|Year : 2009 | Volume
| Issue : 2 | Page : 55-61
Autofluorescence and diffuse reflectance spectroscopic analysis of oral premalignancy and malignancy
S Jayachandran1, Virender Gombra1, S Ganesan2, Sivabalan2
1 Department of Oral Medicine and Radiology, Tamil Nadu Government Dental College and Hospital, Chennai, India
2 Department of Medical Physics, Director of Students Affairs, Anna University, Chennai, India
|Date of Web Publication||1-Dec-2009|
Department of Oral Medicine and Radiology, Tamil Nadu Government Dental College and Hospital Chennai - 600003
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Early detection of squamous cell carcinoma in the oral cavity can improve survival. It is often difficult to distinguish neoplastic and premalignant disorders with standard white light illumination. Fast and noninvasive, diagnostic techniques based on fluorescence spectroscopy have the potential to link the biochemical and morphologic properties of tissues to individual patient care. In this study comparison between malignant, premalignant and healthy mucosa groups was done based on autofluorescence and diffuse reflectance spectroscopic analysis. Fluorescence spectroscopy is a new diagnostic modality with the potential to bridge the gap between clinical examination and invasive biopsy.
Keywords: Autofluorescence, malignancy, oral premalignancy
|How to cite this article:|
Jayachandran S, Gombra V, Ganesan S, Sivabalan. Autofluorescence and diffuse reflectance spectroscopic analysis of oral premalignancy and malignancy. J Indian Acad Oral Med Radiol 2009;21:55-61
|How to cite this URL:|
Jayachandran S, Gombra V, Ganesan S, Sivabalan. Autofluorescence and diffuse reflectance spectroscopic analysis of oral premalignancy and malignancy. J Indian Acad Oral Med Radiol [serial online] 2009 [cited 2019 Apr 20];21:55-61. Available from: http://www.jiaomr.in/text.asp?2009/21/2/55/57886
| Introduction|| |
Head and neck squamous cell carcinoma represents a significant and growing public health problem worldwide. Head and neck squamous cell carcinoma is commonly preceded by dysplasia.  Dysplastic lesions often are found in the oral cavity in the form of erythroplakia and leukoplakia. Therefore, early detection of malignancy of the oral cavity is very important for successful treatment and improvement of the survival rate.  Fluorescence spectroscopy is a technique for evaluating the physical and chemical properties of a substance by analyzing the intensity and character of light emitted in the form of fluorescence.
Optical spectroscopy has the potential to detect malignant lesions earlier, before they become macroscopically visible, by probing tissue biochemistry and morphology in vivo in real time. The use of endogenous and exogenous fluorescent markers, with tumor-localizing properties, for the clinical detection of early cancer in vivo has been investigated by several researchers. , Carcinomas, which comprise almost 90% of all human cancers, arise in the epithelium, the superficial layer lining the surfaces of organs and tissues.  During the development of premalignant and malignant changes, epithelial cells undergo transformations that result in modified rates of metabolic activity, cellular proliferation, and/or death. 
Autofluorescence and diffuse reflectance spectroscopy have been studied as noninvasive in vivo tools for the detection of (pre-) malignant tissue alterations. ,,, Autofluorescence of tissues under excitation with light is produced by several endogenous fluorophores. These include fluorophores from tissue matrix molecules and intracellular molecules like collagen, elastin, keratin, FAD and NADH. The presence of disease changes the concentration of these fluorophores, which makes autofluorescence spectroscopy sensitive to tissue alterations. Diffuse reflectance is the result of single and multiple backscattering of the white excitation light. Endogenous autofluorescence has been noticed around 630 nm in tumors. This fluorescence is associated with porphyrins.  It has been demonstrated that, under the range of 325-360 nm excitation wavelength, the emission band at 400-405 nm is mainly attributed to the presence of collagen, while emission at 440-460 nm is mainly due to the presence of nicotinamide adenine dinucleotide (NADH) with an excitation wavelength at 290, 351 nm and emission at 535 nm is due to flavin adinine dinucleotide (FAD) with excitation at 450 nm and 400-405 nm excitation is for porphyrin with emission maxima at 630,690 nm  and emission at 350 nm is for tryptophan with an excitation wavelength at 280 nm [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5].
