ES2526823A1 - Biomarkers, method and kit for the early diagnosis of ductal adenocarcinoma of pancreas (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Biomarkers, method and kit for the early diagnosis of pancreatic ductal adenocarcinoma. Biomarkers, method and kit for the early diagnosis of pancreatic cancer, in particular, pancreatic ductal adenocarcinoma (pdac). (Machine-translation by Google Translate, not legally binding)

Description

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BIOMARCATORS, METHOD AND KIT FOR THE EARLY DIAGNOSIS OF DUCTAL DENGARS OF PANCREAS

FIELD OF THE INVENTION

5 The present invention is within the field of Molecular Biology and Medicine. Specifically, it refers to a method of obtaining useful data for the early diagnosis of pancreatic ductal adenocarcinoma. The early diagnosis is made by analyzing the biomarkers FGF-10, CXCL11, OSM, GPNMB and SCF.

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BACKGROUND OF THE INVENTION

Ductal pancreatic adenocarcinoma (PADC) has one of the highest mortality rates among all types of cancer. The new cases of this cancer reached 44,000 individuals last year (2012) and of them, 37,400 died (15 85%). Men and women have approximately a similar risk of suffering from it (Siegel et al., 2012. C.A. Cancer J. Clin. 62, 10-29). Among the reasons for the poor response to treatment are late diagnosis because in most cases when cancer is detected, metastases and the intrinsic mechanisms of chemotherapy resistance have occurred (Hidalgo, 2010. N. Engl. J 20 Med. 362, 1605-1617; Sakar et al. 2007. Toxicol. Appl. Pharmacol. 224, 326-336). Due to the impact that early diagnosis has on patient survival (Agarwal et al., 2008. Pancreas 36, 15-20), better simple diagnostic tools are necessary for the detection and evaluation of the disease. The serum is the best option to obtain samples where to study the 25 malignant tumors since it can be taken by non-invasive methods. In the case of pancreatic cancer it is especially useful since access to the target organ is very difficult. For these reasons, serum-based cancer biomarkers are the simplest screening tests. To date, the only serum-based biomarker for the PDAC used is the carbohydrate antigen 19-9 (CA), with sensitivity between 70 and 90% and specificity between 70 and 98%, depending on the size of the tumor (Chan et al., 2012. J. Proteomics). However, this biomarker is not detected if the tumor size is less than 2 centimeters and due to lack of expression in 10% of patients. This also occurs in other pathologies. For this and since

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recently, the American Oncology Society does not recommend CA 19-9 for PADC screening tests (Duffy et al., 2010. Ann. Oncl. 21, 441-447)

There is a close relationship between inflammatory processes and carcinogenesis (Mantovani et al., 2008. Nature 454, 436-444; Germano et al., 2008. Cytokine 43, 374379). Pancreatic cancer is characterized by a dense demoplastic reaction, which represents an important barrier that prevents the effective release of chemotherapeutic agents at the main site of the disease. In addition, the microenvironment of the tumor complex nourishes the invasion and metastasis of pancreatic cancer (Shields et al., 2012. Biochem J. 441, 541-542; Nesse et al., 2011. Gut. 60, 861-868; Luo et al ., 2012. Biochim. Biophys Acta. 1826, 170-178). The cytokines released by cancer cells or cells inside the tumor microenvironment are the architects of this reaction. Given the above, we believe that a modulation in cytokine levels could reflect the processes that lead to the development of the disease or chemoresistance.

Antibody-based arrays represent a useful tool for the discovery of cancer biomarkers. This methodology stands out for its ability to offer fast and correct perceptions for the early detection of cancer biomarkers, providing new approaches to cancer therapies (Breman et al., 2010. Nat. Rev. Cancer 10, 605-617)

Therefore, the identification and validation of individual biomarkers or sets of biomarkers for the rapid detection of PDAC (diagnostic biomarkers) would help increase patient survival.

