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ORIGINAL ARTICLE Near infrared fluorescent imaging after intravenous injection of indocyanine green during neck dissection in patients with head and neck cancer: A feasibility study Antoine Digonnet, MD,1* Sophie van Kerckhove, MSc,2 Michel Moreau, MD,3 Esther Willemse, MD,1 Marie Quiriny, MD,1 Bissan Ahmed, MD, PhD,2 Nicolas de Saint Aubain, MD,4 Guy Andry, MD,1 Pierre Bourgeois, MD, PhD2 1 Department of Head and Neck Surgery, Jules Bordet Institute, Universite Libre de Bruxelles, Brussels, Belgium, 2Department of Nuclear Medicine, Jules Bordet Institute, Universite Libre de Bruxelles, Brussels, Belgium, 3Department of Biostatistics, Jules Bordet Institute, Universite Libre de Bruxelles, Brussels, Belgium, 4Department of Pathology, Jules Bordet Institute, Universite Libre de Bruxelles, Belgium. Accepted 20 September 2015 Published online 24 December 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.24331 ABSTRACT: Background. Indocyanine green (ICG) has not been studied during therapeutic lymph node dissections after intravenous injection. The purpose of this study was to explore the distribution of ICG in lymphatic nodes during neck dissection. Methods. Eleven patients requiring neck dissection with or without resection of the primary lesion were included. ICG was intravenously injected at induction time of anesthesia. Imaging was performed before and after surgical resection. Fluorescence was measured in arbitrary units (AUs) in the pathology department. Mixed linear model and generalized estimating equations (GEEs) were used. Results. Mean fluorescence of invaded nodes was 22.6 AUs (SD 5 24.9) and 3.9 AUs (SD 5 8.1) in negative nodes (p 5 .016). After adjustment for the size of the node, the risk of invasion when fluorescence was observed was 12.2 (95% confidence interval [CI] 5 5.3–28.2; p < .0001). Conclusion. This study demonstrates the feasibility of ICG to bring a contrast during surgery between healthy and invaded nodes after i.v. injecC 2015 Wiley Periodicals, Inc. Head Neck 38: E1833–E1837, 2016 tion. V INTRODUCTION To the best of our knowledge, NIR has not yet been studied in therapeutic lymph nodes dissection after intravenous (i.v.) injection. The purpose of this study was to evaluate the feasibility and the distribution of ICG in the lymph nodes after i.v. administration. The nodal status in head and neck squamous cell carcinoma remains one of the most important prognostic factors.1,2 Proper treatment of the neck compartment is a critical issue because leaving behind invaded nodes could lead to earlier recurrence and death. The gold standard for lymph node disease is neck dissection.3 The use of near infrared (NIR) fluorescence light has been recently used to intraoperatively identify lymph nodes, tumors, and vital structures.4 NIR fluorescence using the fluorescent dye indocyanine green (ICG) has been successfully used for sentinel lymph node mapping in head and neck squamous cell carcinoma.5,6 Unfortunately, regarding tumor location, sentinel lymph node mapping is not always technically possible in head and neck cancer. Peripheral vein injection of ICG has shown to be effective to identify macroscopically invaded nodes and primary tumor.7 The optimal timing for carrying out the surgical procedure is between 30 minutes and 2 hours after i.v. ICG administration in head and neck cancer.7 *Corresponding author: A. Digonnet, Department of Head and Neck Surgery, Jules Bordet Institute, Universite Libre de Bruxelles, 1 rue Heger Bordet, 1000 Brussels, Belgium. E-mail: [email protected] Contract grant sponsor: This work was supported by “Les amis de l’institut Bordet” and group Research and Development; “Clinical Applications Fluorescent Imaging” (coordinator: P.B.) KEY WORDS: indocyanine green, lymph node, neck dissection, oncology, fluorescence abstract MATERIALS AND METHODS This clinical trial was approved by the medical ethic committee of the Institute Jules Bordet, Universite Libre de Bruxelles. Patients with a history of renal failure and coronaropathy were excluded. Eleven patients from a single institution were prospectively enrolled. Decision of the neck dissection with or without a