WO2015056195A1

WO2015056195A1 - Use of microrna markers for diagnosis of liver lesions

Info

Publication number: WO2015056195A1
Application number: PCT/IB2014/065342
Authority: WO
Inventors: Krystian JAZDZEWSKI; Kinga DYMECKA; Anna WOJCICKA; Anna KUBIAK; Wojciech GIERLIKOWSKI; Monika MACIAG; Monika KOLANOWSKA; Agnieszka CZAJKA; Marta KOTLAREK; Michal Swierniak
Original assignee: Warszawski Uniwersytet Medyczny
Priority date: 2013-10-15
Filing date: 2014-10-15
Publication date: 2015-04-23
Also published as: PL405648A1

Abstract

The present invention relates to a method for diagnosing liver cancer and distinguishing the hepatocellular carcinoma form colon cancer metastasis to the liver in a patient. The said method comprises: - providing a biological sample from a patient, - determining the amount of one or more miRNAs selected from the group consisting of miR-146a-5p, miR-125b-5p, miR-141-3p, miR-1269 in a biological sample, - making a comparison of the miRNA expression level from a patient to a control from the control group, in which method the patient is diagnosed for cancer and the origin of the cancer is identified, if the level of miRNA expression in the biological sample is altered relative to the expression level observed in samples of biological fluids from healthy patients. The invention also relates to the use of miRNA marker and to diagnostic kit containing markers.

Description

USE OF MICRORNA MARKERS FOR DIAGNOSIS OF LIVER LESIONS

Field of the invention

The present invention relates to the method of diagnosing hepatocellular carcinoma, the use of a microRNA marker for diagnosis of liver lesions, for assessment of disease staging and assessment of patient and/or disease susceptibility to the proposed treatment, as well as the diagnostic kit containing such markers.

Background of the Invention

Liver tumors are among the most commonly diagnosed neoplasms. The most frequently encountered liver pathologies include hepatocellular carcinoma and colorectal cancer (CRC) metastasis to the liver. Hepatocellular carcinoma (HCC) is the fifth most common cancer in humans. In Europe, every year it causes between 1.3 and 5.8 deaths per 100 000 inhabitants (Llovet, J. M., et al. (2003). "Hepatocellular carcinoma." The Lancet 362 (9399): 1907; Point, D. M. , et al. (2005). "Global Cancer Statistics, 2002." CA Cancer J. Clin. 55(2): 74-108). Every year, 500 000 patients worldwide die of HCC, and the number increases. (El-Serag, H. B. et al (1999). "Rising incidence of hepatocellular carcinoma in the United States." New England Journal Of Medicine 340(10): 745-750; Parkin, D. M., et al (2005). "Global Cancer Statistics, 2002." CA Cancer J. Clin. 55(2): 74- 108). At the same time, colorectal cancer (CRC, colorectal carcinoma) is the third most common cancer in the world and it is expected that by 2030. 2.2 million patients per year will develop this type of cancer (World Cancer Research Fund, http ://w w w . wcrf . or /) . Liver metastases are the most common distant lesions associated with CRC (Marshall L. (2008) "Managing Potentially Resectable Metastatic Colon Cancer"). As the early detection of the disease is difficult and the therapy is frequently ineffective, the prognosis for patients with liver cancer is rather poor. Surgical treatment, resection or liver transplantation remain the only possible options that give hope for cure (Mazzaferro, V., et al. (1996). "Liver Transplantation for the Treatment of Small Hepatocellular Carcinomas in Patients with Cirrhosis." N. Engl. J. Med. 334(11): 693-700). However even then, despite the progress of liver surgery and perioperative care, relapse is observed in over 75% of patients within five years of successful tumor resection. More importantly, hepatocellular carcinoma often develops in the cirrhotic liver. Since the large organ imaging is not an easy task, diagnosing the neoplastic lesions in the early stages of their development is often impossible, and thus mortality of patients increases significantly.

For the above reasons, for many years research has been conducted to establish methods of providing early diagnosis to prevent the development of hepatocellular carcinoma, as well as to diagnose and distinguish HCC from other tumors of the liver. In this context, many attempts have been made to lower the risk stratification of hepatocellular carcinoma recurrence. It has been proposed, inter alia, to use the clinical factors such as the level of liver dysfunction, age, tumor size, degree of atypia, vascular invasion, portal vein thrombosis and the presence of micro satellite foci. Unfortunately, none of the diagnostic factors listed above has gained wider recognition (Thorgeirsson, SS (2006) "Genomic decoding of Hepatocellular carcinoma", Gastroenterology 131 (4): 1344- 6). Significantly better results were obtained by using molecular biomarkers, in particular the analysis of gene expression (Thorgeirsson, SS, et al. (2006). "Molecular prognostication of liver cancer: End of the Beginning." Journal of Hepatology 44 (4): 798). Many efforts have been made to develop a set of biomarkers to enable an early detection and allow for risk assessment of the post-surgery tumor recurrence. The microarray studies of the entire genome have identified a set of genes that are good prognostic indicators of a prolonged survival and less frequent appearance of metastatic tumors (Poon, TCW, et al. (2006). "A Tumor Progression Model for Hepatocellular Carcinoma: Bioinformatic Analysis of Genomic Data." Gastroenterology 131 (4): 1262-1270). However, the proposed tissue analyses have one serious limitation that is the gene expression analysis of tissue samples can be performed only after the tumor resection, and therefore this method is not applicable as a preoperative diagnostic assay. The list of genes whose aberrant expression was observed in several cancers, including liver and colon cancer, includes microRNAs. MicroRNAs (miRNAs) are short, approx. 22-nucleotide (e.g. 19-23 nucleotides) RNA molecules, which do not encode proteins themselves, but regulate expression (transcription, translation) of other genes by binding to a specific nucleotide sequence in the 3' untranslated region (3' UTR, 3' untranslated region) of the target mRNA, thereby inhibiting translation in ribosomal complexes or leading to degradation of the transcript. Due to incomplete complementarity between the respective target sequences, one microRNA has the ability to control the expression of hundreds or even thousands of mRNAs. It is estimated that more than half of the expression of protein-coding genes is regulated by miRNA molecules. What is important, the expression of miRNA strictly depends on the tissue type and the developmental stage of the organism, which enables the tight regulation of gene expression depending on the actual cellular requirement for their protein products. Currently, there are over 1000 miRNA sequences deposited in the microRNA repository database (http ://microrna. Sanger . ac .uk) . This large number of sequences identified so far indicates a more complex role of miRNA in the regulation of biological processes than hitherto confirmed.

