Classification of cytological samples from oral potentially malignant lesions through Raman spectroscopy: A pilot study.

The potential of Raman microspectroscopy of exfoliated cells has been demonstrated for oral cancer diagnosis. In this study, brush biopsies were collected from the buccal mucosa/tongue of healthy donors (n = 31) and from oral mucosal dysplastic lesions (n = 31 patients). Raman spectra were acquired and subjected to partial least squares-discriminant analysis (PLS-DA). The patient samples could be differentiated from healthy donor samples with 96% sensitivity and 95% specificity. Furthermore, PLS-DA models were developed based on cytopathological and histopathological assessment. Low and high grade dysplasia could be discriminated with 64% sensitivity and 65% specificity based on cytopathological assessment, while 81% sensitivity and 86% specificity could be achieved when histopathological assessment was within six months of the brush biopsy sampling. Therefore, this explorative study has successfully demonstrated that Raman spectroscopy may have a role in monitoring patients with dysplasia and may reduce the need for multiple biopsies.

Raman spectral cytopathology for cancer diagnostic applications.

Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol takes 2 h 20 min for sample preparation, measurement and data preprocessing and up to 8 h for a complete analysis. A key feature of the protocol is that it uses the same sample preparation procedure as commonly used in diagnostic cytology laboratories (i.e., liquid-based cytology on glass slides), ensuring compatibility with clinical workflows. Our protocol also covers methods to correct for the spectral contribution of glass and sample pretreatment methods to remove contaminants (such as blood and mucus) that can obscure spectral features in the exfoliated cells and lead to variability. The protocol establishes a standardized clinical routine allowing the collection of highly reproducible data for Raman spectral cytopathology for cancer diagnostic applications for cervical and lung cancer and for monitoring suspicious lesions for oral cancer.

The Potential of Raman Spectroscopy in the Diagnosis of Dysplastic and Malignant Oral Lesions.

Early diagnosis, treatment and/or surveillance of oral premalignant lesions are important in preventing progression to oral squamous cell carcinoma (OSCC). The current gold standard is through histopathological diagnosis, which is limited by inter- and intra-observer errors and sampling errors. The objective of this work was to use Raman spectroscopy to discriminate between benign, mild, moderate and severe dysplasia and OSCC in formalin fixed paraffin preserved (FFPP) tissues. The study included 72 different pathologies from which 17 were benign lesions, 20 mildly dysplastic, 20 moderately dysplastic, 10 severely dysplastic and 5 invasive OSCC. The glass substrate and paraffin wax background were digitally removed and PLSDA with LOPO cross-validation was used to differentiate the pathologies. OSCC could be differentiated from the other pathologies with an accuracy of 70%, while the accuracy of the classifier for benign, moderate and severe dysplasia was ~60%. The accuracy of the classifier was lowest for mild dysplasia (~46%). The main discriminating features were increased nucleic acid contributions and decreased protein and lipid contributions in the epithelium and decreased collagen contributions in the connective tissue. Smoking and the presence of inflammation were found to significantly influence the Raman classification with respective accuracies of 76% and 94%.

Biomedical applications of vibrational spectroscopy: Oral cancer diagnostics.

Vibrational spectroscopy, based on either infrared absorption or Raman scattering, has attracted increasing attention for biomedical applications. Proof of concept explorations for diagnosis of oral potentially malignant disorders and cancer are reviewed, and recent advances critically appraised. Specific examples of applications of Raman microspectroscopy for analysis of histological, cytological and saliva samples are presented for illustrative purposes, and the future prospects, ultimately for routine, chairside in vivo screening are discussed.

A pilot study for early detection of oral premalignant diseases using oral cytology and Raman micro-spectroscopy: Assessment of confounding factors.

