Saliva metabolomics: concepts and applications in oral disorders.

OBJECTIVES: The purpose of this review was to present the basic concepts of metabolomics methodology and the use of saliva for diagnostic, prognostic, and predictive strategies. MATERIAL AND METHODS: This review followed the focus in: "saliva metabolomics" and "oral diseases". The authors searched studies on PubMed database. The inclusion criteria were original studies and reviews that assessed metabolomics techniques. A descriptive analysis was performed considering the study design, approach system, clinical steps, and tools for the determination of profile or biomarkers metabolites, and the advantages and disadvantages. RESULTS: Metabolomic analyses use a combination of analytical instrumentation and informatic tools to provide information on metabolite characteristics. In this review we described different technologies applied and the advantages and limitations of each technique. Furthermore, in the literature search, we retrieved 25 studies that investigated saliva metabolites in oral diseases: 8 studies used targeted analysis and 17 untargeted metabolomics approaches. Most studies included patients with periodontal diseases, oral squamous cell carcinoma, and Sjögren Syndrome. The most frequently reported metabolites were glycine, leucine, phenylalanine, dipeptides, linoleic acid, arachidonic acid, tyrosine, choline, taurine, lactate, valine, and proline. CONCLUSIONS: Metabolomics analysis has emerged as a powerful tool for tumor diagnosis and to enhance tumor classification, including salivary gland tumors (SGTs). It also holds promise for developing personalized treatment plans and defining more precise prognostic categories. CLINICAL RELEVANCE: Metabolomics is the most functional and comprehensive technique for monitoring and understanding gene functions and identifying the biochemical state of an organism in response to genetic and environmental changes.

Identification of mutations in canine oral mucosal melanomas by exome sequencing and comparison with human melanomas.

Oral mucosal melanomas (OMMs) are aggressive neoplasms commonly found in dogs but rare in humans. Utilizing whole exome sequencing (WES), which focuses on protein-coding regions to reveal mutation profiles, we conducted a comparative analysis of canine OMM and human melanomas. This study involved DNA extraction, exome enrichment, and sequencing from three canine OMM cell lines (CMGD-2, CMGD-5, TLM-1), five canine OMM frozen samples, a human OMM cell line (MEMO), and a human commercial skin melanoma cell line (SK-MEL-28). The sequencing and subsequent analysis of FASTQ files yielded final variant files, leading to the identification of mutations. Our findings revealed a total of 500 mutated genes in canine OMM, including significant ones such as EP300, FAT4, JAK3, LRP1B, NCOR1, and NOTCH1. Notably, 82 shared mutations were identified between human melanomas and canine OMM genomes. These mutations were categorized based on the gene functions. The identification of these mutations provides critical insights that can pave the way for the development of novel therapeutic strategies for both canine and human OMM, offering hope for more effective treatments in the future.

Targeted saliva metabolomics in Sjögren's syndrome.

OBJECTIVE: Sjögren's Syndrome (SS) is a chronic inflammatory autoimmune exocrinopathy, and although, the role of metabolism in the autoimmune responses has been discussed in diseases such as lupus erythematosus, rheumatoid arthritis, psoriasis and scleroderma. There is a lack of information regarding the metabolic implications of SS. Considering that the disease affects primarily salivary glands; the aim of this study is to evaluate the metabolic changes in the salivary glands' microenvironment using a targeted metabolomics approach. METHODS: The saliva from 10 patients diagnosed with SS by the American-European consensus and 10 healthy volunteers was analyzed in an Ultra-high Performance Liquid Chromatograph Coupled Mass Spectrometry (UPLC-MS). RESULTS: The results showed an increased concentration in SS of metabolites involved in oxidative stress such as lactate, alanine and malate, and amino acids involved in the growth and proliferation of T-cells, such as arginine, leucine valine and isoleucine. CONCLUSIONS: These results revealed that is possible to differentiate the metabolic profile of SS and healthy individuals using a small amount of saliva, which in its turn may reflect the cellular changes observed in the microenvironments of damaged salivary glands from these patients.

Mucosal Cancers Arising in Potentially Malignant Lesions of the Oral Mucosa Are Marjolin Ulcers: New Insights Into Old Concepts.

