Sex differences in the proportion of esophageal squamous cell carcinoma cases attributable to tobacco smoking and alcohol consumption

Objective: Alcohol and tobacco are the two major established environmental factors associated with squamous cell carcinoma of the esophagus (ESCC). However, the prevalence of these exposures differs substantially between men and women. Moreover, the prevalence of smoking has declined in recent years, whereas per capita consumption of alcohol has remained steady in both sexes. Quantifying the burden of ESCC attributable to these causal factors is necessary to inform potential preventive strategies. Methods: We estimated the population attributable fraction (PAF) of ESCC due to smoking and alcohol, using data from an Australian population based case–control study (305 ESCC cases, 1554 controls). Results: Estimated PAF for ESCC were 49% (95% CI: 38–60) and 32% (95% CI: 25–40) due to smoking and heavy alcohol consumption respectively. More than 75% of the ESCC burden in men could be attributed to smokers with heavy alcohol consumption. The highest burden was among ≥30 pack years smokers who also consumed alcohol heavily (>17 drinks/week); this differed significantly between men (PAF 36%, 95% CI 29–44) and women (PAF 5%, 95% CI 2–10). Among women only, low intakes of fruit and vegetables accounted for about 9% of the ESCC burden. Conclusion: The burden of ESCC attributable to smoking combined with heavy alcohol consumption is remarkably high in men. In women, the burden of ESCC due to these factors is lower, and poor nutrition may also play a role.     

The promise of stem cell markers in the diagnosis and therapy of epithelial dysplasia and oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer. Epithelial dysplasia is often initiated in the cells and cell nuclei adjacent to the epithelial cell membrane. Reduced cell–cell adhesions enable cancer cells to detach from the tumor and disseminate to other organs. The mutations in epithelial dysplasia markers such as E‐cadherin and epithelial cell adhesion molecules (CD326) can lead to proliferation, growth and survival of the tumor cells and persistence of numerous malignancies that play a key role in epithelial dysplasia of OSCC. Accordingly, these genes can be considered prognostic markers or potential therapeutic targets for the tailored management of patients with OSCC. The gene expression profile of OSCC stem cells indicates a differential pattern that facilitates establishing a cell signature. Owing to the highly tumorigenic behavior of cancer stem cells and the role of these cells in tumor differentiation, treatment resistance, relapse, and metastasis, we reviewed the role of stem cell markers in epithelial dysplasia and OSCC.     

The treatment with differentiation- and apoptosis-inducing agent, vesnarinone, of a patient with oral squamous cell carcinoma

A patient with histopathological recurrent oral cancer with well-differentiated squamous cell carcinoma, was treated with differentiation- and apoptosis-inducing agent, vesnarinone, per os at a dose of 180 mg/day for 56 days and then at a dose of 60 mg/day for 93 days. The vesnarinone administration caused complete remission of the tumour. It has been found by immunohistochemical staining and PCR-SSCP analysis that the recurrent tumour has wild type p53 gene and relative high level of LeY expression as well as DNA fragmentation in the cancer cells, as assessed by nick-end labelling. These findings suggest that the cure of oral squamous cell carcinoma observed in this case might be associated with induction of differentiation and apoptosis of cancer cells by vesnarinone.     

The enigmatic role of matrix metalloproteinases in epithelial-to-mesenchymal transition of oral squamous cell carcinoma: Implications and nutraceutical aspects

The most prevalent malignancy in the oral cavity is represented by oral squamous cell carcinoma, an aggressive disease mostly detected in low-income communities. This neoplasia is mostly diffused in older men particularly exposed to risk factors such as tobacco, alcohol, and a diet rich in fatty foods and poor in vegetables. In oral squamous cell carcinoma, a wide range of matrix-cleaving proteinases are involved in extracellular matrix remodeling of cancer microenvironment. In particular, matrix metalloproteinases (MMPs) represent the major and most investigated protagonists. Owing to their strong involvement in malignant pathologies, MMPs are considered the most promising new biomarkers in cancer diagnosis and prognosis. The interest in studying MMPs in oral cancer biology is also owing to their prominent role in epithelial-to-mesenchymal transition (EMT). EMT is an intricate process involving different complex pathways. EMT-related proteins are attractive diagnostic biomarkers that characterize the activation of biological events that promote cancer's aggressive expansion. Different antioncogenic natural compounds have been investigated to counteract oral carcinogenesis, with the scope of obtaining better clinical results and lower morbidity. In particular, we describe the role of different nutraceuticals used for the regulation of MMP-related invasion and proliferation of oral cancer cells.     

