WO2015093932A1 - Method for the early diagnosis of hepatocellular carcinoma - Google Patents

Method for the early diagnosis of hepatocellular carcinoma Download PDF

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WO2015093932A1
WO2015093932A1 PCT/MX2014/000207 MX2014000207W WO2015093932A1 WO 2015093932 A1 WO2015093932 A1 WO 2015093932A1 MX 2014000207 W MX2014000207 W MX 2014000207W WO 2015093932 A1 WO2015093932 A1 WO 2015093932A1
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ptgrl
further characterized
protein
expression
ptgr1
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PCT/MX2014/000207
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Julio Isael PÉREZ CARREÓN
Ricardo SÁNCHEZ RODRÍGUEZ
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Instituto Nacional De Medicina Genómica
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90206—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)

Definitions

  • the present invention is inserted within the field of health.
  • the present invention relates to methods of diagnosis and treatment of hepatocellular carcinoma (HCC). Specifically, the present invention relates to methods useful for identifying hepatocellular lesions related to cancer and thereby establishing the early molecular diagnosis of HCC.
  • the proposed molecular diagnostic method is based on the detection of specific biomarkers that are overexpressed in the early stages of this condition, even before the development of HCC.
  • the present invention relates to a method of early diagnosis of CHC by detecting the gene products (mRNA or protein) of prostaglandin reductase 1 (PTGR1) in biological samples.
  • the present invention relates to methods for determining whether a patient with CHC is a candidate to be treated with anti-tumor compounds bioactivated by PTGR1, such as acylfulvenes and their derivatives, among others, using overexpression of PTGR1 in tumors. as enzymatic target of therapy.
  • PTGR1 anti-tumor compounds bioactivated by PTGR1
  • the objective of the present invention is to provide new methods for the early diagnosis of HCC by the detection of specific biomarkers related to Ptgrl and its enzymatic capacities, as well as methods for the selection of individuals with HCC who are candidates for be treated with anti-tumor compounds bioactivated by PTGR1 such as acylfulvenes and their derivatives, among others.
  • CHC Hepatocellular carcinoma
  • CHC chronic liver disease
  • Chronic hepatitis B is the main risk factor for the development of CHC in Asia and Africa
  • chronic hepatitis C is in the US, Europe and Japan.
  • Approximately 80% of liver cancer cases occur in cirrhotic livers.
  • Fibrosis is observed in most chronic liver diseases and precedes the development of cirrhosis.
  • Hepatic fibrogenesis arises as a result of chronic liver damage and tissue repair. It consists of the accumulation of fibroblasts and the progressive deposition of components of the extracellular matrix in the hepatic parenchyma, such as collagen, laminin, fibronectin, among other fibrillar proteins.
  • HBV hepatitis B virus
  • Obesity and diabetes mellitus are factors that may increase the risk of developing CHC in patients with chronic viral hepatitis 4 to 40 times (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 20 3 ).
  • the BCLC classification takes into account several aspects of the disease: the patient's general state of health, the severity of the liver disease and the degree of tumor spread. Patients in stages BCLC 0 (very early) and A (early) have a better prognosis than patients in advanced stages of liver cancer. But only 25% of patients with liver cancer are diagnosed at these stages.
  • the EASL and AASLD guidelines provide recommendations regarding the most appropriate therapy for the treatment of patients at each stage of the BCLC classification. This classification is based only on clinical parameters, since there are no molecular tests yet capable of reliably assessing the individual prognosis of patients with HCC.
  • CHC can be made, by detecting malignant hepatocytes in a liver biopsy or by radiological techniques Enhanced dynamic contrast imaging that highlights the arterialized perfusion of the tumor. Additionally, enhanced contrast ultrasound can generate false positives for the diagnosis of HCC in some patients with cholangiocarcinoma, so its use as the only tool for the diagnosis of HCC is not recommended.
  • MRI or computed tomography studies may be performed. If the findings are characteristic of a malignant tumor, the diagnosis of HCC can be established. If the results are not typical of CHC, dynamic contrast imaging radiological techniques should be applied. If this complementary radiological investigation yields typical CHC results, the diagnosis is confirmed, otherwise, a directed biopsy should be performed (Ul ch Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013) .
  • dysplastic nodule and early CHC poses an important challenge for the pathologist and the medical team, because it depends on the ability to detect specific histological features when cellular dysplasia is still low and therefore undefined and that the portion of tissue contained in the biopsy contains nodular lesions.
  • ⁇ -Fetoprotein is the serological marker that has been used the most, however it has low specificity and rarely high levels are found in the early stages of CHC.
  • AFP is not useful as a tissue marker due to its low sensitivity (25-30%) even in moderately differentiated HCC (The International Consensus Group for Hepatocellular Neoplasia. Hepatology. 2009 Feb; 49 (2): 658-64 ; Forner A, Bruix J. Lancet Oncol 2012; 13: 750-751).
  • glypican 3 Another marker that has been described for the early detection of CHC is glypican 3, which has shown good results in the detection of tumors, little differentiated, however it is not useful to differentiate benign nodules from those with malignant potential because it rises in both cases. Additionally, this marker is not very sensitive to detect well-differentiated CHC (Shafizadeh N, Ferrell LD, Kakar S. Mod Pathol 2008; 21: 1011-1018).
  • HSP70 heat shock protein 70
  • GS glutamine synthetase
  • acylfulvenes are semi-synthetic derivatives of the M and S iludins produced by the fungus Omphalotus olearius. Although iludins have antitumor activity, they have low therapeutic indices since the therapeutic dose is very similar to the toxic dose of the compound.
  • acylfulvenos have therapeutic indexes higher than those of the iludins M and S and the therapeutic doses do not generate toxicity for the patients.
  • acylfulvenes and other similar bioreductive agents have the advantage that they become reactive chemical species, by specific enzymatic activation in the target cells.
  • PTGR1 is one of the enzymes that participates in the catabolism of prostaglandins and some eicosanoids (Tai HH, et al. Prostaglandins Other Lipid Mediat 2002; 68-69: 483-493). They have been assigned alternative names, associated with their molecular function, such as NADP-dependent leukotriene B4 12-hydroxyideshydrogenase (LTB4DH); 15-oxoprostaglandin 13-reductase; NADPH-dependent alkenal / one oxidoreductase; ZADH3 and dithioletiona 1 inducible gene (DIG-1), among others.
  • the human Ptg gene has orthologous genes in several organisms, and specifically they have been described in several mammalian species. Table 1 describes some examples of orthologous Ptgrl genes.
  • This enzyme reduces the unsaturated ⁇ , ⁇ carbonyls, which are produced in the cell under conditions of oxidative stress, such as 4-hydroxy-2-nonenal (4HNE) which is a byproduct of lipid peroxidation.
  • 4HNE and other aldehydes derive from the peroxidation of linoleic acid and arachidonic acid (Spitz DR, et al. Biochem J 990,267: 453-459) and have important cytotoxic effects on cells, ranging from inhibition of DNA synthesis and proteins, until induction of cell death (Chaudhary P, et al. Biochemistry 2010; 49: 6263-6275 and Forman HJ, et al. Arch Biochem Biophys 2008; 477: 183-195).
  • PTGR1 metabolizes 4-HNE in 4-hydroxy nonanal (4-HNA), through 2-alkenal / one oxidoreductase activity, by reducing the double bond ⁇ , ⁇ unsaturated (Dick RA, et al. J Biol Chem 2001; 276 : 40803-40810 and Youn B, et al. J Biol Chem 2006; 281: 40076-40088).
  • the present invention is based on the unexpected fact that the Ptgrl gene is overexpressed both in dysplastic liver nodules, as well as in early and advanced hepatocellular carcinoma (CHC) in rat models of chemical hepatocarcinogenesis using diethylnitrosamine (DEN) as chemical carcinogen
  • DEN diethylnitrosamine
  • the present invention overcomes the deficiencies of the state of the art by providing a new molecular marker and useful methods to establish the early diagnosis of HCC, from stages even prior to the establishment of the neoplasm, by detecting the presence of dysplastic nodules that are precursors of HCC .
  • Ptgrl gene in dysplastic nodules allows it to be used as a molecular biomarker to identify the nodules associated with the development of this cancer.
  • the detection of Ptgrl overexpression in biological samples of liver nodules previously detected by imaging or any other similar technique offers the possibility of establishing a new method of precise molecular diagnosis for HCC, based on the determination of gene overexpression Ptgrl (mRNA), of the protein for which it encodes or of the enzymatic activity of said protein in a biological sample.
  • the overexpression of the Ptgrl gene in dysplastic nodules and in the CHC makes it possible to establish a new method to determine if an individual suffering from CHC is a candidate to be treated with antitumor compounds that can be bioactivated by the PTGR1 enzyme.
  • the invention relates to equipment useful for the early diagnosis of CHC, which comprise means for the detection of Ptgrl gene products in a biological sample.
  • FIG 1A shows the Western blot immunodeteration of Prostaglandin Reductase 1 (PTGR1) and Glutathione S-Transferase P1 (GSTP1) in tumor samples (T) and surrounding non-tumor tissue (NT) obtained in months 8, 9, and 10 of rats submitted to the Solt and Farber protocol or resistant hepatocyte (HR) model.
  • PTGR1 protein was identified as a 33 kDa band expressed only in tumor (T) samples, while GSTP1 is expressed in both T and NT tissue. Actin was used as load control.
  • Figure 1 B shows the immunodetting of PTGR1 by Western blotting in liver protein extracts at different sacrifice times under the Schiffer protocol. It is observed that PTGR1 is present in liver tissue after 12 weeks of treatment with diethylnitrosamine (DEN).
  • DEN diethylnitrosamine
  • Figure 2 shows the 2-alkenal / one oxidoreductase activity of the
  • PTGR1 in liver samples of 6, 12 and 18 weeks of treatment using the Schiffer protocol normal liver (NH) and 6 tumors (T) of the HR model between 9 and 12 months of treatment.
  • An increase in the PTGR1 enzymatic activity is observed, which is statistically significant as of week 18.
  • Figure 3 shows the genetic expression of Ptgrl and Gstpl, a marker of rat hepatocarcinogenesis, determined by quantitative reverse transcription PCR (RT-qPCR) in liver samples of the Schiffer model at 6, 12 and 18 weeks of treatment with DEN , using the Applied Biosystems amplification system with TaqMan Gstpl (Rn00561378_gH), Ptgrl (Rn00593950_m1) and 18S rRNA (Rn03928990) probes.
  • RT-qPCR quantitative reverse transcription PCR
  • Figure 4 shows a Heatmap graph representing the genetic expression of nodules and tumors isolated by laser microdissection and determined with microarrays.
  • Global genetic expression is shown using Affymetrix microarrays in RNA obtained from 4-month nodules, from 9-month tumors (early CHC) and from 17-month tumors (advanced CHC) in hepatocarcinogenesis-induced rats using the HR model.
  • Negative GGT Remodeling (RN) Nodes, GGT Positive (RP) Remodeling Nodes and Positive GGT Persistent Nodes (NP) were analyzed.
  • the lesions identified by their GGT activity are exemplified.
  • a hierarchical grouping of 937 genes with differential expression (1.5 times change with statistical significance p ⁇ 0.05) is described in a heatmap graph (Heatmap).
  • the dendrogram at the center indicates the similarity of the genetic expression profile.
  • the similarity between the different types of nodules and tumors is observed.
  • the samples most similar to each other were the persistent nodule and the early tumor, and these in turn maintain similarity with the advanced tumor.
  • nodules in remodeling are similar to each other in their positive and negative nodular region.
  • the Ptgrl gene is included in the profile of genetic expression that characterizes persistent nodules and tumors.
  • Figure 5 shows the global genetic expression in samples of negative Gamma Glutamyl Transferase (GGT) Remodeling Nodes (Nod R neg), GGT Positive Remodeling Nodes (Nod R pos) and GGT Persistent Node Nodes (Nod P pos) obtained by microdissection and laser capture at 4 months, as well as 9-month tumors (early CHC) and 17-month tumors (advanced CHC) in the HR model.
  • GGT Gamma Glutamyl Transferase
  • Figure 5A shows a color map graph (Heatmap) describing the level of expression of Ptgrl and 5 genes previously reported as markers of hepatocarcinogenesis in rat.
  • the gradients in blue and red denote low expression and overexpression respectively, compared to normal liver. It is observed that Ptgrl is overexpressed only in persistent nodules, early CHC and advanced CHC, unlike the other genes analyzed that express themselves specifically in any type of nodules.
  • Figure 5B shows the genetic expression of Ptgrl and 5 genes previously reported as markers of rat hepatocarcinogenesis, in terms of relative intensity. It is observed that Ptgrl is overexpressed only in persistent nodules, early CHC and advanced CHC, unlike the other genes analyzed.
  • the dashed line marks the level of normal liver expression, the data corresponds to the average and standard deviation. Statistical difference as indicated with lines, ** ** p ⁇ 0.0001, *** p ⁇ 0.001, ** p ⁇ 0.01, * p ⁇ 0.05.
  • Figure 5C shows the genetic expression of Ptgrl and Gstpl determined by RT-qPCR using specific TaqMan probes, in samples from the different stages of CHC development in the HR model.
  • Overexpression of Ptgrl is checked from persistent nodules, unlike Gstpl, which is also overexpressed in remodeling nodules.
  • Statistical difference as follows: ⁇ p ⁇ 0.05 comparing with normal liver, Nod R Neg, Nod R Pos and Nod P Pos. *** p ⁇ 0.001 comparing with normal liver, Nod R Neg, and Nod R Pos. ** p ⁇ 0.01, comparing with Nod P Pos. * P ⁇ 0.05 comparing with normal liver and Nod R Neg.
  • Figure 6 shows the immunohistochemical tests for the detection of PTGR1 in liver samples of the Schiffer model. Photographs with two 50X and 200X magnifications are presented in normal liver (NB), 6, 12 and 18 week samples of DEN treatment (DEN 6, DEN 12, DEN 18) and a tumor of the HR model at 12 months Treatment (Tumor) Overexpression of PTGR1 is observed from week 12 of DEN treatment.
  • Figure 7 shows the immunohistochemical assays for the detection of PTGR1 and Glipicano-3, a marker of. hepatocarcinoma in humans, in samples of CHC of clinical origin.
  • BD-CHC liver tissue without cancer
  • MD-CHC moderately differentiated
  • PD-CHC poorly differentiated
  • Overexpression of PTGR1 is observed in human CHC samples.
  • the scale bar represents 50 ⁇ (200X) or 25 ⁇ (400X).
  • HCC hepatocellular carcinoma
  • HCC hepatic nodules by imaging. Patients with nodules are followed up with imaging at intervals of three or six months, however, the diagnosis with these techniques is inconclusive. Histopathological analysis is also not a useful tool to confirm the diagnosis of CHC in early stages because the characteristics of dysplastic cells in their early stages are not evident with these techniques. Molecular tools offer greater certainty in the diagnosis of HCC in early stages, through the use of biomarkers. Biomarkers known in the state of the art are useful for detecting HCC once the neoplasm has been established, but they are not useful for detecting precursor lesions of HCC in patients at high risk or with liver damage.
  • the present invention provides new molecular methods useful for establishing an early and accurate diagnosis of CHC, by identifying the dysplastic nodules precursors of CHC using specific biomarkers that are expressed in early stages and even from stages prior to the establishment of the neoplasm and that allow to differentiate the hepatic nodules associated with the development of CHC from those that are not associated with the development of CHC.
  • the term “regeneration nodule” refers to the hepatocyte nodule surrounded by a septal fibrosis that does not have cellular atypia
  • the term “dysplastic nodule” refers to the hepatocyte nodule with signs of dysplasia but no malignancy criteria
  • the term “hepatocarcinoma” or CHC refers to the nodule with cytological and histological atypia with malignancy criteria.
  • the other model was based on the protocol described by Schiffer and allowed to study the sequential appearance of cirrhosis and tumorigenesis within 18 weeks.
  • the development of hepatocarcinoma in the cirrhotic tissue of this model is similar to the chronology of the pathological events of human hepatocarcinogenesis.
  • results were obtained confirming the usefulness of prostaglandin reductase 1 (PTGR1) for the early diagnosis of HCC and for the detection of precursor dysplastic nodules of HCC.
  • PTGR1 prostaglandin reductase 1
  • tumor tissue was identified through its activity of Gamma Glutamyl Transferase (GGT) in histological sections, according to the technique described by Rutenburg AM, et al. J Histochem Cytochem 969; 17: 517-526.
  • GGT Gamma Glutamyl Transferase
  • NT non-tumor tissue
  • Hepatocellular tumors reached diameters of more than 5 mm, exhibited anaplasia when stained with hematoxylin-eosin (H&E) and were positive for GGT by histochemistry.
  • H&E hematoxylin-eosin
  • the tumors were distinguished directly in the liver because of its discolored appearance compared to the surrounding non-tumor tissue.
  • livers obtained from the rats treated by the Schiffer protocol were processed with a cryostat. For each section of 16 microns with GGT activity, other sections were collected until 50 mg of tissue for protein extraction or 30 mg of tissue for ribonucleic acid (RNA) extraction was obtained.
  • RNA ribonucleic acid
  • the Schiffer protocol induced multiple nodular lesions positive for GGT.
  • the incidence of these lesions increased in a time-dependent manner; at week 6, foci of altered hepatocytes ( ⁇ 0.5 mm in diameter) were detected without the presence of fibrotic septa; at week 12 nodules ( ⁇ 1 mm in diameter) were identified that were delimited by fibrotic septa and that were positive for fibrillar laminin protein; and at week 18, there was an increase in the proportion of GGT positive tissue with neoplastic lesions that, cumulatively, covered up to 80% of the liver. Rats sacrificed at 12 weeks developed cirrhosis (confirmed with H&E) and dysplastic nodules, while rats sacrificed at 18 weeks presented CHC within the cirrhotic tissue.
  • the gel band was cleaved and processed as previously described in Pérez et al., 2010 Proteome Sci 2010; 8: 27. Proteins that were extracted from the gel were processed on a Plus 4800 MALDI-TOF / TOF mass spectrometer (Applied Biosystems, USA) to obtain the MS / MS spectra and the results were analyzed using ProteinPilot software (Applied Biosystems) . The signals of the mass spectra that were obtained were subjected to a search in the UniProtKB / Swiss-Prot database using the Paragon algorithm, adjusting the search parameters to the alkylation of cysteine with iodoacetamide.
  • the enzyme PTGR1 was identified (RefSeqGenel gene bank 92227 NM_138863.2 NP_620218.1), with a confidence level> 99 % according to the number of concordant peptides (Table 2).
  • a peptide with> 99% confidence contributes 2.0 to ProtScore.
  • any immunoenzymatic technique described in the prior art can be used, including but not limited to: membrane immunodetection (Western blot), immunohistochemistry, immunosorbent assay linked to Enzymes (ELISA) in any of its variants, immunofluorescence, immunocytochemistry and immunoprecipitation, among others.
  • membrane immunodetection Western blot
  • ELISA immunosorbent assay linked to Enzymes
  • PTGR1 is an enzyme that has an NADPH-dependent alkenal / one-oxidoreductase enzyme activity 2, whereby the level of expression or the amount of this enzyme in a given sample can be determined by measuring said enzymatic activity.
  • the activity of PTGR1 can be measured through reactions with substrates that include without limiting aldehydes and unsaturated alpha-beta ketones and nitroalkenes in the presence of NADPH.
  • substrates that include without limiting aldehydes and unsaturated alpha-beta ketones and nitroalkenes in the presence of NADPH.
  • the expression level of the Ptgrl gene can be determined by measuring its mRNA.
  • Ptgrl mRNA can be determined with any molecular technique to detect or quantify nucleic acids including without limitation: RT-PCR, in situ hybridization, hybridization with specific nucleic acid probes and transcript sequencing, among others.
  • Some of the applicable standard techniques for practicing the present invention are described for example in Green M. R. and Sambrook J., Molecular Cloning. A laboratory Manual, Fourth Edition. Cold Spring Harbor Laboratory Press, 2012.
  • Figure 1A shows the expression of PTGR1 and GSTP1 in samples of the HR protocol; PTGR1 was present exclusively in tumor (T) samples at 8, 9, and 10 months, but was not detected in normal liver (HN) samples or in samples of surrounding non-tumor tissue (NT).
  • T tumor
  • HN normal liver
  • NT non-tumor tissue
  • Figure 1 B GSTP1 was found in greater abundance in tumor tissue (T), but it was also found in non-tumor tissue (NT), probably due to the presence of altered hepatocytes.
  • PTGR1 is overexpressed in foci of altered hepatocytes at 6 weeks of DEN treatment, in foci of hepatocytes and nodules at 12 weeks and in nodules and tumors at 18 weeks.
  • the histological location of PTGR1 was in the cytoplasm and in the nucleus of the neoplastic cells in liver tumors.
  • PTGR1 protein in the tissue and confirmed that it is overexpressed in tumor tissue, while maintaining normal levels in surrounding non-tumor tissue (NT) and in normal liver tissue (HN).
  • Nicotinamide Adenine Dinucleotide Phosphate (NADPH) -dependent Nicotinamide Adenine Dinucleotide (NADPH) -dependent enzyme activity was measured in various experimental samples ( Figure 2).
  • the enzymatic activity of PTGR1 was determined by the oxidation of NADPH, measured by its spectrophotometric absorption at 340 nm.
  • the trans-2-nonenal compound was used as a substrate, according to the method described in Dick RA, et al. J Biol Chem 2001; 276: 40803-40810.
  • Enzymatic activity was calculated from the molar extinction coefficient for NADPH (6.2 mM-1 cm-1) expressed as nmol of NADPH / min / mg protein.
  • Figure 2 shows the 2-alkenal / one oxidoreductase activity of PTGR1 in samples of different CHC development times. A time-dependent increase was detected between 6 and 18 weeks, showing the highest enzyme activity at 18 weeks (15 times higher compared to normal liver tissue). Tumor samples (T) in the HR protocol showed 25 times greater enzyme activity compared to normal liver tissue.
  • the overexpression of the PTGR1 protein can be determined by any immunoenzymatic technique described in the state of the art through the use of antibodies specific for this protein or by reactions that reveal the 2-alkenal / one oxidoreductase activity of this enzyme through the use of PTGR1 substrates including without limiting aldehydes and unsaturated alpha-beta ketones and nitroalkenes.
  • RT-qPCR quantitative reverse transcription PCR
  • RNAeasy (Qiagen, Hilden Germany). The concentration and purity of the RNA was determined by spectrophotometry at 260 and 280 nm, as well as its integrity and quality by capillary electrophoresis in an Agilent bioanalyzer, obtaining ratios of RNAr 28S / 18S> 1.7. Total RNA was used to mount the cDNA reactions using the High Capacity Reverse Transcription Kit (Applied Biosystems) with 750 ng of total RNA.
  • qPCR reactions were carried out using the TaqMan gene expression assay in an HT 7900 Fast Real Time PCR system (Applied Biosystem, Mexico), using fluorescein-labeled probes (FAM) (exon-exon limit) for rat Gstpl (Rn0056 378_gH), for Ptgrl (Rn00593950_m1) and for rRNA 18S (Rn03928990) of Applied Biosystems.
  • FAM fluorescein-labeled probes
  • Gstpl and Ptgrl data were normalized against the genetic expression of 8S rRNA using the AACT comparative method.
  • Figure 3 shows the increase in the level of expression of the Ptgrl and Gstpl genes as liver tumors occur in hepatocarcinogenic models.
  • the Ptgrl gene showed expression levels comparable to the Gstpl hepatocarcinogenic marker in liver tumors.
  • the biggest Ptgrl gene expression was presented in tumor samples (T), with values up to 200 times higher compared to those in normal liver (HN) samples.
  • T tumor samples
  • HN normal liver
  • This protocol results in the appearance of two types of preneoplastic nodules characterized by their Gamma Glutamyl Transferase (GGT) activity, as described in Enomoto K. and E. Farber. Cancer Res 42 (1982) 2330-5, remodeling nodules (R) and persistent nodules (P).
  • the remodeling nodules show non-uniform staining of the GGT marker, with negative zones (negative R nodules) and positive zones (positive R nodules), while persistent nodules are stained uniformly with GGT (positive P nodules).
  • RNA From the total RNA the synthesis of the complementary DNA (cDNA) was performed using oligo-dT primers, to then obtain the double stranded DNA i
  • dsDNA biotin-labeled complementary RNA
  • the data generated by the microarray image analysis were normalized using the Bioconductor software, specialized in the analysis of DNA microarrays (http://www.bioconductor.org). The information was integrated into databases with MySQL software. The genes of interest were selected from statistical analysis, hierarchical clustering analysis with Statistical Data Analysis R software (http://www.r-project.org/). Genetic expression profiles were projected for analysis with annotations of genetic expression profiles using GenMAPP software (http://www.genmapp.org/) and Ingenuity Pathways (IPA).
  • GenMAPP http://www.genmapp.org/
  • IPA Ingenuity Pathways
  • Figure 4 shows the genetic expression of the different types of nodules and tumors that develop in experimental models, isolated by laser microdissection.
  • Global genetic expression was determined using Affymetrix microarrays in RNA obtained from 4-month nodules, from 9-month tumors (early CHC) and from 17-month tumors (advanced CHC) in hepatocarcinogenesis-induced rats using the HR model.
  • the nodules in remodeling are similar to each other in their nodular region positive and negative to GGT.
