Relationship between aggressive features of oral squamous cell carcinoma and the immunoexpression of CX3CR1, CX3CL1 and ITGAV.

OBJECTIVE: We aimed to describe the association between CX3CR1, CX3CL1, and ITGAV immunoexpression with PNI and adverse oncologic outcomes in patients with OSCC. STUDY DESIGN: Expression CX3CR1, CX3CL1, and ITGAV was assessed by immunohistochemistry in a cohort of 50 paraffin-embedded resections of OSCC. Survival analysis, Cox, and binary logistic regressions were undertaken to determine the impact on patient survival and predictive value for PNI. RESULTS: CX3CL1 positive nerves exhibited a significant association with tumor budding (TB) (P = .043), whereas nerves positive for ITGAV were associated with PNI (P = .021), T3-T4 tumor size (P = .029), and III-IV stage (P = .044). Cases with ITGAV-positive nerves exhibited an odds ratio of 9.603 (P = .008) for PNI, whereas cases with CX3CL1-positive nerves exhibited and odds ratio of 4.682 (P = .033) for TB. A trend toward decreased 5-year overall survival (P = .078) and 5-year disease-specific survival (P = .09) was observed in relation to ITGAV-positive nerves. However, no independent predictors for poor survival were identified. CONCLUSIONS: The expression of ITGAV was associated with PNI and advanced disease, whereas the expression of CX3CL1 was related to TB, suggesting that ITGAV and CX3CL1 are involved in their respective developments. Therefore, further investigations are encouraged to assess the potential utility of targeted therapies against CX3CL1 receptors in OSCC.

New insights into the role of the oral leukoplakia microenvironment in malignant transformation.

Oral leukoplakia is the most frequent and potentially malignant lesion of the oral cavity. Although dysplasia grading remains the main factor for risk assessment, challenges persist in determining the exact risk of transformation, and the literature has focused on studying alternative biomarkers. The interaction between dysplastic epithelial cells and the microenvironment starts early, and the communication is mainly mediated by lymphocytes, inflammatory factors, fibroblasts, and the extracellular matrix, leading to dysplastic progression. Leukoplakia-infiltrating leukocytes (LILs) and leukoplakia-associated fibroblasts (LAFs) play crucial roles in the dysplastic microenvironment. The immune response is related to intraepithelial T lymphocyte infiltration, mechanisms of immunosuppression coordinated by regulatory T cells, M2 macrophage polarization, and increased numbers of Langerhans cells; in contrast, fibroblastic and extracellular matrix factors are associated with increased numbers of pro-tumorigenic myofibroblasts, increased expression of metalloproteinases vs. decreased expression of TIMPs, and increased expression of chemokines and other inflammatory mediators. The microenvironment offers insights into the progression of leukoplakia to carcinoma, and understanding the complexity of the oral microenvironment in potentially malignant diseases aids in determining the risk of malignant transformation and proposing new therapeutic alternatives.

Barriers to early diagnosis and management of oral cancer in Latin America and the Caribbean.

OBJECTIVE: This study aimed to explore perceived barriers to early diagnosis and management of oral cancer, as well as potential pathways for improvement in Latin America and the Caribbean (LAC). METHODS: This cross-sectional study used a self-administered online questionnaire created via the Research Electronic Data Capture platform. The survey was distributed to health professionals trained in Oral Medicine, Oral Pathology, Oral and Maxillofacial Surgery, and Dentists with clinical and academic expertise in oral potentially malignant disorder (OPMD) and oral cancer. Data obtained were systematically organized and analyzed descriptively using Microsoft Excel. RESULTS: Twenty-three professionals from 21 LAC countries participated. Major barriers included the limited implementation of OPMD and oral cancer control plans (17.4%), low compulsory reporting for OPMD (8.7%) and oral cancer (34.8%), unclear referral pathways for OPMD (34.8%) and oral cancer (43.5%), and a shortage of trained professionals (8.7%). Participants endorsed the utility of online education (100%) and telemedicine (91.3%). CONCLUSION: The survey highlights major perceived barriers to early diagnosis and management of OPMD and oral cancer in LAC, as well as potential avenues for improvement.

Oral squamous cell carcinomas and oral potentially malignant disorders: A Latin American study.

