Clinical label-free biochemical and metabolic fluorescence lifetime endoscopic imaging of precancerous and cancerous oral lesions

Abstract

Introduction: Incomplete head and neck cancer resection occurs in up to 85% of cases, leading to increased odds of local recurrence and regional metastases; thus, image-guided surgical tools for accurate, in situ and fast detection of positive margins during head and neck cancer resection surgery are urgently needed. Oral epithelial dysplasia and cancer development is accompanied by morphological, biochemical, and metabolic tissue and cellular alterations that can modulate the autofluorescence properties of the oral epithelial tissue.

Objective: This study aimed to test the hypothesis that autofluorescence biomarkers of oral precancer and cancer can be clinically imaged and quantified by means of multispectral fluorescence lifetime imaging (FLIM) endoscopy.

Methods: Multispectral autofluorescence lifetime images of precancerous and cancerous lesions from 39 patients were imaged in vivo using a novel multispectral FLIM endoscope and processed to generate widefield maps of biochemical and metabolic autofluorescence biomarkers of oral precancer and cancer.

Results: Statistical analyses applied to the quantified multispectral FLIM endoscopy based autofluorescence biomarkers indicated their potential to provide contrast between precancerous/cancerous vs. healthy oral epithelial tissue.

Conclusion: To the best of our knowledge, this study represents the first demonstration of label-free biochemical and metabolic clinical imaging of precancerous and cancerous oral lesions by means of widefield multispectral autofluorescence lifetime endoscopy. Future studies will focus on demonstrating the capabilities of endogenous multispectral FLIM endoscopy as an image-guided surgical tool for positive margin detection during head and neck cancer resection surgery.

Keywords: Autofluorescence biomarkers; Fluorescence lifetime imaging (FLIM); Oral cancer and dysplasia; Statistical analysis.

Fig. 1.

Schematic depicting the methodology of this study. First, multispectral FLIM endoscopic imaging of oral lesions is performed in a clinical setting. Second, the data is processed yielding multi-parametric FLIM feature images. Finally, statistical analysis is performed on the feature image median value to assess difference in their distributions between precancerous/cancerous and normal groups.

Fig. 2.

Scatter plots of multispectral FLIM feature image median values of moderate dysplastic (MOD-DYS), high grade dysplastic (HG-DYS), and squamous cell carcinoma (SCC) oral lesions (n=40; red points) and their paired contralateral normal tissue (blue points). Mean and standard deviation of the distribution of median values for each population are also shown. P-values resulting from two-tailed paired t-tests are shown on top of each plot.

Fig. 3.

Scatter plots of image median value distributions of absolute fluorescence intensity values for each emission spectral band comparing moderate dysplastic (MOD-DYS), high grade dysplastic (HG-DYS), and squamous cell carcinoma (SCC) oral lesions (n=32; red points) to their paired normal references (blue points). A statistically significant loss in autofluorescence was observed in precancerous/cancerous oral lesions relative to normal in all channels. P-values resulting from two-tailed paired t-tests are shown on top of each plot.

Fig. 4.

Multispectral FLIM feature images or maps of SCC (top panels) and normal (middle panels) tongue tissue from the same patient. Pixel distributions of normal and SCC maps for each feature are also compared (bottom panels). The trends observed in this representative case are consistent with the statistical findings on the feature image median value distributions from precancerous/cancerous vs. normal oral tissue.