Method for co-registration of high-resolution specimen PET-CT with histopathology to improve insight into radiotracer distributions.

Background: As the spatial resolution of positron emission tomography (PET) scanners improves, understanding of radiotracer distributions in tissues at high resolutions is important. Hence, we propose a method for co-registration of high-resolution ex vivo specimen PET images, combined with computed tomography (CT) images, and the corresponding specimen histopathology.

Methods: We applied our co-registration method to breast cancer (BCa) specimens of patients who were preoperatively injected with 0.8 MBq/kg [ $_{}^{18}$ F]fluorodeoxyglucose ([¹⁸F]FDG). The method has two components. First, we used an image acquisition scheme that minimises and tracks tissue deformation: (1) We acquired sub-millimetre (micro)-PET-CT images of ±2 mm-thick lamellas of the fresh specimens, enclosed in tissue cassettes. (2) We acquired micro-CT images of the same lamellas after formalin fixation to visualise tissue deformation. (3) We obtained 1 hematoxylin and eosin (H&E) stained histopathology section per lamella of which we captured a digital whole slide image (WSI). Second, we developed an automatic co-registration algorithm to improve the alignment between the micro-PET-CT images and WSIs, guided by the micro-CT of the fixated lamellas. To estimate the spatial co-registration error, we calculated the distance between corresponding microcalcifications in the micro-CTs and WSIs. The co-registered images allowed to study standardised uptake values (SUVs) of different breast tissues, as identified on the WSIs by a pathologist.

Results: We imaged 22 BCa specimens, 13 cases of invasive carcinoma of no special type (NST), 6 of invasive lobular carcinoma (ILC), and 3 of ductal carcinoma in situ (DCIS). While the cassette framework minimised tissue deformation, the best alignment between the micro-PET-CT images and WSIs was achieved after deformable co-registration. We found an overall average co-registration error of 0.74 ± 0.17 mm between the micro-PET images and WSIs. (Pre)malignant tissue (including NST, ILC, and DCIS) generally showed higher SUVs than healthy tissue (including healthy glandular, connective, and adipose tissue). As expected, inflamed tissue and skin also showed high uptake.

Conclusions: We developed a method to co-register micro-PET-CT images of surgical specimens and WSIs with an accuracy comparable to the spatial resolution of the micro-PET images. While currently, we only applied this method to BCa specimens, we believe this method is applicable to a wide range of specimens and radiotracers, providing insight into distributions of (new) radiotracers in human malignancies at a sub-millimetre resolution.