Intraoperative molecular imaging of colorectal lung metastases with SGM-101: a feasibility study.

PURPOSE: Metastasectomy is a common treatment option for patients with colorectal lung metastases (CLM). Challenges exist with margin assessment and identification of small nodules, especially during minimally invasive surgery. Intraoperative fluorescence imaging has the potential to overcome these challenges. The aim of this study was to assess feasibility of targeting CLM with the carcinoembryonic antigen (CEA) specific fluorescent tracer SGM-101. METHODS: This was a prospective, open-label feasibility study. The primary outcome was the number of CLM that showed a true positive fluorescence signal with SGM-101. Fluorescence positive signal was defined as a signal-to-background ratio (SBR) ≥ 1.5. A secondary endpoint was the CEA expression in the colorectal lung metastases, assessed with the immunohistochemistry, and scored by the total immunostaining score. RESULTS: Thirteen patients were included in this study. Positive fluorescence signal with in vivo, back table, and closed-field bread loaf imaging was observed in 31%, 45%, and 94% of the tumors respectively. Median SBRs for the three imaging modalities were 1.00 (IQR: 1.00-1.53), 1.45 (IQR: 1.00-1.89), and 4.81 (IQR: 2.70-7.41). All tumor lesions had a maximum total immunostaining score for CEA expression of 12/12. CONCLUSION: This study demonstrated the potential of fluorescence imaging of CLM with SGM-101. CEA expression was observed in all tumors, and closed-field imaging showed excellent CEA specific targeting of the tracer to the tumor nodules. The full potential of SGM-101 for in vivo detection of the tracer can be achieved with improved minimal invasive imaging systems and optimal patient selection. TRIAL REGISTRATION: The study was registered in ClinicalTrial.gov under identifier NCT04737213 at February 2021.

Quantification of indocyanine green near-infrared fluorescence bowel perfusion assessment in colorectal surgery.

BACKGROUND: Indocyanine green near-infrared fluorescence bowel perfusion assessment has shown its potential benefit in preventing anastomotic leakage. However, the surgeon's subjective visual interpretation of the fluorescence signal limits the validity and reproducibility of the technique. Therefore, this study aimed to identify objective quantified bowel perfusion patterns in patients undergoing colorectal surgery using a standardized imaging protocol. METHOD: A standardized fluorescence video was recorded. Postoperatively, the fluorescence videos were quantified by drawing contiguous region of interests (ROIs) on the bowel. For each ROI, a time-intensity curve was plotted from which perfusion parameters (n = 10) were derived and analyzed. Furthermore, the inter-observer agreement of the surgeon's subjective interpretation of the fluorescence signal was assessed. RESULTS: Twenty patients who underwent colorectal surgery were included in the study. Based on the quantified time-intensity curves, three different perfusion patterns were identified. Similar for both the ileum and colon, perfusion pattern 1 had a steep inflow that reached its peak fluorescence intensity rapidly, followed by a steep outflow. Perfusion pattern 2 had a relatively flat outflow slope immediately followed by its plateau phase. Perfusion pattern 3 only reached its peak fluorescence intensity after 3 min with a slow inflow gradient preceding it. The inter-observer agreement was poor-moderate (Intraclass Correlation Coefficient (ICC): 0.378, 95% CI 0.210-0.579). CONCLUSION: This study showed that quantification of bowel perfusion is a feasible method to differentiate between different perfusion patterns. In addition, the poor-moderate inter-observer agreement of the subjective interpretation of the fluorescence signal between surgeons emphasizes the need for objective quantification.

Near-infrared fluorescence-guided resection of colorectal liver metastases.