BRIEF DESCRIPTION OF THE INVENTION

A first aspect of the invention relates to the use of the genes (and / or proteins) FGF10, CXCL11, OSM, GPNMB and SCF, for the early diagnosis of pancreatic cancer. The use can be simultaneous or any of the genes (and / or proteins), or any combination thereof can be chosen.

In a preferred embodiment of this aspect of the invention pancreatic cancer is ductal pancreatic adenocarcinoma (PDAC).

Another aspect of the invention relates to a method of obtaining useful data, hereinafter the first method of the invention, for the early diagnosis of pancreatic cancer, which comprises:

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a) Obtain an isolated sample from the individual. b) quantify the expression product of the genes that are selected from the list consisting of: FGF-10, CXCL11, OSM, GPNMB and SCF.

The expression product of all genes can be quantified simultaneously, or any of the genes (and / or proteins), or any combination thereof, can be chosen.

In another preferred embodiment, the method of the invention further comprises:

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

In a preferred embodiment of the method of the invention, steps (b) and / or (c) of the methods described above may be fully or partially automated.

Another aspect of the invention relates to a method of diagnosis of pancreatic cancer which comprises steps (a) - (c) according to the first method of the invention, and further comprises assigning the individual of step (a) to the group of individuals that

15 suffer from pancreatic cancer when they present an amount of expression product of the FGF-10, CXCL11, OSM, GPNMB and SCF genes, greater than the reference amount.

In a preferred embodiment of this aspect of the invention pancreatic cancer is ductal pancreatic adenocarcinoma (PDAC).

In a preferred embodiment of the method of the invention, the isolated biological sample of an individual from step (a) is a blood sample.

In another preferred embodiment, the detection of the amount of expression of any of the FGF-10, CXCL11, OSM, GPNMB and SCF genes is performed by an immunoassay.

In another preferred embodiment, the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay).

Another aspect of the invention relates to an antibody for treating an individual suffering from pancreatic cancer (or alternatively to the use of an antibody in the preparation of a medicament for the treatment of pancreatic cancer), identifiable by a method of the invention. , where the antibody is selected from anti-FGF-10, anti-CXCL11, anti-OSM, anti-GPNM and anti-SCF, or any of its

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combinations Preferably a combination of all the above antibodies is employed. In another preferred embodiment of this aspect of the invention, pancreatic cancer is ductal pancreatic adenocarcinoma (PDAC).

Another aspect of the invention relates to a pharmaceutical composition comprising a modulating agent of at least one of the genes that are selected from FGF-10, CXCL11, OSM; GPNMB and SCF, to treat an individual suffering from pancreatic cancer (or alternatively to the use of a pharmaceutical composition comprising a modulating agent of at least one of the genes selected from FGF-10, CXCL11, OSM, GPNMB and SCF in the preparation of a medicament for the treatment of pancreatic cancer), and more preferably of the ductal adenocarcinoma of the pancreas (PDAC), identifiable by a method of the invention.

Another aspect of the present invention relates to a kit or device, hereafter kit of the invention, comprising the elements necessary to quantify the amount of expression product of the genes that are selected from FGF10, CXCL11, OSM, GPNMB and SCF or any of its combinations.

In a preferred embodiment, the kit of the present invention comprises antibodies that are selected from the list consisting of antibodies: anti-FGF-10, anti-CXCL11, anti-OSM, anti-GPNM and anti-SCF, or any of your combinations Preferably, the kit of the invention simultaneously comprises at least one antibody of each type: anti-FGF-10, anti-CXCL11, anti-OSM, anti-GPNM and anti-SCF.

In another preferred embodiment, the kit of the invention comprises secondary antibodies

or positive and / or negative controls. The kit can also include, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc.

Another aspect of the invention relates to a solid support, or protein chip, comprising at least one of the anti-FGF-10, anti-CXCL11, anti-OSM, anti-GPNM and anti-SCF antibodies, or any of their combinations, to carry out any of the methods of the invention.

