WO2015071876A2 - Use of microrna markers for diagnosis of thyroid tumors and a diagnostic panel containing such markers. - Google Patents

Abstract

The present invention relates to a method for diagnosing thyroid cancer and benign thyroid lesion in a patient in vitro. The said method comprises: - providing a biological sample collected from a patient, - determining the amount of one or more miRNAs selected from the group consisting of miR-146b-5p, miR-146b-3p, miR-22l-5p, miR-221-3p, miR-222-5p, miR-222- 3p, miR-181a-5p and miR-182-5p in a biolgical sample - comparing the obtained microRNA expression level to the control group, in which method the patient is diagnosed for cancer or/and benign lesion 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. The invention also relates to the medical use of abovementioned miRNAs and of diagnostic kits allowing for determination of miRNAs in a biological sample.

Description

Use of microRNA markers for dia nosis of thyroid tumors and a diagnostic panel containing such markers.

Field of the nvention

The present invention relates to the method of diagnosing thyroid cancer and benign thyroid tumors, the use of microRNA marker for diagnosis of thyroid tumor, 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

Malignant thyroid tumors - papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), anaplastic thyroid carcinoma (ATC) and medullary thyroid carcinoma (MTC), of which the most common form, accounting for 85% of all cases, is PTC, constitute a group of cancers with relatively low mortality rates. Early detection allows for successful treatment of patients and for their return to social functioning. It thus seems vitally important to pay particular attention to this group of cancers as their proper management gives high probability of a complete success of therapeutic procedures. Currently used treatment methods include chirurgical resection of the thyroid gland and regional lymph nodes followed by treatment with radioactive iodide (1311).

The number of cancers diagnosed in Poland is constantly growing. Based on the data provided by the National Cancer Registry (Pol. Kr jowy Rejestr Nowotworow, KRN , the number of diagnosed thyroid cancers amounted to 2192 cases in 2010, placing the thyroid cancer on the 23.25^th position among cancers diagnosed in men, and ΙΟ^-Π* 2

position in women. Although, as already mentioned, the aggressiveness and mortality rates of thyroid cancers are believed to be relatively low, the data provided by the Polish Ministry of Health and National Cancer Registry are alarming. In 2009, thyroid cancer was the 10^th cause of cancer-related death among men and I5^m in women and indicate that in contrast to the Western countries, the thyroid cancer - related mortality is still high in Poland (..Cancer in Poland in 2009", ISSN 0867-8251). This situation is caused by the fact that patients are diagnosed in too late and high clinical stages of cancer.

Thyroid cancers show a familial heritability pattern, indicating the existence of genetic predisposition to this cancer (Frich L et al., 2001 Familial occurrence of nonmedullary thyroid cancer a population-based study of 5673 first-degree relatives of thyroid cancer patients from Norway. Cancer Epidemiol Biomarkers Prev 10:113-117). In consequence, the search for molecular markers allowing for estimation of the risk for thyroid cancer and for its diagnosis is constantly on-going. To date, no genetic alterations (mutations) that uniequivocally predispose to thyroid cancer have been identified (Gudmundsson J et al., 2012 Discovery of common variants associated with low TSH levels and thyroid cancer risk. Nat Genet 44:319-322; Gudmundsson J et al., 2009 Common variants on 9q22.33 and 14ql3.3 predispose to thyroid cancer in European populations. Nat Genet 41:460-464; Jendrzejewski J et al, 2012 The polymorphism «944289 predisposes to papillary thyroid carcinoma through a large intergenic noncoding RNA gene of tumor suppressor type. Proc Natl Acad Sci U S A 109:8646-8651) and this fact switched the attention of scientists to the novel class of regulatory genes - microRNAs - whose aberrances might lead to development of cancer.

MicroRNAs (miRNAs, miRs) are short, non-coding RNAs that regulate the expression of protein coding genes binding to complementary sequences in their transcripts and inhibiting further steps of protein synthesis (Bartel DP 2009 MicroRNAs: Target 3

Recognition and Regulatory Functions. Ceil 136:215-233). Increased expression of a microRNA leads to its increased binding with target transcript, resulting in significant downregulation of gene expression. Increased levels of miRs are commonly observed in cancers (Croce, CM. and G.A. Calin, miRNAs, cancer, and stem cell division. Cell, 2005. 122(1); p. 6-7.), leading to deregulation of numerous genes and serious aberrances in numerous cellular processess. It is estimated that the expression of approx 50% of protein- coding genes is regulated by microRNAs. Known miRNAs are deposited in a publicly available repository - miRBase - that is constantly updated with newly discovered microRNAs (Griffiths-Jones, S., miRBase: the microRNA sequence database. Methods Mol Biol, 2006. 342: p. 129-38). The expression of microRNAs is highly tissue-specific. Due to this fact, many of the molecules deposited in miRBase will not be present in the analyzed tissue, which, on the other hand, might express microRNAs that have not yet been identified and deposited in miRBase. For this reason, a comprehensive and reliable study on the influence of microRNAs on gene expression requires information on the complete miRNome of analyzed tissue, and microRNA profiles of most tissue types and their pathologies have not yet been elucidated.