resection of the primary tumor was taken during our multidisciplinary oncologic consultation. Free ICG (0.25 mg/kg; Pulsion Medical System, Belgium) was injected through the radial vein at induction time of anesthesia. The setting in the operating room was performed according to the procedure described by Mieog et al8 and we used a dedicated NIR camera device (PDE, Hamamatsu, Japan). Patients requiring resection of a primary lesion underwent fluorescent tumor inspection before and after resection. After subplatysmal flap elevation, the neck operative field was inspected with the NIR camera to identify potential hotspots, and then the procedure was carried out as previously planned. At the end of the neck dissection, the operative field was re-inspected with the infrared camera, and potential HEAD & NECK—DOI 10.1002/HED APRIL 2016 E1833 DIGONNET ET AL. TABLE 1. Patient characteristics. Patient number Age, y Primary tumor Preoperative TNM classification Resection of the primary 1 2 3 49 63 68 Larynx (glottis) Oropharynx Larynx-esophagus T4N0M0 T2N2b T1N0 and T2NO 55 68 53 60 66 63 44 45 Larynx Piriform sinus Lip Tongue Thyroid Thyroid Melanoma Melanoma T2N0 T4aN3 T2N0 N1 N1a T3N1b IIIb (N1b) IIIb (N1b) Partial laryngectomy Pharyngectomy Eso-pharyngolaryngectomy Total laryngectomy No Resection No No Total thyroidectomy No No 4 5 6 7 8 9 10 11 hotspots in the resected area were harvested. The hotspot identified outside the planned resected area was also harvested. The resected specimen was sent for macroscopic analysis, the number of nodes were counted, and the surface of the nodes were measured (in mm2) according to their great and small diameter, and then the specimen was reexamined under fluorescence and the nodes were recounted. Fluorescence of each resected node was measured in arbitrary units (AUs). Finally, a microscopic analysis was performed for each node to determine its status (invaded or not). Statistical analysis Fluorescence of the nodes was analyzed as a continuous and categorized variable using cutoffs of 6 UAs (6/<6). As the size of the node was not the variable of interest but only an adjustment variable, the analysis was performed in a continuous way. As the unit of analysis was the lymph node and not the patient, and the data are not independent, we used the mixed linear model to test our hypothesis (for continuous variable) and generalized estimating equation (GEE) model (for categorical variable) in order to take into account the correlation structure within patient. Odds ratios (ORs) were derived with their 95% confidence intervals (95% CIs). In the mixed linear model, compound symmetry was used as the type of covariance matrix. In order to have a symmetrical distribution of dependent variables, we used the log-transformation of the fluorescence and the size. In the GEE analyses, empirical instead of model-based SEs were used because they are more robust against misspecification of the correlation structure. The exchangeable covariance matrix was used. Fluorescence was analyzed in univariate and multivariate (adjustment for the node size and histology). RESULTS Patients and tumor characteristics This study was proposed to 13 consecutive patients. Two patients refused the study and 11 patients were E1834 HEAD & NECK—DOI 10.1002/HED APRIL 2016 Size of the resected primary, cm Neck dissection No. of harvested nodes No. of invaded nodes 1.8 3.5 1.3 II–III (bilateral) VI II–III VI 28 8 4 0 2 0 2 II–III–Va (bilateral) V I II–III–Va I retropharyngeal II–III–IV–V III–IV–Vb III–IV–Vb 19 6 2 21 6 57 17 25 0 1 0 1 1 9 2 1 2.2 6 included and injected. The mean age was 58.9 years (range, 44–68 years). Patients, tumors characteristics, and the performed procedure are detailed in Table 1. Six patients underwent resection of the primary lesion with a neck dissection during the same procedure. Among them, 2 patients underwent salvage surgery. Patient number 3 was previously treated by exclusive radiotherapy for glottis cancer and 7 years later he underwent a partial laryngectomy with neck dissection for recurrence. During follow-up, an esophageal cancer was diagnosed and treated by chemoradiotherapy (CRT). One year after CRT, the patient experienced