A 13-Gene DNA Methylation Analysis Using Oral Brushing Specimens as an Indicator of Oral Cancer Risk: A Descriptive Case Report

Analysis of genetic or epigenetic markers from saliva or brushing specimens has been proposed as a diagnostic aid to identify patients at risk of developing oral cancer. However, no reliable non-invasive molecular method for this purpose is commercially available. In the present report, we describe the potential application of a procedure based on a 13-gene DNA methylation analysis using oral brushing samples from a patient affected by oral leukoplakia who developed two metachronous oral carcinomas during the follow-up period. A positive or a negative score was calculated for each brushing sample based on a predefined cut-off value. In this patient, a positive score was detected in the oral leukoplakia diagnosed more than 2 years before the development of oral squamous cell carcinoma and subsequently in clinically healthy mucosa 8 months before the appearance of a secondary tumor. This suggests a potential role of our procedure as an indicator of oral cancer risk

Phylogenomics and systematics of botryllid ascidians, and implications for the evolution of allorecognition

Allorecognition, the ability of an organism to distinguish kin from non-kin, or self from non-self, has been studied extensively in a group of invertebrate chordates, the colonial ascidians called botryllids (Subphylum Tunicata, Class Ascidiacea, Family Styelidae). When two conspecific botryllid colonies come in contact, there are two potential outcomes to an allorecognition reaction: fusion or rejection. The rejection outcome of allorecognition varies by species, and has been classified by type (referred to as R-Type). R-Type is defined according to how far the fusion process progresses before the rejection begins, since the rejection reaction appears as an interference of the fusion process. Here, we map the evolution of R-Types onto an extended and robust phylogeny of the botryllids. In this study, we have reconstructed the largest phylogenomic tree of botryllids, including 97 samples and more than 40 different species, and mapped on it nine of the 13 species for which the R-Type is known. Based on the R-Type known in a single outgroup species (Symplegma reptans), we infer that at least R-Type B and E-like could be ancestral to the Botrylloides/Botryllus group. However, the application of ancestral character state reconstructions does not provide conclusive results since several clades show more than one equiparsimonious R-Type state. Notably, all R-Type A species are clustered together and certainly evolved later than other R-Types. Our phylogenomic tree has been built on 177 nuclear loci and nearly all clades are well supported. Moreover, our phylogenetic analyses also take into account the results of species delimitation analyses based on the mitochondrial COI gene and of careful morphological analyses of the samples. The implementation of this integrated taxonomic approach, combining morphological as well as nuclear and mitochondrial data, has allowed the description of six new species, and the identification of a number of putative unnamed taxa. Thus, our results also demonstrate the existence of an unexplored hidden diversity within botryllids

Shared epigenetic alterations between oral cancer and periodontitis: A preliminary study

Introduction: We recently developed a non-invasive sampling procedure for oral squamous cell carcinoma (OSCC) detection based on DNA methylation analysis of a panel of 13 genes. Oral cancer, as well as acute and chronic inflammatory diseases, may influence the methylation level of several genes in the oral cavity. In the present study, we evaluated the presence of periodontal disease (PD) and the methylation status using our 13-gene panel. Methods: Oral brushing specimens were collected from three different patient groups: 23 gingival OSCC patients, 15 patients affected by PD, and 15 healthy volunteers lacking evidence of PD. DNA methylation analysis was performed and each sample was determined to be positive or negative based on a predefined cut-off value. Results: Positive results were found for 23/23 OSCC patients, 3/15 PD patients, and 0/15 samples from healthy volunteers. The GP1BB and MIR193 genes in the PD group exhibited mean methylation levels similar to OSCC patients. ZAP70 showed different methylation levels among three groups. Conclusion: Preliminary data identified shared epigenetic alterations between PD and OSCC patients in two inflammatory genes (GP1BB and MIR193). This study may help to identify potential links between the two diseases and serve as a starting point for the future research focused on pathogenesis

Beyond head and neck cancer: The relationship between oral microbiota and tumour development in distant organs

An altered oral microbiota has been linked with the development of several oral diseases, such as dental caries, periodontal disease, and oral stomatitis. Moreover, poor oral health has been linked to head and neck cancer, particularly oral cancer. In recent years a growing number of studies indicate that oral microbiota could be involved in the development of primary tumours outside of head and neck region. The aim of this article is to review the recent studies based on high-throughput technology to present evidences of a relationship between oral microbiota and \u201cnon-head and neck tumours.\u201d Oral dysbiosis seem to be more pronounced in patients with tumours of gastrointestinal tract, in particular oesophageal, gastric, pancreatic, and colorectal cancers, paving the way for developing specific oral microbiota test to allow early cancer detection. Regarding other tumour types, the results are promising but highly preliminary and still debated. Currently, there are several factors that limit the generalization of the results, such as the small sample size, the lack of adequate clinical information about patients, the different sequencing techniques used, and biological sample heterogeneity. Although only at the beginning, the analysis of oral microbiota could be the next step in the evolution of cancer therapy and will help clinicians to develop individualised approaches to cancer prevention and treatment

