Correlation of Tumor Hypoxia Metrics Derived from ¹⁸F-Fluoromisonidazole Positron Emission Tomography and Pimonidazole Fluorescence Images of Optically Cleared Brain Tissue

¹⁸F-fluoromisonidazole (FMISO) positron emission tomography (PET) is a widely used noninvasive imaging modality for assessing hypoxia. We describe the first spatial comparison of FMISO PET with an ex vivo reference standard for hypoxia across whole tumor volumes. Eighteen rats were orthotopically implanted with C6 or 9L brain tumors and made to undergo FMISO PET scanning. Whole brains were excised, sliced into 1-mm-thick sections, optically cleared, and fluorescently imaged for pimonidazole using an in vivo imaging system. FMISO maximum tumor uptake, maximum tumor-to-cerebellar uptake (TCmax), and hypoxic fraction (extracted 110 minutes after FMISO injection) were correlated with analogous metrics derived from pimonidazole fluorescence images. FMISO SUVmax was not significantly different between C6 and 9L brain tumors (P = 0.70), whereas FMISO TCmax and hypoxic fraction were significantly greater for C6 tumors (P < 0.01). FMISO TCmax was significantly correlated with the maximum tumor pimonidazole intensity (ρ = 0.76, P < 0.01), whereas FMISO SUVmax was not. FMISO tumor hypoxic fraction was significantly correlated with the pimonidazole-derived hypoxic fraction (ρ = 0.78, P < 0.01). Given that FMISO TCmax and tumor hypoxic fraction had strong correlations with the pimonidazole reference standard, these metrics may offer more reliable measures of tumor hypoxia than conventional PET uptake metrics (SUVmax). The voxel-wise correlation between FMISO uptake and pimonidazole intensity for a given tumor was strongly dependent on the tumor’s TCmax (ρ = 0.81, P < 0.01) and hypoxic fraction (ρ = 0.85, P < 0.01), indicating PET measurements within individual voxels showed greater correlation with pimonidazole reference standard in tumors with greater hypoxia.