MX2013015064A

MX2013015064A - Method for the early diagnosis of hepatocellular carcinoma.

Info

Publication number: MX2013015064A
Application number: MX2013015064A
Authority: MX
Inventors: Julio Isael Pérez Carreón; Ricardo Sánchez Rodríguez
Original assignee: Inst Nac De Medicina Genómica
Priority date: 2013-12-18
Filing date: 2013-12-18
Publication date: 2015-06-18
Also published as: WO2015093932A1; MX357018B

Abstract

The invention relates to novel methods for the 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

METHOD OF EARLY DIAGNOSIS OF HEPATOCELLULAR CARCINOMA FIELD OF THE INVENTION The present invention is inserted within the field of health.

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 establishing with this 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.

Particularly the present invention relates to a method of early diagnosis of HCC by detecting the gene products (mRNA or protein) of prostaglandin reductase 1 (PTGR1) in biological samples.

Additionally, the present invention relates to methods for determining whether a patient with HCC is candidate to be treated with anti-tumor compounds bioactivated by PTGR1, such as acyl fulvenes and their derivatives, among others, by using overexpression of PTGR1 in tumors. as enzyme target of therapy.

Therefore, the aim of the present invention is to provide new methods for the early diagnosis of HCC by detecting specific biomarkers related to Ptgrl and its enzymatic capacities, as well as methods for the selection of individuals with HCC that are candidates to be treated with antitumor compounds bioactivated by PTGR1 such as acylfulvenes and their derivatives, among others.

BACKGROUND OF THE INVENTION Hepatocellular carcinoma (HCC) is the sixth most common neoplasm in the world and ranks third in cancer-related deaths. HCC is the most common primary tumor of the liver and constitutes 90% of cases. The incidence rates of HCC show a constant increase both in Europe and in the USA; Between 600,000 and 700,000 new cases are diagnosed worldwide each year. The mortality of HCC is very high, approximately 95%, that is, only 5% of those who suffer survive more than 5 years. (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology, Flying Publisher, 2013).

Although the etiology of HCC is multifactorial, it usually develops as a consequence of a chronic liver disease, (mainly the condition of cirrhosis). Chronic hepatitis B is the main risk factor for the development of HCC in Asia and Africa, while chronic hepatitis C is in the US, Europe and Japan. Approximately 80% of cases of liver cancer occur in cirrhotic livers. Fibrosis is seen in most chronic diseases of the liver and precedes the development of cirrhosis. Liver 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 liver parenchyma, such as collagen, laminin, fibronectin, among other fibrillar proteins.

Chronic carriers of the hepatitis B virus (HBV) have a 100-fold increased risk of developing HCC, compared to a population of healthy, non-infected individuals. In developing countries, exposure to aflatoxins increases the risk of developing HCC (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology, Flying Publisher.2013.).

Around the world it is estimated that there are between 130-170 million people infected with the hepatitis C virus, of which 20 to 30% will develop liver cirrhosis. Of these, 3 to 5% of cases will develop HCC each year. In practical terms this means that about one third of cirrhotic patients with hepatitis C will develop HCC (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology, Flying Publisher.2013.).

Frequently, patients with chronic hepatitis C will develop liver cancer, only up to the stage of cirrhosis. The consumption of alcohol or tobacco increases the risk of developing HCC (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology, Flying Publisher.2013.).

Obesity and diabetes mellitus are factors that can increase the risk of developing HCC in patients with chronic viral hepatitis from 4 to 40 times (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology, Flying Publisher. 2013).

When the expected risk of developing HCC is greater than 1.5% per year in patients with hepatitis C and 0.2% in patients with hepatitis B, clinical guidelines suggest that close clinical monitoring be performed, performing ultrasound (US) exams every 6 months and, of every 3 months in patients with cirrhosis that present nodular lesions, due to its high potential for malignancy. The systematic use of ultrasound in patients with a high risk of HCC allows early diagnosis of carcinoma in 30% of patients, who have a reasonable chance of success with curative treatment. However, this monitoring is expensive 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 HCC because it has insufficient sensitivity and specificity (Ulrich Spengleren Mauss, S., et al. (Ed), Hepatology.) Flying Publisher .2013).

Histopathological studies show that HCC develops in several stages in cirrhotic liver tissue. Some hepatocytes can be transformed from some regenerative nodules that give rise to dysplastic nodules (ND) and these preneoplastic lesions can progress from low-grade ND to high-grade ND, and so on, to generate an early HCC and, later, to an advanced HCC ( 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 on a reliable histological diagnosis. (The International Consensus Group for Hepatocellular Neoplasia, Hepatology.2009 Feb; 49 (2): 658-64).

The detection of HCC in early stages is crucial to cure the disease, since the curative treatments available, for example surgical resection, are effective in early stages. The patients who receive the diagnosis in early stages can achieve survival rates of 50-70% at 5 years, through surgical resection, liver transplantation or percutaneous ablation procedures. For intermediate HCC, there are only treatments that can prolong the patient's life a bit like local transarterial embolization and kinase inhibitor therapy (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology.) Flying Publisher.2013 ).

Tumors are classified to stratify patients in relation to their prognosis of survival 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 the CHC and is even used and recommended by the American Association for the Study of Liver Diseases (AASLD) and the Association European Study for the Liver (EASL) (Ulrich Spengler in Mauss, S., et al. (Ed), Hepatology, Flying Publisher.2013).

The BCLC classification takes into account several aspects of the disease: the general health of the patient, 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 in these stages.

The guidelines of the EASL and the AASLD provide recommendations regarding the most appropriate therapy for the treatment of patients in each of the stages of the BCLC classification. This classification is based only on clinical parameters, since there are still no molecular tests capable of reliably assessing the individual prognosis of patients with HCC.

The diagnosis of HCC can be made by detecting malignant hepatocytes in a liver biopsy or by improved dynamic contrast imaging radiological techniques that reveal the arterialized perfusion of the tumor. Additionally, improved 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.

For nodular lesions less 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 every 3 months is suggested.

For lesions larger than 1 cm, magnetic resonance 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 HCC, radiological imaging techniques of dynamic contrast should be applied. If this complementary radiological investigation yields typical HCC results, the diagnosis is confirmed, otherwise a directed biopsy should be performed (Ulrich Spengler in Mauss, S., et al., Ed., Hepatology, Flying Publisher, 2013).

The distinction between a dysplastic nodule and early HCC poses an important challenge for the pathologist and the medical team, because it depends on the ability to detect specific histological features when the cellular dysplasia is still low and therefore undefined and that the portion of tissue contained in the biopsy contains the nodular lesions.

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 to be made in the early stages. A-fetoprotein (AFP) is the most used serological marker, however it has low specificity and high levels are rarely found in early stages of HCC. In addition, AFP is not useful as a tissue marker due to its low sensitivity (25-30%) even in moderately differentiated CHC (The International Consensus Group for Hepatocellular Neoplasia, Hepatology, 2009 Feb; 49 (2): 658-64). FornerA, Bruix J. Lancet Oncol 2012; 13: 750-751).