  • the Ptgrl gene is included in the profile of genetic expression that characterizes persistent nodules and tumors and is not within the differentially expressed genes in remodeling nodules.
  • Figure 5A shows a heat map graph (Heatmap) that describes the level of expression of the A2m, Gpc3, Fat10, Ggt1, Gstpl and Ptgrl genes at the different stages of CHC development in the experimental model.
  • the first five genes have been reported as markers of hepatocarcinogenesis in rats, as described in French, S.W. Exp Mol Pathol. 2010 Apr; 88 (2): 219-24.
  • the Ptgrl gene is the only one that is overexpressed in persistent nodules and tumors but does not over-express in remodeling nodules, unlike the other genes that are overexpressed in any type of nodule or have an inconsistent expression level With the progression of HCC, this specific expression of the Ptgrl gene in persistent nodules allows identifying those nodules that could be precursors of HCC. The determination of the level of expression of the Ptgrl gene allows, therefore, to establish the early diagnosis of HCC and in certain cases to establish the prognosis of an individual to develop HCC.
  • Figure 5B shows the genetic expression of these 6 genes but in terms of relative genetic expression, after normalizing the intensity data of the microarrays. Similarly, it is observed that the Ptgrl gene is overexpressed in persistent nodules and tumors, while maintaining levels normal in nodules in remodeling, which allows to show persistent nodules that could be precursors of CHC.
  • FIG. 5C shows the data of the RT-qPCR where the genetic expression of Ptgrl and Gstpl was confirmed. It is observed that the Gstpl gene is overexpressed both in remodeling nodules and in persistent nodules, while the Ptgrl gene is overexpressed only in persistent nodules, thus allowing to detect those nodules that could develop in CHC and therefore perform a early diagnosis of HCC from stages even before the establishment of the neoplasm.
  • Ptgrl gene expression is elevated in persistent nodules (Nod P Pos), early tumors and advanced tumors, while remaining low in normal liver tissue and remodeling nodules.
  • the nodules and tumors generated by the Solt and Farber protocol are useful for understanding the development of human hepatocarcinoma derived from progenitor cells, as described in Andersen, J. et al. Hepatology 2010 April; 51 (4): 1401-1409.
  • the expression level of the Ptgrl gene was the only one capable of distinguishing tumors and persistent nodules from remodeling nodules (Nod R).
  • Gstpl expression is elevated in both types of preneoplastic nodules, so it is not useful to distinguish persistent nodules that could evolve in cancer.
  • Immunohistochemistry was performed using a mouse polyclonal anti-LT4DH antibody (Abnova) at a 1: 50 dilution.
  • Abnova polyclonal anti-LT4DH antibody
  • a specific immunohistochemical reaction for glypican-3 was carried out on serial sections of the same samples. This protein is a known marker for CHC and is not expressed in benign liver disorders.
  • a mouse monoclonal antibody anti-glipican-3 (1 G12) Cell Marque
  • the Ptgrl gene products can be detected in peripheral blood of individuals with CHC or its derivatives such as serum or plasma.
  • the detection of PTGR1 expression levels in peripheral blood, serum or plasma can be carried out by measuring any of its gene products.
  • serum or plasma can be used any technique known in the state of the art to detect circulating mRNA including without limiting RT-PCR, in situ hybridization, hybridization with specific nucleic acid probes and transcription sequencing, among others.
  • any immunoenzymatic technique known in the state of the art can be used to detect a specific protein including without limiting enzyme-linked immunosorbent assay (ELISA) in any of its variants, immunofluorescence, and immunoprecipitation, among others.
  • ELISA enzyme-linked immunosorbent assay
  • Overexpression of the PTGR1 protein in peripheral blood, serum or plasma can also be determined by measuring the enzymatic activity 2 alkenal / one oxidoreductase dependent on NADPH, through reactions with substrates including without limiting aldehydes and alpha-beta ketones unsaturated and nitroalkenes in the presence of NADPH.
  • PTGR1 in persistent nodules and tumors of animal models and in human CHC samples, is indicative of a molecular change inherent in hepatocellular malignant neoplasms in mammals, so the marker and Diagnostic methods described in the present application are applicable to different classes of mammals, in particular to human individuals.
  • mammal refers to those vertebrate animals with milk-producing mammary glands with which they feed their offspring, including humans and animals of veterinary or livestock interest to the being. human. Some examples of mammals of livestock interest include without limiting the pig, cow, horse, sheep and goat among others.
  • the present invention relates, in one of its aspects, to methods useful for early diagnosis of HCC from stages even prior to the establishment of the neoplasm, by determining the level of expression of the Ptgrl gene or the protein for which they encode a biological sample that can be liver tissue, peripheral blood, serum or plasma. This method is applicable to mammals of livestock or veterinary interest and in particular to human individuals at high risk of CHC.
  • the methods object of the present invention consist in determining the level of expression of the prostaglandin reductase 1 (Ptgrl) gene or of the protein for which it encodes in a previously obtained biological sample, and comparing this level of expression against an expression standard of the Ptgrl gene or its protein in individuals without CHC, where overexpression of the Ptgrl gene or its protein is indicative of the individual suffering from CHC or that he could develop CHC.
  • Ptgrl prostaglandin reductase 1
  • expression standard of Ptgrl is understood as the average expression level of the Ptgrl gene or of the protein for which it encodes detected in samples from a known population of individuals who do not suffer from CHC.
  • overexpression refers to the detectable increase in the expression of the Ptgrl gene or the protein for which it encodes or its enzymatic activity by at least 1, 2, 5 10 , 50 or 100 times more than the expression standard.
  • any methodology described in the state of the art that is useful for determining the level of expression of a gene or the protein for which it encodes can be employed.
  • the expression level of the Ptgrl gene can be determined by measuring either its mRNA, the protein for which it encodes or the enzymatic activity of the protein for which it encodes.
  • Ptgrl mRNA determination can be performed with molecular techniques that include without limitation: RT-qPCR, in situ hybridization, hybridization with specific nucleic acid probes and transcript sequencing, among others;
  • PTGR1 protein can be measured with immunoenzymatic techniques that include without limitation: immunohistochemistry, ELISA and its variations, immunofluorescence, immunocytochemistry and immunoprecipitation; and the enzymatic activity of PTGR1 can be measured with substrates that include without limitation: aldehydes and unsaturated alpha-beta ketones in the presence of NADPH.
  • the determination of the expression level of the Ptgrl gene for the early diagnosis of CHC can be carried out in different types of samples including without limitation: samples of fresh liver tissue, frozen liver tissue, enriched nodular tissue, paraffin-included liver tissue, whole blood , serum or plasma.
  • the expression of the Ptgrl gene can be determined by RT-qPCR in liver tissue samples.
  • the expression of the Ptgrl gene can be determined by RT-qPCR in peripheral blood, serum or plasma samples.
  • Table 3 shows some preferred embodiments of the invention.
  • the present invention is based on the unexpected fact that the Ptgr gene is differentially overexpressed in dysplastic liver nodules, in early CHC and advanced CHC.
  • Another application of this unexpected fact is to determine if an individual suffering from CHC is a candidate to be treated with bioactivated compounds by means of the alkenal / oxidoreductase activity of PTGR1.
  • bioactivation refers to the metabolic conversion of a chemical compound into a more active or toxic derivative within the organism.
  • a compound bioactivated by PTGR1 refers to those chemical compounds that are reduced by the alkenal / oxidoreductase activity of PTGR1 and that generate a more toxic product than the unreduced compound.
  • the level of expression of the Ptgrl gene or the enzyme for which it encodes in liver tissue samples of an individual suffering from CHC it can be defined whether said individual is a candidate to be treated with compounds that are bioactivated by the PTGR1 enzyme, due to that said compounds will be activated in dysplastic nodules or tumor tissue that overexpress this enzyme, thus generating a selective cytotoxicity.
  • the present invention relates to in vitro methods useful for determining whether an individual suffering from CHC is a candidate to be treated with antitumor compounds that are bioactivated by the PTGR1 enzyme.
  • Said method comprises detecting some of the gene products (mRNA or protein) of prostaglandin reductase 1 (Ptgrl) in a sample previously obtained and compare the level of expression in said sample against a standard of expression of PTGR1, where the overexpression of PTGR1 is indicative that the individual is a candidate to be treated with antitumor compounds that are bioactivated by PTGR1 .
  • any methodology described in the state of the art that is useful for determining the level of expression of a gene or the protein for which it encodes can be used.
  • the expression level of the Ptgrl gene can be determined by measuring either its mRNA, the protein for which it encodes or the enzymatic activity of the protein for which it encodes. Ptgrl mRNA determination can be performed with molecular techniques that include without limitation: RT-qPCR, in situ hybridization, hybridization with specific probes. nucleic acids and transcript sequencing, among others; PTGR1 protein can be measured with immunoenzymatic techniques that include without limitation: immunohistochemistry, ELISA and its variations, immunofluorescence, immunocytochemistry and immunoprecipitation; and the enzymatic activity of PTGR1 can be measured with substrates that include without limitation: aldehydes and unsaturated alpha-beta ketones in the presence of NADPH.
  • the determination of the expression level of the Ptgrl gene for the early diagnosis of CHC can be carried out in different types of samples including without limitation: samples of fresh liver tissue, frozen liver tissue, enriched nodular tissue, paraffin-included liver tissue, whole blood , serum or plasma.
  • the expression of Ptgrl is determined by measuring its enzymatic activity.
  • the antitumor compounds that are bioactivated by PTGR1 are analogous compounds of ludin or acylfulvenes.
  • the present invention relates to diagnostic equipment by means of which the methods described in the present application can be performed. These diagnostic kits comprise at least one method for determining the level of Ptgrl expression in a biological sample and a standard for comparing the level of Ptgrl expression. In this sense, diagnostic equipment includes means to determine either the mRNA, the protein or the enzymatic activity of PTGR1.
  • the means contained in the diagnostic equipment described in the present application refer to any type of laboratory reagent or medical reagent that can be used to detect the overexpression of the Ptgrl gene or protein for the one that encodes or the enzymatic activity of the PTGR enzyme.
  • These means may include without limiting polyclonal antibodies, monoclonal antibodies, humanized antibodies, specific probes, specific substrates for PTGR1, and the laboratory reagents necessary to perform standard mRNA detection techniques such as RT-qPCR, in situ hybridization, hybridization.
  • PTGR1 protein such as immunohistochemistry, ELISA and its variations, immunofluorescence, immunocytochemistry and immunoprecipitation and to determine the enzymatic activity of PTGR1 in the presence of compounds with aldehydes and alpha-beta unsaturated ketones or nitroalkenes and in the presence of NADPH
  • mice 20 male Fischer 344 rats between 180g and 200g in weight were used.
  • the animals obtained from the Laboratory Animal Production and Experimentation Unit of the CINVESTAV Mexico City (UPEAL-Cinvestav), were treated according to the institutional guidelines and protocols for handling experimental animals.
  • the rats were maintained with daily light / dark cycles of 12 h and controlled temperature, with food (basic diet) and ad libitum sterilized water.
  • the modified protocol of Solt and Farber consisted of a single intraperitonial administration of 200 mg / kg of DEN, followed by the administration of three intragastric doses of 25 mg / kg of a 1% suspension of 2-acetylaminofluorene (2-AAF) in carboxymethyl cellulose, prepared according to Semple-Roberts E, Int J Cancer 1987; 40: 643-645, on days 7, 8 and 9 after the start of the protocol with DEN and a two-thirds hepatectomy of liver tissue in the day 10 after the start of the protocol.
  • the experimental animals were sacrificed at months 1, 4, 5, 7, 9, 12 or 17, in order to evaluate the development of dysplastic nodules and cancer from their early stages.
  • the rats received intraperitoneal injections of 50 mg / kg of DEN weekly. The number of injections It was 16. Experimental rats were sacrificed at 6, 12, and 18 weeks to collect liver tissue.
  • the animals were slaughtered by exanguination under anesthesia with ether. From each animal 5 ml of blood were obtained by cardiac puncture and the liver tissue was embedded in 2-methyl butane as a cryoprotectant, frozen with liquid nitrogen and stored at -70 ° C for conservation, thus forming a tissue bank for the different essays.
  • tumor tissue was identified based on its GGT activity in histological sections, as described in Rutenburg AM, et al. J Histochem Cytochem 1969; 17: 517-526. Once identified, tumors larger than 5 mm in diameter were separated from the surrounding non-tumor tissue with a scalpel.
  • livers obtained from the rats treated by the Schiffer protocol were processed with a cryostat. For each section of 16 microns with GGT activity, another 10 sections were collected until obtaining 50 mg of tissue for protein extraction or 30 mg of tissue for RNA extraction.
  • liver tissue samples were homogenized in 1-ml lysis buffer containing 50 mM Tris-HCI, 150 mM NaCl, 0.25% Na-deoxycholate, 1 mM EDTA and 1X protease inhibitor cocktail ( ProteoBlock, Fermentas).
  • the total protein was quantified by the Lowry method (RC DC protein assay, BioRad), the samples were prepared in 5X Laemmly buffer. 30 ug of protein extract was separated by 10% SDS-PAGE.
  • the gels were stained with Coomassie colloidal blue G-250 (Bio-Safe, Bio-Rad Laboratories, USA) and the images of the gels were analyzed by band densitometry with the ImageJ software.
  • a solution of 0.5% (v / v) trifluoroacetic acid (TFA) in 50% acetonitrile was used for 10 min at room temperature, the peptides were desalted, concentrated and purified using C-18 ZipTip microcolumns ( Millipore, MA, USA) eluting in 15 ⁇ of 0.1% TFA in 50% acetonitrile.
  • TFA trifluoroacetic acid
  • Protein identification was performed on a Plus mass spectrometer.
  • the liver protein extract (30 ⁇ g) from each sample was separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore) by electrotransfer.
  • the membranes were blocked with 5% skim milk and 0.1% Tween 20 in 1X Tris buffered saline (TBS) for 1 h at room temperature.
  • TBS Tris buffered saline
  • the membranes were incubated overnight at 4 ° C with a polyclonal mouse anti-LTB4DH antibody (Abnova) at a 1: 500 dilution. After washing, the membranes were incubated for 1 h with a secondary anti-mouse antibody conjugated to peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA).
  • Chemiluminescence was obtained by adding the chemiluminescent substrate, luminol, Immobilon Western (Millipore) and the images were obtained with a VersaDoc MP 5000 digital imaging system (BioRad). The same membranes served for immunodetection with an anti-GSTP1 antibody at a 1: 1,000 dilution (Sigma Aldrich) followed by a mouse anti-actin monoclonal antibody (Chemicon International).
  • NADPH-dependent activity 2 alkenal / one oxidoreductase of PTGR1 was determined by continuous measurement of NADPH oxidation through its spectrophotometric absorption at 340 nm, trans-2- nonenal was used as substrate according to the method described in Dick RA, et al. J Biol Chem 2001; 276: 40803-40810.
  • the final reaction consisted of a solution of 0.1 mM of trans-2-nonenal, 0.1 mM of NADPH and 0.2 mg / ml of protein extract in 50 mM sodium / potassium phosphate buffer solution, pH 7.0 at 30 ° C.
  • the trans-2-nonenal was prepared as a concentrated solution (50 mM in ethanol) and then diluted 1: 10 in phosphate buffer solution; the concentration of ethanol in the final reaction was 0.2%.
  • Liver protein extracts were obtained in RIPA buffer solution with a protease inhibitor cocktail.
  • Liver lesions were identified histologically according to their GGT + activity.
  • the different types of nodules and tumors were captured by laser microdissection (LCM) using an Arcturus Laser Microdissector, Veritas 704 and special microdissection lamellae that have a layer of polyethylene naphthalate (PEN Membrane Glass Slides) as It is described in Mena JE, et al; Biochem anal. 2013 Nov 20. pii: S0003-2697 (13) 0055 -4.
  • LCM laser microdissection
  • liver tissue LCM The optimal laser settings for liver tissue LCM were 80 mW of power, 8,000 microseconds duration and an intensity of 100 mV.
  • the cutting laser was adjusted to produce a point of 2 microns in diameter at a power of 20 mW.
  • the tissue caps were placed in a GeneAmp tube (Applied Biosystems, N80 06) containing 320 ⁇ of RLT lysis buffer solution (Qiagen, RNeasy mini kit) supplemented with 1% (v / v) of ⁇ -mercaptoethanol .
  • the cDNA reactions were carried out with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using from 150 to 750 ng of total RNA. For quantitative PCR amplification a 1/10 dilution of the cDNA was used.
  • the reactions were carried out using the TaqMan gene expression assay in an HT 7900 Fast Real Time PCR system (Applied Biosystem, Mexico). Fluorescein-labeled (FAM) probes (exon-exon limit) for rat Gstpl (Rn00561378_gH), PtgM (Rn00593950_m) and 18S rRNA (Rn03928990) were obtained from Applied Biosystems. Gstpl and PtgM data they were normalized against the genetic expression of 18S rRNA using the comparative method ⁇ .
  • FAM Fluorescein-labeled
  • the first cDNA chain was obtained from 200 ng of total RNA using Superscript II reverse transcriptase and oligo-dT primers.
  • the second cDNA chain was synthesized.
  • CRNA was obtained by in vitro transcription and was used as a template for a second cycle of cDNA synthesis with the incorporation of dUTP.
  • the cDNA obtained was fragmented using uracil-DNA glycosylase. The fragments (40-70 meters) were labeled with biotin by a terminal addition reaction of deoxynucleotide.
  • the labeled cDNA was hybridized in the Rat Gene 1.0 ST microarray (Affymetrix Inc.) for 17 hours at 45 ° C.
  • the samples were washed with low astringency (SSPE) and high astringency (100 mM MES, 0.1 M NaCI) buffer solutions and stained with streptavidin-phycoerythrin using the Affymetrix 450 FS450_0007 fluid station protocol.
  • the microarrays were scanned with a GeneChip 3000 7G reader (Affymetrix, Inc.) using Expression Consarch software (Affymetrix, Inc.) to obtain the intensity and quality signals of the scanned microarrays.
  • One-way ANOVA was used to compare differences in gene expression and RNA integrity between samples obtained by CML. Statistical significance was established at p ⁇ 0.05.
  • the tissues were permeabilized with 0.5% Triton X-100 in PBS.
  • the human biopsy sections of CHC were blocked with 1% BSA for 2 hours and then incubated overnight with the primary antibody: either a polyclonal mouse antibody anti-LT4DH (anti-Ptgr-Abnova) at dilution 1: 50 or a mouse monoclonal antibody anti-glipican-3 (1 G12) (Cell Marque) at a 1: 100 dilution.

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Abstract

The invention relates to novel methods for early diagnosis of hepatocellular carcinoma (CHC) by means of the detection of the genic products (mRNA or protein) of prostaglandin reductase 1 (Ptgr1) in biological samples, and to novel methods for determining whether a patient with CHC is a candidate for treatment with antitumoral compounds bioactivated by the Ptgr1.

Description

MÉTODO DE DIAGNÓSTICO TEMPRANO DE CARCINOMA  EARLY CARCINOMA DIAGNOSTIC METHOD
HEPATOCELULAR HEPATOCELLULAR
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se inserta dentro del campo de la salud. The present invention is inserted within the field of health.
De manera general la presente invención se refiere a métodos de diagnóstico y tratamiento del carcinoma hepatocelular (CHC). De forma específica la presente invención se refiere a los métodos útiles para identificar lesiones hepatocelulares relacionadas al cáncer y establecer con esto el diagnóstico molecular temprano del CHC. El método de diagnóstico molecular propuesto se basa en la detección de biomarcadores específicos que se sobre-expresan en las etapas tempranas de este padecimiento, aún antes del desarrollo del CHC.  In general, the present invention relates to methods of diagnosis and treatment of hepatocellular carcinoma (HCC). Specifically, the present invention relates to methods useful for identifying hepatocellular lesions related to cancer and thereby establishing the early molecular diagnosis of HCC. The proposed molecular diagnostic method is based on the detection of specific biomarkers that are overexpressed in the early stages of this condition, even before the development of HCC.
Particularmente la presente invención se refiere a un método de diagnóstico temprano del CHC mediante la detección de los productos génicos (ARNm o proteína) de la prostaglandina reductasa 1 (PTGR1 ) en muestras biológicas.  Particularly the present invention relates to a method of early diagnosis of CHC by detecting the gene products (mRNA or protein) of prostaglandin reductase 1 (PTGR1) in biological samples.
Adicionalmente, la presente invención se refiere a los métodos para determinar si un paciente con CHC es candidato para ser tratado con compuestos antitumorales bioactivados por la PTGR1 , tales como los acilfulvenos y sus derivados, entre otros, utilizando la sobreexpresión de la PTGR1 en los tumores como blanco enzimático de la terapia.  Additionally, the present invention relates to methods for determining whether a patient with CHC is a candidate to be treated with anti-tumor compounds bioactivated by PTGR1, such as acylfulvenes and their derivatives, among others, using overexpression of PTGR1 in tumors. as enzymatic target of therapy.
Por lo tanto, el objetivo de la presente invención es proveer de nuevos métodos para el diagnóstico temprano del CHC mediante la detección de biomarcadores específicos relacionados a la Ptgrl y sus capacidades enzimáticas, así como métodos para la selección de individuos con CHC que sean candidatos de ser tratados con compuestos antitumorales bioactivados por la PTGR1 tales como los acilfulvenos y sus derivados, entre otros. ANTECEDENTES DE LA INVENCIÓN Therefore, the objective of the present invention is to provide new methods for the early diagnosis of HCC by the detection of specific biomarkers related to Ptgrl and its enzymatic capacities, as well as methods for the selection of individuals with HCC who are candidates for be treated with anti-tumor compounds bioactivated by PTGR1 such as acylfulvenes and their derivatives, among others. BACKGROUND OF THE INVENTION
El carcinoma hepatocelular (CHC) constituye la sexta neoplasia más frecuente en el mundo y ocupa el tercer lugar de las muertes relacionadas con el cáncer. El CHC es el tumor primario del hígado más frecuente y constituye el 90% de los casos. Las tasas de incidencia del CHC muestran un constante aumento tanto en Europa, como en los EE.UU; se diagnostican entre 600,000 y 700,000 nuevos casos al año en todo el mundo. La mortalidad del CHC es muy elevada, aproximadamente del 95%, es decir, sólo el 5% de los que la padecen sobreviven más de 5 años. (Ulrich Spengler en Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013). , Hepatocellular carcinoma (CHC) is the sixth most common neoplasm in the world and ranks third in cancer-related deaths. HCC is the most common primary liver tumor and constitutes 90% of cases. The incidence rates of CHC show a steady increase both in Europe and in the US; Between 600,000 and 700,000 new cases are diagnosed each year worldwide. The mortality of CHC is very high, approximately 95%, that is, only 5% of those who suffer from it survive more than 5 years. (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013). ,
Aunque la etiología del CHC es multifactorial, por lo general se desarrolla como consecuencia de una enfermedad hepática crónica, (principalmente la condición de cirrosis). La hepatitis B crónica constituye el principal factor de riesgo para el desarrollo de CHC en Asia y África, mientras que la hepatitis C crónica lo es en EE.UU., Europa y Japón. Aproximadamente el 80% de los casos de cáncer de hígado se presentan en hígados cirróticos. La fibrosis se observa en la mayoría de enfermedades crónicas del hígado y precede el desarrollo de cirrosis. La fibrogénesis hepática surge como consecuencia del daño hepático crónico y de la reparación tisular. Consiste en el acúmulo de fibroblastos y del depósito progresivo de componentes de la matriz extracelular en el parénquima hepático, tales como, colágena, laminina, fibronectina, entre otras proteínas fibrilares.  Although the etiology of CHC is multifactorial, it usually develops as a result of chronic liver disease, (mainly the condition of cirrhosis). Chronic hepatitis B is the main risk factor for the development of CHC in Asia and Africa, while chronic hepatitis C is in the US, Europe and Japan. Approximately 80% of liver cancer cases occur in cirrhotic livers. Fibrosis is observed in most chronic liver diseases and precedes the development of cirrhosis. Hepatic fibrogenesis arises as a result of chronic liver damage and tissue repair. It consists of the accumulation of fibroblasts and the progressive deposition of components of the extracellular matrix in the hepatic parenchyma, such as collagen, laminin, fibronectin, among other fibrillar proteins.
Los portadores crónicos del virus de la hepatitis B (VHB) tienen un riesgo 100 veces mayor de desarrollar CHC, en comparación con una población de individuos sanos no infectados. En países en vías de desarrollo, la exposición a las aflatoxinas aumenta el riesgo de desarrollar CHC (Ulrich Spengler en Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013.).  Chronic carriers of hepatitis B virus (HBV) have a 100-fold greater risk of developing CHC, compared to a population of healthy uninfected individuals. In developing countries, exposure to aflatoxins increases the risk of developing CHC (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013.).
En todo el mundo se estima que existen entre 130-170 millones de personas infectadas con el virus de la hepatitis C, de los cuales del 20 al 30% desarrollarán cirrosis hepática. De éstos, del 3 al 5% de los casos desarrollarán CHC cada año. En términos prácticos esto significa que, aproximadamente un tercio de los pacientes cirróticos con hepatitis C, desarrollarán CHC (Ulrich Spengler en Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013.). Worldwide it is estimated that there are between 130-170 million people infected with the hepatitis C virus, of which 20-30% will develop liver cirrhosis. Of these, 3 to 5% of cases will develop CHC each year. In practical terms this means that approximately one third of the Cirrhotic patients with hepatitis C will develop CHC (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013.).
