Genetic alterations in isocitrate dehydrogenase and other genes in malignant glioma

Abstract

We found mutations of the R132 residue of isocitrate dehydrogenase 1 (IDH1) in the majority of grade II and III astrocytomas and oligodendrogliomas as well as in glioblastomas that develop from these lower grade lesions. Those tumors without mutations in IDH1 often had mutations at the analogous R172 residue of the closely related IDH2 gene. These findings have important implications for the pathogenesis and diagnosis of malignant gliomas.

1 . A method of detecting presence or absence of a somatic mutation in codon 132 in isocitrate dehydrogenase 1 (IDH1) or codon 172 in isocitrate dehydrogenase 2 (IDH2) in a sample from a human subject, comprising:
 assaying the sample for the IDH1 or IDH2 gene or mRNA transcribed from the gene in the human subject or protein translated from the mRNA, to identify residue 132 or a codon for residue 132 of IDH1 or residue 172 or a codon for residue 172 of IDH2, wherein a mutation is identified if the residue or the codon for the residue other than arginine is identified, and wherein if the sample is from a colorectal tumor then the residue or codon for the residue is not for a cysteine at residue 132 of IDH1.   
     
     
         2 . The method of  claim 1  wherein IDH1 gene is assayed and the mutation is identified if a residue or a codon for a residue selected from the group consisting of Histidine, Serine, Cysteine, Leucine, and Glycine is identified in codon or residue 132 of IDH1. 
     
     
         3 . The method of  claim 1  wherein the IDH2 gene is assayed and a mutation is identified if a residue or a codon for a residue selected from the group consisting of Lysine, Glycine, and Methionine is identified in codon or residue 172 of IDH2. 
     
     
         4 . The method of  claim 2  wherein the somatic mutation is R132H in IDH1. 
     
     
         5 . The method of  claim 2  wherein the somatic mutation is R132S in IDH1. 
     
     
         6 . The method of  claim 2  wherein the somatic mutation is R132C in IDH1. 
     
     
         7 . The method of  claim 2  wherein the somatic mutation is R132L in IDH1 
     
     
         8 . The method of  claim 2  wherein the somatic mutation is R132G in IDH1. 
     
     
         9 . The method of  claim 3  wherein the somatic mutation is R172M in IDH2. 
     
     
         10 . The method of  claim 3  wherein the somatic mutation is R172K in IDH2. 
     
     
         11 . The method of  claim 3  wherein the somatic mutation is R172G in IDH2. 
     
     
         12 . The method of  claim 1  wherein the sample is selected from the group consisting of tumor, blood, plasma, serum, cerebrospinal fluid, urine, saliva, and lymph. 
     
     
         13 . The method of  claim 1  wherein the human subject has a tumor selected from the group consisting of diffuse astrocytoma, oligoastrocytoma, anaplastic astrocytoma, anaplastic oligoastrocytoma, oligodendroglioma, glioblastoma multiforme, and anaplastic oligodendroglioma. 
     
     
         14 . The method of  claim 1  wherein the human subject has a glioblastoma multiforme (GBM), and the method further comprises the step of:
 identifying the tumor as likely to be a secondary GBM when the somatic mutation is present or a primary GBM when the somatic mutation is absent. 
 
     
     
         15 . The method of  claim 13  further comprising the step of:
 assigning a more favorable prognosis (longer life expectancy) when the somatic mutation is present or a less favorable prognosis (shorter life expectancy) when the somatic mutation is absent. 
 
     
     
         16 . The method of  claim 1  wherein the step of assaying comprises amplifying at least a portion of:
 the IDH1 gene or cDNA of the IDH1 mRNA, said portion comprising codon 132 or nucleotide 394 or 395 of IDH1 transcript; or 
 the IDH2 gene or cDNA of the IDH2 mRNA, said portion comprising codon 172 or nucleotide 515 of IDH2 transcript. 
 
     
     
         17 . The method of  claim 1  wherein the step of assaying employs an antibody which specifically binds to isocitrate dehydrogenase 1 (IDH1), to isocitrate dehydrogenase 2 (IDH2), or to both IDH1 and IDH2. 
     
     
         18 . The method of  claim 1  wherein the step of assaying employs an antibody which preferentially binds to one or more of R132H, R132C, R132S, R132L, and R132G, of isocitrate dehydrogenase 1 (IDH1) and R172M, R172G, and R172K of IDH2, relative to R132 IDH1 or R172 IDH2. 
     
     
         19 . The method of  claim 1  wherein the step of assaying employs hybridization of an oligonucleotide probe comprising:
 IDH1 codon 132 or nucleotide 394 or 395 of IDH1 transcript plus sufficient adjacent nucleotides of IDH1 to achieve specific hybridization; or 
 IDH2 codon 172 or nucleotide 515 of IDH2 transcript plus sufficient adjacent nucleotides of IDH2 to achieve specific hybridization. 
 
     
     
         20 . The method of  claim 19  wherein the codon is an arginine codon. 
     
     
         21 . The method of  claim 19  wherein the codon is not an arginine codon. 
     
     
         22 . The method of  claim 1  wherein the step of assaying comprises using primer extension to generate a reaction product comprising at least a portion of:
 IDH1 that includes codon 132 or nucleotide 394 or 395 of IDH1 transcript; or 
 IDH2 that includes codon 172 or nucleotide 515 of IDH2 transcript. 
 
     
     
         23 . An isolated antibody which specifically binds to one or more of the following:
 R132H IDH1, or R132C IDH1, or R132S IDH1, or R132L IDH1, or R132G IDH1, but not R132 IDH1;   R172M IDH2, R172G, or R172K of IDH2, but not R172 IDH2;   R132 IDH1; or   R172 IDH2.   
     
     
         24 . The antibody of  claim 23  which is a monoclonal antibody. 
     
     
         25 . The antibody of  claim 23  which is a single chain variable region molecule. 
     
     
         26 . An isolated polynucleotide comprising at least 18 but less than 600 contiguous nucleotide residues of a coding sequence of a human IDH1 or IDH2 protein found in a human tumor, said at least 18 contiguous amino acid residues comprising:
 nucleotides 394 and/or 395 of IDH1, wherein said nucleotides 394 and/or 395 are not C and/or G, respectively;   nucleotide 515 of IDH2, wherein said nucleotide 515 is not a G.   
     
     
         27 . The isolated polynucleotide of  claim 26  wherein the coding sequence is of a human IDH1 protein and the nucleotide 394 is selected from the group consisting of T, A, and G or nucleotide 395 is selected from the group consisting of A and T. 
     
     
         28 . The isolated polynucleotide of  claim 26  wherein the coding sequence is of a human IDH2 protein and the nucleotide 515 is selected from the group consisting of T and A or nucleotide 514 is a G. 
     
     
         1 - 95 . (canceled)