WO2012059617A1 - Method for obtaining useful data for diagnosing pancreatic cancer and for evaluating the response to treatment - Google Patents

Abstract

The invention relates to a method for obtaining useful data for diagnosing an early stage of pancreatic adenocarcinoma, as well for evaluating the response to the treatment of said disease, making it possible to establish an individual recognition pattern (qualitative and quantitative), that differs depending on the stage of the disease, and is modified post-treatment, making it possible to establish patient groups, as well as an early diagnosis. The kit includes the means necessary to carry out the method described in the invention.

Description

 METHOD OF OBTAINING USEFUL DATA FOR THE DIAGNOSIS OF CANCER OF PANCREAS AND TO EVALUATE THE RESPONSE TO TREATMENT.

The present invention is within medicine and molecular biology, and refers to a method of obtaining useful data for the diagnosis at an early stage of pancreatic adenocarcinoma, as well as for evaluating the response to the treatment of said disease, which It allows the establishment of a specific individual (qualitative and quantitative) recognition pattern, which is different depending on the disease status and is modified post-treatment, allowing the establishment of groups of patients, as well as early diagnosis.

STATE OF THE PREVIOUS TECHNIQUE

Pancreatic cancer is among the top five causes of cancer death in the developed world with a survival rate of 19% at 1 year. 95% of malignant tumors of the pancreas are infiltrating ductal adenocarcinoma. In most cases it is diagnosed in a very advanced state and even patients with localized resectable tumors have metastases. Tumors that surround or compress large arterial structures such as the branches of the celiac trunk or the superior mesenteric artery are considered unresectable. Surgical intervention remains the central treatment, although it must be complemented with other options to increase survival time. In this sense, chemotherapy and radiation treatment are used either in combination with surgery, or as a definitive treatment in cases of advanced, unresectable tumors (Yeo et al. Cancer of the Pancreas. Cancer: principies and practice of oncology Vincent T. DeVita, Jr., Samuel Hellman, Steven A. Rosenberg; 7th Edition 2005). Thus, in advanced disease, the administration of gemcitabine is the standard agent, combining it with cytostatics to improve the response.

The molecular level knowledge of the signaling pathways involved in cell transformation has allowed the development of new cancer treatments. Deregulation of signaling through the EGFR epidermal growth factor receptor is involved in the carcinogenesis process. Various types of cancer tend to express high levels of EGFR and their activation initiates a signaling cascade whose components are potential new targets for cancer treatment. Molecules such as erlotinib, a tyrosine kinase inhibitor at the intracellular level, act by preventing the autophosphorylation of the EGFR receptor and subsequent signal transduction.

 EGFR inhibitors appear to show maximum activity in lung cancer patients with activating mutations in the cytoplasmic domain. This suggests that patients in whom the tumors present certain mutations of this receptor, would benefit from a better clinical outcome. This fact would indicate that the study of the presence of these mutations in patients could be used as a guideline for the application of therapy for those who responded better to EGFR inhibitors (3-5). However, this is not entirely true, since not all patients with detected mutations respond to treatment, while patients without such mutations are responders. In addition to EGFR, other molecules seem to have a role in the onset and progression of cancer. Thus the activation of the K-ras oncogene together with the inactivation of tumor suppressor genes (p53, DPC4, p16, and BRCA2) has been associated with the development of pancreatic cancer (Orchekowski et al., 2005. Cancer Res 65: 1 1 193-202).

 On the other hand, certain genes are overexpressed in adenocarcinoma of the pancreas, among them claudin 4, fascin, Hsp47, mesothelin, Muc4, PSCA, S100A4. These data indicate that a study of the gene expression profile provides a unique opportunity to improve the knowledge of the diagnosis and evolution of this lethal tumor and provide an improvement in patient treatment guidelines, both at the level of search for appropriate biomarkers that reflect a effective response as at the level of application of the best treatment at the individual level to each patient according to the biomarkers they manifest.

On the other hand, genetic alterations result in tumor cells producing different molecules that are involved in neoplastic transformation and tumor progression. These molecules often cause growth-stimulating autocrine and paracrine signals, secreted peptide growth factors, cytokines and hormones. Increased levels of these soluble molecules can be used for cancer diagnosis and therapeutic management. Expression of cancer-associated proteins, such as HER2, has been detected by circulating tumor cells in the blood. These cells have prognostic value in metastatic disease, and its presence increases, after a cycle of treatment, in patients likely to be ineffective. These results indicate the value of using analytics that allow the detection in the blood of cancer patients or circulating cancer cells or the molecules they can produce, and that are involved in the carcinogenic process. This procedure is less invasive to monitor both the evolution of the disease and the response to therapy or surgery. In this way, these target molecules can help characterize the biology of the tumorigenesis, the progression of the disease and can also identify biomarkers to monitor the treatment of cancer patients.

 One way to detect these molecules is to use specific antibodies, to detect their presence in the serum of cancer patients, so in patients with bladder cancer, the presence of p33ING1 and cyclin D2 has been associated with loss of cycle regulation. mobile; angiostatin and epidermal growth factor with antiangiogenic response; osteopontin and CXCR4 with metastatic risk. The serum protein profile that can be detected using antibodies may be characteristic of the disease (Orchekowski et al., 2005. Cancer Res 65: 1 1 193-202).

