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Tumour heterogeneity in oral cancer is attributed to the presence of cancer stem cells (CSCs). CSCs are the most migratory and metastatic cellular subpopulation within tumours. Assessment of CSC markers as significant predictors of lymph node metastasis may prove valuable in the clinical setting. Furthermore, analysis of this panel of putative stem cell markers in oral dysplasia may additionally inform of the likelihood for oral potentially malignant disorders (OPMDs) to progress to oral squamous cell carcinoma (OSCC). The present study aims to assess the significance of CSC markers in the progression of OPMDs to OSCC and assessment of lymph node metastasis in OSCC. CD44 and ALDH1 were assessed immunohistochemically in 25 normal, 30 OPMDs, and 24 OSCCs. CD44 is a membranous marker and ALDH1 is a cytoplasmic marker. The immunohistochemical expression of these markers were compared between OPMDs with and without dysplasia, as well as between low-risk and high-risk dysplasias. Similarly, expression was compared between OSCC with and without lymph node metastasis and among grades of OSCC. Positive CD44 expression was seen in all normal mucosal tissues. The expression decreased from normal epithelium to OPMDs but increased in OSCC. CD44 expression was positive in 21 cases of OSCC (87.5%) and reduced from well-differentiated to poorly differentiated OSCC. CD44 staining index was higher in OSCC without lymph node metastasis (3.59) when compared with OSCC with lymph node metastasis (1.33). There was a statistically significant difference observed in the ALDH1 staining index among three groups (p

Background The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism essential to ensure the accuracy of chromosome segregation during mitosis. Our aim was to evaluate the expression of SAC proteins in oral carcinogenesis, and to assess their potential in predicting malignant transformation of oral leukoplakia. Material and Methods We analysed the immunoexpression of BubR1, Mad2, Bub3, and Spindly proteins in 64 oral biopsies from 52 oral leukoplakias and 12 normal tissues. Univariate and multivariate analysis were performed to evaluate predictive factors for malignant transformation (MT). Results We observed that BubR1 and Mad2 were more highly expressed in high dysplasia grade lesions than in low grade or normal tissues (P

Introduction. Identification of persons with a high oncological risk to squamous cell carcinoma of the head and neck region is an urgent problem for the early diagnosis of this disease. The activity of circulating proteasomes can be a criterion for predicting the risk of the larynx and oral cavity cancers in patients with precancerous diseases of the upper respiratory and gastrointestinal tracts. The aim of the study is to investigate the chymotrypsin-like and caspase-like activities of circulating serum proteasomes depending on the localization of precancerous and neoplastic diseases of the larynx and oral cavity. Material and Methods. The study population consisted of 35 patients with histologically verified HNSCC (T1–3N0–3M0), 15 patients with chronic hyperplastic laryngitis (CHL) and oral leukoplakia, and 10 healthy volunteers who did not have chronic upper respiratory tract diseases in the acute stage. The median age of the patients was 53 ± 5.3 years. Results. An increase in the studied proteasome activities was found in the blood serum of patients with malignant tumors as compared with patients with chronic hyperplastic diseases associated with precancerous changes, as well as in the larynx and oral cavity cancers groups as compared with healthy donors. At the same time, depending on the localization of the pathological process, it was shown that only the chymotrypsin-like activity of the circulating pool of proteasomes significantly differs both in the groups of oral cancer leukoplakia, and in the groups of laryngeal cancer chronic hyperplastic laryngitis with dysplastic epithelial lesions. In addition, differences were found between chymotrypsin-like and caspase-like activities of circulating serum proteasomes in patients with chronic hyperplastic laryngitis with oral dysplasia and leukoplakia. Conclusion. The results obtained indicate that the determination of the CTp activity of the circulating pool of proteasomes can be used as a criterion for predicting the risk of the larynx and oral cavity cancers in patients with precancerous diseases of the larynx and oral cavity.

Background To analyse the clinical and histological characteristics from a series of oral leukoplakias, leukoerythroplakias, erythroplakias and actinic cheilitis diagnosed in a 14-year period. Methods The files were reviewed and all cases diagnosed as leukoplakia, leukoerythroplakia, erythroplakia and actinic cheilitis were selected. Clinical information was obtained from the biopsy submission forms, and histological review was performed in all cases. Results Final sample included 953 lesions, mostly affecting females (534, 56%), and 87.5% of the patients were 41 to 80 years old. The most commonly affected regions were the lower lip (20.1%), tongue (18.1%) and buccal mucosa (16.9%). Leukoplakias, actinic cheilitis, leukoerythroplakias and erythroplakias represented, respectively, 74.6%, 15.2%, 9.3% and 0.8% of the sample. Most cases presented no dysplasia (42.1%) or mild dysplasia (33.5%). Lesions in the tongue, floor of mouth and lower lip, as well as lesions that presented hyperparakeratosis, showed higher frequencies of moderate dysplasia and severe dysplasia/carcinoma in situ. The most common histological criteria were the increase in number and size of nucleoli, loss of polarity of the basal cells and variations in cellular size and shape. Classification by the binary system showed that 7% were high-risk lesions. Conclusion All histological criteria for classification of oral epithelial dysplasia recommended by the World Health Organization showed increased frequency as grading increased. Additional criteria seem to be useful in grading oral epithelial dysplasia, such as the presence of normal and abnormal superficial mitotic figures and endophytic epithelial proliferation.

Background The psychosocial impact of receiving the diagnosis of oral epithelial dysplasia, which presents up to 3.5% increased annual risk of mouth cancer, remains unknown. Using validated instruments, the present study aimed to investigate the prevalence and existing correlations between anxiety, depression and dental anxiety symptoms and burden on oral health-related quality of life. Methods A clinical cohort of 82 patients with oral dysplasia was asked to complete the Hospital Anxiety and Depression Scale, the Modified Dental Anxiety Scale and the shortened version of the Oral Health Impact Profile. Spearman's correlation coefficient and regression analyses were performed. Results The participants' scores were in keeping with the presence of anxiety, depression and emotional distress symptoms in 30%, 16% and 26%, respectively. However, 69% experienced anxiety related to procedures that may be required as part of long-term management of oral dysplasia (e.g. local anaesthetic injection). The oral health-related quality of life scores showed 41.5% reporting a recent daily problem due to their oral or dental health. Significant correlations [p >0.05] were found among and between all of the used instruments. Being a female with oral dysplasia also predicted increased odds of indicating higher anxiety and dental anxiety scores than males [p >0.05]. Conclusion Oral dysplasia can adversely impact on the psychosocial well-being of affected persons. Establishing a causal relationship between the measured variables may, however, be challenging and would need further longitudinal studies.

Epithelial-mesenchymal transition (EMT) enhances the invasive ability, providing an explanation for the mechanism of tumor metastasis. The stiffness of the stromal extracellular matrix collagen can regulate the epithelial cell growth and migration in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Objectives: to study the epithelial expression of Vimentin as a marker for EMT and to elucidate the nature of collagen in OED and OSCC and finally to correlate EMT to collagen quality in these lesions. Material and methods: the area fraction of immunohistochemical epithelial expression of Vimentin and color polarization of picrosirius red stained collagen fibers were detected in ten specimens of each grade of OED and OSCC as well as normal oral mucosa followed by statistical analysis. Results: a statistically significant increase in the median area fractions of epithelial Vimentin expression and yellow-green fibers were detected as the grade of OED and OSCC progressed. Spearman’s correlation coefficient revealed a statistically significant direct correlation between epithelial Vimentin expression and yellow-green fibers in these lesions. Conclusion: there was a statistically significant increase in EMT and decrease in the collagen quality with the advance of OED and dedifferentiation of the neoplasm. The significant inverse correlation between EMT and collagen quality indicates the role of EMT in stromal collagen quality in these lesions and aid in their prognosis.

Background Oral cancer represents the sixth most common cancer in the world and is associated with 40-50% survival at 5 years. Within oral malignancies, oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, which, according to histopathological criteria, are referred to as oral dysplasia and their diagnosis are associated with higher rates of malignant transformation towards cancer. We recently reported that aberrant activation of the Wnt/β‑catenin pathway is due to overexpression of Wnt ligands in oral dysplasia. However, the expression of other regulators of this pathway, namely components of the β-catenin destruction complex has not been explored in oral dysplasia. Material and Methods Using immunohistochemical analyses, we evaluated nuclear expression of β‑catenin and its association with Wnt3a and Wnt5a. Likewise, components of the β-catenin destruction complex, including Adenomatous Polyposis Coli (APC), Axin and Glycogen Synthase Kinase 3 beta (GSK-3β) were also evaluated in oral dysplasia and OSCC biopsies. Results We found that moderate and severe dysplasia samples, which harbored increased expression of nuclear β‑catenin, depicted augmented cytoplasmic expression of GSK‑3β, Axin and APC, in comparison with OSCC samples. Also, GSK-3β was found nuclear in mild dysplasia and OSCC samples, when compared with other study samples. Conclusions Cytoplasmic levels of components of the β-catenin destruction complex are increased in oral dysplasia and might be responsible of augmented nuclear β‑catenin. Key words:Oral cancer, oral dysplasia, β-Catenin, Wnt ligands, destruction complex.

