Spectroscopic detection of precancerous changes in the oral cavity

Background and Motivation
According to a February 2002 Fact Sheet from the National Center for Disease Control and Prevention1, oral cancer accounts for 2-4% of all cancers diagnosed annually in the United States, occurring primarily among people over the age of 40. Much more alarming is the fact that only one-half of those persons diagnosed with oral cancer are living five years after the diagnosis [1]. In fact, in the past 16 years the overall U.S. survival rate from oral cancer has not improved. Considering the accessibility of the oral cavity for screening, the very well established risk factors, and in a number of cases, the presence of a visible pre-cancerous lesion, what is preventing the effective management of this disease? The answer lies in the limited nature of the current detection and diagnostic tools.

Typically, a dentist or other health professional screens the oral cavity for disease by gross examination. If a suspicious site is encountered, it may be biopsied and examined by a pathologist using a microscope. A qualitative assessment is made about the health of the tissue, yet the usefulness of this diagnosis is confounded by inter- and intra-observer variability in grading [2], the qualitative nature of the markers used for assessment [3], the limited value of this assessment to guide treatment [4,5], the limited number of biopsies that can be taken, and issues concerning how representative the sample is of the stage of the disease in the suspicious area as a whole6. The field cancerization effect, in which the entire area of the oral cavity is exposed to a carcinogen such as tobacco, thereby causing the appearance of many different cancerous clones, also necessitates appropriate attention to examining the oral cavity as a whole7. Spectroscopy may prove an effective tool for probing biological tissue and deriving information not available, not easily attainable, or that compliments that gained by existing clinical methods.

Figure 1. H&E stained slide from a biopsy taken of the lateral tongue.

Project Goals and Current Work
The goals of this project are the following: 1) To establish spectroscopic methods for accurate diagnosis of oral dysplasia, an early stage in cancer progression characterized by nuclear atypia and disorganization in cellular morphology, as assessed by the combination of diffuse reflectance, light scattering, and fluorescence spectroscopy, and 2) To develop a clear understanding of the changes in organization and concentration of these chromophores that give rise to the observed fluorescence signal, particularly for the case of porphyrins. Initial work in our laboratory has shown the promise as well as underscored the challenges that remain in the advancement of this technology8. Figure 2A-C show results from an early pilot study in which the diffuse reflectance, intrinsic fluorescence, and light scattering signal were used to distinguish normal from diseased sites.

A spectroscopic diagnosis for each site, based on the consensus classification from all three methods, demonstrated a specificity and sensitivity of 96% and 96%, respectively, for distinguishing normal from abnormal tissue.

Figure 2. These plots show the separation between normal, dysplastic, and cancerous tissue in the oral cavity based on the respective parameters extracted from each of the three spectral modalities. A decision line for distinguishing the three groups based on logistic regression is also plotted8. A). A plot of the intercept versus slope for a fit to the reduced scattering parameter curve (derived from the diffuse reflectance signal) , B) A plot of the NADH and collagen contribution at 340nm excitation using the intrinsic fluorescence signal, and C) A plot of nuclear size standard deviation versus the percentage of enlarged nuclei, information obtained from the light scattering (single) signal.

The laboratory maintains close collaborations with several physicians and pathologists, as they are critical in the successful collection and interpretation of our clinical data. Currently, we have clinical instruments located at Boston Medical Center and the Veteran’s Administration Hospital, Jamaica Plains, and we are in the process of collecting in vivo reflectance and fluorescence spectra. The study participants include patients who have benign lesions (i.e. inflamed tissue), are at a greater risk for developing oral cancer (smokers), present with a suspicious lesion, and those with fully developed cancer who are undergoing surgery to remove the diseased tissue. Data is also being collected from healthy volunteers who have no history of smoking or drinking. We are collecting spectra from sites that will also be biopsied so that microscopic analysis by a pathologist or other analyses, such as of DNA content, can be carried out. This will allow us to assign a category, such as dysplasia, to that particular site. Quantitative limits will be assigned for the parameters obtained from the intrinsic fluorescence, diffuse reflectance, and single-scattering data, respectively, which separate healthy from unhealthy tissue with the highest sensitivity and specificity. This will provide us with a diagnostic algorithm to make diagnoses in real-time based on our spectroscopic evaluation of a tissue site.

Recent Publications

1. 1. CDC fact sheet: http://www.cdc.gov/OralHealth/factsheets/oc-facts.htm (accessed April, 2003).
2. Sudbø, J.; Bryne, M.; Johannessen, A.C.; Kildal, W.; Danielsen, H.E.; Reith, A. "Comparison of histological grading and large-scale genomic status (DNA ploidy) as prognostic tools in oral dysplasia." J. Pathol. 2001,194 (3), 303-310.
3. van der Waal, I.; Schepman, K.P.; van der Meij, E.H.; Smeele, L.E. "Oral leukoplakia: a clinicopathological review." Oral Oncol. 1997, 33(5), 291-301.
4. Tradati, N.; Grigolat, R.; Calabrese, L.; Costa, L.; Giugliano, G.; Morelli, F.; Scully, C.; Boyle, P.; Chiesa, F. "Oral leuokoplakias: to treat or not?" Oral Oncol. 1997, 33(5), 317-321.
5. Zhang, L.; Poh, C.F.; Lam, W.L.; Epstein, J. B.; Cheng, X.; Zhang, X.; Priddy, R.; Lovas, J.; Le, N. D.; Rosin, M.P. "Impact of localized treatment in reducing risk of progression of low-grade oral dysplasia: molecular evidence of incomplete resection." Oral Oncol. 2001, 37(6), 505-512.
6. Warnakulasuriya, S. "Editorial: Histological grading of oral epithelial dysplasia: revisited." J. Path. 2001,194, 294-297.
7. Thomson, P.J. "Field change and oral cancer: new evidence for widespread carcinogenesis?" Int. J. Oral Max. Surg. 2002, 31, 262-266.
8. Muller, M.G.; Valdez, T.A.; Georgakoudi, I.; Backman, V.; Fuentes, C.; Kabani, S.; Laver, N.; Wang, Z.M.; Boone, C.W.; Dasari, R.R.; Shapshay, S.M.; Feld, M.S. "Spectroscopic detection and evaluation of morphologic and biochemical changes in early human oral carcinoma." Cancer 2003, 97 (7), 1681-1692.