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.

A Novel Preclinical In Vitro 3D Model of Oral Carcinogenesis for Biomarker Discovery and Drug Testing.

This study aimed to develop an in vitro three-dimensional (3D) cell culture model of oral carcinogenesis for the rapid, scalable testing of chemotherapeutic agents. Spheroids of normal (HOK) and dysplastic (DOK) human oral keratinocytes were cultured and treated with 4-nitroquinoline-1-oxide (4NQO). A 3D invasion assay using Matrigel was performed to validate the model. RNA was extracted and subjected to transcriptomic analysis to validate the model and assess carcinogen-induced changes. The VEGF inhibitors pazopanib and lenvatinib were tested in the model and were validated by a 3D invasion assay, which demonstrated that changes induced by the carcinogen in spheroids were consistent with a malignant phenotype. Further validation was obtained by bioinformatic analyses, which showed the enrichment of pathways associated with hallmarks of cancer and VEGF signalling. Overexpression of common genes associated with tobacco-induced oral squamous cell carcinoma (OSCC), such as MMP1, MMP3, MMP9, YAP1, CYP1A1, and CYP1B1, was also observed. Pazopanib and lenvatinib inhibited the invasion of transformed spheroids. In summary, we successfully established a 3D spheroid model of oral carcinogenesis for biomarker discovery and drug testing. This model is a validated preclinical model for OSCC development and would be suitable for testing a range of chemotherapeutic agents.

Immunoexpression of oral brush biopsy enhances the accuracy of diagnosis for oral lichen planus and lichenoid lesions.

BACKGROUND: This study assessed the efficacy of using oral liquid-based brush cytology (OLBC) coupled with immunostained cytology-derived cell-blocks, quantified using machine-learning, in the diagnosis of oral lichen planus (OLP). METHODS: Eighty-two patients diagnosed clinically with either OLP or oral lichenoid lesion (OLL) were included. OLBC samples were obtained from all patients before undergoing surgical biopsy. Liquid-based cytology slides and cell-blocks were prepared and assessed by cytomorphology and immunocytochemistry for four antibodies (Ki-67, BAX, NF-κB-p65, and AMACR). For comparison purposes, a sub-group of 31 matched surgical biopsy samples were selected randomly and assessed by immunohistochemistry. Patients were categorized according to their definitive diagnoses into OLP, OLL, and clinically lichenoid, but histopathologically dysplastic lesions (OEDL). Machine-learning was utilized to provide automated quantification of positively stained protein expression. RESULTS: Cytomorphological assessment was associated with an accuracy of 77.27% in the distinction between OLP/OLL and OEDL. A strong concordance of 92.5% (κ = 0.84) of immunostaining patterns was evident between cell-blocks and tissue sections using machine-learning. A diagnostic index using a Ki-67-based model was 100% accurate in detecting lichenoid cases with epithelial dysplasia. A BAX-based model demonstrated an accuracy of 92.16%. The accuracy of cytomorphological assessment was greatly improved when it was combined with BAX immunoreactivity (95%). CONCLUSIONS: Cell-blocks prepared from OLBC are reliable and minimally-invasive alternatives to surgical biopsies to diagnose OLLs with epithelial dysplasia when combined with Ki-67 immunostaining. Machine-learning has a promising role in the automated quantification of immunostained protein expression.

Prevalence of the JP2 genotype of Aggregatibacter actinomycetemcomitans in the world population: a systematic review.

