A hybrid radioactive and fluorescence approach is more than the sum of its parts; outcome of a phase II randomized sentinel node trial in prostate cancer patients.

OBJECTIVE: To determine the diagnostic accuracy of the hybrid tracer indocyanine green (ICG)-Technetium-99 m(99mTc)-nanocolloid compared to sequential tracers of 99mTc-nanocolloid and free-ICG in detecting tumor-positive lymph nodes (LN) during primary surgery in prostate cancer (PCa) patients. INTRODUCTION: Image-guided surgery strategies can help visualize individual lymphatic drainage patterns and sentinel lymph nodes (SLNs) in PCa patients. For lymphatic mapping radioactive, fluorescent and hybrid tracers are being clinically exploited. In this prospective randomized phase II trial, we made a head-to-head comparison between ICG-99mTc-nanocolloid (hybrid group) and 99mTc-nanocolloid and subsequent free-ICG injection (sequential group). METHODS: PCa patients with a >5% risk of lymphatic involvement according to the 2012 Briganti nomogram and planned for prostatectomy were included and randomized (1:1) between ultrasound-guided intraprostatic tracer administration of ICG-99mTc-nanocolloid (n = 69) or 99mTc-nanocolloid (n = 69) 5 h before surgery. Preoperative lymphoscintigraphy and SPECT/CT were performed to define the locations of the SLNs. Additionally, all participants in the sequential group received an injection of free-ICG at time of surgery. Subsequently, all (S)LNs were dissected using fluorescence guidance followed by an extended pelvic lymph node dissection (ePLND). The primary outcome was the total number of surgically removed (S)LNs and tumor-positive (S)LNs. RESULTS: The total number of surgically removed (S)LN packages was 701 and 733 in the hybrid and sequential groups, respectively (p = 0.727). The total number of fluorescent LNs retrieved was 310 and 665 nodes in the hybrid and sequential groups, respectively (p < 0.001). However, no statistically significant difference was observed in the corresponding number of tumor-positive nodes among the groups (44 vs. 33; p = 0.470). Consequently, the rate of tumor-positive fluorescent LNs was higher in the hybrid group (7.4%) compared to the sequential group (2.6%; p = 0.002), indicating an enhanced positive predictive value for the hybrid approach. There was no difference in complications within 90 days after surgery (p = 0.78). CONCLUSIONS: The hybrid tracer ICG-99mTc-nanocolloid improved the positive predictive value for tumor-bearing LNs while minimizing the number of fluorescent nodes compared to the sequential tracer approach. Consequently, the hybrid tracer ICG-99mTc-nanocolloid enables the most reliable and minimal invasive method for LN staging in PCa patients.

The impact of drainage pathways on the detection of nodal metastases in prostate cancer: a phase II randomized comparison of intratumoral vs intraprostatic tracer injection for sentinel node detection.

INTRODUCTION: Previous studies indicated that location and amount of detected sentinel lymph nodes (SLNs) in prostate cancer (PCa) are influenced where SLN-tracer is deposited within the prostate. To validate whether intratumoral (IT) tracer injection helps to increase identification of tumor-positive lymph nodes (LNs) better than intraprostatic (IP) tracer injection, a prospective randomized phase II trial was performed. METHODS: PCa patients with a > 5% risk of lymphatic involvement were randomized between ultrasound-guided transrectal injection of indocyanine green-[99mTc]Tc-nanocolloid in 2 depots of 1 mL in the tumor (n = 55, IT-group) or in 4 depots of 0.5 mL in the peripheral zone of the prostate (n = 58, IP-group). Preoperative lymphoscintigraphy and SPECT/CT were used to define the location of the SLNs. SLNs were dissected using combination of radio- and fluorescence-guidance, followed by extended pelvic LN dissection and robot-assisted radical prostatectomy. Outcome measurements were number of tumor-bearing SNs, tumor-bearing LNs, removed nodes, number of patients with nodal metastases, and metastasis-free survival (MFS) of 4-7-year follow-up data. RESULTS: IT-injection did not result in significant difference of removed SLNs (5.0 vs 6.0, p = 0.317) and histologically positive SLNs (28 vs 22, p = 0.571). However, in IT-group, the SLN-positive nodes were 73.7% of total positive nodes compared to 37.3% in IP-group (p = 0.015). Moreover, significantly more node-positive patients were found in IT-group (42% vs 24%, p = 0.045), which did not result in worse MFS. In two patients (3.6%) from whom the IT-tracer injection only partly covered intraprostatic tumor spread, nodal metastases in ePLND without tumor-positive SNs were yielded. CONCLUSIONS: The percentage-positive SLNs found after IT-injection were significantly higher compared to IP-injection. Significantly more node-positive patients were found using IT-injection, which did not affect MFS. IT-injection failed to detect nodal metastases from non-index satellite lesions. Therefore, we suggest to combine IT- and IP-tracer injections in men with visible tumor on imaging.

