This study demonstrated many of the fluorescence spectroscopy characteristics that can be used to identify carcinoma of the upper aerodigestive tract. The results highlight the additional information that can be obtained from time-resolved parameters and is the first study to use the Laguerre expansion coefficients for head and neck carcinoma. The lifetime values at 440-470 nm, attributed to the NADH biomolecule, demonstrated the most significant difference between normal and malignant tissue. The second order Laguerre coefficient was beneficial at both the 370-400 nm band (collagen), and the 440-470 nm band (NADH).
Previous spectrally-resolved studies have shown reduced overall intensity and a wavelength shift to longer wavelengths in malignant lesions of the head and neck.10,11
These findings were consistent with the results from this study. Multiple explanations have been suggested for this phenomenon. A few of the possible causes include increased loss of collagen crosslinking, thickening of the epithelium, and increased hemoglobin absorption.10
Data using steady-state techniques, or spectrally-resolved fluoroscopy, in the head and neck are well described. However, time-resolved fluorescence spectroscopy has not received the same amount of attention. Chen et al used lifetime measurements to distinguish normal oral mucosa from premalignant lesions.12
They were successfully able to distinguish all premalignant lesions from normal mucosa using the lifetime measurements at 633 nm.
A previous study by the authors using the hamster buccal pouch model found that TR-LIFS could reliably distinguish dysplastic and malignant lesions from normal mucosa.13
The most diagnostic emission wavelengths in that study were 380 nm, 460 nm, and 635 nm. The fluorescence at these wavelengths are believed to originate from collagen, NADH, and porphyrin, respectively. Unlike the animal model, in this human study there was no significant intensity peak at 635 nm. Betz et al believes that the porphyrin is synthesized by microbes living on the necrotic surface of ulcerated tumors and is not reliable as a diagnostic indicator.
In this study, the lifetime measurements at the 440-470 nm band were significantly different between the malignant and normal mucosa. The Laguerre expansion coefficients (LEC-2) were significantly different at both the 370-400 nm and the 440-470 nm bands. Adding the information gleaned from the Laguerre expansion coefficients contributes to the diagnostic capabilities of lifetime fluorescence spectroscopy.
The small sample size included in this study limits the ability to generalize this data to all patients with head and neck carcinoma. The purpose of this initial pilot study was to identify some of the obvious differences in lifetime fluorescence between normal and malignant tissue of the head and neck and confirm in our human patients the findings found in our previous hamster cheek pouch research. One challenge encountered in this study was the varying spectroscopic values among normal tissue in different subsites of the upper aerodigestive tract. Variability has been seen even among different sites of the oral cavity.14
This lack of uniformity presents difficulty in creating a normal range of values across all subsites. When creating diagnostic algorithms in the future, criteria will vary depending on the anatomical site of interest.
An additional limitation in this study was the lack of normal healthy controls. The authors wanted to compare histologic results with fluoroscopic findings and obtaining biopsies in normal healthy volunteers would be difficult to justify. The ‘controls,’ or ‘normal’ tissue used in this study were biopsy samples taken from the surrounding tissue. As mentioned previously, lifetime fluorescence is sensitive to the biological microenvironment. This microenvironment could be affected by infection, inflammation, and contact exposures such as tobacco and alcohol. In this study population, many patients had a strong tobacco history and one-third had prior external beam radiation. Although histologically ‘normal,’ many of these surrounding areas had exposure to tobacco and radiation. The effects of tobacco exposure and radiotherapy on the microenvironment and its impact on lifetime fluorescence have not been elucidated. Future studies comparing histologically normal tissue between smokers and nonsmokers, as well as a data set of normal healthy volunteers, could help answer some of these questions.
This study adds to the current fluorescence spectroscopy studies by revealing the significance of the shorter lifetime between 440-470 nm in malignant tissues of the aerodigestive tract. Also, we introduce the concept of using the Laguerre expansion coefficient in fluorescence spectroscopy for head and neck cancer. Time-resolved fluorescence spectroscopy provides additional information to spectrally-resolved techniques and warrants further development to achieve its potential as a non-invasive diagnostic instrument for head and neck squamous cell carcinoma. Current limitations include lack of access to the relatively expensive fiberoptic probe and increased data to provide definitive diagnostic algorithms.