PURPOSE: To investigate the global prevalence of the JP2 genotype of Aggregatibacter actinomycetemcomitans (Aa). METHODS: A comprehensive electronic search of databases, PUBMED, MEDLINE, EMBASE, BIOSIS, and SCOPUS, was conducted up to August 2021. All published articles and studies were considered, excluding animal studies, editorials, personal opinions, letters to editor, conference abstracts, posters, and those studies without full text. The primary objective of this systematic review was to determine the presence of the JP2 genotype of Aa in the world population. RESULTS: A total of 295 articles were identified, of which 62 were preselected, and 51 were finally included in this review. Due to variable study designs and high heterogeneity, a meta-analysis was not conducted. A total of 9744 subjects were screened for the presence of the JP2 genotype of Aa worldwide, and only 621 cases were found positive. CONCLUSIONS: A relatively high presence of JP2 genotype of Aa was found in subjects from South America, North America, and Africa. There were no studies estimating the presence of the JP2 genotype of Aa in the Oceania region. The heterogeneity and quality of the included publications suggest that caution should be exercised when interpreting the data and that there remains an important need for additional evidence. CLINICAL RELEVANCE: Periodontitis is a highly prevalent inflammatory oral disorder with substantial aesthetic, functional, and psychological implications for patients. The JP2 genotype of Aa is implicated in the pathogenesis of periodontitis. To the best of our knowledge, there is a lack of systematic reviews estimating the presence of the JP2 genotype of Aa in the global population. We identified a relatively high presence of the JP2 genotype of Aa in specific geographic areas of the world, and we propose that cross-sectional and longitudinal studies are lacking in the Oceania region and need to be conducted to estimate the presence of the JP2 genotype of Aa in this region.

Oral lichen planus has a very low malignant transformation rate: A systematic review and meta-analysis using strict diagnostic and inclusion criteria.

BACKGROUND: The malignant transformation (MT) potential of oral lichen planus (OLP) has sparked heated debates for almost a century, despite the fact that global figures of OLP prevalence and oral cancer incidence do not support an association mathematically. In this study, we performed a systematic review and meta-analysis, using strict inclusion criteria, to more precisely assess the malignant potential rate of OLP and the influence of associated risk factors. METHODS: All reports that documented MT of OLP and published in the English language until January 2020 were included if they met the following strict criteria: (a) the presence of a properly verified OLP diagnosis, (b) a clear description of the cancerous lesion developing at the same site as the verified OLP lesion; and (c) a follow-up period of a minimum of 6 months prior to carcinoma development. RESULTS: Thirty-three studies were included in this analysis with a total of 12 838 OLP patients. Of these, 151 cases were initially considered to have progressed to carcinoma (1.2%). However, after applying strict criteria, only 56 cases were considered to have undergone MT from OLP (0.44%). The risk of MT was significantly higher among OLP patients who smoked (OR = 4.62), consumed alcohol (OR = 3.22), were seropositive for HCV (OR = 3.77) and/or displayed a red OLP subtype (OR = 0.37). CONCLUSIONS: Our results suggest that the reported OLP malignant transformation rates are exaggerated, and these do not reflect the actual clinical course of the disease according to strict clinical and histopathological criteria.

A machine-learning algorithm for the reliable identification of oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is a relatively common oral disorder which shares clinical and histopathological features with other lichenoid lesions, leading to considerable inter-observer disagreement. This negatively impacts understanding of the pathogenesis and malignant transformation potential of this condition. METHODS: Artificial intelligence was employed to create a machine-learning artificial neural network to identify and quantify mononuclear cells and granulocytes within the inflammatory infiltrates in digitized hematoxylin and eosin microscopic slides. Twenty-four regions of interest were extracted from OLP cases for learning purposes and validated on a retrospective cohort of 130 cases. All cases were related to patients with confirmed diagnoses of OLP, oral lichenoid lesions (OLLs), or oral epithelial dysplasia (OED) with lichenoid host response. RESULTS: The number of inflammatory cells was statistically significantly higher in OLP compared to OLLs or OED with lichenoid host response (p < 0.0005). The proposed machine-learning method was reliably capable of detecting OLP cases based on the number of inflammatory cells and the number of mononuclear cells with an area under the curve of 0.982 and 0.988, respectively. Identifying a cut-off point between OLP and other lichenoid conditions based on the number of mononuclear cells showed a sensitivity of 100% and an accuracy of 94.62%. CONCLUSION: Artificial intelligence has shown promising outcomes and provides a robust approach to enhance the accuracy of anatomical pathologists in accurately diagnosing OLP using features of disease pathogenesis.

Three-Dimensional Cell Culture Models to Investigate Oral Carcinogenesis: A Scoping Review.