Pelvic lymph node distribution and metastases of prostate and bladder cancer: a systematic literature review and template proposal.

PURPOSE: To systematically review the relevant literature that evaluates the LN topographical distribution and propose a uniform template. METHODS: A bibliographic search of PubMed/Medline, Embase and SCOPUS was performed for studies reporting data of LN imaging and/or nodal resection. RESULTS: 101 and 26 articles met the inclusion criteria for PCa and BCa, respectively. In PCa, the most common locations of positive LNs for surgical and imaging studies were external iliac (both 38 studies), followed by obturator (38 and 37, respectively). Similarly, in BCa, the most common location of positive nodes for surgical and imaging studies were external iliac (19 and 4, respectively), followed by obturator (15 and 3 studies, respectively). In PCa, median percentages of positive external iliac nodes/patient were 12.2% and 11.6% for surgical and imaging studies, respectively while corresponding rates for BCa were 3.9% and 17.6%. There were high risks of bias across studies as well as high heterogeneity in the definition of the anatomic boundaries of lymphadenectomy templates. CONCLUSIONS: This review highlights the lack of detailed information on exact LN templates and metastases location, which in turn hinders generation of high-quality evidence on optimal lymphadenectomy templates. Our proposed template is applicable for both imaging and surgical description and could facilitate the translation of anatomical location from imaging to surgical resection.

Interventional nuclear medicine: a focus on radioguided intervention and surgery.

Within interventional nuclear medicine (iNM) a prominent role is allocated for the sub-discipline of radioguided surgery. Unique for this discipline is the fact that an increasing number of clinical indications (e.g. lymphatic mapping, local tumor demarcation and/or tumor receptor targeted applications) have been adopted into routine care. The clinical integration is further strengthened by technical innovations in chemistry and engineering that enhance the translational potential of radioguided procedures in iNM. Together, these features not only ensure ongoing expansion of iNM but also warrant a lasting clinical impact for the sub-discipline of radioguided surgery.

The value of periprostatic fascia thickness and fascia preservation as prognostic factors of erectile function after nerve-sparing robot-assisted radical prostatectomy.

PURPOSE: To determine the correlation of preoperative fascia thickness (FT) and intraoperative fascia preservation (FP) with erectile function (EF) after nerve-sparing robot-assisted radical prostatectomy (RARP). METHODS: Our analysis included 106 patients, with localized prostate cancer and no erectile dysfunction (ED) before RARP, assessed with preoperative 3 Tesla (3 T) multiparametric magnetic resonance imaging (MRI). FP score was defined as the extent of FP from the base to the apex of the prostate, quantitatively assessed by the surgeon. Median fascia thickness (MFT) per patient was defined as the sum of the median FT of 12 MRI regions. Preserved MFT (pMFT) was the sum of the saved MFT. The percentage of pFMT (ppMFT) was also calculated. Fascia surface (FS) was measured on MRI and it was combined with FP score resulting in preserved FS (pFS) and percentage of pFS (ppFS). RESULTS: FP score, pMFT, ppMFT, pFS and ppFS were significantly lower (p < 0.0001) in patients with ED. In the multivariate regression analysis, lower FP score [odds ratio (OR) 0.721, p = 0.03] and lower ppMFT (OR 0.001, p = 0.027) were independent predictors of ED. ROC analysis showed the highest area under the curve for ppMFT (0.787) and FP score (0.767) followed by pMFT (0.755) and ppFS (0.743). CONCLUSIONS: MRI-determined periprostatic FT combined with intraoperative FP score are correlated to postprostatectomy EF. Based on the hypothesis that a thicker fascia forms a protective layer for the nerves, we recommend assessing FT preoperatively to counsel men for the odds of preserving EF after RARP.

Navigation of Fluorescence Cameras during Soft Tissue Surgery-Is it Possible to Use a Single Navigation Setup for Various Open and Laparoscopic Urological Surgery Applications?