Another marker that has been described for the early detection of HCC is glypican 3, which has shown good results in the detection of poorly differentiated tumors, 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 HCC (Shafizadeh N, Ferrell LD, Kakar S. od Pathol 2008; 21: 1011-1018).

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 HCC, where a positive result for two of any of these three markers confirms the presence of HCC 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).

Some genes or signatures of genes have also been described, whose expression functions as a marker for the diagnosis of HCC, but until now none has been validated clinically. The American patent application No. 2008/0038736, which is incorporated by reference, describes methods to establish whether a hepatic nodule is a dysplastic nodule or an early HCC, by determining the expression of at least three, of any of the following selected markers TERT, GPC3, gankyrin, survivin, TOP2A, LYVE1, Ecaderin, IGFBP3, PDGFRA, TGFA, cielin D1 and HGF, however, this method detects developing CHC and is not able to detect it from the stage in which dysplastic nodules appear.

On the other hand, the US patent application No.2011 / 0306513 cited here as reference, describes serum markers for the diagnosis of HCC, however determinations are made comparing serum from normal individuals with serum from patients with liver cancer without correlation of the beginning of the disease, so it is not viable to establish an early diagnosis of liver cancer.

Based on the current state of the art, the need for new methods for the early diagnosis of HCC through the detection of specific biomarkers in tissue and serum, capable of detecting the disease from early stages or even from the appearance of dysplastic nodules.

With regard to the treatments available for CHC, it has been proven that only successful therapeutic results are achieved when 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.

Among the existing antitumor compounds, acylfulvenes have been described, which are semisynthetic derivatives of the M and S illudins produced by the fungus Omphalotus olearius. Although iludins have antitumor activity, they have low therapeutic rates since the therapeutic dose is very similar to the toxic dose of the compound.

The acilfulvenos have therapeutic indexes superior to those of the iludinas M and S and the therapeutic doses do not generate toxicity for the patients. In addition, acyl-fumes and other similar bio-reducing agents have the advantage that they are converted into reactive chemical species by a specific enzymatic activation in the target cells.

The US patent application No. 2008/0306147, which is incorporated by reference, describes analogous compounds of iludin useful for inhibiting tumor growth, mainly of solid tumors, however it does not specify its usefulness for HCC nor does it describe a reliable method to determine which patients with HCC could benefit from these compounds.

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 hydroxymethylacylfulvene.

PTGR1 is one of the enzymes involved in the catabolism of prostaglandins and some eicosanoids (Tai HH, et al., Prostaglandins Other Lipid Mediat 2002; 68-69: 483-493). It has been assigned alternative names, associated with its molecular function, such as leukotriene B4 12-hydroxideshydrogenase-dependent NADP (LTB4DH); 15-oxoprostaglandin 13-reductase; alkene / ona oxidoreductase dependent on NADPH; ZADH3 and gene inducible by dithiolethione 1 (DIG-1), among others. The human Ptgrl gene has orthologous genes in several organisms, and specifically have been described in several mammalian species. Some examples of orthologous Ptgrl genes are described in Table 1.

Table 1. Orthologous genes of the Ptgrl gene in mammals This enzyme reduces the unsaturated α, β-carbonyls that 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 are derived from the peroxidation of linoleic acid and arachidonic acid (Spitz DR, et al., Biochem J 1990; 267: 453-459) and have important cytotoxic effects on cells, ranging from inhibition of synthesis of DNA and proteins, up to the 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 the activity 2-alquenal / ona oxidoreductase, by reducing the double bond a, b unsaturated (Dick RA, et al., J Biol Chem 2001; 276 : 40803-40810 and Youn B, et al., J Biol Chem 2006; 281: 40076-40088).

On the other hand it has been described that the Ptgrl gene is induced by dithiolethias (Primiano T, et al Carcinogenesis 1998; 19: 999-1005 and Primiano T, et al., Carcinogenesis 1996; 17: 2297-2303), a well-known class of cancer chemopreventive agents, which induce the production of detoxification enzymes of carcinogens, such as: NQ01, SGT, EPHX, GCL, and UGT, through the activation of the Nrf2 pathway (Zhang Y, Munday R. Mol Cancer 2008 Ther; 7: 3470-3479). All these observations support the idea that PTGR1 acts as a cytoprotective enzyme that has both antioxidant and anti-inflammatory functions.

BRIEF DESCRIPTION OF THE INVENTION The present invention is based on the unexpected fact that the Ptgrl gene is overexpressed both in dysplastic hepatic nodules, and in early and advanced hepatocellular carcinoma (HCC) in models of chemical hepatocarcinogenesis in rat using diethylnitrosamine (DEN) as chemical carcinogen. Overexpression of PTGR1 in experimentally induced lesions correlates with overexpression of PTGR1 in HCC samples in human individuals.

The present invention overcomes the deficiencies of the state of the art by providing a new molecular marker and useful methods for establishing 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. .

The over-expression 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 the overexpression of Ptgrl in biological samples of hepatic 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 the overexpression of the gene Ptgrl (mRNA), of the protein for which it encodes or of the enzymatic activity of said protein in a biological sample.

On the other hand, the over-expression of the Ptgrl gene in dysplastic nodules and in the HCC allows establishing a new method to determine if an individual suffering from HCC is a candidate to be treated with antitumor compounds susceptible to being bioactivated by the PTGR1 enzyme.

In another aspect, the invention relates to equipment useful for the early diagnosis of HCC, which comprise means for the detection of the gene products of Ptgrl in a biological sample.

BRIEF DESCRIPTION OF THE FIGURES Figure 1A shows immunodetection by Western blot of Prostaglandin Reductase 1 (PTGR1) and Glutathione S-Transferase P1 (GSTP1) in tumor samples (T) and surrounding non-tumoral tissue (NT) obtained in months 8, 9, and 10 of rats subjected to the Solt and Farber protocol or resistant hepatocyte (HR) model. The PTGR1 protein was identified as a 33 kDa band expressed only in the tumor samples (T), while the GSTP1 is expressed in both T and NT tissue. Actin was used as charge control.

Figure 1B shows the immunodetection of PTGR1 by Western blot in hepatic protein extracts at different times of sacrifice under the Schiffer protocol. It is observed that PTGR1 is present in the liver tissue after 12 weeks of treatment with diethylnitrosamine (DEN).

Figure 2 shows the 2-alkene / ona oxidoreductase activity of the PTGR1 in liver samples of 6, 12 and 18 weeks of treatment using the Schiffer protocol, normal liver (HN) and 6 tumors (T) of the HR model between 9 and 12 months of treatment. An increase in enzymatic activity of PTGR1 is observed, which is statistically significant starting from week 18. Statistical difference as indicated **** p < 0.0001 compared with HN, 6, 12 and 18 weeks, *** p < 0.001 compared with HN, ** p < 0.01 compared with 6 weeks.