Three-dimensional (3-D) cell culture models, such as spheroids, organoids, and organotypic cultures, are more physiologically representative of the human tumor microenvironment (TME) than traditional two-dimensional (2-D) cell culture models. They have been used as in vitro models to investigate various aspects of oral cancer but, to date, have not be widely used in investigations of the process of oral carcinogenesis. The aim of this scoping review was to evaluate the use of 3-D cell cultures in oral squamous cell carcinoma (OSCC) research, with a particular emphasis on oral carcinogenesis studies. Databases (PubMed, Scopus, and Web of Science) were systematically searched to identify research applying 3-D cell culture techniques to cells from normal, dysplastic, and malignant oral mucosae. A total of 119 studies were included for qualitative analysis including 53 studies utilizing spheroids, 62 utilizing organotypic cultures, and 4 using organoids. We found that 3-D oral carcinogenesis studies had been limited to just two organotypic culture models and that to date, spheroids and organoids had not been utilized for this purpose. Spheroid culture was most frequently used as a tumorosphere forming assay and the organoids cultured from human OSCCs most often used in drug sensitivity testing. These results indicate that there are significant opportunities to utilize 3-D cell culture to explore the development of oral cancer, particularly as the physiological relevance of these models continues to improve.

Malignant transformation rate of oral leukoplakia in an Australian population.

OBJECTIVES: Oral leukoplakia (OLK) is one of the most common oral potentially malignant lesions (OPMD) and is reported to undergo malignant transformation (MT) to oral squamous cell carcinoma (OSCC) at rates of between 0.13% and 34%. This study seeks to determine the proportion of OLK lesions that develop into OSCC in an Australian population and assess the risk factors associated with this transformation. METHODS: The study is a retrospective audit of patients from a private oral medicine clinic, diagnosed with OLK using clinical and histopathological data between 2006 and 2014. Patients were cross-matched with Cancer Registry data for OSCC, and the rate and time to malignant transformation were determined. RESULTS: Oral leukoplakia patients with histopathological confirmation of their lesions underwent MT at a rate of 1.49% (3/202), with an average time to MT (TMT) of 5.2 years. When patients without histopathological confirmation were assessed, the MT rate was slightly less (1.30%; 4.9 years TMT). Patients who transformed were more likely to be older females with a history of smoking and alcohol use, with OLK present on the tongue or floor of mouth. The rate of oral epithelial dysplasia (OED) in the transformed group was surprisingly low (one third). CONCLUSIONS: Oral leukoplakia is at a moderate risk of malignant transformation which can be reduced by careful management. Current tools for identifying high-risk OLK, including histopathological assessment of OED, may not capture all lesions that undergo MT and should be supplemented by unbiased molecular biomarkers.

Oral lichenoid dysplasia and not oral lichen planus undergoes malignant transformation at high rates.

OBJECTIVES: Oral potentially malignant disorders (OPMD) include a variety of mucosal lesions such as oral lichen planus (OLP), oral lichenoid lesions (OLL) and oral lichenoid dysplasia (OLD). Their rate of malignant transformation ranges from 0% to 34% and is dependent on OPMD type, lesion site and a range of risk factors. This study seeks to determine the proportion of oral lichenoid conditions that transform into oral squamous cell carcinoma (OSCC) in an Australian population. METHODS: The study is a retrospective audit of patients from a private oral medicine clinic, diagnosed with OLP, OLL or OLD using clinical and histopathological data between 2006 and 2014. Patients were cross-matched with Cancer Registry data for OSCC, and the rate and time to malignant transformation determined. RESULTS: OLP and OLL patients displayed a low risk of malignant transformation; 0.49% (1/206) for OLP and 0% (0/31) for OLL. In contrast, OLD patients, all of whom presented clinically as OLP, were at much higher risk with 6.81% (3/44) developing OSCC over an average time of 4.6 years (±2.4 SD). Rates of smoking and alcohol consumption were no higher in OLD patients compared to others. CONCLUSIONS: Compared with other oral lichenoid conditions, OLD lesions are at a particularly high risk of malignant transformation and should be managed based on the presence of dysplasia and not the lichenoid inflammatory infiltrate. OLP demonstrates a relatively low rate of malignant transformation. Diagnostic histopathology is important for discriminating OLP from OLD.