 These molecules reach the bloodstream both by secretion or excretion and by degradation mechanisms including apoptosis or tissue necrosis. Neovascularization, autocrine and paracrine tumor cell pathways, are contributing factors that these antigenic targets reach circulation and can be detected in blood (Burczynski et al., 2006. J Mol Diagn. 8:51-61; Golub et al. , 1999. Science 286: 531-537; Smirnov et al., 2005. Cancer Researc. 65: 4993-4997).

In the case of pancreatic cancer, in a study on the expression of prostate stem cell antigen (PSCA) mRNA was detected by RT-PCR in the blood of 7 of 1 1 patients with pancreatic cancer (63.6%) , and was not observed in the blood of healthy individuals. Other genetic alterations characteristic of pancreatic cancer, such as K-ras mutation that is present in 80-90% of cases, can be detected in the plasma of these patients by the same technique, with potential application in disease monitoring. In this line, the viability of measuring in peripheral blood molecules that allow to know the evolution of pancreatic cancer was the demonstration that the levels of mRNA transcripts of the tumor necrosis factor alpha (TN F-α) in peripheral blood were elevated in the 10 Patients with pancreatic cancer studied compared to 9 healthy controls (p <0.05) measured by Q-RT-PCR and TNF-α mRNA transcripts were reduced to a level similar to the controls after tumor excision.

 Current technology allows to determine the molecular profile of the disease, using microarrays capable of simultaneously measuring the expression levels of thousands of genes. The expression can be measured at the mRNA level, for this there are the microarrays that use probes with cDNA or synthetic oligonucleotides or measure the proteins using as specific "probes" antibodies. In the case of measuring the expression levels of the genes at the mRNA level, one can start from total RNA or mRNA isolated from the tissue or cell sample. Both in the case of starting from total RNA and mRNA, a preliminary step of obtaining complementary DNA (cDNA) is performed, and then an in vitro transcription is performed to obtain biotin-labeled cRNA proportional to the original mRNA expressed. in the tissue. This biotinylated cRNA is fragmented and hybridized specifically with the panel of cDNA or synthetic oligonucleotide probes present in the microarray under specific conditions, the one that has not hybridized is eliminated and the amount of biotinylated cRNA for each gene is placed manifestly by adding a fluorophore that is usually Cy5 bound to streptavidin. Fluorescence is detected with a scanner, which converts it into numerical data and a database is created in which we can have fluorescence intensity values directly related to the expression level of each particular gene. This allows to visualize the interactions of thousands of genes simultaneously.

In recent years, gene expression profiles based on microarrays have been used for the diagnosis of various diseases including cancer, although studies have been limited to certain types of this disease, which has allowed classifying the different types of cancer in clinically relevant subgroups, in a more precise manner and before severe clinical manifestations appear, thereby increasing the possibility of a better early diagnosis, better monitoring of clinical evolution and the response to different treatments with regarding conventional methods or in conjunction with them. In this way, by analyzing the gene expression profile, it is possible to predict the response to chemotherapy or agents directed against cancer molecular targets (Burczynski et al., 2006. J Mol Diagn. 8:51-61; Golub et al., 1999. Science 286: 531-537; Smirnov et al., 2005. Cancer Researc. 65: 4993-4997; Burczynski et al., 2005. Clin Cancer Res 1 1: 1 181-9). The molecular profile can be used as an additional tool within of clinical examinations to determine the sensitivity to a particular treatment, and thus reduce unnecessary treatments.

 Peripheral blood is a type of tissue essential for clinical research due to its critical role in the immune and metabolic response, and its easy collection, which is essential for the discovery of biological markers of disease. Thus, the application of peripheral blood microarray technology can provide new insights into the variations in global genetic expression specifically associated with the disease (Burczynski et al., 2005. Clin Cancer Res 1 1: 1 181-9; Feezor et al., 2004. Physiol. Genomics 19: 247-254; Samuel et al., 2003. ASCO Molecular Therapeutics Symposium Nov 2002 San Diego. BMC Cancer 3: 3; Jazaeri et al., 2005. Cancer Clin Res. 1 1: 6300-10; McPhail et al., 2005. 10: 1485-1487). Isolation of total blood RNA is possible without risk of degradation, and although it was initially found that there were differences in expression due to overabundance of hemoglobin mRNA, once techniques are available that eliminate RNA from the erythrocyte fraction systems Blood RNA collection are suitable for studies that require many samples, monitoring over time or multicentre. It has been shown that the globin mRNA reduction method manages to increase the data quality of the stabilized RNA samples with less intragroup variation and a detection rate of expressed genes similar to that of blood mononuclear cells. The use of peripheral blood allows an increase in the identification of biomarkers based on the RNA expression profile and can help in what is already known as pharmacogenomics. The use of peripheral blood mononuclear cells for transcriptome analysis has proven its value in assessing the genetic signature associated with the disease and related to drug response. The availability of DNA microarrays is accelerating the discovery of cancer targets (Andrea et al., 2006. Nature 439: 353-357). RT-PCR has been used to detect mRNA transcripts of breast cancer or associated with epithelium such as cytokeratins, EGFR, mamoglobulin, MUC-1, beta-HCG, c-Met, GalNac-T, MAGE-3.