INTRODUCTION: Several disparate mucocutaneous diseases present oral mucosal lesions that have been classically labeled as "pre-cancerous", "pre-malignant", or "potentially malignant". These include oral lichen planus, dyskeratosis congenita, tertiary syphilitic glossitis chronic graft-versus-host-disease, and oral discoid lupus erythematosus. There is much confusion in literature regarding the real malignant potential of these oral lesions in relation to the incidence of squamous cell carcinoma. OBJECTIVES: We tried to unify the occurrence of squamous cell carcinoma in some oral mucosal diseases into the classic concept of "Marjolin ulcer". METHODS: We analyzed the most relevant published evidence of the occurrence of squamous cell carcinoma arising in oral lichen planus, dyskeratosis congenita, tertiary syphilitic glossitis chronic graft-versus-host-disease, and oral discoid lupus erythematosus, and tried to establish a logical link between them. RESULTS: Reported cases of squamous cell carcinoma occurring in oral lesions of these diseases seem to appear in old standing, scarring lesions. CONCLUSIONS: Oral lichen planus, dyskeratosis congenita, tertiary syphilitic glossitis, chronic graft-versushost-disease, and oral discoid lupus erythematosus are not "pre-malignant diseases", their long-lasting mucosal scars are prone to the development of squamous cell carcinomas. In this sense, this tumor can be considered a mucosal type of Marjolin ulcer.

Molecular Aspects of Mucoepidermoid Carcinoma and Adenoid Cystic Carcinoma of the Salivary Gland.

BACKGROUND: Salivary gland tumors (SGTs) are rare and highly heterogeneous lesions, making diagnosis a challenging activity. In addition, the small number of studies and samples evaluated difficults the determination of prognosis and diagnosis. Despite the solid advances achieved by research, there is still an intense need to investigate biomarkers for diagnosis, prognosis and that explain the evolution and progression of SGTs. METHODS: We performed a comprehensive literature review of the molecular alterations focusing on the most frequent malignant SGTs: mucoepidermoid carcinoma and adenoid cystic carcinoma. RESULTS: Due to the importance of biomarkers in the tumorigenenic process, this review aimed to address the mechanisms involved and to describe molecular and biomarker pathways to better understand some aspects of the pathophysiology of salivary gland tumorigenesis. CONCLUSIONS: Molecular analysis is essential not only to improve the diagnosis and prognosis of the tumors but also to identify novel driver pathways in the precision medicine scenario.

TGFß signaling pathway in salivary gland tumors.

OBJECTIVE: Pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are the most prevalent salivary gland tumors. Their pathogenesis has been recently associated with complex molecular cascades, including the TGFß signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFß signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFß cascade. DESIGN: Thirteen PA, 17 MEC, 13 ACC, and 10 non-neoplastic salivary gland samples were analyzed by real-time RT-PCR. RESULTS: Cases of PA presented increased TGFB1, LTPB1, c-MYC, and FBN1 expressions, whereas SMAD2 expression was decreased when compared to non-neoplastic tissue. MEC patients displayed increased expressions of TGFB1, ITGB6, FBN1, and c-MYC and decreased expressions of SMAD2 and SMAD4. ACC cases exhibited elevated expressions of the investigated genes except TGFB1. The present results suggest that decreased expression of SMAD2 and SMAD4 does not impede the transcriptional regulation of c-MYC, especially in PA and MEC. Increased expressions of ITGB6, TGFB1, LTBP1, and FBN1 appear to be related to the regulation of the TGFß signaling pathway in these tumors. Additionally, we observed a higher expression of SMAD4 in ACC and a raised expression of ITGB6 and lowered expression of SMAD2 in MEC. CONCLUSIONS: Our study demonstrated the differential expression of TGFß cascade members in salivary gland tumors such as SMAD2/SMAD4 and c-MYC as well as the participation of ITGB6, TGFB1, LTBP1, and FBN1, contributing to the understanding of the mechanisms involved in tumor progression.

Oral lupus erythematosus: Immunohistochemical evaluation of CD1a, CD21, CD123, and langerin expression in dendritic cells.

