A fluorescent photoimmunoconjugate for imaging of cholesteatoma

Abstract

Cholesteatoma is a potentially serious complication of chronic ear infections and requires surgical intervention for definitive management. Long-term complications include a frequent need for repeat surgical intervention for disease recurrence, and techniques to improve efficacy of single-stage surgery are an important area of continued research. This study investigates a novel application of the photosensitizer immune conjugate (PIC) cetuximab-benzoporphyrin derivative (Cet-BPD) for in vitro localization of human cholesteatoma tissue, coupled with an in vivo safety study for middle ear application of Cet-BPD in a murine model. In fresh human cholesteatoma tissues, Cet-BPD demonstrates selective localization to the hyperplastic squamous cell tissue associated with cholesteatoma, without localizing to other tissues such as middle ear mucosa. Applied to the murine middle ear, Cet-BPD does not demonstrate any deleterious effect on murine hearing when assessed by any of auditory brainstem response (ABR) thresholds, distortion product otoacoustic emission thresholds, or ABR wave I amplitudes. These findings demonstrate the technical promise and encouraging safety profile for the use of PICs for intraoperative localization and treatment of cholesteatoma.

Figure 1

Graphic representation of typical cholesteatoma progression in the middle ear space, beginning with (A) attic retraction pocket of the pars flaccida portion of the tympanic membrane followed by (B) gradual progression into the middle ear and mastoid space with erosion of ossicles and potentially other bony structures as well. Of note, cholesteatoma can also commonly be acquired from a tympanic membrane perforation, or may also be congenital in origin. Illustration Chris Gralapp.

Figure 2

Cet-BPD PIC is not ototoxic. (A) Schematic timeline of the experimental protocol. (B) Schematic of ABR waveform; ABR wave I amplitude is measured between P1 and N1. (C) ABR thresholds and (D) DPOAE thresholds are plotted as a function of frequency for Cet-BPD group (red, N = 6 mice) and control group that received phosphate-buffered saline (gray; N = 5 mice). Data are represented as means + SEM. (E) ABR wave I amplitude, normalized to the amplitude of ABR wave I at 80 dB in the same animal, is plotted as a function of stimulus level for three representative frequencies for Cet-BPD group (red, N = 6 mice) and control group that received phosphate-buffered saline (gray; N = 5 mice). Data are represented as means + SEM.

Figure 3

Cet-BPD PIC selectively localizes to human cholesteatoma and skin. (A) Cet-BPD PIC localizes to the epidermal region of fresh human skin, and (B) fresh human cholesteatoma tissue, but not (C) fresh middle ear mucosa tissue. (D) Cet-AF647 applied to epidermal region of fixed human skin, (E) fixed cholesteatoma tissue, and (F) fixed middle ear mucosa tissue confirms the pattern observed with Cet-BPD applied to fresh tissues.