Simultaneous mapping of pan and sentinel lymph nodes for real-time image-guided surgery

Abstract

The resection of regional lymph nodes in the basin of a primary tumor is of paramount importance in surgical oncology. Although sentinel lymph node mapping is now the standard of care in breast cancer and melanoma, over 20% of patients require a completion lymphadenectomy. Yet, there is currently no technology available that can image all lymph nodes in the body in real time, or assess both the sentinel node and all nodes simultaneously. In this study, we report an optical fluorescence technology that is capable of simultaneous mapping of pan lymph nodes (PLNs) and sentinel lymph nodes (SLNs) in the same subject. We developed near-infrared fluorophores, which have fluorescence emission maxima either at 700 nm or at 800 nm. One was injected intravenously for identification of all regional lymph nodes in a basin, and the other was injected locally for identification of the SLN. Using the dual-channel FLARE intraoperative imaging system, we could identify and resect all PLNs and SLNs simultaneously. The technology we describe enables simultaneous, real-time visualization of both PLNs and SLNs in the same subject.

Keywords: Near-infrared fluorescence; cervical cancer.; completion lymphadenectomy; image-guided surgery; pan lymph node mapping; sentinel lymph node mapping.

Figure 1

Chemical Structures and Optical Properties of Lymph Node Contrast Agents: Shown are chemical structures (top) and optical properties (bottom) of 700 nm (left) and 800 nm (right) lymph node contrast agents. FL, fluorescence; MW, molecular weight.

Figure 2

Pan Lymph Node Imaging in Rats: 50 nmol ESNF14 (A) or 20 nmol ZW800-3a (B) was injected intravenously into SD rats 4 h prior to imaging. In the merged image, pseudo-colored red and green were used for 700 nm and 800 nm channels, respectively. LNs are identified with arrowheads. (C) SBR, expressed as the LN to muscle (Mu) ratio (mean ± SEM) for each agent in all major LN basins 4 h post- injection of 50 nmol ESNF series and MB, or 20 nmol ZW800-3a and ICG. Controls were injected with saline alone. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post-test (*P < 0.05, **P < 0.01, ***P < 0.001). (D) Kinetics of pan lymph node NIR fluorescence. SBR, expressed as the LN to muscle (Mu) ratio (mean ± SEM) for each agent over time. (E) Biodistribution of intravenously injected NIR fluorophores 4 h post-injection. Data are from N = 3 rats per condition. Ad, white adipose; Bl, bladder; Du, duodenum; He, heart; In, intestine; Ki, kidney; Li, liver; Lu, lung; LN, lymph node. Scale bars = 1 cm.

Figure 3

Pan Lymph Node Imaging in Pigs: (A) Inguinal LNs (top), gastric LNs (2nd row), mesenteric LNs (3rd row) and mediastinal LNs (bottom) after intravenous injection of 640 nmol ZW800-3a into 35 kg Yorkshire pigs 4 h prior to imaging. Shown are representative images from N = 3 pigs, and pseudo-colored green was used for merged image. LNs are identified with arrowheads. Es, esophagus; Je, jejunum; Lu, lung; LN, lymph node; Pa, pancreas; St, stomach. Scale bars = 1 cm. (B) SBR, expressed as the LN to muscle (Mu) ratio (mean ± SEM) for each agent in all major LN basins 4 h after intravenous injection of 1.4 µmol ESNF series or 640 nmol ZW800-3a. Data are from N = 3 pigs per condition. Statistical analysis of ZW800-3a vs. each ESNF dye was performed using one-way ANOVA followed by Tukey's multiple comparison post-test (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Kinetics of pan lymph node NIR fluorescence. SBR, expressed as the LN to muscle (black squares), liver (white diamonds), or kidney (white circles) ratio (mean ± SEM) for each agent over time. N = 3 pigs per data point.

Figure 4

Simultaneous Pan and Sentinel Lymph Node Mapping in Pig and Histological Analysis: (A) Sentinel lymph node mapping using 5 nmol of the 700 nm NIR fluorophore ESNF14 injected into the uterine cervix 5 min prior to imaging and pan lymph node mapping of the pelvic LNs using 640 nmol ZW800-3a injected intravenously 4 h prior to imaging in a 35 kg Yorkshire pig. Images are representative of N = 3 pigs, and pseudo-colored red and green were used in merged image for 700 nm and 800 nm channels, respectively. LNs are identified with arrowheads. The SLN is identified with a red broken circle. Bl, bladder; Ov, ovary; Re, rectum. Dashed circle = SLN; arrowheads = pelvic LNs. Scale bar = 1 cm. (B) Histological analysis NIR fluorescence within pelvic LNs. Control LNs (top row) are from uninjected pigs. Pan lymph node (PLN; middle row) and sentinel lymph node (SLN; bottom row) are from Figure 4A. For each are shown hematoxylin and eosin (H&E) brightfield images (left column), consecutive histological sections viewed under 700 nm (2^nd column) and 800 nm (3^rd column) NIR fluorescence, and a pseudocolored merge of the three (right column). NIR fluorescence images have identical exposure times and normalizations. Scale bars = 100 µm. LF, lymphoid follicle; S, subcapsular sinus.