BACKGROUND: Dendritic cells participate in the pathophysiology of lupus erythematosus (LE), which are studied in systemic and cutaneous forms; however, little is known about their oral manifestations. METHODS: The expressions of dendritic cell markers (including CD1a, CD21, CD123, and langerin) were investigated by immunohistochemistry technique. Sixty intraoral and lower lip LE lesions, and additional 10 control samples were collected from 2003 to 2019. They were topographically analyzed in the epithelium (EP), lamina propria (LP), epithelial junction (JUN), and deep perivascular (PV) areas. RESULTS: The expression of CD1a was decreased in the EP (p = 0.003) and increased in the deep PV area (p = 0.002). Langerin immunostaining showed no significant decrease in EP (p = 0.944); however, it increased in LP (p = 0.012) and JUN (p = 0.006). CD21 was expressed in only two specimens (EP, p = 0.012; LP, p < 0.001; deep PV area, p = 0.018). CD123 expression increased in all topographies (EP, p < 0.005; LP, p < 0.001, JUN, p < 0.001; deep PV, p < 0.001). The comparison between vermilion and intraoral mucosa LE lesions suggested that sun-exposed sites showed higher expression of CD123 (EP, p = 0.024; LP, p = 0.047; JUN, p = 0.001). CONCLUSIONS: CD1a, langerin, and CD123 expressions were detected coincidently surrounding the inflammatory infiltrate in oral LE, suggesting that these cells may play an important role in immune response. Interestingly, plasmacytoid dendritic cells showed increased CD123 expression in sun-exposed site lesions, which point out a possible function in their pathogenesis. Further studies are needed to confirm this hypothesis.

Histopathologic findings in ointment pseudo-cheilitis: An alert to dermatopathologists.

Ointment pseudo-cheilitis is a recently recognized distinctive type of self-induced cheilitis. Lesions consist of a variable amount of crusts adhered to the vermilion. These crusts consist of dried saliva and dead cells mixed with applied medications attached to the lip surface. Patients are typically severely anxious or depressed; the condition impacts quality of life. Ointment pseudo-cheilitis is frequently misdiagnosed as exfoliative cheilitis or cheilitis glandularis. Biopsy reports are often non-revealing because there are no established histopathological criteria for this disease, and clinicians usually do not formulate the correct diagnostic hypothesis. Here, we present the histopathological findings of four cases of ointment pseudo-cheilitis. The most consistent finding was the presence of laminated parakeratotic material detached from the epithelium in biopsies that are devoid of other significant diagnostic changes. This material at the lip surface possibly represents physiologic labial desquamation mixed with dried saliva and applied medication. With this report, we intend to alert dermatopathologists to the diagnosis of ointment pseudo-cheilitis if they receive biopsies from patients who present clinically exuberant labial lesions that show only minimal histopathological changes.

Targeted saliva metabolomics in Sjogren's syndrome

Abstract Objective: Sjögren's Syndrome (SS) is a chronic inflammatory autoimmune exocrinopathy, and although, the role of metabolism in the autoimmune responses has been discussed in diseases such as lupus erythematosus, rheumatoid arthritis, psoriasis and scleroderma. There is a lack of information regarding the metabolic implications of SS. Considering that the disease affects primarily salivary glands; the aim of this study is to evaluate the metabolic changes in the salivary glands' microenvironment using a targeted metabolomics approach. Methods: The saliva from 10 patients diagnosed with SS by the American-European consensus and 10 healthy volunteers was analyzed in an Ultra-high Performance Liquid Chromatograph Coupled Mass Spectrometry (UPLCMS). Results: The results showed an increased concentration in SS of metabolites involved in oxidative stress such as lactate, alanine and malate, and amino acids involved in the growth and proliferation of T-cells, such as arginine, leucine valine and isoleucine. Conclusions: These results revealed that is possible to differentiate the metabolic profile of SS and healthy individuals using a small amount of saliva, which in its turn may reflect the cellular changes observed in the microenvironments of damaged salivary glands from these patients.

Oral lichen planus: case series and experience in a tertiary dermatology service in Brazil

Abstract Background: Lichen planus is an inflammatory disease that can affect both the skin and mucous membranes, including the oral mucosa. There is very little original Brazilian dermatology literature about oral lichen planus. Objective: To describe the clinical, pathological, and treatment data of 201 patients diagnosed with oral lichen planus followed at the Stomatology Outpatient Clinic of Hospital das Clínicas, Universidade de São Paulo, from 2003 to 2021. Method: The patients demographic profile, the morpho-topographic features of the lesions, the treatment employed, and the possible presence of squamous cell carcinoma were analyzed. Results: The disease was more common in women over 50 years of age, tending to be chronic, with a large number of cases showing cicatricial sequelae in the mucosa. Topical treatment with potent corticosteroids was shown to be effective in the vast majority of cases. Squamous cell carcinoma in oral lichen planus cicatricial sequelae was observed in eight cases. Study limitations: Retrospective study of medical records, with gaps regarding the filling out of data; unequal observation time among the studied cases. Conclusions: This is the largest Brazilian dermatology series on oral lichen planus. The response to topical corticoid therapy was excellent in the vast majority of cases. The high prevalence of atrophic lesions, demonstrating the chronicity and tissue destruction potential of this disease, may explain the large number of cases of squamous cell carcinoma.

