HEPATOFLUO: A prospective monocentric study assessing the benefits of indocyanine green (ICG) fluorescence for hepatic surgery.

BACKGROUND AND OBJECTIVES: Fluorescence imaging using indocyanine green (ICG) is undergoing extensive development. This study aimed to assess the merits of ICG in regard to hepatic surgery. METHODS: Patients with liver lesions that required a resection were eligible. They received an injection of ICG the day before the surgery. Step 1 allowed assessment of use of the medical device under surgical conditions. Steps 2 and 3 assessed the capacity of the MD to detect known tumorous lesions and to spot a predefined area of the liver following injection of ICG into the portal vein (ICGp). RESULTS: The 1st step allowed for validation of the MD use with three patients. Between 04-2013 and 04-2015, 45 pts were included (40 eligible) in steps 2 and 3. All of the tumorous lesions (95/119) exhibited fluorescence. Four new metastasis were detected in 3 pts, and two missing metastases in 1 pt. False positive were 22%. The maximal depth for detection by fluorescence was 13 mm. Injection of ICGp allowed the corresponding anatomical area to be identified in 16/20 patients. CONCLUSION: This study confirmed that intraoperative fluorescence is a helpful and relevant tool for the liver surgeon (NCT 01738217).

Use of methylene blue and near-infrared fluorescence in thyroid and parathyroid surgery.

PURPOSE: Intraoperative localisation and preservation of parathyroid glands improves outcomes following thyroid and parathyroid surgery. This can be facilitated by fluorescent imaging and methylene blue; a fluorophore is thought to be taken up avidly by parathyroid glands. This preliminary study aims to identify the optimum dose of methylene blue (MB), fluorescent patterns of thyroid and parathyroid glands and develop a protocol for the use of intravenous MB emitted fluorescence to enable parathyroid identification. METHODS: This is a phase 1b, interventional study (NCT02089542) involving 41 patients undergoing thyroid and/or parathyroid surgery. After exposure of the thyroid and/or parathyroid gland(s), intravenous boluses of between 0.05 and 0.5 mg/kg of MB were injected. Fluobeam® (a hand held fluorescence real-time imager) was used to record fluorescence from the operating field prior and up to 10 min following administration. RESULTS: The optimum dose of MB to visualise thyroid and parathyroid glands was 0.4 mg/kg body weight. The median time to onset of fluorescence was 23 and 22 s and the median time to peak fluorescence was 41.5 and 40 s, respectively. The peak fluorescence for thyroid and parathyroid glands compared to muscle were 2.6 and 4.3, respectively. Parathyroid auto-fluorescence prior to methylene blue injection was commonly observed. CONCLUSIONS: A clinical protocol for detection of fluorescence from MB during thyroid and parathyroid surgery is presented. Parathyroids (especially enlarged glands) fluoresce more intensely than thyroid glands. Auto-fluorescence may aid parathyroid detection, but MB fluorescence is needed to demonstrate viability.

Near infrared fluorescence imaging of rabbit thyroid and parathyroid glands.

BACKGROUND: Near infrared fluorescence imaging using intravenous methylene blue (MB) is a novel technique that has potential to aid the parathyroid gland (PG) localization during thyroid and parathyroid surgery. The aim of this study was to examine MB fluorescence in the rabbit neck and determine the influence of MB dose and time following administration on fluorescence from thyroid and PGs. METHODS: Thyroid and external PGs were exposed in six New Zealand white rabbits under anesthesia. Varying doses of MB (0.025-3 mg/kg) were injected through the marginal ear vein. Near infrared fluorescence from exposed tissues was recorded at different time intervals (10-74 min) using Fluobeam 700. Specimens of identified glands were then resected for histologic assessment. RESULTS: Histology confirmed accurate identification of all excised thyroid and PGs; these were the only neck structures to demonstrate significant fluorescence. The parathyroid demonstrated lower fluorescence intensities and reduced washout times at all MB doses compared with the thyroid gland. A dose of 0.1 mg/kg MB was adequate to identify fluorescence; this also delineated the blood supply of the external PGs. CONCLUSIONS: The study demonstrates that near infrared fluorescence with intravenous MB helps differentiate between thyroid and PGs in the rabbit. This has potential to improve outcomes in thyroid and parathyroid surgery by increasing the accuracy of parathyroid identification; however, the findings require replication in human surgery. The use of low doses of MB may also avoid the side effects associated with currently used doses in humans (3-7 mg/kg).