However, miRNA expression is disrupted in numerous diseases, including many cancers. The high specificity of expression profiles makes microRNAs an important diagnostic tools, allowing for assessment of the severity of a disease or for monitoring the response to therapies. Importantly, cancer cells secrete microRNA to the blood, from which it can be isolated and characterized (Chen X., et al., Characterization of miRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases, Cell Research 2008. 18: 997-1006). As miRNA molecules are short, they are relatively resistant to endonucleases and thus can be detected and measured in peripheral blood. The changes of microRNA expression in tumor cell can be observed in blood. This fact is of great importance for the genetic diagnostic applications, as it enables the development of specific and non-invasive diagnostic tests that are based solely on changes observed in patients' blood. It has been known for several years that miRNA expression is frequently deregulated in solid tumors and can be used for the diagnostic purpose (Croce, CM, et al. (2005). "miRNAs, cancer, and stem cell division." Cell 122 (1): 6 -7). The correlation between specific genes (mRNA) or expressed proteins and resistance or susceptibility of tumor samples to the proposed treatment was mentioned in US 2004/0214203 and US 2006/0160114. For example, U.S. Patent US 2008/0076674 and WO 2008/046911 disclose the relation between the presence of some miRNAs and the presence and/or the risk of breast cancer. WO 2008/154098 describes a number of miRNAs whose expression shows differences between samples from tumor and these from healthy tissues. The WO 2009/080437 description discloses a method of sample analysis, which involves identification and analysis of miRNA profiles, particularly in samples from the affected tumor tissue, in order to determine the phenotype of the patient or the disease, especially cancer, with regard to the resistance or susceptibility of the patient or the disease to therapy, especially cancer therapy. Patent WO 2007/081720 description discloses a method for the diagnosis, prognosis and treatment of lung cancer based on the miRNA levels measured in tissues. This method consists of measurements of the amount of at least one microRNA in a test sample and comparison of the result to the control sample, followed by the estimation of lung cancer risk.

Although research on solid tissue samples has been widely carried out over the last decade, the use of miRNA expression in patient's plasma for cancer diagnosis has become the subject of studies in a few recent years. For example, the description of WO 2009/143379 relates to a method of detection, classification, diagnosis, prognosis and/or monitoring of the response to treatment of a disease or disorder, and the method is based on determining the quantity, in particular the changes of miRNA levels in a biological sample comprising the sample of physiological fluids. However, it does not relate to any specific disease markers, especially cancer markers. Some of the early work on the determination of the plasma levels of specific miRNA that are related to the occurrence of malignancies has been conducted by the group of Professor Lawrie (Lawrie CH, et al., Detection of elevated levels of tumor-associated miRNAs in Serum of Patients with diffuse large B -cell lymphoma, British Journal of Hematology, 2008 141, 672-675). The authors performed an analysis of the selected miRNAs in the plasma of 60 patients suffering from Diffuse Large B Cell Lymphoma (DLBCL) and in the plasma of 43 healthy controls. The studies showed that the plasma levels miR-155, miR-210 and miR-21 are elevated in cancer patients compared to the levels observed in healthy subjects. Subsequent studies have shown that miRNA miR-92 is significantly elevated in the plasma of patients with acute myeloid leukemias (Tanaka M. et al., Down-regulation of miR-92 in human plasma is a novel marker for acute leukemia Patients, PLoS ONE 2009 4 (5): 5532-5537), and the level of miR-21, miR-92 and miR-93 enables early detection of ovarian cancer (Resnick, A., et al., 2009). Mitchell et al. (Mitchell, PS, et al. (2008). "Circulating miRNAs as stable blood-based markers for cancer detection." Proceedings of the National Academy of Sciences 105 (30): 10513-10518.) demonstrated that miR-141 may be used as a marker (indicator) to identify cancer patients with 60% sensitivity and 100% specificity. WO 2009/015357 (EP-2181332B 1) discloses methods for diagnosis and prognosis of cancer and its impact on adverse pregnancy outcomes by determining the amount of one or more miRNAs from exosomes associated with cancer or adverse pregnancy outcomes. WO 2010/145035 describes miRNAs that can be applied as markers to detect and monitor the treatment of patients with kidney cancer. Another patent WO 2011/080315 provides specific miRNAs that are associated with prostate cancer.

Still, no miRNAs whose aberrant expression could serve as a prognostic marker for hepatocellular carcinoma have yet been described. Moreover there are no microRNAs described that could be used for the diagnosis of hepatocellular carcinoma, and allow for differentiation between HCC and colon cancer metastases to the liver, based on measurements in biological sample comprising the physiological fluids.