This study demonstrates the efficacy of Raman micro-spectroscopy of oral cytological samples for differentiating dysplastic, potentially malignant lesions from those of normal, healthy donors. Cells were collected using brush biopsy from healthy donors (n = 20) and patients attending a Dysplasia Clinic (n = 20). Donors were sampled at four different sites (buccal mucosa, tongue, alveolus, gingiva), to ensure matched normal sites for all lesions, while patient samples were taken from clinically evident, histologically verified dysplastic lesions. Spectra were acquired from the nucleus and cytoplasm of individual cells of all samples and subjected to partial least squares-discriminant analysis. Discriminative sensitivities of 94% and 86% and specificity of 85% were achieved for the cytoplasm and nucleus, respectively, largely based on lipidic contributions of dysplastic cells. Alveolar/gingival samples were differentiated from tongue/buccal samples, indicating that anatomical site is potentially a confounding factor, while age, gender, smoking and alcohol consumption were confirmed not to be.

Raman microspectroscopic study for the detection of oral field cancerisation using brush biopsy samples.

Field cancerisation (FC) is potentially an underlying cause of poor treatment outcomes of oral squamous cell carcinoma (OSCC). To explore the phenomenon using Raman microspectroscopy, brush biopsies from the buccal mucosa, tongue, gingiva and alveolus of healthy donors (n = 40) and from potentially malignant lesions (PML) of Dysplasia Clinic patients (n = 40) were examined. Contralateral normal samples (n = 38) were also collected from the patients. Raman spectra were acquired from the nucleus and cytoplasm of each cell, and subjected to partial least squares-discriminant analysis (PLS-DA). High discriminatory accuracy for donor and PML samples was achieved for both cytopalmic and nuclear data sets. Notably, contralateral normal (patient) samples were also accurately discriminated from donor samples and contralateral normal samples from patients with multiple lesions showed a similar spectral profile to PML samples, strongly indicating a FC effect. These findings support the potential of Raman microspectroscopy as a screening tool for PML using oral exfoliated cells.

Can ethanol affect the cell structure? A dynamic molecular and Raman spectroscopy study.

The role that tobacco consumption plays in the etiology of oral cancer carcinogenesis, and of alcohol consumption acting as a co-factor, have been well established. However, in recent years, the contribution of alcohol consumption alone to oral cancer has been proposed. In fact, a high percentage of patients who develop oral cancer have both habits (tobacco and alcohol consumption), and other small patient groups only consume alcohol or do not have any other identifiable bad habits. In the present study we demonstrate, using a combination of dynamic molecular modelling and Raman spectroscopy, that ethanol has a significant effect on oral cells in vitro, mainly interacting with the lipids of the cell membrane, changing their conformation. Thus, it is possible to conclude that ethanol can affect the cell permeability, and by consequence serve as a possible trigger in oral carcinogenesis.

Oral lymphangiectasias and Crohn's disease: two case reports.

Lymphangiectasias, or acquired lymphangiomas, are rare in the oral cavity, more typically occurring on the skin or the genital area and, to our knowledge, have not been reported previously in association with Crohn's disease. Lymphangiectasias can occur at any age and develop secondary to chronic obstruction of the lymphatics. This differentiates them from congenital lymphangiomas, which are congenital malformations of the lymphatic system. We present 2 cases of oral lymphangiectasias associated with Crohn's disease, one of which was treated successfully with cryotherapy.

Raman spectroscopic analysis of oral cells in the high wavenumber region.

Raman spectroscopy can provide a molecular-level signature of the biochemical composition and structure of cells with submicrometer spatial resolution and could be useful to monitor changes in composition for early stage and non-invasive cancer diagnosis, both ex-vivo and in vivo. In particular, the fingerprint spectral region (400-1800cm-1) has been shown to be very promising for optical biopsy purposes. However, limitations for discrimination of dysplastic and inflammatory processes based on the fingerprint region have been demonstrated. In addition, the Raman spectral signal of dysplastic cells is one important source of misdiagnosis of normal versus pathological tissues. The high wavenumber region (2800-3600cm-1) provides more specific information based on NH, OH and CH vibrations and can be used to identify the subtle changes which could be important for discrimination of samples. In this study, we demonstrate the potential of the high-wavenumber spectral region in this context by collecting Raman spectra of nucleolus, nucleus and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (DOK) cell lines and from normal oral epithelial primary cells, in vitro, in water immersion, which were then analyzed by principal components analysis as a method to discriminate the spectra. Analysis was performed before and after digital subtraction of the bulk water signal. In the normal cell line, the three subcellular regions are well differentiated before water subtraction, although the discrimination of the two nuclear regions is less well defined after water subtraction. Comparing the respective subcellular regions of the three cell lines, before water subtraction, the cell lines can be discriminated using sequential PCA and Feature Discriminant Analysis with up to ~100% sensitivity and 97% specificity for the cytoplasm, which is improved to 100% sensitivity and 99% specificity for the nucleus. The results are discussed in terms of discrimination comparing the CH vibrational modes of nucleic acids, proteins and lipids. The potential role of the OH vibrations, considering free water and confined water, in the discrimination of cell cultures and pathological processes are also discussed.