Numerous studies indicate that the use of microRNAs in cancer diagnostics has a great potential, steinming mainly from the fact that microRNA expression profiles are highly informative (He H, Jazdzewski K, et al., 2005 The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci U S A 102: 19075-19080, Lu J et al, 2005 MicroRNA expression profiles classify human cancers. Nature 435:834-838; Baxbarotto E et al., 2008 MicroRNAs and cancer: profile, profile, profile. Int J Cancer 122:969-977). Expression of microRNAs is highly tissue-specific and is altered in numerous pathologies, thus microRNA expression profiles strongly correlate with cancer type and stage of the disease. A relatively small number of microRNAs provides more diagnostic information 4

than a higher number of analyzed mRNAs. As such, microRNA expression can be useful in identification of cancers that cannot be properly classified using histological methods. What is additionally important, changes in microRNA expression are observed at early stages of disease development, therefore their analysis allows for detection of cancer at the initiation stage.

Moreover, cancer cells produce microvesicles that contain their DNA, RNA and proteins. Such microvesicles are secreted to blood, from which they can be easily isolated and determined (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). Since microRNAs are short molecules of approx. 22 nucleotides, they are resistant to endonucleolytic cleavage, what allows for their identification and quantification in blood. If expression of a microRNA is increased in cancer, this increase should also be observed in blood. This phenomenon is particularly important from the perspective of genetic diagnostics, as it allows for elaboration of specific diagnostic tests performed solely in blood. Such tests have already been proposed for numerous cancers, including pancreatic, breast and gastric cancer (Quo et al., Genome-wide screen for aberrantly expressed miRNAs reveals miRNA profile signature in breast cancer Mol Biol Rep. 2013 Mar;40(3):2175-86 ; Liu R et al., 2012 Serum microRNA expression profile as a biomarker in the diagnosis and prognosis of pancreatic cancer. Clin Chem. Mar;58(3):610- 8; Corur A et al., 2012 Determination of plasma microRNA for early detection of gastric cancer. Mol Biol Rep. 2013 Mar,40(3);2091-6). Since microRNA expression is altered at early stages of carcinogenesis, (heir analysis in blood allows for noninvasive monitoring of people with increased risk of cancer development. Early detection of cancer would allow for prompt therapeutic intervention, increasing the chances for complete and successful treatment of the patient. 5

The phenomenon of microRNA deregulation in solid tumors has been known for several years (Croce, C. M. et al. (2005). "miRNAs, cancer, and stem cell division." Cell 122(1): 6-7). The correlation between particular genes (mRNAs) or expressed proteins, and resistance or susceptibility of cancer specimens to the proposed treatment was suggested in patent applications US 2004/0214203 and US 2006/0160114. For example, the descriptions US 2008/0076674 and WO 2008/046911 disclose the relation between 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 derived from tumor and healthy tissue. The WO 2009/080437 description discloses a method of sample analysis, particularly of tissue samples affected by tumor, in order to determine the phenotype of the patient or the disease, especially tumor, with regard to the resistance or susceptibility of the patient or the disease to therapy, especially cancer therapy, which involves identification and analysis of miR A profiles. The WO 2007/081720 patent description discloses a method for the diagnosis, prognosis and treatment of lung cancer based on the miRNA levels measured in tissues. This method consists in measurement 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 fine-needle aspiration biopsies or the patient's plasma has become the subject of studies in a few recent years. For example, the description WO 2009/143379 relates to a method of detection, classification, diagnosis,

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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 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 of mi -155, miR-210 and rniR-21 are elevated in cancer patients compared to the levels observed in healthy subjects. Subsequent studies have shown that 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 20094 (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 an indicator of cancer patients with 60% sensitivity and 100% specificity. WO 2009/015357 (EP- 2181332B1) 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 description WO 2011/080315 provides specific miRNAs that are associated with prostate cancer.

