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
METHODS FOR IN VITRO AND FORECAST IN VITRO DIAGNOSIS OF THE CANCER OF PANCREAS, AND FOR THE DEVELOPMENT OF PHARMACOS AGAINST CANCER OF PANCREAS. Download PDFInfo
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- ES2299281B1 ES2299281B1 ES200302458A ES200302458A ES2299281B1 ES 2299281 B1 ES2299281 B1 ES 2299281B1 ES 200302458 A ES200302458 A ES 200302458A ES 200302458 A ES200302458 A ES 200302458A ES 2299281 B1 ES2299281 B1 ES 2299281B1
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Abstract
Métodos para el diagnóstico in vitro y pronóstico in vitro del cáncer de páncreas, y para el desarrollo de fármacos contra el cáncer de páncreas.Methods for diagnosing in vitro and in vitro prognosis of pancreatic cancer, and for drug development in pancreatic cancer.
La presente invención se refiere a un método in vitro para detectar la presencia de un cáncer de páncreas, en especial un adenocarcinoma ductal de páncreas, en un individuo, para determinar el estadio o la severidad de dicho cáncer en el individuo, o para monitorizar el efecto de la terapia administrada a un individuo que presente dicho cáncer; a la búsqueda, identificación, desarrollo y evaluación de la eficacia de compuestos para terapia de dicho cáncer, con el fin de desarrollar nuevos medicamentos; así como a agentes que inhiben la expresión y/o la actividad de la proteína human AGR2 y/o los efectos de esta expresión.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
Métodos para el diagnóstico in vitro y pronóstico in vitro del cáncer de páncreas, y para el desarrollo de fármacos contra el cáncer de páncreas.Methods for diagnosing in vitro and in vitro prognosis of pancreatic cancer, and for drug development in pancreatic cancer.
La presente invención se refiere a un método in vitro para detectar la presencia de un cáncer de páncreas en un individuo, para determinar el estadio o la severidad de dicho cáncer en el individuo, o para monitorizar el efecto de la terapia administrada a un individuo que presente dicho cáncer; a la búsqueda, identificación, desarrollo y evaluación de la eficacia de compuestos para terapia de dicho cáncer, con el fin de desarrollar nuevos medicamentos; así como a agentes que inhiben la expresión y/o la actividad de las proteína human AGR2 y/o los efectos de esta expresión.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.
El cáncer de páncreas fue la causa de más de 220.000 muertes en el mundo, y de más de 3.600 en España, durante el año 2.000 (GLOBOCAN). El comportamiento clínico del cáncer de páncreas es homogéneo y siempre infausto, sin diferencias notables en la supervivencia según el estadio. El número de pacientes con cáncer de páncreas de buen pronóstico es insignificante. Una posible explicación es que incluso en pacientes con tumores pequeños, encuadrados en el estadio I, la enfermedad está diseminada. Su diagnóstico en una fase inicial, salvo en casos fortuitos, es difícil; un 75% de los pacientes diagnosticados presentan enfermedad avanzada (Estadios III y IV). Se trata de una neoplasia muy agresiva, resistente a los tratamientos citostáticos y sólo entre un 1 y un 4% de los casos permanecen vivos a los cinco años del diagnóstico, siempre que el tumor esté localizado y haya podido ser extirpado en su totalidad (Warshaw Al., y Fernándes del Castillo C., N. Eng. J. Med., 1992, 326:455-465; Ahlgren J.D., Semin. Oncol., 1996, 23:241-250). Es por todo ello que en el caso del cáncer de páncreas, tanto el desarrollo de sistemas de diagnóstico precoz como el de terapias eficaces, resultan cruciales para el control de la enfermedad (Byungwoo R., y col., Cancer Res., 2002, 62:819-826).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).
Muchos de los genes y proteínas implicados en la progresión del cáncer de páncreas son todavía desconocidos; la identificación de genes y proteínas expresados diferencialmente en asociación con el proceso de invasividad tumoral, podría conducir a la identificación de marcadores biológicos y dianas terapéuticas, que podrían tener un gran valor para el diagnóstico, el pronóstico y el tratamiento de esta enfermedad.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.
Inesperadamente, los autores de la presente invención han descubierto, tras laboriosa investigación y empleando diferentes técnicas (DNA-chips y RT-PCR cuantitativa), que la expresión de el gen que codifican para la proteína AGR 2, human anterior gradient 2 (Xenopus laevis) homolog, código GeneBank AF088867 está muy incrementada en los carcinomas de páncreas, en especial en los adenocarcinomas ductales de páncreas, al compararla con la expresión en tejido no tumoral procedente de los mismos individuos, o de individuos afectados de pancreatitis. Estas evidencias convierten a la proteína human AGR2 en una útil diana para el desarrollo de nuevos métodos in vitro de diagnóstico y pronóstico, y para la identificación y desarrollo de compuestos para terapia del cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas.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.
La detección in vitro de niveles altos de expresión del gen human AGR2 o de la proteína codificadas human AGR2 o de cualquier combinación de ellos, en muestras de tejido de páncreas o en otras muestras, preferiblemente suero, de individuos, permitirá la detección precoz del cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas. 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.
El desarrollo de nuevos fármacos dirigidos específicamente contra el gen human AGR2 o contra la proteína codificada human AGR2 o de cualquier combinación de ellos, es una nueva aproximación para tratar el cáncer de páncreas, en especial el adenocarcinoma ductal de páncreas.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.
La presente invención proporciona por tanto un método in vitro de alta sensibilidad para detectar la presencia de un cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas, para determinar el estadio o la severidad de dicho cáncer en el individuo, o para monitorizar el efecto de la terapia administrada a un individuo que presente dicho cáncer, basado en la detección y/o cuantificación de la proteína human AGR2, del mRNA del gen human AGR2 o del correspondiente cDNA del gen en una muestra de dicho individuo. Asimismo, la presente invención proporciona dianas o herramientas para la búsqueda, identificación, desarrollo y evaluación de la eficacia de compuestos para terapia de cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas.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.
