[Precancers of the oral mucosa: clinic, diagnostics]. / Predraki slizistoi obolochki rta: klinika i diagnostika.

OBJECTIVE: The aim of the study. Improving the efficiency of diagnosis and detailing the features of the clinic of «potentially malignant¼ diseases of the oral mucosa. MATERIALS AND METHODS: Clinical and laboratory examination of 124 patients of the department of oral mucosa diseases aged 35 to 80 years, among whom there were 75 women and 49 men, with diseases such as erythroplakia - 12 patients, verrucous leukoplakia - 52 patients, erosive form of leukoplakia - 35 patients, cheilitis Manganotti - 25 patients. Histological and immunohistochemical methods of investigation were used as diagnostics. To assess the proliferative activity of epithelial cells, the determination of the Ki-67 index was used. The synthesis of keratin 15 (K15) in epithelial layers was determined as a diagnostic criterion for the severity of neoplasia. The expression of human papillomavirus type 16 (HPV 16) antigens and p16INK4a protein in epithelial cells was studied, as well as the expression of p53 protein. RESULTS: A high prevalence of p53 mutations was observed in patients with erythroplakia. In leukoplakia, the expression of the Ki-67 protein was detected in the cell nuclei in both the basal and parabasal layers of the multilayer squamous epithelium, in 77% of cases, the expression of the p16INK4a protein in the epithelial nuclei with varying degrees of dysplastic changes was noted, and a positive reaction to HPV16 was also observed in the cell nuclei and cytoplasm of epithelial cells in the basal, parabasal and spiny epithelial layers. The appearance of K15 in the cytoplasm of cells above the basal layer with abrasive precancerous cheilitis was found in 48% of cases. CONCLUSION: To diagnose early manifestations of neoplastic processes in «potentially malignant¼ diseases of the oral mucosa, it is necessary to use both classical histological and immunohistochemical methods of investigation with various markers.

Oral epithelial dysplasia detection and grading in oral leukoplakia using deep learning.

BACKGROUND: The grading of oral epithelial dysplasia is often time-consuming for oral pathologists and the results are poorly reproducible between observers. In this study, we aimed to establish an objective, accurate and useful detection and grading system for oral epithelial dysplasia in the whole-slides of oral leukoplakia. METHODS: Four convolutional neural networks were compared using the image patches from 56 whole-slide of oral leukoplakia labeled by pathologists as the gold standard. Sequentially, feature detection models were trained, validated and tested with 1,000 image patches using the optimal network. Lastly, a comprehensive system named E-MOD-plus was established by combining feature detection models and a multiclass logistic model. RESULTS: EfficientNet-B0 was selected as the optimal network to build feature detection models. In the internal dataset of whole-slide images, the prediction accuracy of E-MOD-plus was 81.3% (95% confidence interval: 71.4-90.5%) and the area under the receiver operating characteristic curve was 0.793 (95% confidence interval: 0.650 to 0.925); in the external dataset of 229 tissue microarray images, the prediction accuracy was 86.5% (95% confidence interval: 82.4-90.0%) and the area under the receiver operating characteristic curve was 0.669 (95% confidence interval: 0.496 to 0.843). CONCLUSIONS: E-MOD-plus was objective and accurate in the detection of pathological features as well as the grading of oral epithelial dysplasia, and had potential to assist pathologists in clinical practice.

Proliferative verrucous leukoplakia: a general dental practitioner-focused clinical review.

Proliferative verrucous leukoplakia (PVL) is a distinct type of oral leukoplakia which has the potential to enlarge or develop into new areas of leukoplakia coupled with areas of a warty surface texture. PVL is usually diagnosed from the fifth decade onwards and is more common in female patients. The most frequent sites involved tend to be gingivae, followed by buccal mucosa and lateral border of tongue. It is one of the oral potentially malignant conditions with a high risk of malignant transformation. It is important for general dental practitioners (GDPs) to identify such lesions to facilitate referral for further investigation and diagnosis. Management is challenging with long-term monitoring and surgical excision when appropriate; however, PVL tends to recur following surgical excision. This article provides an up-to-date review tailored for GDPs on the present knowledge of PVL and illustrates the management challenges with clinical cases.

Global gene expression profile of proliferative verrucous leukoplakia and its underlying biological disease mechanisms.