Oral lichen planus: case series and experience in a tertiary dermatology service in Brazil.

BACKGROUND: Lichen planus is an inflammatory disease that can affect both the skin and mucous membranes, including the oral mucosa. There is very little original Brazilian dermatology literature about oral lichen planus. OBJECTIVE: To describe the clinical, pathological, and treatment data of 201 patients diagnosed with oral lichen planus followed at the Stomatology Outpatient Clinic of Hospital das Clínicas, Universidade de São Paulo, from 2003 to 2021. METHOD: The patients demographic profile, the morpho-topographic features of the lesions, the treatment employed, and the possible presence of squamous cell carcinoma were analyzed. RESULTS: The disease was more common in women over 50 years of age, tending to be chronic, with a large number of cases showing cicatricial sequelae in the mucosa. Topical treatment with potent corticosteroids was shown to be effective in the vast majority of cases. Squamous cell carcinoma in oral lichen planus cicatricial sequelae was observed in eight cases. STUDY LIMITATIONS: Retrospective study of medical records, with gaps regarding the filling out of data; unequal observation time among the studied cases. CONCLUSIONS: This is the largest Brazilian dermatology series on oral lichen planus. The response to topical corticoid therapy was excellent in the vast majority of cases. The high prevalence of atrophic lesions, demonstrating the chronicity and tissue destruction potential of this disease, may explain the large number of cases of squamous cell carcinoma.

Three cases of oral mucosal tuberculosis in patients on tumour-necrosis-factor-α blockers.

We present three cases of oral mucosal lesions caused by Mycobacterium tuberculosis in patients treated with anti-tumour necrosis factor-α for psoriasis or rheumatoid arthritis. Diagnosis of oral mucosal tuberculosis was not easily established in any of the cases. A comparison between these cases and other previously described forms of oral mucosal tuberculosis is presented.

Relevant proteins for the monitoring of engraftment phases after allogeneic hematopoietic stem cell transplantation.

INTRODUCTION: Hematopoietic Stem Cell Transplant (HSCT) has been successfully used as standard therapy for hematological disorders. After conditioning therapy, patients undergoing allogeneic HSCT, present three different phases of engraftment: early pre-engraftment, early post-engraftment, and late engraftment. Severe complications are associated with morbidity, mortality, and malignancies in these phases, which include effects on the oral cavity. OBJECTIVES: The changes in the salivary composition after HSCT may contribute to identifying relevant proteins that could map differences among the phases of diseases, driven for personalized diagnostics and therapy. METHODS: Unstimulated whole saliva was collected from patients submitted to HSCT. The samples were submitted to trypsin digestion for a Mass spectrometry analysis. MaxQuant processed the Data analysis, and the relevant expressed proteins were subjected to pathway and network analyses. RESULTS: Differences were observed in the most identified proteins, specifically in proteins involved with the regulation of body fluid levels and the mucosal immune response. The heatmap showed a list of proteins exclusively expressed during the different phases of HSCT: HBB, KNG1, HSPA, FGB, APOA1, PFN1, PRTN3, TMSB4X, YWHAZ, CAP1, ACTN1, CLU and ALDOA. Bioinformatics analysis implicated pathways involved in protein processing in the endoplasmic reticulum, complement and coagulation cascades, apoptosis signaling, and cholesterol metabolism. CONCLUSION: The compositional changes in saliva reflected the three phases of HSCT and demonstrated the usefulness of proteomics and computational approaches as a revolutionary field in diagnostic methods.

Extraordinary Case: Unique Presentation of an Aggressive Epstein-Barr Virus-Positive Mucocutaneous Ulcer.

ABSTRACT: Epstein-Barr virus-positive mucocutaneous ulcer is a recent and unusual type of lymphoproliferation, mostly associated with various forms of immunosuppression. In most cases, they regress spontaneously, but an increasing number of reports describe a spectral behavior of the lesion, which ranges from a simple ulcer with eosinophilia to aggressive ulcers. In these cases, Epstein-Barr virus-related lymphomas are the main differential diagnosis. We report a unique observation of this rare disease with mandibular involvement. Due to bone erosion, the patient was treated with 6 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) with complete healing of the ulcer on clinical examination and PET-scan control.

Tight junction gene expression in salivary gland tumors.