The role of hypoxia in oral cancer and potentially malignant disorders: a review.

Oral and oropharyngeal cancer are major health problems globally with over 500 000 new cases diagnosed annually. Despite the fact that oral cancer is a preventable disease and has the potential for early detection, the overall survival rate remains at around 50%. Most oral cancer cases are preceded by a group of clinical lesions designated 'potentially malignant disorders'. It is difficult to predict if and when these lesions may transform to malignancy, and in turn it is difficult to agree on appropriate management strategies. Understanding underlying molecular pathways would help in predicting the malignant transformation of oral potentially malignant disorders and ultimately identifying effective methods for early detection and prevention of oral cancer. Reprogramming energy metabolism is an emerging hallmark of cancer that is predominantly controlled by hypoxia-induced genes regulating angiogenesis, tumour vascularization, invasion, drug resistance and metastasis. This review aims to highlight the role of hypoxia in oral carcinogenesis and to suggest future research implications in this arena.

The solution structure of the pentatricopeptide repeat protein PPR10 upon binding atpH RNA.

The pentatricopeptide repeat (PPR) protein family is a large family of RNA-binding proteins that is characterized by tandem arrays of a degenerate 35-amino-acid motif which form an α-solenoid structure. PPR proteins influence the editing, splicing, translation and stability of specific RNAs in mitochondria and chloroplasts ZEA MAYS: PPR10 is amongst the best studied PPR proteins, where sequence-specific binding to two RNA transcripts, ATPH: and PSAJ, HAS BEEN DEMONSTRATED TO FOLLOW: a recognition code where the identity of two amino acids per repeat determines the base-specificity. A recently solved ZmPPR10: PSAJ: complex crystal structure suggested a homodimeric complex with considerably fewer sequence-specific protein-RNA contacts than inferred PREVIOUSLY: Here we describe the solution structure of the ZmPPR10: ATPH: complex using size-exclusion chromatography-coupled synchrotron small-angle X-ray scattering (SEC-SY-SAXS). Our results support prior evidence that PPR10 binds RNA as a monomer, and that it does so in a manner that is commensurate with a canonical and predictable RNA-binding mode across much of the RNA-protein interface.

The design and structural characterization of a synthetic pentatricopeptide repeat protein.

Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid α-hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to the de novo design of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2 Šresolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.

The promises of PCSK9 inhibition.

PURPOSE OF REVIEW: In the past 10 years, the LDL receptor inhibitor proprotein convertase subtilisin kexin type 9 (PCSK9) has emerged as a validated target for lowering plasma LDL cholesterol levels. Here we review the most recent reports on PCSK9 out of a total of 500 publications published in print or online before March 2013 and indexed on PubMed. RECENT FINDINGS: All published in 2012, phase I and II clinical trials demonstrate that fully human monoclonal antibodies targeting PCSK9 dramatically reduce LDL-C and enable patients to reach their target goals, without severe or serious safety issues. SUMMARY: This review summarizes the discovery of PCSK9, its original mode of action as a secreted inhibitor of the LDL receptor, as well as its genetic regulation by statins. We then focus on the major results from the 2012 phase I and II PCSK9 inhibitor clinical trials. We also review the recent in-vivo studies demonstrating the potential cardiovascular benefits of long-term PCSK9 inhibition and discuss its potential side-effects.

Genome-wide characterization of the mutational landscape of proliferative verrucous leukoplakia.

OBJECTIVES: Proliferative verrucous leukoplakia (PVL) is a rare but highly aggressive variant of oral leukoplakia that almost inevitably progresses to oral squamous cell carcinoma (OSCC). The aims of this study were to perform whole exome sequencing of a cohort of patients diagnosed with PVL and identify potential mutational profiles and pathways in this disorder. STUDY DESIGN: A total of 12 oral cavity mucosal biopsies from 6 patients with oral lesions clinically compatible with PVL were used. Of these, 9 were diagnosed as dysplasia, 1 OSCC, and 2 hyperkeratosis/hyperplasia. Exome sequencing used the Ion AmpliSeq Exome platform. Ion Reporter software was used for variant calling, annotation, and filtering. Analysis and visualization of somatic mutations was carried out using the MAFtools R package. RESULTS: Following exome sequencing and mutational profiling, we analyzed the profiles for cancer associated genes and signatures. Genes previously associated with OSCC, including HYDIN, MUC16, MAML3, CDKN2A, FAT1, and CASP8, were mutated in multiple samples. Several DNA damage repair genes including PARP1 were mutated in PVL samples. NOTCH and Hippo pathways were the most frequently impacted by mutation. CONCLUSIONS: This genome wide characterization of premalignant PVL identifies both known and potentially novel oncogenic mechanisms in this disorder.

