Intraoperative near-infrared imaging of surgical wounds after tumor resections can detect residual disease

Abstract

Purpose: Surgical resection remains the most effective therapy for solid tumors worldwide. The most important prognostic indicator for cure following cancer surgery is a complete resection with no residual disease. However, intraoperative detection of retained cancer cells after surgery is challenging, and residual disease continues to be the most common cause of local failure. We hypothesized that visual enhancement of tumors using near-infrared imaging could potentially identify tumor deposits in the wound after resection.

Experimental design: A small animal model of surgery and retained disease was developed. Residual tumor deposits in the wound were targeted using an U.S. Food and Drug Administration-approved imaging agent, indocyanine green, by the enhanced permeability and retention effect. A novel handheld spectrometer was used to optically visualize retained disease after surgery.

Results: We found residual disease using near-infrared imaging during surgery that was not visible to the naked eye or micro-CT. Furthermore, examination of tumor nodules was remarkably precise in delineating margins from normal surrounding tissues. This approach was most successful for tumors with increased neovasculature.

Conclusions: The results suggest that near-infrared examination of the surgical wound after curative resection can potentially enable the surgeon to locate residual disease. The data in this study is the basis of an ongoing Phase I/II clinical trial in patients who undergo resection for lung and breast cancer.

©2012 AACR

Figures

Figure 1

A surgical model for local recurrence of disease following surgery. Syngeneic immunocompetent mice were injected with 1.5e6 TC1 tumor cells into the right flank (not over the liver) and developed large tumors (>800 mm3). When the tumors measured 800mm3, they were either partially resected with positive margins or completely removed. In the partial resection group, the surgeon used a #15 scalpel to sharply divide the tumor and leave the smallest possible residual nodule (<5%) that was technically feasible (ranged 2 to 4 mm in diameter). This tumor deposit was typically left attached to either the skin or underlying muscle. (A) A representative animal from each group is shown. Two independent observers first visually inspected the wound, and then palpated the wound in order to determine which animals had residual disease versus curative surgery. Animals (n=20) without obvious tumor to independent observers underwent microCT scans to determine if residual disease could be radiographically located. White arrows designate flank tumors. (B) Following surgical intervention, all animals with (i) positive margins and (ii) total resections were monitored for recurrence of their flank tumors. Tumor volume vs time and Kaplan Meier survival curves for a typical flank TC1 (n=20) experiment are depicted.

Figure 1

A surgical model for local recurrence of disease following surgery. Syngeneic immunocompetent mice were injected with 1.5e6 TC1 tumor cells into the right flank (not over the liver) and developed large tumors (>800 mm3). When the tumors measured 800mm3, they were either partially resected with positive margins or completely removed. In the partial resection group, the surgeon used a #15 scalpel to sharply divide the tumor and leave the smallest possible residual nodule (<5%) that was technically feasible (ranged 2 to 4 mm in diameter). This tumor deposit was typically left attached to either the skin or underlying muscle. (A) A representative animal from each group is shown. Two independent observers first visually inspected the wound, and then palpated the wound in order to determine which animals had residual disease versus curative surgery. Animals (n=20) without obvious tumor to independent observers underwent microCT scans to determine if residual disease could be radiographically located. White arrows designate flank tumors. (B) Following surgical intervention, all animals with (i) positive margins and (ii) total resections were monitored for recurrence of their flank tumors. Tumor volume vs time and Kaplan Meier survival curves for a typical flank TC1 (n=20) experiment are depicted.

Figure 2

Residual disease in the surgical bed fluoresces after incomplete resection. C57bL/6 mice were injected with 1.5e6 TC1 cells into the right flank. Once tumors reached 800mm3, animals were intravenously injected with 7.5 mg/kg ICG intravenously. Twenty-four hours later, the surgeon sharply removed 95% of the tumor with minimal residual disease. Two independent observers were then designated to locate suspicious margins. If residual disease was detected, the animal was eliminated from the study. The remaining animals had their surgical bed imaged by the Li-Cor Pearl® Impulse. The surgical field was then arbitrarily assigned a virtual grid and three readings using the NIR hand held machine were recorded and averaged in each sector. The mean background value was calculated from this data. If a region of high fluorescence was detected, serial readings were performed circumferentially at 2 mm intervals surrounding the suspicious location. (A) A representative C57bl/6 mouse with a flank TC1 is depicted. After surgical resection, the residual nodule could not be visualized by two independent observers or with assistance from the Li-Cor Pearl® Impulse. The hand held device rapidly detected the lesion and a typical map of the surgical wound is presented. (B) The mean fluorescence from the residual nodules (n=54) and the peritumoral tissue 4 mm from the margin is shown in the bubble graph. The fold difference between the background signal, 4mm margin and actual residual nodule are represented in the bar graph. (C) Duplicate experiments were performed in mice bearing AE17-GFP tumors in order to evaluate if ICG expression was occurring in residual tumors versus leaking into surrounding tissues following the surgery.