Salivary gland neoplasms comprise a heterogeneous group of lesions with multiple histological subtypes, each with distinct growth patterns, resulting in a spectrum of tumor-specific prognoses; pleomorphic adenoma (PA) and mucoepidermoid carcinoma (MEC) are the most common representatives of these neoplasms. Many studies have associated specific profiles of membrane and adhesion molecules in salivary gland tissues; these profiles appear to be relevant in tumor biology as well as be interpreted as fingerprints for tumor classification, diagnostic prognostic and therapeutic targets. One of these membrane molecule complexes are the tight junctions, composed by various proteins, in which claudins are protagonists. The aim of this study was to evaluate the expressions of genes that encode tight junction proteins (CLDN-1, -3, -4, -5, -7, and -11, occludin [OCLN], zonula occludens [TJP1, TJP2, and TJP3] and junctional adhesion molecule A [F11R]) in MEC and PA using real time RT-PCR. We observed high expression of CLDN-1 and -7 and low expression of CLDN-3, -11 and TJP2 in MEC compared to PA. PA samples demonstrated high OCLN expression when compared to MEC. CRTC1::MAML2 fusion was detected in 12 of 20 (60.0%) MEC samples and was associated with CLDN7 expression, while the absence of fusion was associated with high histological grade. Increased CLDN5 expression was associated with submandibular gland tumors. This study demonstrated differential expressions of genes encoding tight junction constituent proteins and their associations with tumor characteristics, suggesting their potential future role as diagnostic and prognostic markers.

The Role of Reflectance Confocal Microscopy in the Evaluation of Pigmented Oral Lesions and Their Relationship With Histopathological Aspects.

ABSTRACT: Oral pigmentations are a heterogeneous group and can be the result of physiological activity of oral mucosal melanocytes, secondary to exogenous causes, associated with systemic or local diseases, or due to proliferative activity of melanocytes. Their diagnosis is critical because these lesions can be markers of internal diseases or, in the case of melanocytic proliferative processes, they may represent a malignant neoplasm. In the past decade, the use of reflectance confocal microscopy, a noninvasive imaging tool, has aided the analysis of such lesions, but the establishment of firm criteria in their evaluation is still lacking. This study evaluated a series of 19 cases of pigmented oral lesions and correlated the reflectance confocal microscopy findings with histopathological classical criteria. We found 13 cases of melanotic macule, 1 of them associated with Peutz-Jeghers syndrome and 2 with Laugier-Hunzinker syndrome; 1 melanocytic nevus; 2 lentigo maligna; 2 pigmented actinic cheilitis; and 1 case of postinflammatory pigmentation secondary to a lupus erythematosus oral discoid lesion. The main difference between benign and malignant lesions was the presence of atypical proliferation in lentigo maligna. Langerhans cells with thick dendritic processes, which may be present in other benign and inflammatory pigmentations is one of the main reasons for diagnostic pitfalls.

Epithelial-mesenchymal transition related to bone invasion in oral squamous cell carcinoma.

INTRODUCTION: Bone invasion is an important prognostic factor in oral squamous cell carcinoma, leading to a lower survival rate and the use of aggressive treatment approaches. Epithelial-mesenchymal transition (EMT) is possibly involved in this process, because it is often related to mechanisms of cell motility and invasiveness. This study examined whether a panel of epithelial-mesenchymal markers are present in cases of oral squamous cell carcinoma with bone invasion and whether these proteins have any relationship with patients' clinical-pathological parameters and prognostic factors. METHODS: Immunohistochemical analysis of E-cadherin, twist, vimentin, TGFß1, and periostin was performed in paraffin-embedded samples of 62 oral squamous cell carcinoma cases. RESULTS: The analysis revealed that most cases (66%) presented with a dominant tumor infiltrative pattern in bone tissue, associated with lower survival rates, when compared with cases with a dominant erosive invasion pattern (P = 0.048). Twenty-seven cases (43%) expressed markers that were compatible with total or partial EMT at the tumor-bone interface. There was no association between evidence of total or partial EMT and other demographic or prognostic features. E-cadherin-positive cases were associated with tobacco smoking (P = 0.022); vimentin-positive cases correlated with tumors under 4 cm (P = 0.043). Twistexpression was observed in tumors with a dominant infiltrative pattern (P = 0.041) and was associated with the absence of periostin (P = 0.031). CONCLUSION: We observed evidence of total or partial EMT in oral squamous cell carcinoma bone invasion. The transcription factor twist appears to be involved in bone invasion and disease progression.