WO2015047071A1 - In vitro test for early diagnosis and staging of liver fibrosis - Google Patents

In vitro test for early diagnosis and staging of liver fibrosis Download PDF

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WO2015047071A1
WO2015047071A1 PCT/MX2014/000147 MX2014000147W WO2015047071A1 WO 2015047071 A1 WO2015047071 A1 WO 2015047071A1 MX 2014000147 W MX2014000147 W MX 2014000147W WO 2015047071 A1 WO2015047071 A1 WO 2015047071A1
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liver
fibrosis
staging
patients
protein
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PCT/MX2014/000147
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Spanish (es)
French (fr)
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María del Carmen GARCÍA DE LEÓN MÉNDEZ
Alains HERNÁNDEZ CEQUÉRA
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Universidad Nacional Autónoma de México
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Definitions

  • the present invention relates to in vitro tests for diagnosis of diseases that occur with fibrosis, more specifically it relates to tests to detect the process of fibrosis in the liver and more specifically to the use of non-invasive biomarkers for diagnosis and staging of liver fibrosis
  • the liver plays a unique role as the metabolic center of the organism. Its average weight in adult individuals is approximately 1,400 + 270 g, without significant gender-related differences. It is made up of five different types of cells that occupy about 80% of their volume. The remaining 20% corresponds to the extracellular spaces and components of the extracellular matrix (Rojkind M, Greenwel P. The extracellular matrix and the liver. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Shashter DA, Shafritz DA, (ed) The Liver: Biology and Pathology, Third Ed. Raven Press, New York 1994, pp 843-868).
  • the liver tissue is organized microscopically in lobes formed by plaques consisting of cells that extend from the portal area in a linear fashion to the central vein; the spaces that cross the septa are the hepatic sinusoids, separated from the hepatocytes by the perisinusoidal space of Disse.
  • the hepatic sinusoids have an average diameter of 5 to 7 ⁇ , and lead the mixed blood, from the terminal branches of the hepatic artery and the portal vein to the terminal branching of the hepatic vein.
  • Sinusoids represent the only form of capillary tube in the liver and have a continuous but fenestrated endothelial lining, Kupffer cells, dendritic cells within of light, a perisinusoidal space (Disse space) and absence of a continuous basement membrane.
  • SEC sinusoidal endothelial cells
  • the absence of a basement membrane facilitates the rapid exchange of blood components with hepatocytes.
  • Hepatic sinusoids connect the portal spaces with the terminal branches of the hepatic vein (central veins).
  • the endothelial cells of the hepatic artery are elongated and arranged longitudinally, while those of the portal and central veins are polygonal, flattened and have microvilli (Kmiec Z., 2001. Cooperation of liver cells in health and disease. Adv Anat Embryol CelI Biol 161: lll-XIII: 1-151).
  • the sinusoidal endothelium whose structure is designated as the liver filter, is probably the main target of oxidative stress, this allows the free exchange of proteins and other nutrients between hepatocytes and blood, in addition to isolating them from most blood cells, of platelets and larger colloidal particles, such as chylomicrons and viruses (Cogger, VC, Muller, M, Fraser, R, Mclean, AJ, Khan, J, Le Couteur, DG. 2004. The effects of oxidative stress on the liver sieve. J Hepatol. 41: 370-376)
  • Fibrosis is a common response of the liver to chronic injuries caused by a variety of attacks, such as metabolic diseases, viral infections, alcohol intake, drugs, autoimmune attack on hepatocytes, bile ducts and abnormalities congenital
  • ECM extracellular matrix
  • the components of the ECM are similar to those present in normal liver (collagen and others) but quantitatively increased, with the generation of fibrosis, the normal structure of the matrix present in the subendothelial space is transformed into a matrix of interstitial type with high content of fibrillar collagen, product of paracrine activation of stellar cells (HSC; Friedman SL. 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem.
  • the normal liver allows the free exchange of molecules and nutrients, between the liver cells and blood flow, due to the porous constitution of sinusoidal endothelial cells (SEC) and the shortage of MEC in the perisinusoidal space; therefore, the capiiarization of the sinusoid powerfully impairs this exchange.
  • SEC sinusoidal endothelial cells
  • any quantitative or qualitative modification of the MEC will influence the microenvironment and cellular interactions, producing changes in the hepatic stellar cell phenotype (HSC) and deterioration of the function of the hepatocyte
  • HSC hepatic stellar cell phenotype
  • Fibrosis itself is an important biological event, product of the imbalance between the synthesis and degradation of the ECM molecules, which, when associated with other organ processes such as architectural deformation and vascular redistribution, produces serious consequences for liver function.
  • fibrosis promotes the development of cirrhosis which, in the absence of timely and adequate treatment, usually leads to a fatal outcome.
  • Cirrhosis, together with other chronic liver diseases represents at national level, the second cause of death of individuals of productive age, which has a significant impact on both public health and the economy, involving significant costs in hospitalization, treatment and absence from work (SINAIS. General functions report of the Ministry of Health, 2008. League:
  • the main biological targets of free radicals and ROS are proteins, lipids and DNA. Peroxidation occurs mainly in the plasma membrane, altering its physical properties and as a consequence its biological functions. Hepatocytes and Kupffer cells are a source of ROS; these compounds exert paracrine stimulation on HSCs, whose activity in vivo is amplified by the loss of antioxidants, as occurs typically in liver disease. Over-expression in the HSCs of the cytochrome p450 2E1 enzyme, whose activity generates ROS, stimulates the expression of the collagen I gene, which is attenuated by antioxidants. Damaged SECs stimulate the production of a cellular variant of fibronectin (EIIIA isoform) that has an activating effect on HSCs.
  • EIIIA isoform a cellular variant of fibronectin
  • SEC converts the latent transforming growth factor- ⁇ ((TGF- ⁇ ) to its active form through the action of plasmin, a process that finally triggers the formation of the scar consisting of complex MEC fibers, contributing to the loss of hepatocyte microvilli and sinusoid capillarization with the consequent deterioration of liver function (Friedman SL. 2000 Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 275 (4): 2247-2250).
  • TGF- ⁇ latent transforming growth factor- ⁇
  • HAI histological activity index
  • the first component (periportal and / or bridge necrosis) evaluated on a scale of 0-10.
  • the following two components are scored 0-4.
  • the combination of these three markers indicates the amount of inflammation in the liver.
  • the fourth component shows the amount of scarring in the liver and is scored as: F0 (no scars), F1 (portal fibrosis without septa), F3 (numerous septa without cirrhosis) and F4 (Cirrhosis or advanced scars in the liver) (Sebastiani G, Alberti A. Non invasive fibrosis biomarkers reduce but not substitute the need for liver biopsy. World J Gastroenterol2006; 21; 12 (23): 3682-94).
  • the Metavir scoring system has been specially designed to assess liver status in people infected with Hepatitis C Virus, HCV.
  • the index includes the use of the sum of the score assigned to the degree of inflammatory activity observed in the sample (0-4; being, 0 without activity and 3 or 4 the activity considered serious) in addition to that provided by staging, which represents the amount of fibrosis: 0 (no scars), 1 (minimal scars), 2 (scarring has occurred and extends outside the areas that contain blood vessels), 3 (fibrosis bridges extending and connecting with other areas that contain fibrosis ) and 4 (cirrhosis) (Bedossa P, Poynard T. METAVIR Cooperative Study Group. An algorithm for the grading of activity in chronic hepatitis C. Hepatol; 24: 289-293).
  • AASLD American Association for the Study of Liver Diseases
  • liver biopsy On the other hand, it is a histological evaluation that depends strictly on the pathologist's experience (observer error). There are risks associated with obtaining liver biopsy, ranging from pain (84%) and hypotension as the most frequent, to peritoneal bleeding (0.5%) and damage to the biliary system as the most serious complications, although with a significantly low level of mortality and morbidity (0.09-0.12-%) (Piccinino F, Sagnelli E, Pasquale G and cois. 1986. Complications following percutaneous liver biopsy. A multicenter retrospective study on 68 276 biopsies J. Hepatol. 2: 165-73; Rockey DC, Caldwell SH, Goodman ZD et al. 2009. Liver biopsy. Hepatol.
  • Biomarkers of liver fibrosis Due to the drawbacks of liver biopsy in recent years, the interest in identifying and describing liver fibrosis through the use of non-invasive markers has been increasing (Plebani M, and Burlina A. 1991. Biochemical Markers of Hepatic Fibrosis, Clin Biochem. 24, 219-239). The search for specific biomarkers of liver fibrosis has been applied in tissue and serum research, and these have been used to understand the fundamental biological processes and their interrelationships, to be used as a tool in diagnostic and therapeutic development. However, there are few non-invasive biomarkers (located in serum or biological fluids), which indicate with certainty the fibrogenic activity.
  • fibrosis can be determined non-invasively in two ways, one of which is based on the quantification of biomarkers in serum (biological approach) and the second measuring the stiffness of the liver (physical approach), both forms are finally complementary. Hardness is a characteristic property, exclusive to the liver parenchyma, while serum biomarkers may indicate an association with the stage of fibrosis. Serum biomarkers of hepatic fibrosis offer an attractive and cost-effective alternative for both the patient and the doctor. In addition to not being invasive, they practically do not cause complications, sampling errors are few or zero and have the advantage that measurements can be carried out repeatedly, thus allowing dynamic disease control (Zhou K, Lu LG 2009. Assessment of fibrosis in chronic liver diseases, Journal of Digestive Diseases, 10 (1): 7-14).
  • Serum biomarkers have been valued primarily for their ability to determine the stage of fibrosis. Two types of them have been proposed: the direct ones, which reflect the deposition or elimination of the extracellular matrix in the liver; and the indirect ones, which include molecules released into the blood induced by inflammation, synthesized, regulated or excreted by the organ, as a product of the processes commonly altered as a result of impaired liver function (Gressner Olav A., Ralf Weismün, Gressner Axel M., 2007. Biomarkers of liver fibrosis: Clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Cl ⁇ nica Chimica Acta. 381: 107-113)
  • biomarkers encompass the different fragments of the components of the ECM produced by stellar cells (HSC) and other liver cells during the liver matrix remodeling process (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis J Gastrointestin Liver Dis. 5 (2): 149-159), including glycoproteins such as hyaluric acid, laminin and YLK-40; collagen, (pro-collagen III, type IV collagen), and matrix metalloproteases (MMPs) and their inhibitors (TIMPs)
  • HSC stellar cells
  • MMPs matrix metalloproteases
  • PICP pro-collagen type I
  • PIIINP pro Type III collagen
  • fibrils I and III
  • the collagen is integrated into the MEC.
  • levels of type I collagen can increase up to eight times, in addition, the ratio l / lll also changes, from 1: 1 in the healthy liver, to 1: 2 in the cirrhotic (Gressner Olav A., Ralf Weismaschinen , Gressner Axel M., 2007.
  • Biomarkers of liver fibrosis Clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Cl ⁇ nica Chimica Acta. 381: 107-113).
  • PIIINP is an important component of connective tissue; its relative concentration in the basement membrane is higher during hepatic fibrogenesis followed by an increase in its serum levels.
  • PIIINP levels correlate with aminotransferase levels, reflecting the degree of fibrosis, but unfortunately, it is not specific since it also rises in acromegaly, pulmonary fibrosis, chronic pancreatitis, and rheumatic diseases (Lieber CS, Weiss DG, Paronetto F. 2008.
  • Type IV collagen is an essential component of hepatic ECM. Unlike type I and III collagen that are processed by proteolysis, this molecule is deposited intact in the matrix and its presence in the serum directly reflects its degradation. Therefore, tests to detect fragments of type IV collagen (NC1 and PIVNP) in serum are used more frequently in practice. These have a positive correlation with the degree of Liver fibrosis in patients with chronic viral hepatitis and alcoholic liver disease functioning as sensitive indicators of the presence of cirrhosis in hemochromatosis.
  • stage F2 In hepatitis C, the cut-off point in stage F2 (110 ng / ml) was established for diagnosis and to predict stage F3 (130 ng / ml) (Murawaki Y, Koda M, Okamoto K et al. 2001. Diagnostic valué of serum type IV collagen test in comparison with piateiet count for predicting the fibrotic stage in patients with chronic hepatitis. J Gastroenterol Hepatol. 16: 777-781) (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis. J Gastrointestin Liver Dis. .15 (2): 149-159).
  • TGF- ⁇ Transforming growth factor ⁇ 1
  • TGF- ⁇ Transforming growth factor ⁇ 1
  • ⁇ 1, ⁇ 2 and ⁇ 3 Three isoforms of this cytokine ( ⁇ 1, ⁇ 2 and ⁇ 3) have been identified, but only la- ⁇ has been linked to hepatic fibrogenesis.
  • TGF- ⁇ It is commonly known as a central component of fibrogenic response in wounds and as a regulatory envelope for different diseases. The correlation between TGF- ⁇ levels and fibrosis progression is considerably accepted (Kanzler S, Baumann M, Schirmacher P, Dries V, Bayer E, Gerken G, Dienes HP, Lohse AW. 2001.
  • Hyaluronic acid It is a glycosaminoglycan component of the MEC synthesized by HSC. Under normal circumstances, hepatic SECs are the structures that directly intervene in their uptake and degradation. Elevated levels of HA may be due to decreased elimination or increased production, these levels have been detected in the serum of patients with liver disease of different etiologies and in particular in those with cirrhosis (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis J Gastrointestin Liver Dis. 15 (2): 149-159).
  • HA was selected as the best marker for fibrosis, with an area under the curve (AUC) of 0.97 (Lydatakis H, Hager IP, Kostadelou E, Mpousmpoulas S, Pappas S, Diamantis I. 2006. Non-invasive markers to predict the liver fibrosis in non alcoholic fatty liver disease. Liver Int. 26: 864-871).
  • Protein 4 associated with Microfibrils It is a collagen binding molecule that contains at its C-terminal end a fibrinogen-like domain and a ligand binding motif located at the N-terminal end of the protein. It participates in the innate immune response and allows free gaseous exchange in the lungs through its binding to the collagen region of surfactant proteins (SP-A and SP-D). Its N-terminal region includes a cysteine residue and an Arg-Gly-Asp (RGD) sequence, which is a reason for cell adhesion for several members of the integrin family (Greenlee K J., Werb Z, and Kheradmand F. 2007.
  • AST Aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALT and AST can also be elevated although to a lesser extent due to muscular, renal and cardiac problems (Giboney PT. 2005. Mildly elevated liver transaminase levéis in the asymptomatic patient. Am Fam. Physician. 71 (6): 1105-1110) .
  • liver disease of a determined etiology there is a wide overlap between those who, for diagnosis, depend exclusively on the AST / ALT relationship, for example, patients suffering from both hepatitis C and have a history of alcohol abuse.
  • a technical review of the AGA reports that serum ALT has daytime variation, and can be modified day by day, even with exercise, it also points out that serum AST levels can be up to 15% higher in men black with respect to white race (Green RM, Flamm S. 2002. AGA technical review on the evaluation of liver chemistry tests. Gastroenterology. 123: 1367-84).
  • Thrombocytopenia is a valuable biomarker for advanced liver diseases, it may be related to mechanisms such as hypersplenism, HCV myelosuppression, decreased thrombopoietin production, development of autoimmune processes, however, joint evaluation of the AST / ALT and PLT ratio has a high diagnostic value for cirrhosis (70-90%) (Giannini E, Risso D, Botta F et al. 2003. Validity and clinical utility of the aspartate aminotransferase-alanine aminotransferase ratio in assessing disease severity and prognosis in patients with hepatitis C virus-related chronic liver disease. Arch Intern Med. 163: 218-224).
  • Prothrombin time It is an index that reflects the liver's ability to synthesize, and therefore is one of the initial indicators of cirrhosis.
  • prothrombin time PLT, AST / ALT ratio and alkaline phosphatase levels, were predictive of cirrhosis.
  • prothrombin time correlated with the presence and size of esophageal varices.
  • Prothrombin time is a component of different indices (Croquet V, Vuillemin E, Ternisien C et al. 2002. Prothrombin index is an direct marker of severe liver fibrosis. Eur J Gastroenterol Hepatol. 14: 1133-141)
  • Direct and indirect biomarkers can be used alone or in combination, to produce composite scores.
  • the calculation of such scores may be relatively simple or may be based on complicated formulas (for example, Fibrotest / Fibrosure).
  • Fibrotest / Fibrosure Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non-invasive markers for hepatic fibrosis. BMC Gastroenterol. 11: 91); FibroTest (patented by Biopredictive, Paris, France) was the first multicomponent that combined the data resulting from various tests (Imbert-Bismut F, Ratziu V, Pieroni L, et al. 2001. Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study. Lancet.
  • Table 2 serological indices of multicomponents for liver fibrosis.
  • GGT apolipoprotein A1
  • hyaluronic acid urea PGA index
  • It combines the measurement of the prothrombin time index, levels of ⁇ -glutamyltransferase and apolipoprotein A1. Subsequently, the determination of a2-macroglobulin was added, which resulted in PGAA improving its performance.
  • the PGA index is related, in chronic liver diseases, to both inflammation and fibrosis (p ⁇ 0.01, p ⁇ 0.05, respectively).
  • APRI It is the index that results from the AST-Platelet ratio, this is calculated as follows:
  • the Foms index It is based on 4 routine clinical variables: age, platelet count, cholesterol levels, and glutamyltransferase (Forns X et al, 2002). With this method, patients with mild fibrosis (F0-F1) can be distinguished from those with severe fibrosis (F4), but it is less accurate to distinguish patients with intermediate grades from F2 to F4. The Forns index has been validated in other cohorts, as a tool for predicting the response to therapy HCV (Rossi E, Adams LA, Bulsara M, Jeffrey GP. 2007: Assessing liver fibrosis withserum marker models. Clin Biochem Rev. 28 (1): 3-10).
  • HepaScore This index combines age, gender, serum concentrations of bilirubin, ⁇ glutamyltransferase, hyaluronic acid and Y2-macroglobulin in a score of 0.00 to 1.00.
  • the HepaScore can be automated using a single analyzer (Guechot J, Lasnier E, Sturm N, Paris A, Zarski JP. 2010.
  • ANRS HC EP 23 Fibrostar study group Automation of the Hepascore and validation as a biochemical index of liver fibrosis in patients with chronic hepatitis C from the ANRS HC EP 23 Fibrostar cohort Clin Chim Acta. 411 (1-2): 86-91).
  • FibroTest and FibroSURE are identical tests for the evaluation of the degree of fibrosis and necro-inflammatory activity, marketed under different names in Europe and America.
  • the FibroTest score is obtained by accessing a license from a website and calculating it based on the patient's age, sex, serum haptoglobin concentration, a2-macroglobulin, apolipoprotein A1, ⁇ -glutamyltransferase and bilirubin (Imbert- Bismut F, Messous D, Thibault V, Myers RB, Python A, Thabut D, Devers L, Hainque B, Mercadier A, Poynard T. 2004.
  • the values for the sensitivity and specificity of FibroTest are based on the detection of severe primary fibrosis ranging between 75 and 85%, respectively (Lu LG, Zeng MD, Mao YM, Li JQ, Qiu DK, Fang JY, Cao AP, Wan MB, Li CZ, Ye J, Cai X, Chen CW, Wang JY, Wu SM, Zhu JS, Zhou XQ. 2003. Relationship between clinical and pathologic findings in patients with chronic liver diseases. World J Gastroenterol. 9 (12): 2796-2800).
  • liver specific liver specific, and their results can be influenced by comorbid conditions that require a more critical interpretation of the data.
  • each biomarker is compared with the benefit exhibited by one or more panels, in addition to limiting its evaluation to a pathological condition (eg, alcoholic liver disease), and the exact contribution of each biomarker just described is difficult because of this.
  • a pathological condition eg, alcoholic liver disease
  • phosphoproteomics can improve the knowledge of the pathogenesis of liver fibrosis, using the phosphorylated profiles of the proteins that participate in the signaling pathways of this process, as demonstrated by (Younossi ZM, Baranova A, Stepanova M, Page S , Calvert VS, Afendy A, Goodman Z, Chandhoke V, Liotta L, Petricoin E. 2010. Phosphoproteomic biomarkers predicting histologic nonalcoholic steatohepatitis and fibrosis. J Proteome Res. 9 (6): 3218-24), whose results suggest that biomarkers Phosphoproteics could potentially be used in a clinical setting to identify patients with NASH. In addition, they provide information about the metabolic pathways that may be involved in the pathogenesis of the disease.
  • non-invasive tests are not able to differentiate with certainty early stages of fibrosis. In fact, most of these tests can distinguish mainly cirrhosis from fibrosis. minimum To date, non-invasive tests for the diagnosis of significant fibrosis (F> 2) cannot replace the biopsy. Therefore, the current utility of non-invasive diagnosis remains limited, since it only allows the physician to reduce the population of patients who are candidates for liver biopsy.
  • fibrogenesis process is a common response of the liver, when there is a chronic injury caused by a variety of attacks, as part of the development of the disease (Friedman SL. 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury J Biol Chem. 275 (4): 2247-2250), which has hindered the proper search for specific biomarkers, and has become a challenge in translational hepatology.
  • hepatic fibrosis there is therefore an urgent need to have specific markers of hepatic fibrosis, through non-invasive methods for various reasons, one of them is the existence of more than 170 million patients infected with hepatitis C virus (HCV) throughout the world, which correspond to 3% of the world's population.
  • HCV hepatitis C virus
  • liver cirrhosis frequently used is the liver's response to the hepatotoxic drug carbon tetrachloride (ICC 4 ), which induces peroxidation of hepatocyte membrane lipids and depending on the dose, exposure time, or The age of the animals used can result in the regression of the process, with the almost complete recovery of the liver (Friedman SL. 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 275 (4): 2247-2250).
  • ICC 4 hepatotoxic drug carbon tetrachloride
  • Covalent binding of methyl trichloride to cellular proteins is considered the first step between sequential events that produce lipid peroxidation of the membrane, whose end point is cellular necrosis (Zimmerman, HJ, 1999. Hepatotoxicity: The Adverse Effects of Drugs and Other Chemicals on the Liver, Drug- and chemical-induced cholestasis.Toxicol Lett. 8; 105 (1): 39-46; Rosenberg W, Burt A, Becka M, Voelker M, Arthur MJP. 2000. Automated assays of serum markers of liver fibrosis predict histologic hepatic fibrosis. Hepatology. 32: 183A).
  • TAA Thioacetamide
  • Microarrays The technologies used to search for biomarkers include traditional in vitro analyzes of the variations and expression of DNA, RNA and proteins, and the quantification of metabolites, as well as in vivo measurements of biological processes in humans and animals, using morphological images and functional technologies. The importance of the measurements is correlated with its prediction capacity for the points clinically relevant, such as the prognosis of the disease, and the response to therapy, to name a few examples.
  • DNA microarray technology emerged as a powerful tool for the analysis of mRNA transcript levels expressed in various situations.
  • the oligonucleotides can be printed on a chip by photolithographic methods.
  • DNA arrays are used to analyze gene expression, monitoring the levels of thousands of them simultaneously allowing to obtain information about the sample being worked on (Schena M, Shalon D, Davis RW, Brown PO. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 270 (5235): 467-70).
  • PCR products can also be seen in the DNA microarray chips. Although their values do not always correlate, information on mRNA expression levels and the corresponding protein abundance (or its activity) are certainly useful in genomic analysis (Tomizaki in-ya, Kenji Usui and Hisakazu Mihara, 2010. Protein-protein interactions and selection: array-based techniques for screening disease-associated biomarkers in predictive / early diagnosis, doi: 10.1111 / j. 1742-4658).
  • RNA is isolated from each sample followed by various steps to enrich and in some cases to amplify cRNAs.
  • This material, cDNA or in the case of cRNA amplification, is directly or indirectly labeled generating a cDNA in a reverse transcription reaction with labeled nucleotides and hybridizes in a microarray, the change in gene expression is determined by means of the quantification of the fluorescence emitted by each point of the microarray (hybrid formed) (Harm van Bakel and Frank CP. Holstege, 2008. A tutorial for DNA Microarray Expression Profiling. Cell press: 22-28).
  • This data can be analyzed using the programs and databases that have been designed to contain the information resulting from the expression of the microarrays, among the best known are SAM (Significance Analysis of Microarrays, Flex Array), DAVID (The Datábase for Annotation, Visualized and Integrated Discovery), Ingenuity, Ace View to name a few.
  • SAM Signal Analysis of Microarrays, Flex Array
  • DAVID The Datábase for Annotation, Visualized and Integrated Discovery
  • Ingenuity Ace View to name a few.
  • Microarray technology is capable of simultaneously detecting the relative expression levels of a large number of genes and gene fragments; Such experiments can help identify the links between specific phenotypes and related patterns of gene expression.
  • NAFLD nonalcoholic fatty liver disease
  • Gene expression related to hepatocellular carcinoma (HCC) has been studied using this technique since this pathology (HCC) is associated with different liver diseases including NASH, for example, a study carried out by Kim and cois, 2004 (Kim JW, Ye Q, Forgues M, Chen Y, Budhu A, Sime J, Hofseth LJ, Kaul R, Wang XW. 2004. Cancer-associated molecular signature in the tissue samples of patients with cirrhosis. Hepatology.
  • liver disease 39: 518-27 revealed specific changes in gene expression in liver samples of people with end-stage cirrhosis, indicating that different chronic liver diseases may represent a "precondition" of liver tissue for malignancy, Smith and cois. (Smith MW, Yue ZN, Korth MJ, Do HA, Boix L, Fausto N, Bruix J, Carithers RL Jr, Katze MG. 2003. Hepatitis C virus and liver disease: global transcriptional profiling and Identification of potential markers. Hepatology. 38: 1458-67) using the mism or approach identified a set of genes with normal physiological variations in the human liver. This group of genes related to tumorigenesis were selected from a differentially expressed gene system in cirrhosis, which led to the identification of 132 molecules as potential markers.
  • the importance of the present invention is that the design of the panel of non-invasive biomarkers for diagnosis and staging of the hepatic fibrosis process, was obtained from an experimental model and from there it became a successful application in the diagnosis and staging of human disease
  • An objective of the present invention is to overcome all the inconveniences that arise in order to diagnose liver fibrosis through the use of a panel of non-invasive biomarkers, even being able to establish the diagnosis during the early phases of liver disease.
  • Another objective of the present invention is to allow the staging of liver fibrosis, through the use of the panel of non-invasive biomarkers, which will enable the establishment of the most effective treatment.
  • biomarker panel of the present invention is to allow the treating physician of the liver disease that develops with fibrosis, to have non-invasive scientific elements to evaluate the effectiveness of the applied treatment, also allowing the monitoring and evolution of the condition Brief description of the figures:
  • Figure 1 shows the flow chart followed during the development of expression microarrays from the total RNA obtained from the liver of all animals.
  • polyadenylated cDNA chain (AAAAA3 '), synthesized by reverse transcription (black)
  • Biotin-labeled RNA obtained from double stranded cDNA
  • FIGs 2a, 2b, 2c and 2d show representative images of liver tissue sections from normal animals, fixed with buffered formalin and included in paraffin, stained with H&E (2a, 2b) and RS (2c, 2d).
  • Hepatocytes are polygonal, with granular cytoplasm and central nucleus, round and with prominent nucleoli.
  • Sinusoids have been upholstered by elongated cells (endothelial and Kupffer cells), close to hepatocytes, leaving the Disse space as an almost virtual region. (The magnitude of the amplification is detailed in each image)
  • Figures 3a, 3b, 3c and 3d show representative images of liver tissue cuts from animals of Phase I of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (3a, 3b) and Sirius Red ( 3c, 3d).
  • H&E histoneum
  • 3c, 3d Sirius Red
  • the onset of structural alteration is observed due to the presence of periportal fibrosis bands (bf) and multiple small steatosis vesicles (e) with nuclei that retain their central position (n).
  • Figures 4a, 4b, 4c, and 4d show representative images of liver tissue sections from animals of Phase II of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (4a, 4b) and RS (4c , 4d).
  • H&E periportal fibrosis
  • steatosis lagoons e
  • rounded vesicles large and small, are observed in centrolobular hepatocytes.
  • centrolobular hepatocytes In the cytoplasm of hepatocytes there are empty areas with displacement of the nuclei to the periphery (n).
  • Figures 5a, 5b, 5c and 5d show representative images of liver tissue sections from animals of Phase III of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (5a, 5b) and RS (5c, 5 d).
  • H&E H&E
  • RS RS
  • FIGS. 6a, 6b, 6c and 6c show representative images of liver tissue sections from animals of Phase IV of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (6a, 6b) and RS (6c, 6d).
  • the liver architecture presents a cirrhotic state with areas of focal necrosis, minimal steatosis (e), regeneration nodules surrounded by bands of fibrotic tissue (bf) of smaller thickness, ranging from portal vein to centrolobular vein.
  • Figures 7a, 7b, 7c and 7d show representative images of liver tissue sections from Phase V animals of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (7a, 7b) and RS (7c, 7d).
  • H&E (7a, 7b) and RS (7c, 7d) stained with H&E (7a, 7b) and RS (7c, 7d).
  • the hepatic architecture presents a cirrhotic state with no presence of steatosis, regeneration nodules surrounded by thin bands of fibrotic tissue (bf) that go from portal vein to centrolobular vein.
  • bf fibrotic tissue
  • Figure 8 is a graphical representation of the determination of the average collagen concentration for each Phase.
  • Figures 9a, 9b and 9c show the analysis of the intensity of expression in the microarray of the genes selected as the candidates to be part of the fibrosis diagnostic molecular panel.
  • Figure 10 is a representation of the validation procedure followed to obtain the proteins that make up the Molecular Panel for the Diagnosis of Fibrosis, with an emphasis on early fibrosis.
  • Figures 12a and 12b show the graphs resulting from the statistical analysis (t-test, for (12a). ELISA and (12b). Roe curve for MFAP-4.
  • Figures 13a and 13b show the graphs resulting from the statistical analysis (t-test, for (13a). ELISA and (13b). Roe curve for FBN1.
  • Figures 14a and 14b show the graphs resulting from the statistical analysis (t-test, for (14a). ELISA and (14b). Roe curve for ALDH1A3.
  • Figures 15a and 15b show the graphs resulting from the statistical analysis (t-test, for (15a). ELISA and (15b). Roe curve for CYP26A1.
  • Figures 16a and 16b show the graphs resulting from the statistical analysis (t-test, for (16a). ELISA and (16b). Roe curve for MGP.
  • Figures 17a and 17b show the graphs resulting from the statistical analysis (t-test, for (17a). ELISA and (17b). Roe curve for C7.
  • Figure 18a and 18b show the graphs resulting from the statistical analysis (t-test, for (18a). ELISA and (18b). Roe curve for PLA2G7.
  • Figure 19 is a graphic representation of the combination of the results obtained with the 7 panel molecules. Where each line represents the average of the values obtained for each protein, using the ELISA test using the serum of the patients of each of the analyzed groups, Ctl (healthy subjects), F1 (Initial fibrosis), F4 (Cirrhosis), FP (Pulmonary Fibrosis) and AHA (Amibian liver abscess).
  • the present invention relates to the construction of a panel of non-invasive molecular biomarkers for diagnosis and staging of the fibrosis process that occurs in chronic liver disease, obtained and selected from the altered expression of various proteins, observed in the course of the induction of fibrosis-cirrhosis in experimental models (ICC 4 and TAA) developed to reproduce the disease, which can be used to determine the diagnosis and / or prognosis at different stages of the liver fibrosis process.
