IDH1 TARGETED MUTATION ANALYSIS WITH REFLEX TO IDH2 MUT
Label Name: IDH1
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6491 
Last Review:  3/16/2017 2:54:17 PM
Specimen Type
  Tissue
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years CHECK WITH LABORATORY 1  ML
Collection Notes
  All:
  • Fresh tissue: 200 mg tissue. Tissue should be frozen within 1 hour of collection and sent to the laboratory on dry ice. Please contact the Clinical Molecular Diagnostics Laboratory for help shipping samples.

    Formalin Fixed Paraffin Embedded Tissue: The laboratory can receive either a paraffin embedded tissue block or four freshly cut (within one week)5uM thick unstained slides containing 3 to 20 square mm of tissue. Unstained slides should be accompanied by an H&E stained slide for histologic evaluation.

 
Storage
  Tissue should be frozen on dry ice.
Transport
  Tissue should be sent frozen on dry ice.

Turn Around Time -  Routine: 14 days   Stat: N/A
Reference Values
IDH1
Methodology
  This assay uses PCR amplification followed by Sanger DNA sequencing to detect point mutations in exon 4 of the IDH1 gene, with reflex testing to detect point mutations in exon 4 of the IDH2 gene for all IDH1 negative cases. An H&E stained slide for each case is first evaluated to identify the regions of greatest tumor content. These regions are then macro-dissected from adjacent unstained formalin-fixed paraffin-embedded sections and used to prepare genomic DNA. The protein coding and flanking intronic sequences of IDH1 exon 4 (containing codon 132), and, if reflex testing is performed, IDH2 exon 4 (containing codon 172) are amplified in duplicate from this purified genomic DNA by PCR. The primers used in these PCR reactions contain M13 universal primer "tails" at their 5' ends, and have 3' ends that are complementary to their genomic target sequence. The resulting PCR products are treated with an exonuclease/ phosphatase mixture (ExoSAP-IT) to remove excess PCR primers and nucleotides. These purified DNA amplicons are then sequenced using universal M13 forward and reverse sequencing primers (M13 Forward/-20 and M13 Reverse/-27) and the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The products of the completed sequencing reactions are purified with the Big Dye XTerminator Purification Kit and resolved using the ABI 3130xl Genetic Analyzer. Data is analyzed using the ABI Data Collection software v3.0, Sequencing Analysis software 5.2 and SeqScape software v2.6. Sequences are compared to the reference DNA sequence for the IDH1 and IDH2 genes. (GenBank Accession IDH1: NM_005896.2; IDH2: NM_002168.2)

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
   
   
Clinical Significance and Interpretive Data
    BACKGROUND

Isocitrate dehydrogenase 1 (IDH1) maps to chromosome 2q33.3, and contains 10 exons (1). IDH1 localizes to the cytoplasm and peroxisomes, and acts as a NADP-dependent protein that catalyzes decarboxylation of isocitrate into alpha-ketoglutarate. The pathway produces NADPH, which likely provides cellular protection from oxidative damage. The homologous isocitrate dehydrogenase 2 (IDH2) maps to chromosome 15q26.1 and contains 11 exons (2). IDH2 is the only protein homologous to IDH1 that also utilizes NADP; however IDH2 localizes to the mitochondria. IDH2 plays an important role in controlling the mitochondrial redox balance, and in providing protection from oxidative damage similar to IDH1.

Several recent studies have identified a genetic alteration in the IDH1 or IDH2 genes of malignant brain tumors. Originally, a genome-wide study examining glioblastoma multiformes (GBM; WHO grade IV astrocytoma) isolated recurrent mutations in the conserved active site of IDH1 in 12% of GBMs (3). A broad examination of IDH1 across a large spectrum of brain tumor types showed the majority of grade II and III astrocytomas and oligodendrogliomas also carried IDH1 mutations (4), while primary gliobastoma and low grade astrocytic tumors demonstrated an absence or low frequency of IDH1 mutations. Further analysis of IDH1 and the homologous IDH2 gene in almost 500 brain tumors correlated the previous IDH1 findings, and identified novel IDH2 mutations in a subset of astrocytomas and oligodendrogliomas lacking IDH1 mutations (5).

All IDH alterations are point mutations resulting in a missense change at codon 132 of IDH1, and codon 172 of IDH2. These two residues are analogous between genes, and occur in exon 4 at a highly conserved region of the isocitrate binding site. The mutation is thought to down-regulate or even eliminate enzyme activity, leading to increased cellular oxidative stress and damage.