CD200 as a Potential New Player in Inflammation during Rotator Cuff Tendon Injury/Repair: An In Vitro Model

Rotator cuff tendon (RCT) disease results from multifactorial mechanisms, in which inflammation plays a key role. Pro-inflammatory cytokines and tendon stem cell/progenitor cells (TSPCs) have been shown to participate in the inflammatory response. However, the underlying molecular mechanism is still not clear. In this study, flow cytometry analyses of different subpopulations of RCT-derived TSPCs demonstrate that after three days of administration, TNFα alone or in combination with IFNγ significantly decreases the percentage of CD146+CD49d+ and CD146+CD49f+ but not CD146+CD109+ TSPCs populations. In parallel, the same pro-inflammatory cytokines upregulate the expression of CD200 in the CD146+ TSPCs population. Additionally, the TNFα/IFNγ combination modulates the protein expression of STAT1, STAT3, and MMP9, but not fibromodulin. At the gene level, IRF1, CAAT (CAAT/EBPbeta), and DOK2 but not NF-κb, TGRF2 (TGFBR2), and RAS-GAP are modulated. In conclusion, although our study has several important limitations, the results highlight a new potential role of CD200 in regulating inflammation during tendon injuries. In addition, the genes analyzed here might be new potential players in the inflammatory response of TSPCs

Expression profiling of microRNAs and isomiRs in conventional central chondrosarcoma

Conventional central chondrosarcoma (CCC) is a malignant bone tumor that is characterized by the production of chondroid tissue. Since radiation therapy and chemotherapy have limited effects on CCC, treatment of most patients depends on surgical resection. This study aimed to identify the expression profiles of microRNAs (miRNAs) and isomiRs in CCC tissues to highlight their possible participation to the regulation of pathways critical for the formation and growth of this type of tumor. Our study analyzed miRNAs and isomiRs from Grade I (GI), Grade II (GII), and Grade III (GIII) histologically validated CCC tissue samples. While the different histological grades shared a similar expression profile for the top abundant miRNAs, we found several microRNAs and isomiRs showing a strong different modulation in GII + GIII vs GI grade samples and their involvement in tumor biology could be consistently hypothesized. We then in silico validated these differently expressed miRNAs in a larger chondrosarcoma public dataset and confirmed the expression trend for 17 out of 34 miRNAs. Our results clearly suggests that the contribution of miRNA deregulation, and their targeted pathways, to the progression of CCC could be relevant and strongly indicates that when studying miRNA deregulation in tumors, not only the canonical miRNAs, but the whole set of corresponding isomiRs should be taken in account. Improving understanding of the precise roles of miRNAs and isomiRs over the course of central chondrosarcoma progression could help identifying possible targets for precision medicine therapeutic intervention

An Evolutionary Cancer Epigenetic Approach Revealed DNA Hypermethylation of Ultra-Conserved Non-Coding Elements in Squamous Cell Carcinoma of Different Mammalian Species

BACKGROUND: Ultra-conserved non-coding elements (UCNEs) are genomic sequences that exhibit > 95% sequence identity between humans, mammals, birds, reptiles, and fish. Recent findings reported their functional role in cancer. The aim of this study was to evaluate the DNA methylation modifications of UNCEs in squamous cell carcinoma (SCC) from different mammal species. METHODS: Fifty SCCs from 26 humans, 17 cats, 3 dogs, 1 horse, 1 bovine, 1 badger, and 1 porcupine were investigated. Fourteen feline stomatitis and normal samples from 36 healthy human donors, 7 cats, 5 dogs, 5 horses, 2 bovines and 1 badger were collected as normal controls. Bisulfite next generation sequencing evaluated the DNA methylation level from seven UCNEs (uc.160, uc.283, uc.416, uc.339, uc.270, uc.299, and uc.328). RESULTS: 57/59 CpGs were significantly different according to the Kruskal-Wallis test (p < 0.05) comparing normal samples with SCC. A common DNA hypermethylation pattern was observed in SCCs from all the species evaluated in this study, with an increasing trend of hypermethylation starting from normal mucosa, through stomatitis to SCC. CONCLUSIONS: Our findings indicate that UCNEs are hypermethylated in human SCC, and this behavior is also conserved among different species of mammals

