In vivo fluorescence imaging of gastrointestinal stromal tumors using fluorophore-conjugated anti-KIT antibody

Abstract

Background: Gastrointestinal stromal tumors (GISTs) are frequently characterized by KIT overexpression. Tumor-free margins and complete cytoreduction of disease are mainstays of treatment. We hypothesized that fluorescently labeled anti-KIT antibodies can label GIST in vivo.

Methods: KIT K641E(+/-) transgenic mice that spontaneously develop cecal GISTs were used in this study, with C57BL/6 mice serving as controls. Alexa 488 fluorophore-conjugated anti-KIT antibodies were delivered via the tail vein 24 h prior to fluorescence imaging. Following fluorescence laparoscopy, mice were sacrificed. The gastrointestinal tracts were grossly examined for tumors followed by fluorescence imaging. Tumors were harvested for histologic confirmation.

Results: KIT K641E(+/-) mice and C57BL/6 control mice received anti-KIT antibody or isotope control antibody. Fluorescence laparoscopy had a high tumor signal-to-background noise ratio. Upon blinded review of intravital fluorescence and bright light images, there were 2 false-positive and 0 false-negative results. The accuracy was 92 %. The sensitivity, specificity, positive and negative predictive values were 100, 87, 85, and 100 %, respectively, for the combined modalities.

Conclusions: In this study, we present a method for in vivo fluorescence labeling of GIST in a murine model. Several translatable applications include: laparoscopic staging; visualization of peritoneal metastases; assessment of margin status; endoscopic differentiation of GISTs from other benign submucosal tumors; and longitudinal surveillance of disease response. This novel approach has clear clinical applications that warrant further research and development.

FIG. 1

Transgenic mouse model of GIST. a Image of a KIT K641E^+/− mouse at necropsy demonstrating the presence of a cecal GIST. Upon magnification (b), the cecal tumor is visualized (arrow). c Hematoxylin and eosin staining confirms the presence of a spindle cell neoplasm. Positive KIT immunostaining (d) is consistent with a GIST

FIG. 2

Fluorescence laparoscopy detects Alexa 488-labeled GISTs. Fluorophore-conjugated anti-KIT antibodies combined with fluorescence laparoscopy permitted enhanced detection and localization of cecal GISTs in the transgenic mouse model. Laparoscopic images of a cecal GIST from the same mouse labeled with anti-KIT antibodies demonstrate different fluorescence signal intensities of the (a) background and (b) 488-labeled GIST. c Enhanced contrast between tumor and normal bowel (e.g., background) is quantified. Signal intensity is calculated using ImageJ from laparoscopic images

FIG. 3

In vivo fluorescence labeling of GIST with anti-KIT Alexa 488 antibody vs. IgG isotype Alexa 488 antibody. a (i) and (iii) are wholebody images of 2 transgenic mice with fluorescently labeled cecal GISTs. The circles identify the location of tumors. (ii) and (iv) are magnified images of these circled cecal GISTs which better illustrate the specificity of the Alexa 488-conjugated antibody in labeling GISTs. (v) KIT staining (brown) of the cecal GIST, with corresponding magnified view (vi), from the K641E^+/− mouse labeled with Alexa 488 [imaged in (i) and (ii)] confirms the presence of a GIST. (i/iii) Intravital images of 2 transgenic mice with fluorescently labeled cecal GISTs (circles) were assessed using an OV-100 Imager. (ii/iv) Magnified images of cecal GISTs illustrate the specificity of Alexa 488-conjugated antibody in labeling GISTs. (v/vi) Photomicrographs (v, 2× and vi, 20×) of KIT immunostaining for the cecal tumors imaged in panels i/ii. b Intravital images taken of wild-type mice injected with (i) anti-KIT Alexa 488 antibody or (ii) IgG isotype control antibody. Given that wild-type mice do not develop cecal GISTs, these mice serve as a negative control in order to test the specificity of the antibody. Fluorescence signal is not seen at the cecum (white arrows). Transgenic mice were injected with either anti-KIT-Alexa 488 antibody or IgG isotype control-Alexa 488 antibody. Injection of the IgG antibody served as a negative control for the anti-KIT-Alexa 488 antibody. (iv) The cecal GIST is not fluorescently labeled in this mouse. (iii) In contrast, anti-KIT-Alexa 488 antibody labels the cecal GIST as evidenced by the presence of bright fluorescence signal