Frecuentemente, los pacientes con hepatitis C crónica desarrollarán cáncer de hígado, sólo hasta la etapa de cirrosis. El consumo de alcohol o tabaco incrementan el riesgo de desarrollar CHC (Ulrich Spengler en Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013 ).  Frequently, patients with chronic hepatitis C will develop liver cancer, only until the stage of cirrhosis. The consumption of alcohol or tobacco increases the risk of developing CHC (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013).
La obesidad y la diabetes mellitus son factores que pueden incrementar de 4 a 40 veces el riesgo de desarrollar CHC en pacientes con hepatitis viral crónica (Ulrich Spengler en Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 20 3).  Obesity and diabetes mellitus are factors that may increase the risk of developing CHC in patients with chronic viral hepatitis 4 to 40 times (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 20 3 ).
Cuando el riesgo esperado de desarrollo de CHC es mayor del 1.5% por año en pacientes con hepatitis C y del 0.2% en pacientes con  When the expected risk of CHC development is greater than 1.5% per year in patients with hepatitis C and 0.2% in patients with
clínicas sugieren que se realice una vigilancia clínica
Figure imgf000005_0001
clinics suggest that clinical surveillance be performed
Figure imgf000005_0001
exámenes de ultrasonido (US) cada 6 meses y, de cada 3 meses en pacientes con cirrosis que presentan lesiones nodulares, por su elevado potencial de malignidad. El uso sistemático del ultrasonido en los pacientes con un alto riesgo de CHC permite diagnosticar el carcinoma temprano en el 30% de los pacientes, quienes tienen una posibilidad razonable de tener éxito con el tratamiento curativo. Sin embargo, esta vigilancia resulta costosa y no siempre se tiene acceso a la tecnología y al personal médico necesarios. En tanto, la determinación de alfa-fetoproteína (AFP) se ha dejado de utilizar y recomendar para la detección de CHC porque presenta una sensibilidad y especificidad insuficientes (Ulrich Spengler en Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013). Ultrasound exams (US) every 6 months and, every 3 months in patients with cirrhosis who have nodular lesions, due to their high potential for malignancy. The systematic use of ultrasound in patients with a high risk of CHC makes it possible to diagnose carcinoma early in 30% of patients, who have a reasonable chance of succeeding with curative treatment. However, this surveillance is costly and the necessary technology and medical personnel are not always available. Meanwhile, the determination of alpha-fetoprotein (AFP) has been discontinued and recommended for the detection of CHC because it has insufficient sensitivity and specificity (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013).
Los estudios histopatológicos muestran que el CHC se desarrolla en varias etapas en el tejido hepático cirrótico. De algunos nodulos regenerativos pueden transformarse hepatocitos que dan lugar a nodulos displásicos (ND) y estas lesiones preneoplásicas pueden progresar de ND de bajo grado a un ND de alto grado y así sucesivamente, hasta generar un CHC temprano y, posteriormente a un CHC avanzado (El-Serag and Rudolph, Gastroenterology. 2007 Jun; 132(7):2557-76). Estos nodulos se encuentran en una amplia gama de diagnósticos, algunos benignos y otros con potencial maligno, que sólo se pueden definir por sus características histológicas, y por tanto su manejo clínico depende de un diagnóstico histológico confiable. (The International Consensus Group for Hepatocellular Neoplasia. Hepatology. 2009 Feb;49(2):658-64). Histopathological studies show that CHC develops in several stages in cirrhotic liver tissue. From some regenerative nodules hepatocytes that give rise to dysplastic nodules (ND) can be transformed and these preneoplastic lesions can progress from low-grade ND to high-grade ND and so on, until generating an early CHC and, later, an advanced CHC ( El-Serag and Rudolph, Gastroenterology. 2007 Jun; 132 (7): 2557-76). These nodules are found in a wide range of diagnoses, some benign and others with malignant potential, which can only be defined by their histological characteristics, and therefore their clinical management depends of a reliable histological diagnosis. (The International Consensus Group for Hepatocellular Neoplasia. Hepatology. 2009 Feb; 49 (2): 658-64).
La detección del CHC en etapas tempranas es determinante para curar la enfermedad, ya que los tratamientos curativos disponibles, por ejemplo la resección quirúrgica, son eficaces en etapas tempranas. Los pacientes que reciben el diagnóstico en etapas tempranas pueden lograr tasas de supervivencia de 50-70% a 5 años, mediante resección quirúrgica, trasplante de hígado o procedimientos de ablación percutánea. Para el CHC intermedio sólo existen tratamientos que pueden prolongar un poco la vida del paciente como la embolización transarterial local y la terapia con inhibidores de cinasas (Ulrich Spengler en Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013).  The detection of CHC in early stages is decisive to cure the disease, since the available curative treatments, for example surgical resection, are effective in early stages. Patients who receive the diagnosis in early stages can achieve survival rates of 50-70% at 5 years, by surgical resection, liver transplantation or percutaneous ablation procedures. For intermediate CHC, there are only treatments that can prolong the patient's life a bit, such as local transarterial embolization and kinase inhibitor therapy (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013 ).
Los tumores se clasifican para estratificar a los pacientes en relación con su pronóstico de supervivencia y para seleccionar la mejor opción terapéutica para cada estadio del tumor. La clasificación de Barcelona (Barcelona Clinic Liver Cáncer, BCLC) ha sido adoptada como el estándar internacional para el CHC e incluso la usa y recomienda, la Asociación Americana para el Estudio de Enfermedades Hepáticas (AASLD, por sus siglas en inglés) y la Asociación Europea para el Estudio del Hígado (EASL, por sus siglas en inglés) (Ulrich Spengler en Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013).  Tumors are classified to stratify patients in relation to their survival prognosis and to select the best therapeutic option for each stage of the tumor. The Barcelona classification (Barcelona Clinic Liver Cancer, BCLC) has been adopted as the international standard for CHC and even uses and recommends it, the American Association for the Study of Liver Diseases (AASLD) and the Association European for the Study of the Liver (EASL) (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013).
La clasificación BCLC toma en cuenta varios aspectos de la enfermedad: el estado general de salud del paciente, la gravedad de la enfermedad hepática y el grado de propagación del tumor. Los pacientes en etapas BCLC 0 (muy temprana) y A (temprana) tienen mejor pronóstico que los pacientes en etapas avanzadas del cáncer de hígado. Pero sólo 25% de los pacientes con cáncer de hígado se diagnostican en estas etapas.  The BCLC classification takes into account several aspects of the disease: the patient's general state of health, the severity of the liver disease and the degree of tumor spread. Patients in stages BCLC 0 (very early) and A (early) have a better prognosis than patients in advanced stages of liver cancer. But only 25% of patients with liver cancer are diagnosed at these stages.
Las guías de la EASL y de la AASLD proveen recomendaciones respecto a la terapia más adecuada para el tratamiento de pacientes en cada una de las etapas de la clasificación BCLC. Esta clasificación se basa solamente en parámetros clínicos, ya que aún no existen pruebas moleculares capaces de evaluar de forma fiable el pronóstico individual de los pacientes con CHC.  The EASL and AASLD guidelines provide recommendations regarding the most appropriate therapy for the treatment of patients at each stage of the BCLC classification. This classification is based only on clinical parameters, since there are no molecular tests yet capable of reliably assessing the individual prognosis of patients with HCC.
El diagnóstico de CHC se puede realizar, mediante la detección de hepatocitos malignos en una biopsia de hígado o mediante técnicas radiológicas de imagen de contraste dinámico mejorado que ponen de manifiesto la perfusión arterializada del tumor. Adicionalmente el ultrasonido de contraste mejorado puede generar falsos positivos para el diagnóstico de CHC en algunos pacientes con colangiocarcinoma, por lo que no se recomienda su uso como única herramienta para el diagnóstico de CHC. The diagnosis of CHC can be made, by detecting malignant hepatocytes in a liver biopsy or by radiological techniques Enhanced dynamic contrast imaging that highlights the arterialized perfusion of the tumor. Additionally, enhanced contrast ultrasound can generate false positives for the diagnosis of HCC in some patients with cholangiocarcinoma, so its use as the only tool for the diagnosis of HCC is not recommended.
Para las lesiones nodulares inferiores a 1 cm, no se recomienda el análisis de imagen detallado debido a que la mayoría de las lesiones representarán nodulos en regeneración, en lugar de nodulos displásicos y CHC. Sin embargo, se sugiere el seguimiento clínico con técnicas de imagen cada 3 meses.  For nodular lesions smaller than 1 cm, detailed image analysis is not recommended because most lesions will represent nodules in regeneration, rather than dysplastic nodules and HCC. However, clinical follow-up with imaging techniques is suggested every 3 months.
Para lesiones mayores de 1 cm, se pueden realizar estudios de resonancia magnética o tomografía computarizada. Si los hallazgos son característicos de un tumor maligno se puede establecer el diagnóstico de CHC. Si los resultados no son típicos del CHC, se deben aplicar técnicas radiológicas de imagen de contraste dinámico. Si esta investigación radiológica complementaria arroja resultados típicos de CHC, se confirma el diagnóstico, de lo contrario, se debe realizar una biopsia dirigida (Ul ch Spengler en Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013).  For lesions larger than 1 cm, MRI or computed tomography studies may be performed. If the findings are characteristic of a malignant tumor, the diagnosis of HCC can be established. If the results are not typical of CHC, dynamic contrast imaging radiological techniques should be applied. If this complementary radiological investigation yields typical CHC results, the diagnosis is confirmed, otherwise, a directed biopsy should be performed (Ul ch Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013) .
La distinción entre un nodulo displásico y CHC temprano plantea un reto importante para el patólogo y el equipo médico, debido a que depende de la habilidad para detectar las características histológicas específicas cuando la displasia celular es aún baja y por tanto poco definida y de que la porción de tejido contenido en la biopsia contenga las lesiones nodulares.  The distinction between a dysplastic nodule and early CHC poses an important challenge for the pathologist and the medical team, because it depends on the ability to detect specific histological features when cellular dysplasia is still low and therefore undefined and that the portion of tissue contained in the biopsy contains nodular lesions.
Para compensar las deficiencias del análisis histopatológico, se han buscado marcadores biológicos asociados al desarrollo del CHC que permitan realizar un diagnóstico más certero en etapas tempranas. La α-fetoproteina (AFP) es el marcador serológico que más se ha utilizado, sin embargo presenta una baja especificidad y rara vez se encuentran niveles elevados en etapas tempranas del CHC. Además, la AFP no es útil como un marcador tisular debido a su baja sensibilidad (25-30%) aún en el CHC moderadamente diferenciado (The International Consensus Group for Hepatocellular Neoplasia. Hepatology. 2009 Feb;49(2):658-64; Forner A, Bruix J. Lancet Oncol 2012;13:750-751 ). Otro marcador que ha sido descrito para la detección temprana de CHC es el glipicano 3, que ha mostrado buenos resultados en la detección de tumores , poco diferenciados, sin embargo no es útil para diferenciar nodulos benignos de los que tienen potencial maligno porque se eleva en ambos casos. Adicionalmente este marcador es poco sensible para detectar CHC bien diferenciado (Shafizadeh N, Ferrell LD, Kakar S. Mod Pathol 2008;21 :1011-1018). To compensate for the deficiencies of the histopathological analysis, biological markers associated with the development of HCC have been sought to allow a more accurate diagnosis in early stages. Α-Fetoprotein (AFP) is the serological marker that has been used the most, however it has low specificity and rarely high levels are found in the early stages of CHC. In addition, AFP is not useful as a tissue marker due to its low sensitivity (25-30%) even in moderately differentiated HCC (The International Consensus Group for Hepatocellular Neoplasia. Hepatology. 2009 Feb; 49 (2): 658-64 ; Forner A, Bruix J. Lancet Oncol 2012; 13: 750-751). Another marker that has been described for the early detection of CHC is glypican 3, which has shown good results in the detection of tumors, little differentiated, however it is not useful to differentiate benign nodules from those with malignant potential because it rises in both cases. Additionally, this marker is not very sensitive to detect well-differentiated CHC (Shafizadeh N, Ferrell LD, Kakar S. Mod Pathol 2008; 21: 1011-1018).
Se ha propuesto la inmunodetección histológica de tres proteínas; glipicano 3, la proteína de choque térmico 70 (HSP70) y la glutamina sintetasa (GS) para diferenciar los nodulos displásicos del CHC temprano, en donde un resultado positivo para dos de cualquiera de estos tres marcadores, confirma la presencia de CHC con una sensibilidad del 72% y una especificidad del 100%. (Ulrich Spengler en Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013; The International Consensus Group for Hepatocellular Neoplasia. Hepatology. 2009 Feb;49(2):658-64).  The histological immunodetection of three proteins has been proposed; glypican 3, heat shock protein 70 (HSP70) and glutamine synthetase (GS) to differentiate dysplastic nodules from early CHC, where a positive result for two of any of these three markers confirms the presence of CHC with a sensitivity of 72% and a specificity of 100%. (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology. Flying Publisher. 2013; The International Consensus Group for Hepatocellular Neoplasia. Hepatology. 2009 Feb; 49 (2): 658-64).
También se han descrito algunos genes o firmas de genes, cuya expresión funciona como marcador para el diagnóstico de CHC pero hasta el momento ninguno ha sido validado clínicamente. La solicitud de patente norteamericana No. 2008/0038736, que se incorpora como referencia, describe métodos para establecer si un nodulo hepático es un nodulo displásico o un CHC temprano, mediante la determinación de la expresión de al menos tres, de alguno de los siguientes marcadores seleccionados TERT, GPC3, gankirina, survivina, TOP2A, LYVE1 , Ecaderina, IGFBP3, PDGFRA, TGFA, ciclina D1 y HGF, sin embargo, este método detecta el CHC en desarrollo y no es capaz de detectarlo desde la etapa en la que aparecen nodulos displásicos.  Some genes or gene signatures have also been described, whose expression functions as a marker for the diagnosis of HCC but so far none has been clinically validated. US Patent Application No. 2008/0038736, which is incorporated by reference, describes methods for establishing whether a hepatic nodule is a dysplastic nodule or an early CHC, by determining the expression of at least three, of any of the following Selected markers TERT, GPC3, gankirin, survivin, TOP2A, LYVE1, Ecaderina, IGFBP3, PDGFRA, TGFA, cyclin D1 and HGF, however, this method detects the developing CHC and is not able to detect it from the stage in which they appear dysplastic nodules
Por otra parte, la solicitud de patente norteamericana No. 2011/0306513 citada aquí como referencia, describe marcadores séricos para el diagnóstico de CHC, sin embargo las determinaciones se realizan comparando suero de individuos normales con suero de pacientes con cáncer de hígado sin correlación respecto del inicio del padecimiento, por lo que no es viable para establecer un diagnóstico temprano del cáncer hepático.  On the other hand, the US patent application No. 2011/0306513 cited here as a reference, describes serum markers for the diagnosis of CHC, however the determinations are made by comparing serum of normal individuals with serum of liver cancer patients without correlation with respect to of the onset of the condition, so it is not feasible to establish an early diagnosis of liver cancer.
Con base en el estado de la técnica actual, es evidente la necesidad de contar con nuevos métodos, para el diagnóstico temprano del CHC mediante la detección de biom arcadores específicos en tejido y en suero, capaces de detectar el padecimiento desde etapas tempranas o aún desde la aparición de nodulos displásicos. Based on the state of the art, the need for new methods is evident, for the early diagnosis of HCC by means of detection of specific biom archers in tissue and serum, capable of detecting the condition from early stages or even from the appearance of dysplastic nodules.
En lo que se refiere a los tratamientos disponibles para CHC, se ha comprobado que solamente se consiguen resultados terapéuticos exitosos, cuando el CHC se detecta en etapas tempranas. Otro problema relacionado con la práctica clínica de pacientes con CHC es la ausencia de criterios confiables para guiar la selección de un tratamiento específico.  Regarding the treatments available for CHC, it has been proven that only successful therapeutic results are achieved, when the CHC is detected in early stages. Another problem related to the clinical practice of patients with HCC is the absence of reliable criteria to guide the selection of a specific treatment.
Entre los compuestos antitumorales existentes, se han descrito los acilfulvenos, que son derivados sem ¡sintéticos de las iludinas M y S producidas por el hongo Omphalotus olearius. Si bien las iludinas tienen actividad antitumoral, presentan bajos índices terapéuticos ya que la dosis terapéutica es muy similar a la dosis tóxica del compuesto.  Among the existing antitumor compounds, acylfulvenes have been described, which are semi-synthetic derivatives of the M and S iludins produced by the fungus Omphalotus olearius. Although iludins have antitumor activity, they have low therapeutic indices since the therapeutic dose is very similar to the toxic dose of the compound.
Los acilfulvenos tienen índices terapéuticos superiores a los de las iludinas M y S y las dosis terapéuticas no generan toxicidad para los pacientes. Además, los acilfulvenos y de otros agentes biorreductores similares tienen la ventaja de que se convierten en especies químicas reactivas, mediante una activación enzimática específica en las células blanco.  The acylfulvenos have therapeutic indexes higher than those of the iludins M and S and the therapeutic doses do not generate toxicity for the patients. In addition, acylfulvenes and other similar bioreductive agents have the advantage that they become reactive chemical species, by specific enzymatic activation in the target cells.
La solicitud de patente norteamericana No. 2008/0306147, que se incorpora como referencia, describe compuestos análogos de la iludina útiles para inhibir el crecimiento tumoral, principalmente de tumores sólidos, sin embargo no especifica su utilidad para el CHC y tampoco describe un método fiable para determinar qué pacientes con CHC podrían beneficiarse con esos compuestos.  US Patent Application No. 2008/0306147, which is incorporated by reference, describes analogous iludin compounds useful for inhibiting tumor growth, mainly of solid tumors, however it does not specify its usefulness for CHC and also does not describe a reliable method. to determine which patients with CHC could benefit from these compounds.
Por otro lado se ha descrito en Yu X. et al. J Pharmacol Exp Ther. 2012 Nov;343(2):426-33, que la inducción de la PTGR1 en líneas celulares de cáncer de hígado y de colon, incrementa la susceptibilidad de las células tumorales al hidroximetilacilfulveno.  On the other hand it has been described in Yu X. et al. J Pharmacol Exp Ther. 2012 Nov; 343 (2): 426-33, that the induction of PTGR1 in liver and colon cancer cell lines, increases the susceptibility of tumor cells to hydroxymethylacetylvene.
La PTGR1 es una de las enzimas que participa en el catabolismo de prostaglandinas y algunos eicosanoides (Tai HH, et al. Prostaglandins Other Lipid Mediat 2002;68-69:483-493). Se le han asignado nombres alternativos, asociados a su función molecular, como el leucotrieno B4 12-hidroxideshidrogenasa dependiente de NADP (LTB4DH); 15-oxoprostaglandina 13-reductasa; alquenal/ona oxidorreductasa dependiente de NADPH; ZADH3 y gen inducible por ditioletiona 1 (DIG-1 ), entre otros. El gen Ptg humano tiene genes ortólogos en varios organismos, y específicamente han sido descritos en varias especies de mamíferos. En la Tabla 1 se describen algunos ejemplos de genes ortólogos de Ptgrl . PTGR1 is one of the enzymes that participates in the catabolism of prostaglandins and some eicosanoids (Tai HH, et al. Prostaglandins Other Lipid Mediat 2002; 68-69: 483-493). They have been assigned alternative names, associated with their molecular function, such as NADP-dependent leukotriene B4 12-hydroxyideshydrogenase (LTB4DH); 15-oxoprostaglandin 13-reductase; NADPH-dependent alkenal / one oxidoreductase; ZADH3 and dithioletiona 1 inducible gene (DIG-1), among others. The human Ptg gene has orthologous genes in several organisms, and specifically they have been described in several mammalian species. Table 1 describes some examples of orthologous Ptgrl genes.
Tabla 1. Genes ortólogos del gen Ptgrl en mamíferos  Table 1. Orthologous genes of the Ptgrl gene in mammals
Figure imgf000010_0001
Figure imgf000010_0001
Esta enzima reduce los carbonilos α,β insaturados, que se producen en la célula bajo condiciones de estrés oxidativo, como el 4-hidroxi-2-nonenal (4HNE) que es un subproducto de la peroxidación de lípidos. El 4HNE y otros aldehidos derivan de la peroxidación de ácido linoléico y del ácido araquidónico (Spitz DR, et al. Biochem J 990,267:453-459) y tienen importantes efectos citotoxicos sobre las células, que van desde la inhibición de la síntesis de DNA y proteínas, hasta la inducción de la muerte celular (Chaudhary P, et al. Biochemistry 2010;49:6263- 6275 y Forman HJ, et al. Arch Biochem Biophys 2008;477:183-195). This enzyme reduces the unsaturated α, β carbonyls, which are produced in the cell under conditions of oxidative stress, such as 4-hydroxy-2-nonenal (4HNE) which is a byproduct of lipid peroxidation. 4HNE and other aldehydes derive from the peroxidation of linoleic acid and arachidonic acid (Spitz DR, et al. Biochem J 990,267: 453-459) and have important cytotoxic effects on cells, ranging from inhibition of DNA synthesis and proteins, until induction of cell death (Chaudhary P, et al. Biochemistry 2010; 49: 6263-6275 and Forman HJ, et al. Arch Biochem Biophys 2008; 477: 183-195).
La PTGR1 metaboliza el 4-HNE en 4-hidroxi nonanal (4-HNA), mediante la actividad 2-alquenal/ona oxidorreductasa, al reducir el doble énlace α,β insaturado (Dick RA, et al. J Biol Chem 2001 ;276:40803-40810 y Youn B, et al. J Biol Chem 2006;281 :40076-40088).  PTGR1 metabolizes 4-HNE in 4-hydroxy nonanal (4-HNA), through 2-alkenal / one oxidoreductase activity, by reducing the double bond α, β unsaturated (Dick RA, et al. J Biol Chem 2001; 276 : 40803-40810 and Youn B, et al. J Biol Chem 2006; 281: 40076-40088).
Por otra parte se ha descrito que el gen Ptgrl es inducido por ditioletionas (Primiano T, et al. Carcinogenesis 1998;19:999-1005 y Primiano T, et al. Carcinogénesis 996; 7:2297-2303), una clase bien conocida de agentes quimiopreventores de cáncer, que inducen la producción de enzimas de detoxificacion de carcinógenos, como: NQ01 , SGT, EPHX, GCL, y UGT, a través de la activación de la vía Nrf2 (Zhang Y, Munday R. Mol Cáncer Ther 2008;7:3470-3479). Todas estas observaciones apoyan la idea de que la PTGR1 actúa como una enzima citoprotectora que tiene tanto funciones antioxidantes, como anti-inflamatorias. On the other hand, it has been described that the Ptgrl gene is induced by dithioletionas (Primiano T, et al. Carcinogenesis 1998; 19: 999-1005 and Primiano T, et al. Carcinogenesis 996; 7: 2297-2303), a well known class of cancer chemopreventing agents, which induce the production of enzymes from detoxification of carcinogens, such as: NQ01, SGT, EPHX, GCL, and UGT, through the activation of the Nrf2 pathway (Zhang Y, Munday R. Mol Cancer Ther 2008; 7: 3470-3479). All these observations support the idea that PTGR1 acts as a cytoprotective enzyme that has both antioxidant and anti-inflammatory functions.
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BREVE DESCRIPCIÓN DE LA INVENCIÓN BRIEF DESCRIPTION OF THE INVENTION
La presente invención se basa en el hecho inesperado de que el gen Ptgrl se encuentra sobre-expresado tanto en nodulos hepáticos displásicos, como en el carcinoma hepatocelular (CHC) temprano y avanzado en modelos de hepatocarcinogénesis química en rata empleando la dietilnitrosamina (DEN) como carcinógeno químico. La sobre-expresión de la PTGR1 en las lesiones inducidas experimentalmente correlaciona con la sobre-expresión de PTGR1 en muestras de CHC en individuos humanos. The present invention is based on the unexpected fact that the Ptgrl gene is overexpressed both in dysplastic liver nodules, as well as in early and advanced hepatocellular carcinoma (CHC) in rat models of chemical hepatocarcinogenesis using diethylnitrosamine (DEN) as chemical carcinogen Overexpression of PTGR1 in experimentally induced lesions correlates with overexpression of PTGR1 in CHC samples in human individuals.
La presente invención supera las deficiencias del estado de la técnica al proveer un nuevo marcador molecular y métodos útiles para establecer el diagnóstico temprano del CHC, desde etapas incluso previas al establecimiento de la neoplasia, al detectar la presencia de nodulos displásicos que son precursores del CHC.  The present invention overcomes the deficiencies of the state of the art by providing a new molecular marker and useful methods to establish the early diagnosis of HCC, from stages even prior to the establishment of the neoplasm, by detecting the presence of dysplastic nodules that are precursors of HCC .
La sobre-expresión del gen Ptgrl en nodulos displásicos, permite utilizarlo como un biomarcador molecular para identificar los nodulos asociados al desarrollo de este cáncer. La detección de la sobreexpresión de Ptgrl en muestras biológicas de nodulos hepáticos previamente detectados por imagenología o cualquier otra técnica similar, brinda la posibilidad de establecer un nuevo método de diagnóstico molecular preciso para el CHC, basado en la determinación de la sobre-expresión del gen Ptgrl (ARNm), de la proteína para la que codifica o de la actividad enzimática de dicha proteína en una muestra biológica.  Overexpression of the Ptgrl gene in dysplastic nodules allows it to be used as a molecular biomarker to identify the nodules associated with the development of this cancer. The detection of Ptgrl overexpression in biological samples of liver nodules previously detected by imaging or any other similar technique, offers the possibility of establishing a new method of precise molecular diagnosis for HCC, based on the determination of gene overexpression Ptgrl (mRNA), of the protein for which it encodes or of the enzymatic activity of said protein in a biological sample.
