Naiara G. Bediaga

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.

Epigenetic biomarkers in lung cancer

Lung cancer mortality is strongly associated with the predominant diagnosis of late stage lesions that hampers effective therapy. Molecular biomarkers for early lung cancer detection is an unmet public health need and the lung cancer research community worldwide is putting a lot of effort to utilise major lung cancer population programmes in order to develop such molecular tools. The study of cancer epigenetics in the last decade has radically altered our views in cancer pathogenesis, providing new insights in biomarker development for risk assessment, early detection and therapeutic stratification. DNA methylation and miRNAs have rapidly emerged as potential biomarkers in body fluids showing promise to assist the clinical management of lung cancer. These new developments are exemplified in this review, demonstrating the huge potential of clinical cancer epigenetics, but also critically discussing the necessary validation steps to bring epigenetic biomarkers towards clinical implementation and the weaknesses of current biomarker studies.

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.

GSTT1 and GSTM1 gene copy number analysis in paraffin-embedded tissue using quantitative real-time PCR.

GSTT1 and GSTM1 genes possess an inherited deletion associated with a lack of enzyme activity. The heterozygous condition of this deletion is difficult to determine in low-quality DNA with existing PCR protocols. We designed and validated a multiplex real-time PCR assay by adapting the DeltaDeltaCt relative quantification method for the analysis of GSTT1 and GSTM1 markers to accurately differentiate the three genotypes ( *1/1, *1/0, and *0/0) in degraded DNA from formalin-fixed paraffin-embedded tissue. Gene copy number values obtained provide for unambiguous homozygous and heterozygous differentiation. The efficacy shown by the PCR assay endorses its usefulness for complete genotyping of glutathione S-transferases in archival tissues.

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.

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.

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.

Epigenetics and neurodegeneration: Blood tissue as a surrogate for brain in methylation DNA studies

High anxiety is the base not only for depression development but also to impulsive aggression manifestation. In the previous study we revealed the differences in neurohumoral status in animals with submissive and dominant behavioral types. Hypothalamic pituitary adrenal axis hyperactivity, the increase in noradrenaline and the decrease in serotonin levels in limbicocortical regions were observed in submissive male rats (high anxiety). Due to these results, we studied an interrelation between anxiety level and aggressiveness index and its components. The research involved 138 participants: 121 young men aged 18 to 22 years and 17 male adolescents within the age range 15-16 years. They were asked to answer Buss-Durkee Hostility Inventory, Spielberger State-Trait Anxiety Inventory and Eysenck Personality Inventory. The anxiety level was assessed in points. The aggressiveness index, physical, verbal and indirect aggressions were estimated in a percentage of the maximum level. No correlation between the anxiety level and the aggressiveness index was found in whole group of young men. Whole group was separated into three subgroups depending on anxiety level: with high, moderate and low anxiety levels. Strong positive correlation between anxiety level and aggression index in men with high anxiety level and negative correlation between these two parameters in men with low anxiety level were revealed. In last subgroup the correlation was statistically insignificant. In men with moderate anxiety level no correlation between anxiety level and aggression index was observed. This interrelation may be taken into account in anxiety treatment and in the prevention of impulsive aggression manifestation. In whole group of male adolescents no correlation between anxiety and aggressiveness index was found. Obtained data indicate the necessity of participants division depending on anxiety level and using the closed age groups to study the mechanisms of aggression development.T hypothesis of the present study is that N-methyl D-aspartate (NMDA) receptor antagonist and 5-hydroxy tryptamine (5HT1a) agonist will restore brain serotonin levels and can have role in the treatment of depression. Depression hypothesis is majorly based on neurochemical alteration. The neurotransmitters like serotonin, norepinephrine, dopamine and glutamate have a strong interlink. The mice were divided into 7 groups consisting of 6 animals each. Depression was induced with P-chlorophenylalanine (PCPA). On first day 300 mg/kg and on alternative days 100 mg/kg was administered to all the groups. The drug treatment memantine, 8-OH DPAT and their combination was continued for 14 days. Fluoxetine was used as a positive control. Behavioral screening, neurotransmitters and neurochemical were measured in different regions of control and serotonin depleted mice brain to assess the anti-depressant property of the drugs. The histopathological examination was also carried out. The results have shown that, depletion of brain serotonin level with PCPA treatment and resulted in reduced locomotion, anxiety behavior and ambulation with no alteration in rearing and grooming behaviors. The NMDA antagonist memantine, 5-HT1a agonist 8-OH DPAT and their combination have shown no significant change in locomotion. Whereas memantine 40 mg/kg have shown increased anxiolytic activity. The mice treated with memantine and its combination with 8-OH DPAT has shown significant change in force swim test. In neurotransmitters, the memantine and 8-OH DPAT group exhibited significant reduction in glutamate and aspartate levels, whereas GABA levels were increased. Histopathological report shows focal areas of degeneration in cerebrum region with PCPA treatment and drug treatments restored the morphology of the brain. Hence, it can be concluded that, memantine, 8-OH DPAT and their combination have better result in improving the depressive symptoms induced with PCPA than the positive control fluoxetine.C is an oral 1, 5-benzodiazepine used worldwide for the treatment of many types of Epilepsies, although it is currently only approved for Lennox–Gastaut syndrome in the USA. This anticonvulsant and anxiolytic therapeutic has repeatedly demonstrated great efficacy and a high safety profile in refractory epilepsy as well as in a few monotherapy trials in both children and adults. Clobazam allosterically activates the GABAA receptor, and it binds less to subunits that mediate sedative effects than other benzodiazepines. It acts quickly, maintaining a therapeutic effect for a long duration due to its active metabolite, N-desmethylclobazam. Dosage is between 5 mg and 40 mg a day, depending on patient weight, efficacy, and tolerability. Efficacy tolerance has not been a problem in the best studies. Clobazam has provided many benefits to epileptic patients. It should be used by clinicians early as an adjuvant therapy in the treatment of refractory epilepsy and even considered as monotherapy in a broad spectrum of epilepsy syndromes.BACKGROUND: Cerebral venous thrombosis (CVT) is a disease with high potential of disability and high rates of mortality. It affects females more than males. The estimated annual incidence of CVT is 3 to 4 cases per 1 million, but the incidence rate is much higher in Iran. It particularly increases in Ramadan and Zilhijjah months of the Islamic calendar. We hypothesized that the use of contraceptive components especially during the month of Ramadan is associated with an increase in the risk of CVT in women.Quetiapine is a novel antipsychotic drug. However, there is limited clinical evidence regarding prescribing patterns for quetiapine when used as maintenance treatment for bipolar disorder. Thirty-six albino rats were divided into 3 equal groups: control normal group [1] without exposure to chronic restraint for 6 hours daily/21 days, group [2] received DMSO 5% (v:v), as a solvent of quetiapine, with exposure to chronic restraint for 6 hours daily/21 days and group [3] received quetiapine 10 mg/kg/day ip for 3 weeks during exposure to chronic restraint for 6 hours daily/21 days. Intraperitoneal (ip) administration of quetiapine at a dose of 10 mg/kg/day for 3 weeks significantly (p<0.05) reduces the duration of immobility recorded by the forced swimming test (FST) and significantly (p<0.05) increases the contents of GABA neurotransmitter in hippocampus homogenates. The present study adds a positive implication of quetiapine, as an antipsychotic drug, on both the immobility and the reduction of GABA content in hippocampus of albino rats exposed restraint model for 21 days.Single i. m. injection of amitriptyline in rats in high doses of 10-30 mg/kg causes a weak an- tidepressive effect because only in 1.3-1.7 times decrease immobilization in Porsolt test. In the specified doses amitriptyline exhibits appreciable sedative effect because in 3-6 times decrease horizontal activity, and also in 2-2.5 times decrease vertical activity of rats in the open field test. Combined single i.m. injection of amitriptyline in a small dose of 3 mg/kg and high dose of 30 mg/kg with phenylephine in threshold, noneffective alone dose of 0.02 mg/kg, causes the ma- ximal antidepressive effect because decreases immobilization in Porsolt test, accordingly, in 3 and 4.6 times, but does not produce side sedative effect in the open field test. The basis of the mec- hanism of potentiation of antidepressive action and elimination of sedative action of amitriptyline is the stimulation of gastric mucosa afferents by phenylephine.

