George Themelis

Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results

The prognosis in advanced-stage ovarian cancer remains poor. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and thereby improve prognosis. The overexpression of folate receptor-α (FR-α) in 90–95% of epithelial ovarian cancers prompted the investigation of intraoperative tumor-specific fluorescence imaging in ovarian cancer surgery using an FR-α–targeted fluorescent agent. In patients with ovarian cancer, intraoperative tumor-specific fluorescence imaging with an FR-α–targeted fluorescent agent showcased the potential applications in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery.

Real-time intraoperative fluorescence imaging system using light-absorption correction

We present a novel fluorescence imaging system developed for real-time interventional imaging applications. The system implements a correction scheme that improves the accuracy of epi-illumination fluorescence images for light intensity variation in tissues. The implementation is based on the use of three cameras operating in parallel, utilizing a common lens, which allows for the concurrent collection of color, fluorescence, and light attenuation images at the excitation wavelength from the same field of view. The correction is based on a ratio approach of fluorescence over light attenuation images. Color images and video is used for surgical guidance and for registration with the corrected fluorescence images. We showcase the performance metrics of this system on phantoms and animals, and discuss the advantages over conventional epi-illumination systems developed for real-time applications and the limits of validity of corrected epi-illumination fluorescence imaging.

Intraoperative near-infrared fluorescence imaging for sentinel lymph node detection in vulvar cancer: First clinical results

OBJECTIVE Disadvantages of the combined sentinel lymph node (SLN) procedure with radiocolloid and blue dye in vulvar cancer are the preoperative injections of radioactive tracer in the vulva, posing a painful burden on the patient. Intraoperative transcutaneous imaging of a peritumorally injected fluorescent tracer may lead to a one-step procedure, while maintaining high sensitivity. Aim of this pilot study was to investigate the applicability of intraoperative fluorescence imaging for SLN detection and transcutaneous lymphatic mapping in vulvar cancer. METHODS Ten patients with early stage squamous cell carcinoma of the vulva underwent the standard SLN procedure. Additionally, a mixture of 1 mL patent blue and 1 mL indocyanin green (ICG; 0.5 mg/mL) was injected immediately prior to surgery, with the patient under anesthesia. Color and fluorescence images and videos of lymph flow were acquired using a custom-made intraoperative fluorescence camera system. The distance between skin and femoral artery was determined on preoperative CT-scan as a measure for subcutaneous adipose tissue. RESULTS In 10 patients, SLNs were detected in 16 groins (4 unilateral; 6 midline tumors). Transcutaneous lymphatic mapping was possible in five patients (5 of 16 groins), and was limited to lean patients, with a maximal distance between femoral artery and skin of 24 mm, as determined on CT. In total, 29 SLNs were detected by radiocolloid, of which 26 were also detected by fluorescence and 21 were blue. CONCLUSIONS These first clinical results indicate that intraoperative transcutaneous lymphatic mapping using fluorescence is technically feasible in a subgroup of lean vulvar cancer patients.

Intraoperative Multispectral Fluorescence Imaging for the Detection of the Sentinel Lymph Node in Cervical Cancer: A Novel Concept

PurposeReal-time intraoperative near-infrared fluorescence (NIRF) imaging is a promising technique for lymphatic mapping and sentinel lymph node (SLN) detection. The purpose of this technical feasibility pilot study was to evaluate the applicability of NIRF imaging with indocyanin green (ICG) for the detection of the SLN in cervical cancer.ProceduresIn ten patients with early stage cervical cancer, a mixture of patent blue and ICG was injected into the cervix uteri during surgery. Real-time color and fluorescence videos and images were acquired using a custom-made multispectral fluorescence camera system.ResultsReal-time fluorescence lymphatic mapping was observed in vivo in six patients; a total of nine SLNs were detected, of which one (11%) contained metastases. Ex vivo fluorescence imaging revealed the remaining fluorescent signal in 11 of 197 non-sentinel LNs (5%), of which one contained metastatic tumor tissue. None of the non-fluorescent LNs contained metastases.ConclusionsWe conclude that lymphatic mapping and detection of the SLN in cervical cancer using intraoperative NIRF imaging is technically feasible. However, the technique needs to be refined for full applicability in cervical cancer in terms of sensitivity and specificity.

Multispectral imaging using multiple-bandpass filters

We present a method for multispectral imaging. This method uses color CCD cameras with a multiple-bandpass filter, which modifies the spectral response of the cameras used and enables concurrent acquisition of multiple images at defined spectral bands. We experimentally demonstrate methodological feasibility using two color CCD cameras and a polychroic mirror to simultaneously capture eight spectral bands. We discuss how the method developed is well suited for multispectral applications of transient phenomena or for real-time measurements.

