A novel imaging system permits real-time in vivo tumor bed assessment after resection of naturally occurring sarcomas in dogs

Abstract

Background: Treatment of soft tissue sarcoma (STS) includes complete tumor excision. However, in some patients, residual sarcoma cells remain in the tumor bed. We previously described a novel hand-held imaging device prototype that uses molecular imaging to detect microscopic residual cancer in mice during surgery.

Questions/purposes: To test this device in a clinical trial of dogs with naturally occurring sarcomas, we asked: (1) Are any adverse clinical or laboratory effects observed after intravenous administration of the fluorescent probes? (2) Do canine sarcomas exhibit fluorescence after administration of the cathepsin-activated probe? (3) Is the tumor-to-background ratio sufficient to distinguish tumor from tumor bed? And (4) can residual fluorescence be detected in the tumor bed during surgery and does this correlate with a positive margin?

Methods: We studied nine dogs undergoing treatment for 10 STS or mast cell tumors. Dogs received an intravenous injection of VM249, a fluorescent probe that becomes optically active in the presence of cathepsin proteases. After injection, tumors were removed by wide resection. The tumor bed was imaged using the novel imaging device to search for residual fluorescence. We determined correlations between tissue fluorescence and histopathology, cathepsin protease expression, and development of recurrent disease. Minimum followup was 9 months (mean, 12 months; range, 9-15 months).

Results: Fluorescence was apparent from all 10 tumors and ranged from 3 × 10(7) to 1 × 10(9) counts/millisecond/cm(2). During intraoperative imaging, normal skeletal muscle showed no residual fluorescence. Histopathologic assessment of surgical margins correlated with intraoperative imaging in nine of 10 cases; in the other case, there was no residual fluorescence, but tumor was found at the margin on histologic examination. No animals had recurrent disease at 9 to 15 months.

Conclusions: These initial findings suggest this imaging system might be useful to intraoperatively detect residual tumor after wide resections.

Clinical relevance: The ability to assess the tumor bed intraoperatively for residual disease has the potential to improve local control.

Fig. 1A–B

(A) A diagram illustrates the optical layout of the intraoperative imaging device. Light enters the device through the fiber bundle where it is reflected through an excitation filter (ex filter) and reflected with a dichroic mirror (DM) onto the tumor sample/tumor bed where emitted light returns through the emission filter (em filter) to the detector. Several lenses (L) are used to relay the fluorescence image at no magnification into a charge-coupled device (CCD) where the fluorescence emission of individual cancer cells is mapped into 2 to 4 pixels. (B) A photograph shows the device in use in a dog with a chest wall STS.

Fig. 2A–F

Representative intraoperative imaging of the dogs is shown. Most of the dogs showed increased NIR fluorescence from tumor compared to images from the tumor bed. (A) An image-normalized intensity histogram shows distinct separation between (B) tumor and (C) tumor bed images. In contrast, in Dog 4 with an MCT, (D) an image-normalized intensity histogram shows a separation between (E) tumor and (F) tumor bed that is not as distinct; (F) a small area of residual fluorescence (arrow) was detected in the tumor bed after resection that was greater than the threshold of 80% of the minimum signal from the tumor.

Fig. 3A–E

Multiple cathepsin proteases that activate VM249 are upregulated in tumors compared to normal muscle as detected by quantitative real-time PCR: (A) cathepsin B, (B) cathepsin K, (C) cathepsin L, and (D) cathepsin S. (E) A heat map for a variety of cathepsins in canine STS and skeletal muscle is shown. Red indicates high levels of cathepsin expression whereas blue indicates low levels of cathepsin expression. CTSD = cathepsin D, CTSF = cathepsin F, etc.