In the case of pancreatic cancer, protein microarrays have been studied for the detection of serum markers (Orchekowski et al., 2005. 65: 1 1 193-202), as well as 158 expressed genes have been identified by means of cDNA microarrays. in the neoplastic epithelium with greater expression (greater than double, p <0.01) in 10 pancreatic cancers compared with 10 samples of non-tumor pancreas (Logsdon et al., 2003. Cancer Res 63: 2649-57). Researchers at the University of Ulm, taking advantage of the knowledge of previous studies of the genetic expression profile of pancreatic cancer, designed a diagnostic array of cDNA specially designed for differential diagnosis of pancreatic tumors based on the expression profile of biopsies obtained with fine needle aspiration, since more than 90% of pancreatic tumors represent ductal adenocarcinomas; According to these authors, the results allowed differentiating ductal adenocarcinomas from non-malignant tumors of the pancreas with 95% specificity (Buchloz et al., 2005. Clin Cancer Res. Nov 15; 1 1 (22): 8048-54). Likewise, comparative studies of the microarray analyzes used in pancreatic cancer have been carried out in order to find genes and biomarkers that could be used in new diagnostic and therapeutic strategies (Brandt et al., 2004. Pancreatology 4: 587-597) .

 It is important, therefore, to find biomarkers that can be used for the early diagnosis of pancreatic cancer.

DESCRIPTION OF THE INVENTION

The present invention provides a method of obtaining useful data for the early diagnosis of pancreatic cancer, as well as for evaluating the response to the treatment of said disease, allowing the establishment of groups of patients.

 Thus, a first aspect of the invention relates to the use of one of the genes selected from the list comprising HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, to obtain useful data in the diagnosis, prognosis, or monitoring of pancreatic cancer. In a preferred embodiment of this aspect of the invention, the genes are used simultaneously.

 Another aspect of the invention relates to a method of obtaining useful data for the diagnosis, prognosis and monitoring of pancreatic cancer, hereinafter the first method of the invention, comprising:

a) obtain an isolated biological sample from an individual, and b) detect the amount of expression of at least one of the genes that are selected from the list comprising HLAG, ARFGAP1, S100A2, SLC25A23, KLRG 1, GZMB, GFOD 1 , GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, in the biological sample isolated from (a).

 In a preferred embodiment, the first method of the invention further comprises:

 c) compare the quantities obtained in step (b) with a reference quantity.

 In another preferred embodiment of this aspect of the invention the isolated biological sample of an individual from step (a) is obtained from peripheral blood, and / or comprises peripheral blood cells (PBCs).

 In another preferred embodiment, the first method of the invention further comprises assigning the individual according to step (a) to the group of individuals to whom a peripheral blood sample has been taken seven days after a surgical intervention for pancreatic cancer, when it presents an amount of expression product of the HBD, RPS5, BNIP3L, PCDH9 and FKBP1B genes, or any combination thereof, detected in step (b) greater and statistically significant compared to a reference amount (obtained from the control group individuals with pancreatic cancer before surgery).

 In another preferred embodiment of this aspect of the invention, the detection of the expression product of at least one gene that is selected from the list comprising: HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20 , MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, is performed by RT-PCR.

 In another preferred embodiment of this aspect of the invention, the detection of the expression product of at least one gene that is selected from the list comprising: HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20 , MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, is performed using a microarray.

 Another aspect of the invention relates to a method of diagnosis, prognosis or monitoring of pancreatic cancer, hereinafter second method of the invention, comprising:

a) obtain an isolated biological sample from an individual, and b) detect the amount of expression of at least one of the genes that are selected from the list comprising HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI , GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, in the biological sample isolated from (a),

 c) compare the quantities obtained in step (b) with a reference quantity, and

 d) assign the individual of step (a) to the group of individuals with pancreatic cancer when they have an expression level twice as high as that of healthy individuals.

 The reference amount is obtained from the constitutive expression values of the gene, in a group of healthy individuals, or from the expression of the gene in the group of individuals before undergoing surgical intervention. In particular, in the present invention, peripheral blood samples were obtained before surgery (TO) and seven days after it was performed (T7d).

 The steps (b) and / or (c) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the quantity in step (b) or the computerized comparison in step (c).

 In another preferred embodiment of this aspect of the invention the isolated biological sample of an individual from step (a) is obtained from peripheral blood, and / or comprises peripheral blood cells (PBCs).

 In another preferred embodiment of this aspect of the invention, the detection of the expression product of at least one gene that is selected from the list comprising: HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20 , MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, is performed by RT-PCR.

 In another preferred embodiment of this aspect of the invention, the detection of the expression product of at least one gene that is selected from the list comprising: HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20 , MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, is performed using a microarray.

The term "diagnosis", as used in the present invention, to the ability to discriminate between individuals affected or not by adenocarcinoma of the pancreas. In turn, according to the method of the present invention, other subclassifications could be established within this principal, thus facilitating the choice and establishment of appropriate therapeutic regimens. This discrimination, as understood by one skilled in the art, is not intended to be correct in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly. The amount that is statistically significant can be established by a person skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the significance value P, Student test or discriminant functions. Fisher, non-parametric measurements of Mann Wh itney, Spearman correlation, logistic regression, linear regression, area under the ROC curve (AUC). Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferably, the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention makes it possible to correctly detect the disease differentially at least 60%, more preferably at least 70%, much more preferably at least 80%, or even much more preferably at least 90 % of the subjects of a certain group or population analyzed.

 An "isolated biological sample" includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art. Preferably, the isolated biological sample comprises peripheral blood cells (PBCs).

 The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The term "individual" is not intended to be limiting in any aspect, and may be of any age, sex and physical condition.

