Dual-tracer PET of viable tumor volume and hypoxia for identification of necrosis-containing radio-resistant Sub-volumes.

Introduction: Positron emission tomography (PET) using hypoxia-selective tracers like FAZA may guide radiation dose-escalation approaches. However, poor resolution combined with slow tracer retention in relatively inaccessible target cells and slow clearance of unbound tracer results in low-contrast images, and areas where viable hypoxic tracer retaining cells and necrosis (no tracer) are intermixed may pass unnoticed during image thresholding. Here we hypothesized that a clinical feasible one-day dual tracer approach that combines a short-lived (e.g., ¹¹C labeled) metabolic tracer that provides voxel-wise information on viable tissue volume (preferably independently of tumor microenvironment) and a hypoxia marker, may limit threshold-based errors. Material and methods:¹¹C-acetate and ¹¹C-methionine uptake was quantified in tumor cell lines under tumor microenvironment-mimicking conditions of high/low O₂ (21%/0%) and pH (7.4/6.7). Next, tumor-bearing mice were administered FAZA and sacrificed 1 h (mimics a clinical low-contrast image scenario) or 4 h (high contrast) later. In addition, all mice were administered pimonidazole (hypoxia) and ¹⁴C-methionine 1 h prior to sacrifice. Tumor tissue sections were analyzed using dual-tracer autoradiography. Finally, FAZA, or FAZA normalized to ¹⁴C-methionine retention (to adjust for differences in viable tissue volume) was compared to hypoxic fraction (deduced from immune-histological analysis of pimonidazole; ground truth) in PET-mimicking macroscopic pixels with variable extent of necrosis/hypoxia. Results/conclusions: Low pH stimulated ¹¹C-acetate retention in many cell lines, and uptake was further modified by anoxia, compromising its usefulness as a universal marker of viable tumor volume. In contrast, ¹¹C-methionine was largely unaffected by the in vitro microenvironment and was further tested in mice. Necrosis increased the risk of missing hypoxia-containing pixels during thresholding and hypoxic fraction and FAZA signal correlated poorly in the low contrast-scenario. Voxel-based normalization to ¹⁴C-methionine increased the likelihood of detecting voxels harboring hypoxic cells profoundly, but did not consistently improve the correlation between the density of hypoxic cells and tracer signal.