Por otra parte, la sobre-expresión del gen Ptgrl en nodulos displásicos y en el CHC permite establecer un nuevo método para determinar si un individuo que sufre de CHC, es candidato para ser tratado con compuestos antitumorales susceptibles de ser bioactivados por la enzima PTGR1.  On the other hand, the overexpression of the Ptgrl gene in dysplastic nodules and in the CHC makes it possible to establish a new method to determine if an individual suffering from CHC is a candidate to be treated with antitumor compounds that can be bioactivated by the PTGR1 enzyme.
En otro aspecto, la invención se refiere a equipos útiles para el diagnóstico temprano de CHC, los cuales comprenden medios para la detección de los productos génicos de Ptgrl en una muestra biológica. BREVE DESCRIPCIÓN DE LAS FIGURAS In another aspect, the invention relates to equipment useful for the early diagnosis of CHC, which comprise means for the detection of Ptgrl gene products in a biological sample. BRIEF DESCRIPTION OF THE FIGURES
La Figura 1A expone la inmunodeteeción por Western blot de la Prostaglandina Reductasa 1 (PTGR1 ) y la Glutation S-Transferasa P1 (GSTP1 ) en muestras tumorales (T) y tejido no tumoral circundante (NT) obtenidas en los meses 8, 9, y 10 de ratas sometidas al protocolo de Solt y Farber o modelo del hepatocito resistente (HR). La proteína PTGR1 fue identificada como una banda de 33 kDa expresada únicamente en las muestras de tumor (T), mientras que la GSTP1 se expresa tanto en T como en tejido NT. Se utilizó actina como control de carga. Figure 1A shows the Western blot immunodeteration of Prostaglandin Reductase 1 (PTGR1) and Glutathione S-Transferase P1 (GSTP1) in tumor samples (T) and surrounding non-tumor tissue (NT) obtained in months 8, 9, and 10 of rats submitted to the Solt and Farber protocol or resistant hepatocyte (HR) model. PTGR1 protein was identified as a 33 kDa band expressed only in tumor (T) samples, while GSTP1 is expressed in both T and NT tissue. Actin was used as load control.
La Figura 1 B exhibe la inmunodeteeción de PTGR1 por Western blot en extractos de proteína hepática a diferentes tiempos de sacrificio bajo el protocolo de Schiffer. Se observa que la PTGR1 se encuentra presente en el tejido hepático tras 12 semanas de tratamiento con dietilnitrosamina (DEN).  Figure 1 B shows the immunodetting of PTGR1 by Western blotting in liver protein extracts at different sacrifice times under the Schiffer protocol. It is observed that PTGR1 is present in liver tissue after 12 weeks of treatment with diethylnitrosamine (DEN).
La Figura 2 expone la actividad 2-alquenal/ona oxidorreductasa de la Figure 2 shows the 2-alkenal / one oxidoreductase activity of the
PTGR1 en muestras hepáticas de 6, 12 y 18 semanas de tratamiento mediante el protocolo de Schiffer, hígado normal (HN) y 6 tumores (T) del modelo del HR de entre 9 y 12 meses de tratamiento. Se observa un aumento de la actividad enzimática de la PTGR1 que es estadísticamente significativo a partir de la semana 18. Diferencia estadística según se indica ****p< 0.0001 comparada con HN, 6, 12 y 18 semanas, ***p<0.001 comparada con HN, **p<0.01 comparada con 6 semanas. PTGR1 in liver samples of 6, 12 and 18 weeks of treatment using the Schiffer protocol, normal liver (NH) and 6 tumors (T) of the HR model between 9 and 12 months of treatment. An increase in the PTGR1 enzymatic activity is observed, which is statistically significant as of week 18. Statistical difference as indicated **** p <0.0001 compared to NH, 6, 12 and 18 weeks, * ** p < 0.001 compared to HN, * * p <0.01 compared to 6 weeks.
La Figura 3 expone la expresión genética de Ptgrl y Gstpl , un marcador de hepatocarcinogénesis en rata, determinadas mediante PCR cuantitativo de transcripción reversa (RT-qPCR) en muestras hepáticas del modelo de Schiffer a las 6, 12 y 18 semanas de tratamiento con DEN, empleando el sistema de amplificación de Applied Biosystems con las sondas TaqMan Gstpl (Rn00561378_gH), Ptgrl (Rn00593950_m1 ) y 18S rRNA (Rn03928990).  Figure 3 shows the genetic expression of Ptgrl and Gstpl, a marker of rat hepatocarcinogenesis, determined by quantitative reverse transcription PCR (RT-qPCR) in liver samples of the Schiffer model at 6, 12 and 18 weeks of treatment with DEN , using the Applied Biosystems amplification system with TaqMan Gstpl (Rn00561378_gH), Ptgrl (Rn00593950_m1) and 18S rRNA (Rn03928990) probes.
Adicionalmente se presenta la expresión genética de 6 tumores (T) del modelo del HR de entre 9 y 12 meses de tratamiento. Los datos se calcularon como expresión relativa utilizando el método ΔΔ0Τ. Se empleó la expresión del Acido Ribonucleico ribosomal (ARNr) 18S como gen constitutivo y la del hígado normal (HN) como referencia control. Diferencia estadística ***p<0.001 comparada con el HN, **p<0.01 comparada con el HN, *p<0.05 comparada con 6 semanas,††p<0.01 comparada con 6 semanas,†p<0.05 comparada con 6, 12 y 18 semanas. Additionally, the genetic expression of 6 tumors (T) of the HR model between 9 and 12 months of treatment is presented. Data were calculated as relative expression using the ΔΔ0Τ method. The expression of Ribosomal Ribonucleic Acid (rRNA) 18S was used as the constitutive gene and that of the liver normal (HN) as control reference. Statistical difference *** p <0.001 compared to NH, * * p <0.01 compared to NH, * p <0.05 compared to 6 weeks, †† p <0.01 compared to 6 weeks, † p <0.05 compared to 6, 12 and 18 weeks.
La Figura 4 muestra un gráfico de mapa de calor (Heatmap) que representa la expresión genética de nodulos y tumores aislados mediante microdisección láser y determinada con microarreglos. Se muestra la expresión genética global usando microarreglos Affymetrix en ARN obtenido de nodulos de 4 meses, de tumores de 9 meses (CHC temprano) y de tumores de 17 meses (CHC avanzado) en ratas inducidas a hepatocarcinogenesis usando el modelo del HR. Se analizaron Nodulos en Remodelación GGT negativos (RN), Nodulos en Remodelación GGT positivos (RP) y Nodulos Persistentes GGT positivos (NP). A la izquierda de la Figura se ejemplifican las lesiones identificadas por su actividad GGT. A la derecha se describe en un gráfico de mapa de calor (Heatmap) un agrupamiento jerárquico de 937 genes con expresión diferencial (1.5 veces de cambio con significancia estadística p<0.05). El dendrograma al centro indica la similitud del perfil de expresión genético.  Figure 4 shows a Heatmap graph representing the genetic expression of nodules and tumors isolated by laser microdissection and determined with microarrays. Global genetic expression is shown using Affymetrix microarrays in RNA obtained from 4-month nodules, from 9-month tumors (early CHC) and from 17-month tumors (advanced CHC) in hepatocarcinogenesis-induced rats using the HR model. Negative GGT Remodeling (RN) Nodes, GGT Positive (RP) Remodeling Nodes and Positive GGT Persistent Nodes (NP) were analyzed. To the left of the Figure, the lesions identified by their GGT activity are exemplified. On the right, a hierarchical grouping of 937 genes with differential expression (1.5 times change with statistical significance p <0.05) is described in a heatmap graph (Heatmap). The dendrogram at the center indicates the similarity of the genetic expression profile.
Se observa la similitud entre los distintos tipos de nodulos y tumores. Las muestras con mayor similitud entre si fueron el nodulo persistente y el tumor temprano, y éstos a su vez mantienen similitud con el tumor avanzado. Por otro lado los nodulos en remodelación son similares entre si en su región nodular positiva y negativa. El gen Ptgrl está incluido dentro del perfil de expresión genética que caracteriza a los nodulos persistentes y a los tumores.  The similarity between the different types of nodules and tumors is observed. The samples most similar to each other were the persistent nodule and the early tumor, and these in turn maintain similarity with the advanced tumor. On the other hand, nodules in remodeling are similar to each other in their positive and negative nodular region. The Ptgrl gene is included in the profile of genetic expression that characterizes persistent nodules and tumors.
La Figura 5 expone la expresión genética global en muestras de Nodulos en Remodelación Gamma Glutamil Transferasa (GGT) negativos (Nod R neg), Nodulos en Remodelación GGT positivos (Nod R pos) y Nodulos Persistentes GGT positivos (Nod P pos) obtenidos por microdisección y captura láser a los 4 meses, así como de tumores de 9 meses (CHC temprano) y de tumores de 17 meses (CHC avanzado) en el modelo del HR.  Figure 5 shows the global genetic expression in samples of negative Gamma Glutamyl Transferase (GGT) Remodeling Nodes (Nod R neg), GGT Positive Remodeling Nodes (Nod R pos) and GGT Persistent Node Nodes (Nod P pos) obtained by microdissection and laser capture at 4 months, as well as 9-month tumors (early CHC) and 17-month tumors (advanced CHC) in the HR model.
La Figura 5A muestra un gráfico de mapa de color (Heatmap) que describe el nivel de expresión de Ptgrl y 5 genes previamente reportados como marcadores de hepatocarcinogénesis en rata. Los gradientes en azul y rojo denotan la baja expresión y la sobre-expresión respectivamente, comparada con el hígado normal. Se observa que la Ptgrl se sobre-expresa solo en nodulos persistentes, CHC temprano y CHC avanzado, a diferencia de los demás genes analizados que se expresan ¡nespecíficamente en cualquier tipo de nodulos. Figure 5A shows a color map graph (Heatmap) describing the level of expression of Ptgrl and 5 genes previously reported as markers of hepatocarcinogenesis in rat. The gradients in blue and red denote low expression and overexpression respectively, compared to normal liver. It is observed that Ptgrl is overexpressed only in persistent nodules, early CHC and advanced CHC, unlike the other genes analyzed that express themselves specifically in any type of nodules.
La Figura 5B expone la expresión genética de Ptgrl y 5 genes previamente reportados como marcadores de hepatocarcinogénesis en rata, en términos de intensidad relativa. Se observa que Ptgrl se sobre-expresa solo en nodulos persistentes, CHC temprano y CHC avanzado, a diferencia de los demás genes analizados. La línea discontinua marca el nivel de expresión del hígado normal, los datos corresponden al promedio y desviación estándar. Diferencia estadística según se indica con líneas, ****p< 0.0001 , ***p<0.001 , **p<0.01 , *p<0.05. Figure 5B shows the genetic expression of Ptgrl and 5 genes previously reported as markers of rat hepatocarcinogenesis, in terms of relative intensity. It is observed that Ptgrl is overexpressed only in persistent nodules, early CHC and advanced CHC, unlike the other genes analyzed. The dashed line marks the level of normal liver expression, the data corresponds to the average and standard deviation. Statistical difference as indicated with lines, ** ** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05.
La Figura 5C muestra la expresión genética de Ptgrl y Gstpl determinada por RT-qPCR utilizando sondas TaqMan específicas, en muestras de los diferentes estadios de desarrollo del CHC en el modelo del HR. Se comprueba la sobre-expresión de Ptgrl a partir de nodulos persistentes, a diferencia del Gstpl que se sobre-expresa también en nodulos en remodelación. Diferencia estadística como sigue:†p<0.05 comparando con hígado normal, Nod R Neg, Nod R Pos y Nod P Pos. ***p<0.001 comparando con hígado normal, Nod R Neg, y Nod R Pos. **p<0.01 , comparando con Nod P Pos. *p<0.05 comparando con hígado normal y Nod R Neg.  Figure 5C shows the genetic expression of Ptgrl and Gstpl determined by RT-qPCR using specific TaqMan probes, in samples from the different stages of CHC development in the HR model. Overexpression of Ptgrl is checked from persistent nodules, unlike Gstpl, which is also overexpressed in remodeling nodules. Statistical difference as follows: † p <0.05 comparing with normal liver, Nod R Neg, Nod R Pos and Nod P Pos. *** p <0.001 comparing with normal liver, Nod R Neg, and Nod R Pos. ** p < 0.01, comparing with Nod P Pos. * P <0.05 comparing with normal liver and Nod R Neg.
La Figura 6 expone los ensayos inmunohistoquímicos para la detección de la PTGR1 en muestras de hígado del modelo de Schiffer. Se presentan fotografías con dos aumentos 50X y 200X en muestras de hígado normal (HN), 6, 12 y 18 semanas de tratamiento con DEN (DEN 6, DEN 12, DEN 18) y de un tumor del modelo del HR a los 12 meses de tratamiento (Tumor) Se observa la sobre- expresión de la PTGR1 a partir de la semana 12 de tratamiento con DEN.  Figure 6 shows the immunohistochemical tests for the detection of PTGR1 in liver samples of the Schiffer model. Photographs with two 50X and 200X magnifications are presented in normal liver (NB), 6, 12 and 18 week samples of DEN treatment (DEN 6, DEN 12, DEN 18) and a tumor of the HR model at 12 months Treatment (Tumor) Overexpression of PTGR1 is observed from week 12 of DEN treatment.
La Figura 7 muestra los ensayos de ¡nmunohistoquímica para la detección de PTGR1 y Glipicano-3, un marcador de. hepatocarcinoma en humanos, en muestras de CHC de origen clínico. Imágenes representativas de un tejido hepático sin cáncer (No-cáncer), de hepatocarcinoma bien diferenciado (BD- CHC), moderadamente diferenciado (MD-CHC) y poco diferenciado (PD-CHC). Se observa la sobre-expresión de la PTGR1 en las muestras de CHC de humanos. La barra de escala representa 50μιτι (200X) ó 25 μηη (400X). Figure 7 shows the immunohistochemical assays for the detection of PTGR1 and Glipicano-3, a marker of. hepatocarcinoma in humans, in samples of CHC of clinical origin. Representative images of a liver tissue without cancer (Non-cancer), of well differentiated hepatocarcinoma (BD-CHC), moderately differentiated (MD-CHC) and poorly differentiated (PD-CHC). Overexpression of PTGR1 is observed in human CHC samples. The scale bar represents 50μιτι (200X) or 25 μηη (400X).
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
La detección y diagnóstico temprano de un cáncer en un individuo es un factor que determina el éxito del tratamiento. Con las herramientas diagnósticas actuales es poco frecuente detectar el carcinoma hepatocelular (CHC) en etapas tempranas y no es posible detectarlo en etapas previas al establecimiento de la neoplasia, a pesar de que por su evolución se presentan cambios celulares específicos. Los tratamientos disponibles para el CHC, tales como la resección y la ablación percutánea, sólo son efectivos cuando se detecta el desarrollo de la enfermedad en etapas tempranas. The early detection and diagnosis of a cancer in an individual is a factor that determines the success of the treatment. With the current diagnostic tools it is rare to detect hepatocellular carcinoma (HCC) in early stages and it is not possible to detect it in stages prior to the establishment of the neoplasm, although due to its evolution there are specific cellular changes. The treatments available for HCC, such as resection and percutaneous ablation, are only effective when early disease development is detected.
Actualmente el diagnóstico temprano del CHC se basa en la detección de nodulos hepáticos mediante imagenología. A los pacientes con nodulos, se les da un seguimiento con imagenología a intervalos de tres o seis meses, sin embargo, el diagnóstico con estas técnicas no es concluyente. El análisis histopatológico tampoco constituye una herramienta útil para confirmar el diagnóstico de CHC en etapas tempranas porque las características de las células displásicas en sus etapas iniciales no son evidentes con estas técnicas. Las herramientas moleculares ofrecen una mayor certeza en el diagnóstico del CHC en etapas tempranas, a través del uso de biomarcadores. Los biomarcadores conocidos en el estado de la técnica son útiles para detectar el CHC una vez que se ha establecido la neoplasia, pero no son útiles para detectar lesiones precursoras del CHC en los pacientes con alto riesgo o con daño hepático.  Currently the early diagnosis of HCC is based on the detection of hepatic nodules by imaging. Patients with nodules are followed up with imaging at intervals of three or six months, however, the diagnosis with these techniques is inconclusive. Histopathological analysis is also not a useful tool to confirm the diagnosis of CHC in early stages because the characteristics of dysplastic cells in their early stages are not evident with these techniques. Molecular tools offer greater certainty in the diagnosis of HCC in early stages, through the use of biomarkers. Biomarkers known in the state of the art are useful for detecting HCC once the neoplasm has been established, but they are not useful for detecting precursor lesions of HCC in patients at high risk or with liver damage.
La presente invención proporciona nuevos métodos moleculares útiles para establecer un diagnóstico temprano y preciso del CHC, mediante la identificación de los nodulos displásicos precursores del desarrollo de CHC empleando biomarcadores específicos que se expresan en etapas tempranas e incluso desde etapas anteriores al establecimiento de la neoplasia y que permiten diferenciar a los nodulos hepáticos asociados al desarrollo de CHC de aquellos que no están asociados al desarrollo de CHC.  The present invention provides new molecular methods useful for establishing an early and accurate diagnosis of CHC, by identifying the dysplastic nodules precursors of CHC using specific biomarkers that are expressed in early stages and even from stages prior to the establishment of the neoplasm and that allow to differentiate the hepatic nodules associated with the development of CHC from those that are not associated with the development of CHC.
Como se usa en la descripción de la presente invención, el término "nodulo de regeneración" se refiere al nodulo de hepatocitos rodeado de una fibrosis septal que no presenta atipia celular, el término "nodulo displásico" se refiere al nodulo de hepatocitos con signos de displasia pero sin criterios de malignidad; y el término "hepatocarcinoma" o CHC se refiere al nodulo con atipias citológicas e histológicas con criterios de malignidad. As used in the description of the present invention, the term "regeneration nodule" refers to the hepatocyte nodule surrounded by a septal fibrosis that does not have cellular atypia, the term "dysplastic nodule" refers to the hepatocyte nodule with signs of dysplasia but no malignancy criteria; and the term "hepatocarcinoma" or CHC refers to the nodule with cytological and histological atypia with malignancy criteria.
Para el desarrollo de la presente invención se emplearon dos modelos experimentales en animales para estudiar el desarrollo del CHC. Uno de ellos se basa en un protocolo de Solt y Farber también conocido como modelo del hepatocito resistente (HR), con el que se estudiaron las alteraciones del CHC desde la aparición de nodulos de hepatocitos hasta su progresión en cáncer, en un período de 12 meses.  For the development of the present invention two experimental models in animals were used to study the development of CHC. One of them is based on a protocol of Solt and Farber also known as a model of the resistant hepatocyte (HR), with which CHC alterations were studied from the appearance of hepatocyte nodules until their progression in cancer, in a period of 12 months
El otro modelo se basó en el protocolo descrito por Schiffer y permitió estudiar la aparición secuencial de la cirrosis y la tumorigénesis en un plazo de 18 semanas. El desarrollo del hepatocarcinoma en el tejido cirrótico de este modelo es similar a la cronología de los eventos patológicos de la hepatocarcinogénesis humana. En ambos modelos se obtuvieron resultados que confirman la utilidad de la prostaglandina reductasa 1 (PTGR1 ) para el diagnóstico temprano del CHC y para la detección de nodulos displásicos precursores del CHC.  The other model was based on the protocol described by Schiffer and allowed to study the sequential appearance of cirrhosis and tumorigenesis within 18 weeks. The development of hepatocarcinoma in the cirrhotic tissue of this model is similar to the chronology of the pathological events of human hepatocarcinogenesis. In both models, results were obtained confirming the usefulness of prostaglandin reductase 1 (PTGR1) for the early diagnosis of HCC and for the detection of precursor dysplastic nodules of HCC.
En las muestras de tejido hepático, se identificó el tejido tumoral a través de su actividad de Gamma Glutamil Transferasa (GGT) en secciones histológicas, de acuerdo con la técnica descrita por Rutenburg AM, et al. J Histochem Cytochem 969;17:517-526. Para el protocolo Jel HR, los tumores (T) mayores de 5 mm de diámetro se separaron del tejido congelado junto con el tejido no tumoral circundante (NT). Mediante este protocolo, en el primer mes, se indujeron nodulos de hepatocitos de hasta 1 mm de diámetro positivos para el marcador GGT que, en conjunto, representó un 4.8% del área total del hígado. Algunos de estos nodulos alcanzaron diámetros de hasta 3 mm después de 5 meses y mostraron una proliferación celular aumentada de acuerdo con el mareaje para la proteína Ki-67.  In liver tissue samples, tumor tissue was identified through its activity of Gamma Glutamyl Transferase (GGT) in histological sections, according to the technique described by Rutenburg AM, et al. J Histochem Cytochem 969; 17: 517-526. For the Jel HR protocol, tumors (T) larger than 5 mm in diameter were separated from the frozen tissue along with the surrounding non-tumor tissue (NT). Through this protocol, in the first month, hepatocyte nodules of up to 1 mm in diameter were induced positive for the GGT marker which, together, represented 4.8% of the total liver area. Some of these nodules reached diameters of up to 3 mm after 5 months and showed an increased cell proliferation according to the Ki-67 protein screening.
Cuando los animales fueron sacrificados entre los 7 y 9 meses, el hígado presentó CHC con tamaños que dependían del tiempo de sacrificio: Los tumores hepatocelulares alcanzaron diámetros de más de 5 mm, exhibieron anaplasia al ser teñidos con hematoxilina-eosina (H&E) y fueron positivos para GGT por histoquímica. Además, los tumores fueron distinguidos directamente en el hígado por su apariencia descolorida en comparación con el tejido circundante no tumoral. When the animals were sacrificed between 7 and 9 months, the liver presented CHC with sizes that depended on the time of sacrifice: Hepatocellular tumors reached diameters of more than 5 mm, exhibited anaplasia when stained with hematoxylin-eosin (H&E) and were positive for GGT by histochemistry. In addition, the tumors were distinguished directly in the liver because of its discolored appearance compared to the surrounding non-tumor tissue.
Los hígados obtenidos de las ratas tratadas mediante el protocolo de Schiffer se procesaron con un criostato. Por cada sección de 16 mieras con actividad de GGT, se colectaron otras secciones hasta obtener 50 mg de tejido para la extracción de proteínas ó 30 mg de tejido para la extracción de ácido ribonucleico (ARN).  The livers obtained from the rats treated by the Schiffer protocol were processed with a cryostat. For each section of 16 microns with GGT activity, other sections were collected until 50 mg of tissue for protein extraction or 30 mg of tissue for ribonucleic acid (RNA) extraction was obtained.
El protocolo de Schiffer indujo múltiples lesiones nodulares positivas para GGT. La incidencia de estas lesiones aumentó de manera dependiente del tiempo; en la semana 6, se detectaron focos de hepatocitos alterados (<0.5 mm de diámetro) sin la presencia de septos fibróticos; en la semana 12 se identificaron nodulos (<1 mm de diámetro) que se encontraban delimitados por septos fibróticos y que fueron positivos a la proteína fibrilar laminina; y en la semana 18, se presentó un aumento en la proporción de tejido positivo a GGT con lesiones neoplásicas que, de manera acumulativa, abarcaron hasta el 80% del hígado. Las ratas sacrificadas a las 12 semanas desarrollaron cirrosis (confirmada con H&E) y nodulos displásicos, mientras que las ratas sacrificadas a las 18 semanas presentaron CHC dentro del tejido cirrótico.  The Schiffer protocol induced multiple nodular lesions positive for GGT. The incidence of these lesions increased in a time-dependent manner; at week 6, foci of altered hepatocytes (<0.5 mm in diameter) were detected without the presence of fibrotic septa; at week 12 nodules (<1 mm in diameter) were identified that were delimited by fibrotic septa and that were positive for fibrillar laminin protein; and at week 18, there was an increase in the proportion of GGT positive tissue with neoplastic lesions that, cumulatively, covered up to 80% of the liver. Rats sacrificed at 12 weeks developed cirrhosis (confirmed with H&E) and dysplastic nodules, while rats sacrificed at 18 weeks presented CHC within the cirrhotic tissue.
Con el propósito de identificar las diferencias de expresión de las proteínas entre el tejido hepático tumoral y el tejido hepático normal, se analizaron las proteínas mediante electroforesis en gel de poliacrilamida con dodecilsulfato de sodio (SDS-PAGE). Los perfiles de las proteínas del tejido hepático normal (HN) y las del tejido no tumoral (NT) fueron similares entre sí, mientras que los de las proteínas del tejido tumoral (T) revelaron bandas distintas, en particular se detectó una banda de aproximadamente 33 kDa en el tejido tumoral (T).  In order to identify differences in protein expression between tumor liver tissue and normal liver tissue, proteins were analyzed by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE). The profiles of normal liver tissue (HN) and non-tumor tissue (NT) proteins were similar to each other, while those of tumor tissue (T) proteins revealed different bands, in particular a band of approximately 33 kDa in the tumor tissue (T).