Figure 3 shows the genetic expression of Ptgrl and Gstpl, a marker of hepatocarcinogenesis in rat, 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 the TaqMan Gstpl (Rn00561378_gH), Ptgrl (Rn00593950_m1) and 18S rRNA (Rn03928990) probes.

Additionally, the genetic expression of 6 tumors (T) of the HR model is presented between 9 and 12 months of treatment. The data were calculated as a relative expression using the DDOT method. The expression of ribosomal ribosomal acid (rRNA) 18S was used as the constitutive gene and that of the normal liver (HN) as a control reference. Statistical difference *** p < 0.001 compared to HN, ** p < 0.01 compared to the HN, * p < 0.05 compared to 6 weeks, †† p < 0.01 compared to 6 weeks, † p < 0.05 compared to 6, 12 and 18 weeks.

Figure 4 shows a heat map graph (Heatmap) representing the genetic expression of nodules and tumors isolated by laser microdissection and determined with microarrays. Global gene expression is shown using Affymetrix microarrays in RNA obtained from 4-month nodules, 9-month tumors (early HCC) and 17-month tumors (advanced HCC) in rats induced hepatocarcinogenesis using the HR model. Nodules in Negative GGT Remodeling (RN), Positive GGT Removal Nodules (RP) and Persistent GGT Nodules (NP) were analyzed. To the left of the figure are exemplified the injuries identified by their activity GGT On the right, a hierarchical clustering of 937 genes with differential expression (1.5 times change with statistical significance p <0.05) is described on a Heatmap graph. The dendrogram to the center indicates the similarity of the gene expression profile.

The similarity between the different types of nodules and tumors is observed.

The samples with greater similarity between them were the persistent node 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 regions. The Ptgrl gene is included within the gene expression profile that characterizes persistent nodules and tumors.

Figure 5 shows the global gene expression in samples of Gamma Negative Glutamyl Transferase Removal (GGT) (Nod R neg), GGT Positive Removal Nodules (Nod R pos) and Persistent GGT Nodules (Nod P pos) obtained by microdissection and laser capture at 4 months, as well as tumors of 9 months (early HCC) and tumors of 17 months (advanced HCC) in the HR model.

Figure 5A shows a color map chart (Heatmap) that describes the level of expression of Ptgrl and 5 genes previously reported as markers of hepatocarcinogenesis in rats. 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 HCC and advanced HCC, unlike the other analyzed genes that are expressed non-specifically in any type of nodules.

Figure 5B shows the genetic expression of Ptgrl and 5 genes previously reported as markers of hepatocarcinogenesis in rats, in terms of relative intensity. It is observed that Ptgrl is over-expressed only in persistent nodules, early HCC and advanced HCC, unlike the other genes analyzed. The broken line marks the level of normal liver expression, the data correspond to the average and standard deviation. Statistical difference as indicated by 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 of the different stages of development of HCC in the HR model. The over-expression of Ptgrl from persistent nodules is checked, unlike the Gstpl that is also expressed in nodules in remodeling. 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 assays for the detection of PTGR1 in liver samples of the Schiffer model. Photographs are presented with two magnifications 50X and 200X in samples of normal liver (HN), 6, 12 and 18 weeks of treatment with DEN (DEN 6, DEN 12, DEN 18) and a tumor model HR at 12 months of treatment (Tumor) Overexpression of PTGR1 is observed from week 12 of treatment with DEN.

Figure 7 shows the immunohistochemical assays for the detection of PTGR1 and Glipican-3, a marker of hepatocarcinoma in humans, in HCC samples of clinical origin. Representative images of 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 dqmiti (200X) or 25 mm (400X).

DETAILED DESCRIPTION OF THE INVENTION The detection and early diagnosis of a cancer in an individual is a factor that determines the success of the treatment. With the current diagnostic tools, it is not very frequent 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, specific cellular changes occur. The treatments available for HCC, such as resection and percutaneous ablation, are only effective when the development of the disease is detected in the early stages.

Currently, early diagnosis of HCC is based on the detection of liver 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 not conclusive. Histopathological analysis is not a useful tool to confirm the diagnosis of HCC in early stages because the characteristics of dysplastic cells in their initial stages are not evident with these techniques. Molecular tools offer greater certainty in the diagnosis of HCC in the early stages, through the use of biomarkers. The 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 HCC precursor lesions 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 HCC, by identifying dysplastic nodules precursors of HCC development using specific biomarkers that are expressed in early stages and even from previous stages to the establishment of neoplasia and that differentiate the hepatic nodules associated with the development of HCC from those that are not associated with the development of HCC.

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 present cellular atypia, the term "dysplastic nodule" refers to the hepatocyte nodule with signs of dysplasia but without criteria of malignancy; and the term "hepatocarcinoma" or HCC refers to the nodule with cytological and histological atypia with criteria of malignancy.

For the development of the present invention, two experimental models in animals were used to study the development of HCC. One of them is based on a Solt and Farber protocol also known as the resistant hepatocyte (HR) model, with which alterations in HCC were studied from the appearance of hepatocyte nodules to its progression in cancer, over a period of 12 months. months The other model was based on the protocol described by Schiffer and allowed to study the sequential appearance of cirrhosis and tumorigenesis within a period of 18 weeks. The development of hepatocarcinoma in the cirrhotic tissue of this model is similar to the chronology of the pathological events of hepatocarcinogenesis human In both models, results were obtained that confirm the usefulness of prostaglandin reductase 1 (PTGR1) for the early diagnosis of HCC and for the detection of dysplastic nodules precursors of HCC.

In the liver tissue samples, the tumor tissue was identified through its Gamma Glutamyl Transferase (GGT) activity in histological sections, according to the technique described by Rutenburg AM, et al. J Histochem Cytochem 1969; 17: 517-526. For the HR protocol, tumors (T) greater than 5 mm in diameter were separated from the frozen tissue together with the surrounding non-tumor tissue (NT). Through this protocol, in the first month, nodules of hepatocytes up to 1 mm in diameter were induced that were positive for the GGT marker, which together represented 4.8% of the total area of the liver. Some of these nodules reached diameters of up to 3 mm after 5 months and showed increased cell proliferation according to the labeling for the Ki-67 protein.

When the animals were sacrificed between 7 and 9 months, the liver presented HCC 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 by their discolored appearance compared to the non-tumor surrounding tissue.

The livers obtained from the rats treated by the Schiffer protocol were processed with a cryostat. For each section of 16 micras with GGT activity, other sections were collected until obtaining 50 mg of tissue for the extraction of proteins or 30 mg of tissue for the extraction of ribonucleic acid (RNA).

The Schiffer protocol induced multiple nodular lesions positive for GGT. The incidence of these injuries increased in a time-dependent manner; in 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 to the fibrin laminin protein; and in week 18, there was an increase in the proportion of tissue positive for GGT with neoplastic lesions that cumulatively covered up to 80% of the liver. Rats killed at 12 weeks developed cirrhosis (confirmed with H & E) and dysplastic nodules, whereas rats sacrificed at 18 weeks presented HCC within the cirrhotic tissue.