BACKGROUND: The fundamental principle of oncologic surgery is the complete resection of malignant cells. However, small tumors are often difficult to find during surgery using conventional techniques. The objectives of this study were to determine if optical imaging, using a contrast agent already approved for other indications, could improve hepatic metastasectomy with curative intent, to optimize dose and timing, and to determine the mechanism of contrast agent accumulation. METHODS: The high tissue penetration of near-infrared (NIR) light was exploited by use of the FLARE (Fluorescence-Assisted Resection and Exploration) image-guided surgery system and the NIR fluorophore indocyanine green in a clinical trial of 40 patients undergoing hepatic resection for colorectal cancer metastases. RESULTS: A total of 71 superficially located (< 6.2 mm beneath the liver capsule) colorectal liver metastases were identified and resected using NIR fluorescence imaging. Median tumor-to-liver ratio was 7.0 (range, 1.9-18.7) and no significant differences between time points or doses were found. Indocyanine green fluorescence was seen as a rim around the tumor, which is shown to be entrapment around cytokeratin 7-positive hepatocytes compressed by the tumor. Importantly, in 5 of 40 patients (12.5%, 95% confidence interval = 5.0-26.6), additional small and superficially located lesions were detected using NIR fluorescence, and were otherwise undetectable by preoperative computed tomography, intraoperative ultrasound, visual inspection, and palpation. CONCLUSIONS: NIR fluorescence imaging, even when used with a nontargeted, clinically available NIR fluorophore, is complementary to conventional imaging and able to identify missed lesions by other modalities.

Ex vivo sentinel node mapping in colon cancer combining blue dye staining and fluorescence imaging.

BACKGROUND: The sentinel lymph node procedure has been proposed to improve nodal staging in colon cancer patients. The aim of this study was to assess the added value of near-infrared (NIR) fluorescence imaging to conventional blue dye staining for ex vivo sentinel lymph node mapping. MATERIALS AND METHODS: We included 22 consecutive patients undergoing surgery for colon cancer. After tumor resection, we submucosally injected a premixed cocktail of the near-infrared lymphatic tracer HSA800 and blue dye around the tumor for detection of sentinel lymph nodes. We used the Mini-FLARE imaging system for fluorescence imaging. RESULTS: In 95% of patients, we identified at least one sentinel lymph node. Overall, a total of 77 sentinel lymph nodes were identified, 77 of which were fluorescent (100%) and 70 of which were blue (91%). Sentinel lymph nodes that were located deeper in the mesenteric fat could easily be located by NIR fluorescence. In four of five patients with lymph node metastases, tumor cells were present in at least one of the sentinel lymph nodes. CONCLUSIONS: This study shows the successful use and added value of the NIR fluorescence tracer HSA800 to conventional blue dye for the ex vivo sentinel lymph node procedure in colon cancer.

Intraoperative prediction of postoperative flap outcome using the near-infrared fluorophore methylene blue.

Methylene blue (MB) is a near-infrared fluorophore that provides a stable visual map of skin perfusion after intravenous injection. We explored the capability of MB to predict submental flap postoperative outcome using a single intraoperative measurement. Submental flaps were created in N = 15 pigs and imaged using the FLARE imaging system immediately after surgery and at 72 hours. Using the first 3 pigs, optimal MB dosing was found to be 2.0 mg/kg. Training and validation sets of 6 pigs each were then used for receiver operating characteristic analysis. In the training set, a contrast-to-background ratio (CBR) threshold of 1.24 provided the highest sensitivity and specificity to predict tissue necrosis at 72 hours. In the validation set, this threshold provided a prediction sensitivity of 95.3% and a specificity of 98.0%. We demonstrate that a single intraoperative near-infrared measurement can predict submental flap outcome at 72 hours.

Quantitative assessment of nipple perfusion with near-infrared fluorescence imaging.

Preserving the nipple-areolar complex with a nipple-sparing mastectomy improves cosmesis compared with skin-sparing mastectomy. However, complications such as necrosis of the nipple-areolar complex significantly affect cosmetic outcome. Many factors influence nipple-areolar perfusion, and no consensus currently exists on optimal incisional choice. This study evaluates 2 nipple-sparing mastectomy incision models using near-infrared fluorescence to assess perfusion quantitatively. The periareolar and radial incisions were compared with 2 control models in Yorkshire pigs (N = 6). Methylene blue and indocyanine green were injected intravenously, and near-infrared fluorescence images were recorded at 3 time points: before surgery, immediately after (0 hour), and 3 days postoperatively. Contrast-to-background ratio was used to assess perfusion. At 72 hours, radial incisions showed a statistically significantly higher perfusion compared with periareolar incisions (P < 0.05). Based on our findings, radial incisions for nipple-sparing mastectomy may be preferable due to higher perfusion; however, clinical trials are necessary for further assessment.