  • the genes that suffer alterations throughout the inductive process of liver fibrosis were identified and validated and correlated with the metabolic pathways in which they intervene, obtaining a molecular profile to be used in the development of a prognostic test and / or diagnosis for liver fibrosis.
  • the experimental model briefly consisted of: a) Liver fibrosis was induced in male rats through intraperitoneal injection of CCI 4 .
  • liver tissue samples were obtained for microarray processing, for histopathological analysis and quantitative determination of collagen deposition.
  • RNA was obtained from the samples and from it the cDNA was synthesized for hybridization in the microarray.
  • the data obtained from the microarray were analyzed in the SAM and DAVID programs.
  • the molecular profile was formed, based on which the molecular biomarker panel of the present invention was developed, which is used as a non-invasive method for presumptive diagnosis and staging of liver fibrosis.
  • Wistar rats Male, 150-200 g in weight, fed ad libitum with water and purine were used and the animals were managed following the guidelines set by the Council of International Organizations of Medical Sciences and the Official Mexican Standard (NOM-062- ZOO-1999). Cirrhosis was induced according to what was previously established (Pérez-Tamayo R., Montfort I., González E., 1987. Collagenolytic activity in experimental cirrhosis of the liver. Exp Mol Pathol.
  • Phase IV 30 days after hepatotoxic suspension (6 months).
  • Phase V 180 days after hepatotoxic suspension (11 months).
  • RNA With the total RNA extracted by each stage, at least 3 i individual microarrays were made, with which the genetic pattern shown by the analyzed RNA was obtained, during the period of induction (Fl), progression and establishment of cirrhosis (Phases II and III) and fibrosis recovery (Phases IV and V), as a result of the time the sample was taken, and compared with the microarrays obtained from the control animals (2 per phase).
  • RNA was obtained from 100 mg of tissue, by extraction with TRIZOL, according to the manufacturer's instructions (Invitrogen).
  • qPCR Real Time PCR
  • the qPCR is one of the methods used to validate the selected genes as candidates.
  • the cDNA was obtained from the reverse transcription of the total RNA obtained from the liver samples of each of the animals (experimental and controls) that formed the model, using the Finy Enzyme "Inphusion" retrotranscription Kit (Affimetrix).
  • Figure 1 shows the flow chart followed during the development of expression microarrays from the total RNA obtained from the liver of each and every animal slaughtered according to the scheme mentioned above.
  • the total RNA (gray) is represented, in the second the first polyadenylated cDNA chain (AAAAA3 ') in black, synthesized by reverse transcription;
  • the double strand of synthesized polyadenylated cDNA black
  • the following line shows the process of obtaining the biotinylated (black) RNA (gray) from the double stranded cDNA by in vitro transcription with the use of biotinylated nbonucleotides and T7 RNA polymerase, the result of transcription is indicated in the following line (single chain RNA with uracil tails at its 5 'end, labeled with biotin), these single RNA chains are fragmented with RNAse H which they are applied to the chip where the microarray of the rat liver genes (> 27,000
  • FIG. 2a, 2b, 2c, 2d, 3a, 3b, 3c, 3d, 4a, 4b, 4c, 4d, 5a, 5b, 5c, 5d, 6a, 6b, 6c, 6d, 7a, 7b, 7c and 7d show by microphotographs, the results of the histopathological analysis performed on the liver sections of each group, which were fixed with buffered formalin (pH7.0) and stained with H&E) for a direct observation of the different structures present in the liver tissue in each Phase of the model and with Sirius Red (RS), special staining that allows distinguishing, by microscopic observation with polarized light, the two predominant types of collagen present in the fibrosis bands, since each of them when observed by This technique emits a different color on a dark background, the
  • Figures 2a, 2b, 2c and 2d show a normal control with representative images of liver tissue sections from normal animals, fixed with buffered formalin and included in paraffin, stained with H&E (2a, 2b) and RS (2c, 2d) .
  • Hepatocytes are polygonal, with granular cytoplasm and central nucleus, round and with prominent nucleoli.
  • Sinusoids have been upholstered by elongated cells (endothelial and Kupffer cells), close to hepatocytes, leaving the Disse space as an almost virtual region. The magnitude of the amplification is Refer in each picture.
  • Figures 3a, 3b, 3c and 3d show representative images of liver tissue sections from animals in Phase I of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (3a, 3b) and Sirius Red (3c , 3d).
  • H&E H&E
  • Sirius Red 3c , 3d
  • the onset of structural alteration is observed due to the presence of periportal fibrosis bands (bf) and multiple small steatosis vesicles (e) with nuclei that retain their central position (n).
  • the magnitude of the amplification is detailed in each image.
  • Figures 4a, 4b, 4c and 4d correspond to Phase II, where representative images of liver tissue cuts from animals in Phase II of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (4a,) are seen. 4b) and RS (4c, 4d).
  • areas with thick and thin bands of periportal fibrosis (bf), steatosis lagoons (e) with rounded vesicles, large and small are observed in centrolobular hepatocytes.
  • n The magnitude of the amplification is detailed in each image.
  • Figures 5a, 5b, 5c and 5d correspond to Phase III, showing representative images of liver tissue cuts from animals in Phase III of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (5a, 5b) and RS (5c, 5d).
  • H&E H&E
  • RS RS
  • FIGS. 6a, 6b, 6c and 6d show representative images of liver tissue sections from animals in Phase IV of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (6a, 6b) and RS (6c, 6d ).
  • the liver architecture represents a cirrhotic stage with areas of focal necrosis, minimal steatosis (e), regeneration nodules surrounded by bands of fibrous tissue (bf) of smaller thickness, ranging from portal vein to centrolobular vein.
  • Figures 7a, 7b, 7c and 7d show representative images of liver tissue sections from Phase V animals of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (7a, b7) and RS (7c, 7d).
  • H&E (7a, b7) and RS (7c, 7d) stained with H&E (7a, b7) and RS (7c, 7d).
  • the hepatic architecture presents a cirrhotic state with no presence of steatosis, regeneration nodules surrounded by thin bands of fibrotic tissue (bf) that go from portal vein to centrolobular vein.
  • bf fibrotic tissue
  • the magnitude of the amplification is detailed in each image.
  • the concentration remains constant in the control animals without treatment, but not in those who receive the hepatotoxic, the deposited ECM increases gradually as the administration of the drug remained constant, as a clear result in response to chronic damage ( Phases I, II, III), we can also note that once the toxic is suspended (Phases IV and V), the fibrosis slowly decreases showing a long-term recovery effect, since according to our experience, the organ does not achieve Total recovery (Garc ⁇ a de León Mar ⁇ a del Carmen, Irmgard Montfort, Eusebio Tello Montes, Rosario López Vancell, Alfonso Olivos Garc ⁇ a, Augusto González Canto, Mario Nequiz-Avenda ⁇ o, Ruy Pérez-Tamayo.
  • V repressed genes were found.
  • the 239 genes are grouped into 187 clusters, and only 37 of them presented significant values (p ⁇ 0.05) taking into account the value of 'ES' and 'FE.
  • These clusters encompass genes involved from organ development processes to metabolic processes.
  • Lipid metabolism various enzymes, phospholipases and other 9 retinoic acid and aa,
  • Figures 9a, 9b and 9c show the analysis of the intensity of expression in the microarray, which identifies some genes that are over-expressed by preferential way in some of the stages of fibrosis. These genes were selected as the candidate genes to be part of the diagnostic molecular panel.
  • a graph of 6 bars is shown for each of the selected genes, where the first bar corresponds to the value of the average intensity of the gene shown by all samples corresponding to the control animals.
  • the second bar shows the same value but in this case of the animals corresponding to Phase I; the third the average intensity of the animals of Phase II, the fourth bar the same but of animals of Phase III, the fifth bar corresponds to Phase IV and the sixth and last bar to Phase V.
  • Each of these bars are repeated for each gene in the three figures 9a, 9b and 9c, because for each phase the corresponding gene analysis was performed and the results were grouped according to the intensity range.
  • Tables 5a, b and c show the values found for each of the candidate genes. In addition to the requirements for their choice, the final molecules selected as markers met the following characteristics:
  • the ELISA test was performed, using specific monoclonal antibodies for each of the selected proteins and revealing the reaction with an antibody.
  • HRP horseradish peroxidase
  • DAB diamino benzidine
  • the levels of each protein in two serum groups were determined: a) Patients infected with the hepatitis C virus who present with liver fibrosis, in two stages of the disease (with Knodel Fl and F-4 index) and; b) Patients with idiopathic pulmonary fibrosis; The latter with the purpose of establishing the specificity of the organ that we are looking for in our panel, the test was also applied to sera from patients with acute liver disease (amoebic liver abscess) to rule out that they could also be detected in a liver condition other than fibrosis (chronic disease) and with this, select those molecules that will show us the qualities of specificity, sensitivity, positive and negative predictive value suitable, to be part of the panel sought.
  • the Knodell index was developed to generate a numerical liver damage score with values ranging from F0 to F4, with liver biopsy samples obtained from patients with asymptomatic active chronic hepatitis.
  • Biopsies were classified into four categories: periportal necrosis, intralobular necrosis, portal inflammation with fibrosis and cirrhosis (Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplówitz N, Kieman TW, Wollman J. 1981.
  • Tables 6a and 6b show the characteristics of the patients separated according to the Knodell index, with liver disease whose sera were used for the determinations and subsequent validation of the 20 proteins selected as candidates to integrate the final panel for diagnosis and Staging of fibrotic liver disease, object of the present invention.
  • Table 6a Patients separated by Knodell index, Phase 0-1.
  • Figure 10 shows step by step the diagram followed by the tests performed for the validation by ELISA and Immunohistochemistry to the 20 candidate genes, using sera from patients with different diseases from which the genes that gave us the best results were identified and thus build the Biomarker panel for diagnosis and staging of liver fibrosis object of the present invention.
  • Sensitivity Positive Value / Positive Value + Negative False
  • Specificity Negative Value / Negative Value / Positive False
  • the value of the area under the resulting ROC curve indicates whether the comparisons we make are excellent (0.97, 1), good (0.75, 0.9), regular (0.6, 0.75) or bad (0.5, 6), in addition to this curve we It provides a cut-off point at which, to be taken into account, the sensitivity and specificity must be close to 100%.
  • Figures 12a, 12b, 13a, 13b 14a, 14b 15a, 15b, 16a, 16b, 17a, 17b, 18a and 18b show the graphs obtained with the 7 genes selected to integrate the biomarked panel for diagnosis and staging of fibrosis of the liver, object of the present invention, where each of the values obtained indicates the importance of the molecule and its feasibility to be part of a diagnostic group.
  • Figure 12b shows the representation of the 3 ROC curves obtained with the different comparisons made between the results obtained with the different serum groups of patients in the ELISA test with each protein and whose results are shown at the bottom of the graphs, the first column corresponds to Ctls vs F1 and was used as an example to describe each of the values referred to in subsequent columns.
  • MFAP-4 is an extracellular matrix protein that participates in the innate immune response and allows free gaseous exchange in the lungs through its binding to the collagen region of surfactant proteins (SP-A and SP-D).
  • the first column at the bottom of the graphs of the ROC curves corresponds to each of the comparisons made between the groups for this protein, in the second row of this column the results of the comparison of the group of sera of normal individuals Ctls vs sera of fibrosis patients with Knodell F1 index are observed, the second column corresponds to the AUC, area under the curve, with a value of 0.99, which means that this Comparison is perfect or excellent to discriminate healthy individuals from those with incipient fibrosis, the third column corresponds to the cut-off point with a value of 0.097, this means that from this value everything that is above will be fibrotic and everything that being below is healthy; the fourth and fifth columns refer to the sensitivity and specificity at this cut-off point, corresponding to these characteristics the values of 100 and 94% respectively, which means that this test at the cut-off point of 0.097, 100% of individuals are positive cases, this is really sick cases and 94% are negative cases, this is really healthy cases.
  • the last two columns refer to the positive and negative predictive values which correspond to the values of 75 and 100%, data that tell us that an individual has a 75% chance of having the disease if the result of their diagnostic test with this protein it is positive; and 100% probability of not having the disease if the result of the diagnostic test is negative.
  • Figure 13a and 13b illustrate the behavior of the FBN1 molecule in the tests, it is a structural and support protein, which regulates the maturation of osteoblasts by controlling the bioavailability and levels of BMP bone morphogenetic proteins, or Bone Morphogenetic Proteins (BMPs) growth factors that belong to the family of transforming growth factors (TGF- ⁇ ), a super family of proteins with the ability to strongly induce the formation of new bone, cartilage and connective tissue and TGF-beta.
  • BMPs Bone Morphogenetic Proteins
  • ALDH1A3 is an enzyme that helps the detoxification of aldehydes in the cell, this protein has significant differences between the groups compared when we look at the graph corresponding to the results obtained with the ELISA Figure 14a, it has a gradual increase between the group of incipient fibrosis and cirrhosis (p ⁇ 0.001) and also presents significant differences with respect to the FP and AA groups (p ⁇ 0.001); In the data obtained from the ROC curve, Figure 14b identifies that the S, E and NPV are high> 96%, while the PPV result in 83%. These data allow us to take this molecule as part of our panel, because in an individual analysis it can provide us with data that reinforce the diagnosis, in addition to being a highly specific protein for liver disease.
  • CYP cytochromes have oxygenase activity and commonly catalyze redox reactions. This group of enzymes are found in elevated levels in the liver, where they play an important role in the metabolism of endogenous toxic drugs and compounds.
  • Figure 15a we can observe in Figure 15a that comparisons between groups despite having some individuals with expressions above the mean (F1) and a high standard deviation, have a strong difference between them, with an increase in their levels in cirrhotic patients (p ⁇ 0.001). The data obtained also indicate a difference with respect to patients with IPF and AA (p ⁇ 0.001).
  • the highest values found for this protein in the ROC curve Figure 15b are for the comparison between Ctls vs F4 being 100% for sensitivity, specificity, PPV and NPV, in patients with this stage of the disease.
  • MGP is a protein associated with the organic matrix of bone and cartilage. It is thought to act as an inhibitor of bone formation.
  • the ELISA of this protein Figure 16a shows significant differences between the groups compared, there are individuals above the average (in F1) and below it (in F4) but the differences are clear (p ⁇ 0.001) and this can be reinforced with the values obtained from the ROC curve Figure 16b, being 100% perfect in sensitivity, specificity, VPP and VPPN. Finally, the protein also shows clear differences in the groups of patients with liver disease with respect to the IPF and AA groups (p ⁇ 0.001).
  • the C7 protein is part of the membrane attack complex (MAC) of the complement system, with a key role in innate and adaptive immunity.
  • MAC membrane attack complex
  • the result of the ELISA Figure 17a for this protein only gave significant value in patients with initial liver fibrosis F1 (p ⁇ 0.001) so it can be used to detect liver fibrosis at this stage and this is corroborated by the data obtained from the curve ROC Figure 17b, where for the comparison Ctls vs Fl, the sensitivity, VPP and VPN specificity values are 100%.
  • Phospholipases including PLA2G7, are a group of enzymes that hydrolyse phospholipids producing fatty acids and other lipophilic molecules. that have diverse biological functions, among which we can mention inflammation, cell growth and signaling among others.
  • this protein shows a lower expression in the serum of patients with Fl, compared to the levels of expression detected in the serum of healthy individuals data that is significant (p ⁇ 0.001) and in patients with F4 is undetectable at least by this technique.
  • the proteins selected to integrate the Biomarker panel for Diagnosis and Staging of liver fibrosis are: MFAP-4, FBN1, ALDH1A3, CYP26A1; MGP, C7 and PLA2G7. Some of them did not show detectable titres in patients with cirrhosis such as C7 and PLA2G7, but these two molecules showed very good ELISA values when performing the test with sera corresponding to the FO-I stage, which is very useful for staging the process.
  • Table 7 shows the average absorbance values obtained by the ELISA test for each of the panel proteins with the sera of the patients of each group analyzed, the crosses represent non-significant values for the individual protein in these groups of patients
  • Figure 19 shows the graph resulting from the integration of the average values obtained by ELISA, shown in Table 7.
  • Table 8 shows the Multicomponent Serological Index, composed of the 7 proteins, in this table we can see the cut-off values defined for each of the proteins, determined by analyzing the results obtained by ELISA with the sera of patients with liver disease in stage F1 or F4, the arrows represent the increase (up) or decrease (down) in the expression of the protein with respect to the controls.
  • PLA2G7 0.175 l X According to the combination of the results of the test for each patient (within the ranges shown in table 8) in the determination of the entire panel, performed in the serum, it will be possible to define at what stage of the fibrosis process It finds and therefore its degree of improvement or deterioration.
  • This invention provides important information that will allow a better understanding of the molecular mechanisms and interrelations that occur during the liver fibrosis process, in addition to providing both the doctor and the patient with a non-invasive tool for the diagnosis, staging and monitoring of liver fibrosis.

Abstract

The invention relates to a novel test for the in vitro diagnosis of fibrosis during liver disease, using the determination of non-invasive biomarkers which allow the opportune and early detection of the process which determines the degree of damage of the organ. The panel of non-invasive biomarkers of the present invention also allows liver fibrosis to be staged, facilitating the most appropriate choice of treatment for the subject.

Description

PRUEBA IN VITRO PARA DIAGNÓSTICO TEMPRANO Y ESTADIFICACIÓN DE FIBROSIS HEPÁTICA  IN VITRO TEST FOR EARLY DIAGNOSIS AND STABILITY OF HEPTIC FIBROSIS
Campo técnico de la invención  Technical Field of the Invention
La presente invención se relaciona con las pruebas in vitro para diagnóstico de enfermedades que cursan con fibrosis, más específicamente se relaciona con las pruebas para detectar el proceso de fibrosis en el hígado y más específicamente con el uso de biomarcadores no invasivos para diagnóstico y estadificación de la fibrosis hepática. The present invention relates to in vitro tests for diagnosis of diseases that occur with fibrosis, more specifically it relates to tests to detect the process of fibrosis in the liver and more specifically to the use of non-invasive biomarkers for diagnosis and staging of liver fibrosis
Antecedentes de la invención Background of the invention
Anatomía del hígado. El hígado desempeña un papel único como centro metabólico del organismo. Su peso promedio en individuos adultos es de aproximadamente de 1 ,400+270 g, sin diferencias significativas relacionadas con el género. Se compone de cinco tipos distintos de células que ocupan cerca del 80% de su volumen. El 20% restante corresponde a los espacios extracelulares y los componentes de la matriz extracelular (Rojkind M, Greenwel P. The extracellular matrix and the liver. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Shashter DA, Shafritz DA, (ed). The Liver: Biology and Pathology. Third Ed. Raven Press ,New York 1994, pp 843-868). Anatomy of the liver The liver plays a unique role as the metabolic center of the organism. Its average weight in adult individuals is approximately 1,400 + 270 g, without significant gender-related differences. It is made up of five different types of cells that occupy about 80% of their volume. The remaining 20% corresponds to the extracellular spaces and components of the extracellular matrix (Rojkind M, Greenwel P. The extracellular matrix and the liver. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Shashter DA, Shafritz DA, (ed) The Liver: Biology and Pathology, Third Ed. Raven Press, New York 1994, pp 843-868).
El tejido hepático se organiza a nivel microscópico en lóbulos formados por placas constituidas por células que se extienden desde la zona porta en forma lineal a la vena central; los espacios que atraviesan los tabiques son los sinusoides hepáticos, separados de los hepatocitos por el espacio perisinusoidal de Disse. Los sinusoides hepáticos, tienen un diámetro promedio de 5 a 7 μηι, y conducen la sangre mezclada, de las ramificaciones terminales de la arteria hepática y de la vena porta a la ramificación terminal de la vena hepática. Los sinusoides representan la única forma de tubo capilar en el hígado y tienen un revestimiento endotelial continuo, pero fenestrado, células de Kupffer, células dendríticas dentro de la luz, un espacio perisinusoidal (espacio de Disse) y ausencia de una membrana basal continua. The liver tissue is organized microscopically in lobes formed by plaques consisting of cells that extend from the portal area in a linear fashion to the central vein; the spaces that cross the septa are the hepatic sinusoids, separated from the hepatocytes by the perisinusoidal space of Disse. The hepatic sinusoids, have an average diameter of 5 to 7 μηι, and lead the mixed blood, from the terminal branches of the hepatic artery and the portal vein to the terminal branching of the hepatic vein. Sinusoids represent the only form of capillary tube in the liver and have a continuous but fenestrated endothelial lining, Kupffer cells, dendritic cells within of light, a perisinusoidal space (Disse space) and absence of a continuous basement membrane.
Los sinusoides y el espacio de Disse se comunican a través de las ventanas o fenestras de las células endoteliales sinusoidales (SEC). La ausencia de membrana basai facilita el rápido intercambio de componentes de la sangre con los hepatocitos. Los sinusoides hepáticos conectan los espacios porta con las ramas terminales de la vena hepática (venas centrales). Las células endoteliales de la arteria hepática son alargadas y arregladas longitudinalmente, mientras que las de las venas porta y central son poligonales, aplanadas y poseen microvellosidades (Kmiec Z., 2001. Cooperation of liver cells in health and disease. Adv Anat Embryol CelI Biol. 161 : lll-XIII: 1 -151). The sinusoids and the Disse space communicate through the windows or fenestras of the sinusoidal endothelial cells (SEC). The absence of a basement membrane facilitates the rapid exchange of blood components with hepatocytes. Hepatic sinusoids connect the portal spaces with the terminal branches of the hepatic vein (central veins). The endothelial cells of the hepatic artery are elongated and arranged longitudinally, while those of the portal and central veins are polygonal, flattened and have microvilli (Kmiec Z., 2001. Cooperation of liver cells in health and disease. Adv Anat Embryol CelI Biol 161: lll-XIII: 1-151).
El endotelio sinusoidal cuya estructura es señalada como el filtro del hígado, es probablemente él blanco principal del estrés oxidativo, este permite el libre intercambio de proteínas y otros nutrientes entre los hepatocitos y la sangre, además de aislarlos de la mayoría de las células sanguíneas, de las plaquetas y de partículas coloidales más grandes, como los quilomicrones y los virus (Cogger, VC, Muller, M, Fraser, R, Mclean, AJ, Khan, J, Le Couteur, DG. 2004. The effects of oxidative stress on the liver sieve. J Hepatol. 41 : 370-376) The sinusoidal endothelium whose structure is designated as the liver filter, is probably the main target of oxidative stress, this allows the free exchange of proteins and other nutrients between hepatocytes and blood, in addition to isolating them from most blood cells, of platelets and larger colloidal particles, such as chylomicrons and viruses (Cogger, VC, Muller, M, Fraser, R, Mclean, AJ, Khan, J, Le Couteur, DG. 2004. The effects of oxidative stress on the liver sieve. J Hepatol. 41: 370-376)
Fisiología de la fibrosis: La fibrosis es una respuesta común del hígado, a lesiones crónicas producidas por una variedad de agresiones, como enfermedades metabólicas, infecciones virales, abuso en la ingesta de alcohol, drogas, ataque autoinmune a los hepatocitos, conductos biliares y anormalidades congénitas. Physiology of fibrosis: Fibrosis is a common response of the liver to chronic injuries caused by a variety of attacks, such as metabolic diseases, viral infections, alcohol intake, drugs, autoimmune attack on hepatocytes, bile ducts and abnormalities congenital
En el espacio de Disse del hígado normal, se puede observar en contacto directo con la lámina basal, a un conjunto organizado de proteínas denominado matriz extracelular (MEC) que constituye alrededor de 0.5 % del peso total del hígado. Sostén para las células parenquimatosas que además de reforzar la arquitectura del órgano, hace posible el intercambio de moléculas entre los hepatocitos, en un flujo semicontinuo debido a su composición no fibrilar, lo que resulta fundamental para el mantenimiento de las funciones diferenciadas de todas las células residentes en el hígado (Geerts A. 2001. History heterogeneity developmental biology and functions of quiescent hepatic stellate cells. Semin Liv Dis. 21 :311-335; Friedman S. 2003. Liver fibrosis-from bench to beside. J of Hepatol. 38:s38-s53) In the Disse space of the normal liver, an organized set of proteins called extracellular matrix (ECM) that constitutes about 0.5% of the total liver weight can be observed in direct contact with the basal lamina. Support for parenchymal cells that, in addition to strengthening the architecture of the organ, makes it possible to exchange molecules between hepatocytes, in a semi-continuous flow due to their non-fibrillar composition, which is essential for maintaining the differentiated functions of all liver-resident cells (Geerts A. 2001. History heterogeneity developmental biology and functions of quiescent hepatic stellate cells. Semin Liv Dis. 21: 311-335; Friedman S. 2003. Liver fibrosis-from bench to beside. J of Hepatol .38: s38-s53)
En el hígado fibrótico, los componentes de la MEC son similares a los presentes en hígado normal (colágena y otros) pero incrementados cuantitativamente, con la generación de fibrosis, la estructura normal de la matriz presente en el espacio subendotelial se transforma en una matriz de tipo intersticial con alto contenido de colágena fibrilar, producto de la activación paracrina de las células estelares (HSC; Friedman SL. 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 275(4): 2247-2250), inducida a su vez, por la activación de las células de Kupffer, con la consecuente sobreexpresión y redistribución de cantidades relativas de estas proteínas, inicialmente en el espacio porta y/o vena central, lo que conduce al desarrollo de conexiones fibrosas de una estructura vascular a otra, acompañadas de la pérdida tanto de la naturaleza fenestrada del endotelio sinusoidal (capiiarización) como de las microvellosidades en los hepatocitos, lo que incide, no solo en la expansión de la MEC, sino en la interrupción de la vascularización normal del lóbulo hepático, contribuyendo al deterioro de la función del órgano (Bedossa P., Paradis V., 2003. Liver extracellular matrix in health and disease. J Pathol. 200: 504-515). In the fibrotic liver, the components of the ECM are similar to those present in normal liver (collagen and others) but quantitatively increased, with the generation of fibrosis, the normal structure of the matrix present in the subendothelial space is transformed into a matrix of interstitial type with high content of fibrillar collagen, product of paracrine activation of stellar cells (HSC; Friedman SL. 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 275 (4): 2247 -2250), in turn induced by the activation of Kupffer cells, with the consequent overexpression and redistribution of relative amounts of these proteins, initially in the portal space and / or central vein, which leads to the development of fibrous connections from one vascular structure to another, accompanied by the loss of both the fenestrated nature of the sinusoidal endothelium (capiiarization) and the microvillus s in hepatocytes, which affects, not only in the expansion of ECM, but in the disruption of normal vascularization of the hepatic lobe, contributing to the deterioration of organ function (Bedossa P., Paradis V., 2003. Liver extracellular matrix in health and disease. J Pathol. 200: 504-515).
El hígado normal, permite el libre intercambio de moléculas y nutrientes, entre las células del hígado y el flujo sanguíneo, debido a la constitución porosa de las células endoteliales sinusoidales (SEC) y la escasez de MEC en el espacio perisinusoidal; por lo tanto, la capiiarización del sinusoide deteriora poderosamente este intercambio. Además, debido a las conexiones directas entre las células y su entorno, cualquier modificación cuantitativa o cualitativa de la MEC influenciará el microambiente y las interacciones celulares, produciendo cambios en el fenotipo de las células estelares hepáticas (HSC) y el deterioro de la función del hepatocito. Estos cambios ilustran el papel principal de la MEC en el hígado, no solo como armazón para su arquitectura, sino también como una red continua entre las células que permite, vía sus propios receptores, el intercambio continuo de señales entre ellas (Bedossa P., Paradis V., 2003. Liver extracellular matrix in health and disease. J Pathol. 200: 504-515). The normal liver, allows the free exchange of molecules and nutrients, between the liver cells and blood flow, due to the porous constitution of sinusoidal endothelial cells (SEC) and the shortage of MEC in the perisinusoidal space; therefore, the capiiarization of the sinusoid powerfully impairs this exchange. In addition, due to the direct connections between the cells and their environment, any quantitative or qualitative modification of the MEC will influence the microenvironment and cellular interactions, producing changes in the hepatic stellar cell phenotype (HSC) and deterioration of the function of the hepatocyte These changes illustrate the main role of ECM in the liver, not only as a framework for its architecture, but also as a continuous network between cells that allows, via their own receptors, the continuous exchange of signals among them (Bedossa P., Paradis V., 2003. Liver extracellular matrix in health and disease. J Pathol. 200: 504-515).
Por sí misma la fibrosis es un evento biológico importante, producto del desequilibrio entre la síntesis y degradación de las moléculas de la MEC, que al asociarse con otros procesos del órgano como la deformación arquitectónica y redistribución vascular, produce graves consecuencias para la función hepática. A largo plazo, la fibrosis promueve el desarrollo de la cirrosis que, en ausencia de tratamiento oportuno y adecuado, suele llevar a un desenlace fatal. La cirrosis, conjuntamente con otras enfermedades crónicas hepáticas, representa a nivel nacional, la segunda causa de muerte de individuos en edad productiva, lo que tiene un impacto importante tanto en la salud pública como en la economía, implicando costos significativos en hospitalización, tratamiento y ausencia laboral (SINAIS. Informe de funciones generales de la Secretaría de Salud, 2008. Liga: Fibrosis itself is an important biological event, product of the imbalance between the synthesis and degradation of the ECM molecules, which, when associated with other organ processes such as architectural deformation and vascular redistribution, produces serious consequences for liver function. In the long term, fibrosis promotes the development of cirrhosis which, in the absence of timely and adequate treatment, usually leads to a fatal outcome. Cirrhosis, together with other chronic liver diseases, represents at national level, the second cause of death of individuals of productive age, which has a significant impact on both public health and the economy, involving significant costs in hospitalization, treatment and absence from work (SINAIS. General functions report of the Ministry of Health, 2008. League:
(http://www.sinais.salud.gob.mx/mortalidad/index.html) (http://www.sinais.salud.gob.mx/mortalidad/index.html)
En el estudio de la fibrosis, se ha puesto atención especial en su asociación frecuente con el incremento de la peroxidación de lípidos y/o la alteración del sistema anti-oxidante. La ruptura oxidativa de los lípidos de la membrana puede deberse a su interacción con gran variedad de radicales libres caracterizada por el incremento de especies reactivas de oxígeno (ROS) y radicales orgánicos intermedios durante el proceso de propagación (reacción en cadena). Este proceso, mediado por radicales libres, está implicado frecuentemente en el cambio del equilibrio oxido-reductor intracelular hacia la oxidación, lo que bioquímicamente se define como estrés oxidativo (Poli G., Parola M. 1997. Oxidative damage and fibrogenesis. Free Radicáis Biol Med. 22 (1/2): 287-305). In the study of fibrosis, special attention has been given to its frequent association with the increase in lipid peroxidation and / or the alteration of the anti-oxidant system. The oxidative breakdown of membrane lipids may be due to their interaction with a wide variety of free radicals characterized by the increase in reactive oxygen species (ROS) and intermediate organic radicals during the propagation process (chain reaction). This process, mediated by free radicals, is frequently involved in the change of the intracellular oxide-reducing balance towards oxidation, which is biochemically defined as oxidative stress (Poly G., Parola M. 1997. Oxidative damage and fibrogenesis. Free Radicáis Biol Med. 22 (1/2): 287-305).