Previous studies have shown that miRNAs are frequently deregulated in liver tumors and can be used to distinguish HCC from benign tumors (Murakami, Y., et al. (2005). "Comprehensive analysis of miRNA expression patterns in hepatocellular carcinoma and non- tumorous tissues. "Oncogene 25 (17): 2537; Ladeiro, Y., et al. (2008). "The miRNA profiling in hepatocellular tumors is associated with clinical features and oncogene / tumor suppressor gene mutations." Hepatology 47 (6): 1955-1963, but the results of these studies did not even make a suggestion towards a high specificity of such assays, nor did they mention the possibility to use them in the analysis of biological fluids.

It is known that three of the already known miRNAs (miR-224, miR-122a, miR- 422a) have altered expression in all liver tumors, other two are specific for hepatocellular adenomas (miR-200c, miR-203), and another two for HCC (miR-lOb, miR-222). On the other hand, WO 2009/156507 describes a method to establish a treatment regimen for subjects suffering from a viral infection of the liver, and the method uses the fact that the miR-122 level is reduced in relation to the control group, indicating the lack of susceptibility of a subject to antiviral therapy. In addition, expression of miR-125b appears to be connected to the successful outcome of HCC (Wenxi Li 2008).

All the above studies analyzed miRNA expression in tumor tissue, but none of the previously disclosed methods referred to the diagnostic use of miRs based on the level of these genes in peripheral blood plasma. Although miRNAs appear particularly useful for characterization of liver tumors and tumor progress prediction, their concentration in the peripheral blood or other tissue fluids is not known, and their use in plasma-based diagnostic and prognostic test for HCC has been never considered.

At the same time it is known CRC is accompanied by a decreased expression of numerous microRNAs, particularly miR-21 miR-31, miR-17-3p, miR-17-5p (Rohr et al. (2013) "High-throughput miRNA and mRNA sequencing of paired colorectal normal, tumor and metastasis tissues and Bioinformatic modeling of miRNA- 1 therapeutic applications "PLoS One. 2013 Jul 2; 8 (7): e67461, Sche et al. (2013)" Deep Sequencing of the MicroRNA transcriptome in colorectal Cancer "PLoS One. 2013 Jun 18; 8 (6): e66165). Based on the expression of microRNAs it is thus possible to differentiate between healthy and cancerous tissue. Studies have also demonstrated that the abovementioned microRNA can be determined in the blood of patients with CRC and therefore can be used as marker to distinguish CRC patients from the group of healthy individuals. Applicability of the microRNA expression level estimation to diagnose CRC metastases in other tissues has not been studied to date. The summary of the invention

The present inventors have surprisingly found that the expression level of one or more miRNA selected from miR-146a-5p, miR-125b-5p, miR-141-3p, and miR-1269a, preferably selected from miR-146a-5p, miR-125b-5p, and miR- 1269a, and most preferably selected from miR-146a-5p and miR-125b-5p in a tissue, and in biological fluids, such as plasma of peripheral blood, urine, exudates, biopsy washings or intercellular fluids, allows for unequivocal diagnosis of neoplastic lesions in the liver, differentiation, as well as the assessment of response to treatment of such changes. Use of a set of markers provided by the present invention allows for the development of specific and highly sensitive assay that is useful for the diagnosis of hepatocellular carcinoma and metastatic colorectal cancer (CRC) to the liver.

Determination of expression levels of specific microRNAs has been carried out using well-known hybridization methods, such as Taqman probes, diagnostic arrays made on glass, plastic or gold-covered surface, comprising microRNA set of miR-146a-5p and miR-125b-5p, optionally supplemented with additional sets using miR-141-3p, miR-1269a. Application of such test provides a unique opportunity to distinguish patients with hepatocellular carcinoma from healthy individuals, without the need for any invasive and thus significant risk related treatments, for example, the necessity of collecting a tissue sample from a patient, as well as allows to distinguish patients with liver cirrhosis from the patients in whom the tumorigenic process was initiated on the basis of cirrhotic liver, as well as patients with hepatocellular carcinoma (HCC) from patients with colorectal cancer (CRC) metastases to the liver. The present invention provides the possibility of a noninvasive monitoring of the health status of a patient at risk of liver cancer (HCC), and provides the possibility to diagnose malignancy at an early stage, therefore allowing for a very significant reduction of mortality caused by the tumor.

The present invention relates to a method allowing for diagnosis of liver cancer and for distinguishing hepatocellular carcinoma from colon cancer metastasis to the liver. The said method comprises:

- collecting of the biological sample from a patient, - determining the amount of one or more miRNAs selected from the group consisting of miR-146a-5p, miR-125b-5p, miR-141-3p, miR-1269a in a biological sample,

- comparing the patient's miRNA expression level to the control group,

in which method the patient is diagnosed for tumor type and the origin of tumor if the level of miRNA expression in the biological sample is altered relative to the expression level observed in samples of biological fluids from healthy subjects. Preferably, the biological sample is a biological fluid. In a preferred embodiment a sample of the biological fluid is a whole blood sample, serum, plasma, urine, saliva, lymph, tears, pleural effusion, mucus ascites fluid, derived from the respiratory system such as bronchial secretions, amniotic fluid, cerebrospinal fluid, milk fluid derived from cysts such as, for example, ovarian cysts or any other liquid from the tissue, including ascites, and particularly preferably blood plasma.

Preferably, the miRNA is selected from the group comprising miR-146a-5p, miR- 125b-5p and miR- 1269a, and most preferably from the group comprising miR-146a-5p and miR-125b-5p.