Another aspect of the invention relates to a solid support, or DNA chip, comprising oligonucleotides or single channel microarrays designed from a known sequence or an mRNA of at least one of the FGF-10, CXCL11, OSM genes, GPNM and SCF. Preferably, it comprises the oligonucleotides capable of detecting the mRNA of all the FGF-10, CXCL11, OSM, GPNM and SCF genes.

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DESCRIPTION OF THE INVENTION

The authors of the present invention have analyzed the members of the cytokine family in healthy individuals, in individuals suffering from untreated PDAC and in individuals suffering from PDAC who have undergone treatment with Gemcitabine and Erlotinib. They have found a series of markers that allow them to diagnose individuals with PDAC early. Thus the present invention provides a

10 method of obtaining useful data for the early diagnosis of individuals with PDAC.

Therefore, a first aspect of the invention relates to the use of the genes (and / or proteins) FGF-10, CXCL11, OSM, GPNMB and SCF, for the early diagnosis of pancreatic cancer. The use can be simultaneous or you can choose any of the

15 genes (and / or proteins), or any combination thereof.

In a preferred embodiment of this aspect of the invention pancreatic cancer is ductal pancreatic adenocarcinoma (PDAC).

Another aspect of the invention relates to a method of obtaining useful data, hereinafter the first method of the invention, for the early diagnosis of

20 pancreatic cancer, comprising:

d) Obtain an isolated sample from the individual. e) quantify the expression product of the genes that are selected from the list consisting of: FGF-10, CXCL11, OSM, GPNMB and SCF.

The expression product of all genes can be quantified simultaneously, or

25 any of the genes (and / or proteins), or any combination thereof can be chosen.

In another preferred embodiment, the method of the invention further comprises:

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

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In a preferred embodiment of the method of the invention, steps (b) and / or (c) of the methods described above can be fully or partially automated, for example, by means of a robotic sensor device for detecting the amount in step (b) or the computerized comparison in step (c).

A preferred embodiment of this aspect of the invention relates to a method of diagnosis of pancreatic cancer that comprises steps (a) - (c) of the first method of the invention, and further comprises assigning the individual from step (a) to group of individuals suffering from pancreatic cancer when they present an amount of expression product of the FGF-10, CXCL11, OSM, GPNMB and SCF genes, greater than the reference amount. It can present an amount of expression product of any of the FGF-10, CXCL11, OSM, GPNMB and SCF genes, or all of them simultaneously, greater than the reference amount.

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.

In a preferred embodiment of the method of the invention, the isolated biological sample of an individual from step (a) is a blood sample, and even more preferably serum.

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 amount of expression product of FGF-10, CXCL11, OSM, GPNMB and SCF can be detected by any means known in the state of the art. The authors of the present invention have shown that the detection of the amount or concentration of antibodies against these cytokines semiquantitatively or quantitatively allows early diagnosis of individuals suffering from ductal adenocarcinoma of the pancreas.

The levels of gene expression will give a certain gene expression profile. The term "expression level", also called "gene product amount" or "amount of expression product" refers to the biochemical material, either RNA or protein, resulting from the expression of a gene. Sometimes a measure of the amount of gene product is used to infer how active a gene is. "Gene expression profile" means the gene profile obtained after quantification of the mRNA and / or protein produced by the genes of interest or biomarkers, that is, by the FGF-10, CXCL11, OSM, GPNMB and SCF genes, 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.

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The measurement of the quantity or concentration of expression product of these genes, preferably semi-quantitatively or quantitatively, can be carried out directly or indirectly. The direct measure refers to the measure of the quantity

or the concentration of the gene expression product, based on a signal that is obtained directly from the transcripts of said genes, or from proteins, and that is directly correlated with the number of RNA molecules or proteins produced by the 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, ‘‘ label ’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 or the amount of antibodies, as well as any other value or parameter related to them or that may be derived from them. 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 amount of gene expression products or the biological sample to be analyzed, also called the biological problem sample, with a quantity of gene expression products of one or more desirable reference samples. 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.

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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 without limiting ourselves, 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 the disease.

