Clinical translation of ex vivo sentinel lymph node mapping for colorectal cancer using invisible near-infrared fluorescence light

Abstract

Background: Sentinel lymph node (SLN) mapping in colorectal cancer may have prognostic and therapeutic significance; however, currently available techniques are not optimal. We hypothesized that the combination of invisible near-infrared (NIR) fluorescent light and ex vivo injection could solve remaining problems of SLN mapping in colorectal cancer.

Methods: The FLARE imaging system was used for real-time identification of SLNs after injection of the NIR lymphatic tracer HSA800 in the colon and rectum of (n = 4) pigs. A total of 32 SLN mappings were performed in vivo and ex vivo after oncologic resection using an identical injection technique. Guided by these results, SLN mappings were performed in ex vivo tissue specimens of 24 consecutive colorectal cancer patients undergoing resection.

Results: Lymph flow could be followed in real-time from the injection site to the SLN using NIR fluorescence. In pigs, the SLN was identified in 32 of 32 (100%) of SLN mappings under both in vivo and ex vivo conditions. Clinically, SLNs were identified in all patients (n = 24) using the ex vivo strategy within 5 min after injection of fluorescent tracer. Also, 9 patients showed lymph node involvement (N1 disease). In 1 patient, a 3-mm mesenteric metastasis was found adjacent to a tumor-negative SLN.

Conclusions: The current pilot study shows proof of principle that ex vivo NIR fluorescence-guided SLN mapping can provide high-sensitivity, rapid, and accurate identification of SLNs in colon and rectum. This creates an experimental platform to test optimized, non-FDA-approved NIR fluorescent lymphatic tracers in a clinical setting.

Fig. 1

FLARE image-guided surgery system. The dichroic mirror directs light below and above 650 nm to a color video camera and NIR fluorescence camera, respectively, permitting simultaneous acquisition of color video and NIR fluorescence images. The surgeon’s monitor includes a pseudocolored (lime green) merge of the 2 images

Fig. 2

In vivo and ex vivo SLN mapping in colon and rectum. a Identification of the SLN (arrows) in pig colon after in vivo (top row) or ex vivo (bottom row) injection (arrowheads) of 0.1 cc of 10 μM HSA800. Shown are the color video (left), NIR fluorescence (middle), and pseudocolored (lime green) merge of the 2 (right). b Ex vivo identification of the SLN (arrow) after injection (arrowhead) of 0.1 cc of 10 μM HSA800 (top row). Resected negative (−) and positive (+) nodes in rectum (bottom row). Shown are the color video (left), NIR fluorescence (middle), and pseudocolored (lime green) merge of the 2 (right). Note accumulation of the dye at the sinus entry site. c H&E histological staining of the (+) tissue section (100× magnification) from b

Fig. 3

NIR fluorescence-guided SLN mapping in patients with colorectal cancer. a Ex vivo identification of a SLN (arrow) in the mesentery of the right hemicolon of a patient with a cecal adenocarcinoma after submucosal peritumoral injection (arrowhead) of 1 cc of 50 μM HSA800. Shown are the color video (left), NIR fluorescence (middle), and pseudocolored (lime green) merge of the 2 (right). b Ex vivo identification of a SLN (arrow) in the mesorectum of a patient with a rectal adenocarcinoma after submucosal peritumoral injection (arrowhead) of 1 cc of 50 μM HSA800. After 48 h fixation in 2% buffered formalin, tissue specimen was sliced and imaged. Shown are the color video (left), NIR fluorescence (middle), and pseudocolored (lime green) merge of the 2 (right)

Fig. 4

Palpable metastatic lesion identified in the mesenteric fat. H&E histological staining of a mesenteric metastasis (25× magnification) not identified as a SLN. No nodal tissue was observed