Los principales blancos biológicos de los radicales libres y las ROS son las proteínas, los lípidos y el DNA. La peroxidación ocurre principalmente en la membrana plasmática alterando sus propiedades físicas y como consecuencia sus funciones biológicas. Los hepatocitos y las células de Kupffer son una fuente de ROS; estos compuestos ejercen una estimulación paracrina sobre las HSCs, cuya actividad in vivo se amplifica por la pérdida de antioxidantes, como ocurre típicamente en la enfermedad hepática. La sobre-expresión en las HSCs de la enzima citocromo p450 2E1 , cuya actividad genera ROS, estimula la expresión del gen de colágena I, la cual es atenuada por antioxidantes. Las SEC dañadas estimulan la producción de una variante celular de fibronectina (isoforma EIIIA) que tiene un efecto activador sobre las HSCs, adicionalmente, las SEC convierten el factor de crecimiento transformante-βΐ latente (TGF-βΙ) a su forma activa a través de la acción de la plasmina, proceso que finalmente desencadena la formación de la cicatriz constituida por fibras complejas de MEC, contribuyendo a la pérdida de las microvellosidades de los hepatocitos y la capilarización del sinusoide con el consiguiente deterioro de la función hepática (Friedman SL. 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 275(4): 2247-2250). The main biological targets of free radicals and ROS are proteins, lipids and DNA. Peroxidation occurs mainly in the plasma membrane, altering its physical properties and as a consequence its biological functions. Hepatocytes and Kupffer cells are a source of ROS; these compounds exert paracrine stimulation on HSCs, whose activity in vivo is amplified by the loss of antioxidants, as occurs typically in liver disease. Over-expression in the HSCs of the cytochrome p450 2E1 enzyme, whose activity generates ROS, stimulates the expression of the collagen I gene, which is attenuated by antioxidants. Damaged SECs stimulate the production of a cellular variant of fibronectin (EIIIA isoform) that has an activating effect on HSCs. Additionally, SEC converts the latent transforming growth factor-β ((TGF-βΙ) to its active form through the action of plasmin, a process that finally triggers the formation of the scar consisting of complex MEC fibers, contributing to the loss of hepatocyte microvilli and sinusoid capillarization with the consequent deterioration of liver function (Friedman SL. 2000 Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 275 (4): 2247-2250).
Las manifestaciones clínicas de la fibrosis pueden variar extensamente, desde la ausencia de sintomatología hasta la instauración de la insuficiencia hepática. En promedio, un 40% de pacientes con cirrosis son asintomáticos y pueden permanecer así por más de una década, sin embargo el deterioro progresivo es inevitable una vez que se presentan complicaciones tales como insuficiencia hepática, ascitis, várices esofágicas, encefalopatía, etc. La mortalidad en estos pacientes alcanza en 5 años hasta un 50% y el 70% de estas muertes aproximadamente, son atribuibles directamente a la enfermedad hepática (Milani S., Herbst H., Schuppan D., Grappone C, Pellegrini G., Pinzani M., Casini A., Calabró A., Ciando G., Stefanini F., Burroughs A.K., Surrenti C. 1994. Differential expression of matrix metalloproteinase-1 and -2 genes in normal and fibrotic human liver. Am J Pathol. 144: 528-537). The clinical manifestations of fibrosis can vary widely, from the absence of symptoms to the establishment of liver failure. On average, 40% of patients with cirrhosis are asymptomatic and may remain so for more than a decade, however progressive deterioration is inevitable once complications such as liver failure, ascites, esophageal varices, encephalopathy, etc. occur. Mortality in these patients reaches up to 50% in 5 years and approximately 70% of these deaths are directly attributable to liver disease (Milani S., Herbst H., Schuppan D., Grappone C, Pellegrini G., Pinzani M., Casini A., Calabró A., Ciando G., Stefanini F., Burroughs AK, Surrenti C. 1994. Differential expression of matrix metalloproteinase-1 and -2 genes in normal and fibrotic human liver. Am J Pathol. 144 : 528-537).
La biopsia hepática ha sido considerada durante los últimos 50 años como el método estándar de oro para clasificar la fibrosis; ya que ha permitido a los médicos obtener información diagnóstica, no solo acerca de la fibrosis sino de otros procesos de daño, tales como la necrosis, inflamación, esteatosis, depósitos de hierro o cobre. El método Knodell comenzó a utilizarse en 1981 , a partir de su publicación donde se informaba de un procedimiento para la evaluación cuantitativa de las alteraciones histológicas producidas por la hepatitis crónica, con el cual se cuantifican, la inflamación portal, la necrosis, la actividad periportal, la necrosis lobulillar y la fibrosis. Como los informes convencionales de biopsias no proporcionaban criterios claros de valoración convincentes y concluyentes para su análisis estadístico, Knodell y sus colegas establecieron la construcción un "índice de actividad histológica" (HAI). Se compone de cuatro números asignados de forma individual que conforman una sola puntuación. El primer componente (necrosis periportal y/o en puente) evaluado en una escala de 0-10. Los dos componentes siguientes (degeneración intralobular e inflamación portal) se puntúan 0-4. La combinación de estos tres marcadores indica la cantidad de inflamación en el hígado. El cuarto componente muestra la cantidad de cicatrices en el hígado y se puntúa como: F0 (sin cicatrices), F1 (fibrosis portal sin septos), F3 (numerosos septos sin cirrosis) y F4 (Cirrosis o cicatrices avanzadas en el hígado) (Sebastiani G, Alberti A. Non invasive fibrosis biomarkers reduce but not substitute the need for liver biopsy. World J Gastroenterol2006; 21 ; 12(23):3682-94). Liver biopsy has been considered for the past 50 years as the gold standard method for classifying fibrosis; since it has allowed doctors to obtain diagnostic information, not only about fibrosis but other damage processes, such as necrosis, inflammation, steatosis, iron or copper deposits. The Knodell method began to be used in 1981, from its publication where a procedure for the quantitative evaluation of the histological alterations produced by chronic hepatitis was reported, with which quantify, portal inflammation, necrosis, periportal activity, lobular necrosis and fibrosis. Since conventional biopsy reports did not provide clear and convincing clear criteria for statistical analysis, Knodell and his colleagues established the construction of a "histological activity index" (HAI). It consists of four individually assigned numbers that make up a single score. The first component (periportal and / or bridge necrosis) evaluated on a scale of 0-10. The following two components (intralobular degeneration and portal inflammation) are scored 0-4. The combination of these three markers indicates the amount of inflammation in the liver. The fourth component shows the amount of scarring in the liver and is scored as: F0 (no scars), F1 (portal fibrosis without septa), F3 (numerous septa without cirrhosis) and F4 (Cirrhosis or advanced scars in the liver) (Sebastiani G, Alberti A. Non invasive fibrosis biomarkers reduce but not substitute the need for liver biopsy. World J Gastroenterol2006; 21; 12 (23): 3682-94).
En la actualidad los sistemas de puntuación más utilizados para evaluar la biopsia hepática son, Knodell, Ishak y Metavir. El sistema de puntuación Metavir ha sido especialmente diseñado para evaluar el estado del hígado en personas infectadas con el Virus de la Hepatitis C, VHC. El índice incluye el empleo de la suma de la puntuación asignada al grado de actividad inflamatoria observada en la muestra (0-4; siendo, 0 sin actividad y 3 o 4 la actividad considerada grave) además de la proporcionada por la estadificación, que representa la cantidad de fibrosis: 0 (sin cicatrices), 1 (cicatrices mínimas), 2 (la cicatrización ha ocurrido y se extiende fuera de las áreas que contienen vasos sanguíneos), 3 (puentes de fibrosis extendiéndose y conectándose con otras áreas que contienen fibrosis) y 4 (cirrosis) (Bedossa P, Poynard T. METAVIR Cooperative Study Group. An algorithm for the grading of activity in chronic hepatitis C. Hepatol; 24: 289-293). Currently, the scoring systems most used to evaluate liver biopsy are Knodell, Ishak and Metavir. The Metavir scoring system has been specially designed to assess liver status in people infected with Hepatitis C Virus, HCV. The index includes the use of the sum of the score assigned to the degree of inflammatory activity observed in the sample (0-4; being, 0 without activity and 3 or 4 the activity considered serious) in addition to that provided by staging, which represents the amount of fibrosis: 0 (no scars), 1 (minimal scars), 2 (scarring has occurred and extends outside the areas that contain blood vessels), 3 (fibrosis bridges extending and connecting with other areas that contain fibrosis ) and 4 (cirrhosis) (Bedossa P, Poynard T. METAVIR Cooperative Study Group. An algorithm for the grading of activity in chronic hepatitis C. Hepatol; 24: 289-293).
La asociación americana para el estudio de las enfermedades hepáticas (AASLD) recomienda "para que una biopsia pueda ser considerada como apropiada, ésta debe ser tomada con aguja calibre 16, medir de 2-3 cm de longitud y contener por lo menos 11 tractos portales completos que permitan una adecuada clasificación histológica del parénquima"; sin embargo, pocas muestras percutáneas en la práctica clínica cumplen estos criterios, como lo demuestra el trabajo de Regev y colaboradores, publicado en el 2002, en el que señalaron que la biopsia tenía una alta tasa de error de muestreo interindividual, en base al análisis realizado en muestras de pacientes con hepatitis C crónica, tomadas de ambos lóbulos hepáticos (derecho e izquierdo), el cual mostraba diferencias en gradación histológica y estadificación (33.1%) en una gran proporción de ellos, sin embargo, las discrepancias en más de una etapa o grado fueron poco frecuentes (Regev A, Berho M, Jeffers LJ, y cois. Sampling error and intraobserver variation in liver biopsy in patients with chronic HCV infection. Am J Gastroenterology 2002; 97:2614-2618). Esta variabilidad se incrementa en muestras de biopsias pequeñas (1.5 cm) hasta en un 60.2% y para las de 1 cm en un 86.6% (p<0.001) (Colloredo G, Guido M, Sonzogni A, y cois. Impact of liver biopsy size on histological evaluation of chronic viral hepatitis: the smaller the sample, the milder the disease. J. Hepatol. 2003; 39:239-44) y la diferencia media en las biopsias fue de 2.4 + 2.1 para la actividad necroinflamatoria y 0.6 ± 0.9 para la fibrosis con una r=0,53, p <0,01) y (r=0,62, p 0,0001) respectivamente (Siddique I, El-Naga HA, Madda JP y cois. 2003. Sampling variability on percutaneous liver biopsy in patients with chronic hepatitis C virus infection. Scand J Gastroenterol. 38(4):427-32). Otros estudios han demostrado discordancia del 30% en la estadificación histopatológica durante el análisis de biopsias de hígado de paciente con enfermedad de hígado graso no alcohólico (EHGNA) tomadas de los lóbulos hepáticos derecho e izquierdo, la obtención de una muestra de tamaño adecuado (> 2 cm de longitud, con > 10 tractos portales), reduce en gran medida el sesgo (considerando que se obtiene alrededor de 1/50,000 de la masa hepática) (Bedossa P, Dargere D, and Paradis V. 2003. Sampling Variability of Liver Fibrosis in Chronic Hepatitis C. Hepatol. 38: 1449-1457). Inconvenientes como su carácter invasivo, la poca calidad de la muestra y tamaño del tejido (coeficiente de variación de 45-35%), que la hacen irreproducible en función de la longitud. The American Association for the Study of Liver Diseases (AASLD) recommends "for a biopsy to be considered appropriate, it must be taken with a 16 gauge needle, measure 2-3 cm in length and contain at least 11 portal tracts complete that allow an adequate histological classification of the parenchyma "; however, few samples Percutaneous clinical practice meets these criteria, as evidenced by the work of Regev and collaborators, published in 2002, in which they indicated that the biopsy had a high interindividual sampling error rate, based on the analysis performed on patient samples with chronic hepatitis C, taken from both liver lobes (right and left), which showed differences in histological grading and staging (33.1%) in a large proportion of them, however, discrepancies in more than one stage or grade were little frequent (Regev A, Berho M, Jeffers LJ, and cois. Sampling error and intraobserver variation in liver biopsy in patients with chronic HCV infection. Am J Gastroenterology 2002; 97: 2614-2618). This variability is increased in small biopsy samples (1.5 cm) up to 60.2% and for 1 cm samples in 86.6% (p <0.001) (Colloredo G, Guido M, Sonzogni A, and cois. Impact of liver biopsy size on histological evaluation of chronic viral hepatitis: the smaller the sample, the milder the disease. J. Hepatol. 2003; 39: 239-44) and the average difference in biopsies was 2.4 + 2.1 for necroinflammatory activity and 0.6 ± 0.9 for fibrosis with a r = 0.53, p <0.01) and (r = 0.62, p 0.0001) respectively (Siddique I, El-Naga HA, Madda JP et al. 2003. Sampling variability on percutaneous liver biopsy in patients with chronic hepatitis C virus infection, Scand J Gastroenterol 38 (4): 427-32). Other studies have shown 30% disagreement in histopathological staging during the analysis of liver biopsies of patients with non-alcoholic fatty liver disease (EHGNA) taken from the right and left hepatic lobes, obtaining a sample of adequate size (> 2 cm in length, with> 10 portal tracts), greatly reduces bias (considering that about 1 / 50,000 of the liver mass is obtained) (Bedossa P, Dargere D, and Paradis V. 2003. Sampling Variability of Liver Fibrosis in Chronic Hepatitis C. Hepatol. 38: 1449-1457). Disadvantages such as its invasive character, the poor quality of the sample and tissue size (coefficient of variation of 45-35%), which make it irreproducible depending on the length.
Por otra parte, es una evaluación histológica que depende estrictamente de la experiencia del patólogo (error del observador). Existen riesgos asociados a la obtención de la biopsia hepática, que van desde el dolor (84%) e hipotensión como los más frecuentes, hasta el sangrado peritoneal (0.5%) y el daño al sistema biliar como las complicaciones más graves, aunque con un nivel de mortalidad y morbilidad significativamente bajo (0.09-0.12-%) (Piccinino F, Sagnelli E, Pasquale G y cois. 1986. Complications following percutaneous liver biopsy. A multicentre retrospective study on 68 276 biopsies. J. Hepatol. 2: 165-73; Rockey DC, Caldwell SH, Goodman ZD y cois. 2009. Liver biopsy. Hepatol. 49(3): 1017-44; Terjung B, Lemnitzer i, Dumouiin FL y cois. 2003. Bleeding complications after percutaneous liver biopsy. An analysis of risk factors. Digestión, 67(3): 138-45), sustentando así, las consideraciones éticas que implican su obtención evitando que sean tomadas biopsias múltiples del mismo paciente. Finalmente, los resultados generados tienden a ser representativos cuando se trata de una enfermedad relativamente avanzada (Gressner OA, Weiskirchen R, Gressner AM. 2007. Biomarkers of liver fibrosis: clinical translation of molecular pathogenesis or based on liver dependent malfunction tests. Clin Chim Acta; 381 :107- 13; Iredale J.P. 2007. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest. 117(3):539-48). Las consideraciones a favor y en contra se resumen en la Tabla 1. On the other hand, it is a histological evaluation that depends strictly on the pathologist's experience (observer error). There are risks associated with obtaining liver biopsy, ranging from pain (84%) and hypotension as the most frequent, to peritoneal bleeding (0.5%) and damage to the biliary system as the most serious complications, although with a significantly low level of mortality and morbidity (0.09-0.12-%) (Piccinino F, Sagnelli E, Pasquale G and cois. 1986. Complications following percutaneous liver biopsy. A multicenter retrospective study on 68 276 biopsies J. Hepatol. 2: 165-73; Rockey DC, Caldwell SH, Goodman ZD et al. 2009. Liver biopsy. Hepatol. 49 (3): 1017-44; Terjung B, Lemnitzer i, Dumouiin FL et al. 2003. Bleeding complications after percutaneous liver biopsy.An analysis of risk factors.Digestion, 67 (3): 138-45), thus supporting the ethical considerations that imply obtaining them preventing multiple biopsies from being taken from the same patient. Finally, the results generated tend to be representative when it is a relatively advanced disease (Gressner OA, Weiskirchen R, Gressner AM. 2007. Biomarkers of liver fibrosis: clinical translation of molecular pathogenesis or based on liver dependent malfunction tests. Clin Chim Acta ; 381: 107-13; Iredale JP 2007. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest. 117 (3): 539-48). The considerations for and against are summarized in Table 1.
Tabla 1.- Ventajas y desventajas de la biopsia hepática. Table 1.- Advantages and disadvantages of liver biopsy.
Ventajas Desventajas Advantages Disadvantages
Estándar de oro para el diagnóstico Prueba altamente invasiva  Gold standard for diagnosis Highly invasive test
Valor diagnóstico confirmatorio Complicaciones incluyen la mortalidad  Confirmatory diagnostic value Complications include mortality
Sugerencia etiológica Error de muestreo significativo Diagnóstico diferencial Alto costo  Etiological suggestion Significant sampling error Differential diagnosis High cost
Evaluación del grado y estadio Variación inter-observador  Assessment of grade and stage Inter-observer variation
Decisión terapéutica (elegibilidad) Consideraciones Éticas para obtener mayor número de muestras  Therapeutic decision (eligibility) Ethical considerations to obtain a larger number of samples
Evaluación del tratamiento (eficacia)  Treatment evaluation (efficacy)
Comparación en el seguimiento de los  Comparison in the follow-up of
pacientes no tratados y tratados Biomarcadores de fibrosis hepática: Debido a los inconvenientes de la biopsia hepática en los últimos años, el interés por identificar y describir a la fibrosis hepática mediante el uso de marcadores no invasivos ha ido en aumento (Plebani M, and Burlina A. 1991. Biochemical Markers of Hepatic Fibrosis. Clin Biochem. 24, 219-239). La búsqueda de biomarcadores específicos de fibrosis hepática se ha aplicado en la investigación sobre tejidos y suero, y estos han sido empleados para comprender los procesos biológicos fundamentales y sus ¡nterrelaciones, para ser usados como herramienta en el desarrollo diagnóstico y terapéutico. No obstante, son escasos los biomarcadores no invasivos (localizados en suero o líquidos biológicos), que indiquen con certeza la actividad fibrogénica. untreated and treated patients Biomarkers of liver fibrosis: Due to the drawbacks of liver biopsy in recent years, the interest in identifying and describing liver fibrosis through the use of non-invasive markers has been increasing (Plebani M, and Burlina A. 1991. Biochemical Markers of Hepatic Fibrosis, Clin Biochem. 24, 219-239). The search for specific biomarkers of liver fibrosis has been applied in tissue and serum research, and these have been used to understand the fundamental biological processes and their interrelationships, to be used as a tool in diagnostic and therapeutic development. However, there are few non-invasive biomarkers (located in serum or biological fluids), which indicate with certainty the fibrogenic activity.
Actualmente la fibrosis puede ser determinada de manera no invasiva mediante dos formas, una de ellas se basa en la cuantificación de biomarcadores en suero (aproximación biológica) y la segunda midiendo la rigidez del hígado (aproximación física), ambas formas finalmente resultan complementarias. La dureza es una propiedad característica, privativa del parénquima hepático, mientras que los biomarcadores en suero podrían indicar una asociación con el estadio de la fibrosis. Los biomarcadores séricos de fibrosis hepática ofrecen una alternativa atractiva y rentable tanto para el paciente como para el médico. Además de no ser invasivos, prácticamente no provocan complicaciones, los errores de muestreo son pocos o nulos y tienen la ventaja de que las mediciones pueden llevarse a cabo repetidamente, permitiendo por lo tanto, el control dinámico de la enfermedad (Zhou K, Lu LG. 2009. Assessment of fibrosis in chronic liver diseases. Journal of Digestive Diseases. 10(1):7-14). Currently, fibrosis can be determined non-invasively in two ways, one of which is based on the quantification of biomarkers in serum (biological approach) and the second measuring the stiffness of the liver (physical approach), both forms are finally complementary. Hardness is a characteristic property, exclusive to the liver parenchyma, while serum biomarkers may indicate an association with the stage of fibrosis. Serum biomarkers of hepatic fibrosis offer an attractive and cost-effective alternative for both the patient and the doctor. In addition to not being invasive, they practically do not cause complications, sampling errors are few or zero and have the advantage that measurements can be carried out repeatedly, thus allowing dynamic disease control (Zhou K, Lu LG 2009. Assessment of fibrosis in chronic liver diseases, Journal of Digestive Diseases, 10 (1): 7-14).
El valor diagnóstico de los biomarcadores séricos para fibrosis hepática se ha explorado en numerosos estudios, y con base en las necesidades clínicas y de investigación, el marcador ideal debe reunir características que permitan la supervisión de la progresión de la enfermedad o su regresión, como parte de la historia natural del padecimiento hepático o como resultado de los regímenes de tratamiento; entre ellas debe exhibir: The diagnostic value of serum biomarkers for hepatic fibrosis has been explored in numerous studies, and based on clinical and research needs, the ideal marker should have characteristics that allow monitoring of disease progression or regression, as part of the natural history of liver disease or as a result of treatment regimens; among them it must exhibit:
• alta sensibilidad y especificidad que permitan identificar diferentes etapas de fibrosis. • disponibilidad, seguridad, economía y reproducibilidad. • high sensitivity and specificity to identify different stages of fibrosis. • availability, security, economy and reproducibility.
• capacidad para diferenciar la fibrosis de otros trastornos inflamatorios hepáticos, es decir, evitar falsos positivos.  • ability to differentiate fibrosis from other inflammatory liver disorders, that is, avoid false positives.
Actualmente no existe un biomarcador ideal para la fibrosis, aunque se han identificado varias moléculas o algoritmos como indicadores útiles cuando se manejan combinados (Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non- invasive markers for hepatic fibrosis. BMC Gastroenterol. 11:91). Algunos de los nuevos métodos no invasivos han sido evaluados mediante el análisis del área bajo la curva (AUROC), tomando a la biopsia como referencia. Sin embargo, al momento no existe una prueba que exhiba una AUROC >90, que le permita ser tomado como el marcador no invasivo de elección. There is currently no ideal biomarker for fibrosis, although several molecules or algorithms have been identified as useful indicators when managed in combination (Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non-invasive markers for hepatic fibrosis. BMC Gastroenterol 11:91). Some of the new non-invasive methods have been evaluated by analyzing the area under the curve (AUROC), taking biopsy as a reference. However, at the moment there is no test that exhibits an AUROC> 90, which allows it to be taken as the non-invasive marker of choice.
Los biomarcadores en suero han sido valorados principalmente por su capacidad para determinar el estadio de la fibrosis. Se han propuesto dos tipos de ellos: los directos, que reflejan el depósito o la eliminación de la matriz extraceluiar en el hígado; y los indirectos, que incluyen moléculas liberadas a la sangre inducidas por la inflamación, sintetizadas, reguladas o excretadas por el órgano, como producto de los procesos alterados comúnmente a consecuencia del deterioro de la función hepática (Gressner Olav A., Ralf Weiskirchen, Gressner Axel M., 2007. Biomarkers of liver fibrosis: Clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Clínica Chimica Acta. 381 : 107-113) Serum biomarkers have been valued primarily for their ability to determine the stage of fibrosis. Two types of them have been proposed: the direct ones, which reflect the deposition or elimination of the extracellular matrix in the liver; and the indirect ones, which include molecules released into the blood induced by inflammation, synthesized, regulated or excreted by the organ, as a product of the processes commonly altered as a result of impaired liver function (Gressner Olav A., Ralf Weiskirchen, Gressner Axel M., 2007. Biomarkers of liver fibrosis: Clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Clínica Chimica Acta. 381: 107-113)
A la fecha, los biomarcadores directos engloban a los diferentes fragmentos de los componentes de la MEC producidos por las células estelares (HSC) y otras células del hígado durante el proceso de remodelación de la matriz hepática (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis. J Gastrointestin Liver Dis. 5(2): 149-159), entre ellos están incluidas glicoproteínas como el ácido hialuró ico, laminina y YLK-40; colágenas, (pro-colágena III, la colágena tipo IV), y metaloproteasas de matriz (MMPs) y sus inhibidores (TIMPs) To date, direct biomarkers encompass the different fragments of the components of the ECM produced by stellar cells (HSC) and other liver cells during the liver matrix remodeling process (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis J Gastrointestin Liver Dis. 5 (2): 149-159), including glycoproteins such as hyaluric acid, laminin and YLK-40; collagen, (pro-collagen III, type IV collagen), and matrix metalloproteases (MMPs) and their inhibitors (TIMPs)
Biomarcadores séricos más sobresalientes o novedosos: Péptido carboxilo- terminal de la pro-colágena tipo I (PICP) y el péptido amino-terminal de la pro- colágena tipo III (PIIINP): En el hígado humano sano los tipos de colágena más abundantes, son los formadores de fibrillas (I y III). En su forma madura, la colágena está integrada a la MEC. Durante la fibrogénesis los niveles de colágena tipo I pueden aumentar hasta ocho veces, además, la relación tipo l/lll también cambia, de 1 :1 en el hígado sano, a 1 :2 en el cirrótico (Gressner Olav A., Ralf Weiskirchen, Gressner Axel M., 2007. Biomarkers of liver fibrosis: Clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Clínica Chimica Acta. 381 : 107-113). El PIIINP es un componente importante del tejido conectivo; su concentración relativa en la membrana basal es mayor durante la fibrogénesis hepática seguida por un aumento de sus niveles en suero. En la hepatitis aguda, los niveles de PIIINP correlacionan con los niveles de aminotransferasa, reflejando el grado de fibrosis, pero desafortunadamente, no es específico ya que también se eleva en la acromegalia, fibrosis pulmonar, pancreatitis crónica, y enfermedades reumáticas (Lieber CS, Weiss DG, Paronetto F. 2008. Veterans Affairs Cooperative Study 391 Group: Valué of fibrosis markers for staging liver fibrosis ¡n patients with precirrhotic alcoholic liver disease. Alcohol Clin Exp Res. 32(6): 1031 -9). Los niveles séricos de PICP en pacientes con infección crónica leve por el ; virus de hepatitis C, no se diferencian de los detectados en individuos sanos, sólo se elevan en pacientes (50%) con enfermedad moderadamente avanzada o crónica avanzada, incluyendo pacientes con cirrosis hepática, y debido a que no existe una correlación entre los niveles de PICP y PIIINP en suero no permite la detección potencial de todos los casos de fibrosis. (Jarcuska P, Janicko M, Veseliny E, Jarcuska P, Skladany L. 2010. Circulating markers of liver fibrosis progression. Clínica Chimica Acta. 411 (ISIS):! 009-1017) además, el uso combinado de ambas moléculas para establecer el grado de fibrosis basado en su determinación sérica, no es confiable. Most outstanding or novel serum biomarkers: Carboxyl-terminal peptide of pro-collagen type I (PICP) and the amino-terminal peptide of the pro Type III collagen (PIIINP): In the healthy human liver, the most abundant types of collagen are the fibrils (I and III). In its mature form, the collagen is integrated into the MEC. During fibrogenesis, levels of type I collagen can increase up to eight times, in addition, the ratio l / lll also changes, from 1: 1 in the healthy liver, to 1: 2 in the cirrhotic (Gressner Olav A., Ralf Weiskirchen , Gressner Axel M., 2007. Biomarkers of liver fibrosis: Clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Clínica Chimica Acta. 381: 107-113). PIIINP is an important component of connective tissue; its relative concentration in the basement membrane is higher during hepatic fibrogenesis followed by an increase in its serum levels. In acute hepatitis, PIIINP levels correlate with aminotransferase levels, reflecting the degree of fibrosis, but unfortunately, it is not specific since it also rises in acromegaly, pulmonary fibrosis, chronic pancreatitis, and rheumatic diseases (Lieber CS, Weiss DG, Paronetto F. 2008. Veterans Affairs Cooperative Study 391 Group: Valué of fibrosis markers for staging liver fibrosis in patients with precirrhotic alcoholic liver disease Alcohol Clin Exp Res. 32 (6): 1031-9). Serum levels of PICP in patients with mild chronic infection; Hepatitis C virus, do not differ from those detected in healthy individuals, only rise in patients (50%) with moderately advanced or chronic advanced disease, including patients with liver cirrhosis, and because there is no correlation between levels of PICP and PIIINP in serum does not allow the potential detection of all cases of fibrosis. (Jarcuska P, Janicko M, Veseliny E, Jarcuska P, Skladany L. 2010. Circulating markers of liver fibrosis progression. Clínica Chimica Acta. 411 (ISIS):! 009-1017) in addition, the combined use of both molecules to establish the Fibrosis grade based on your serum determination, is not reliable.
Colágena tipo IV es un componente esencial de la MEC hepática. A diferencia de las colágenas tipo I y III que son procesadas por proteólisis, esta molécula se deposita intacta en la matriz y su presencia en el suero refleja directamente su degradación. Por lo tanto, los ensayos para detectar los fragmentos de colágena tipo IV (NC1 y PIVNP) en el suero, se utilizan con más frecuencia en la práctica. Estos tienen una correlación positiva con el grado de fibrosis hepática en pacientes con hepatitis viral crónica y enfermedad hepática alcohólica funcionando como indicadores sensibles a la presencia de cirrosis en la hemocromatosis. En la hepatitis C, se estableció para el diagnóstico el punto de corte en la etapa F2 (110 ng/ml) y para predecir etapa F3 (130 ng/ml) (Murawaki Y, Koda M, Okamoto K et al. 2001. Diagnostic valué of serum type IV collagen test in comparison with piateiet count for predicting the fibrotic stage in patients with chronic hepatitis. J Gastroenterol Hepatol. 16: 777-781) (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis. J Gastrointestin Liver Dis. 15(2): 149-159). Type IV collagen is an essential component of hepatic ECM. Unlike type I and III collagen that are processed by proteolysis, this molecule is deposited intact in the matrix and its presence in the serum directly reflects its degradation. Therefore, tests to detect fragments of type IV collagen (NC1 and PIVNP) in serum are used more frequently in practice. These have a positive correlation with the degree of Liver fibrosis in patients with chronic viral hepatitis and alcoholic liver disease functioning as sensitive indicators of the presence of cirrhosis in hemochromatosis. In hepatitis C, the cut-off point in stage F2 (110 ng / ml) was established for diagnosis and to predict stage F3 (130 ng / ml) (Murawaki Y, Koda M, Okamoto K et al. 2001. Diagnostic valué of serum type IV collagen test in comparison with piateiet count for predicting the fibrotic stage in patients with chronic hepatitis. J Gastroenterol Hepatol. 16: 777-781) (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis. J Gastrointestin Liver Dis. .15 (2): 149-159).
