Resected tumor tissues were processed using standard clinical techniques. IDH1 R132H analysis was confirmed by immunohistochemistry and DNA sequencing as previously described (Yip et al., 2012 (link); Agarwal et al., 2013 (link)). Paraffin sections of the intracranial tumor specimens were stained with IDH1 R132H mutation-specific antibodies (1:50; H09 clone, Dianova). The IDH forward primer (5′-ACC AAA TGG CAC CAT ACG A-3′) and reverse primer (5′-GCA AAATCA CAT TAT TGC CAA C-3′) were designed to amplify exon 4 (codon R132) of the IDH gene.
H09 clone
Manufactured by Dianova
Sourced in Germany
The H09 clone is a laboratory equipment product designed for specific scientific applications. It serves a core function within the research environment. No further details are provided to maintain an unbiased and factual approach.
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7 protocols using h09 clone
1
IDH1 R132H Mutation Analysis in Gliomas
2
Molecular Profiling of CNS Tumors
3
Comprehensive Molecular Profiling of CNS Tumors
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The operative tissue samples were processed using standard clinical techniques. The IDH1 mutation status was assessed by immunohistochemistry analysis using the H09 clone (Dianova, Hamburg, Germany) generated against the R132H mutant of IDH1. Fluorescent in situ hybridization testing was performed for assessing 1p/19q codeletion. The TERT promoter mutation status was assigned by sequencing. All the cases were evaluated and re-classified according to the WHO CNS5 criteria by two experienced pathologists who were blinded to the findings of imaging. The final decision was made by a third pathologist in case of any discordance in the results.
4
Profiling IDH1/IDH2 Mutations in Surgical Specimens
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Surgical specimens were analyzed by two pathologists (H.T. and A.K.) and diagnosed according to the WHO classification [19 ]. IDH1 R132H immunohistochemical (IHC) analysis (H09 clone, Dianova, Hamburg, Germany; 1:100) was performed in formalin-fixed, paraffin imbedded section using the avidin-biotin-peroxide method (Vector, Burlingame, CA, USA) with diaminobenzidine as the chromogen and counterstained with hematoxylin.
For cases showing negative staining for IDH1 R132H, DNA sequencing for IDH1 and IDH2 was analyzed. Genomic DNA was extracted from paraffin-embedded sections, and as described previously [20 (link),21 (link)], PCR amplification was performed by using primer sets (forward: 5’-CGGTCTTCAGAGAAGCCATT-3’ , and reverse 5’-TTCATACCTTGCTTAATGGGTGT-3’) at codon 132 for the IDH1 gene and (forward: 5’-AATTTTAGGACCCCCGTCTG-3’ , and reverse 5’-CTGCAGAGACAAGAGGATGG-3’) at codon 172 for the IDH2 gene. The PCR products were then sequenced on a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with a Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) in accordance with the manufacturer’s instructions.
For cases showing negative staining for IDH1 R132H, DNA sequencing for IDH1 and IDH2 was analyzed. Genomic DNA was extracted from paraffin-embedded sections, and as described previously [20 (link),21 (link)], PCR amplification was performed by using primer sets (forward: 5’-CGGTCTTCAGAGAAGCCATT-3’ , and reverse 5’-TTCATACCTTGCTTAATGGGTGT-3’) at codon 132 for the IDH1 gene and (forward: 5’-AATTTTAGGACCCCCGTCTG-3’ , and reverse 5’-CTGCAGAGACAAGAGGATGG-3’) at codon 172 for the IDH2 gene. The PCR products were then sequenced on a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with a Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) in accordance with the manufacturer’s instructions.
5
Immunohistochemical Analysis of IDH1 (R132H)
6
Naïve IDH1/2 wild-type GBM Analysis
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From the archives of the Department of Human Pathology of Adult and Evolutive Age (University of Messina, Messina, Italy) and the Department of Medical, Surgical Sciences, and Advanced Technologies ‘G.F. Ingrassia’ (University of Catania, Catania, Italy), 40 consecutive patients (26 men, 14 women; mean age, 55.85 years; range, 35–73 years) surgically treated for naȉve IDH1/2 wild-type GBM were included in the present analysis. Initially, during routine pathology diagnostics, IDH1/2 status was analyzed by immunohistochemistry utilizing mouse monoclonal antibody IDH1 R132H (work dilution 1:50, clone H09, Dianova GmbH, catalogue n. 075874). Furtherly, the IDH1/2 wild type status on the same casuistry was verified utilizing IDH1/2 mutation detection kit for real-time PCR (EntroGen, product code IDH-RT38). For all cases, primary as well as recurrent neoplasms were available and histologically reviewed by two independent observers according to World Health Organization (WHO) 2016 criteria. Clinical characteristics of each patient, including age, sex, MGMT promoter methylation status assessed by quantitative polymerase chain reaction, disease-free interval, and overall survival were available from the medical records of our institution.
7
IDH1 Mutation and 1p/19q Codeletion Assessment
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IDH1 mutation status was assessed by immunohistochemistry using an antibody specific for the R132H mutation (clone H09, 1:100 dilution; Dianova). Consequently, for the purpose of this study, the designation of a tumour as 'IDH1 positive' refers only to the R132H mutation. A cell was considered positive if it demonstrated dark cytoplasmic staining with or without dark nuclear staining. This method has been shown to have high sensitivity and specificity when compared to polymerase chain reaction.[8 (link)]
The status of chromosomes 1p and 19q was assessed by fluorescence in situ hybridization (FISH) on paraffin-embedded tissue with human probes localizing to 1p32, 1q25, 19q13, and 19p13. The test was conducted by counting the number of probe signals within 40 tumour cells for each of the two probes and calculating the average probe number per cell. Tumours containing a 1p36/1q25 ratio less than 0.88 and a 19q13/19p13 ratio less than 0.74 were considered to be deleted for 1p36 and 19q13, respectively.
The status of chromosomes 1p and 19q was assessed by fluorescence in situ hybridization (FISH) on paraffin-embedded tissue with human probes localizing to 1p32, 1q25, 19q13, and 19p13. The test was conducted by counting the number of probe signals within 40 tumour cells for each of the two probes and calculating the average probe number per cell. Tumours containing a 1p36/1q25 ratio less than 0.88 and a 19q13/19p13 ratio less than 0.74 were considered to be deleted for 1p36 and 19q13, respectively.
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