Robot-assisted thoracic surgery for stages IIB-IVA non-small cell lung cancer: retrospective study of feasibility and outcome.

Robot-assisted thoracic surgery (RATS) for higher stages non-small cell lung carcinoma (NSCLC) remains controversial. This study reports the feasibility of RATS in patients with stages IIB-IVA NSCLC. A single-institute, retrospective study was conducted with patients undergoing RATS for stages IIB-IVA NSCLC, from January 2015 until January 2020. Unforeseen N2 disease was excluded. Data were collected from the Dutch Lung Cancer Audit database. Conversion rate, radical (R0) resection rate, local recurrence rate and complications were analyzed, as were risk factors for conversion. RATS was performed in 95 patients with NSCLC clinical or pathological stages IIB (N = 51), IIIA (N = 39), IIIB (N = 2) and IVA (N = 3). 10.5% had received neoadjuvant chemoradiotherapy. Pathological staging was T3 in 33.7% and T4 in 34.7%. RATS was completed in 77.9% with a radical resection rate of 94.8%. Lobectomy was performed in 67.4% of the total resections. Conversion was for strategic (18.9%) and emergency (3.2%) reasons. Pneumonectomy (p = 0.001), squamous cell carcinoma (p < 0.001), additional resection of adjacent structures (p = 0.025) and neoadjuvant chemoradiation (p = 0.017) were independent risk factors for conversion. Major post-operative complications occurred in ten patients (10.5%) including an in-hospital mortality of 2.1% (n = 2). Median recurrence-free survival was estimated at 39.4 months (CI 16.4-62.5). Two- and 5-year recurrence-free survival rates were 53.8% and 36.7%, respectively. This study concludes that RATS is safe and feasible in higher staged NSCLC tumors after exclusion of unforeseen N2 disease. It brings new perspective on the potential of RATS in higher stages, dealing with larger and more invasive tumors.

Data-Driven Identification of Targets for Fluorescence-Guided Surgery in Non-Small Cell Lung Cancer.

PURPOSE: Intraoperative identification of lung tumors can be challenging. Tumor-targeted fluorescence-guided surgery can provide surgeons with a tool for real-time intraoperative tumor detection. This study evaluated cell surface biomarkers, partially selected via data-driven selection software, as potential targets for fluorescence-guided surgery in non-small cell lung cancers: adenocarcinomas (ADC), adenocarcinomas in situ (AIS), and squamous cell carcinomas (SCC). PROCEDURES: Formalin-fixed paraffin-embedded tissue slides of resection specimens from 15 patients with ADC and 15 patients with SCC were used and compared to healthy tissue. Molecular targets were selected based on two strategies: (1) a data-driven selection using > 275 multi-omics databases, literature, and experimental evidence; and (2) the availability of a fluorescent targeting ligand in advanced stages of clinical development. The selected targets were carbonic anhydrase 9 (CAIX), collagen type XVII alpha 1 chain (collagen XVII), glucose transporter 1 (GLUT1), G protein-coupled receptor 87 (GPR87), transmembrane protease serine 4 (TMPRSS4), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), folate receptor alpha (FRα), integrin αvß6 (αvß6), and urokinase-type plasminogen activator receptor (uPAR). Tumor expression of these targets was assessed by immunohistochemical staining. A total immunostaining score (TIS, range 0-12), combining the percentage and intensity of stained cells, was calculated. The most promising targets in ADC were explored in six AIS tissue slides to explore its potential in non-palpable lesions. RESULTS: Statistically significant differences in TIS between healthy lung and tumor tissue for ADC samples were found for CEA, EpCAM, FRα, αvß6, CAIX, collagen XVII, GLUT-1, and TMPRSS4, and of these, CEA, CAIX, and collagen XVII were also found in AIS. For SCC, EpCAM, uPAR, CAIX, collagen XVII, and GLUT-1 were found to be overexpressed. CONCLUSIONS: EpCAM, CAIX, and Collagen XVII were identified using concomitant use of data-driven selection software and clinical evidence as promising targets for intraoperative fluorescence imaging for both major subtypes of non-small cell lung carcinomas.