Factor de crecimiento transformante β1 (TGF-βΙ); Es una citocina pleiotrópica implicada en la regulación del crecimiento tisular, diferenciación, producción de MEC y la respuesta inmune. Se han identificado tres isoformas de esta citocina (β1 , β2 y β3), pero sólo la-βΐ ha sido vinculada a la fibrogénesis hepática. El TGF-β! es conocido comúnmente como componente central de respuesta fibrogénica en las heridas y como un sobre regulador de diferentes enfermedades. La correlación entre los niveles de TGF-βΙ y la progresión de la fibrosis es aceptada considerablemente (Kanzler S, Baumann M, Schirmacher P, Dries V, Bayer E, Gerken G, Dienes HP, Lohse AW. 2001. Prediction of progressive liver fibrosis in hepatitis C infection by serum and tissue levéis of transforming growth factorbeta. J Viral Hepat. 8(6):430-7) (Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non-invasive markers for hepatic fibrosis. BMC Gastroenterol. 11 :91). Transforming growth factor β1 (TGF-βΙ); It is a pleiotropic cytokine involved in the regulation of tissue growth, differentiation, ECM production and the immune response. Three isoforms of this cytokine (β1, β2 and β3) have been identified, but only la-βΐ has been linked to hepatic fibrogenesis. TGF-β! It is commonly known as a central component of fibrogenic response in wounds and as a regulatory envelope for different diseases. The correlation between TGF-βΙ levels and fibrosis progression is considerably accepted (Kanzler S, Baumann M, Schirmacher P, Dries V, Bayer E, Gerken G, Dienes HP, Lohse AW. 2001. Prediction of progressive liver fibrosis in hepatitis C infection by serum and tissue levéis of transforming growth factorbeta. J Viral Hepat. 8 (6): 430-7) (Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non-invasive markers for hepatic fibrosis. BMC Gastroenterol. 11: 91).
Ácido hialurónico (HA): Es un glicosaminoglicano componente de la MEC sintetizado por las HSC. En circunstancias normales las SEC hepáticas son las estructuras que intervienen directamente en su captación y degradación. Los niveles elevados de HA pueden deberse a su eliminación disminuida o al aumento de su producción, estos niveles han sido detectados en el suero de pacientes con enfermedad hepática de etiologías diferentes y en particular en aquellos con cirrosis (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis. J Gastrointestin Liver Dis. 15(2):149-159). En un estudio realizado en pacientes con enfermedad de hígado graso no alcohólico (NAFLD), el HA fue seleccionado como el mejor marcador para fibrosis, con un área bajo la curva (AUC) de 0,97 (Lydatakis H, Hager IP, Kostadelou E, Mpousmpoulas S, Pappas S, Diamantis I. 2006. Non- invasive markers to predict the liver fibrosis in non alcoholic fatty liver disease. Liver Int. 26:864-871). Sin embargo, su valor predictivo negativo es mucho más alto (98- 100%) que el valor predictivo positivo (61%), concluyéndose que la utilidad principal de este marcador, reside en que dependiendo de su nivel sérico, es posible descartar fibrosis avanzada y cirrosis, (Gressner Olav A., Ralf Weiskirchen, Gressner Axel M. 2007. Biomarkers of liver fibrosis: Clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Clínica Chimica Acta 381 : 107-113). Hyaluronic acid (HA): It is a glycosaminoglycan component of the MEC synthesized by HSC. Under normal circumstances, hepatic SECs are the structures that directly intervene in their uptake and degradation. Elevated levels of HA may be due to decreased elimination or increased production, these levels have been detected in the serum of patients with liver disease of different etiologies and in particular in those with cirrhosis (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis J Gastrointestin Liver Dis. 15 (2): 149-159). In a study in patients with non-alcoholic fatty liver disease (NAFLD), HA was selected as the best marker for fibrosis, with an area under the curve (AUC) of 0.97 (Lydatakis H, Hager IP, Kostadelou E, Mpousmpoulas S, Pappas S, Diamantis I. 2006. Non-invasive markers to predict the liver fibrosis in non alcoholic fatty liver disease. Liver Int. 26: 864-871). However, its negative predictive value is much higher (98-100%) than the positive predictive value (61%), concluding that the main utility of this marker is that depending on its serum level, it is possible to rule out advanced fibrosis and cirrhosis, (Gressner Olav A., Ralf Weiskirchen, Gressner Axel M. 2007. Biomarkers of liver fibrosis: Clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Clínica Chimica Acta 381: 107-113).
Proteína 4 asociada a Microfibrillas (MFAP-4): Es una molécula de unión a colágena que contiene en su extremo C-terminal un dominio tipo fibrinógeno y un motivo de unión a ¡ntegrína situado en el extremo N terminal de la proteína. Participa en la respuesta inmune innata y permite el libre intercambio gaseoso en los pulmones a través de su unión a la región colagénica de proteínas surfactantes (SP-A y SP-D). Su región N-terminal, incluye un residuo de cisteína y una secuencia Arg-Gly-Asp (RGD), que es un motivo de adhesión celular para varios miembros de la familia de las integrinas (Greenlee K J., Werb Z, and Kheradmand F. 2007. Matrix Metalloproteinases in Lung: Múltiple, Multifarious, and Multifaceted. Physiol Rev. 87: 69-98,). En un estudio reciente la MFAP-4 mostró niveles séricos de diagnóstico de alta precisión para la predicción de enfermedades no hepáticas contra cirrosis (AUROC = 0,97, p <0,0001), así como el estadio F0 en comparación con F4 (AUROC = 0,84, p <0,0001), y etapas F0 a F3 versus F4 (AUROC = 0,76, p< 0.0001) (Adams L A, George J, Bugianesi E, Rossi E, De Boer W B, Van der Poorten D, Ching H L I, Bulsara M, and Jeffrey G P. 2011. Complex non-invasive fibrosis models are more accurate than simple models in non-alcoholic fatty liver disease. Journal of Gastroenterology and Hepatology. 26: 1536-1543); (Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non-invasive markers for hepatic fibrosis. BMC Gastroenterol. 11 :91). Esta proteína además figura entre las proteínas hepato-especificas, obtenida de nuestro modelo experimental de fibrosis en ratas, como el biomarcador ideal en suero, objeto de nuestra solicitud. Los llamados biomarcadores indirectos, son determinados en ensayos rutinarios de laboratorio, entre ellos se encuentran, los tiempos de protrombina, conteo de plaquetas, determinación de transaminasas (ALT y AST) los cuales indican alteración hepática. Protein 4 associated with Microfibrils (MFAP-4): It is a collagen binding molecule that contains at its C-terminal end a fibrinogen-like domain and a ligand binding motif located at the N-terminal end of the protein. It participates in the innate immune response and allows free gaseous exchange in the lungs through its binding to the collagen region of surfactant proteins (SP-A and SP-D). Its N-terminal region includes a cysteine residue and an Arg-Gly-Asp (RGD) sequence, which is a reason for cell adhesion for several members of the integrin family (Greenlee K J., Werb Z, and Kheradmand F. 2007. Matrix Metalloproteinases in Lung: Multiple, Multifarious, and Multifaceted. Physiol Rev. 87: 69-98,). In a recent study, MFAP-4 showed high-precision diagnostic serum levels for the prediction of non-hepatic diseases against cirrhosis (AUROC = 0.97, p <0.0001), as well as stage F0 compared to F4 (AUROC = 0.84, p <0.0001), and stages F0 to F3 versus F4 (AUROC = 0.76, p <0.0001) (Adams LA, George J, Bugianesi E, Rossi E, De Boer WB, Van der Poorten D, Ching HLI, Bulsara M, and Jeffrey G P. 2011. Complex non-invasive fibrosis models are more accurate than simple models in non-alcoholic fatty liver disease. Journal of Gastroenterology and Hepatology. 26: 1536-1543); (Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non-invasive markers for hepatic fibrosis. BMC Gastroenterol. 11: 91). This protein also appears among the hepato-specific proteins, obtained from our experimental model of fibrosis in rats, as the ideal biomarker in serum, object of our application. The so-called indirect biomarkers are determined in routine laboratory tests, including prothrombin times, platelet count, transaminase determination (ALT and AST) which indicate liver impairment.
Aspartato aminotransferasa (AST) y alanina aminotransferasa (ALT); Son enzimas hepáticas excretadas al torrente sanguíneo por los hepatocitos dañados. El valor predictivo de la relación AST / ALT se ha validado en la enfermedad hepática no alcohólica, hepatitis viral crónica, colangitis esclerosante primaria, y cirrosis biliar primaria (Haukeland JW, Schreiner LT, Lorgen I, Frigstad SO, Bang C, Raknerud N, Konopski Z. 2008. AST/ALT ratio provides prognostic information independently of Child-Pugh class, gender and age in non-alcoholic cirrhosis. Scand J Gastroenterol. 43(10): 1241 -1248). En algunas formas de hepatitis aguda y crónica, y/o esteatosis esta relación es≤ 1 , mientras que en la hepatitis alcohólica, una relación AST / ALT es a menudo >2, mientras que estas proporciones son solo sugerentes para ciertas etiologías hepáticas, existe una superposición entre los grupos que dependen de la relación AST / ALT exclusivamente, al hacer un diagnóstico, por ejemplo, en pacientes con hepatitis C con abuso del alcohol. Además la ALT y la AST también pueden elevarse aunque en menor medida por problemas musculares, renales y cardiacos (Giboney PT. 2005. Mildly elevated liver transaminase levéis in the asymptomatic patient. Am Fam. Physician. 71(6):1105- 1110). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT); They are liver enzymes excreted to the bloodstream by damaged hepatocytes. The predictive value of the AST / ALT relationship has been validated in non-alcoholic liver disease, chronic viral hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis (Haukeland JW, Schreiner LT, Lorgen I, Frigstad SO, Bang C, Raknerud N, Konopski Z. 2008. AST / ALT ratio provides prognostic information independently of Child-Pugh class, gender and age in non-alcoholic cirrhosis. Scand J Gastroenterol. 43 (10): 1241-1248). In some forms of acute and chronic hepatitis, and / or steatosis this ratio is ≤ 1, while in alcoholic hepatitis, an AST / ALT ratio is often> 2, while these proportions are only suggestive for certain hepatic etiologies, there is an overlap between the groups that depend on the AST / ALT relationship exclusively, when making a diagnosis, for example, in patients with hepatitis C with alcohol abuse. In addition, ALT and AST can also be elevated although to a lesser extent due to muscular, renal and cardiac problems (Giboney PT. 2005. Mildly elevated liver transaminase levéis in the asymptomatic patient. Am Fam. Physician. 71 (6): 1105-1110) .
Si bien estas proporciones son sugestivas de enfermedad hepática de etiología determinada, existe un amplio solapamiento entre aquéllas que, para el diagnóstico dependen exclusivamente de la relación AST/ALT, por ejemplo, pacientes que padecen tanto hepatitis C y tienen antecedentes de abuso de alcohol. Una revisión técnica de la AGA (Asociación Americana de Gastroenterología) reporta que la ALT sérica tiene variación diurna, y puede modificarse día a día, incluso con el ejercicio, también señala que los niveles séricos de AST pueden ser hasta 15% mayores en los hombres de raza negra con respecto a los de raza blanca (Green RM, Flamm S. 2002. AGA technical review on the evaluation of liver chemistry tests. Gastroenterology. 123:1367-84). Recuento de plaquetas (PLT): La trombocitopenia es un biomarcador valioso para enfermedades hepáticas avanzadas, puede estar relacionada con mecanismos tales como hiperesplenismo, mielosupresión por VHC, disminución de la producción de trombopoyetina, desarrollo de procesos autoinmunes, sin embargo, la evaluación conjunta de la relación AST/ALT y PLT tiene un valor diagnóstico alto para la cirrosis (70-90%) (Giannini E, Risso D, Botta F et al. 2003. Validity and clinical utility of the aspartate aminotransferase-alanine aminotransferase ratio in assessing disease severity and prognosis ¡n patients with hepatitis C virus-related chronic liver disease. Arch Intern Med. 163: 218-224). Although these proportions are suggestive of liver disease of a determined etiology, there is a wide overlap between those who, for diagnosis, depend exclusively on the AST / ALT relationship, for example, patients suffering from both hepatitis C and have a history of alcohol abuse. A technical review of the AGA (American Gastroenterology Association) reports that serum ALT has daytime variation, and can be modified day by day, even with exercise, it also points out that serum AST levels can be up to 15% higher in men black with respect to white race (Green RM, Flamm S. 2002. AGA technical review on the evaluation of liver chemistry tests. Gastroenterology. 123: 1367-84). Platelet count (PLT): Thrombocytopenia is a valuable biomarker for advanced liver diseases, it may be related to mechanisms such as hypersplenism, HCV myelosuppression, decreased thrombopoietin production, development of autoimmune processes, however, joint evaluation of the AST / ALT and PLT ratio has a high diagnostic value for cirrhosis (70-90%) (Giannini E, Risso D, Botta F et al. 2003. Validity and clinical utility of the aspartate aminotransferase-alanine aminotransferase ratio in assessing disease severity and prognosis in patients with hepatitis C virus-related chronic liver disease. Arch Intern Med. 163: 218-224).
Tiempo de Protrombina: Es un índice que refleja la capacidad de síntesis del hígado, y por lo tanto es uno de los indicadores iniciales de cirrosis. En un estudio retrospectivo con pacientes HALT-C (Hepatitis C Antiviral Long-term Treatment Against Cirrhosis), el tiempo de protrombina, la PLT, la relación AST/ALT y los niveles de fosfatasa alcalina, resultaron predictivos de cirrosis. En otro estudio, el tiempo de protrombina correlacionaba con la presencia y el tamaño de las varices esofágicas. El tiempo de protrombina es componente de diferentes índices (Croquet V, Vuillemin E, Ternisien C et al. 2002. Prothrombin index is an ¡ndirect marker of severe liver fibrosis. Eur J Gastroenterol Hepatol. 14: 1133- 141) Prothrombin time: It is an index that reflects the liver's ability to synthesize, and therefore is one of the initial indicators of cirrhosis. In a retrospective study with HALT-C patients (Hepatitis C Antiviral Long-term Treatment Against Cirrhosis), prothrombin time, PLT, AST / ALT ratio and alkaline phosphatase levels, were predictive of cirrhosis. In another study, prothrombin time correlated with the presence and size of esophageal varices. Prothrombin time is a component of different indices (Croquet V, Vuillemin E, Ternisien C et al. 2002. Prothrombin index is an direct marker of severe liver fibrosis. Eur J Gastroenterol Hepatol. 14: 1133-141)
Los biomarcadores directos e indirectos se pueden usar solos o combinados, para producir puntajes compuestos. El cálculo de tales puntuaciones puede ser relativamente simple o puede estar basado en fórmulas complicadas (por ejemplo, Fibrotest/Fibrosure). (Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non- invasive markers for hepatic fibrosis. BMC Gastroenterol. 11 :91); el FibroTest (patentado por Biopredictive, París, Francia) fue el primer multicomponente que combinó los datos resultantes de diversas pruebas (Imbert-Bismut F, Ratziu V, Pieroni L, et al. 2001. Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study. Lancet. 357: 1069-1075) (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis. J Gastrointestin Liver Dis. 15(2): 149- 159). También han sido propuestos otros índices, 4 de ellos están patentados (Fibrotest; Biopredictive, Houilles, France, US Patent Application Serial No. 09/687,459), Actitest, NASHtest, FibroMax) y son comerciales y algunos otros se encuentran en estudio. Un resumen de estos se presentan en la Tabla 2. Direct and indirect biomarkers can be used alone or in combination, to produce composite scores. The calculation of such scores may be relatively simple or may be based on complicated formulas (for example, Fibrotest / Fibrosure). (Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non-invasive markers for hepatic fibrosis. BMC Gastroenterol. 11: 91); FibroTest (patented by Biopredictive, Paris, France) was the first multicomponent that combined the data resulting from various tests (Imbert-Bismut F, Ratziu V, Pieroni L, et al. 2001. Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study. Lancet. 357: 1069-1075) (Grigorescu M. 2006. Noninvasive Biochemical Markers of Liver Fibrosis. J Gastrointestin Liver Dis. 15 (2): 149-159). Other indexes have also been proposed, 4 of them are patented (Fibrotest; Biopredictive, Houilles, France, US Patent Application Serial No. 09 / 687,459), Actitest, NASHtest, FibroMax) and are commercial and some others are under study. A summary of these are presented in Table 2.
Tabla 2 - índices serológicos de multicomponentes para fibrosis hepática. Table 2 - serological indices of multicomponents for liver fibrosis.
Estudio Prueba serológica Sensibilidad (%) Especificidad (%)Study Serological test Sensitivity (%) Specificity (%)
APRI AST /plaquetas 89 75 APRI AST / platelets 89 75
PGA Protrombina, GGT, 91 81  PGA Prothrombin, GGT, 91 81
apolipoproteína A1  apolipoprotein A1
PGAA Tiempos de Protrombina, 79 89  PGAA Prothrombin Times, 79 89
GGT, apolipoproteína A1,  GGT, apolipoprotein A1,
a2-macroglobulina  a2-macroglobulin
Forns Edad, Plaquetas, GGT, colesterol 94 51  Age Forns, Platelets, GGT, Cholesterol 94 51
FibroTest Edad, genero, GGT, Bilirrubinas,  FibroTest Age, gender, GGT, Bilirubins,
a2-macroglobulina, apolipoproteína A1, 75 85 haptoglobina  a2-macroglobulin, apolipoprotein A1, 75 85 haptoglobin
Hepascore Edad, genero, bilirrubina, GGT, 63 89  Hepascore Age, gender, bilirubin, GGT, 63 89
ácido hialurónico, Y2-macroglobu!ina  hyaluronic acid, Y2-macroglobu! ina
FIB-4 Plaquetas , ALT, AST, Edad 70 74  FIB-4 Platelets, ALT, AST, Age 70 74
Fibrolndex Plaquetas, AST, GGT 78 74  Fibrolndex Platelets, AST, GGT 78 74
FibroMeter Plaquetas , AST, Edad, 81 84  FibroMeter Platelets, AST, Age, 81 84
Y2-macroglobulina,  Y2-macroglobulin,
Tiempos de Protrombina,  Prothrombin times,
ácido hialurónico, urea índice PGA; Combina la medición del índice de tiempo de protrombina, niveles de γ-glutamiltransferasa y apolipoproteína A1. Posteriormente se adicionó la determinación de a2-macroglobulina, lo que dio lugar a la PGAA mejorando su rendimiento. El índice PGA está relacionado, en las enfermedades crónicas hepáticas, tanto con la inflamación, como la fibrosis (p <0.01 , p <0.05, respectivamente). Sin embargo, su precisión en general es relativamente baja (Lu LG, Zeng MD, Mao YM, Li JQ, Qiu DK, Fang JY, Cao AP, Wan MB, Li CZ, Ye J, Cai X, Chen CW, Wang JY, Wu SM, Zhu JS, Zhou XQ. 2003. Relationship between clinical and pathologic findings in patients with chronic liver diseases. World J Gastroenterol. 9(12):2796-2800) (Nguyen-Khac E, Chatelain D, Tramier B, Decrombecque C, Robert B, Joly JP, Brevet M, Grignon P, Lion S, Le Page L, Dupas JL. 2008. Assessment of asymptomatic liver fibrosis in alcoholic patients using FibroScan: prospective comparison with seven non-invasive laboratory tests. Aliment Pharmacoi Ther. 28(10): 1188-98). hyaluronic acid, urea PGA index; It combines the measurement of the prothrombin time index, levels of γ-glutamyltransferase and apolipoprotein A1. Subsequently, the determination of a2-macroglobulin was added, which resulted in PGAA improving its performance. The PGA index is related, in chronic liver diseases, to both inflammation and fibrosis (p <0.01, p <0.05, respectively). However, its overall accuracy is relatively low (Lu LG, Zeng MD, Mao YM, Li JQ, Qiu DK, Fang JY, Cao AP, Wan MB, Li CZ, Ye J, Cai X, Chen CW, Wang JY, Wu SM, Zhu JS, Zhou XQ. 2003. Relationship between clinical and pathologic findings in patients with chronic liver diseases. World J Gastroenterol. 9 (12): 2796-2800) (Nguyen-Khac E, Chatelain D, Tramier B, Decrombecque C, Robert B, Joly JP, Brevet M, Grignon P, Lion S, Le Page L, Dupas JL. 2008. Assessment of asymptomatic liver fibrosis in alcoholic patients using FibroScan: prospective comparison with seven non-invasive laboratory tests. Aliment Pharmacoi Ther. 28 (10): 1188-98).
APRI: Es el índice que resulta de la relación de AST-Plaquetas, este se calcula como sigue: APRI: It is the index that results from the AST-Platelet ratio, this is calculated as follows:
(AST / límite superior del rango normal *)  (AST / upper limit of normal range *)
PLT (109/L) x 100  PLT (109 / L) x 100
* depende del valor de referencia de cada laboratorio. * depends on the reference value of each laboratory.
Previamente, ha sido validado, en pacientes coinfectados con VIH/VHC, como un marcador sustituto de fibrosis hepática significativa. Recientemente se ha empleado para determinar fibrosis avanzada en pacientes con HIV; sin embargo, el resultado de un meta-análisis sugirió que el APRI puede identificar, la relación hepatitis C-fibrosis, solo con un grado de precisión moderada (63.74 %, p<0.01) (Lin ZH, Xin YN, Dong QJ, Wang Q, Jiang XJ, Zhan SH, Sun Y, Xuan SY. 2011. Performance of the aspartate aminotransferase-to-platelet ratio index for the staging of hepatitis C-related fibrosis: an updated meta-analysis. Hepatology. 53(3):726-36) (Wenwen Jin, Zhonghua Lin, Yongning Xin, Xiangjun Jiang, Quanjiang Dong and Shiying Xuan. 2012. Diagnostic accuracy of the aspartate aminotransferase-to- platelet ratio index for the prediction of hepatitis B-related fibrosis: a leading meta- analysis. BMC Gastroenterology. 12: 14). Previously, it has been validated, in patients co-infected with HIV / HCV, as a substitute marker for significant hepatic fibrosis. Recently it has been used to determine advanced fibrosis in patients with HIV; However, the result of a meta-analysis suggested that APRI can identify, the hepatitis C-fibrosis ratio, only with a moderate degree of accuracy (63.74%, p <0.01) (Lin ZH, Xin YN, Dong QJ, Wang Q, Jiang XJ, Zhan SH, Sun Y, Xuan SY. 2011. Performance of the aspartate aminotransferase-to-platelet ratio index for the staging of hepatitis C-related fibrosis: an updated meta-analysis. Hepatology. 53 (3): 726-36) (Wenwen Jin, Zhonghua Lin, Yongning Xin, Xiangjun Jiang, Quanjiang Dong and Shiying Xuan. 2012. Diagnostic accuracy of the aspartate aminotransferase-to-platelet ratio index for the prediction of hepatitis B-related fibrosis: a leading meta - analysis BMC Gastroenterology 12: 14).
El índice de Foms: Se basa en 4 variables clínicas de rutina: edad, recuento de plaquetas, niveles de colesterol, y y glutamiltransferasa (Forns X et al, 2002). Con este método se puede diferenciar a los pacientes con Fibrosis leve (F0-F1) de aquellos con fibrosis grave (F4), pero es menos preciso para distinguir a los pacientes con grados intermedios de F2 a F4. El índice Forns ha sido validado en otras cohortes, como una herramienta de predicción de la respuesta a la terapia anti-VHC (Rossi E, Adams LA, Bulsara M, Jeffrey GP. 2007: Assessing liver fibrosis withserum marker models. Clin Biochem Rev. 28(1):3-10). The Foms index: It is based on 4 routine clinical variables: age, platelet count, cholesterol levels, and glutamyltransferase (Forns X et al, 2002). With this method, patients with mild fibrosis (F0-F1) can be distinguished from those with severe fibrosis (F4), but it is less accurate to distinguish patients with intermediate grades from F2 to F4. The Forns index has been validated in other cohorts, as a tool for predicting the response to therapy HCV (Rossi E, Adams LA, Bulsara M, Jeffrey GP. 2007: Assessing liver fibrosis withserum marker models. Clin Biochem Rev. 28 (1): 3-10).
HepaScore; Este índice combina la edad, el género, las concentraciones séricas de bilirrubina, γ glutamiltransferasa, ácido hialurónico y Y2-macroglobulina en una puntuación de 0.00 a 1.00. En un estudio realizado en 512 pacientes con hepatitis C crónica, el HepaScore mostró valores predictivos favorables para la identificación de fibrosis significativa (AUROC = 0.81), fibrosis severa (AUROC = 0.82) y cirrosis (AUROC = 0.88). Es importante destacar que el HepaScore se puede automatizar utilizando un analizador único (Guechot J, Lasnier E, Sturm N, París A, Zarski JP. 2010. ANRS HC EP 23 Fibrostar study group: Automation of the Hepascore and validation as a biochemical index of liver fibrosis in patients with chronic hepatitis C from the ANRS HC EP 23 Fibrostar cohort. Clin Chim Acta. 411(1-2):86-91). HepaScore; This index combines age, gender, serum concentrations of bilirubin, γ glutamyltransferase, hyaluronic acid and Y2-macroglobulin in a score of 0.00 to 1.00. In a study in 512 patients with chronic hepatitis C, the HepaScore showed favorable predictive values for the identification of significant fibrosis (AUROC = 0.81), severe fibrosis (AUROC = 0.82) and cirrhosis (AUROC = 0.88). Importantly, the HepaScore can be automated using a single analyzer (Guechot J, Lasnier E, Sturm N, Paris A, Zarski JP. 2010. ANRS HC EP 23 Fibrostar study group: Automation of the Hepascore and validation as a biochemical index of liver fibrosis in patients with chronic hepatitis C from the ANRS HC EP 23 Fibrostar cohort Clin Chim Acta. 411 (1-2): 86-91).
FibroTest y FibroSURE; Son pruebas idénticas para la evaluación del grado de fibrosis y actividad necro-inflamatoria, comercializadas con diferentes nombres en Europa y América. La puntuación del FibroTest se obtiene mediante el acceso a una licencia de un sitio web y calculándose con base en la edad del paciente, sexo, concentración de haptoglobina sérica, a2-macroglobul¡na, apolipoproteína A1, γ- glutamiltransferasa y bilirrubina (Imbert-Bismut F, Messous D, Thibault V, Myers RB, Pitón A, Thabut D, Devers L, Hainque B, Mercadier A, Poynard T. 2004. Intra- laboratory analytical variability of biochemical markers of fibrosis (Fibrotest) and activity (Actitest) and reference ranges in healthy blood donors. Clin Chem Lab Med. 42(3):323-333). Con estos datos se genera la puntuación que se correlaciona con el grado de daño hepático. Debido a la variabilidad de los componentes del ensayo y el tipo de analizadores empleados, el FibroTest sólo puede realizarse en laboratorios validados (Friedrich-Rust M, Rosenberg W, Parkes J, Herrmann E, Zeuzem S, Sarrazin C. 2010. Comparison of ELF, FibroTest and FibroScan for the non-invasive assessment of liver fibrosis. BMC Gastroenterology. 10:103). Un estudio en 74 pacientes que agrupan a: 36 con VHC, 10 con VHB, y 28 con cirrosis biliar primaria (Koda M, Matunaga Y, Kawakami M, Kishimoto Y, Suou T, Murawaki Y. 2007. Fibrolndex, a practical index for predicting significant fibrosis in patients with chronic hepatitis C. Hepatology. 45:297-306) mostró un AUROC de 0.69 y 0.91 para el diagnóstico de fibrosis significativa (F≥2) y cirrosis hepática. Los valores para la sensibilidad y especificidad de FibroTest se basan en la detección de fibrosis primaria grave que oscila entre 75 y 85%, respectivamente (Lu LG, Zeng MD, Mao YM, Li JQ, Qiu DK, Fang JY, Cao AP, Wan MB, Li CZ, Ye J, Cai X, Chen CW, Wang JY, Wu SM, Zhu JS, Zhou XQ. 2003. Relationship between clinical and pathologic findings in patients with chronic liver diseases. World J Gastroenterol. 9(12):2796-2800). FibroTest and FibroSURE; They are identical tests for the evaluation of the degree of fibrosis and necro-inflammatory activity, marketed under different names in Europe and America. The FibroTest score is obtained by accessing a license from a website and calculating it based on the patient's age, sex, serum haptoglobin concentration, a2-macroglobulin, apolipoprotein A1, γ-glutamyltransferase and bilirubin (Imbert- Bismut F, Messous D, Thibault V, Myers RB, Python A, Thabut D, Devers L, Hainque B, Mercadier A, Poynard T. 2004. Intra-laboratory analytical variability of biochemical markers of fibrosis (Fibrotest) and activity (Actitest) and reference ranges in healthy blood donors. Clin Chem Lab Med. 42 (3): 323-333). With this data, the score that correlates with the degree of liver damage is generated. Due to the variability of the test components and the type of analyzers used, FibroTest can only be performed in validated laboratories (Friedrich-Rust M, Rosenberg W, Parkes J, Herrmann E, Zeuzem S, Sarrazin C. 2010. Comparison of ELF , FibroTest and FibroScan for the non-invasive assessment of liver fibrosis. BMC Gastroenterology. 10: 103). A study in 74 patients that group: 36 with HCV, 10 with HBV, and 28 with primary biliary cirrhosis (Koda M, Matunaga Y, Kawakami M, Kishimoto Y, Suou T, Murawaki Y. 2007. Fibrolndex, a practical index for predicting significant fibrosis in patients with chronic hepatitis C. Hepatology. 45: 297-306) showed an AUROC of 0.69 and 0.91 for the diagnosis of significant fibrosis (F≥2) and liver cirrhosis. The values for the sensitivity and specificity of FibroTest are based on the detection of severe primary fibrosis ranging between 75 and 85%, respectively (Lu LG, Zeng MD, Mao YM, Li JQ, Qiu DK, Fang JY, Cao AP, Wan MB, Li CZ, Ye J, Cai X, Chen CW, Wang JY, Wu SM, Zhu JS, Zhou XQ. 2003. Relationship between clinical and pathologic findings in patients with chronic liver diseases. World J Gastroenterol. 9 (12): 2796-2800).
Ventajas y desventajas de los biomarcadores actuales: Con relación al uso del análisis de biomarcadores séricos entre las ventajas, se incluyen: su alta aplicabilidad (>95%), la reproducibilidad inter-laboratorio y su disponibilidad generalizada (fácil distribución). Advantages and disadvantages of current biomarkers: With regard to the use of serum biomarker analysis among the advantages, they include: its high applicability (> 95%), inter-laboratory reproducibility and its widespread availability (easy distribution).
Entre las principales desventajas encontramos, que ninguno de ellos es hígado específico, y sus resultados pueden ser influenciados por condiciones comórbidas que requieren de una interpretación más crítica de los datos. Among the main disadvantages we find that none of them is liver specific, and their results can be influenced by comorbid conditions that require a more critical interpretation of the data.
Con el Fibro Test y el Hepa Score, por ejemplo, se obtienen falsos positivos cuando se realizan en pacientes con Síndrome de Gilbert o con hemolisis, debido al estado de hiperbilirrubinemia (Poynard T, Munteanu M, Imbert-Bismut F, et al. 2004. Prospective analysis of discordant results between biochemical markers and biopsy in patients with chronic hepatitis C. Clin Chem. 10:10), así mismo en pruebas como el APRI, índice Forns o Fibrometer, la hepatitis aguda puede producir falsos positivos ya que en todos ellos se evalúan los niveles de aminotransferasas y éstas enzimas también se elevan en otras enfermedades no hepáticas. With the Fibro Test and the Hepa Score, for example, false positives are obtained when performed in patients with Gilbert's syndrome or with hemolysis, due to the state of hyperbilirubinemia (Poynard T, Munteanu M, Imbert-Bismut F, et al. 2004 Prospective analysis of discordant results between biochemical markers and biopsy in patients with chronic hepatitis C. Clin Chem. 10:10), as well as in tests such as APRI, Forns index or Fibrometer, acute hepatitis can produce false positives since in all they assess the levels of aminotransferases and these enzymes also rise in other non-hepatic diseases.
