Clinical Translation of Ex Vivo Sentinel Lymph Node Mapping for Colorectal Cancer Using Invisible Near-Infrared Fluorescence Light

The preparation of HSA800 (IRDye 800CW conjugated to human serum albumin) has been described in detail previously.30 The ratio of fluorophore (IRDye 800CW; Li-Cor, Lincoln, NE) to albumin (HSA) was 3:1, estimated using the extinction coefficients of HSA (ε_280nm = 32,900 M⁻¹ cm⁻¹) and CW800 (ε_778nm = 240,000 M⁻¹ cm⁻¹) in phosphate-buffered saline (PBS). Peak absorbance and emission of HSA800 were 778 and 795 nm, respectively. A stock solution of 10 μM (in pig) or 50 μM (in human) HSA800 in PBS, pH 7.4 was used for all injections.

Preclinical SLN mapping was performed using the FLARE image-guided surgery system as described in detail previously.29 The system is shown schematically in Fig. 1. It consists of 2 wavelength isolated light sources: a “white” light source, generating 40,000 lx of 400–650 nm light and a “near-infrared” light source, generating 12.5-mW/cm² 760-nm light over a 15-cm field of view. Color video and NIR fluorescence images are simultaneously acquired and displayed in real time using custom optics and software that separate the color video and NIR fluorescence images.29 A pseudocolored (lime green) merged image of the color video and NIR fluorescence images is also displayed. Clinical SLN mapping was performed using a miniaturized version of FLARE (Mini-FLARE), which is described in detail elsewhere (Troyan et al., manuscript in preparation), with settings of 20,000 lx of 400–650 nm “white” light and 7 mW/cm² of 760-nm NIR light.

Animals were housed in an AAALAC-certified facility staffed by full-time veterinarians and were studied under the supervision of an approved institutional protocol. This protocol adhered to the “Guide for the Care and Use of Laboratory Animals,” NRC 1996. Four female Yorkshire pigs were purchased at 35 kg from E.M. Parsons and Sons (Hadley, MA). All animals acclimated to the animal facility for at least 48 h prior to experimentation. Anesthesia was induced with 4.4-mg/kg intramuscular Telazol (Fort Dodge Labs, Fort Dodge, IA). The animal was then intubated, and anesthesia was maintained with 2% isoflurane (Baxter Healthcare Corporation, Deerfield, IL). Electrocardiogram (ECG), heart rate, oxygen saturation, and body temperature were monitored throughout the experiment. At the end of each experiment, euthanasia was induced by rapid intravenous injection of 10 mL of Fatal-Plus (Vortech Pharmaceuticals, Dearborn, MI), a method consistent with the recommendation of the Panel on Euthanasia of the American Veterinary Medical Association.

SLN mapping was performed using a 1-cc syringe equipped with a 27-gauge, 0.5-in. needle. Each injection consisted of 0.1 cc of HSA800 solution injected subserosally, with the needle bevel facing upward. The injection site was then gently massaged to stimulate lymphatic flow through increased hydrostatic pressure.

All animals received multiple injections at separate locations in the colon and rectum. For each condition (in vivo and ex vivo), 8 injections were performed in colon and 8 injections were performed in the rectum, for a total of 32 injections. The NIR fluorescence signal was monitored using the FLARE system, and the SLN identified by watching uninterrupted lymphatic flow reach the first lymph node in real-time. After successful identification, the suspected lymph node was resected under fluorescence guidance, fixed in 2% paraformaldehyde for 12 h, frozen in LN₂ using Tissue-Tek OCT, cryosectioned at 10 μm, and analyzed by hematoxylin and eosin (H&E) staining.

After the in vivo injections, a subtotal colectomy and a total mesorectal excision (TME) were performed. The resected bowel specimens were placed on a table and at locations that were not injected in vivo, 0.1 cc of HSA800 was injected subserosally, identical to the technique used for in vivo injections. Tissue samples were also collected and processed for histology as described previously.

Time needed to identify the SLN was compared between in vivo and ex vivo conditions using an unpaired t test. Assumption of equal variances was confirmed by Levene’s test. Statistical analyses, performed using SPSS software version 17.0 (SPSS Inc., Chicago, IL), were 2-tailed, and P < .05 was considered significant.