Finalmente, la invención proporciona agentes caracterizados porque inhiben la expresión y/o la actividad de la proteína human AGR2, para el tratamiento del cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas.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.
La presente invención tiene como objeto principal el desarrollo de un método in vitro para detectar la presencia del cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas, para determinar el estadio o la severidad de dicho cáncer en el individuo, o para monitorizar el efecto de la terapia administrada a un individuo que presente dicho cáncer.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.
Un segundo objeto de la presente invención es un método in vitro para buscar, identificar, desarrollar y evaluar la eficacia de compuestos para la terapia del cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas.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.
Un objeto adicional de la invención reside en el
uso de secuencias derivadas del gen human AGR2, para el diagnóstico
y pronóstico in vitro del cáncer de páncreas, en especial
del adenocarcinoma ductal de páncreas, así como para la búsqueda,
identificación, desarrollo y evaluación de la eficacia de compuestos
para la terapia de dicho
cáncer.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.
Otro objeto de la presente invención consiste en proporcionar agentes caracterizados porque inhiben la expresión y/o la actividad de la proteína human AGR2 para el tratamiento del cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas.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.
Por último, es también objeto de la invención una composición farmacéutica que comprenda uno o varios agentes terapéuticos junto con un excipiente farmacéuticamente aceptable, para el tratamiento del cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas.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.
Figura 1: Curva de desnaturalización del producto de la PCR del gen diana, human AGR2 (Tm = 77ºC) y del gen de referencia, ribI10 (Tm=84ºC), en experimentos de medida de la expresión génica por RT-PCR cuantitativa en tiempo real, en muestras de páncreas.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.
Figura 2: Cálculo de la eficiencia de amplificación de las reacciones de PCR de human AGR2, en experimentos de medida de la expresión génica por RT-PCR cuantitativa en tiempo real, en muestras de páncreas.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.
Figura 3: Cálculo de la eficiencia de amplificación de las reacciones de PCR de ribI10, en experimentos de medida de la expresión génica por RT-PCR cuantitativa en tiempo real, en muestras de páncreas.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.
Para facilitar la comprensión de la presente solicitud de patente, exponemos a continuación el significado de algunos términos y expresiones en el contexto de la invención:To facilitate the understanding of this patent application, we explain below the meaning of Some terms and expressions in the context of the invention:
Los términos "sujeto" o "individuo" se refieren a miembros de especies de animales mamíferos, e incluye, pero no se limita, a animales domésticos, primates y humanos; el sujeto es preferiblemente un ser humano, masculino o femenino, de cualquier edad o raza.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.
El término "cáncer" se refiere a la enfermedad que se caracteriza por un crecimiento anormal o descontrolado de las células, capaces de invadir tejidos adyacentes y diseminarse a órganos lejanos.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.
El término "carcinoma" se refiere al tejido que resulta del crecimiento celular anormal o descontrolado.The term "carcinoma" refers to tissue that results from abnormal or uncontrolled cell growth.
Los términos "cáncer de páncreas", "cáncer en el páncreas" o "adenocarcinoma de páncreas" se refieren a cualquier desorden proliferativo maligno de células del páncreas.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.
El término "tumor" se refiere a cualquier masa anormal de tejido producto de un proceso neoplásico, benigno (no canceroso) o maligno (canceroso).The term "tumor" refers to any abnormal tissue mass resulting from a benign neoplastic process (not cancerous) or malignant (cancerous).
El término "gen" se refiere a una región de una cadena molecular de desoxirribonucleótidos que codifica una proteína y que puede representar una porción de la secuencia codificante o la secuencia codificante completa.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.
El término "DNA" se refiere al ácido desoxirribonucleico. Una secuencia de DNA es una secuencia de desoxirribonucleótidos.The term "DNA" refers to acid deoxyribonucleic. A DNA sequence is a sequence of deoxyribonucleotides.
El término "RNA" se refiere al ácido ribonucleico. Una secuencia de RNA es una secuencia de ribonucleótidos.The term "RNA" refers to acid ribonucleic. An RNA sequence is a sequence of ribonucleotides
El término "mRNA" se refiere al ácido ribonucleico mensajero, que es la fracción del RNA total que se traduce a proteínas.The term "mRNA" refers to acid messenger ribonucleic, which is the fraction of total RNA that is Translates to proteins.
El término "cDNA" se refiere a una secuencia de nucleótidos, complementaria de una secuencia de mRNA.The term "cDNA" refers to a nucleotide sequence, complementary to a sequence of mRNA
La frase "mRNA transcrito de" se refiere a la transcripción del gen (DNA) en mRNA, como primer paso para que el gen se exprese y traduzca a proteína.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.
El término "secuencia de nucleótidos" o "secuencia nucleotídica" se refiere indistintamente a una secuencia de ribonucleótidos (RNA) o de desoxirribonucleótidos (DNA).The term "nucleotide sequence" or "nucleotide sequence" refers interchangeably to a ribonucleotide (RNA) or deoxyribonucleotide sequence (DNA).
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El término "proteína" se refiere a una cadena molecular de aminoácidos, unidos por enlaces covalentes o no covalentes. El término incluye todas las formas de modificaciones post-traduccionales, por ejemplo glicosilación, fosforilación o acetilación.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.
Los términos "péptido" y "polipéptido" se refieren a cadenas moleculares de aminoácidos que representan un fragmento proteico. Los términos "proteína" y "péptido", se usan indistintamente.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.
