Improved intraoperative identification of close margins in oral squamous cell carcinoma resections using a dual aperture fluorescence ratio approach: first in-human results.

Significance: Surgical excision is the main treatment for solid tumors in oral squamous cell carcinomas, where wide local excision (achieving a healthy tissue margin of >5 mm around the excised tumor) is the goal as it results in reduced local recurrence rates and improved overall survival. Aim: No clinical methods are available to assess the complete surgical margin intraoperatively while the patient is still on the operating table; and while recent intraoperative back-bench fluorescence-guided surgery approaches have shown promise for detecting "positive" inadequate margins (<1 mm), they have had limited success in the detection of "close" inadequate margins (1 to 5 mm). Here, a dual aperture fluorescence ratio (dAFR) approach was evaluated as a means of improving detection of close margins. Approach: The approach was evaluated on surgical specimens from patients who were administered a tumor-specific fluorescent imaging agent (cetuximab-800CW) prior to surgery. The dAFR approach was compared directly against standard wide-field fluorescence imaging and pathology measurements of margin thickness in specimens from three patients and a total of 12 margin locations (1 positive, 5 close, and 6 clear margins). Results: The area under the receiver operating characteristic curve, representing the ability to detect close compared to clear margins (>5 mm) was found to be 1.0 and 0.57 for dAFR and sAF, respectively. Improvements in dAFR were found to be statistically significant (p<0.02). Conclusions: These results provide evidence that the dAFR approach potentially improves detection of close surgical margins.

EGFR-targeted fluorescence molecular imaging for intraoperative margin assessment in oral cancer patients: a phase II trial.

Inadequate surgical margins occur frequently in oral squamous cell carcinoma surgery. Fluorescence molecular imaging (FMI) has been explored for intraoperative margin assessment, but data are limited to phase-I studies. In this single-arm phase-II study (NCT03134846), our primary endpoints were to determine the sensitivity, specificity and positive predictive value of cetuximab-800CW for tumor-positive margins detection. Secondary endpoints were safety, close margin detection rate and intrinsic cetuximab-800CW fluorescence. In 65 patients with 66 tumors, cetuximab-800CW was well-tolerated. Fluorescent spots identified in the surgical margin with signal-to-background ratios (SBR) of ≥2 identify tumor-positive margins with 100% sensitivity, 85.9% specificity, 58.3% positive predictive value, and 100% negative predictive value. An SBR of ≥1.5 identifies close margins with 70.3% sensitivity, 76.1% specificity, 60.5% positive predictive value, and 83.1% negative predictive value. Performing frozen section analysis aimed at the fluorescent spots with an SBR of ≥1.5 enables safe, intraoperative adjustment of surgical margins.

Towards in vivo characterization of thyroid nodules suspicious for malignancy using multispectral optoacoustic tomography.

PURPOSE: Patient-tailored management of thyroid nodules requires improved risk of malignancy stratification by accurate preoperative nodule assessment, aiming to personalize decisions concerning diagnostics and treatment. Here, we perform an exploratory pilot study to identify possible patterns on multispectral optoacoustic tomography (MSOT) for thyroid malignancy stratification. For the first time, we directly correlate MSOT images with histopathology data on a detailed level. METHODS: We use recently enhanced data processing and image reconstruction methods for MSOT to provide next-level image quality by means of improved spatial resolution and spectral contrast. We examine optoacoustic features in thyroid nodules associated with vascular patterns and correlate these directly with reference histopathology. RESULTS: Our methods show the ability to resolve blood vessels with diameters of 250 µm at depths of up to 2 cm. The vessel diameters derived on MSOT showed an excellent correlation (R2-score of 0.9426) with the vessel diameters on histopathology. Subsequently, we identify features of malignancy observable in MSOT, such as intranodular microvascularity and extrathyroidal extension verified by histopathology. Despite these promising features in selected patients, we could not determine statistically relevant differences between benign and malignant thyroid nodules based on mean oxygen saturation in thyroid nodules. Thus, we illustrate general imaging artifacts of the whole field of optoacoustic imaging that reduce image fidelity and distort spectral contrast, which impedes quantification of chromophore presence based on mean concentrations. CONCLUSION: We recommend examining optoacoustic features in addition to chromophore quantification to rank malignancy risk. We present optoacoustic images of thyroid nodules with the highest spatial resolution and spectral contrast to date, directly correlated to histopathology, pushing the clinical translation of MSOT.

