Background: Accurate and rapid intraoperative tumor margin assessment remains a major challenge in surgical oncology. Current gold-standard methods, such as frozen section histology, are time-consuming, operator-dependent, and prone to misclassification, which limits their clinical utility. Objective: To develop and evaluate a novel hyperspectral imaging (HSI) workflow that integrates deep learning with three-dimensional (3D) tumor modeling for real-time, label-free tumor margin delineation in head and neck squamous cell carcinoma (HNSCC). Methods: Freshly resected HNSCC samples were snap-frozen and imaged ex vivo from multiple perspectives using a standardized HSI protocol, resulting in a 3D model derived from HSI. Each sample was serially sectioned, stained, and annotated by pathologists to create high-resolution 3D histological reconstructions. The volumetric histological models were co-registered with the HSI data (n = 712 Datacubes), enabling voxel-wise projection of tumor segmentation maps from the HSI-derived 3D model onto the corresponding histological ground truth. Three deep learning models were trained and validated on these datasets to differentiate tumor from non-tumor regions with high spatial precision. Results: This work demonstrates strong potential for the proposed HSI system, with an overall classification accuracy of 0.98 and a tumor sensitivity of 0.93, underscoring the system’s ability to reliably detect tumor regions and showing high concordance with histopathological findings. Conclusion: The integration of HSI with deep learning and 3D tumor modeling offers a promising approach for precise, real-time intraoperative tumor margin assessment in HNSCC. This novel workflow has the potential to improve surgical precision and patient outcomes by providing rapid, label-free tissue differentiation.

Background: In vivo visualization of malignant tumors remains the main challenge during head and neck cancer surgery. This can result in inadequate tumor margin assessment and incomplete tumor resection, adversely affecting patient outcomes. Hyperspectral imaging (HSI) is a promising approach to address this issue. However, its application in surgery has been limited by the lack of medically approved HSI devices compliant with MDR regulations, as well as challenges regarding the integration into the surgical workflow. Methods: In this feasibility study, we employed endoscopic HSI during surgery to visualize the tumor sites of 12 head and neck cancer patients. We optimized the HSI workflow to minimize time required during surgery and to reduce the adaptation period needed for surgeons to adjust to the new workflow. Additionally, we implemented data processing to enable real-time classification and visualization of HSI within the intraoperative setting. HSI evaluation was conducted using principal component analysis and k-means clustering, with this clustering validated through comparison with expert annotations. Results: Our complete HSI workflow requires two to three minutes, with each HSI measurement—including evaluation and visualization—taking less than 10 s, achieving an accuracy of 79%, sensitivity of 72%, and specificity of 84%. Medical personnel became proficient with the HSI system after two surgeries. Conclusions: This study presents an HSI workflow for in vivo tissue differentiation during head and neck cancer surgery, providing accurate and visually accessible results within minimal time. This approach enhances the in vivo evaluation of tumor margins, leading to more clear margins and, consequently, improved patient outcomes.