PURPOSE: Real-time visualization fluorescence imaging can guide surgeons during tissue resection. Unfortunately tissue induced signal attenuation limits the value of this technique to superficial applications. By positioning the fluorescence camera via a dedicated navigation setup we reasoned that the technology could be made compatible with deeper lesions, increasing its impact on clinical care. Such an impact would benefit from the ability to implement the navigation technology in different surgical settings. For that reason we evaluated whether a single fluorescence camera could be navigated toward targeted lesions during open and laparoscopic surgery. MATERIALS AND METHODS: A fluorescence camera with scopes available for open and laparoscopic procedures was integrated with a navigation platform. Lymph nodes identified on SPECT/CT (single photon emission computerized tomography/computerized tomography) or free-hand single photon emission computerized tomography acted as navigation targets and were displayed as augmented overlays in the fluorescence camera video feed. The accuracy of this setup was evaluated in a phantom study of 4 scans per single photon emission computerized tomography imaging modality. This was followed by 4 first in human translations into sentinel lymph node biopsy procedures for penile (open surgery) and prostate (laparoscopic surgery) cancer. RESULTS: Overall the phantom studies revealed a tool-target distance accuracy of 2.1 mm for SPECT/CT and 3.2 mm for freehand single photon emission computerized tomography, and an augmented reality registration accuracy of 1.1 and 2.2 mm, respectively. Subsequently open and laparoscopic navigation efforts were accurate enough to localize the fluorescence signals of the targeted tissues in vivo. CONCLUSIONS: The phantom and human studies performed suggested that the single navigation setup is applicable in various open and laparoscopic urological surgery applications. Further evaluation in larger patient groups with a greater variety of malignancies is recommended to strengthen these results.

(Near-Infrared) Fluorescence-Guided Surgery Under Ambient Light Conditions: A Next Step to Embedment of the Technology in Clinical Routine.

BACKGROUND AND PURPOSE: In open surgery procedures, after temporarily dimming the lights in the operation theatre, the Photo Dynamic Eye (PDE) fluorescence camera has, amongst others, been used for fluorescence-guided sentinel node (SN) biopsy procedures. To improve the clinical utility and logistics of fluorescence-guided surgery, we developed and evaluated a prototype modified PDE (m-PDE) fluorescence camera system. METHODS: The m-PDE works under ambient light conditions and includes a white light mode and a pseudo-green-colored fluorescence mode (including a gray-scaled anatomical background). Twenty-seven patients scheduled for SN biopsy for (head and neck) melanoma (n = 16), oral cavity (n = 6), or penile (n = 5) cancer were included. The number and location of SNs were determined following an indocyanine green-(99m)Tc-nanocolloid injection and preoperative imaging. Intraoperatively, fluorescence guidance was used to visualize the SNs. The m-PDE and conventional PDE were compared head-to-head in a phantom study, and in seven patients. In the remaining 20 patients, only the m-PDE was evaluated. RESULTS: Phantom study: The m-PDE was superior over the conventional PDE, with a detection sensitivity of 1.20 × 10(-11) M (vs. 3.08 × 10(-9) M) ICG in human serum albumin. In the head-to-head clinical comparison (n = 7), the m-PDE was also superior: (i) SN visualization: 100 versus 81.4 %; (ii) transcutaneous SN visualization: 40.7 versus 22.2 %; and (iii) lymphatic duct visualization: 7.4 versus 0 %. Findings were further underlined in the 20 additionally included patients. CONCLUSION: The m-PDE enhanced fluorescence imaging properties compared with its predecessor, and provides a next step towards routine integration of real-time fluorescence guidance in open surgery.

Multimodal hybrid imaging agents for sentinel node mapping as a means to (re)connect nuclear medicine to advances made in robot-assisted surgery.

PURPOSE: Radical prostatectomy and complementary extended pelvic lymph node dissection (ePLND) of sentinel lymph nodes (SNs) and non-sentinel lymph nodes (LNs) at risk of containing metastases are increasingly being performed using high-tech robot-assisted approaches. Although this technological evolution has clear advantages, the physical nature of robotic systems limits the integrated use of routine radioguided surgery technologies. Hence, engineering effort in robotics are focused on the integration of fluorescence guidance technologies. Using the hybrid SN tracer indocyanine green-(99m)Tc-nanocolloid (radioactive and fluorescent), for the first time in combination with a robot-integrated laparoscope, we investigated whether the robot-assisted approach affects the accuracy of fluorescence detection of SNs identified preoperatively using nuclear medicine. METHODS: The study included 55 patients (Briganti nomogram-based risk >5 % on LN metastases) scheduled for robot-assisted radical prostatectomy, SN biopsy and ePLND. Following indocyanine green-(99m)Tc-nanocolloid injection, preoperative nuclear imaging (lymphoscintigraphy and SPECT/CT) was used to locate the SN(s). The fluorescence laparoscope was used intraoperatively to identify the SN(s) with standard fluorescence settings (in 50 patients) and with customized settings (in 5 patients). The number and location of the SNs, the radioactive, fluorescence (both in vivo and ex vivo) and tumour status of the resected SNs/LNs, and postoperative complications were recorded and analysed. RESULTS: Combined, preoperative lymphoscintigraphy and SPECT/CT imaging identified 212 SNs (median 4 per patient). Intraoperative fluorescence imaging using standard fluorescence settings visualized 80.4 % (148/184 SNs; 50 patients; ex vivo 97.8 %). This increased to 85.7 % (12/14 SNs; 5 patients; ex vivo 100 %) with customized fluorescence settings. SPECT/CT images provided guidance towards the residual SNs. Ex vivo all removed SNs were radioactive. SNs were tumour-positive in 25.4 % of patients (14/55; false-negative rate 7 %, 1/14 patients). In ten patients, the SN was the only tumour-positive LN. Surgical complications were minimal. CONCLUSION: Directly linking 3D preoperative nuclear imaging information on SNs to a robot-integrated fluorescence laparoscope improved the surgeon's use of the technology and did not influence the sensitivity or morbidity of the procedure. To our surprise, however, the detection rates with the current fluorescence camera did not improve.