The characteristics of the cytokines that are the object of the study are described below:

The FGF-10 or fibroblast growth factor 10 (KGF-2: keratinocyte growth factor 2) gene is found on chromosome 5 (5p13-p12). This factor has been described as a promoter of morphogenesis in primary stages of organogenesis as well as a regulator of the proliferation of pancreatic epithelial progenitor cells (Bhushan et al., 2001. Development 128, 5109-5117). Recently, a link between FGF10 / FGFR2-IIIb signaling and the migration and invasion of pancreatic cancer cells through the induction of type 1 membrane matrix metalloproteinases and transforming the growth factor TGF-β1 has also been described. which is an important regulator of mesenchymal epithelial transition (Nomura et al., 2008. Br. J. Cancer 99, 305-313).

In the context of the present invention, FGF-10 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 8, and which would comprise 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 FGF-10 protein. Among said nucleic acid molecules is the collection in the sequence SEQ ID NO: 1.

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The CXCL11 or hemokine (C-X-C motif) ligand 11 (C-X-C motif chemokine 11; beta gene

5 R1; interferon gamma-inducible protein 9; interferon-inducible T-cell alpha chemoattractant; small inducible cytokine B11; small inducible cytokine subfamily B (Cys-X-Cys), member 11; small inducible cytokine subfamily B (Cys-X-Cys), member 9B; Small-inducible cytokine B11, H174, I-TAC, IP-9, IP9, SCYB11, SCYB9B, b-R1) is located on chromosome 4 (4q21.2). CXCL11 is a chemokine that interacts

10 specifically with the CXCR3 receiver. Stimulates phosphorylation of the MAPK kinase pathways leading to the proliferation and prevention of apoptosis (Miekus et al., 2010. Folia Histochem. Cytobiol. 48, 104-111) In addition, its role in various types of tumorigenesis has been described cancerous (Lo et al., 2010. Am. J. Pathol. 176, 24352446; Furuya et al., 2011. Gynecol. Oncol. 122, 648-655). Although here we present,

15 for the first time, CXCL11 as a possible biomarker in patients with PDAC, has recently been proposed as a serum biomarker for prostate adenocarcinoma (Klee et al., 2012. Clin. Chem. 58, 599-609)

In the context of the present invention, CXCL11 is also defined by a nucleotide or polynucleotide sequence, which constitutes the protein coding sequence.

20 collected in SEQ ID NO: 9, and which 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

30 nucleic acids possess the activity and structural characteristics of the CXCL11 protein. Among said nucleic acid molecules is the collection in the sequence SEQ ID NO: 2.

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The WHO gene or oncostatin M (CXC motif chemokine 11; beta-R1; interferon gammainducible protein 9; interferon-inducible T-cell alpha chemoattractant; small inducible cytokine B11; small inducible cytokine subfamily B (Cys-X-Cys), member 11 ; small inducible cytokine subfamily B (Cys-X-Cys), member 9B; small-inducible cytokine B11, H174, I-TAC, IP-9, IP9, SCYB11, SCYB9B, b-R1) is found on chromosome 4 ( 4q21.2). Oncostatin M belongs to the same family as interleukin-6 and stimulates several functions involved in wound repair. Although initially described as a leukemia cell growth inhibitor (Zarling et al., 1986. Proc. Nat. Acad. Sci. USA. 83, 9739-43), it has recently been proven that it can house a double role, also as a promoter in proliferation, cell migration and invasion (Fossey et al. 2011, BMC Cancer 11, 125; Li et al., 2011. Int. J. Mol. Med. 28, 101-108; Winder et al., 2011 J. Pathol. 225, 448-462; Tiffen et al., 2008. Mol. Endocrinol. 22, 2677-2688). Once OMS joins its receptor, it promotes reciprocal phosphorylation and activation of the Janus kinase (JAK) / STAT family pathway. Several transcriptional objectives of STAT3 are important contributors to the biology of PDAC (Corcoran et al., 2011. Cancer Res. 71, 5020-5029)

In the context of the present invention, WHO is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 10, and which would comprise various 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%, 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 OMS protein. Among said nucleic acid molecules is the collection in the sequence SEQ ID NO: 3.