According to one preferred aspect of the present invention, the amount of at least two miRNAs is determined at the same time, particularly the amount of miR-141-3p, miR- 1269a.

Preferably, the patient is a human.

The invention also relates to the use of a miRNA marker to diagnose lesion in the liver, to evaluate the degree of the disease severity and assessment of the susceptibility of the patient and/or disease to the proposed treatment. The said marker:

- is isolated from a patient's biological sample

- the concentration of the marker in the sample is determined,

- the methods of miRNA quantification are used to measure microRNA expression in the samples,

- the result of microRNA expression is analyzed, wherein the microRNA marker is selected from the group comprising miR-146a-5p, miR-125b-5p, miR-141-3p, miR-1269a and is characterized by altered expression level compared to the control group, indicating a high risk of lesion in the liver.

Preferably, the marker sequence is selected from the group comprising sequences of microRNA molecules deposited in miRBase database (http://www.mirbase.org): miR- 146a-5p, miR-125b-5p, miR-141-3p, miR-1269a.

The liver lesion is a tumor, preferably selected from the group consisting of hepatocellular carcinoma (HCC), metastatic colon cancer to the liver (CRCM), and focal nodular hyperplasia (FNH).

In another preferred embodiment of the present invention, the expression profile of miR-146a and/or miR-125b is a marker for the differentiation degree and malignancy, and the lowered level of miR-146a is a marker of tumor progression toward a more malignant stage.

Preferably, the expression profile of miR-146a and miR-125b is a marker of the type of liver tumor, and the said tumor is selected from the group consisting of hepatocellular carcinoma (HCC), liver metastasis of colon cancer (CRCM), and focal nodular hyperplasia (FNH).

Preferably, the expression profile of the miR-141-3p is a marker of liver tumor type, and the said tumor is selected from the group consisting of hepatocellular carcinoma (HCC) and metastasis of colon cancer to the liver.

Preferably, the expression profile of the miR- 1269a is a marker of hepatocellular carcinoma (HCC).

According to another preferred embodiment of the invention, diversified expression of microRNA miR-146a and miR-125b in body fluids is a marker for liver cirrhosis. Preferably, the present invention also refers to the application where a change in miR- 146a expression is a marker for patient and/or liver lesions susceptibility to the proposed treatment, in particular preferably cirrhosis. The invention also provides a diagnostic kit for the assessment of diagnosis of a lesion in the liver, assessment of disease staging and assessment of patient and/or disease susceptibility to the proposed treatment, comprising: a. at least one "forward" amplification primer

b. at least one "reverse" amplification primer

c. geno typing probe

wherein the primers are designed in such a way that they enable specific amplification of the nucleotide sequences of miR-146a-5p, miR-125b-5p, miR-141-3p, miR- 1269a, allowing for determination of the amount of selected miRNA in a biological sample.

The biological sample is a biological fluid, preferably such as whole blood, serum, plasma, urine, saliva, lymph, tears, pleural effusion, mucus ascites fluid, derived from the respiratory system such as bronchial secretions, amniotic fluid, cerebrospinal fluid, milk, liquid from cysts such as, for example, ovarian cysts or any other liquid from the tissue, including ascites, particularly preferably blood plasma.

According to a preferred embodiment of the invention, the miRNA is selected from the group consisting of miR-146a-5p, and miR-125b-5p.

Preferably, the kit allows for simultaneous measurement of the amount of the at least two miRNAs, in particular preferably miR-141-3p and miR-1269. The term "microRNA" or "miRNA" or "miR" refers to small, non-coding RNA sequences containing from 15 to 30 nucleotides that can be determined in a biological sample. As used herein, the term "biological sample" refers to any sample comprising bio-molecules, and/or a sample from a patient. Examples of bio-molecules comprise DNA, RNA, including mRNA and miRNA, or protein. Examples of biological samples are any cells, groups of cell fragments from patients, which may be used in the practice of the present invention. Such samples may be tissue samples of both solid as well as biological fluids.

The term "biological fluid" refers to any fluid derived from a patient. Such fluids include, but are not limited to, blood and its fractions, serum, plasma, urine, saliva, lymph, tears, pleural effusion, mucus, ascites fluid, fluid from the respiratory system such as bronchial secretions, amniotic fluid, cerebrospinal fluid, milk, liquid from cysts, such as, for example, ovarian cysts, or any other liquid from the tissue, including ascites.

The term "tumor" or "neoplasm" refers to or describes the condition of the patient, particularly a human, which is associated with the irregular growth of cells and includes both "benign neoplasm", described as hyperproliferation of cells, incapable of forming an aggressive, metastasizing tumor in vivo, and "malignant neoplasm" or "cancer" characterized by cells with multiple cellular and biochemical anomalies which are capable of forming a systemic disease, creating, for example, tumor metastasis to distant organs. Examples of cancer include, but are not limited to, hepatocellular carcinoma, metastatic colon cancer to the liver, and the like.

The present invention is illustrated in the following figures, where: Fig. 1 shows a variable microRNA expression in tumor samples (T) and control (N), both in HCC (upper panel - microRNAs with increased expression in the tumor, middle panel - microRNAs with reduced expression in tumor) and the CRC metastasis to the liver (lower panel). The graphs show microRNAs with high expression in tissue samples and a significantly altered expression in tumor relative to control tissue.

