Assessing breast cancer margins ex vivo using aqueous quantum-dot-molecular probes

Abstract

Positive margins have been a critical issue that hinders the success of breast- conserving surgery. The incidence of positive margins is estimated to range from 20% to as high as 60%. Currently, there is no effective intraoperative method for margin assessment. It would be desirable if there is a rapid and reliable breast cancer margin assessment tool in the operating room so that further surgery can be continued if necessary to reduce re-excision rate. In this study, we seek to develop a sensitive and specific molecular probe to help surgeons assess if the surgical margin is clean. The molecular probe consists of the unique aqueous quantum dots developed in our laboratory conjugated with antibodies specific to breast cancer markers such as Tn-antigen. Excised tumors from tumor-bearing nude mice were used to demonstrate the method. AQD-Tn mAb probe proved to be sensitive and specific to identify cancer area quantitatively without being affected by the heterogeneity of the tissue. The integrity of the surgical specimen was not affected by the AQD treatment. Furthermore, AQD-Tn mAb method could determine margin status within 30 minutes of tumor excision, indicating its potential as an accurate intraoperative margin assessment method.

Figure 1

Immunofluroscent staining of HT29 cells for Tn antigen expression. (a) HT29 cells stained with AQD-Tn mAb complexes; (b) negative control, PEG activated AQD without antibody. Blue: nuclei, red: Tn antigen expression.

Figure 2

Background signal from normal tissues when excited at 460 nm. There were 2 emission cut-off wavelengths: 509 nm and 610 nm. (a) Kidney; (b) liver; (c) muscle; (d) integrated fluorescent intensity depended on the emission cut-off wavelength. Dash-line indicates background signal.

Figure 3

The proposed process of margin determination using AQD-Tn mAb probe.

Figure 4

Blocking study for nonspecific staining using liver. (a) no blocking—0 min; (b) 5 min blocking; (c) 10 min blocking; (d) 15 min blocking; (e) quantification of the integrated fluorescent intensity versus time. Dash-line indicates acceptable background signal.

Figure 5

Animal tumor imaging. Top panel: fluorescent imaging using IVIS system. Bottom panel: bright field images of the same tumor. Two orientations of the tumor were imaged: (a) ventral side; (b) dorsal side. (c) The integrated fluorescent intensity was quantified using IVIS software for 3 regions of the tumor. Dash-line indicates the cut-off between normal and cancer areas.

Figure 6

H&E stained sections correlated to the regions (square box) of the examined tumor. (a) Dorsal side; (b) Ventral side. Cancer cells were absent in region 2 of the dorsal side. Cancer cells were detected in region 2 of the ventral side by AQD-Tn mAb probe and confirmed by H&E stained section.

Figure 7

Whole mouse tumor imaging. (a) Ventral site where the tumor was exposed. (b) Dorsal site, tumor was underneath the skin. Other organs had negative signal, indicating AQD-Tn mAb prove was specific and sensitive to the tumor.

Figure 8

Interference examination of AQD-Tn mAb probe on standard pathological evaluations. (a) Control tumors; (b) AQD-treated tumors. Different markers were evaluated such as Tn antigen, p53, and ki67. No interference with the following pathological examination was found.