The detection of the quantity of the expression product of the genes selected from the list comprising: HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B , or any combination thereof, can be carried out by any means known in the state of the art. The authors of the present invention have demonstrated that the detection of the quantity or concentration of these expression products in a semi-quantitative or quantitative manner makes it possible to differentiate between Different stages of pancreatic cancer. In this way, a differential diagnosis can be established in individuals affected by pancreatic cancer, which allows them to subclassify them.

 The measurement of the amount or concentration, preferably semi-quantitatively or quantitatively, can be carried out directly or indirectly. Direct measurement refers to the measure of the quantity or concentration of the gene expression product, based on a signal that is obtained directly from the transcripts of said genes, or from the proteins to which they are translated, and which is directly correlated with the number of RNA molecules or proteins produced by genes. Said signal - which we can also refer to as an intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of said products. The indirect measurement includes the measurement obtained from a secondary component or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products).

 The term "quantity", as used in the description, refers to, but is not limited to, the absolute or relative quantity of gene expression products, as well as any other value or parameter related thereto or that can be derived from these. Said values or parameters comprise signal intensity values obtained from any of the physical or chemical properties of said expression products obtained by direct measurement. Additionally, said values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.

The term "comparison", as used in the description, refers to, but is not limited to, the comparison of the quantity of expression products of the HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1 genes. GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and / or FKBP1B of the biological sample to be analyzed, also called the biological problem sample, with a quantity of the expression products of the HLAG, ARFGAP1, S100A2, SLC25A23 genes , KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and / or FKBP1B of one or more desirable reference samples described elsewhere in this description. The reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. The comparison described in section (c) of the method of the present invention can be performed manually or assisted by a computer.

The gene expression products are going to give a certain gene expression profile. "Gene expression profile" means the gene profile obtained after quantification of mRNA and / or protein produced by the genes of interest or biomarkers, that is, by the genes H LAG, ARFGAP 1, S 1 00A2, SLC25A23, KLRG 1, GZM B, GFOD 1, GPI, GPR56, M UC20, MAP4K1, H BD, RPS5, BN I P3L, PCDH9 and FKBP1 B, in an isolated biological sample. The expression profile of the genes is preferably performed by determining the level of mRNA derived from its transcription, after extracting the total RNA present in the isolated biological sample, which can be performed by protocols known in the state of the art. The level of mRNA derived from the transcription of the HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B genes can be determined, for example, , but not limited to, by amplification by polymerase chain reaction (PCR), retrotranscription in combination with the polymerase chain reaction (RT-PCR), quantitative RT-PCR, retrotranscription in combination with the chain reaction of the ligase (RT-LCR), or any other nucleic acid amplification method; serial analysis of gene expression (SAGE, SuperSAGE); DNA chips made with oligonucleotides deposited by any mechanism; DNA microarrays made with oligonucleotides synthesized in situ by photolithography or by any other mechanism; in situ hybridization using specific probes marked with any method of marking; by electrophoresis gels; by membrane transfer and hybridization with a specific probe; by nuclear magnetic resonance or any other diagnostic imaging technique using paramagnetic nanoparticles or any other type of detectable nanoparticles functionalized with antibodies or by any other means. The gene expression profile could also be obtained by the detection and / or quantification of the proteins resulting from the translation of the mRNA derived from the transcription of the HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20 genes. , MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, for example, but not limited to, western blot immunodetection. Quantitative detection of the expression of the HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B genes can be performed more preferably by real-time PCR ( RT-PCR or RTqPCR). The real-time detection of the amplified products can be carried out by means of the use of fluorescent molecules that are intercalated in the double-stranded DNA or by hybridization with different types of probes.

 The term "reference quantity", as used in the description, refers to the absolute or relative quantity (to the reference gene) of expression products of the HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB genes, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and / or FKBP1B that allows to discriminate a certain stage of pancreatic cancer from other stages, or other diseases. Suitable reference amounts can be determined by the method of the present invention from a reference sample that can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. Thus, for example, but not limited to, the reference sample may be the negative controls, that is, the amounts detected by the method of the invention in samples of individuals not suffering from pancreatic cancer.

 The reference amount will be, for example, in the case of differentiation between patients affected by pancreatic cancer from healthy individuals, the constitutive expression of the gene in a control group of healthy individuals. However, in the case of subclassification of patients affected by pancreatic cancer based on their manifestations, the control group will consist of a group of patients with pancreatic cancer who did not have that clinical manifestation.

 Thus, the sample or reference samples can be, for example, obtained from the blood cells of a patient with pancreatic cancer, in a certain clinical phase. In another preferred embodiment of this aspect of the present invention, the reference amount is obtained from a reference sample. The reference amount can also be obtained, for example, from the limits of normal distribution of an amount found in samples obtained from a population of individuals with pancreatic cancer in different phases, by means of well-known statistical techniques.

The HLAG gene, major histocompatibility class I antigen, involved in the presentation of antigens to the immune system. Its amino acid sequence is found with access number in GenBank (NCBI) NP_0021 18; and / or in SEQ ID NO: 1). In the context of the present invention, the HLAG gene is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 1, and which would comprise various variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 1, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the KMP1 1 protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 002721.