Circumorificial plasmacytosis/plasma cell orificial mucositis: a case series and a review of the literature.

Circumorificial plasmacytosis is a rare plasma cell proliferative disorder of the orificial mucous membranes. The etiology is unknown, and there are no reported effective treatments to date. We report three cases of idiopathic circumorificial plasmacytosis with varying clinical presentations and responses to treatment, including a first reported case of resolution with adalimumab therapy.

Raman micro-spectroscopy for rapid screening of oral squamous cell carcinoma.

Raman spectroscopy can provide a molecular-level fingerprint of the biochemical composition and structure of cells with excellent spatial resolution and could be useful to monitor changes in composition for dysplasia and early, non-invasive cancer diagnosis (carcinoma in situ), both ex-vivo and in vivo. In this study, we demonstrate this potential by collecting Raman spectra of the nucleoli, nuclei and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (pre-cancerous, DOK) cell lines and from normal oral epithelial primary cell cultures, in vitro, which were then analysed by principal component analysis (PCA) as a multivariate statistical method to discriminate the spectra. Results show significant discrimination between cancer and normal cell lines. Furthermore, the dysplastic and cancer cell lines could be discriminated based on the spectral profiles of the cytoplasmic regions. The principal component loading plot, which elucidates the biochemical features responsible for the discrimination, showed significant contributions of nucleic acid and proteins for nucleolar and nuclear sites and variation in features of lipids for the cytoplasmic area. This technique may provide a rapid screening method and have potential use in the diagnosis of dysplasia and early, non-invasive oral cancer, the treatment of which involves much less extensive and complex surgery and a reduction in associated co-morbidity for the patient.

Genotyping Candida albicans from Candida leukoplakia and non-Candida leukoplakia shows no enrichment of multilocus sequence typing clades but enrichment of ABC genotype C in Candida leukoplakia.

Oral leukoplakias are histopathologically-diagnosed as Candida leukoplakia or non-Candida leukoplakia by the presence or absence of hyphae in the superficial epithelium. Candida leukoplakia lesions have significantly increased malignant potential. Candida albicans is the most prevalent fungal species associated with oral leukoplakia and may contribute to malignant transformation of Candida leukoplakia. To date, no detailed population analysis of C. albicans isolates from oral leukoplakia patients has been undertaken. This study investigated whether specific C. albicans genotypes were associated with Candida leukoplakia and non-Candida leukoplakia in a cohort of Irish patients. Patients with histopathologically-defined Candida leukoplakia (n = 31) or non-Candida leukoplakia (n = 47) were screened for Candida species by culture of oral rinse and lesional swab samples. Selected C. albicans isolates from Candida leukoplakia patients (n = 25), non-Candida leukoplakia patients (n = 19) and oral carriage isolates from age and sex matched healthy subjects without leukoplakia (n = 34) were subjected to multilocus sequence typing (MLST) and ABC genotyping. MLST revealed that the clade distribution of C. albicans from both Candida leukoplakia and non-Candida leukoplakia lesions overlapped with the corresponding clade distributions of oral carriage isolates and global reference isolates from the MLST database indicating no enrichment of leukoplakia-associated clones. Oral leukoplakia isolates were significantly enriched with ABC genotype C (12/44, 27.3%), particularly Candida leukoplakia isolates (9/25, 36%), relative to oral carriage isolates (3/34, 8.8%). Genotype C oral leukoplakia isolates were distributed in MLST clades 1,3,4,5,8,9 and 15, whereas genotype C oral carriage isolates were distributed in MLST clades 4 and 11.