Fluorescence guided surgery and tracer-dose, fact or fiction?

INTRODUCTION: Fluorescence guidance is an upcoming methodology to improve surgical accuracy. Challenging herein is the identification of the minimum dose at which the tracer can be detected with a clinical-grade fluorescence camera. Using a hybrid tracer such as indocyanine green (ICG)-(99m)Tc-nanocolloid, it has become possible to determine the accumulation of tracer and correlate this to intraoperative fluorescence-based identification rates. In the current study, we determined the lower detection limit of tracer at which intraoperative fluorescence guidance was still feasible. METHODS: Size exclusion chromatography (SEC) provided a laboratory set-up to analyze the chemical content and to simulate the migratory behavior of ICG-nanocolloid in tissue. Tracer accumulation and intraoperative fluorescence detection findings were derived from a retrospective analysis of 20 head-and-neck melanoma patients, 40 penile and 20 prostate cancer patients scheduled for sentinel node (SN) biopsy using ICG-(99m)Tc-nanocolloid. In these patients, following tracer injection, single photon emission computed tomography fused with computed tomography (SPECT/CT) was used to identify the SN(s). The percentage injected dose (% ID), the amount of ICG (in nmol), and the concentration of ICG in the SNs (in µM) was assessed for SNs detected on SPECT/CT and correlated with the intraoperative fluorescence imaging findings. RESULTS: SEC determined that in the hybrid tracer formulation, 41 % (standard deviation: 12 %) of ICG was present in nanocolloid-bound form. In the SNs detected using fluorescence guidance a median of 0.88 % ID was present, compared to a median of 0.25 % ID in the non-fluorescent SNs (p-value < 0.001). The % ID values could be correlated to the amount ICG in a SN (range: 0.003-10.8 nmol) and the concentration of ICG in a SN (range: 0.006-64.6 µM). DISCUSSION: The ability to provide intraoperative fluorescence guidance is dependent on the amount and concentration of the fluorescent dye accumulated in the lesion(s) of interest. Our findings indicate that intraoperative fluorescence detection with ICG is possible above a µM concentration.

First-in-human evaluation of a hybrid modality that allows combined radio- and (near-infrared) fluorescence tracing during surgery.

PURPOSE: The clinical introduction of the hybrid tracer indocyanine green (ICG)-(99m)Tc-nanocolloid, composed of a radioactive and a near-infrared (NIR) fluorescence component, has created the need for surgical (imaging) modalities that allow for simultaneous detection of both signals. This study describes the first-in-human use of a prototype opto-nuclear probe during sentinel node (SN) biopsy using ICG-(99m)Tc-nanocolloid. METHODS: To allow for fluorescence tracing, a derivative of the conventional gamma probe technology was generated in which two optical fibers were integrated to allow for excitation (785 nm) and emission signal collection (> 810 nm). The ability of this opto-nuclear probe to detect the fluorescence signal of the hybrid tracer ICG-(99m)Tc-nanocolloid was firstly determined ex vivo in (non)SNs samples obtained from 41 patients who underwent hybrid tracer-based SN biopsy in the head and neck or urogenital area. In an in vivo proof-of-concept study in nine of these 41 patients, SNs were localized using combined gamma and fluorescence tracing with the opto-nuclear probe. Fluorescence tracing was performed in a similar manner as gamma tracing and under ambient light conditions. RESULTS: Ex vivo, the gamma tracing option of the opto-nuclear probe correctly identified the SN in all 150 evaluated (non)SN samples. Ex vivo fluorescence tracing in the low-sensitivity mode correctly identified 71.7% of the samples. This increased to 98.9% when fluorescence tracing was performed in the high-sensitivity mode. In vivo fluorescence tracing (high-sensitivity mode) accurately identified the SNs in all nine patients (20 SNs evaluated; 100%). CONCLUSION: This study demonstrates the first-in-human evaluation of a hybrid modality capable of detecting both gamma and fluorescence signals during a surgical procedure. Fluorescence tracing could be performed in ambient light.

Advances in sentinel node dissection in prostate cancer from a technical perspective.