Based on this correlation of endogenous fluorophors and dysplastic and malignant lesions this study was conducted to evaluate its significance as a noninvasive diagnostic modality. We employed in vivo spectroscopy for measuring autofluorescence at 280 nm, 325 nm, 405 nm, 450 nm excitation wavelength along with diffuse reflectance study of normal, clinically diagnosed premalignant and malignant lesions of the oral cavity followed by comparison of spectroscopic results between three groups based on histopathologically diagnosed malignant lesions and clinical diagnosis or histopathologic diagnosis of premalignant lesions which was done if indicated.
| Aims and Objectives|| |
- To evaluate whether autofluorescence is capable of providing a higher contrast between a lesion and healthy tissue.
- To evaluate the difference in fluorescence intensity of emission wavelength spectra at 280 nm, 325 nm, 405 nm and 450 nm for healthy, premalignant and malignant lesions.
- To evaluate whether diffuse reflectance spectroscopy is capable of providing a higher contrast between a malignant, premalignant lesion and healthy tissue.
| Materials and Methods|| |
Autofluorescence and diffuse reflectance spectra were collected from 20 patients with a known or suspected premalignant lesion, 20 patients with a known or suspected malignant oral cavity lesion reporting to the Department of Oral Medicine and Radiology of Tamil Nadu Government Dental College and Hospital, Chennai -600003 with complaint of an oral lesion and 20 healthy volunteers with no clinically observable lesions were recruited to participate after they had given their informed consent. This study was approved by ethical committee of the Tamil Nadu Government Dental College and Hospital. The following parameters were used in the establishment of the diagnosis: The lesions were visually inspected and palpated in the head, neck, oral, and pharyngeal regions. The procedure involved digital palpation of neck node regions, bimanual palpation of the floor of mouth and tongue, and inspection with palpation and observation of the oral and pharyngeal mucosa with an adequate light source, and mouth mirrors were used for the examination. The social, familial, and medical history was documented with risk behaviors (tobacco chewing, betel quid chewing, smoking and alcohol usage), a history of head and neck radiotherapy, familial history of head and neck cancer, and a personal history of cancer was taken and recorded and patients were reassured and counseling was given along with advice to quit their habit if present.
Based on history and clinical examination clinical diagnosis of premalignant lesion including non-scrapable white lesion with habit of smoking suggestive of leukoplakia, oral submucous fibrosis, oral submucous fibrosis with lichenoid reaction or lichen planus, based on clinical features diagnosis of malignant lesion of oral cavity was given.
A monochromator with a 150-W ozone-free Xenon lamp provided the excitation light. The desired excitation wavelength and the emission spectrum were selected by PC-controlled monochromator. The excitation light was guided to illuminate samples by one arm of a Y-type quartz fiber bundle, and the emission fluorescence was collected by another arm of the fiber bundle and directed to the photo multiplier detector. The signal was then amplified and displayed on the computer monitor. Any computer that meets the required specifications can be used as the controlling (external) computer. All Fluromax - 2 functions were controlled by the DataMax software which communicates between a PC-compatible computer and the Fluromax-2 [Figure 6] and [Figure 7]. The DataMax software enables to specify the experimental parameters, acquire and display data, manage files, process data, specify the hardware components, control the spectrometers and supply high voltage to the signal detector. The optical fiber probe was disinfected with 2.4% gluteraldehyde solution, rinsed with phosphate-buffered saline, and the tip of the probe was sterilized in autoclave before using.
All spectroscopic studies were done in the Division of Medical Physics of the department of physics of Anna University Chennai-600025. The procedure was explained to all the patients before the spectroscopic study. The probe was placed in gentle contact with the oral mucosa. The measurements were performed in a completely darkened room to prevent stray light from entering the spectrograph. All patients and volunteers rinsed their mouth for 1 min with a 0.9% saline solution in order to minimize the influence of consumed food and beverages.
In this study excitation for autofluorescence spectroscopy was done with ultraviolet light excitation at 280 nm, 325 nm and visible light excitation at 405 nm and 450 nm. The resulting emission spectra were recorded from 325 nm to 70 0nm in 1-mm increments. For diffuse reflectance measurements - Diffuse reflectance spectra in the range of 270-700 nm were analyzed using a model based on light diffusion theory to extract the absorption and reduced scattering coefficients of tissue. Following spectroscopic measurements, biopsy was done for all sites interrogated with the fiber-optic probe. Histopathology examination was done in the Department of Oral Pathology of Tamil Nadu Government Dental College. Biopsies were always performed after the spectra had been acquired so as not to influence the spectra. Histopathology was considered for thick, homogenous, speckled leukoplakia, OSMF with lichenoid reaction, erosive and erythematous lesions and malignant lesions, and for other premalignant lesions diagnosis was obtained by clinical inspection. However, all dysplastic and malignant lesions were histologically proven. Based on histopathologic examination diagnosis of premalignant lesions was specified as dyspastic or no dysplastic changes; and all malignant lesions were specified as mild, moderate or well-differentiated squamous cell carcinoma.
