miRNA FINGERPRINT IN THE DIAGNOSIS OF WILMS' TUMOUR

Abstract

MicroRNAs (miRNA) are a recently discovered class of small non-coding RNAs (17-14 nucleotides). Due to their function as regulators of gene expression they play a critical role both in physiological and in pathological processes, such as cancer. The present invention provides novel methods for diagnosing a state of health based on the determination of specific miRNAs that have altered expression levels in different conditions, e.g. disease states compared to healthy controls.

1 . A method of treating a patient suspected to be suffering from Wilms' tumor, comprising the steps:
 (a) obtaining a blood sample from the patient;   (b) separating the blood sample into a blood cell sample and a plasma or serum sample;   (c) extracting total RNA from the cells of the blood cell sample;   (d) determining an expression profile of a predetermined set of miRNAs in the total RNA extracted from said blood cell sample;   (e) comparing said expression profile to a reference expression profile, wherein the reference expression profile is determined using a blood cell sample from at least one healthy patient,   (f) diagnosing Wilms' tumor when the expression profile is altered compared to the reference profile; and   (g) treating the patient using at least one of radiotherapy and chemotherapy if the expression profile is altered compared to the reference profile.   
     
     
         2 . The method according to  claim 1 , wherein the expression profile is determined from miRNAs selected from  FIG. 3 . 
     
     
         3 . The method according to  claim 1 , wherein the expression profile is determined from miRNAs selected from  FIG. 4 . 
     
     
         4 . The method according to  claim 1 , wherein the predetermined set of miRNAs comprises at least one set of miRNAs listed in  FIG. 5 . 
     
     
         5 . The method according to  claim 1 , wherein the predetermined set of miRNAs comprises at least 1, 7, 10, 15, 20, 25, 30, 35, 40, 50, 75, or 100 of miRNAs. 
     
     
         6 . The method according to  claim 1 , wherein the expression profile is determined by nucleic acid hybridization, nucleic acid amplification, polymerase extension, sequencing, mass spectroscopy, flow cytometry or any combinations thereof. 
     
     
         7 . The method according to  claim 1 , wherein the predetermined set of miRNAs comprises at least one of hsa-miR-197 and hsa-miR-126. 
     
     
         8 . The method according to  claim 7 , wherein the predetermined set of miRNAs comprises hsa-miR-197. 
     
     
         9 . The method according to  claim 7 , wherein Wilms' tumor is diagnosed when hsa-miR-197 is upregulated and/or hsa-miR-126 is downregulated. 
     
     
         10 . The method according to  claim 1 , wherein the predetermined set of miRNAs comprises hsa-miR-17, hsa-miR-18a, hsa-miR-20a, or combinations thereof. 
     
     
         11 . The method according to  claim 10 , wherein Wilms' tumor is diagnosed when hsa-miR-17, hsa-miR-18a, and/or hsa-miR-20a is downregulated. 
     
     
         12 . The method according to  claim 1 , wherein the total RNA extracted from the blood cell sample is guarded against degradation. 
     
     
         13 . The method according to  claim 12 , wherein the total RNA extracted from the blood cell sample is guarded against degradation using special collection tubes or additives. 
     
     
         14 . The method of  claim 1 , wherein the reference expression profile is determined using blood cell samples from a plurality of healthy subjects. 
     
     
         15 . A method of determining the occurrence or presence of Wilms' tumor, monitoring the course of Wilms' tumor, staging of Wilms' tumor, measuring the response of a patient with Wilms' tumor to therapeutic intervention, and/or segmentation of a subject suffering from Wilms' tumor in a subject in need thereof, which comprises specifically detecting the level of hsa-miR-197 in a total RNA sample extracted from the blood cells of a sample from said subject wherein a level of hsa-miR-197 that is upregulated compared to the level of hsa-miR-197 in a reference profile indicates the presence of Wilms' tumor; and wherein the reference profile is determined using a blood cell sample from at least one healthy patient. 
     
     
         16 . A Wilms' tumor detection system comprising
 (a) means for obtaining a blood sample from the patient;   (b) means for separating the blood sample into a blood cell sample and a plasma or serum sample;   (c) means for extracting total RNA from the cells of the blood cell sample;   (d) means for determining an expression profile of a predetermined set of miRNAs in the total RNA extracted from said blood cell sample; and   (e) means for determining a reference profile using a blood cell sample from at least one healthy patient;   wherein Wilms' tumor is detected when the expression profile is altered compared to the reference profile.   
     
     
         17 . A method of selectively treating Wilms' tumor, comprising the steps:
 (a) selecting a patient for treatment on the basis of the patient having an altered miRNA expression profile compared to a reference miRNA expression profile, wherein the patient's expression profile is determined by measuring expression of a predetermined set of miRNAs in total RNA extracted from a blood cell sample, and the reference expression profile is determined by measuring expression of a predetermined set of miRNAs in total RNA extracted from a blood cell sample from at least one healthy patient; and   (b) selectively administering at least one of radiotherapy and chemotherapy to the patient.   
     
     
         18 . The method according to  claim 17 , wherein the predetermined set of miRNAs comprises at least one of hsa-miR-197 and hsa-miR-126. 
     
     
         19 . The method according to  claim 18 , wherein the predetermined set of miRNAs comprises hsa-miR-197. 
     
     
         20 . The method according to  claim 18 , wherein hsa-miR-197 is upregulated and/or hsa-miR-126 is downregulated. 
     
     
         21 . The method according to  claim 17 , wherein the predetermined set of miRNAs comprises hsa-miR-17, hsa-miR-18a, hsa-miR-20a, or combinations thereof. 
     
     
         22 . The method according to  claim 21 , wherein at least one of hsa-miR-17, hsa-miR-18a, and hsa-miR-20a is downregulated.