Detection of oral squamous cell carcinoma with ex vivo fluorescence confocal microscopy: Sensitivity and specificity compared to histopathology

Real‐time microscopic imaging of freshly excised tissue enables a rapid bedside‐pathology. A possible application of interest is the detection of oral squamous cell carcinomas (OSCCs). The aim of this study was to analyze the sensitivity and specificity of ex vivo fluorescence confocal microscopy (FCM) for OSCCs and to compare confocal images visually and qualitatively with gold standard histopathology. Two hundred eighty ex vivo FCM images were prospectively collected and evaluated immediately after excision. Every confocal image was blindly assessed for the presence or absence of malignancy by two clinicians and one pathologist. The results were compared with conventional histopathology with hematoxylin and eosin staining. OSCCs were detected with a very high sensitivity of 0.991, specificity of 0.9527, positive predictive value of 0.9322 and negative predictive value of 0.9938. The results demonstrate the potential of ex vivo FCM in fresh tissue for rapid real‐time surgical pathology.     

LCK, survivin and PI-3K in the molecular biomarker profiling of oral lichen planus and oral squamous cell carcinoma

T cell signaling is critical in oral lichen planus (OLP) based on the pathogenesis of this chronic inflammatory autoimmune mucocutaneous lesion. Lck plays a key role in T cell signaling; ultimately this signaling affects other targets such as PI-3K. Excessive activity in PI-3K inhibits apoptosis and promotes uncontrolled cell growth. Molecular biomarker profiling in OLP, Chronic Interface Mucosities (CIM), Epithelial Dysplasia (EpD) and Oral Squamous Cell Carcinoma (SCCA) with application of the principle of biomarker voting may represent a new frontier in the diagnosis, assessment and the arguable debate of OLP transformation to cancer. The presence of Lck, PI-3K and Survivin, a cancer specific anti-apoptotic protein was assessed, using immunohistochemistry and tissue micro-array on patient samples, in OLP, SCCA, CIM and EpD. Lck expression was very high in 78.6 % of OLP patients compared to 3.7% in SCCA; PI-3K was high in 63% of SCCA, 100% of EpD, and 35.7% OLP cases. Survivin was high in 64.3% of OLP cases, 96.3% of SCCA, and 100% of EpD. CIM cases may be slightly different molecularly to OLP. Taken together, our data suggest that biomarker protein voting can be effectively used to isolate high-risk OLP cases. Specifically, we show data with four remarkable cases demonstrating that molecular factors are predictive of histopathology. We conclude that it is safer to treat OLP as premalignant lesions, to adopt aggressive treatment measure in histopathologic described well and moderately differentiated SCCA, and to monitor progress of these diseases molecularly using individualized auto-proteomic approach. The use of Lck inhibitors in OLP management needs to be investigated in the future.     

Cytostatic effect of inostamycin, an inhibitor of cytidine 5'-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): inositol transferase, on oral squamous cell carcinoma cell lines.

Inostamycin, which was recently isolated from Streptomyces sp. MH816-AF15 as an inhibitor of cytidine 5'-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): inositol transferase, caused a G1-phase accumulation in the cell cycle of small cell lung carcinomas. To investigate whether the cytostatic effect of inostamycin is restricted to lung carcinoma cell lines or applicable to other type of cells, we tested five oral squamous cell carcinoma (SCC) cell lines. Cell growth was suppressed in 62.5--125 ng/ml inostamycin in the culture medium in all oral cancer cell lines tested, with non-viable cells being <1%, indicating inostamycin is cytostatic on SCC cell lines. Decrease in cyclin D1 mRNA and protein expression due to the inostamycin treatment was accompanied by suppression of phosphorylated retinoblastoma susceptibility gene product (pRB-P) levels. Moreover, flow cytometric analysis showed that inostamycin induced an increase in G1/G0 cells (1.2--3.2 fold) over 24 h. These results suggest that inostamycin is a useful agent for tumour dormant cytostatic therapy for oral SCC.     