El término "anticuerpo" se refiere a una glucoproteína que exhibe una actividad de unión específica por una molécula diana, a la que se denomina "antígeno". El término "anticuerpo" comprende anticuerpos monoclonales, o anticuerpos policlonales, intactos, o fragmentos de ellos; e incluye anticuerpos humanos, humanizados y de origen no humano. Los "anticuerpos monoclonales" son poblaciones homogéneas de anticuerpos, altamente específicos, que están dirigidos contra un único sitio o "determinante" antigénico. Los "anticuerpos policlonales" incluyen poblaciones heterogéneas de anticuerpos, que están dirigidos contra diferentes determinantes antigénicos.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
El término "epítopo", tal como se utiliza en la presente invención, se refiere a un determinante antigénico de una proteína, que es la secuencia de aminoácidos de la proteína que un anticuerpo específico reconoce.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.
El término "fase sólida", tal como se utiliza en la presente invención, se refiere a una matriz no acuosa a la que se puede unir el anticuerpo. Ejemplos de materiales para fase sólida incluyen vidrio, polisacáridos, por ejemplo agarosa, poliacrilamida, poliestireno, alcohol polivinílico y siliconas. Ejemplos de formas de fase sólida son el pocillo de una placa de ensayo o una columna de purificación.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.
Los términos "oligonucleótido" y "oligonucleótido cebador" se utilizan indistintamente y se refieren, tal como se utiliza en la presente invención, a secuencias nucleotídicas, que son complementarias de una secuencia nucleotídica del gen human AGR2. Cada cebador hibrida con su secuencia nucleotídica diana y actúa como un sitio de inicio para la polimerización nucleotídica catalizada por la DNA polimerasa, la RNA polimerasa o la transcriptasa inversa.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.
El término "sonda", tal como se utiliza en la presente invención, se refiere a una secuencia nucleotídica complementaria de una secuencia nucleotídica derivada del gen human AGR2 que se puede utilizar para detectar esa secuencia nucleotídica derivada de dicho gen.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.
El término "diana terapéutica" se refiere a secuencias nucleotídicas o peptídicas, contra las que se puede diseñar y aplicar clínicamente un fármaco o compuesto terapéutico.The term "therapeutic target" refers to nucleotide or peptide sequences, against which you can Clinically design and apply a drug or compound therapeutic.
El término "antagonista" se refiere a cualquier molécula que inhiba la actividad biológica de la molécula antagonizada. Ejemplos de moléculas antagonistas incluyen, entre otros, proteínas, péptidos, variaciones de secuencia de péptidos naturales y pequeñas moléculas orgánicas (de peso molecular inferior a 500 daltons).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).
La presente invención se basa en el descubrimiento de que tanto la expresión del gen human AGR2, como la concentración de la proteína human AGR2 se ven incrementadas en el adenocarcinoma ductal de páncreas de un individuo.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.
En este sentido, la presente invención proporciona, en primer lugar, un método in vitro para detectar la presencia de un cáncer de páncreas, en especial de un adenocarcinoma ductal de páncreas, en un individuo, para determinar el estadio o la severidad de dicho cáncer en el individuo, o para monitorizar el efecto de la terapia administrada a un individuo que presente dicho cáncer, que comprende: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) la detección y/o cuantificación de la proteína human AGR2, del mRNA del gen human AGR2 o del correspondiente cDNA de dicho gen en una muestra de dicho individuo, ya) 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) la comparación de la cantidad de proteína human AGR2 , de la cantidad de mRNA del gen human AGR2, o de la cantidad del correspondiente cDNA del gen detectada en una muestra de un individuo, con la cantidad de proteína human AGR2, con la cantidad del mRNA del gen human AGR2 o con la cantidad del correspondiente cDNA del gen detectada en las muestras de individuos control o en muestras anteriores del mismo individuo o con los valores normales de referencia.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.
El método proporcionado por la presente invención es de alta sensibilidad y especificidad, y se basa en que sujetos o individuos diagnosticados de cáncer de páncreas, en especial de adenocarcinoma ductal de páncreas, presentan niveles elevados de mRNA transcrito del gen human AGR2, o concentraciones elevadas de la proteína codificada por el gen human AGR2 (proteína human AGR2), en comparación con los correspondientes niveles en muestras procedentes de sujetos sin historial clínico de este carcinoma.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.
El presente método comprende una etapa de obtención de la muestra del individuo. Se puede trabajar con distintas muestras fluidas como, por ejemplo: orina, sangre, plasma, suero, líquido pleural, líquido ascítico, líquido sinovial, bilis, semen, exudado de flujo gástrico o líquido cefalorraquídeo. La muestra también puede ser tejido de páncreas que se puede obtener por cualquier método convencional, preferiblemente resección quirúrgica.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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Las muestras pueden ser obtenidas de sujetos previamente diagnosticados, o no diagnosticados, de un determinado tipo de cáncer; o también de un sujeto en tratamiento, o que ha sido tratado previamente contra un cáncer, en particular contra el cáncer de páncreas.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.
El presente método comprende además una etapa de extracción de la muestra, ya sea para obtener el extracto de proteínas de ésta, o bien para obtener el extracto de RNA total. Uno de estos dos extractos representa el material de trabajo para la siguiente fase. Los protocolos de extracción de la proteína total o del RNA total son bien conocidos por el experto en la materia (Chomczynski P. y col., Anal. Biochern., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532).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).
Cualquier ensayo convencional se puede utilizar en el marco de la invención para detectar un cáncer, siempre que mida in vitro el nivel de mRNA transcrito del gen human AGR2, o su cDNA complementario, o la concentración de la proteína human AGR2, en muestras recogidas de los individuos a analizar y de individuos control.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.
Así pues, esta invención proporciona un método para detectar la presencia de un cáncer de páncreas, en especial de un adenocarcinoma ductal de páncreas, en un individuo, para determinar el estadio o la severidad de dicho cáncer en el individuo, o para monitorizar el efecto de la terapia administrada a un individuo que presente dicho cáncer, basado bien en la medida de la concentración de la proteína human AGR2, o bien en la medida del nivel de expresión del gen human AGR2.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.