BACKGROUND: Proliferative verrucous leukoplakia (PVL) is a rare and enigmatic oral potentially malignant disorder which almost invariably results in oral squamous cell carcinoma (OSCC). The aims of this project were to use transcriptome profiling to characterise PVL gene expression patterns for biomarker identification and gain insight into the molecular aetiopathogenesis of PVL. METHODS: Forty-three oral cavity mucosal biopsies from 32 patients with oral lesions clinically compatible with either PVL or non-PVL conventional oral leukoplakia (OLK) underwent transcriptome profiling by RNA sequencing. Data was analysed by hierarchical clustering, differential gene expression, functional enrichment and network analysis, sparse partial least squares discriminant analysis sPLS-DA, and immune cell phenotypic estimation. RESULTS: We found 464 genes significantly differentially expressed at least 2-fold between PVL and non-PVL OLK (193 up and 271 down). HOX genes, including HOXA1 and HOXB7, keratin-associated proteins (KRTAPs) and olfactory receptor G proteins (OR) were significantly upregulated in PVL. Other upregulated genes in PVL included FOS, WNT16 and IFNA1. Pathway analysis showed that there was a significant downregulation of connective tissue signalling in PVL. Classifying multivariate models based upon 22 genes discriminated PVL from non-PVL OLK. Bioinformatic profiling showed that immune cell profiles in PVL and OLK were similar except that fibroblast markers were reduced in PVL. CONCLUSION: These results demonstrate that PVL and conventional OLK are molecularly distinct with upregulation of many cancer-associated genes. They provide insight into the pathogenesis of PVL and show that biomarker based molecular diagnostics is feasible to discriminate and inform diagnosis and management.

Transient infantile lingual leukoplakia: An underrecognized cause of white tongues in infancy.

We have observed a distinct phenomenon of transient oral lingual leukoplakia in infancy and report 22 healthy infants with gray-white plaques on the dorsal tongue with sparing of the tip from four medical centers in three countries. The onset of the eruption ranged from 1 week to 7 months of life and resolved in 19 patients (86%, with 3 patients lost to follow-up). None of the eight patients examined at 1 year of age had residual findings. We believe this is a common entity that can be distinguished from oral candidiasis on clinical and/or laboratory examination and name this entity "transient infantile lingual leukoplakia."

Proliferative verrucous leukoplakia/multifocal leukoplakia in patients with and without oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) and proliferative verrucous leukoplakia (PVL) are established as oral potentially malignant disorders. Dual pathology of the two conditions is not commonly encountered in clinical practice. This study aims to present a case series of multifocal leukoplakia in patients with and without OSF to outline the clinical behavior and challenges in the management of this high-risk group in clinical practice. MATERIAL AND METHODS: We retrospectively analyzed cases of six Indian patients (four with OSF) managed over a period of 5.5 to 13 years at the Government Dental College, Nagpur. Patient data consisting of age, gender, medical history, habits, clinical findings, and biopsy reports were recorded at the initial visit. During follow-up visits, the clinicopathological data were reassessed. When surgical intervention failed to arrest the disease or when surgery was contraindicated metronomic therapy with Folitrax 15 mg once a week and Celecoxib 100mg twice daily was initiated. RESULTS: All patients developed PVL after the initial pathology diagnosis of OSF or oral leukoplakia. Initial lesions were either homogenous or non-homogenous leukoplakia. All patients developed multiple recurrences, regional or systemic metastasis. Despite thorough interventions, the patients died of, or with the disease. CONCLUSIONS: The occurrence of two or more oral potentially malignant disorders poses challenges in patient management and possibly presents a higher risk of malignant transformation. More clinical trials are necessary to assess the benefits of metronomic therapy for patients diagnosed with aggressive PVL concurrently found with OSF.

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.

[Research progress in the diagnosis and management of proliferative verrucous leukoplakia].

Proliferative verrucous leukoplakia (PVL) is one of the oral potentially malignant disorders (OPMD) with the highest malignant potential. PVL tends to be easily misdiagnosed owing to the resemblance in clinical manifestations between PVL and other diseases such as oral leukoplakia or oral lichen planus. PVL is considered as a special type of oral leukoplakia by some scholars, which is characterized by its tendency of recurrence and metastasis, along with its high risk of malignant transformation. So far, the accurate clinic diagnosis and management of PVL are still intractable due to the lack of definite histopathological definition, unified diagnostic criteria and effective treatment modalities. This review aims to provide the clinical practitioners with a series of advices on the clinical diagnosis and management of PVL by systematically reviewing the diagnostic logistics, therapeutic strategies, malignant transformation detection based on tremendous relevant data and evidence-based medicine.