Figure 2

Residual disease in the surgical bed fluoresces after incomplete resection. C57bL/6 mice were injected with 1.5e6 TC1 cells into the right flank. Once tumors reached 800mm3, animals were intravenously injected with 7.5 mg/kg ICG intravenously. Twenty-four hours later, the surgeon sharply removed 95% of the tumor with minimal residual disease. Two independent observers were then designated to locate suspicious margins. If residual disease was detected, the animal was eliminated from the study. The remaining animals had their surgical bed imaged by the Li-Cor Pearl® Impulse. The surgical field was then arbitrarily assigned a virtual grid and three readings using the NIR hand held machine were recorded and averaged in each sector. The mean background value was calculated from this data. If a region of high fluorescence was detected, serial readings were performed circumferentially at 2 mm intervals surrounding the suspicious location. (A) A representative C57bl/6 mouse with a flank TC1 is depicted. After surgical resection, the residual nodule could not be visualized by two independent observers or with assistance from the Li-Cor Pearl® Impulse. The hand held device rapidly detected the lesion and a typical map of the surgical wound is presented. (B) The mean fluorescence from the residual nodules (n=54) and the peritumoral tissue 4 mm from the margin is shown in the bubble graph. The fold difference between the background signal, 4mm margin and actual residual nodule are represented in the bar graph. (C) Duplicate experiments were performed in mice bearing AE17-GFP tumors in order to evaluate if ICG expression was occurring in residual tumors versus leaking into surrounding tissues following the surgery.

Figure 2

Residual disease in the surgical bed fluoresces after incomplete resection. C57bL/6 mice were injected with 1.5e6 TC1 cells into the right flank. Once tumors reached 800mm3, animals were intravenously injected with 7.5 mg/kg ICG intravenously. Twenty-four hours later, the surgeon sharply removed 95% of the tumor with minimal residual disease. Two independent observers were then designated to locate suspicious margins. If residual disease was detected, the animal was eliminated from the study. The remaining animals had their surgical bed imaged by the Li-Cor Pearl® Impulse. The surgical field was then arbitrarily assigned a virtual grid and three readings using the NIR hand held machine were recorded and averaged in each sector. The mean background value was calculated from this data. If a region of high fluorescence was detected, serial readings were performed circumferentially at 2 mm intervals surrounding the suspicious location. (A) A representative C57bl/6 mouse with a flank TC1 is depicted. After surgical resection, the residual nodule could not be visualized by two independent observers or with assistance from the Li-Cor Pearl® Impulse. The hand held device rapidly detected the lesion and a typical map of the surgical wound is presented. (B) The mean fluorescence from the residual nodules (n=54) and the peritumoral tissue 4 mm from the margin is shown in the bubble graph. The fold difference between the background signal, 4mm margin and actual residual nodule are represented in the bar graph. (C) Duplicate experiments were performed in mice bearing AE17-GFP tumors in order to evaluate if ICG expression was occurring in residual tumors versus leaking into surrounding tissues following the surgery.

Figure 3

Residual tumor deposits have elevated fluorescent signals ex vivo. Following intraoperative imaging, residual nodules that were detected by the NIR imaging system were harvested. (A) A representative tumor nodule (right) is detected adjacent to a 25 gauge hypodermic needle (outer diameter 0.51 mm). Additional peritumoral adipose tissue was harvested for comparison (left). Li-Cor Pearl® Impulse imaging and NIR imaging from the hand held device was performed on all specimens once they were removed from the experimental animals. (B) Ex vivo fluorescence of the background tissue, peritumoral tissue and the residual nodule. H&E staining was used to confirm residual cancer cells in all experiments. (C) In vivo fluorescence of murine cancerous and non-cancerous tissues was compared to the fluorescence ex vivo after surgery.