Still, no miRNAs whose aberrant expression could serve as a marker of thyroid cancer and that could be used for elaboration of sensitive and specific test for the diagnosis of thyroid cancer in biological samples comprising biological fluids or fine-needle aspiration biopsy specimens, have been identified to date. Previous studies revealed deregulation of a number of miRs in thyroid carcinogenesis, indicating their potential use in differentiating between PTC and normal thyroid tissue (He H, Jazdzewski K_» et al., 2005 The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci U S A 102:19075-19080; Swiemiak M et al In-depth characterization of the microRNA transcriptome in normal thyroid and papillary thyroid carcinoma. J Clin Endocrinol Metab, 2013 Aug;98(8):E1401-9; Chen YT, Kitabayashi N, Zhou XK, Fahey TJ 3rd, Scognamiglio T. MicroRNA analysis as a potential diagnostic tool for papillary thyroid 7

carcinoma. Mod Pathol. 2008;21:1139-1146). Other studies have shown the usefulness of miRNAs for classification of other lesions in thyroid gland, including follicular thyroid carcinoma (FTC) ( ikiforova MN, Tseng GC, Steward D, Diorio D, Nikiforov YE. MicroRNA expression profiling of thyroid tumors: biological significance and diagnostic utility. J Clin Endocrinol Metab. 2008;93:1600-1608; Weber F, Teresi RE, Broelsch CE, Frilling A, Eng C. A limited set of human MicroRNA is deregulated in follicular thyroid carcinoma. J Clin Endocrinol Metab. 2006;91:3384-359). However, neither of the abovementioned studies analyzed the possible use of microRNAs for differentiation between particular types of thyroid cancer and benign tumors (FTC vs PTC vs MTC vs ATC vs adenoma vs MNG). The results of these studies do not suggest high specificity of such measurements and do not estimate the possibility of their use in the analysis of biological fluids.

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 thyroid tumors and the prediction of disease progress, their concentration in the peripheral blood or other tissue fluids is not known, and their use in plasma-based diagnostic and prognostic test for thyroid tumors has been never considered. The use of microRNAs for diagnosis of thyroid lesions was proposed in WO 2012/009499, but this use is limited solely to the use of miR-122 for differentiation between benign tumors and follicular thyroid carcinoma (FTC) in material derived from intraoperative tissue resection or fine needle aspiration biopsy.

Background of the invention

In 2006, a multicentre study was performed in Poland aimed at verification of repeatability, reliability and incompatibility of histopathological diagnosis of thyroid cancer (Lange D, Sporny S, Sygut J, ulig A, Jarzab M, Kula D, Jarzab B. Histopathological diagnosis of thyroid cancer in a multicenter trial, Endokrynologia Polska 2006 Jul-Aug;57(4):336-42). This examination involved evaluation of 690 specimens by 40 pathologists from 25 centers in Poland; the specimens have been previously evaluated 8

by four experts in the above field. The examination showed that more than 21% of all diagnoses given by the physicians participating in the examination were inconsistent with the reference diagnosis. Furthermore, at more than 33% of the examined specimens, the diagnosis of 15% of pathologists differed from the reference diagnosis, and in case of 10% of pathologists, the percentage of non-compliance diagnoses reached 50%. The most common discrepancy was classifying benign lesions as malignant. Therefore, taking into consideration that 2192 patients were diagnosed with thyroid cancer in 2010 (National Cancer Registry), 460 patients could have undergone thyroid resection only due to incorrect diagnosis. Ensuring the proper diagnostics, leading to reduction in the percentage of resections of benign lesions, and allowing for removal of malignant lesions in their early stages that will allow avoiding the removal of the whole thyroid gland, will have a significant impact on the daily operations of the patients and their quick and complete recovery. An initial diagnosis of thyroid cancer is carried out based on the ultrasounds examination, underlying the decision on the performance of the fine-needle aspiration biopsy. During the biopsy, small tissue fragment is collected and is subsequently subjected to histopathological examination. The serious impediment in the proper assessment of biopsy material is the fact that the collection of tissue often results in its destruction. In addition, despite of the fact that puncture is performed under ultrasounds guidance, it is very difficult to collect the material directly from the tumor, the consequence of which is a false-negative result of histopathological examination.