The most important feature of sentinel node biopsy for prostate cancer procedure is that staging can be improved. Sentinel nodes might be found outside the extended pelvic lymph node dissection template what renders the sentinel node additive of extended pelvic lymph node dissection. At the same time, staging within the template can be further refined. We reviewed the literature regarding the sentinel node biopsy procedure for prostate cancer. PubMed and Embase were searched for all English-language publications from January 1999 to September 2014 by using the keywords as "prostate cancer" and "sentinel lymph node" plus "biopsy" "dissection" and/or "procedure." The present review discusses step-by-step sentinel node biopsy for prostate cancer. Topics of discussion are: (i) preoperative sentinel node mapping (tracers and imaging); (ii) intraoperative sentinel node identification (surgical procedure and outcome); and (iii) novelties to improve sentinel node identification (pre- and intraoperative approaches). Conventional sentinel node mapping is carried out after the injection of a (99m) Tc-based tracer and subsequent preoperative imaging; for example, lymphoscintigraphy and single-photon emission computed tomography/computed tomography. This approach allowed the detection of sentinel nodes outside the extended lymph node dissection template in 3.6-36% of men with intermediate- and high-risk prostate cancer. Hereby, an overall false negative rate of sentinel nodes was reported between 0% and 24.4%. To further refine the intraoperative sampling procedure, novel imaging methods such as fluorescence imaging have been introduced. Prospective randomized comparison studies are required to confirm the added benefit of sentinel template directed nodal dissection. A proper and obtainable end-point of such a study could be the number of removed positive nodes for carrying out nodal dissection with or without sentinel template directed dissection. Similarly, the clinical impact of novel imaging technologies requires further investigation.

Quantifying the Impact of Signal-to-background Ratios on Surgical Discrimination of Fluorescent Lesions.

PURPOSE: Surgical fluorescence guidance has gained popularity in various settings, e.g., minimally invasive robot-assisted laparoscopic surgery. In pursuit of novel receptor-targeted tracers, the field of fluorescence-guided surgery is currently moving toward increasingly lower signal intensities. This highlights the importance of understanding the impact of low fluorescence intensities on clinical decision making. This study uses kinematics to investigate the impact of signal-to-background ratios (SBR) on surgical performance. METHODS: Using a custom grid exercise containing hidden fluorescent targets, a da Vinci Xi robot with Firefly fluorescence endoscope and ProGrasp and Maryland forceps instruments, we studied how the participants' (N = 16) actions were influenced by the fluorescent SBR. To monitor the surgeon's actions, the surgical instrument tip was tracked using a custom video-based tracking framework. The digitized instrument tracks were then subjected to multi-parametric kinematic analysis, allowing for the isolation of various metrics (e.g., velocity, jerkiness, tortuosity). These were incorporated in scores for dexterity (Dx), decision making (DM), overall performance (PS) and proficiency. All were related to the SBR values. RESULTS: Multi-parametric analysis showed that task completion time, time spent in fluorescence-imaging mode and total pathlength are metrics that are directly related to the SBR. Below SBR 1.5, these values substantially increased, and handling errors became more frequent. The difference in Dx and DM between the targets that gave SBR < 1.50 and SBR > 1.50, indicates that the latter group generally yields a 2.5-fold higher Dx value and a threefold higher DM value. As these values provide the basis for the PS score, proficiency could only be achieved at SBR > 1.55. CONCLUSION: By tracking the surgical instruments we were able to, for the first time, quantitatively and objectively assess how the instrument positioning is impacted by fluorescent SBR. Our findings suggest that in ideal situations a minimum SBR of 1.5 is required to discriminate fluorescent lesions, a substantially lower value than the SBR 2 often reported in literature.

Evaluation of the Hybrid Tracer Indocyanine Green- 99m Tc-Nanocolloid for Sentinel Node Biopsy in Bladder Cancer-A Prospective Pilot Study.

RATIONALE: In muscle-invasive bladder cancer (MIBC), lymph node invasion has proven to be an independent predictor of disease recurrence and cancer-specific survival. We evaluated the feasibility of targeting the sentinel node (SN) for biopsy in MIBC patients using the hybrid tracer indocyanine green (ICG)- 99m Tc-nanocolloid for simultaneous radioguidance and fluorescence guidance. METHODS: Twenty histologically confirmed cN0M0 MIBC patients (mean age, 63.3 years; range, 30-82 years), scheduled for radical cystectomy with SN biopsy and extended pelvic lymph node dissection (ePLND), were prospectively included. Twelve patients were operated on following neoadjuvant chemotherapy. The patients received lymphoscintigraphy as well as SPECT/CT after 4 transurethral injections of ICG- 99m Tc-nanocolloid (mean, 208 MBq; range, 172-229 MBq) around the tumor/scar in the detrusor muscle of the bladder on the day before radical cystectomy. Sentinel node resection was performed under radioguidance and fluorescence guidance. RESULTS: Nineteen patients could be analyzed. On preoperative imaging, SNs could be identified in 10 patients (53%; mean, 1.6 SN/patient), which revealed drainage pathways outside the ePLND in 20% of the patients. Interesting to note is that 2 patients (10%) with preoperative nonvisualization displayed fluorescent and radioactive SNs during surgery. Location of the primary tumor near the left lateral side of the bladder seemed to be a factor for nonvisualization. Nodal harvesting with ePLND varied among patients (mean, 23.3). Histopathology confirmed tumor-positive nodes in 4 (21%) of all patients. In the 2 patients where an SN could be identified, the ePLND specimens were tumor-negative. All patients with tumor-positive nodes had advanced disease (stage III). CONCLUSION: Sentinel node biopsy in bladder cancer using the hybrid tracer ICG- 99m Tc-nanocolloid is feasible, and preoperative imaging is predictive for the ability to perform SN biopsy in 83% of the patients who displayed an SN. In patients with a successful preoperative SN mapping using lymphoscintigraphy and SPECT/CT, the intraoperative SN guidance and detection were effective, even outside the ePLND area. As such, this study underscores the critical role that preoperative imaging plays in challenging image-guided surgery applications.