Autofluorescence data were analyzed to determine which excitation and emission wavelengths showed significant difference between the three groups. Statistical analysis was done based on peak fluorescence intensity seen in spectrograph by taking into account the ratio of two peaks seen within one spectrograph at 280 nm, 325 nm and 405nm excitation wavelength. Comparison of fluorescence intensity was done at 450 nm excitation because at this wavelength no evidence of significant peak in emission was seen. Comparison of fluorescence intensity was done for diffuse reflectance for three groups. All tests were done utilizing one-way ANOVA test followed by post hoc tests for multiple comparisons between groups.
| Results|| |
This study enrolled 60 subjects which included 20 normal, 20 clinically diagnosed premalignant lesions and 20 clinically diagnosed malignant lesions of the oral cavity. Age range for malignant group was 36 to 75 years, for premalignant group was 31 to 62 years and for normal mucosa, the range was 28 to 42 years. Our study group for malignant lesions included five females and 15 males, premalignant lesions included two females and 18 males and the normal mucosa group included eight females and 12 males.
Excitation in ultraviolet spectrum was given at 280 nm and 325 nm and excitation at visible light spectrum was given at 405 nm and 450 nm. A continuous excitation at ultraviolet and visible light was given to asses diffuse reflectance. All spectrographs were compared between malignant lesions, premalignant lesions and normal mucosa for autofluorescence and diffuse reflectance.
Comparison of autofluorescence spectrum at 280 nm excitation
Shape of emission spectrum for malignant, premalignant and normal mucosa was almost similar with peak in intensity seen at 340 nm and 468 nm. Variability in fluorescence intensity was seen in the spectrum of premalignant, malignant lesions and normal mucosa. Average of emission peak ratio at 340 nm (+5 / -5 nm) and 468 nm (+5 / -5nm) was considered as R1 for excitation at 280 nm. ANOVA test revealed significant difference between R1 of the three groups with P value of .000 suggestive of 99.9% significant difference. This was followed by post hoc analysis which revealed a significant difference between carcinoma and premalignant lesions with P value of .004 suggestive of 99% significant difference, and P value .000 was present between carcinoma and normal group suggestive of 99.9% significant difference. R1 value was higher for carcinoma than it decreases for premalignant lesions and than for normal mucosa.
Comparison of autofluorescence spectrum at 325 nm excitation
Shape of spectrum for malignant, premalignant and normal mucosa was almost similar with peak in intensity seen at 468 nm and at 560 nm. Variability in fluorescence intensity was seen in spectrum of premalignant, malignant lesions and normal mucosa. Average of emission peak ratio at 468 nm (+5 /-5nm) and 560nm (+5/-5nm) was considered as R2 for excitation at 325 nm. Significant difference was seen at 325 nm excitation. ANOVA test revealed 99% significant difference between the three groups with P value of 0.002. This was followed by post hoc tests which showed a significant difference between carcinoma and premalignant lesions with a P value of 0.01 suggestive of 99% significant difference, and P value 0.004 was present between the carcinoma and normal group suggestive of 99% significant difference. R2 value was lowest for carcinoma than it increases for premalignant lesions and than for normal mucosa.
Comparison of autofluorescence spectrum at 405 nm excitation
Shape of spectrum for premalignant and normal mucosa was almost similar with peak in intensity seen at 468 nm and at 636 nm for malignant lesions while premalignant lesions and normal mucosa showed peak at 468 nm only and mild peak was seen at 636 nm for premalignant lesions. Variability in fluorescence intensity was seen in spectrum of premalignant, malignant lesions and normal mucosa. Average of emission peak ratio at 468 nm (+5 / -5nm) and 636 nm (+5/ -5nm) was considered as R3 for excitation at 405 nm. R3 for carcinoma, premalignant lesions and normal mucosa was compared using ANOVA test followed by post hoc test to analyze the significant difference at 405 nm excitation. ANOVA test showed 99.9% significant difference within the three groups. Post hoc tests showed a significant difference between carcinoma and premalignant lesions with a P value of 0.01 suggestive of 99% significant difference, and P value 0.004 was present between the carcinoma and normal group suggestive of 99% significant difference. R3 value was lowest for carcinoma than it increases for premalignant lesions and than for normal mucosa.
