Image-guided oncologic surgery using invisible light: completed pre-clinical development for sentinel lymph node mapping

Abstract

Background: Invisible near-infrared (NIR) fluorescent light permits high sensitivity, real-time image-guidance during oncologic surgery without changing the look of the surgical field. In this study, we complete pre-clinical development of the technology for sentinel lymph node (SLN) mapping using a large animal model of spontaneous melanoma.

Methods: Sinclair swine with spontaneous melanoma metastatic to regional lymph nodes were used because of their similarity to human melanoma. Organic lymphatic tracers tested included FDA-approved indocyanine green adsorbed non-covalently to human serum albumin (HSA), and NIR fluorophore CW800 conjugated covalently to HSA (HSA800). The inorganic/organic hybrid tracer tested was type II NIR quantum dots with an anionic coating. Primary tumors received four peri-tumoral injections of each tracer, with a fluorophore dose of 100 pmol to 1 nmol per injection. SLN mapping and image-guided resection were performed in real-time.

Results: Each of the 3 lymphatic tracers was injected into n = 4 separate primary melanomas in a total of 6 animals. All 12 injections resulted in identification of the SLN(s) and their associated lymphatic channels within 1 minute in 100% of cases, despite highly pigmented skin and black fur. Hydrodynamic diameter had a profound impact on tracer behavior in vivo.

Conclusions: This study completes the pre-clinical development of NIR fluorescence-guided SLN mapping and provides insight into imaging system optimization and tracer choice for future human clinical trials. The technology is likely to eliminate the need for radioactive and colored tracers, permits real-time image guidance throughout the procedure, and assists the pathologist in tissue analysis.

Figure 1. FLARE Revision 2 Intraoperative NIR Fluorescence Imaging System

A. White light and invisible NIR fluorescence excitation light illuminate the surgical field. Reflected white light (i.e., surgical anatomy) is directed to a color video camera, while invisible NIR fluorescent light (from the introduced NIR fluorophore) is directed simultaneously to a NIR camera. The independent light paths for white light (solid arrows) and invisible NIR light (dotted arrows) are shown. B. The FLARE imaging system is completely self-contained and is mounted on an articulated arm that swings over the surgical field. Working distance of the optics is 18″ from the subject. The schematic is drawn to scale relative to a Skytron Model 6001 operating room table. C. Beneath the aluminum cover are the sub-systems for which specifications are provided in Table 1. All parts mount to a rigid, shock-resistant aluminum frame, which ensures alignment of the two cameras.

Figure 2. Physicochemical and Optical Properties of NIR Fluorescent Tracers for Image-Guided SLN Mapping and Resection

A. Organic lymphatic tracers include HSA800 (left) and ICG adsorbed non-covalently to HSA (ICG:HSA; middle). The crystal structure of HSA is shown in white, and the position of NIR fluorophores in green. The three CW800 conjugation sites of HSA800 are adapted from Ohnishi et al. and correspond to Lys162, Lys274, and Lys557. The ICG binding site is presumed to be HSA site I based on the similarity of its chemical structure to other site I ligands. NIR QDs are an inorganic/organic hybrid having a semiconductor metal core and a highly anionic organic coating derived from oligomeric phosphines. All three tracers are drawn to scale, based on their hydrodynamic diameters measured using gel-filtration chromatography. Below each tracer are its physicochemical and optical properties. B. The effect of serum proteins on hydrodynamic diameter was measured using high-resolution gel-filtration with on-line absorbance and fluorescence spectrometry. Lymphatic tracers were analyzed after dilution in PBS (dashed curves) or 100% serum (solid curves). Absorbance at 280 nm (top) is used to show the position of all proteins present. Molecular weight markers M1 (blue dextran; 2 MDa, 43.4 nm HD), M2 (thyroglobulin; 669 kDa, 18.8 nm HD), M3 (γ-globulin; 158 kDa, 11.1 nm HD), M4 (ovalbumin; 44 kDa, 6.1 nm HD), and M5 (myoglobin; 17 kDa, 3.8 nm HD) are shown by arrows. Fluorescence at the peak emission wavelength (bottom) is shown for each tracer.