It should also be mentioned mat in the case of thyroid cancer, except for the papillary carcinoma (PTC), the diagnostic criteria are not clear, and the assessment of specimens itself is technically difficult, further adversely affecting its quality. For example, the microscopic image of well-differentiated follicular carcinoma (FTC) resembles the benign follicular adenoma and the distinction of these two alternations is often impossible without examination of the intra-operative tissue specimen. On the other hand, medullary thyroid carcinoma (MTC) is so infrequent that gaining experience in diagnosing of mis cancer type requires long experience in a large endocrine oncology center. 9

Taking the above into consideration, there is an indisputable demand for the development of method and an applicable test allowing not only for the early detection of tumorous changes in the thyroid gland, but also for their differentiation and estimation of a proper treatment. The possibility of applying the molecular diagnostic panel, which, regardless of the place of collection of the analyzed material allows the detection of tumorigenesis within the thyroid gland, will have very important clinical implications, enabling the detection of tumor at a very early stage and ensuring the reliability of the result. Molecular diagnostic tests will be a perfect complement to the diagnosis of the pathologist, allowing for the verification of the obtained results and minimizing the risk of incorrect classification of the analyzed material. In the future, they will most likely completely replace traditional methods of assessment of tissue material, or will serve a the basic, not complementary, diagnostic tool. As evidenced by, among others, the abovementioned multicenter study, bistopatological assessment is a very subjective method, highly dependent on (he experience of the pathologist.

The present inventors have proposed new diagnostic tests that will surely constitute a significant added value to all diagnosis given by inexperienced pathologists. The results of the test will also be independent of time available for the diagnosis, and of the quality of collected tissu material, ensuring their full objectivity.

The summary of the invention

The present inventors have conducted the experiment of next generation sequencing in which they identified sequences and expression levels of all microRNAs present in thyroid tissue and papillary thyroid carcinoma (Swierniak M et al In-depth characterization of the microRNA transcriptome in normal thyroid and papillary thyroid carcinoma. J Clin Endocrinol Metab, 2013 Aug;98(8):E1401-9). During further analyses of microRNA expression in material derived from other types thyroid cancer and benign tumors; adenoma and multi-nodular goiter (MNO) the inventors have surprisingly found that the level of expression of miR-146b-5p, miR-l46b-3p, miR-221-5p, miR-221-3p, miR-222-5p, miR-222-3p, miR-181a-5p and miR-182-5p in tissue allows unequivocal diagnosis of neoplastic lesions in the thyroid gland, their differentiation as well as the assessment of response of such changes to treatment. Furthermore, the inventors have surprisingly found that such diagnosis can be also made based on material from fine-needle aspiration biopsy and in biological fluids, such as peripheral blood plasma, urine, exudates, biopsy washings or intercellular fluids. 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 thyroid tumors. The proposed diagnostic test allows for differentiation between particular pathologies of the thyroid gland: benign multi-nodular goiter, adenoma, papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), anaplastic thyroid carcinoma (ATC) and medullary thyroid carcinoma (MTC). The importance of this panel lays in the fact that during many procedures in which the material is obtained for patomorphological examination the material is actually collected outside from the tumor, leading to a false negative result of histopathological examination. The use of molecular diagnostic panel that allows for differentiation between tumorous and unaltered thyroid tisssue not depending on the region from which the analyze material was aspirated, will have important clinical implications, allowing for identification of neoplasms at early stages and providing high reliability of the result notwithstanding the actual region from which the tissue specimen was collected.

Detcnnination of the amount of these specific microRNAs is conducted using well- known hybridization methods, such as Taqman probes, diagnostic arrays made on glass, plastic or gold-covered surface, comprising single microRNAs selected among miR-146b- 5p, miR-146b-3p, miR-221-5p, miR-221-3p, miR-222-5p, miR-222-3p, miR-181a-5p and miR-182-5p. Application of the test according to the invention provides a unique opportunity to distinguish patients with thyroid cancer from healthy subjects without the need for any invasive and thus significant risk related treatments, for example, the necessity of collecting a tissue sample from a patient. At the same time, if collection of such a sample is necessary, the test provides an excellent tool contributing to die performance of the pathomorphologist who cannot give a univocal diagnosis based on the examination of fine-needle aspiration biopsy material. The present invention provides the possibility of a non-invasive monitoring of the lealth status of a patient at risk of thyroid tumor, and of diagnosing the process of arcinogenesis at early stages will allow for a significant reduction of mortality and implications caused by this cancer.

The present invention relates to a method allowing for diagnosis of benign thyroid amor and thyroid cancer in patients in vitro and for differentiation between various types f thyroid cancer: papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), edullary thyroid carcinoma (MTC) and anaplastic thyroid carcinoma (ATC) as well as of enign tumors: multi-nodular goiter (MNG) and adenoma. The said method comprises:

- providing a biological sample collected from a patient,

- determining the amount of one or more miRNAs selected from the group consisting of miR-146b-5p, miR-146b-3p, miR-221-5p, miR-221-3p, miR-222-5p, miR-222- 3p, miR-181a-5p i miR-182-5p in a biological sample,

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

a which method the patient is diagnosed for cancer and or benign lesion if the level of liRNA expression in the biological sample is altered relative to the expression level bserved in samples of biological fluids from healthy subjects, and microRNA levels are ifferently altered in different thyroid pathologies.

