Patent Description:
<CIT> discloses a technique for the analysis of global miRNA signatures including a larger panel of miRNAs in various groups of well-characterized colorectal cancers (CRCs). However the disclosed diagnosis method is based on obtaining one or more biological samples from the subject and obtaining expression patterns of one or more MicroRNAs (miRNAs) in the biological samples using a microarray, wherein the one or more miRNAs are either upregulated or downregulated in the tissue sample of the subject suspected of having the CRC and comparison with the sample of healthy subject not suffering from CRC. The method does not involve the comparison with prognostic model and determination of the risk score.

<CIT> discloses biomarkers like miRNA which can be assessed for diagnostic, therapy-related or prognostic methods to identify phenotypes, such as a condition or disease, or the stage or progression of a disease. The method does not involve the comparison with prognostic model and determination of the risk score.

<CIT> discloses a method for predicting the efficacy of an anti-angiogenic treatment alone or in combination with chemotherapy an individual suffering from cancer.

<CIT> provides teaching that miRNAs are differentially expressed in CRC and metastases, however the particular microRNA species which refers. to the invention were not disclosed.

It is known from "Abstract <NUM>: The role of miR-135b in clinical outcome and metastasis in colorectal cancer, URL:https://cancerres. aacIjournals. org/content /<NUM>/15_Supplement/<NUM>; that the expression level is important with respect to clinical outcome of colorectal cancer and metastasis, but discloses only correlation of miR-135b.

<NPL> discloses that marker miR-<NUM> is important in the development of colorectal cancer.

<NPL> discloses that miR-<NUM> was identified as colon cancer growth and metastases suppressing microRNA which functioning as a tumor suppressor.

All mentioned disclosures do not provide teaching for method of detecting of the presence of micrometastases and an increased risk of recurrence of colorectal cancer after a radical surgical treatment.

<NPL> discloses prognostic tool to effectively classify patients with stage II colon cancer into groups at low and high risk of disease recurrence. This tool is based on a six-miRNA signatures being miR-<NUM>-5p, miR-20a-5p, miR-103a-3p, miR-106b-5p, miR-<NUM>-5p and miR-<NUM>.

The prognosis of colorectal cancer in I and II stage is usually good, nevertheless, approx. <NUM>% of them will develop metastases in distant organs (cancer dissemination) within a few years after undergoing a radical operation. At this stage the disease becomes incurable. Applying chemotherapy or immunotherapy after a surgical procedure could prevent a part of recurrences of patients at high risk of cancer dissemination.

Notwithstanding the above, on the basis of the analysis of clinical and microscopic features, it is impossible to distinguish patients with a high risk of cancer dissemination. Due to the knowledge of molecular basics of the appearance and development of tumours, it is currently known that clinical course of a tumour can be conditioned by genes' expression. A test conducted according to the invention consists of a molecular analysis of a fragment of resected tumour in terms of its ability to form metastases.

The method is based on detection in the samples of total RNA obtained from the tumour tissue of short RNA fragments (MicroRNA), and analysing their levels using molecular biology techniques, such as for instance: polymerase chain reaction with the use of reverse transcriptase (RT-PCR), Nano-String, or deep sequencing of transcriptome techniques. The use of the abovementioned method will enable the determination of individual risk of tumour dissemination more precisely, and in consequence, it will be possible to isolate a group of patients with adverse prognosis, among whom an adjuvant therapy could be applied (adjuvant chemotherapy, immunotherapy, or elective treatment).

Adjuvant therapy, which is applied after a surgery, inactivates microscopic metastasis, which are undetectable with the use of currently available diagnostic methods, and thereby it prevents incurable recurrence of the tumour. <NUM>,<NUM> Europeans receive surgical treatment due to colorectal cancer annually, and one in four will experience tumour dissemination. On the one hand, the implementation of a prognostic test indicating patients with an increased risk of recurrence to clinical practice and subjecting them to a post-operative adjuvant therapy could improve the results of treatment in this group, and on the other hand, enable the avoidance of a toxic and unnecessary treatment of patients with good prognosis.

At the current clinical practice, post-operative chemotherapy is not applied in patients who have I-IIA stage of a disease (T2N0-T3N0), due to the fact that <NUM>% of them do not experience tumour recurrence. It is believed that an empirical use of chemotherapy constitutes overtreatment in this group of CC patients. However, such an approach results in the lack of cure in <NUM>% of patients who have an increased risk of recurrence, and who would benefit from post-operative chemotherapy whereby their risk of recurrence would be decreased. Therefore, forgoing adjuvant chemotherapy among these patients is a suboptimal behaviour.

