Comparison and evaluation of immunohistochemical, and molecular methods on the detection of IDH1 mutations in gliomas

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Comparison and evaluation of immunohistochemical, and molecular methods on the detection of IDH1 mutations in gliomas

 

Author: Choi, Hon Keung Kenny
Title: Comparison and evaluation of immunohistochemical, and molecular methods on the detection of IDH1 mutations in gliomas
Degree: M.Sc.
Year: 2012
Subject: Gliomas -- Genetic aspects.
Mutation (Biology)
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xvii, 126 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2524037
URI: http://theses.lib.polyu.edu.hk/handle/200/6629
Abstract: IDH1 gene mutations have been identified in more than 70 % of WHO grade II and III gliomas. The most frequent mutation leads to a specific amino acid change from arginine to histidine at codon 132 (R132H, 395G>A). IDH1 mutated tumors show a better prognosis than IDH1 non-mutated tumors. In this study, we first compared the detection methods in IDH1 mutation including immunohistochemistry, allele specific PCR and DNA sequencing. In the second part, we performed the proof of principle that the point mutation of IDH1 at codon 132 can be detected by PCR based-allele specific hybridization on glass slides. We performed a retrospective study of 22 samples with pilocytic astrocytoma (n=2), anaplastic astrocytoma (n=4), Grade III Oligodendroglioma (n=4), Grade III Oligoastrocytoma (n=3), Glioblastoma multiforme (GBM, Grade IV astrocytoma) (n=8) and atypical case (n=1). DNA sequencing was performed as a gold standard reference to show the exactly sequence of IDH1 gene fragment. A fragment of exon 4 spanning the sequence encoding the catalytic domain of IDH1, including codon 132, was amplified and sequenced. Immunohistochemistry was performed with mouse monoclonal mIDH1 R132H antibody. DNA was extracted from formalin fixed paraffin embedded (FFPE) sections following microdissection, and from whole sections. Allele specific PCR was performed using 2 forward primers (one of which was allele specific primer for the mutant variant producing a smaller PCR product size of 266bp, specific for the mutant variant only) and a common reverse primer. IDH1 mutation were found in 9/22 cases (41%) by DNA sequencing. R132H mutations was found in 7/9 cases (78%) by immunohistochemistry. The result of allele specific PCR with microdissection positively correlated with DNA sequencing. However, two of the samples from whole sections without microdissection shown false negative result. Other mutation (R132S) was found by DNA sequencing in 2/9 cases (22%). mIDH1 R132H immunohistochemistry was found in the 7 cases presenting the R132H mutation from DNA sequencing (sensitivity 7/7 cases, 100% only for R132H mutation). None of the cases presenting wild-type IDH1 gene were stained (specificity 13/13, 100%). Our results demonstrated that immunohistochemistry using the mIDH1 R132H antibody and allele specific PCR with microdissection are highly sensitive detection methods for the most frequent mutation R132H of the IDH1 gene. In the second part, we tried to prove the principle that the point mutation of IDH1 R132H can be detected by PCR based-allele specific hybridization on glass slides. Diagnositic discrimination was demonstrated between wild type and mutant variant of biotinylated synthetic oligonucleotides with immobilized C 18-5'-oligonucleotide R132H allele specific probe on agarose glass slide at 50 °C hybridization incubated overnight with 60 °C washing temperature, the optimized concentrations of biotinylated synthetic oligonucleotide probes (M and Wt) and C18-5'-o1igonucleotide R132H allele specific probe were 10umol and O.1mM respectively. It showed better discriminative results while compared to that of on traditional silylated glass slide. In conclusion, IHC is a rapid, sensitive and inexpensive routine screening test. However, DNA sequencing is still a gold standard for all the point mutation detection. And PCR-based allele-specific amplification followed by allele-specific on agarose glass slide has been shown in this study to be an alternative molecular detection method that can be used in the routine histopathology laboratory.

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