In order to identify the protein expression differences between the hepatic tumor tissue and the normal liver tissue, the proteins were analyzed by polyacrylamide gel electrophoresis with sodium dodecylsulfate (SDS-PAGE). The protein profiles of the normal liver tissue (HN) and those of the non-tumor tissue (NT) were similar to each other, while those of the proteins of the tumor tissue (T) revealed distinct bands, in particular a band of approximately 33 kDa in the tumor tissue (T).

To identify the proteins present in the 33 kDa band, the gel band was excised and processed as previously described in Perez et al., 2010 Proteome Sci 2010; 8: 27. The proteins that were extracted from the gel were processed in a Plus 4800 MALDI-TOF / TOF mass spectrometer (Applied Biosystems, USA) to obtain the MS / MS spectra and the results were analyzed using the 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.

When analyzing the 33 kDa band that was expressed in the samples of tumor tissue but not in those of normal liver tissue, the PTGR1 enzyme was identified (Gene Bank RefSeqGenel 92227 NM_138863.2 NP_620218.1), with a level of confidence > 99% according to the number of concordant peptides (Table 2).

Table 2. Identification of PTGR1 in rat hepatic tumors.

Statistics of peptides identified for PTGR1 8 The position occupied by the protein in the list of proteins identified at 33 kDa according to the ProtScore value. 0 Peptides identified with 99% confidence. c Measure calculated from the evidence of peptides identified according to the confidence value.

A peptide with > 99% confidence contributes 2.0 to ProtScore. d Percentage of amino acids of the peptides identified with respect to the sequence of the protein.

To determine the overexpression of PTGR1 at different stages of CHC development, experiments were carried out using different Molecular techniques, useful to highlight the expression level 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 identification and specific quantification of proteins.

To determine the level of expression or amount of the protein PTGR1 in a given sample can be used any immunoenzymatic technique described in the state of the art, including without limitation: membrane immunodetection (Western blot), immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) in any of its variants, immunofluorescence, immunocytochemistry and immunoprecipitation, among others. Some of the standard techniques applicable to practice the present invention are described, for example, in Green M. R. and Sambrook J., Molecular Cloning. A laboratory Manual, Fourth Edition. Coid Spring Harbor Laboratory Press, 2012 and in Greenfield E.A., Antibodies. A laboratory Manual Coid Spring Harbor Laboratory Press, 2013.

PTGR1 is an enzyme that has an NADPH-dependent alkene / oxa reductase enzymatic activity 2, so the level of expression or 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 including without limiting alpha-beta unsaturated aldehydes and ketones and nitroalkenes in the presence of NADPH. Some of the standard techniques applicable to practice the present invention are described for example in Dick R.A. et al. J Biol Chem., 2001 Nov 2; 276 (44): 40803-10. The level of expression of the Ptgrl gene can be determined by measuring its mRNA. The determination of Ptgrl mRNA can be performed 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 transcription sequencing, among others. Some of the standard techniques applicable to practice the present invention are described, for example, in Green M. R. and Sambrook J., Molecular Cloning. A laboratory Manual, Fourth Edition. Coid Spring Harbor Laboratory Press, 2012.

To determine the levels of the 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 antibody anti-LTB4DH (Abnova) at a dilution of 1: 500 As a marker of hepatocarcinogenesis, the immunodetection of Glutathione S-Transferase P1 (GSTP1) was used, which is recognized as a tumor marker according to that described in Sakai M. and Muramatsu M. Biochem Biophys Res Commun. 2007 Jun 8; 357 (3): 575-8 and b-actin immunodetection was used as load control.

Figure 1A shows the expression of PTGR1 and GSTP1 in samples of the HR protocol; PTGR1 was present exclusively in tumor samples (T) at 8, 9, and 10 months, but was not detected in normal liver samples (HN) or in samples of surrounding non-tumor tissue (NT). In the Schiffer model, over-expression of PTGR1 was observed in samples of rats killed at 12 and 18 weeks (Figure 1B). The GSTP1 found in greater abundance in the tumor tissue (T), but it was also found in the non-tumoral (NT) tissue, probably, due to the presence of altered hepatocytes.

Immunohistochemistry experiments were performed to determine the presence and cellular localization of PTGR1 in tissue sections (Figure 6), using a rabbit anti-PTGR1 polyclonal antibody (Novus Biologicals) at a dilution of 1:25. The primary antibodies were detected using the avidin-biotin technique with the LSAB + HRP Kit (Dako Corporation, California, USA).

As shown in Figure 6, PTGR1 was over-expressed in foci of altered hepatocytes at 6 weeks of treatment with DEN, in foci of hepatocytes and nodules at 12 weeks and in nodules and tumors at 18 weeks. The histological localization of the PTGR1 was in the cytoplasm and in the nucleus of the neoplastic cells in the hepatic tumors.

The results demonstrated the presence of the 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).

To confirm the overexpression of PTGR1 and determine if the protein is in its active form, the enzyme activity 2 alkene / ona-dependent Nicotinamide Adenine Dinucleotide Phosphate (NADPH) (EC 1.3.1.74) 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 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-alkene / ona oxidoreductase activity of the PTGR1 in samples of different times of CHC development. A time-dependent increase between 6 and 18 weeks was detected, presenting the highest enzymatic activity at 18 weeks (15 times higher compared to normal liver tissue). Tumor (T) samples in the HR protocol showed a 25-fold greater enzyme activity compared to normal liver tissue.

According to the above, in one embodiment of the invention 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-alkene / ona oxidoreductase activity of this enzyme through the use of substrates of PTGR1 including without limiting aldehydes and alpha-beta unsaturated ketones and nitroalkenes.

To detect the over-expression of the Ptgrl gene, RT-qPCR experiments (quantitative reverse transcription PCR) were performed to detect the mRNA in samples of different times of CHC development (Figure 3).

From the liver tissue the total RNA was obtained using RNAeasy columns (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 proportions of rRNA 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.

Once the cDNA was obtained, the qPCR reactions were carried out using the TaqMan gene expression assay in a HT 7900 Fast Real Time PCR system (Applied Biosystem, Mexico), using fluorescein-labeled probes (FAM) (limit exon-exon) for rat Gstpl (Rn00561378_gH), for Ptgrl (Rn00593950_m1) and for 18S rRNA (Rn03928990) from Applied Biosystems. The Gstpl and Ptgrl data were normalized against the 18S rRNA gene expression using the DDOT comparative method.

Figure 3 shows the increase in the expression level of the genes Ptgrl and Gstpl as liver tumors occur in models of hepatocarcinogenesis. The Ptgrl gene presented expression levels comparable to the hepatocarcinogenesis marker Gstpl in hepatic tumors. The highest expression of the Ptgrl gene was present in the tumor samples (T), with values up to 200 times higher compared to the samples of normal liver (HN). In tumors of the Schiffer model of rats killed between 12 and 18 weeks, the level of expression of the Ptgrl gene was 17 to 19 times higher than in the HN.

The Solty Farber protocol (HR) allowed to evaluate the evolution of HCC from the appearance of preneoplastic lesions to its progression in cancer. This protocol results in the appearance of two types of preneoplastic nodes 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 a non-uniform staining of the GGT marker, with negative zones (negative R nodes) and positive zones (positive R nodes), while persistent nodules are stained uniformly with GGT (positive P nodes).

To determine the expression of the Ptgrl gene in these different types of nodules and tumors that are generated during the development of HCC, the laser microdissection technique was used, linked to the expression analysis (Figures 4 and 5), as described in Mena JE , et al. Anal Biochem. 2013 Nov 20. pii: S0003-2697 (13) 00551 -4, which allows the tissue of each type of lesion to be precisely separated and the mRNA extracted to carry out gene expression analyzes.

From the samples obtained by laser microdissection, global gene expression was determined in each type of tissue using GeneChip Rat Gene 1.0 ST microRNAs from Affimetrix, which allow measuring the expression of more than 28,000 transcripts in rat according to that described in Mena JE, et al. Anal Biochem. 2013 Nov 20. pii: S0003-2697 (13) 00551 -4. Samples of negative R-nodes, positive R-nodes and positive P-nodules were analyzed, all of them from 4 months of development, as well as tumor samples from 9 months (early HCC), tumors of 17 months (advanced HCC) and normal liver.

The synthesis of complementary DNA (cDNA) using oligo-dT primers was performed from the total RNA, to obtain the double-stranded DNA (dsDNA). With the dsDNA, biotin-labeled complementary RNA (cRNA) was obtained by in vitro transcription, which was purified and hybridized with the GeneChip Rat Gene 1.0 ST microarray of Affimetrix.

The data generated by the image analysis of the microarray was normalized using the Bioconductor software, specialized in the analysis of DNA microarrays (http: bwww.bioconductor.org). The information was integrated into databases with MySQL software. The genes of interest were selected from the statistical analysis, hierarchical clustering analysis with the software Statistical Data Analysis R (http://www.r-project.org/). The gene expression profiles were projected for analysis with annotations of gene expression profiles using GenMAPP software (http://www.genmapp.org/) and Ingenuity Pathways (IPA).

Figure 4 shows the genetic expression of the different types of nodules and tumors that develop in the experimental models, isolated by laser microdissection. Global gene expression was determined using Affymetrix microarrays in RNA obtained from 4-month nodules, 9-month tumors (early HCC) and 17-month tumors (advanced HCC) in rats induced hepatocarcinogenesis using the HR model.

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. Nodules in Negative GGT Remodeling (RN), Positive GGT Removal Nodules (RP) and Persistent GGT Nodules (NP) were analyzed (Figure 4).

We analyzed the data obtained from the microarray 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 graphic with a hierarchical grouping was integrated. The dendrogram (Figure 4) indicates the similarity of the gene expression profile between the different types of nodules and tumors.

According to the data of global gene expression, the persistent nodules and early tumors showed a close similarity, and these in turn maintain similarity with the advanced tumor. On the other hand, the remodeling nodules are similar to each other in their positive and negative nodular regions.

GGT The Ptgrl gene is included within the genetic expression profile that characterizes persistent nodules and tumors and is not found within the genes differentially expressed in the nodules undergoing remodeling.

These data show that persistent nodules are closely related to early and advanced tumors according to their genetic expression, which may indicate that they are precursor nodules of HCC, unlike remodeling nodules that do not have this similarity with Early and advanced CHC. The fact that the Ptgrl gene is It is found to be overexpressed in persistent nodules and early or advanced CHC but not in nodules in remodeling. It is characterized as a useful marker for the early diagnosis of HCC and for the identification of precursor nodules of HCC.

Figure 5A shows a heat map graph (Heatmap) that describes the expression level of the A2m, Gpc3, Fat10, Ggt1, Gstpl and Ptgrl genes in the different stages of HCC development in the experimental model. The first five genes have been reported as markers of hepatocarcinogenesis in rat, as described in French, S.W. Exp Mol Pathol. 2010 Apr; 88 (2): 219-24. The Ptgrl gene is the only one that is over-expressed in persistent nodules and tumors but is not over-expressed in remodeling nodules, unlike the other genes that are over-expressed in any type of nodule or present an inconsistent level of expression with the progression of CHC. 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 gene expression, after normalizing the intensity data of the microarrays. It is also observed that the Ptgrl gene is over-expressed in persistent nodules and tumors, while maintaining levels normal in remodeling nodules, which makes it possible to highlight the persistent nodules that could be precursors of HCC.

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 development of the CHC obtained by microdissection and laser capture. Figure 5C shows the data of the RT-qPCR where the gene expression of Ptgrl and Gstpl was confirmed. It is observed that the Gstpl gene is overexpressed both in nodules in remodeling and in persistent nodules, while the Ptgrl gene is overexpressed only in persistent nodules, thus allowing to detect those nodules that could develop in HCC and therefore perform a early diagnosis of HCC from stages even prior to the establishment of the neoplasm.

Ptgrl gene expression is elevated in persistent type nodules (Nod P Pos), early tumors, and advanced tumors, while remaining low in normal liver tissue and in 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 level of expression of the Ptgrl gene was the only one capable of distinguishing tumors and persistent type nodules from the remodeling type nodules (Nod R). The expression of Gstpl is elevated in both types of preneoplastic nodules, so it is not useful to distinguish persistent nodules that could evolve in cancer.

The results of genetic expression at different stages of CHC development confirm the usefulness of Ptgrl as a marker of early diagnosis 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 its expression is maintained at nl levels in nl liver tissue and in remodeling type nodules.

To determine if the results obtained in the experimental models are extrapolar to other mammals and in particular to human individuals, the expression level of PTGR1 was analyzed in hepatic biopsies included in paraffin and resection samples of 12 cases of HCC of human individuals, by immunohistochemistry (Figure 7). As a control of pathology not associated with cancer, the parenchyma region of a case of hepatic fibrosis was used. All samples were stained with H & E for routine histological diagnosis. According to the histopathological study, the HCC 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 cellular differentiation .

Immunohistochemistry was perfd using a mouse anti-LT4DH polyclonal antibody (Abnova) at a 1:50 dilution. To verify the neoplastic nature of HCC, a specific immunohistochemical reaction for glypican-3 was carried out in serial sections of the same samples. This protein is a known marker for HCC and is not expressed in liver disorders benign For this, an anti-glipican-3 (1G12) mouse monoclonal antibody (Cell Marque) was used at a 1: 100 dilution.

Immunohistochemistry showed that PTGR1 is over-expressed in all HCC samples, while non-cancerous liver tissue showed low intensity of expression (Figure 7). The staining levels were independent of the histological pattern and the degree of anaplasia.

These results confirmed that PTGR1 is over-expressed in tissue sections with HCC, while its levels of expression were negative or basal in non-tumor tissue sections.