In the case of patients with T4N0 feature, the risk of post-operative cancer dissemination is estimated at approximately <NUM>%. The role of post-operative chemotherapy in this group is controversial, nevertheless this form of treatment is applied due to empirical grounds. This means, however, that <NUM>% of patients having T4N0 stage of disease, who would not have had the recurrence, are subjected to toxic treatment with no clinical benefit.

Currently, available diagnostic method, which enables precise differentiation among people suffering from colorectal cancer in the stage of I-II of a subgroup of patients with an increased risk of metastatic development need to be improved in a way such that the qualification of patients to post-operative chemotherapy on the basis of individually estimated risk of cancer recurrence would be possible. The elaborated assay aims to identify such patients and direct them to proper treatment, which should contribute to the improvement of their results. This approach corresponds to the current paradigm of personalised medicine.

The object of invention is to provide an alternative method based on differential expression of microRNA markers, with which to prognosticate on distant metastasis free survival of colorectal cancer. More particular, the invention provides an assay aimed in evaluation of the risk of recurrence for people suffering from colorectal cancer in the stage I and IIA (T2-T3N0M0), who are subjected to radical surgical treatment, based on the assessment of selected MicroRNA (miRNA) expression.

The study demonstrated expression of <NUM>% of the <NUM> miRNAs tested, the tested miRNA were included in screening audiometer TaqMan Low Density Arrays A+B3 of LifeTechnologies. The miRNA, of which expression correlated with the tendency to form distant metastasis, was indicated.

On the grounds of the obtained results, a predictive test was prepared - signatures of miRNA expression, which are given by the linear regression formula: logit(RS), by which it is possible to calculate risk index, which is a marker of micrometastases risk and recurrence of colorectal cancer after a post-operative therapy.

As an example, the risk index, which is calculated according to the algorithm weighting the predicting influence of selected miRNA expression for signature <NUM> of miRNA expression, is highly connected with distant metastasis free survival (DMFS): odds ratio for survival free from tumour recurrence (HR) is <NUM> (<NUM>% CI <NUM>-<NUM>); p<<NUM>, with sensitivity and specificity of detection of metastases <NUM>% and <NUM>%, respectively. Three-year survival rate without metastases for patients with high and low risk of recurrence, which is determined by the aforesaid test was <NUM>% and <NUM>%, respectively. Negative and positive predictive value of the test was <NUM>% and <NUM>%, respectively. In a multifactorial model, to which also tumour's stage was included (pT2 vs. pT3), as well as the stage of its histological differentiation, the risk index turned out to be the only independent predictive factor (HR=<NUM>,<NUM> [<NUM>% CI <NUM>-<NUM>]).

The predictive value of expression signature was validated positively internally in a leave-one-out analysis, in which sensitivity and specificity of prediction of recurrence was estimated on <NUM>% and <NUM>%, respectively. The risk index was also highly connected with overall survival (HR=<NUM> [<NUM>% CI <NUM>-<NUM>]; p=<NUM>. 24E-<NUM>).

Predictive power of occurring of distant metastases by the predictive test, which was prepared by us (signature of miRNA expression), significantly exceeds the predictive value of conventional clinical-pathological factors of people suffering from colorectal cancer of I and IIA stage. At the same time, there are no useful solutions of this kind on the market.

Test's parameters: Negative and positive predictive value - <NUM>% and <NUM>% respectively (a "training" group), and <NUM>% and <NUM>% respectively for internal validation leave-one-out. Odds ratio (HR) of recurrence for patients with High and Low result of risk index was <NUM> (<NUM>% CI <NUM>-<NUM>) in this validation.

The purpose of this invention is to make a diagnostic device accessible to patients and doctors, which facilitates the selection of appropriate treatment concerning adjuvant systemic therapy for people suffering from colorectal cancer, who were subjected to a radical surgical treatment (tumour resection) in the I-IIIa stage. On the grounds of the test results, patients with a low risk of recurrence would not be subjected to additional treatment, and patients with a high risk of recurrence would be offered an adjuvant therapy (adjuvant chemotherapy, immunotherapy, or targeted therapies).

The subject of the invention is a method of determining distant metastases free survival in colon cancer patients after surgical treatment consisting of the determination of the quantity of microRNAs comprising isolation of RNA from colorectal cancer tissue and quantification of microRNA, characterised in that <NUM>-microRNAs: hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>-5p, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM> are quantified by quantitative reverse transcriptase polymerase chain reaction (known as RT-PCR) or hybridisation method, such as Nanostring technology and further the obtained microRNA expression values are input to the microRNA expression values in the recurrence risk prediction model and the risk score RS of recurrence of colorectal cancer is calculated as logit(RS) function including a weighting factor of the contribution of each analysed microRNA according to formula :logit(RS) = <NUM>,<NUM> + <NUM> × hsa-miR-<NUM> + <NUM> × hsa-miR-<NUM> - <NUM> × hsa-miR-<NUM> - <NUM> × hsa-miR-<NUM> + <NUM> × hsa-miR-<NUM> - <NUM>,<NUM> × hsa-miR-<NUM>-5p - <NUM>,<NUM> × hsa-miR-<NUM> - <NUM>,<NUM> × hsa-miR-<NUM> + <NUM> × hsa-miR-<NUM> - <NUM>,<NUM> × hsa-miR-<NUM>.

