Genotype-targeted local therapy of glioma

Abstract

Aggressive neurosurgical resection to achieve sustained local control is essential for prolonging survival in patients with lower-grade glioma. However, progression in many of these patients is characterized by local regrowth. Most lower-grade gliomas harbor isocitrate dehydrogenase 1 (IDH1) or IDH2 mutations, which sensitize to metabolism-altering agents. To improve local control of IDH mutant gliomas while avoiding systemic toxicity associated with metabolic therapies, we developed a precision intraoperative treatment that couples a rapid multiplexed genotyping tool with a sustained release microparticle (MP) drug delivery system containing an IDH-directed nicotinamide phosphoribosyltransferase (NAMPT) inhibitor (GMX-1778). We validated our genetic diagnostic tool on clinically annotated tumor specimens. GMX-1778 MPs showed mutant IDH genotype-specific toxicity in vitro and in vivo, inducing regression of orthotopic IDH mutant glioma murine models. Our strategy enables immediate intraoperative genotyping and local application of a genotype-specific treatment in surgical scenarios where local tumor control is paramount and systemic toxicity is therapeutically limiting.

Keywords: glioma; intraoperative diagnostics; local therapy; metabolic therapeutics.

Fig. 1.

IDH1-mutated diffuse astrocytomas shows local disease progression. (A) This representative patient with diffuse astrocytoma illustrates disease progression adjacent to the initial lesion. (B) Retrospective analysis of 130 patients with IDH-mutated diffuse astrocytomas reveals local progression within 2 cm of the initial lesion in 81.8% of patients. (C) Median progression-free survival was 4.7 y for local failure vs. 5.1 y for distal failure.

Fig. 2.

Proposed intraoperative workflow for genotype-targeted therapy of glioma. Current surgical treatment strategy for patients with nonenhancing brain lesions is maximal resection. Here, we establish a modern paradigm for establishing a molecular diagnosis of a tumor specimen (red arrow) to guide the delivery of a genotype-directed local therapy at the surgical margin during neurosurgical resection (blue arrow) to target the surrounding infiltrative disease (Bottom).

Fig. 3.

MP formulation for sustained release of NAMPTis shows selective in vitro activity against IDH-mutated cells. SEM revealed (A) drug crystals in the formulation with highest drug loading and (B) pure MP composition in a formulation with 4.5% drug loading. (C) Mean particle size is 3.4 ± 1.6 μm as measured by SEM imaging followed by processing by ImageJ software. (D) GMX-1778 MPs were dispersed in artificial cerebrospinal fluid and placed at 37 °C. At various time points, the particles were separated by centrifugation (1,500 × g for 10 min). The supernatant was collected for HPLC analysis, and the particles were suspended in fresh release medium. Data are represented as mean ± SD (n = 3). (E) Treatment of MGG152, an IDH1 R132H-mutated human glioma cell line, with MPs containing a total of 1 µM GMX-1778 or 1 µM FK866 reveals a time-dependent decrease in cell viability compared with cells treated with similarly prepared compound-free MPs over 72 h (96.3 ± 0.2% for GMX-1778, n = 3 and 80.5 ± 3.4% for FK866, n = 3). HPLC analysis of GMX-1778 in the media of cells treated with the sustained release MP formulation revealed concentrations of 40.4 ± 2.3 nM at 24 h and 63.8 ± 3.7 nM at 72 h. (F) The time-dependent activity of the GMX-1778 MP formulation was noted in an additional IDH1 R132C-mutated chondrosarcoma cell line HT1080 (red; n = 3) and IDH1 R132H-mutated glioma cell line MGG119 (brown) compared with the IDH wild-type glioblastoma cell lines U87 (dark blue; n = 3) and Hs683 (light blue; n = 3). mt, mutant; wt, wild type.

Fig. 4.

Method for detection of recurrent mutations noted in glioma allows for rapid molecular genotyping to guide intraoperative decision for use of targeted local therapy. (A) The rapid genotyping assay utilizes Taqman-based probes for fluorescence-based detection of mutant alleles with clamping of the wild-type allele amplicon via peptide nucleic acid oligonucleotides, allowing for detection of tumor variants to an allelic fraction of 1% within 30 min. (B) Validation of genotyping assay correlates mutation call for IDH1 R132, TERT promoter variants, H3F3A K27M, and BRAF V600E in 87 brain tumor specimens from a clinically annotated database. EGFR, epidermal growth factor receptor; GBM, glioblastoma; IHC, immunohistochemistry; MET, tyrosine-protein kinase Met; MGMT, O6-methylguanine DNA methyltransferase; PNA, peptide nucleic acid; Rn, normalized reporter value.

Fig. 5.

Intratumoral implantation of sustained release MP formulation shows in vivo activity and increased survival in IDH-mutated orthotopic model. (A) To assess in vivo activity of MP formulation, U87- or MGG152-expressing luciferase (2 × 10⁵ cells) was intracerebrally implanted in SCID mice. Bioluminescence imaging was performed 15 d after implantation to establish a baseline. MPs containing a mass of GMX-1778 to reach a concentration of 5 µM in a sphere with radius 2 mm were implanted at that time point. Bioluminescence imaging was performed on a weekly basis. No significant difference in survival was noted with GMX-1778 MP implantation in the IDH wild-type U87 glioblastoma orthotopic model (n = 4 for blank and n = 5 for GMX-1778 MPs). PLGA, poly(lactide-coglycolide). GMX-1778 MPs (B) significantly decreased tumor growth and (C) increased survival in the orthotopic MGG152 model (58.5 d, n = 6 for blank vs. 79.0 d, n = 8 for GMX-1778 MPs; P < 0.01; Cox proportional hazard ratio). wt, wild type.