Raman exfoliative cytology for prognosis prediction in oral cancers: A proof of concept study

Oral cancer is associated with high rates of recurrence, attributable to field cancerization. Early detection of advanced field changes that can potentially progress to carcinoma can facilitate timely intervention and can lead to improved prognosis. Previous in vivo studies have successfully detected advanced field effects in oral cancers. Raman exfoliative cytology has previously shown to differentiate normal, oral pre‐cancer and cancers. The present study explores Raman‐exfoliative‐cytology‐based detection of field effects. Exfoliated cells were collected from tumor (n = 16) and contralateral‐normal appearing mucosa (n = 16) of oral cancer patients, and healthy tobacco habitués (n = 20). After spectral acquisition, specimens were Pap‐stained for cytological evaluation. Data analysis, by Principal Component Analysis and Principal Component‐Linear Discriminant Analysis, indicate several spectral‐misclassifications between contralateral normal and tumor, which were investigated and correlated with spectral, cytological and clinical outcomes. A qualitative analysis by grouping patients with number of misclassifications with tumor (Group 1: 0, Group 2: 1 and Group 3: >1) was explored. Group 3 with highest misclassifications showed spectral and cytological similarity to tumor group — one patient was a case of early inoperable residual disease, despite clear margins on histopathology. Thus, these misclassifications could be indicative of cancer field changes, and can prospectively help to identify patients susceptible to recurrences .     

Cytometric analysis of oral scrapings of patients with oral lichen planus

A. Acha‐Sagredo, Y. Jiménez, J. V. Bagán, M. A. Echebarria‐Goicouria and J. M. Aguirre‐Utric analysis of oral scrapings of patients with oral lichen planus Objective: Aneuploidy has been associated with malignant and premalignant oral lesions. In the past few years, its application in oral precancerous lesions and its prognostic meaning have been controversial issues. The aim of our study was to characterize alterations in DNA content by automated DNA image cytometry in oral scrapings of patients with oral lichen planus. Methods: Cytological samples from 40 patients clinicopathologically diagnosed with oral lichen planus were analysed by DNA image cytometry. Results: All the cases were classified as diploid, showing a predominant population of cells with normal DNA content (DNA index, 0.85–1.15). Atrophic/erosive lesions showed a higher percentage of tetraploid cells when compared with reticular/papular lesions but this was not statistically significant (P = 0.09). Conclusions: Aneuploidy does not seem a common event in oral lichen planus lesions. However, we consider that the use of DNA image cytometry of oral scrapings may be an easy and helpful methodology in the follow‐up of patients with these lesions.     

The impact of liquid-based oral cytology on the diagnosis of oral squamous dysplasia and carcinoma

OBJECTIVE: Even though diagnostic oral exfoliative cytology is a useful, economical and practical tool in the diagnosis of oral dysplasia and carcinoma, it is not yet extensively used. The results of conventional exfoliative and liquid-based diagnostic cytology in oral potentially malignant lesions (PML) are herein reported and compared with the histological diagnosis. METHODS: Either conventional (89) or liquid-based (384) exfoliative cytology was used for the diagnosis of oral dysplasia/carcinoma in 473 subjects and the results were compared with scalpel biopsy histology. Cells were collected using a Cytobrush device for conventional smears and with a dermatological curette for the liquid-based cytology. The 'curette technique' also allowed for the collection of 'accidental' tissue fragments, utilized as microbiopsies. RESULTS: Histological diagnosis was squamous carcinoma in 96 of 473 cases, high-grade dysplasia (oral intraepithelial neoplasia two to three) in 24 and other lesions in 353 cases. The smears in the conventional cytology group were inadequate in 12.4%, with an 85.7% sensitivity and a 95.9% specificity. There were 8.8% of inadequate specimens in the liquid-based cytology group; sensitivity was 95.1% and specificity was 99.0%. CONCLUSIONS: Although conventional cytology is useful when diagnosing oral PML (better sensitivity and predictive positive value if compared with the cervical smear test with similar specificity) and can improve the accuracy of histological diagnosis, liquid-based cytology gives better results, as it not only enhances both sensitivity and specificity, but also provides material for further investigation (AgNORs, DNA, microbiopsies, etc.).     