The second subject of the invention is a method of determining distant metastases free survival in colon cancer patients after surgical treatment consisting of the determination of the quantity of microRNAs comprising isolation of RNA from colorectal cancer tissue and quantification of microRNA, characterised in that <NUM>-microRNAs: hsa-miR-135b, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM> are quantified by quantitative reverse transcriptase polymerase chain reaction or hybridisation method, such as Nanostring technology, and further the obtained microRNA expression values are input to the microRNA expression values in the recurrence risk prediction model and the risk score RS of recurrence of colorectal cancer is calculated as logit(RS) function including a weighting factor of the contribution of each analysed microRNA according to formula: logit(RS) = -<NUM>,<NUM> - <NUM> × hsa. miR-135b + <NUM> × hsa. miR-<NUM> - <NUM> × hsa. miR-<NUM> - <NUM> × hsa. miR-<NUM> + <NUM> × hsa. miR-<NUM> + <NUM>,<NUM> × hsa. miR-<NUM> + <NUM>,<NUM> × hsa. miR-<NUM> - <NUM>,<NUM> × hsa. miR-<NUM> + <NUM> × hsa.

The third subject of the invention is a method of determining distant metastases free survival in colon cancer patients after surgical treatment consisting of the determination of the quantity of microRNAs comprising isolation of RNA from colorectal cancer tissue and quantification of microRNA, characterised in that <NUM>-microRNAs: hsa-miR-135b, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM> are quantified by quantitative reverse transcriptase polymerase chain reaction or hybridisation method, such as Nanostring technology, and further the obtained microRNA expression values are input to the microRNA expression values in the recurrence risk prediction model and the risk score RS of recurrence of colorectal cancer is calculated as logit(RS) function including a weighting factor of the contribution of each analysed microRNA according to formula: logit(RS) = -<NUM>,<NUM> - <NUM> × hsa-miR-135b + <NUM> × hsa-miR-<NUM> - <NUM> × hsa-miR-<NUM> - <NUM> × hsa-miR-<NUM> + <NUM> × hsa-miR-<NUM> + <NUM>,<NUM> × hsa-miR-<NUM> + <NUM> × hsa-miR-<NUM>;.

The forth subject of the invention is a method of determining distant metastases free survival in colon cancer patients after surgical treatment consisting of the determination of the quantity of microRNAs comprising isolation of RNA from colorectal cancer tissue and quantification of microRNA, characterised in that <NUM>-microRNAs: hsa. miR-<NUM>, hsa. miR-135b, hsa. miR-<NUM>, hsa. miR-<NUM>, hsa. miR-<NUM> are quantified by quantitative reverse transcriptase polymerase chain reaction or hybridisation method, such as Nanostring technology and further the obtained microRNA expression values are input to the microRNA expression values in the recurrence risk prediction model and the risk score RS of recurrence of colorectal cancer is calculated as logit(RS) function including a weighting factor of the contribution of each analysed microRNA according to formula: logit (RS) = -<NUM> - <NUM> × hsa-miR-135b + <NUM> × hsa-miR-<NUM> - <NUM> × hsa-miR-<NUM> - <NUM> × hsa-miR-<NUM> + <NUM> × hsa-miR-<NUM>.

The method wherein normalization of expression of each miRNA from the formulas of RS or colon cancer-specific miRNAs, is carried out by the referencing of the prognostic and colon cancer-specific miRNAs to the geometric average of the expression of all or selected miRNAs from the following group of miRNAs: hsa. miR-<NUM>, hsa. miR-<NUM>, hsa. miR-200c, hsa. miR-26a, hsa. let-<NUM>, hsa. miR-27a, hsa. miR-<NUM>, hsa. miR-<NUM>-3p, hsa. miR-<NUM>, hsa. miR-1274B, hsa. miR-<NUM>, hsa. miR-1274A, hsa. miR-<NUM>, hsa. miR-<NUM>, hsa. miR-<NUM>, hsa. miR-30e-3p, hsa. miR-<NUM>, hsa.

The advantages of the method according to the invention includes:.

The invention is illustrated by the following examples of performance, which at the same time do not constitute its limitation.