Salivary metabolomics for oral leukoplakia with and without dysplasia.

PURPOSE: Oral leukoplakia (OL) is a common potentially malignant oral disorder. Therefore, there is a need for simple screening methods for OL before its transformation into oral cancer. Furthermore, because invasive open biopsy is the sole method to determine if an OL lesion is dysplastic, there is also a clinical need for non-invasive methods to differentiate dysplastic OL from non-dysplastic OL. This study aimed to identify salivary metabolites that can help differentiate patients with OL from healthy controls (HC) and also dysplastic OL from non-dysplastic OL. MATERIAL & METHODS: Whole unstimulated saliva samples were collected from patients with OL (n = 30) and HCs (n = 29). The OL group included nine patients with dysplastic OL and 20 with non-dysplastic OL. Hydrophilic metabolites in the saliva samples were comprehensively analyzed through capillary electrophoresis mass spectrometry. To evaluate the discrimination ability of a combination of multiple markers, a multiple logistic regression (MLR) model was developed to differentiate patients with OL from HCs and dysplastic OL from non-dysplastic OL. RESULTS: Twenty-eight metabolites were evidently different between patients with OL and HCs. Finally, three metabolites (guanine, carnitine, and N-acetylputrescine) were selected to develop the MLR model, which resulted in a high area under curve (AUC) of the receiver operating characteristic (ROC) to differentiate patients with OL from HCs (AUC = 0.946, p < 0.001, 95% confidential interval [CI] = 0.889- 1.000). Similarly, two metabolites were evidently different between patients with dysplastic and non-dysplastic OL. Finally, only one metabolite (7-methylguanine) was selected in the MLR model, which revealed a moderate discrimination ability for dysplastic and non-dysplastic OL (AUC = 0761, p = 0.027, 95% CI = 0.551-0.972). CONCLUSION: Our candidate salivary metabolites showed potential not only to discriminate OL from HC, but also to discriminate dysplastic OL from non-dysplastic OL.

A paradigm shift in the prevention and diagnosis of oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a widespread disease with only 50%-60% 5-year survival. Individuals with potentially malignant precursor lesions are at high risk. METHODS: Survival could be increased by effective, affordable, and simple screening methods, along with a shift from incisional tissue biopsies to non-invasive brush biopsies for cytology diagnosis, which are easy to perform in primary care. Along with the explainable, fast, and objective artificial intelligence characterisation of cells through deep learning, an easy-to-use, rapid, and cost-effective methodology for finding high-risk lesions is achievable. The collection of cytology samples offers the further opportunity of explorative genomic analysis. RESULTS: Our prospective multicentre study of patients with leukoplakia yields a vast number of oral keratinocytes. In addition to cytopathological analysis, whole-slide imaging and the training of deep neural networks, samples are analysed according to a single-cell RNA sequencing protocol, enabling mapping of the entire keratinocyte transcriptome. Mapping the changes in the genetic profile, based on mRNA expression, facilitates the identification of biomarkers that predict cancer transformation. CONCLUSION: This position paper highlights non-invasive methods for identifying patients with oral mucosal lesions at risk of malignant transformation. Reliable non-invasive methods for screening at-risk individuals bring the early diagnosis of OSCC within reach. The use of biomarkers to decide on a targeted therapy is most likely to improve the outcome. With the large-scale collection of samples following patients over time, combined with genomic analysis and modern machine-learning-based approaches for finding patterns in data, this path holds great promise.

Identification of Proteomic Biomarkers in Proliferative Verrucous Leukoplakia through Liquid Chromatography With Tandem Mass Spectrometry.