Para identificar las proteínas presentes en la banda de 33 kDa, se escindió la banda del gel y se procesó de acuerdo a lo descrito previamente en Pérez et al., 2010 Proteome Sci 2010;8:27. Las proteínas que se extrajeron del gel se procesaron en un espectrómetro de masas Plus 4800 MALDI-TOF/TOF (Applied Biosystems, EE.UU.) para obtener los espectros MS/MS y los resultados se analizaron utilizando el software ProteinPilot (Applied Biosystems). Las señales de los espectros de masas que se obtuvieron se sometieron a una búsqueda en la base de datos UniProtKB/Swiss-Prot utilizando el algoritmo de Paragon, ajustando los parámetros de la búsqueda a la alquilación de cisteína con yodoacetamida. To identify the proteins present in the 33 kDa band, the gel band was cleaved and processed as previously described in Pérez et al., 2010 Proteome Sci 2010; 8: 27. Proteins that were extracted from the gel were processed on a Plus 4800 MALDI-TOF / TOF mass spectrometer (Applied Biosystems, USA) to obtain the MS / MS spectra and the results were analyzed using ProteinPilot software (Applied Biosystems) . The signals of the mass spectra that were obtained were subjected to a search in the UniProtKB / Swiss-Prot database using the Paragon algorithm, adjusting the search parameters to the alkylation of cysteine with iodoacetamide.
Al analizar la banda de 33 kDa que se expresó en las muestras de tejido tumoral pero no en las de tejido hepático normal se identificó la enzima PTGR1 (Banco de genes RefSeqGenel 92227 NM_138863.2 NP_620218.1 ), con un nivel de confianza >99% de acuerdo con el número de péptidos concordantes (Tabla 2).  When analyzing the 33 kDa band that was expressed in tumor tissue samples but not in those of normal liver tissue, the enzyme PTGR1 was identified (RefSeqGenel gene bank 92227 NM_138863.2 NP_620218.1), with a confidence level> 99 % according to the number of concordant peptides (Table 2).
Tabla 2. Identificación de PTGR1 en tumores hepáticos de rata.  Table 2. Identification of PTGR1 in rat liver tumors.
Estadística de péptidos Identificados para la PTGR1  Peptide Statistics Identified for PTGR1
Figure imgf000020_0001
Figure imgf000020_0001
a La posición que ocupó la proteína en la lista de proteínas identificadas a 33 kDa de acuerdo al valor ProtScore.  a The position occupied by the protein in the list of proteins identified at 33 kDa according to the ProtScore value.
D Péptidos identificados con 99% de confianza. D Peptides identified with 99% confidence.
c Medida calculada a partir de la evidencia de péptidos identificados según el valor de confianza.  c Measure calculated from the evidence of peptides identified according to the confidence value.
Un péptído con >99% de confianza contribuye en 2.0 al ProtScore.  A peptide with> 99% confidence contributes 2.0 to ProtScore.
d Porcentaje de aminoácidos de los péptidos identificados respecto a la secuencia de la protelna.  d Percentage of amino acids of the peptides identified with respect to the prothena sequence.
Para determinar la sobre-expresión de la PTGR1 en los diferentes estadios del desarrollo del CHC se realizaron experimentos empleando diversas técnicas moleculares, útiles para poner de manifiesto el nivel de expresión de la PTGR1. El nivel de una proteína en particular se puede determinar mediante cualquiera de las metodologías descritas en el estado de la técnica para la identificación y cuantificación específica de proteínas. To determine the overexpression of PTGR1 at different stages of the development of CHC, experiments were carried out using various molecular techniques, useful to show the level of expression of PTGR1. The level of a particular protein can be determined by any of the methodologies described in the state of the art for the specific identification and quantification of proteins.
Para determinar el nivel de expresión o la cantidad de la proteína PTGR1 en una muestra determinada se pueden emplear cualquier técnica inmunoenzimática descrita en el estado de la técnica, incluyendo sin limitar: inmunodetección en membrana (Western blot), inmunohistoquímica, ensayo por inmunoabsorción ligado a enzimas (ELISA por sus siglas en inglés) en cualquiera de sus variantes, inmunofluorescencia, inmunocitoquímica e inmunoprecipitación, entre otras. Algunas de las técnicas estándar aplicables para poner en práctica la presente invención se describen por ejemplo en Green M. R. y Sambrook J., Molecular Cloning. A laboratory Manual, Fourth Edition. Cold Spring Harbor Laboratory Press, 2012 y en Greenfield E. A., Antibodies. A laboratory Manual Cold Spring Harbor Laboratory Press, 2013. To determine the level of expression or the amount of PTGR1 protein in a given sample, any immunoenzymatic technique described in the prior art can be used, including but not limited to: membrane immunodetection (Western blot), immunohistochemistry, immunosorbent assay linked to Enzymes (ELISA) in any of its variants, immunofluorescence, immunocytochemistry and immunoprecipitation, among others. Some of the applicable standard techniques for practicing the present invention are described for example in Green MR and Sambrook J., Molecular Cloning. A laboratory Manual, Fourth Edition. Cold Spring Harbor Laboratory Press, 2012 and in Greenfield EA, Antibodies. A laboratory Manual Cold Spring Harbor Laboratory Press, 2013.
La PTGR1 es una enzima que presenta una actividad enzimática 2 alquenal/ona oxidoreductasa dependiente de NADPH, por lo que el nivel de expresión o la cantidad de esta enzima en una muestra determinada se puede determinar midiendo dicha actividad enzimática.  PTGR1 is an enzyme that has an NADPH-dependent alkenal / one-oxidoreductase enzyme activity 2, whereby the level of expression or the amount of this enzyme in a given sample can be determined by measuring said enzymatic activity.
La actividad de la PTGR1 se puede medir a través de reacciones con substratos que incluyen sin limitar aldehidos y cetonas alfa-beta insaturados y nitroalquenos en presencia de NADPH. Algunas de las técnicas estándar aplicables para poner en práctica la presente invención se describen por ejemplo en Dick R A. et al. J Biol Chem., 2001 nov 2; 276(44):40803-10. El nivel de expresión del gen Ptgrl puede determinarse al medir su ARNm. La determinación del ARNm de Ptgrl puede realizarse con cualquier técnica molecular para detectar o cuantificar ácidos nucleicos incluyendo sin limitar: RT-PCR, hibridación in situ, hibridación con sondas específicas de ácidos nucleicos y secuenciación de transcritos, entre otros. Algunas de las técnicas estándar aplicables para poner en práctica la presente invención se describen por ejemplo en Green M. R. y Sambrook J., Molecular Cloning. A laboratory Manual, Fourth Edition. Cold Spring Harbor Laboratory Press, 2012.  The activity of PTGR1 can be measured through reactions with substrates that include without limiting aldehydes and unsaturated alpha-beta ketones and nitroalkenes in the presence of NADPH. Some of the applicable standard techniques for practicing the present invention are described for example in Dick R A. et al. J Biol Chem., 2001 Nov 2; 276 (44): 40803-10. The expression level of the Ptgrl gene can be determined by measuring its mRNA. Ptgrl mRNA can be determined with any molecular technique to detect or quantify nucleic acids including without limitation: RT-PCR, in situ hybridization, hybridization with specific nucleic acid probes and transcript sequencing, among others. Some of the applicable standard techniques for practicing the present invention are described for example in Green M. R. and Sambrook J., Molecular Cloning. A laboratory Manual, Fourth Edition. Cold Spring Harbor Laboratory Press, 2012.
Para determinar los niveles de la proteína PTGR1 en los distintos tipos de tejido hepático derivados de los modelos experimentales, se empleó la técnica de Western blot (Figura 1 ), empleando un anticuerpo policlonal de ratón anti-LTB4DH (Abnova) a una dilución 1 :500. Como marcador de hepatocarcinogénesis se utilizó la inmunodetección de la Glutation S-Transferasa P1 (GSTP1 ), la cual es reconocida como un marcador tumoral de acuerdo con lo descrito en Sakai M. y Muramatsu M. Biochem Biophys Res Commun. 2007 Jun 8;357(3):575-8 y como control de carga se utilizó la inmunodetección de β-actina.  To determine the levels of PTGR1 protein in the different types of liver tissue derived from the experimental models, the Western blot technique was used (Figure 1), using a mouse polyclonal anti-LTB4DH antibody (Abnova) at dilution 1: 500 As an hepatocarcinogenic marker, the immunodetection of Glutathione S-Transferase P1 (GSTP1) was used, which is recognized as a tumor marker in accordance with that described in Sakai M. and Muramatsu M. Biochem Biophys Res Commun. 2007 Jun 8; 357 (3): 575-8 and β-actin immunodetection was used as load control.
En la Figura 1A se muestra la expresión de PTGR1 y GSTP1 en muestras del protocolo del HR; la PTGR1 estuvo presente de manera exclusiva en las muestras de tumor (T) a los 8, 9, y 10 meses, pero no fue detectada en muestras de hígado normal (HN) ni en muestras de tejido no tumoral circundante (NT). En el modelo de Schiffer se observó la sobre-expresión de la PTGR1 en muestras de ratas sacrificadas a las 12 y 18 semanas (Figura 1 B). La GSTP1 se encontró en mayor abundancia en el tejido tumoral (T), pero también se encontró en el tejido No tumoral (NT), probablemente, debido a la presencia de hepatocitos alterados. Figure 1A shows the expression of PTGR1 and GSTP1 in samples of the HR protocol; PTGR1 was present exclusively in tumor (T) samples at 8, 9, and 10 months, but was not detected in normal liver (HN) samples or in samples of surrounding non-tumor tissue (NT). In The Schiffer model showed the overexpression of PTGR1 in samples of rats sacrificed at 12 and 18 weeks (Figure 1 B). GSTP1 was found in greater abundance in tumor tissue (T), but it was also found in non-tumor tissue (NT), probably due to the presence of altered hepatocytes.
Se realizaron experimentos de inmunohistoquímica para determinar la presencia y localización celular de la PTGR1 en cortes de tejido (Figura 6), empleando un anticuerpo policlonal de conejo anti-PTGR1 (Novus Biologicals) a una dilución de 1 :25. Los anticuerpos primarios se detectaron utilizando la técnica de avidina-biotina con el Kit LSAB + HRP (Dako Corporation, California, EE.UU.).  Immunohistochemical experiments were performed to determine the presence and cellular location of PTGR1 in tissue sections (Figure 6), using a rabbit polyclonal antibody anti-PTGR1 (Novus Biologicals) at a dilution of 1: 25. Primary antibodies were detected using the avidin-biotin technique with the LSAB + HRP Kit (Dako Corporation, California, USA).
Como se muestra en la Figura 6, la PTGR1 se sobre-expresa en focos de hepatocitos alterados a las 6 semanas del tratamiento con DEN, en focos de hepatocitos y nodulos a las 12 semanas y en nodulos y tumores a las 18 semanas. La localización histológica de la PTGR1 fue en el citoplasma y en el núcleo de las células neoplásicas en los tumores hepáticos.  As shown in Figure 6, PTGR1 is overexpressed in foci of altered hepatocytes at 6 weeks of DEN treatment, in foci of hepatocytes and nodules at 12 weeks and in nodules and tumors at 18 weeks. The histological location of PTGR1 was in the cytoplasm and in the nucleus of the neoplastic cells in liver tumors.
Los resultados demostraron la presencia de la proteína PTGR1 en el tejido y confirmaron que se sobre-expresa en tejido tumoral, mientras que mantiene niveles normales en tejido no-tumoral circundante (NT) y en el tejido hepático normal (HN).  The results demonstrated the presence of PTGR1 protein in the tissue and confirmed that it is overexpressed in tumor tissue, while maintaining normal levels in surrounding non-tumor tissue (NT) and in normal liver tissue (HN).
Para confirmar la sobre-expresión de la PTGR1 y determinar si la proteína se encuentra en su forma activa, se midió la actividad enzimática 2 alquenal/ona oxidoreductasa dependiente de Nicotinamida Adenina Dinucleótido Fosfato (NADPH) (EC 1.3.1.74) en diversas muestras experimentales (Figura 2).  To confirm the overexpression of PTGR1 and determine if the protein is in its active form, Nicotinamide Adenine Dinucleotide Phosphate (NADPH) -dependent Nicotinamide Adenine Dinucleotide (NADPH) -dependent enzyme activity was measured in various experimental samples (Figure 2).
La actividad enzimática de la PTGR1 se determinó por la oxidación de NADPH, medida por su absorción espectrofotométrica a 340 nm. Se empleó el compuesto trans-2-nonenal como sustrato, de acuerdo con el método descrito en Dick RA, et al. J Biol Chem 2001 ;276:40803-40810. La actividad enzimática se calculó a partir del coeficiente de extinción molar para el NADPH (6.2 mM-1 cm-1 ) expresado como nmol de NADPH/min/mg de proteína.  The enzymatic activity of PTGR1 was determined by the oxidation of NADPH, measured by its spectrophotometric absorption at 340 nm. The trans-2-nonenal compound was used as a substrate, according to the method described in Dick RA, et al. J Biol Chem 2001; 276: 40803-40810. Enzymatic activity was calculated from the molar extinction coefficient for NADPH (6.2 mM-1 cm-1) expressed as nmol of NADPH / min / mg protein.
La Figura 2 muestra la actividad 2-alquenál/ona oxidoreductasa de la PTGR1 en muestras de diferentes tiempos de desarrollo del CHC. Se detectó un incremento dependiente del tiempo entre las 6 y 18 semanas, presentando la mayor actividad enzimática a las 18 semanas (15 veces mayor en comparación al tejido hepático normal). Las muestras de tumor (T) en el protocolo del HR mostraron una actividad enzimática 25 veces mayor en comparación con el tejido hepático normal. Figure 2 shows the 2-alkenal / one oxidoreductase activity of PTGR1 in samples of different CHC development times. A time-dependent increase was detected between 6 and 18 weeks, showing the highest enzyme activity at 18 weeks (15 times higher compared to normal liver tissue). Tumor samples (T) in the HR protocol showed 25 times greater enzyme activity compared to normal liver tissue.
De acuerdo con lo anterior, en una modalidad de la invención la sobre- expresión de la proteína PTGR1 puede determinarse mediante cualquier técnica inmunoenzimática descrita en el estado de la técnica a través del uso de anticuerpos específicos para esta proteína o mediante reacciones que ponen de manifiesto la actividad 2-alquenal/ona oxidoreductasa de esta enzima a través del uso de sustratos de la PTGR1 incluyendo sin limitar aldehidos y cetonas alfa-beta insaturados y nitroalquenos.  In accordance with the foregoing, in an embodiment of the invention the overexpression of the PTGR1 protein can be determined by any immunoenzymatic technique described in the state of the art through the use of antibodies specific for this protein or by reactions that reveal the 2-alkenal / one oxidoreductase activity of this enzyme through the use of PTGR1 substrates including without limiting aldehydes and unsaturated alpha-beta ketones and nitroalkenes.
Para detectar la sobre-expresión del gen Ptgr se realizaron experimentos de RT-qPCR (PCR cuantitativo de transcripción reversa, por sus siglas en inglés) para detectar el ARNm en muestras de diferentes tiempos de desarrollo del CHC (Figura 3).  To detect overexpression of the Ptgr gene, RT-qPCR (quantitative reverse transcription PCR) experiments were performed to detect mRNA in samples of different times of CHC development (Figure 3).
A partir del tejido hepático se obtuvo el ARN total empleando columnas From the liver tissue the total RNA was obtained using columns
RNAeasy (Qiagen, Hilden Germany). Se determinó la concentración y pureza del ARN mediante espectrofotometría a 260 ,y 280 nm, así como su integridad y calidad mediante electroforesis capilar en un bioanalizador Agilent, obteniendo proporciones de RNAr 28S/18S>1.7. El ARN total se empleó para montar las reacciones de ADNc usando el kit de Transcripción Reversa de Alta Capacidad (Applied Biosystems) con 750 ng de ARN total. RNAeasy (Qiagen, Hilden Germany). The concentration and purity of the RNA was determined by spectrophotometry at 260 and 280 nm, as well as its integrity and quality by capillary electrophoresis in an Agilent bioanalyzer, obtaining ratios of RNAr 28S / 18S> 1.7. Total RNA was used to mount the cDNA reactions using the High Capacity Reverse Transcription Kit (Applied Biosystems) with 750 ng of total RNA.
Una vez obtenido el ADNc se llevaron a cabo las reacciones de qPCR utilizando el ensayo de expresión génica TaqMan en un sistema HT 7900 Fast Real Time PCR (Applied Biosystem, México), empleando sondas marcadas con fluoresceína (FAM) (límite exón-exón) para Gstpl de rata (Rn0056 378_gH), para Ptgrl (Rn00593950_m1 ) y para ARNr 18S (Rn03928990) de Applied Biosystems. Los datos de Gstpl y Ptgrl se normalizaron contra la expresión genética de ARNr 8S usando el método comparativo AACT.  Once the cDNA was obtained, qPCR reactions were carried out using the TaqMan gene expression assay in an HT 7900 Fast Real Time PCR system (Applied Biosystem, Mexico), using fluorescein-labeled probes (FAM) (exon-exon limit) for rat Gstpl (Rn0056 378_gH), for Ptgrl (Rn00593950_m1) and for rRNA 18S (Rn03928990) of Applied Biosystems. Gstpl and Ptgrl data were normalized against the genetic expression of 8S rRNA using the AACT comparative method.
La Figura 3 muestra el incremento del nivel de expresión de los genes Ptgrl y Gstpl conforme se producen tumores hepáticos en los modelos de hepatocarcinogénesis. El gen Ptgrl presentó niveles de expresión comparables al marcador de hepatocarcinogénesis Gstpl en los tumores hepáticos. La mayor expresión del gen Ptgrl se presentó en las muestras de tumor (T), con valores hasta 200 veces mayores comparados a los de las muestras de hígado normal (HN). En los tumores del modelo de Schiffer de ratas sacrificadas entre 12 y 18 semanas, el nivel de expresión del gen Ptgrl fue de 17 a 19 veces que mayor que en el HN. Figure 3 shows the increase in the level of expression of the Ptgrl and Gstpl genes as liver tumors occur in hepatocarcinogenic models. The Ptgrl gene showed expression levels comparable to the Gstpl hepatocarcinogenic marker in liver tumors. The biggest Ptgrl gene expression was presented in tumor samples (T), with values up to 200 times higher compared to those in normal liver (HN) samples. In tumors of the Schiffer model of rats sacrificed between 12 and 18 weeks, the level of expression of the Ptgrl gene was 17 to 19 times higher than in the HN.
El protocolo de Solt y Farber (HR) permitió evaluar la evolución del CHC desde la aparición de lesiones preneoplásicas hasta su progresión en cáncer. Este protocolo da lugar a la aparición de dos tipos de nodulos preneoplásicos caracterizados por su actividad Gamma Glutamil Transferasa (GGT), como se describe en Enomoto K. y E. Farber. Cáncer Res 42 (1982) 2330-5, los nodulos en remodelación (R) y los nodulos persistentes (P). Los nodulos en remodelación muestran una tinción no uniforme al marcador GGT, con zonas negativas (nodulos R negativos) y zonas positivas (nodulos R positivos), mientras que los nodulos persistentes se tiñen de manera uniforme con GGT (nodulos P positivos).  The protocol of Solt and Farber (HR) allowed to evaluate the evolution of CHC from the appearance of preneoplastic lesions to its progression in cancer. This protocol results in the appearance of two types of preneoplastic nodules characterized by their Gamma Glutamyl Transferase (GGT) activity, as described in Enomoto K. and E. Farber. Cancer Res 42 (1982) 2330-5, remodeling nodules (R) and persistent nodules (P). The remodeling nodules show non-uniform staining of the GGT marker, with negative zones (negative R nodules) and positive zones (positive R nodules), while persistent nodules are stained uniformly with GGT (positive P nodules).
Para determinar la expresión del gen Ptgrl en estos diferentes tipos de nodulos y tumores que se generan durante el desarrollo del CHC se empleó la técnica de microdisección láser ligada al análisis de expresión (Figuras 4 y 5), de acuerdo con lo descrito en Mena J.E., et al. Anal Biochem. 2013 Nov 20. pii: S0003-2697(13)00551 -4, la cual permite separar de manera precisa el tejido de cada tipo de lesión y extraer el ARNm para realizar los análisis de expresión genética.  To determine the expression of the Ptgrl gene in these different types of nodules and tumors that are generated during the development of the CHC, the laser microdissection technique linked to the expression analysis was used (Figures 4 and 5), according to what is described in Mena JE , et al. Biochem anal. 2013 Nov 20. pii: S0003-2697 (13) 00551 -4, which allows the tissue to be precisely separated from each type of lesion and to extract the mRNA for genetic expression analysis.
■ A partir de las muestras obtenidas mediante microdisección láser, se determinó la expresión genética global en cada tipo de tejido utilizando microarreglos GeneChip Rat Gene 1.0 ST de Affimetrix, que permiten medir la expresión de más de 28,000 transcritos en rata de acuerdo con lo descrito en Mena J.E., et al. Anal Biochem. 2013 Nov 20. pii: S0003-2697( 3)00551 -4. Se analizaron muestras de nodulos R negativos, nodulos R positivos y nodulos P positivos, todos estos de 4 meses de desarrollo, así como muestras de tumores de 9 meses (CHC temprano), tumores de 17 meses (CHC avanzado) e hígado normal. ■ From the samples obtained by laser microdissection, the global genetic expression in each type of tissue was determined using Affimetrix GeneChip Rat Gene 1.0 ST microarrays, which allow the expression of more than 28,000 rat transcripts to be measured in accordance with what is described in Mena JE, et al. Biochem anal. 2013 Nov 20. pii: S0003-2697 (3) 00551 -4. Samples of R-negative nodules, R-positive nodules and P-positive nodules were analyzed, all these of 4 months of development, as well as samples of 9-month tumors (early CHC), 17-month tumors (advanced CHC) and normal liver.
A partir del ARN total se realizó la síntesis del ADN complementario (cDNA) usando iniciadores oligo-dT, para luego obtener el ADN de cadena doble i From the total RNA the synthesis of the complementary DNA (cDNA) was performed using oligo-dT primers, to then obtain the double stranded DNA i
(dsDNA). Con el dsDNA se obtuvo ARN complementario (cRNA) marcado con biotina mediante transcripción in vitro, que se purificó e hibridó con el microarreglo GeneChip Rat Gene 1.0 ST de Affimetrix.  (dsDNA). With the dsDNA, biotin-labeled complementary RNA (cRNA) was obtained by in vitro transcription, which was purified and hybridized with the Affimetrix GeneChip Rat Gene 1.0 ST microarray.
Los datos generados por el análisis de imagen del microarreglo se normalizaron utilizando el software Bioconductor, especializado en el análisis de microarreglos de ADN (http://www.bioconductor.org). La información fue integrada en bases de datos con el software MySQL. Los genes de interés fueron seleccionados a partir del análisis estadístico, análisis de agrupamiento jerárquico con el software Statistical Data Análisis R (http://www.r-project.org/). Los perfiles de expresión genética fueron proyectados para su análisis con anotaciones de perfiles de expresión genética usando el software GenMAPP (http://www.genmapp.org/) e Ingenuity Pathways (IPA).  The data generated by the microarray image analysis were normalized using the Bioconductor software, specialized in the analysis of DNA microarrays (http://www.bioconductor.org). The information was integrated into databases with MySQL software. The genes of interest were selected from statistical analysis, hierarchical clustering analysis with Statistical Data Analysis R software (http://www.r-project.org/). Genetic expression profiles were projected for analysis with annotations of genetic expression profiles using GenMAPP software (http://www.genmapp.org/) and Ingenuity Pathways (IPA).
En la Figura 4 se muestra la expresión genética de los diferentes tipos de nodulos y tumores que se desarrollan en los modelos experimentales, aislados por microdisección láser. Se determinó la expresión genética global usando microarreglos Affymetrix en ARN obtenido de nodulos de 4 meses, de tumores de 9 meses (CHC temprano) y de tumores de 17 meses (CHC avanzado) en ratas inducidas a hepatocarcinogenesis usando el modelo del HR.  Figure 4 shows the genetic expression of the different types of nodules and tumors that develop in experimental models, isolated by laser microdissection. Global genetic expression was determined using Affymetrix microarrays in RNA obtained from 4-month nodules, from 9-month tumors (early CHC) and from 17-month tumors (advanced CHC) in hepatocarcinogenesis-induced rats using the HR model.
Los distintos tipos de nodulos fueron identificados de acuerdo con su actividad GGT, como se describe en Enomoto K. y E. Farber. Cáncer Res 42 (1982) 2330-5 y separados mediante microdisección y captura láser. Se analizaron Nodulos en Remodelación GGT negativos (RN), Nodulos en Remodelación GGT positivos (RP) y Nodulos Persistentes GGT positivos (NP) (Figura 4).  The different types of nodules were identified according to their GGT activity, as described in Enomoto K. and E. Farber. Cancer Res 42 (1982) 2330-5 and separated by microdissection and laser capture. Negative GGT Remodeling (RN) Nodes, GGT Positive (RP) Remodeling Nodes and Positive GGT Persistent Nodes (NP) were analyzed (Figure 4).