Characterizing biochemical and morphological variations of clinically relevant anatomical locations of oral tissue in vivo with hybrid Raman spectroscopy and optical coherence tomography technique

This study aims to characterize biochemical and morphological variations of the clinically relevant anatomical locations of in vivo oral tissue (ie, alveolar process, lateral tongue and floor of the mouth) by using hybrid Raman spectroscopy (RS) and optical coherence tomography (OCT) technique. A total of 1049 in vivo fingerprint (FP: 800‐1800 cm^?1) and high wavenumber (HW: 2800‐3600 cm^?1) Raman spectra were acquired from different oral tissue (alveolar process = 331, lateral tongue = 339 and floor of mouth = 379) of 26 normal subjects in the oral cavity under the OCT imaging guidance. The total Raman dataset were split into 2 parts: 80% for training and 20% for testing. Tissue optical attenuation coefficients of alveolar process, lateral tongue and the floor of the mouth were derived from OCT images, revealing the inter‐anatomical morphological differences; while RS uncovers subtle FP/HW Raman spectral differences among different oral tissues that can be attributed to the differences in inter‐ and intra‐cellular proteins, lipids, DNA and water structures and conformations, enlightening biochemical variability of different oral tissues at the molecular level. Partial least squares‐discriminant analysis implemented on the training dataset show that the integrated tissue optical attenuation coefficients and FP/HW Raman spectra provide diagnostic sensitivities of 99.6%, 82.3%, 50.2%, and specificities of 97.0%, 75.1%, 92.1%, respectively, which are superior to using either RS (sensitivities of 90.2%, 77.5%, 48.8%, and specificities of 95.8%, 72.1%, 88.8%) or optical attenuation coefficients derived from OCT (sensitivities of 75.0%, 78.2%, 47.2%, and specificities of 96.2%, 67.7%, 85.0%) for the differentiation among alveolar process, lateral tongue and the floor of the mouth. Furthermore, the diagnostic algorithms applied to the independent testing dataset based on hybrid RS‐OCT technique gives predictive diagnostic sensitivities of 100%, 76.5%, 51.3%, and specificities of 95.1%, 77.6%, 89.6%, respectively, for the classifications among alveolar process, lateral tongue and the floor of the mouth, which performs much better than either RS or optical attenuation coefficient derived from OCT imaging. This work suggests that inter‐anatomical morphological and biochemical variability are significant which should be considered as an important parameter in the interpretation and rendering of hybrid RS‐OCT technique for oral tissue diagnosis and characterization.      

Differentiating smokers and nonsmokers based on Raman spectroscopy of oral fluid and advanced statistics for forensic applications

Raman spectroscopy has proven to be a valuable tool for analyzing various types of forensic evidence such as traces of body fluids. In this work, Raman spectroscopy was employed as a nondestructive technique for the analysis of dry traces of oral fluid to differentiate between smoker and nonsmoker donors with the aid of advanced statistical tools. A total of 32 oral fluid samples were collected from donors of differing gender, age and race and were subjected to Raman spectroscopic analysis. A genetic algorithm was used to determine eight spectral regions that contribute the most to the differentiation of smokers and nonsmokers. Thereafter, a classification model was developed based on the artificial neural network that showed 100% accuracy after external validation. The developed approach demonstrates great potential for the differentiation of smokers and nonsmokers based on the analysis of dry traces of oral fluid.     

Oral lesions in elderly population: a biopsy survey using 2250 histopathological records

Objective: To perform an oral biopsy survey focused on the incidence of oral lesions in Brazilian elderly patients. Methods: A total of 17 329 oral biopsy records were analysed and divided into two age groups: elderly patients, ≥60 years old; and non‐elderly patients, <60 years old. Information about sex, race, age and histopathological diagnosis (categorised in non‐neoplastic and neoplastic lesions) was collected. Differences of diagnosis incidence were tested by comparison between two proportions (binomial test). Results: The incidence of epithelial malignant neoplasms and pre‐malignant lesions in the elderly group was higher than non‐elderly group, as well as autoimmune diseases and salivary gland tumours. The three most prevalent lesions in the elderly group were inflammatory fibrous hyperplasia, squamous cell carcinoma, and fibroma. Conclusion: The distribution of oral diseases using biopsies allows greater accuracy in data about oral health of elderly patients, especially when considering malignant and pre‐malignant lesions.     

