Characterization of R132H mutation-specific IDH1 antibody binding in brain tumors

Abstract

Heterozygous point mutations of isocitrate dehydrogenase (IDH)1 codon 132 are frequent in grade II and III gliomas. Recently, we reported an antibody specific for the IDH1R132H mutation. Here we investigate the capability of this antibody to differentiate wild type and mutated IDH1 protein in central nervous system (CNS) tumors by Western blot and immunohistochemistry. Results of protein analysis are correlated to sequencing data. In Western blot, anti-IDH1R132H mouse monoclonal antibody mIDH1R132H detected a specific band only in mutated tumors. Immunohistochemistry of 345 primary brain tumors demonstrated a strong cytoplasmic and weaker nuclear staining in 122 cases. Correlation with direct sequencing of 186 cases resulted in consensus of 177 cases. Genetic retesting of cases with conflicting findings resulted in a match of 186/186 cases, with all discrepancies resolving in favor of immunohistochemistry. Intriguing is the ability of mIDH1R132H to detect single infiltrating tumor cells. The very high frequency and the distribution of this mutation among specific brain tumor entities allow the highly sensitive and specific discrimination of various tumors by immunohistochemistry, such as anaplastic astrocytoma from primary glioblastoma or diffuse astrocytoma World Health Organization (WHO) grade II from pilocytic astrocytoma or ependymoma. Noteworthy is the discrimination of the infiltrating edge of tumors with IDH1 mutation from reactive gliosis.

Figure 1

Representative Western blot of IDH1R132H‐mutated and wild type (wt) gliomas demonstrating a highly specific band at the predicted molecular weight of 46 kD only in mutated tumors (left panel). On the right panel, the same blot has been incubated with an antibody (rIDH1) detecting both mutated and wt IDH1. Abbreviations: IDH = isocitrate dehydrogenase; A III = astrocytoma World Health Organization (WHO) grade III; OA III = oligoastrocytoma WHO grade III; GBM = glioblastoma.

Figure 2

A. Lysates of tumors with IDH1R132C or IDH1R132G mutation are not detected by mIDH1R132H. One case with IDH2 mutation is also not detected. Right panel shows same blot incubated with rIDH1 to demonstrate presence of IDH1. B. HEK 293T cells overexpressing all variants of IDH1 mutations so far detected show that mIDH1R132H only detects R132H mutation (left panel). Double bands at ∼57 and ∼48 represent IDH1R132H with and without FLAG tag because of two start of translation sites in the plasmid used for overexpression. Additional weaker nonspecific bands are detected at ∼72 kD and ∼95 kD. Right panel shows re‐incubation of the same blot with rIDH1 for loading control. Abbreviations: IDH = isocitrate dehydrogenase; O II = oligodendroglioma World Health Organization (WHO) grade II; A II = astrocytoma WHO grade II; OA III = oligoastrocytoma WHO grade III; wt = wild type.

Figure 3

A. Astrocytoma World Health Organization (WHO) grade II (A II) with cytoplasmic as well as nuclear binding of mIDH1R132H (×200). B. Double immunohistochemistry of glial fibrillary acidic protein (red) and mIDH1R132H (brown) of infiltration zone of anaplastic oligodendroglioma showing that reactive astrocytes (red) are not bound by mIDH1R132H (×200). C. IDH1R132G‐mutated A II does not exhibit mIDH1R132H binding (×200). D. Infiltration zone of diffuse astrocytoma with tumor cells forming long uni‐ to bipolar processes (×200). E. Infiltration zone of oligodendroglioma WHO grade III. Although the tumor seems sharply demarcated to normal brain (right), single infiltrating tumor cells can be seen (×200). F. Tumor cells in the infiltration zone of anaplastic astrocytoma showing perivascular accumulation (×200). G. Positively stained tumor cells in close proximity to neurons as an example of perineuronal satellitosis (×400). H. Identification of loose subpial spread of tumor cells distant from tumor bulk by mIDH1R132H (×100). Abbreviation: IDH = isocitrate dehydrogenase.

Figure 4

A. High magnification of astrocytoma World Health Organization (WHO) grade II showing mIDH1R132H positive tumor cells with uni‐ to bipolar processes (×400). B. In 7 of 31 astrocytoma WHO grade III, fine stellar processes in the majority of tumor cells were seen (×200). C. Gemistocytes often showed a clearing of antibody binding in central cytoplasm (×400). D. mIDH1R132H binding of oligodendroglial tumor bulk. Cellular processes were generally not observed (×200). E. mIDH1R132H showed no binding in pilocytic astrocytoma tumor cells (×200). F. High magnification of a different case of pilocytic astrocytoma showing positive macrophages. Tumor cells show no binding of mIDH1R132H (×400). G. mIDH1R132H did not bind ependymoma, here WHO III (×200). H. The majority of meningiomas showed nonspecific staining of fibers usually close to blood vessels or around small groups of tumor cells (×200). Abbreviation: IDH = isocitrate dehydrogenase.