Fig. 2 shows a dilution curve for the tested microRNA. As shown in the figure, the expression of miR-122 and miR-lOa in plasma is 18-fold lower than the expression of miR-93 reference gene (the difference in Ct = 9 cycles)

Fig. 3 shows differential expression of microRNA miR-125b and miR-146a in patients with liver cirrhosis and in patients without liver cirrhosis, Mann -Whitney U test, p = 6x10- 6 and p = 1x10-9, respectively. The charts show the median, first and third quartile, and minimum and maximum values.

Fig. 4 shows differential expression of miR-146a in patients with liver cirrhosis and in patients without liver cirrhosis, t-test, p = 0.012. The graphs show the mean, standard error and 95% confidence interval.

Fig. 5 shows the expression of miR-146a in the tissue fluid 125b (in this study: blood plasma) of patients with hepatocellular carcinoma (HCC) with tumor grade Gl, G2 or G3 (p = 0.047). The charts show the median, first and third quartile, and minimum and maximum value

Fig. 6 shows the analysis of the expression of miR-125b and miR-146a in the blood plasma of patients with hepatocellular carcinoma (HCC), metastatic colon cancer to the liver (CRCM), and focal nodular hyperplasia (FNH) and tissue fluid of healthy volunteers. Fig. 7 shows the number of patients (dark gray) or healthy volunteers (light gray), (in percentage), in which the analyzed microRNA, miR-125b or miR-146a is expressed in the tissue fluid (in this study: blood plasma)

Fig. 8 shows the number of patients with liver cirrhosis (light gray) or patients with liver cirrhosis and HCC (dark gray), (in percentage), in which the analyzed microRNA, miR- 125b or miR-146a is expressed in the tissue fluid (in this study: blood plasma)

The invention is illustrated in more detail in the examples of practical embodiment, which are provided for illustrative purposes without limiting the scope of the invention as defined in the appended claims. The background of the invention is the measurement of expression of at least one of the aforementioned markers (microRNA). The expression level is compared to the value for the control group and the specific values are correlated with the high or the low risk of disease. Inventors of the present invention revealed that the increased expression of particular microRNA is the marker sensu stricto of the high risk of occurrence of the liver tumor.

Examples

Characteristics of the diagnostic panel used in the studies.

To confirm the proposed diagnosis method and the markers, the expression of selected markers has been performed in biological samples derived from 434 patients, including:

- 103 patients with primary liver tumors, including 82 patients with malignant neoplasm (hepatocellular carcinoma, n = 48, intrahepatic cholangiocarcinoma, n = 34) and 21 patients with benign tumors (haemangioma cavernosum, n = 9, focal nodular hyperplasia, FNH, n = 12)

-209 patients with metastasis of colorectal cancer to the liver, n = 209,

- 91 patients with metastases of other cancers, and

- 122 healthy patients without identified cancerous changes. Tests were conducted using a biological fluid, which was peripheral blood, and in addition, for patients who underwent surgery, using solid-tissue samples obtained during liver resection.

Determination of effective methods for the isolation of plasma microRNA

MicroRNA expression analysis in serum is an extremely demanding and time- consuming task. Commonly used methods for the isolation of microRNAs, such as TRIzol, do not allow the isolation of microRNAs with sufficient quantity and purity. Therefore, the inventors have developed a method for the efficient isolation of microRNA. After testing of various commercially available methods and kits for isolation of RNA, microRNA isolation from plasma was carried out using the mirVana Paris Kit from Invitrogen, according to the protocol for the isolation of RNA from tissues and liquid cultures. RNA was isolated from 500 μΐ of plasma.

Identification of microRNA expression changes characteristic for hepatocellular carcinoma

MicroRNA expression measurements were carried out by standard procedures using a reverse transcription kit and Taqman probes from LifeTechnologies. The reverse transcription reaction was performed in a total volume of lOul with 2ul of RNA template isolated from blood plasma under the following conditions: 16 ° C-30 min, 42 ° C-50 min, 85 ° C-5 min, 4 ° C-∞. Amplification reaction was performed in a total volume of 10 ul using 1.6 ul of the reverse transcription reaction product.

The results confirmed that the process of liver carcinogenesis is accompanied by a decrease in expression of numerous microRNAs, including miR-146a-5p (0.74x), miR- 10a-5p (0.27x), miR-200a-3p (0.21x ), miR-200b-3p (0.21x), miR-199a-3p (0.19x), miR- 199a-5p (0.17x), miR-30a-5p (0.51x), miR-125b- 5p (0.81x), miR-127-3p (0.49x), miR- 142-5p (0.49x) (Fig. 1, top panel). At the same time, the inventors observed a significant increase in the expression of microRNA miR-1269 (99.9x), miR-183-5p (5.6x), miR-224- 5p in tumor tissue. (3.9x), miR-452-5p (3.75x), miR-182-5p (3.6x), miR-10b-5p (2.92x), miR-222-3p (1.88x) miR-221-3p (1.87x), miR-32-5p (1.8x) (Fig. 1, middle panel). The analyses also showed significant differences in microRNA expression between the CRC metastasis to liver and surrounding liver tissue. MicroRNAs of the highest expression included miR-552 (186x), miR-196a-5p (161x), miR-196b-5p (102x), miR-135b (65x), miR-141-3p (62x), miR-200a-5p (42x), miR-147b (39x), miR-429 (21x), miR-1246 (38x), miR-183-5p (29x), miR-34c-5p (26.5x), miR-200b-5p (24.5x).