Preferably, the biological sample is a tissue specimen, particularly preferably a ine-needle aspiration biopsy or a biological fluid. In a preferred embodiment a sample of le biological fluid is a whole blood sample, serum, plasma, urine, saliva_* lymph, tears, leural effusion, mucus ascites fluid, derived from the respiratory system such as bronchial ecretions, amniotic fluid, cerebrospinal fluid, milk, fluid derived from cysts such as, for xample, ovarian cysts or any other liquid from the tissue, including ascites, and articularly preferably blood plasma.

Preferably, the miRNA is selected from the group comprising raiR-146b-5p, 146b- p, miR-221-3p and miR-181a-5p.

According the method according to the invention, the amount of at least one miRNA i determined The invention also relates to the use of a miRNA marker to diagnose lesion in the hyroid, 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 he samples with the use of specific primers for reverse transcription and amplification nimers and probes or by hybridization to specific probes or by next generation sequencing

• the result of microRNA expression is analyzed,

vherein the microRNA marker is selected from the group comprising miR-146b-5p, miR- .46b-3p, miR-221-Sp, miR-221-3p, miR-222-5p, miR-222-3p, miR-181a-5p and miR-182- ⁽p, and their length isoforms, and the altered expression level of a miRNA compared to !ontrol group indicates a high risk for thyroid lesion. Preferably, the marker sequence is elected from the group comprising miR-146b-5p, miR-146b-3p, miR-221*3p and miR- 81a-5p. The sequences of said microRNAs are deposited in the microRNA database niRBase mtto://www.mirbase.org).

The lesion within the thyroid gland is a multi-nodular goiter (MNG) or a benign umor - adenoma. Alternatively, the thyroid lesion is a thyroid cancer, such as papillary hyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), anaplastic thyroid carcinoma ATC) and medullary thyroid carcinoma (MTC).

According to another preferred embodiment of the invention, expression of raiR- 46b-5p or miR-146b-3p or miR-22l-5p or miR-221-3p or miR-222-5p or miR-222-3p or niR-18la-5p or miR-182-Sp is a marker for differentiation and malignancy of cancer and Defeased expression of miRNA is a marker of tumor progression towards a more nalignant state.

Preferably, the expression profile of the miR-146b-5p, miR-146b-3p, miR-221-5p, oiR-221-3p, miR-222-5p, miR-222-3p, miR-181a-5p or miR-182-5p is a marker of hyroid cancer type, and the cancer is chosen from the group comprising papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), anaplastic thyroid carcinoma (ATC) and medullary thyroid carcinoma (MTC).

Preferably, the present invention also refers to the application in which expression profile of miR-1 6¾-5p or miR-146b-3p or miR-221-5p or miR-221-3p or miR-222-5p or miR-222-3p or miR-181a-5p or miR-182-5p is a marker for a type of thyroid lesion which is selected from the group comprising adenoma and thyroid cancers: papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), anaplastic thyroid carcinoma (ATC) and medullary thyroid carcinoma (MTC), as well as a multi-nodular goiter (MNG).

The invention also provides a diagnostic kit for the assessment of diagnosis of a thyroid lesion, 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. genotyping probe or an immobilized probe for microRNA detection or the resources for sample analysis commonly used for analysis of short RNAs in next generation sequencing experiment,

wherein the primers are designed in such a way that they enable specific amplification of the nucleotide sequences of miR-146b-5p, miR-146b-3p, miR-221-5p, miR-221-3p, miR- 222-5p, miR-222-3p, miR-181a-5p and miR-182-5p and allow for determination of the amount of at least one of selected miR As in a biological sample.

Preferably, the diagnostic kit according to the invention contains primers designed to allow for amplification and measurement of all microRNAs among miR-146b-5p, miR- 146b-3p, miR-221-5p, miR-221-3p, miR-222-5p, miR-222-3p, miR-181a-5p and miR-182- Sp in a biological sample, or provides resources allowing for the analysis of these microRNAs by next generation sequencing.

The biological sample is a tissue sample or 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 sucb 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-146¾-5p, 146b-3p, miR-221-3p and raiR-181a-5p.

