Evaluation of Usage of Different Diagnostic Aids for Oral Cancer by Oral and Maxillofacial Surgeons: An Original Research.

Objective: The purpose of this study was to assess how oral and maxillofacial surgeons used various diagnostic tools for oral cancer. Materials and Methods: A cross-sectional methodology was used, and a standardized questionnaire was given to oral and maxillofacial surgeons randomly chosen sample. The questionnaire gathered information on demographics and the use of diagnostic tools. Data analysis methods included Chi-square testing and descriptive statistics. Results: The study included 200 oral and maxillofacial surgeons in total. The most often used diagnostic tool (95%) was visual inspection, followed by toluidine blue staining (48%) and brush biopsy (32%). Less frequently used were newer methods like optical coherence tomography (12.5%) and autofluorescence imaging (15%). No significant correlations between demographic factors and patterns of use of diagnostic tools were found by Chi-square tests. Conclusion: The results show that oral and maxillofacial surgeons frequently use brush biopsy, toluidine blue staining, and ocular evaluation. However, there is a need for more widespread adoption of cutting-edge technologies. By removing obstacles and offering training opportunities, one can increase the use of diagnostic tools, improving patient outcomes and the diagnosis of oral cancer.

Diagnosis of differentiated dysplasia as a variant of oral epithelial dysplasia.

OBJECTIVES: The World Health Organization's definition of oral epithelial dysplasia includes differentiated dysplasia, which is defined by purely architectural abnormalities of oral mucosa without cytological changes. We analysed differentiated dysplasia's frequency, progression risk and correlation with oral brush cytology. MATERIALS AND METHODS: Cytoarchitectural criteria and expression patterns of keratin 13/17 and ki67 were studied in oral biopsies clinically diagnosed with leukoplakia. Biopsies were assessed for dysplasia and its grade. Available brush cytology findings were obtained from clinical records. RESULTS: We included 159 biopsies from 112 patients (33% differentiated dysplasia; 27% keratosis without dysplasia; oral epithelial dysplasia with atypia of mild, moderate and severe degree including invasive cancers in 9%, 8% and 7%, respectively). Keratin 13 loss and keratin 17 gain were higher in differentiated-dysplasia cases (p < 0.0001), which had the highest hypergranulosis frequency. Keratin 17 expression was associated with higher malignant-transformation rates (p = 0.0028). The transformation rate and time were comparable between dysplasia with atypia and differentiated-dysplasia cases, which had higher progression rates and shorter time periods than keratosis cases without dysplasia (p = 0.08). Cytology prior to differentiated dysplasia all indicated normal oral mucosa. CONCLUSIONS: Keratin 17 but not oral brush cytology can help identify patients with differentiated dysplasia with higher risk for malignant transformation.

Culture expansion of primary human nasal epithelial cells (NEC) isolated with a nasal scraping spoon.

OBJECTIVE: To obtain high-purity nasal epithelial cells (NEC) while avoiding the irritation experienced by patients during nasal biopsies. METHODS: This prospective, observational study enrolled patients undergoing surgical treatment for nasal septum deviation. After general anaesthesia, a novel nasal scraping spoon was used to collect epithelial cells from the mid-part of the inferior turbinate. The cells were evenly plated on six-well plates coated with rat tail collagen. The morphology and growth of the cells were observed at different time-points using an inverted phase-contrast microscope. Immunofluorescent staining of cytokeratin 18 was used to identify NEC. Ki67 staining was used to check cell viability. RESULTS: This study collected samples from 19 patients during a short procedure. No postoperative complications were observed. Cell samples ranging from 8.31 × 105 to 2.04 × 106 cells/sample were obtained. The culture model was suitable for primary NEC culture as demonstrated by the faster proliferation (5-7 days). There was no fungal or bacterial contamination. Immunofluorescent staining confirmed the presence and proliferative activity of NEC in the cultures. CONCLUSION: A novel nasal scraping spoon provided an easy sampling method, avoided nasal injuries and psychological barriers to sampling and sufficient viable NEC to establish primary cultures.

Assessment of brush biopsy findings and salivary LDH levels in oral mucosal lesions of tobacco users.