OBJECTIVE: To determine the frequency of oral squamous cell carcinoma (OSCC) associated or not with oral potentially malignant disorders (OPMD), and the epidemiological profile and traditional risk factors in Latin America. METHODS: A retrospective observational study was conducted in 17 Latin American centres. There were included cases of OSCC, analysing age, gender, OSCC and their association with previous OPMD. Clinicopathological variables were retrieved. The condition of sequential-OSCC versus OSCC-de novo (OSCC-dn) was analysed concerning the aforementioned variables. Quantitative variables were analysed using Student's t-test, and qualitative variables with chi-square. RESULTS: In total, 2705 OSCC were included with a mean age of 62.8 years old. 55.8% were men. 53.75% of the patients were smokers and 38% were common drinkers. The lateral tongue border was the most affected site (24.65%). There were regional variations in OPMD, being leukoplakia the most frequent. Of the overall 2705 OSCC cases, 81.4% corresponded to OSCC-dn, while s-OSCC were 18.6%. Regarding lip vermillion SCC, 35.7% corresponded to de novo lip SCC and 64.3% were associated with previous OPMD. CONCLUSIONS: In Latin America, OSCC-dn seems to be more frequent with regional variations of some clinical and histopathological features. Further prospective studies are needed to analyse this phenomenon.

CR5/CCL5 axis is linked to a poor outcome, and inhibition reduces metastasis in oral squamous cell carcinoma.

PURPOSE: The CCR5/CCL5 axis is essential for interactions between malignant cells and microenvironment components, promoting tumor progression in oral squamous cell carcinoma (OSCC). This study aims to evaluate the association of CCL5 and CCR5 with the behavior of oral cancer and assess the therapeutic potential of a CCR5 antagonist. METHODS: A retrospective study to analyze CCR5 and CCL5 expression on paraffin-embedded tissues was performed. In cell lines, rhCCL5 was added to induce CCR5-related pathways, and Maraviroc and shRNA against CCR5 were used to neutralize the receptor. Finally, an in vivo murine orthotopic xenograft model of tongue cancer was used to evaluate Maraviroc as an oncologic therapy. After 15 days, the mice were killed, and the primary tumors and cervical lymph nodes were analyzed. RESULTS: The expression of CCR5 was associated with clinical stage and metastasis, and CCL5 was related to overall survival. Adding rhCCL5 induced cell proliferation, while shRNA and Maraviroc reduced it in a dose-dependent manner. Maraviroc treatment also increased apoptosis and modified cytoskeletal organization. In vivo, Maraviroc reduced neck metastasis. CONCLUSIONS: The effects of CCR5 antagonists in OSCC have been poorly studied, and this study reports in vitro and in vivo evidence for the effects of Maraviroc in OSCC. Our results suggest that the CCR5/CCL5 axis plays a role in oral cancer behavior, and that its inhibition is a promising new therapy alternative.

Overexpression of heat-shock protein 47 impacts survival of patients with oral squamous cell carcinoma.

BACKGROUND: The expression of heat-shock protein 47 (HSP47) has been linked to collagen synthesis control and implicated in fibrotic disorders, but more recent studies have demonstrated its role in solid tumors. In this study, we explored the prognostic impact of HSP47 in oral squamous cell carcinomas (OSCC) and determined the in vitro effects of its loss-of-function on viability, proliferation, migration, invasion, and resistance to cisplatin of OSCC cells. METHODS: The HSP47 expression in tumor samples was assessed by immunohistochemistry in two independent cohorts totaling 339 patients with OSCC, and protein levels were associated with clinicopathological features and survival outcomes. The OSCC cell lines HSC3 and SCC9 were transduced with lentivirus expressing short hairpin RNA to stably silence HSP47 and used in assays to measure cellular viability, proliferation, migration, and invasion. RESULTS: HSP47 was overexpressed in OSCC samples, and its overexpression was significantly and independently associated with poor disease-specific survival and shortened disease-free survival in both OSCC cohorts. The knockdown of HSP47 showed no effects on cell viability or cisplatin sensitivity, but impaired significantly proliferation, migration, and invasion of OSCC cells, with stronger effects on SCC9 cells. CONCLUSION: Our results show a significant prognostic impact of HSP47 overexpression in OSCC and reveal that HSP47 inhibition impairs the proliferation, migration, and invasion of OSCC cells. HSP47 may represent a potential therapeutic target for OSCC.