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)‐FTIR and HT‐Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.     

Adaptive Boosting (AdaBoost)‐based multiwavelength spatial frequency domain imaging and characterization for ex vivo human colorectal tissue assessment

The current gold standard diagnostic test for colorectal cancer remains histological inspections of endoluminal neoplasia in biopsy specimens. However, biopsy site selection requires visual inspection of the bowel, typically with a white‐light endoscope. Therefore, this technique is poorly suited to detect small or innocuous‐appearing lesions. We hypothesize that an alternative modality—multiwavelength spatial frequency domain imaging (SFDI)—would be able to differentiate various colorectal neoplasia from normal tissue. In this ex vivo study of human colorectal tissues, we report the optical absorption and scattering signatures of normal, adenomatous polyp and cancer specimens. An abnormal vs. normal adaptive boosting (AdaBoost) classifier is trained to dichotomize tissue based on SFDI imaging characteristics, and an area under the receiver operating characteristic (ROC) curve (AUC) of 0.95 is achieved. We conclude that AdaBoost‐based multiwavelength SFDI can differentiate abnormal from normal colorectal tissues, potentially improving endoluminal screening of the distal gastrointestinal tract in the future.     

Superpixel Raman spectroscopy for rapid skin cancer margin assessment

Spontaneous Raman micro‐spectroscopy has been demonstrated great potential in delineating tumor margins; however, it is limited by slow acquisition speed. We describe a superpixel acquisition approach that can expedite acquisition between ~×100 and ×10 000, as compared to point‐by‐point scanning by trading off spatial resolution. We present the first demonstration of superpixel acquisition on rapid discrimination of basal cell carcinoma tumor from eight patients undergoing Mohs micrographic surgery. Results have been demonstrated high discriminant power for tumor vs normal skin based on the biochemical differences between nucleus, collagen, keratin and ceramide. We further perform raster‐scanned superpixel Raman imaging on positive and negative margin samples. Our results indicate superpixel acquisition can facilitate the use of Raman microspectroscopy as a rapid and specific tool for tumor margin assessment.     

Fluorescence fingerprints of oral bacteria

The rapid detection and identification of microorganisms is one of the most important factors in many cases of ill health. The purpose of this study was to determine the fluorescence characteristics of seven oral bacteria using emission spectra with the aim of distinguishing between the bacteria, and to compare fluorescence imaging methods for the direct assessment of oral bacteria. Fluorescence images of each bacterium were obtained under a 405‐nm light source using a two‐filter system. The emissions of all samples were measured with a fluorescence spectrometer. The complete fluorescence data set collected for each sample employed a three‐dimensional data cube. The differences in the autofluorescence characteristics of the seven oral bacteria were determined by principal components analysis (PCA). The fluorescence images of the oral bacteria varied with the genus and the filter system. The three‐dimensional excitation‐emission matrix fluorescence spectra exhibited distinctive fluorescence features associated with intracellular fluorophores. The seven bacteria could be clearly differentiated on the PCA score plot. The findings of this study indicate that oral bacteria can be identified based on their autofluorescence characteristics. Fluorescence spectroscopy coupled with PCA can be used to detect and classify oral bacteria.      

Automated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins,cephalosporins, and fluoroquinolones using specific Raman marker bands

A Raman‐based, strain‐independent, semi‐automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch‐wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third‐generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug‐resistant Gram‐negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long‐term comparability of Raman measurements.     

Measurement of cartilage sub-component distributions through the surface by Raman spectroscopy-based multivariate analysis

Articular cartilage posesses unique material properties due to a complex depth-dependent composition of sub-components. Raman spectroscopy has proven valuable in quantifying this composition through cartilage cross-sections. However, cross-sectioning requires tissue destruction and is not practical in situ. In this work, Raman spectroscopy-based multivariate curve resolution (MCR) was employed in porcine cartilage samples (n = 12) to measure collagen, glycosaminoglycan, and water distributions through the surface for the first time; these were compared against cross-section standards. Through the surface Raman measurements proved reliable in predicting composition distribution up to a depth of approximately 0.5 mm. A fructose-based optical clearing agent (OCA) was also used in an attempt to further improve depth of resolution of this measurement method. However, it did not; mainly due to a high-spectral overlap with the Raman spectra of main cartilage sub-components. This measurement technique potentially could be used in situ, to better understand the etiology of joint diseases such as osteoarthritis (OA).     