Introduction: The oral brush cytology is an alternative method developed to improve the efficacy of conventional cytology in oral potentially malignant disorder (OPMD), and salivary lactate dehydrogenase (LDH) which is a cytoplasmic enzyme has been widely used as a marker for diagnosing various diseases. The purpose of the study is to evaluate the brush biopsy findings and salivary LDH levels for the early diagnosis of premalignant and malignant lesions of the oral cavity. Materials and Methods: Patients with deleterious habits including tobacco-related lesions such as leukoplakia, tobacco pouch keratosis, and oral cancer were included in the study. For each patient, saliva sample was collected, brush biopsy was done and smears were prepared. Collected saliva samples were analysed for salivary LDH levels and prepared smears were analysed for dysplastic changes and statistical analysis was performed. Results: Out of 80 samples, 30 were leukoplakia, 45 were tobacco pouch keratosis and 5 were oral cancer, and 13 samples showed positive dysplastic changes, 26 samples showed atypical dysplastic changes and 41 samples showed no signs of dysplastic changes and concluded as negative. On comparing the results of brush biopsy findings and salivary LDH levels, the mean salivary LDH value for positive dysplasia was elevated and the P value was statistically significant (P value: 0.00). Conclusion: Brush biopsy showed good potential in detecting premalignant lesions and salivary LDH levels showed a marked increase which can be used as a diagnostic biomarker and serve as a potent diagnostic aid for early detection of malignancy.

Assessment of Ploidy Status in Oral Potentially Malignant Disorders - A Systematic Review.

Malignant and potentially malignant epithelial lesions are often associated with various abnormalities such as epithelial dysplasia, abnormal DNA content, loss of heterozygosity, and chromosomal number aberrations. Screening and early detection of such abnormalities facilitates proper care and also helps to prevent further progression of potentially malignant lesions to malignancy. In such way, the presence of DNA aneuploidy in oral potentially malignant disorders (OPMDs) may serve as an indicator for the malignant transforming potential. Various assessment methods have been proposed to find the DNA ploidy status of cells. This current systematic review is mainly designed to assess the importance of ploidy status in OPMD while measuring the feasibility of using this biomarker for evaluating the hazard of malignant transformation. As an upshot of this systematic review, we can conclude that use of DNA ploidy status can serve as an independent bio-marker for predicting the malignant transformation of lesions. Furthermore, as a future scope the use of DNA ploidy analysis in normal mucosa of smokers will help to assess the malignancy risk and this technique might also help to predict the genetic predisposition of patients with malignancy.

Biobanked tracheal basal cells retain the capacity to differentiate.

Objective: While airway epithelial biorepositories have established roles in the study of bronchial progenitor stem (basal) cells, the utility of a bank of tracheal basal cells from pediatric patients, who have or are suspected of having an airway disease, has not been established. In vitro study of these cells can enhance options for tracheal restoration, graft design, and disease modeling. Development of a functional epithelium in these settings is a key measure. The aim of this study was the creation a tracheal basal cell biorepository and assessment of recovered cells. Methods: Pediatric patients undergoing bronchoscopy were identified and endotracheal brush (N = 29) biopsies were collected. Cells were cultured using the modified conditional reprogramming culture (mCRC) method. Samples producing colonies by day 14 were passaged and cryopreserved. To explore differentiation potential, cells were thawed and differentiated using the air-liquid interface (ALI) method. Results: No adverse events were associated with biopsy collection. Of 29 brush biopsies, 16 (55%) were successfully cultured to passage 1/cryopreserved. Samples with higher initial cell yields were more likely to achieve this benchmark. Ten unique donors were then thawed for analysis of differentiation. The average age was 2.2 ± 2.2 years with five donors (50%) having laryngotracheal pathology. Nine donors (90%) demonstrated differentiation capacity at 21 days of culture, as indicated by detection of ciliated cells (ACT+) and mucous cells (MUC5B+). Conclusion: Pediatric tracheal basal cells can be successfully collected and cryopreserved. Recovered cells retain the ability to differentiate into epithelial cell types in vitro. Level of Evidence: Level 3.

Evaluation of Static DNA Ploidy Analysis Using Conventional Brush Biopsy-Based Cytology Samples as an Adjuvant Diagnostic Tool for the Detection of a Malignant Transformation in Potentially Oral Malignant Diseases: A Prospective Study.

BACKGROUND: The accuracy of DNA image cytometry as an investigation method for potentially malignant disorders of the oral cavity is currently still a subject of controversy, due to inconsistently applied definitions of DNA aneuploidy, small cohorts and different application techniques of the method. The aim of this study was to examine the accuracy of the method as a supplementary diagnostic tool in addition to the cytological examination using internationally consented definitions for DNA aneuploidy. METHODS: A total of 602 samples from 467 patients with various oral lesions were included in this prospective study. Brush biopsies from each patient were first cytologically examined and categorized by a pathologist, second evaluated using DNA image cytometry, and finally compared to either histological biopsy result or clinical outcome. RESULTS: Using the standard definition of DNA aneuploidy, we achieved a sensitivity of 93.5%, a positive predictive value for the detection of malignant cells of 98.0%, and an area under the curve of 0.96 of DNA ploidy analysis for the detection of severe oral epithelial dysplasia, carcinoma in situ or oral squamous cell carcinoma. Importantly, using logistic regression and a two-step model, we were able to describe the increased association between DNA-ICM and the detection of malignant cells (OR = 201.6) as a secondary predictor in addition to cytology (OR = 11.90). CONCLUSION: In summary, this study has shown that DNA ploidy analysis based on conventional specimens of oral brush biopsies is a highly sensitive, non-invasive, patient-friendly method that should be considered as an additional diagnostic tool for detecting malignant changes in the oral cavity.

Retrospective evaluation of the oral brush biopsy in daily dental routine - an effective way of early cancer detection.

OBJECTIVES: Oral brush biopsies are a well researched index for early detection of oral cancer in specialised centers. But the performance of the exfoliative biopsy is not yet researched in daily dental routine. METHODS: Private dentists and private oral surgeons in Germany took brush biopsies out of 814 suspicious lesions from 670 patients using the Orcellex brush while regular dental appointments. The analyses of the biopsies were performed by the Cytological Laboratory of Bonn (CLB) using liquid-based cytology. RESULTS: The final results were 74 oral squamous cell carcinomas and one verrucous carcinoma, histological proven, 232 cases of leukoplakia, 242 cases of lichen planus, 17 cases of erythroplakia, 259 cases of benign inflammatory, traumatic or hyperplastic oral lesions. The sensitivity for the detection of cancer cells using brush biopsy archived 100%, the specificity for the detection of non-neoplastic cells was 86.5%. The positive predictive value was 43.1%, the negative predicative value was at 100%. CONCLUSION: The oral brush biopsy seems to be a sufficient tool for early cancer detection in private dental offices. CLINICAL RELEVANCE: Generally, practicing dentists do not see various oral squamous cell carcinomas in their careers, so the experience in identifying oral squamous cell carcinomas as such is very low. The brush biopsy might help them in cases of doubt to prevent tumors from expansive growth.

Oral brush biopsy using liquid-based cytology is a reliable tool for oral cancer screening: A cost-utility analysis: Oral brush biopsy for oral cancer screening.

BACKGROUND: This study aimed to assess the diagnostic utility and associated cost of oral liquid-based brush cytology (OLBC) in the diagnosis of oral cancer and oral potentially malignant disorders (OPMDs). METHODS: A total of 284 patients with oral mucosal lesions were included. OLBC samples were collected from all patients immediately before undergoing surgical biopsies. A liquid-based cytology slide was prepared from each OLBC sample for cytological evaluation using the modified 2014 Bethesda cytology system. The results and the cost were compared with the histopathological outcomes. RESULTS: The level of agreement between the two approaches was very good (weighted kappa = 0.824). The accuracy of OLBC in differentiating between the different diagnostic groups was 91.69%, whereas the associated sensitivity and specificity were 79.23% and 94.81%, respectively. The estimated cost of each OLBC sample was at least 26% less than the cost of a single biopsy and more than 42% less in cases of multiple biopsied lesions. CONCLUSIONS: The proposed modifications of the Bethesda system can be adopted as a standardized system for oral cytological assessment. Our findings support OLBC as a reliable adjunct to surgical biopsy in the diagnosis of OPMDs. This tool has potential for oral cancer-finding and surveillance programs.