El rendimiento de cada biomarcador, se compara con el beneficio que exhibe uno o más paneles, además de limitar su evaluación a una condición patológica (ejemplo, enfermedad hepática alcohólica), y se dificulta debido a esto, la contribución exacta de cada biomarcador recién descrito (Park SH, Kim CH, Kim DJ, Suk KT, Cheong JY, Cho SW, Hwang SG, Lee YJ, Cho M, Yang JM, Kim YB. 2011. Usefulness of múltiple biomarkers for the prediction of significant fibrosis in chronic hepatitis B. J Clin Gastroenterol. 45(4):361-5). The performance of each biomarker is compared with the benefit exhibited by one or more panels, in addition to limiting its evaluation to a pathological condition (eg, alcoholic liver disease), and the exact contribution of each biomarker just described is difficult because of this. (Park SH, Kim CH, Kim DJ, Suk KT, Cheong JY, Cho SW, Hwang SG, Lee YJ, Cho M, Yang JM, Kim YB. 2011. Usefulness of multiple biomarkers for the prediction of significant fibrosis in chronic hepatitis B. J Clin Gastroenterol. 45 (4): 361-5).
La aparición de tecnologías nuevas tales como la proteómica, que evalúa patrones de proteínas o glicoproteínas por espectroscopia de masas utilizando muestras de suero, han mostrado una aplicabilidad limitada, al no poder discriminar la etiología de las enfermedades no hepáticas. Por ejemplo, Callewaert N et al., 2004 (Callewaert N, Vlierberghe HV, Hecke AV y cois. 2004. Non invasive diagnosis of liver cirrhosis using DNA sequence-based total serum protein glycomics. Nat Med. 10:429-434) desarrollaron un estudio con suero de pacientes con enfermedad hepática crónica en base a la presencia de proteínas totales N- glicosiladas (GlycoCirrhoTest y GlycoFibro Test), al tratar de distinguir cirrosis con la combinación de las dos pruebas, se obtuvo una sensibilidad de 79% y especificidad del 86%. Por otra parte, las mismas modificaciones en las proteínas séricas (glicosilacíón) aparecen de forma continua en todas las enfermedades hepáticas, (Blomme B, Van Steenkiste C, Callewaert N, Van Vlierberghe H.2009. Alteration of protein glycosylation in liver diseases. J Hepatol. 50(3):592-603), por lo que es necesario ampliar estos estudios prospectivos para determinar la aplicación clínica de estas técnicas. Además la fosfoproteómica puede mejorar el conocimiento de la patogenia de la fibrosis hepática, utilizando los perfiles fosforilados de las proteínas que participan en las vías de señalización de este proceso, como ha sido demostrado por (Younossi ZM, Baranova A, Stepanova M, Page S, Calvert VS, Afendy A, Goodman Z, Chandhoke V, Liotta L, Petricoin E. 2010. Phosphoproteomic biomarkers predicting histologic nonalcoholic steatohepatitis and fibrosis. J Proteome Res. 9(6): 3218-24), cuyos resultados sugieren que los biomarcadores fosfoproteicos podrían ser utilizados potencialmente en un entorno clínico para identificar a los pacientes con NASH. Además, proporcionan información acerca de las vías metabólicas que pueden estar implicadas en la patogenia de la enfermedad. The emergence of new technologies such as proteomics, which evaluates protein or glycoprotein patterns by mass spectroscopy using serum samples, have shown limited applicability, as they cannot discriminate the etiology of non-hepatic diseases. For example, Callewaert N et al., 2004 (Callewaert N, Vlierberghe HV, Hecke AV et al. 2004. Non invasive diagnosis of liver cirrhosis using DNA sequence-based total serum protein glycomics. Nat Med. 10: 429-434) developed A serum study of patients with chronic liver disease based on the presence of total N-glycosylated proteins (GlycoCirrhoTest and GlycoFibro Test), when trying to distinguish cirrhosis with the combination of the two tests, a sensitivity of 79% and specificity were obtained of 86%. On the other hand, the same modifications in serum proteins (glycosylation) appear continuously in all liver diseases, (Blomme B, Van Steenkiste C, Callewaert N, Van Vlierberghe H.2009. Alteration of protein glycosylation in liver diseases. J Hepatol 50 (3): 592-603), so it is necessary to expand these prospective studies to determine the clinical application of these techniques. In addition, phosphoproteomics can improve the knowledge of the pathogenesis of liver fibrosis, using the phosphorylated profiles of the proteins that participate in the signaling pathways of this process, as demonstrated by (Younossi ZM, Baranova A, Stepanova M, Page S , Calvert VS, Afendy A, Goodman Z, Chandhoke V, Liotta L, Petricoin E. 2010. Phosphoproteomic biomarkers predicting histologic nonalcoholic steatohepatitis and fibrosis. J Proteome Res. 9 (6): 3218-24), whose results suggest that biomarkers Phosphoproteics could potentially be used in a clinical setting to identify patients with NASH. In addition, they provide information about the metabolic pathways that may be involved in the pathogenesis of the disease.
Es importante destacar que la mayoría de las pruebas no invasivas, no son capaces de diferenciar con certeza etapas tempranas de la fibrosis. De hecho, la mayoría de estas pruebas pueden distinguir principalmente cirrosis de fibrosis mínima. A la fecha, las pruebas no invasivas para el diagnóstico de fibrosis significativa (F>2) no pueden sustituir a la biopsia. Por lo tanto, la utilidad actual del diagnóstico no invasivo sigue siendo limitado, ya que solamente permite al médico reducir la población de pacientes candidatos a una biopsia hepática. It is important to highlight that most non-invasive tests are not able to differentiate with certainty early stages of fibrosis. In fact, most of these tests can distinguish mainly cirrhosis from fibrosis. minimum To date, non-invasive tests for the diagnosis of significant fibrosis (F> 2) cannot replace the biopsy. Therefore, the current utility of non-invasive diagnosis remains limited, since it only allows the physician to reduce the population of patients who are candidates for liver biopsy.
E! manejo exitoso dei tratamiento en las enfermedades crónicas del hígado depende de la estad ificación correcta de la fibrosis. Con el fin de proporcionar los medios para el diagnóstico y seguimiento de la enfermedad y su respuesta a la terapia, se requiere de la realización de pruebas reproducibles y no invasivas. El proceso de fibrogénesis es una respuesta común del hígado, cuando se presenta una lesión crónica producida por una variedad de agresiones, como parte del desarrollo de la enfermedad (Friedman SL. 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 275(4): 2247- 2250), lo que ha dificultado la búsqueda adecuada de biomarcadores específicos, y se ha convertido en un reto en la hepatología traslacional. AND! Successful management of treatment in chronic liver diseases depends on the correct staging of fibrosis. In order to provide the means for the diagnosis and monitoring of the disease and its response to therapy, reproducible and non-invasive tests are required. The fibrogenesis process is a common response of the liver, when there is a chronic injury caused by a variety of attacks, as part of the development of the disease (Friedman SL. 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury J Biol Chem. 275 (4): 2247-2250), which has hindered the proper search for specific biomarkers, and has become a challenge in translational hepatology.
Prevalece por lo tanto una necesidad urgente de disponer de marcadores de fibrosis hepática específicos, mediante métodos no invasivos por diversas razones, una de ellas es la existencia de más de 170 millones de pacientes infectados con el virus de la hepatitis C (HCV) en todo el mundo, que corresponden a un 3% de la población mundial. Por otra parte, actualmente no se conoce un ensayo sérico estándar, estudio de imagen, o análisis virológico, que pueda distinguir a aquéllos individuos que estén en riesgo de padecer fibrosis progresiva, sin ser expuestos a los riesgos potenciales, tanto por inconveniencia y costo de la biopsia hepática y de su interpretación. Si existiera un análisis confiable, no invasor, para excluir certeramente la posibilidad de desarrollar fibrosis significativa, tales pacientes podrían ser monitoreados regularmente para confirmar la ausencia de progresión de la fibrosis. Sobre todo con la expectativa del desarrollo de terapias antifibróticas que abre la necesidad de la supervisión temprana y la regulación de la respuesta a dichas terapias para establecer su eficacia y optimizar su dosificación. Esta necesidad de supervisión rebasa la posibilidad de realizar la biopsia hepática percutánea o transyugular (Gressner Olav A., Ralf Weiskirchen, Gressner Axel M., 2007. Biomarkers of liver fibrosis: Clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Clínica Chimica Acta. 381 : 107-113). There is therefore an urgent need to have specific markers of hepatic fibrosis, through non-invasive methods for various reasons, one of them is the existence of more than 170 million patients infected with hepatitis C virus (HCV) throughout the world, which correspond to 3% of the world's population. On the other hand, there is currently no known standard serum test, imaging study, or virological analysis, which can distinguish those individuals who are at risk of progressive fibrosis, without being exposed to potential risks, both for inconvenience and cost of liver biopsy and its interpretation. If there is a reliable, non-invasive analysis to accurately exclude the possibility of developing significant fibrosis, such patients could be monitored regularly to confirm the absence of fibrosis progression. Especially with the expectation of the development of antifibrotic therapies that opens the need for early supervision and regulation of the response to these therapies to establish their effectiveness and optimize their dosage. This need for supervision exceeds the possibility of performing percutaneous or transjugular liver biopsy (Gressner Olav A., Ralf Weiskirchen, Gressner Axel M., 2007. Biomarkers of liver fibrosis: Clinical translation of molecular pathogenesis or based on liver-dependent malfunction tests. Chimica Clinic Acta. 381: 107-113).
Tanto los avances de la biología molecular, como el desarrollo de modelos de ratones transgénicos, y la utilización de cultivos celulares de diferentes tipos, han permitido la determinación de funciones patogénicas específicas de células individuales durante los procesos fibróticos. The advances in molecular biology, as well as the development of transgenic mouse models, and the use of cell cultures of different types, have allowed the determination of specific pathogenic functions of individual cells during fibrotic processes.
No existe un modelo experimental que reproduzca exactamente la fibrosis hepática humana por etiología; no obstante, cada uno de los modelos desarrollados ha servido para acentuar nuestra comprensión de los mecanismos patogénicos de la fibrosis en el órgano. Se han obtenido resultados importantes derivados de diversos modelos, el mejor ejemplo, es el esclarecimiento de la función de las células estelares hepáticas en el proceso de fibrogénesis. La implicación de éstas células en este proceso fue observada constantemente en los modelos experimentales, sin importar si el estímulo fibrogénico fuese alimenticio, hepatotóxico o inmunológico. Los mecanismos celulares y moleculares de la activación de las HSC entre otros, han comenzado a ser explorados en diversos modelos. Otro ejemplo es el papel de TGF-β en la fibrogénesis del hígado (Schuppan D., Ruehl M., Somasundaram R., Hahn E.G., 2001. Matrix as a modulator of hepatic Fibrogénesis. Semin Liver Dis. 21 (3): 351-372); (Tsukamoto H, Matsuoka M, French SW. 1990 Experimental models of hepatic fibrosis: a review. Semin Liver Dis. : 56-65) There is no experimental model that exactly reproduces human liver fibrosis by etiology; However, each of the models developed has served to accentuate our understanding of the pathogenic mechanisms of fibrosis in the organ. Important results derived from various models have been obtained, the best example is to clarify the role of hepatic stellar cells in the fibrogenesis process. The involvement of these cells in this process was constantly observed in experimental models, regardless of whether the fibrogenic stimulus was nutritional, hepatotoxic or immunological. The cellular and molecular mechanisms of HSC activation among others, have begun to be explored in various models. Another example is the role of TGF-β in liver fibrogenesis (Schuppan D., Ruehl M., Somasundaram R., Hahn EG, 2001. Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis. 21 (3): 351 -372); (Tsukamoto H, Matsuoka M, French SW. 1990 Experimental models of hepatic fibrosis: a review. Semin Liver Dis.: 56-65)
Un modelo experimental de cirrosis hepática empleado con frecuencia es la respuesta del hígado a la droga hepatotóxica tetracloruro de carbono (CCI4), la cual induce peroxidación de los lípidos de la membrana del hepatocito y dependiendo de la dosis, tiempo de exposición, o la edad de los animales empleados, puede resultar en la regresión del proceso, con la recuperación casi completa del hígado (Friedman SL. 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 275(4): 2247-2250). La cirrosis experimental inducida por CCU parece reproducir superficialmente las características principales de la enfermedad humana, el hígado es nodular, hay indicios de hipertensión portal y la arquitectura normal es substituida por nodulos de células hepáticas en regeneración, rodeadas por tabiques de tejido conjuntivo con proliferación de conductos biliares y desarrollo de anastomosis porta-cava tal como ocurre en el hombre. Es un potente hepatotóxico, el cual, con una sola dosis conduce rápidamente a la necrosis y esteatosis graves. La necrosis hepática causada por el CCI4 se debe a la producción de radicales libres generados a partir de la vía del citocromo P450 (CYP2E1). La unión covalente de tricloruro de metilo a proteínas celulares está considerada como el primer paso entre los eventos secuenciales que producen la peroxidación lipídica de la membrana, cuyo punto final es la necrosis celular (Zimmerman, H. J., 1999. Hepatotoxicity: The Adverse Effects of Drugs and Other Chemicals on the Liver, Drug- and chemical-induced cholestasis. Toxicol Lett. 8; 105(1 ): 39-46; Rosenberg W, Burt A, Becka M, Voelker M, Arthur MJP. 2000. Automated assays of serum markers of liver fibrosis predict histologic hepatic fibrosis. Hepatology. 32: 183A). An experimental model of liver cirrhosis frequently used is the liver's response to the hepatotoxic drug carbon tetrachloride (ICC 4 ), which induces peroxidation of hepatocyte membrane lipids and depending on the dose, exposure time, or The age of the animals used can result in the regression of the process, with the almost complete recovery of the liver (Friedman SL. 2000. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 275 (4): 2247-2250). Experimental cirrhosis induced by CCU seems to superficially reproduce the main characteristics of human disease, the liver is nodular, there are indications of portal hypertension and the normal architecture is replaced by nodules of regenerating liver cells, surrounded by connective tissue septa with proliferation of bile ducts and development of porta-cava anastomosis as occurs in man. It is a potent hepatotoxic, which, with a single dose, quickly leads to severe necrosis and steatosis. Hepatic necrosis caused by ITC 4 is due to the production of free radicals generated from the cytochrome P450 pathway (CYP2E1). Covalent binding of methyl trichloride to cellular proteins is considered the first step between sequential events that produce lipid peroxidation of the membrane, whose end point is cellular necrosis (Zimmerman, HJ, 1999. Hepatotoxicity: The Adverse Effects of Drugs and Other Chemicals on the Liver, Drug- and chemical-induced cholestasis.Toxicol Lett. 8; 105 (1): 39-46; Rosenberg W, Burt A, Becka M, Voelker M, Arthur MJP. 2000. Automated assays of serum markers of liver fibrosis predict histologic hepatic fibrosis. Hepatology. 32: 183A).
La tioacetamida (TAA) es otro hepatotóxico potente que requiere ser activado metabólicamente por diversas oxidasas. La TAA aparentemente se convierte en óxido de tioacetamida que a su vez se transforma en un metabolito tóxico activo que se une covalentemente a moléculas tisulares, induciendo la necrosis (Zimmerman, H. J., 1999. Hepatotoxicity: The Adverse Effects of Drugs and Other Chemicals on the Liver, Drug- and chemical-induced cholestasis. Toxicol Lett. 8; 105(1):39-46). Thioacetamide (TAA) is another potent hepatotoxic that needs to be metabolically activated by various oxidases. TAA apparently converts to thioacetamide oxide which in turn transforms into an active toxic metabolite that covalently binds to tissue molecules, inducing necrosis (Zimmerman, HJ, 1999. Hepatotoxicity: The Adverse Effects of Drugs and Other Chemicals on the Liver, Drug- and chemical-induced cholestasis Toxicol Lett. 8; 105 (1): 39-46).
La disponibilidad de los modelos animales es crucial para el estudio de la fibrosis hepática. The availability of animal models is crucial for the study of liver fibrosis.
Microarreglos: Las tecnologías utilizadas para la búsqueda de biomarcadores incluyen análisis tradicionales ¡n vitro de las variaciones y expresión del DNA, RNA y proteínas, y la cuantificacíón de metabolitos, así como las mediciones in vivo de los procesos biológicos en seres humanos y animales, utilizando imágenes morfológicas y tecnologías funcionales. La importancia de las mediciones se correlaciona con su capacidad de predicción para los puntos clínicamente relevantes, tales como el pronóstico de la enfermedad, y la respuesta a la terapéutica, por mencionar algunos ejemplos. Microarrays: The technologies used to search for biomarkers include traditional in vitro analyzes of the variations and expression of DNA, RNA and proteins, and the quantification of metabolites, as well as in vivo measurements of biological processes in humans and animals, using morphological images and functional technologies. The importance of the measurements is correlated with its prediction capacity for the points clinically relevant, such as the prognosis of the disease, and the response to therapy, to name a few examples.
A finales de 1990, surgió la tecnología de microarreglos de DNA como una poderosa herramienta para el análisis de los niveles de los transcritos de RNAm expresados en diversas situaciones. Los oligonucleótidos pueden ser impresos en un chip por métodos fotolitográficos. Los arreglos de DNA son utilizados para analizar la expresión de genes, monitorizándose los niveles de miles de ellos de forma simultánea permitiendo obtener información acerca de la muestra que se esté trabajando (Schena M, Shalon D, Davis RW, Brown PO. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 270 (5235): 467-70). In the late 1990s, DNA microarray technology emerged as a powerful tool for the analysis of mRNA transcript levels expressed in various situations. The oligonucleotides can be printed on a chip by photolithographic methods. DNA arrays are used to analyze gene expression, monitoring the levels of thousands of them simultaneously allowing to obtain information about the sample being worked on (Schena M, Shalon D, Davis RW, Brown PO. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. 270 (5235): 467-70).
Los productos de PCR también pueden ser vistos en los chips de microarreglos de DNA. A pesar de que sus valores no siempre se correlacionan, la información sobre los niveles de expresión del RNAm y la abundancia de la proteína correspondiente (o su actividad) son sin duda útiles en el análisis genómico (Tomizaki in-ya, Kenji Usui and Hisakazu Mihara, 2010. Protein-protein interactions and selection: array-based techniques for screening disease-associated biomarkers in predictive/early diagnosis, doi: 10.1111/j. 1742-4658). PCR products can also be seen in the DNA microarray chips. Although their values do not always correlate, information on mRNA expression levels and the corresponding protein abundance (or its activity) are certainly useful in genomic analysis (Tomizaki in-ya, Kenji Usui and Hisakazu Mihara, 2010. Protein-protein interactions and selection: array-based techniques for screening disease-associated biomarkers in predictive / early diagnosis, doi: 10.1111 / j. 1742-4658).
El RNA total se aisla de cada muestra seguida de diversos pasos para enriquecer y en algunos casos para amplificar cRNAs. Este material, cDNA o en el caso de la amplificación cRNA, se etiqueta directa o indirectamente generando un cDNA en una reacción de transcripción inversa con nucleótidos marcados y se hibridan en un microarreglo, el cambio en la expresión de los genes se determina por medio de la cuantificación de la fluorescencia emitida por cada punto del microarreglo (híbrido formado) (Harm van Bakel and Frank CP. Holstege, 2008. A Tutorial for DNA Microarray Expression Profiling. Cell press: 22-28). Total RNA is isolated from each sample followed by various steps to enrich and in some cases to amplify cRNAs. This material, cDNA or in the case of cRNA amplification, is directly or indirectly labeled generating a cDNA in a reverse transcription reaction with labeled nucleotides and hybridizes in a microarray, the change in gene expression is determined by means of the quantification of the fluorescence emitted by each point of the microarray (hybrid formed) (Harm van Bakel and Frank CP. Holstege, 2008. A Tutorial for DNA Microarray Expression Profiling. Cell press: 22-28).
Estos datos pueden analizarse utilizando los programas y bases de datos que se han diseñado para contener la información resultante de la expresión de los microarreglos, entre las más conocidas se encuentran SAM (Significance Analysis of Microarrays, Flex Array), DAVID (The Datábase for Annotation, Visualizaron and Integrated Discovery), Ingenuity, Ace View por mencionar algunas. La tecnología de microarreglos es capaz de detectar simultáneamente los niveles de expresión relativos de un gran número de genes y fragmentos génicos; tales experimentos pueden ayudar a identificar los vínculos entre fenotipos específicos y patrones relacionados de la expresión génica. This data can be analyzed using the programs and databases that have been designed to contain the information resulting from the expression of the microarrays, among the best known are SAM (Significance Analysis of Microarrays, Flex Array), DAVID (The Datábase for Annotation, Visualized and Integrated Discovery), Ingenuity, Ace View to name a few. Microarray technology is capable of simultaneously detecting the relative expression levels of a large number of genes and gene fragments; Such experiments can help identify the links between specific phenotypes and related patterns of gene expression.
Un estudio de expresión génica en la enfermedad de hígado graso no alcohólico (NAFLD) indicó la sobreexpresión significativa del receptor de FAS en los hepatocitos de enfermos con esteatohepatitis no alcohólica (NASH) acompañado por la activación de caspasas y un aumento en cantidad de células apoptóticas (TUNEL positivas), así como la activación del factor de transcripción NF-kB que se correlaciona con la presencia de inflamación hepática y fibrosis, pero no con esteatosis (Ribeiro PS, Cortez-Pinto H, Solá S, Castro RE, Ramalho RM, Baptista A, Moura MC, Camilo ME, Rodrigues CM. 2004. Hepatocyte apoptosis, expression of death receptors, and activation of NF-kappaB ¡n the Iiver of nonalcoholic and alcoholic steatohepatitis patients. Am J Gastroenterol. 99: 1708- 17). La expresión génica relacionada con el carcinoma hepatocelular (HCC) ha sido estudiada mediante esta técnica ya que esta patología (HCC, está asociada con diferentes enfermedades hepáticas incluyendo el NASH. Por ejemplo, un estudio llevado a cabo por Kim y cois, 2004 (Kim JW, Ye Q, Forgues M, Chen Y, Budhu A, Sime J, Hofseth LJ, Kaul R, Wang XW. 2004. Cancer-associated molecular signature in the tissue samples of patients with cirrhosis. Hepatology. 39: 518-27) reveló cambios específicos en la expresión génica en muestras de hígado de personas con cirrosis en etapa terminal, indicando que distintas enfermedades crónicas hepáticas pueden representar una "condición previa" del tejido hepático para la malignidad, Smith y cois. (Smith MW, Yue ZN, Korth MJ, Do HA, Boix L, Fausto N, Bruix J, Carithers RL Jr, Katze MG. 2003. Hepatitis C virus and liver disease: global transcriptional profiling and Identification of potential markers. Hepatology. 38: 1458-67) utilizando el mismo enfoque identificaron un conjunto de genes con variaciones fisiológicas normales en el hígado humano. Este grupo de genes relacionados con la tumorigénesis fueron seleccionados a partir de un sistema de genes diferencialmente expresados en la cirrosis, lo que condujo a la identificación de 132 moléculas como marcadoras potenciales. A study of gene expression in nonalcoholic fatty liver disease (NAFLD) indicated significant overexpression of the FAS receptor in hepatocytes of patients with non-alcoholic steatohepatitis (NASH) accompanied by caspases activation and an increase in the amount of apoptotic cells (TUNEL positive), as well as the activation of the transcription factor NF-kB that correlates with the presence of liver inflammation and fibrosis, but not with steatosis (Ribeiro PS, Cortez-Pinto H, Solá S, Castro RE, Ramalho RM, Baptista A, Moura MC, Camilo ME, Rodrigues CM. 2004. Hepatocyte apoptosis, expression of death receptors, and activation of NF-kappaB in the Iiver of nonalcoholic and alcoholic steatohepatitis patients. Am J Gastroenterol. 99: 1708-17). Gene expression related to hepatocellular carcinoma (HCC) has been studied using this technique since this pathology (HCC) is associated with different liver diseases including NASH, for example, a study carried out by Kim and cois, 2004 (Kim JW, Ye Q, Forgues M, Chen Y, Budhu A, Sime J, Hofseth LJ, Kaul R, Wang XW. 2004. Cancer-associated molecular signature in the tissue samples of patients with cirrhosis. Hepatology. 39: 518-27) revealed specific changes in gene expression in liver samples of people with end-stage cirrhosis, indicating that different chronic liver diseases may represent a "precondition" of liver tissue for malignancy, Smith and cois. (Smith MW, Yue ZN, Korth MJ, Do HA, Boix L, Fausto N, Bruix J, Carithers RL Jr, Katze MG. 2003. Hepatitis C virus and liver disease: global transcriptional profiling and Identification of potential markers. Hepatology. 38: 1458-67) using the mism or approach identified a set of genes with normal physiological variations in the human liver. This group of genes related to tumorigenesis were selected from a differentially expressed gene system in cirrhosis, which led to the identification of 132 molecules as potential markers.
La mayor parte de los estudios realizados con esta herramienta han sido efectuados a partir de muestras humanas, pocos en modelos animales (Walters Kathie-Anne, Michael A Joyce, Jill C Thompson, Sean Proll, James Wallace, María W Smith, Jeff Furlong, D Lome Tyrrelland Michael G Katze. 2006. Application of functional genomics to the chimeric mouse model of HCV infection: optimization of microarray protocols and genomics analysis. Virology Journal. 3:37). Most of the studies carried out with this tool have been made from human samples, few in animal models (Walters Kathie-Anne, Michael A Joyce, Jill C Thompson, Sean Proll, James Wallace, Maria W Smith, Jeff Furlong, D Lome Tyrrelland Michael G. Katze. 2006. Application of functional genomics to the chimeric mouse model of HCV infection: optimization of microarray protocols and genomics analysis. Virology Journal. 3:37).
Sumario de la Invención Summary of the Invention
La importancia de la presente invención radica en que el diseño del panel de biomarcadores no invasivos para diagnóstico y estadificación del proceso de fibrosis hepática, fue obtenido a partir de un modelo experimental y de ahí pasó a ser una exitosa aplicación en el diagnóstico y estadificación de la enfermedad humana. The importance of the present invention is that the design of the panel of non-invasive biomarkers for diagnosis and staging of the hepatic fibrosis process, was obtained from an experimental model and from there it became a successful application in the diagnosis and staging of human disease
Un objetivo de la presente invención es subsanar todos los inconvenientes que se presentan para poder diagnosticar la fibrosis hepática mediante el uso de un panel de biomarcadores no invasivos, pudiendo inclusive establecer el diagnóstico durante las fases tempranas de la enfermedad hepática. An objective of the present invention is to overcome all the inconveniences that arise in order to diagnose liver fibrosis through the use of a panel of non-invasive biomarkers, even being able to establish the diagnosis during the early phases of liver disease.
Otro objetivo de la presente invención, es permitir la estadificación de la fibrosis hepática, mediante el uso del panel de biomarcadores no invasivos, lo que facultará para establecer el tratamiento más efectivo. Another objective of the present invention is to allow the staging of liver fibrosis, through the use of the panel of non-invasive biomarkers, which will enable the establishment of the most effective treatment.
Asimismo otro más de los aspectos del uso del panel de biomarcadores de la presente invención, es permitir al médico tratante de la enfermedad hepática que cursa con fibrosis, contar con elementos científicos no invasivos para evaluar la efectividad del tratamiento aplicado, posibilitando además el seguimiento y evolución del padecimiento. Breve descripción de las figuras: Likewise, another aspect of the use of the biomarker panel of the present invention is to allow the treating physician of the liver disease that develops with fibrosis, to have non-invasive scientific elements to evaluate the effectiveness of the applied treatment, also allowing the monitoring and evolution of the condition Brief description of the figures:
La Figura 1 , muestra el diagrama de flujo seguido durante el desarrollo de los microarreglos de expresión a partir del RNA total obtenido del hígado de todos los animales.  Figure 1 shows the flow chart followed during the development of expression microarrays from the total RNA obtained from the liver of all animals.
