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S. Stübinger et al. (eds.)Lasers in Oral and Maxillofacial Surgeryhttps://doi.org/10.1007/978-3-030-29604-9_12
12. Laser Applications and Autofluorescence
Abstract
The diagnostic pathway for oral suspicious lesions usually starts with the clinical examination based on inspection and palpation of the oral mucosa. Such a phase is strongly related to the experience of the operator. Moreover, oral epithelial dysplasia and early oral carcinomas may already be present within areas of macroscopically intact oral mucosa. A great interest for techniques potentially improving the diagnostic accuracy has developed in several fields of surgical oncology in order to increase the specificity and sensitivity of the conventional diagnostic pathway. The development of noninvasive methods for real-time screening of neoplastic changes in oral cavity may be associated with the improvement of patients’ quality of life and survival rate. The analysis of tissue autofluorescence (AF) for improving sensitivity and specificity in cancer diagnosis has been proposed for different organs, including colon, lung, cervix, and esophagus. Particularly, there are several evidences supporting the effectiveness of this technique in head and neck cancer diagnosis. Autofluorescence shows high specificity and sensitivity for oral cancer and precancerous lesions: 72.4% and 63.79%, respectively. It can also provide valuable information for diagnosis, for planning of margin resection in surgical excision, and for monitoring the therapeutic response during follow-up. Direct visual fluorescence examination (DVFE) is based on the action of irradiation of specific wavelengths, between 375 and 440 nm, which excites some natural fluorochromes which show fluorescence in the range of the green color. The analysis of the lesions with AF tools must be performed in a dark environment to avoid the interference of white light wavelengths and to improve the quality of recorded images. Healthy oral mucosa emits fluorescence, detectable as green light. Cell and tissues within dysplastic and malignant lesions display modifications of the amount, distribution, and chemical–physical properties of the endogenous fluorophores. This results in an autofluorescence pattern variation that can be potentially used at diagnostic level. Loss of autofluorescence (LAF) seems to increase in correspondence to the progression of dysplasia, and altered tissue appears dark (brown to black). LAF in dysplasia and carcinoma seems to be connected to different mechanisms, such as altered metabolic activity of dysplastic keratinocytes, altered structure of subepithelial collagen, and absorbance of light by increased blood circulation due to inflammatory phenomena in dysplastic tissue and cancer. AF can be used for guiding incisional biopsy and in the excision to identify the resection margins.
Autofluorescence (AF)FluorophoresOral cancer diagnosisNoninvasive tools for cancer diagnosisPotentially malignant oral disorder surgical treatmentBiopsy of oral precancerous lesions
12.1 Noninvasive Methods in Diagnostics and Surgical Oncology
Oral cancer is a common malignant tumor. The 5-year survival rate of this neoplasm is low (approximately 50%), the delayed diagnosis being among the most important reasons. It is therefore important to emphasize the role of early diagnosis and treatment [1].
The diagnostic pathway for oral suspicious lesions usually starts with the clinical examination based on inspection and palpation of the oral mucosa. Such a phase is strongly related to the experience of the operator. Moreover, oral epithelial dysplasia and early oral carcinomas may already be present within areas of macroscopically intact oral mucosa.
A great interest for techniques potentially improving the diagnostic accuracy has developed in several fields of surgical oncology in order to increase the specificity and sensitivity of the conventional diagnostic pathway. The development of noninvasive methods for real-time screening of neoplastic changes in oral cavity may be associated with the improvement of patients’ quality of life and survival rate.
Principles underlying the functioning of noninvasive visual diagnostic tools for oral cancer and dysplastic lesions are very different, being based on diverse specific cellular and tissue characteristics. Most common tools within such a context are chemiluminescence (CL), toluidine blue (TL), and chemiluminescence associated with toluidine blue (CLTB). Among these, the use of autofluorescence (AF) has recently attracted the interest of researchers.
The analysis of tissue autofluorescence for improving sensitivity and specificity in cancer diagnosis has been proposed for different organs, including colon, lung, cervix, and esophagus. Particularly, there are several evidences supporting the effectiveness of this technique in head and neck cancer diagnosis. Autofluorescence shows high specificity and sensitivity for oral cancer and precancerous lesions: 72.4% and 63.79%, respectively. It can also provide valuable information for diagnosis, for planning of margin resection in surgical excision, and for monitoring the therapeutic response during follow-up [2].
