Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

Abstract

Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16 . Ten animals showed no residual tumor cells in the wound bed (mean SBR<2 , P<0.001 ). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15 , and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

Fig. 1

Monte-Carlo simulation of light transport. (a) Schematic of tissue geometry used in the simulation. (b) Total absorbed excitation light in the tissue, expressed in a logarithmic scale. The $x$ axis denotes radial coordinate while the $y$ axis denotes depth. (c) Detected fluorescence on the surface of the tissue expressed as a function of radial distance from the center of the tissue. (d) Fluorescence intensity detected beyond the tumor edge (inset). Radial fluorescence intensity fit to a photon diffusion model. Linear fit denotes that the radial distribution of light fits a diffusion model.

Fig. 2

Representative resection of a 7-cm perivascular wall tumor in a 17-kg Brittany spaniel. (a) The canine was injected with $3\mathrm{mg}/\mathrm{kg}$ of indocyanine green (ICG) 24 h prior to surgery. During the operation, the tumor was fluorescent in vivo. Note that the surgeon’s hand appears to be fluorescent because of strong reflection from the latex gloves. We have found that the use of nonlatex black gloves solves this issue. (b) After removing the sarcoma, the wound bed was examined and did not demonstrate any residual tumor cells. (c) Ex vivo, the tumor was fluorescent. The near-infrared (NIR) signal emitted from the sarcoma extended up to 6 mm into the surrounding tissues that did not contain microscopic evidence of tumor cells or ICG. Scale bars are approximate.

Fig. 3

Representative resection of a 4-cm sarcoma from the right antebrachium of a 13 kg mixed breed dog. (a) Again, the tumor was fluorescent in vivo and confirmed to be a perivascular wall tumor by H&E. (b) After removing the sarcoma, the wound bed was interrogated for residual tumor cells. Although grossly there did not appear to be cancer cells, spectroscopy and optical imaging rapidly brought attention to an area on the skin that contained a positive surgical margin. This was confirmed by pathology. (c) Ex vivo, the tumor was fluorescent and the NIR signal was strong throughout the entire specimen and part of the surrounding normal tissues. Scale bars are approximate.

Fig. 4

Surgical removal of a 4.5-cm undifferentiated sarcoma from the right carpus of a 30 kg husky. (a) In vivo, the tumor was fluorescent and saturated the spectrometer and CCD imaging apparatus. (b) After removing the tumor, spectroscopy and CCD imaging identified a concerning region along the superior flap of skin, indicated by the white arrow. This tissue was biopsied and confirmed to contain residual tumor cells. Skin appears to be fluorescent due to specular reflection. Placing a blue sterile lining around the wound eliminates this background. However, visibility of the skin can be useful, because it provides context for the surgical wound in relation to canine extremities. (c) After removing the tumor, the specimen was uniformly fluorescent along the entire surgical border. Scale bars are approximate.

Fig. 5

(a) In 15 canine subjects, the mean fluorescence from fat, muscle, blood vessels, fascia, and the tumors were $5897\pm 3181$, $684\pm 376$, $5781\pm 2821$, $3601\pm 1741$, and $\mathrm{58,522}\pm 871$, respectively. (b) After removing the primary tumor, the mean fluorescence from the wound bed was dependent on the presence of residual tumor cells. In the cases with histologically proven negative margins, the mean fluorescence was $6885\pm 3804$. In the five cases with residual tumor in the wound, the mean fluorescent intensity was $\mathrm{51,546}\pm 2931$. The signal-to-background ratio in the wounds with the negative margins versus positive margins was 1.8 versus 16.3, respectively.

Fig. 6

The NIR signal around the sarcomas typically extended beyond the tumor pseudocapsule into the fat and muscle. The spectral signal from these normal tissues dissipated when the tissue was removed from its in situ location. H&E did not demonstrate tumor cells outside the tumor pseudocapsule nor did immunofluorescence identify ICG particles outside the pseudocapsule.

Fig. 7

A 57-year-old male with an 11-cm right lateral flank/chest wall sarcoma underwent surgical resection with NIR imaging. (a) The tumor did not appear to invade into the underlying rib case on T1- and T2-weighted MRI images. FDG-PET demonstrated increased FDG uptake (SUV 8.8). (b) In surgery, the tumor was fluorescent with an S/N ratio of 10.3. (c) After removing the tumor, the wound bed did not have residual tumor cells. (d) The tumor was fluorescent ex vivo and the NIR emission signal did extend beyond the pseudocapsule. In follow-up, the patient remains disease free at over 1 year.