Oral leukoplakia (OL) is the most common oral potentially malignant disorder, with a global prevalence of 2%-3%, variable malignant transformation rate and incompletely understood aetiology. Considering the subjectivity in oral dysplasia grading, other evaluation methods have been tested as predictors of malignant transformation. DNA ploidy status and loss of heterozygosity signatures have been shown to be good predictive markers of malignant transformation. However, effective markers to predict which lesions will progress to invasive carcinoma and by which mechanisms remain unclear. Recent evidence suggests that dysplasia progression to carcinoma occurs through neutral clonal evolution (i.e. randomly). We focus on the genetic basis of OL, encompassing the gross chromosomal alterations and single-gene mutations, and discuss such alterations in the context of aetiology, clinical presentation and progression. The deeper we understand the genetic basis of OL, the more we approach a better comprehension of the complex and poorly understood process of oral carcinogenesis.

Background Stathmin is an intracellular phosphoprotein that controls the microtubule dynamics by further regulating proper attachment and alignment of chromosomes in a dividing cell. Thus, any mutation or aberrantly expressed protein that reduces the fidelity of spindle assembly will enhance chromosomal instability contributing to aneuploidy. Oral Squamous Cell Carcinoma is an extensively studied malignancy that occurs due to accumulated genetic changes due to carcinogens. The current study is done to evaluate the stathmin role and its expression in OSCC and Oral epithelial dysplasia (OED). Objective The aim of the present study is to evaluate the role of stathmin in OSCC and Oral dysplasia and also to correlate the expression of Stathmin with respect to the different histopathological grades of OED and OSCC. Materials and methods 30 neutral buffered formalin fixed, paraffin embedded (FFPE) tissues of Oral Leukoplakia/OED and 30 FFPE tissues of OSCC were subjected to immunohistochemistry with stathmin antibody. Five fields of each case with 300 cells were examined and a mean percentage of positive-stained slides were determined. The percentages were recorded accordingly with their respective histological grades. The results were analysed statistically. Results The results of the present study demonstrated higher mean values of stathmin in tissues with OSCC (2.50) compared to leukoplakia (2.11) and normal tissues (0.00) with a high level of statistical significance (0.0001). There is also an increase in the percentage levels of stathmin with increase in the histological grade of differentiation in OSCC as well as leukoplakia. Conclusion The present study found a statistical correlation between increased grades of the disease with expression levels of stathmin. This confirms that stathmin expression can contribute to disease progression and that stathmin might have a potential role as an early diagnostic biomarker and can be a therapeutic target for OSCC. .

Objectives: Previous studies have demonstrated that microRNA-21 (miR-21) and total antioxidant capacity (TAC) could be potential diagnostic biomarkers for oral squamous cell carcinoma. Their diagnostic potential in the early stages of carcinogenesis is not clear yet. This study was conducted to determine the salivary levels of miR-21 and TAC in patients with oral hyperplasia and dysplasia. Methods: We assessed expression of miR-21 and TAC in whole unstimulated saliva samples of 30 patients with oral mucosal lesions demonstrating hyperplastic or dysplastic changes and 30 healthy individuals with normal mucosa. Biopsy was taken from the lesions for histopathologic assessment. Statistical analysis was performed with IBM® SPSS® (P ≤ 0.05) and ROC curve analysis was conducted with MedCalc. Results: miR-21 expression varied among the studied groups with significant difference. However, TAC expression varied only between mucosal lesions and normal mucosa with significant difference. Diagnostic accuracy, sensitivity, specificity, positive predictive value and negative predictive value were higher in miR-21 than TAC. Conclusions: Salivary miR-21 are more accurate in detecting oral dysplasia than salivary TAC. Salivary miR-21 could be potential diagnostic biomarker for screening and early detection of oral cancer. More studies are required to validate miR-21.

Introdução: A queilite actínica (QA) é uma desordem potencialmente maligna associada à exposição crônica à luz solar como principal fator etiológico. A QA tem como principal sítio de acometimento o vermelhão do lábio inferior, apresentando características clínicas marcantes. Porém, a análise histopatológica desta lesão merece um enfoque especial pelas características bastante expressivas para o potencial de malignidade. Assim, para essa análise, são elencados dois sistemas principais: o sistema da Organização Mundial da Saúde (OMS), e o binário. Objetivo: Realizar uma revisão da literatura para demonstrar os pontos avaliados e o valor preditivo dos sistemas de gradação histológica em QA. Metodologia: Trata-se de um estudo de revisão bibliográfica, utilizando artigos da base de dados da SCIELO e PUBMED, encontrados com o uso dos descritores: “actinic cheilitis”, “WHO system”, “binary system”, “oral potentially malignant disorders”, “histological features in actinic cheilitis”, fazendo uso do operador booleano “AND”. Conclusão: Diante da revisão realizada, percebeu-se a importância da adoção dos sistemas de gradação histológica da OMS, e o binário para uma análise minuciosa de lesões de QA, que apresentam potencial de malignidade. Ademais, constatou-se o favorecimento do sistema binário na redução da subjetividade entre os patologistas quanto à gradação das avaliações histopatológicas. Descritores: Queilite; Patologia Bucal; Neoplasias Labiais; Classificação; Diagnóstico. Referências Lopes MLDS, Silva Junior FLS, Lima KC, Oliveira PT, Silveira EJD. Clinicopathological profile and management of 161 cases of actinic cheilitis. An Bras Dermatol. 2015;90(4):347-50. Arnaud RR, Soares MSM, Paiva MAF, Figueiredo CRLV, Santos MGC, Lira CC. Queilite actínica: avaliação histopatológica de 44 casos. Rev Odontol UNESP. 2014;43(6):384-89. Pilati S, Bianco BC, Vieira D, Modolo F. Histopathologic features in actinic cheilitis by the comparison of grading dysplasia systems. Oral Dis. 2017 Mar;23(2):219-224. Pilati S, Bianco BC, Vieira D, Modolo F. Histopathologic features in actinic cheilitis by the comparison of grading dysplasia systems. Oral Dis. 2017;23(2):219-24. Maia HCM, Pinto ANS, Pereira JS, Medeiros AMC, Silveira EJD, Miguel MCC. Lesões orais potencialmente malignas: correlações clínico-patológicas. Einstein. 2016;14(1): 35-40. Savage NW, McKay C, Faulkner C. Actinic cheilitis in dental practice. Aust Dent J. 2010; 55(Suppl 1):78-84. Vieira RA, Minicucci EM, Marques ME, Marques SA. Actinic cheilitis and squamous cell carcinoma of the lip: clinical, histopathological and immunogenetic aspects. An Bras Dermatol. 2012; 87(1):105-14. Dancyger A, Heard V, Huang B, Suley C, Tang D, Ariyawardana A. Malignant transformation of actinic cheilitis: A systematic review of observational studies. J Investig Clin Dent. 2018; 9(4):e12343. de Santana Sarmento DJ, da Costa Miguel MC, Queiroz LM, Godoy GP, da Silveira EJ. Actinic cheilitis: clinicopathologic profile and association with degree of dysplasia. Int J Dermatol. 2014; 53(4):466-72. Wood NH, Khammissa R, Meyerov R, Lemmer J, Feller L. Actinic cheilitis: a case report and a review of the literature. Eur J Dent. 2011; 5(1):101-6. Mello FW, Melo G, Modolo F, Rivero ER. Actinic cheilitis and lip squamous cell carcinoma: Literature review and new data from Brazil. J Clin Exp Dent. 2019;11(1):e62-9. Warnakulasuriya S, Johnson NW, van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med. 2007;36(10):575-80. Paiva MAF, Soares MSM, Figueiredo CRLV, Luna AH, Oliveira VEN, Brasil Júnior O. Associação entre displasia e inflamação em queilite actínica. J Bras Patol Med Lab. 2012; 48(6):455-58. Warnakulasuriya S, Reibel J, Bouquot J, Dabelsteen E. Oral epithelial dysplasia classification systems: predictive value, utility, weaknesses and scope for improvement. J Oral Pathol Med. 2008;37(3):127-33. Câmara PR, Dutra SN, Takahama Júnior A, Fontes K, Azevedo RS. A comparative study using WHO and binary oral epithelial dysplasia grading systems in actinic cheilitis. Oral Dis. 2016; 22(6):523-9. Kujan O, Oliver RJ, Khattab A, Roberts SA, Thakker N, Sloan P. Evaluation of a new binary system of grading oral epithelial dysplasia for prediction of malignant transformation. Oral Oncol. 2006;42(10):987-93. Nagata G, Santana T, Queiroz A, Caramez RH, Trierveiler M. Evaluation of epithelial dysplasia adjacent to lip squamous cell carcinoma indicates that the degree of dysplasia is not associated with the occurrence of invasive carcinoma in this site. J Cutan Pathol. 2018. doi: 10.1111/cup.13270. Mello FW, Miguel AFP, Dutra KL, Porporatti AL, Warnakulasuriya S, Guerra ENS, Rivero ERC. Prevalence of oral potentially malignant disorders: A systematic review and meta-analysis. J Oral Pathol Med. 2018;47(7):633-40. Izumo T. Oral premalignant lesions: from the pathological viewpoint. Int J Clin Oncol. 2011;16(1):15-26. Kujan O, Khattab A, Oliver RJ, Roberts SA, Thakker N, Sloan P. Why oral histopathology suffers inter-observer variability on grading oral epithelial dysplasia: an attempt to understand the sources of variation. Oral Oncol. 2007; 43(3):224-31. El-Naggar AK, Chan JKC, Grandis JR, Takata T, Slootweg PJ. World Health Organization Classification of Head and Neck Tumours. WHO/IARC Classification of Tumours 2017; 4th ed. Lyon, France: IARC Press. R SA, B N P, Hegde U, K U, G S, G K, Sil S. Inter- and Intra-Observer Variability in Diagnosis of Oral Dysplasia. Asian Pac J Cancer Prev. 2017;18(12):3251-54. Olinici D, Cotrutz CE, Mihali CV, Grecu VB, Botez EA, Stoica L, Onofrei P, Condurache O, Dimitriu DC. The ultrastructural features of the premalignant oral lesions. Rom J Morphol Embryol. 2018;59(1):243-48.