Due to its over-expression in dysplastic nodules and hepatic tumor tissue, the gene products of Ptgrl, mRNA or protein can be detected in peripheral blood of individuals with HCC or its derivatives such as serum or plasma. The detection of expression levels of PTGR1 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 peripheral blood, serum or plasma, any technique known in the state of the art can be used to detect circulating mRNA including without limitation RT-PCR, in situ hybridization, hybridization with specific probes of nucleic acids and sequencing of transcribed, among others.

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 limitation assay by enzyme-linked immunosorbent assay (ELISA) in any of its variants, immunofluorescence, and immunoprecipitation, among others.

Overexpression of the PTGR1 protein in peripheral blood, serum or plasma can also be determined by measuring NADPH-dependent alkene / oxa reductase enzymatic activity 2 through reactions with substrates including without limiting alpha-beta aldehydes and ketones unsaturated and nitroalkenes in the presence of NADPH.

Overexpression of PTGR1 in persistent nodules and tumors of animal models and in HCC samples from humans is indicative of an inherent molecular change to hepatocellular malignancies in mammals, so the marker and the The diagnostic methods described in the present application are applicable to different kinds of mammals, in particular to human individuals.

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 young, including humans and animals of veterinary or livestock interest for humans. Some examples of mammals of cattle interest include without limitation the pig, the cow, the horse, the sheep and the goat among others.

Some examples of mammals of veterinary interest include without limiting the dog and the cat among others. Based on the above description, the present invention relates, in one of its aspects, to useful methods for perform the early diagnosis of HCC from stages even prior to the establishment of the neoplasm, by determining the expression level of the Ptgrl gene or the protein for which they encode a biological sample that can be hepatic tissue, peripheral blood, serum or plasma. Said method is applicable to mammals of cattle or veterinary interest and in particular to human individuals with high risk of suffering from HCC.

The methods object of the present invention consist of determining the level of expression of the gene of prostaglandin reductase 1 (Ptgrl) or of the protein for which it codes 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 HCC, where the over-expression of the Ptgrl gene or its protein is indicative that the individual suffers from HCC or that he or she could develop HCC.

For the purpose of the present invention, the term "expression standard" of Ptgrl is understood to mean the average expression level of the Ptgrl gene or of the protein for which it encodes detected in samples of a known population of individuals not suffering from HCC. As used in the description of the present invention, the term "over-expression" refers to the detectable increase in the expression of the Ptgrl gene or the protein for which it encodes or its enzymatic activity in at least 1, 2, 5 , 50 or 100 times more with respect to the expression standard.

For the practice 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.

The level of expression 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. The determination of Ptgrl mRNA can be carried out with molecular techniques that include, but are not limited to: RT-qPCR, in situ hybridization, hybridization with specific nucleic acid probes and transcription sequencing, among others; the 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 including without limitation: aldehydes and alpha-beta unsaturated ketones in the presence of NADPH.

The determination of the expression level of the Ptgrl gene for the early diagnosis of HCC can be carried out in different types of samples including without limitation: samples of fresh liver tissue, frozen liver tissue, enriched nodular tissue, paraffin embedded liver tissue, whole blood , serum or plasma.

In a preferred embodiment of the invention, the expression of the Ptgrl gene can be determined by RT-qPCR in liver tissue samples.

In another preferred embodiment of the invention, 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.

Table 3. Preferred embodiments of the invention Sample Preservation Determination Methods Location Immunohistochemistry hi ló i Immunofluorescence Immunological techniques Protein detection Western blot ELISA Mass spectrometry Freezing RT-PCR in situ hybridization Messenger RNA DNA microarrays RNAseq Enzymatic enzyme Biopsy Enzymatic activity Liver-dependent NADPH reaction Spectrophotometry . Fluorometric Location Histopathological immunohistochemistry Immunofluorescence Chemical fixation e Inclusion in Immunological techniques for a Western blot Protein detection ELISA Mass spectrometry RT-PCR media preservation of messenger in situ hybridization RNA (RNA later, DNA R A save microarrays) RNAseq Enzymatic enzyme NADPH dependent reaction Enzymatic activity Spectrophotometry Fluorometric Serum or plasma Freezing Messenger RNA -j circulating RNA ",. ,,. , J Immunological techniques Protein detection? ° r l Western blot As mentioned above, the present invention is based on the unexpected fact that the Ptgrl gene is differentially overexpressed in dysplastic hepatic nodules, in early HCC and advanced HCC.

Another application of this unexpected fact is to determine if an individual suffering from HCC is a candidate to be treated with bioactivated compounds through the alkene / oxidoreductase activity of PTGR1.

As used in the present description, the term "bioactivation" refers to the metabolic conversion of a chemical compound to 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 / ona oxidoreductase activity of PTGR1 and that generate a more toxic product than the unreduced compound.

By determining the expression level of the Ptgrl gene or the enzyme for which it encodes liver tissue samples from an individual suffering from HCC, it can be defined whether said individual is a candidate to be treated with compounds that are bioactivated by the enzyme PTGR1, due to that said compounds will be activated in dysplastic nodules or tumor tissue that over-express this enzyme, thus generating a selective cytotoxicity.

In the state of the technique, various compounds have been described that can be bioactivated in the organism and that can therefore serve as chemotherapeutic agents. The international patent applications WO / 1991/04754, WO / 1998/03458 and WO / 2007/019308 describe analogues of iludine 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 the hydroxymethylacylfulvene functions as a substrate for the enzyme PTGR1, which catalyzes its reduction towards biologically active intermediates.

Therefore, in another embodiment, the present invention relates to in vitro methods useful for determining whether an individual suffering from HCC is a candidate for treatment with antitumor compounds that are bioactivated by the enzyme PTGR1. Said method comprises detecting some of the gene products (mRNA or protein) of prostaglandin reductase 1 (Ptgrl) in a biological sample previously obtained and comparing the level of expression in said sample against an expression standard of PTGR1, where overexpression of PTGR1 is indicative that the individual is a candidate to be treated with antitumor compounds that are bioactivated by PTGR1.

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 codes can be used.

The level of expression 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. The determination of Ptgrl mRNA can be performed with molecular techniques that include without limitation: RT-qPCR, in situ hybridization, hybridization with specific nucleic acid probes and sequencing of transcribed, among others; PTGR1 protein can be measured with immunoenzymatic techniques including but not limited to: immunohistochemistry, ELISA and its variations, immunofluorescence, immunocytochemistry and immunoprecipitation; and the enzymatic activity of PTGR1 can be measured with substrates including without limitation: aldehydes and alpha-beta unsaturated ketones in the presence of NADPH.

Some of the standard techniques applicable to practice the present invention are described, for example, in Green M. R. and Sambrook J., Molecular Cloning. A laboratory Manual, Fourth Edition. Coid Spring Harbor Laboratory Press, 2012 and in Greenfield E.A., Antibodies. A laboratory Manual Coid Spring Harbor Laboratory Press, 2013.

In a preferred embodiment of the invention, the expression of Ptgrl is determined by measuring its enzymatic activity.