Extracellular vesicles from rat-bone-marrow mesenchymal stromal/stem cells improve tendon repair in rat Achilles tendon injury model in dose-dependent manner: A pilot study

Mesenchymal stromal/stem cells (MSCs) are increasingly employed for tissue regeneration, largely mediated through paracrine actions. Currently, extracellular vesicles (EVs) released by MSCs are major mediators of these paracrine effects. We evaluated whether rat-bone-marrow-MSC-derived EVs (rBMSCs-EVs) can ameliorate tendon injury in an in vivo rat model. Pro-collagen1A2 and MMP14 protein are expressed in rBMSC-EVs, and are important factors for extracellular-matrix tendon-remodeling. In addition, we found pro-collagen1A2 in rBMSC-EV surface-membranes by dot blot. In vitro on cells isolated from Achilles tendons, utilized as rBMSC -EVs recipient cells, EVs at both low and high doses induce migration of tenocytes; at higher concentration, they induce proliferation and increase expression of Collagen type I in tenocytes. Pretreatment with trypsin abrogate the effect of EVs on cell proliferation and migration, and the expression of collagen I. When either low- or high-dose rBMSCs-EVs were injected into a rat-Achilles tendon injury-model (immediately after damage), at 30 days, rBMSC-EVs were found to have accelerated the remodeling stage of tendon repair in a dose-dependent manner. At histology and histomorphology evaluation, high doses of rBMSCs-EVs produced better restoration of tendon architecture, with optimal tendon-fiber alignment and lower vascularity. Higher EV-concentrations demonstrated greater expression of collagen type I and lower expression of collagen type III. BMSC-EVs hold promise as a novel cell-free modality for the management of tendon injuries

The use of ciclosporin in paediatric inflammatory bowel disease: an Italian experience

Aim: To asses the efficacy and safety of ciclosporin in a paediatric population with inflammatory bowel disease. Patients and methods: Twenty-three Italian children treated with ciclosporin were studied retrospectively. The indications for treatment were severe unresponsive colitis, chronic active colitis or severe fistulizing Crohn's disease. The treatment duration, follow-up and causes of drug discontinuation were assessed. Results: Sixteen patients were treated intravenously for a mean time of 10 +/- 7 days (1-24 days) and 19 orally for a mean time of 133 days (17-660 days). The mean follow-up of all patients was 13.2 months. Ciclosporin was totally ineffective, being discontinued for surgery, in nine of 23 patients (39%); it was discontinued for partial response in three patients (13%). During treatment, clinical remission was achieved in eight children (35%) and maintained after drug withdrawal in four (17%). In severe unresponsive colitis, urgent colectomy was avoided in 12 (85%) of 14 patients who tolerated the drug. Side-effects appeared in six of 23 patients (26%), and three (13%) required ciclosporin to be discontinued due to neurotoxicity. Conclusions: Ciclosporin shows disappointing long-term results in the treatment of refractory inflammatory bowel disease, but can play an important role in preventing urgent surgery in unresponsive severe colitis. Severe side-effects can occur

Pre-operative evaluation of dna methylation profile in oral squamous cell carcinoma can predict tumor aggressive potential

Background: Prognosis of oral squamous cell carcinoma (OSCC) is difficult to exactly assess on pre-operative biopsies. Since OSCC DNA methylation profile has proved to be a useful pre-operative diagnostic tool, the aim of the present study was to evaluate the prognostic impact of DNA methylation profile to discriminate OSCC with high and low aggressive potential. Methods: 36 OSCC cases underwent neoplastic cells collection by gentle brushing of the lesion, before performing a pre-operative biopsy. The CpG islands methylation status of 13 gene (ZAP70, ITGA4, KIF1A, PARP15, EPHX3, NTM, LRRTM1, FLI1, MiR193, LINC00599, MiR296, TERT, GP1BB) was studied by bisulfite Next Generation Sequencing (NGS). A Cox proportional hazards model via likelihood-based component-wise boosting was used to evaluate the prognostic power of the CpG sites. Results: The boosting estimation identified five CpGs with prognostic significance: EPHX3-24, EPHX3-26, ITGA4-3, ITGA4-4, and MiR193-3. The combination of significant CpGs provided promising results for adverse events prediction (Brier score = 0.080, C-index = 0.802 and AUC = 0.850). ITGA4 had a strong prognostic power in patients with early OSCC. Conclusions: These data confirm that the study of methylation profile provides new insights into the molecular mechanisms of OSCC and can allow a better OSCC prognostic stratification even before surgery