Comparison of autofluorescence spectrum at 450 nm excitation
Shape of spectrum for malignant, premalignant and normal mucosa was almost similar with peak in intensity seen at 493 nm which was just followed by a dip at 487 nm. Variability in fluorescence intensity was seen in the spectrum of premalignant, malignant lesions and normal mucosa. Fluorescence spectroscopy at 450 nm excitation revealed peak at 468 nm with no evidence of any other peak. Statistical analysis done by comparing the fluorescence intensity between the carcinoma group, premalignant lesions and normal mucosa using ANOVA test revealed 99% significant difference within groups with P value of 0.004. Post hoc tests revealed significant difference between carcinoma and normal mucosa with P value of 0.03. Fluorescent intensity was highest for normal mucosa and it decreased for premalignant lesions followed by malignant lesions.
Comparison of diffuse reflectance emission spectrum
Shape of spectrograph was almost similar for malignant, premalignant and normal mucosa with dip seen in all emission spectra at 468 nm. Variability in intensity of emission was seen for malignant lesions, premalignant lesions as well as normal mucosa. Emission spectrum intensity comparison was done for carcinoma group, premalignant group and normal mucosa group using ANOVA test and post hoc test. ANOVA test revealed 99% significant difference between the three groups with P value 0.006 and post hoc tests revealed significant difference between carcinoma group and normal mucosa group with P value of 0.004, suggestive of 99% significant difference between both groups. With higher intensity was observed for carcinoma group than it was decreasing from premalignant group and further decrease in intensity was observed for normal mucosa group.
| Discussion|| |
This study identified a significant difference between malignant lesions, premalignant lesions and normal mucosa with fluorescence spectroscopy and diffuse reflectance.
At 280 nm excitation emission peak was seen at 340 nm and mild peak was seen at 468 nm. This emission at 340 nm was due to tryptophan as proteins absorb light near 280 nm and fluorescence emission maxima range from 320 nm to 350 nm. The tryptophan residues of proteins generally account for about 90% of the total fluorescence from proteins.  Ratio of emission at 340 nm (+/-5nm) and 468 nm (+/-5nm) was considered as R1. One-way ANOVA test revealed significant difference of 99.9% between the three groups for R1. Comparison of fluorescence spectrograph between the three groups revealed decreasing intensity from premalignant to normal to carcinoma group suggestive of increased tryptophan in premalignant compared to malignant group and tryptophan in normal mucosa is more than malignant mucosa.
At 325 nm excitation peak in emission was seen at 400 nm, 468 nm and mild peak at 560 nm. It has been demonstrated that, in the range of 325-360 nm excitation wavelength, the emission band at 400-405 nm is mainly attributed to the presence of collagen.  One-way ANOVA test was done for ratio at 400 nm (+/-5nm) and 468 nm (+/-5nm) which was considered as R2 and revealed significant difference of 99% with decrease in R2 was observed from carcinoma group to premalignant group to normal mucosa group. Comparison of fluorescence intensity between the three groups revealed decreasing intensity from normal to premalignant to malignant group suggestive of decrease in collagen with transformation from normal to premalignant to malignant lesions. Collagen cross-link decomposition, associated with neoplasia development, contributes to the reduction of fluorescence signal intensity. Muller et al. have shown that oral cavity carcinoma is accompanied by an increase in epithelial thickness, which reduces the depth of penetration of excitation light that reaches stromal collagen, thus reducing its contribution to the total signal. 
At 405 nm excitation spectra emission was attributed with mild peak at 468 nm followed by peak at 636 nm. Peak at 636 nm was more pronounced for the carcinoma group, than it was of almost nil for premalignant group and normal mucosa group. Comparison of spectrographs between averages of three groups also revealed significant peak at 636 nm seen only in carcinoma group. R3 was considered as ratio between 468 nm and 636 nm. R3 was compared by one-way ANOVA for the three groups which revealed significant difference of 99.9% between groups. There was decrease in R3 observed from cancer group to premalignant group followed by normal mucosa group. 405 nm excitation is commonly used in autofluoroscence spectroscopy.  It was found that the fluorescence excited in the wavelength range from 375 to 405 nm could be well fitted by the fluorescence excited at 355 nm and 435 nm that are dominated by NADH and FAD signals, respectively. In particular, the results show that with 405 nm excitation, the NADH and FAD fluorescence almost reach a balanced level. The autofluorescence spectrum excited at 405 nm can be used for accurate estimation of the redox ratio.  The most prominent difference found between dysplastic or malignant and healthy or benign mucosa was the porphyrin-like peak centered at 635 nm. Ingrams et al. have equated the presence of a certain concentration of this compound in cells with the presence of dysplasia or malignancy. They also noted that they cannot be sure whether these porphyrin-like substances correspond to dysplasia or malignancy because of accumulation porphyrinIX due to a lack of ferrochelate in tumor cells, or because of bacteria that produce porphyrins. The latter would make the method less reliable, since bacteria can live at benign lesions as well. 