Analysis of laryngeal brush biopsy-based cytology results in patients of the 4th Military Teaching Hospital and Polyclinic in Wroclaw in years 2019-2020.

<b>Introduction:</b> Cytological examination of exfoliated epithelial cells of the uterine cervix, oral cavity, or rectum has been successfully used in the diagnostics of pathological conditions of these organs for many years. In these cases, the test material is collected from the available regions. </br></br> <b>Aim:</b> The aim of the study consisted in the analysis of cytological smears of laryngeal epithelial cells from patients hospitalized at the Department of Otolaryngology, Head and Neck Surgery of the 4th Military Teaching Hospital and Polyclinic in Wroclaw in years 2019-2020. The analysis was aimed at demonstrating whether representative laryngeal epithelial material could be obtained from brush biopsies. </br></br> <b>Material and methods:</b> The study was carried out in 92 subjects aged between 26 and 85 years, including 34 women (37.0%), from whom material for cytological examination had been collected from the larynx in the course of microsurgical procedures carried out using the Kleinsasser laryngeal instrument set in 2019-2020. </br></br> <b>Results: </b>Analysis was performed on 90 out of 92 cell smears (97.8%). Two smears were not qualified for analysis due to ille-gibility. The smears were assessed using a proprietary scale consisting in a modification of the Bethesda system. Abnormal results of cytological examinations were obtained in a majority of cases. HSILs with invasive features were the most common abnormal results of cytological examinations. </br></br> <b>Conclusions:</b> Laryngeal epithelial cells can be successfully evaluated by means of cytological examination. Abnormal presen-tation of cytological smear is frequently hypercellular, with inflammatory cells being observed less frequently. No statistically significant relationship was observed between the results of the cytological examination and the overall quality of the smear, number of cells, number of erythrocytes, or the severity of inflammation.

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.

Brush swab as a noninvasive surrogate for tissue biopsies in epigenomic profiling of oral cancer.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has poor survival rates. There is a pressing need to develop more precise risk assessment methods to tailor clinical treatment. Epigenome-wide association studies in OSCC have not produced a viable biomarker. These studies have relied on methylation array platforms, which are limited in their ability to profile the methylome. In this study, we use MethylCap-Seq (MC-Seq), a comprehensive methylation quantification technique, and brush swab samples, to develop a noninvasive, readily translatable approach to profile the methylome in OSCC patients. METHODS: Three OSCC patients underwent collection of cancer and contralateral normal tissue and brush swab biopsies, totaling 4 samples for each patient. Epigenome-wide DNA methylation quantification was performed using the SureSelectXT Methyl-Seq platform. DNA quality and methylation site resolution were compared between brush swab and tissue samples. Correlation and methylation value difference were determined for brush swabs vs. tissues for each respective patient and site (i.e., cancer or normal). Correlations were calculated between cancer and normal tissues and brush swab samples for each patient to determine the robustness of DNA methylation marks using brush swabs in clinical biomarker studies. RESULTS: There were no significant differences in DNA yield between tissue and brush swab samples. Mapping efficiency exceeded 90% across all samples, with no differences between tissue and brush swabs. The average number of CpG sites with at least 10x depth of coverage was 2,716,674 for brush swabs and 2,903,261 for tissues. Matched tissue and brush swabs had excellent correlation (r = 0.913 for cancer samples and r = 0.951 for normal samples). The methylation profile of the top 1000 CpGs was significantly different between cancer and normal samples (mean p-value = 0.00021) but not different between tissues and brush swabs (mean p-value = 0.11). CONCLUSIONS: Our results demonstrate that MC-Seq is an efficient platform for epigenome profiling in cancer biomarker studies, with broader methylome coverage than array-based platforms. Brush swab biopsy provides adequate DNA yield for MC-Seq, and taken together, our findings set the stage for development of a non-invasive methylome quantification technique for oral cancer with high translational potential.

Noninvasive Acquisition of Oral Mucosal Epithelial miRNA and Bacteria DNA/RNA from a Single Site.