 The ARFGAP1 or ADP-ribosylation factor GTPase activating protein 1 gene (RP1 1 - 261 N 1 1 .1, ARF 1 GAP, H RI H FB2281, MGC39924), encodes a GTPase activating protein that interacts with the factor of ribosylation ARF1 (Weimer et al., 2008. J Cell Biol. Nov 17; 183 (4) 725-35). Its amino acid sequence is found with access number in GenBank (NCBI) NP_060679; and / or SEQ ID NO: 2.

 In the context of the present invention, ARFGAP1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 2, and which would comprise various variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 2, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the protein ARFGAP1. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 018209.2.

 The S100A2 gene, or "S100 calcium binding protein A2" (RP1 1-49N14.8, CAN 19, MGC1 1 1539, S100L), is a gene that codes for a S100 family protein, involved in the regulation of processes such as cell cycle and differentiation. An altered expression of this gene has been detected in breast cancer. Under-expression of this gene is associated with poor tumor differentiation and reduced survival in laryngeal carcinoma (Almadori et al., 2009. J Otolaryngol Head Neck Surg. Feb; 38 (1): 16-22).

 In the context of the present invention, S100A2 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein found in the Ban Ban Gen (NCBI) NP_005969 and / or in SEQ ID NO: 3, and which would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 3, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the S100A2 protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 005978.3.

 The SLC25A23 or "solute carríer family 25 (mitochondrial carrier; phosphate carrier), member 23" gene (APC2, MCSC2, MGC2615, SCaMC-3j. It is a mitochondrial transporter of Ca-dependent solutes. It can act as an ATP-Mg / Pi exchanger that mediates the transport of Mg-ATP in phosphate exchange, catalyzing the net level or efflux of adenine nucleotides inside or outside the mitochondria (Bassi et al., 2005. Gene. Jan 31; 345 (2): 173 -82 Epub 2005 Jan 7.).

In the context of the present invention, SLC25A23 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence that It has an access number in GenBank (NCBI) NP_077008 and / or in SEQ ID NO: 4, and which would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 4, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the SLC25A23 protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 024103.2.

 The KLRG1 gene or "killer cell lectin-like receptor subfamily G, member l", (2F1, CLEC15A, MAFA, MAFA-2F1, MAFA-L, MAFA-LIKE, MGC13600, plays an inhibitory role of "natural killer" cells (NK) (Schwartzkopff et al., 2007. J Immunol. Jul 15; 179 (2): 1022-9)

 In the context of the present invention, KLRG1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_077008 and / or in SEQ ID NO: 5, and that it would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 5,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 5, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the KLRG1 protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 005810.3. The GZMB gene or "granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1", (CCPI, CGL-1, CGL1, CSP-B, CSPB, CTLA1, CTSGL1, HLP, SECTJ is involved in induction of apoptosis This gene encodes the enzyme Granzima B, necessary to direct cell lysis in cell-mediated immune responses. It appears to be linked to an activation of caspases responsible for apoptosis (Rotonda et al., 2001. Chem Biol. Apr; 8 ( 4): 357-68).

 In the context of the present invention, GZMB is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_004122 and / or in SEQ ID NO: 6, and that it would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 6, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the GZMB protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 004131 .3.

 The GFOD1 gene or "glucose-fructose oxidoreductase domain containing 1", is involved in induction of apoptosis. This gene encodes the enzyme Granzima B, necessary to direct cell lysis in cell-mediated immune responses. It seems to be linked to an activation of caspases responsible for apoptosis (Rotonda et al., 2001. Chem Biol. Apr; 8 (4): 357-68).

 In the context of the present invention, GFOD1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_061861 and / or in SEQ ID NO: 7, and that it would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7,

b) nucleic acid molecules whose complementary hybrid chain with the a) polynucleotide sequence,

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 7, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the GFOD1 protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 018988.2.

 The GPI or "glucose-6-phosphate isomerase" gene encodes a dimeric enzyme that catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6- phosphate, it can also act as a tumor-secreted cytokine and as an angiogenic factor ( AMF) that stimulates the motility of the endothelial cell (25).

 In the context of the present invention, GPI (AMF, DKFZp686C13233, GNPI, NLK, PGI, PHI, SA-36, SA36) is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence that meets the number of Access in GenBank (NCBI) NP_000166, NP_001 171651.1 and / or in SEQ ID NO: 8, and which would include various variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 8, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the GPI protein. Among these nucleic acid molecules is the collection in the GenBank (NCBI) sequence NM_000175.3 or NM_001 184722.1.

The GPR56 or "G protein-coupled receptor 56" gene (UNQ540 / PRO1083, BFPP, DKFZp781 L1398, TM7LN4, TM7XN1) G protein-linked receptor whose overexpression can suppress tumor growth and metastasis (Huang et al., 2008. Mol Cell Biochem. Jan; 308 (1-2): 133-9. Epub 2007 Oct 12). In the context of the present invention, GPR56 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_002057 and / or in SEQ ID NO: 9, and that it would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 9,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 9, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the GPR56 protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 201524.1.

 The MUC20 gene or "mucin 20, cell surface associated", (UNQ2782 / PR07170, FLJ 14408, FLJ53153, KIAA1359, MUC-20) codes for a mucin, glycoprotein that forms an insoluble mucous barrier (Higuchi et al., 2004. Mol Cell Biol. Sep; 24 (17): 7456-68). This gene appears to reduce the transient activation of MAPK induced by hepatocyte growth factor (HGF). It also inhibits the proliferation of MMP1 and MMP9 expression induced by HGF (Higuchi et al., 2004. Mol Cell Biol. Sep; 24 (17): 7456-68).