The object illustrating the essence of application are the results of miRNA expression in tumour tissue of <NUM> patients suffering from colorectal cancer in stage T2-3N0, who were subjected to radical surgical treatment and who had metastases after a surgery (tumour recurrence) or who recovered completely (without tumour recurrence). (Table <NUM>). The study was conducted on sections of tumour, which were preserved in formalin and submerged in paraffin wax.

754miRNA expression was analysed with the use of qRT-PCR method. A proprietary method of normalisation of miRNA expression for the purpose of examination of miRNA expression in biological material of people suffering from colorectal cancer was elaborated (Table <NUM>). A proper normalisation is a necessary, integral element of diagnostic testing, based on miRNA expression assessment in order to determine the prognosis of patients who have colorectal cancer. On the grounds of normalised data, miRNA was identified (Table <NUM> and Table <NUM>), which can contribute to defining prognoses of patients suffering from colorectal cancer. It was stated that expressions miR-<NUM> and miR-<NUM> are connected with an increased risk of colorectal cancer dissemination in stage T2-T3N0, after the amendment of multiple testing. Moreover, a group of several microRNA was extracted, whose expression identifies tumour tissue (specific expression for tumour tissue), or identifies normal colorectal tissue (specific expression for colon) (Table <NUM>). The inclusion of testing towards these markers constitutes an integral element of diagnostic testing, based on miRNA expression assessment in order to determine the prognosis of patients who have colorectal cancer. It was determined that signature based on expression <NUM> miRNA, <NUM> miRNA, <NUM> miRNA, or <NUM> miRNA can be a potentially clinically useful predictive marker in this group of patients in relation to cancer dissemination risk.

The study has obtained a permission of the Independent Bioethics Committee for Scientific Research at the Medical University of Gdańsk. The analysis involved patients suffering from colorectal cancer (RJG) in stage pT2-3N0, who were subjected to surgical treatment between <NUM> and <NUM>, and who had metastases (tumour recurrence), or who recovered completely (without tumour recurrence). In case of the group of patients without cancer recurrence, the minimal survival time free from disease recurrence was <NUM> years. Both groups were balanced in terms of clinical and patho-morphological prognostic factors. Patients who had local or nodal recurrence were not included in the study. All patients were subjected to pathological radical hemicolectomy or sigmoidectomy while having lymph node dissection. In order to avoid the effect of hidden metastases to lymph nodes, in all the cases, at least <NUM> lymph nodes were removed. None of the patients received adjuvant or neoadjuvant chemotherapy or radiotherapy.

The examined material consisted of sections of removed tumour, which were preserved in formalin and submerged in paraffin wax (FFPE). All tissue blocks were assessed in terms of the confirmation of diagnosis and evaluation of the content percentage of tumour tissue by estimation of a microscopic slide subj ected to haematoxylin and eosin stain (HE) by a patho-morphologist. FFPE block of the highest content of tumour pattern was selected for molecular analyses. In order to further diminish tissue elements' content, which are not included in tumour tissue, elements of normal mucosa of colon, as well as necrotic tissue were excised from FFPE block with the use of macrodissection. After macrodissection, the microscopic slide contained at least <NUM>% of tumour pattern. In order to avoid contamination, each tissue block was cut with the use of a new blade of a microtome.

Four fragments of the thickness of <NUM>, cut from FFPE block were applied to RNA isolation using RecoverAll Kit (Ambion). RNA concentration was assessed in NanoDrop®. Reverse transcription was conducted with the use of <NUM> ng RNA and TaqManMiRNA RT kit (Applied Biosystems), as well as primers stem-loop pool A and B (Megaplex™ PrimerPools, Human Pools Set v3. <NUM>, Applied Biosystems), as recommended by the manufacturer. Each pool of primers consists of specific primers towards <NUM> miRNA, therefore, in order to obtain cDNA for <NUM> miRNA, the reverse transcription was conducted in two reactions containing in (i) primers of pool A, and in (ii) primers of pool B. With the use of the obtained cDNA, primers specific for miRNA, fluorescent probes TaqMan, and polymerase with the activity of <NUM>' of nuclease, reactions qRT-PCR were carried out in microfluidic cards (TaqMan® ArrayMicrofluidic Cards, Applied Biosystems) in cycler HT <NUM> (Applied Biosystems), in conditions recommended by the manufacturer (Applied Biosystems). Raw expression values (Ct) were obtained in application SDS. <NUM> (Applied Biosystems).

