Amelia Acha-Sagredo

DNA methylation epigenotypes in breast cancer molecular subtypes

IntroductionIdentification of gene expression-based breast cancer subtypes is considered a critical means of prognostication. Genetic mutations along with epigenetic alterations contribute to gene-expression changes occurring in breast cancer. So far, these epigenetic contributions to sporadic breast cancer subtypes have not been well characterized, and only a limited understanding exists of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and to deliver specific epigenotypes associated with particular breast cancer subtypes.MethodsBy using a microarray approach, we analyzed DNA methylation in regulatory regions of 806 cancer-related genes in 28 breast cancer paired samples. We subsequently performed substantial technical and biologic validation by pyrosequencing, investigating the top qualifying 19 CpG regions in independent cohorts encompassing 47 basal-like, 44 ERBB2+ overexpressing, 48 luminal A, and 48 luminal B paired breast cancer/adjacent tissues. With the all-subset selection method, we identified the most subtype-predictive methylation profiles in multivariable logistic regression analysis.ResultsThe approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identified novel subtype-specific epigenotypes that clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors.ConclusionsOur results provide evidence that well-defined DNA methylation profiles enable breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer.

Oral cancer and polymorphism of ethanol metabolising genes

Oral cancer is the sixth most common cancer worldwide and a major health problem in some parts of the world. Epidemiological studies have shown that habitual alcohol consumption could be a risk factor in oral carcinogenesis, although the true involvement of alcohol is unknown. Via alcohol dehydrogenase (ADH) and cytochrome P450 oxidase (CYP) alcohol is metabolized to acetaldehyde, a highly toxic compound, which plays an important role in carcinogenesis. Subsequently, and during the metabolizing process, acetaldehyde becomes acetate by acetaldehyde dehydrogenase (ALDH). Therefore, acetaldehyde levels are determined mainly by the action of ADH, CYP and ALDH. Recently, several studies have found that certain polymorphisms of genes encoding these enzymes confer a higher or lower metabolic activity and therefore different risk for certain malignancies such as oral cancer. In this review, we analyze the polymorphisms of alcohol metabolising enzymes in relation susceptibility to an oral cancer.

A microRNA-based prediction algorithm for diagnosis of non-small lung cell carcinoma in minimal biopsy material

Background:Diagnosis is jeopardised when limited biopsy material is available or histological quality compromised. Here we developed and validated a prediction algorithm based on microRNA (miRNA) expression that can assist clinical diagnosis of lung cancer in minimal biopsy material to improve clinical management.Methods:Discovery utilised Taqman Low Density Arrays (754 miRNAs) in 20 non-small cell lung cancer (NSCLC) tumour/normal pairs. In an independent set of 40 NSCLC patients, 28 miRNA targets were validated using qRT–PCR. A prediction algorithm based on eight miRNA targets was validated blindly in a third independent set of 47 NSCLC patients. The panel was also tested in formalin-fixed paraffin-embedded (FFPE) specimens from 20 NSCLC patients. The genomic methylation status of highly deregulated miRNAs was investigated by pyrosequencing.Results:In the final, frozen validation set the panel had very high sensitivity (97.5%), specificity (96.3%) and ROC-AUC (0.99, P=10−15). The panel provided 100% sensitivity and 95% specificity in FFPE tissue (ROC-AUC=0.97 (P=10−6)). DNA methylation abnormalities contribute little to the deregulation of the miRNAs tested.Conclusion:The developed prediction algorithm is a valuable potential biomarker for assisting lung cancer diagnosis in minimal biopsy material. A prospective validation is required to measure the enhancement of diagnostic accuracy of our current clinical practice.

Role of cytochrome P‐450 genetic polymorphisms in oral carcinogenesis

Oral cancer is one of the most frequent head and neck cancers, and epidemiological studies have shown that smoking is a major risk factor in this pathology. However, as not all smokers develop oral cancer, some individuals must be more susceptible to develop this disease. This individual susceptibility has been related to different genetic variants in metabolizing enzymes. The cytochrome P-450 (CYP) family of enzymes metabolizes tobacco-related carcinogens producing reactive metabolites, which could cause DNA damage. Because of their functional role in the metabolism of tobacco-related compounds, the genetic polymorphisms found in the genes that code for CYP enzymes have been suggested to modulate oral cancer risk and contribute to individual susceptibility. In this review, we analyze and update the available evidence in the literature regarding the polymorphisms of CYP genes in relation to the susceptibility of developing oral cancer.

Polymorphisms in alcohol and tobacco metabolism genes in head and neck cancer in the Basque Country

BACKGROUND The Basque Country has one of the highest rates of head and neck squamous cell carcinoma (HNSCC) in Europe, although tobacco and alcohol consumption are not high when compared to other European countries where HNSCC incidence is lower. Our aim was to determine the role of genetic variation with regard to the metabolism of alcohol and carcinogens from tobacco smoke in the Basque Country. METHODS Fourteen polymorphisms in alcohol or tobacco metabolism genes were genotyped in 84 HNSCC patients and 242 healthy individuals from the Basque Country. RESULTS ADH1B histidine allele (rs1229984), CYP2E1 rs3813867 heterozygous genotype, and GSTT1 deletion conferred protection against HNSCC (OR: 0.318 [0.04-0.75], OR: 0.13 [0.02-0.94], and OR: 0.12 [0.02-0.60], respectively) while GSTP1 (rs1695) Val/Val genotype was related to an increased risk (OR: 4.12 [1.11-15.31]). Regarding alcohol and tobacco habits, GSTT1 deletion was associated with tobacco usage, while the 3 polymorphisms tested in ALDH2 were associated with alcohol consumption. However, genotypic distributions of these 7 SNPs did not differ from those observed for other Caucasian populations where HNSCC incidence is lower. CONCLUSIONS The identified genotypic variations in alcohol and tobacco metabolizing genes only by themselves do not seem to be responsible for the higher incidence of HNSCC observed in the Basque Country.