RNA total (gris) obtenido del hígado de los animales  Total RNA (gray) obtained from animal liver
1a. cadena de cDNA poliadenilado (AAAAA3'), sintetizado por transcripción inversa (negro) 1 a . polyadenylated cDNA chain (AAAAA3 '), synthesized by reverse transcription (black)
cDNA de doble cadena final poliadenilado (negro)  Polyadenylated final double chain cDNA (black)
RNA (gris) marcado con biotina obtenido a partir del cDNA de doble cadena Biotin-labeled RNA (gray) obtained from double stranded cDNA
RNA (gris) fragmentado marcado con biotina (negro) Fragmented (gray) RNA labeled with biotin (black)
Proceso de hibridación en el Chip  Chip Hybridization Process
Lavado del Chip  Chip Wash
Revelado con Estreptavidina  Revealed with Streptavidin
Escaneo del chip y análisis final de los resultados  Chip scanning and final analysis of the results
Las Figuras 2a, 2b, 2c y 2d muestran imágenes representativas de cortes de tejido hepático provenientes de animales normales, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (2a, 2b) y RS (2c, 2d). Los hepatocitos son poligonales, con citoplasma granular y núcleo central, redondo y con nucléolo prominente. Los sinusoides se hayan tapizados por células elongadas (células endoteliales y de Kupffer), próximas a los hepatocitos, quedando el espacio de Disse como una región casi virtual. (La magnitud de la amplificación se detalla en cada imagen) Figures 2a, 2b, 2c and 2d show representative images of liver tissue sections from normal animals, fixed with buffered formalin and included in paraffin, stained with H&E (2a, 2b) and RS (2c, 2d). Hepatocytes are polygonal, with granular cytoplasm and central nucleus, round and with prominent nucleoli. Sinusoids have been upholstered by elongated cells (endothelial and Kupffer cells), close to hepatocytes, leaving the Disse space as an almost virtual region. (The magnitude of the amplification is detailed in each image)
Las Figuras 3a, 3b, 3c y 3d muestran imágenes representativas de cortes de tejido hepático provenientes de animales de la Fase I del Modelo Experimental, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (3a, 3b) y Rojo de Sirio (3c, 3d). En este grupo de animales se observa el inicio de alteración estructural debido a la presencia de bandas de fibrosis periportal (bf) y múltiples vesículas pequeñas de esteatosis (e) con núcleos que conservan su posición central (n). (La magnitud de la amplificación se detalla en cada imagen) Las Figuras 4a, 4b, 4c, y 4d muestran imágenes representativas de cortes de tejido hepático provenientes de animales de la Fase II del Modelo Experimental, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (4a, 4b) y RS (4c, 4d). En este grupo de animales se observan zonas con bandas gruesas y delgadas de fibrosis periportal (bf), lagunas de esteatosis (e) con vesículas redondeadas, grandes y pequeñas en hepatocitos centrolobulillares. En el citoplasma de los hepatocitos hay zonas vacías con desplazamiento de los núcleos a la periferia (n). (La magnitud de la amplificación se detalla en cada imagen) Figures 3a, 3b, 3c and 3d show representative images of liver tissue cuts from animals of Phase I of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (3a, 3b) and Sirius Red ( 3c, 3d). In this group of animals the onset of structural alteration is observed due to the presence of periportal fibrosis bands (bf) and multiple small steatosis vesicles (e) with nuclei that retain their central position (n). (The magnitude of the amplification is detailed in each image) Figures 4a, 4b, 4c, and 4d show representative images of liver tissue sections from animals of Phase II of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (4a, 4b) and RS (4c , 4d). In this group of animals, areas with thick and thin bands of periportal fibrosis (bf), steatosis lagoons (e) with rounded vesicles, large and small, are observed in centrolobular hepatocytes. In the cytoplasm of hepatocytes there are empty areas with displacement of the nuclei to the periphery (n). (The magnitude of the amplification is detailed in each image)
Las Figuras 5a, 5b, 5c y 5d muestran imágenes representativas de cortes de tejido hepático provenientes de animales de la Fase III del Modelo Experimental, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (5a, 5b) y RS (5c, 5d). En este grupo de animales ya es aparente la presencia de cirrosis, con nodulos de regeneración rodeados por bandas gruesas de tejido fibrótico que van de vena porta a vena centrolobulillar y hepatocitos binucleados (bn) de mayor tamaño. (La magnitud de la amplificación se detalla en cada imagen) Figures 5a, 5b, 5c and 5d show representative images of liver tissue sections from animals of Phase III of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (5a, 5b) and RS (5c, 5 d). In this group of animals the presence of cirrhosis is already apparent, with regeneration nodules surrounded by thick bands of fibrotic tissue that go from portal vein to centrolobular vein and larger binucleated hepatocytes (bn). (The magnitude of the amplification is detailed in each image)
Las Figuras 6a, 6b, 6c y 6c muestran imágenes representativas de cortes de tejido hepático provenientes de animales de la Fase IV del Modelo Experimental, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (6a, 6b) y RS (6c, 6d). En este grupo de animales la arquitectura hepática presenta un estado cirrótico con zonas de necrosis focal, esteatosis mínima (e), nodulos de regeneración rodeados por bandas de tejido fibrótico (bf) de menor grosor, que van de vena porta a vena centrolobulillar. Hepatocitos con hipercromatismo nuclear, enucleación (an), binucleación (bn) y pérdida de la relación núcleo-citoplasma. (La magnitud de la amplificación se detalla en cada imagen) Figures 6a, 6b, 6c and 6c show representative images of liver tissue sections from animals of Phase IV of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (6a, 6b) and RS (6c, 6d). In this group of animals the liver architecture presents a cirrhotic state with areas of focal necrosis, minimal steatosis (e), regeneration nodules surrounded by bands of fibrotic tissue (bf) of smaller thickness, ranging from portal vein to centrolobular vein. Hepatocytes with nuclear hyperchromatism, enucleation (an), binucleation (bn) and loss of the nucleus-cytoplasm relationship. (The magnitude of the amplification is detailed in each image)
Las Figuras 7a, 7b, 7c y 7d muestran imágenes representativas de cortes de tejido hepático provenientes de animales de la Fase V del Modelo Experimental, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (7a, 7b) y RS (7c, 7d). En este grupo de animales la arquitectura hepática presenta un estado cirrótico con nula presencia de esteatosis, nodulos de regeneración rodeados por bandas finas de tejido fibrótico (bf) que van de vena porta a vena centrolobulillar. Se observa (an) enucleación, binucleación (bn) hipercromatismo nuclear, y pérdida de la relación núcleo-citoplasma. (La magnitud de la amplificación se detalla en cada imagen) Figures 7a, 7b, 7c and 7d show representative images of liver tissue sections from Phase V animals of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (7a, 7b) and RS (7c, 7d). In this group of animals the hepatic architecture presents a cirrhotic state with no presence of steatosis, regeneration nodules surrounded by thin bands of fibrotic tissue (bf) that go from portal vein to centrolobular vein. There is (an) enucleation, binucleation (bn) nuclear hyperchromatism, and loss of the nucleus-cytoplasm relationship. (The magnitude of the amplification is detailed in each image)
La Figura 8 es una representación gráfica de la determinación de la concentración de colágena promedio por cada Fase. Figure 8 is a graphical representation of the determination of the average collagen concentration for each Phase.
Las Figuras 9a, 9b y 9c muestran el análisis de la intensidad de expresión en el microarreglo de los genes seleccionados como los candidatos para formar parte del panel molecular de diagnóstico de fibrosis. Figures 9a, 9b and 9c show the analysis of the intensity of expression in the microarray of the genes selected as the candidates to be part of the fibrosis diagnostic molecular panel.
La Figura 10 es una representación del procedimiento de validación seguido para obtener las proteínas que integran el Panel Molecular para el Diagnóstico de Fibrosis, con énfasis en fibrosis temprana. Figure 10 is a representation of the validation procedure followed to obtain the proteins that make up the Molecular Panel for the Diagnosis of Fibrosis, with an emphasis on early fibrosis.
La Figura 11 muestra la interpretación de la Curva ROC Figure 11 shows the interpretation of the ROC Curve
Las Figuras 12a y 12b muestran las gráficas resultantes del análisis estadístico (prueba de t, para el (12a). ELISA y (12b). Curva Roe para MFAP-4. Figures 12a and 12b show the graphs resulting from the statistical analysis (t-test, for (12a). ELISA and (12b). Roe curve for MFAP-4.
Las Figuras 13a y 13b muestran las gráficas resultantes del análisis estadístico (prueba de t, para el (13a). ELISA y (13b). Curva Roe para FBN1. Figures 13a and 13b show the graphs resulting from the statistical analysis (t-test, for (13a). ELISA and (13b). Roe curve for FBN1.
Las Figuras 14a y 14b muestran las gráficas resultantes del análisis estadístico (prueba de t, para el (14a). ELISA y (14b). Curva Roe para ALDH1A3. Figures 14a and 14b show the graphs resulting from the statistical analysis (t-test, for (14a). ELISA and (14b). Roe curve for ALDH1A3.
Las Figuras 15a y 15b muestran las gráficas resultantes del análisis estadístico (prueba de t, para el (15a). ELISA y (15b). Curva Roe para CYP26A1. Figures 15a and 15b show the graphs resulting from the statistical analysis (t-test, for (15a). ELISA and (15b). Roe curve for CYP26A1.
Las Figuras 16a y 16b muestran las gráficas resultantes del análisis estadístico (prueba de t, para el (16a). ELISA y (16b). Curva Roe para MGP. Las Figuras 17a y 17b muestran las gráficas resultantes del análisis estadístico (prueba de t, para el (17a). ELISA y (17b). Curva Roe para C7. Figures 16a and 16b show the graphs resulting from the statistical analysis (t-test, for (16a). ELISA and (16b). Roe curve for MGP. Figures 17a and 17b show the graphs resulting from the statistical analysis (t-test, for (17a). ELISA and (17b). Roe curve for C7.
La Figura 18a y 18b muestran las gráficas resultantes del análisis estadístico (prueba de t, para el (18a). ELISA y (18b). Curva Roe para PLA2G7. Figure 18a and 18b show the graphs resulting from the statistical analysis (t-test, for (18a). ELISA and (18b). Roe curve for PLA2G7.
La Figura 19 es una representación gráfica de la combinación de los resultados obtenidos con las 7 moléculas del panel. Donde cada línea representa el promedio de los valores obtenidos para cada proteína, mediante la prueba de ELISA empleando el suero de los pacientes de cada uno de los grupos analizados, Ctl (sujetos sanos), F1 (Fibrosis inicial), F4 (Cirrosis), FP (Fibrosis Pulmonar) y AHA (Absceso hepático amibiano). Figure 19 is a graphic representation of the combination of the results obtained with the 7 panel molecules. Where each line represents the average of the values obtained for each protein, using the ELISA test using the serum of the patients of each of the analyzed groups, Ctl (healthy subjects), F1 (Initial fibrosis), F4 (Cirrhosis), FP (Pulmonary Fibrosis) and AHA (Amibian liver abscess).
Descripción detallada de la invención. Detailed description of the invention.
La presente invención se refiere a la construcción de un panel de biomarcadores moleculares no invasivos para diagnóstico y estadificación del proceso de fibrosis que se presenta en la enfermedad hepática crónica, obtenido y seleccionado a partir de la expresión alterada de diversas proteínas, observada en el transcurso de la inducción de fibrosis-cirrosis en modelos experimentales (CCI4 y TAA) desarrollados para reproducir la enfermedad, que puede ser empleado para determinar el diagnóstico y/o pronóstico en las diferentes etapas del proceso de fibrosis hepática. En la presente invención se identificaron y validaron los genes que sufren alteraciones a lo largo del proceso inductivo de fibrosis hepática y se correlacionaron con las vías metabólicas en las que intervienen, obteniéndose un perfil molecular para ser utilizado en el desarrollo de una prueba pronostica y/o diagnóstica para fibrosis hepática. Las proteínas cuyos genes sufrieron alteraciones a lo largo del desarrollo de los modelos experimentales, se seleccionaron como candidatas y las más significativas, se validaron obteniéndose un perfil molecular que puede ser empleado como una prueba pronostica y/o diagnóstica no invasiva para fibrosis hepática. The present invention relates to the construction of a panel of non-invasive molecular biomarkers for diagnosis and staging of the fibrosis process that occurs in chronic liver disease, obtained and selected from the altered expression of various proteins, observed in the course of the induction of fibrosis-cirrhosis in experimental models (ICC 4 and TAA) developed to reproduce the disease, which can be used to determine the diagnosis and / or prognosis at different stages of the liver fibrosis process. In the present invention the genes that suffer alterations throughout the inductive process of liver fibrosis were identified and validated and correlated with the metabolic pathways in which they intervene, obtaining a molecular profile to be used in the development of a prognostic test and / or diagnosis for liver fibrosis. The proteins whose genes underwent alterations throughout the development of the experimental models, were selected as candidates and the most significant, were validated obtaining a molecular profile that can be used as a non-invasive prognostic and / or diagnostic test for liver fibrosis.
El modelo experimental brevemente consistió de: a) La fibrosis hepática se indujo en ratas machos a través de la inyección intraperitoneal de CCI4. The experimental model briefly consisted of: a) Liver fibrosis was induced in male rats through intraperitoneal injection of CCI 4 .
b) Posteriormente se obtuvieron muestras de tejido hepático para el procesamiento de microarreglos, para el análisis histopatológico y determinación cuantitativa del depósito de colágena. b) Subsequently, liver tissue samples were obtained for microarray processing, for histopathological analysis and quantitative determination of collagen deposition.
c) De las muestras se obtuvo el RNA y a partir de éste se sintetizó el cDNA para la hibridación en el microarreglo. Los datos obtenidos del microarreglo, se analizaron en los programas SAM y DAVID. c) RNA was obtained from the samples and from it the cDNA was synthesized for hybridization in the microarray. The data obtained from the microarray were analyzed in the SAM and DAVID programs.
d) Se validaron los genes seleccionados en el análisis de expresión. d) The genes selected in the expression analysis were validated.
e) Empleando el método de ELISA, se determinó el grado de expresión de los productos de traducción de 21 genes candidatos, seleccionados a partir del análisis del microarreglos de expresión, en el suero obtenido de pacientes con enfermedad hepática crónica del Departamento de Gastroenterología Hospital de Especialidades del IMSS (n= 30), Fibrosis pulmonar idiopática (FPI, n=32) donados por el Dr. Moisés Selman Lama, División de Investigación del Instituto Nacional de Enfermedades Respiratorias, y sueros de individuos normales (n=100) que acudieron al banco de Sangre del Hospital General de México, para disponer de controles sanos. e) Using the ELISA method, the degree of expression of the translation products of 21 candidate genes, selected from the analysis of the expression microarrays, in the serum obtained from patients with chronic liver disease of the Department of Gastroenterology Hospital of IMSS Specialties (n = 30), Idiopathic Pulmonary Fibrosis (IPF, n = 32) donated by Dr. Moisés Selman Lama, Research Division of the National Institute of Respiratory Diseases, and sera of normal individuals (n = 100) who attended to the Blood Bank of the General Hospital of Mexico, to have healthy controls.
f) Se determinó la presencia y localización celular de los productos de traducción de los 21 genes candidatos, mediante un ensayo inmunohistoquímico sobre cortes de tejido hepático (n=30), incluidos en parafina, correspondientes a muestras de pacientes con diferente grado de daño, que provienen del archivo de la unidad de Patología del Hospital General de México. f) The presence and cellular localization of the translation products of the 21 candidate genes was determined by an immunohistochemical test on liver tissue sections (n = 30), included in paraffin, corresponding to samples of patients with different degree of damage, that come from the archive of the Pathology unit of the General Hospital of Mexico.
g) Se validaron clínicamente, utilizando los datos obtenidos del ELISA para construir las Curvas ROC, utilizando el programa estadístico SPSS 10.5, para establecer la especificidad y sensibilidad de las moléculas determinadas, analizando el área bajo la curva (AUC), obtener los valores presuntivos positivos y negativos de cada proteína analizada. g) They were clinically validated, using the data obtained from the ELISA to construct the ROC Curves, using the SPSS 10.5 statistical program, to establish the specificity and sensitivity of the determined molecules, analyzing the area under the curve (AUC), obtaining the presumptive values positive and negative of each protein analyzed.
h) Se constituyó el perfil molecular, con base en el cual se elaboró el panel de biomarcadores moleculares de la presente invención, que es utilizado como método no invasivo para el diagnóstico presuntivo y estad ificación de la fibrosis hepática. h) The molecular profile was formed, based on which the molecular biomarker panel of the present invention was developed, which is used as a non-invasive method for presumptive diagnosis and staging of liver fibrosis.
Se emplearon ratas Wistar, machos, de 150-200 g de peso, alimentadas ad libitum con agua y purina y los animales se manejaron siguiendo los lineamientos marcados por el Consejo de Organizaciones Internacionales de Ciencias Médicas y la Norma Oficial Mexicana (NOM-062-ZOO-1999). La cirrosis fue inducida de acuerdo a lo establecido previamente (Pérez-Tamayo R., Montfort I., González E., 1987. Collagenolytic activity in experimental cirrhosis of the liver. Exp Mol Pathol. 47: 300-308) (García de León María del Carmen, Irmgard Montfort, Eusebio Tello Montes, Rosario López Vancell, Alfonso Olivos García, Augusto González Canto, Mario Nequiz-Avendaño, Ruy Pérez-Tamayo. 2006. Hepatocyte production of modulators of extracellular liver matrix in normal and cirrhotic rat liver. Experimental and Molecular Pathology. 80: 97-108). Cada animal fue inyectado vía intraperitoneal (i.p.) con 0.25 mi de CCI4 al 33 % en aceite de olivo, dos veces por semana, durante 20 semanas. Se trabajó con tres grupos de animales: el primero designado como grupo experimental constituido por 25 animales que recibieron el tratamiento con el hepatotóxico; el segundo que se utilizó como grupo control con 15 animales sin tratamiento. Además, un tercer grupo que recibió solamente aceite de oliva cuyo análisis indicó un comportamiento idéntico a nivel hepático a los animales no tratados. Wistar rats, male, 150-200 g in weight, fed ad libitum with water and purine were used and the animals were managed following the guidelines set by the Council of International Organizations of Medical Sciences and the Official Mexican Standard (NOM-062- ZOO-1999). Cirrhosis was induced according to what was previously established (Pérez-Tamayo R., Montfort I., González E., 1987. Collagenolytic activity in experimental cirrhosis of the liver. Exp Mol Pathol. 47: 300-308) (García de León María del Carmen, Irmgard Montfort, Eusebio Tello Montes, Rosario López Vancell, Alfonso Olivos García, Augusto González Canto, Mario Nequiz-Avendaño, Ruy Pérez-Tamayo. 2006. Hepatocyte production of modulators of extracellular liver matrix in normal and cirrhotic rat liver. Experimental and Molecular Pathology. 80: 97-108). Each animal was injected intraperitoneally (ip) with 0.25 ml of CCI 4 at 33% in olive oil, twice a week, for 20 weeks. We worked with three groups of animals: the first designated as an experimental group consisting of 25 animals that received hepatotoxic treatment; the second that was used as a control group with 15 animals without treatment. In addition, a third group that received only olive oil whose analysis indicated identical behavior at the liver level to untreated animals.
Fase I, al 1er mes, 8 inyecciones.  Phase I, at the 1st month, 8 injections.
Fase II, 21 2 meses, 20 inyecciones. Phase II, 2 1 2 months, 20 injections.
Fase III, 5 meses, 40 inyecciones.  Phase III, 5 months, 40 injections.
Fase IV, 30 días posteriores a la suspensión del hepatotóxico (6 meses). Fase V, 180 días posteriores a la suspensión de hepatotóxico (11 meses).  Phase IV, 30 days after hepatotoxic suspension (6 months). Phase V, 180 days after hepatotoxic suspension (11 months).
Al término de cada Fase se tomaron muestras de tejido hepático de cada uno de los animales correspondientes, mismo lóbulo en todos ellos y suero (5 animales), igualmente, de los grupos mantenidos sin tratamiento les fueron tomadas muestras en a 3 animales al término de cada Fase. Las muestras se dividieron en tres fragmentos pequeños, el primero se empleó para la extracción del RNA total, empleado para la elaboración de los microarreglos; con el segundo fragmento una vez fijado e incluido en parafina, y de estos se cortaron las muestras para el análisis histopatológico con tinciones de hematoxilina y eosina (H&E) y rojo de Sirio (RS), y el tercer fragmento se utilizó para el cálculo del contenido de colágena (mediante la técnica de cuantificación de Hidroxi-Prolina (OH-prol, Woessner J F, Jr. 1961. The determination of hydroxyproline ¡n tissue and protein samples containing small proportions of this ¡mino acid. Arch. Biochem. Biophys. 93:440-7), con el fin de monitorear la evolución de la fibrosis en el modelo. At the end of each Phase samples of liver tissue were taken from each of the corresponding animals, same lobe in all of them and serum (5 animals), likewise, from the groups maintained without treatment samples were taken in 3 animals at the end of Each Phase The samples were divided into three small fragments, the first one was used for the extraction of the total RNA, used for the elaboration of the microarrays; with the second fragment once fixed and included in paraffin, and of these the samples were cut for histopathological analysis with stains of hematoxylin and eosin (H&E) and Sirius red (RS), and the third fragment was used for the calculation of collagen content (using the Hydroxy-Proline quantification technique (OH-prol, Woessner JF, Jr. 1961. The determination of hydroxyproline ¡n tissue and protein samples containing small proportions of this mino acid. Arch. Biochem. Biophys. 93: 440 -7), in order to monitor the evolution of fibrosis in the model.
Estas mismas muestras sirvieron para realizar la validación de los posibles i genes alterados (RT-PCR en tiempo real, ELISA e inmunohistoquímica), seleccionados en el análisis de micro arreglos. These same samples were used to validate the possible altered genes (real-time RT-PCR, ELISA and immunohistochemistry), selected in the microarray analysis.
Determinación de la concentración de colágena: La determinación de la concentración de colágena, es una forma práctica de medir la cantidad de MEC principalmente colágena tipo I y III depositadas en el hígado de los animales tratados con CCI4, fue realizada mediante la técnica de Hidroxi-Prolina (OH-prol) (Woessner J F, Jr. 1961. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. Biophys. 93:440-7) empleando muestras de 100 mg de tejido hepático deshidratado i (experimentales n=5, controles n=3; de cada Fase). Determination of the concentration of collagen: The determination of the concentration of collagen, is a practical way to measure the amount of MEC mainly collagen type I and III deposited in the liver of animals treated with CCI 4 , was performed using the Hydroxy technique -Prolina (OH-prol) (Woessner JF, Jr. 1961. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. Biophys. 93: 440-7) using 100 mg samples of dehydrated liver tissue i (experimental n = 5, controls n = 3; of each Phase).
Se preparó una curva estándar de OH-Pro, para extrapolar los datos obtenidos con los animales. Todas las reacciones se leyeron en espectrofotómetro a 557nm. Los datos obtenidos se aplicaron a la siguiente fórmula con la cual obtuvimos la concentración de colágena en miligramos, por mg de tejido. mg Col= (pg de OH-prol / 1000) X 7.42 A standard OH-Pro curve was prepared to extrapolate the data obtained with the animals. All reactions were read by spectrophotometer at 557 nm. The data obtained were applied to the following formula with which we obtained the concentration of collagen in milligrams, per mg of tissue. mg Col = (pg OH-prol / 1000) X 7.42
RNA: Con el RNA total extraído por cada etapa, se realizaron por lo menos 3 i microarreglos individuales, con los cuales se obtuvo el patrón genético mostrado por el RNA analizado, durante el período de inducción (F-l), progresión e instauración de la cirrosis (Fases II y III) y recuperación de la fibrosis (Fases IV y V), como resultado del momento en el que fue tomada la muestra, y se compararon con los microarreglos obtenidos de los animales control (2 por fase). El RNA se obtuvo a partir de 100 mg de tejido, mediante la extracción con TRIZOL, de acuerdo a las indicaciones del fabricante (Invitrogen). RNA: With the total RNA extracted by each stage, at least 3 i individual microarrays were made, with which the genetic pattern shown by the analyzed RNA was obtained, during the period of induction (Fl), progression and establishment of cirrhosis (Phases II and III) and fibrosis recovery (Phases IV and V), as a result of the time the sample was taken, and compared with the microarrays obtained from the control animals (2 per phase). RNA was obtained from 100 mg of tissue, by extraction with TRIZOL, according to the manufacturer's instructions (Invitrogen).
Microarreglos de Expresión; (GeneChip Rat gene 1.0 ST Array, Affimetrix). Todo el trabajo experimental de microarreglos, se realizó en la Unidad de Medicina Genomica del Hospital General de México a cargo del Dr. Jaime Berumen Campos. Con el RNA extraído de 100pg de tejido de cada rata, se obtuvo su cDNA marcado y este se aplicó en un CHIP (uno por rata), del tipo GeneChip Rat gene 1.0 ST Array de Affimetrix, que consta de 27,342 genes de rata en total. Expression Microarrays; (GeneChip Rat gene 1.0 ST Array, Affimetrix). All the experimental work of microarrays was carried out in the Genomic Medicine Unit of the General Hospital of Mexico by Dr. Jaime Berumen Campos. With the RNA extracted from 100pg of tissue from each rat, its labeled cDNA was obtained and this was applied in a CHIP (one per rat), of the GeneChip Rat gene 1.0 ST Array type of Affimetrix, which consists of 27,342 rat genes in total .
PCR Tiempo Real (qPCR); El qPCR es uno de los métodos empleados para validar los genes seleccionados como candidatos. Para realizar el qPCR se obtuvo el cDNA a partir de la transcripción reversa del RNA total obtenido de las muestras de hígado de cada uno de los animales (experimentales y controles) que conformaron el modelo, empleando el Kit de retrotrancripción "Inphusion" marca Finy Enzyme (Affimetrix). Real Time PCR (qPCR); The qPCR is one of the methods used to validate the selected genes as candidates. To perform the qPCR, the cDNA was obtained from the reverse transcription of the total RNA obtained from the liver samples of each of the animals (experimental and controls) that formed the model, using the Finy Enzyme "Inphusion" retrotranscription Kit (Affimetrix).
La Figura 1 muestra el diagrama de flujo seguido durante el desarrollo de los microarreglos de expresión a partir del RNA total obtenido del hígado de todos y cada uno de los animales sacrificados de acuerdo al esquema mencionado anteriormente. En la primera línea se representa el RNA total (gris), en la segunda la primera cadena de cDNA poliadenilado (AAAAA3') en negro, sintetizado por transcripción inversa; en la siguiente, la doble cadena de cDNA poliadenilado sintetizado (negro), la línea siguiente nos muestra el proceso de obtención del RNA (gris) marcado con biotina (negro) a partir del cDNA de doble cadena mediante transcripción in vitro con el empleo de nbonucleótidos biotinilados y RNA polimerasa T7, el resultado de la transcripción se indica en la siguiente línea (RNA de cadena simple con colas de uracilos en su extremo 5', marcado con biotina), estas cadenas de RNA simple se fragmentan con RNAsa H los cuales son aplicados al chip donde se encuentran el microarreglo de los genes de hígado de rata (>27,000) con los cuales se hibridaron estos fragmentos. El chip se reveló con el reactivo de estreptavidina, se lavó (flechas contiguas), y en el último paso el chip se escanea para su análisis. Figure 1 shows the flow chart followed during the development of expression microarrays from the total RNA obtained from the liver of each and every animal slaughtered according to the scheme mentioned above. In the first line the total RNA (gray) is represented, in the second the first polyadenylated cDNA chain (AAAAA3 ') in black, synthesized by reverse transcription; In the following, the double strand of synthesized polyadenylated cDNA (black), the following line shows the process of obtaining the biotinylated (black) RNA (gray) from the double stranded cDNA by in vitro transcription with the use of biotinylated nbonucleotides and T7 RNA polymerase, the result of transcription is indicated in the following line (single chain RNA with uracil tails at its 5 'end, labeled with biotin), these single RNA chains are fragmented with RNAse H which they are applied to the chip where the microarray of the rat liver genes (> 27,000) are found with which these fragments were hybridized. The chip was revealed with The streptavidin reagent was washed (adjacent arrows), and in the last step the chip is scanned for analysis.
El análisis histopatológico del hígado de los animales confirmó la presencia gradual de fibrosis hepática desde la primera etapa del modelo, además de las características esperadas para cada fase del modelo experimental: Fase I, fibrosis portal moderada e irregular, en la Fase II, regeneración nodular del parénquima (RNP), y Fase III cirrosis clara. Las muestras obtenidas en las Fases IV y V, también mostraron cirrosis, solo que en estas fases las bandas fibrosas que se observan son más estrechas, con menos células y la RNP menos evidente. Histopathological analysis of the liver of the animals confirmed the gradual presence of liver fibrosis from the first stage of the model, in addition to the expected characteristics for each phase of the experimental model: Phase I, moderate and irregular portal fibrosis, in Phase II, nodular regeneration of the parenchyma (RNP), and Phase III clear cirrhosis. The samples obtained in Phases IV and V also showed cirrhosis, only that in these phases the fibrous bands observed are narrower, with fewer cells and less obvious RNP.
Al realizar la observación de los cortes histológicos del modelo experimental se advierten diferencias marcadas entre los controles y los animales experimentales en cuanto al depósito de matriz extracelular, la cual aumenta en las Fases I, II y III y disminuye gradualmente conforme se desarrolla el proceso. Las Figuras 2a, 2b, 2c, 2d, 3a, 3b, 3c, 3d, 4a, 4b, 4c, 4d, 5a, 5b, 5c, 5d, 6a, 6b, 6c, 6d, 7a, 7b, 7c y 7d muestran mediante microfotografías, los resultados del análisis histopatológico realizado a los cortes de hígado de cada grupo, los cuales fueron fijados con formol amortiguado (pH7.0) y teñidos con H&E) para una observación directa de las diferentes estructuras presentes en el tejido hepático en cada Fase del modelo y con Rojo de Sirio (RS), tinción especial que permite distinguir, mediante la observación con microscopio con luz polarizada, los dos tipos de colágena predominantes presentes en las bandas de fibrosis, ya que cada una de ellas al ser observadas mediante esta técnica, emite un color distinto sobre un fondo oscuro, la colágena tipo I muestra bandas color rojo y la colágena tipo III en verde, si hay una mezcla equivalente se ven las bandas en color amarillo. La Figuras 2a, 2b, 2c y 2d muestran un control normal con imágenes representativas de cortes de tejido hepático provenientes de animales normales, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (2a, 2b) y RS (2c, 2d). Los hepatocitos son poligonales, con citoplasma granular y núcleo central, redondo y con nucléolo prominente. Los sinusoides se hayan tapizados por células elongadas (células endoteliales y de Kupffer), próximas a los hepatocitos, quedando el espacio de Disse como una región casi virtual. La magnitud de la amplificación se refiere en cada imagen. Las Figuras 3a, 3b, 3c y 3d muestran imágenes representativas de cortes de tejido hepático provenientes de animales en Fase I del Modelo Experimental, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (3a, 3b) y Rojo de Sirio (3c, 3d). En este grupo de animales se observa el inicio de alteración estructural debido a la presencia de bandas de fibrosis periportal (bf) y múltiples vesículas pequeñas de esteatosis (e) con núcleos que conservan su posición central (n). La magnitud de la amplificación se detalla en cada imagen. Las Figuras 4a, 4b, 4c y 4d corresponden a la Fase II, donde se aprecian imágenes representativas de cortes de tejido hepático provenientes de animales en Fase II del Modelo Experimental, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (4a, 4b) y RS (4c, 4d). En este grupo de animales se observan zonas con bandas gruesas y delgadas de fibrosis periportal (bf), lagunas de esteatosis (e) con vesículas redondeadas, grandes y pequeñas en hepatocitos centrolobulillares. En el citoplasma de los hepatocitos hay zonas vacías con desplazamiento de los núcleos a la periferia (n). La magnitud de la amplificación se detalla en cada imagen. Las Figuras 5a, 5b, 5c y 5d corresponden a la Fase III, apreciándose imágenes representativas de cortes de tejido hepático provenientes de animales en Fase III del Modelo Experimental, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (5a, 5b) y RS (5c, 5d). En este grupo de animales ya es aparente la presencia de cirrosis, con nodulos de regeneración rodeados por bandas gruesas de tejido fibrótico que van de vena porta a vena centrolobulillar, hepatocitos binucleados (bn) de mayor tamaño. La magnitud de la amplificación se detalla en cada imagen. Las Figuras 6a, 6b, 6c y 6d muestran imágenes representativas de cortes de tejido hepático provenientes de animales en Fase IV del Modelo Experimental, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (6a, 6b) y RS (6c, 6d). En este grupo de animales la arquitectura hepática representa un estadio cirrótico con zonas de necrosis focal, esteatosis mínima (e), nodulos de regeneración rodeados por bandas de tejido fibroso (bf) de menor grosor, que van de vena porta a vena centrolobulillar. Hepatocitos con hipercromatismo nuclear, enucleación (an), binucleación (bn) y pérdida de la relación núcleo-citoplasma. La magnitud de la amplificación se detalla en cada imagen. Finalmente, las Figuras 7a, 7b, 7c y 7d muestran imágenes representativas de cortes de tejido hepático provenientes de animales en Fase V del Modelo Experimental, fijados con formol amortiguado e incluidos en parafina, teñidos con H&E (7a, b7) y RS (7c, 7d). En este grupo de animales la arquitectura hepática presenta un estado cirrótico con nula presencia de esteatosis, nodulos de regeneración rodeados por bandas finas de tejido fibrótico (bf) que van de vena porta a vena centrolobulillar. Se observa (an) enucleación, binucleación (bn) hipercromatismo nuclear, y pérdida de la relación núcleo-citoplasma. La magnitud de la amplificación se detalla en cada imagen. When observing the histological sections of the experimental model, there are marked differences between controls and experimental animals in terms of extracellular matrix deposition, which increases in Phases I, II and III and gradually decreases as the process develops. Figures 2a, 2b, 2c, 2d, 3a, 3b, 3c, 3d, 4a, 4b, 4c, 4d, 5a, 5b, 5c, 5d, 6a, 6b, 6c, 6d, 7a, 7b, 7c and 7d show by microphotographs, the results of the histopathological analysis performed on the liver sections of each group, which were fixed with buffered formalin (pH7.0) and stained with H&E) for a direct observation of the different structures present in the liver tissue in each Phase of the model and with Sirius Red (RS), special staining that allows distinguishing, by microscopic observation with polarized light, the two predominant types of collagen present in the fibrosis bands, since each of them when observed by This technique emits a different color on a dark background, the type I collagen shows red bands and the type III collagen in green, if there is an equivalent mixture the bands are seen in yellow. Figures 2a, 2b, 2c and 2d show a normal control with representative images of liver tissue sections from normal animals, fixed with buffered formalin and included in paraffin, stained with H&E (2a, 2b) and RS (2c, 2d) . Hepatocytes are polygonal, with granular cytoplasm and central nucleus, round and with prominent nucleoli. Sinusoids have been upholstered by elongated cells (endothelial and Kupffer cells), close to hepatocytes, leaving the Disse space as an almost virtual region. The magnitude of the amplification is Refer in each picture. Figures 3a, 3b, 3c and 3d show representative images of liver tissue sections from animals in Phase I of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (3a, 3b) and Sirius Red (3c , 3d). In this group of animals the onset of structural alteration is observed due to the presence of periportal fibrosis bands (bf) and multiple small steatosis vesicles (e) with nuclei that retain their central position (n). The magnitude of the amplification is detailed in each image. Figures 4a, 4b, 4c and 4d correspond to Phase II, where representative images of liver tissue cuts from animals in Phase II of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (4a,) are seen. 4b) and RS (4c, 4d). In this group of animals, areas with thick and thin bands of periportal fibrosis (bf), steatosis lagoons (e) with rounded vesicles, large and small, are observed in centrolobular hepatocytes. In the cytoplasm of hepatocytes there are empty areas with displacement of the nuclei to the periphery (n). The magnitude of the amplification is detailed in each image. Figures 5a, 5b, 5c and 5d correspond to Phase III, showing representative images of liver tissue cuts from animals in Phase III of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (5a, 5b) and RS (5c, 5d). In this group of animals the presence of cirrhosis is already apparent, with regeneration nodules surrounded by thick bands of fibrotic tissue that go from portal vein to centrolobular vein, binucleated hepatocytes (bn) of larger size. The magnitude of the amplification is detailed in each image. Figures 6a, 6b, 6c and 6d show representative images of liver tissue sections from animals in Phase IV of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (6a, 6b) and RS (6c, 6d ). In this group of animals the liver architecture represents a cirrhotic stage with areas of focal necrosis, minimal steatosis (e), regeneration nodules surrounded by bands of fibrous tissue (bf) of smaller thickness, ranging from portal vein to centrolobular vein. Hepatocytes with nuclear hyperchromatism, enucleation (an), binucleation (bn) and loss of the nucleus-cytoplasm relationship. The magnitude of the amplification is detailed in each image. Finally, Figures 7a, 7b, 7c and 7d show representative images of liver tissue sections from Phase V animals of the Experimental Model, fixed with buffered formalin and included in paraffin, stained with H&E (7a, b7) and RS (7c, 7d). In this group of animals the hepatic architecture presents a cirrhotic state with no presence of steatosis, regeneration nodules surrounded by thin bands of fibrotic tissue (bf) that go from portal vein to centrolobular vein. There is (an) enucleation, binucleation (bn) nuclear hyperchromatism, and loss of the nucleus-cytoplasm relationship. The magnitude of the amplification is detailed in each image.