12.2 Autofluorescence: Background
The extent and nature of structural and biochemical changes taking place during the transformation from normal to precancerous state to oral cancer are poorly understood.
The native cellular fluorescence is the innate capacities of tissues to absorb and transmit light. It has been well recognized since many years that several subcellular components, called fluorophores, are capable of emitting light of specific wavelengths different from that of an exciting radiation.
Fluorophores can be classified into endogenous and exogenous. Endogenous fluorophores, either intracellular or extracellular, are present in several biologic tissues, being responsible for the phenomenon of autofluorescence (AF). Autofluorescence is a peculiar visual property of some tissues depending on the concentration and distribution of specific fluorophores.
In the literature, the use of this property in differentiating normal from neoplastic oral mucosa is widely reported [3].
The technique of AF detection is based on illumination of suspicious lesions with monochromatic light, followed by the recording of fluorescent spectra emitted by endogenous tissue fluorophores. The presence of disease results in alteration in concentration of fluorophores as well as in alteration of light scattering and absorption properties of tissue, nuclear size changes and distribution, collagen content, and epithelial thickness, which leads to spectral variations [4].
It has been hypothesized that fluorophores of oral human tissues are some proteins (e.g., structural proteins, collagen, elastin; coat proteins, keratin) and several coenzymes involved in cellular metabolism, including nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Such molecules are stimulated by wavelengths between blue and violet/ultraviolet light.
The reduced form of NADH and the oxidized form of FAD are important fluorophores that are good indicators of cellular metabolism. It has been shown that fluorescence intensity due to NADH increases with dysplastic progression, while that of FAD decreases. Maximum NADH fluorescence occurs at 340-nm excitation and 45-nm emission, and FAD occurs at 450-nm excitation and 515-nm emission [5].
Another source of AF originates in the submucosal collagen cross-links which have been demonstrated to decrease in the immediate vicinity of malignant or premalignant lesions.
Loss of collagen fluorescence is generally attributed to changes in its biochemistry, possibly due to the breakdown of the extracellular matrix by dysplastic cells.
Collagen cross-links and basal lamina of tissue affected by cancer or epithelial dysplasia are destroyed, and glucose is highly consumed in malignant tissue even in an aerobic environment. On the contrary, the concentration of FAD decreases in epithelial dysplastic tissues [6].
One hypothesis is that matrix metalloproteinase (MMP) expression in stromal cells and the consequent remodeling of the extracellular matrix are induced by altered signaling from dysplastic epithelial cells. Collagen yields maximum fluorescence at 340-nm excitation and 420-nm emission and has significant fluorescence when excited between 410 and 470 nm [5].
12.3 Autofluorescence: A Diagnostic Support in Oral Cancer and Precancerous Lesions
Oral squamous cell carcinoma (OSCC) has an incidence of more than 500,000 cases per year worldwide.
The most important prognostic factor in influencing the disease-specific survival rate is the tumor stage at diagnosis.
The 5-year relative survival rate is 64.3%. However, survival rates for OSCC are highly stage-dependent, with 83.7% of people alive 5 years after diagnosis when a localized cancer is diagnosed and 64.2% and 38.5% of people alive 5 years after diagnosis when regional and distant metastases are diagnosed, respectively. Approximately 70% of all new cases are diagnosed at a late stage, underscoring the importance of early detection and prevention [7].
The diagnostic pathway for oral suspicious lesions usually starts with the conventional objective examination (COE) based on inspection and palpation of the oral mucosa with the support of an incandescent light available on the dental chair. It is well known that COE mainly depends on a subjective interpretation, which is a consequence of the experience of the operator.
Oral epithelial dysplasia (OED) is often observed in the tissue surrounding oral squamous cell carcinoma (OSCC), and it is reportedly associated with a malignant transformation rate of 2.2–38.1% [8].
Moreover, epithelial dysplasia and early oral cancer can be located within the context of oral potentially malignant disorders such as leukoplakia, erythroplakia, submucous fibrosis, and oral lichen planus as well in areas of apparently healthy mucosa.
The gold standard for the diagnosis of oral dysplastic and neoplastic malignant lesions is the histological examination. Incisional or excisional biopsy techniques are the most reliable methods to collect a surgical specimen suitable for microscopic evaluation. However, despite the little invasivity of such techniques, they still have some disadvantages in terms of morbidity and possible artifacts induced by the method of collection.
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