IDH1/IDH2 Mutation Analysis by Pyrosequencing, Paraffin
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
Supporting a diagnosis of grade II or III astrocytoma, oligodendroglioma, oligoastrocytoma, or secondary glioblastoma
Stratifying prognosis of gliomas
Additional Tests Lists test(s) that are always performed, at an additional charge, with the initial test(s)
|Test ID||Reporting Name||Available Separately||Always Performed|
|60254||AP Special Studies Review||No||Yes|
Testing Algorithm Delineates situation(s) when tests are added to the initial order. This includes reflex and additional tests.
This test is performed in conjunction with 60254 AP Special Studies Review and 70011 Slide Review. Additional testing may be performed after review by pathologist. Upon approval from the requesting clinician, 60254 AP Special Studies Review could be changed to 70012 Pathology Consultation if they determine this is more appropriate.
Special Instructions and Forms Describes specimen collection and preparation information, test algorithms, and other information pertinent to test. Also includes pertinent information and consent forms to be used when requesting a particular test
Polymerase Chain Reaction (PCR) and Pyrosequencing
(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
IDH1/IDH2 Genes, Known Mutations
Isocitrate Dehydrogenase 1
Isocitrate Dehydrogenase 2
Isocitrate Dehydrogenase 1
Isocitrate Dehydrogenase 2
Specimen Type Describes the specimen type needed for testing
Specimen Required Defines the optimal specimen. This field describes the type of specimen required to perform the test and the preferred volume to complete testing. The volume allows automated processing, fastest throughput and, when indicated, repeat or reflex testing.
A pathology/diagnostic report including a brief history is required. If available, include IDH1 mutation (R132H) immunostain results.
Container/Tube: Surgical Pathology Packaging Kit (Supply T554) requested, but not required
Preferred: Formalin-fixed, paraffin-embedded (FFPE) tissue block with a minimum of 40% tumor cell population
Alternate: Unstained slides with a minimum of 40% tumor population; slides may be stained and/or scraped
1. Process all fresh or frozen specimens into FFPE blocks prior to submission.
2. If submitting slides, a minimum of five, 4- to 5-micron thick, unstained slides are required.
1. A quality specimen is essential for evaluation. Submit only tissue containing tumor cells; minimal tissue is required for evaluation.
2. Special stains performed outside Mayo Medical Laboratories and included with the case may be repeated and charged at the reviewing pathologist's discretion. Testing requested by referring physician may not be performed if deemed unnecessary by Mayo Clinic pathologist.
Forms: If not ordering electronically, submit a Pathology/Cytology Request Form (Supply T246) with the specimen.
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
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
Method Description Describes how the test is performed and provides a method-specific reference
Paraffin-embedded tissue is deparaffinized, lysed, and digested. Genomic DNA is then extracted from the sample using either a phenol-chloroform method or the QIAamp DNA FFPE Tissue kit (Qiagen). The DNA is PCR amplified using primers specific for regions surrounding IDH1 codon 132 and IDH2 codon 172. Controls are run with each specimen to assess possible contamination issues and overall test performance. Samples are pyrosequenced and pyrograms are analyzed for the presence or absence of IDH1 codon 132 and IDH2 codon 172 mutations.(Qiagen DNA FFPE Tissue Handbook, Qiagen PyroMark Gold Q24 reagents Handbook; unpublished Mayo method)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday; 8 a.m.-5 p.m.
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
1 week/7 days
Performing Laboratory Location The location of the laboratory that performs the test
Test Classification Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer's instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR), Investigation Use Only (IUO) product, or a Research Use Only (RUO) product.
This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.
CPT Code Information Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
81403-IDH1 (isocitrate dehydrogenase 1 [NADP+], soluble) (eg, glioma), common exon 4 variants (eg, R132H, R132C)
81403-IDH2 (isocitrate dehydrogenase 2 [NADP+], mitochondrial) (eg, glioma), common exon 4 variants (eg, R140W, R172M)
LOINC® Code Information Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the result codes returned for this test or profile.
|Result ID||Reporting Name||LOINC Code|
|61207||IDH1/IDH2 Genes, Known Mutations||In Process|