recurrences on both sites leading to a total esophageal laryngectomy. Patient number 4 was previously treated by radiotherapy for epiglottic lesion; he experienced a first recurrence treated by transoral robotic surgery (epiglotectomy). The last recurrence was again supraglottic and treated by total laryngectomy. The 5 remaining patients were operated on for lymph nodes metastasis. Patient number 5 had a T4aN3 of the piriform sinus. He refused total laryngectomy and was enrolled to receive induction chemotherapy. After 2 cycles of docetaxel, cisplatin, and 5-fluorouracil, he experienced a persistence of nodes in level V. He underwent level V dissection and was sent for concurrent CRT. No adverse reactions or complications related to the ICG injection occurred during the current study. Timing of fluorescence Table 2 details the timing (Delta) between ICG and the initial inspection with the fluorescence camera. Two patients (8-9) underwent initial inspection after 2 hours and were those who required 2 separate incisions to perform the planned neck dissections. Using GEE analysis, fluorescence (AU) was not correlated to the Delta (minutes; p 5 .64). Perioperative imaging Before neck dissection, the lymph node compartment was subjectively fluorescent in all patients with the exception of 1 patient (patient 3). An example of intraoperative fluorescence is shown in Figure 1. Fluorescent examination of the resected area showed no residual INDOCYANINE TABLE 2. Timing between injection time and initial inspection. Patient number 1 2 3 4 5 6 7 8 9 10 11 Injection time 9 h 38 12 h 35 14 h 22 9 h 14 11 h 17 14 h 13 12 h 00 9 h 10 11 h 30 10 h 31 11 h 48 Initial inspection 11 h 22 13 h 59 16 h 02 11 h 05 12 h 05 14 h 45 12 h 30 10 h 56–13 h 28 13 h 45 11 h 30 13 h 05 GREEN IN THERAPEUTIC LYMPH NODE DISSECTION TABLE 3. Sensitivity and specificity. Delta, min 104 84 100 111 48 32 30 106–258 165 59 77 fluorescence, thus, no additional lymph node was harvested in this field. One patient had a hotspot outside the planned resection area and 1 had 2 hotspots. Additional resection revealed 1 benign lymph node, 1 fatty tissue, and a muscle fragment. Regarding primary tumor examination, thyroidectomies were excluded because margin evaluation was less relevant. Primary tumor examination (n 5 5) revealed fluorescence in all patients with the exception of 1 patient (patient 3). Fluorescent imaging after resection of the primary lesion showed residual fluorescence in 1 patient (patient 6) and frozen section confirmed margin involvement leading to complementary resection. Margins were Node status Positive Negative 13 4 17 41 135 ICG imaging Positive Negative 54 139 Abbreviation: ICG, indocyanine green. Sensitivity: 13 of 17 5 76.5%. Specificity: 135 of 176 5 76.7%. clear after the second resection and no residual fluorescence was found. The remaining 3 patients without residual fluorescence after resection of the primary tumor had immediately clear margins. Histopathologic analysis Fluorescence imaging did not modify the lymph node count in the department of pathology. Lymph nodes Across the 11 patients, 193 lymph nodes were harvested with an average number of 17.5 lymph nodes (SD 5 16.1) per patient and a median of 13. Five patients (45.4%) had <10 resected nodes, 2 (18.2%) had between 10 and 20 resected nodes, and 4 (36.4%) had >20 resected nodes. Among the 193 lymph nodes, 17 were positive (8.8%), 54 (28%) were fluorescent (AUs 6). The mean fluorescence and size of the 193 lymph nodes were respectively 5.7 UAs (SD 5 12.1) and 94.6 mm2 (SD 5 126.5). In invaded lymph nodes, the mean fluorescence was 22.6 UAs (SD 5 24.9) and 3.9 UAs (SD 5 8.1) in negative lymph nodes (p 5 .016). For the size, we had 238.2 mm2 (SD 5 244.6) and 78.6 mm2 (SD 5 94.5), respectively, with p 5 .011. The median fluorescence in positive lymph nodes was 11 AUs (range, 0–86 AUs). The median size of metastatic deposits that were ICG-positive was 120 mm2 (range, 20–900 mm2). We observed a correlation between amount of ICG uptake and the size of the metastatic nodes (p 5 .02). Sensitivity and specificity of the test with ICG were 76.5% (13 of 17) and 76.7% (135 of 176), respectively (Table 3). In univariate analysis (GEE), a