In a preferred embodiment of the invention, the antitumor compounds that are bioactivated by PTGR1 are analogous compounds of iludine 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 carried out. These diagnostic equipment comprise at least one method to determine the level of expression of Ptgrl in a biological sample and a standard of comparison of the expression level of Ptgrl. In this sense, the diagnostic kits comprise means to determine either the mRNA, the protein or the enzymatic activity of the PTGR1. As used in the present invention, the means containing the diagnostic equipment described in the present application refers to any type of laboratory reagent or medical reagent that can be used to detect the overexpression of the Ptgrl gene or the protein for the one that encodes or the enzymatic activity of the PTGR1 enzyme. These means may include without limitation polyclonal antibodies, monoclonal antibodies, humanized antibodies, specific probes, substrates specific for PTGR1, and laboratory reagents necessary to perform standard techniques for the detection of mRNAs such as RT-qPCR, in situ hybridization, hybridization with specific probes of nucleic acids and sequencing of transcripts, 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 This invention is further illustrated by the following examples, which are not considered in any sense as limitations imposed on the scope thereof. On the contrary, these are to clearly understand that you can resort to several other modalities, modifications and equivalents of them.

EXAMPLES EXAMPLE 1 Animal models for the development of hepatocarcinogenesis.

Development of animal models of hepatocarcinogenesis.

Two models of experimental hepatocarcinogenesis in rat were used. The first one 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 alternative 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 cirrhosis induced with diethylnitrosamine (DEN).

For each model, 20 Fischer 344 male rats were used between 180g and 200g of weight. The animals obtained from the Laboratory Animal Production and Experimentation Unit of the CINVESTAV Ciudad de México (UPEAL-Cinvestav), were treated in accordance with the guidelines and institutional protocols for the management of experimental animals. The rats were maintained with daily light / dark cycles of 12 h and controlled temperature, with food (basic diet) and water sterilized ad libitum.

The modified protocol of Solt and Farber consisted of a single intraperitoneal 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 hepatectomy of two thirds of the liver tissue on day 10 after the start of the protocol. 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.

For the Schiffer protocol, the rats received intraperitoneal injections of 50 mg / kg of DEN weekly. The number of injections was 16. Experimental rats were sacrificed at 6, 12, and 18 weeks to collect the liver tissue.

An additional group of adult rats did not receive any treatment and was sacrificed in parallel to the experimental group to be used as a control group where the liver was normal (HN).

The sacrifice of the animals was carried out by exsanguination under ether anesthesia. From each animal, 5 ml of blood was obtained by cardiac puncture and the liver tissue was embedded in 2-methyl butane as cryoprotectant, frozen with liquid nitrogen and stored at -70 ° C for its conservation, thus forming a tissue bank for the different essays.

In the livers obtained from the modified Solt and Farber protocol, 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 greater than 5 mm in diameter were separated from the surrounding non-tumoral tissue with a scalpel.

The livers obtained from the rats treated by the Schiffer protocol were processed with a cryostat. For each 16-micron section with GGT activity, another 10 sections were collected to obtain 50 mg of tissue for protein extraction or 30 mg of tissue for RNA extraction.

EXAMPLE 2 Identification of PTGR1 in tumors by mass spectrometry. 50 mg of the liver tissue samples were homogenized in 1 ml of lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 0.25% Na-deoxycholate, 1 mM EDTA and 1X cocktail of protease inhibitors (ProteoBlock, Fermentas). The total protein was quantified by the Lowry method (RC assay of DC proteins, BioRad), the samples were prepared in 5X Laemmly buffer. 30 ug of protein extract were separated by 10% SDS-PAGE. The gels were stained with colloidal Coomassie blue G-250 (Bio-Safe, Bio-Rad Laboratories, USA) and the images of the gels were analyzed by band densitometry with ImageJ software.

The differential bands were excised from the gels and processed for the identification of proteins according to what was previously described in Perez et al., 2010 Proteome Sci 2010; 8: 27. The gel fragments were destained twice with 500 ml of a 50 mM solution of ammonium bicarbonate / 50% (v / v) acetonitrile at 50 ° C for 5 minutes, then dehydrated with 100 pl 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. For the extraction of peptides a 0.5% (v / v) solution of 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 ml of 0.1% TFA in 50% acetonitrile.

Protein identification was performed on a Plus 4800 MALDI-TOF / TOF mass spectrometer (Applied Biosystems, USA). The MS / MS spectra were analyzed using the 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.

EXAMPLE 3 Immunodetection of proteins by Western blot.

The hepatic protein extract (30 pg) of each sample were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore) by electroblotting. The membranes were blocked with 5% skimmed milk and 0.1% Tween 20 in 1X Tris buffer solution (TBS) for 1 hr at room temperature. The membranes were incubated overnight at 4 ° C with a mouse anti-LTB4DH polyclonal antibody (Abnova) at a 1: 500 dilution.

After washing, the membranes were incubated for 1 h with a secondary anti-mouse antibody conjugated with peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA). The chemiluminescence was obtained by adding the chemiluminescent substrate, luminol, Immobilon Western (Millipore) and the images were obtained with a digital image system VersaDoc MP 5000 (BioRad). The same membranes served for immunodetection with an anti-GSTP1 antibody at a 1: 1,000 dilution (Sigma Aldrich) followed by an anti-actin mouse monoclonal antibody (Chemicon International).

EXAMPLE 4 Activity 2 alkennal / ona oxidoreductase of PTGR1 in tumors.

The NADPH-dependent alkene / oxidoreductase activity 2 of PTGR1 was determined by continuously measuring the oxidation of NADPH 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 trans-2-nonenal, 0.1 mM NADPH and 0.2 mg / ml protein extract in 50 mM sodium phosphate / potassium buffer, 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; the ethanol concentration in the final reaction was 0.2%. Protein extracts from the liver were obtained in RIPA buffer with a protease inhibitor cocktail.

The NADPH oxidation rate was monitored at 340 nm for 10 minutes in 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 of protein.

EXAMPLE 5 Gene expression of Ptgrl in hepatic tumors.

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). is described in Mena JE, et al. Anal Biochem. 2013 Nov 20. pii: S0003-2697 (13) 00551 -4.

The tissue of interest was captured with a lid (CapSure Macro LCM Caps, Applied Biosystems) that becomes adhesive by means of infrared laser pulses. The optimal laser settings for the LCM of liver tissue were 80 mW of power, 8,000 microseconds duration and an intensity of 100 mV. He Cutting laser was adjusted to produce a point 2 microns in diameter at a power of 20 mW. The covers with the tissue were placed in a GeneAmp tube (Applied Biosystems, N8010611) containing 320 ml of RLT lysis buffer (Qiagen, RNeasy mini kit) supplemented with 1% (v / v) of b-mercaptoethanol.

From the tissue of interest obtained by LCM the total RNA was extracted by the isolation method in RNAeasy columns (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 proportions of RNAr28S / 18S > 1.7.