The progression of tumor depends on the level of Bcl-2 which protects the cells from undergoing apoptosis, and its level should be more than the level of the death inducer. So, our Aim: To investigate chemopreventive effect of olive oil in epithelial alteration which induced in hamster buccal pouch. Methods: Epithelial dysplasia will occur by the application of DMBA on alternative days for 8 weeks on hamster buccal pouch. Hematoxylin and Eosin (H&E) and Streptavidin-biotin immunoperoxidase staining techniques were used to detect the expression of Bcl-2 in three groups (G1 negative control or normal group, G2 DMBA group and G3 olive oil chemoprevention group). Evaluation of the immunostaining was done by using computer image analyzer system. Results: H & E stained histological section in G1 with normal mucosa of HBP intact, and continuous epithelium of thin keratinized stratified squamous epithelium while in G2 there were incidence of severe epithelial dysplasia with hyperkeratosis & G3 reduced dysplasia incidence and severity as compared to G2. Bcl-2 immunohistochemical expression in G1 in basal & suprabasal layers while in G2 & G3 located in all epithelial layers with statistically highly significant difference (P G1, G3) and (G3>G1). Conclusion: Olive oil plays an important role in reduction and prevention of transformation of normal oral epithelium into epithelial dysplasia. The liquid form of olive oil may be considered as a new line of chemoprevention in oral dysplasia and the Bcl-2 expression evidenced this reduction of abnormality in epithelial dysplasia.

Potentially malignant lesions, commonly referred to as dysplasia, are associated with malignant transformation by mechanisms that remain unclear. We recently reported that increased Wnt secretion promotes the nuclear accumulation of β-catenin and expression of target genes in oral dysplasia. However, the mechanisms accounting for nuclear re-localization of β-catenin in oral dysplasia remain unclear. In this study, we show that endosomal sequestration of the β-catenin destruction complex allows nuclear accumulation of β-catenin in oral dysplasia, and that these events depended on the endocytic protein Rab5. Tissue immunofluorescence analysis showed aberrant accumulation of enlarged early endosomes in oral dysplasia biopsies, when compared with healthy oral mucosa. These observations were confirmed in cell culture models, by comparing dysplastic oral keratinocytes (DOK) and non-dysplastic oral keratinocytes (OKF6). Intriguingly, DOK depicted higher levels of active Rab5, a critical regulator of early endosomes, when compared with OKF6. Increased Rab5 activity in DOK was necessary for nuclear localization of β-catenin and Tcf/Lef-dependent transcription, as shown by expression of dominant negative and constitutively active mutants of Rab5, along with immunofluorescence, subcellular fractionation, transcription, and protease protection assays. Mechanistically, elevated Rab5 activity in DOK accounted for endosomal sequestration of components of the destruction complex, including GSK3β, Axin, and adenomatous polyposis coli (APC), as observed in Rab5 dominant negative experiments. In agreement with these in vitro observations, tissue immunofluorescence analysis showed increased co-localization of GSK3β, APC, and Axin, with early endosome antigen 1- and Rab5-positive early endosomes in clinical samples of oral dysplasia. Collectively, these data indicate that increased Rab5 activity and endosomal sequestration of the β-catenin destruction complex leads to stabilization and nuclear accumulation of β-catenin in oral dysplasia.

Oral leukoplakia is the most common oral potentially malignant disorder with a malignant transformation rate into oral squamous cell carcinoma of 1–3% annually. The presence and grade of World Health Organization defined dysplasia is an important histological marker to assess the risk for malignant transformation, but is not sufficiently accurate to personalize treatment and surveillance. Differentiated dysplasia, known from differentiated vulvar intraepithelial neoplasia, is hitherto not used in oral dysplasia grading. We hypothesized that assessing differentiated dysplasia besides World Health Organization defined (classic) dysplasia will improve risk assessment of malignant transformation of oral leukoplakia. We investigated a retrospective cohort consisting of 84 oral leukoplakia patients. Biopsies were assessed for dysplasia presence and grade, and the expression of keratins 13 (CK13) and 17, known to be dysregulated in dysplastic vulvar mucosa. In dysplastic oral lesions, differentiated dysplasia is as common as classic dysplasia. In 25 out of 84 (30%) patients, squamous cell carcinoma of the upper aerodigestive tract developed during follow-up. Considering only classic dysplasia, 11 out of 56 (20%) patients with nondysplastic lesions progressed. With the incorporation of differentiated dysplasia, only 2 out of 30 (7%) patients with nondysplastic lesions progressed. The risk of progression increased from 3.26 (Hazard ratio, p = 0.002) when only classic dysplasia is considered to 7.43 (Hazard ratio, p = 0.001) when classic and differentiated dysplasia are combined. Loss of CK13, combined with presence of dysplasia, is associated with greater risk of malignant progression (p = 0.006). This study demonstrates that differentiated dysplasia should be recognized as a separate type of dysplasia in the oral mucosa and that its distinction from classic dysplasia is of pathological and clinical significance since it is a strong (co)prognostic histopathological marker for oral malignant transformation. In oral lesions without dysplasia and retained CK13 staining the risk for progression is very low.

Objectives: Oral potentially malignant disorders (OPMDs) are lesions that may undergo malignant transformation to oral cancer. The early diagnosis and surveillance of OPMDs reduce the morbidity and mortality of patients. Diagnostic techniques based on medical images analysis have been developed to diagnose clinical conditions. This systematic review and meta-analysis aimed to evaluate the efficacy of imaging-based techniques compared to the gold standard of histopathology to assess their ability to correctly identify the presence of OPMDs. Design: Literature searches of free text and MeSH terms were performed using MedLine (PubMed), Scopus, Google Scholar, and the Cochrane Library (from 2000 to 30 June 2020). The keywords used in the search strategy were: (“oral screening devices” or “autofluorescence” or “chemiluminescence” or “optical imaging” or “imaging technique”) and (“oral dysplasia” or “oral malignant lesions” or “oral precancerosis”). Results: The search strategy identified 1282 potential articles. After analyzing the results and applying the eligibility criteria, the remaining 43 papers were included in the qualitative synthesis, and 34 of these were included in the meta-analysis. Conclusions: None of the analyzed techniques based on assessing oral images can replace the biopsy. Further studies are needed to explore the role of techniques-based imaging analysis to identify an early noninvasive screening method.

Introduction Clinical management of oral potentially malignant disorders relies on accurate histopathological assessment of the presence and grade of oral epithelial dysplasia. Whilst adjunctive laboratory tests have provided useful prognostic information, none are in widespread clinical use. This study was performed to assess the clinical utility of two contemporary oral epithelial dysplasia grading systems. Methods Patients were identified from a clinical database. Oral epithelial dysplasia grading was performed by three oral and maxillofacial pathologists blinded to clinical outcome using the WHO 2017 system and a binary classification. The primary outcome measure was the development of oral squamous cell carcinoma, termed 'malignant transformation'. Results 131 cases satisfied the inclusion criteria, of which 23 underwent malignant transformation. There was substantial inter-rater agreement between the study pathologists for both grading systems, measured using kappa statistics (κ = 0.753-0.784). However, there was only moderate agreement between the consensus WHO 2017 dysplasia grade for the study against the original grade assigned by a pool of six pathologists in the context of the clinical service (κ = 0.491). Higher grade categories correlated with an increased risk of developing cancer using both grading systems. Conclusion This study demonstrates that the WHO 2017 and binary grading systems are reproducible between calibrated pathologists and that consensus reporting is likely to improve the consistency of grading. The WHO and binary systems were prognostically comparable. We recommend that institutions implement consensus oral epithelial dysplasia grading and prospectively audit the effectiveness of risk stratifying their patients with oral potentially malignant disorders. (249 words).

BACKGROUND Oral epithelial dysplasia (OED) is the presence of cells of an abnormal type within a tissue, which may signify a stage preceding the development of cancer. Our aim was to determine the interrelation between the expression of multiple molecular markers and the histological features of oral dysplasia. METHODS Fifteen samples of OED (five for each severity degree) were analyzed through software assisted image cytometry nuclear morphology. p53 (wildtype and mutated form), Bax and Bcl2 expression was immunohistochemically determined, and the gene expression of MMP1, MMP2,MMP9 and hTERT was determined by RT-PCR. The mean, standard deviation, ANOVA and Fisher´s exact test (p

Simple Summary Complete resection of dysplastic and malignant tissue improves overall survival and delays cancer recurrence in oral cancer patients; however, intraoperative surgical margin assessment is limited to visual inspection and palpation, making it difficult to achieve total resection. There is currently no tool capable of providing real-time, accurate, and continuous margin-assessment guidance during oral cancer resection surgery. Multispectral autofluorescence lifetime imaging (maFLIM) is a label-free imaging modality that enables quantifying a plurality of metabolic and compositional autofluorescence biomarkers of oral dysplasia and cancer. We have developed and validated a machine-learning assisted computer aided detection (CAD) system for automated discrimination of dysplastic and cancerous from healthy oral tissue based on in vivo widefield maFLIM endoscopy data. This CAD system can be potentially embedded into maFLIM endoscopes to enable continuous in situ detection of positive margins during oral cancer resection surgery, thus facilitating maximal tumor resection and improving surgical outcomes for oral cancer patients. Abstract Multispectral autofluorescence lifetime imaging (maFLIM) can be used to clinically image a plurality of metabolic and biochemical autofluorescence biomarkers of oral epithelial dysplasia and cancer. This study tested the hypothesis that maFLIM-derived autofluorescence biomarkers can be used in machine-learning (ML) models to discriminate dysplastic and cancerous from healthy oral tissue. Clinical widefield maFLIM endoscopy imaging of cancerous and dysplastic oral lesions was performed at two clinical centers. Endoscopic maFLIM images from 34 patients acquired at one of the clinical centers were used to optimize ML models for automated discrimination of dysplastic and cancerous from healthy oral tissue. A computer-aided detection system was developed and applied to a set of endoscopic maFLIM images from 23 patients acquired at the other clinical center, and its performance was quantified in terms of the area under the receiver operating characteristic curve (ROC-AUC). Discrimination of dysplastic and cancerous from healthy oral tissue was achieved with an ROC-AUC of 0.81. This study demonstrates the capabilities of widefield maFLIM endoscopy to clinically image autofluorescence biomarkers that can be used in ML models to discriminate dysplastic and cancerous from healthy oral tissue. Widefield maFLIM endoscopy thus holds potential for automated in situ detection of oral dysplasia and cancer.

Background The prognosis of human cancer depends on the deregulations of many molecular patterns. In recent years, a great interest in the intracellular signaling mechanisms related to nitric oxide (NO)-induced carcinogenesis has appeared, as one of the most preeminent prognostic markers for many types of neoplasms. In this study, we identify the levels of iNOS and nitrotyrosine in the sample of normal oral mucosa (NOM), oral leukoplakia (OL), and oral squamous cell carcinoma (OSCC). Methods Quantitative polymerase chain reactions (qPCRs) were utilized to detect the NOS2 levels in fresh-frozen tissue samples of NOM (n = 6), OL (n = 20), and OSCC (n = 15). Moreover, the immunohistochemical method was used to examine the levels of iNOS and nitrotyrosine in 85 cases of OSCC (39 cases without metastases and 46 with metastases), 42 cases of OL, and 16 cases of NOM. Results There are rising tendencies in the iNOS mRNA and protein levels during human oral carcinogenesis. Similar findings were obtained in the nitrotyrosine staining. Furthermore, iNOS and nitrotyrosine immunostaining are associated with several clinical-pathological features of OSCC (site, presence of metastasis, staging, recidivism, and survival). Conclusions The NO-signaling pathway plays a vital role in the development and progression of human oral dysplastic and neoplastic diseases. Nitrotyrosine was a significant marker for the discrimination of OSCC prognosis and survival.

Background: Light-based detection agents using autofluorescence may be helpful in the detection of early dysplasia, which would otherwise be misdiagnosed as nondysplastic by conventional oral examination (COE) with white light. Visually-enhanced lesion scope (VELscope) is one of such an aid used for the purpose. A meta-analysis was carried out on the sensitivity and specificity of VELscope that would enable in providing evidence of its usage in oral dysplasia. Materials and Methods: MeSH terms such as auto florescence in oral dysplasia, VELscope, Oral ID, Identifi, in a different medical database such as PubMed, Cochrane, EBSCO, and Google scholar was carried out by four research associates. The total articles available were 242, of which, 230 were excluded based on strict criteria of randomized control trials and proper design. Finally, only 12 studies were chosen for the present analysis. Of 1643 patients from 12 studies, 1264 patients had undergone the autofluorescence examination after the COE. Only 774 patients have shown the loss of fluorescence with VELScope examination and 487 had retained the fluorescence. Biopsy was performed on 1176 patients after the autofluorescence examination in the areas where there was the loss of fluorescence. The available data were subjected to software Review Manager for analysis. Results and Discussion: Of the 11 studies analyzed, majority of them showed that the autofluorescence device were sensitive enough > 0.70; however, the values of sensitivity and specificities varied significantly. With the VELscope the diagnostic performance of the inexpert examiner was not improved, obtaining a sensitivity of 0.40 (95% of confidence interval [CI]: 0.406–0.773) and a specificity of 0.80 (95% CI: 0.614–0.923). Conclusion: The new technique may help as an adjunct to histopathology in detecting the dysplasia initially and stop further progression to the carcinoma.

It is believed that the majority of oral cancers develop from oral potentially malignant lesions (OPML). Though they can be easily detected during screening, risk stratification is difficult. During screening clinicians often find it difficult to distinguish OPMLs from benign lesions, and predicting OPML at risk of malignant transformation is particularly challenging. DNA aneuploidy has been known to be a marker of malignancy in a number of sites including the oral cavity. We performed a systematic review to evaluate the effectiveness of DNA-ICM using brushings in differentiating OPMLs from benign/inflammatory lesions during screening and in predicting malignant transformation. MEDLINE, Pubmed, EMBASE electronic databases were systematically searched using a combination of keywords and subject headings. A total of 11 articles satisfied our inclusion criteria. These studies reported a wide range of sensitivity (16-96.4%) and specificity (90-100%) due to the differences in study design, definitions of high risk or low risk lesions and DNA-ICM protocol used. No long-term longitudinal studies were identified to assess the role of DNA-ICM using brushings in predicting malignant transformation. No studies evaluated the role of DNA-ICM in community screening settings. A number of studies combined DNA-ICM with other techniques like cytology or argyrophilic nucleolar organizer region counts leading to improved test results. In spite of DNA aneuploidy being accepted as a marker of malignancy, there is limited evidence of DNA-ICM using brushings being successful as an adjunct oral cancer screening tool. Longitudinal studies and large community screening studies need to be undertaken to draw stronger conclusion.

Objectives To map the genomic pathways of patients with oral leukoplakia (OLK) which transformed to cancer (progressive) and those which did not (non-progressive), and to compare their exomic profiles. Materials and methods Whole exome sequencing was performed on 42 sequential samples from five progressive and eight non-progressive patients. Association of genomic variant frequencies with progression or lesion severity were analysed by non-parametric tests (Kruskal-Wallis and Mann-Whitney-Wilcoxon) and multivariate sparse partial least squares discriminant analysis (sPLS-DA). Enrichment analysis was used to characterise the effect of mutations upon biological pathways. Confirmatory studies used qPCR and immunohistochemistry. Results Using sPLS-DA, the variant frequency of a small number of genes could be used to classify the samples based on lesion severity or progressive status. Enrichment analysis showed that DNA damage repair gene related pathways were highly impacted in lesions which progressed to cancer. Multivariate analysis of a set of 148 DNA damage repair genes could be used to classify progressive lesions using mutation frequency. BRCA1, BRCA 2 and other double strand break (DSB) repair Fanconi anaemia (FA)/BRCA pathway genes were prominent contributors to this classification. Conclusion Patients with progressive and non-progressive OLK can be differentiated using the frequency of exomic variants, particularly in DNA damage repair pathway genes. To our knowledge, this is the first report of FA/BRCA (DSB) pathway involvement in malignant transformation of OLK to oral squamous cell carcinoma (OSCC).

Abnormal epithelial stratification is a sign of oral dysplasia and hence evaluation of surface characteristics of oral epithelial region can help in detection of cancerous progression. Surface characteristics can be better visualised by Scanning Electron Microscopy (SEM) in comparison to light microscopy. In our study we have developed automated image processing algorithms i.e. Gaussian with median filtering and Gradient filtering, using MATLAB 2016b, to segment the surface characteristics i.e. the ridges and pits in the SEM images of oral tissue of normal (13 samples) and Oral Submucous Fibrosis (OSF) (36 samples) subjects. After segmentation, quantitative measurement of the parameters like area, thickness and textural features like entropy, contrast and range filter of ridges as well as area of pit and the ratio of area of ridge vs. area of pit was done. Statistical significant differences were obtained in between normal and OSF study groups for thickness (p=0.0107), entropy (p

Objective The molecular mechanisms underlying the development of dysplasia in leukoplakia are unknown. We used RNA sequencing to examine the molecular and biological pathway differences in oral leukoplakia with and without oral epithelial dysplasia. Materials and methods Excisional biopsy specimens (25) were taken from 24 patients with oral leukoplakia diagnosed histopathologically as either oral epithelial dysplasia (13) or epithelial hyperplasia and keratosis without dysplasia (12). Transcriptome analysis used RNA sequencing, differential expression and hierarchical clustering. Biological signalling was examined by gene ontology, pathway and protein-protein interaction analysis. Results Differential expression analysis showed distinction between the two groups identifying 47 genes as altered in leukoplakia with dysplasia, including SAA1, SAA2, KRT31, KRT37, KRT76, ROBO2, DNAJB5 and DNAJA4. Using hierarchical clustering, dysplastic leukoplakia readily segregated from leukoplakia without dysplasia. Pathway and ontology enrichment analysis provided evidence that downregulation of extracellular matrix (ECM) pathways was a feature of dysplastic lesions. Conclusion Our results suggest that there are detectable changes in the molecular profile of oral leukoplakia exhibiting dysplasia including downregulated ECM as a distinguishing feature of dysplastic lesions. This suggests that reactive changes in stroma may be an early manifestation of dysplastic development. Our study also demonstrates the feasibility of detecting such molecular changes in oral leukoplakia, providing avenues for further investigation of molecular mechanisms of oral dysplasia.

Review: The backbone of clonal evolution model claims that every individual malignant cell has the potential to initiate a new neoplasm. Yet, the cancer stem cell theory confirms that only a few number of stem-like cancer cells can initiate a new neoplasm with all clones of the original neoplasm. CD133 is the most commonly used marker to identify the CSCs from different malignancies. Among the important mediators of the inflammatory pathways is COX-2, which has been found to be elevated in several human cancers. The strong association of different types of cancer with some chronic inflammatory diseases confirms the influential role of inflammation in carcinogenesis.Aim of study: The present study aimed to find the possible correlation between the stemness (CD133+) and the inflammatory modulator (COX-2) in different grades of oral epithelial dysplasias which might help in predicting the oral cancer development.Material and Methods: Immunohistochemical expression of CD133 and COX-2 was evaluated in 5 histologically normal samples of normal oral mucosa and 30 samples of oral dysplasia.Results: For both CD133 and COX-2, there were a statistically significant difference between the different grades of oral dysplasias (P-value

Over the past few decades, the artificial intelligence is being employed in diverse fields like pattern classification, image processing, object identification, recommender systems, speech recognition, etc. Machine learning has made it possible to develop intelligent systems through training that equip machines to handle different tasks, exactly on the analogy similar to humans. In medical field, machine learning algorithms are being used for prediction, early detection and prognosis of various diseases. These algorithms suffer a certain threshold due to their inability to handle large amount of data. Deep learning based techniques are emerging as efficient tools and can easily overcome the above difficulties in processing data related to medical imaging that includes mammographs, CT scans, MRIs and histopathology slide images. Deep learning has already achieved greater accuracy in early detection, diagnosis and prognosis of various diseases especially in cancer. Dysplasia is considered to be a pathway that leads to cancer. So, in order to diagnose oral cancer at its early stage, it is highly recommended to firstly detect dysplastic cells in the oral epithelial squamous layer. In our research work, we have proposed a deep learning based framework (convolutional neural network) to classify images of dysplastic cells from oral squamous epithelium layer. The proposed framework has classified the images of dysplastic cells into four different classes, namely normal cells, mild dysplastic cells, moderate dysplastic cells and severe dysplastic cells. The dataset undertaken for analysis consists of 2557 images of epithelial squamous cells of the oral cavity taken from 52 patients. Results show that on training the proposed framework gave an accuracy of 94.6% whereas, in testing it gave an accuracy of 90.22%. The results produced by our framework has also been tested and validated by comparing the manual results recorded by the medical experts working in this area.

Objectives To identify human papillomavirus (HPV), Epstein–Barr virus (EBV), and Candida albicans in oral leukoplakia with different degrees of dysplasia. Materials and methods An observational, cross‐sectional, descriptive study was performed using 30 formalin‐fixed and paraffin‐embedded tissues from patients with clinical suspicion of leukoplakia and confirmed diagnosis of oral dysplasia. Histological analyses were performed by two pathologists (interobserver) and dysplasias were classified as mild, moderate, or severe. Conventional PCR was used to detect HPV and EBV viruses and C. albicans. To determine the association between each microorganism with different degrees of dysplasia a Chi‐square test was employed. Results The tongue was the most common site for leukoplakias (71.4%) in females with a mean age of 50 years (ranging between 30 to 50 years old; 57.1%). EBV was the most frequently detected (73.3%), followed by HPV (43.3%), mainly of type 16 (40%), and C. albicans (23.3%). Significant differences were observed between degrees of dysplasia and HPV presence (p = 0.005). In lesions positive for HPV, EBV, and C. albicans the most frequent histological changes were hyperkeratosis, irregular interpapillary ridges, and loss of basal stratum cell polarity. Conclusion Co‐infection with human papillomavirus, Epstein Barr virus, and Candida albicans in oral leukoplakia could be associated with dysplastic changes.

Most oral cancers arise from oral potentially malignant lesions, which show varying grades of dysplasia. Risk of progression increases with increasing grade of dysplasia; however, risk prediction among oral low-grade dysplasia (LGD), that is, mild and moderate dysplasia can be challenging as only 5%–15% transform. Moreover, grading of dysplasia is subjective and varies with the area of the lesion being biopsied. To date, no biomarkers or tools are used clinically to triage oral LGDs. This study uses a combination of DNA ploidy and chromatin organization (CO) scores from cells obtained from lesion brushings to identify oral LGDs at high-risk of progression. A total of 130 lesion brushings from patients with oral LGDs were selected of which 16 (12.3%) lesions progressed to severe dysplasia or cancer. DNA ploidy and CO scores were analyzed from nuclear features measured by our in-house DNA image cytometry (DNA-ICM) system and used to classify brushings into low-risk and high-risk. A total of 57 samples were classified as high-risk of which 13 were progressors. High-risk DNA brushing was significant for progression (P = 0.001) and grade of dysplasia (P = 0.004). Multivariate analysis showed high-risk DNA brushing showed 5.1- to 8-fold increased risk of progression, a stronger predictor than dysplasia grading and lesion clinical features. DNA-ICM can serve as a non-invasive, high-throughput tool to identify high-risk lesions several years before transformation. This will help clinicians focus on such lesions whereas low-risk lesions may be spared from unnecessary biopsies. Prevention Relevance: DNA ploidy and chromatin organization of cells collected from oral potentially malignant lesions (OPMLs) can identify lesions at high-risk of progression several years prior. This non-invasive test would enable clinicians to triage high-risk (OPMLs) for closer follow-up while low-risk lesions can undergo less frequent biopsies reducing burden on healthcare resources.

It is presumed that the vast majority of cancers develop from a pre-cancerous or dysplastic state. Thus, understanding dysplasia at a clinical and molecular level is critical to our ability to decrease the overall incidence of squamous cell carcinoma of the oral cavity. For multiple reasons, our current appreciation of dysplasia is limited; this chapter describes clinical detection and treatment with a special emphasis on future directions.

Oral cancer is one of the most common cancers worldwide. Despite easy access to the oral cavity and significant advances in treatment, the morbidity and mortality rates for oral cancer patients are still very high, mainly due to late-stage diagnosis when treatment is less successful. Oral cancer has also been found to be the most expensive cancer to treat in the United States. Early diagnosis of oral cancer can significantly improve patient survival rate and reduce medical costs. There is an urgent unmet need for an accurate and sensitive molecular-based diagnostic tool for early oral cancer detection. Fourier transform infrared spectroscopy has gained increasing attention in cancer research due to its ability to elucidate qualitative and quantitative information of biochemical content and molecular-level structural changes in complex biological systems. The diagnosis of a disease is based on biochemical changes underlying the disease pathology rather than morphological changes of the tissue. It is a versatile method that can work with tissues, cells, or body fluids. In this review article, we aim to summarize the studies of infrared spectroscopy in oral cancer research and detection. It provides early evidence to support the potential application of infrared spectroscopy as a diagnostic tool for oral potentially malignant and malignant lesions. The challenges and opportunities in clinical translation are also discussed.

Background Dysplasia is defined as the presence of cells of an abnormal type within any tissue, which may signify a stage preceding the development of cancer. It can be categorized as mild/moderate/severe and carcinoma in situ. Objective To determine the incidence of oral dysplasia and the incidence of focal invasion following surgical excision of severe dysplasia and carcinoma in situ. Methods Histopathology reports for all patients diagnosed with dysplasia of the oral cavity at the Norfolk and Norwich University Hospital between the periods of May 2017 to May 2019 were analyzed. Data on site of lesion, grade of dysplasia, and presence of focal invasion were recorded. Results A total of 130 patients had 150 biopsies with a diagnosis of dysplasia. Eighty-two had mild (55%), 27 had moderate (18%), and 41 had severe or carcinoma in situ (27%). Patients with a diagnosis of severe dysplasia or carcinoma in situ (n = 41) had excision of the lesion, of which 9 (21.9%) showed focal invasion, changing their diagnosis to oral squamous cell carcinoma. Of these, 3 underwent further surgery following our local multidisciplinary team meeting. Conclusions Histopathology is essential in the management of these cases, as it defines the treatment modality required. If invasion is identified on histopathologic examination following excision, further excision may be required to deem the procedure as curative and reduce risk of recurrence. Hence all patients with a diagnosis of carcinoma in situ should be told of the risk of invasion and need for further surgery.

OBJECTIVES We performed a systematic review to evaluate the published biomarkers related to oral leukoplakia (OL), aiming to identify the biomarkers that indicate any future risk of cancer in patients with oral leukoplakia. METHODS A search strategy was developed for three main electronic databases: PubMed, Cochrane Library, and EBSCO, and also for Google Scholar, until February 28, 2020. The study selection was performed in a two-phase process aiming at studies assessing tissue biomarkers for "malignant transformation of OL." Risk of bias analysis of included studies was performed using the Quality in Prognosis Studies Tool. RESULTS From 3,130 articles initially identified by searching databases, a total of 46 studies were included in this systematic review, with a combined sample of 3,783 patients, of whom 1,047 presented with malignant transformation of a previously diagnosed OL as reported by the authors. The cancer incidence in the whole group was 27.6% (range: 5.4% to 54.1%). The studies were derived from different geographic areas, including Asia (n = 21), Europe (n = 15), North America (n = 9), and Oceania (n = 1). There were 49 different molecular biomarkers evaluated in the 46 included studies: p53 and podoplanin proteins were the most frequently reported, followed by abnormalities at particular chromosomal loci (e.g., LOH). Risk of bias analysis revealed concerns associated with "measurement of prognostic factor," "study confounding" and "statistical analysis and reporting." CONCLUSIONS Substantial heterogeneity and lack of standardized reporting of data among the studies were identified. The most promising biomarkers reported to have a significant association with the malignant transformation in OL included podoplanin and chromosomal loci abnormalities. A critical examination of the follow-up studies on OL published so far indicated that tissue biomarkers that could predict the risk of oral cancer in patients with OL are still in a discovery phase.

Objectives Numerous clinical and histopathological characteristics have been associated with malignant transformation (MT) of oral leukoplakia (OL), including classic and differentiated epithelial dysplasia, but MT predictions remain suboptimal. The objective of this study was to determine the annual MT rate of OL and to identify clinicopathological risk factors associated with MT. Patients and methods 170 patients with OL were included in this retrospective cohort study, 117 females and 53 males. Follow-up ranged from 12 to 219 months (median 54). The analyzed variables included age, gender, smoking habits, clinical presentation, subsite, size and treatment. In a subgroup of 140 patients, histopathological diagnoses were reviewed with regard to the presence of dysplasia, discerning both classic dysplasia and differentiated dysplasia. Results MT occurred in 23% of the patients, resulting in an annual MT rate of 4.9% (95% CI: 3.5 – 6.6) which remained consistent. High-risk subsite (tongue and floor of mouth) was the only clinical predictor for MT (Hazard Ratio = 2.7, 95% CI: 1.3 – 5.5, p = 0.007). In 140 patients, classic dysplasia (Hazard Ratio = 7.2, 95% CI: 1.6 – 33.1, p = 0.012) and differentiated dysplasia (Hazard Ratio = 6.6, 95% CI: 1.2 – 25.4, p = 0.026) were predictors for MT. Binary grading between dysplasia and no dysplasia was significant for predicting MT (Hazard Ratio = 6.4, 95% CI: 1.5 – 27.5, p = 0.013). Conclusion Since annual MT rate of OL remains stable during follow-up, regular long-term or even life-long follow-up is advocated. Specific oral subsites and epithelial dysplasia are predictors for MT of OL.

OBJECTIVE Recurrent disease occurs in a significant proportion of early-stage oral squamous cell carcinoma (OSCC) despite removal of the entire tumor with clear surgical margins. Our study sought to identify clinicopathologic factors that increase the risk of locoregional recurrence in T1N0 OSCC. STUDY DESIGN We conducted an observational retrospective analysis of 65 cases of T1N0 OSCC over a period of 13 years. For each case, we examined the clinical presentation, histopathologic appearance, and treatment characteristics of interest to evaluate the association between these variables and locoregional recurrence. RESULTS The 5-year and 10-year locoregional recurrence rates were 29.2% and 33.8%, respectively. Individuals with prior oral dysplasia had significantly higher odds for locoregional recurrence (P < .01) and reduced 5-year disease-free survival rates (P < .01). OSCC of the tongue and floor of the mouth had lower recurrence odds than carcinomas of the gingiva, buccal mucosa, and palate (P < .05). Histologic grade (P = .80), depth of invasion (P = .82), and elective neck dissection (P = .80) did not appear to influence locoregional recurrence for T1N0 tumors. CONCLUSIONS Given the morbidity and mortality associated with OSCC, understanding of the clinicopathologic factors associated with recurrent disease may lead to improved treatment and follow-up protocols for a subset of early-stage patients.

Squamous cell carcinoma makes up 90% of cases of oral cancer. However, a myriad of premalignant, inflammatory, and immune-based conditions can manifest as oral mucosal lesions. Biopsy of these lesions shares many of the principles of cutaneous lesions. Biopsy of oral mucosal lesions is a procedure that is safely performed in most cases in the outpatient ambulatory setting using local anesthesia. Special considerations should be taken depending on the presumed diagnosis based on physical examination. Its clinical relevance depends on a sound clinicopathologic assessment of the patient's condition. This article reviews specific considerations for biopsy of oral mucosal lesions.

We demonstrate that structured illumination microscopy has the potential to enhance fluorescence lifetime imaging microscopy (FLIM) as an early detection method for oral squamous cell carcinoma. FLIM can be used to monitor or detect changes in the fluorescence lifetime of metabolic cofactors (e.g. NADH and FAD) associated with the onset of carcinogenesis. However, out of focus fluorescence often interferes with this lifetime measurement. Structured illumination fluorescence lifetime imaging (SI-FLIM) addresses this by providing depth-resolved lifetime measurements, and applied to oral mucosa, can localize the collected signal to the epithelium. In this study, the hamster model of oral carcinogenesis was used to evaluate SI-FLIM in premalignant and malignant oral mucosa. Cheek pouches were imaged in vivo and correlated to histopathological diagnoses. The potential of NADH fluorescence signal and lifetime, as measured by widefield FLIM and SI-FLIM, to differentiate dysplasia (pre-malignancy) from normal tissue was evaluated. ROC analysis was carried out with the task of discriminating between normal tissue and mild dysplasia, when changes in fluorescence characteristics are localized to the epithelium only. The results demonstrate that SI-FLIM (AUC = 0.83) is a significantly better (p-value = 0.031) marker for mild dysplasia when compared to widefield FLIM (AUC = 0.63).

The DNA cytosine deaminase APOBEC3B (A3B) is a newly recognized endogenous source of mutations in a range of human tumors, including head/neck cancer. A3B inflicts C-to-T and C-to-G base substitutions in 5′-TCA/T trinucleotide motifs, contributes to accelerated rates of tumor development, and affects clinical outcomes in a variety of cancer types. High-risk human papillomavirus (HPV) infection causes A3B overexpression, and HPV-positive cervical and head/neck cancers are among tumor types with the highest degree of APOBEC signature mutations. A3B overexpression in HPV-positive tumor types is caused by the viral E6/E7 oncoproteins and may be an early off-to-on switch in tumorigenesis. In comparison, less is known about the molecular mechanisms responsible for A3B overexpression in HPV-negative head/neck cancers. Here, we utilize an immunohistochemical approach to determine whether A3B is turned from off-to-on or if it undergoes a more gradual transition to overexpression in HPV-negative head/neck cancers. As positive controls, almost all HPV-positive oral epithelial dysplasias and oropharyngeal cancers showed high levels of nuclear A3B staining regardless of diagnosis. As negative controls, A3B levels were low in phenotypically normal epithelium adjacent to cancer and oral epithelial hyperplasias. Interestingly, HPV-negative and low-grade oral epithelial dysplasias showed intermediate A3B levels, while high-grade oral dysplasias showed high A3B levels similar to oral squamous cell carcinomas. A3B levels were highest in grade 2 and grade 3 oral squamous cell carcinomas. In addition, a strong positive association was found between nuclear A3B and Ki67 scores suggesting a linkage to the cell cycle. Overall, these results support a model in which gradual activation of A3B expression occurs during HPV-negative tumor development and suggest that A3B overexpression may provide a marker for advanced grade oral dysplasia and cancer.

Background and Objectives: Stathmin is a phosphoprotein, which in its phosphorylated/unphosphorylated states plays a major role in polymerization/depolymerization of microtubules, respectively. Assembly of microtubules is an important aspect of cell division called mitosis. Hinderance in the function of stathmin could lead to damage in the mitotic process resulting in aneuploidy which is common manifestation of malignancies. Hence, stathmin could be used as a tumor marker for oral dysplasias and cancers. The purpose of the study is to compare the expression of stathmin in normal subjects to the patients with oral leukoplakia and to correlate its expression with different histopathological grades of oral leukoplakia This is the first ever study conducted to examine the expression of stathmin in oral dysplasia. Methodology: Thirty histopathologically confirmed cases of oral dysplasia were selected for the study. These tissues were evaluated immunohistochemically for stathmin. To enumerate the stathmin stained cells, 300 cells were examined manually in at least 5 areas and a mean percentage of positive–stained slides were determined. Then, each sample was assigned to one of the following staining scores: (0) – (

Oral carcinogenesis is a complex and multifactorial process that involves cumulative genetic and molecular alterations, leading to uncontrolled cell proliferation, impaired DNA repair and defective cell death. At the early stages, the onset of potentially malignant lesions in the oral mucosa, or oral dysplasia, is associated with higher rates of malignant progression towards carcinoma in situ and invasive carcinoma. Efforts have been made to get insights about signaling pathways that are deregulated in oral dysplasia, as these could be translated into novel markers and might represent promising therapeutic targets. In this context, recent evidence underscored the relevance of the Wnt/β-catenin signaling pathway in oral dysplasia, as this pathway is progressively “switched on” through the different grades of dysplasia (mild, moderate and severe dysplasia), with the consequent nuclear translocation of β-catenin and expression of target genes associated with the maintenance of representative traits of oral dysplasia, namely cell proliferation and viability. Intriguingly, recent studies provide an unanticipated connection between active β-catenin signaling and deregulated endosome trafficking in oral dysplasia, highlighting the relevance of endocytic components in oral carcinogenesis. This review summarizes evidence about the role of the Wnt/β-catenin signaling pathway and the underlying mechanisms that account for its aberrant activation in oral carcinogenesis.

Background Current evidence on diagnostic value of aneuploidy with DNA image cytometry (ICM) using brushings for oral potentially malignant disorders (OPMD) is limited by sample size and inconsistent classification criteria of aneuploidy. This study aimed to explore the optimal cut‐off values of DNA content and evaluate the diagnostic accuracy of DNA‐ICM for detecting dysplasia and/or carcinoma in OPMD. Materials and Methods A total of 401 consecutive patients with OPMD were enrolled in this prospective diagnostic study. Brushing and biopsy sample form each patient was processed by DNA‐ICM and histological examination respectively. Results When the optimal cut‐off of at least one aneuploid cell with DNA index (DI) ≥2.3, the area under the curves (AUC) was 0.735 and positive predictive value was 92.7% for detecting dysplasia within OPMD. When the optimal cut‐off of at least one aneuploid cell with DI ≥ 3.5, the AUC was 0.851 and negative predictive values was 96.8% for detecting carcinoma within OPMD. Importantly, multivariate analysis revealed that aneuploidy with DI ≥ 2.3 in OPMD was significantly associated with dysplasia risk (adjusted OR, 5.52; 95%CI, 2.90‐10.51; P, This largest‐scale diagnostic study optimized the criteria of aneuploidy cytology for noninvasive detection of oral dysplasia and carcinoma within OPMD. DNA aneuploidy in OPMD was an independent marker that strongly associated with oral dysplasia/carcinoma. Our findings suggest that DNA‐ICM may serve as a useful noninvasive adjunctive tool for oral cancer and OPMD screening.

Oral dysplasia is a pre-malignant stage of oral epithelial carcinomas, e.g., oral squamous cell carcinoma, where significant changes in tissue layers and cells can be observed under the microscope. However, malignancy can be reverted or cured using proper medication or surgery if the grade of malignancy is assessed properly. The assessment of correct grade is therefore critical in patient management as it can change the treatment decisions and prognosis for the dysplastic lesion. This assessment is highly challenging due to considerable inter- and intraobserver variability in pathologists’ agreement, which highlights the need for an automated grading system that can predict more accurate and reliable grade. Recent advancements have made it possible for digital pathology (DP) and artificial intelligence (AI) to join forces from the digitization of tissue slides into images and using those images to train and predict more accurate grades using complex AI models. In this regard, we propose a novel morphometric approach exploiting the architectural features in dysplastic lesions i.e., irregular epithelial stratification where we measure the widths of different layers of the epithelium from the boundary layer i.e., keratin projecting inwards to the epithelium and basal layers to the rest of the tissue section from a clinically significant viewpoint.

Oral cancer is one of the most common malignant tumors. There are 354,864 new cases and 177,384 death per year globally according to Globocan 2018 report. Most of the cases are in low- and middle-income countries that lack trained specialists and health services, of which India accounts for approximately one-third of the new cases and two-fifth deaths. Point-of-care oral screening tool to enable early diagnosis is urgently needed. We developed a dual-mode intraoral oral cancer screening platform and an automatic classification algorithm for oral dysplasia and malignancy images using deep learning.

Subjectivity in oral dysplasia grading has prompted evaluation of molecular‑based tests to predict malignant transformation. Aneuploidy detected by DNA image‑based cytometry (ICM) is currently the best predictor but fails to detect certain high risk lesions. A novel multiplex fluorescence in situ hybridization (FISH) panel was used to explore possible explanations by detecting aneuploidy at the single cell level. FISH was compared to reference standard DNA ICM in 19 oral lesions with epithelial dysplasia and used to characterize the cellular architecture. Copy number variation at 3q28, 7p11.2, 8q24.3, 11q13.3 and 20q13.12 and matched chromosome specific loci were assessed by dual‑color FISH to assess numerical and spatial patterns of copy number increase and gene amplification. FISH revealed wide variation in copy number at different loci. Only low level copy number gain was present and often in only a small proportion of cells, although usually with all or all but one locus (9/12). Four cases showed gene amplification, one at two loci. Some probes revealed an internal presumed clonal structure within lesions not apparent in routine histological examination. Both methods produced similar diagnostic results with concordance in detection of aneuploidy by both methods in 17 out of 19 samples (89%). We have shown that oral dysplastic lesions may contain very few aneuploid cells at a cellular level, high copy number gain is rare and changes appear to arise from large chromosomal fragment duplications. Single stem lines are relatively homogeneous for loci with copy number gain but there is a subclonal structure revealed by gene amplification in some lesions.

Background Oral cancer associated with high risk (HPV-HR) human papilloma virus (HPV) has been increasing. HPV-HR has been associated with epithelial dysplasia, however, little information exists on its frequency in epithelial hyperplasia lesions. The aim of this study is to compare HPV genotypes in dysplastic and hyperplastic lesions of oral cavity. Material and Methods Two hundred and fifty oral lesions: 131 dysplasia and 119 hyperplasia from two regions of Colombia were evaluated. One hundred seventy-four coming from urban area and 104 from a high risk population to oral cancer from a rural area. HPV was identified by qPCR and Twenty-four HPVs genotypes were evaluated by Luminex® technology. Logistic regressions were performed to establish the associations between HPV infections with oral dysplasia. Results Twenty-eight percent (70/250) of the samples were positives for any HPV and HPV-HRs were more frequently than low risk HPVs. HPV-16 was the most detected genotype (16%) followed by HPV-31, 53, 18 and 45. HPV, HPV-HRs and HPV-16 were only associated with dysplasia in urban area; OR 3.28 (CI 95% 1.49-7.17), OR 7.94 (CI 95% 2.97-21.2) and OR 5.90 (CI 95% 2.05-17). Individuals in rural area showed more HPV and HPV-HRs infection in hyperplasic lesions than urban population. The majority of HPV+ lesions had multi-type of HPV (52/70) and the urban individuals showed more genotypes than rural population. Conclusions HPV-.HRs are frequently found in hyperplastic and dysplastic epithelial lesions. HPV-HRs and HPV-16 were associated with dysplasia in urban population. Rural high risk population and urban population differ in the frequency and variety of HPV genotypes. Key words:Human papilloma virus, epithelial dysplasia, epithelial hyperplasia, HPV-genotypes.

Objective The 4-nitroquinoline 1-oxide (4-NQO) model for carcinogenesis has been used to investigate cancer stem cells (CSC), but no study has addressed the role of the p75 neurotrophin receptor (p75NTR) in 4-NQO-induced oral dysplasia and oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the immunohistochemistry profile of the p75NTR during 4-NQO-induced oral carcinogenesis in rats and to verify whether this profile has an association with proliferating cell nuclear antigen (PCNA) immunolabeling. Design For 28 weeks, rats were exposed to 4-NQO, which was diluted in the drinking water. After 3, 5, 7, 16, and 28 weeks, the animals were euthanized and their tongues were histologically analyzed using p75NTR and PCNA immunolabeling. Results In animals without 4-NQO exposure, the p75NTR and PCNA were expressed only in the basal epithelial layer and in a clustered manner. The oral epithelium showed dysplasia and a significant increase in the number of p75NTR- and PCNA-positive cells, which were localized mainly in the basal and suprabasal epithelial layers during weeks 5–16 of 4-NQO exposure. When the epithelium invaded the lamina propria and well-differentiated OSCC began, the p75NTR-positive cell frequency drastically decreased in epithelial cords and nests, showing a negative correlation with PCNA expression. p75NTR immunolabeling during 4-NQO-induced carcinogenesis was similar to that described for human head and neck dysplasia and neoplasia. Conclusions p75NTR immunolabeling observed in 4-NQO-induced oral dysplastic and OSCC lesions were related to the early phases of oral carcinogenesis and may help predict cell dysplasia and malignant transformation.

The development of dysplastic changes in oral epithelial lesions is a potential long-term complication after hematopoietic stem cell transplantation (HSCT). This may be related to mechanisms including radiation and chemotherapy regimens, chronic graft-versus-host disease (cGVHD), inflammation, and prolonged immunosuppression. The current case describes a 54-year-old male with multiple myeloma treated by autologous and allogenic HSCT followed by development of cGVHD (mouth, skin and the eyes) with the complaint of diffuse white lesions on the buccal mucosa, tongue, and palate. A biopsy performed with histopathological analysis revealed moderate to severe epithelial dysplasia with hyperkeratosis, positive for p16INK4A as a surrogate marker for human papillomavirus (HPV). Our finding suggests a possible association of oral dysplasia and HPV in patients after receiving allogenic HSCT with the necessity of more clinical follow-ups for those patients that may be at a higher risk for the development of oral dysplasia associated with HPV.

Objectives The aim of this study was to identify an association or link between cyclin D1 and p27KIP1 protein expression and dysplastic changes or progression. Study Design Oral mucosal biopsies with a diagnosis of non-neoplastic tissue (gingivitis) (n = 10), mild to moderate oral epithelial dysplasia (n = 12), and oral squamous cell carcinoma (n = 11) were evaluated by using immunohistochemistry. Scanning software was used to determine cyclin D1 and p27KIP1 intensity of expression, location, and pattern. Results A significant increase in expression of cyclin D1 and a decrease in expression of p27KIP1 proteins were identified in oral epithelial dysplasia and less differentiated oral squamous cell carcinoma (OSCC). There was a more diffuse distribution of cyclin D1 protein expression extending from the basal cell layer into the prickle cell layers in epithelial dysplasia and extending within all epithelial layers in OSCC. Cases of oral epithelial dysplasia had moderate infrequent expression of p27KIP1. There were no p27KIP1-positive cells in OSCC. The percentage of cells with both nuclear and cytoplasmic cyclin D1 staining was higher in OSCC specimens than control groups and oral epithelial dysplasia. Conclusions The expression of both cyclin D1 and p27KIP1 correlated with the grade of oral epithelial dysplasia and degree of OSCC differentiation. The results obtained will be verified through a basic follow-up of the cases to determine the prognosis/progression of oral dysplasia.

Background: Oral premalignant lesions (OPL) include a wide range of lesions including leukoplakia, dysplastic leukoplakia, erythroplakia, oral submucous fibrosis, dysplastic lichenoid lesions, and oral lichen planus. Proper diagnosis and management of oral premalignant lesions (OPL) are very important; as 17% of these lesions have been reported to be transformed into malignant lesions within first 7 years of their diagnosis. Vitamin A has always been known for its chemopreventive effect in several diseases through the modulation of cell proliferation and, it is also known for being a potent antioxidant and anti-inflammatory agent. Any alterations in p53 protein results in its accumulation in the cell nuclei; may play a critical role in carcinogenesis, including oral premalignant lesions and oral malignant lesions, i.e., p53 protein is one of the determinants in the progression of oral dysplasia to invasive malignancy.Methods: Fifteen patients having oral premalignant lesions of mild and moderate dysplasia; diagnosed both clinically and histologically, were given a chemopreventive therapy in the form of βeta- carotene (Vit. A) for four months. The levels of p53 protein immunostaining were measured pre- and post-treatment.Results: The mean value of optical staining density of P53 in pre-treatment specimen was (59.78 ± 10.22) compared to (39.74 ± 3.36) in post-treatment cases; this value was higher in pre-treatment cases than post-treatment cases. The p-value was 0.0003.Thus when statistically compared the difference is considered statistically significant.Conclusion: The expression of P53 protein in (OPL) is found to be inversely related to the clinical as well as histopathological response to beta-carotene supplementation.

We studied the progression from dysplasia to invasive carcinoma and subsequent second primaries or locoregional recurrences in 11 patients with recurrent squamous cell carcinoma (SCC). Between one and six samples were sequenced/patient. DNA samples were prepared, and libraries multiplexed to between 40 and 80 samples/lane of an Illumina HiSeq 3000 and sequenced with 2 × 100 bp paired end sequencing. Copy number data were generated by CNAnorm (Bioconductor package). Samples of recurrent SCC showed unique patterns of descent when compared with earlier samples from the primary tumour, and three main patterns emerged. In four patients there was convincing evidence that the later lesion was descended directly from cells from the first, and in a further four there were no detectable genomic events between the two lesions. Three patients had some shared events between the early and later lesions, but although there were enough differences to deduce that the two lesions had a shared ancestor, they were not directly descended from each other. We present the patients’ characteristics in detail, including the overall survival in each group. There was a distinct genomic pattern after a second episode of SCC in all the groups. A larger study that uses similar methods and a longer duration could provide reliable conclusions with respect to survival. With the use of new techniques, genomic data can be available to clinical teams during the planning of treatment.