The clinical evaluation of the oral status in Alzheimer-type dementia patients

doi: 10.1111/j.1741‐2358.2010.00401.x
The clinical evaluation of the oral status in Alzheimer‐type dementia patients Background: To evaluate the oral health status in patients with Alzheimer dementia (AD), and the association of the disease severity with the oral findings. Methods: The study was conducted on the study group (31 AD patients) and the control group (47 healthy volunteers) from the Neurology Department of the Institute. Cognitive status was evaluated with the MMSE scoring system. Oral parameters, such as decaying, missing, filled teeth (DMFT) index and teeth present (PT) were evaluated in the patients. Oral hygiene status, denture status and mucosal lesions, including denture‐induced stomatitis were also investigated. Results: The study was conducted on the study group (31 AD patients) and the control group (47 healthy volunteers). Tooth brushing and denture cleaning were irregular in 22 of 31 (70%) patients with AD. The ratio of the subjects who forgot to remove their denture during the night was significantly higher in the study group than in the control group (p < 0.001). In this study, decreased cognitive functions in AD patients have been demonstrated to result in a deterioration of denture care and increased denture‐related mucosal lesions. Conclusions: These findings were considered due to decreased denture care including the non‐removal of the denture in the night in the patients with decreased cognitive functions.     

Comparison of the Cytobrush®, dermatological curette and oral CDx® brush test as methods for obtaining samples of RNA for molecular analysis of oral cytology

M. D. Reboiras‐López, M. Pérez‐Sayáns, J. M. Somoza‐Martín, P. Gayoso‐Diz, F. Barros‐Angueira, J. M. Gándara‐Rey and A. García‐García Comparison of the Cytobrush^®, dermatological curette and oral CDx^® brush test as methods for obtaining samples of RNA for molecular analysis of oral cytology Objective: Interest in oral exfoliative cytology has increased with the availability of molecular markers that may lead to the earlier diagnosis of oral squamous cell carcinoma. This research aims to compare the efficacy of three different instruments (Cytobrush, curette and Oral CDx brush) in providing adequate material for molecular analysis. Methods: One hundred and four cytological samples obtained from volunteer healthy subjects were analysed using all three instruments. The clinical and demographical variables under study were age, sex and smoking habits. The three instruments were compared for their ability to obtain adequate samples and for the amount of RNA obtained using quantitative real‐time polymerase chain reaction (PCR‐qRT) analysis of the Abelson (ABL) housekeeping gene. Results: RNA of the ABL gene has been quantified by number of copies. Adequate samples were more likely to be obtained with a curette (90.6%) or Oral CDx (80.0%) than a Cytobrush (48.6%); P < 0.001. Similarly, the RNA quantification was 17.64 ± 21.10 with a curette, 16.04 ± 15.81 with Oral CDx and 6.82 ± 6.71 with a Cytobrush. There were statistically significant differences between the Cytobrush and curette (P = 0.008) and between the Cytobrush and OralCDx (P = 0.034). There was no difference according to the demographical variables. Conclusions: Oral exfoliative cytology is a simple, non‐invasive technique that provides sufficient RNA to perform studies on gene expression. Although material was obtained with all three instruments, adequate samples were more likely to be obtained with the curette or Oral CDx than with a Cytobrush. The Oral CDx is a less aggressive instrument than the curette, so could be a useful tool in a clinical setting.     