Proliferative verrucous leukoplakia (PVL) is an oral potentially malignant disorder associated with high risk of malignant transformation. Currently, there is no treatment available, and restrictive follow-up of patients is crucial for a better prognosis. Oral leukoplakia (OL) shares some clinical and microscopic features with PVL but exhibits different clinical manifestations and a lower rate of malignant transformation. This study aimed to investigate the proteomic profile of PVL in tissue and saliva samples to identify potential diagnostic biomarkers with therapeutic implications. Tissue and saliva samples obtained from patients with PVL were compared with those from patients with oral OL and controls. Label-free liquid chromatography with tandem mass spectrometry was employed, followed by qualitative and quantitative analyses, to identify differentially expressed proteins. Potential biomarkers were identified and further validated using immunohistochemistry. Staining intensity scan analyses were performed on tissue samples from patients with PVL, patients with OL, and controls from Brazil, Spain, and Finland. The study revealed differences in the immune system, cell cycle, DNA regulation, apoptosis pathways, and the whole proteome of PVL samples. In addition, liquid chromatography with tandem mass spectrometry analyses showed that calreticulin (CALR), receptor of activated protein C kinase 1 (RACK1), and 14-3-3 Tau-protein (YWHAQ) were highly expressed in PVL samples. Immunohistochemistry validation confirmed increased CARL expression in PVL compared with OL. Conversely, RACK1 and YWHA were highly expressed in oral potentially malignant disorder compared to the control group. Furthermore, significant differences in CALR and RACK1 expression were observed in the OL group when comparing samples with and without oral epithelial dysplasia, unlike the PVL. This research provides insights into the molecular mechanisms underlying these conditions and highlights potential targets for future diagnostic and therapeutic approaches.

[White lesions of the oral mucosa: leukoplakia]. / Witte afwijking van het mondslijmvlies: leukoplakie.

White lesions of the oral mucosa may be caused by various disorders. In most instances of white lesions, diagnoses can be made solely on clinical grounds. When the clinical diagnosis is not compatible with a known disease, the term leukoplakia is used. This is of importance since the yearly malignant transformation rate of oral leukoplakia into a squamous cell carcinoma is 2-4%. The presence and degree of epithelial dysplasia is the most important predictor for malignant transformation.

Proliferative verrucous/multifocal leukoplakia: Updates and literature review "case report".

RATIONALE: Proliferative verrucous leukoplakia (PVL) is a multifocal, slowly evolving lesion that resists all types of treatment and has a high propensity for malignant transformation into oral squamous cell carcinoma. Lack of awareness and acquaintance with white lesions of the oral cavity makes it difficult to diagnose. Besides being rare, PVL significantly aggressive, so clinicians need to be aware of it carefully. Therefore, it is recommended to have the earliest possible diagnosis and total excision of this lesion. We report this case to present typical clinical and histologic features of PVL so a For the purpose of sensitizing clinician. PATIENT CONCERN: A 61-year-old female came to the clinic concerning of recurring painless, white patch on the tongue 2 months ago, associated with oropharyngeal dryness. DIAGNOSES: This case satisfies these major and minor criteria to diagnosed PVL. INTERVENTION: An excisional biopsy of the lesion was done to check for the presence of dysplasia, as lesions were persisting. Hemostasis was achieved with single interrupted sutures. OUTCOME: no recurrence has been observed since excisional 1 year follow-up. LESSON: The key feature is early detection, precisely in cases of PVL it is critical for better treatment outcomes, lifesaving, quality-of-life enhancement. To detect and treat any potential pathologies, clinicians should meticulously examine the oral cavity and patients have to be aware and informed of the importance of regular screenings. This lesion is resistant to the presently available treatment modalities; therefore, total excision with free surgical margins is critical combined with a lifelong follow-up.

Reactive and Nonreactive White Lesions of the Oral Mucosa.

White lesions in the oral cavity may be diverse in etiology and may present with significant clinical and sometimes histologic overlap between categories, making accurate diagnosis difficult at times. Although white lesions of immune and infectious etiology are covered in another article, this article discusses the differential diagnosis between developmental, reactive, idiopathic, premalignant, and malignant white lesions focusing on clinical features of each category.

Automated immunohistochemical quantification of hypoxia biomarkers shows correlation with dysplastic epithelial changes.

BACKGROUND: Dysregulation of the hypoxia-aerobic system has been postulated in various malignancies. Nonetheless, the contribution of hypoxia to oral carcinogenesis is yet to be elucidated. Understanding this mechanism is important for improving diagnostic tools and targeted therapies. This study aimed to assess the dysregulation of hypoxia-related factors during different stages of oral squamous cell carcinoma (OSCC) development. METHODS: Ninety-two patients diagnosed clinically with oral leukoplakia or OSCC were included and classified according to their histopathological diagnoses. A panel of seven hypoxia-related antibodies were used for immunohistochemical staining of each case. Automated quantification of immunostaining was used for objective reporting. Microvessel density was also assessed. RESULTS: Significant associations were reported for non-dysplastic epithelial changes and malignancy for Glut1, HIF-1α, vascular endothelial growth factor, and signal transducer and activator of transcription 3(p < 0.005). Similarly, microvessel density significantly increased with the severity of epithelial disorders. A multiple regression model including the H-score of HIF-1α and microvessel density could statistically significantly predict the grade of epithelial disorder (p < 0.005). The associated diagnostic accuracy of this approach was 88%. CONCLUSIONS: Hypoxia-associated events are observed during early epithelial dysplastic changes and have a potential role in oral carcinogenesis. The level of hypoxia may assist in stratifying the severity of epithelial changes among patients with oral leukoplakia.

