Cancers of oral cavities are one of the most common malignancies in India and other south-Asian countries. Tobacco habits are the main etiological factors for oral cancer. Identification of premalignant lesions is required for improving survival rates related to oral cancer. Optical spectroscopy methods are projected as alternative/adjunct for cancer diagnosis. Earlier studies have demonstrated the feasibility of classifying normal, premalignant, and malignant oral ex-vivo tissues. We intend to evaluate potentials of Raman spectroscopy in detecting premalignant conditions. Spectra were recorded from premalignant patches, contralateral normal (opposite to tumor site), and cancerous sites of subjects with oral cancers and also from age-matched healthy subjects with and without tobacco habits. A total of 861 spectra from 104 subjects were recorded using a fiber-optic probe-coupled HE-785 Raman spectrometer. Spectral differences in the 1200- to 1800-cm-1 region were subjected to unsupervised principal component analysis and supervised linear discriminant analysis followed by validation with leave-one-out and an independent test data set. Results suggest that premalignant conditions can be objectively discriminated with both normal and cancerous sites as well as from healthy controls with and without tobacco habits. Findings of the study further support efficacy of Raman spectroscopic approaches in oral-cancer applications.

Occurrence of metachronous and synchronous secondary tumors in oral cavities has been associated with poor prognosis and decreased 5-year disease-free survival rates. The origin of secondary tumors in the oral cavity has been primarily attributed to cancer field effects (CFE) or malignancy-associated changes (MAC) in uninvolved areas. Classification of normal, cancerous and pre-cancerous oral lesions by in vivo Raman spectroscopy (RS) has already been demonstrated. In the present study, MAC/CFE in oral buccal mucosa were explored. In vivo Raman spectra from 84 subjects (722 spectra) under five categories - cancer and contralateral normal (opposite side of tumor), healthy controls (no tobacco habit, no cancer), habitués healthy controls (tobacco habit, no cancer) and non-habitués contralateral normal (no tobacco habit with cancer) were acquired. Mean and difference spectra suggest that loss of lipids and additional features representing proteins and DNA are characteristics of all pathological conditions, with respect to healthy controls. Spectral data were analyzed by PC-LDA followed by leave-one-out cross-validation. Results suggest that Raman characteristics of mucosa of healthy controls are exclusive, while those of habitués healthy controls are similar to those of contralateral normal mucosa. It was observed that the cluster of non-habitués contralateral normal mucosa is different from habitués healthy controls, suggesting that malignancy associated changes can be identified and also indicating that transformation of uninvolved oral mucosa due to tobacco habit or malignancy is different. The findings of the study demonstrate the potential of RS in identifying early transformation changes in oral mucosa and the efficacy of this approach in oral cancer applications.

Oral squamous cell carcinoma is one of among the top 10 malignancies. Optical spectroscopy, including Raman, is being actively pursued as alternative/adjunct for cancer diagnosis. Earlier studies have demonstrated the feasibility of classifying normal, premalignant, and malignant oral ex vivo tissues. Spectral features showed predominance of lipids and proteins in normal and cancer conditions, respectively, which were attributed to membrane lipids and surface proteins. In view of recent developments in deep tissue Raman spectroscopy, we have recorded Raman spectra from superior and inferior surfaces of 10 normal oral tissues on intact, as well as incised, biopsies after separation of epithelium from connective tissue. Spectral variations and similarities among different groups were explored by unsupervised (principal component analysis) and supervised (linear discriminant analysis, factorial discriminant analysis) methodologies. Clusters of spectra from superior and inferior surfaces of intact tissues show a high overlap; whereas spectra from separated epithelium and connective tissue sections yielded clear clusters, though they also overlap on clusters of intact tissues. Spectra of all four groups of normal tissues gave exclusive clusters when tested against malignant spectra. Thus, this study demonstrates that spectra recorded from the superior surface of an intact tissue may have contributions from deeper layers but has no bearing from the classification of a malignant tissues point of view.

Biophysical techniques play an important role in detecting physiological alterations during pathogenesis. Raman spectroscopy has shown immense potential in identifying several diseased conditions, including oral cancers. Classification of normal, inflammatory, premalignant and malignant conditions has been demonstrated using ex vivo Raman spectroscopy. Feasibility of recording in vivo spectra in clinically implementable time has also been shown. Translation of this technology to clinics requires extensive validation of methodologies, building of robust models and testing the same under stringent conditions as well as on diverse populations. In this context, the ability of Raman spectroscopy in identifying subtle changes in oral mucosa with increasing age, and the influence of these aging related changes on classification with tobacco-related pathological changes was evaluated. A total of 451 spectra from 62 subjects were recorded from buccal mucosa of healthy subjects of 4 different age groups (aged 20-60 years). Also, 478 spectra from 85 subjects belonging to 4 different categories, tobacco exposed mucosa, contralateral normal (opposite side of tumor), premalignant patches and tumors on buccal mucosa were recorded using fiber optic probe-coupled commercial Raman spectrometer. Differences in spectra were explored by unsupervised Principal Component Analysis (PCA) and supervised Linear Discriminant Analysis (LDA), followed by Leave one out cross validation. Results indicate feasibility of classifying early and late age groups. Also, clear classification is observed between healthy and pathological groups, thus inherent heterogeneity in healthy groups seems to have no bearing on classification of normal with abnormal conditions. Findings of the study indicate high sensitivity of Raman spectroscopy in detecting subtle mucosal changes, further supporting efficacy of Raman spectroscopic approaches in oral cancer applications. Prospectively, more vigorous validation studies of Raman methodology would enable routine clinical applications.

Findings provide proof-of-concept for Raman spectroscopy-based identification of sites that have higher propensity to progress to carcinomas before becoming clinically apparent. Prospective validation of Raman spectroscopy by including additional oral cavity subsites and use of multifiber bundles may improve rate of identification of recurrence-prone subjects.