All FFPE blocks selected for molecular analyses, were also examined in terms of expression of marker proteins for MMR (mismatchrepair) with the use of immune-histochemical methods (IHC) in tissue microarrays (TMAs), which was covered with two tissue fragments, <NUM> diameter each. After cutting slides of the thickness of <NUM>, TMA was stained in Dakoautostaine on the presence of proteins: MLH1 (clone ES05, Dako, ready to use), MSH2 (G219-<NUM>, Cell Marque, <NUM>:<NUM>) and MSH6 (clone EP49, Dako, ready to use). A complete lack of stain reaction (IHC) for MLH1, MSH2, or MSH6 tumour tissue, with preservation of staining in surrounding stroma, was regarded as a determinant of instability of microsatellites (MSI).

The samples of normal colorectal mucosa (NCM) were collected from the area of an operating margin of a microscopic slide, free from tumour in the histopathologic examination.

The primary endpoint was time off from distant metastasis (metastasis-free survival (MFS)). The number of miRNA expression was specified at least in <NUM>%, <NUM>%, <NUM>% and <NUM>% of the samples in both analysed groups (' with recurrence of cancer ' or ' non-recurrence of cancer '). MiRNA with signal amplification (Ct ≥ <NUM>) in fewer than <NUM>% of the samples was included in the prognostic analyses. Missing values of expression (Ct ≥ <NUM>) were imputed in the model "EM-based of the missing data mechanism" [<NUM>]. Ct values for miRNA were normalized against the geometric mean for the Ct value of U6 RNA, RNU44, RNU48, and <NUM> miRNAs, which expression level is the most stable in all tested samples-Norm Finder application (Appendix A, table <NUM>.

Expression of miRNA was received by means of formula <NUM> ^-(ΔCt) [<NUM>, <NUM>], these values of expression were used to determine times of differences in expression and <NUM>% of confidence ranges. MiRNA expression, specified by ΔCt method, was applied in the subsequent analyses. Lilliefors test was used to verify the hypothesis of normality of the distribution of the signal. Single-factorial analyses were carried out to compare the standardized expression values of miRNA between groups of patients with the use of the nonparametric Mann-Whitney test, and in the analysis of Cox's univariate linear regression, against DMFS values. p values were corrected on multiples of tests with the use of Benjamin-Hochberg's algorithm. All statistical inference was based on threshold of significance alpha = <NUM>. Reoccurrence free survival curves (DMFS) were generated using the Kaplan-Meier method. Clinical prognostic value of each miRNA was analysed in the Cox's polyamorous test, to which the following variables were included: the expression of miRNA (as-ΔCt), as well as degree of advancement T and histological differentiation. Negative sign ΔCt allowed for the intuitive interpretation of HR value, for example, in cases where a high expression of miRNA was associated with a high risk of recurrence, the resulting HR value was greater than <NUM>. T feature was expressed in a binary method (pT2 vs. pT3) and degree of histological malignancy as <NUM>, <NUM> or <NUM>, respectively. Multi-factorial linear regression model was constructed to determine an expression signature, which might characterise the relationship between miRNA expression and the result of treatment (reoccurrence vs. non-recurrence). A risk index was calculated for each patient (recurrence Score (RS)) according to the equation from the model of logistic linear regression. Due to a large number of potential variables, the regression model included miRNA, whose expression was associated with DMFS on the significance level p- value < <NUM> (univariate analyses) [<NUM>].

The number of coefficients of the model was specified using the algorithm "forward selection scheme", with the use of a proprietary modulation method based on the Bayesian Information Criterion (BIC), R<NUM> and the probability of value ratio p-value that indicates the difference in relation to the model with a smaller number of coefficients. The impact of the individual coefficients was measured with the use of Wald's test. The optimal cut-off threshold RS was found by maximising the positive predictive value (PPV) with a negative predictive value restriction (negative predictive value (NPV)) above <NUM>. In addition, regulated parameters of the created model were sought using the LASSO regression model.

A prognostic value of the obtained model was verified by "internal leave-one-out cross-validation" method. A clinical value of the obtained model was also reviewed by means of the Cox's multi-factorial model of the linear regression, to which the following variables were entered: degree of advancement T and histological differentiation. A study involving <NUM> patients with colorectal cancer, among whom <NUM> patients experienced reoccurrence of cancer in the form of distant metastasis- 'cases', and <NUM> patients, who did not experience reoccurrence of cancer -'controls ', has <NUM>% power to detect the difference in survival between groups of "High" and "Low" risk of recurrence of cancer, which is specified according to the size of the test HR = <NUM> with an I error type alpha = <NUM>.

In order to find the miRNA, whose expression is specific to colorectal cancer, the data from expression Ct were confounded into the categories of: (I) no signal of amplification: Ct ≥ <NUM>, (ii) expression: Ct <<NUM>. Confounded data were compared between samples of the tumour and normal tissue of the colon by means of the McNamara's test. In order to compare, the expression was normalized as in the section above. The expression miRNA obtained by means of ΔCt method was compared between the groups via T-test for paired data.