Intraoperative fluorescence imaging of peritoneal dissemination of ovarian carcinomas. A preclinical study.

OBJECTIVE: Improvement of the management and outcome of ovarian cancers may require intraoperative detection and therapeutic intervention to treat minimal residual disease after complete surgery. The aim of this study was to validate the importance of fluorescence in the peroperative detection of human ovarian adenocarcinoma cells and to determine its efficiency in detecting infra millimetric tumor metastases. METHODS: A fluorescent RAFT-(cRGD)4 tracer molecule (AngioStamp®) was used. The tracer is based on a biomarker, which has a very high affinity for the α(v)ß3 integrin, which is overexpressed in a large ratio of cancer cells and neovessel endothelial cells during angiogenesis. Infrared fluorescence was visualized with Fluobeam®, an open fluorescent imaging system that could potentially be used in peroperative conditions in the future. RESULTS: This novel technique allowed the specific detection of residual tumor deposits and inframillimetric metastases, smaller than 500µm, which were resected under fluorescent guidance. AngioStamp® was able to detect all types of cell lines, derived from human ovarian adenocarcinomas, before or after chemotherapy treatment in animals. The effectiveness of AngioStamp® for the detection of various human ovarian adenocarcinomas was assessed on 10 different fragments of tumor, implanted subcutaneously in nude mice. All implanted tumor fragments were visualized by AngioStamp®. CONCLUSIONS: The high rate of recurrence after apparently complete surgery and/or complete clinical response to chemotherapy implies that most patients have undetected minimal residual disease. Novel techniques such as laparoscopic or laparotomic fluorescence may prove to be crucial in reassessing the definition of primary outcome in ovarian cancer management.

Fluorescence-guided surgery for cancer patients: a proof of concept study on human xenografts in mice and spontaneous tumors in pets.

Surgery is often the first treatment option for patients with cancer. Patient survival essentially depends on the completeness of tumor resection. This is a major challenge, particularly in cases of peritoneal carcinomatosis, where tumors are widely disseminated in the large peritoneal cavity. Any development to help surgeons visualize these residual cells would improve the completeness of the surgery. For non-disseminated tumors, imaging could be used to ensure that the tumor margins and the draining lymph nodes are free of tumor deposits. Near-infrared fluorescence imaging has been shown to be one of the most convenient imaging modalities. Our aim was to evaluate the efficacy of a near-infrared fluorescent probe targeting the αvß3 integrins (Angiostamp™) for intraoperative detection of tumors using the Fluobeam® device. We determined whether different human tumor nodules from various origins could be detected in xenograft mouse models using both cancer cell lines and patient-derived tumor cells. We found that xenografts could be imaged by fluorescent staining irrespective of their integrin expression levels. This suggests imaging of the associated angiogenesis of the tumor and a broader potential utilization of Angiostamp™. We therefore performed a veterinary clinical trial in cats and dogs with local tumors or with spontaneous disseminated peritoneal carcinomatosis. Our results demonstrate that the probe can specifically visualize both breast and ovarian nodules, and suggest that Angiostamp™ is a powerful fluorescent contrast agent that could be used in both human and veterinary clinical trials for intraoperative detection of tumors.

Multimodal In Vivo Imaging of Tumorigenesis and Response to Chemotherapy in a Transgenic Mouse Model of Mammary Cancer.

