Acquisition and annotation in high resolution in vivo digital biopsy by confocal microscopy for diagnosis in oral precancer and cancer

Abstract

Introduction: Scanned fibre endomicroscopes are full point-scanning confocal microscopes with submicron lateral resolution with an optical slice thickness thin enough to isolate individual cell layers, allow active positioning of the optical slice in the z-axis and collection of megapixel images. Here we present descriptive findings and a brief atlas of an acquisition and annotation protocol high resolution in vivo capture of oral mucosal pathology including oral squamous cell carcinoma and dysplasia using a fluorescence scanned fibre endomicroscope with 3 topical fluorescent imaging agents: fluorescein, acriflavine and PARPi-FL.

Methods: Digital biopsy was successfully performed via an acquisition protocol in seventy-one patients presenting for investigation of oral mucosal abnormalities using a miniaturized, handheld scanned fibre endoscope. Multiple imaging agents were utilized and multiple time points sampled. Fifty-nine patients had a matched histopathology correlating in location with imaging. The images were annotated back to macrographic location using a purpose-built software, MouthMap™.

Results: Acquisition and annotation of cellular level resolved images was demonstrated with all 3 topical agents. Descriptive observations between clinically or histologically normal oral mucosa showed regular intranuclear distance, a regular nuclear profile and fluorescent homogeneity. This was dependent on the intraoral location and type of epithelium being observed. Key features of malignancy were a loss of intranuclear distance, disordered nuclear clustering and irregular nuclear fluorescence intensity and size. Perinuclear fluorescent granules were seen in the absence of irregular nuclear features in lichenoid inflammation.

Discussion: High resolution oral biopsy allows for painless and rapid capture of multiple mucosal sites, resulting in more data points to increase diagnostic precision. High resolution digital micrographs can be easily compared serially across multiple time points utilizing an annotation software. In the present study we have demonstrated realization of a high-resolution digital biopsy protocol of the oral mucosa for utility in the diagnosis of oral cancer and precancer..

Keywords: OSCC (oral squamous cell carcinoma); confocal; digital health; dysplasia; endomicroscope; fluorescence; oral cancer; oral potentially malignancy disorders (OPMDs).

Figure 1

(A) Software interface of the InVivage® demonstrating laser and capture settings on the left-hand side and location and patient selection on the right-hand side panel (B) Handheld intraoral probe and command handpiece allows the user to adjust settings without de-gloving (C) The portable InVivage® unit can be transported alongside a conventional dental chair.

Figure 2

MouthMap™ interface showing macrographs allocated into the 20 intraoral locations.

Figure 3

Site specific micrographs of the right lateral surface of the tongue visualized in MouthMap™ demonstrating time stamp, location, and laser setting image macros. Image captures are 475µm (W) x 475µm (H).

Figure 4

Fluorescence images capture with molecule-targeted 1µM topical PARPi-FL (A) and pan-cytoarchitectural fluorescent agents topical 0.1% fluorescein (B) and 0.1% acriflavine (C). Images are 335µm (W) x 475µm (H).

Figure 5

(A) Bacterial plaque and pellicle debris visualized on the gingiva; (B) Candidal hyphae observed in verrucous hyperplasia of the buccal mucosa with 0.1% fluorescein. Images are 422 µm (W) x 400µm (H).

Figure 6

Regional mucosal variation with the InVivage® utilizing topical 0.1% fluorescein (A) keratinized epithelium of the hard palate, (B) specialized epithelium with papillae of the dorsal tongue, (C) transitional epithelium of the lateral tongue, (D) non-keratinized epithelium of the soft palate, (E) gingival epithelium, (F) buccal mucosa with fluorescein. Images are 475µm (W) x 400µm (H).

Figure 7

Oral squamous cell carcinoma of the left edentulous maxillary alveolus (A) Micrograph using topical 0.1% fluorescein [282µm (W) x 475µm (H)], (B) Micrograph using 0.1% acriflavine [282µm (W) x 475µm (H)], (C) Digital macrograph of red and white lesion on left maxillary alveolus, inset upper right corner is enlargement of area using magnifying visual tool in MouthMap ™ (D) H&E-stained histopathology (500µms scale bar).

Figure 8

Micrography over time with topical 0.1% acriflavine demonstrating oral squamous cell carcinoma at Visit 1 and subsequent clinically normal non-keratinized mucosa adjacent to radial forearm free flap following resection. Images are 475µm (W) x 475µm (H).

Figure 9

Macroscopic and micrographic images with topical 0.1% acriflavine demonstrating mild dysplasia over time. Macrographic photos, with right upper corner magnification viewed in MouthMap™, taken pre scalpel biopsy which was performed at Visit 1. Micrographically, the cellular outline and a regular intranuclear distance can be observed despite variability in nuclear size and fluorescence intensity. Images are 475µm (W) x 475µm (H).

Figure 10

Mild dysplasia of the gingiva between the upper left central and lateral incisor (A) visualized with 1µM topical PARPi-FL demonstrating positive nuclei [275µm (W) x 475µm (H)] (B) visualized with topical 0.1% acriflavine [275µm (W) x 475µm (H)] (C) macrographic photo with right upper corner magnification views in MouthMap™ and (D) H&E histopathology (100 µm scale bar).

Figure 11

High-grade dysplasia of the lower left alveolus [lower panels 475µm (W) x 475µm (H)] and the contralateral clinically normal appearing mucosa [upper panels 475µm (W) x 300µm (H)]. Visualized using topical 1µM PARPi-FL, 0.1% fluorescein and 0.1% acriflavine.

Figure 12

(A) Hyperkeratosis of the upper right gingiva (clinically non-dysplastic leukoplakia) with 0.1% topical fluorescein 475µm (W) x 475µm (H). Note the regularity of cellular dimensions despite variable fluorescent signal intensities. (B) Macrographic photo with right upper corner magnification views in MouthMap™. (C) H&E slide (scale bar 200µm).

Figure 13

Micrographs of hyperkeratosis and hyperplasia observed with 0.1% fluorescein immediately before scalpel biopsy (performed at Visit 1) and corresponding macrographic photographs. Micrographs demonstrate consistent nuclei size and regular cellular outlines. The lesion resolved 6 months later showing clinically normal lateral tongue mucosa.

Figure 14

Perinuclear granules observed in lesions showing oral lichenoid inflammation, observed with both (A) topical 0.1% fluorescein and (B) topical 0.1% acriflavine.