Multi-wavelength fluorescence imaging with a da Vinci Firefly-a technical look behind the scenes.

The field of fluorescence-guided surgery builds on colored fluorescent tracers that have become available for different clinical applications. Combined use of complementary fluorescent emissions can allow visualization of different anatomical structures (e.g. tumor, lymphatics and nerves) in the same patient. With the aim to assess the requirements for multi-color fluorescence guidance under in vivo conditions, we thoroughly characterized two FDA-approved laparoscopic Firefly camera systems available on the da Vinci Si or da Vinci Xi surgical robot. In this process, we studied the cameras' performance with respect to the photophysical properties of the FDA-approved dyes Fluorescein and ICG. Our findings indicate that multi-wavelength fluorescence imaging of Fluorescein and ICG is possible using clinical-grade fluorescence laparoscopes, but critical factors for success include the photophysical dye properties, imaging system performance and the amount of accumulated dye. When comparing the camera performance, the Xi system provided more effective excitation (adaptions in the light source) and higher detection sensitivity (chip-on-a-tip and/or enhanced image processing) for both Fluorescein and ICG. Both systems can readily be used for multi-wavelength fluorescence imaging of Fluorescein and ICG under clinically relevant conditions. With that, another step has been made towards the routine implementation of multi-wavelength image-guided surgery concepts.

Technologic (R)Evolution Leads to Detection of More Sentinel Nodes in Patients with Melanoma in the Head and Neck Region.

Sentinel lymph node (SN) biopsy (SNB) has proven to be a valuable tool for staging melanoma patients. Since its introduction in the early 1990s, this procedure has undergone several technologic refinements, including the introduction of SPECT/CT, as well as radioguidance and fluorescence guidance. The purpose of the current study was to evaluate the effect of this technologic evolution on SNB in the head and neck region. The primary endpoint was the false-negative (FN) rate. Secondary endpoints were number of harvested SNs, overall operation time, operation time per harvested SN, and postoperative complications. Methods: A retrospective database was queried for cutaneous head and neck melanoma patients who underwent SNB at The Netherlands Cancer Institute between 1993 and 2016. The implementation of new detection techniques was divided into 4 groups: 1993-2005, with preoperative lymphoscintigraphy and intraoperative use of both a γ-ray detection probe and patent blue (n = 30); 2006-2007, with addition of preoperative road maps based on SPECT/CT (n = 15); 2008-2009, with intraoperative use of a portable γ-camera (n = 40); and 2010-2016, with addition of near-infrared fluorescence guidance (n = 192). Results: In total, 277 patients were included. At least 1 SN was identified in all patients. A tumor-positive SN was found in 59 patients (21.3%): 10 in group 1 (33.3%), 3 in group 2 (20.0%), 6 in group 3 (15.0%), and 40 in group 4 (20.8%). Regional recurrences in patients with tumor-negative SNs resulted in an overall FN rate of 11.9% (group 1, 16.7%; group 2, 0%; group 3, 14.3%; group 4, 11.1%). The number of harvested nodes increased with advancing technologies (P = 0.003), whereas Breslow thickness and operation time per harvested SN decreased (P = 0.003 and P = 0.017, respectively). There was no significant difference in percentage of tumor-positive SNs, overall operation time, and complication rate between the different groups. Conclusion: The use of advanced detection technologies led to a higher number of identified SNs without an increase in overall operation time, possibly indicating an improved surgical efficiency. Operation time per harvested SN decreased; the average FN rate remained 11.9% and was unchanged over 23 y. There was no significant change in postoperative complication rate.

Can Intraoperative Fluorescence Imaging Identify All Lesions While the Road Map Created by Preoperative Nuclear Imaging Is Masked?

