Mutant IDH1 and thrombosis in gliomas

Abstract

Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90 % in wild-type versus 2-6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

Keywords: D-2-hydroxyglutarate; Glioma; Isocitrate dehydrogenase; Platelet; Thrombosis; Tissue factor.

Fig. 1

Microthrombi and IDH1/2 mutations in gliomas. Frequency of intratumoral microthrombi according to IDH1/2 mutation status in discovery (left) and validation (right) cohorts, in all WHO grade II-IV gliomas (a & b), grade II-III (c & d), grade IV GBM (e & f), and necrotic gliomas (g & h). P < 0.001 in panels a–b and e–h; P = 0.02 in panel c and 0.0034 in panel d. All P values were calculated by Fisher’s exact test.

Fig. 2

Coagulation-associated genes according to IDH1/2 status in gliomas. Expression of key genes known to be involved in clotting and glioma-associated thrombosis [21, 22, 33, 45, 47, 62] (Supplemental Table 5) was assessed via Illumina HiSeq 2000 RNA Sequencing platform in grade II-IV TCGA gliomas. Each row represents a single tumor, columns indicate either IDH1/2 status (aqua = IDH2 mutant, red = IDH1 mutant, black = IDH1/2 wild-type); WHO grade (peach = II, green = III, purple = IV); or gene expression as a ratio of the individual tumor mRNA relative to the mean of all IDH1/2 wild-type tumors for that gene (blue = downregulated, yellow = upregulated, white = no change). Genes in the pink shaded area are procoagulant; genes in the light blue shaded area are anti-coagulant. N = 196 IDH1/2 wild-type and 234 mutant IDH1/2 gliomas.

Fig. 3

TF by IDH1/2 status in gliomas. (a) Mean increase in methylation beta value at each CpG site in the F3 promoter of mutant IDH1/2 gliomas, relative to wild-type gliomas. CpG sites are grouped according to their location within and around the CpG island of the F3 promoter. *P < 0.01 in all 3 grades of glioma. See Supplemental Table 6 for additional details. (b) Representative H&E photomicrographs of IDH1/2 wild-type and mutant GBMs from the TMA cohort, and their corresponding tissue factor IHC. Scale bar in lower right panel = 50 µm. (c) TF IHC scores according to IDH1/2 mutation status in high-grade (III-IV) gliomas. (d) Preoperative TF-MP activity, measured in pg Factor Xa generation per ml in platelet-free plasma, obtained from the arterial blood of patients with IDH1/2 wild-type and mutant gliomas (see Table 2). Dotted line represents the cutoff for “strong TF activity” in cancer patients, as published previously [19]. In (c) and (d), each point represents a single tumor, and red triangles in (d) represent patients who developed VTE. Bars = mean ± SD.

Fig. 4

Platelet activity, calcium, and clotting time in the presence of mutant IDH1 and D-2-HG. Washed human platelets (2 × 10⁸/mL) from 3 distinct donors were stimulated by (a) 1 µg/mL collagen or (b) 0.1 U/mL thrombin in the presence or absence of D-2-HG. Platelet aggregation was measured by turbidity (Chronolog) with stirring (1000 rpm). (c) Platelets were labeled with Fura2-AM and iCa⁺⁺ levels were determined via 340nm/380nm excitation ratio under the specified conditions. (d) Washed human platelets were stimulated by thrombin or the calcium ionophore A23187 in the presence or absence of D-2-HG. (e) Human serum levels of free Ca⁺⁺ in the presence of D-2-HG (note, SD bars small and not visible). (f) Clotting time of human blood in the presence of D-2-HG. Data are expressed as clotting time beyond matched untreated controls (mean baseline = 190 seconds) ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (g) Free Ca⁺⁺ levels in serum from mice bearing flank xenografts of human gliomas with and without R132H IDH1. (h) Tail vein bleeding times for mice bearing flank xenografts of human gliomas with and without R132H IDH1.

Fig. 5

Postulated mechanism of mutant IDH1/2 suppression of local and systemic thrombosis. (a) Wild-type IDH1/2 glioma cells produce tissue factor-containing microparticles (TF-MPs, blue dots), which promote microthrombus formation within the tumor (yellow and brown objects = platelets). TF-MPs from the glioma also circulate throughout the body, increasing the risk of DVTs and PEs. (b) In contrast, mutant IDH1/2 gliomas make very little TF, and consequently release very little TF-MPs, but do produce D-2-HG (pink triangles). In the local tumor vascular bed, where permeability increases in higher-grade tumors (arrows), the reduced numbers of TF-MPs, plus the antiplatelet activity of D-2-HG, combine to prevent intratumoral thrombosis. In the systemic circulation, D-2-HG is not present at sufficient concentrations to inhibit peripheral platelet aggregation, but the lack of glioma-derived TF-MPs still results in low risk for VTE.