Expression analysis of the set of microRNAs in the serum of patients with liver tumors and in control groups

The expression of microRNAs was measured. Measurements of the expression were performed as described above. The results were compared with the results of the prior art, inter alia, indicating the important role of the expression level of miR-125b in conditioning the outcome of hepatocellular carcinoma and the role of polymorphism in the miR-146a in the development of genetic predisposition to the onset and development of cancer. For the studies aimed at identification of liver tumor- specific microRNA alterations in serum, miR-146a-5p, miR-125b-5p, miR-141-3p, miR-1269 were chosen, due to their relatively high expression levels in tissue, allowing for correct measurement, and due to the difference in their expression between tumor and control tissue. The selected microRNAs were subjected to further analysis. The results were normalized and the proper comparative assessment of markers was performed.

Selection of the appropriate reference gene

Comparison of gene expression between different samples requires accurate normalization of their expression that equalizes the differences resulting from variable amounts of available template, i.e. the starting amount of RNA in the sample, allowing for comparison of the results obtained in various systems. The concentration of microRNA isolated from a single sample of plasma is extremely low, about 10 ng / μΐ. Such low concentration values limit the possibility of accurate and precise spectrophotometric determination, even with the use of specifically dedicated spectrophotometers such as the Nanodrop. Therefore, for a reliable and objective comparison of expression of the selected microRNAs, the present inventors carried out the search to identify the reference gene, which would allow for normalization of the results of microRNA expression analysis. The transcript of such gene should be detectable in serum and its expression should not be altered in liver pathologies.

MicroRNA expression is most commonly normalized to the expression of small nucleolar RNA genes, such as U66 or U43, due to their stable expression, which does not depend on the ongoing disease process in a given tissue. However, the measurements performed using specific Taqman probes revealed that the expression of these genes is undetectable in plasma. The genes routinely used as references for measuring mRNA expression such as HPRT, TBP or GUSB, proved to be indeterminable either.

Based on the literature data, two microRNAs: miR-191 and miR-93, and small nucleolar RNA U44 were chosen, as their presence in serum is known in the art, for example from the publication de Kok JB et al. (2005) "Normalization of gene expression measurements in tumor tissues: comparison of 13 Endogenous control genes "Lab Invest. Jan; 85 (1): 154-9. The genes expression was measured in the plasma of patients with HCC, metastatic colon cancer to liver and in plasma collected from healthy volunteers. The studies allowed for identifying a molecule that exhibits the least variable expression between the groups. Analysis of the results carried out in the NormFinder demonstrated that miR-93 shows the highest stability of serum levels (stability value = 0.093). Therefore miR-93 was used for normalization and for further analysis of the results obtained for the tested microRNAs.

Analysis of expression of selected microRNAs in serum

In the first step, the use of RNA and cDNA dilution curves (standard curves)allowed for verification of amplification efficiency of the selected microRNAs, allowing to perform accurate measurements of their expression. The data are shown in Fig. 2. The analysis demonstrated that despite their significant over-expression in the liver tissue, the expression of miR-122 and miR-lOa in the plasma is very low. Therefore it is not possible to measure the amount of these molecules with appropriate accuracy.

Then the expression of miR-125b and miR-146a was analyzed in the plasma of patients with hepatocellular carcinoma (HCC), metastatic colon cancer to the liver (CRCM), and focal nodular hyperplasia (FNH) and in the plasma of healthy volunteers. This analysis revealed a high diversity of microRNA expression depending on the ongoing disease process.

Based on these studies, the present inventors much to their surprise have found that the expression profile of miR-125b and miR-146a in the plasma enables a clear distinction between patients with cirrhosis and the patients who do not suffer from this disease. As mentioned above, cirrhosis is one of the main factors of pathogenesis of HCC. Liver cirrhosis is an important factor that increases the risk of developing hepatocellular carcinoma, even up to 10% in case of decompensated HBV caused cirrhosis and up to 5% in case of liver cirrhosis caused by hepatitis C virus. Therefore, it is vital to develop methods for monitoring the changes in liver tissue that are causative factor of its fibrosis.

The present inventors have found that the differential expression levels of microRNA miR-146a and miR-125b in body tissue allow for distinguishing patients with liver cirrhosis from patients without the diagnosed cirrhosis, irrespective of the presence of a tumor or tumor type. The results of the analysis are shown in Fig. 3.

Further studies have shown that the expression of miR-146a is statistically significantly elevated in tissue fluid, such as blood plasma, in patients with hepatocellular carcinoma and cirrhosis, irrespective of tumor stage (n = 17) compared to patients without liver cirrhosis (n = 21). The differential expression of miR-146a in patients with liver cirrhosis and in patients without liver cirrhosis is illustrated in Fig. 4.

Based on the obtained results it was also found that the expression profile of miR- 146a allows for distinguishing tumors with the lowest grade from less differentiated tumors. Furthermore, the studies have demonstrated that the expression of both microRNA miR-146a and miR-125b in the tissue fluid, such as blood plasma, is varied, and depends on the degree of differentiation and malignancy. The classification was based on WHO guidelines, according to which Gl indicates the presence of well-differentiated cells in tumor, G2 - intermediately differentiated (transitional stage), G3 - poorly differentiated. Tumors classified as Gl are characterized by the lowest degree of malignancy.

Based on the performed studies it has been also demonstrated that patients with hepatocellular carcinoma classified according to the WHO rank as Gl are characterized by statistically significantly higher expression of miR-146a compared to patients classified as G2 and G3 (Kruskall-Wallis Kruskal-Wallis one-way analysis of variance). The results shown in Fig. 5 confirm this conclusion.

According to the performed studies, down-regulation of miR-146a indicates the progression of tumor toward a more malignant stage Thus, the measurements of the expression of miR-146a and its changes enable prediction of the course of hepatocellular carcinoma, and also allow for determination of the susceptibility of the patient and/or tumor to the proposed treatment.

