A Pilot Series Demonstrating Fluorescence Staining of Laryngeal Papilloma Using 5‐Aminolevulinic Acid

Recurrent laryngeal papillomas are the most common benign neoplasms of the larynx in both children and adults and may result in a potentially life-threatening disease in the event of complete respiratory obstruction. Many treatment modalities have been evaluated, but none with curative intent. 1.-3. Thus far, laser treatment to remove papillomas in conjunction with interferon therapy has been the best course of management. 4.-7. A possible new method for achieving improved demarcation of laryngeal papilloma during laser treatment, aimed at improved long-term results, is fluorescence staining of papillomas using 5-aminolevulinic acid (5-ALA). 5-ALA selectively induces the accumulation of protoporphyrin IX (PPIX) fluorescence in different kinds of tumors. 8. This technique is reported to be a powerful tool for the detection of malignancies located in the urinary bladder, 9. the brain, 10. the oral cavity, 11. and the larynx. 12. It was found to be easy to use and free of side effects when topically applied. 12. This article reports the first clinical study of the use of topically administered 5-ALA for fluorescence staining of laryngeal papilloma. In this study, 10 patients (mean age, 54.9 y; range, 29–79 y; 6 mens, 4 women) with laryngeal papillomas and 4 patients (mean age, 41 y; range, 19–62 y; 2 men, 2 women) with benign lesions were investigated. Informed consent was obtained from all patients. 5-ALA was applied 1 to 2 hours before intraoperative fluorescence examination. 5-Aminolevulinic acid was topically applied to the patient's larynx by inhalation. For this purpose, 30 mg 5-ALA hydrochloride (Medac, Hamburg, Germany) were dissolved in 5 mL 0.9% NaCl solution (0.6%) and nebulized with a Pari nebulizer (Pari, Starnberg, Germany). Inhalation was performed at rapid pace with intermittent vocalization, which has been reported to maximize total laryngeal aerosol deposition during inhalation. 14. During direct laryngoscopy, fluorescence excitation (λex = 375–440 nm) was achieved using a xenon short-arc lamp equipped with a special filter system (D-Light/AF, art. No. 20133201, Storz, Tuttlingen, Germany). A foot switch allowed changing between white light illumination and fluorescence excitation light (λex = 375–440 nm). For fluorescence imaging, the main portion of the remitted excitation light was blocked using a special endoscope with an integrated 440-nm long-pass filter. A small portion of the remitted light was allowed to pass through to improve fluorescence imaging quality. Video images were obtained using a target integrating color charge coupled device camera (Telecam SL PAL, art. No. 20212020, Storz), which could be mounted on the endoscope. White light and fluorescence findings were recorded on videotape for documentation purposes (S-VHS, Panasonic, Osaka, Japan). To allow fluorescence-guided laser resections, the microscope was equipped with a fluorescence detection filter (λ = 440 nm long-pass). For video documentation, the charge coupled device camera could be attached to the microscope with a C-mount adapter. Biopsies were taken from papillomas and normal tissue under fluorescence guidance. To allow correlation with histology, the intensity of red fluorescence at the biopsied site, which was depicted on a monitor, was classified as strong, weak, or negative by the surgeon (a.l.) in all 10 patients. In addition to fluorescence imaging, spectroscopic measurements were performed on all 14 patients to quantify the fluorescence contrast. Fluorescence spectra were taken from papillomas as well as from corresponding normal tissue. For the spectral measurements, an optical multichannel analyzer (S 2000, Ocean-Optics, Dunedin, FL) was attached to the endoscope instead of the charge coupled device camera. A (70/30) beamsplitter provided the surgeon with a fluorescence image of sufficient quality to allow correct positioning of the endoscope. To obtain reproducible results, the measurements were made in a fixed geometrical setup using a resection loop protruding exactly 12 mm from the front end of the endoscope and defining the distance from the tissue. Spectra (wavelength range, 450–750 nm) were recorded from a central spot (2 mm diameter) of the endoscopic fluorescence image and corrected for background, which was recorded before each series of measurements. One to 2 hours after 5-ALA application, papillomas in the patients' larynges could be clearly distinguished from adjacent normal epithelium on fluorescence images. Papillomas showed a strong red fluorescence and normal mucosa appeared in a bluish color because of the remitted excitation light (Fig. 1., Fig. 2.). However, in the case of strong bleeding, fluorescence detection was severely impeded