PURPOSE: Transgenic mice expressing the polyoma middle T oncoprotein (PyMT) in the mammary epithelium were explored by multimodal imaging to monitor longitudinally spontaneous tumor growth and response to chemotherapy. PROCEDURES: Positron emission tomography (PET) with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), single photon emission tomography (SPECT) with [(99m)Tc]TcO4 ([(99m)Tc]TEC), X-ray computed tomography, and fluorescent confocal endomicroscopy (FCE) images were acquired during tumor progression in female PyMT mice. Imaging with [(18)F]FDG and [(99m)Tc]TEC was also performed in untreated, doxorubicin-treated, and docetaxel-treated PyMT mice. Total tumor volumes were quantified. Tumors were collected and macroscopic and histological examinations were performed. RESULTS: All PyMT mice developed multifocal tumors of the mammary epithelium that became palpable at 8 weeks of age (W8). Computed tomography (CT) detected tumors at W14, while a clear tumoral uptake of [(99m)Tc]TEC and [(18)F]FDG was present as early as W6 and W8, respectively. No contrast between mammary tumors and surrounding tissue was observed at any stage with [(18)F]FLT. FCE detected an angiogenic switch at W10. Lung metastases were not clearly evidenced by imaging. Doxorubicin and docetaxel treatments delayed tumor growth, as shown by [(18)F]FDG and [(99m)Tc]TEC, but tumor growth resumed upon treatment discontinuation. Tumor growth fitted an exponential model with time constant rates of 0.315, 0.145, and 0.212 week(-1) in untreated, doxorubicin, and docetaxel groups, respectively. CONCLUSIONS: Molecular imaging of mammary tumors in PyMT is precocious, precise, and predictive. [(18)F]FDG-PET and [(99m)Tc]TEC SPECT monitor tumor response to chemotherapy.

A palm-sized high-sensitivity near-infrared fluorescence imager for laparotomy surgery.

In laparotomy surgery guided by near-infrared fluorescence imaging, the access to the field of operation is limited by the illumination and/or the imaging field. The side of cavities or organs such as the liver or the heart cannot be examined with the systems available on the market, which are too large and too heavy. In this article, we describe and evaluate a palm sized probe, whose properties, weight, size and sensitivity are adapted for guiding laparotomy surgery. Different experiments have been performed to determine its main characteristics, both on the illumination and imaging sides. The device has been tested for fluorescent molecular probe imaging in preclinical procedures, to prove its ability to be used in cancer nodule detection during surgery. This system is now CE certified for clinical procedures and Indocyanine Green imaging has been performed during clinical investigations: lymphedema and surgical resection of liver metastases of colorectal cancers.

Targeted imaging of α(v)ß(3) expressing sarcoma tumor cells in vivo in pre-operative setting using near infrared: a potential tool to reduce incomplete surgical resection.

Tumor size and location along with efficacy of pre-operative imaging are limiting factors for optimal surgical excision for osteosarcoma. Our general hypothesis is that targeting αvß3 integrin-rich osteosarcoma neoangiogenesis should provide improved delivery of diagnostic compounds and assist surgeons intra operatively using near-infrared imaging techniques. We evaluated in an orthotopic metastatic osteosarcoma in rats the potential of AngioStamp™ targeting αvß3 integrins and detected intra operatively by near infrared (NIR) illumination (Fluobeam™) as a novel, intra operative imaging technique. To determine the potential of this association in improving tumor and metastasis detection, we compared the quality and sensitivity of tumor/metastasis margin delineation and tumor resection using intra-operative NIR imaging to the ones guided by pre-operative imaging (i.e., MRI subsequently confirmed by histopathological analysis). Chemotherapy being essential in osteosarcoma treatment, we evaluated the capacity of AngioStamp™ to specifically localize to the tumor after chemotherapy treatment. We showed a significantly lesser extent of healthy tissue resection after surgical excision when assessing tumor margin intra operatively using AngioStamp™/Fluobeam™ association compared to pre-operative MRI post-operatively confirmed by histopathological analysis (p<0.01). Importantly, intra-operative NIR illumination of lungs revealed more metastases than were detected by CT Scan or under intra-operative white light examination (p<0.01). Importantly, chemotherapy did not alter AngioStamp™ tumor specific targeting nor the sensitivity of tumor detection. Our preclinical data confirm the potential of intra-operative imaging for improved primary tumor and lung metastasis excision. Based on these promising results, we now propose to evaluate this approach as a mean to improve surgical excision while maintaining tumor control in other sarcoma or tumors overexpressing αvß3 integrins.