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.

PD-1/PD-L1, Treg-related proteins, and tumour-infiltrating lymphocytes are associated with the development of oral squamous cell carcinoma.

Cancer immunomodulation has been implicated in the development of oral squamous cell carcinoma (OSCC), however the role of specific immunomodulatory proteins is not completely understood, particularly in the early stages of the disease. Oral potentially malignant disorders such as leukoplakia commonly precede OSCC but not all will undergo malignant transformation. The aim of this study was to evaluate the diagnostic utility of specific immunomodulator proteins and their role in the progression of OSCC. Samples from 101 patients were included in the study. Cases were classified based on histopathology into four groups: non-dysplastic epithelial hyperplasia/keratosis, low-grade dysplasia, high-grade dysplasia, and OSCC. The PD-1/PD-L1 pathway, as well as regulatory T cell (Treg)-related proteins including FOXP3, TGF-ß, IL-6, and IL-10 were immunohistochemically quantified. The number of tumour-infiltrating lymphocytes (TILs) was also assessed for each case. Multinominal regression models were undertaken to assess the significance of each protein in predicting the histopathological grade of oral epithelial disorders, and three diagnostic models were assessed. Significant positive associations were found between the immunoreactive score of each protein and the histopathological grade, p<0.05 suggesting that the PD-1/PD-L1 pathway, Treg-related proteins, and TILs are associated with the development of OSCC. Diagnostic models using the investigated proteins and TILs predicted the grade of oral epithelial disorder, p<0.05. The associated accuracy of this approach was 84.92%. Our findings support the notion that immunomodulation events may play a role in evading the immune system and contributing to potential malignant transformation of oral epithelial disorders. Our data also provide supporting evidence for the potential application of immune checkpoint inhibitors in the chemoprevention of OSCC. Further longitudinal studies to assess individual T-cell populations within the immune microenvironment of various oral potentially malignant disorders are warranted.

Effect of post-curing light exposure time on the physico-mechanical properties and cytotoxicity of 3D-printed denture base material.

OBJECTIVE: This study investigated the effect of post curing light exposure time on the physico-mechanical properties and cytotoxicity of a 3D-printed PMMA-based denture material in comparison to a conventional heat-cured alternative as a control. METHODS: 3D-printed specimens were fabricated followed by post-curing for 0, 5, 10 or 20 min at 200 W and light wavelength range of 390-540 nm. Heat-cured specimens were fabricated using a standard protocol. Specimens were placed in artificial saliva at 37 â„ƒ for 48 h (immediate groups) and 6 months (aged group), then evaluated flexural strength/modulus, fracture toughness, microhardness, and degree of conversion. Water sorption and solubility was assessed after 28 days. Flexural strength, flexural modulus, and fracture toughness were tested through three-point bending tests, while the surface hardness was tested using Vickers's test. Fractured specimens were viewed by scanning electron microscope (SEM). Cytotoxicity in term of cell viability was evaluated using human oral fibroblasts. RESULTS: Flexural strength/modulus, fracture toughness and surface hardness significantly improved with the increase in light curing time up to 20 min. The same pattern of improvement was found with degree of conversion, water sorption, solubility, and cell viability. There was no significant difference (p < 0.01) between heat-cured material and 3D specimens post-cured for 20 min in term of flexural strength/modulus, surface hardness, and degree of conversion at the two-storage time points. SIGNIFICANCE: Generally, the physico-mechanical properties of the 3D-printed denture base material improve as post curing time increases up to 20 min which exhibited comparable performance as the conventional heat-cured control.