because of the absorption characteristics of heme. Papillomas showed a much higher accumulation of PPIX than did adjacent healthy tissue, represented by differences in peak heights at 635 and 700 nm (Fig. 3). Macroscopically visible contrasts in protoporphyrin IX fluorescence (fluorescence excitation, λex = 375–440 nm) between papilloma and healthy tissues. A. Papilloma of the right vocal cord, which was clearly visible under white light examination. B. The image under fluorescence excitation with additional papillomas on the left vocal cord and the right vocal cord, which were confirmed by histopathology. A. Recurrent papilloma on the right vocal cord under white light illumination. B. The protoporphyrin IX red fluorescence findings of the same papilloma under fluorescence excitation (fluorescence excitation, λex = 375–440 nm). The performed screening with 5-aminolevulinic acid revealed a suspected red fluorescence area on the left vocal cord and both processes vocales. The histological diagnosis of the biopsies was papilloma. Thus we were able to detect additional papillomas in this patient. The suspicious tissue in the anterior commissure (fluorescence-negative) turned out to be vocal polyps due to earlier operation. Mean spectra of papilloma and adjacent normal laryngeal mucosa after application of 5-aminolevulinic acid in four patients (fluorescence excitation, λex = 375–440 nm). In 3 of the 10 patients, additional areas of papillomas were detected microscopically using 5-ALA–induced PPIX fluorescence in contrast to white light examination. The findings were confirmed by routine histology. The investigation of other benign laryngeal lesions, e.g., nodules and polyps (n = 4), revealed no specific PPIX-induced red fluorescence. This study shows that laryngeal papillomas selectively accumulate PPIX after topical application of 5-ALA. High subjective contrasts of papillomas (red) to healthy mucosa (blue) after 5-ALA application may be due to changed enzyme activity patterns in highly proliferating tissue 8. and therefore higher accumulation of PPIX. The strong red fluorescence, which originates from PPIX accumulation, can easily be distinguished from the weak green autofluorescence signal of healthy tissue, which is overlaid by remitted blue-violet excitation light. In 3 of 10 patients from our initial group, a fluorescence control check of the resection sites revealed additional findings of papillomas that were overlooked during white light examination. This might offer improved intraoperative and postoperative results of laser resection. However, we must await follow-up studies concerning the recurrence of papillomas after fluorescence-aided resection to answer this question. Other benign lesions of the larynx, such as nodules and polyps (n = 4), revealed no specific 5-ALA–induced red PPIX fluorescence. The local application of 5-ALA combines the advantages of using a naturally occurring substance that is nontoxic when topically applied with a rapid metabolization of systemically induced PPIX, which minimizes undesired photosensitization of the skin. After systemic administration of hematoporphyrin derivative, patients are required to stay away from sunlight for several days. The presented procedure is highly reproducible and easy to perform. 5-ALA is an interesting and potentially valuable drug. As well as being fluorescent, it may also become cytotoxic when excited by light of a certain wavelength and intensity and is therefore frequently used for photodynamic therapy. There is a potential role for simultaneous detection and photodynamic therapy treatment of laryngeal papilloma. 2., 15. In addition, there is an application for this technology in the treatment of children with recurrent respiratory papilloma. There is no substantial difference in children compared with adults in the application of 5-ALA to the larynx before surgery for recurrent respiratory papilloma. The equipment necessary for the detection of papillomas consists of a xenon short-arc lamp, an endoscope with a camera, or adaptation of the charge-coupled device camera to the microscope with a C-mount. The additional time needed to perform fluorescence spectroscopy and imaging is 5 to 10 minutes. Fluorescence staining of laryngeal papilloma after topical application of 5-ALA is a promising new procedure for improved detection of papillomas during laser treatment. The method is noninvasive, free of side effects, reproducible, and easy to execute. The objective of further investigations will be the evaluation of this sensitive and clinically reliable method for fluorescence-guided laser resection of laryngeal papillomas after topical and systemic (per os) 5-ALA application as well as for photodynamic therapy. In addition, follow up studies concerning papilloma recurrences are planned.