Digital analysis of immunostaining and tissue distribution of the R132H mutant form of isocitrate dehydrogenase 1 in diffuse low or high-grade adult astrocytomas

Background: Diffuse gliomas are common primary tumors of the central nervous system. The current diagnosis of such lesions, which include astrocytomas, depends on molecular features, including the presence of mutation in the enzyme isocitrate dehydrogenase(IDH). The wild isoforms of IDH-1(cytosolic) and 2(mitochondrial) convert isocitrate into α-ketoglutarate(αCG), promoting the reduction of NADP+ to NADPH. Both αCG and NADPH are antioxidants. The most common mutant form of IDH1(R132H) has a gain of function in which αCG is converted into D2-hydroxyglutarate, an oncogenic compound that consumes NADPH. Thus, a functional relationship is observed between IDH1, IDH2 and IDH1R132H. Objective: To perform neuropathological characterization of diffuse astrocytomas in adults as well as to investigate the immunostaining and possible alteration in tissue distribution of IDH1, IDH2 and IDH1R132H, considering the presence of the R132H mutation and histological grade. Methods: We evaluated 25 surgical specimens from adults with diffuse gliomas, which were molecularly and immunophenotypically characterized as astrocytomas of low- or high-grade (2 (n=10) or 4 (n=15), respectively), according to the World Health Organization current criteria. We also analyzed the immunostaining pattern of IDH1, IDH2 and IDH1R132H, as well as their tissue distribution (mean % of positivity assessed by using ImageJ® software), considering the R132H mutation and the lesion histological grade. Results: Regarding age at diagnosis(< or ≥55 years) and R132H immunopositivity, the lesions were classified as: astrocytoma, IDH-mutant, grade 2 (5/25); astrocytoma, not otherwise specified (NOS), grade 2 (5/25); glioblastoma, IDH-wildtype, grade 4 (9/25) or astrocytoma, NOS, grade 4 (6/25). IDH1, IDH2 and IDH1R132H showed a diffuse cytoplasmic staining pattern, regardless of grade. When comparing low- and high-grade lesions, the tissue distribution of IDH1 was lower in the former (14.20±1.70% and 16.40±1.60%, respectively; p=0.0051), no difference being observed for IDH2 (15.10±1.80% and 14.90±2.0%; p=0.6773) and R132H (5.20±5.60% and 1.80±4.80%; p=0.1234). Specifically, low-grade lesions with (5/10) or without (5/10) the mutation showed no differences regarding tissue distribution of IDH1 (14.40±2.30% and 14.10±0.90%, respectively; p=0.2963) or IDH2(15.60±1.0% and 14.70±2.40%; p=0.8345). Irrespective of the histological grade, there was correlation neither between tissue expressions of R132H and IDH1 nor between R132H and IDH2. Conclusion: In the present series, IDH1R132H was more frequent in grade 2 than in grade 4 lesions. Furthermore, our data suggest that (1) such mutation does not interfere with tissue expression of wild-type IDH1 and IDH2, and (2) the wild-type IDH1 allele would ensure protein synthesis at physiological levels.