Effects of fermented wheat germ extract on oral cancer cells and research of biomarkers for diagnosis and prognosis of oral cancer

Oral squamous cell carcinoma (OSCC) represents one of the most aggressive types of cancer. The disease occurs when the accumulation of multiple genetic mutations in the oral epithelial cells leads to an irreversible damage of DNA and the cells lose their normal life cycle. The prognosis correlates to several factors and an early diagnosis, certainly, improves the outcome. The treatment strategy for OSCC incorporates both the surgical and oncologic approaches. There are two main challenges of the current research in cancer treatment: the first one is the development of more personalized and effective therapies, since not all tumors of the same stage respond to the therapy in the same way, and the second one is the setup of a more targeted therapy, that can affect only the cancer cells, without destroying healthy ones. Many efforts are made to find compounds that can support and improve the cancer therapy, and great attention is focused on some of natural products, known to have beneficial properties on the human organism. The aim of this thesis is to present results deriving from a research directed to investigate a possible use of a natural compound, Fermented Wheat Germ Extract (FWGE), for the treatment of Oral squamous cell carcinoma (OSCC). In order to summarize the scientific evidence of the use of FWGE for treatment of cancer cells, a systematic review of the literature was performed. Sixteen articles were included in the final qualitative analysis. Various types of cancer cells treated with FWGE have been analyzed, showing mainly cytotoxic effects, alteration of the cell cycle, antiproliferative effects, and induction of apoptosis. After that, a series of in vitro experiments, including MTT assay, invasion and migration assays were performed to investigate the effects of the treatment of OSCC cells (HSC-3, SAS and SCC-25) with different concentrations of FWGE. The inhibitory effect on viability 2 of OSCC cells, exerted by chemotherapeutic drugs (cisplatin and 5-fluorouracil) and the combination of these with FWGE, was also evaluated. The results showed a significant reduction of cells viability after treatment with FWGE. Regarding migration and invasion capacity, the HSC-3 cells resulted to be the most sensitive to the treatment with FWGE. The combination of chemotherapeutic drugs and FWGE at 10mg/ml led to a significantly higher decrease in cell viability. A secondary purpose of this thesis regarded the investigation of prognostic meaning of certain mutations and expression of proteins characterizing OSCC. Firstly, a histologic and bioinformatic analysis of Musashi 2 (MSI2) expression was performed and its correlation with clinic-pathologic and prognostic features of OSCC evaluated. Musashi-2 is an RNA-binding protein, playing a fundamental role in the oncogenesis of several cancers. A bioinformatic analysis was performed on data downloaded from The Cancer Genome Atlas (TCGA) database. The MSI2 expression data were analysed for their correlation with clinic-pathological and prognostic features. In addition, an immunohistochemical evaluation of MSI2 expression on 108 OSCC samples included in a tissue microarray and 13 healthy mucosae samples was performed. 