Figure 3

Residual tumor deposits have elevated fluorescent signals ex vivo. Following intraoperative imaging, residual nodules that were detected by the NIR imaging system were harvested. (A) A representative tumor nodule (right) is detected adjacent to a 25 gauge hypodermic needle (outer diameter 0.51 mm). Additional peritumoral adipose tissue was harvested for comparison (left). Li-Cor Pearl® Impulse imaging and NIR imaging from the hand held device was performed on all specimens once they were removed from the experimental animals. (B) Ex vivo fluorescence of the background tissue, peritumoral tissue and the residual nodule. H&E staining was used to confirm residual cancer cells in all experiments. (C) In vivo fluorescence of murine cancerous and non-cancerous tissues was compared to the fluorescence ex vivo after surgery.

Figure 3

Residual tumor deposits have elevated fluorescent signals ex vivo. Following intraoperative imaging, residual nodules that were detected by the NIR imaging system were harvested. (A) A representative tumor nodule (right) is detected adjacent to a 25 gauge hypodermic needle (outer diameter 0.51 mm). Additional peritumoral adipose tissue was harvested for comparison (left). Li-Cor Pearl® Impulse imaging and NIR imaging from the hand held device was performed on all specimens once they were removed from the experimental animals. (B) Ex vivo fluorescence of the background tissue, peritumoral tissue and the residual nodule. H&E staining was used to confirm residual cancer cells in all experiments. (C) In vivo fluorescence of murine cancerous and non-cancerous tissues was compared to the fluorescence ex vivo after surgery.

Figure 4

(A) Intraoperative detection of residual nodules after surgical resection prevents tumor relapses. In two separate experiments, syngeneic mice were injected with TC1 or AB12 tumor cells into the flank. Once tumors reached 800mm3, the animals were injected with ICG via tail vein and then partially resected 24 hours later. Again, two independent observers examined all animals and eliminated any mice with obvious residual disease. These animals were then chosen to undergo imaging by the hand held NIR spectroscopic device or to have their wounds closed without any further procedures. If the hand held device detected a signal that was 5 times higher than the background for the surgical bed, the abnormal tissue was removed. A Kaplan Meier survival curve for the flank TC1 (n=25) experiment is depicted. (B) Animals were injected with TC1 or AB12 cell lines and tumors were allowed to grow to 800mm3. The tumors were imaged by the hand held imaging device in 5 locations and averaged. Tumors were harvested, sectioned and underwent a microvascular density assay using monoclonal CD31 staining. ** indicates a p value of < 0.01.

Figure 4

(A) Intraoperative detection of residual nodules after surgical resection prevents tumor relapses. In two separate experiments, syngeneic mice were injected with TC1 or AB12 tumor cells into the flank. Once tumors reached 800mm3, the animals were injected with ICG via tail vein and then partially resected 24 hours later. Again, two independent observers examined all animals and eliminated any mice with obvious residual disease. These animals were then chosen to undergo imaging by the hand held NIR spectroscopic device or to have their wounds closed without any further procedures. If the hand held device detected a signal that was 5 times higher than the background for the surgical bed, the abnormal tissue was removed. A Kaplan Meier survival curve for the flank TC1 (n=25) experiment is depicted. (B) Animals were injected with TC1 or AB12 cell lines and tumors were allowed to grow to 800mm3. The tumors were imaged by the hand held imaging device in 5 locations and averaged. Tumors were harvested, sectioned and underwent a microvascular density assay using monoclonal CD31 staining. ** indicates a p value of < 0.01.

Figure 5

A canine with a spontaneous abdominal wall sarcoma underwent image guided surgery. After ICG injection, (A) a canine abdominal was opened to reveal a well demarcated soft tissue sarcoma. The primary tumor was imaged in 8 radial directions from the center of the tumor and fluorescence averaged. The tumor bed was imaged prior to wound closure. (B) After resection, ex vivo fluorescence measurements were again performed.

Figure 5

A canine with a spontaneous abdominal wall sarcoma underwent image guided surgery. After ICG injection, (A) a canine abdominal was opened to reveal a well demarcated soft tissue sarcoma. The primary tumor was imaged in 8 radial directions from the center of the tumor and fluorescence averaged. The tumor bed was imaged prior to wound closure. (B) After resection, ex vivo fluorescence measurements were again performed.