Simultaneous quantification of nucleoproteins and comparison of methyl green-pyronin Y and Feulgen staining in sections of oral squamous cell carcinoma,dysplastic lesions and normal mucosa

A fundamental difference between normal cells and tumor cells is the proliferative activity of the nucleus and nucleolus, which increases progressively from normal to oral dysplastic mucosa to oral squamous cell carcinoma (OSCC). This activity is evaluated routinely using hematoxylin and eosin (H & E) staining, but in some cases, inter-observer variability occurs among pathologists. We evaluated cellular proliferation by staining sections with the methyl green-pyronin Y procedure and the Feulgen reaction. We also compared the efficacy of methyl green-pyronin Y and Feulgen staining for studying nuclear and nucleolar features in oral dysplastic mucosa and in different grades of OSCC. Sections cut from formalin fixed, paraffin embedded blocks of five normal mucosa, 15 dysplastic mucosa, 10 well-differentiated OSCC, 10 moderately differentiated OSCC and five poorly differentiated OSCC cases were stained with Hematoxylin and Eosin, methyl green-pyronin Y and the Feulgen reaction. The mean diameters of the nuclei and number of nucleoli showed significant differences. A progressive increase in diameter of the nucleus and number of nucleoli was observed from normal mucosa through poorly differentiated OSCC. We observed that methyl green-pyronin Y stain is more useful than Feulgen and hematoxylin and eosin for simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine examination.     

Identification and expression analysis of an actin gene from the soft tick, Ornithodoros moubata (Acari: Argasidae)

Actin genes are found in all living organisms and highly conserved in various animals as shown by numerous studies on actin gene expression and function. Because of this ubiquitous nature of actin, it is often used as an internal control in gene expression studies. To clarify the suitability of actin gene as an internal control in soft ticks, isolation and expression analyses of an actin gene from Ornithodoros moubata was performed. An actin gene of Ornithodoros moubata (OmAct2, GenBank accession no. AB208021) with 1,131 bp and 376 amino acid residues was identified. The homology of OmAct2 with other arthropod actin genes was greater than 80% in nucleotides and 99% in amino acids. OmAct2 gene was classified as a cytoskeletal actin type by absence of muscle-specific amino acids commonly found in insects and ubiquitous expression in all stages and both sexes. Southern blot revealed that O. moubata has four to seven actin genes. In addition, actin expression analyzed by real-time PCR before and after blood feeding was not significantly different indicating OmAct2 is an appropriate internal control for the analysis of gene expression in these ticks.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.     

Associations between oral hygiene habits,diet, tobacco and alcohol and risk of oral cancer: A case–control study from India

ObjectiveThis study examines the association between the incidence of oral cancer in India and oral hygiene habits, diet, chewing and smoking tobacco, and drinking alcohol. We also assessed the effects of oral hygiene habits with oral cancer risk among chewers versus never chewers.MethodsA hospital-based case–control study was conducted in Pune, India, based on face-to-face interviews, anthropometry, and intra-oral examinations conducted for 187 oral cancer cases and 240 controls.ResultsPoor oral hygiene score was associated with a significant risk of oral cancer (adjusted OR = 6.98; 95%CI 3.72–13.05). When stratified by tobacco-chewing habit, the poor oral hygiene score was a significant risk factor only among ever tobacco chewers (adjusted OR = 14.74; 95%CI 6.49–33.46) compared with never chewers (adjusted OR = 0.71; 95%CI 0.14–3.63). Dental check-ups only at the time of pain by ever-chewers with poor oral hygiene was associated with an elevated risk (adjusted OR = 4.22; 95%CI 2.44–7.29), while consumption of green, yellow, and cruciferous vegetables and citrus fruits was protective. A linear dose–response association was observed between oral cancer and chewing tobacco in terms of age at initiation, duration, and frequency of chewing per day (P < 0.001). Smoking more than 10 bidis/cigarettes per day (adjusted OR = 2.74; 95%CI 1.28–5.89) and for a duration >25 years (adjusted OR = 2.31; 95%CI 1.14–4.71) elevated the risk of oral cancer.ConclusionGood oral hygiene habits – as characterized by healthy gums, brushing more than once daily, use of toothpaste, annual dental check-ups, and a minimal number of missing teeth – can reduce the risk of oral cancer significantly. In addition to refraining from chewing/smoking tobacco, a diet adequate in fruits and vegetables may protect against the disease.     

