What can be expected from laser induced fluorescence?
K.K. Wang, M.W.K. Song, A. Norbasch, F. Prendergast (Rochester, MN)
The detection of early cancers is increasing in importance given the large number of pre-malignant diseases being identified through screening programs and genetic testing. Laser induced fluorescence (LIF) refers to a technique which studies the differences in the concentration of fluorescent compounds within a tissue to aid in determination of the malignant potential of tissue. Fluorescence occurs when light is absorbed by a compound causing it to achieve a higher energy state. The fluorescent compound will then re-emit the absorbed energy as a different wavelength or color of light. Most tissues contain several different fluorescent compounds which include the amino acid tryptophan, the oxidation-reduction mediator NADH, as well as connective tissue components elastin and collagen. A more complete list of these compounds as well as their absorption maximum is shown in Table I.
As can be appreciated from this list, these molecules are probably present in most tissues regardless of their malignant potential. However, it has been hypothesized that the amount of fluorophores between normal and abnormal tissues is different.
The technique used in LIF involves a laser which can stimulate the fluorescent molecules to emit light. A filtered light source can be used in place of a laser for cutaneous neoplasms since there is no difficulty in delivering light to the target lesion. If the lesion to be examined must be visualized through an endoscope, a high powered light source such as a laser is generally used to deliver adequate light through optical fibers to the tissue. Although the laser power used for these lasers is very large, the lasers are usually activated in very short pulses which decrease the amount of energy delivered to the tissue. The pulsed laser light stimulates the fluorescent compounds within the tissue which emit fluorescent light which in turn can be captured by other optical fibers and resolved into their component colors using a spectroscope. The resulting spectra can be displayed as individual graphs or combined into an image. The graphs give more quantitative information regarding the fluorescent compounds while an image can be assigned colors to represent areas suspicious for neoplasms and be more easily applied clinically. A typical spectra of tissue autofluorescence is shown on Figure 1.
Figure 1. A typical spectra of tissue autofluorescence.
This spectra was obtained from normal human esophagus using pulsed ultraviolet light from a nitrogen laser to excite the endogenous compounds within the tissue to fluoresce. The fluorescent light is shown with the intensity displayed on the vertical (y) axis and the wavelength on the horizontal (x) axis. Neoplastic tissue appears to have a much more decreased peak intensity and a more symmetric appearing curve shape.
Origins of fluorescence
At the current time, the origin of the fluorescent signals used for clinical studies has yet to be determined. It is clear that some of the patterns of fluorescence are due to hemoglobin absorption of the fluorescence signal. In fact, much of the difference between the curves generated from neoplastic tissue as compared to normal tissue is due to the hypervascularity found in cancers. The increased hemoglobin in the tissue absorbs the fluorescent light causing the peak fluorescence to be decreased in cancers. Some investigators feel that the pattern of fluorescence found in dysplasia or early carcinomas are due to the decreased detection of the prominent fluorescent signal from collagen which is covered by a thicker layer of pre-malignant mucosa. However, there is some evidence that fluorescence patterns are unique to neoplastic cells and not just a phenomenon of tissue structure.
We have been able to demonstrate using cell cultures of neoplastic cells and their non-neoplastic primary cultures that there are intrinsic differences in the fluorescent content of the cells . These patterns are not only different between normal and neoplastic cells lines but are different between neoplastic cells from different cell origins. This suggests that there may be potential to recognize neoplastic cells based upon their unique fluorescent signature.
This fluorescent excitation-emission matrix (Figure 2) illustrates the components of fluorescence found in cultured squamous cell carcinoma cells. The vertical axis is the wavelength light that is sent into the tissue while the horizontal axis represents the spectra of the light that is released through fluorescence from the tissue. The contour lines represent the relative intensity of the fluorescence. The areas of highest intensity are the fluorescence peaks of biomolecules such as porphyrins, NADH, and FAD.
Figure 2. Fluorescent excitation emission matrix.
Figure 3 represents the same type of cell but is a non-neoplastic primary culture. The fluorescence signal from this type of cell is distinctly different from that of the neoplastic cell even though both derive from squamous cells. It is the hope of fluorescence imaging that these differences can be accentuated and used to detect neoplastic lesions within the gastrointestinal tract.
Figure 3. Non neoplastic primary culture.