En el caso de que lo que se pretenda detectar sea la proteína human AGR2, el método de la invención comprende una primera etapa de puesta en contacto del extracto de proteínas de la muestra con una composición de uno o más anticuerpos específicos contra uno o más epítopos de la proteína human AGR2 y una segunda etapa de cuantificación de los complejos formados por los anticuerpos y la proteína human AGR2.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.
Existe una amplia variedad de ensayos inmunológicos disponibles para detectar y cuantificar la formación de complejos específicos antígeno-anticuerpo; numerosos ensayos de unión de proteínas, competitivos y no competitivos, han sido previamente descritos y un gran número de estos ensayos está disponible comercialmente.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.
Así, la proteína human AGR2 se puede cuantificar con anticuerpos como, por ejemplo: anticuerpos monoclonales, policlonales, intactos o fragmentos recombinantes de ellos, combibodies y fragmentos Fab o scFv de anticuerpos, específicos contra la proteína human AGR2; siendo estos anticuerpos humanos, humanizados o de origen no humano. Los anticuerpos que se emplean en estos ensayos pueden estar marcados o no; los anticuerpos no marcados se pueden utilizar en ensayos de aglutinación; los anticuerpos marcados se pueden utilizar en una amplia variedad de ensayos. Las moléculas marcadoras que se pueden utilizar para marcar los anticuerpos incluyen radionucleótidos, enzimas, fluoróforos, reactivos quimioluminiscentes, sustratos enzimáticos o cofactores, inhibidores enzimáticos, partículas, colorantes y derivados.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.
Existe una amplia variedad de ensayos bien conocidos, que se pueden utilizar en la presente invención, que utilizan anticuerpos no marcados (anticuerpo primario) y anticuerpos marcados (anticuerpo secundario); entre estas técnicas se incluyen el Western-blot o transferencia Western, ELISA (Enzyme-Linked inmunosorbent assay o ensayo inmunoabsorvente ligado a enzima), RIA (Radioinmunoassay o Radioinmunoensayo), EIA competitivo (Competitive enzyme immunoassay o Inmunoensayo enzimático competitivo), DAS-ELISA (Double antibody sándwich-ELISA o ensayo ELISA sándwich con doble anticuerpo), técnicas petroleoquímicas e inmunohistoquímicas, técnicas basadas en el empleo de biochips o microarrays de proteínas que incluyan anticuerpos específicos o ensayos basados en precipitación coloidal en formatos tales como dipsticks. Otras maneras para detectar y cuantificar la proteína human AGR2, incluyen técnicas de cromatografía de afinidad, ensayos de unión a ligando o ensayos de unión a lectina.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.
El inmunoensayo preferido en el método de la invención es un ensayo ELISA sándwich con doble anticuerpo (DAS-ELISA). En este inmunoensayo se puede utilizar cualquier anticuerpo o combinación de anticuerpos, específicos contra uno o más epítopos de la proteína human AGR2. Como ejemplo de uno de los muchos posibles formatos de este ensayo, un anticuerpo, monoclonal o policlonal, o un fragmento de este anticuerpo, o una combinación de anticuerpos, que recubren una fase sólida, se ponen en contacto con la muestra a analizar, y se incuban durante un tiempo y en condiciones apropiados para formar los complejos antígeno-anticuerpo. Después de un lavado en condiciones apropiadas para eliminar los complejos no específicos, se incuba con los complejos antígeno-anticuerpo, en condiciones y tiempo apropiados, un reactivo indicador, que comprende un anticuerpo monoclonal o policlonal, o un fragmento de este anticuerpo, o una combinación de estos anticuerpos, unidos a un compuesto generador de una señal. La presencia de la proteína human AGR2 en la muestra a analizar, se detecta y cuantifica, en caso de que exista, midiendo la señal generada. La cantidad de proteína human AGR2 presente en la muestra analizar es proporcional a esa señal.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.
En el caso de que se pretenda detectar el mRNA o el cDNA correspondiente al gen human AGR2 y no las proteínas, el método de la invención para detectar in vitro el carcinoma posee etapas diferentes. Así, una vez obtenida la muestra y extraído el RNA total, según el método de la invención, se realiza la detección del mRNA o del correspondiente cDNA del gen human AGR2, que comprende una primera etapa de amplificación del extracto de RNA total o del correspondiente cDNA sintetizado por transcripción inversa a partir del mRNA, y una segunda etapa de cuantificación del producto de la amplificación del mRNA o del cDNA del human AGR2.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.
Un ejemplo de amplificación del mRNA, consiste en retrotranscribir el mRNA en cDNA (RT), seguido de la Reacción en Cadena de la polimerasa (PCR), usando oligonucleótidos cebadores, siendo las secuencias de los cebadores utilizados 5'-AGAAATTGGCAGAGCAGTTTGTC-3' (SEQ ID NO 1) y 5'-GCCATCAGGAGAAAGGTGTTTG-3' (SEQ ID NO 2) para human AGR2; la PCR es una técnica de amplificación de una determinada secuencia nucleotídica (diana) contenida en una mezcla de secuencias nucleotídicas. En la PCR, se utiliza un exceso de una pareja de oligonucleótidos cebadores, que hibridan con las hebras complementarias de la secuencia nucleotídica diana. A continuación, una enzima con actividad polimerasa (DNA Taq Polimerasa) extiende cada cebador, utilizando como molde la secuencia nucleotídica diana. Los productos de la extensión se convierten entonces en secuencias dianas, tras la disociación de la hebra diana original. Nuevas moléculas de cebador hibridan y la polimerasa las extiende; el ciclo se repite para aumentar exponencialmente el número de secuencias diana. Esta técnica está descrita en las patentes US 4683195 y US 4683202. Se han descrito previamente muchos métodos para detectar y cuantificar los productos de la amplificación por PCR, de los que cualquiera puede ser usado en esta invención. En un método preferido de la invención, el producto amplificado se detecta por electroforesis en gel de agarosa, de la manera siguiente: cinco microlitros del producto de la amplificación se someten a una separación por electroforesis en un gel de agarosa a una concentración del 2%, en un tampón TBE 0,5x a 100 vdc, durante una hora. Tras la electroforesis, el gel se tiñe con bromuro de etidio y el producto de la amplificación se visualiza al iluminar el gel con luz ultravioleta (uv); como alternativa a la tinción, y realización preferida, se puede transferir el producto amplificado a una membrana de nailon por técnicas de Southern blotting o transferencia Southern, para ser detectado con una sonda específica del cDNA del gen human AGR2 convenientemente marcada.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.