fluorescence of 6 AUs or more is associated with a 10.3-fold (95% CI 5 4.8– 22.1) increased risk to have a positive node (Table 4). After adjustment for the size of the lymph node, the OR for invasion in case of fluorescence was 14.1 (95% CI 5 4.6–42.8). Preoperative workup, and fluorescent and invaded lymph nodes FIGURE 1. Perioperative view using near infrared (NIR) imaging for patients 2 and 10 improved the quality of the image compared to patient 2 assessing the existence of a learning curve (a fluorescent node is seen between the grasping forceps). Table 5 details the number of suspicious nodes found on preoperative staging, the number of invaded nodes that were ICG fluorescent, and the total amount of invaded nodes. Table 5 demonstrates that there were 3 invaded nodes detected by ICG but were not detected at preoperative workup (patient 9). In the same patients, there were 4 invaded nodes that were not ICG fluorescent. HEAD & NECK—DOI 10.1002/HED APRIL 2016 E1835 DIGONNET ET AL. TABLE 4. Univariate analysis (generalized estimating equation). Variables Fluorescence 6 Positive node number (%) OR Lower 95% CI Upper 95% CI p value 13 (7.3) 10.3 4.8 22.1 < .0001 Abbreviations: OR, odds ratio; 95% CI, 95% confidence interval. In patient 11, we also saw that we had a false-positive result on positron emission tomography-CT, this was not the case with MRI, ultrasonography, or ICG. DISCUSSION The use of ICG in human oncology started in 2000 in breast cancer.9 Further studies demonstrate the ability of ICG to bind tumor tissues.10,11 ICG was first described in head and neck oncology after peritumoral injection for sentinel lymph node mapping.5,6 Anatomic constraints of head and neck cancer localization, particularly below the oropharynx, complicated the routine use of this procedure. In 2013, Yokoyama et al7 demonstrated the property of ICG to bind head and neck tumoral tissue after i.v. injection. The optimal timing for surgery was found to be between 30 minutes and 2 hours after i.v. injection. In line with this result, we decided to perform the injection at the induction time of the anesthesia. In our series, 2 patients underwent the initial fluorescence inspection after 2 hours; however, we did not find a decreased fluorescence in those patients compared with the rest of the population. The optimal timing, set by Yokohama et al,7 used subjective scales of fluorescence, which may have led to a decrease in precision. Regarding the administered preparation, some authors have mixed ICG with human serum albumin to obtain better retention in the lymph nodes.12 We decided to inject free ICG because there is no argument for the superiority of mixed ICG to serum albumin compared to free ICG.13 The principal finding was the correlation between node involvement and fluorescence magnitude. In their study, Yokoyama et al7 demonstrated the ability of NIR imaging to distinguish cancer from normal surrounding tissue. However, they only dealt with macroscopic lesion identifiable by sight or palpation. In the present study, we found that fluorescence was associated with a 14.1-fold risk of invasion regardless of the size of the node. In summary, fluorescence was correlated to invasion status and, to a very small extent, to the size of the node. According to our knowledge, this is the first study to bring out a perioperative contrast between invaded and healthy nodes after i.v. injection of ICG. The second finding was the observation of an ICG distribution in healthy nodes resulting in subjective fluorescence. This result allows delimitation between the nodes and the surrounding tissues. However, in this preliminary study, NIR imaging did not increase lymph node yield during neck dissection because visualization of the resection site demonstrated no residual fluorescence. Surprisingly, we observed discordance between subjective fluorescence and objective measurement of the healthy nodes’ AU value (mean, 3.9 UAs). This result may be explained by the fact that fluorescence was measured in the department of pathology. This condition has probably led to a decrease in the measure of fluorescence. Because tissue layers are thin in the neck compartment, NIR imaging may also help