Milton V. Marshall

Lymphatic imaging in humans with near-infrared fluorescence

While the lymphatic system is increasingly associated with diseases of prevalence, study of these diseases is difficult owing to the paucity of imaging techniques with the sensitivity and temporal resolution to discriminate lymphatic function. Herein, we review the known, pertinent features of the human lymphatic system in health and disease and set the context for a number of emerging studies that use near-infrared fluorescence imaging to non-invasively assess tumor draining lymphatic basins in cancer patients, intraoperatively guide resection of first draining lymph nodes, and to interrogate the difference between normal and aberrant lymphatic structure and function.

Near-Infrared Fluorescence Imaging in Humans with Indocyanine Green: A Review and Update.

Near-infrared (NIR) fluorescence imaging clinical studies have been reported in the literature with six different devices that employ various doses of indocyanine green (ICG) as a non-specific contrast agent. To date, clinical applications range from (i) angiography, intraoperative assessment of vessel patency, and tumor/metastasis delineation following intravenous administration of ICG, and (ii) imaging lymphatic architecture and function following subcutaneous and intradermal ICG administration. In the latter case, NIR fluorescence imaging may enable new discoveries associated with lymphatic function due to (i) a unique niche that is not met by any other conventional imaging technology and (ii) its exquisite sensitivity enabling high spatial and temporal resolution. Herein, we (i) review the basics of clinical NIR fluorescence imaging, (ii) survey the literature on clinical application of investigational devices using ICG fluorescent contrast, (iii) provide an update of non-invasive dynamic lymphatic imaging conducted with our FDPM device, and finally, (iv) comment on the future NIR fluorescence imaging for non-invasive and intraoperative use given recent demonstrations showing capabilities for imaging following microdose administration of contrast agent.

New Horizons for Imaging Lymphatic Function

In this review, we provide a comprehensive summary of noninvasive imaging modalities used clinically for the diagnosis of lymphatic diseases, new imaging agents for assessing lymphatic architecture and cancer status of lymph nodes, and emerging near‐infrared (NIR) fluorescent optical imaging technologies and agents for functional lymphatic imaging. Given the promise of NIR optical imaging, we provide example results of functional lymphatic imaging in mice, swine, and humans, showing the ability of this technology to quantify lymph velocity and frequencies of propulsion resulting from the contractility of lymphatic structures.

Assessment of Lymphatic Contractile Function After Manual Lymphatic Drainage Using Near-Infrared Fluorescence Imaging

OBJECTIVE To investigate the feasibility of assessing the efficacy of manual lymphatic drainage (MLD), a method for lymphedema (LE) management, by using near-infrared (NIR) fluorescence imaging. DESIGN Exploratory pilot study. SETTING Primary care unit. PARTICIPANTS Subjects (N=10; age, 18-68y) with a diagnosis of grade I or II LE and 12 healthy control subjects (age, 22-59y). INTERVENTION Indocyanine green (25 μg in 0.1 mL each) was injected intradermally in bilateral arms or legs of subjects. Diffused excitation light illuminated the limbs, and NIR fluorescence images were collected by using custom-built imaging systems. Subjects received MLD therapy, and imaging was performed pre- and posttherapy. MAIN OUTCOME MEASURES Apparent lymph velocities and periods between lymphatic propulsion events were computed from fluorescence images. Data collected pre- and post-MLD were compared and evaluated for differences. RESULTS By comparing pre-MLD lymphatic contractile function against post-MLD lymphatic function, results showed that average apparent lymph velocity increased in both the symptomatic (+23%) and asymptomatic (+25%) limbs of subjects with LE and control limbs (+28%) of healthy subjects. The average lymphatic propulsion period decreased in symptomatic (-9%) and asymptomatic (-20%) limbs of subjects with LE, as well as in control limbs (-23%). CONCLUSIONS We showed that NIR fluorescence imaging could be used to quantify immediate improvement of lymphatic contractile function after MLD.

Direct evidence of lymphatic function improvement after advanced pneumatic compression device treatment of lymphedema

Lymphedema affects up to 50% of all breast cancer survivors. Management with pneumatic compression devices (PCDs) is controversial, owing to the lack of methods to directly assess benefit. This pilot study employed an investigational, near-infrared (NIR) fluorescence imaging technique to evaluate lymphatic response to PCD therapy in normal control and breast cancer-related lymphedema (BCRL) subjects. Lymphatic propulsion rate, apparent lymph velocity, and lymphatic vessel recruitment were measured before, during, and after advanced PCD therapy. Lymphatic function improved in all control subjects and all asymptomatic arms of BCRL subjects. Lymphatic function improved in 4 of 6 BCRL affected arms, improvement defined as proximal movement of dye after therapy. NIR fluorescence lymphatic imaging may be useful to directly evaluate lymphatic response to therapy. These results suggest that PCDs can stimulate lymphatic function and may be an effective method to manage BCRL, warranting future clinical trials.

Seeing it through: translational validation of new medical imaging modalities.

Medical imaging is an invaluable tool for diagnosis, surgical guidance, and assessment of treatment efficacy. The Network for Translational Research (NTR) for Optical Imaging consists of four research groups working to “bridge the gap” between lab discovery and clinical use of fluorescence- and photoacoustic-based imaging devices used with imaging biomarkers. While the groups are using different modalities, all the groups face similar challenges when attempting to validate these systems for FDA approval and, ultimately, clinical use. Validation steps taken, as well as future needs, are described here. The group hopes to provide translational validation guidance for itself, as well as other researchers.