Label-Free Proteomics of Oral Mucosa Tissue to Identify Potential Biomarkers That Can Flag Predilection of Precancerous Lesions to Oral Cell Carcinoma: A Preliminary Study.

Oral squamous cell carcinomas are mostly preceded by precancerous lesions such as leukoplakia and erythroplakia. Our study is aimed at identifying potential biomarker proteins in precancerous lesions of leukoplakia and erythroplakia that can flag their transformation to oral cancer. Four biological replicate samples from clinical phenotypes of healthy control, leukoplakia, erythroplakia, and oral carcinoma were annotated based on clinical screening and histopathological evaluation of buccal mucosa tissue. Differentially expressed proteins were delineated using a label-free quantitative proteomic experiment done on an Orbitrap Fusion Tribrid mass spectrometer in three technical replicate sets of samples. Raw files were processed using MaxQuant version 2.0.1.0, and downstream analysis was done via Perseus version 1.6.15.0. Validation included functional annotation based on biological processes and pathways using the ClueGO plug-in of Cytoscape. Hierarchical clustering and principal component analysis were performed using the ClustVis tool. Across control, leukoplakia, and cancer, L-lactate dehydrogenase A chain, plectin, and WD repeat-containing protein 1 were upregulated, whereas thioredoxin 1 and spectrin alpha chain, nonerythrocytic 1 were downregulated. Across control, erythroplakia, and cancer, L-lactate dehydrogenase A chain was upregulated whereas aldehyde dehydrogenase 2, peroxiredoxin 1, heat shock 70 kDa protein 1B, and spectrin alpha chain, nonerythrocytic 1 were downregulated. We found that proteins involved in leukoplakia were associated with alteration in cytoskeletal disruption and glycolysis, while in erythroplakia, they were associated with alteration in response to oxidative stress and glycolysis across phenotypes. Hierarchical clustering subgrouped half of precancerous samples under the main branch of the control and the remaining half under carcinoma. Similarly, principal component analysis identified segregated clusters of control, precancerous lesions, and cancer, but erythroplakia phenotypes, in particular, overlapped more with the cancer cluster. Qualitative and quantitative protein signatures across control, precancer, and cancer phenotypes explain possible functional outcomes that dictate malignant transformation to oral carcinoma.

Wolf in sheep's clothing - Oral proliferative verrucous leukoplakia: Progression with longitudinal molecular insights.

BACKGROUND: Oral proliferative verrucous leukoplakia (OPVL) is a chronic form of oral leukoplakia that progresses to a multifocal disease with confluent, exophytic and proliferative features. The clinical differential diagnosis for OPVL includes frictional keratosis, leukoplakia, chronic hyperplastic candidiasis, squamous papilloma, verrucous hyperplasia, verrucous carcinoma and squamous cell carcinoma. In this study, we aimed to delineate the dynamic changes in molecular signature during OPVL progression. We compare to a cohort of oral cavity keratinizing squamous cell carcinoma (OSCC) patients covering the spectrum of verrucous carcinoma to invasive squamous cell carcinoma including cytologically bland cuniculatum variant. METHODS: Samples from a large OPVL lesion that exhibited a histopathologic continuum of OPVL progression. RESULTS: Canonical hotspot TERT promoter mutations were identified in all patients. TERT C228T was dominant and mutually exclusive with TERT C250T. In patients with TERT C250T, there was concurrent PI3 point mutation. TP53 mutations were also consistently found (8/10). At the protein level, p53 was abnormal, with loss of function and gain of function. CONCLUSIONS: OPVL is a pathology that shows proximity to the gene expression profile of OSCC, highlighting signatures in common that can be important targets for drug treatment, as well as in the development of diagnostic and prognostic strategies for this disease.