The study concerned <NUM> patients, including <NUM> with distant metastasis and <NUM> without metastasis during at least four years of observation, with an average time of observation of <NUM> and <NUM> years, respectively. The outbreaks of metastasis were located , inter alia, in the lungs, liver, bone and skin. There were no statistically significant differences noted between the groups with and without relapse of the disease with the exception of the disease advancement level (more patients with I stage of advancement were included in the control group; Table <NUM>. Out of <NUM> analysed microRNAs, <NUM> (<NUM>%) of the Ct <<NUM> within the limits of <NUM>-<NUM>% were considered as a non-expression.

The expression of <NUM> miRNAs (<NUM>%) was reported in at least <NUM>% of the samples, <NUM> miRNAs (<NUM>%) in at least <NUM>% of the samples, <NUM> miRNAs (<NUM>%) in at least <NUM>% of the samples and <NUM> miRNAs (<NUM>%) in all the samples. The expression of <NUM> miRNAs (<NUM>%) was present in more than <NUM>% of the evaluated samples. These miRNAs were evaluated and underwent further analyses. The quality of RNA was typical for FFPE blocks, while Ct values for individual RNA which were used in the standardisation process are shown in Table <NUM>.

Unifactorial model of Cox's regression has shown a statistically significant relationship between a low expression of miR-<NUM> and miR-<NUM> and a shorter survival, free from distant metastasis (distant metastasis free survival, DMFS) (after correction for multiple comparisons, p=<NUM>). Within Cox's multi-factorial prognostic model, which included expression of miRNA, pT and degree of histological malignancy, the low expression of miR-<NUM> (p= <NUM>; HR=<NUM>,<NUM> [<NUM>% CI: <NUM>,<NUM>-<NUM>]) and pT (p=<NUM>; HR = <NUM>,<NUM> [<NUM>% CI: <NUM>-<NUM>,<NUM>]) correlated independently with DMFS. The comparison of medium expressions of miRNA for groups of patients with relapsed disease, and without disease recurrence did not show significant differences after applying the correction on multiple repetitions. In order to identify specific microRNAs potentially involved in the process of metastasis, we searched for the microRNAs whose level of expression was simultaneously correlating with DMFS, (unadjusted Cox test; p <<NUM>) and differed between the groups of patients with relapsed disease and those without recurrence of disease (the unadjusted U test; p <<NUM>,<NUM>). Both conditions met the following miRNA miR-<NUM>, miR-<NUM>, miR-<NUM>, miR-<NUM>, miR-<NUM>, miR-<NUM>, miR-422a, miR-<NUM>, miR-<NUM>, miR-<NUM>, miR-<NUM>, miR-26a-<NUM>, miR-<NUM>, miR-<NUM> and miR-<NUM> (table <NUM>). MiRNA with a prognostic significance or a potentially prognostic significance in colon cancer showed mostly a lower expression in patients with relapsed disease, except miR-<NUM>, which underwent an increased expression. A complete list of miRNA, whose expression correlated with DMFS (Cox's test), and comparison of miRNA expression between groups of patients with relapsed disease and those without disease recurrence (U test) has been shown in Table <NUM>.

By applying the algorithm, which generates the prognostic model of expression of miRNA, it was possible to create a signature expression of the five microRNAs (<NUM>-miRNA <NUM> signature) undergoing a greater expression (miR-<NUM>, miR-135b and miR539) or less expressive (miR-<NUM> and miR-<NUM>) in patients with colon cancer and relapse after the surgical treatment. The contribution of each miRNA along with "weight factors" allowed for the formation the following signature: logit (RS) =-<NUM>-<NUM> × miR-135b <NUM> × miR-<NUM>-<NUM> x miR-<NUM>-<NUM> × miR-<NUM><NUM> x miR-<NUM>. The cut off value for the index of risk was: threshold valued RS = <NUM>. The risk index of the presented signature was noted to be strongly associated with DMFS (HR = <NUM> [<NUM>% CI <NUM>,<NUM>-<NUM>,<NUM>] p< <NUM>), upon sensitivity and specificity of the test at the level of <NUM>% and <NUM>% (<FIG>). Average DMFS in the "high risk" group is estimated at <NUM> months and has not been reached for a group of "low risk". The three-year DMFS for prognostic groups of "high risk" and "Low-risk" was estimated at <NUM>% and <NUM>% respectively. Negative predictive value (NPV) and positive predictive value (PPV) were estimated at <NUM>% and <NUM>% respectively, and measure of the value of the test different from distribution of chances equalled p=<NUM>. 28e-<NUM> (chi-squared test). In Cox's multivariate analysis, covering the odds ratio (risk factor), the degree of clinical malice (combined grades G1 and G2 against grade <NUM>) and advancement level (pT2 pT3 v), the risk ratio was the only variable significantly correlating with DMFS (HR = <NUM>,<NUM> [<NUM>% CI <NUM>,<NUM>-<NUM>,<NUM>]. Then, the signature of expression of miRNA underwent cross-validation according to "leave-one-out" method, reaching the sensitivity and specificity of <NUM>% and <NUM>%. The risk coefficient was also correlating with time of survival (HR=<NUM>,<NUM> [<NUM>% CI <NUM>,<NUM>-<NUM>,<NUM>]; p=<NUM>,24E-<NUM>).