 In the context of the present invention, MUC20 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_002057 and / or in SEQ ID NO: 10, and that it would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, a 98% or 99% with SEQ ID NO: 10, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the MUC20 protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 152673.

 The MAP4K1 gene or "mitogen-activated protein kinase kinase kinase kinase 1", (HPK1) codes for a mucin, glycoprotein that forms an insoluble mucosal barrier (Higuchi et al., 2004. Mol Cell Biol. Sep; 24 (17): 7456-68). This gene appears to reduce the transient activation of MAPK induced by hepatocyte growth factor (HGF). It also inhibits the proliferation of MMP1 and MMP9 expression induced by HGF (Higuchi et al., 2004. Mol Cell Biol. Sep; 24 (17): 7456-68).

 In the context of the present invention, MAP4K1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_002057 and / or in SEQ ID NO: 1 1 , and that would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 1,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 1 1, and wherein the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the MAP4K1 protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 007181 .3.

 The HBD or "hemoglobin, delta" gene is involved in the transport of oxygen from the lung to other peripheral tissues.

 In the context of the present invention, HBD is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_002057 and / or in SEQ ID NO: 12, and that it would include several variants from:

a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 12, b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 12, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the HBD protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 000519.3.

 The RPS5 or "ribosomal protein S5" gene encodes a ribosomal protein whose variable expression has been detected, in addition to pancreatic cancer, in colorectal cancer, although it has not been related to the severity of the disease (Campagna et al. , 2008. Int J Clin Exp Pathol 1: 32-43, Frigerio et al., 1995. Biochim Biophys Acta 1262: 64-68).

 In the context of the present invention, RPS5 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_002057 and / or in SEQ ID NO: 13, and that it would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 13,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 13, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the RPS5 protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 031902.3.

The BN I P3L or "BCL2 / adenovirus E1B 19kDa interacting protein 3-like" gene (BN I P3a, N IX) can function as a tumor suppressor. I nhibits BNIP3-induced apoptosis (Fei et al., 2004. Cancer Cell. Dec; 6 (6): 597-609; Mellor et al., 2007. Cancer Metastasis Rev. Dec; 26 (3-4): 553-66).

 In the context of the present invention, BNIP3L is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_004322 and / or in SEQ ID NO: 14, and that it would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 14, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the BN I P3L protein. Among these nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 004331 .2.

 The PCDH9 or "protocadherin 9" gene (BNIP3a, NIX) is a gene that encodes a calcium-dependent cell adhesion protein (protocadherin) and altered in pancreatic cancer (Hidalgo, 2010. Pancreatic Cancer. N Engl J Med 362 : 1605-17).

 In the context of the present invention, PCDH9 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_065136 and / or in SEQ ID NO: 15, and that it would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 15,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 15, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the protein PCDH9. Among these nucleic acid molecules is the collection in the GenBank (NCBI) sequence NM 020403.4 or NM 203487.2.

 The FKBP1 B or "FK506 binding protein 1B, 12.6 kDa" gene, (FKBP12.6, FKBP1 L, OTK4, PKBP1 L, PPIase) this gene encodes a member protein of the immunophilin family, involved in immunoregulation and processes basic cell phones such as protein folding and their passage through membranes. This protein is a cis-trans prolyl isomerase that binds to immunosuppressant FK506 and rapamycin.

In the context of the present invention, FKBP1 B is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence found with access number in GenBank (NCBI) NP_004107 and / or in SEQ ID NO: 16 , and that would include several variants from:

 a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 16,

 b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

 c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 16, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the FKBP1 B protein. Among said nucleic acid molecules is the collection in the GenBank sequence (NCBI) NM 0041 16.2

 In the sense used in this description, the term "variant" refers to a protein substantially homologous to any of the proteins HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5 , BN I P3L, PCDH9 and / or FKBP 1 B. In general, a variant includes additions, deletions or amino acid substitutions. The term "variant" also includes proteins resulting from posttranslational modifications such as, but not limited to, glycosylation, methylation phosphorylation or acylation.

As used herein, a protein is "substantially homologous" to any of the proteins HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and / or FKBP1 B, when its amino acid sequence shows a good alignment with the sequence amino acid SEQ ID NO: 1, SEQ ID NO: 2, SEO ID NO: 3, SEO ID NO: 4, SEO ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively as described above; that is, when its amino acid sequence has an identity degree with respect to the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, of at least 50%, typically of at least 80%, advantageously of at least 85%, preferably of at least 90%, more preferably, at least 95%, and, even more preferably, at least 99%. The sequences homologous to any of the HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BN I P3L, PCDH9 and / or FKBP 1 B proteins can easily be identified by a person skilled in the art, for example, with the help of an appropriate computer program to compare sequences.

 The term "functionally equivalent", as used herein, means that the proteins or fragment / s of the protein / s in question essentially maintain the biological or immunological properties described herein. Said capacity can be determined by conventional methods.

 In another preferred embodiment, the detection of the amount of any of the proteins LAG, ARFGAP1, S 100A2, SLC25A23, KLRG1, GZM B, G FO D1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and / or FKBP1 B is performed by an immunoassay. The term "immunoassay," as used herein, refers to any analytical technique that is based on the reaction of conjugation of an antibody with an antigen. Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot, enzyme-linked immunosorbent assay (ELISA), linear immunoassay (LIA), radioimmunoassay (RIA), immunofluorescence, x-map or protein chips .