Comparison of narrow band and fluorescence molecular imaging to improve intraoperative tumour margin assessment in oral cancer surgery.

OBJECTIVE: New techniques have emerged to aid in preventing inadequate margins in oral squamous cell carcinoma (OSCC) surgery, but studies comparing different techniques are lacking. Here, we compared narrow band imaging (NBI) with fluorescence molecular imaging (FMI), to study which intraoperative technique best assesses the mucosal tumour margins. MATERIALS AND METHODS: NBI was performed in vivo and borders were marked with three sutures. For FMI, patients received 75 mg of unlabelled cetuximab followed by 15 mg cetuximab-800CW intravenously-two days prior to surgery. The FMI borders were defined on the excised specimen. The NBI borders were correlated with the FMI outline and histopathology. RESULTS: Sixteen patients were included, resulting in 31 NBI and 30 FMI measurements. The mucosal border was delineated within 1 mm of the tumour border in 4/31 (13 %) of NBI and in 16/30 (53 %) FMI cases (p = 0.0008), and within 5 mm in 23/31 (74 %) of NBI and in 29/30 (97 %) of FMI cases (p = 0.0048). The median distance between the tumour border and the imaging border was significantly greater for NBI (3.2 mm, range -6.1 to 12.8 mm) than for FMI (0.9 mm, range -3.0 to 7.4 mm; p = 0.028). Submucosal extension and previous irradiation reduced NBI accuracy. CONCLUSION: Ex vivo FMI performed more accurately than in vivo NBI in mucosal margin assessment, mainly because NBI cannot detect submucosal extension. NBI adequately identified the mucosal margin especially in early-stage and not previously irradiated tumours, and may therefore be preferable in these tumours for practical and cost-related reasons.

A Guideline for Clinicians Performing Clinical Studies with Fluorescence Imaging.

Fluorescence imaging is an emerging imaging technique that has shown many benefits for clinical care. Currently, the field is in rapid clinical translation, and an unprecedented number of clinical trials are performed. Clinicians are inundated with numerous opportunities and combinations of different imaging modalities. To streamline this process, a multidisciplinary approach is needed with drug discovery, software and systems engineering, and translational medicine. Here, we discuss the main constituents of a uniform fluorescence imaging protocol to match the clinical need and ensure consistent study designs and reliable data collection in clinical trials. In an era in which the potential of fluorescence imaging has become evident, consistent conduct of studies, data analysis, and data interpretation is essential for implementation into the standard of care.

Epidermal Growth Factor Receptor-Targeted Fluorescence Molecular Imaging for Postoperative Lymph Node Assessment in Patients with Oral Cancer.

In most oral cancer patients, surgical treatment includes resection of the primary tumor combined with excision of lymph nodes (LNs), either for staging or for treatment. All LNs harvested during surgery require tissue processing and subsequent microscopic histopathologic assessment to determine the nodal stage. In this study, we investigated the use of the fluorescent tracer cetuximab-800CW to discriminate between tumor-positive and tumor-negative LNs before histopathologic examination. Here, we report a retrospective ad hoc analysis of a clinical trial designed to evaluate the resection margin in patients with oral squamous cell carcinoma (NCT02415881). Methods: Two days before surgery, patients were intravenously administered 75 mg of cetuximab followed by 15 mg of cetuximab-800CW, an epidermal growth factor receptor-targeting fluorescent tracer. Fluorescence images of excised, formalin-fixed LNs were obtained and correlated with histopathologic assessment. Results: Fluorescence molecular imaging of 514 LNs (61 pathologically positive nodes) could detect tumor-positive LNs ex vivo with 100% sensitivity and 86.8% specificity (area under the curve, 0.98). In this cohort, the number of LNs that required microscopic assessment was decreased by 77.4%, without missing any metastases. Additionally, in 7.5% of the LNs false-positive on fluorescence imaging, we identified metastases missed by standard histopathologic analysis. Conclusion: Our findings suggest that epidermal growth factor receptor-targeted fluorescence molecular imaging can aid in the detection of LN metastases in the ex vivo setting in oral cancer patients. This image-guided concept can improve the efficacy of postoperative LN examination and identify additional metastases, thus safeguarding appropriate postoperative therapy and potentially improving prognosis.

