Mirna expression signature in the classification of thyroid tumors

Abstract

The present invention provides a method for classification of thyroid tumors through the analysis of the expression patterns of specific microRNAs in fine needle aspiration samples. Thyroid tumor classification according to a microRNA expression signature allows optimization of diagnosis and treatment, as well as determination of signature-specific therapy.

1 . A method of classifying a thyroid lesion sample as malignant or benign, comprising:
 a. providing RNA extracted from a thyroid lesion sample obtained from a human subject using fine need aspiration (FNA);   b. obtaining by real time polymerase chain reaction (PCR) performed on the RNA an expression profile comprising expression levels of miRNAs comprising hsa-miR-31-5p (SEQ ID NO: 5, 6, or 7), hsa-miR-222-3p (SEQ ID NO: 1 or 2), hsa-miR-146b-5p (SEQ ID NO: 10 or 11), MID-16582 (SEQ ID NO: 25), hsa-miR-342-3p (SEQ ID NO: 17 or 18), hsa-miR-125b-5p (SEQ ID NO: 9), hsa-miR-375 (SEQ ID NO: 8), hsa-miR-486-5p (SEQ ID NO: 22), hsa-miR-551b-3p (SEQ ID NO: 3 or 4), hsa-miR-152-3p (SEQ ID NO: 12 or 13), hsa-miR-138-5p (SEQ ID NO: 19, 20, or 21), hsa-miR-23a-3p (SEQ ID NO: 26), and hsa-miR-574-3p (SEQ ID NO: 36 or 37); wherein the PCR comprises contacting the RNA with forward and reverse primers for each of the miRNAs, wherein each forward primer comprises 15-21 nucleotides identical to one of the miRNAs; and wherein the forward primers comprise SEQ ID NO: 317;   c. applying a classifier algorithm to the expression profile; wherein the classifier algorithm compares the expression profile to a reference value; and   d. classifying the thyroid lesion as benign or malignant based on the result from the classifier algorithm.   
     
     
         2 . The method of  claim 1 , wherein the thyroid lesion has been classified as Bethesda III, IV or V according to the Bethesda system. 
     
     
         3 . The method of  claim 1 , wherein said classifier algorithm is a machine-learning algorithm. 
     
     
         4 . The method of  claim 1 , wherein said classifier algorithm is a multi-step classifier. 
     
     
         5 . The method of  claim 4 , wherein the classifier algorithm comprises at least one linear discriminant analysis (LDA) classifier. 
     
     
         6 . The method of  claim 5 , wherein the classifier algorithm comprises at least one LDA classifier combined with a KNN classifier. 
     
     
         7 . The method of  claim 1 , wherein following step (b), the method further comprises a step of obtaining a ratio between the expression levels of at least one pair of microRNAs; and wherein in step (c) said classifier algorithm is applied to any one of the microRNA expression profile, said ratio of at least one pair of microRNAs, or to a combination thereof. 
     
     
         8 . The method of  claim 1 , wherein said algorithm further combines at least one of clinical or genetic data from said sample. 
     
     
         9 . The method of  claim 1 , further comprising the step of administering a differential treatment to said subject if said thyroid lesion is classified as benign or malignant. 
     
     
         10 . The method of  claim 9 , wherein said lesion is classified as malignant and said treatment is any one of surgery, chemotherapy, radiotherapy, hormone therapy, or any other recommended treatment. 
     
     
         11 . The method of  claim 1 , wherein said classifying further includes a step of eliminating a sample classified as medullary malignant carcinoma. 
     
     
         12 . The method of  claim 1 , wherein said classification has a negative predictive value of between 84 and 96%.