By applying the algorithm, which generates the prognostic model of expression of miRNA, it was possible to create a signature of expression of seven microRNAs (signature <NUM>-miRNA), which either underwent greater expression (miR-<NUM>, miR-135b and miR539) or were less expressive (miR-<NUM> and miR-<NUM>, miR-<NUM>, miR-<NUM>) in patients with colon cancer and disease relapse after the treatment. The contribution of each miRNA along with "weight factors" allowed the formation of the following signatures: logit(RS) = -<NUM>,<NUM> - <NUM> × miR-135b + <NUM> × miR-<NUM> - <NUM> × miR-<NUM> - <NUM> × miR-<NUM> + <NUM> × miR-<NUM> + <NUM>,<NUM> × miR-<NUM> + <NUM> × miR-<NUM>. The cut off value for the index of risk was: threshold valued RS = <NUM>. The index of risk of presented signature was strongly associated with DMFS (HR = <NUM>,<NUM> [<NUM>% CI <NUM>,<NUM>-<NUM>,<NUM>] p <<NUM>), with the sensitivity and specificity of the test at the level of <NUM>% and <NUM>% (<FIG>). Average DMFS in the "high risk" group amounted to <NUM> months and has not been reached for a group of "low risk". The three-year DMFS for prognostic groups of "high risk" and "Low-risk" amounted to <NUM>% and <NUM>% respectively. Negative predictive value (NPV) and positive predictive value (PPV) were calculated at <NUM>% and <NUM>% respectively, and the measure of the value of the test different from distribution chances equalled p=<NUM>. 47e-<NUM> (chi-squared test). Subsequently, the signature expression of miRNA was subjected to cross-validation according to "leave-one-out" method, reaching the sensitivity and the specificity of <NUM>% and <NUM>% level.

By applying the algorithm, which generates the prognostic model of expression of miRNA, it was possible to create a signature of expression of nine microRNAs (<NUM>-miRNA signature), which could either be more expressive (miR-<NUM>, miR-135b, miR-<NUM> and miR-<NUM>) or less expressive (miR-<NUM>, miR-<NUM>, miR-<NUM>, miR-<NUM> and miR-<NUM>) in patients with colon cancer and a disease relapse after the surgical treatment. The contribution of each miRNA along with "weight factors" allowed the formation of the following signature: logit(RS) = -<NUM>,<NUM> - <NUM> × miR-135b + <NUM> × miR-<NUM> - <NUM> × miR-<NUM> - <NUM> × miR-<NUM> + <NUM> × miR-<NUM> + <NUM>,<NUM> × miR-<NUM> + <NUM>,<NUM> × miR-<NUM> - <NUM>,<NUM> × miR-<NUM> + <NUM> × miR-<NUM>. The cut-off value for the index of risk amounted to: threshold valued RS = <NUM>. The index of risk of the presented signature was strongly associated with DMFS (HR = <NUM>,<NUM> [<NUM>% CI <NUM>-<NUM>,<NUM>] p<<NUM>), with the sensitivity and specificity of the test at the level of <NUM>% and <NUM>% (<FIG>). The average DMFS in the "high risk" group amounted to <NUM> months and has not been reached by a group of "low risk". The three-year DMFS for prognostic groups of "high risk" and "Low-risk", amounted to <NUM>% and <NUM>% respectively. Negative predictive value (NPV) and positive predictive value (PPV), amounted to <NUM>% and <NUM>% respectively, and the measure of the value of the test different from distribution of chances equalled p=<NUM>. 33e-<NUM> (chi-squared test). Then, the signature of expression of miRNA underwent cross-validation according to "leave-one-out" method, reaching the sensitivity and the specificity of <NUM>% and <NUM>% levels.