Excitation at 450 nm revealed no evidence of significant peak. So, no evidence of FAD emission was seen in any of the group as FAD shows emission maxima at 535 nm when excitation maxima is 450 nm. Hence intensity for the carcinoma group, premalignant group and normal mucosa group was compared using one-way ANOVA test which revealed 99% significant difference between groups followed by post hoc test which showed significant difference between the normal and carcinoma group. Increasing order of fluorescence intensity was seen from the carcinoma group to premalignant group to normal group and excitation at 450 nm was considered for FAD with emission maxima at 515-535 nm. Although our study was inconsistent with emission maxima our observation at 450 nm excitation was consistent with Richahrds-Kortum et al. They found that the fluorescence intensity was lower for neoplastic lesions than for normal mucosa.  Comparison of fluorescence spectra between the three groups also revealed decrease in fluorescence intensity from normal to premalignant to malignant lesions.
Spectroscopy study for diffuse reflectance showed a significant difference between the three groups with decrease in fluorescence intensity from carcinoma to premalignant to normal mucosa group. Higher fluorescence intensity in lesions than normal mucosa could be due to hyperkeratosis and increased epithelial thickness. Although our results were inconsistent with previous studies which show that there was decrease in fluorescence intensity from normal to premalignant to malignant group which could be attributed to increase in absorption and less reflectance because of increase in vascularity in the premalignant and malignant group. There was no evidence of significant difference between kerartinized and non-keratinized epithelium results within the carcinoma group.
There are several possible explanations for the large intra- and inter-subject variability in total fluorescence intensity. Fluorescence intensity can be influenced by intersubject variability in the amount of blood, with absorption leading to a wavelength-dependent decrease in fluorescence intensity. Besides the biological variation, varying experimental circumstances can influence the total fluorescence intensity. Also, our study sample was comparatively small and within the group itself there was variability in oral lesions like the carcinoma group included two verrucous carcinoma, 13 well-differentiated and five moderately differentiated squamous cell carcinoma and the premalignant group consisted of two OSMF, two OSMF with lichenoid reactions, two dysplastic leukoplakia, one erosive lichen planus and 13 leukoplakia cases.
Diagnostic hurdles and limitations
The results obtained in the various investigations performed to date demonstrate the exciting potential for the application of this new technology to the diagnosis of neoplasia in the oral cavity. However, before this technique can be brought to the general public, several major obstacles must be overcome. First, larger clinical trials need to be performed to confirm the preliminary results obtained in the initial clinical trials. Second, the optimal excitation and emission wavelengths needed to differentiate between normal and abnormal tissue at each oral cavity location must be ascertained. It is important to note that, to date, the only investigations using complete EEMs (i.e., analysis at all wavelengths within a given range) have been performed in vitro. Because there are substantial differences between the results obtained in vitro and in vivo, clinical investigations using complete EEMs are needed to assess the optimal excitation and emission wavelengths. First, all the probes described in the literature so far require direct contact with the mucosal surface to obtain a spectroscopic reading. This will make application to lesions in the oropharynx, nasopharynx, and larynx more difficult without general anesthesia. Second, the monochromatic light signal penetrates only about 500 mm deep into the tissue. Thus, this technique is capable of analyzing only the most superficial portions of oral lesions. This will limit the application of this technique for the detection of submucosal disease. 
| Conclusion|| |
The current study used fluorescence and reflectance spectra of the oral cavity to extract quantitative biochemical and morphologic features associated with carcinoma progression. The rich source of information and diagnostic potential provided by noninvasive spectroscopic techniques allows us to understand more fully the changes that take place during the onset and progression of neoplasia. The results of our study demonstrated that autofluorescence due to tryptophan; collagen, NADH, FAD, porphyrin and diffuse reflectance changes has excellent diagnostic potential. Because this technique can discriminate between malignant, premalignant epithelial lesions and healthy mucosa, it could be used as a diagnostic modality to differentiate between malignant and premalignant epithelial lesions and to define margins of resection for various surgical procedures. Additionally, this technique could also be used to monitor the response of tissues to various therapeutic interventions.
However, the results of our study on autofluorescence and diffuse reflectance characteristics in the diagnosis of malignant and premalignant epithelial lesions can be validated with more studies involving large samples and with more homogenous samples.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]