Fluorescent Molecular Imaging Can Improve Intraoperative Sentinel Margin Detection in Oral Squamous Cell Carcinoma

Abstract

In head and neck cancer, a major limitation of current intraoperative margin analysis is the ability to detect areas most likely to be positive based on specimen palpation, especially for larger specimens where sampling error limits detection of positive margins. This study aims to prospectively examine the clinical value of fluorescent molecular imaging to accurately identify "the sentinel margin," the point on a specimen at which the tumor lies closest to the resected edge in real-time during frozen section analysis. Methods: Eighteen patients with oral squamous cell carcinoma were enrolled into a prospective clinical trial and infused intravenously with 50 mg of panitumumab-IRDye800CW 1-5 d before surgery. Resected specimens were imaged in a closed-field near-infrared optical imaging system in near real-time, and custom-designed software was used to identify locations of highest fluorescence on deep and peripheral margins. The surgeon identified the sentinel margin masked to optical specimen mapping, and then the regions of highest fluorescence were identified and marked for frozen analysis. Final pathology based on specimen reconstruction was used as reference standard. Results: Resected specimens were imaged in the operating room, and fluorescence had a higher interobserver agreement with pathology (Cohen κ value 0.96) than the surgeon (Cohen κ value of 0.82) for the location of the closest margin. Plotting margin distance at the predicted sentinel margin location of each observer versus the actual closest margin distance at pathology demonstrated best correlation between fluorescence and pathology (R² = 0.98) with surgeon (R² = 0.75). Conclusion: Fluorescence imaging can improve identification of the sentinel margin in head and neck cancer resections, holding promise for rapid identification of positive margins and improved oncologic outcomes.

Keywords: fluorescent image-guided surgery; head and neck cancer; oral squamous cell carcinoma; surgical oncology; tumor margins.

Graphical abstract

FIGURE 1.

Study workflow is demonstrated in representative patient 18 with right-sided retromolar trigone squamous cell carcinoma. Patient was infused intravenously with 50 mg of panitumumab-IRDye800CW 3 d before surgery. From left to right, workflow shows lesion in vivo and then ex vivo, examining mucosal (A) and deep surfaces of resection (B). On both surfaces, surgeon and fluorescence were in agreement as to location of sentinel margin. Sampled tissue from these locations underwent closed-field fluorescent imaging and then hematoxylin and eosin (H&E) staining to evaluate margin distance. (C) Mucosal surface fluorescent sentinel margin analysis in this patient as demonstrated by a mask manually fitted around periphery of specimen within 1 mm of resection edge followed by graph showing the raw fluorescence data of each point along mask in 8-bit gray scale format enabling isolation of area of highest fluorescence on periphery (represented by red star). (D) Deep surface fluorescent sentinel margin analysis as demonstrated using 3-dimensional signal-mapping tool to scale and isolate area of highest fluorescence intensity (represented by red star and arrow).

FIGURE 2.

Correlation between distance at predicted sentinel margin by observer with distance of closest margin on final pathology.

FIGURE 3.

(A) Specimen evaluation workflow demonstrating partial glossectomy resection in situ, ex vivo specimen imaged in brightfield and closed-field fluorescence, followed by a depiction of specimen bread-loafed at final pathology. (B) Main centrally located tumor came closest to specimen edge superiorly, at a point accurately identified by both fluorescence and surgeon to be closest margin for this tumor (3 mm). Smaller separate focus of tumor was not macroscopically visible and registered as third highest region of fluorescent signal on peripheral margin. Blue dots outline tumor boundaries on mucosal surface. BF = brightfield; FLU = fluorescence; H&E = hematoxylin and eosin.

FIGURE 4.

(A) Magnified view of 2 bread loafs demonstrating margin distance of 3 mm, where surgeon and fluorescence predicted sentinel margin on main tumor, compared with margin distance of 1 mm where a sperate secondary focus of tumor came close to anterior specimen edge. (B) Slides confirming presence of squamous cell carcinoma and EGFR expression at point on each tumor focus closest to margin edge. In main tumor, EGFR expression was quantified at 81.2% compared with 43.2% at secondary focus. +ve = positive; H&E = hematoxylin and eosin.

FIGURE 5.

Cases in which fluorescence sentinel margin identification demonstrated improved identification of true closest margin when compared with conventional analysis technique by surgeon. (A) Patient 1 with a pT3 right lateral tongue squamous cell carcinoma infused 2 d before surgery. (B) Patient 2 with pT4a right lateral tongue squamous cell carcinoma infused 1 d before surgery. (C) Patient 13 with a pT4a right retromolar squamous cell carcinoma infused 2 d before surgery. BF = brightfield; Flu = fluorescence; H&E = hematoxylin and eosin.