in an individual, to determine the stage or severity of said cancer in the individual, or to monitor the effect of therapy administered to an individual presenting said cancer, which understands: a) the detection and / or quantification of the human AGR2 protein, from the human AGR2 gene mRNA or from the corresponding cDNA of at least one of said genes in a sample of said individual, and b) comparison of the amount of protein human AGR2, of the amount of mRNA of the human AGR2 gene or of the amount of the corresponding cDNA of the gene detected in a sample of an individual, with the amount of human protein AGR2 with the amount of the mRNA of the human AGR2 gene or with the amount of corresponding cDNA of the gene detected in the samples of control individuals or in previous samples of the same individual or with normal reference values. 2. Method according to claim 1 wherein the Pancreatic cancer is a ductal adenocarcinoma of the pancreas. 3. Method according to claim 1 wherein said sample is from pancreas tissue. 4. Method according to claim 3 wherein said sample of pancreas tissue to be analyzed is obtained by any conventional method, preferably resection surgical 5. Method according to claim 1 wherein said sample is a sample of urine, blood, plasma, serum, aspirated gastric juice, bile or semen. 6. Method according to claim 1, wherein said sample to be analyzed is obtained from an individual who is not You have previously diagnosed a cancer. 7. Method according to claim 1, wherein said sample to be analyzed is obtained from an individual who has been previously diagnosed a cancer, preferably in the pancreas. 8. Method according to claim 1, wherein said sample to be analyzed is obtained from an individual in treatment, or who has been previously treated, against cancer, preferably in the pancreas. 9. Method according to claim 1 characterized in that it comprises carrying out a sample extraction, either to obtain a protein extract or to obtain an extract consisting of total RNA. 10. Method according to claim 9 characterized in that the detection of the human AGR2 protein comprises a first step of contacting the sample protein extract with a composition of one or more specific antibodies against one or more epitopes of the human AGR2 protein and a second stage of quantification of the complexes formed by antibodies and the human AGR2 protein. Method according to claim 10 characterized in that said antibodies comprise monoclonal, polyclonal, intact antibodies or recombinant fragments thereof, combibodies and Fab or scFv fragments of antibodies, specific against the human AGR2 protein; these antibodies being human, humanized or of non-human origin. 12. Method according to claims 10 or 11 characterized in that for the quantification of complexes formed by antibodies and the human AGR2 protein, techniques selected from the group consisting of: Western-blot, ELISA (Enzyme-Linked Immunosorbent Assay or immunosorbent assay) are used linked to enzyme), RIA (Radioimmunoassay or Radioimmunoassay), Competitive EIA (Competitive Enzyme Immunoassay or Competitive Enzyme Immunoassay), DAS-ELISA (Double antibody Sandwich-ELISA or ELISA sandwich with Double Antibody), immunocytochemical, immunohistochemical and immunohistochemical techniques the use of biochips or microarrays of proteins that include specific antibodies, tests based on colloidal gold precipitation in formats such as dipsticks ; or by affinity chromatography techniques, ligand binding assays or binding assays
to lectin. 13. Method according to claim 9 characterized in that the detection of the mRNA or the corresponding cDNA of the human AGR2 gene comprises a first stage of amplification of the mRNA present in the total RNA extract, or of the corresponding cDNA synthesized by reverse transcription of the mRNA, and a second stage of quantification of the product of the amplification of the mRNA or the cDNA of the human AGR2 gene. 14. Method according to claim 13 characterized in that the amplification is performed qualitatively or quantitatively, by RT-PCR using oligonucleotide primers, the sequences of the primers being used to amplify the sequence of the human gene AGR2 SEQ ID NO 1 and SEQ ID NO 2.

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15. Method according to claim 9 characterized in that the detection is carried out with specific probes of the mRNA or the corresponding cDNA of the human AGR2 gene, by techniques such as Northern blot or Northern blotting. 16. Method according to claim 9 characterized in that the detection of the mRNA is carried out by means of real-time quantitative RT-PCR (Q-PCR). 17. Use of nucleotide or peptide sequences derived from the human AGR2 gene to detect in vitro the presence of pancreatic cancer, especially a pancreatic ductal adenocarcinoma, to determine in vitro the stage or severity of said cancer in the individual, or to monitor in vitro the effect of the therapy administered to an individual presenting said cancer. 18. In vitro method for identifying and evaluating the efficacy of compounds for therapy of a pancreatic cancer, especially of a ductal adenocarcinoma of the pancreas, comprising: a) contact a cell culture tumor, with the candidate compound, under the conditions and during the appropriate time to allow them to interact, b) detect and quantify the levels of expression of the human AGR2 gene or at least one of the human protein AGR2 and c) compare these expression levels with the of control cultures of untreated tumor cells with the candidate compound. 19. Use of nucleotide or peptide sequences derived from the human AGR2 gene, in search methods, identification, development and evaluation of the effectiveness of compounds for therapy of pancreatic cancer, especially a ductal adenocarcinoma of the pancreas.