Determinación de la concentración de colágena: El cálculo del contenido de colágena, fue realizado con el fin de monitorear la evolución de la fibrosis en el modelo. La Figura 8, muestra la concentración de colágena promedio por cada Fase, datos mostrados en la Tabla 3, la cual fue de 19, 34, 51 , 35 y 26 mg de colágena/mg tejido respectivamente. Determination of collagen concentration: The calculation of collagen content was performed in order to monitor the evolution of fibrosis in the model. Figure 8 shows the average collagen concentration per Phase, data shown in Table 3, which was 19, 34, 51, 35 and 26 mg of collagen / mg tissue respectively.
Tabla 3, Concentración de colágena en el hígado de rata del modelo Table 3, Concentration of collagen in the rat liver of the model
Fase Controles n=3) Experimentales (n=5) Phase Controls n = 3) Experimental (n = 5)
I 11.83864±3.288 19.715±3.651  I 11,83864 ± 3,288 19,715 ± 3,651
II 12.45516±4.610 34.031 ±8.668  II 12.45516 ± 4.610 34.031 ± 8.668
III 15.765±1.903 51.922 ±11.657  III 15,765 ± 1,903 51,922 ± 11,657
IV 15.318±4.339 35.309 ±0.187  IV 15,318 ± 4,339 35,309 ± 0.187
V 15.026±0.368 26.826±7.324  V 15,026 ± 0.368 26,826 ± 7,324
Como se puede observar, la concentración se mantiene constante en los animales controles sin tratamiento, no así en aquéllos que reciben el hepatotóxico, la MEC depositada aumenta paulatinamente conforme la administración del fármaco permaneció constante, como un claro resultado en respuesta a un daño crónico (Fases I, II, III), también podemos notar que una vez que se suspende el tóxico (Fases IV y V), la fibrosis disminuye lentamente mostrando un efecto de recuperación a largo plazo, ya que según nuestra experiencia, el órgano no logra una recuperación total, (García de León María del Carmen, Irmgard Montfort, Eusebio Tello Montes, Rosario López Vancell, Alfonso Olivos García, Augusto González Canto, Mario Nequiz-Avendaño, Ruy Pérez-Tamayo. 2006. Hepatocyte production of modulators of extracellular liver matrix ¡n normal and cirrhotic rat liver. Experimental and Molecular Pathology. 80: 97-108). Los resultados anteriores nos muestran el estado histopatológico en que se encuentra el hígado en las diferentes etapas analizadas, y de este modo nos permitió correlacionar los resultados con el análisis que se realizó con la tecnología de microarreglos. As can be seen, the concentration remains constant in the control animals without treatment, but not in those who receive the hepatotoxic, the deposited ECM increases gradually as the administration of the drug remained constant, as a clear result in response to chronic damage ( Phases I, II, III), we can also note that once the toxic is suspended (Phases IV and V), the fibrosis slowly decreases showing a long-term recovery effect, since according to our experience, the organ does not achieve Total recovery (García de León María del Carmen, Irmgard Montfort, Eusebio Tello Montes, Rosario López Vancell, Alfonso Olivos García, Augusto González Canto, Mario Nequiz-Avendaño, Ruy Pérez-Tamayo. 2006. Hepatocyte production of modulators of extracellular liver matrix ¡n normal and cirrhotic rat liver. Experimental and Molecular Pathology. 80: 97-108). The previous results show us the histopathological state in which the liver is in the different stages analyzed, and thus allowed us to correlate the results with the analysis that was performed with microarray technology.
Microarreglos; Una vez terminados los microarreglos del modelo de CCI4 (un Chip para cada animal experimental (n=3) y control (n=2) por fase), se realizó el análisis empleando el programa estadístico SAM que permite diferenciar los genes que sufren un cambio en su expresión. Microarrays; Once the CCI4 model microarrays were completed (one Chip for each experimental animal (n = 3) and control (n = 2) per phase), the analysis was performed using the SAM statistical program that allows differentiating the genes that undergo a change in his expression.
Se seleccionaron 239 genes expresados 1.5 veces más por arriba del grupo control correspondiendo 56 a la Fase I, 62 a la Fase II, 93 a la Fase III, 14 a la Fase239 genes expressed 1.5 times above the control group were selected corresponding 56 to Phase I, 62 to Phase II, 93 to Phase III, 14 to Phase
IV y 10 a la Fase V, donde también se seleccionaron 4 genes reprimidos. La mayor cantidad de genes sobre-expresados se encuentran en la Fase III y solo en la FaseIV and 10 to Phase V, where 4 repressed genes were also selected. The most overexpressed genes are found in Phase III and only in Phase
V se encontraron genes reprimidos. V repressed genes were found.
Estos 239 genes se sometieron a análisis estadísticos; los datos reportados fueron examinados finalmente por medio del programa DAVID, que es una base que integra los resultados obtenidos de microarreglos, de acuerdo a las siguientes características: un valor de p menor a 0.05, valores de Fold enrichment o taza de enriquecimiento (FE) y enrichment score o enriquecimiento (ES) altos, datos que nos indican cuantas veces esta enriquecido nuestro cluster respecto al genoma total. These 239 genes were subjected to statistical analysis; The reported data were finally examined through the DAVID program, which is a base that integrates the results obtained from microarrays, according to the following characteristics: a value of p less than 0.05, Fold enrichment or enrichment cup (FE) values and enrichment score or enrichment (ES) high, data that indicate how many times our cluster is enriched with respect to the total genome.
De acuerdo a DAVID los 239 genes están agrupados en 187 clústeres, y sólo 37 de ellos presentaron valores significativos (p< 0.05) tomándose en cuenta el valor del 'ES' y el 'FE. Estos clústeres abarcan genes involucrados desde procesos de desarrollo de órganos hasta procesos metabólicos.  According to DAVID, the 239 genes are grouped into 187 clusters, and only 37 of them presented significant values (p <0.05) taking into account the value of 'ES' and 'FE. These clusters encompass genes involved from organ development processes to metabolic processes.
Al analizar con más detalle en Access e IPA, encontramos que solo 50 genes correspondían a los genes alterados con más frecuencia en estos procesos, así que se decidió seleccionar aquellos que tuvieran un valor de cambio por arriba del control, con funciones involucradas con el proceso de fibrosis y que no tuvieran relación con otras patología que cursaran con este proceso en otros órganos, estos genes se resumen en la Tabla 4 los cuales fueron distribuidos en 5 procesos fundamentales, relacionados con: Matriz extracelular, inmunidad, proliferación celular, metabolismo de lípidos y otros. When analyzing in more detail in Access and IPA, we found that only 50 genes corresponded to the most frequently altered genes in these processes, so it was decided to select those that had a change value above the control, with functions involved with the process of fibrosis and that they didn't have In relation to other pathologies that will be carried out with this process in other organs, these genes are summarized in Table 4 which were distributed in 5 fundamental processes, related to: Extracellular matrix, immunity, cell proliferation, lipid metabolism and others.
Finalmente tratando de establecer los genes candidatos que se pudiesen integrar a un panel detectable en fluidos biológicos, se eligieron solamente aquellos que cumplieran con los siguientes requisitos: 1) Genes extracelulares o que de alguna manera puedan encontrase en el espacio extracelular; 2) Con un valor de cambio mayor que el del control; 3) Relacionados con el proceso de fibrosis y; 4) Con valor de p significativa. De esta forma se seleccionaron 20 genes candidatos para ser validados como marcadores para fibrosis hepática. Finally, trying to establish the candidate genes that could be integrated into a detectable panel in biological fluids, only those that met the following requirements were chosen: 1) Extracellular genes or that in some way can be found in the extracellular space; 2) With an exchange value greater than that of the control; 3) Related to the fibrosis process and; 4) With significant p value. In this way, 20 candidate genes were selected to be validated as markers for liver fibrosis.
Tabla 4. Genes alterados con más frecuencia. Table 4. Most frequently altered genes.
Procesos Relacionados Tipo de Moléculas #GenesRelated Processes Type of Molecules #Genes
MEC. Diversos Tipos 17MEC. Various Types 17
Inmunidad. MHC; Complemento, Etc 6Immunity. MHC; Complement, Etc 6
Proliferación Celular. Factores de Transcripción, 8 Cell Proliferation Transcription Factors, 8
Cinasas Dependientes de  Dependent Kinases of
Ciclinas, Etc  Cyclines, Etc
Metabolismo de lípidos, Enzimas Diversas, Fosfolipasas y Otras 9 ácido retinoico y aa,  Lipid metabolism, various enzymes, phospholipases and other 9 retinoic acid and aa,
transporte de iones,  ion transport,
vías de señalización.  signaling paths
Adhesión celular y Integrinas y Receptores de Moléculas 4 señalización de Adhesión  Cell adhesion and Integrins and Molecule Receptors 4 Adhesion signaling
Fibrogénesis Factores de Crecimiento y Receptores 4 Fibrogenesis Growth Factors and Receptors 4
Respuesta a estrés y diversas 2 transporte de drogas Stress response and various 2 drug transport
Total: 50  Total: 50
Las Figuras 9a, 9b y 9c muestran el análisis de la intensidad de expresión en el microarreglo, donde se identifican algunos genes que se sobre expresan de manera preferencial en alguna de las etapas de la fibrosis. Estos genes fueron seleccionados como los genes candidatos para formar parte del panel molecular de diagnóstico. En las Figuras 9a, 9b y 9c se muestra una gráfica de 6 barras para cada uno de los genes seleccionados, donde la primera barra corresponde al valor de la intensidad promedio del gen mostrado por todas las muestras correspondientes a los animales control. La segunda barra muestra el mismo valor pero en este caso de los animales correspondientes a la Fase I; la tercera el promedio de intensidad de los animales de la Fase II, la cuarta barra lo mismo pero de animales de la Fase III, la quinta barra corresponde a la Fase IV y la sexta y última barra a la Fase V. Cada una de estas barras se repite para cada gen en las tres figuras 9a, 9b y 9c, pues para cada fase se realizó el análisis del gen correspondiente y los resultados se agruparon según el rango de intensidad. Figures 9a, 9b and 9c show the analysis of the intensity of expression in the microarray, which identifies some genes that are over-expressed by preferential way in some of the stages of fibrosis. These genes were selected as the candidate genes to be part of the diagnostic molecular panel. In Figures 9a, 9b and 9c a graph of 6 bars is shown for each of the selected genes, where the first bar corresponds to the value of the average intensity of the gene shown by all samples corresponding to the control animals. The second bar shows the same value but in this case of the animals corresponding to Phase I; the third the average intensity of the animals of Phase II, the fourth bar the same but of animals of Phase III, the fifth bar corresponds to Phase IV and the sixth and last bar to Phase V. Each of these bars are repeated for each gene in the three figures 9a, 9b and 9c, because for each phase the corresponding gene analysis was performed and the results were grouped according to the intensity range.
Muchos de ellos siguen un comportamiento similar al esperado para el modelo experimental, aumento paulatino en su expresión en las 3 primeras fases del proceso y disminución gradual en las correspondientes a la recuperación, como C7, Cyp26a1 , Mfap4 que sobresalen como los mayormente expresados y otros que siguen el mismo comportamiento pero de una manera más discreta como Lpl. Estos datos son importantes para poder establecer un perfil con una o varias moléculas que puedan ayudarnos a detectar estadios tempranos de Fibrosis Hepática. Many of them follow a behavior similar to that expected for the experimental model, gradual increase in its expression in the first 3 phases of the process and gradual decrease in those corresponding to recovery, such as C7, Cyp26a1, Mfap4 that stand out as the most expressed and others They follow the same behavior but in a more discreet way like Lpl. These data are important to establish a profile with one or several molecules that can help us detect early stages of liver fibrosis.
Las tablas 5a, b y c muestran los valores encontrados para cada uno de los genes candidatos. Además de los requerimientos para su elección, las moléculas finales seleccionadas como marcadores reunieron las siguientes características:Tables 5a, b and c show the values found for each of the candidate genes. In addition to the requirements for their choice, the final molecules selected as markers met the following characteristics:
• Alta sensibilidad y especificidad que permitan identificar diferentes etapas de fibrosis. • High sensitivity and specificity to identify different stages of fibrosis.
• Disponibilidad, seguridad, economía y reproducibilidad.  • Availability, security, economy and reproducibility.
• Capacidad para diferenciar la fibrosis de otros trastornos inflamatorios hepáticos, es decir, evitar falsos positivos.  • Ability to differentiate fibrosis from other inflammatory liver disorders, that is, avoid false positives.
• Permitir la supervisión de la progresión de la enfermedad o su regresión, para ser utilizadas como un panel de diagnóstico y seguimiento que haga posible la sustitución de la biopsia hepática. (Zhou K, Lu LG. 2009. Assessment of fibrosis in chronic liver diseases. Journal of Digestive Diseases. 10(1):7-14; Adams L A, George J, Bugianesi E, Rossi E, De Boer W B, Van der Poorten D, Ching H L I, Bulsara M, and Jeffrey G P. 2011. Complex non-invasive fibrosis models are more accurate than simple models in non-alcoholic fatty liver disease. Journal of Gastroenterology and Hepatology 26 1536-1543; Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non-invasive markers for hepatic fibrosis. BMC Gastroenterol. 11 :91) • Allow the monitoring of the progression of the disease or its regression, to be used as a diagnostic and follow-up panel that makes it possible to replace the liver biopsy. (Zhou K, Lu LG. 2009. Assessment of fibrosis in chronic liver diseases. Journal of Digestive Diseases. 10 (1): 7-14; Adams LA, George J, Bugianesi E, Rossi E, De Boer WB, Van der Poorten D, Ching HLI, Bulsara M, and Jeffrey G P. 2011. Complex non-invasive fibrosis models are more accurate than simple models in non-alcoholic fatty liver disease Journal of Gastroenterology and Hepatology 26 1536-1543; Baranova A, Lal P, Birerdinc A, Younossi ZM. 2011. Non-invasive markers for hepatic fibrosis. BMC Gastroenterol. 11: 91)
Tabla 5-a: Genes candidatos graficados en la Figura 9a Table 5-a: Candidate genes plotted in Figure 9a
Gen Ctl * F-l * F-ll * F-lll * F-IV * F-V *Gen Ctl * Fl * F-ll * F-lll * F-IV * FV *
Postn 8.612 19.025 8.280 14.215 9.668 8.218 Post 8,612 19,025 8,280 14,215 9,668 8,218
Pla2g7 18.301 51.865 40.806 87.046 39.727 22.531  Pla2g7 18.301 51.865 40.806 87.046 39.727 22.531
Fabp4 18.580 88.193 25.020 124.711 28.347 21.923  Fabp4 18,580 88,193 25,020 124,711 28,347 21,923
Pdgfd 21.191 23.578 27.109 43.072 25.860 22.112  Pdgfd 21,191 23,578 27,109 43,072 25,860 22,112
Igsf 10 25.423 44.348 42.231 69.448 29.647 32.250  Igsf 10 25,423 44,348 42,231 69,448 29,647 32,250
Myof 33.968 49.092 52.237 67.585 42.491 34.712  Myof 33,968 49,092 52,237 67,585 42,491 34,712
Tgfb3 35.912 73.569 63.949 83.736 46.593 32.634  Tgfb3 35,912 73,569 63,949 83,736 46,593 32,634
* Promedios  * Averages
Tabla 5-b genes candidatos graficados en la Figura 9b Table 5-b candidate genes plotted in Figure 9b
Gen- Ctl * F-l * F-ll * F-lll * F-IV * F-V * Gen- Ctl * Fl * F-ll * F-lll * F-IV * FV *
Pla2g2a 45.252 143.365 132.178 112.762 48.102 47.859  Pla2g2a 45,252 143,365 132,178 112,762 48,102 47,859
Lpl 48.498 157.041 159.112 216.656 117.539 85.056  Lpl 48,498 157,041 159,112 216,656 117,539 85,056
Aldh1a3 49.625 71.001 82.140 95.704 75.863 59.559  Aldh1a3 49,625 71,001 82,140 95,704 75,863 59,559
Cyp26a1 50.048 54.622 307.140 301.227 189.297 106.768  Cyp26a1 50.048 54.622 307.140 301.227 189.297 106.768
Adamts2 64.804 110.699 78.575 118.671 71.567 77.197  Adamts2 64,804 110,699 78,575 118,671 71,567 77,197
Fbn1 72.315 109.798 111.869 1 7.627 93.812 62.813  Fbn1 72,315 109,798 111,869 1 7,627 93,812 62,813
Mfap4 73.581 229.273 183.518 325.298 178.377 176.669  Mfap4 73,581 229,273 183,518 325,298 178,377 176,669
Abcg2 74.324 100.202 150.020 121.734 108.000 67.515  Abcg2 74,324 100,202 150,020 121,734 108,000 67,515
Fbln5 86.042 109.077 149.007 180.630 117.653 106.447  Fbln5 86.042 109.077 149.007 180.630 117.653 106.447
C7 90.614 348.715 457.100 610.127 282.482 187.556  C7 90.614 348.715 457.100 610.127 282.482 187.556
* Promedios Tabla 5-c genes candidatos graficados en la Figura 9c * Averages Table 5-c candidate genes plotted in Figure 9c
Gen Ctl * F-l * F-ll * F-lll * F-IV * F-V * Gen Ctl * Fl * F-ll * F-lll * F-IV * FV *
Mgp 129.357 310.584 379.531 468.256 276.846 227.482 Mgp 129,357 310,584 379,531 468,256 276,846 227,482
Pld3 172.442 227.2913 220.301 301.219 191.413 200.577  Pld3 172.442 227.2913 220.301 301.219 191.413 200.577
Igf p7 696.488 946.730 982.922 1422.863 870.308 863.146  Igf p7 696.488 946.730 982.922 1422.863 870.308 863.146
* Promedios  * Averages
Una vez seleccionados los genes candidatos para integrar el panel molecular de biomarcadores para diagnóstico y estadificación de la fibrosis del hígado, se realizó la prueba de ELISA, empleando en ella anticuerpos monoclonales específicos para cada una de las proteínas seleccionadas y revelando la reacción con un anticuerpo secundario acoplado a peroxidasa de rábano (HRP), empleando diamino bencidina (DAB) como sustrato para la enzima. Once the candidate genes were selected to integrate the biomarker molecular panel for diagnosis and staging of liver fibrosis, the ELISA test was performed, using specific monoclonal antibodies for each of the selected proteins and revealing the reaction with an antibody. secondary to horseradish peroxidase (HRP), using diamino benzidine (DAB) as a substrate for the enzyme.
Para discriminar la proteína en el suero de pacientes con patologías similares pero en órganos distintos, se determinaron los niveles de cada proteínas en dos grupos de suero: a) Pacientes infectados con el virus de la hepatitis C que cursan con fibrosis hepática, en dos estadios de la enfermedad (con índice de Knodel F-l y F-4) y; b) Pacientes con fibrosis pulmonar idiopática; estos últimos con la finalidad de establecer la especificidad de órgano que buscamos en nuestro panel, además se aplicó también la prueba a sueros de pacientes con enfermedad hepática aguda (absceso hepático amibiano) para descartar que pudiesen ser detectadas también en un padecimiento hepático diferente a la fibrosis (enfermedad crónica) y con esto, seleccionar aquéllas moléculas que nos mostraran las cualidades de especificidad, sensibilidad, valor predictivo positivo y negativo idóneas, para formar parte del panel buscado. To discriminate the protein in the serum of patients with similar pathologies but in different organs, the levels of each protein in two serum groups were determined: a) Patients infected with the hepatitis C virus who present with liver fibrosis, in two stages of the disease (with Knodel Fl and F-4 index) and; b) Patients with idiopathic pulmonary fibrosis; The latter with the purpose of establishing the specificity of the organ that we are looking for in our panel, the test was also applied to sera from patients with acute liver disease (amoebic liver abscess) to rule out that they could also be detected in a liver condition other than fibrosis (chronic disease) and with this, select those molecules that will show us the qualities of specificity, sensitivity, positive and negative predictive value suitable, to be part of the panel sought.
El índice Knodell fue desarrollado para generar una puntuación numérica de daño hepático con valores que van desde F0 hasta F4, con muestras de biopsia hepática obtenidas a partir de pacientes con hepatitis crónica activa asintomática. Las biopsias se clasificaron en cuatro categorías: necrosis periportal, necrosis intralobular, inflamación portal con fibrosis y cirrosis (Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplówitz N, Kieman TW, Wollman J. 1981. Formulation and application of numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology. 1 : 431-435) donde F0 es ausencia de enfermedad. The Knodell index was developed to generate a numerical liver damage score with values ranging from F0 to F4, with liver biopsy samples obtained from patients with asymptomatic active chronic hepatitis. Biopsies were classified into four categories: periportal necrosis, intralobular necrosis, portal inflammation with fibrosis and cirrhosis (Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplówitz N, Kieman TW, Wollman J. 1981. Formulation and application of numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1: 431-435) where F0 is absence of disease.
En las Tablas 6a y 6b se muestran las características de los pacientes separados de acuerdo ai índice de Knodell, con enfermedad hepática cuyos sueros fueron empleados para las determinaciones y la subsecuente validación de las 20 proteínas seleccionadas como candidatas para integrar el panel final para diagnóstico y estadificación de la enfermedad fibrótica del hígado, objeto de la presente invención. Tables 6a and 6b show the characteristics of the patients separated according to the Knodell index, with liver disease whose sera were used for the determinations and subsequent validation of the 20 proteins selected as candidates to integrate the final panel for diagnosis and Staging of fibrotic liver disease, object of the present invention.
Tabla 6a. Pacientes separados por índice de Knodell, Fase 0-1. Table 6a. Patients separated by Knodell index, Phase 0-1.
FASE Num Hombres Mujeres PHASE Num Men Women
0-1 15 6 9  0-1 15 6 9
Num Pac Edad Sexo IMC Diagnóstico Knodell/METAVIR p35 33 M 25.15 VHC (1) 1 Num Pac Age Sex BMI Diagnosis Knodell / METAVIR p35 33 M 25.15 HCV (1) 1
p10 34 F 29.01 VHC (1) 1  p10 34 F 29.01 HCV (1) 1
p14 40 M 32.9 VHC 0  p14 40 M 32.9 HCV 0
p02 40 M 19.7 VHC 1  p02 40 M 19.7 HCV 1
p39 40 M 37.24 VHC(1B) 1  p39 40 M 37.24 HCV (1B) 1
p33 43 F 32 VHC(1 B) 0  p33 43 F 32 HCV (1 B) 0
p21 45 F 25.11 VHC(2B) 0  p21 45 F 25.11 VHC (2B) 0
p 1 53 F 31.41 VHC(1A) 1  p 1 53 F 31.41 HCV (1A) 1
p38 55 F 26.03 VHC 0  p38 55 F 26.03 HCV 0
p09 58 M 28.25 VHC(2B) 0  p09 58 M 28.25 VHC (2B) 0
p27 61 M 30.3 VHC 0  p27 61 M 30.3 HCV 0
p06 61 F 29.66 VHC(2A,2C) 1  p06 61 F 29.66 HCV (2A, 2C) 1
p08 65 F 23.1 VHC(1) 1  p08 65 F 23.1 HCV (1) 1
p34 68 F 25.3 VHC(1B) 0  p34 68 F 25.3 HCV (1B) 0
p01 75 F 25.27 VHC 1 Tabla 6b. Pacientes separados por índice de Knodell, Fase 4. p01 75 F 25.27 HCV 1 Table 6b Patients separated by Knodell index, Phase 4.
FASE Num Hombres Mujeres PHASE Num Men Women
4 15 6 9  4 15 6 9
Num Pac Edad Sexo IMC Diagnóstico Knodell/METAVIR p25 32 M 27.33 VHC (1A) 4 Num Pac Age Sex BMI Diagnosis Knodell / METAVIR p25 32 M 27.33 HCV (1A) 4
p32 38 M peso 76 VHC (1 B) 4  p32 38 M weight 76 VHC (1 B) 4
p13 45 F 34.3 VHC (1B) 4  p13 45 F 34.3 HCV (1B) 4
p20 47 M 32.83 VHC (1) 4  p20 47 M 32.83 HCV (1) 4
p03 49 F 31.6 VHC (1A) 4  p03 49 F 31.6 HCV (1A) 4
p17 49 M 35.3 VHC ( A) 4  p17 49 M 35.3 HCV (A) 4
p29 51 F 24 VHC (2) 4  p29 51 F 24 VHC (2) 4
p23 53 F 22.36 VHC (1A) 4  p23 53 F 22.36 HCV (1A) 4
p16 58 F 27.33 VHC (2B) 4  p16 58 F 27.33 HCV (2B) 4
p22 60 F 29.68 VHC(1A) 4  p22 60 F 29.68 HCV (1A) 4
p28 62 F 27.34 VHC (1) 4  p28 62 F 27.34 HCV (1) 4
p04 63 F 23.19 VHC(1A,1 B) 4  p04 63 F 23.19 HCV (1A, 1 B) 4
p24 63 M 23.8 VHC(2B) 4  p24 63 M 23.8 HCV (2B) 4
p37 66 M 24.15 VHC(2A, 2C) 4  p37 66 M 24.15 VHC (2A, 2C) 4
p18 70 F 27.33 VHC(2A, 2C) 4  p18 70 F 27.33 HCV (2A, 2C) 4
La Figura 10 muestra paso a paso el diagrama seguido de las pruebas realizadas para la validación por ELISA e Inmunohistoquímica a los 20 genes candidatos, empleando sueros de pacientes con diferentes enfermedades de donde se identificaron los genes que nos dieron los mejores resultados y así construir el panel de biomarcadores para diagnóstico y estadificación de la fibrosis del hígado objeto de la presente invención. Figure 10 shows step by step the diagram followed by the tests performed for the validation by ELISA and Immunohistochemistry to the 20 candidate genes, using sera from patients with different diseases from which the genes that gave us the best results were identified and thus build the Biomarker panel for diagnosis and staging of liver fibrosis object of the present invention.
Una vez obtenido el resultado con cada una de ellas y para definir las características ideales mencionadas para un biomarcador, se determinó la Sensibilidad (Sensibilidad = Valor Positivo/ Valor Positivo + Falso Negativo), Especificidad (Especificidad = Valor Negativo/Valor Negativo/Falso Positivo) lo que nos indica las propiedades o capacidades de esta para clasificar un estado de enfermedad y salud (enfermos y no enfermos) así como los Valores Predictivos positivos y negativos, y para ello los datos fueron sometidos al análisis estadístico para conocer su sensibilidad y especificidad, realizando las comparaciones necesarias entre los diferentes grupos a los que les fue aplicada la prueba, mediante la elaboración de las Curvas ROC (Receiver Operating Characteristic) respectivas y de acuerdo a estas, obtener los valores predictivos positivo (VPP) y negativo (VPN), lo que se muestra en la Figura 11. La interpretación de las curvasOnce the result was obtained with each of them and to define the ideal characteristics mentioned for a biomarker, Sensitivity was determined (Sensitivity = Positive Value / Positive Value + Negative False), Specificity (Specificity = Negative Value / Negative Value / Positive False) which indicates the properties or capabilities of this to classify a state of disease and health (sick and non-sick) as well as positive and negative Predictive Values, and for this Data were subjected to statistical analysis to know their sensitivity and specificity, making the necessary comparisons between the different groups to which the test was applied, by preparing the respective ROC (Receiver Operating Characteristic) Curves and according to these, obtain the positive predictive values (PPV) and negative (NPV), which is shown in Figure 11. The interpretation of the curves
ROC se realizó en base a los siguientes valores del área bajo la curva: (Fogarty J,ROC was performed based on the following values of the area under the curve: (Fogarty J,
Hudson S E., Atkeson C G., Avraham D I, Forlizzi J., Kiesler S, Lee J C, and Yang J. 2005. Predicting Human Interruptibility with Sensors. ACM Transactions on Computer-Human Interaction, 12(1): 119-146.) Hudson S E., Atkeson C G., Avraham D I, Forlizzi J., Kiesler S, Lee J C, and Yang J. 2005. Predicting Human Interruptibility with Sensors. ACM Transactions on Computer-Human Interaction, 12 (1): 119-146.)
[0.5, 6): Test malo. [0.5, 6): Bad test.
[0.6, 0.75): Test regular.  [0.6, 0.75): Regular test.
[0.75, 0.9): Test bueno.  [0.75, 0.9): Good test.
[0.9, 0.97): Test muy bueno.  [0.9, 0.97): Very good test.
[0.97, 1): Test excelente.  [0.97, 1): Excellent test.
