IDH1/IDH2 Mutation Analysis by Pyrosequencing, Paraffin
Supporting a diagnosis of grade II or III astrocytoma, oligodendroglioma, oligoastrocytoma, or secondary glioblastoma
Stratifying prognosis of gliomas
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Adult World Health Organization (WHO) grade II and III astrocytomas, oligodendrogliomas and oligoastrocytomas, and secondary glioblastomas (GBM) have been shown to harbor IDH1 and IDH2 mutations.(1-5) These missense mutations most frequently involve the arginine amino acid at IDH1 position 132 (R132) and at IDH2 position 172 (R172). The most frequent IDH1 amino acid alteration accounting for over 90% mutations is R132H, in addition to R132C, R132S, R132G, R132L, and R132V.(1) For IDH2, R172K, R172G, R172M, and R172W mutations have also been reported.(4,5) IDH proteins are nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate dehydrogenases that catalyze the oxidative decarboxylation of isocitrate to produce alpha-ketoglutarate. IDH1 and IDH2 mutations appear to be an early event in the development of these tumors and impair the enzyme activity,(3-4) resulting in loss of the ability to catalyse conversion of isocitrate to alpha-ketoglutarate. However, the enzyme acquires a neomorphic activity and is able to catalyze the NADPH-reduction of alpha-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). These mutations appear to have prognostic significance with increased overall survival(1,4) and have been found to be associated with a younger age among adult diffuse astrocytomas, WHO grade III astrocytomas,(4) and GBM patients.(1-3) Of note, IDH1 mutations are only rarely reported among pilocytic astrocytomas,(2-4) primary GBM,(1,2) supratentorial primitive neuroectodermal tumors,(2) and pleomorphic xanthoastrocytomas,(4) and are absent in pediatric diffuse astrocytomas, ependymomas, medulloblastomas, primitive neuroectodermal tumors, and dysembryoblastic tumors.(3,4)
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
The presence of an IDH1 or IDH2 mutation supports a diagnosis of grade II or III astrocytoma, oligodendroglioma, oligoastrocytoma, or secondary glioblastoma (GBM) in the context of other corroborating pathologic features.
IDH1 codon 132 and IDH2 codon 172 mutations have been identified in more than 70% of brain tumors diagnosed as grade II and III astrocytoma, oligodendroglioma, oligoastrocytoma, and secondary GBM. These mutations are rarely found in other brain tumors and nonbrain tumors. The ordering physician is responsible for the diagnosis and management of disease and decisions based on the data provided.
A negative result does not exclude the presence of a brain tumor.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
Reliable results are dependent on adequate specimen collection and processing. This test has been validated on formalin-fixed, paraffin-embedded tissues; other types of fixatives are discouraged. Improper treatment of tissues, such as decalcification, may cause PCR failure. False-negative results may occur in heterozygous tumor specimens when tumor cells comprise <40% of the cell population. Tumor cells are routinely enriched by macrodissection to avoid false-negative results.
Clinical diagnosis and therapy should not be based solely on this assay. The results should be considered in conjunction with clinical information, histologic evaluation, and additional diagnostic tests.
This test is designed to detect mutations in codon 132 of the IDH1 gene and codon 172 of the IDH2 gene and does not detect mutations in other areas of these genes.
In a validation study performed in our laboratory, 61 tumor specimens and 6 normal brain tissue specimens were assessed for IDH1 and IDH2 mutations. The tumor specimens included 18 low-grade oligoastrocytoma or oligodendroglioma, 19 high-grade oligoastrocytoma or oligodendroglioma, 6 low-grade astrocytoma, 8 pilocytic astrocytoma, 5 glioblastoma multiforma/grade IV oligoastrocytoma, and the glioma tumor type was unavailable for 5.
One specimen could not be analyzed by pyrosequencing for IDH1 mutations due to an inadequate amount of PCR product. Of the remaining 66 specimens, 29 were wild-type, and 37 had mutations of codon 132 the IDH1 gene. Of the 37 specimens showing mutations, 34 had the most common Arg132His (CGT>CAT) mutation, 2 had an Arg132Gly (CGT>GGT) mutation, and 1 had an Arg132Ser (CGT>AGT) mutation. All results were confirmed by Sanger sequencing resulting in 100% concordance between the 2 methods.
When evaluating the same specimens for IDH2 gene mutations, pyrosequencing detected an Arg172Lys (AGG->AAG) mutation in 3 specimens with all other specimens lacking an IDH2 mutation. Sanger sequencing detected 2 of 3 mutations identified by pyrosequencing, and confirmed the wild-type sequence in all other specimens, which resulted in a concordance of 98% between the 2 methods.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. Parsons DW, Jones S, Zhang X, et al: An integrated genomic analysis of human glioblastoma multiforme. Science 2008;321:1807-1812
2. Balss J, Meyer J, Mueller W, et al: Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol 2008;116:597-602
3. Ichimura K, Pearson DM, Kocialkowski S, et al: IDH1 mutations are present in the majority of common adult gliomas but rare in primary glioblastomas. Neuro Oncol 2009;11:341-347
4. Yan H, Parsons DW, Jin G, et al: IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009;360:765-773
5. Hartmann C, Meyer J, Balss J, et al: Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta Neuropathol 2009;118:469-474