The GPNMB or glycoprotein (transmembrane) nmb (UNQ1725 / PRO9925, HGFIN, NMB, glycoprotein NMB; glycoprotein nmb-like protein; osteoactivin; transmembrane gene

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HGFIN glycoprotein; Transmembrane glycoprotein, NMB NQ1725 / PRO9925, HGFIN, NMB) is found on chromosome 7 (7p15). GPNMB glycoprotein, also known as osteoactivin (OA), DC-HIL or HGFIN is a type 1 transmembrane protein, which was initially described as low at undetectable level in malignant cells (Weterman et al., 1995. Int. J Cancer 60, 73-81) However, more recent studies have described GPNMB / osteoactivin as a promoter of metastasis and invasion (Rose et al., 2010. PLos One 5, 12093) in some cancers such as melanoma (Tomihari et al., 2010. Cancer Res 70, 5778-5787; Tse et al., 2006. Clin. Cancer Res. 12, 1373-1382), uveal melanoma (Williams et al., 2010. Melanoma res. 10 20, 184 -190), glioma (Kuan et al., 2006. Clin. Cancer Res. 12, 1970-1982), hepatocellular carcinoma (Onaga et al., 2003. J. Hepatol. 39, 779-785 and breast cancer, in which has also been described as a prognostic indicator of recurrence (Rose et al., 2010. Clin. Cancer Res. 16, 2147-2156). Recently an induction of GPNMB in the spinal cord of pacien has been described tees with lateral sclerosis

15 amyotrophic as an inducer of motor neuron degeneration (Tanaka et al., 2012. Sci. Rep. 2, 573).

In the context of the present invention, GPNMB is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 11 or SEQ ID NO: 12, and which would comprise various

20 variants from:

a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 or 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: 11 or SEQ ID NO: 12, and in which the polypeptide

30 encoded by said nucleic acids possesses the activity and structural characteristics of the GPNMB protein. Among said nucleic acid molecules is the collection in the sequence SEQ ID NO: 4 or in SEQ ID NO: 5.

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The SCF or KIT ligand gene (KITLG, FPH2, KL-1, Kitl, MGF, SCF, SF, SHEP7, c-Kit ligand; familial progressive hyperpigmentation 2; ligand kit; mast cell growth factor; steel factor; stem cell factor) It is found on chromosome 12 (12q22). SCF is the main ligand for the tyrosine kinase receptor c-kit (KIT). Their unions support proliferation, differentiation and survival in KIT-expressing cells, both normal cells and tumor cells, including pancreatic cancer cells (Yasuda et al., 2006. Mol. Cancer 5, 46) through the activation of the pathways (PI3K) / Akt; MAPK and STAT (Yasuda et al., 2007. Dig. Dis. Sci. 52, 2292-2300). Previous analyzes have described high levels of stem cell factor (FCM) in colorectal cancer sera and PDAC (Mroczko et al., 2005. Clin. Chem. Lab. Med. 43, 146-150; Mroczko et al., 2004. Clin. Chem. Lab. Med. 42, 256-260; Mroczko et al., 2005. Int.J. Colorectal Dis. 22,33-38). .

In the context of the present invention, SCF is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 13 or in SEQ ID NO: 14, and which would comprise various variants from:

a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 13 or 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: 13 or with the SEQ ID NO: 14, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the SCF protein. Among said nucleic acid molecules is the collection in the sequence SEQ ID NO: 6 or in SEQ ID NO.

7.

As PDAC has multiple genetic and epigenetic alterations and involves several signaling pathways, a combination of CA 19-9 with various biomarkers could represent a new approach to establish new diagnostic biomarkers. FGF-10, CXCL11, OSM, GPNMB and SCF have been selected by the authors of the present invention for an early diagnosis of the disease.