Inhibition of Vascular Inflammation by Apolipoprotein A-IV.

Background: Apolipoprotein (apo) A-IV, the third most abundant apolipoprotein in human high density lipoproteins (HDLs), inhibits intestinal and systemic inflammation. This study asks if apoA-IV also inhibits acute vascular inflammation. Methods: Inflammation was induced in New Zealand White rabbits by placing a non-occlusive silastic collar around the common carotid artery. A single 1 mg/kg intravenous infusion of lipid-free apoA-IV or saline (control) was administered to the animals 24 h before collar insertion. The animals were euthanised 24 h post-collar insertion. Human coronary artery cells (HCAECs) were pre-incubated with reconstituted HDLs containing apoA-IV complexed with phosphatidylcholine, (A-IV)rHDLs, then activated by incubation with tumour necrosis factor (TNF)-α. Cell surface vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in the TNF-α-activated HCAECs was quantified by flow cytometry. VCAM-1, ICAM-1 and 3ß-hydroxysteroid-Δ24 reductase (DHCR24) mRNA levels were quantified by real time PCR. Results: Apolipoprotein ApoA-IV treatment significantly decreased collar-induced endothelial expression of VCAM-1, ICAM-1 and neutrophil infiltration into the arterial intima by 67.6 ± 9.9% (p < 0.01), 75.4 ± 6.9% (p < 0.01) and 74.4 ± 8.5% (p < 0.05), respectively. It also increased endothelial expression of DHCR24 by 2.6-fold (p < 0.05). Pre-incubation of HCAECs with (A-IV)rHDLs prior to stimulation with TNF-α inhibited VCAM-1 and ICAM-1 protein levels by 62.2 ± 12.1% and 33.7 ± 5.7%, respectively. VCAM-1 and ICAM-1 mRNA levels were decreased by 55.8 ± 7.2% and 49.6 ± 7.9%, respectively, while DHCR24 mRNA expression increased by threefold. Transfection of HCAECs with DHCR24 siRNA attenuated the anti-inflammatory effects of (A-IV)rHDLs. Pre-incubation of TNF-α-activated HCAECs with (A-IV)rHDLs also inhibited nuclear translocation of the p65 subunit of nuclear factor-κB (NF-κB), and decreased IκBα phosphorylation. Conclusion: These results indicate that apoA-IV inhibits vascular inflammation in vitro and in vivo by inhibiting NF-κB activation in a DHCR24-dependent manner.

Lichenoid dysplasia is not a distinct pathological entity.

OBJECTIVES: Oral Lichenoid Dysplasia (OLD) is a controversial histological term applied to lesions that display features of oral lichen planus (OLP) and oral epithelial dysplasia (OED). In this study we investigated the molecular profiles of OLD, OLP and OED to determine whether OLD exists as a distinct pathological entity. MATERIALS AND METHODS: Samples from patients presenting with lesions diagnosed histologically as OLP, OLD or OED underwent RNA sequencing followed by differential gene expression, functional enrichment and network analysis, sparse partial least squares discriminant analysis, and immune cell phenotypic estimation. RESULTS: Unsupervised clustering demonstrated a group of genes with high expression in OLP and OLD, and low expression in OED, predominantly involved in inflammatory processes. Many genes were significantly differentially expressed between either OLD or OLP and OED, but few between OLD and OLP. Functional enrichment showed significant pathways and ontologies related to inflammatory signalling and immune response between OLD or OLP and OED. Broad commonality was found between OLP and OLD in upregulation of specific immune system pathways. Classifying models discriminated histologically diagnosed OLD from OED based upon molecular data alone. Bioinformatic profiling showed that immune cell populations in OLP and OLD were consistent, and distinct from OED. CONCLUSION: Molecular data shows that OLD is not a distinct pathological entity. Its transcriptomic and immunophenotypic profile is similar to OLP and distinct from OED. We recommend that oral lichenoid dysplasia not be used as a distinct pathological entity. Our data further supports exclusion of dysplasia in diagnosis of OLP.

Exome sequencing of oral leukoplakia and oral squamous cell carcinoma implicates DNA damage repair gene defects in malignant transformation.

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, BRCA2 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).