Whole-exome sequencing of oral epithelial dysplasia samples reveals an association with new genes.

The genetic basis of oral epithelial (OED) is unknown, and there is no reliable method for evaluating the risk of malignant transformation. Somatic mutations are responsible for the transformation of dysplastic mucosa to invasive cancer. In addition, these genomic variations could represent objective markers of the potential for malignant transformation. We performed whole-exome sequencing of 10 OED samples from Brazilian and Chilean patients. Using public genetic repositories, we identified 41 deleterious variants that could produce high-impact changes in the amino acid structures of 38 genes. In addition, the variants were filtered according to normal skin and Native American genome profiles. Finally, 13 genes harboring 15 variants were found to be exclusively related to OED. High-grade epithelial dysplasia samples showed a tendency to accumulate highly deleterious variants. We observed that 62% of 13 OED genes identified in our study were also found in head and neck squamous cell carcinoma. Among the shared genes, eight were not identified in oral squamous cell carcinoma. To our knowledge, we have described for the first time 13 genes that are found in OED in a Latin American population, of which five genes have already been observed in oral squamous cell carcinoma. Through this study, we identified genes that may be related to basal biological functions in OED.

HER2 classification in breast cancer cells: A new explainable machine learning application for immunohistochemistry.

The immunohistochemical (IHC) evaluation of epidermal growth factor 2 (HER2) for the diagnosis of breast cancer is still qualitative with a high degree of inter-observer variability, and thus requires the incorporation of complementary techniques such as fluorescent in situ hybridization (FISH) to resolve the diagnosis. Implementing automatic algorithms to classify IHC biomarkers is crucial for typifying the tumor and deciding on therapy for each patient with better performance. The present study aims to demonstrate that, using an explainable Machine Learning (ML) model for the classification of HER2 photomicrographs, it is possible to determine criteria to improve the value of IHC analysis. We trained a logistic regression-based supervised ML model with 393 IHC microscopy images from 131 patients, to discriminate between upregulated and normal expression of the HER2 protein. Pathologists' diagnoses (IHC only) vs. the final diagnosis complemented with FISH (IHC + FISH) were used as training outputs. Basic performance metrics and receiver operating characteristic curve analysis were used together with an explainability algorithm based on Shapley Additive exPlanations (SHAP) values to understand training differences. The model could discriminate amplified IHC from normal expression with better performance when the training output was the IHC + FISH final diagnosis (IHC vs. IHC + FISH: area under the curve, 0.94 vs. 0.81). This may be explained by the increased analytical impact of the membrane distribution criteria over the global intensity of the signal, according to SHAP value interpretation. The classification model improved its performance when the training input was the final diagnosis, downplaying the weighting of the intensity of the IHC signal, suggesting that to improve pathological diagnosis before FISH consultation, it is necessary to emphasize subcellular patterns of staining.

Whole-exome sequencing of oral epithelial dysplasia samples reveals an association with new genes

Abstract The genetic basis of oral epithelial (OED) is unknown, and there is no reliable method for evaluating the risk of malignant transformation. Somatic mutations are responsible for the transformation of dysplastic mucosa to invasive cancer. In addition, these genomic variations could represent objective markers of the potential for malignant transformation. We performed whole-exome sequencing of 10 OED samples from Brazilian and Chilean patients. Using public genetic repositories, we identified 41 deleterious variants that could produce high-impact changes in the amino acid structures of 38 genes. In addition, the variants were filtered according to normal skin and Native American genome profiles. Finally, 13 genes harboring 15 variants were found to be exclusively related to OED. High-grade epithelial dysplasia samples showed a tendency to accumulate highly deleterious variants. We observed that 62% of 13 OED genes identified in our study were also found in head and neck squamous cell carcinoma. Among the shared genes, eight were not identified in oral squamous cell carcinoma. To our knowledge, we have described for the first time 13 genes that are found in OED in a Latin American population, of which five genes have already been observed in oral squamous cell carcinoma. Through this study, we identified genes that may be related to basal biological functions in OED.

Connecting multiple microenvironment proteomes uncovers the biology in head and neck cancer.