LIF was first applied to human colonic mucosa in order to differentiate between colonic polyps and normal mucosa . An ultraviolet nitrogen laser was used to produce a tissue spectra similar to that illustrated in esophageal tissue. The spectrum was processed using regression analysis techniques to produce a score which could differentiate the polyps from normal in all cases except for one hyperplastic polyp. Since this study, a number have been published illustrating that LIF can differentiate between normal mucosa and colonic neoplasms [3-7]. Investigators have shown that LIF has a sensitivity of 80%, specificity of 92%, positive predictive value of 82%, and negative predictive value of 91% for distinguishing polyps . One difficulty with this analysis is the lack of real time usability of these instruments. More areas of tissue can be sampled with LIF as compared to endoscopic biopsies, however, the results are only available after analysis.
LIF has also been applied to pulmonary lesions to detect early neoplasms [8-10]. Although early studies suggested that exogenous fluorophores such as hematoporphyrin derivative could be administered to enhance detection of pulmonary neoplasms, it has been found that unenhanced autofluorescence patterns seem to be able to detect neoplasms without need of exogenous fluorophores. An imaging system has been created similar to Figure 4 to take advantage of tissue autofluorescence.
This system has been created for the respiratory tract and allows viewing both white light and fluorescent images sequentially. The fluorescent image can be superimposed on the white light image to allow the endoscopist visualize the areas of concern. In a comparison between this fluorescent imaging system and normal white light bronchoscopy, it was found that both techniques had similar specificity (94%) but the fluorescence imaging system was more sensitive (73% compared with 48%) in detecting carcinoma and dysplastic lesions in the lung [8, 10].
Similar work has been done to detect occult lung cancer using hematoporphyrin derivative . Hematoporphyrin derivative was injected intravenously 72 hours prior to bronchoscopy in two patients with occult lung cancer determined by positive sputum cytology and a negative chest x-ray. Laser induced fluorescence was produced with a krypton laser (407-413 nm) and viewed directly though an eyepiece using a filter in the 620-730 nm range. The investigators were able to detect very small 2 mm areas of lung cancer that were invisible to the eye in white light by using this strategy.
Figure 4. Autofluorescence imaging system.
LIF has also been applied to esophageal cancer. We have utilized a pulsed N2-laser coupled optically to a probe to perform LIF on resected esophageal specimens containing normal mucosa, Barrett's esophagus (BE), and adenocarcinoma . A section of tissue was examined using an optical probe consisting of a central fiber for delivering the excitation energy and a 6 fiber bundle surrounding the central fiber for detection of the fluorescence. Fluorescence readings were taken from 54 normal esophageal sections and 32 sections of adenocarcinoma. A fluorescence index obtained from the tumor sections was 0.68 ± 0.01 compared with 0.51 ± 0.01 for the normal sections (p < 0.001). This index was based on curve shape rather than absolute values of fluorescence. This technique had a sensitivity of 81 % and a specificity of 100% for detection of malignant tissue. The positive predictive value was 100% and the negative predictive value was 90% for an overall accuracy of 93%. Other investigators have used a similar LIF system to detect the presence of esophageal malignancy . These investigators used a linear discriminate analysis to classify malignant tissue with a specificity of 98% with a sensitivity of 100% after sampling 26 malignant and 108 normal sites. Preliminary results utilizing LIF to examine Barrett's esophagus yield more mixed results . The technique appears to be able to classify low grade dysplasia as benign in 96% of cases but classified tissue with mixed high grade dysplasia and low grade dysplasia as pre-malignant in only 28% of the cases. Unfortunately, tissue is often mixed in nature with microscopic areas of high grade dysplasia intertwined with areas of low grade dysplasia. These studies demonstrate that LIF has potential in performing an "optical biopsy" of the mucosa. This could help direct histological biopsies and save cost in the evaluation of patients with long segments of Barrett's esophagus.
At this time, there has not been a fluorescent imaging system developed for the esophagus. The current imaging systems have been designed for the lung because of technical aspects dealing with the need to illuminate a larger mucosal surface. This technology will undoubtedly be developed for the esophagus and may prove to be an advancement in the detection of dysplasia or carcinoma in Barrett's esophagus. This type of instrument would also be an excellent tool to monitor the results of treatment of Barrett's esophagus.
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