El valor del área bajo la curva ROC resultante, nos indica si las comparaciones que realizamos son excelentes (0.97, 1), buenas (0.75, 0.9), regulares (0.6, 0.75) o malas (0.5, 6), además esta curva nos proporciona un punto de corte en el cual, para ser tomadas en cuenta, la sensibilidad y especificidad deben ser cercanas al 100%. The value of the area under the resulting ROC curve, indicates whether the comparisons we make are excellent (0.97, 1), good (0.75, 0.9), regular (0.6, 0.75) or bad (0.5, 6), in addition to this curve we It provides a cut-off point at which, to be taken into account, the sensitivity and specificity must be close to 100%.
Mientras que los valores predictivos son útiles para determinar, para cada paciente, la probabilidad condicional de estar o no enfermo y estos pueden calcularse mediante cuadros bimatriciales formados por los datos generados por el punto de corte donde tendremos datos positivos, negativos y los falsos positivos y negativos, los valores óptimos que deben buscarse en los VPP y VPN deben ser cercanos o iguales al 100%. Las Figuras 12a, 12b, 13a, 13b 14a, 14b 15a, 15b, 16a, 16b, 17a, 17b, 18a y 18b, muestran las gráficas obtenidas con los 7 genes seleccionados para integrar el panel de biomarcadoes para diagnóstico y estadificación de la fibrosis del hígado, objeto de la presente invención, donde cada uno de los valores obtenidos indica la importancia de la molécula y su factibilidad para ser parte de un grupo diagnóstico. Todas ellas tienen una alta sensibilidad y especificidad, el diagnóstico y estadificación de la fibrosis estará sujeto a los resultados presentados por estas moléculas en una prueba de ELISA, con lo que se determina el estadio de la fibrosis del paciente, de acuerdo al análisis en conjunto de valores obtenidos con la prueba completa. While the predictive values are useful to determine, for each patient, the conditional probability of being or not sick and these can be calculated using bimatricial tables formed by the data generated by the cut-off point where we will have positive, negative and false positive data and negative, the optimal values that must be sought in the VPP and VPN must be close to or equal to 100%. Figures 12a, 12b, 13a, 13b 14a, 14b 15a, 15b, 16a, 16b, 17a, 17b, 18a and 18b, show the graphs obtained with the 7 genes selected to integrate the biomarked panel for diagnosis and staging of fibrosis of the liver, object of the present invention, where each of the values obtained indicates the importance of the molecule and its feasibility to be part of a diagnostic group. All of them have high sensitivity and specificity, the diagnosis and staging of fibrosis will be subject to the results presented by these molecules in an ELISA test, which determines the stage of the patient's fibrosis, according to the analysis as a whole of values obtained with the complete test.
Verdadero Positivo  True Positive
Valor Predictivo Positivo = —  Positive Predictive Value = -
Valor Positivo + Falso Positivo  Positive Value + Positive False
Valor en la realidad Value in reality
P N  P N
Verdaderos Falsos  True False
Predicción positivos positivos  Positive Positive Prediction
outcome Falsos Verdaderos  outcome False True
n'  n '
negativos negativos  negative negatives
N  N
Ejemplo: Example:
Valor en la realidad Value in reality
P N Total  P N Total
Predicción p' 18 1 P' outcome n' 7 29 N'  Prediction p '18 1 P' outcome n '7 29 N'
P N  P N
Sensibilidad % Especificidad % VPP % VPN %  Sensitivity% Specificity% VPP% VPN%
72 96 94 80 Ejemplos de uso 72 96 94 80 Usage Examples
Para confirmar la utilidad de cada una de los biomarcadores para diagnóstico y estadificación de la fibrosis hepática, su utilizaron sueros de pacientes infectados con el virus de la hepatitis C que cursan con fibrosis hepática, en dos estadios de la enfermedad (con índice de Knodell FO-I 15 en total (6 varones y 9 mujeres) y F-4 igualmente 15 en total (6 varones y 9 mujeres)  To confirm the usefulness of each of the biomarkers for diagnosis and staging of hepatic fibrosis, their sera were used from patients infected with hepatitis C virus who present with hepatic fibrosis, in two stages of the disease (with Knodell FO index -I 15 in total (6 men and 9 women) and F-4 also 15 in total (6 men and 9 women)
Ejemplo de uso 1. Biomarcador MFAP-4 Example of use 1. Biomarker MFAP-4
Se realizaron las diferentes comparaciones de los grupos de fibrosis y cirrosis respecto a los controles encontrando una alta significancia entre ellos como se puede apreciarse en la Figura 12a, con valores de p < 0.0001 , lo cual nos confirma que esta proteína puede diferenciar entre etapas tempranas, tardías y terminales (cirrosis) del proceso fibrótico, incluso podría ser empleada con enfermos en remisión ya que sí observamos la gráfica de intensidades, obtenida en el microarreglo cabría la posibilidad de obtener datos en este estadio; la última comparación realizada entre F1 y F4 vs FPI nos indica que esta proteína además es hígado específica ya que los resultados son altamente significativos (pO.0001) con estos sueros, por otra parte la comparación entre F1 , F4 vs AA al ser significativa nos dice que esta proteína además no se expresa en otras patologías hepáticas que no cursen con fibrosis (patologías hepáticas agudas), características que la hacen una candidata ideal para formar parte del panel de diagnóstico.  The different comparisons of the fibrosis and cirrhosis groups with respect to the controls were made, finding a high significance among them as can be seen in Figure 12a, with values of p <0.0001, which confirms that this protein can differentiate between early stages , late and terminal (cirrhosis) of the fibrotic process, could even be used with patients in remission since we do observe the intensity graph, obtained in the microarray, it would be possible to obtain data at this stage; The last comparison between F1 and F4 vs FPI indicates that this protein is also a specific liver since the results are highly significant (pO.0001) with these sera, on the other hand the comparison between F1, F4 vs AA being significant He says that this protein is also not expressed in other liver diseases that do not have fibrosis (acute liver diseases), characteristics that make it an ideal candidate to be part of the diagnostic panel.
La Figura 12b muestra la representación de las 3 curvas ROC obtenidas con las diferentes comparaciones realizadas entre los resultados conseguidos con los distintos grupos de suero de pacientes en la prueba de ELISA con cada proteína y cuyos resultados se muestran al pie de las gráficas, la primera columna corresponde a Ctls vs F1 y se utilizó como ejemplo para describir cada uno de los valores que se refieren en las columnas subsecuentes. MFAP-4 es una proteína de matriz extracelular que participa en la respuesta inmune innata y permite el libre intercambio gaseoso en los pulmones a través de su unión a la región colagénica de proteínas surfactantes (SP-A y SP-D). La primera columna al pie de las gráficas de las curvas ROC (Figura 12b), corresponde a cada una de las comparaciones realizadas entre los grupos para esta proteína, en la segunda fila de esta columna se observan los resultados de la comparación del grupo de sueros de individuos normales Ctls vs sueros de pacientes con fibrosis con índice Knodell F1 , la segunda columna corresponde al AUC, área bajo la curva, con un valor de 0.99, lo que quiere decir que esta comparación es perfecta o excelente para discriminar a individuos sanos de los que tienen fibrosis incipiente, la tercera columna corresponde al punto de corte con valor de 0.097, esto significa que a partir de este valor todo lo que este por arriba será fibrótico y todo lo que esté por debajo es sano; la cuarta y quinta columnas se refieren a la sensibilidad y especificidad en este punto de corte, correspondiendo a estas características los valores de 100 y 94 % respectivamente, lo que quiere decir que esta prueba en el punto de corte de 0.097, el 100 % de los individuos son casos positivos, esto es los casos realmente enfermos y que el 94 % son casos negativos, esto es los casos realmente sanos. Las últimas dos columnas se refieren a los valores predictivos positivos y negativos los cuales corresponden a los valores de 75 y 100 %, datos que nos dicen que un individuo tiene un 75 % de la probabilidad de tener la enfermedad si el resultado de su prueba diagnóstica con esta proteína es positiva; y el 100 % de probabilidad de no tener la enfermedad si el resultado de la prueba diagnóstica es negativa. Figure 12b shows the representation of the 3 ROC curves obtained with the different comparisons made between the results obtained with the different serum groups of patients in the ELISA test with each protein and whose results are shown at the bottom of the graphs, the first column corresponds to Ctls vs F1 and was used as an example to describe each of the values referred to in subsequent columns. MFAP-4 is an extracellular matrix protein that participates in the innate immune response and allows free gaseous exchange in the lungs through its binding to the collagen region of surfactant proteins (SP-A and SP-D). The first column at the bottom of the graphs of the ROC curves (Figure 12b), corresponds to each of the comparisons made between the groups for this protein, in the second row of this column the results of the comparison of the group of sera of normal individuals Ctls vs sera of fibrosis patients with Knodell F1 index are observed, the second column corresponds to the AUC, area under the curve, with a value of 0.99, which means that this Comparison is perfect or excellent to discriminate healthy individuals from those with incipient fibrosis, the third column corresponds to the cut-off point with a value of 0.097, this means that from this value everything that is above will be fibrotic and everything that being below is healthy; the fourth and fifth columns refer to the sensitivity and specificity at this cut-off point, corresponding to these characteristics the values of 100 and 94% respectively, which means that this test at the cut-off point of 0.097, 100% of individuals are positive cases, this is really sick cases and 94% are negative cases, this is really healthy cases. The last two columns refer to the positive and negative predictive values which correspond to the values of 75 and 100%, data that tell us that an individual has a 75% chance of having the disease if the result of their diagnostic test with this protein it is positive; and 100% probability of not having the disease if the result of the diagnostic test is negative.
En esta misma Figura 12a, se observa cómo es la expresión de la proteína MFAP-4 determinada por la técnica de ELISA en los sueros de 5 grupos, el primero corresponde al grupo control, el segundo al grupo de fibrosis incipiente o mínima (F1), el tercero al grupo de cirrosis (F4) estos dos grupos corresponden a nuestro objeto de estudio, el grupo 4 corresponde a pacientes con fibrosis pulmonar idiopática (FPI) y el 5 grupo corresponde a sueros de pacientes con absceso hepático amibiano (AHA). Los valores mostrados al pie de la Figura 12a corresponden al valor de p en una prueba de T con respecto a los controles de cada uno de los grupos mencionados. In this same Figure 12a, it is observed how the expression of the MFAP-4 protein is determined by the ELISA technique in the sera of 5 groups, the first corresponds to the control group, the second to the group of incipient or minimal fibrosis (F1) , the third to the cirrhosis group (F4) these two groups correspond to our object of study, group 4 corresponds to patients with idiopathic pulmonary fibrosis (IPF) and the group 5 corresponds to sera from patients with amibial liver abscess (AHA). The values shown at the bottom of Figure 12a correspond to the value of p in a T test with respect to the controls of each of the groups mentioned.
Ejemplo de uso 2. Biomarcador FBN1 Example of use 2. Biomarker FBN1
La Figura 13a y 13b ilustran el comportamiento de la molécula de FBN1 en las pruebas, se trata de una proteína estructural y de soporte, que regula la maduración de osteoblastos mediante el control de la biodisponibilidad y los niveles de BMP proteínas morfo genéticas del hueso, o Bone Morphogenetic Proteins (BMPs) factores de crecimiento que pertenecen a la familia de los factores de crecimiento transformantes (TGF-β ), una súper familia de proteínas con la capacidad de inducir fuertemente la formación de hueso nuevo, cartílago y tejido conjuntivo y TGF-beta. Figure 13a and 13b illustrate the behavior of the FBN1 molecule in the tests, it is a structural and support protein, which regulates the maturation of osteoblasts by controlling the bioavailability and levels of BMP bone morphogenetic proteins, or Bone Morphogenetic Proteins (BMPs) growth factors that belong to the family of transforming growth factors (TGF-β), a super family of proteins with the ability to strongly induce the formation of new bone, cartilage and connective tissue and TGF-beta.
Las diferentes comparaciones entre los grupos probados con diferencias significativas con un valor de p<0.001 , Figura 13a, esta proteína se expresa mayoritariamente en los pacientes con fibrosis incipiente y presenta una disminución en su expresión en el grupo correspondiente a los enfermos cirróticos y tanto F1 y F4 son diferencialmente expresados respecto a los grupos de fibrosis pulmonar y Absceso hepático amibiano. Los valores generados por la curva ROC Figura 13b hacen de esta molécula un marcador perfecto para diferenciar etapas tempranas y avanzadas de fibrosis, con especificidad, sensibilidad, VPP y VPN del 100 %  The different comparisons between the groups tested with significant differences with a value of p <0.001, Figure 13a, this protein is mostly expressed in patients with incipient fibrosis and presents a decrease in its expression in the group corresponding to cirrhotic patients and both F1 and F4 are differentially expressed with respect to the groups of pulmonary fibrosis and amoebic liver abscess. The values generated by the ROC curve Figure 13b make this molecule a perfect marker to differentiate early and advanced stages of fibrosis, with specificity, sensitivity, PPV and NPV of 100%
Ejemplo de uso 3. Biomarcador ALDH1A3 Example of use 3. Biomarker ALDH1A3
La ALDH1A3 es una enzima que ayuda a la detoxificación de aldehidos en la célula, esta proteína presenta diferencias significativas entre los grupos comparados cuando observamos la gráfica correspondiente a los resultados obtenidos con el ELISA Figura 14a, tiene un aumento gradual entre el grupo de fibrosis incipiente y cirrosis (p<0.001) y también presenta diferencias significativas con respecto a los grupos de FP y AA (p<0.001); en los datos obtenidos de la curva ROC Figura 14b se identifica que la S, E y VPN son altos > 96%, mientras que los VPP resultan en un 83 %. Estos datos nos permiten tomar esta molécula como parte de nuestro panel, pues en un análisis individual nos puede proporcionar datos que refuercen el diagnóstico, además que mostró ser una proteína altamente específica para enfermedad hepática.  ALDH1A3 is an enzyme that helps the detoxification of aldehydes in the cell, this protein has significant differences between the groups compared when we look at the graph corresponding to the results obtained with the ELISA Figure 14a, it has a gradual increase between the group of incipient fibrosis and cirrhosis (p <0.001) and also presents significant differences with respect to the FP and AA groups (p <0.001); In the data obtained from the ROC curve, Figure 14b identifies that the S, E and NPV are high> 96%, while the PPV result in 83%. These data allow us to take this molecule as part of our panel, because in an individual analysis it can provide us with data that reinforce the diagnosis, in addition to being a highly specific protein for liver disease.
Ejemplo de uso 4. Biomarcador CYP26A1 Example of use 4. Biomarker CYP26A1
Los citocromos CYPs tienen actividad de oxigenasa y comúnmente catalizan reacciones redox. Este grupo de enzimas se encuentran en niveles elevados en el hígado, donde tienen un papel importante en el metabolismo de fármacos y compuestos tóxicos endógenos. Podemos observar en la Figura 15a que las comparaciones entre los grupos a pesar de tener algunos individuos con expresiones por arriba de la media (F1) y una desviación estándar alta, tienen fuerte diferencia entre ellos, con un aumento de sus niveles en los pacientes cirróticos (p< 0.001). Los datos obtenidos indican también diferencia con respecto a los pacientes con FPI y AA (p< 0.001). Los valores más altos encontrados para esta proteína en la curva ROC Figura 15b son para la comparación entre Ctls vs F4 siendo el 100 % para la sensibilidad, especificidad, VPP y VPN, en enfermos con este estadio de la enfermedad. CYP cytochromes have oxygenase activity and commonly catalyze redox reactions. This group of enzymes are found in elevated levels in the liver, where they play an important role in the metabolism of endogenous toxic drugs and compounds. We can observe in Figure 15a that comparisons between groups despite having some individuals with expressions above the mean (F1) and a high standard deviation, have a strong difference between them, with an increase in their levels in cirrhotic patients (p <0.001). The data obtained also indicate a difference with respect to patients with IPF and AA (p <0.001). The highest values found for this protein in the ROC curve Figure 15b are for the comparison between Ctls vs F4 being 100% for sensitivity, specificity, PPV and NPV, in patients with this stage of the disease.
Ejemplo de uso 5. Biomarcador MGP Example of use 5. MGP biomarker
La MGP es una proteína asociada con la matriz orgánica del hueso y el cartílago. Se piensa que actúa como un inhibidor de la formación ósea. El ELISA de esta proteína Figura 16a muestra diferencias significativas entre los grupos comparados, existen individuos por arriba de la media (en F1) y por debajo de ella (en F4) pero las diferencias son claras (p<0.001) y esto puede reforzarse con los valores obtenidos de la curva ROC Figura 16b al ser perfectos 100 % en sensibilidad, especificidad, VPP y VPPN. Finalmente la proteína también muestra diferencias claras de los grupos de pacientes con enfermedad hepática con respecto a los grupos de FPI y AA (p<0.001).  MGP is a protein associated with the organic matrix of bone and cartilage. It is thought to act as an inhibitor of bone formation. The ELISA of this protein Figure 16a shows significant differences between the groups compared, there are individuals above the average (in F1) and below it (in F4) but the differences are clear (p <0.001) and this can be reinforced with the values obtained from the ROC curve Figure 16b, being 100% perfect in sensitivity, specificity, VPP and VPPN. Finally, the protein also shows clear differences in the groups of patients with liver disease with respect to the IPF and AA groups (p <0.001).
Ejemplo de uso 6. Biomarcador C7 Example of use 6. Biomarker C7
La proteína C7 forma parte del complejo de ataque a la membrana (MAC) del sistema del complemento, con un papel clave en la inmunidad innata y adaptativa. El resultado del ELISA Figura 17a para esta proteína solo dio valor significativo en los pacientes con fibrosis hepática inicial F1 (p<0.001) por lo que puede utilizarse para detectar la fibrosis hepática en este estadio y esto es corroborado por los datos obtenidos de la curva ROC Figura 17b, donde para la comparación Ctls vs Fl, los valores de sensibilidad, especificidad VPP y VPN son del 100 %.  The C7 protein is part of the membrane attack complex (MAC) of the complement system, with a key role in innate and adaptive immunity. The result of the ELISA Figure 17a for this protein only gave significant value in patients with initial liver fibrosis F1 (p <0.001) so it can be used to detect liver fibrosis at this stage and this is corroborated by the data obtained from the curve ROC Figure 17b, where for the comparison Ctls vs Fl, the sensitivity, VPP and VPN specificity values are 100%.
Ejemplo de uso 7. Biomarcador PLA2G7. Example of use 7. Biomarker PLA2G7.
Las fosfolipasas entre ellas la PLA2G7, son un grupo de enzimas que hidrolizan los fosfolípidos produciendo ácidos grasos y otras moléculas lipofílicas que tienen diversas funciones biológicas, entre las que se puede mencionar la inflamación, crecimiento celular y señalización entre otras. En el análisis de ELISA Figura 18a esta proteína presenta una expresión menor en el suero de los pacientes con Fl, en comparación a los niveles de expresión detectados en el suero de individuos sanos dato que es significativo (p<0.001) y en los pacientes con F4 es indetectable al menos por esta técnica. Los datos obtenidos de esta comparación con la curva ROC Figura 18b hacen a esta molécula de mediana fuerza, pero utilizada en conjunto con el resto del panel de biomarcadores objeto de la presente invención, sus resultados son importantes para el diagnóstico, principalmente si pensamos en la estadificación y detección de fibrosis temprana. Phospholipases, including PLA2G7, are a group of enzymes that hydrolyse phospholipids producing fatty acids and other lipophilic molecules. that have diverse biological functions, among which we can mention inflammation, cell growth and signaling among others. In the ELISA analysis Figure 18a this protein shows a lower expression in the serum of patients with Fl, compared to the levels of expression detected in the serum of healthy individuals data that is significant (p <0.001) and in patients with F4 is undetectable at least by this technique. The data obtained from this comparison with the ROC curve Figure 18b make this medium-strength molecule, but used in conjunction with the rest of the biomarker panel object of the present invention, its results are important for diagnosis, especially if we think of the staging and detection of early fibrosis.
Las proteínas seleccionadas para integrar el panel de Biomarcadores para Diagnóstico y Estadificación de la fibrosis hepática son: MFAP-4, FBN1 , ALDH1A3, CYP26A1 ; MGP, C7 y PLA2G7. Algunas de ellas no mostraron títulos detectables en pacientes con cirrosis como son C7 y PLA2G7, pero estas dos moléculas mostraron valores de ELISA muy buenos al realizar la prueba con sueros correspondientes al estadio FO-I, lo que es muy útil para la estadificación del proceso fibrótico. The proteins selected to integrate the Biomarker panel for Diagnosis and Staging of liver fibrosis are: MFAP-4, FBN1, ALDH1A3, CYP26A1; MGP, C7 and PLA2G7. Some of them did not show detectable titres in patients with cirrhosis such as C7 and PLA2G7, but these two molecules showed very good ELISA values when performing the test with sera corresponding to the FO-I stage, which is very useful for staging the process. fibrotic
El empleo de los sueros de paciente con fibrosis pulmonar idiopática y los de absceso hepático amibiano, además de sueros de individuos normales, como controles, nos permitió seleccionar las moléculas que sólo se detectan claramente en la enfermedad hepática crónica que se acompaña de fibrosis, lo cual puede darle a nuestro panel la característica de hígado específico. The use of sera from patients with idiopathic pulmonary fibrosis and those with amibian liver abscess, in addition to sera from normal individuals, as controls, allowed us to select the molecules that are only clearly detected in the chronic liver disease that is accompanied by fibrosis, which can give our panel the specific liver characteristic.
En la tabla 7 se indican los valores de absorbancia promedio obtenidos mediante la prueba de ELISA para cada una de las proteínas del panel con los sueros de los pacientes de cada grupo analizado, las cruces representan valores no significativos para la proteína individual en estos grupos de pacientes. Finalmente en la Figura 19 se muestran la gráfica resultante de la integración de los valores promedio obtenidos por ELISA, mostrados en la tabla 7. Es importante decir que al tener este panel el beneficio directo al paciente con enfermedad hepática crónica, se traduce en la posibilidad de proporcionarle un diagnóstico preciso sin necesidad de una biopsia, además brindar al médico la posibilidad de un seguimiento más frecuente y acertado del paciente, y con la detección temprana del proceso, una mejor expectativa de vida para los individuos que inician el padecimiento y a quienes actualmente es imposible detectar en etapas tempranas. Table 7 shows the average absorbance values obtained by the ELISA test for each of the panel proteins with the sera of the patients of each group analyzed, the crosses represent non-significant values for the individual protein in these groups of patients Finally, Figure 19 shows the graph resulting from the integration of the average values obtained by ELISA, shown in Table 7. It is important to say that having this panel the direct benefit to the patient with chronic liver disease, it translates into the possibility to provide a diagnosis precise without the need for a biopsy, in addition to providing the doctor with the possibility of a more frequent and successful follow-up of the patient, and with the early detection of the process, a better life expectancy for the individuals who initiate the disease and to whom it is currently impossible to detect in early stages
Tabla 7: Valores de absorbancia. Table 7: Absorbance values.
Ctls F1 F4 FP AHACtls F1 F4 FP AHA
Mfap4 0.0714 0.1778 0.3549 0.1035 0.0874 Mfap4 0.0714 0.1778 0.3549 0.1035 0.0874
C7 0.0512 0.1911 0.1676 X 0.0768  C7 0.0512 0.1911 0.1676 X 0.0768
MGP 0.0874 0.1872 0.2402 0.1029 0.0794  MGP 0.0874 0.1872 0.2402 0.1029 0.0794
Aldh1a3 0.0539 0.1588 0.1936 0.1154 0.1021  Aldh1a3 0.0539 0.1588 0.1936 0.1154 0.1021
FBN1 0.0489 0.1515 0.1186 0.0742 0.0645  FBN1 0.0489 0.1515 0.1186 0.0742 0.0645
CYP26A1 0.1179 0.1504 0.1896 0.0942 0.0916  CYP26A1 0.1179 0.1504 0.1896 0.0942 0.0916
Pla2g7 0.2299 0.17638 0.2039 X 0.0730  Pla2g7 0.2299 0.17638 0.2039 X 0.0730
La Tabla 8 muestra el índice Serológico Multicomponente, integrado por las 7 proteínas, en esta tabla podemos observar los valores de corte definidos para cada una de las proteínas, determinados mediante el análisis de los resultados obtenidos por ELISA con los sueros de pacientes con enfermedad hepática en estadio F1 o F4, las flechas, representan el incremento (hacia arriba) o disminución (hacia abajo) en la expresión de la proteína respecto a los controles. Table 8 shows the Multicomponent Serological Index, composed of the 7 proteins, in this table we can see the cut-off values defined for each of the proteins, determined by analyzing the results obtained by ELISA with the sera of patients with liver disease in stage F1 or F4, the arrows represent the increase (up) or decrease (down) in the expression of the protein with respect to the controls.
Tabla 8: índice Serológico Multicomponente Table 8: Multicomponent Serological Index
Molécula F1-F0 (Abs) F4 (Abs) F1-F0 (Abs) F4 (Abs) molecule
MFAP4 0.097 t 0.154 †  MFAP4 0.097 t 0.154 †
FBN1 0.095 t 0.08 i  FBN1 0.095 t 0.08 i
ALDH1A3 0.114 t 0.126 t  ALDH1A3 0.114 t 0.126 t
CYP26A1 0.129 X 0.134 †  CYP26A1 0.129 X 0.134 †
MGP 0.139 † 0.135 †  MGP 0.139 † 0.135 †
C7 0.119 t X  C7 0.119 t X
PLA2G7 0.175 l X De acuerdo a la combinación de los resultados que arroje la prueba para cada paciente (dentro de los rangos mostrados en la tabla 8) en la determinación de todo el panel, realizado en el suero, será posible definir en qué estadio del proceso de fibrosis se encuentra y por ende su grado de mejoría o deterioro. PLA2G7 0.175 l X According to the combination of the results of the test for each patient (within the ranges shown in table 8) in the determination of the entire panel, performed in the serum, it will be possible to define at what stage of the fibrosis process It finds and therefore its degree of improvement or deterioration.
Esta invención proporciona información importante que permitirá un mejor entendimiento de los mecanismos e interrelaciones moleculares que ocurren durante el proceso de fibrosis hepática, además de proporcionar tanto al médico como al paciente una herramienta no invasiva para el diagnóstico, estadificación y seguimiento de la fibrosis hepática. This invention provides important information that will allow a better understanding of the molecular mechanisms and interrelations that occur during the liver fibrosis process, in addition to providing both the doctor and the patient with a non-invasive tool for the diagnosis, staging and monitoring of liver fibrosis.
Agradecimientos: Este trabajo fue realizado con el financiamiento proporcionado por la DGAPA; PAPIIT # IN 208107/ IN 205210/ IT201213; SEP-CONACyT 84837 Acknowledgments: This work was carried out with the financing provided by the DGAPA; PAPIIT # IN 208107 / IN 205210 / IT201213; SEP-CONACyT 84837

Claims

REIVINDICACIONES
1. Un panel de biomarcadores no invasivos para diagnóstico y estadificación de la enfermedad hepática que curse con fibrosis, que consiste de las siguientes proteínas: MFAP-4, FBN1 , ALDH1A3, CYP26A1 ; MGP, C7 y PLA2G7. 1. A panel of non-invasive biomarkers for diagnosis and staging of liver disease that occurs with fibrosis, consisting of the following proteins: MFAP-4, FBN1, ALDH1A3, CYP26A1; MGP, C7 and PLA2G7.
2. El panel de biomarcadores de conformidad con la reivindicación 1, caracterizado porque la proteína MFAP-4 permite diferenciar entre etapas tempranas, tardías y terminales (cirrosis) del proceso fibrótico hepático. 2. The biomarker panel according to claim 1, characterized in that the MFAP-4 protein allows to differentiate between early, late and terminal stages (cirrhosis) of the hepatic fibrotic process.
3. El panel de biomarcadores de conformidad con la reivindicación 1 , caracterizado porque la proteína FBN1 permite diferenciar etapas tempranas y avanzadas del proceso fibrótico del hígado. 3. The biomarker panel according to claim 1, characterized in that the FBN1 protein makes it possible to differentiate early and advanced stages of the liver's fibrotic process.
4. El panel de biomarcadores de conformidad con la reivindicación 1 , caracterizado porque la proteína ALDH1A3 permite detectar que existe un proceso fibrótico en el hígado. 4. The biomarker panel according to claim 1, characterized in that the ALDH1A3 protein makes it possible to detect that there is a fibrotic process in the liver.
5. El panel de biomarcadores de conformidad con la reivindicación 1 , caracterizado porque la proteína CYP26A1 permite detectar el estadio cirrótico de la enfermedad hepática. 5. The biomarker panel according to claim 1, characterized in that the CYP26A1 protein allows the cirrhotic stage of liver disease to be detected.
6. El panel de biomarcadores de conformidad con la reivindicación 1 , caracterizado porque la proteína MGP permite detectar un proceso fibrótico en el hígado. 6. The biomarker panel according to claim 1, characterized in that the MGP protein allows to detect a fibrotic process in the liver.
7. El panel de biomarcadores de conformidad con la reivindicación 1 , caracterizado porque la proteína C7 permite detectar estadios iniciales de fibrosis hepática. 7. The biomarker panel according to claim 1, characterized in that the C7 protein allows to detect initial stages of liver fibrosis.
8. El panel de biomarcadores de conformidad con la reivindicación 1 , caracterizado porque la proteína PLA2G7 permite la estadificación y la detección de fibrosis hepática temprana. 8. The biomarker panel according to claim 1, characterized in that the PLA2G7 protein allows staging and detection of early liver fibrosis.
9. Una prueba in vitro para diagnóstico y estadificación de la enfermedad hepática que curse con fibrosis, que consiste de las siguientes proteínas: MFAP-4, FBN1 , ALDH1A3, CYP26A1 ; MGP, C7 y PLA2G7, presentes en el suero humano. 9. An in vitro test for diagnosis and staging of liver disease with fibrosis, which consists of the following proteins: MFAP-4, FBN1, ALDH1A3, CYP26A1; MGP, C7 and PLA2G7, present in human serum.
10. La prueba para diagnóstico y estadificación de la reivindicación 9 caracterizada porque está montada en un sistema para diagnóstico in vitro que permita la detección de las moléculas mencionadas mediante el uso de una pequeña cantidad de suero, con el empleo de anticuerpos específicos, en pruebas in vitro, tales como ELISA, WESTERN BLOT, BIOPLEX, y de cualquier método que permita determinar los niveles séricos de las proteínas incluidas en el presente panel. 10. The diagnostic and staging test of claim 9 characterized in that it is mounted in an in vitro diagnostic system that allows the detection of said molecules by using a small amount of serum, with the use of specific antibodies, in tests in vitro, such as ELISA, WESTERN BLOT, BIOPLEX, and any method that allows to determine the serum levels of the proteins included in this panel.
11. La prueba para diagnóstico y estadificación de la reivindicación 9, caracterizada por que su aplicación permite la detección del proceso fibrótico hepático en diferentes etapas de la enfermedad hepática incluyendo etapas iniciales de la enfermedad. 11. The diagnostic and staging test of claim 9, characterized in that its application allows the detection of the hepatic fibrotic process at different stages of liver disease including initial stages of the disease.
12. La prueba para diagnóstico y estadificación de la reivindicación 9, caracterizada porque permite establecer la estadificación del grado de fibrosis hepática para elegir el tratamiento más adecuado. 12. The diagnostic and staging test of claim 9, characterized in that it allows staging the degree of liver fibrosis to choose the most appropriate treatment.
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