The poor prognosis of head and neck cancer (HNC) is associated with metastasis within the lymph nodes (LNs). Herein, the proteome of 140 multisite samples from a 59-HNC patient cohort, including primary and matched LN-negative or -positive tissues, saliva, and blood cells, reveals insights into the biology and potential metastasis biomarkers that may assist in clinical decision-making. Protein profiles are strictly associated with immune modulation across datasets, and this provides the basis for investigating immune markers associated with metastasis. The proteome of LN metastatic cells recapitulates the proteome of the primary tumor sites. Conversely, the LN microenvironment proteome highlights the candidate prognostic markers. By integrating prioritized peptide, protein, and transcript levels with machine learning models, we identify nodal metastasis signatures in blood and saliva. We present a proteomic characterization wiring multiple sites in HNC, thus providing a promising basis for understanding tumoral biology and identifying metastasis-associated signatures.

CD49b Targeting Inhibits Tumor Growth and Boosts Anti-tumor Immunity.

The suppressive function of T-regulatory cells (Tregs) can have a detrimental effect on immune responses against tumor cells. Within the Treg cells subset, a new non-classical population has been reported, which expresses high levels of CD49b molecule and, depending on their activation status, can also express the canonical Tregs transcription factor Foxp3. In this report, we sought to characterize Tregs subsets in a murine melanoma model and disrupt the CD49b/CD29 axis by administering an anti-CD29 antibody in tumor-bearing mice. Our data shows that whereas in the draining lymph nodes, the Tr1 cells subset composes <5% of CD4+ T cells, in the tumor, they reach ∼30% of CD4+ T cells. Furthermore, Tr1 cells share the expression of suppressive molecules, such as Nrp-1, PD-1, and CD73, which are highly expressed on Tr1 cells found in tumor-infiltrating leukocytes (TILs). Regardless of the phenotypic similarities with cTreg cells, Tr1 cells display a low proliferative activity, as shown in the kinetics and the incorporation of 5-bromodeoxyuridine (BrdU) experiments. With the intent to impact on Tr1 cells, we administered anti-CD29 antibody into tumor mice, observing that the treatment effectively inhibits tumor growth. This effect is at least mediated by the enrichment of pro-inflammatory T cells, including IFN-γ+ cTreg and IFN-γ+ Tr1 cells (with reduced expression of IL-10), plus Th1 and Tc cells. In this study, we present Tr1 cell characterization in tumor-bearing animals and introduce CD29 as a target for tumor therapy, supported by a meta-analysis indicating that CD29 is present in human biopsies.

Therapeutic Perspectives of HIV-Associated Chemokine Receptor (CCR5 and CXCR4) Antagonists in Carcinomas.

The interaction between malignant cells and the tumor microenvironment is critical for tumor progression, and the chemokine ligand/receptor axes play a crucial role in this process. The CXCR4/CXCL12 and CCR5/CCL5 axes, both related to HIV, have been associated with the early (epithelial-mesenchymal transition and invasion) and late events (migration and metastasis) of cancer progression. In addition, these axes can also modulate the immune response against tumors. Thus, antagonists against the receptors of these axes have been proposed in cancer therapy. Although preclinical studies have shown promising results, clinical trials are needed to include these drugs in the oncological treatment protocols. New alternatives for these antagonists, such as dual CXCR4/CCR5 antagonists or combined therapy in association with immunotherapy, need to be studied in cancer therapy.

Trophoblast cell surface antigen 2 expression predicts outcome in oral squamous cell carcinomas.

BACKGROUND: Trophoblast cell surface antigen 2 (TROP2) has unclear clinical role in oral squamous cell carcinomas (OSCC). Here, we investigated the association of TROP2 immunoexpression with clinicopathological parameters and survival of OSCC patients. SUBJECTS AND METHODS: Cancer-specific survival (CSS) and disease-free survival (DFS) were assessed in a cohort composed of 266 OSCC. An independent cohort with 88 OSCC samples matched with the normal oral tissue, as well as 17 metastatic lymph nodes, was used for validation. RESULTS: Multivariate analysis showed TROP2 as an independent marker of favorable prognosis for both CSS (HR: 0.60, 95% CI: 0.40-0.90, p = .01) and DFS (HR: 0.57, 95% CI: 0.36-0.89, p = .01). Furthermore, TROP2 protein expression was significantly higher in morphologically normal tissues compared to primary tumors (p < .0001) and lymph node metastases (p = .001), and it was significantly associated with CSS (HR: 0.26, 95% CI: 0.09-0.74, p = .008) in the validation cohort. A pooled mRNA analysis performed on the Oncomine™ database confirmed the underexpression in OSCC compared with normal tissues (p = .014). CONCLUSIONS: In summary, our results point to a favorable prognostic significance of TROP2 overexpression in a large cohort of oral cancer patients, suggesting it as an attractive clinical marker.