Furthermore, it was surprisingly found that the profile of the expression of miR- 146a and miR-125b enables the discrimination between patients with various liver tumors. As illustrated in Fig. 6, the expression of miR-125b and miR-146a is different in the plasma of patients with hepatocellular carcinoma (HCC), metastatic colon cancer to the liver (CRCM), focal nodular hyperplasia (FNH) and healthy volunteers.

Development of a diagnostic test for the detection of liver tumors

Based on the expression profile of miR-146a and miR-125b in plasma, a sensitive and specific diagnostic test, allowing for early detection of liver tumors, was developed. The test involves determination of microRNA expression using specific primers and probes complementary to the microRNA sequence. Studies were conducted using tissue fluid (such as blood plasma) collected from 328 patients, including 206 patients with various liver and biliary tract tumors: hepatocellular carcinoma (HCC, n = 36), cholangiocarcinoma (CCC, n = 30) gallbladder cancer (GC, n = 16), metastatic colon cancer to the liver (CRC, n = 124) and metastasis of other tumors ("Other", n = 37) and from 122 healthy volunteers. In all cases, expression levels of miR-146a and miR-125b were determined independently.

Statistical analysis was performed using Statistica Software. Normal distribution was tested using the Shapiro-Wilk and comparisons between groups were performed using the Kruskall - Wallis test. Analysis of the results showed that miR-125b expression profile allows to distinguish patients with hepatocellular carcinoma (HCC) from patients with metastatic colorectal cancer to the liver (CRCM) (p = 0.017), patients with cholangiocarcinoma (CCC) from gallbladder cancer patients (GC) (p = 1.6x10-9) and CRCM patients from the control group (p = 4.7x10-11). For the group of patients pointed out above, the expression levels of miR-125b were determined using samples of biological fluid. The obtained results are shown in Table 1.

Table 1. Expression profile of microRNA miR-125b in patients with hepatocellular carcinoma (HCC), metastatic colorectal cancer to the liver (CRCM) (p = 0.017), cholangiocarcinoma (CCC), other cancers (Other) (p = 0.03), and the control group (control) (p = 4.7x10-11).

The studies on the miR-125b showed that miR-125b expression profile enables to differentiate patients with hepatocellular carcinoma (HCC) from patients with metastatic colorectal cancer to the liver (CRCM) (p = 0.017), patients with cholangiocarcinoma (CCC) from patients with other malignancies (Other) (p = 0.03) and CRCM patients from the control group (Control) (p = 4.7x10-11). Similarly as described above for miR-125b, for the indicated group of patients miR-146a expression levels were determined in biological fluid samples. The obtained results are shown in Table 2

Table 2. microRNA miR-146a expression profile in patients with hepatocellular carcinoma (HCC), metastatic colorectal cancer to the liver (CRCM) (p = 0.017), cholangiocarcinoma (CCC), other cancers (Other) (p = 0.014 ) and control group (Control)

The studies clearly indicate that the profile of expression of microRNA miR-146a allows to distinguish patients with CRCM (p = 1x10-6) from patients with metastases of other tumors (p = 0.014), and from patients in the control group. Further, the inventors have found that miR-146a and miR-125b expression profiles together allow to distinguish patients with CRCM from the control group (p <0.0001). As shown in Fig. 7 the expression levels of microRNA miR-125b and miR-146a allow for precise distinguishing patients with liver tumors from healthy volunteers. Furthermore, simultaneous analysis of expression of both microRNAs and application of a cut-off value equal to 0.024 for miR- 125b and 0.7 for miR-146a (2-ACt value normalized to the expression of miR-93) results in obtaining of a diagnostic test with a sensitivity of 90%, positive predictive value PPV) of 75% and negative predictive value NPV of 63%. These results confirm that microRNA expression measurements allow for development of a diagnostic panel that can be used as a tool to distinguish patients with liver tumors from healthy subjects.

In addition, the expression profiles of microRNAs miR-146a and miR-125b allow distinguishing patients with cirrhotic liver from patients with cirrhotic liver and hepatocellular carcinoma. The devised test, measuring the biomarkers expression level would be a non-invasive tool to identify the moment at which liver cancer starts developing in cirrhotic liver. The inventors performed a study illustrating how the assay works and provided the data to confirm its usability as shown in Fig. 8.

The obtained results show that simultaneous analysis of expression of both microRNAs and the application of a cut-off value of 0.05 for miR-125b and 0.5 for miR- 146a (2-ACt value normalized to the expression of miR-93) results in diagnostic test of specificity of 100%, positive predictive value (PPV) of 95% and negative predictive value (NPV) of 100%. The results show that the diagnostic panel based on the measurement of the expression of microRNA miR-125b and miR-146a allows distinguishing patients with liver cirrhosis from patients in whom liver cirrhosis progressed towards hepatocellular carcinoma. Thus, the tool gives opportunity to monitor and diagnose this tumor, otherwise difficult to detect in diagnostic imaging at an early stage.

Claims

1. A method of diagnosing hepatocellular carcinoma and distinguishing hepatocellular carcinoma form colon cancer metastasis to the liver in a patient, which method comprises: providing a biological sample from a patient,

determining the amount of one or more miRNAs selected from the group comprising miR-146a-5p, miR-125b-5p, miR-141-3p, miR-1269a in a biological sample, comparing the miRNA expression level to the control group,

in which method the patient is diagnosed for tumor type and the origin of tumor if the level of miRNA expression in the biological sample is altered relative to the expression level observed in samples of biological fluids from healthy subjects.