Abstract 4133: MicroRNAs for early detection of lung cancer

Introduction: Early detection of lung cancer by screening of high risk populations (identified by epidemiological and life-style factors) has the potential to save many lives. However, effective screening is reliant on minimally invasive techniques, such as CT screening, bronchioalveolar lavage (BAL) and blood tests, and the identification of suitable biomarkers. CT screening is effective in reducing mortality, but generates a large proportion of indeterminate nodules that must be further characterised. MicroRNAs (miRNA) have great potential as biomarkers due to their tissue-specific and cancer-specific expression patterns. We have identified tumour-specific miRNAs for non-small cell lung cancer (NSCLC), using a combination of screening on TaqMan microRNA TLDA cards and validation with qRTPCR assays, with the aim of utilising these as biomarkers in the early detection setting. Methods: Our sample group consisted of 31 frozen samples from 20 Liverpool Lung Project (LLP) NSCLC patients, including 10 adenocarcinomas (Ad), 10 squamous cell carcinomas (SCC) & matched normal tissue. Two further validation sets consisted of equal numbers of Ad and SCC tumour/normal pairs (124 in total). MiRNA was prepared from tumour and normal specimens using Qiagen MicroRNeasy kits. Reverse transcription and pre-amplification was performed using Applied Biosystems MegaPlex Pools and miRNAs were quantified on a 7900HT Real-Time PCR System with TaqMan Array Human MiRNA Card Set v3.0 (covering 754 human miRNAs). Ct values were exported using SDS v2.3 data and RQ Manager software and further analysed in Bioconductor. Validation qRTPCR was performed with individual miRNA assays, following reverse transcription with MegaPlex pools. Results: When Benjamin-Hoechst-adjusted-p value 4.0 fold-change in the cancer group. A subset of 22 miRNAs including miR-34a, miR-96, let-7g and miR-183 was identified with the greatest expression in tumours. Differential expression of all 22 miRNAs was confirmed in an independent set of 24 tumour/normal pairs. Using these 22 validated miRNAs we performed discriminative modelling and identified a model based on just 8 markers that gave a specificity of 100% and a sensitivity of 98%. This panel was validated, with 97% specificity and 91% sensitivity, in a 2nd independent sample set containing 48 tumours and paired normal samples. Conclusion: A number of miRNAs was identified that showed good discriminatory power individually, but greatest sensitivity and specificity when combined as an 8 member panel. The lung cancer specific miRNAs we have identified provide a potential source of early detection biomarkers. Their applicability to minimally-invasive samples is being evaluated in a range of samples including plasma and bronchial lavage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4133. doi:1538-7445.AM2012-4133