Se analizaron los datos obtenidos del microarreglo para seleccionar aquellos genes con expresión diferencial (1.5 veces de cambio con significancia estadística p<0.05). Con los datos de 937 genes expresados de manera diferencial se integró un gráfico de mapa de calor (heatmap) con agrupamiento jerárquico. El dendrograma (Figura 4) indica la similitud del perfil de expresión genético entre los diferentes tipos de nodulos y tumores.  The data obtained from the microarray were analyzed to select those genes with differential expression (1.5 times of change with statistical significance p <0.05). With the data of 937 differentially expressed genes, a heatmap graph with hierarchical grouping was integrated. The dendrogram (Figure 4) indicates the similarity of the profile of genetic expression between different types of nodules and tumors.
De acuerdo con los datos de expresión genética global, los nodulos persistentes y los tumores tempranos presentaron una estrecha similitud, y estos a su vez mantienen similitud con el tumor avanzado. Por otro lado los nodulos en remodelación son similares entre si en su región nodular positiva y negativa a GGT. El gen Ptgrl está incluido dentro del perfil de expresión genética que caracteriza a los nodulos persistentes y los tumores y no se encuentra dentro de los genes diferencialmente expresados en los nodulos en remodelación. According to the global genetic expression data, persistent nodules and early tumors showed a close similarity, and these in turn they maintain similarity with the advanced tumor. On the other hand the nodules in remodeling are similar to each other in their nodular region positive and negative to GGT. The Ptgrl gene is included in the profile of genetic expression that characterizes persistent nodules and tumors and is not within the differentially expressed genes in remodeling nodules.
Estos datos muestran que los nodulos persistentes se encuentran estrechamente relacionados con los tumores temprano y avanzado de acuerdo con su expresión genética lo cual puede ser indicativo de que se trata de nodulos precursores del CHC a diferencia de los nodulos en remodelación que no guardan dicha similitud con el CHC temprano y avanzado. El hecho de que el gen Ptgrl se encuentra sobre-expresado en nodulos persistentes y CHC temprano o avanzado pero no en nodulos en remodelación, lo caracteriza como un marcador útil para el diagnóstico temprano de CHC y para la identificación de nodulos precursores del CHC.  These data show that persistent nodules are closely related to early and advanced tumors according to their genetic expression, which may be indicative of the fact that they are precursor nodules of CHC, unlike remodeling nodules that do not bear such similarity to the early and advanced CHC. The fact that the Ptgrl gene is overexpressed in persistent nodules and early or advanced HCC but not in nodules in remodeling, characterizes it as a useful marker for the early diagnosis of HCC and for the identification of precursor nodules of HCC.
En la Figura 5A se muestra un gráfico de mapa de calor (Heatmap) que describe el nivel de expresión de los genes A2m, Gpc3, Fat10, Ggt1 , Gstpl y Ptgrl en las distintas etapas de desarrollo del CHC en el modelo experimental. Los primeros cinco genes han sido reportados como marcadores de hepatocarcinogénesis en rata, como se describe en French, S.W. Exp Mol Pathol. 2010 Apr;88(2):219-24. El gen Ptgrl es el único que se sobre-expresa en nodulos persistentes y tumores pero no se sobre-expresa en nodulos en remodelación, a diferencia de los otros genes que se sobre-expresan en cualquier tipo de nodulo o presentan un nivel de expresión inconsistente con la progresión del CHC, Esta expresión específica del gen Ptgrl en nodulos persistentes permite identificar aquellos nodulos que podrían ser precursores del CHC. La determinación del nivel de expresión del gen Ptgrl permite, por lo tanto, establecer el diagnóstico temprano del CHC y en ciertos casos establecer el pronóstico de un individuo para desarrollar CHC.  Figure 5A shows a heat map graph (Heatmap) that describes the level of expression of the A2m, Gpc3, Fat10, Ggt1, Gstpl and Ptgrl genes at the different stages of CHC development in the experimental model. The first five genes have been reported as markers of hepatocarcinogenesis in rats, as described in French, S.W. Exp Mol Pathol. 2010 Apr; 88 (2): 219-24. The Ptgrl gene is the only one that is overexpressed in persistent nodules and tumors but does not over-express in remodeling nodules, unlike the other genes that are overexpressed in any type of nodule or have an inconsistent expression level With the progression of HCC, this specific expression of the Ptgrl gene in persistent nodules allows identifying those nodules that could be precursors of HCC. The determination of the level of expression of the Ptgrl gene allows, therefore, to establish the early diagnosis of HCC and in certain cases to establish the prognosis of an individual to develop HCC.
En la Figura 5B se muestra la expresión genética de estos 6 genes pero en términos de expresión genética relativa, después de normalizar los datos de intensidad de los microarreglos. De igual forma se observa que el gen Ptgrl se sobre-expresa en nodulos persistentesy tumores, mientras que mantiene niveles normales en nodulos en remodelación, lo que permite poner de manifiesto a los nodulos persistentes que pudieran ser precursores del CHC. Figure 5B shows the genetic expression of these 6 genes but in terms of relative genetic expression, after normalizing the intensity data of the microarrays. Similarly, it is observed that the Ptgrl gene is overexpressed in persistent nodules and tumors, while maintaining levels normal in nodules in remodeling, which allows to show persistent nodules that could be precursors of CHC.
Los datos de expresión de Ptgrl y Gstpl se confirmaron mediante RT-qPCR utilizando las sondas TaqMan específicas, en las muestras de los diferentes estadios de desarrollo del CHC obtenidas por microdisección y captura láser. En la Figura 5C se muestran los datos del RT-qPCR en donde se confirmó la expresión genética de Ptgrl y Gstpl . Se observa que el gen Gstpl se sobre- expresa tanto en nodulos en remodelación como en nodulos persistentes, mientras que el gen Ptgrl se sobre-expresa solo en nodulos persistentes, permitiendo así detectar aquellos nodulos que pudieran desarrollar en CHC y realizar por lo tanto un diagnóstico temprano del CHC desde etapas incluso anteriores al establecimiento de la neoplasia.  The expression data of Ptgrl and Gstpl were confirmed by RT-qPCR using the specific TaqMan probes, in the samples of the different stages of CHC development obtained by microdissection and laser capture. Figure 5C shows the data of the RT-qPCR where the genetic expression of Ptgrl and Gstpl was confirmed. It is observed that the Gstpl gene is overexpressed both in remodeling nodules and in persistent nodules, while the Ptgrl gene is overexpressed only in persistent nodules, thus allowing to detect those nodules that could develop in CHC and therefore perform a early diagnosis of HCC from stages even before the establishment of the neoplasm.
La expresión del gen Ptgrl se encuentra elevada en los nodulos tipo persistentes (Nod P Pos), los tumores tempranos y los tumores avanzados, mientras que permanece en nivel bajo en el tejido hepático normal y en los nodulos en remodelación. Los nodulos y tumores generados mediante el protocolo de Solt y Farber son útiles para entender el desarrollo del hepatocarcinoma humano derivado de células progenitoras, como se describe en Andersen, J. et al. Hepatology. 2010 April; 51 (4): 1401-1409. El nivel de expresión del gen Ptgrl fue el único capaz de distinguir los tumores y los nodulos tipo persistentes respecto de los nodulos tipo remodelación (Nod R). La expresión de Gstpl se encuentra elevada en los dos tipos de nodulos preneoplásicos por lo que no es útil para distinguir los nodulos persistentes que pudieran evolucionar en cáncer.  Ptgrl gene expression is elevated in persistent nodules (Nod P Pos), early tumors and advanced tumors, while remaining low in normal liver tissue and remodeling nodules. The nodules and tumors generated by the Solt and Farber protocol are useful for understanding the development of human hepatocarcinoma derived from progenitor cells, as described in Andersen, J. et al. Hepatology 2010 April; 51 (4): 1401-1409. The expression level of the Ptgrl gene was the only one capable of distinguishing tumors and persistent nodules from remodeling nodules (Nod R). Gstpl expression is elevated in both types of preneoplastic nodules, so it is not useful to distinguish persistent nodules that could evolve in cancer.
Los resultados de expresión genética en los distintos estadios del desarrollo de CHC confirman la utilidad de Ptgrl como marcador de diagnóstico temprano de este padecimiento, en etapas aún anteriores al establecimiento de la neoplasia, debido a que se encuentra sobre-expresado en nodulos persistentes, en tumores tempranos y en tumores avanzados, mientras que su expresión se mantiene en niveles normales en el tejido hepático normal y en nodulos tipo remodelación. Para determinar si los resultados obtenidos en los modelos experimentales son extrapolables a otros mamíferos y en particular a individuos humanos, se analizó el nivel de expresión de la PTGR1 en biopsias hepáticas incluidas en parafina y muestras de resección de 12 casos de CHC de individuos humanos, mediante inmunohistoquímica (Figura 7). Como control de patología no asociada a cáncer se utilizó la región del parénquima de un caso de fibrosis hepática. Todas las muestras fueron sometidas a tinción con H&E para el diagnóstico histológico de rutina. De acuerdo al estudio histopatológico las muestras de CHC se agruparon en: bien diferenciado (n=2), moderadamente diferenciado (n=6) y poco diferenciado (n=4), de acuerdo con la etapa del tumor y el grado de diferenciación celular. The results of genetic expression in the different stages of the development of CHC confirm the usefulness of Ptgrl as an early diagnostic marker of this condition, in stages even before the establishment of the neoplasm, because it is over-expressed in persistent nodules, in early tumors and in advanced tumors, while their expression is maintained at normal levels in normal liver tissue and remodeling nodules. To determine whether the results obtained in the experimental models are extrapolated to other mammals and in particular to human individuals, the level of expression of PTGR1 was analyzed in liver biopsies included in paraffin and resection samples from 12 cases of CHC from human individuals, by immunohistochemistry (Figure 7). As a control of pathology not associated with cancer, the parenchyma region of a case of liver fibrosis was used. All samples were stained with H&E for routine histological diagnosis. According to the histopathological study, the CHC samples were grouped into: well differentiated (n = 2), moderately differentiated (n = 6) and poorly differentiated (n = 4), according to the stage of the tumor and the degree of cell differentiation .
La inmunohistoquímica se realizó empleando un anticuerpo policlonal de ratón anti-LT4DH (Abnova) a una dilución 1 :50. Para verificar la naturaleza neoplásica del CHC, se llevó a cabo una reacción inmunohistoquímica específica para glipicano-3 en secciones seriadas de las mismas muestras. Esta proteína es un marcador conocido para el CHC y no se expresa en los trastornos hepáticos benignos. Para esto se empleó un anticuerpo monoclonal de ratón anti-glipican-3 (1 G12) (Cell Marque) a una dilución 1 :100.  Immunohistochemistry was performed using a mouse polyclonal anti-LT4DH antibody (Abnova) at a 1: 50 dilution. To verify the neoplastic nature of CHC, a specific immunohistochemical reaction for glypican-3 was carried out on serial sections of the same samples. This protein is a known marker for CHC and is not expressed in benign liver disorders. For this, a mouse monoclonal antibody anti-glipican-3 (1 G12) (Cell Marque) was used at a 1: 100 dilution.
Mediante la reacción de inmunohistoquímica se puso de manifiesto que la PTGR1 se encuentra sobre-expresada en todas las muestras de CHC, mientras que el tejido hepático no canceroso presentó baja intensidad de expresión (Figura 7). Los niveles de tinción fueron independientes del patrón histológico y del grado de anaplasia.  Through the immunohistochemical reaction it was shown that PTGR1 is overexpressed in all CHC samples, while non-cancerous liver tissue showed low expression intensity (Figure 7). Staining levels were independent of histological pattern and degree of anaplasia.
Estos resultados confirmaron que la PTGR1 se encuentra sobre-expresada en cortes de tejido con CHC, mientras que sus niveles de expresión fueron negativos o básales en cortes de tejido no tumoral.  These results confirmed that PTGR1 is overexpressed in tissue sections with CHC, while its expression levels were negative or basal in non-tumor tissue sections.
Debido a su sobre-expresión en nodulos displásicos y tejido tumoral hepático, los productos génicos de la Ptgrl , ARNm o proteína, pueden detectarse en sangre periférica de individuos con CHC o sus derivados como son el suero o plasma. La detección de los niveles de expresión de la PTGR1 en sangre periférica, suero o plasma puede llevarse a cabo midiendo cualquiera de sus productos génicos. Para determinar la sobreexpresión del gen Ptgrl en sangre periférica, suero o plasma puede emplearse cualquier técnica conocida en el estado de la técnica para detectar ARNm circulante incluyendo sin limitar RT- PCR, hibridación in situ, hibridación con sondas específicas de ácidos nucleicos y secuenciación de transcritos, entre otros. Due to their overexpression in dysplastic nodules and liver tumor tissue, the Ptgrl gene products, mRNA or protein, can be detected in peripheral blood of individuals with CHC or its derivatives such as serum or plasma. The detection of PTGR1 expression levels in peripheral blood, serum or plasma can be carried out by measuring any of its gene products. To determine the overexpression of the Ptgrl gene in blood Peripheral, serum or plasma can be used any technique known in the state of the art to detect circulating mRNA including without limiting RT-PCR, in situ hybridization, hybridization with specific nucleic acid probes and transcription sequencing, among others.
Para determinar la sobreexpresión de la proteína PTGR1 en sangre periférica, suero o plasma puede emplearse cualquier técnica inmunoenzimática conocida en el estado de la técnica para detectar una proteína específica incluyendo sin limitar ensayo por inmunoabsorcion ligado a enzimas (ELISA por sus siglas en inglés) en cualquiera de sus variantes, inmunofluorescencia, e inmunoprecipitación, entre otras.  To determine the overexpression of the PTGR1 protein in peripheral blood, serum or plasma, any immunoenzymatic technique known in the state of the art can be used to detect a specific protein including without limiting enzyme-linked immunosorbent assay (ELISA) in any of its variants, immunofluorescence, and immunoprecipitation, among others.
La sobre-expresión de la proteína PTGR1 en sangre periférica, suero o plasma también puede determinarse mediante la medición de la actividad enzimática 2 alquenal/ona oxidoreductasa dependiente de NADPH, a través de reacciones con substratos que incluyen sin limitar aldehidos y cetonas alfa-beta insaturados y nitroalquenos en presencia de NADPH.  Overexpression of the PTGR1 protein in peripheral blood, serum or plasma can also be determined by measuring the enzymatic activity 2 alkenal / one oxidoreductase dependent on NADPH, through reactions with substrates including without limiting aldehydes and alpha-beta ketones unsaturated and nitroalkenes in the presence of NADPH.
La sobre-expresión de la PTGR1 en nodulos persistentes y tumores de los modelos animales y en muestras de CHC de humanos, es indicativo de que se trata de un cambio molecular inherente a las neoplasias malignas hepatocelulares en mamíferos, por lo que el marcador y los métodos de diagnóstico descritos en la presente solicitud son aplicables a distintas clases de mamíferos, en particular a individuos humanos.  The overexpression of PTGR1 in persistent nodules and tumors of animal models and in human CHC samples, is indicative of a molecular change inherent in hepatocellular malignant neoplasms in mammals, so the marker and Diagnostic methods described in the present application are applicable to different classes of mammals, in particular to human individuals.
Como se usa en la descripción de la presente invención, el término "mamífero" se refiere a aquellos animales vertebrados con glándulas mamarias productoras de leche con las que alimentan a sus crías, incluyendo al ser humano y animales de interés veterinario o ganadero para el ser humano. Algunos ejemplos de mamíferos de interés ganadero incluyen sin limitar el cerdo, la vaca, el caballo, el borrego y la cabra entre otros.  As used in the description of the present invention, the term "mammal" refers to those vertebrate animals with milk-producing mammary glands with which they feed their offspring, including humans and animals of veterinary or livestock interest to the being. human. Some examples of mammals of livestock interest include without limiting the pig, cow, horse, sheep and goat among others.
Algunos ejemplos de mamíferos de interés veterinario incluyen sin limitar el perro y el gato entre otros. Con base en la descripción anterior, la presente invención se refiere, en uno de sus aspectos, a métodos útiles para realizar el diagnóstico temprano del CHC desde etapas incluso anteriores al establecimiento de la neoplasia, mediante la determinación del nivel de expresión del gen Ptgrl o la proteína para la que codificaen una muestra biológica que puede ser tejido hepático, sangre periférica, suero o plasma. Dicho método es aplicable a mamíferos de interés ganadero o veterinario y en particular a individuos humanos con riesgo elevado de padecer CHC. Some examples of mammals of veterinary interest include without limiting the dog and cat among others. Based on the above description, the present invention relates, in one of its aspects, to methods useful for early diagnosis of HCC from stages even prior to the establishment of the neoplasm, by determining the level of expression of the Ptgrl gene or the protein for which they encode a biological sample that can be liver tissue, peripheral blood, serum or plasma. This method is applicable to mammals of livestock or veterinary interest and in particular to human individuals at high risk of CHC.
Los métodos objeto de la presente invención consisten en determinar el nivel de expresión del gen de la prostaglandina reductasa 1 (Ptgrl ) o de la proteína para la que codifica en una muestra biológica previamente obtenida, y comparar este nivel de expresión contra un estándar de expresión del gen Ptgrl o su proteína en individuos sin CHC, en donde la sobre-expresión del gen Ptgrl o su proteína es indicativa de que el individuo sufre de CHC o de que podría desarrollar CHC.  The methods object of the present invention consist in determining the level of expression of the prostaglandin reductase 1 (Ptgrl) gene or of the protein for which it encodes in a previously obtained biological sample, and comparing this level of expression against an expression standard of the Ptgrl gene or its protein in individuals without CHC, where overexpression of the Ptgrl gene or its protein is indicative of the individual suffering from CHC or that he could develop CHC.
Para el objeto de la presente invención se entiende por "estándar de expresión" de Ptgrl , el nivel de expresión medio del gen Ptgrl o de la proteína para la que codifica detectado en muestras de una población conocida de individuos que no padecen CHC. Como se usa en la descripción de la presente invención, el término "sobre-expresión" se refiere al aumento detectable de la expresión del gen Ptgrl o la proteína para la que codifica o su actividad enzimática en por lo menos 1 , 2, 5 10, 50 ó 100 veces más con respecto del estándar de expresión.  For the purpose of the present invention, "expression standard" of Ptgrl is understood as the average expression level of the Ptgrl gene or of the protein for which it encodes detected in samples from a known population of individuals who do not suffer from CHC. As used in the description of the present invention, the term "overexpression" refers to the detectable increase in the expression of the Ptgrl gene or the protein for which it encodes or its enzymatic activity by at least 1, 2, 5 10 , 50 or 100 times more than the expression standard.
Para la puesta en práctica de la presente invención, puede emplearse cualquier metodología descrita en el estado de la técnica que sea útil para determinar el nivel de expresión de un gen o la proteína para la que codifica.  For the implementation of the present invention, any methodology described in the state of the art that is useful for determining the level of expression of a gene or the protein for which it encodes can be employed.
El nivel de expresión del gen Ptgrl puede determinarse al medir ya sea su ARNm, la proteína para la que codifica o la actividad enzimática de la proteína para la que codifica. La determinación del ARNm de Ptgrl puede realizarse con técnicas moleculares que incluyen sin limitar: RT-qPCR, hibridación in situ, hibridación con sondas específicas de ácidos nucleicos y secuenciación de transcritos, entre otros; la proteína PTGR1 puede medirse con técnicas ¡nmunoenzimáticas que incluyen sin limitar: inmunohistoquímica, ELISA y sus variaciones, inmunofluorescencia, inmunocitoquímica e inmunoprecipitación; y la actividad enzimática de la PTGR1 puede medirse con substratos que incluyen sin limitar: aldehidos y cetonas alfa-beta insaturados en presencia de NADPH. La determinación del nivel de expresión del gen Ptgrl para el diagnóstico temprano de CHC puede llevarse a cabo en distintos tipos de muestras incluyendo sin limitar: muestras de tejido hepático fresco, tejido hepático congelado, tejido nodular enriquecido, tejido hepático incluido en parafina, sangre total, suero o plasma. The expression level of the Ptgrl gene can be determined by measuring either its mRNA, the protein for which it encodes or the enzymatic activity of the protein for which it encodes. Ptgrl mRNA determination can be performed with molecular techniques that include without limitation: RT-qPCR, in situ hybridization, hybridization with specific nucleic acid probes and transcript sequencing, among others; PTGR1 protein can be measured with immunoenzymatic techniques that include without limitation: immunohistochemistry, ELISA and its variations, immunofluorescence, immunocytochemistry and immunoprecipitation; and the enzymatic activity of PTGR1 can be measured with substrates that include without limitation: aldehydes and unsaturated alpha-beta ketones in the presence of NADPH. The determination of the expression level of the Ptgrl gene for the early diagnosis of CHC can be carried out in different types of samples including without limitation: samples of fresh liver tissue, frozen liver tissue, enriched nodular tissue, paraffin-included liver tissue, whole blood , serum or plasma.
En una modalidad preferida de la invención, la expresión del gen Ptgrl se puede determinar mediante RT-qPCR en muestras de tejido hepático.  In a preferred embodiment of the invention, the expression of the Ptgrl gene can be determined by RT-qPCR in liver tissue samples.
En otra modalidad preferida de la invención, la expresión del gen Ptgrl se puede determinar mediante RT-qPCR en muestras de sangre periférica, suero o plasma.  In another preferred embodiment of the invention, the expression of the Ptgrl gene can be determined by RT-qPCR in peripheral blood, serum or plasma samples.
La Tabla 3 muestra algunas modalidades preferidas de la invención. Table 3 shows some preferred embodiments of the invention.
Tabla 3. Modalidades preferidas de la invención Table 3. Preferred Modalities of the Invention
Muestra Preservación Determinación Métodos  Sample Preservation Determination Methods
Localización Imnunohistoquímica histopatológica ínmunofluorescencia  Immunohistochemical histopathological immunofluorescence location
"' Técnicas inmunoiógicas Western blot "' Western blot immunogenic techniques
Detección ele proteina  Protein Detection
ELISA  ELISA
Espectrometría de masas Mass spectrometry
Congelación RT-PCR RT-PCR freezing
hibridación in si tu  hybridization in if you
ARN mensajero  Messenger RNA
microarreglos ADN  DNA microarrays
RNAseq  RNAseq
Enzayo enziraático Enzymatic Enzyme
Biopsia Reacción NADPH dependiente  Biopsy NADPH dependent reaction
Actividad enzimática  Enzymatic activity
hepática Espectro fotometría  hepatic photometry spectrum
{.Píiioramétna  {. Píiioramétna
Figure imgf000032_0001
Figure imgf000032_0001
Medios de Γ RT-PCR  Γ RT-PCR media
preservación de hibridación in sim  preservation of hybridization in sim
ARN mensajero  Messenger RNA
AR (RNA later, microarreglos ADN  AR (RNA later, DNA microarrays
RNA save) RNAse  RNA save) RNAse
Enzayo enzimática Enzymatic Enzyme
Reacción NADPH dependiente NADPH dependent reaction
Actividad enzimática 4 Enzymatic Activity 4
Espectrot tomeiria  Tomeiria spectrot
Fhiorométria  Phyometrometry
Suero o  Serum or
Congelación  Freezing
plasma  plasma
mensajero circulante  circulating messenger
Técnicas inmunoiógicas Detección ele proteina Immunogenic techniques Protein detection
Western blot Western blot
Figure imgf000032_0002
Como se ha mencionado anteriormente, la presente invención se basa en el hecho inesperado de que el gen Ptgr se encuentra sobre-expresado de manera diferencial en nodulos hepáticos displásicos, en el CHC temprano y el CHC avanzado.
Figure imgf000032_0002
As mentioned above, the present invention is based on the unexpected fact that the Ptgr gene is differentially overexpressed in dysplastic liver nodules, in early CHC and advanced CHC.
Otra de las aplicaciones de este hecho inesperado consiste en determinar si un individuo que sufre de CHC, es candidato a ser tratado con compuestos bioactivados mediante la actividad alquenal/ona oxidorreductasa de la PTGR1 .  Another application of this unexpected fact is to determine if an individual suffering from CHC is a candidate to be treated with bioactivated compounds by means of the alkenal / oxidoreductase activity of PTGR1.
Como se usa en la presente descripción, el término "bioactivación" se refiere a la conversión metabolica de un compuesto químico en un derivado más activo o tóxico dentro del organismo. En el caso particular de la presente invención, un compuesto bioactivado por la PTGR1 se refiere a aquellos compuestos químicos que son reducidos mediante la actividad alquenal/ona oxidorreductasa de la PTGR1 y que generan un producto más toxico que el compuesto sin reducir.  As used herein, the term "bioactivation" refers to the metabolic conversion of a chemical compound into a more active or toxic derivative within the organism. In the particular case of the present invention, a compound bioactivated by PTGR1 refers to those chemical compounds that are reduced by the alkenal / oxidoreductase activity of PTGR1 and that generate a more toxic product than the unreduced compound.
Al determinar el nivel de expresión del gen Ptgrl o la enzima para la que codifica en muestras de tejido hepático de un individuo que padece CHC, puede definirse si dicho individuo es candidato a ser tratado con compuestos que son bioactivados por la enzima PTGR1 , debido a que dichos compuestos serán activados en los nodulos displásicos o el tejido tumoral que sobre-expresan esta enzima, generando así una citotoxicidad selectiva.  By determining the level of expression of the Ptgrl gene or the enzyme for which it encodes in liver tissue samples of an individual suffering from CHC, it can be defined whether said individual is a candidate to be treated with compounds that are bioactivated by the PTGR1 enzyme, due to that said compounds will be activated in dysplastic nodules or tumor tissue that overexpress this enzyme, thus generating a selective cytotoxicity.