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In another preferred embodiment, the detection of the amount of expression of any of the FGF-10, CXCL11, OSM, GPNMB and SCF genes 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 an immunoreactive (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 marker compound. 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 FGF-10, CXCL11, OSM, GPNMB and SCF. 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.

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such as alkaline phosphatase or peroxidase, radioactive isotopes such as or 35S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, auto-radiography, fluorimetry, or metallography respectively.

Another aspect of the invention relates to an antibody for treating an individual suffering from pancreatic cancer (or alternatively to the use of an antibody in the preparation of a medicament for the treatment of pancreatic cancer), identifiable by a method of the invention, wherein the antibody is selected from anti-FGF-10, anti-CXCL11, anti-OSM, anti-GPNM and anti-SCF, or any combination thereof. Preferably a combination of all the above antibodies is employed.

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Another aspect of the invention relates to a pharmaceutical composition comprising a modulating agent of at least one of the genes that are selected from FGF-10, CXCL11, OSM; GPNMB and SCF, to treat an individual suffering from pancreatic cancer (or alternatively to the use of a pharmaceutical composition comprising a modulating agent of at least one of the genes selected from FGF-10, CXCL11, OSM; GPNMB and SCF in the preparation of a medicament for the treatment of pancreatic cancer), identifiable by a method of the invention. Preferably, the composition comprises one or more modulating agents of all FGF-10, CXCL11, OSM genes; GPNMB and SCF simultaneously. - Even more preferably, the pharmaceutical composition further comprises another active ingredient. In another more preferred embodiment, the modulating agent is an antibody that is selected from anti-FGF-10, anti-CXCL11, anti-OSM, anti-GPNM and anti-SCF, or any combination thereof.

Another aspect of the present invention relates to a kit or device, hereafter kit of the invention, comprising the elements necessary to quantify the amount of expression product of the genes that are selected from FGF10, CXCL11, OSM, GPNMB and SCF or any of its combinations.

In a preferred embodiment, the kit of the present invention comprises antibodies that are selected from the list consisting of antibodies: anti-FGF-10, anti-CXCL11, anti-OSM, anti-GPNM and anti-SCF, or any of your combinations Preferably, the kit of the invention simultaneously comprises at least one antibody of each type: anti-FGF-10, anti-CXCL11, anti-OSM, anti-GPNM and anti-SCF.

In another preferred embodiment, the kit of the invention comprises secondary antibodies

or positive and / or negative controls. The kit can also include, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc.

On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit further comprises instructions for carrying out the methods of the invention.

Another aspect of the invention relates to a solid support, or protein chip, comprising at least one of the anti-FGF-10 /, anti-CXCL11, anti-OSM, anti-GPNM and anti-SCF antibodies, or any of its combinations, to carry out any of the methods of the invention.

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Another aspect of the invention relates to a solid support, or DNA chip, comprising oligonucleotides or single channel microarrays designed from a known sequence or an mRNA of at least one of the genes. Preferably, it comprises the oligonucleotides capable of detecting the mRNA of all the FGF-10, CXCL11, OSM, GPNM and SCF genes.

Thus, for example, oligonucleotide sequences are constructed on the surface of the chip by sequential elongation of a growing chain with a single nucleotide using photolithography. Thus, the oligonucleotides are anchored at the 3 'end by a method of selective activation of nucleotides, protected by a photolabile reagent, by the selective incidence of light through a photomask. The photomask can be physical or virtual.

Thus, oligonucleotide probes can be between 10 and 100 nucleotides, more preferably, between 20 and 70 nucleotides, and even more preferably, between 24 and 30 nucleotides. For the quantification of gene expression, preferably about 40 oligonucleotides per gene are used.

Synthesis in situ on a solid support (for example, glass) could be done using ink-jet technology, which requires longer probes. The supports could be, but are not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy. The probe is each of the chip samples. The target is the sample to be analyzed: messenger RNA, total RNA, a PCR fragment, etc.