The term ,,microRNA" or„miRNA" or„miR" refers to short, non-coding RNA sequences of the length of 15-30 nucleotides that can be determined in a biological sample The term "biological sample" used herein refers to any sample containing biomolecules and/or a specimen derived from a patient. Exemplary biomolecules comprise DNA, RNA, including mRNA and miRNA, or proteins. Exemplary biological samples constitute any cells, groups of cells of cellular fragments derived from patients that can be used in practical implementation of the present invention. Such specimens can constitute solid tissue samples, in particular, but not limited to, samples obtained during fine-needle aspiration biopsies, 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 irregular growth of cells, characterized by cells with multiple cellular and biochemical anomalies that in their malignant state are capable of forming a systemic disease, creating, for example, tumor metastasis to distant organs. Examples of malignant tumors include, but are not limited to, papillary, follicular, anaplastic and medullary thyroid carcinoma.

The term ''benign tumor" or "benign neoplasm" refers to or describes the condition of the patient, particularly a human, which is associated with irregular growth of cells and includes alterations connected with hyperproliferation of cells, incapable of forming aggressive, metastizing cancer in vivo. Examples of benign lesions include, but are not limited to, adenoma and multi-nodular goiter. The term "control group'Vderived from control group" relates to the group of healthy subjects constituting a reference for the study patients and any material derived from the healthy subjects, respectively. In particular this term relates to thyroid samples taken from patients in whom no carcinogenic process was diagnosed.

The present invention is illustrated in the following figures, where:

Fig. 1 shows the expression of microRNAs in tumor and control samples. The expression is given in RPM (reads per milion) units;

Fig. 2 shows a dilution curve for the chosen reference gene - TJ 4 - analyzed in fine-needle aspiration samples;

Fig. 3 shows dilution curves for the reference microRNAs: miR-93 and miR-195 analyzed in plasma samples;

Fig. 4 shows amplification curves of two from the analyzed microRNAs: miR-146b and miR-221 (fine-needle aspiration biopsy material);

Fig. 5 shows amplification curves of two from the analyzed microRNAs: miR-146b and miR-221 (plasma material);

Fig. 6 shows graphs illustrating that a single microRNA from the families of miR-146, miR-181, miR-182, miR-221, miR-222 allows for distinguishing between cancer and control samples.

The invention is illustrated below 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 basis 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 microRNAs is the marker sensu stricto of the high risk of occurrence of the thyroid tumor. Examples

Characteristics of the diagnostic panel used In the studies.

To confirm the proposed diagnostic method and the markers, the expression of selected markers has been performed in biological samples derived from 104 patients and 396 subjects constituting a control group.

Tests were conducted in material from fine-needle aspiration biopsies and biological fluid, which was peripheral blood, and in addition, for patients who underwent surgery, in a solid-tissue samples obtained during thyroid resection.

Determination of effective methods for isolation of microRNA from fine-needle aspiration biopsies

Analysis of microRNA expression in fine-needle aspiration biopsy material is a very demanding task, due to small amount of the extracted material. Thus, the present inventors tested several methods of microRNA isolation including commercially availblc RNA extraction kits. As a result, microRNA isolation from fine-needle aspiration biopsy was conducted by passing 500 μΐ of TRIzol reagent through the biopsy needle.

Determination of effective methods for isolation of microRNA from plasma

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 present 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.

Determination of microRNA expression profiles characteristic for thyroid tumors

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 or with 1 μΐ of RNA template isolated from fine-needle º ºspiration biopsy, under the following conditions: 16 ^º C-30 min, 42 ^º C-50 min, 85 ^º C-5 min, 4 ^º C-oo. Amplification reaction was performed in a total volume of 10 ul using 1.6 ul of the reverse transcription reaction product

The results confirmed mat the process of thyroid carcinogenesis is accompanied by altered expression of numerous miRNAs, including decreased expression of miR-1179, miR-7-2-3p, miR-144-3p, miR-451a in tumor compared to non-tmorous tisse samples. At the same time, the inventors observed a significant increase in the expression of microRNA miR-146b-3p, miR~146b-5p, miR-221-3p, miR-222-3p, miR-34a-3p, miR-187-3p, miR-181a, miR-181b. A set comprising of miR-146b-Sp, miR-146b-3p, miR-221-5p, miR- 221-3p, miR-222-5p, miR-222-3p, miR-181a-5p and miR-182-5p was chosen for further analysis.

Analysis of expression of the set of microRNAs in One-needle aspiration biopsies from patients with thyroid tumors and control group

The expression of microRNAs was measured. Measurements of the expression were performed as described above. In the studies allowing for identification of thyroid-tumor specific miR A expression changes in fine-needle aspiration biopsy miR-146b-5p, miR- 146b-3p, miR-221-5p, miR-221-3p, miR-222-5p, miR-222-3p, miR-181a-5p and miR-182- Sp were chosen due to their high expression in tissue, allowing for correct measurement, and due to the differences in expression between tumor and control tissue. Expression levels were normalized to the expression of small nuclear U44 gene.