2. The method according to claim 1, characterized in that the amount of one or more miRNA selected from the group comprising miR-146a-5p, miR-125b-5p and miR- 1269a is determined.

3. The method according to claim 1, characterized in that the amount of one or more miRNA selected from the group comprising miR-146a-5p and miR-125b-5p is determined.

4. The method according to any of claims 1-3, characterized in that the biological sample is a biological fluid.

5. The method according to claim 4, characterized in that the sample of biological fluid is whole blood, serum, plasma, urine, saliva, lymph, tears, pleural effusion, mucus ascites fluid, derived from the respiratory system such as bronchial secretions, amniotic fluid, cerebrospinal fluid, milk, liquid from the cyst, such as, for example, ovarian cysts or any other liquid from the tissue, including ascites.

6. The method according to claim 5, characterized in that the biological fluid sample is blood plasma.

7. The method according to any one of claims. 1-6, characterized in that the amount of at least two miRNA is determined simultaneously.

8. The method according to claim 7, characterized in that the amount of miR-141-3p and miR- 1269a is determined simultaneously.

9. The method according to any one of claims 1-7, characterized in that the patient is a human.

10. Use of a miRNA marker for diagnosis of a lesion in the liver, assessment of disease staging and assessment of patient and/or disease susceptibility to the proposed treatment which marker

- is isolated from a patient's biological sample

- the concentration of the marker in the sample is determined,

-the amount of miRNA is analyzed with quantitative methods of measuring the

microRNAs' expression in the samples with the use of specific primers for reverse transcription, and primers and probes for the amplification or by hybridization to specific probes

- the outcome of microRNA expression result is analyzed,

characterized in that the microRNA marker is selected from the group comprising miR-146a-5p, miR-125b-5p, miR-141-3p, miR-1269a and is characterized by altered expression level compared to the control group, indicating a high risk of lesion in the liver.

12. Use according to claim 11, characterized in that the marker sequence is selected from the group comprising sequences of microRNA molecules deposited in miRBase database under the number of miR-146a-5p and miR-125b-5p.

13. Use according to any of claims 10-12, characterized in that the lesion in the liver is a tumor.

14. Use according to claim 13, characterized in that the tumor is selected from the group consisting of hepatocellular carcinoma (HCC), metastatic colon cancer to the liver

(CRCM), focal nodular hyperplasia (FNH) and metastases of other cancers to the liver.

15. Use according to any of claims 10-14, characterized in that the profile of expression of miR- 146a and/or the profile of the expression of miR- 125b is a marker for degree of differentiation and malignancy of tumor.

16. Use according to any of claims 10-15, characterized in that the reduction of the miR- 146a expression is a marker of tumor progression toward a more malignant state.

17. Use according to any of claims 10-15, characterized in that the profile of expression of miR-146a and miR-125b is a type of liver tumor marker, the said tumor is selected from the group comprising hepatocellular carcinoma (HCC), metastatic colon cancer to the liver (CRCM), and focal nodular hyperplasia (FNH).

18. Use according to any of claims 10-14, characterized in that the profile of expression of miR-141-3p is a marker of liver tumor type, the said tumor is selected from the group comprising hepatocellular carcinoma (HCC) and metastasis of colon cancer to the liver.

19. Use according to any of claims 10-14, characterized in that the profile of expression of miR-1269a is a marker of hepatocellular carcinoma (HCC).

20. Use according to any of claims 10-12, characterized in that the altered expression of microRNA miR-146a and miR-125b in a body fluid is a marker for liver cirrhosis.

21. Use according to any of claims 10-20, characterized in that the change in the expression of miR-146a is a marker of the susceptibility of the patient and/or lesions in the liver to the proposed treatment.

22. Use according to claim 21, characterized in that the lesion in the liver is cirrhosis.

23. A diagnostic kit for the assessment of diagnosis of a lesion in the liver, assessment of disease stage and assessment of patient and/or disease susceptibility to the proposed treatment comprising:

a. at least one "forward" amplification primer

b. at least one "reverse" amplification primer

c. probe for the detection of microRNA or immobilized probe for the detection of microRNA,

wherein the primers are designed in such a way that they enable specific amplification of the nucleotide sequences of miR-146a-5p, miR-125b-5p, miR-141-3p, miR-1269a, allowing for determination of the amount of one of selected miRNA in a biological sample.

24. A diagnostic kit according to claim 23, characterized in that the biological sample is a biological fluid.

25. A diagnostic kit according to claim 24, characterized in that the sample of biological fluid is whole blood, serum, plasma, urine, saliva, lymph, tears, pleural effusion, mucus ascites fluid, derived from the respiratory system such as bronchial secretions, amniotic fluid, cerebrospinal fluid, milk, liquid from cysts such as, for example, ovarian cysts or any other liquid from the tissue, including ascites.

26. A diagnostic kit according to claim 25, characterized in that the biological fluid sample is blood plasma.

27. A diagnostic kit according to any one of claims 23-26, characterized in that the miRNA is selected from the group comprising miR-146a-5p, miR-125b-5p and miR- 1269a.

28. A diagnostic kit according to any one of claims 23-27, characterized in that the miRNA is selected from the group comprising miR-146a-5p and miR-125b-5p.

29. A diagnostic kit according to any one of claims 23-28, characterized in that the amount of at least two miRNAs is measured simultaneously.

30. A diagnostic kit according to claim 29, characterized in that the amount of miR-141- 3p, and miR- 1269a is measured simultaneously.