En el estado de la técnica se han descrito diversos compuestos que pueden ser bioactivados en el organismo y que por lo tanto pueden servir como agentes quimioterapéuticos. Las solicitudes de patente internacional WO/1991/04754, WO/ 998/03458 y WO/2007/019308 describen análogos de iludina y de acilfulvenos útiles como agentes antitumorales. Por ejemplo, en Yu X. et al. J Pharmacol Exp Ther. 2012 Nov;343(2):426-33, se describe que el hidroximetilacilfulveno funciona como substrato para la enzima PTGR1 , la que cataliza su reducción hacia intermediarios biológicamente activos.  Various compounds have been described in the state of the art that can be bioactivated in the organism and therefore can serve as chemotherapeutic agents. International patent applications WO / 1991/04754, WO / 998/03458 and WO / 2007/019308 describe analogs of iludin and acylfulvenes useful as antitumor agents. For example, in Yu X. et al. J Pharmacol Exp Ther. 2012 Nov; 343 (2): 426-33, it is described that hydroxymethylacilfulvene functions as a substrate for the PTGR1 enzyme, which catalyzes its reduction towards biologically active intermediates.
Por lo tanto, en otra modalidad, la presente invención se refiere a métodos in vitro útiles para determinar si un individuo que sufre de CHC, es candidato para ser tratado con compuestos antitumorales que son bioactivados por la enzima PTGR1 . Dicho método comprende detectar alguno de los productos génicos (ARNm o proteína) de la prostaglandina reductasa 1 (Ptgrl ) en una muestra biológica previamente obtenida y comparar el nivel de expresión en dicha muestra contra un estándar de expresión de la PTGR1 , en donde la sobre-expresión de la PTGR1 es indicativa de que el individuo es candidato de ser tratado con compuestos antitumorales que son bioactivados por la PTGR1. Therefore, in another embodiment, the present invention relates to in vitro methods useful for determining whether an individual suffering from CHC is a candidate to be treated with antitumor compounds that are bioactivated by the PTGR1 enzyme. Said method comprises detecting some of the gene products (mRNA or protein) of prostaglandin reductase 1 (Ptgrl) in a sample previously obtained and compare the level of expression in said sample against a standard of expression of PTGR1, where the overexpression of PTGR1 is indicative that the individual is a candidate to be treated with antitumor compounds that are bioactivated by PTGR1 .
Para la puesta en práctica de esta modalidad de la invención, puede emplearse cualquier metodología descrita en el estado de la técnica que sea útil para determinar el nivel de expresión de un gen o la proteína para la que codifica.  For the implementation of this embodiment of the invention, any methodology described in the state of the art that is useful for determining the level of expression of a gene or the protein for which it encodes can be used.
El nivel de expresión del gen Ptgrl puede determinarse al medir ya sea su ARNm, la proteína para la que codifica o la actividad enzimática de la proteína para la que codifica. La determinación del ARNm de Ptgrl puede realizarse con técnicas moleculares que incluyen sin limitar: RT-qPCR, hibridación in situ, hibridación con sondas específicas de. ácidos nucleicos y secuenciación de transcritos, entre otros; la proteína PTGR1 puede medirse con técnicas inmunoenzimáticas que incluyen sin limitar: inmunohistoquímica, ELISA y sus variaciones, inmunofluorescencia, inmunocitoquímica e inmunoprecipitación; y la actividad enzimática de la PTGR1 puede medirse con substratos que incluyen sin limitar: aldehidos y cetonas alfa-beta insaturados en presencia de NADPH.  The expression level of the Ptgrl gene can be determined by measuring either its mRNA, the protein for which it encodes or the enzymatic activity of the protein for which it encodes. Ptgrl mRNA determination can be performed with molecular techniques that include without limitation: RT-qPCR, in situ hybridization, hybridization with specific probes. nucleic acids and transcript sequencing, among others; PTGR1 protein can be measured with immunoenzymatic techniques that include without limitation: immunohistochemistry, ELISA and its variations, immunofluorescence, immunocytochemistry and immunoprecipitation; and the enzymatic activity of PTGR1 can be measured with substrates that include without limitation: aldehydes and unsaturated alpha-beta ketones in the presence of NADPH.
Algunas de las técnicas estándar aplicables para poner en práctica la presente invención se describen por ejemplo en Green M. R. y Sambrook J., Molecular Cloning. A laboratory Manual, Fourth Edition. Cold Spring Harbor Laboratory Press, 2012 y en Greenfield E. A., Antibodies. A laboratory Manual Cold Spring Harbor Laboratory Press, 20 3.  Some of the applicable standard techniques for practicing the present invention are described for example in Green M. R. and Sambrook J., Molecular Cloning. A laboratory Manual, Fourth Edition. Cold Spring Harbor Laboratory Press, 2012 and in Greenfield E. A., Antibodies. A laboratory Manual Cold Spring Harbor Laboratory Press, 20 3.
La determinación del nivel de expresión del gen Ptgrl para el diagnóstico temprano de CHC puede llevarse a cabo en distintos tipos de muestras incluyendo sin limitar: muestras de tejido hepático fresco, tejido hepático congelado, tejido nodular enriquecido, tejido hepático incluido en parafina, sangre total, suero o plasma.  The determination of the expression level of the Ptgrl gene for the early diagnosis of CHC can be carried out in different types of samples including without limitation: samples of fresh liver tissue, frozen liver tissue, enriched nodular tissue, paraffin-included liver tissue, whole blood , serum or plasma.
En una modalidad preferida de la invención, la expresión de Ptgrl se determina mediante la medición de su actividad enzimática.  In a preferred embodiment of the invention, the expression of Ptgrl is determined by measuring its enzymatic activity.
En una modalidad preferida de la invención, los compuestos antitumorales que son bioactivados por la PTGR1 son compuestos análogos de la ¡ludina o de los acilfulvenos. En otra modalidad, la presente invención se refiere a equipos de diagnóstico mediante los cuales se puede efectuar los métodos descritos en la presente solicitud. Estos equipos de diagnóstico comprenden al menos un método para determinar el nivel de expresión de Ptgrl en una muestra biológica y un estándar de comparación del nivel de expresión de Ptgrl . En este sentido, los equipos de diagnóstico comprenden medios para determinar ya sea el ARNm, la proteína o la actividad enzimática de la PTGR1. Como se utiliza en la presente invención, los medios que contiene el equipo de diagnóstico descrito en la presente solicitud se refieren a cualquier tipo de reactivo de laboratorio o reactivo médico que puede emplearse para detectar la sobre-expresión del gen Ptgrl o de la proteína para la que codifica o la actividad enzimática de la enzima PTGR . Estos medios pueden incluir sin limitar anticuerpos policlonales, anticuerpos monoclonales, anticuerpos humanizados, sondas específicas, sustratos específicos para la PTGR1 , y los reactivos de laboratorio necesarios para realizar técnicas estándar para la detección de ÁRNm tales como RT-qPCR, hibridación in situ, hibridación con sondas específicas de ácidos nucleicos y secuenciación de transcritos, entre otros; para la detección de la proteína PTGR1 tales como inmunohistoquímica, ELISA y sus variaciones, inmunofluorescencia, inmunocitoquímica e inmunoprecipitación y para determinar la actividad enzimática de la PTGR1 en presencia de compuestos con aldehidos y cetonas alfa-beta insaturados o nitroalquenos y en presencia de NADPH In a preferred embodiment of the invention, the antitumor compounds that are bioactivated by PTGR1 are analogous compounds of ludin or acylfulvenes. In another embodiment, the present invention relates to diagnostic equipment by means of which the methods described in the present application can be performed. These diagnostic kits comprise at least one method for determining the level of Ptgrl expression in a biological sample and a standard for comparing the level of Ptgrl expression. In this sense, diagnostic equipment includes means to determine either the mRNA, the protein or the enzymatic activity of PTGR1. As used in the present invention, the means contained in the diagnostic equipment described in the present application refer to any type of laboratory reagent or medical reagent that can be used to detect the overexpression of the Ptgrl gene or protein for the one that encodes or the enzymatic activity of the PTGR enzyme. These means may include without limiting polyclonal antibodies, monoclonal antibodies, humanized antibodies, specific probes, specific substrates for PTGR1, and the laboratory reagents necessary to perform standard mRNA detection techniques such as RT-qPCR, in situ hybridization, hybridization. with specific nucleic acid probes and transcript sequencing, among others; for the detection of PTGR1 protein such as immunohistochemistry, ELISA and its variations, immunofluorescence, immunocytochemistry and immunoprecipitation and to determine the enzymatic activity of PTGR1 in the presence of compounds with aldehydes and alpha-beta unsaturated ketones or nitroalkenes and in the presence of NADPH
Esta invención se ¡lustra adicionalmente por los siguientes ejemplos, que no se consideran en ningún sentido como limitaciones impuestas sobre el alcance de las mismas. Por el contrario, éstos son para entender claramente que se puede recurrir a varias otras modalidades, modificaciones y equivalentes de las mismas. EJEMPLOS EJEMPLO 1 This invention is further illustrated by the following examples, which are not considered in any way as limitations imposed on the scope thereof. On the contrary, these are to clearly understand that several other modalities, modifications and equivalents thereof can be used. EXAMPLES EXAMPLE 1
Modelos animales para el desarrollo de hepatocarcinogénesis  Animal models for the development of hepatocarcinogenesis
Desarrollo de modelos animales de hepatocarcinogénesis.  Development of animal models of hepatocarcinogenesis.
Se emplearon dos modelos de hepatocarcinogénesis experimental en rata. El primero se desarrolló de acuerdo a la descripción realizada en Marche-Cova A, et al. Arch Med Res 1995;26 Spec No:S169-173 y Carrasco-Legleu CE, et al. Int J Cáncer 2004;108:488-492, el cual es un protocolo alterno al modelo descrito originalmente por Solt D. y Farber E. en Nature 1976;263:701 -703. El segundo modelo está basado en la descripción realizada por Schiffer E, et al. en Hepatology 2005;41 :307-314 y es un modelo de hepatocarcinogénesis asociado a cirrosis inducido con dietilnitrosamina (DEN).  Two models of experimental hepatocarcinogenesis were used in rats. The first was developed according to the description made in Marche-Cova A, et al. Arch Med Res 1995; 26 Spec No: S169-173 and Carrasco-Legleu CE, et al. Int J Cancer 2004; 108: 488-492, which is an alternate protocol to the model originally described by Solt D. and Farber E. in Nature 1976; 263: 701-703. The second model is based on the description made by Schiffer E, et al. in Hepatology 2005; 41: 307-314 and is a model of hepatocarcinogenesis associated with diethylnitrosamine-induced cirrhosis (DEN).
Para cada modelo se emplearon 20 ratas macho Fischer 344 de entre 180g y 200g de peso. Los animales obtenidos de la Unidad de Producción y Experimentación de Animales de Laboratorio del CINVESTAV Ciudad de México (UPEAL-Cinvestav), fueron tratados de acuerdo con las guías y protocolos institucionales para el manejo de animales de experimentación. Las ratas fueron mantenidas con ciclos diarios de luz / oscuridad de 12 h y temperatura controlada, con alimento (dieta básica) y agua esterilizada ad libitum.  For each model, 20 male Fischer 344 rats between 180g and 200g in weight were used. The animals obtained from the Laboratory Animal Production and Experimentation Unit of the CINVESTAV Mexico City (UPEAL-Cinvestav), were treated according to the institutional guidelines and protocols for handling experimental animals. The rats were maintained with daily light / dark cycles of 12 h and controlled temperature, with food (basic diet) and ad libitum sterilized water.
El protocolo modificado de Solt y Farber consistió en una administración única intraperitonial de 200 mg/kg de DEN, seguida de la administración de tres dosis intragástricas de 25 mg/kg de una suspensión al 1 % de 2- acetilaminofluoreno (2-AAF) en carboximetil celulosa, preparada de acuerdo con Semple-Roberts E, Int J Cáncer 1987;40:643-645, en los días 7, 8 y 9 después del inicio del protocolo con DEN y una hepatectomía de dos tercios del tejido hepático en el día 10 después del inicio del protocolo. Los animales de experimentación fueron sacrificados a los meses 1 , 4, 5, 7, 9, 12 ó 17, a fin de poder evaluar el desarrollo de los nodulos displásicos y del cáncer desde sus etapas tempranas.  The modified protocol of Solt and Farber consisted of a single intraperitonial administration of 200 mg / kg of DEN, followed by the administration of three intragastric doses of 25 mg / kg of a 1% suspension of 2-acetylaminofluorene (2-AAF) in carboxymethyl cellulose, prepared according to Semple-Roberts E, Int J Cancer 1987; 40: 643-645, on days 7, 8 and 9 after the start of the protocol with DEN and a two-thirds hepatectomy of liver tissue in the day 10 after the start of the protocol. The experimental animals were sacrificed at months 1, 4, 5, 7, 9, 12 or 17, in order to evaluate the development of dysplastic nodules and cancer from their early stages.
Para el protocolo de Schiffer, las ratas recibieron inyecciones intraperitoneales de 50 mg/kg de DEN semanalmente. El número de inyecciones fue de 16. Las ratas de experimentación se sacrificaron a las 6, 12, y 18 semanas para recolectar el tejido hepático. For the Schiffer protocol, the rats received intraperitoneal injections of 50 mg / kg of DEN weekly. The number of injections It was 16. Experimental rats were sacrificed at 6, 12, and 18 weeks to collect liver tissue.
Un grupo adicional de ratas adultas no recibió ningún tratamiento y se sacrificó de forma paralela al grupo experimental para ser empleado como grupo control donde el hígado fue normal (HN) .  An additional group of adult rats received no treatment and was sacrificed in parallel to the experimental group to be used as a control group where the liver was normal (NH).
El sacrificio de los animales se llevó a cabo mediante exanguinación bajo anestesia con éter. De cada animal se obtuvieron 5 mi de sangre por punción cardiaca y el tejido hepático fue embebido en 2-metil butano como crioprotector, congelado con nitrógeno líquido y almacenado a -70°C para su conservación, formando así un banco de tejidos para los diferentes ensayos.  The animals were slaughtered by exanguination under anesthesia with ether. From each animal 5 ml of blood were obtained by cardiac puncture and the liver tissue was embedded in 2-methyl butane as a cryoprotectant, frozen with liquid nitrogen and stored at -70 ° C for conservation, thus forming a tissue bank for the different essays.
En los hígados obtenidos a partir del protocolo modificado de Solt y Farber, se identificó tejido tumoral con base en su actividad de GGT en secciones histológicas, como se describe en Rutenburg AM, et al. J Histochem Cytochem 1969;17:517-526. Una vez identificados, los tumores mayores de 5 mm de diámetro se separaron del tejido no tumoral circundante con un bisturí.  In the livers obtained from the modified protocol of Solt and Farber, tumor tissue was identified based on its GGT activity in histological sections, as described in Rutenburg AM, et al. J Histochem Cytochem 1969; 17: 517-526. Once identified, tumors larger than 5 mm in diameter were separated from the surrounding non-tumor tissue with a scalpel.
Los hígados obtenidos de las ratas tratadas mediante el protocolo de Schiffer se procesaron con un criostato. Por cada sección de 16 mieras con actividad de GGT, se colectaron otras 10 secciones hasta obtener 50 mg de tejido para la extracción de proteínas ó 30 mg de tejido para la extracción de RNA.  The livers obtained from the rats treated by the Schiffer protocol were processed with a cryostat. For each section of 16 microns with GGT activity, another 10 sections were collected until obtaining 50 mg of tissue for protein extraction or 30 mg of tissue for RNA extraction.
EJEMPLO 2 EXAMPLE 2
Identificación de PTGR1 en tumores mediante espectrometría de masas Identification of PTGR1 in tumors by mass spectrometry
Se homogeneizaron 50 mg de las muestras de tejido hepático en 1 - ml de buffer de lisis que contenía 50 mM Tris-HCI, 150 mM de NaCI, 0.25% de Na- desoxicolato, 1 mM EDTA y 1X de coctel de inhibidores de proteasas (ProteoBlock, Fermentas). La proteína total se cuantificó por el método de Lowry (RC ensayo de proteínas DC, BioRad), las muestras se prepararon en buffer 5X Laemmly. 30 ug de extracto de proteína fueron separados por SDS-PAGE al 10%. Los geles se tiñeron con azul de Coomassie coloidal G-250 (Bio-Safe, Bio-Rad Laboratories, EE.UU.) y las imágenes de los geles se analizaron por densitometría de bandas con el software ImageJ. Las bandas diferenciales fueron escindidas de los geles y se procesaron para la identificación de proteínas de acuerdo a lo descrito previamente en Pérez et al., 2010 Proteome Sci 2010;8:27. Los fragmentos de gel se destiñeron dos veces con 500 μΙ de una solución de 50 mM de bicarbonato de amonio / 50% (v / v) acetonitrilo a 50°C por 5 minutos, después se deshidrató con 100 μΙ de acetonitrilo por 5 min a temperatura ambiente. Las piezas de gel se dejaron secar y las proteínas fueron digeridas en el gel durante 10 minutos con una solución de tripsina (25 ng/l, Promega, EE.UU.). A continuación los geles fueron incubados con una solución 5 mM de bicarbonato de amonio durante toda la noche a 37°C. 50 mg of the liver tissue samples were homogenized in 1-ml lysis buffer containing 50 mM Tris-HCI, 150 mM NaCl, 0.25% Na-deoxycholate, 1 mM EDTA and 1X protease inhibitor cocktail ( ProteoBlock, Fermentas). The total protein was quantified by the Lowry method (RC DC protein assay, BioRad), the samples were prepared in 5X Laemmly buffer. 30 ug of protein extract was separated by 10% SDS-PAGE. The gels were stained with Coomassie colloidal blue G-250 (Bio-Safe, Bio-Rad Laboratories, USA) and the images of the gels were analyzed by band densitometry with the ImageJ software. Differential bands were cleaved from the gels and processed for protein identification as previously described in Pérez et al., 2010 Proteome Sci 2010; 8: 27. The gel fragments were stained twice with 500 μΙ of a 50 mM solution of ammonium bicarbonate / 50% (v / v) acetonitrile at 50 ° C for 5 minutes, then dehydrated with 100 μΙ of acetonitrile for 5 min at room temperature. The gel pieces were allowed to dry and the proteins were digested in the gel for 10 minutes with a trypsin solution (25 ng / l, Promega, USA). The gels were then incubated with a 5 mM solution of ammonium bicarbonate overnight at 37 ° C.
Para la extracción de péptidos se utilizó una solución de 0.5% (v/v) de ácido trifluoroacético (TFA) en acetonitrilo al 50% durante 10 min a temperatura ambiente, los péptidos se desalaron, concentraron y purificaron empleando microcolumnas C-18 ZipTip (Millipore, MA, EE.UU.) eluyendo en 15 μΙ de TFA al 0.1 % en acetonitrilo al 50%.  For the extraction of peptides a solution of 0.5% (v / v) trifluoroacetic acid (TFA) in 50% acetonitrile was used for 10 min at room temperature, the peptides were desalted, concentrated and purified using C-18 ZipTip microcolumns ( Millipore, MA, USA) eluting in 15 μΙ of 0.1% TFA in 50% acetonitrile.
La identificación de proteínas se realizó en un espectrómetro de masas Plus Protein identification was performed on a Plus mass spectrometer.
4800 MALDI-TOF/TOF (Applied Biosystems, EE.UU.). Los espectros MS/MS se analizaron utilizando el software ProteinPilot (Applied Biosystems) y las listas de las señales obtenidas de los espectros de masas se sometieron a una búsqueda en la base de datos UniProtKB/Swiss-Prot utilizando el algoritmo de Paragon, los parámetros de la búsqueda se ajustaron para la alquilación de cisteína con yodoacetamida. 4800 MALDI-TOF / TOF (Applied Biosystems, USA). The MS / MS spectra were analyzed using ProteinPilot software (Applied Biosystems) and the lists of the signals obtained from the mass spectra were subjected to a search in the UniProtKB / Swiss-Prot database using the Paragon algorithm, the parameters of the search were adjusted for the alkylation of cysteine with iodoacetamide.
EJEMPLO 3 EXAMPLE 3
Inmunodetección de proteínas por Western blot  Immunodetection of proteins by Western blot
El extracto de proteína hepática (30 μg) de cada muestra fueron separadas por SDS-PAGE al 10% y transferidas a membranas de PVDF (Millipore) mediante electrotransferencia. Las membranas se bloquearon con leche descremada al 5% y Tween 20 al 0.1 % en solución salina amortiguadora con Tris al 1X (TBS) durante 1 h a temperatura ambiente. Las membranas fueron incubadas toda la noche a 4°C con un anticuerpo policlonal de ratón anti-LTB4DH (Abnova) a una dilución 1 :500. Después del lavado, las membranas se incubaron durante 1 h con un anticuerpo secundario anti-ratón conjugado con peroxidasa (Jackson ImmunoResearch Laboratories, West Grove, PA). La quimioluminiscencia se obtuvo al adicionar el sustrato quimioluminisente, luminol, Immobilon Western (Millipore) y las imágenes se obtuvieron con un sistema de imagen digital VersaDoc MP 5000 (BioRad). Las mismas membranas sirvieron para la inmunodetección con un anticuerpo anti-GSTP1 a una dilución 1 :1 ,000 (Sigma Aldrich) seguido de un anticuerpo monoclonal de ratón anti-actina (Chemicon International). The liver protein extract (30 μg) from each sample was separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore) by electrotransfer. The membranes were blocked with 5% skim milk and 0.1% Tween 20 in 1X Tris buffered saline (TBS) for 1 h at room temperature. The membranes were incubated overnight at 4 ° C with a polyclonal mouse anti-LTB4DH antibody (Abnova) at a 1: 500 dilution. After washing, the membranes were incubated for 1 h with a secondary anti-mouse antibody conjugated to peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA). Chemiluminescence was obtained by adding the chemiluminescent substrate, luminol, Immobilon Western (Millipore) and the images were obtained with a VersaDoc MP 5000 digital imaging system (BioRad). The same membranes served for immunodetection with an anti-GSTP1 antibody at a 1: 1,000 dilution (Sigma Aldrich) followed by a mouse anti-actin monoclonal antibody (Chemicon International).
EJEMPLO 4 EXAMPLE 4
Actividad 2 aiquenal/ona oxidoreductasa de PTGR1 en tumores  PTGR1 aiquenal / one oxidoreductase activity 2 in tumors
La actividad 2 alquenal/ona oxidoreductasa dependiente de NADPH de la PTGR1 , se determinó mediante la medición continua de la oxidación de NADPH a través de su absorción espectrofotométrica a 340 nm, se utilizó el trans-2- nonenal como sustrato de acuerdo con el método descrito en Dick RA, et al. J Biol Chem 2001 ;276:40803-40810.  The NADPH-dependent activity 2 alkenal / one oxidoreductase of PTGR1 was determined by continuous measurement of NADPH oxidation through its spectrophotometric absorption at 340 nm, trans-2- nonenal was used as substrate according to the method described in Dick RA, et al. J Biol Chem 2001; 276: 40803-40810.
La reacción final consistió en una solución de 0.1 mM de trans-2-nonenal, 0.1 mM de NADPH y 0.2 mg/ml de extracto de proteína en solución amortiguadora de fosfato de sodio/potasio 50 mM, pH 7.0 a 30°C. El trans-2-nonenal se preparó como una solución concentrada (50 mM en etanol) y luego se diluyó 1 :10 en solución amortiguadora de fosfatos; la concentración de etanol en la reacción final fue de 0.2%. Los extractos de proteína del hígado se obtuvieron en solución amortiguadora RIPA con un coctel inhibidor de proteasas.  The final reaction consisted of a solution of 0.1 mM of trans-2-nonenal, 0.1 mM of NADPH and 0.2 mg / ml of protein extract in 50 mM sodium / potassium phosphate buffer solution, pH 7.0 at 30 ° C. The trans-2-nonenal was prepared as a concentrated solution (50 mM in ethanol) and then diluted 1: 10 in phosphate buffer solution; the concentration of ethanol in the final reaction was 0.2%. Liver protein extracts were obtained in RIPA buffer solution with a protease inhibitor cocktail.
Se monitoreó la tasa de oxidación de NADPH a 340 nm durante 10 minutos en 2 condiciones consecutivas: sin el sustrato trans-2-nonenal y después de adicionar el sustrato durante otros 10 minutos. Se obtuvo la diferencia en la absorbancia por minuto y se calculó la actividad enzimática a partir del coeficiente de extinción molar para el NADPH (6.2 mM-1 cm-1 ) expresado como nmol de NADPH/min/mg de proteína. EJEMPLO 5 The oxidation rate of NADPH at 340 nm was monitored for 10 minutes under 2 consecutive conditions: without the trans-2-nonenal substrate and after adding the substrate for another 10 minutes. The difference in absorbance per minute was obtained and the enzymatic activity was calculated from the molar extinction coefficient for NADPH (6.2 mM-1 cm -1) expressed as nmol of NADPH / min / mg protein. EXAMPLE 5
Expresión génica de PtgM en tumores hepáticos PtgM gene expression in liver tumors
Las lesiones hepáticas fueron identificadas histológicamente según su actividad GGT+. Los diferentes tipos de nodulos y tumores fueron capturados mediante microdisección láser (LCM, por sus siglas en inglés) empleando un Microdisector Láser Arcturus, Veritas 704 y laminillas especiales para microdisección que poseen una capa de polietileno naftalato (PEN Membrane Glass Slides) tal y como se describe en Mena JE, et al; Anal Biochem. 2013 Nov 20. pii: S0003-2697(13)0055 -4. Liver lesions were identified histologically according to their GGT + activity. The different types of nodules and tumors were captured by laser microdissection (LCM) using an Arcturus Laser Microdissector, Veritas 704 and special microdissection lamellae that have a layer of polyethylene naphthalate (PEN Membrane Glass Slides) as It is described in Mena JE, et al; Biochem anal. 2013 Nov 20. pii: S0003-2697 (13) 0055 -4.
