Sentinel lymph node mapping of invasive urinary bladder cancer in animal models using invisible light

Abstract

Objectives: With conventional methodology, sentinel lymph node (SLN) mapping of invasive urinary bladder cancer is technically challenging. This study was performed to determine the utility of invisible, near-infrared fluorescent (NIRF) light for patient-specific SLN mapping, in real time under complete image guidance.

Methods: Lymphatic tracers, injection volume, NIRF excitation fluence rate, light collection of emitted fluorescence, and degree of bladder distension were systematically optimized in normal dogs and pigs. SLN mapping was then performed in pet dogs with naturally occurring invasive transitional cell carcinoma (InvTCC) of the urinary bladder, which closely mimics the human disease.

Results: NIRF albumin (hydrodynamic diameter [HD], 7.4 nm) and NIRF quantum dots (15-20 nm HD) injected into the bladder wall resulted in identification of draining lymph nodes (LNs) in under 3 min. In both species, considerable variability in the lymphatic drainage was observed among individuals. Optimal SLN mapping was achieved with the use of superficial, serosal injection of NIRF tracer, with the bladder distended to an intraluminal pressure of 20-40 cm H(2)O. In dogs with InvTCC, NIRF tracers identified SLNs that were confirmed histologically to harbor metastases.

Conclusions: The use of invisible NIRF light permits real-time, patient-specific identification of SLNs that drain bladder cancer. Intraluminal bladder pressure is a key parameter that needs to be controlled for optimal results.

Fig. 1

Real-time intraoperative near-infrared (NIR) fluorescence imaging system. Light paths for color video (solid lines) and NIR fluorescence (dotted lines) are indicated. The system has two wavelength-isolated light sources of 400- to 700-nm “white” light and 725- to 775-nm NIR fluorescence excitation light, which generate fluence rates of 0.5 mW/cm² and 5 mW/cm², respectively. The 400- to 700-nm white light permits normal visualization of the surgical field without producing background fluorescence that would otherwise occur with full-spectrum room lights and surgical lights.

Fig. 2

Near-infrared (NIR) fluorescent sentinel lymph node (SLN) mapping in the pig. (A) NIR fluorescence imaging immediately after injection (Inj.) into the bladder wall; lymphatic channels (arrowheads) are identified within 10 s, and fluorescence in nodes within 30 s to 3 min (bottom). Shown are the color video image, NIR fluorescence image, and a pseudocolored (lime green) merge of the two for each condition. (B) Postresection, ex vivo processing of the SLN. (C) Isosulfan blue injection into the bladder wall.

Fig. 2

Near-infrared (NIR) fluorescent sentinel lymph node (SLN) mapping in the pig. (A) NIR fluorescence imaging immediately after injection (Inj.) into the bladder wall; lymphatic channels (arrowheads) are identified within 10 s, and fluorescence in nodes within 30 s to 3 min (bottom). Shown are the color video image, NIR fluorescence image, and a pseudocolored (lime green) merge of the two for each condition. (B) Postresection, ex vivo processing of the SLN. (C) Isosulfan blue injection into the bladder wall.

Fig. 2

Near-infrared (NIR) fluorescent sentinel lymph node (SLN) mapping in the pig. (A) NIR fluorescence imaging immediately after injection (Inj.) into the bladder wall; lymphatic channels (arrowheads) are identified within 10 s, and fluorescence in nodes within 30 s to 3 min (bottom). Shown are the color video image, NIR fluorescence image, and a pseudocolored (lime green) merge of the two for each condition. (B) Postresection, ex vivo processing of the SLN. (C) Isosulfan blue injection into the bladder wall.

Fig. 3

Photomicrographs (hematoxylin-eosin stain; original magnification: ×200) of resected sentinel lymph nodes (SLNs). Histology was used to confirm that fluorescing structures viewed intraoperatively were lymph nodes. (A) Histologic section of SLN removed from a normal laboratory dog. The lymphoid follicle (LF) and subcapsular sinus (S) are labeled. (B) Histological section of SLN removed from a dog with invasive transitional cell cancer (InvTCC). Neoplastic cells fill the subcapsular sinus (S). Lymphoid follicle (LF) is also labeled.

Fig. 4

The effect of intraluminal bladder pressure on sentinel lymph node (SLN) mapping. Intraluminal bladder pressure was varied from 10 cm H₂0 (top) to 45 cm H₂O (bottom), with only intermediate pressures (middle), resulting in free flow of the tracer from the injection site to the SLN. Shown are the color video image, near-infrared fluorescence image, and a pseudocolored (lime green) merge of the two, for each condition.