Outcomes from the first mouth cancer awareness and clinical check-up day in the Dublin Dental University Hospital.

PURPOSE OF THE STUDY: To increase public awareness about mouth cancer, the Dublin Dental University Hospital (DDUH) hosted an awareness day and free mouth check-up in September 2010. The messages of information, self-examination and risk management, and the importance of early detection, were available to all attendees. The role of general dental and medical practitioners in examination of the mouth was stressed. MATERIAL AND METHODS: A questionnaire regarding knowledge about the causes of and risk factors for mouth cancer, and a clinical check-up, were completed. RESULTS: A total of 1,661 individuals (675 male, 986 female) were examined. The mean age was 59.6 years. Just over one-third (36.5%) of those examined required no action, and slightly less (30%) were advised to return to their general dental or medical practitioner (GDP/GMP). Some 21% were advised about self-examination of the mouth, and 8% about smoking cessation. Of the remainder, 52 people (3.5%) were sent for a second opinion. Of these, 30 individuals were referred for further investigation, including biopsy in 27 cases. Following biopsy, five individuals were diagnosed with carcinoma in situ or carcinoma. CONCLUSIONS: The diagnosis of five people with mouth cancers, who may not otherwise have been identified for early treatment, highlights the need for regular mouth examination. It is inappropriate that such an exercise would remain the preserve of the dental teaching hospitals, and it is vital that all dentists take on the responsibility for regular mouth checks for all of their patients. More should be done to encourage those identified as high risk to visit their dentist. There is a need for recognition of the additional resources required for the detection and timely management of such cancers.

Oral and neck examination for early detection of oral cancer--a practical guide.

Cancer of the head and neck region presents a challenge since, unlike other areas of the body, the boundaries are not always easy to delineate. The functional morbidity associated with head and neck cancer and its treatment are considerable. Head and neck cancer is described as cancer of the lip, mouth, tongue, tonsil, pharynx (unspecified), salivary gland, hypopharynx, larynx and other. Oral cancer refers to cancers of the lip, tongue, gingivae, floor of the mouth, palate (hard and soft), maxilla, vestibule and retromolar area up to the anterior pillar of the fauces (tonsil). When patients present with oral cancer, over 60% of them have regional (lymph node) and sometimes distant (metastatic) spread. The overall five-year survival rates for oral cancer average at between 50 and 80%, depending on the stage of the disease, varying from 86% for stage I to 12-16% for stage IV. The incidence of 'field cancerisation'/unstable oral epithelium is high (17%), and even after successful treatment our patients need to be monitored for dental care and further disease. Unlike other areas in the body, the oral epithelium is readily accessible for examination and even self-examination. Dentists and dental hygienists are effective clinicians in the examination of the oral cavity for mouth cancer. An oral and neck examination must be part of every dental examination. An examination protocol is suggested here, which is similar to, but more detailed than, the standardised oral examination method recommended by the World Health Organisation, and consistent with those protocols followed by the Centres for Disease Control and Prevention and the National Institutes of Health.

Patch testing for food-associated allergies in orofacial granulomatosis.

BACKGROUND: Food-associated allergies, especially to benzoates and cinnamon-related compounds, have been associated with orofacial granulomatosis and both standard and urticarial patch testing have been used to detect such allergies. Elimination diets have also been shown to be effective in some patients. OBJECTIVES: To compare the results of standard and urticarial patch testing in a cohort of patients with orofacial granulomatosis. MATERIALS AND METHODS: Records of 120 cases seen in two hospitals were retrieved and examined for patch test details. RESULTS: Standard patch testing was much less likely to detect allergy to benzoates and cinnamon compounds (7%) than urticarial tests (55%). All urticarial tests that were positive had shown a reaction by 60 min. CONCLUSIONS: Both standard and urticarial patch tests are required to detect food allergies in orofacial granulomatosis. The difficulties of patient self-recording of urticarial tests can be eliminated by retaining patients in the testing unit for professional reading of patches at 60 min.