241 patients’ data from TCGA were included in the final analysis. No DNA mutations were detected for the MSI2 gene, but a hyper methylated condition of the gene emerged. MSI2 mRNA expression correlated with Grading (p = 0.009) and overall survival (p = 0.045), but not with disease free survival (p = 0.549). Males presented a higher MSI2 mRNA expression than females. The immunohistochemical evaluation revealed a weak expression of MSI2 in both OSCC samples and in healthy oral mucosae. In addition, MSI2 expression directly correlated with Cyclin-D1 expression (p = 0.022). However, no correlation has been detected with prognostic outcomes (overall and disease free survival). The role of MSI2 expression in OSCC seems to be not so closely correlated with prognosis, as in other human neoplasms. 3 The correlation with Cyclin-D1 expression suggests an indirect role that MSI2 might have in the proliferation of OSCC cells, but further studies are needed to confirm such results. Secondly, the role of programmed death ligand 1 (PD‐L1) in the tumour immunity and its potential function as a marker for OSCC prognosis were investigated through a metaanalysis. The studies were identified by searching PubMed, SCOPUS, Web of Science and were assessed by two of the authors. After the selection process, 11 articles met eligibility criteria and were included in the meta‐analysis. Quality assessment of studies was performed according to the REMARK guidelines, and the risk of biases across studies was investigated through Q and I2 tests. Meta‐analysis was performed to investigate the association between the PD‐L1 expression either overall survival (OS), disease‐free survival (DFS), diseasespecific survival (DSS), gender and lymph node metastasis. A total of 1060 patients were analysed in the 11 studies included in the meta‐analysis. Pooled analysis revealed that the expression of PD‐L1 did not correlate with poor OS (HR, 0.60; 95% CI: [0.33, 1.10]; P = 0.10), DFS (HR, 0.62; 95% CI: [0.21, 1.88]; P = 0.40), DSS (HR, 2.05; 95% CI: [0.53, 7.86]; P = 0.29 and lymph node metastasis (HR, 1.15; 95% CI: [0.74, 1.81]; P = 0.53). Furthermore, results of the meta‐analysis showed that high expression of PD‐L1 is two times more frequent in female patients (OR, 0.5; 95% CI: [0.36, 0.69]; P < 0.0001) compared to males. For all the three outcomes analysed, a high rate of heterogeneity was detected (I2 > 50%). High PD‐L1 expression did not correlate with poor prognosis of patients suffering for oral squamous cell carcinoma. Studies published on the topic showed a significant variation in results, limiting the use of PD‐L1 expression by immunohistochemistry as prognostic biomarker in clinical practice. Lastly, the role of the tumour-suppressor gene TP53 was evaluated in different head and neck squamous cell carcinoma (HNSCC). A systematic bioinformatics appraisal of TP53 mutations was performed on 415 HNSCC cases available on The Cancer Genome Atlas (TCGA). The following features were analysed and correlated with known 4 clinicopathological variables: mutational profile of TP53, location (within secondary structure and predicted domains of p53 protein) and well-known hotspot mutations. Interactome–genome–transcriptome network analysis highlighted different gene networks. An algorithm was generated to develop a new prognostic classification system based on patients’ overall survival. TP53 mutations in HNSCCs exhibited distinct differences in different anatomical sites. The mutational profile of TP53 was an independent prognostic factor in HNSCC. High risk of death mutations, identified by our novel classification algorithm, was an independent prognostic factor in TCGA HNSCC database. Finally, network analysis suggested that distinct p53 molecular pathways exist in a site- and mutation-specific manner. The mutational profile of TP53 may serve as an independent prognostic factor in HNSCC patients, and is associated with distinctive site-specific biological networks