Near-infrared fluorescence imaging of lymphatics in head and neck lymphedema

Treatment of lymphatic disease is complicated and controversial, due in part to the limited understanding of the lymphatic system. Lymphedema (LE) is a frequent complication after surgical resection and radiation treatment in cancer survivors, and is especially debilitating in regions where treatment options are limited. Although some extremity LE can be effectively treated with manual lymphatic drainage (MLD) therapy or compression devices to direct proximal lymph transport, head and neck LE is more challenging, due to complicated geometry and complex lymphatic structure in head and neck region. Herein, we describe the compassionate use of an investigatory technique of near-infrared (NIR) fluorescence imaging to understand the lymphatic anatomy and function, and to help direct MLD in a patient with head and neck LE. Immediately after 9 intradermal injections of 25 μg indocyanine green each around the face and neck region, NIR fluorescence images were collected using a custom-built imaging system with diffused excitation light illumination. These images were then used to direct MLD therapy. In addition, 3-dimensional (3D) surface profilometry was used to monitor response to therapy. NIR fluorescence images of functioning lymphatic vessels and abnormal structures were obtained. Precise geometries of facial structures were obtained using 3D profilometry, and detection of small changes in edema between therapy sessions was achieved. NIR fluorescence imaging provides a mapping of lymphatic architecture to direct MLD therapy and thus improve treatment efficacy in the head and neck LE, while 3D profilometry allowed longitudinal assessment of edema to evaluate the efficacy of therapy.

Preclinical characterization and validation of a dual-labeled trastuzumab-based imaging agent for diagnosing breast cancer

OBJECTIVE The combination of both nuclear and fluorescent reporters provides unique opportunities for noninvasive nuclear imaging with subsequent fluorescence image-guided resection and pathology. Our objective was to synthesize and optimize a dual-labeled trastuzumab-based imaging agent that can be used to validate an optical imaging agent with potential use in identifying tumor metastases in human epidermal growth factor receptor 2 (HER2) positive breast cancer patients. METHODS [(111)In]-DTPA-trastuzumab-IRDye 800 was synthesized by a three-step procedure. Purity, stability, immunoreactivity, internalization and biodistribution were explored in HER2+ SKBR-3 cells. Biodistribution of [(111)In]-DTPA-trastuzumab-IRDye 800 was performed in a SKBR-3 xenograft model. RESULTS [(111)In]-DTPA-trastuzumab-IRDye 800 demonstrated high purity by both chemical and fluorometric determinations. Both flow cytometry and the Lindmo assay demonstrated a high binding affinity of [(111)In]-DTPA-trastuzumab-IRDye 800 to HER2-overexpressing cells. The dual-labeled conjugate was stable in PBS, but not in serum after 24 h at 37 °C. Larger molecules (>150 kD) were seen after a 24 h-incubation in human serum. Biodistribution studies revealed tumor-specific accumulation of [(111)In]-DTPA-trastuzumab-IRDye 800 in SKBR-3 tumors, and tumor uptakes at 24 and 48 h were (12.42±1.72)% and (9.96±1.05)%, respectively, following intravenous administration. The tumor-to-muscle ratio was 9.13±1.68 at 24 h, and increased to 12.79±2.13 at 48 h. Liver and kidney showed marked uptake of the dual-labeled imaging agent. CONCLUSIONS [(111)In]-DTPA-trastuzumab-IRDye 800 is an effective diagnostic biomarker that can be used to validate dual-labeled, molecularly targeted imaging agents and can allow these agents to be translated into clinical practice for identifying HER2+ lesions.

Validation of ALFIA: A platform for quantifying near-infrared fluorescent images of lymphatic propulsion in humans

Recently, we demonstrated near-infrared (NIR) fluorescence imaging for quantifying real-time lymphatic propulsion in humans following intradermal injections of microdose amounts of indocyanine green. However computational methods for image analysis are underdeveloped, hindering the translation and clinical adaptation of NIR fluorescent lymphatic imaging. In our initial work we used ImageJ and custom MatLab programs to manually identify lymphatic vessels and individual propulsion events using the temporal transit of the fluorescent dye. In addition, we extracted the apparent velocities of contractile propagation and time periods between propulsion events. Extensive time and effort were required to analyze the 6-8 gigabytes of NIR fluorescent images obtained for each subject. To alleviate this bottleneck, we commenced development of ALFIA, an integrated software platform which will permit automated, near real-time analysis of lymphatic function using NIR fluorescent imaging. However, prior to automation, the base algorithms calculating the apparent velocity and period must be validated to verify that they produce results consistent with the proof-of-concept programs. To do this, both methods were used to analyze NIR fluorescent images of two subjects and the number of propulsive events identified, the average apparent velocities, and the average periods for each subject were compared. Paired Students t-tests indicate that the differences between their average results are not significant. With the base algorithms validated, further development and automation of ALFIA can be realized, significantly reducing the amount of user interaction required, and potentially enabling the near real-time, clinical evaluation of NIR fluorescent lymphatic imaging.

NIR Fluorescence Imaging for in vivo Assessment of Normal and Diseased Lymphatics

Near-infrared fluorescence imaging with microdose indocyanine green was used to visualize the normal and diseased lymphatic structure and quantify the lymphatic function in vivo. Lymphatic function was significantly improved after manual lymphatic drainage.