A histochemical comparison of methyl green-pyronin,and hematoxylin and eosin for detecting apoptotic cells in oral squamous cell carcinoma,oral leukoplakia,oral submucous fibrosis and normal oral mucosa

Abstract

Analysis of apoptotic cells in oral pathological states could be useful for determining the rates of tissue turnover, which would help determine prognosis. The use of histochemical stains such as hematoxylin and eosin (H & E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for detecting apoptotic cells. We compared the efficacy of MGP and H & E for detecting apoptotic cells in oral squamous cell carcinoma (OSCC), oral leukoplakia (OL), oral submucous fibrosis (OSMF) and normal oral mucosa (NOM). Ten cases each of OSCC, OSMF, OL and NOM were retrieved from the archives and two serial sections were stained, one with H & E and the other with MGP. Apoptotic cells were identified at 100 x magnification and the apoptotic index was calculated. Apoptotic cells were distinguished more readily in MGP stained sections than in those stained with H & E. Also, the apoptotic cell count was greater in OSCC compared to OL, OSMF and NOM. We concluded that MGP staining can be used as a routine, cost-effective method for detecting apoptotic cells.     

Platycodin D,a triterpenoid saponin from Platycodon grandiflorum,suppresses the growth and invasion of human oral squamous cell carcinoma cells via the NF‐κB pathway

This work was undertaken to explore the effects of platycodin D, a triterpenoid saponin from Platycodon grandiflorum, on the growth and invasiveness of human oral squamous cell carcinoma (OSCC). Platycodin D caused a significant, concentration‐dependent inhibition of cell viability and induced significant apoptosis in OSCC cells. Moreover, platycodin D significantly inhibited OSCC cell invasion. At the molecular level, platycodin D increased the amounts of IκBα protein and reduced the expression of phosphorylated NF‐κB p65, MMP‐2, and MMP‐9. Ectopic expression of constitutively active NF‐κB p65 prevented platycodin D‐mediated induction of apoptosis and suppression of invasion in OSCC cells. In vivo studies confirmed that platycodin D retarded the growth of subcutaneous SCC‐4 xenograft tumors and reduced phosphorylation of NF‐κB p65. Altogether, platycodin D shows inhibitory activity on OSCC growth and invasion through inactivation of the NF‐κB pathway and might provide therapeutic benefits in the treatment of OSCC.     

Seasonal variation in diagnosis of cutaneous invasive melanoma and cutaneous squamous cell carcinoma: A nationwide study in the Netherlands

BackgroundCurrently, there is no study that has reported on the seasonal trends of skin cancer in the Netherlands. This study aimed to investigate seasonal variation in diagnosis of cutaneous melanoma (CM) and cutaneous squamous cell carcinoma (cSCC) focusing on different subgroups.MethodsCM diagnosed from 2001 till 2019 and cSCCs from 2001 till 2015 were selected from the Netherlands Cancer Registry. The monthly distribution of CM and cSCC diagnoses were evaluated. Summer-to-winter ratios (SWRs) were calculated overall and stratified by patient and tumour characteristics.ResultsSignificant increases in melanoma incidence were noted over the summer months (SWR 1.39 (CI 1.37–1.40)). This increase was less apparent for cSCCs, as higher incidence rates were observed in the months September-November (SWR 1.13 (CI 1.12–1.14)). The seasonal variation of CM was greater in women and younger people, in superficial spreading melanoma and lentigo maligna melanoma, for the extremities, in thinner lesions, and for stage I at diagnosis. The seasonal variation of cSCC was similar for both sexes, most marked in patients 45‐69 and ≥ 70, and for the extremities.ConclusionsOur findings showed a pronounced seasonal variation in the diagnosis of CM with a peak in the summer months. For cSCC, no evident peak was observed, but an increase in diagnosis was noted in fall. Both CM and cSCC showed strong seasonal effects for the extremities.     