Impact of head and neck radiotherapy on the longevity of dental adhesive restorations: A systematic review and meta-analysis.

STATEMENT OF PROBLEM: Established restorative protocols for patients after head and neck radiotherapy are lacking, increasing the failure rates of dental adhesive restorations. PURPOSE: The purpose of this systematic review and meta-analysis was to analyze the evidence regarding the impact of head and neck radiotherapy on the longevity of dental adhesive restorations. MATERIAL AND METHODS: A search was performed using PubMed, Scopus, and Embase in May 2018 (updated in November 2020). Data extraction was performed regarding the percentage of restoration failure among dental adhesive materials, including glass ionomer cements, resin-modified glass ionomer cements, and composite resins. Risk of bias was assessed by the meta-analysis of statistics assessment and review instrument (MAStARI). Confidence in cumulative evidence was evaluated by the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) protocol. RESULTS: Four studies met the inclusion criteria. All included studies were classified as having a moderate risk of bias and reported results regarding class V restorations. Overall, composite resins presented lower failure rates at 2 years (30%) when compared with resin-modified glass ionomer (41%) and glass ionomer cements (57%). Meta-analysis showed that the risk of failure with glass ionomer cements was greater than with resin-modified glass ionomer cements (RR: 1.71, P<.001). Composite resins presented lower risk of failure when compared with glass ionomer (RR: 2.29, P<.001) and resin-modified glass ionomer cements (RR: 1.30, P=.03). Three studies reported results regarding fluoride compliance, which had a negative effect on the survival rates of glass ionomer and resin-modified glass ionomer cements and a positive effect on composite resin restorations. CONCLUSIONS: The results suggest that composite resin restorations associated with fluoride gel compliance seems to be the best alternative for restoring class V lesions in patients after head and neck radiotherapy. However, the results showed moderate certainty of evidence, which justifies the need for more randomized clinical trials regarding this subject.

Stress induced phosphoprotein 1 overexpression controls proliferation, migration and invasion and is associated with poor survival in oral squamous cell carcinoma.

Objective: Although there have been remarkable achievements in the molecular landscape of oral squamous cell carcinoma (OSCC) in recent years, bringing advances in the understanding of its pathogenesis, development and progression, little has been applied in the prognosis and choosing the optimal treatment. In this study, we explored the influence of the stress induced phosphoprotein 1 (STIP1), which is frequently reported to be highly expressed in many cancers, in OSCCs. Methods: STIP1 expression was assessed in the TCGA database and in two independent cohorts by immunohistochemistry. Knockdown strategy was applied in OSCC cell lines to determine the impact of STIP1 on viability, proliferation, migration and invasion. The zebrafish model was applied for studying tumor formation and metastasis in vivo. The association of STIP1 and miR-218-5p was explored by bioinformatics and mimics transfection. Results: STIP1 was highly expressed in OSCCs and significantly associated with shortened survival and higher risk of recurrence. STIP1 down-regulation decreased proliferation, migration and invasion of tumor cells, and reduced the number of metastases in the Zebrafish model. STIP1 and miR-218-5p were inversely expressed, and the transfection of miR-218-5p mimics into OSCC cells decreased STIP1 levels as well as proliferation, migration and invasion. Conclusion: Our findings show that STIP1 overexpression, which is inversely associated with miR-218-5p levels, contributes to OSCC aggressiveness by controlling proliferation, migration and invasion and is a determinant of poor prognosis.

Optogenetic control of cancer cell survival in ChR2-transfected HeLa cells.