Imaging the Bio-Distribution of Fluorescent Probes Using Multispectral Epi-Illumination Cryoslicing Imaging

PurposeThe increasing availability of fluorescent probes for in vivo optical imaging enables the interrogation of complex biological processes in small animals serving as models for human-like tissue function and disease. However, the validation of probe bio-distribution during their development or the study of different disease models, in support of in vivo imaging studies, is not straightforward.ProceduresThe imaging system developed consists of a customized multispectral planar imager that has been adapted on a commercial cryomicrotome and provides a powerful modality for ex vivo imaging of small animals.ResultsThe ability to capture 3D anatomical (color) and fluorescence volumetric distributions of multiple fluorescent markers in high resolution is showcased.ConclusionsServing both as a method for accurately imaging the bio-distribution of multiple fluorescent agents inside organisms and as a modality for the validation of non-invasive methods, multispectral cryoslicing imaging offers useful insights to ex vivo optical imaging of molecular probes.

Real-time near infrared fluorescence (NIRF) intra-operative imaging in ovarian cancer using an αvβ3-integrin targeted agent

BACKGROUND In ovarian cancer, optimal cytoreductive surgery is of the utmost importance for long-term survival. The ability to visualize minuscule tumor deposits is important to ensure complete resection of the tumor. The purpose of our study was to estimate the in vivo sensitivity, specificity and diagnostic accuracy of an intra-operative fluorescence imaging system combined with an α(v)β(3)-integrin targeted near-infrared fluorescent probe. METHOD Tumor bearing mice were injected intravenously with a fluorescent probe targeting α(v)β(3) integrins. Fluorescent spots and non-fluorescent tissue were identified and resected. Standard histopathology and fluorescence microscopy were used as gold-standard for tumor detection. RESULTS Fifty-eight samples excised with support of intra-operative image-guided surgery were analyzed. The mean target to background ratio was 2.2 (SD 0.5). The calculated sensitivity of the imaging system was 95%, and the specificity was 88% with a diagnostic accuracy of 96.5%. CONCLUSION Near-infrared image-guided surgery in this model has a high diagnostic accuracy and a fair target to background ratio that supports the development towards clinical translation of α(v)β(3)-integrin targeted imaging.

In vivo imaging of CT26 mouse tumours by using cmHsp70.1 monoclonal antibody.

The major stress‐inducible heat shock protein 70 (Hsp70) is frequently present on the cell surface of human tumours, but not on normal cells. Herein, the binding characteristics of the cmHsp70.1 mouse monoclonal antibody (mAb) were evaluated in vitro and in a syngeneic tumour mouse model. More than 50% of the CT26 mouse colon carcinoma cells express Hsp70 on their cell surface at 4°C. After a temperature shift to 37°C, the cmHsp70.1‐fluorescein isothiocyanate mAb translocates into early endosomes and lysosomes. Intraoperative and near‐infrared fluorescence imaging revealed an enrichment of Cy5.5‐conjugated mAb cmHsp70.1, but not an identically labelled IgG1 isotype‐matched control, in i.p. and s.c. located CT26 tumours, as soon as 30 min. after i.v. injection into the tail vein. Due to the rapid turnover rate of membrane‐bound Hsp70, the fluorescence‐labelled cmHsp70.1 mAb became endocytosed and accumulated in the tumour, reaching a maximum after 24 hrs and remained detectable at least up to 96 hrs after a single i.v. injection. The tumour‐selective internalization of mAb cmHsp70.1 at the physiological temperature of 37°C might enable a targeted uptake of toxins or radionuclides into Hsp70 membrane‐positive tumours. The anti‐tumoral activity of the cmHsp70.1 mAb is further supported by its capacity to mediate antibody‐dependent cytotoxicity.

Multimodal Molecular Imaging of Integrin αvβ3 for In Vivo Detection of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. Late detection of then nonresectable or metastasized tumors emphasizes the need for novel imaging approaches. Here, we report on so far nonexploited potentials of αvβ3 integrin–targeted molecular imaging technologies for detection of PDAC using genetically engineered mouse models. Methods: Immunohistochemistry and Western blot were used for characterization of αvβ3 expression in murine and human PDAC. We applied IntegriSense 680 fluorescence molecular tomography, intraoperative fluorescence imaging, and 68Ga-NODAGA-RGD PET for αvβ3 integrin molecular in vivo imaging of spontaneous PDAC occurring in Ptf1a+/Cre;Kras+/LSL-G12D;p53LoxP/LoxP mice. (NODAGA is 1,4,7-triazacyclononane-1,4-bis[acetic acid]-7-[2-glutaric acid] and RGD is arginine-glycine-aspartic acid.) Results: αvβ3 integrin is expressed in tumor cells of human and murine PDAC. IntegriSense fluorescence molecular tomography and 68Ga-NODAGA-RGD PET enabled faithful visualization of PDAC. Furthermore, intraoperative optical imaging with IntegriSense 680 allowed good delineation of tumor borders. Conclusion: Imaging approaches targeting αvβ3 integrin expand the potential of molecular imaging for identification of αvβ3-positive PDAC with potential implications in early detection, fluorescence-guided surgery, and therapy monitoring.

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid. Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9. The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.