Intraoperative near-infrared imaging can distinguish cancer from normal tissue but not inflammation

Abstract

Introduction: Defining tumor from non-tumor tissue is one of the major challenges of cancer surgery. Surgeons depend on visual and tactile clues to select which tissues should be removed from a patient. Recently, we and others have hypothesized near-infrared (NIR) imaging can be used during surgery to differentiate tumors from normal tissue.

Methods: We enrolled 8 canines and 5 humans undergoing cancer surgery for NIR imaging. The patients were injected with indocyanine green (ICG), an FDA approved non-receptor specific NIR dye that accumulates in hyperpermeable tissues, 16-24 hours prior to surgery. During surgery, NIR imaging was used to discriminate the tumor from non-tumor tissue.

Results: NIR imaging identified all tumors with a mean signal-to-background ratio of 6.7. Optical images were useful during surgery in discriminating normal tissue from cancer. In 3 canine cases and 1 human case, the tissue surrounding the tumor was inflamed due to obstruction of the vascular supply due to mass effect. In these instances, NIR imaging could not distinguish tumor tissue from tissue that was congested, edematous and did not contain cancer.

Conclusions: This study shows that NIR imaging can identify tumors from normal tissues, provides excellent tissue contrast, and it facilitates the resection of tumors. However, in situations where there is significant peritumoral inflammation, NIR imaging with ICG is not helpful. This suggests that non-targeted NIR dyes that accumulate in hyperpermeable tissues will have significant limitations in the future, and receptor-specific NIR dyes may be necessary to overcome this problem.

Figure 1. Preclinical evidence for NIR tumor labeling to detect primary and metastatic tumor deposits.

(A) Five cancer cell types were injected into the flank of syngeneic mice. Once established (200 mm3), animals were dosed with 7.5 mg/kg of ICG and imaged. Tumors were harvested, imaged and stained for CD31 (Marked with black arrows). Histology images taken at 200x magnification. (B) C57bl/6 mice (n = 21) were injected with LLC cells in their flanks on Day 0. Starting on Day 12, the animals were euthanized, dosed with 7.5 mg/kg ICG 24 hours earlier and their thoracic cavities opened. Observers determined if the metastatic tumor nodules were visible in the lung. NIR imaging was then used to detect disease that was not visible to the un-assisted human eye. Histology images taken at 100x.

Figure 2. Representative intraoperative image of a canine lung cancer.

(A) Signal-to-background ratio of tumor to surrounding normal lung tissue in situ and ex vivo in 8 canines. All values are reported in arbitrary units (a.u.). ^†Due to the large size of this tumor, no measurements of normal lung fluorescence could be obtained ex vivo. (B) After opening the chest, the tumor was visualized in the chest. The tumor was well-circumscribed and was highly fluorescent (signal-to-background ratio 11.3). The tumor lies in the caudad position and the hilum of the lung is cranial. (C) Ex vivo, the tumor was fluorescent (SBR 12.7) and the margins of the tumor were well-defined. (D) H&E confirmed a lung adenocarcinoma with 2+ CD31 staining. The signal-to-background ratio (SBR) was higher ex vivo than in situ because of superior control of the surrounding environment such as lighting conditions, exposure and lack of motion. Although the fluorescence from the tumors did not significantly change, the background fluorescence from the normal lung was reduced when the environment could be better controlled. Microscopically, the tumor microvascular density (MVD) did not seem to impact the degree of fluorescence.

Figure 3. Intraoperative imaging of surgical margins during pulmonary resection.

(A) In situ, the tumor can be visualized and palpated. (B) Stitches mark the tumor margin at 5 mm intervals from the palpable tumor edge. (C) The spectrometer was used to measure NIR fluorescence (805 nm) at each location on the specimen and develop a heat map. The heat map predicted the tumor margins assessed by the surgeon and the pathologist. In a second case with significant peritumoral inflammation, (D) intraoperative images demonstrated a tumor in a pulmonary lobe as it was retracted from the canine chest. (E) Ex vivo, the pulmonary lobe could be seen fluorescing, however, it was difficult by brightfield or fluorescence to discriminate the margins between tumor tissue and inflammatory lung tissue. Spectroscopy demonstrated some difference in the fluorescence from the tumor versus congested tissue, but clinically this was tedious and not practical. The surgeon also had difficulty in identifying tumor from non-tumor tissue by manual palpation because the lung was congested and edematous.

Figure 4. Intraoperative imaging of surgical margins during pulmonary resection.

(A) Intraoperatively, the animal underwent right thoracotomy and palpation of the primary tumor. Intraoperative image of the pulmonary lobe as it is retracted from the canine chest revealed the dorsal portion of the lobe has significant compression (ie. atelectasis) from tumor obstruction. The ventral portion of the lung retained its normal appearance. Spectroscopic analysis of the tumor was performed in situ. All sites were recorded in triplicate and averaged. The graph shows the inflammatory tissue was highly fluorescent and could not be distinguished from tumor. (B) H&E demonstrated normal alveolar parenchyma (left panel), congestion lung with neutrophils and fibrotic plugs (middle panel) and tumor (right panel) from representative biopsies of the pulmonary lobe.

Figure 5. Two representative human tumors.

Computed tomography (CT) scan and positron emission tomography (PET) scan demonstrated an anterior mediastinal mass and a lung nodule in two patients. Patients were injected with ICG, and then underwent resection of their tumors. Ex vivo, NIR imaging demonstrated the tumors were highly fluorescent and the surrounding organ had minimal background noise. The optical images were easy to interpret by the surgeon and facilitated the identification of the tumor. Spectroscopy demonstrated a SBR of 8.1 for the thymoma and 7.9 for the carcinoid. The tumors were discrete and well-circumscribed and had minimal peritumoral inflammation.

Figure 6. Representative photos of a patient with an 8 cm left upper lobe squamous cell carcinoma of the lung.

(A) The CAT scan and PET scan demonstrate the tumor in the periphery of the lung with surrounding post-obstructive collapse of the lung. Once the tumor was removed, it is hard to distinguish tumor from inflammation in the left upper pulmonary lobe. NIR imaging shows similar fluorescence from both areas. (B) Histologically, the areas of inflammation that fluoresced did not contain tumor cells but had congestion, edema, fibrotic plugs and inflammatory cells.