By applying the algorithm, which generates the prognostic model of expression of miRNA, it was possible to create a signature of expression of ten microRNAs (<NUM>-miRNA signature), which could be either more expressive (miR-<NUM>, miR-135b, miR-<NUM>, miR-<NUM>, miR-<NUM>-<NUM> p) or less expressive (miR-<NUM>, miR-<NUM>, miR-<NUM>, miR-<NUM> and miR-<NUM>, miR-<NUM>) in patients with colon cancer and a disease relapse after the surgical treatment. The contribution of each miRNA along with "weight factors", allowed the formation of the following signature: logit(RS) = <NUM>,<NUM> + <NUM> × miR-<NUM> + <NUM> × miR-<NUM> - <NUM> × miR-<NUM> - <NUM> × miR-<NUM> + <NUM> × miR-<NUM> - <NUM>,<NUM> × miR-<NUM>-5p - <NUM>,<NUM> × miR-<NUM> - <NUM>,<NUM> × miR-<NUM> + <NUM> × miR-<NUM> - <NUM>,<NUM> × miR-<NUM>. The cut-off value for the index of risk was: threshold valued RS = <NUM>. The index of risk of the presented signature was strongly associated with DMFS (HR = <NUM>,<NUM> [<NUM>% CI <NUM>-<NUM>,<NUM>] p<<NUM>), with the sensitivity and specificity of the test at the level of <NUM>% and <NUM>% (<FIG>). The average DMFS in the "high risk" group amounted to <NUM> months and was not reached by a group of "low risk". The three-year DMFS for prognostic groups of "high risk" and "Low-risk" amounted to <NUM>% and <NUM>% respectively. Negative predictive value (NPV) and positive predictive value (PPV) amounted to <NUM>% and <NUM>% respectively, and the measure of the value of the test different from distribution of chances equalled p=<NUM>. 13e-<NUM> (chi-squared test). Then, the signature of expression of miRNA underwent cross-validation according to "leave-one-out" method, reaching the sensitivity and specificity at the levels of <NUM>% and <NUM>%.

For each of the microRNA the value of "stability of expression" was calculated, using the NormFinder algorithm (Andersen et al, <NUM>). The expression of miRNA in FFPE tissues was noted in a wide range of values of Ct: Ct <NUM>-<NUM>. For this reason, it is important to incorporate into the miTNS panel standardization miRNAs, expressed in different expression scopes. In our standardization strategy, <NUM> miRNAs with stable expression between groups of colon cancer samples from patients with relapse and without relapse were included in the standardization panel. (Table <NUM>). Selected MiRNAs belong to one of three groups of the expression level: standardization miRNAs with a low expression: average value of the Ct ><NUM>; standardization miRNAs with the average value of the expression, the average value of Ct ><NUM>-<NUM>; standardization miRNAs with a high value of expression, average value of Ct ≤ <NUM>. For each group, <NUM> miRNAs with the lowest value the stability of expression were selected in accordance with the NormFinder algorithm, separately for the pool A (<NUM> standardization miRNAs) and pool B (<NUM> standardization miRNAs).

MiRNA expression was compared in both, cancer tissue and healthy tissue of the large intestine. By comparing the digitalised values of expression <NUM> miRNAs were identified with specific expression for colorectal cancer for example, miR-<NUM>, miR-<NUM>, miR-18b, miR-302a, miR-<NUM>-<NUM> p, miR-<NUM>-<NUM> p, miR-<NUM>-<NUM> p, miR-<NUM> (p <<NUM>) and only two miRNAs with specific expression for cancer-free large intestine: miR-<NUM>-<NUM> p and miR-<NUM> (p <<NUM>) (Table <NUM>).

Claim 1:
A method of determining distant metastases free survival in colon cancer patients after surgical treatment consisting of determination of the quantity of microRNAs comprising isolation of RNA from colorectal cancer tissue, quantification of microRNA, characterised in that
<NUM>-microRNAs: hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>-5p, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM>, hsa-miR-<NUM> are quantified by quantitative reverse transcriptase polymerase chain reaction or hybridisation method, such as Nanostring technology, and further the obtained microRNA expression values are input to the microRNA expression values in the recurrence risk prediction model and the risk score RS of recurrence of colorectal cancer is calculated as logit(RS) function including a weighting factor of the contribution of each analysed microRNA according to formula: logit (RS)= <NUM>,<NUM> + <NUM> × hsa-miR-<NUM> + <NUM> × hsa-miR-<NUM> - <NUM> × hsa-miR-<NUM> - <NUM> × hsa-miR-<NUM> + <NUM> × hsa-miR-<NUM> - <NUM>,<NUM> × hsa-miR-<NUM>-5p - <NUM>,<NUM> × hsa-miR-<NUM> - <NUM>,<NUM> × hsa-miR-<NUM> + <NUM> × hsa-miR-<NUM> - <NUM>,<NUM> × hsa-miR-<NUM>.