Tracers can help visualize the lymphatic drainage patterns and sentinel nodes (SNs) of individual prostate cancer patients. To determine the role of nuclear medicine in surgical guidance, in particular the positional guidance of a SPECT/CT-based 3-dimensional imaging road map, in this process we studied to what extent fluorescence guidance underestimated the number of target lesions relative to radioguidance. Methods: SPECT/CT imaging was performed after intraprostatic tracer administration of either indocyanine green (ICG)-99mTc-nanocolloid (hybrid-tracer group) or 99mTc-nanocolloid to create a road map that depicted all SNs. Patients who received 99mTc-nanocolloid were injected with "free" ICG immediately before surgery. Before unmasking, fluorescence guidance was used for intraoperative SN identification. This was followed by extended pelvic lymph node dissection (ePLND). After unmasking of the SPECT/CT images, the number of missed SNs was recorded and their resection was pursued when there was no risk of intraoperative complications. Results: Preoperative SPECT/CT revealed no differences in the SN identification rate between ICG-99mTc-nanocolloid and 99mTc-nanocolloid. However, fluorescence guidance allowed intraoperative removal of all SNs in only 40% of patients in the hybrid-tracer group and 20% of patients in the free-ICG group. Overall, 75.9% of the intraoperatively resected SNs in the hybrid-tracer group and 51.8% of the SNs in the free-ICG group were removed solely under fluorescence guidance. During ePLND, 22 additional SNs were resected (7 in the hybrid-tracer group and 15 in the free-ICG group). After unmasking, 18 remaining SNs were identified (6 in the hybrid group and 12 in the free-ICG group). In the free-ICG group, ex vivo evaluation of the excised specimens revealed that 14 SNs removed under ePLND or after unmasking contained radioactivity but no fluorescence. Conclusion: The preoperative imaging road map provided by SPECT/CT enhanced the detection of prostate SNs in more ectopic locations in 17 of the 25 patients, and the hybrid tracer ICG-99mTc-nanocolloid was shown to outperform free ICG. Overall, fluorescence-guided pelvic nodal surgery underestimated the number of SNs in 60%-80% of patients.

Fluorescence background quenching as a means to increase Signal to Background ratio - a proof of concept during Nerve Imaging.

Introduction: Adequate signal to background ratios are critical for the implementation of fluorescence-guided surgery technologies. While local tracer administrations help to reduce the chance of systemic side effects, reduced spatial migration and non-specific tracer diffusion can impair the discrimination between the tissue of interest and the background. To combat background signals associated with local tracer administration, we explored a pretargeting concept aimed at quenching non-specific fluorescence signals. The efficacy of this concept was evaluated in an in vivo neuronal tracing set-up. Methods: Neuronal tracing was achieved using a wheat germ agglutinin (WGA) lectin. functionalized with an azide-containing Cy5 dye (N3-Cy5-WGA). A Cy7 quencher dye (Cy7-DBCO) was subsequently used to yield Cy7-Cy5-WGA, a compound wherein the Cy5 emission is quenched by Förster resonance energy transfer to Cy7. The photophysical properties of N3-Cy5-WGA and Cy7-Cy5-WGA were evaluated together with deactivation kinetics in situ, in vitro (Schwannoma cell culture), ex vivo (muscle tissue from mice; used for dose optimization), and in vivo (nervus ischiadicus in THY-1 YFP mice).Results:In situ, conjugation of Cy7-DBCO to N3-Cy5-WGA resulted in >90% reduction of the Cy5 fluorescence signal intensity at 30 minutes after addition of the quencher. In cells, pretargeting with the N3-Cy5-WGA lectin yielded membranous staining, which could efficiently be deactivated by Cy7-DBCO over the course of 30 minutes (91% Cy5 signal decrease). In ex vivo muscle tissue, administration of Cy7-DBCO at the site where N3-Cy5-WGA was injected induced 80-90% quenching of the Cy5-related signal after 10-20 minutes, while the Cy7-related signal remained stable over time. In vivo,Cy7-DBCO effectively quenched the non-specific background signal up to 73% within 5 minutes, resulting in a 50% increase in the signal-to-background ratio between the nerve and injection site. Conclusion: The presented pretargeted fluorescence-quenching technology allowed fast and effective reduction of the background signal at the injection site, while preserving in vivo nerve visualization. While this proof-of-principle study was focused on imaging of nerves using a fluorescent WGA-lectin, the same concept could in the future also apply to applications such as sentinel node imaging.

Hybrid Indocyanine Green-99mTc-nanocolloid for Single-photon Emission Computed Tomography and Combined Radio- and Fluorescence-guided Sentinel Node Biopsy in Penile Cancer: Results of 740 Inguinal Basins Assessed at a Single Institution.

