For cancer surgery to be considered fully successful, a tumor must be completely removed with no diseased tissue left behind. Tumor margin analysis in excised tissue samples is a widely-used assessment of whether a tumor was fully removed. Traditionally, margin analysis has been based on subjective evaluations of tissue differences in white light. In many cases, however, differences in cancerous and non-cancerous tissue are very difficult to discern in white light. For some cancers like head and neck squamous cell carcinoma, “positive margin” rates – rates of cancerous tissue (likely) being left behind – are as high as 40% .
Novel methods are needed to reduce positive margin rates and provide better clinical outcomes. Fluorescent dyes conjugated to tumor-specific monoclonal antibodies are emerging as a visual aid for tumor margin analysis and are proving to be particularly useful in cancers where positive margins still dominate clinical outcomes.
Evaluating Tumor Margins in Excised Tissue Samples With IRDye® 800CW-Cetuximab Fluorescent Images
Previous blog posts (Optical Probe Specificity and Dual-Modality Labeling with IRDye Near-Infrared Fluorescent Dyes and The Pivotal Role of Validation in Optical Probe Development) have highlighted in vivo and in situ clinical applications of IRDye 800CW-cetuximab. In an article published Rosenthal et.al. in Clinical Cancer Research, fluorescent contrast agents were shown to also improve visualization of cancer margins in excised tissue samples.
In this first-in-human study, 12 patients scheduled to have squamous-cell carcinoma tumors removed from the head or neck were given an infusion of IRDye 800-cetuximab prior to surgery. Fresh tumor tissue sections were imaged ex vivo with the LI-COR Pearl® Impulse imager to “determine the ability of tumor fluorescence to differentiate tumor from normal tissue and identification of positive margins” . Like the intraoperative in situ results, the histopathological ex vivo results were also promising. The authors noted that “Fluorescence in histologically confirmed tumor tissue was significantly greater (P<0.001) than negative epithelial margins, muscle, and skin for each dose” .
Rosenthal and colleagues also utilized the Odyssey® imaging platform (LI-COR Biosciences) to quantify fluorescence in slide-mounted tissue sections after imaging with Pearl Impulse imager within the surgery suite. The fluorescent images taken in the Odyssey imager were correlated with routine H&E (hematoxylin and eosin) stains to compare IRDye 800CW-cetuximab against established pathological standards, corroborating the results of the Pearl Impulse scans.
The authors concluded “Here we demonstrate for the first time that … cetuximab-IRDye 800CW can be safely administered as a tumor-specific contrast agent,” and that “The use of real-time fluorescence imaging during ablative procedures to delineate tumor margins has the potential to reduce morbidity, improve locoregional control and reduce operative time “.
Cetuximab-IRDye 800CW in the Clinic Part 2: Enhanced Pathological Assessment with Fluorescent Probes
In a 2016 article published in The Journal of Pathology: Clinical Research, Warram et.al. utilized IRDye 800CW-cetuximab to address the lack of “tools to consistently discriminate tumor and normal tissue in real-time” for pathological assessments of tumor margins in head and neck squamous cell carcinoma (HNSSC) . In this proof-of-principle study, the authors tested fluorescent assessment of diseased tissue margins against standard histological methods. 80 tumor margin assessments were collected from post-resection wound beds of 20 mice with SCC1-luc tumors after administration of IRDye 800CW-cetuximab.
The results were significant: fluorescent images improved pathologist prediction of positive tumor margins from 21/39 (49%) to 33/39 (85%), or a 36% increase in sensitivity in positive tumor margin predictions. The authors noted false negative margin predictions lead to a 90% 5-year post resection mortality rate, demonstrating the magnitude of impact that fluorescent-guided tumor margin analysis may have on patient outcomes.
Figure 1: Fluorescent analysis of primary tumor specimen . Circles represent positive or negative biopsy-confirmed cancer cells, showing the distribution and specificity of IRDye 800CW to diseased cells.
Figure 2: Demonstration of how specificity translates into margin classification in excised tissue samples .
The authors ultimately concluded that “The ability of fluorescence assessment to localize diseases in these margins was sensitive and specific with a NPV of 87%, which was superior to both surgical assessment (58%) and pathological assessment (66%)” . The authors also noted that “This report provides evidence that tumor-specific fluorescence can be used by the surgeon or pathologist to guide sampling for frozen sections” . Although the current research does not suggest that fluorescence is a bona fide replacement for current methods, “Fluorescence-guided pathology can … be easily implemented into the clinical care workflow and used in adjunct to fluorescence-guided surgery to help guide the pathologist when assessing margins for both intraoperative assessment and staging” .
In certain cancers like head and neck squamous cell carcinoma, even the most effective treatment still has relatively high rates of failure. Novel methods are needed to reduce the failure rate and provide better clinical outcomes.
Fluorescent dyes conjugated to tumor-specific monoclonal antibodies are emerging as a promising visual aid for tumor analysis. Rosenthal et.al. and Warram et.al. showed how fluorescent dye-antibody conjugates can enhance tissue assessments, also demonstrating the versatility of fluorescent probes for both in situ and in vitro assessments.
- Rosenthal, E.L., et al. Safety and Tumor-specifity of Cetuximab-IRDye800 for Surgical Navigation in Head and Neck Cancer. Clin Cancer Res 2015, Aug; 21(16):3658-3666. doi: 10.1158/1078-0432.CCR-14-3284.
- Warram, J. M., de Boer, E., van Dam, G. M., Moore, L. S., Bevans, S. L., Walsh, E. M., & Young, E. S., et.al. (2016, March 2). Fluorescence Imaging to Localize Head and Neck Squamous Cell Carcinoma for Enhanced Pathological Assessment. Journal of Pathological Cancer Research, 2(2), 104-112. doi:10.1002/cjp2.40