El tejido de interés fue capturado con una tapa (CapSure Macro LCM Caps, The tissue of interest was captured with a cap (CapSure Macro LCM Caps,
Applied Biosystems) que se toma adhesivo mediante pulsos de láser infrarrojo. Los ajustes del láser óptimas para la LCM de tejido hepático fue de 80 mW de potencia, 8,000 microsegundos duración y una intensidad del 100 mV. El láser de corte se ajustó para producir un punto de 2 mieras de diámetro a una potencia de 20 mW. Las -tapas con el tejido se colocaron en un tubo de GeneAmp (Applied Biosystems, N80 06 ) que contenía 320 μΙ de solución amortiguadora de lisis RLT (Qiagen, RNeasy mini kit) suplementado con 1 % (v/v) de β-mercaptoetanol. Applied Biosystems) which is taken adhesive using infrared laser pulses. The optimal laser settings for liver tissue LCM were 80 mW of power, 8,000 microseconds duration and an intensity of 100 mV. The cutting laser was adjusted to produce a point of 2 microns in diameter at a power of 20 mW. The tissue caps were placed in a GeneAmp tube (Applied Biosystems, N80 06) containing 320 μΙ of RLT lysis buffer solution (Qiagen, RNeasy mini kit) supplemented with 1% (v / v) of β-mercaptoethanol .
Del tejido de interés obtenido por LCM se extrajo el ARN total mediante el método de aislamiento en columnas RNAeasy (Qiagen, Hilden Germany). La concentración de ARN y su pureza fue determinada espectrofotométricamente a 260 y 280 nm empleando un espectrómetro UV. La integridad y calidad del ARN fue verificada por electroforesis capilar en un bioanalizador Agilent 2100 obteniendo proporciones de RNAr 28S/18S > 1.7.  From the tissue of interest obtained by LCM, total RNA was extracted by the RNAeasy column isolation method (Qiagen, Hilden Germany). The concentration of RNA and its purity was determined spectrophotometrically at 260 and 280 nm using a UV spectrometer. The integrity and quality of the RNA was verified by capillary electrophoresis in an Agilent 2100 bioanalyzer obtaining ratios of RNAr 28S / 18S> 1.7.
Las reacciones de ADNc se llevaron a cabo con el kit de Transcripción Reversa de ADNc de Alta Capacidad (Applied Biosystems) empleando desde 150 a 750 ng de ARN total. Para la amplificación por PCR cuantitativo se empleó una dilución 1/10 del ADNc.  The cDNA reactions were carried out with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using from 150 to 750 ng of total RNA. For quantitative PCR amplification a 1/10 dilution of the cDNA was used.
Las reacciones se llevaron a cabo utilizando el ensayo de expresión génica TaqMan en un sistema HT 7900 Fast Real Time PCR (Applied Biosystem, México). Las sondas marcadas con Fluoresceína (FAM) (límite exón-exón) para Gstpl de rata (Rn00561378_gH), PtgM (Rn00593950_m ) y ARNr 18S (Rn03928990) se obtuvieron de Applied Biosystems. Los datos de Gstpl y PtgM se normalizaron contra la expresión genética de ARNr 18S usando el método comparativo ΔΔΟί. The reactions were carried out using the TaqMan gene expression assay in an HT 7900 Fast Real Time PCR system (Applied Biosystem, Mexico). Fluorescein-labeled (FAM) probes (exon-exon limit) for rat Gstpl (Rn00561378_gH), PtgM (Rn00593950_m) and 18S rRNA (Rn03928990) were obtained from Applied Biosystems. Gstpl and PtgM data they were normalized against the genetic expression of 18S rRNA using the comparative method ΔΔΟί.
EJEMPLO 6 EXAMPLE 6
Análisis de expresión genética con microarreglos Genetic expression analysis with microarrays
A partir de 200 ng de ARN total se obtuvo la primera cadena de ADNc usando transcriptasa reversa Superscript II e iniciadores oligo-dT. Se sintetizó la segunda cadena de ADNc. Se obtuvo el ARNc por transcripción in vitro y se utilizó como molde para un segundo ciclo de síntesis de ADNc con la incorporación de dUTP. El ADNc obtenido se fragmentó utilizando uracil-DNA glicosilasa. Los fragmentos (40-70meros) fueron marcadas con biotina por una reacción de adición terminal de desoxinucleótido. The first cDNA chain was obtained from 200 ng of total RNA using Superscript II reverse transcriptase and oligo-dT primers. The second cDNA chain was synthesized. CRNA was obtained by in vitro transcription and was used as a template for a second cycle of cDNA synthesis with the incorporation of dUTP. The cDNA obtained was fragmented using uracil-DNA glycosylase. The fragments (40-70 meters) were labeled with biotin by a terminal addition reaction of deoxynucleotide.
El ADNc marcado se híbrido en el microarreglo Gene 1 ,0 ST de rata (Affymetrix Inc.) durante 17 horas a 45°C. Las muestras se lavaron con soluciones amortiguadoras de baja astringencia (SSPE) y alta astringencia (100 mM MES, 0.1 M NaCI) y se tiñeron con estreptavidina-ficoeritrina usando el protocolo de la estación de fluidos Affymetrix 450 FS450_0007. Los microarreglos se escanearon con un lector GeneChip 3000 7G (Affymetrix, Inc.) empleando el software Expression Consolé ( Affymetrix , Inc. ) para obtener las señales de intensidad y calidad de los microarreglos escaneados. Se utilizó el ANOVA de una vía para comparar las diferencias en la expresión génica y la integridad del ARN entre las muestras obtenidas por LMC. La significancia estadística se estableció en p<0.05.  The labeled cDNA was hybridized in the Rat Gene 1.0 ST microarray (Affymetrix Inc.) for 17 hours at 45 ° C. The samples were washed with low astringency (SSPE) and high astringency (100 mM MES, 0.1 M NaCI) buffer solutions and stained with streptavidin-phycoerythrin using the Affymetrix 450 FS450_0007 fluid station protocol. The microarrays were scanned with a GeneChip 3000 7G reader (Affymetrix, Inc.) using Expression Consolé software (Affymetrix, Inc.) to obtain the intensity and quality signals of the scanned microarrays. One-way ANOVA was used to compare differences in gene expression and RNA integrity between samples obtained by CML. Statistical significance was established at p <0.05.
EJEMPLO 7 EXAMPLE 7
Detección Inmunohistoquímica de PTGR1 en tumores Immunohistochemical detection of PTGR1 in tumors
Las secciones histológicas de tejido hepático de rata se incubaron con un anticuerpo policlonal de conejo anti-PTGR1 (Novus Biologicals) a una dilución de 1 :25 y un anticuerpo policlonal de conejo anti-GSTP (Dako) a una dilución 1 : 100. Los anticuerpos primarios se detectaron utilizando la técnica de avidina-biotina con el Kit LSAB + HRP (Dako Corporation, California, EE.UU.). No se observó tinción cuando el anticuerpo primario fue sustituido con el control isotipo de ratón. EJEMPLO 8 Histological sections of rat liver tissue were incubated with a polyclonal rabbit anti-PTGR1 antibody (Novus Biologicals) at a dilution of 1: 25 and a rabbit polyclonal antibody anti-GSTP (Dako) at a dilution 1: 100. Primary antibodies were detected using the avidin-biotin technique with the LSAB + HRP Kit (Dako Corporation, California, USA). No staining was observed when the primary antibody was replaced with the mouse isotype control. EXAMPLE 8
Detección de PTGR1 en muestras clínicas de CHC PTGR1 detection in clinical samples of CHC
Se obtuvieron biopsias hepáticas incluidas en parafina y muestras de resección de 12 casos de CHC (6 mujeres, 6 varones, edad media 54.4 años, de entre 17-70 años de edad) del Centro de cancerología del Estado de Veracruz, México. Como control de patología no asociada a cáncer se utilizó la región del parénquima de un caso de fibrosis hepática. Todas las muestras fueron sometidas a tinción con hematoxilina-eosina (H&E) para el diagnóstico histológico de rutina. De acuerdo al estudio histopatológico las muestras de CHC se agruparon de la siguiente manera de acuerdo con la etapa del tumor y el grado de diferenciación celular: bien diferenciado (n=2), moderadamente diferenciado (n=6) y poco diferenciado (n=4). Hepatic biopsies included in paraffin and resection samples were obtained from 12 cases of CHC (6 women, 6 men, mean age 54.4 years, between 17-70 years of age) from the Cancer Center of the State of Veracruz, Mexico. As a control of pathology not associated with cancer, the parenchyma region of a case of liver fibrosis was used. All samples were stained with hematoxylin-eosin (H&E) for routine histological diagnosis. According to the histopathological study, the CHC samples were grouped as follows according to the stage of the tumor and the degree of cell differentiation: well differentiated (n = 2), moderately differentiated (n = 6) and poorly differentiated (n = 4).
A partir de las biopsias hepáticas embebidas en parafina se obtuvieron cortes seriados de 5 pmde espesor. Los cortes fueron desparafinados y rehidratados por lavados secuenciales con xileno y etanol a diferentes concentraciones. La recuperación de antígeno se llevó a cabo por calentamiento con una solución amortiguadora de citrato de sodio (citrato de sodio 10 mM y Tween 20 al 0.05%, pH 6.0) (Dako Corporation, California, EE.UU.) fijando las secciones seriadas con acetona fría.  From the liver biopsies embedded in paraffin, serial cuts of 5 pm thick were obtained. The cuts were dewaxed and rehydrated by sequential washes with xylene and ethanol at different concentrations. Antigen recovery was carried out by heating with a sodium citrate buffer solution (10 mM sodium citrate and 0.05% Tween 20, pH 6.0) (Dako Corporation, California, USA) by fixing the serial sections with cold acetone
Los tejidos se permeabilizaron con Tritón X-100 al 0.5% en PBS. Las secciones de biopsias humanas de CHC fueron bloqueadas con BSA al 1 % durante 2 horas y después se incubaron durante toda la noche con el anticuerpo primario: ya sea un anticuerpo policlonal de ratón anti-LT4DH (anti- Ptgr - Abnova) a una dilución 1 :50 o un anticuerpo monoclonal de ratón anti-glipican-3 (1 G12) (Cell Marque) a una dilución 1 : 100.  The tissues were permeabilized with 0.5% Triton X-100 in PBS. The human biopsy sections of CHC were blocked with 1% BSA for 2 hours and then incubated overnight with the primary antibody: either a polyclonal mouse antibody anti-LT4DH (anti-Ptgr-Abnova) at dilution 1: 50 or a mouse monoclonal antibody anti-glipican-3 (1 G12) (Cell Marque) at a 1: 100 dilution.
Debe entenderse que lo precedente se relaciona solamente a modalidades preferidas de la presente invención y que numerosas modificaciones o alteraciones pueden hacerse en ésta sin apartarse del espíritu y alcance de la invención como se establece en las reivindicaciones anexas.  It should be understood that the foregoing relates only to preferred embodiments of the present invention and that numerous modifications or alterations can be made therein without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims

NOVEDAD DE LA INVENCIÓN REIVINDICACIONES NEWS OF THE INVENTION CLAIMS
1. - Un método in vitro útil para el diagnóstico temprano de carcinoma hepatocelular en un mamífero, dicho método caracterizado porque comprende: (a) medir el nivel de expresión del gen Ptgrl o de la proteína para la que codifica en una muestra biológica previamente obtenida y (b) comparar el nivel de expresión en dicha muestra contra un estándar de expresión del gen Ptgrl o de la proteína para la que codifica, en donde la sobre-expresión del gen Ptgrl o de la proteína para la que codifica es indicativa de que el individuo sufre de CHC o de que podría desarrollar CHC. 1. - An in vitro method useful for the early diagnosis of hepatocellular carcinoma in a mammal, said method characterized in that it comprises: (a) measuring the expression level of the Ptgrl gene or the protein for which it encodes in a previously obtained biological sample and (b) compare the level of expression in said sample against an expression standard of the Ptgrl gene or the protein for which it encodes, where the overexpression of the Ptgrl gene or the protein for which it encodes is indicative that the individual suffers from HCC or might develop HCC.
2. - El método de conformidad con la reivindicación 1 , caracterizado además porque el nivel de expresión de Ptgrl se determina mediante la medición de su ARNm. 2. - The method according to claim 1, further characterized in that the expression level of Ptgrl is determined by measuring its mRNA.
3.- El método de conformidad con la reivindicación 2, caracterizado además porque el ARNm se determina mediante cualquiera de las metodologías seleccionadas del grupo que comprende: RT-PCR, hibridación in situ, hibridación con sondas específicas de ácidos nucleicos y secuenciación de transcritos. 3.- The method according to claim 2, further characterized in that the mRNA is determined by any of the methodologies selected from the group comprising: RT-PCR, in situ hybridization, hybridization with specific nucleic acid probes and transcript sequencing.
4. - El método de conformidad con la reivindicación 1 , caracterizado además porque el nivel de expresión de Ptgrl se determina mediante la medición de la proteína PTGRL 4. - The method according to claim 1, further characterized in that the expression level of Ptgrl is determined by measuring the PTGRL protein
5. - El método de conformidad con la reivindicación 4, caracterizado además porque la proteína PTGR1 se determina mediante cualquiera de las metodologías seleccionadas del grupo que comprende: ¡nmunohistoquímica, ELISA y sus variantes, inmunofluorescencia, inmunocitoquímica e inmunoprecipitación. 5. - The method according to claim 4, further characterized in that the PTGR1 protein is determined by any of the methodologies selected from the group comprising: immunohistochemistry, ELISA and its variants, immunofluorescence, immunocytochemistry and immunoprecipitation.
6. - El método de conformidad con la reivindicación 1 , caracterizado además porque el nivel de expresión de Ptgrl se determina mediante la medición de la actividad enzimática de la enzima PTGR1 . 6. - The method according to claim 1, further characterized in that the expression level of Ptgrl is determined by measuring the enzymatic activity of the PTGR1 enzyme.
7. - El método de conformidad con la reivindicación 6, caracterizado además porque la actividad enzimática de la PTGR1 se determina mediante cualquiera de las metodologías seleccionadas del grupo que comprende: reacciones con aldehidos y cetonas alfa-beta insaturados o nitroalquenos en presencia de NADPH. 7. - The method according to claim 6, further characterized in that the enzymatic activity of PTGR1 is determined by any of the methodologies selected from the group comprising: reactions with aldehydes and alpha-beta unsaturated ketones or nitroalkenes in the presence of NADPH.
8. - El método de conformidad con cualquiera de las reivindicaciones 1 a 7, caracterizado además porque el nivel de expresión de Ptgrl se determina en una muestra de tejido hepático. 8. - The method according to any of claims 1 to 7, further characterized in that the expression level of Ptgrl is determined in a liver tissue sample.
9. - El método de conformidad con cualquiera de las reivindicaciones 1 a 7, caracterizado además porque el nivel de expresión de PTGR1 se determina en una muestra de suero, plasma o sangre periférica total. 9. - The method according to any of claims 1 to 7, further characterized in that the expression level of PTGR1 is determined in a serum, plasma or total peripheral blood sample.
10. - El método de conformidad con cualquiera de las reivindicaciones 1 a 9, caracterizado además porque el mamífero es un humano. 10. - The method according to any of claims 1 to 9, further characterized in that the mammal is a human.
1 - El método de conformidad con cualquiera de las reivindicaciones 1 a 9, caracterizado además porque el mamífero es un animal doméstico o de granja, preferiblemente un perro, un gato o un caballo o una vaca. 1 - The method according to any of claims 1 to 9, further characterized in that the mammal is a domestic or farm animal, preferably a dog, a cat or a horse or a cow.
12. - Un método in vitro útil para determinar si un mamífero que sufre de carcinoma hepatocelular es candidato a ser tratado con compuestos que son bioactivados por la PTGR1 , dicho método caracterizado porque comprende: (a) medir el nivel de expresión del gen Ptgrl o de la proteína para la que codifica en una muestra biológica previamente obtenida y (b) comparar el nivel de expresión en dicha muestra contra un estándar de expresión del gen Ptgrl o de la proteína para la que codifica, en donde la sobre-expresión del gen Ptgrl o de la proteína para la que codifica es indicativa de que el mamífero es candidato a ser tratado con compuestos que son bioactivados por la PTGR1. 12. - A useful in vitro method to determine if a mammal suffering from hepatocellular carcinoma is a candidate to be treated with compounds that are bioactivated by PTGR1, said method characterized in that it comprises: (a) measuring the expression level of the Ptgrl gene or of the protein for which it codes in a previously obtained biological sample and (b) compare the level of expression in said sample against an expression standard of the Ptgrl gene or the protein for which it codes, where the over-expression of the gene Ptgrl or the protein it encodes is indicative that the mammal is a candidate to be treated with compounds that are bioactivated by PTGR1.
13. - El método de conformidad con la reivindicación 12 caracterizado además porque el compuesto que es bioactivado por la PTGR1 se selecciona del grupo que comprende a los derivados de la iludina, los derivados de los acilfulvenos o los nitroalquenos. 13. - The method according to claim 12 further characterized in that the compound that is bioactivated by PTGR1 is selected from the group comprising iludin derivatives, acylfulvene derivatives or nitroalkenes.
14. - El método de conformidad con cualquiera de las reivindicaciones 12 y 13 caracterizado además porque el nivel de expresión de Ptgrl se determina mediante la medición de la actividad enzimática de la PTGR1. 14. - The method according to any of claims 12 and 13 further characterized in that the expression level of Ptgrl is determined by measuring the enzymatic activity of PTGR1.
15.- El método de conformidad con la reivindicación 14, caracterizado además porque la actividad enzimática de la PTGR1 se determina mediante cualquiera de las metodologías seleccionadas del grupo que comprende: reacciones con aldehidos y cetonas alfa-beta insaturados o nitroalquenos en presencia de NADPH. 15.- The method according to claim 14, further characterized in that the enzymatic activity of PTGR1 is determined by any of the methodologies selected from the group comprising: reactions with aldehydes and alpha-beta unsaturated ketones or nitroalkenes in the presence of NADPH.
16. - El método de conformidad con cualquiera de las reivindicaciones 12 y 13, caracterizado además porque el nivel de expresión de Ptgrl se determina mediante la medición de su ARNm. 16. - The method according to any of claims 12 and 13, further characterized in that the expression level of Ptgrl is determined by measuring its mRNA.
17. - El método de conformidad con la reivindicación 16, caracterizado además porque el ARNm se determina mediante cualquiera de las metodologías seleccionadas del grupo que comprende: RT-PCR, hibridación in situ, hibridación con sondas específicas de ácidos nucleicos y secuenciación de transcritos. 17. - The method according to claim 16, further characterized in that the mRNA is determined by any of the methodologies selected from the group comprising: RT-PCR, in situ hybridization, hybridization with specific nucleic acid probes and sequencing of transcripts.
18.- El método de conformidad con cualquiera de las reivindicaciones 12 y 18.- The method according to any of claims 12 and
13, caracterizado además porque el nivel de expresión de Ptgrl se determina mediante la medición de la proteína PTGRL 13, further characterized in that the expression level of Ptgrl is determined by measuring the PTGRL protein
19.- El método de conformidad con la reivindicación 18, caracterizado además porque la proteína PTGR1 se determina mediante cualquiera de las metodologías seleccionadas del grupo que comprende: inmunohistoquímica, ELISA y sus variantes, inmunofluorescencia, inmunocitoquímica e inmunoprecipitación. 19.- The method according to claim 18, further characterized in that the PTGR1 protein is determined by any of the methodologies selected from the group comprising: immunohistochemistry, ELISA and its variants, immunofluorescence, immunocytochemistry and immunoprecipitation.
20 - El método de conformidad con cualquiera de las reivindicaciones 12 a 19, caracterizado además porque el nivel de expresión de Ptgrl se determina en una muestra de tejido hepático. 20 - The method according to any of claims 12 to 19, further characterized in that the expression level of Ptgrl is determined in a liver tissue sample.
21 .- El método de conformidad con cualquiera de las reivindicaciones 12 a 19, caracterizado además porque el nivel de expresión de Ptgrl se determina en una muestra de suero, plasma o sangre periférica total. 21.- The method according to any of claims 12 to 19, further characterized in that the expression level of Ptgrl is determined in a serum, plasma or total peripheral blood sample.
22.- El método de conformidad con cualquiera de las reivindicaciones 12 a22.- The method according to any of claims 12 to
21 , caracterizado además porque el mamífero es un humano. 21, further characterized because the mammal is a human.
23.- El método de conformidad con cualquiera de las reivindicaciones 12 a 21 , caracterizado además porque el mamífero es un animal doméstico o de granja, preferiblemente un perro, un gato o un caballo o una vaca. 23.- The method according to any of claims 12 to 21, further characterized in that the mammal is a domestic or farm animal, preferably a dog, a cat or a horse or a cow.
24.- Un equipo de diagnóstico útil para el diagnóstico temprano del carcinoma hepatocelular en un mamífero caracterizado porque comprende al menos: a) medios para determinar el nivel de expresión del gen Ptgrl o de la proteína para la que codifica en una muestra biológica y b) un estándar de expresión del gen Ptgrl o de la proteína para la que codifica para comparar el nivel de expresión. 24.- A diagnostic kit useful for the early diagnosis of hepatocellular carcinoma in a mammal characterized in that it comprises at least: a) means to determine the level of expression of the Ptgrl gene or the protein for which it codes in a biological sample and b) an expression standard of the Ptgrl gene or the protein for which it codes to compare the expression level.
25.- El equipo de la reivindicación 24 caracterizado además porque los medios para determinar el nivel de expresión del gen Ptgrl o de la proteína para la que codifica son medios necesarios para determinar el ARNm de Ptgrl mediante cualquiera de RT-PCR, hibridación in situ, hibridación con sondas específicas de ácidos nucleicos y secuenciación de transcritos. 25.- The equipment of claim 24 further characterized in that the means for determining the level of expression of the Ptgrl gene or the protein for which it encodes are necessary means for determining the Ptgrl mRNA by means of any of RT-PCR, in situ hybridization , hybridization with specific nucleic acid probes and transcript sequencing.
26 - El equipo de la reivindicación 24 caracterizado además porque los medios para determinar el nivel de expresión del gen Ptgrl o de la proteína para la que codifica son medios necesarios para determinar la proteína PTGR1 mediante cualquiera de inmunohistoquímica, ELISA y sus variantes, inmunofluorescencia, inmunocitoquímica e inmunoprecipitación. 26 - The equipment of claim 24 further characterized in that the means for determining the level of expression of the Ptgrl gene or the protein for which it encodes are necessary means for determining the PTGR1 protein by means of any of immunohistochemistry, ELISA and its variants, immunofluorescence, Immunocytochemistry and immunoprecipitation.
27. - El equipo de la reivindicación 24 caracterizado además porque los medios para determinar el nivel de expresión del gen Ptgrl o de la proteína para la que codifica son medios necesarios para determinar la actividad enzimática de la PTGR1 mediante cualquier reacción con aldehidos y cetonas alfa-beta insaturados o nitroalquenos en presencia de NADPH. 27. - The equipment of claim 24 further characterized in that the means for determining the level of expression of the Ptgrl gene or the protein for which it encodes are necessary means for determining the enzymatic activity of PTGR1 through any reaction with aldehydes and alpha ketones. -beta unsaturated or nitroalkenes in the presence of NADPH.
28. - El equipo de conformidad con cualquiera de las reivindicaciones 24 a 27, caracterizado además porque el nivel de expresión del gen Ptgrl o de la proteína para la que codifica se determina en una muestra de tejido hepático. 28. - The equipment according to any of claims 24 to 27, further characterized in that the expression level of the Ptgrl gene or the protein for which it encodes is determined in a sample of liver tissue.
29. - El equipo de conformidad con cualquiera de las reivindicaciones 24 a 27, caracterizado además porque el nivel de expresión del gen Ptgrl o de la proteína para la que codifica se determina en una muestra de suero, plasma o sangre periférica total. 29. - The equipment according to any of claims 24 to 27, further characterized in that the expression level of the Ptgrl gene or the protein for which it encodes is determined in a sample of serum, plasma or total peripheral blood.
30. - El equipo de conformidad con cualquiera de las reivindicaciones 24 a 29, caracterizado además porque el mamífero es un humano. 30. - The equipment according to any of claims 24 to 29, further characterized in that the mammal is a human.
31 . - El equipo de conformidad con cualquiera de las reivindicaciones 24 a 29, caracterizado además porque el mamífero es un animal doméstico o de granja, preferiblemente un perro, un gato o un caballo o una vaca. 31. - The equipment according to any of claims 24 to 29, further characterized in that the mammal is a domestic or farm animal, preferably a dog, a cat or a horse or a cow.
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