Optogenetics is a molecular biological technique involving transfection of cells with photosensitive proteins and the subsequent study of their biological effects. The aim of this study was to evaluate the effect of blue light on the survival of HeLa cells, transfected with channelrhodopsin-2 (ChR2). HeLa wild-type cells were transfected with a plasmid that contained the gene for ChR2. Transfection and channel function were evaluated by real-time polymerase chain reaction (RT-PCR), fluorescence imaging using green fluorescent protein (GFP) and flow cytometry for intracellular calcium changes using a Fura Red probe. We developed a platform for optogenetic stimulation for use within the cell culture incubator. Different stimulation procedures using blue light (467 nm) were applied for up to 24 h. Cell survival was determined by flow cytometry using propidium iodide and rhodamine probes. Change in cell survival showed a statistically significant (p < 0.05) inverse association with the frequency and time of application of the light stimulus. This change seemed to be associated with the ChR2 cis-trans-isomerization cycle. Cell death was associated with high concentrations of calcium in the cytoplasm and stimulation intervals less than the period of isomerization. It is possible to transfect HeLa cells with ChR2 and control their survival under blue light stimulation. We suggest that this practice should be considered in the future development of optogenetic systems in biological or biomedical research.

Plasmacytoma and plasma cell myeloma affecting the jaws: A multi-institutional collaborative study.

BACKGROUND: Plasma cell neoplasms are characterized by the proliferation of a single clone of plasma cells with production of a monoclonal immunoglobulin. They can manifest as a single lesion (plasmacytoma) or as multiple lesions (multiple myeloma). METHODS: Paraffin-embedded tissue blocks of patients microscopically diagnosed with plasma cell neoplasms in the jaws were retrieved from five pathology files. Data including clinical, radiographic, microscopic and immunohistochemical findings, treatment employed and follow-up status were retrieved from the pathology reports. RESULTS: Fifty-two cases were retrieved (mean age: 59.4 years) without sex predilection. The mandible was the most affected site (67.3%), usually associated with pain and/or paresthesia (53.8%). Lesions in other bones besides the jaws were reported for 24 patients (46.2%). Radiographically, tumours usually presented as poorly defined osteolytic lesions with unilocular or multilocular images, while microscopy revealed diffuse proliferation of neoplastic plasma cells with nuclear displacement and abundant eosinophilic cytoplasm. Two cases were classified as anaplastic, and amyloid deposits were found in two other cases. Immunohistochemistry was positive for plasma cell markers and negative for CD20 and CD3, and monoclonality for kappa light chain predominated. The overall survival rate after 5 years of follow-up was 26.6%. CONCLUSION: Plasma cell neoplasms are aggressive tumours with a poor prognosis and involvement of the jaws may be the first complaint of the patient. Thus, oral pathologists, head and neck surgeons and dentists should be aware of their clinical, radiographic and microscopic manifestations.

SPINK7 expression changes accompanied by HER2, P53 and RB1 can be relevant in predicting oral squamous cell carcinoma at a molecular level.

The oral squamous cell carcinoma (OSCC), which has a high morbidity rate, affects patients worldwide. Changes in SPINK7 in precancerous lesions could promote oncogenesis. Our aim was to evaluate SPINK7 as a potential molecular biomarker which predicts OSCC stages, compared to: HER2, TP53, RB1, NFKB and CYP4B1. This study used oral biopsies from three patient groups: dysplasia (n = 33), less invasive (n = 28) and highly invasive OSCC (n = 18). The control group consisted of clinically suspicious cases later to be confirmed as normal mucosa (n = 20). Gene levels of SPINK7, P53, RB, NFKB and CYP4B1 were quantified by qPCR. SPINK7 levels were correlated with a cohort of 330 patients from the TCGA. Also, SPINK7, HER2, TP53, and RB1, were evaluated by immunohistofluorescence. One-way Kruskal-Wallis test and Dunn's post-hoc with a p < 0.05 significance was used to analyze data. In OSCC, the SPINK7 expression had down regulated while P53, RB, NFKB and CYP4B1 had up regulated (p < 0.001). SPINK7 had also diminished in TCGA patients (p = 2.10e-6). In less invasive OSCC, SPINK7 and HER2 proteins had decreased while TP53 and RB1 had increased with respect to the other groups (p < 0.05). The changes of SPINK7 accompanied by HER2, P53 and RB1 can be used to classify the molecular stage of OSCC lesions allowing a diagnosis at molecular and histopathological levels.