Quantitative characterization of duodenal gastrinoma autofluorescence using multiphoton microscopy

Abstract

Background: Duodenal gastrinomas (DGASTs) are neuroendocrine tumors that develop in the submucosa of the duodenum and produce the hormone gastrin. Surgical resection of DGASTs is complicated by the small size of these tumors and the tendency for them to develop diffusely in the duodenum. Endoscopic mucosal resection of DGASTs is an increasingly popular method for treating this disease due to its low complication rate but suffers from poor rates of pathologically negative margins. Multiphoton microscopy can capture high-resolution images of biological tissue with contrast generated from endogenous fluorescence (autofluorescence [AF]) through two-photon excited fluorescence (2PEF). Second harmonic generation is another popular method of generating image contrast with multiphoton microscopy (MPM) and is a lightscattering phenomenon that occurs predominantly from structures such as collagen in biological samples. Some molecules that contribute to AF change in abundance from processes related to the cancer disease process (e.g., metabolic changes, oxidative stress, and angiogenesis). Study Design/Materials and Methods: MPM was used to image 12 separate patient samples of formalin-fixed and paraffin-embedded duodenal gastrinoma slides with a second-harmonic generation (SHG) channel and four 2PEF channels. The excitation and emission profiles of each 2PEF channel were tuned to capture signal dominated by distinct fluorophores with well-characterized fluorescent spectra and known connections to the physiologic changes that arise in cancerous tissue. Results: We found that there was a significant difference in the relative abundance of signal generated in the 2PEF channels for regions of DGASTs in comparison to the neighboring tissues of the duodenum. Data generated from texture feature extraction of the MPM images were used in linear discriminant analysis models to create classifiers for tumor versus all other tissue types before and after principal component analysis (PCA). PCA improved the classifier accuracy and reduced the number of features required to achieve maximum accuracy. The linear discriminant classifier after PCA distinguished between tumor and other tissue types with an accuracy of 90.6%−93.8%. Conclusions: These results suggest that multiphoton microscopy 2PEF and SHG imaging is a promising label-free method for discriminating between DGASTs and normal duodenal tissue which has implications for future applications of in vivo assessment of resection margins with endoscopic MPM.

Mueller matrix polarization imaging of gastrinoma shows promise for tumor localization

“…All specimens were placed in 10% neutral-buffered formalin prior to paraffin embedding. A total of 11 different specimens satisfied the inclusion criteria as described below and were used for this study 14 .…”

“…Previous studies have found that a majority of duodenal gastrinomas (DGASTs) were localized between Brunner's Glands (BGs) 12 . BGs are a suspected precursor to DGASTs due to the presence of gastrinexpressing endocrine cells within the duodenal BGs that exhibit similar characteristics to that of a tumor 14 . While conventional whole-body imaging techniques allow for detection of gastrinomas when patients present with symptoms, these methods can only reliably detect large (>2cm) lesions.…”

“…MPM quantifies tissue fluorescence and other non-linear processes such as second harmonic generation at high resolution and contrast, as the use of excitation of light of longer wavelengths in MPM allows for deeper tissue sample penetration depth, which is essential for probing through tissue layers deep within the small intestine. 18 Fluorescence imaging with tumor-targeted contrast agents is a promising technique as a safe intraoperative imaging approach for wide-field localization 14 . OCT reconstructs depth-resolved microstructural maps of tissues with a spatial resolution at the µm level, which can assist in detecting early-stage cancers of premalignant lesions that exhibit extracellular and microstructural disruptions 19 .…”