Post-orthodontic position of lower incisors and gingival recession : a retrospective study

To evaluate if changes in lower incisor position following orthodontic treatment are correlated with development of gingival recessions. Pre- and post-treatment digital models and lateral cephalograms of 22 subjects were collected retrospectively. The clinical crown length, gingival scallop, and papilla height of the central lower incisor were measured along with the cephalometric incisor?s inclination, the distance from the mandibular plane, and the distance between the Infradentale and Menton points. Statistical correlations between gingival and cephalometric variables were studied. In addition, two groups were defined based on the post-treatment incisor inclination value (?normal? or ?proclined?) and compared. The incisor inclination was correlated with the change in gingival scallop and papilla height. Moreover, there was a statistically significant difference in clinical crown height and gingival scallop between the ?normal? group and the ?proclined? group. Changes in lower incisor position, especially an excessive proclination, after orthodontic treatment may play a role in the development of gingival recession

Retrospective Analysis of Clinical and Radiologic Data Regarding Zygomatic Implant Rehabilitation with a Long-Term Follow-Up

Background: Zygomatic implants have been introduced to rehabilitate edentulous patients with severely atrophic maxillae. Their use has been reported by several studies, describing high overall survival rates at medium-long follow-up. The aim of this study was to retrospectively analyze if a few patient-related and implant-related features are correlated with implant success or the onset of complications. Materials and methods: Data of patients treated with zygomatic implants between May 2005 and November 2012 at three private clinics were collected and retrospectively analyzed. For each implant, the following data were collected: implant length, insertion path, ridge atrophy and sinus characteristics (width, pneumatization, thickness of mucosae, patency of sinus ostium). General patient characteristics and health status data were also recorded. The outcomes evaluated were implant failure, infective complications, early neurologic complications and overall complications. Results: A total of 33 patients (14 men, 17 women, mean age 59.1) that received 67 zygomatic implants were included in the study. The mean duration of the follow-up was of 141.6 months (min 109; max 198). In this period, a total of 16 (23.88%) implants in 8 (24.24%) patients were removed and 17 (51.51%) patients with 36 (53.73%) implants reported complications. Immediate loading resulted in a significantly lower risk of complications compared with the two-stage prosthetic rehabilitation (OR: 0.04, p = 0.002). A thickness of the sinus mucosa > 3 mm emerged to be correlated with a greater occurrence of infective complications (OR: 3.39, p = 0.019). Severe and extreme pneumatization of the sinus was significantly correlated with the incidence of overall complications (p = 0.037) and implant failure (p = 0.044). A large sinus width was predisposed to a higher risk of neurologic complications, infective complications and implant failure (p = 0.036, p = 0.032, p = 0.04, respectively). Conclusions: zygomatic implants are an alternative procedure for atrophic ridge rehabilitation when a conventional implant placement is not possible. Several clinical and anatomical factors can have a significant role in complication occurrence

Addition of the tumour-stroma ratio to the 8th edition American Joint Committee on Cancer staging system improves survival prediction for patients with oral tongue squamous cell carcinoma

Aims One of the objectives of current research is to customise the treatment of cancer patients. The achievement of this objective requires stratification of patients based on the most significant prognostic factors. The aims of this study were to evaluate the prognostic value of the tumour-stroma ratio (TSR), defined as the proportion of tumour cells relative to surrounding stroma, in patients with oral tongue squamous cell carcinoma (OTSCC), and to develop a prognostic nomogram based on the most significant clinicopathological features. Methods and results Clinicopathological data of 211 patients treated at 'Ospedali Riuniti' General Hospital (Ancona, Italy) for OTSCC were collected. One hundred and thirty-nine patients were restaged according to the 8th edition American Joint Committee on Cancer (AJCC) staging system. Evaluation of the TSR was performed on haematoxylin and eosin-stained slides, and correlation with survival outcomes was evaluated. In addition, with the aim of integrating the independent value of the TSR with the 8th edition AJCC staging system, a prognostic nomogram for OTSCC has been developed. OTSCC with a low TSR (i.e. a high proportion of stroma and a low proportion of tumour cells) was shown to have negative prognostic value in terms of disease-specific survival, with a hazard ratio (HR) of 1.883 and a 95% confidence interval (CI) of 1.033-3.432 (P = 0.039), and overall survival (HR = 1.747, 95% CI 0.967-3.154;P = 0.044), independently of other histological and clinical parameters. For the cohort of 139 patients restaged according to the 8th edition AJCC staging system, variables correlating with a poor prognosis were: the TSR, perineural invasion, and sex. The nomogram built on these parameters showed good predictive capacity, outperforming the 8th edition AJCC staging system in stratifying disease-specific survival in OTSCC patients. Conclusions Including the TSR in the predictive model could improve risk stratification of OTSCC patients and aid in making treatment decisions.Peer reviewe

The Effects of Adjuvant Fermented Wheat Germ Extract on Cancer Cell Lines: A Systematic Review

Fermented wheat germ extract (FWGE; trade name AVEMAR) is a natural compound derived from industrial fermentation of wheat germ. Its potential anticancer properties has emerged from recent studies. The aim of this systematic review is to summarize the data available in the scientific literature concerning the in vitro activity of FWGE on malignant cells. A systematic review of English articles in electronic databases has been performed. The primary outcomes of the review regarded types of cancer cell lines subjected to the investigation and the main results concerning cell viability, proliferation, and apoptosis observed within the studies. Sixteen articles were included in the final qualitative analysis. Various types of cancer cells treated with FWGE have been analyzed, showing mainly cytotoxic effects, alteration of the cell cycle, antiproliferative effects, and induction of apoptosis. FWGE can be a promising drug component in cancer treatment; however, further in vitro and in vivo studies are necessary to prove its effectiveness and safety in humans