BACKGROUND: Sentinel node (SN) biopsy in penile cancer (PeCa) is typically performed using 99mTc-nanocolloid and blue dye. Recent reports suggested that the hybrid (radioactive and fluorescent) tracer indocyanine green (ICG)-99mTc-nanocolloid may improve intraoperative optical SN identification. OBJECTIVE: The current study aimed to confirm the reliability of ICG-99mTc-nanocolloid and to assess whether blue dye is still of added value. DESIGN, SETTING, AND PARTICIPANTS: A total of 400 ≥T1G2N0 PeCa patients were staged with SN biopsy at a single European centre. SNs were preoperatively identified with lymphoscintigraphy and single-photon emission computed tomography. Intraoperatively, SNs were detected via gamma tracing, blue staining, and fluorescence imaging. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: All patients (n=400, 740 groins) received ICG-99mTc-nanocolloid. Intraoperative SN identification rates were retrospectively evaluated. In those patients who received ICG-99mTc-nanocolloid and blue dye (n=266, 492 groins), SN visualisation rates were compared using the McNemar test. RESULTS AND LIMITATIONS: In total, 740 groins were assessed. No tracer-related (allergic) reactions were reported. All preoperatively defined SNs (n=1163) were localised intraoperatively. Of all excised SNs, 98% were detectable with gamma probe and 96% were visible with fluorescence imaging. In the analysis of the patients who received ICG-99mTc-nanocolloid and blue dye, fluorescence imaging yielded a 39% higher SN detection rate than blue dye (95% confidence interval 36-43%, p<0.001). Of the SNs that were tumour positive, 100% were intraoperatively visualised by fluorescence imaging, whereas merely 84% of the positive nodes stained blue. CONCLUSIONS: This study confirms that ICG-99mTc-nanocolloid is a reliable SN tracer for PeCa that significantly improves optical SN detection over blue dye. PATIENT SUMMARY: Hybrid indocyanine green (ICG)-99mTc-nanocolloid is a safe and reliable sentinel node (SN) tracer, as established in this large series of 400 penile cancer patients (740 groins). It enables accurate pre- and intraoperative SN identification and significantly improves SN detection rate compared with blue dye, without staining the surgical field or the need for an additional injection.

Head-to-head comparison of the hybrid tracer indocyanine green-99mTc-nanocolloid with 99mTc-Senti-Scint using sentinel node lymphoscintigraphy and single-photon emission computed tomography combined with computer tomography in melanoma.

OBJECTIVE: The hybrid tracer indocyanine green (ICG)-Tc-nanocolloid has been introduced for sentinel node imaging. However, until now, a comparison of this tracer with other radiocolloids with a larger particle size has not been effectuated. Based on a head-to-head evaluation in patients with melanoma, we have compared ICG-Tc-nanocolloid (particle size 5-80 nm) with Tc-Senti-Scint (particle size 100-600 nm) to establish differences in drainage pattern and sentinel node localization using lymphoscintigraphy and single-photon emission computed tomography combined with computer tomography (SPECT-CT) in melanoma patients scheduled for sentinel node biopsy. METHODS: Twenty-five patients (mean age: 56.9 years, range: 25-79 years) with a melanoma scheduled for SLN biopsy prior to (re)excision of the primary lesion (scar) were prospectively included following a two-day procedure. The first day, after Tc-Senti-Scint injection in four intradermal depots around the primary lesion or scar, early/delayed lymphoscintigraphy and SPECT-CT images were acquired. The injection sites were marked. The second day, after assessing lymph node radioactivity using planar scintigraphy, ICG-Tc-nanocolloid was injected at the previously marked skin points and imaging was performed. The paired planar and SPECT-CT images of both tracers were evaluated with respect to drainage patterns, SLN visualization and non-SLN appearing. RESULTS: Twenty-four out of 25 patients were evaluable. SLN visualization on a patient basis was 100% for ICG-Tc-nanocolloid and 96% for Tc-Senti-Scint, whereas uptake in non-SLNs was found in, respectively, 71% (17/24) and 61% (14/23). Concordance in drainage to 45 lymph node basins was 91%. Discordant drainage was found for two melanomas in the head-and-neck and one in the clavicular area. Unique lymph node basins were seen in 44/45 (98%) for ICG-Tc-nanocolloid and 42/45 (93%) for Tc-Senti-Scint. Concerning identified SLNs, the number was similar for both tracers (n = 58); however, more non-SLNs (65 vs 50) were visualized with ICG-Tc-nanocolloid than with Tc-Senti-Scint. CONCLUSION: A slightly higher SLN visualization accompanied by a tendency to depict more non-SLNs was found for ICG-Tc-nanocolloid. Excepting the head and neck area, an overall high concordance in drainage was found for both radiotracers. With an additional value for the hybrid tracer due to the combination of preoperative imaging and the additional visual signal in the operation room, added by the fluorescent component of the hybrid tracer, there was a preference for ICG-Tc-nanocolloid.