Another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of any of the methods of the invention (of the first or second method of the invention ).

Another aspect of the invention relates to a transmissible signal comprising program instructions capable of having a computer perform the steps of any of the methods of the invention.

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

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Analysis of the ROC curves for each of the five serum cytokines, found that they were differentially expressed among patients with PADC. The ROC curves summarize the accuracy of cytokines in the prediction of patients with PDAC. The area under the curve (ABC) is the average sensitivity of the biomarker. A biomarker with non-predictive values would have an ABC of 0.5, while a biomarker with a perfect ability to predict the disease would have an ABC of 1. A shows the values of ABC, cut-off value, sensitivity and specificity for FGF- 10; shows the ABC values, cut-off value, sensitivity and specificity for I-CXCL11; C shows the ABC values, cut-off value, sensitivity and specificity for OSM; D shows the ABC values, cut-off value, sensitivity and specificity for the GPNMB; E shows the ABC values, cut-off value, sensitivity and specificity for SCF; F shows the values of ABC, cut-off value, sensitivity and specificity for the five cytokines combined. The cut-off values (intensity signal values) selected were those with both the highest sensitivity and specificity.

EXAMPLES 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.

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The study involved 39 patients. 3 different groups were established:

1) 12 healthy patients, as a control group. 2) 14 patients with PDAC who had not received any treatment 5 (referred to as pre-treated). 3) 13 patients with PDAC under 2 weeks of treatment with the combined Gemcitabine + Erlotinib therapy (referred to as post-treated)

All patients were diagnosed with PDAC at the Virgen de las Nieves Hospital (Granada, Spain) between 2008 and 2011. All the information of the patients was recorded.

10 patients, including gender, age, degree of disease and symptoms. The average age of the patients was 66 years (range 41-79 years) with a ratio between men and women of 50:50. The clinical classification of patients with pancreatic adenocarcinoma was as follows: state III (28%) and state IV (72%; Table 1).

Table 1. Clinicopathological characteristics of the study population (n = 14)

Age of diagnosis, years (mean ± SD) 66 ± 10.5

Gender M: 50% F: 50%

Disease stage III (28%) IV (72%)

Type of chemotherapy Gemcitabine + Erlotinib

Clinical response PR (14.29%) SD (21.43%) PD (64.29%)

Survival time, months (mean ± SD) 12.6 ± 12.6

CEA level [µg / l] (mean ± SD) 2219 ± 5017

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Healthy: 0-37

AC level 19-9 [U / l] (mean ± SD) 899 ± 3185 Healthy: 0-5

PR: partial response; SD: stable disease; PD: progressive disease; SD: standard deviation.

Table 1. Clinical-pathological characteristics of the study population (n = 14). Information collected from all PDAC patients, participants in the study.

5 Origin of the samples.

Blood samples were collected after obtaining approval from relevant ethical committees and consents signed by the patients. A total of 39 serum samples were collected from 2009 to 2011, using the standard procedures of the Oncology Service of the Virgen de las Nieves Hospital (Granada,

10 Spain). Blood samples were obtained from patients diagnosed with PDAC at the start of the study and 2 weeks after the start of therapy (Gemcitabine + Erlotinib) and also from healthy individuals. The serum was obtained after centrifugation of the blood at 1500 rpm for 10 minutes at 4 ° C. The samples were alicu

15 otadas and stored at -80ºC.

Cytokine antibody assay

The serum-soluble proteins of patients with PADC were measured using an array of biotin-labeled human antibodies (Human Antibody L-series 507 Array (RayBiotech, Norcross, GA, USA), according to the recommended protocols. samples were biotinylated, antibodies were immobilized at specific sites on glass slides, incubation of arrays with biological samples consisted of the binding of cytokines to their corresponding antibodies, signals were visualized using streptavidin-HRP conjugates and colorimetry.

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final intensities were measured as the original intensities minus the background. The data were normalized to the positive control for each individual result.