Hereditary gingival fibromatosis associated with the missense mutation of the KCNK4 gene

Gingival fibromatosis (GF) is a rare oral condition characterized by proliferative fibrous overgrowth of the attached gingiva, marginal gingiva, and interdental papilla, typically presenting during the growth period, causing aesthetic, functional, and masticatory disturbances of the oral cavity and psychological discomfort.1 GF can present as either hereditary gingival fibromatosis (HGF), which may appear as an isolated entity or as part of a genetic disease or syndrome, or as idiopathic gingival fibromatosis.2 HGF has a variable clinical presentation. In some cases, it shows only minimal involvement, characterized by enlargement of the tuberosity area and buccal gingiva around the mandibular molars; however, in severe forms, it can involve both maxillary and mandibular gingivae. In the severe form, the gingival enlargement can be so massive that it may cover the crowns of the primary and permanentHereditary gingival fibromatosis (HGF) is a rare oral condition that may appear as an isolated entity or as part of a genetic disease or syndrome. Molecular and biochemical mechanisms that trigger this pathologic process are not completely understood. In this article, we present a rare case of hereditary gingival fibromatosis in conjunction with a syndromic phenotype, associated with a rare missense mutation of the KCNK4 gene. This mutation induces a change in the structure of the TRAAK channel belonging to the 2-pore potassium channels. The gain of function promoted by the mutation could represent the pathogenetic basis of gingival fibromatosis. (Oral Surg Oral Med Oral Pathol Oral Radiol 2021;131:e175-e182

Comparative cost-analysis for removable complete dentures fabricated with conventional, partial, and complete digital workflows

Statement of problem: Comparative cost-analysis related to different manufacturing workflows for removable complete denture fabrication is seldom performed before the adoption of a new technology. Purpose: The purpose of this study was to compare the clinical and laboratory costs of removable complete dentures fabricated with a conventional (workflow C), a partial digital (workflow M), and a complete digital (workflow D) workflow and to calculate the break-even points for the implementation of digital technologies in complete denture fabrication. Material and methods: Clinical and laboratory costs for each of the investigated workflows and the manufacturing options related to denture base and denture teeth fabrication were collected from 10 private Italian dental laboratories and clinics. The selected variables included the clinical and laboratory manufacturing time needed to complete each workflow (opportunity cost); costs for materials, labor, packaging, and shipping; and capital and fixed costs for software and hardware, including maintenance fees. The effect of manufacturing workflows and their options on the outcomes of interest was investigated by using generalized estimated equations models (α=.05). Cost minimization and sensitivity analysis were also performed, and break-even points were calculated for the equipment capital costs related to the implementation of workflows M and D. Results: From a laboratory standpoint, workflows M and D and related manufacturing options significantly (P<.001) reduced manufacturing time (5.90 to 6.95 hours and 6.30 to 7.35 hours, respectively), and therefore the opportunity cost of each denture compared with workflow C. Workflow M allowed variable costs savings between 81 and 169 USD, while workflow D allowed for an additional saving of 34 USD. The sensitivity analysis showed that the break-even point related to the capital investment for the equipment needed to implement workflows M and D could be reached, depending on the manufacturing options adopted, between 170 and 933 dentures for workflow M and between 73 and 534 dentures for workflow D. From a clinical standpoint, workflows C and M were almost identical. Conversely, workflow D, which included intraoral scanning, required 1 fewer appointment, saving 0.6 hours of chairside time and about 14 USD for materials compared with M. Conclusions: Digital workflows (partial and complete digital workflows) were more efficient and cost-effective than the conventional method of fabricating removable complete dentures, with workflow D showing the lowest opportunity and variable costs and break-even point. Savings increased when stock denture teeth were replaced with milled denture teeth and still further with the adoption of 3-dimensionally (3D) printed denture teeth. Milling equipment and materials for denture base fabrication were more expensive than those for 3D-printing. Milling monobloc dentures reduced opportunity and labor costs but increased material cost