A total of 24 consecutive patients undergoing curative-intended surgery for colorectal cancer were included. This study was approved by the Leiden University Medical Center Medical Ethics Committee and performed in accordance with the ethical standards of the Helsinki Declaration of 1975. All patients underwent a standard oncological resection including lymphadenectomy. In cases of colon cancer, a segmental colonic resection was performed; in cases of rectal cancer, a resection following the principles of the total mesorectal excision (TME) was performed. Directly following resection, bowel segments were delivered fresh to the department of pathology, where the specimen was opened anti-mesenterically by the pathologist. In rectal cancer, surgical quality audit protocol prohibits altering the perirectal fat in any way. Consequently, in rectal cancer, the specimen was opened no further than the rectosigmoid junction. Then, 1 cc of 50-μM HSA800 was injected submucosally circumferentially with a 5-mm margin around the tumor, and the injection site was massaged for 5 min. After 5 min, the specimen was inspected using Mini-FLARE. Any fluorescent hotspots were either marked with a suture or directly resected for separate fixation in 2% buffered formalin. The specimen was fixed using 2% buffered formalin for 24 h (colon) or 48 h (rectum). After fixation, fluorescent nodes were identified under direct NIR fluorescence guidance with the Mini-FLARE, and the specimen was processed following the standard clinical protocol. For rectal cancer, the specimen was processed using the slicing technique as described by Quirke et al.33 All lymph nodes (sentinel and nonsentinel) were paraffin embedded and 4-μm sections were H&E stained and subsequently analyzed by microscope for the presence of tumor cells.

In both in vivo and ex vivo conditions, all injections led to a successful identification of a SLN (Table 1). Shortly after massaging the injection site, lymphatic flow was visualized on the NIR fluorescence image (Fig. 2). In all cases, lymphatic flow terminated, without interruption, in 1 or more lymph nodes.

In the colon, we found no difference (Table 1) in the time needed to identify the SLN between in vivo (56.4 seconds on average) and ex vivo (58.8 seconds on average) conditions (t = −0.111, P = .913). In the rectum, however, ex vivo injection led to a significantly faster identification time for the SLN (26.8 seconds on average) compared with in vivo (54.1 seconds; t = 2.942, P = .011). Mean time between resection of the bowel and ex vivo injection was 25 min (range 10–57 min). All resected specimens (n = 32) were analyzed by H&E histology and confirmed to be lymph node tissue (Fig. 2c).

Patient and tumor characteristics are listed in Table 2.

In a preliminary set of experiments on n = 6 clinical specimens, it was found that the subserosal injection technique that worked so well with normal pig specimens did not provide reliable results with human tumor specimens (data not shown). Injecting subserosally and circumferentially around human tumors resulted in a larger injection site, which outshined lymph nodes close to the tumor. To overcome this problem, a submucosal injection technique was used.16,34 It was also found that because of the thickness of human specimens versus normal pig specimens, a high concentration (50 μM) of HSA800 was optimal.

Using this optimized strategy, in all 24 consecutive patients, the procedure led to a successful detection of SLNs. An example of the NIR fluorescence images of the procedure in colon cancer is shown in Fig. 3a. In rectal cancer, quality control dictates that the specimen is not altered prior to inking, fixation, and slicing. As shown in Fig. 3b, in rectal cancer, nodes were harvested successfully after mesorectal slicing. Based on prefixation and postfixation images, the formalin fixation process did not appear to alter NIR fluorescence of HSA800. On average, per specimen, 3.0 SLNs (range 1–5 SLNs) were identified by NIR fluorescence (Table 2). On average, a total of 15.9 lymph nodes (range 8–31 lymph nodes) were harvested per specimen. Histological analysis showed that 9 of 24 patients had lymph node metastases. In all but 1 of those cases, at least 1 of the SLNs contained tumor cells. In 1 patient, however, a small tumor deposit (3 mm) was found in the mesentery, whereas the SLN was tumor negative. Although no clear lymphatic tissue could be identified (Fig. 4), pathological guidelines (WHO Classification, 6th edition) require that this lesion be classified as a lymph node metastasis.