p53 mutation is rare in oral mucosa brushings from patients previously treated for a head and neck squamous cell carcinoma

Mutations of the tumour suppressor gene p53 are common in human cancer, and seem to be an early event in head and neck squamous cell carcinomas. The aim of our study was to determine the status of the tumour suppressor gene p53 in the oral mucosa of patients previously treated for a head and neck squamous cell carcinoma, at risk of developing an oral squamous cell carcinoma, but without oral clinical lesions. Oral brushings from 87 patients were sequenced with matched genomic DNA. No mutations were found in exons 5, 7 and 8, whereas in exon 6 silent mutations (n=6) and a polymorphism (n=7) were found. Mutation of the tumour suppressor gene p53 does not seem to be a frequent event in patients at risk but without oral lesions.

Global DNA methylation: uncommon event in oral lichenoid disease

OBJECTIVES Accumulating evidence indicates that aberrant DNA methylation is closely related to oral carcinogenesis, and it has been shown that methylation changes might be used as prognostic biomarker in oral squamous cell carcinoma. Oral lichenoid disease (OLD) is the most common oral potentially malignant disorder in our region. The aim of this study was to perform the first wide DNA methylation study in OLD in order to investigate the relevance of DNA methylation changes in this premalignant disorder. MATERIALS AND METHODS Two different Illumina microarray platforms, namely the GoldenGate Cancer Panel I and the HumanMethylation27 DNA Analysis BeadChip, were utilized in the discovery phase to interrogate the methylation profile of 59 OLD cases and 9 healthy individuals. Top-ranked genes were further validated by pyrosequencing in a second sample set consisting of 160 OLD and 65 controls. RESULTS Our results show that the frequency of aberrant DNA methylation is rare in OLD, and this finding was further corroborated by pyrosequencing in the biological validation. CONCLUSIONS These findings reinforce the notion that molecular alterations associated with oral carcinogenesis do not seem to be common events in OLD, which in turn might explain the low rate of malignization of this disorder.

Genomewide miRNA profiling of oral lichenoid disorders and oral squamous cell carcinoma

OBJECTIVE To dissect the aberrant microRNA profile of oral lichenoid disorders (OLD) by analyzing the larger set of OLD samples tested so far. MATERIALS AND METHODS MicroRNA expression profiles were assessed using TLDA card in 32 samples (16 OLD, 8 OSCC, and 8 control). The findings were validated using RT-qPCR in an independent cohort of 91 samples. RESULTS We identified 20 differentially expressed microRNAs in OLD, of which several are functionally related to cell proliferation, response to organic substances, or immune processes. Further validation of the top-ranked microRNAs revealed that they were all aberrantly expressed in OLD. CONCLUSION We have identified a new microRNA signature associated with OLD that may provide a meaningful basis for better understanding the physiopathology of the disease. In addition, we validated seven microRNAs whose expression was shown to be higher in OLD tissue in comparison with the control and OSCC tissues.

Oral Candida colonization in patients with chronic periodontitis. Is there any relationship

BACKGROUND Candida can be implicated in the pathology of chronic periodontitis. AIMS To analyze the oral Candida carriage in patients suffering from chronic periodontitis (CP) and its correlation with the severity of this condition. METHODS Microbiological samples were taken from 155 patients using the oral rinse (OR) technique and by using paper points in the periodontal pockets (GPP). These patients were divided into 3 groups: 89 patients without CP (control), 47 with moderate CP, and 19 with severe CP. Samples were cultured in a Candida chromogenic agar for Candida. Species were identified by microbiological and molecular methods. RESULTS Candida was isolated in the OR of 45 (50.6%), 21 (44.7%), and 11 (57.9%) patients, respectively, and in the GPP of 32 (36%), 14 (29.2%), and 10 (42.6%) patients from the control, moderate CP and severe CP groups, respectively. Candida was isolated more frequently and in a greater burden in OR than in GPP (p<0.01). Candida albicans was the most prevalent species. GPP of patients with CP had poor fungal biodiversity (p<0.01). CONCLUSIONS Colonization by Candida was present in the samples of patients without CP, and with both moderate and severe CP. Nonetheless, patients with severe CP had a higher rate of Candida colonization, especially by C. albicans.

Toll-like receptor 2 rs4696480 polymorphism and risk of oral cancer and oral potentially malignant disorder

OBJECTIVES The aim of this study was to identify the possible association between TLR polymorphisms and an increased risk of developing head and neck cancer, including oral (OSCC) and laryngeal squamous cell carcinoma (LSCC), and oral potentially malignant disorders, such as oral lichenoid disease (OLD), including oral lichen planus (OLP) and oral lichenoid lesions (OLL). DESIGN This case-control study included 40 OSCC, 35 LSCC, 175 OLD (129 OLP and 46 OLL) patients and 89 healthy controls, all of them from the Basque Country, Spain. Genetic polymorphisms in TLR1, TLR2, TLR4, TLR6, TLR9, and TLR10 were genotyped by TaqMan® assays or pyrosequencing. RESULTS The chi-square analysis showed that the variant A of the SNP TLR2-rs4696480 polymorphism significantly increased the risk of OSCC (p=0.03) and OLL (p=0.02). CONCLUSIONS The TLR2-rs4696480 polymorphism may be relevant to OSCC and OLL susceptibility in this population encouraging further studies on the TLR2 pathway and its possible association with this group of oral potentially malignant disorders and oral cancer. This may also prove the use of TLR polymorphisms as risk markers for oral and laryngeal cancer.