En otro ejemplo la detección del mRNA se realiza transfiriendo el mRNA a una membrana de nailon, mediante técnicas de transferencia como por ejemplo Northern-blot o transferencia Northern, y detectándolo con sondas específicas del mRNA o del correspondiente cDNA del gen human AGR2.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.
En una realización particular la amplificación y cuantificación del mRNA correspondiente al gen human AGR2 se realiza a la vez mediante RT-PCR cuantitativa a tiempo real (Q-PCR).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).
El paso final del método de la invención para detectar in vitro la presencia del cáncer, en una muestra procedente de un individuo, comprende comparar la cantidad de proteína human AGR2, la cantidad de mRNA del gen human AGR2 o la cantidad del correspondiente cDNA del gen detectada en una muestra de un individuo, con la cantidad de proteína human AGR2, la cantidad de mRNA del gen human AGR2, o la cantidad del correspondiente cDNA del gen detectada en las muestras de sujetos control o en muestras anteriores no tumorales del mismo individuo, o con los valores normales de referencia.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.
En su segundo objeto, la invención también proporciona un método in vitro para identificar y evaluar la eficacia de agentes para terapia del cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas, que comprende: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) poner en contacto un cultivo de células tumorales, con el compuesto candidato, en las condiciones y durante el tiempo apropiados para permitir que interaccionen,a) contact a cell culture tumor, with the candidate compound, under the conditions and during the appropriate time to allow them to interact,
b) detectar y cuantificar los niveles de expresión del gen human AGR2 o de al menos una de las proteínas human AGR2, yb) detect and quantify the levels of expression of the human AGR2 gene or at least one of the proteins human AGR2, and
c) comparar dichos niveles de expresión con los de cultivos control de células tumorales sin tratar con el compuesto candidato.c) compare these expression levels with the of control cultures of untreated tumor cells with the candidate compound.
La cuantificación de los niveles de expresión del gen human AGR2, o la proteína human AGR2 se realizan de modo semejante a como se indica en el método de la invención para detectar in vitro la presencia de un cáncer de páncreas, en especial de un adenocarcinoma ductal de páncreas, en un individuo.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.
Cuando un agente disminuye los niveles de expresión génica y/o proteica de la proteína human AGR2, o revierte los efectos de dicha expresión elevada, preferiblemente disminuyendo los niveles de proliferación celular, este agente se convierte en candidato para la terapia del cáncer.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.
Por tanto, otro objeto de la invención se refiere al uso de secuencias nucleotídicas o peptídicas derivadas del gen human AGR2, en métodos de búsqueda, identificación, desarrollo y evaluación de la eficacia de compuestos para terapia del cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas. Resaltar la importancia adquirida últimamente por los métodos de screening de fármacos basados en el binding, competitivo o no, de la molécula potencial fármaco a la diana terapéutica.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.
Otro objeto adicional de la invención se refiere al uso de secuencias nucleotídicas o peptídicas derivadas del gen human AGR2 para detectar la presencia del cáncer de páncreas, en especial del adenocarcinoma ductal de páncreas, para determinar el estadio o la severidad de dicho cáncer en el individuo, o para monitorizar el efecto de la terapia administrada a un individuo que presente dicho cáncer.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.
Otro objeto de la invención consiste en proporcionar agentes caracterizados porque inhiben la expresión y/o la actividad de la proteína human AGR2. Estos agentes, que se pueden identificar y evaluar según la presente invención, pueden ser seleccionados del grupo formado por: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) un anticuerpo, o combinación de anticuerpos, específicos contra uno o más epitopos presentes en la proteína human AGR2, preferiblemente un anticuerpo monoclonal humano o humanizado; pudiendo ser también un fragmento del anticuerpo, un anticuerpo de cadena sencilla o un anticuerpo anti-idiotipo,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) agentes citotóxicos, tales como toxinas, moléculas con átomos radiactivos, o agentes quimio-terapéuticos, entre los que se incluyen, sin limitación, pequeñas moléculas orgánicas e inorgánicas, péptidos, fosfopéptidos, moléculas antisentido, ribozimas, siRNAs, moléculas de triple hélice, etc., que inhiben la expresión y/o la actividad de la proteína human AGR2 yb) 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) compuestos antagonistas de la proteína human AGR2 que inhiben una o más de las funciones de la proteína human AGR2.c) human protein antagonist compounds AGR2 that inhibit one or more of the functions of the human protein AGR2.
Por último, constituye también un objeto de la presente invención una composición farmacéutica que comprende una cantidad terapéuticamente eficaz de uno o varios agentes de los mencionados anteriormente junto con uno o más excipientes y/o sustancias transportadoras. Además dicha composición puede contener cualquier otro ingrediente activo que inhiba la función de la proteína human 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.