Analysis of expression of the set of microRNA in the plasma of patients with thyroid tumors and in control groups

The expression of microRNAs was measured. Measurements of the expression were performed as described above. In the studies allowing for identification of thyroid-tumor specific miRNA expression changes in serum miR-146b-5p, miR-146b-3p, mlR-221-5p, miR-221-3p, miR-222-5p, miR-222-3p, miR-181a-5p and miR-182-5p 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 miCFoR As were subjected to further analysis leadi g to generation of normalized results allowing for (be proper comparative assessment of markers.

Selection of the appropriate reference gene for the plasma-based analyses

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/ul. Such low concentration values limit the possibility of accurate and precise spectrophotometry 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 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 thyroid 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, unaltered by 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 HFRT, TOP or GUSB, proved to be indeterminable either. Based on the literature data, two microRNAs miR-93 i miR-195, were chosen, as their presence in serum is known in the art (de Kok JB et al., (2005) ..Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes" Lab Invest. Jan;85(l): 154-9.

The expression of genes was measured in the plasma of patients with thyroid tumors and control group. The studies allowed for identifying a molecule that exhibits the least variable expression between the groups. Analysis of the results carried out in the NonnFlnder 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 microR As.

Analysis of expression of selected microRNAs in the fine-needle aspiration biopsy material

In the first step, amplification of the selected microRNAs was analyzed using the RNA and cDNA dilution curves (standard curves) in order to verify its proper efficiency, allowing for accurate measurement of microRNA expression. The data for miR-146b and miR-221 are shown in Pig.4.

Based on these studies, the present inventors much to their surprise have found that the expression profile of single microRNAs from the list including miR-146b-5p, raiR- 146b-3p, miR_'221-Sp, miR-221 -3p, miR-222-5p, miR-222-3p, miR-181a-5p and miR-182- 5p in the fine-needle aspiration biopsy material allows a clear distinction between patients with thyroid cancer from patients with benign tumors and from control subjects. As already mentioned, diagnostic criteria for thyroid cancers are ambiguous and the examination of biopsies is technically difficult itself, what additionally negatively influences its quality. For example, the microscopic image of a well-differentiated follicular thyroid carcinoma (FTC) resembles a benign follicular adenoma, and distinguishing between the two lesions is frequently impossible without examination of die intra-operative tissue specimen. On the other hand, medullary thyroid carcinoma (MTC) is so infrequent that gaining experience in diagnosing of mis cancer type requires long experience in a large endocrine oncology center. Thus, the possibility of using the proposed molecular diagnostic panel that allows for identification of cancerous process within the thyroid gland notwithstanding the region from which the analyzed material was collected, will have important clinical implications allowing for detection of cancer at its early stages and ensuring high reliability of the obtained result.

Analysis of expression of selected microRNAs In serum

In die 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 for mJR-146b and miR-221 are shown in Fig.5.

Based on these studies, the present inventors much to their surprise have found that the expression profile of single microR As from the list including miR-146b-5p, miR- 146b-3p, miR-221 -5p, miR-221 -3p, miR-222-Sp, miR-222-3p, miR-181a-5p and miR-182- Sp in the plasma enables for distinguishing patients with thyroid cancer from patients with benign tumors, and from control subjects. This fact allows for elaboration of a unique diagnostic panel allowing for non-invasive prognostics and diagnostics of thyroid tumors, without the need of obtaining material from the thyroid gland.

De elopment of a diagnostic test (set) for the detection of thyroid tumors

Based on the expression profile of a single microRNA from the list comprising miR- 146b-5p, miR-146b-3p, miR-221 -5p, miR-221-3p, miR-222-5p, miR-222-3p, miR-181a-5p or miR-182-5p in plasma, a sensitive and specific diagnostic test, allowing for early detection of thyroid tumors, was developed and tested. The test involves determination of microRNA expression using specific primers and probes complementary to the microRNA sequence.

Studies were conducted using fine-needle aspiration material or tissue fluid (such as blood plasma) collected from 104 patients and 396 control subjects.

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 the expression of microRNAs miR-146b-5p or miR-146b-3p or miR-221 -5p or miR-221 -3p or miR-222-Sp or miR-222- 3p or miR-181a-5p or miR-182-5p allows to distinguish patients with thyroid cancer from patients with benign lesions and from the control group. Figure 6 illustrates that a single microRNA allows for distinction of tumorous specimens from control tissue, i.e. thyroid samples collected from subjects in whom no thyroid tumorigenesis process was diagnosed.