Simultaneous assessment of luminal integrity and vascular perfusion of the gastrointestinal tract using dual-channel near-infrared fluorescence.

Anastomotic complications such as stenosis and leakage in the gastrointestinal (GI) tract can cause high patient morbidity and mortality. To identify the potential preconditions of these complications intraoperatively, we explored the use of two 700 nm near-infrared (NIR) fluorophores administered intraluminally: (1) chlorella, an over-the-counter herbal supplement containing high concentrations of chlorophyll, and (2) methylene blue (MB). In parallel, we administered the 800 nm NIR fluorophore indocyanine green (ICG) intravenously to assess vascular function. Dual-channel, real-time intraoperative imaging and quantitation of the contrast to background ratio (CBR) were performed under normal conditions or after anastomosis or leakage of the stomach and intestines in 35 kg Yorkshire pigs using the Fluorescence-Assisted Resection and Exploration (FLARE) imaging system. Luminal integrity could be assessed with relatively high sensitivity with either chlorella or MB, although chlorella provided significantly higher CBR. ICG angiography provided assessment of blood perfusion of normal, ischemic, and anastomotic areas of the GI tract. Used simultaneously, 700 nm (chlorella or MB) and 800 nm (ICG) NIR fluorescence permitted independent assessment of luminal integrity and vascular perfusion of the GI tract intraoperatively and in real time. This technology has the potential to identify critical complications, such as anastomotic leakage, intraoperatively, when correction is still possible.

Randomized comparison of near-infrared fluorescence imaging using indocyanine green and 99(m) technetium with or without patent blue for the sentinel lymph node procedure in breast cancer patients.

BACKGROUND: Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has the potential to improve sentinel lymph node (SLN) mapping of breast cancer. We performed a randomized clinical trial to assess the value of blue dyes when used in combination with NIR fluorescence. We also preliminarily examined the possibility of performing SLN mapping without radiotracers. METHODS: Clinical trial subjects were 24 consecutive breast cancer patients scheduled to undergo SLN biopsy. All patients received standard of care using 99(m) technetium-nanocolloid and received 1.6 mL of 500 µM ICG injected periareolarly. Patients were randomly assigned to undergo SLN biopsy with or without patent blue. To assess the need for radiocolloids to localize the SLN or SLNs, the surgeon did not use the handheld gamma probe during the first 15 min after the axillary skin incision. RESULTS: SLN mapping was successful in 23 of the 24 patients. No significant difference was found in signal-to-background ratio between the groups with and without patent blue (8.3 ± 3.8 vs. 10.3 ± 5.7, respectively, P = 0.32). In both groups, 100 % of SLNs were radioactive and fluorescent, and in the patent blue group, only 84 % of SLNs were stained blue. In 25 % of patients, the use of the gamma probe was necessary to localize the SLN within the first 15 min. CONCLUSIONS: This study shows that there is no benefit of using patent blue for SLN mapping in breast cancer patients when using NIR fluorescence and 99(m) technetium-nanocolloid. NIR fluorescence imaging outperformed patent blue in all patients.

Optimization of near-infrared fluorescent sentinel lymph node mapping for vulvar cancer.

OBJECTIVES: Near-infrared fluorescence imaging has the potential to improve sentinel lymph node mapping in vulvar cancer, which was assessed in the current study. Furthermore, dose optimization of indocyanine green adsorbed to human serum albumin was performed. STUDY DESIGN: Nine vulvar cancer patients underwent the standard sentinel lymph node procedure using (99m)technetium-nancolloid and patent blue. In addition, intraoperative imaging was performed after peritumoral injection of 1.6 mL of 500, 750, or 1000 µM of indocyanine green adsorbed to human serum albumin. RESULTS: Near-infrared fluorescence sentinel lymph node mapping was successful in all patients. A total of 14 sentinel lymph nodes (average, 1.6; range, 1-4) were detected: 14 radioactive (100%), 11 blue (79%), and 14 near-infrared fluorescent (100%). CONCLUSION: This study demonstrates feasibility and accuracy of sentinel lymph node mapping using indocyanine green adsorbed to human serum albumin. Considering safety, cost, and pharmacy preferences, an indocyanine green adsorbed to human serum albumin concentration of 500 µM appears optimal for sentinel lymph node mapping in vulvar cancer.