Near-infrared optical guided surgery of highly infiltrative fibrosarcomas in cats using an anti-αvß3 integrin molecular probe.

We investigated how near-infrared imaging could improve highly infiltrative spontaneous fibrosarcoma surgery in 12 cats in a clinical veterinary phase. We used an RGD-based nanoprobe at different doses and times before surgery and a portable clinical grade imaging system. All tumours were labelled by the tracer and had an overall tumour-to-healthy tissue ratio of 14±1 during surgery. No false negatives were found, and the percentage of tumour cells was linearly correlated with the fluorescence intensity. All cats recovered well and were submitted to long-term follow-up that is currently on-going 1year after the beginning of the study.

Lipid nanoparticle vectorization of indocyanine green improves fluorescence imaging for tumor diagnosis and lymph node resection.

Fluorescence imaging is opening a new era in image-guided surgery and other medical applications. The only FDA approved contrast agent in the near infrared is IndoCyanine Green (ICG), which despites its low toxicity, displays poor chemical and optical properties for long-term and sensitive imaging applications in human. Lipid nanoparticles are investigated for improving ICG optical properties and in vivo fluorescence imaging sensitivity. 30 nm diameter lipid nanoparticles (LNP) are loaded with ICG. Their characterization and use for tumor and lymph node imaging are described. Nano-formulation benefits dye optical properties (6 times improved brightness) and chemical stability (>6 months at 4 degrees C in aqueous buffer). More importantly, LNP vectorization allows never reported sensitive and prolonged (>1 day) labeling of tumors and lymph nodes. Composed of human-use approved ingredients, this novel ICG nanometric formulation is foreseen to expand rapidly the field of clinical fluorescence imaging applications.

Cellular uptake of (99m)TcN-NOET in human leukaemic HL-60 cells is related to calcium channel activation and cell proliferation.

PURPOSE: A major goal of nuclear oncology is the development of new radiolabelled tracers as proliferation markers. Intracellular calcium waves play a fundamental role in the course of the cell cycle. These waves occur in non-excitable tumour cells via store-operated calcium channels (SOCCs). Bis(N-ethoxy, N-ethyldithiocarbamato) nitrido technetium (V)-99m ((99m)TcN-NOET) has been shown to interact with L-type voltage-operated calcium channels (VOCCs) in cultured cardiomyocytes. Considering the analogy between VOCCs and SOCCs, we sought to determine whether (99m)TcN-NOET also binds to activated SOCCs in tumour cells in order to clarify the potential value of this tracer as a proliferation marker. METHODS: Uptake kinetics of (99m)TcN-NOET were measured in human leukaemic HL-60 cells over 60 min and the effect of several calcium channel modulators on 1-min tracer uptake was studied. The uptake kinetics of (99m)TcN-NOET were compared both with the variations of cytosolic free calcium concentration measured by indo-1/AM and with the variations in the SG2M cellular proliferation index. RESULTS: All calcium channel inhibitors significantly decreased the cellular uptake of (99m)TcN-NOET whereas the activator thapsigargin induced a significant 10% increase. In parallel, SOCC activation by thapsigargin, as measured using the indo-1/AM probe, was inhibited by nicardipine. These results indicate that the uptake of (99m)TcN-NOET is related to the activation of SOCCs. Finally, a correlation was observed between the tracer uptake and variations in the proliferation index SG2M. CONCLUSION: The uptake of (99m)TcN-NOET seems to be related to SOCC activation and to cell proliferation in HL-60 cells. These results indicate that (99m)TcN-NOET might be a marker of cell proliferation.