Prevalence of oral mucosal lesions in institutionalized elderly people in Mashhad, Northeast Iran

doi: 10.1111/j.1741‐2358.2011.00588.x
Prevalence of oral mucosal lesions in institutionalized elderly people in Mashhad, Northeast Iran Objective: The aim of this study was to determine the prevalence of oral mucosal lesions in institutionalized elderly people in Mashhad, northeast of Iran. Background: This study was conducted to assess the prevalence of oral lesions in the institutionalized elderly. Methods: In this survey, we studied all of the nursing homes in Mashhad; a total of five encompassing 237 residents. The subjects were examined and lesions recorded in a designed checklist. T‐test, chi‐square and Fisher’s exact tests were used for the analysis. Results: The mean age of the patients was 79.59 ± 8.88. Ninety‐eight per cent of cases had at least one oral mucosal lesion. The most common lesions were fissured tongue (66.5%), atrophic glossitis (48.8%), sublingual varicosity (42%) and xerostomia (38%). There was no significant difference in the prevalence of denture‐related lesions (DRLs) between men and women (p > 0.05). Xerostomia was more prevalent in 70–79‐year‐old than in 60–69‐year‐old subjects. No case of oral malignant lesion was detected. Discussion: These findings revealed a higher prevalence of oral mucosal lesions in Mashhad city compared with other studies conducted in other major cities in Iran and abroad. The results emphasise the necessity of national programmes towards oral health promotion.     

Estimation of salivary nitric oxide in oral precancer patients

Abstract

The role of nitric oxide (NO) in the initiation, promotion and progression of cancer has been the subject of speculation and conflicting reports in the literature. The high incidence of oral cancer and precancer has been linked to tobacco chewing and smoking habits; NO is considered an indicator of tobacco-related diseases. We compared salivary NO levels in oral precancer and normal patients. Unstimulated whole saliva was collected from 15 patients with oral precancer (group 1) and 15 healthy age and sex matched subjects (group 2). Salivary nitrite levels were estimated using a colorimetric method and a spectrophotometer. The salivary nitrite concentration of group 2 (median = 4.21 μg/ml) was significantly less than for group 1 (median = 12.91 μg/ml). We have added evidence concerning involvement of NO in the pathogenesis of oral cancer, but whether it is a potentially carcinogenic agent at the concentration at which it is present in oral precancer patients requires further evaluation.     

Are the barriers to good oral hygiene in nursing homes within the nurses or the patients?

doi: 10.1111/j.1741‐2358.2011.00554.x
Are the barriers to good oral hygiene in nursing homes within the nurses or the patients? Objective: To explore nursing home patients’ oral hygiene and their nurses’ assessments of barriers to improvement. Background: In nursing homes, nurses are responsible for patients’ oral hygiene. Materials and methods: This study assessed the oral hygiene of 358 patients in 11 Norwegian nursing homes. 494 nurses in the same nursing homes participated in a questionnaire study. Results: More than 40% of patients had unacceptable oral hygiene. ‘More than 10 teeth’ gave OR = 2, 1 (p = 0.013) and ‘resist being helped’ OR = 2.5 (p = 0.018) for unacceptable oral hygiene. Eighty percent of the nurses believed knowledge of oral health was important, and 9.1% often considered taking care of patients’ teeth unpleasant. Half of the nurses reported lack of time to give regular oral care, and 97% experienced resistant behaviour in patients. Resistant behaviour often left oral care undone. Twenty‐one percent of the nurses had considered making legal decisions about use of force or restraints to overcome resistance to teeth cleaning. Conclusion: Oral hygiene in the nursing homes needed to be improved. Resistant behaviour is a major barrier. To overcome this barrier nurses’ education, organisational strategies to provide more time for oral care, and coping with resistant behaviour in patients are important factors.     

Altered serum and salivary C-reactive protein levels in patients with oral premalignant lesions and oral squamous cell carcinoma

Chronic inflammation is associated with cancer development. C-reactive protein (CRP), an acute phase protein synthesized primarily in the liver, is a marker for inflammation and for the progression of many cancers. We compared serum and salivary CRP levels in 20 normal individuals, 20 patients with oral premalignant lesions and 20 patients with oral squamous cell carcinoma (OSCC) to assess its efficacy as a prognostic indicator for OSCC. Saliva and blood samples were obtained and evaluated for CRP levels. Mean CRP levels were higher in patients with oral premalignant lesions compared to controls. CRP levels in OSCC patients were elevated and were associated with advanced tumor stages.