Near-infrared fluorescence imaging of liver metastases in rats using indocyanine green.

BACKGROUND: Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) is a promising technique to obtain real-time assessment of the extent and number of colorectal liver metastases during surgery. The current study aims to optimize dosage and timing of ICG administration. MATERIALS AND METHODS: Liver tumors were induced in 18 male WAG/Rij rats by subcapsular inoculation of CC531 rat colorectal cancer cells into three distinct liver lobes. Rats were divided in two groups: imaging after 24 and 48 h or 72 and 96 h after intravenous ICG administration. In each time group, rats were allocated to three dose groups: 0.04, 0.08, or 0.16 mg ICG. Intraoperative imaging and ex vivo measurements were performed using the Mini-FLARE imaging system and confirmed by fluorescence microscopy. Fluorescence intensity was quantified using the Mini-FLARE software and the difference between tumor signal and liver signal (tumor-to-liver ratio; TLR) was calculated. RESULTS: In all 18 rats, all colorectal liver metastases (n = 34), some as small as 1.2 mm, were identified using ICG and the Mini-FLARE imaging system. Average tumor-to-liver ratio (TLR) over all groups was 3.0 ± 1.2. TLR was significantly higher in the 72 h time group compared with other time points. ICG dose did not significantly influence TLR, but a trend was found favoring the 0.08 mg dose group. Fluorescence microscopy demonstrated a clear fluorescent rim around the tumor. CONCLUSIONS: This study demonstrates that colorectal cancer liver metastases can be clearly identified during surgery using ICG and the Mini-FLARE imaging system, with optimal timing of 72 h post-injection and an optimal dose of 0.08 mg (0.25 mg/kg) ICG. NIR fluorescence imaging has the potential to improve intraoperative detection of micrometastases and, thus, the completeness of resection.

Fundamentals and developments in fluorescence-guided cancer surgery.

Fluorescence-guided surgery using tumour-targeted imaging agents has emerged over the past decade as a promising and effective method of intraoperative cancer detection. An impressive number of fluorescently labelled antibodies, peptides, particles and other molecules related to cancer hallmarks have been developed for the illumination of target lesions. New approaches are being implemented to translate these imaging agents into the clinic, although only a few have made it past early-phase clinical trials. For this translational process to succeed, target selection, imaging agents and their related detection systems and clinical implementation have to operate in perfect harmony to enable real-time intraoperative visualization that can benefit patients. Herein, we review key aspects of this imaging cascade and focus on imaging approaches and methods that have helped to shed new light onto the field of intraoperative fluorescence-guided cancer surgery with the singular goal of improving patient outcomes.

Fluorescence-guided surgery in colorectal cancer; A review on clinical results and future perspectives.

BACKGROUND: Colorectal cancer is the fourth most diagnosed malignancy worldwide and surgery is one of the cornerstones of the treatment strategy. Near-infrared (NIR) fluorescence imaging is a new and upcoming technique, which uses an NIR fluorescent agent combined with a specialised camera that can detect light in the NIR range. It aims for more precise surgery with improved oncological outcomes and a reduction in complications by improving discrimination between different structures. METHODS: A systematic search was conducted in the Embase, Medline and Cochrane databases with search terms corresponding to 'fluorescence-guided surgery', 'colorectal surgery', and 'colorectal cancer' to identify all relevant trials. RESULTS: The following clinical applications of fluorescence guided surgery for colorectal cancer were identified and discussed: (1) tumour imaging, (2) sentinel lymph node imaging, (3) imaging of distant metastases, (4) imaging of vital structures, (5) imaging of perfusion. Both experimental and FDA/EMA approved fluorescent agents are debated. Furthermore, promising future modalities are discussed. CONCLUSION: Fluorescence-guided surgery for colorectal cancer is a rapidly evolving field. The first studies show additional value of this technique regarding change in surgical management. Future trials should focus on patient related outcomes such as complication rates, disease free survival, and overall survival.

Near-Infrared Fluorescence Tumor-Targeted Imaging in Lung Cancer: A Systematic Review.

Lung cancer is the most common cancer type worldwide, with non-small cell lung cancer (NSCLC) being the most common subtype. Non-disseminated NSCLC is mainly treated with surgical resection. The intraoperative detection of lung cancer can be challenging, since small and deeply located pulmonary nodules can be invisible under white light. Due to the increasing use of minimally invasive surgical techniques, tactile information is often reduced. Therefore, several intraoperative imaging techniques have been tested to localize pulmonary nodules, of which near-infrared (NIR) fluorescence is an emerging modality. In this systematic review, the available literature on fluorescence imaging of lung cancers is presented, which shows that NIR fluorescence-guided lung surgery has the potential to identify the tumor during surgery, detect additional lesions and prevent tumor-positive resection margins.

Use of fluorescence imaging and indocyanine green during laparoscopic cholecystectomy: Results of an international Delphi survey.

BACKGROUND: Published empirical data have increasingly suggested that using near-infrared fluorescence cholangiography during laparoscopic cholecystectomy markedly increases biliary anatomy visualization. The technology is rapidly evolving, and different equipment and doses may be used. We aimed to identify areas of consensus and nonconsensus in the use of incisionless near-infrared fluorescent cholangiography during laparoscopic cholecystectomy. METHODS: A 2-round Delphi survey was conducted among 28 international experts in minimally invasive surgery and near-infrared fluorescent cholangiography in 2020, during which respondents voted on 62 statements on patient preparation and contraindications (n = 12); on indocyanine green administration (n = 14); on potential advantages and uses of near-infrared fluorescent cholangiography (n = 18); comparing near-infrared fluorescent cholangiography with intraoperative x-ray cholangiography (n = 7); and on potential disadvantages of and required training for near-infrared fluorescent cholangiography (n = 11). RESULTS: Expert consensus strongly supports near-infrared fluorescent cholangiography superiority over white light for the visualization of biliary structures and reduction of laparoscopic cholecystectomy risks. It also offers other advantages like enhancing anatomic visualization in obese patients and those with moderate to severe inflammation. Regarding indocyanine green administration, consensus was reached that dosing should be on a milligrams/kilogram basis, rather than as an absolute dose, and that doses >0.05 mg/kg are necessary. Although there is no consensus on the optimum preoperative timing of indocyanine green injections, the majority of participants consider it important to administer indocyanine green at least 45 minutes before the procedure to decrease the light intensity of the liver. CONCLUSION: Near-infrared fluorescent cholangiography experts strongly agree on its effectiveness and safety during laparoscopic cholecystectomy and that it should be used routinely, but further research is necessary to establish optimum timing and doses for indocyanine green.

Randomized, double-blind comparison of indocyanine green with or without albumin premixing for near-infrared fluorescence imaging of sentinel lymph nodes in breast cancer patients.