Los excipientes, sustancias transportadoras y sustancias auxiliares tienen que ser farmacéuticamente y farmacológicamente tolerables, de modo que puedan ser combinados con otros componentes de la formulación o preparación y no ejerzan efectos adversos en el organismo tratado. Las composiciones farmacéuticas o formulaciones incluyen aquellas que son adecuadas para la administración oral o parenteral (incluyendo subcutánea, intradérmica, intramuscular e intravenosa), aunque la mejor vía de administración depende del estado del paciente. Las formulaciones pueden ser en forma de dosis sencillas. Las formulaciones se preparan de acuerdo con métodos conocidos en el campo de la farmacología. Las cantidades de sustancias activas para administrarse pueden variar en función de las particularidades de la terapia.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.
Los siguientes ejemplos ilustran la invención.The following examples illustrate the invention.
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Microarrays. Se utilizaron los
microarrays GeneChip Test 3 (Affymetrix, Santa Clara), que permiten
testar la calidad del RNA, previamente al análisis de expresión con
el array GeneChip Human Genome U133A (Affymetrix, Santa Clara), que
representa 13.220 secuencias completas de genes anotados; el gen
human AGR2 está representado en el microarray por el set de sondas
209173_at; cada set de sondas está compuesto por 11
oligonucleótidos sentido de 25 nucleótidos de longitud, diseñados en
base a la secuencia Hs. 12802 de Unigene, o AB007860 de
GeneBank. 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.
Muestras. Las muestras estudiadas procedían de biopsias clasificadas clínicamente, obtenidas por resección quirúrgica: Biopsias de tejido pancreático no tumoral, procedentes de individuos que presentaban adenocarcinoma ductal de páncreas ("no tumoral") (n = 1); biopsias de tejido pancreático no tumoral, pero inflamado, procedentes de individuos sin adenocarcinoma ductal de páncreas, pero con pancreatitis crónica ("pancreatitis crónica") (n = 1); biopsias de tejido pancreático no tumoral, pero inflamado, procedentes de individuos que presentaban adenocarcinoma ductal de páncreas y pancreatitis ("pancreatitis") (n = 1); biopsias de tejido pancreático tumoral , procedentes de pacientes que presentaban adenocarcinoma ductal de páncreas (n = 10) en uno de los siguientes estadios: Estadio I, tumor limitado al páncreas, Estadio III, tumor extendido a ganglios linfáticos regionales y Estadio IVB, existe metástasis en tejidos u órganos distantes. Todas las muestras fueron clasificadas clínica e histológicamente (grado y estadio) en el Hospital Central de Asturias, el mismo hospital donde las muestras habían sido recogidas, siguiendo los preceptos de la declaración de Helsinki. Las muestras se congelaron en nitrógeno líquido inmediatamente tras su extracción y se mantuvieron a -80ºC hasta el momento de su análisis. 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.
De cada tipo de tumor se recibieron los siguientes casos:For each type of tumor the following cases:
- - Tumor estadio I:- Stadium tumor I:
- 7 muestras7 samples
- - Tumor estadio III:- Stadium tumor III:
- 1 muestra1 sample
- - Tumor estadio IV:- Stadium tumor IV:
- 2 muestras2 samples
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El análisis se llevó a cabo con RNA total procedente de sujetos individuales. Las 13 muestras que fueron analizadas se describen en la Tabla 1.The analysis was carried out with total RNA from individual subjects. The 13 samples that were analyzed are described in Table 1.
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El RNA total de cada una de las biopsias se obtuvo homogenizando el tejido en TRizol® Reagent (Life Technologies), siguiendo las recomendaciones del proveedor. El RNA total resultante se limpió con el kit Rneasy (QIAGEN) (Chomczynski P. et al., Anal. Biochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532). De cada preparación de RNA total se usaron 10 \mug como material de partida para la síntesis de la primera hebra de cDNA con la enzima transcriptasa inversa SuperScript^{TM} II RNase (Life Technologies), usando como cebador un oligonucleótido oligo-dT que contenía la secuencia del promotor de la RNA polimerasa del fago T7. La segunda hebra de cDNA se sintetizó utilizando los enzimas DNA polimerasa I de E. coli (Invitrogen Life Technologies), DNA ligasa de E. coli (Invitrogen Life Technologies), Rnasa H de E. coli (Invitrogen Life Technologies), y DNA polimerasa del fago T4 (Invitrogen Life Technologies). El cRNA marcado con biotina se sintetizó usando el kit ENZO BioArray^{TM} HighYield^{TM} Transcript Labeling Kit (Enzo Diagnostics Inc). Después de la transcripción in vitro, se eliminaron los nucleótidos no incorporados usando las columnas Rneasy (QIAGEN).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).
Se fragmentaron 15 \mug de cada cRNA biotinilado a 94ºC durante 35 minutos en una solución tampón que contenía 40 mM Tris-Acetato (pH 8.1), 100 mM KOAc y 30 mM MgOAc. El cRNA fragmentado se mezcló con buffer de hibridación (100 mM MES, 1M NaCl, 20 mM EDTA, 0.01% Tween 20) y se calentó a 99º durante 5 minutos y posteriormente a 45º durante 5 minutos, para a continuación ser cargado en el array de Affymetrix. El primer array en el que se realizó la hibridación fue el Test 3 de Affymetrix. Este array permite testar la calidad del RNA previo al análisis de expresión en el Affymetrix® GeneChip® Human Genome 133 A (HG-U133A).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).
Para la hibridación, los arrays se incubaron en un horno rotatorio a 45º durante 16 horas y con una rotación constante de 60 rpm.For hybridization, arrays were incubated in a 45º rotary kiln for 16 hours and with a rotation 60 rpm constant.