In another preferred embodiment, the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay). The ELISA is based on the premise that uni nm or norreactive (antigen or antibody) can be immobilized on a solid support, then bringing that system into contact with a fluid phase containing the complementary reagent that can bind to a compound marker. There are different types of ELISA: direct ELISA, indirect ELISA or sandwich ELISA.

The term "marker compound", as used herein, refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and quantification of the amount of antibodies against to the antigens HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and / or FKBP1 B. The marker compound is selected from the list comprising radioisotopes enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and / or quantified directly. This marker compound can bind to the antibody directly, or through another compound. Some examples of marker compounds that bind directly are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as ³² P or ³⁵ S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, auto-radiography , fluorimetry, or metallography respectively.

 In another preferred embodiment, the second method of the invention further comprises assigning the individual according to step (a) to the group of individuals surgically operated and at which seven days after it is taken, a peripheral blood sample is taken when a quantity of product is present. of expression of the HBD, RPS5, BNIP3L, PCDH9 and FKBP1B genes, or any combination thereof, detected in step (b) greater and statistically significant compared to a reference amount (obtained from the control group individuals with cancer of pancreas before surgery).

In turn, in accordance with the methods of the present invention, other subclassifications could be established within this principal, thus facilitating the choice and establishment of suitable therapeutic regimens. This discrimination, as understood by one skilled in the art, is not intended to be correct in 1% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly. The quantity that is significantly statistical can be established by a person skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the p-value, Student's test or functions. discriminators of Fisher Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferably, the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention makes it possible to correctly detect the disease differentially by at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a certain group or population analyzed.

 Current chemotherapy is very effective in the early stages. The method of the present invention allows early diagnosis, and therefore, significantly anticipates the start of treatment.

 Another aspect of the invention relates to a method of monitoring the evolution of pancreatic cancer, hereinafter the third method of the invention, comprising:

 a) take an isolated biological sample from an individual,

 b) detect the amount of expression product of at least one of the genes that are selected from the list comprising HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, H BD , RPS5, BN I P3L, PCDH9 and FKBP 1 B, or any combination thereof, in the biological sample isolated from (a).

 c) compare the quantities obtained in step (b) with a reference quantity, and

 d) repeat at least twice the sequence of steps (a) - (c) in samples obtained from the same individual according to step (a), not simultaneously.

 In another preferred embodiment of this aspect of the invention the isolated biological sample of an individual from step (a) is obtained from peripheral blood, and / or comprises peripheral blood cells (PBCs).

 In another preferred embodiment of this aspect of the invention, the detection of the expression product of at least one gene that is selected from the list comprising: HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20 , MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, is performed by RT-PCR.

In another preferred embodiment of this aspect of the invention, the detection of the expression product of at least one gene that is selected from the list comprising: HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, is performed using a microarray.

 The term "evolution monitoring", as used in the present description, refers to the monitoring of disease development, such as, but not limited to, the evaluation of the response to a particular cancer treatment of pancreas, or surgery. Therefore, in a preferred embodiment of this aspect of the invention, the follow-up is carried out post-treatment.

 In a preferred embodiment, the detection of expression products is performed at least two, preferably three, four, five, six, seven, eight, nine, ten, eleven, twelve thirteen, fourteen, fifteen, and even more preferably the Sixteen HLAG, ARFGAP1, S 100A2, SLC25A23, KLRG 1, GZMB, GFOD 1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1 B genes. In another more preferred embodiment, the detection of the products of HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B genes are performed simultaneously.

 Another aspect of the present invention relates to a kit or device, hereafter kit or device of the invention, comprising the elements necessary to analyze the amount of the expression product of the genes in the list comprising: HLAG, ARFGAP1 , S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof.

 More preferably it comprises the means necessary to compare the amount detected in step (b) with a reference amount.

 Even more preferably, the kit of the present invention comprises the elements necessary to carry out any of the methods of the present invention.

Said kit may contain all those reagents necessary to analyze the quantity quantity of the expression product of the genes HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, by any of the methods described above in this document. The kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand the kit It can include all the supports and containers necessary for its implementation and optimization. Preferably, the kit further comprises instructions for carrying out any of the methods of the invention.

 Another aspect of the invention relates to the use of the kit or device of the invention for obtaining useful data in the diagnosis, prognosis or monitoring of pancreatic cancer.

 The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).

 The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding , chemically or biochemically modified.

 Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Visualization of the 3 tubes with RNA stabilizer for each patient and time.

Fig. 2. Gene expression. Overexpression greater than twice compared to healthy volunteers (> 2-fold, p <0.05) and under-expression. (A) Before the intervention. (B) 7 days after surgery.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which shows the specificity and effectiveness of the methods of the invention to obtain useful data in the diagnosis of Chagas disease, and for the diagnosis and monitoring of said disease.