Because IDH mutations are associated with secondary glioblastomas and rarely seen in primary tumors, they are likely indicative of an early event in malignant glioma formation. Testing of several tumors from individual patients with progressive gliomas has shown the same IDH1 mutation present in both low- and high-grade tumor samples (5). Furthermore, IDH mutations may alter the glial stem cells that differentiate into both astrocytes and oligodendrocytes, and are the stem cell population for GBM5. IDH altered tumors represent a molecularly distinct subset of gliomas that are yet to be fully characterized, and may have unique therapeutic targets.

CLINICAL UTILITY

IDH mutated gliomas are a biologically distinct subgroup that occur in younger patients and have a significantly better clinical prognosis than IDH intact tumors. They also carry a higher frequency of TP53 mutations and a lower frequency of other common molecular alterations seen in GBMs (3). Patients whose tumors have an IDH mutation averaged an overall survival of 39 months, as compared to 13.5 months for IDH-intact GBM patients. For anaplastic astrocytomas, the presence of an IDH mutation increased overall survival to 65 months as compared to 19 months for IDH wild-type patients (5). The average age of onset decreases an average of 10 years for mutated IDH patients when compared to wild-type patients within the same tumor types (5).

INTERPRETATION

Sequence variants will be compared to those referenced by the available literature and recorded in the MDL database. Individual known polymorphisms may not be reported. Sequence changes will be reported as: (1) sequence variation is previously reported as a known mutation, (2) sequence variation is previously unreported and is of unknown significance, (3) sequence variation is previously unreported and is likely not significant, or (4) sequence variant is previously reported as a benign polymorphism. At the discretion of the director, the significance of a sequence change may be speculated upon based on the type of variant, its position in the gene, and its effect on the amino acid sequence/protein. (5) In the case of an indeterminate sample result due to a variant detected in both forward and reverse reactions below the assay sensitivity of ~15%, at the discretion of the laboratory director the sample may be subject to additional/alternative testing to confirm/rule out a mutation.

REFERENCES

1. McKusick, VA. (1986) #147700 ISOCITRTE DEHYDROGENASE 1; IDH1. OMIM, created 1986. http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=147700.

2. McKusick, VA. (1986) #147650 ISOCITRTE DEHYDROGENASE 2; IDH2. OMIM, created 1986. http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=147650.

3. Parsons DW, et. al. (2008) An integrated genomic analysis of human glioblastoma mutiforme. Science Express. Sep 26;321(5897):1807-12.

4. Balss J, et. al. (2008) Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol. 116:597-602.

5. Yan H, et. al. (2009) IDH1 and IDH2 gene mutations play a fundamental role in astrocytoma and oligodendroglioma development. N Engl J Med. 360(8):765-73.

6. ACMG Laboratory Practice Committee Working Group. ACMG recommendations for standards for interpretation of sequence variations. Genetics in Medicine. Vol 2. 2000.

   
Indications
    The IDH-dependent pathway of tumorigenesis can be seen in gliomas of astrocytic, oligodendroglial, mixed oligo-astrocytic differentiation, and secondary glioblastomas which progressed from these lower grade tumors. Primary and pediatric GBMs, ependymomas, and pilocyticatrocytomas have been shown to carry a low frequency or absence of IDH mutations and should not be considered for IDH testing.
   
Contraindications
    None
   
Limitations
    Multiple factors contribute to prognosis in patients with primary CNS neoplasms. Thus, this assay is intended for use as an aid in developing patient specific prognostic predictions but is not a substitute for a complete pathologic and clinical evaluation, or physician's judgment and clinical experience.

The sensitivity and specificity of DNA sequencing is high for the detection of nucleotide base changes, small deletions, and insertions in the regions analyzed. This assay may not detect an acquired mutation which is present below the 15% detection limit (i.e., mutant cell population of < 30%). Only amino acids 42-138 of the IDH1 gene and amino acids 126-178 of the IDH2 gene were examined. Changes outside of this region will not be detected. The presence of a mutant population containing a large deletion, duplication, insertion, aberrant splicing, or sequence alteration adversely affecting primer binding may not be identified using these methods. Mutations or polymorphisms in the DNA oligonucleotide primer binding regions, poor DNA quality, insufficient DNA quantity or the presence of PCR inhibitors can result in uninterpretable or (rarely) inaccurate results.
   
Test Synonyms
  Synonym(s):  BRAIN TUMOR
Synonym(s):  ISOCITRATE DEHYDROGENASE
Synonym(s): IDH1
Synonym(s): IDH2
Molecular Diagnostics Laboratory
(MDX)

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

Address: 
 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

Performing Times: