Fully Automated Image-Based Multiplexing of Serial PET/CT Imaging for Facilitating Comprehensive Disease Phenotyping

Combined PET/CT imaging provides critical insights into both anatomic and molecular processes, yet traditional single‐tracer approaches limit multidimensional disease phenotyping; to address this, we developed the PET Unified Multitracer Alignment (PUMA) framework—an open‐source, postprocessing tool that multiplexes serial PET/CT scans for comprehensive voxelwise tissue characterization. Methods: PUMA utilizes artificial intelligence–based CT segmentation from multiorgan objective segmentation to generate multilabel maps of 24 body regions, guiding a 2-step registration: affine alignment followed by symmetric diffeomorphic registration. Tracer images are then normalized and assigned to red–green–blue channels for simultaneous visualization of up to 3 tracers. The framework was evaluated on longitudinal PET/CT scans from 114 subjects across multiple centers and vendors. Rigid, affine, and deformable registration methods were compared for optimal coregistration. Performance was assessed using the Dice similarity coefficient for organ alignment and absolute percentage differences in organ intensity and tumor SUV_mean. Results: Deformable registration consistently achieved superior alignment, with Dice similarity coefficient values exceeding 0.90 in 60% of organs while maintaining organ intensity differences below 3%; similarly, SUV_mean differences for tumors were minimal at 1.6% ± 0.9%, confirming that PUMA preserves quantitative PET data while enabling robust spatial multiplexing. Conclusion: PUMA provides a vendor-independent solution for postacquisition multiplexing of serial PET/CT images, integrating complementary tracer data voxelwise into a composite image without modifying clinical protocols. This enhances multidimensional disease phenotyping and supports better diagnostic and therapeutic decisions using serial multitracer PET/CT imaging.