El lavado y tinción de cada array se llevó a cabo en la Estación de Fluidos de Affymetrix®. Se usó un programa de lavado y tinción que incluía: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 ciclos de lavado con SSPE-T 6x (0.9 m NaCl, 60 mM NaH_{2}PO4, 6 mM EDTA, 0.01% Tween 20) a 25º,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 °,
- --
- 4x15 ciclos con 0.1 mM MES, 0.1M NaCl, 0.01% Tween 20 a 50º,4x15 cycles with 0.1 mM MONTH, 0.1M NaCl, 0.01% Tween 20 at 50º,
- --
- Tinción del cRNA biotinilado con un conjugado estreptavidina-ficoeritrina (10 \mug/mlMolecular Probes)Staining of biotinylated cRNA with a streptavidin-phycoerythrin conjugate (10 \ mug / mlMolecular Probes)
- --
- 10x4 ciclos de lavado con SSPE-T a 25º10x4 wash cycles with SSPE-T at 25º
- --
- Tinción con un anticuerpo anti-estreptavidina durante 10 minutosStaining with an antibody anti-streptavidin for 10 minutes
- --
- Tinción un conjugado estreptavidina-ficoeritrina (1 mg/ml, Molecular Probes) durante 10 minutosStaining a conjugate Streptavidin-phycoerythrin (1 mg / ml, Molecular Probes) for 10 minutes
- --
- 15x4 ciclos de lavado con SSPE-T a 30º15x4 wash cycles with SSPE-T at 30º
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Los arrays se escanearon a 560 nm usando un microscopio confocal que utiliza emisión láser (Agilent GeneArray Scanner). El análisis de las lecturas de intensidad se realizó con el software Microarray Suite 5.0. Para la comparación de arrays, éstos fueron escalados a una intensidad total de 100.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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El análisis diferencial de la expresión del gen human AGR2 en las biopsias de tejido pancreático tumoral, con respecto a las biopsias de tejido pancreático no tumoral, con o sin pancreatitis, se realizó a partir de los datos de comparación de arrays obtenidos utilizando el software de Affymetrix. Los parámetros que se tuvieron en cuenta (en el orden en el que aparecen en la lista) fueron: i) Detección. Indica si el transcrito está Presente (P), Ausente (A) o Marginal (M), ii) Cambio: Indica si la expresión de un determinado transcrito Aumenta (I), Decrece (D), No Cambia (NC), Aumenta Marginalmente (MI), o Decrece Marginalmente (MD), iii) Signal Loq Ratio (SLR): Indica el nivel de cambio de expresión entre la línea base (control) y una muestra problema. Este cambio se expresa como el log_{2} del ratio (fold change o número de veces que la expresión del gen está elevada o reprimida en la muestra problema-tumoral frente a la muestra control-no tumoral). Se considera significativo un valor de SLR de 1 (equivalente a un fold change de 2), para transcritos cuya expresión aumenta frente al control y de -1, para transcritos cuya expresión disminuye frente al control.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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El análisis diferencial de la expresión del gen human AGR2 en los estadios tumorales con respecto al control no tumoral PA36, o al control con pancreatitis (PA46), demostró que los niveles de expresión del gen human AGR2 en los estadios tumorales estaban incrementados en todos los tumores analizados, salvo en 2 de las 7 biopsias de adenocarcinoma ductal de páncreas de estadio I. Los incrementos eran consistentemente superiores al comparar los tumores frente al control con pancreatitis, con incrementos de más de 100 veces (SLR=6,7 y SLR=11,5), que al compararlos frente al control no tumoral PA36, con incrementos que oscilaban entre 3 y 60 veces (SLR=1,4 y SLR=5,7) (Tablas 16 y 17).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).
Estos resultados demostraron que la expresión del gen human AGR2 estaba incrementada en la gran mayoría de los adenocarcinomas ductales de páncreas analizados, tanto frente a biopsias de tejido no tumoral PA36, como frente a biopsias de pancreatitis crónica.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.
El método empleado consiste en la transcripción inversa del mRNA a cDNA y su posterior amplificación en un equipo LightCycler (Roche), utilizando SYBR Green para la detección del producto amplificado. La cuantificación se realiza en tiempo real y permite calcular la expresión relativa de la secuencia en diferentes muestras en la fase de amplificación lineal de la reacción.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.
Muestras. Las muestras estudiadas en el análisis de expresión de los genes human AGR2s procedían de biopsias clasificadas clínicamente, obtenidas por resección quirúgica: Biopsias de tejido pancreático no tumoral, procedentes de individuos que presentaban adenocarcinoma ductal de páncreas ("no tumoral") (n = 1); biopsias de tejido pancreático no tumoral, pero inflamado, procedentes de individuos sin adenocarcinoma ductal de páncreas, pero con pancreatitis crónica (n = 1) ("pancreatitis crónica"); biopsias de tejido pancreático tumoral, procedentes de pacientes que presentaban adenocarcinoma ductal de páncreas (n = 5) en uno de los siguientes estadios: Estadio I, tumor limitado al páncreas y Estadio IV, existe metástasis en tejidos u órganos distantes. 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.
Todas las muestras fueron clasificadas histológicamente (grado y estadio) en el Hospital Central de Asturias, el mismo hospital donde las muestras habían sido recogidas siguiendo los preceptos de la Declaración de Helsinki. Las muestras se congelaron en nitrógeno líquido inmediatamente tras su extracción y se mantuvieron a -80ºC hasta el momento de su análisis.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.