Intraoperative imaging in pathology-assisted surgery.

The pathological assessment of surgical specimens during surgery can reduce the incidence of positive resection margins, which otherwise can result in additional surgeries or aggressive therapeutic regimens. To improve patient outcomes, intraoperative spectroscopic, fluorescence-based, structural, optoacoustic and radiological imaging techniques are being tested on freshly excised tissue. The specific clinical setting and tumour type largely determine whether endogenous or exogenous contrast is to be detected and whether the tumour specificity of the detected biomarker, image resolution, image-acquisition times or penetration depth are to be prioritized. In this Perspective, we describe current clinical standards for intraoperative tissue analysis and discuss how intraoperative imaging is being implemented. We also discuss potential implementations of intraoperative pathology-assisted surgery for clinical decision-making.

VEGF-Targeted Multispectral Optoacoustic Tomography and Fluorescence Molecular Imaging in Human Carotid Atherosclerotic Plaques.

Vulnerable atherosclerotic carotid plaques are prone to rupture, resulting in ischemic strokes. In contrast to radiological imaging techniques, molecular imaging techniques have the potential to assess plaque vulnerability by visualizing diseases-specific biomarkers. A risk factor for rupture is intra-plaque neovascularization, which is characterized by overexpression of vascular endothelial growth factor-A (VEGF-A). Here, we study if administration of bevacizumab-800CW, a near-infrared tracer targeting VEGF-A, is safe and if molecular assessment of atherosclerotic carotid plaques in vivo is possible using multispectral optoacoustic tomography (MSOT). Healthy volunteers and patients with symptomatic carotid artery stenosis scheduled for carotid artery endarterectomy were imaged with MSOT. Secondly, patients were imaged two days after intravenous administration of 4.5 bevacizumab-800CW. Ex vivo fluorescence molecular imaging of the surgically removed plaque specimen was performed and correlated with histopathology. In this first-in-human MSOT and fluorescence molecular imaging study, we show that administration of 4.5 mg bevacizumab-800CW appeared to be safe in five patients and accumulated in the carotid atherosclerotic plaque. Although we could visualize the carotid bifurcation area in all subjects using MSOT, bevacizumab-800CW-resolved signal could not be detected with MSOT in the patients. Future studies should evaluate tracer safety, higher doses of bevacizumab-800CW or develop dedicated contrast agents for carotid atherosclerotic plaque assessment using MSOT.

Improving oral cavity cancer diagnosis and treatment with fluorescence molecular imaging.

Early diagnosis and radical surgical excision of oral squamous cell carcinomas are essential for achieving optimal treatment outcomes. To date, diagnostic tools that rely on anatomical anomalies provide limited information and resolution in clinical practice. As a result, oral cancer is often detected in an advanced stage. Also, no reliable real-time intraoperative tools are readily available for the evaluation of surgical resection margins. Fluorescence imaging visualises biological processes that occur in early carcinogenesis and could, therefore, enable detection of small tumours in early stages. Furthermore, due to the high sensitivity and spatial resolution, fluorescence imaging could assist in resection margin assessment during surgery. In this review, we discuss several techniques that employ fluorescence for early diagnosis and surgical guidance in oral squamous cell carcinoma and present future perspectives on the potential of fluorescence imaging in oral cancer in the near future.