to identify residual invaded nodes outside the resected area once the neck dissection is completed. However, per-operative fluorescence remains subjective because AUs are measured postoperatively. If the device imaging seemed to be simple to handle and a few were time-consuming for the surgical procedure, we believe that a learning curve is associated with the perioperative setting of the device and interpretation of fluorescence (Figure 1). In the future, we could elaborate a subjective scale of fluorescence and compare it to objective UA measurements. The third finding was the potential relationship between fluorescence and margin status. Indeed, in our series, 5 patients underwent resection for primary head and neck squamous cell carcinoma. Four patients had fluorescent tumors and imaging of the resected field revealed residual fluorescence in 1 patient. Correlation with frozen section biopsy showed only invasion in the patient with residual TABLE 5. Number of suspicious nodes found on preoperative staging, number of invaded nodes indocyanine green fluorescent, and the total amount of invaded nodes. Patient number 1 2 3 4 5 6 7 8 9 10 11 No. of suspicious nodes (imaging) CT MRI PET-CT 0 2 0 0 1 0 Ultrasonography 2 0 0 1 3 1 2 1 2 2 2 1 2 1 FNA 1 1 FNA Abbreviations: PET, positron emission tomography; ICG, indocyanine green; FNA, fine-needle aspiration. E1836 HEAD & NECK—DOI 10.1002/HED APRIL 2016 No. of suspicious nodes (preoperative) No. of invaded nodes ICG1 No. of invaded nodes 0 2 0 0 1 0 1 3 2 2 2 0 2 0 0 1 0 1 1 5 2 1 0 2 0 0 1 0 1 1 9 2 1 INDOCYANINE fluorescence. The 3 remaining patients had healthy margins at frozen section examination. This finding, if confirmed by a larger series, could establish that NIR fluorescence imaging has the potential to help improve tumor resection compared with current intraoperative methods. In a recent study, Martirosyan et al14 studied animal models of glioblastoma. They performed tumor resection with the use of NIR laser confocal endomicroscopy with ICG. They found that ICG provides striking distinguishing features of normal brain and tumor regions, providing definitive tumor boarder delimitation. In our series, 1 patient (patient 3) did not experience node and primary tumor fluorescence after i.v. injection of ICG. This patient underwent 2 sessions of radiotherapy and 1 surgery with node dissection before our last procedure. We can assume that those previous treatments might have impaired the biological property of tissue and lymphatic vessels leading to a decreased fluorescence. Imaging of the operative field after neck dissection did not reveal residual nodes in the resected area, but hotspots were found outside of those limits. Resection of those hotspots provides poor information because we found 1 node, 1 fatty fragment, and 1 muscle piece. These results could be interpreted by a nonspecific tissue fixation increasing with time because imaging was performed at the end of the procedure. In conclusion, NIR imaging was easily achievable without disturbing the surgical procedure. NIR imaging after i.v. injection of ICG offers the ability to visualize the neck node compartments during surgery. Fluorescence magnitude was correlated to node invasion and brings a contrast between healthy and invading nodes after i.v. injection. Regarding the primary lesion, NIR imaging could help to define the tumor border and guide the surgical resection. GREEN IN THERAPEUTIC LYMPH NODE DISSECTION Larger and homogenous series are required to define the optimal role of NIR fluorescence in head and neck cancer and its potential routine utilization. REFERENCES 1. Clayton CE, Marsh KA, Dyson A, et al. Ultrahigh-gradient acceleration of injected eletrons by laser-excited relativistic electron plasma waves. Phys Rev Lett 1993;70:37–40. 2. Layland MK, Sessions DG, Lenox J. The influence of lymph node metastasis in the treatment of squamous cell carcinoma of the oral cavity, oropharynx, larynx, and hypopharynx: N0 versus N1. Laryngoscope 2005;115: 629–639. 3. Ferlito A, Robbins KT, Shah JP, et al. Proposal for a rational classification of neck dissections. Head Neck 2011;33:445–450. 4. 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