Liquid Biopsies in Head and Neck Cancer: Current State and Future Challenges.

Head and neck cancers are the seventh most frequent malignancy worldwide, consisting of a heterogeneous group of cancers that develop in the oral cavity, pharynx, and larynx, with head and neck squamous cell carcinoma (HNSCC) being the most common pathology. Due to limitations with screening and physical examination, HNSCC often presents in advanced disease states and is thus associated with poor survival. In this setting, liquid biopsies, or obtaining patient bodily fluid samples for cancer diagnosis and prognosis, may play a dramatic role in optimizing care for HNSCC patients. In recent years, there have been dramatic advancements in investigations focused on optimizing and implementing liquid biopsies in general, and specifically for HNSCC patients. Moving forward, there remain significant challenges in liquid biopsy technological development, as well as opportunities for the development of HNSCC liquid biopsy clinical trials and treatment paradigms. In this review, we discuss the current state of liquid biopsy technologies via circulating tumor cells, circulating tumor DNA and exosomes, approaches in head and neck cancer, challenges to optimization and application of liquid biopsies for clinical study, and future prospects for this field of research as it applies to head and neck cancer.

Intraoperative Margin Assessment in Oral and Oropharyngeal Cancer Using Label-Free Fluorescence Lifetime Imaging and Machine Learning.

OBJECTIVE: To demonstrate the diagnostic ability of label-free, point-scanning, fiber-based Fluorescence Lifetime Imaging (FLIm) as a means of intraoperative guidance during oral and oropharyngeal cancer removal surgery. METHODS: FLIm point-measurements acquired from 53 patients (n = 67893 pre-resection in vivo, n = 89695 post-resection ex vivo) undergoing oral or oropharyngeal cancer removal surgery were used for analysis. Discrimination of healthy tissue and cancer was investigated using various FLIm-derived parameter sets and classifiers (Support Vector Machine, Random Forests, CNN). Classifier output for the acquired set of point-measurements was visualized through an interpolation-based approach to generate a probabilistic heatmap of cancer within the surgical field. Classifier output for dysplasia at the resection margins was also investigated. RESULTS: Statistically significant change (P 0.01) between healthy and cancer was observed in vivo for the acquired FLIm signal parameters (e.g., average lifetime) linked with metabolic activity. Superior classification was achieved at the tissue region level using the Random Forests method (ROC-AUC: 0.88). Classifier output for dysplasia (% probability of cancer) was observed to lie between that of cancer and healthy tissue, highlighting FLIm's ability to distinguish various conditions. CONCLUSION: The developed approach demonstrates the potential of FLIm for fast, reliable intraoperative margin assessment without the need for contrast agents. SIGNIFICANCE: Fiber-based FLIm has the potential to be used as a diagnostic tool during cancer resection surgery, including Transoral Robotic Surgery (TORS), helping ensure complete resections and improve the survival rate of oral and oropharyngeal cancer patients.

Probe-based fluorescence dosimetry of an antibody-dye conjugate to identify head and neck cancer as a first step to fluorescence-guided tissue preselection for pathological assessment.

BACKGROUND: Despite the rapid growth of fluorescence imaging, accurate sampling of tissue sections remains challenging. Development of novel technologies to improve intraoperative assessment of tissue is needed. METHODS: A novel contact probe-based fluorescence dosimeter device, optimized for IRDye800CW quantification, was developed. After evaluation of the device in a phantom setup, its clinical value was defined ex vivo in patients with head and neck squamous cell carcinoma who received panitumumab-IRDye800CW. RESULTS: Ten patients were enrolled with a total of 216 data points obtained. Final histopathology showed tumor in 119 spots and normal tissue in 97 spots. Fluorescence-to-excitation ratios in tumor tissue were more than three times higher than those in normal tissue. The area under the curve was 0.86 (95% CI: 0.81-0.91) for tumor detection. CONCLUSIONS: Fluorescence-guided tissue preselection using a fluorescence dosimeter could have substantial impact on tissue sampling for frozen section analysis and potentially reduce sampling errors.

Intraoperative Molecular Imaging for ex vivo Assessment of Peripheral Margins in Oral Squamous Cell Carcinoma.

Objective: Complete surgical resection is the standard of care for treatment of oral cancer although the positive margin rate remains 15-30%. Tissue sampling from the resected specimen and from the wound bed for frozen section analysis (FSA) remains the mainstay for intraoperative margin assessment but is subject to sampling error and can require the processing of multiple samples. We sought to understand if an ex vivo imaging strategy using a tumor-targeted fluorescently labeled antibody could accurately identify the closest peripheral margin on the mucosal surface of resected tumor specimen, so that this "sentinel margin" could be used to guide pathological sampling. Materials and Methods: Twenty-nine patients with oral squamous cell carcinoma scheduled for surgical resection were consented for the study and received systemic administration of a tumor-targeted fluorescently labeled antibody (Panitumumab IRDye800CW). After surgical resection, the tumor specimen was imaged using a closed-field fluorescent imaging device. Relevant pathological data was available for five patients on retrospective review. For each of these five patients, two regions of highest fluorescence intensity at the peripheral margin and one region of lowest fluorescence intensity were identified, and results were correlated with histology to determine if the region of highest fluorescence intensity along the mucosal margin (i.e., the sentinel margin) was truly the closest margin. Results: Imaging acquisition of the mucosal surface of the specimen immediately after surgery took 30 s. In all of the specimens, the region of highest fluorescence at the specimen edge had a significantly smaller margin distance than other sampled regions. The average margin distance at the closest, "sentinel," margin was 3.2 mm compared to a margin distance of 8.0 mm at other regions (p < 0.0001). Conclusions: This proof-of-concept study suggests that, when combined with routine FSA, ex vivo fluorescent specimen imaging can be used to identify the closest surgical margin on the specimen. This approach may reduce sampling error of intraoperative evaluation, which should ultimately improve the ability of the surgeon to identify the sentinel margin. This rapid sentinel margin identification improves the surgeon's orientation to areas most likely to be positive in the surgical wound bed and may expedite pathology workflow.