Many digestive tract microbes live adhered to tract epithelium. Work in recent years has brought the realization that these microbes and the host epithelial cells certainly must interact and that this interaction has an effect on both. One way to understand the interaction is to measure which genes are expressed in the epithelial cells and what bacteria are present. Even more informative would be to also determine what genes the bacteria express. Presented is a method to noninvasively isolate oral mucosal epithelium so to provide purified miRNA that can be used to profile miRNA expression specifically in the epithelium. miRNA is a major regulator of cell functions. Simultaneously, DNA and RNA from bacteria at the same site can be isolated to allow characterization of bacteria that coat the epithelial cells and extracellular matrix. This provides insight on the interaction between host and bacteria.

Comparison between two cell collecting methods for liquid-based brush biopsies: a consecutive and retrospective study.

BACKGROUND: This study compares two different cell collectors, the Orcellex Brush (rigid brush) and the Cytobrush GT (nylon brush), using liquid-based cytology. A comparison of their obtainment procedures was also considered. The aim was to determine the diagnostic accuracy for detection of malignancy in oral brush biopsies. PICO-Statement: In this consecutive and retrospective study we had as population of interests, patients with oral lesions, the intervention was the brush biopsy with two different cell collectors and the control was healthy oral mucosa. The outcome of the study was to compare both cell collectors. METHODS: From 2009 to 2018, 2018 patients with oral lesions were studied using the nylon brush (666 cases) and rigid brush (1352 cases). In the first cohort five smears per patient were taken with the nylon brush, while each patient received one smear with the rigid brush in the second cohort. These were further processed in a liquid-based procedure. Cytological evaluations were categorised into 'negative', which were considered as negative, whereas 'doubtful', 'suspicious' and 'positive' cytological results were overall considered as positive for malignancy in comparison to the final histological diagnoses. Additionally, the clinical expenditure for each collector was estimated. RESULTS: 2018 clinically and histologically proven diagnoses were established, including 181 cases of squamous cell carcinomas, 524 lichen, 454 leukoplakias, 34 erythroplakias and 825 other benign lesions. The sensitivity and specificity of the nylon brush was 93.8% (95% CI 91.6-95.5%) and 94.2% (95% CI 91.8-95.5%) respectively, whereas it was 95.6% (95% CI 94.4-96.6%) and 84.9% (95% CI 83.8-87.5%) for the rigid brush. The temporal advantage using the plastic brushes was 4× higher in comparison to the nylon brush. The risk suffering from a malignant oral lesion when the result of the brushes was positive, suspicious, or doubtful was significantly high for both tests (nylon brush OR: 246.3; rigid brush OR: 121.5). CONCLUSIONS: Both systems have a similar sensitivity, although only the rigid brush achieved a satisfactory specificity. Additional methods, such as DNA image cytometry, should also be considered to improve the specificity. Furthermore, the rigid brush proved to be more effective at taking a sufficient number of cells, whilst also being quicker and presenting less stress for the patient.

Oral cancer diagnostics: An overview.

Cancer was first mentioned in medicine texts by Egyptians. Ancient Indians studied oral cancer in great detail under Susruta. Cancer has continued to be a challenge to physicians from ancient times to the present. Over the years, cancer underwent a shift in management from radical surgeries toward a more preventive approach. Early diagnosis is vital in reducing cancer-associated mortality especially with oral cancer. Even though the mainstay of oral cancer diagnosis still continues to be a trained clinician and histopathologic examination of malignant tissues. Translating innovation in technological advancements in diagnostic aids for oral cancer will require both improved decision-making and a commitment toward optimizing cost, skills, turnover time between capturing data and obtaining a useful result. The present review describes the conventional to most advanced diagnostic modalities used as oral cancer diagnostics. It also includes the new technologies available and the future trends in oral cancer diagnostics.

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.

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.

Current evidence on DNA aneuploidy cytology in noninvasive detection of oral cancer.

DNA-aneuploidy cytology as a promising noninvasive tool in diagnosing oral precancer and cancer has been proposed in 2015. In this letter, we identified 9 studies on DNA aneuploidy cytology with special emphasis on using fresh tissue sample in detection of oral precancer and cancer. Evidence was updated as follows, for detection of OSCC in general oral lesions, the pooled sensitivity and specificity was 84.8 and 99.0 respectively; for discrimination of dysplasia and OSCC form oral lesions, the sensitivity and specificity was 75.7 and 76.8 respectively. On the whole, current evidence on the theme is not robust, and multicenter prospective studies are needed to consolidate the evidence.