Back-Table Fluorescence-Guided Imaging for Circumferential Resection Margin Evaluation Using Bevacizumab-800CW in Patients with Locally Advanced Rectal Cancer.

Negative circumferential resection margins (CRM) are the cornerstone for the curative treatment of locally advanced rectal cancer (LARC). However, in up to 18.6% of patients, tumor-positive resection margins are detected on histopathology. In this proof-of-concept study, we investigated the feasibility of optical molecular imaging as a tool for evaluating the CRM directly after surgical resection to improve tumor-negative CRM rates. Methods: LARC patients treated with neoadjuvant chemoradiotherapy received an intravenous bolus injection of 4.5 mg of bevacizumab-800CW, a fluorescent tracer targeting vascular endothelial growth factor A, 2-3 d before surgery (ClinicalTrials.gov identifier: NCT01972373). First, for evaluation of the CRM status, back-table fluorescence-guided imaging (FGI) of the fresh surgical resection specimens (n = 8) was performed. These results were correlated with histopathology results. Second, for determination of the sensitivity and specificity of bevacizumab-800CW for tumor detection, a mean fluorescence intensity cutoff value was determined from the formalin-fixed tissue slices (n = 42; 17 patients). Local bevacizumab-800CW accumulation was evaluated by fluorescence microscopy. Results: Back-table FGI correctly identified a tumor-positive CRM by high fluorescence intensities in 1 of 2 patients (50%) with a tumor-positive CRM. For the other patient, low fluorescence intensities were shown, although (sub)millimeter tumor deposits were present less than 1 mm from the CRM. FGI correctly identified 5 of 6 tumor-negative CRM (83%). The 1 patient with false-positive findings had a marginal negative CRM of only 1.4 mm. Receiver operating characteristic curve analysis of the fluorescence intensities of formalin-fixed tissue slices yielded an optimal mean fluorescence intensity cutoff value for tumor detection of 5,775 (sensitivity of 96.19% and specificity of 80.39%). Bevacizumab-800CW enabled a clear differentiation between tumor and normal tissue up to a microscopic level, with a tumor-to-background ratio of 4.7 ± 2.5 (mean ± SD). Conclusion: In this proof-of-concept study, we showed the potential of back-table FGI for evaluating the CRM status in LARC patients. Optimization of this technique with adaptation of standard operating procedures could change perioperative decision making with regard to extending resections or applying intraoperative radiation therapy in the case of positive CRM.

Effect of adjuvant radiotherapy on the local recurrence of oral squamous cell carcinoma with perineural invasion: A systematic review.

OBJECTIVES OF THE REVIEW: The decision whether to include postoperative radiotherapy on patients with oral squamous cell carcinoma depends on the risk of local recurrence. The objectives of this study were to systematically review literature on whether perineural invasion in oral squamous cell carcinoma patients is associated with higher local recurrence rates and whether local recurrence is influenced by the administration of postoperative radiotherapy in patients presenting with perineural invasion. TYPE OF REVIEW: Systematic review. SEARCH STRATEGY: Embase, PubMed, Web Of Science. EVALUATION METHOD: The databases above were searched for studies that analysed: the treatment of oral squamous cell carcinoma patients with perineural invasion, local recurrence and postoperative radiotherapy. The data of seven studies were analysed qualitatively. RESULTS: The overall quality of the studies was moderate to low. There was no evidence of the effect of postoperative radiotherapy on local recurrence rates in patients presenting with perineural invasion. Some evidence suggests that local recurrence rates may increase in cases of multifocal perineural invasion, especially if nerves >1 mm are involved but these data should be interpreted with caution due to the low-quality evidence. CONCLUSIONS: High-quality evidence regarding the prognostic value of perineural invasion and the impact of postoperative radiotherapy in patients presenting with perineural invasion is lacking in the literature, making it difficult to select a postoperative strategy for early-stage tumours.