Statistic analysis

All statistics and data analyzes were performed using the IBM SPSS software

5 Statistic 20 or the statistical language R. Quality analysis was developed using the “ArrayQualityMetrics” pack in R, to eliminate any possible outliers (Kauffmann et al., 2009. Bioinformatics 25, 415-416) Like cytokines in all the groups did not present a normal distribution, the Mann-Whitney test was used to highlight the differences between groups (p <0.05). In addition, the exchange rate was calculated

10 (fold change, FC). The cytokine exchange rate values were checked to indicate their relative levels of expression. Neither FC ≤ 1.5 nor FC ≥ 1.5 in the intensity signal between the groups were considered relevant. The area under the curve (ABC) of the receiver's operating characteristic (ROC) of each marker was calculated to assess its diagnostic significance representing the true positive rate

15 (sensitivity) against the false positive rate (1-specificity). In addition, a marker combination index was generated and the ROC curve was also plotted for this combination. The cut-off values were selected to improve both sensitivity and specificity. Additionally, box diagrams showing the modulation of the expression of those modified common cytokines were represented

20 significantly between post-treated versus pre-treated and between pre-treated and control.

RESULTS

Analysis of diagnostic serum biomarkers in patients with PDAC and healthy (control).

25 In order to find markers that would help the early detection of PDAC, extensive screening with an array of antibodies to 507 human cytokines was performed to identify those cytokines differentially expressed between healthy individuals and those suffering from PDAC. The clinical pathological characteristics of patients with PDAC are summarized in Table 1. As shown in the table.

30 2, there are 5 overexpressed cytokines in patients with untreated PDAC when compared with healthy volunteers (p <0.05). FGF-10, CXCL11, OSM, GPNMB and SCF cytokines could represent an altered serum profile in patients with PDAC.

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Table 2: Significantly overexpressed cytokines in PDAC patients.

ID

P-Value Log FC FC

FGF-10

0.040 1.10 2.15

CXCL11

0.046 0.93 1.91

OSM

0.040 0.96 1.95

GPNMB

0.019 1.36 2.56

SCF

0.022 1.51 2.85

Table 2. The 5 cytokines significantly overexpressed. The differences were obtained by the Mann-Whitney test (p <0.05). Their relative expression levels are given by their corresponding HR. Each FC≥1.5 or ≤1 / 1.5 at the signal strength between

5 groups were considered relevant.

Sensitivity and specificity analysis of serum biomarkers for early diagnosis of PDAC.

To determine whether these 5 cytokines could be used as markers for the early detection of PDAC, the diagnostic value (the ability to differentiate between health and disease) of each biomarker was evaluated, alone and in combination. The sensitivity and specificity for each biomarker were analyzed using the area method under the receiver operating characteristics curve (ROC). Curves are based on cytokine levels in healthy individuals and individuals before and after treatment. Each candidate biomarker 15 was independently tested and investigated (Figure 1A-E). All possible new markers showed AUC values between 0.74 and 0.78 to differentiate healthy patients and patients with PDAC. The areas under the ROC curve for the FGF-10, CXCL11, OSM, GPNMB and SCF cytokines were 0.744, 0.737, 0.744, 0.776 and 0.772 respectively. Osteoactivin stood out for developing the highest sensitivity for the prediction of PDAC. Next, to determine if these 5 cytokines as a whole could be used as a group of biomarkers for PDAC detection, a combination of these 5 markers was analyzed to obtain an integrated ROC curve. The combined group of these cytokines significantly improved the ability of all biomarkers separately in isolation to distinguish

25 patients with pancreatic cancer from healthy controls, the AUC value improved to 0.841 (Figure 1F).

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To determine the sensitivity and specificity of each biomarker, those cut-off points were established that provide the best points of renunciation between the highest sensitivity and the highest specificity (Figure 1). All showed a specificity of 75% and a sensitivity with a range between 69-77%. The group of the 5 cytokines showed the best performance to detect PDAC in serum samples, also reflecting a sensitivity of 77% but a specificity of 83%, higher than separately.

Claims (1)

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