MATERIALS AND METHODS

Patients and Methods

Study approved by the Clinical Research Ethics Committee of the Virgen de las Nieves University Hospital in Granada, in which four patients with adenocarcinoma of the pancreas have been studied compared with a group of five healthy volunteers. Peripheral blood samples were obtained before surgery (T0) and seven days after it was performed (T7d) using for each patient and sampling time, three tubes containing an RNA stabilizer. Once extracted, this RNA is purified by checking its quality in a bioanalyzer, so that the RNA samples whose ratio 28S / 1 8S is not of the order of 1, 5 are discarded. Then, the reverse transcription reaction is carried out, as well as to mark the cRNAs obtained with Cy5-streptavidin, proceeding to hybridize these in the human complete genome microarrays of the CodeLink system. Each microarray is performed in duplicate by loading 2 μg of each patient's cRNA, to compare it with 2 μg of healthy volunteers' cRNA. Once the hybridization was finished during a period of 12h and at 37 ° C, the reading of the microarrays was carried out on a laser scanner, quantifying and normalizing the values obtained by means of the CodeLink 5.0 software.

From each patient participating in the study, 18 ml of peripheral blood were obtained before surgery (T0) and 7 days after surgery (T7d) distributed as follows: 12 ml for obtaining RNA, 6 ml for a study of serum biochemistry of the parameters lipase, amylase, γ-glutamyl transferase (GGT) and alanin-L-transferase (ALT = GPT) as indicators of pancreatic functionality, and 6 ml for performing a complete blood count as well as leukocyte formula. The gene expression profile was studied using bioarrays of the CodeLink system (Applied Microarrays, Tempe, AZ, USA) from total peripheral blood RNA obtained with Paxgene Blood RNA Tubes (PreAnalytix, Qiagen, The Netherlands) and purified with Paxgene Blood RNA kit (Qiagen, The Netherlands). The quality of the RNA obtained was measured by microfluidics in a bioanalyzer (Experion, Bio-Rad, Richmond, VI, USES). The fluorescence of the bioarrays was determined on a GenePix-4000B (Axon I nstruments) scanner. The data was processed with the software of the CodeLink 5.0 platform (GE Healthcare).

Results

Before surgery (T0), and compared with healthy volunteers, 176 genes have been detected with an overexpression level greater than 2 times (p <0.05), among which are: HLAG, involved in the presentation of antigens to the immune system; ARFGAP1, GTPase activating protein that interacts with the ribosylation factor ARF1; S100A2, involved in the regulation of processes such as cell cycle and differentiation; SLC25A23, Ca-dependent mitochondrial solute transporter; KLRG1, which plays an inhibitory role of "natural killer" cells (N K); GZM B, involved in induction of apoptosis; GFOD1, a glucose-fructose oxidoreductase; GPI, glucose phosphate isomerase that in addition to glycolytic function can act as a cytokine secreted by tumors and as an angiogenic factor; GPR56, G-protein linked receptor whose overexpression can suppress tumor growth and metastasis; MUC20, which codes for a mucin, glycoprotein that forms an insoluble mucous barrier. After the intervention, T7d, compared with T0, 1 1 genes have been detected with an overexpression greater than 2 times (p <0.05), among which are: HBD, hemoglobin delta, involved in the transport of oxygen from the lung to other peripheral tissues; RPS5, which codes for a ribosomal protein whose variable expression has been detected in colorectal cancer, although it has not been related to the severity of the disease.

Claims

 one . The use of one of the genes selected from the list comprising HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any of their combinations, to obtain useful data in the diagnosis, prognosis, or monitoring of pancreatic cancer.

2. A method of obtaining useful data for the diagnosis, prognosis, or monitoring of pancreatic cancer, comprising:

 to. obtain an isolated biological sample from an individual, and b. Detect the amount of expression product of at least one of the genes selected from the list comprising HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, in the biological sample isolated from (a).

3. The method of obtaining useful data for the diagnosis, prognosis or monitoring of pancreatic cancer according to the preceding claim, which further comprises

 C. compare the quantities obtained in step (b) with a reference quantity.

4. A method of diagnosis, prognosis or monitoring of pancreatic cancer, comprising steps (a) - (c) of the method according to claim 3, and further comprising:

 d. Assign the individual in step (a) to the group of individuals with pancreatic cancer when they have an expression level twice as high as in healthy individuals.

5. A method of monitoring the evolution of pancreatic cancer, comprising steps (a) - (c) of the method according to any of claims 3-4, and further:

d. repeat at least twice the sequence of steps (a) - (c) in samples obtained from the same individual according to step (a), not simultaneously.

6. A method comprising the steps (a) - (c) of the method according to any of claims 2-5, which further comprises assigning the individual of (a) to the group of individuals surgically intervened for pancreatic cancer to whom Peripheral blood sample is taken seven days after surgery, when it has an expression level of a gene that is selected from the list comprising: HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, twice superior to that of healthy individuals.

7. The method according to any of claims 2-6, wherein the sample is obtained from peripheral blood.

8. The method according to any of claims 2-7, wherein the detection of the expression product of at least one gene that is selected from the list comprising: HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1, or any combination thereof, is performed by RT-PCR.

9. The method according to any of claims 2-8, wherein the detection of the expression product of at least one gene that is selected from the list comprising: HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof, is performed using a microarray.

10. A kit or device comprising the elements necessary to carry out a method according to any of claims 2-9.

eleven . A kit or device comprises the elements necessary to detect the expression product of a gene that is selected from the list comprising: HLAG, ARFGAP1, S100A2, SLC25A23, KLRG1, GZMB, GFOD1, GPI, GPR56, MUC20, MAP4K1, HBD, RPS5, BNIP3L, PCDH9 and FKBP1B, or any combination thereof.

12. The use of the kit according to any of claims 10-1 1, for obtaining useful data in the diagnosis, prognosis or monitoring of pancreatic cancer.