The cDNA reactions were carried out with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) employing from 150 to 750 ng of total RNA. For the 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). Probes labeled with Fluorescein (FAM) (limit exon-exon) for rat Gstpl (Rn00561378_gH), Ptgrl (Rn00593950_m1) and rRNA 18S (Rn03928990) were obtained from Applied Biosystems. The Gstpl and Ptgrl data were normalized against the 18S rRNA gene expression using the comparative method AACt.

EXAMPLE 6 Analysis of gene expression with microarrets.

From 200 ng of total RNA the first strand of cDNA was obtained using Superscript II reverse transcriptase and oligo-dT primers. The second cDNA chain was synthesized. The cRNA was obtained by in vitro transcription and used as a template for a second cycle of cDNA synthesis with the incorporation of dUTP. The obtained cDNA was fragmented using uracil-DNA glycosylase. Fragments (40-70mers) were labeled with biotin by a terminal addition reaction of deoxynucleotide.

The labeled cDNA was hybridized in the rat 1.0 Gene microarray ST (Affymetrix Inc.) for 17 hours at 45 ° C. Samples were washed with low stringency (SSPE) and high stringency buffer solutions (100 mM MES, 0.1 M NaCl) 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 the Expression Consolé software (Affymetrix, Inc.) to obtain the intensity and quality signals of the scanned microarrays. The one-way ANOVA was used to compare the differences in gene expression and RNA integrity between the samples obtained by CML. Statistical significance was established in p < 0.05.

EXAMPLE 7 Immunohistochemical detection of PTGR1 in tumors The histological sections of rat hepatic tissue were incubated with a polyclonal anti-PTGR1 rabbit antibody (Novus Biologicals) at a dilution of 1:25 and a polyclonal anti-GSTP rabbit antibody (Dako) at a 1: 100 dilution. The 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 substituted with the mouse isotype control.

EXAMPLE 8 Detection of PTGR1 in clinical samples of CHC Hepatic biopsies included in paraffin and resection samples were obtained from 12 cases of HCC (6 women, 6 men, mean age 54.4 years, between 17-70 years of age) from the Cancerology Center of the State of Veracruz, Mexico. As a control of pathology not associated with cancer, the parenchyma region of a case of hepatic fibrosis was used. All samples were stained with hematoxylin-eosin (H &E) for routine histological diagnosis. According to the histopathological study, the HCC samples were grouped according to the stage of the tumor and the degree of cellular differentiation: well differentiated (n = 2), moderately differentiated (n = 6) and poorly differentiated (n = 4).

From the hepatic biopsies embedded in paraffin, serial sections of 5 μm thickness 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 buffer solution of sodium citrate (10 mM sodium citrate and 0.05% Tween 20, pH 6.0) (Dako Corporation, California, USA) fixing the serial sections with cold acetone.

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 anti-LT4DH mouse antibody (anti-Ptgrl-Abnova) at a dilution 1:50 or a mouse monoclonal antibody anti-glipican-3 (1G12) (Cell Marque) at a 1: 100 dilution.

It should be understood that the foregoing relates only to preferred embodiments of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims

NOVELTY OF THE INVENTION CLAIMS

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 level of expression of the Ptgrl gene or the protein for which it encodes a previously obtained biological sample and ( b) comparing the level of expression in said sample against a standard of expression of the Ptgrl gene or of the protein for which it encodes, wherein the overexpression of the Ptgrl gene or of the protein for which it encodes is indicative that the individual suffers from CHC or that you may develop CHC.

2. - The method according to claim 1, further characterized in that the level of expression of Ptgrl is determined by the measurement of its mRNA.

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 probes of nucleic acids and sequencing of transcripts.

4. - The method according to claim 1, further characterized in that the level of expression of Ptgrl is determined by measuring the PTGR1 protein.

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. - The method according to claim 1, further characterized in that the level of expression of Ptgrl is determined by measuring the enzymatic activity of the enzyme PTGR1.

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 alpha-beta unsaturated aldehydes and ketones or nitroalkenes in the presence of NADPH.

8. - The method according to any of claims 1 to 7, further characterized in that the level of expression of Ptgrl is determined in a sample of liver tissue.

9. - The method according to any of claims 1 to 7, further characterized in that the level of expression of PTGR1 is determined in a sample of serum, plasma or total peripheral blood.

10. - The method according to any of claims 1 to 9, further characterized in that the mammal is a human.

11. - 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. - 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 the protein for which it codes in a previously obtained biological sample and (b) compare the level of expression in said sample against a standard of expression of the Ptgrl gene or of the protein for which it encodes, wherein the overexpression of the Ptgrl gene or of the protein for which it encodes is indicative that the mammal is a candidate to be treated with compounds that are bioactivated by PTGR1.

13. - The method according to claim 12 further characterized in that the compound that is bioactivated by the PTGR1 is selected from the group comprising the derivatives of iludine, derivatives of acylfulvenes or nitroalkenes.

14. - The method according to any of claims 12 and 13 further characterized in that the level of expression of Ptgrl is determined by measuring the enzymatic activity of PTGR1.

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. - The method according to any of claims 12 and 13, further characterized in that the level of expression of Ptgrl is determined by the measurement of its mRNA.

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 probes of nucleic acids and sequencing of transcripts.

18. - The method according to any of claims 12 and 13, further characterized in that the level of expression of Ptgrl is determined by measuring the protein PTGR1.

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. - The method according to any of claims 12 to 19, further characterized in that the level of expression of Ptgrl is determined in a sample of liver tissue.

21. - The method according to any of claims 12 to 19, further characterized in that the level of expression of Ptgrl is determined in a sample of serum, plasma or total peripheral blood.

22. - The method according to any of claims 12 to 21, further characterized in that the mammal is a human.

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. - A diagnostic equipment useful for the early diagnosis of hepatocellular carcinoma in a mammal characterized in that it comprises at least: a) means for determining the expression level of the Ptgrl gene or the protein for which it codes in a biological sample and b) a standard of expression of the Ptgrl gene or of the protein for which it codes to compare the level of expression.

25. - The kit 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 to determine the Ptgrl mRNA by any of RT-PCR, in situ hybridization, hybridization with specific nucleic acid probes and transcript sequencing.

26. - The kit of claim 24 further characterized in that the means for determining the expression level of the Ptgrl gene or the protein for which it encodes are necessary means to determine the PTGR1 protein by means of any immunohistochemistry, ELISA and its variants, immunofluorescence, immunocytochemistry and immunoprecipitation.

27. - The equipment of claim 24 further characterized in that the means for determining the expression level of the Ptgrl gene or the protein for the encoding are necessary means to determine the enzymatic activity of PTGR1 by any reaction with aldehydes and alpha-beta unsaturated ketones or nitroalkenes in the presence of NADPH.

28. - The equipment according to any of claims 24 to 27, further characterized in that the level of expression of the Ptgrl gene or the protein for which it encodes is determined in a sample of liver tissue.

29. The equipment according to any of claims 24 to 27, further characterized in that the level of expression of the Ptgrl gene or the protein for which it encodes is determined in a sample of serum, plasma or total peripheral blood.

30. - The equipment according to any of claims 24 to 29, further characterized in that the mammal is a human.

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.