Areca (betel) nut chewing practices of adults and health behaviors of their children in the Freely Associated States,Micronesia: Findings from the Children’s Healthy Living (CHL) Program

BackgroundChewing areca (betel) nut has been deemed carcinogenic. The practice has become a public health concern in Micronesia. The Children's Healthy Living (CHL) Program included an areca (betel) nut questionnaire in a survey of household characteristics in the Freely Associated States (FAS). This paper describes areca (betel) nut chewing practices of adults and the health behaviors of their children.MethodsA cross-section of 1200 children (2–8 year-olds) and their caregivers in Chuuk, Kosrae, Pohnpei, Republic of Palau, Republic of the Marshall Islands (RMI), and Yap were recruited. Socio-demographics, adult areca (betel) nut chewing practices, and other health behaviors of children and adults were assessed. Child anthropometric measurements were collected to estimate weight status.ResultsThe FAS areca (betel) nut chewing prevalence was 42%, ranging from 3% (RMI) to 94% (Yap). Among chewers, 84% added tobacco, 97% added slaked lime, 85% added betel leaf, and 24% mixed the components with alcohol. Among FAS children, 95% practiced daily teeth-brushing and 53% visited the dentist annually. Compared to non-chewing households, areca (betel) nut chewing households were more likely to have very young children enrolled, more highly educated adults, and members that used tobacco and alcohol.ConclusionThe FAS areca (betel) nut chewing prevalence (42%) is above the world prevalence of 10–20%, with wide variability across the islands. The oral health findings in this study may inform future oral cancer prevention programs or policies. Regular monitoring of areca (betel) nut use is needed to measure the impact of such programs or policies.     

Simplex and multiplex real‐time PCR assays for the detection of flagellar (H‐antigen) fliC alleles and intimin (eae) variants associated with enterohaemorrhagic Escherichia coli (EHEC) serotypes O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7

Aims: To develop real‐time PCR assays targeting genes encoding the flagellar antigens (fliC) and intimin subtypes (eae) associated with the five most clinically important serotypes of enterohaemorrhagic Escherichia coli (EHEC), i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7. Methods and Results: Primers and probes specific to fliC_H2, fliC_H7, fliC_H8, fliC_H11, fliC_H28, eae‐β1, eae‐γ1, eae‐ε and eae‐θ were combined in simplex and multiplex 5′‐nuclease PCR assays. The specificity of the assays was assessed on 201 bacterial strains and the sensitivity determined on serially diluted EHEC genomes. The developed PCR assays were found to be highly specific and detected as few as five EHEC genome equivalents per reaction. Furthermore, it was possible to detect the five major EHEC serotypes in cheese samples inoculated at concentration levels of ≤5 CFU per 25 g after overnight enrichment using the PCR assays. Conclusions: The PCR assays developed here were found to be sensitive and specific for the reliable detection of genes encoding the flagellar antigens and intimin variants belonging to the five most clinically relevant EHEC serotypes. Significance and Impact of the Study: Application of real‐time PCR assays should improve the identification of foods contaminated by EHEC and facilitate the molecular typing of these organisms.     

Development and evaluation of a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in pure culture isolates and lacrimal swabs collected from conventionally raised cattle

Aim: To develop a multiplex real‐time PCR assay for the detection and differentiation of Moraxella bovis (M. bovis), M. bovoculi and M. ovis. Methods and Results: The multiplex real‐time PCR assay was validated on three reference strains, 57 pure culture isolates and 45 lacrimal swab samples. All reference strains were identified correctly with no cross‐reactions between species. Sequencing of 53 of the 57 culture isolates confirmed the results obtained with the multiplex real‐time PCR, and the assay had 96·5% (55/57) concordance with a Moraxella spp. multiplex conventional PCR assay on the isolates. Among the lacrimal swab samples, the concordance between the multiplex real‐time PCR and culture was 86·7% (39/45) for M. bovoculi and 75·6% (34/45) for M. bovis. Conclusions: The multiplex real‐time PCR assay is specific and sensitive and can be used directly on lacrimal swab samples. Significance and Impact of Study: The lack of a rapid, specific and sensitive detection method is a barrier for determining the roles of M. bovis, M. bovoculi and M. ovis in infectious bovine keratoconjunctivitis cases, and the developed PCR assay will contribute to improved understanding of the epidemiology and pathogenesis of these three Moraxella species.