ALTERNATIVE SPLICE VARIANT PATTERNS OF HUMAN TELOMERASE REVERSE TRANSCRIPTASE (hTERT) IN THYROID TUMORS TO DISTINGUISH BENIGN FROM MALIGNANT

Abstract

This invention relates, e.g., to a method for determining if a thyroid tumor in a subject is malignant, comprising determining in a sample from the subject the amount of TERT (telomerase reverse transcriptase) mRNA which lacks the β sequence and the amount of TERT mRNA in the sample which comprises the β sequence, wherein a preponderance (e.g., at least about 55%) of TERT mRNA in the sample which comprises the β sequence indicates that the tumor is malignant, and wherein a preponderance of TERT mRNA which lacks the β sequence indicates that the tumor is not malignant.

We claim: 
     
         1 . A method for determining if a thyroid tumor in a subject is malignant, comprising determining in a sample from the subject the amount of TERT (telomerase reverse transcriptase) mRNA that lacks the β sequence and the amount of TERT mRNA in the sample that comprises the β sequence,
 wherein a preponderance of TERT mRNA in the sample that comprises the β sequence indicates that the tumor is malignant, and wherein a preponderance of TERT mRNA that lacks the β sequence indicates that the tumor is not malignant. 
 
     
     
         2 . The method of  claim 1 , wherein the subject is human; the TERT mRNA is hTERT (human telomerase reverse transcriptase) mRNA; and the β sequence is the 182 bp sequence represented by SEQ ID NO:3. 
     
     
         3 . The method of  claim 2 , wherein
 a) a ratio of the amount of
 hTERT mRNA that comprises the sequence of SEQ ID NO:3, compared to the total amount of hTERT mRNA that either comprises or that lacks the sequence of SEQ ID NO:3 
   of at least about 0.55 indicates that the tumor is malignant, and   b) a ratio of the amount of
 hTERT mRNA that lacks the sequence of SEQ ID NO:3, compared to the total amount of hTERT mRNA that either comprises or that lacks the sequence of SEQ ID NO:3 
   of at least about 0.55 indicates that the tumor is not malignant.   
     
     
         4 . The method of  claim 3 , further wherein the amount of mRNA that lacks the α sequence (represented by SEQ ID NO:5) and/or the amount of mRNA that lacks both the α sequence and the β sequence are determined; and wherein,
 a) a ratio of the amount of hTERT mRNA that comprises the sequence of SEQ ID NO:3 compared to the total amount of
 hTERT mRNA that comprises the sequence of SEQ ID NO:3, and 
 hTERT mRNA that lacks the sequence of SEQ ID NO:3, and 
 hTERT mRNA that lacks the α sequence (SEQ ID NO:5) and/or that lacks both SEQ ID NO:3 and SEQ ID NO:5 
 
 of at least about 0.55 indicates that the tumor is malignant, and 
 b) a ratio of the amount of hTERT mRNA that lacks the sequence of SEQ ID NO:3 compared to the total amount of
 hTERT mRNA that comprises the sequence of SEQ ID NO:3, and 
 hTERT mRNA that lacks the sequence of SEQ ID NO:3, and 
   2 hTERT mRNA that lacks the α sequence (SEQ ID NO:5) and/or which lacks both SEQ ID NO:3 and SEQ ID NO:5) 
 
 of at least about 0.55 indicates that the tumor is not malignant. 
 
     
     
         5 . The method of  claim 3 , wherein the thyroid tumor is suspected of being malignant. 
     
     
         6 . The method of  claim 5 , wherein the thyroid tumor is classified as being suspicious for malignancy or as being indeterminate, based on a cytological assay. 
     
     
         7 . The method of  claim 6 , wherein the cytological assay is performed on a sample obtained from a fine needle aspirate (FNA). 
     
     
         8 . The method of  claim 3 , wherein the amount of each of the hTERT mRNAs is determined by a method comprising amplifying mRNA in the sample by reverse transcriptase polymerase chain reaction (RT-PCR) analysis, using suitable PCR primers to amplify each of the mRNA species of interest; and measuring the amounts of the amplified products. 
     
     
         9 . The method of  claim 8 , wherein the amounts of the amplified products are measured by a method comprising
 a) subjecting the amplified products to a sizing procedure and categorizing the amplified products on the basis of their size; and/or   b) hybridizing the amplified products to suitable nucleic acid probes which are specific for the β-sequence or for a control sequence that is present in hTERT mRNAs which either comprise, or lack, the β-sequence.   
     
     
         10 . The method of  claim 3 , wherein the sample is a tissue sample or a fine needle aspirate (FNA), and wherein amount of each of the hTERT mRNAs is determined by a method comprising performing in situ hybridization of the tissue sample, with suitable probes that are specific for the β-sequence or for a control sequence that is present in hTERT mRNAs which either comprise, or lack, the β-sequence. 
     
     
         11 . The method of  claim 3 , wherein the amount of each of the hTERT mRNAs is determined by a method comprising measuring the amounts of polypeptides translated from each of the mRNAs. 
     
     
         12 . The method of  claim 11 , wherein the polypeptides are measured by reacting them with antibodies that are specific for epitopes within the β-sequence or that are specific for control epitopes which are present in polypeptides translated from hTERT mRNAs that either comprise, or lack, the β-sequence, under conditions which are effective for specific interactions of the antibodies and their cognate epitopes. 
     
     
         13 . The method of  claim 3 , wherein the amount of each of the mRNA species is determined by quantitative real time PCR.