RT-PCR Cuantitativa en Tiempo Real. El análisis se llevó a cabo con RNA total procedente de sujetos individuales. Las muestras que fueron analizadas se describen en las Tablas 4. 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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El RNA total de cada una de las biopsias se obtuvo homogenizando el tejido en TRizol® Reagent (Life Technologies), siguiendo las recomendaciones del proveedor. El RNA total resultante se limpió con el kit Rneasy (QIAGEN) (Chomczynski P. et al., Anal. Biochem., 1987, 162: 156; Chomczynski P., Biotechniques, 1993, 15: 532). El RNA se cuantificó espectrofotométricamente y se digirieron 5 \mug de RNA total con DNasa I. Se utilizó 1 pg de RNA tratado con DNAsa como material de partida para la síntesis de la primera hebra de cDNA con la enzima transcriptasa inversa SuperScript^{TM} II RNase (Life Technologies), usando como cebador un oligonucleótido oligo-dT que contenía la secuencia del promotor de la RNA polimerasa del fago T7. Se prepararon alícuotas del cDNA diluido a la concentración de trabajo.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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El cDNA sintetizado se amplificó utilizando cebadores específicos del gen humano AGR2 (SEQ ID NO 1 y SEQ ID NO 2) y cebadores específicos del gen que codifica la proteína ribosomal L10 humana (SEQ ID NO 4 y SEQ ID NO 5). Las reacciones de PCR en tiempo real se prepararon utilizando el kit LightCycler-FastStart DNA master SYBR Green I kit (Roche), siguiendo las instrucciones del fabricante. El programa de amplificación consistía en 1 ciclo de 95ºC durante 10 min ("hot start") seguido de 45 ciclos de 95ºC (desnaturalización) durante 10 seg, 60ºC (anillamiento) durante 5 seg, 72ºC (amplificación y adquisición de fluorescencia) durante 10 seg. El programa de análisis de curvas de desnaturalización consistía en un ciclo de un pulso de 95ºC, 65ºC durante 15 seg, y un pulso de 95ºC durante el paso de amplificación y adquisición.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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En primer lugar se determinó la especificidad de los productos de PCR analizando las curvas de desnaturalización. Posteriormente, como medida relativa de la expresión génica, se calculó la relación entre la abundancia de mRNAs transcritos de human AGR2 y la abundancia de mRNAs transcritos de ribI10 y se normalizó el dato de la relación en cada una de las muestras tumorales en base a los valores de la muestra control. Para calcular la eficiencia de las reacciones de PCR (human AGR2 y ribI10) se construyó, para cada secuencia génica, una curva patrón realizada con diluciones seriadas de cDNA. A las concentraciones de cDNA molde para las reacciones en la curva patrón se les dieron valores arbitrarios 10, 5, 2.5, 1.25 y 0.625. La eficiencia se calculó utilizando la ecuación: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}E = 10 -1 / P
donde E es la eficiencia de amplificación y p es la pendiente de la recta patrón.where E is the amplification efficiency and p is the slope of the standard line.
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La relación de los valores de expresión génica se determinó utilizando la siguiente ecuación, que relaciona los datos experimentales de la amplificación y corrige el error originado por la diferencia de eficiencia de las reacciones de PCR: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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donde E es la eficiencia de amplificación, Cp es el punto de corte (Crossing point), diana es human AGR2, referencia es ribI10, control es la muestra control (no tumoral o pancreatitis crónica) y muestra es la muestra tumoral.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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El análisis de los productos de PCR demostró la amplificación específica de dos productos con temperaturas de desnaturalización cercanas a las esperadas según el cálculo teórico del software de diseño de cebadores, PrimerExpress (Applied Biosystems) (Figuras 1-6).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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De cada una de las 5 diluciones de cDNA se amplificaron dos réplicas y los puntos de corte (Cp) de cada una de ellas se representaron en una gráfica respecto al logaritmo de la concentración de cDNA para construir la recta patrón (Tablas 5-6; figuras 2-3).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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Dos réplicas de cada muestra (sana y tumoral) se amplificaron con los cebadores específicos de human AGR2 y ribI10. A partir de los puntos de corte generados en estas amplificaciones, se calcularon los cambios de expresión del gen human AGR2, en las muestras tumorales respecto al control no tumoral PA36 y al control de pancreatitis PA46, aplicando la ecuación anteriormente descrita. Los datos experimentales y la cuantificación final se describen en las Tablas 7 y 8 para el gen human AGR2.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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Los resultados confirmaron los datos obtenidos midiendo la diferencia de expresión génica con DNA-chips (ejemplo 1), esto es, la expresión del gen human AGR2 estaba fuertemente incrementada en todas las muestras tumorales, excepto PA33, respecto a la muestra no tumoral PA36 y respecto a la muestra con pancreatitis crónica PA46, con un incremento medio de la expresión de 18 veces, con respecto a las muestras no tumorales sin pancreatitis (PA36) y de 19 veces, con respecto a las muestras no tumorales con pancreatitis (PA46) (Tabla 9); no se aprecia diferencia al comparar frente a muestra sana o a muestra con pancreatitis, lo que es diferente al resultado con los DNA-chips en el que el incremento era mayor al comparar con las muestra control con pancreatitis. La sensibilidad y la fiabilidad de la cuantificación son mayores en la técnica de RT-PCR cuantitativa que en la técnica de DNA-chips, que se puede considerar semicuantitativa, por lo que consideramos que este resultado de PCR cuantitativa, en el que la expresión del gen human AGR2 está fuertemente incrementada en todas las muestras tumorales, excepto PA33, respecto a las muestras sanas y respecto a las muestras con pancreatitis, es más correcto.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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Estos resultados confirmaron que el incremento de expresión del gen human AGR2, está asociado al adenocarcinoma ductal de páncreas.These results confirmed that the increase of expression of the human AGR2 gene, is associated with adenocarcinoma ductal pancreas.
Claims (19)
a lectina.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.
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| IACOBUZIO-DONAHUE, C. et al. "Exploration of global gene expression patterns in pancreatic adenocarcinoma using cDNA microarrays". American Journal of Pathology. Abril 2003, vol. 162(4), pp.: 1151-1162, página 1151, resumen; página 1152, columna 1, párrafos 2-3; página 1155, columna 1, párrafo 4; columna 2, párrafo 2; página 1156, tabla-1. * |
| RYU, B. et al. "Relationships and differentially expressed genes among pancreatic cancers examined by large-scale serial analysis of gene expression ". Cancer Research . 2002, vol. 62(3), pp.: 819-826, página 819, resumen; página 822, tabla-3; página 824, columna 1, párrafo 1. * |
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