Figure 2. Physicochemical and Optical Properties of NIR Fluorescent Tracers for Image-Guided SLN Mapping and Resection

A. Organic lymphatic tracers include HSA800 (left) and ICG adsorbed non-covalently to HSA (ICG:HSA; middle). The crystal structure of HSA is shown in white, and the position of NIR fluorophores in green. The three CW800 conjugation sites of HSA800 are adapted from Ohnishi et al. and correspond to Lys162, Lys274, and Lys557. The ICG binding site is presumed to be HSA site I based on the similarity of its chemical structure to other site I ligands. NIR QDs are an inorganic/organic hybrid having a semiconductor metal core and a highly anionic organic coating derived from oligomeric phosphines. All three tracers are drawn to scale, based on their hydrodynamic diameters measured using gel-filtration chromatography. Below each tracer are its physicochemical and optical properties. B. The effect of serum proteins on hydrodynamic diameter was measured using high-resolution gel-filtration with on-line absorbance and fluorescence spectrometry. Lymphatic tracers were analyzed after dilution in PBS (dashed curves) or 100% serum (solid curves). Absorbance at 280 nm (top) is used to show the position of all proteins present. Molecular weight markers M1 (blue dextran; 2 MDa, 43.4 nm HD), M2 (thyroglobulin; 669 kDa, 18.8 nm HD), M3 (γ-globulin; 158 kDa, 11.1 nm HD), M4 (ovalbumin; 44 kDa, 6.1 nm HD), and M5 (myoglobin; 17 kDa, 3.8 nm HD) are shown by arrows. Fluorescence at the peak emission wavelength (bottom) is shown for each tracer.

Figure 2. Physicochemical and Optical Properties of NIR Fluorescent Tracers for Image-Guided SLN Mapping and Resection

A. Organic lymphatic tracers include HSA800 (left) and ICG adsorbed non-covalently to HSA (ICG:HSA; middle). The crystal structure of HSA is shown in white, and the position of NIR fluorophores in green. The three CW800 conjugation sites of HSA800 are adapted from Ohnishi et al. and correspond to Lys162, Lys274, and Lys557. The ICG binding site is presumed to be HSA site I based on the similarity of its chemical structure to other site I ligands. NIR QDs are an inorganic/organic hybrid having a semiconductor metal core and a highly anionic organic coating derived from oligomeric phosphines. All three tracers are drawn to scale, based on their hydrodynamic diameters measured using gel-filtration chromatography. Below each tracer are its physicochemical and optical properties. B. The effect of serum proteins on hydrodynamic diameter was measured using high-resolution gel-filtration with on-line absorbance and fluorescence spectrometry. Lymphatic tracers were analyzed after dilution in PBS (dashed curves) or 100% serum (solid curves). Absorbance at 280 nm (top) is used to show the position of all proteins present. Molecular weight markers M1 (blue dextran; 2 MDa, 43.4 nm HD), M2 (thyroglobulin; 669 kDa, 18.8 nm HD), M3 (γ-globulin; 158 kDa, 11.1 nm HD), M4 (ovalbumin; 44 kDa, 6.1 nm HD), and M5 (myoglobin; 17 kDa, 3.8 nm HD) are shown by arrows. Fluorescence at the peak emission wavelength (bottom) is shown for each tracer.

Figure 3. Real-Time Intraoperative NIR Fluorescent Sentinel Lymph Node Mapping

At T = 0, four peri-tumoral, subcutaneous injections of 1 nmol of HSA800 were made around a primary melanoma on the ventral left torso. Two dominant lymphatic channels, one cranial (Cr) and one caudal (Ca) were found. The caudal channel was followed (T = 5 secs) until two SLNs were identified at T = 15 secs. Images shown include color video (left), NIR fluorescence (middle) and a pseudo-colored (lime green) merge of the two (right). Exposure time (67 msec) and normalizations were the same for all NIR fluorescence images.

Figure 4. Image-Guided Pathological Analysis of Resected SLNs

A. Surgical Pathology. After image-guided resection of the two SLNs and a control LN from the animal in Figure 3, specimens could be re-imaged and trimmed by a surgical pathologist under image guidance. The sinus entry point could also be identified and the LN processed for maximal likelihood of finding metastases. B. Histology. H&E staining of each LN from Figure 4A revealed metastases in both the proximal and distal SLNs, as evidenced by the melanin-containing brown cells with large nuclei, but not the control LN. The dotted white box in each 4X image corresponds to the field shown in the 40X image.