Near-infrared (NIR) fluorescence imaging has the potential to improve sentinel lymph node (SLN) mapping in breast cancer. Indocyanine green (ICG) is currently the only clinically available fluorophore that can be used for SLN mapping. Preclinically, ICG adsorbed to human serum albumin (ICG:HSA) improves its performance as a lymphatic tracer in some anatomical sites. The benefit of ICG:HSA for SLN mapping of breast cancer has not yet been assessed in a clinical trial. We performed a double-blind, randomized study to determine if ICG:HSA has advantages over ICG alone. The primary endpoint was the fluorescence brightness, defined as the signal-to-background ratio (SBR), of identified SLNs. Clinical trial subjects were 18 consecutive breast cancer patients scheduled to undergo SLN biopsy. All patients received standard of care using (99m)Technetium-nanocolloid and patent blue. Patients were randomly assigned to receive 1.6 ml of 500 µM ICG:HSA or ICG that was injected periareolarly directly after patent blue. The Mini-Fluorescence-Assisted Resection and Exploration (Mini-FLARE) imaging system was used for NIR fluorescence detection and quantitation. SLN mapping was successful in all patients. Patient, tumor, and treatment characteristics were equally distributed over the treatment groups. No significant difference was found in SBR between the ICG:HSA group and the ICG alone group (8.4 vs. 11.3, respectively, P = 0.18). In both groups, the average number of detected SLNs was 1.4 ± 0.5 SLNs per patient (P = 0.74). This study shows that there is no direct benefit of premixing ICG with HSA prior to injection for SLN mapping in breast cancer patients, thereby reducing the cost and complexity of the procedure. With these optimized parameters that eliminate the necessity of HSA, larger trials can now be performed to determine patient benefit.

Image-guided tumor resection using real-time near-infrared fluorescence in a syngeneic rat model of primary breast cancer.

Tumor involvement of resection margins is found in a large proportion of patients who undergo breast-conserving surgery. Near-infrared (NIR) fluorescence imaging is an experimental technique to visualize cancer cells during surgery. To determine the accuracy of real-time NIR fluorescence imaging in obtaining tumor-free resection margins, a protease-activatable NIR fluorescence probe and an intraoperative camera system were used in the EMR86 orthotopic syngeneic breast cancer rat model. Influence of concentration, timing and number of tumor cells were tested in the MCR86 rat breast cancer cell line. These variables were significantly associated with NIR fluorescence probe activation. Dosing and tumor size were also significantly associated with fluorescence intensity in the EMR86 rat model, whereas time of imaging was not. Real-time NIR fluorescence guidance of tumor resection resulted in a complete resection of 17 out of 17 tumors with minimal excision of normal healthy tissue (mean minimum and a mean maximum tumor-free margin of 0.2 ± 0.2 mm and 1.3 ± 0.6 mm, respectively). Moreover, the technique enabled identification of remnant tumor tissue in the surgical cavity. Histological analysis revealed that the NIR fluorescence signal was highest at the invasive tumor border and in the stromal compartment of the tumor. In conclusion, NIR fluorescence detection of breast tumor margins was successful in a rat model. This study suggests that clinical introduction of intraoperative NIR fluorescence imaging has the potential to increase the number of complete tumor resections in breast cancer patients undergoing breast-conserving surgery.

Clinical translation of ex vivo sentinel lymph node mapping for colorectal cancer using invisible near-infrared fluorescence light.

BACKGROUND: Sentinel lymph node (SLN) mapping in colorectal cancer may have prognostic and therapeutic significance; however, currently available techniques are not optimal. We hypothesized that the combination of invisible near-infrared (NIR) fluorescent light and ex vivo injection could solve remaining problems of SLN mapping in colorectal cancer. METHODS: The FLARE imaging system was used for real-time identification of SLNs after injection of the NIR lymphatic tracer HSA800 in the colon and rectum of (n = 4) pigs. A total of 32 SLN mappings were performed in vivo and ex vivo after oncologic resection using an identical injection technique. Guided by these results, SLN mappings were performed in ex vivo tissue specimens of 24 consecutive colorectal cancer patients undergoing resection. RESULTS: Lymph flow could be followed in real-time from the injection site to the SLN using NIR fluorescence. In pigs, the SLN was identified in 32 of 32 (100%) of SLN mappings under both in vivo and ex vivo conditions. Clinically, SLNs were identified in all patients (n = 24) using the ex vivo strategy within 5 min after injection of fluorescent tracer. Also, 9 patients showed lymph node involvement (N1 disease). In 1 patient, a 3-mm mesenteric metastasis was found adjacent to a tumor-negative SLN. CONCLUSIONS: The current pilot study shows proof of principle that ex vivo NIR fluorescence-guided SLN mapping can provide high-sensitivity, rapid, and accurate identification of SLNs in colon and rectum. This creates an experimental platform to test optimized, non-FDA-approved NIR fluorescent lymphatic tracers in a clinical setting.