The obtained results show mat analysis of microRNA expression allows for elaboration of a diagnostic test of specificity of 100%, positive predictive value (PPV) of 95% and negative predictive value (NPV) of 100%. Thus, the tool gives opportunity of monitoring and early diagnostics of this tumor.

Claims

22 Claims

1. A method of diagnosing thyroid cancer and benign thyroid lesion in a patient in vitro, which method comprises:

- providing a biological sample from a patient,

- determining the amount of one or more miRNAs selected from the group comprising miR-146b-5p, miR-146b-3p, miR-221-5p, miR-221-3p, miR-222-5p, miR-222-3p, miR-181a~5p and miR-182-5p in a biological sample,

- comparing the miRNA expression level to the level of miR A of the control group, in which method the patient is diagnosed for occurrence of cancer and/or benign lesion 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, wherein the microRNA levels are differently altered in different thyroid pathologies.

2. The method according to claim 1, characterized in that the biological sample is a tissue fragment.

3. The method according to claim 2, characterized in that the biological sample is a fine-needle aspiration biopsy material.

4. The method according to claim 1, characterized in that the biological sample is a biological fluid sample.

5. The method according to claim 4, characterized in that the biological fluid sample 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, 23

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 microRNA is selected from the group comprising miR-146b-5p, I46b-3p, miR-221-3p and miR-181a- Sp.

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

9. The method according to any one of claims 1-8, characterized in that the patient is a human.

10. Use of a miRNA marker for diagnosis of a lesion in the thyroid, 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 methods of microRNA quantification with the use of specific primers for reverse transcription, and primers and probes for the amplification or by hybridization to specific probes or by next generation sequencing

- the result of microRNA expression is analyzed

characterized in that the microRNA marker is selected from the group comprising miR- 146b-5p, mJR-146b-3p, miR-221-5p, miR-221-3p, miR-222-5p, miR-222-3p, miR-181a- 5p, miR-182-5p, and microRNA expression level altered compared to the control group indicates a high risk of thyroid lesion

11. Use according to claim 10, characterized in that the marker sequence is selected from the group comprising miR-146b-5p, raiR-146b-3p, mlR-221-3p and miR-181a-5p.

12. Use according to any of claims 10-11, characterized in that the lesion in the thyroid is a multi-nodular goiter. 24

13. Use according to any of claims 10-11, characterized in that the lesion in the thyroid is tumor.

14. Use according to claim 13, characterized in that the tumor is selected from the group comprising adenoma and papillary (PTC), follicular (FTC), anaplastic (ATC) and medullary (MTC) thyroid carcinoma.

15. Use according to any of claims 10-14, characterized in that the profile of expression of miR-146b-5p or miR-146b-3p or miR-221-5p or miR-221-3p or miR-222-5p or miR-222- 3p or miR-181a-5p or miR-182-5p is a marker of tumor differentiation and malignancy.

16. Use according to any of claims 10-15, characterized in that the profile of expression of miR-146V5p or miR-146b-3p or roiR-221-5p or miR-221-3p or miR-222-5p or miR-222- 3p or miR-181a-5p or miR-182-5p is a marker of thyroid lesion, and the lesion is selected from the group comprising adenoma and thyroid cancers: papillary (PTC), follicular (FTC), anaplastic (ATC) and medullary (MTC) thyroid carcinoma, as well as multinodular goiter (MNG).

17. A diagnostic kit for the assessment of diagnosis of a lesion in the thyroid, 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 or the resources for sample analysis commonly used for analysis of short RNAs in next generation sequencing experiment

wherein the primers are designed in such a way that they enable specific amplification of the nucleotide sequences of miR-146b-5p or miR-146b-3p or miR-221-5p or miR-221-3p or miR-222-5p or miR-222-3p or miR-181a-5p or miR-182-5p and estimation of the amount of at least one of indicated raicroRNAs in a biological sample.

18. A diagnostic kit according to claim 17, characterized in mat the biological sample is a biological fluid. 25

19. A diagnostic kit according to claim 18, characterized in that the sample of biological fluid is a tissue material from intraoperative resection or fine-needle aspiration biopsy or 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.

20. A diagnostic kit according to claim 19, characterized in that the biological fluid sample is blood plasma.

21. A diagnostic kit according to any one of claims 17-20, characterized in that it enables specific amplification and measurement of all microRNAs from the group of miR-146b-5p, miR-146b-3p, miR-221-5p, miR-221-3p, miR-222-5p, miR-222-3p, mJR-181a-5p and miR-182-Sp in a biological sample, or provides the resources for sample analysis commonly used for analysis of short RNAs in next generation sequencing experiment