Epidermal growth factor receptor-targeted immunophotodiagnosis and photoimmunotherapy of oral precancer in vivo

Abstract

Immunophotodiagnosis uses a fluorescence-labeled monoclonal antibody (MAb) that recognizes a tumor-associated antigen to image the fluorescence emitted from the fluorophore-bound MAb that has localized in the tissue. It may be used to diagnose malignant or precancerous lesions, to delineate the margins for tumor resection, or as a feedback mechanism to assess response to treatment. In oral precancer, the epidermal growth factor receptor (EGFR) is overexpressed and could be used as a marker for early detection or as a target for therapy. The goal of this study was to test an anti-EGFR MAb (C225) coupled to either the near-infrared fluorescent dye N,N'-di-carboxypentyl-indodicarbocyanine-5,5'-disulfonic acid for detection or a photochemically active dye (chlorin(e6)) for therapy of early premalignancy in the hamster cheek pouch carcinogenesis model. Fluorescence levels in the carcinogen-treated tissue correlated with the histological stage of the lesions when the C225-N,N'-di-carboxypentyl-indodicarbocyanine-5,5'-disulfonic acid conjugate was used but did not do so with the irrelevant conjugates. Discrete areas of clinically normal mucosa with high fluorescence (hot spots) were subsequently shown by histology to contain dysplastic areas. The best contrast between normal and carcinogen-treated cheek pouches was found at 4-8 days after injection. To test the potential of immunophotodiagnosis as a feedback modality for therapeutic intervention, experiments were conducted with the same MAb conjugated to chlorin(e6) followed by illumination to reduce expression of the EGFR by a photodynamic effect. Subsequent immunophotodiagnosis showed that this treatment led to a significant reduction in fluorescence in the carcinogen-treated cheek pouch compared with nonilluminated areas. This difference between illuminated and dark areas was not seen in the normal cheek pouch. Taken together, the results demonstrate the potential for development of immunophotodiagnosis as a diagnostic tool and as a method of monitoring response to therapy and that the EGFR may be an appropriate target in head and neck cancer.

Fig. 1

UV-visible absorption spectra in 0.1 m NaOH/0.1% SDS. A, C225-Cy5.5, mouse IgG-Cy5.5, and BSA-Cy5.5 conjugates and unconjugated Cy5.5; B, C225-c_e6 and c_e6.

Fig. 2

Fluorescence localization of C225-Cy5.5 with A431 and HCPC-1 cells. Cells were incubated with the conjugate at a final concentration of 0.1 μg of Cy5.5 equivalent in serum-containing medium for 3 h at 37°C (670 nm, bandpass filter excitation; 700 nm, long-pass filter emission). A431 cells with the C225-Cy5.5 conjugate: fluorescence image, a; bright-field image, b. HCPC-1 cells with the C225-Cy5.5 conjugate: fluorescence image, c; bright-field image, d. Bar, 50 μm.

Fig. 3

Fluorescence image of hamster with gross papillary tumor obtained after 12 weeks of painting of the cheek pouch with DBMA. Hamsters were injected with 670 μg of C225-Cy5.5 and imaged 2 days later. a, white-light image of both cheek pouches. b, fluorescence image of normal cheek pouch. c, fluorescence image of carcinogen-treated cheek pouch with papillary tumor (arrow); bar, 5 mm.

Fig. 4

Fluorescence images of normal (a) and DMBA-treated (b) cheek pouches in hamster 6 at 6 days after the injection of the C225-Cy5.5 conjugate. Bar, 5 mm. A fluorescence hot spot (area of increased fluorescence) can be seen within the DMBA-treated cheek pouch (arrow). H&E histology shows (c) normal mucosal epithelium for the DMBA-untreated cheek pouch (bar, 100 μm), (d) mild dysplasia for the DMBA-treated cheek pouch, and (e) moderate dysplasia for the hot spot.

Fig. 5

Time course of mean absolute fluorescence values in the DMBA-treated versus DMBA-untreated cheek pouches after injection of the C225-Cy5.5 conjugate. Values from normal and DMBA-treated cheek pouches are from nine hamsters, whereas values of hot spot fluorescence are from five hamsters. Values are expressed as means ± SE.