Magister Scientiae - MSc / Breast cancer (BC) is the most common cancer in women worldwide, and is the second most common cancer in the world, responsible for more than 500 000 deaths annually. Estimates are that 1 in 8 women will develop BC in their lifetime. In South Africa, BC in women affects about 16.6 % of the population and could see a 78 % increase in cases by 2030. The failure of conventional diagnostic tools to detect BC from an early onset has revealed the need for diagnostic tools that would enable early diagnosis of BC. The current diagnostic tools include breast self-examination, mammography magnetic resonance imaging, ultrasonography and serum biomarkers; BRACA1, BRACA2, HER2. These conventional methods lack sensitivity, specificity and positive predictive value, and some of these diagnostic tools may be expensive and quite invasive. Therefore, novel diagnostic tools such as microRNAs which address the short comings of current methods are required for early diagnosis as well as BC management. MicroRNAs are a class of non-coding RNA molecules, which are important in RNA stability and gene expression. Various methodologies have been employed to identify novel microRNAs for diagnostics such as bioinformatics, also referred to as in silico analysis. The aim of this study is to identify novel microRNAs that can potentially detect BC at its earliest stage.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. It is a result of numerous aetiological factors such as genetic predisposition, smoking, excessive alcohol consumption and viruses such as the human papilloma virus. Due to late diagnosis it has a high mortality and morbidity rates which has remained unchanged over the last 5 decades. Currently no screening is available for high risk patients for better monitoring. Diagnosing OSCC relies on histopathology of biopsy tissue, reviewed for dysplasia and advancing lesions. Although the technique has been used for decades for successful diagnosis it fails to identify the molecular signature of OSCC which appears much before the visual signs. It also falls short in predicting the malignant transformation of pre-malignant oral lesions. Identifying the molecular and genetic changes leading to OSCC lesion will aid in more specific (quantitative) and early diagnosis of the disease reducing the financial burden of treating late-stage OSCC patients on the healthcare system. This study focuses on developing new adjuncts which can be used alongside histopathology for early diagnosis. There is a need to monitor high risk patients through non-invasive methods causing less patient discomfort. We therefore explored the potentials of exosomes which are extracellular vesicles secreted by normal and tumour cells. They can be isolated from body fluids such as blood and saliva. In cancer biology exosomes offer both diagnostic and therapeutic advantage. Their involvement in cell-cell communication indicates their influence in tumour development, progression, metastasis and therapeutic efficacy. Exosomes released by cancerous cells carry numerous biomarkers, which are passed on to healthy cells via microenvironment, causing stromal and angiogenic activation along with immune escape. In this study exosomes were successfully isolated from body fluids (blood, saliva and plasma) and cell line supernatant through ultracentrifugation and characterised by visual and particle size quantification techniques including Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), Zetasizer and Nanosight Tracking Analysis (NTA). Exosomal specific membrane proteins were identified through Western blotting. 5 We report the presence of a potential protein biomarker located exclusively on the outer membrane of cancer exosomes. Since body fluids consist of a heterogeneous population of exosomes derived from multiple cell types, such surface biomarker can potentially be used to isolate OSCC exosomes. Characterisation of exosomal mRNA cargo was done using Agilent Bioanalyzer (for RNA quantity and quality assurance) and reverse transcription-quantitative PCR (RT-qPCR; for gene specific quantitation). Functional significance of exosomes was studied by transfecting normal oral keratinocyte cells with self and cancer-derived exosomes. Through gene-expression microarray and subsequent RT-qPCR verification, we report a panel of differentially expressed genes involved in essential cellular functions being modulated by exosome transfection. A previously developed molecular diagnostic system by our research group called quantitative malignancy index diagnostic system (qMIDS) based on FOXM1 oncogene and its downstream targets was validated on archival formalin fixed paraffin embedded OSCC patient biopsy samples. We report that qMIDS index successfully correlates with the disease stages including dysplasia, tumour and lymph node metastasis. Furthermore, through meta-analysis of 8 OSCC microarray studies we identified a panel of six genes including PLAU, FN1, CDCA5, CRNN, CLEC3B and DUOX1 (q6) which are able to identify two clinically distinct sub-groups of OSCC patient population. Through RT-qPCR the expression of q6 biomarkers was established in 100 OSCC biopsy samples. This information can be of immense importance in developing personalized treatment strategies based on the molecular makeup of the presenting tumour.

Orientador: Luiz Paulo Kowalski / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-18T03:04:55Z (GMT). No. of bitstreams: 1 Francisco_AnaLuciaNoronha_M.pdf: 3407963 bytes, checksum: e0166ff00ce4b9f6c8a317040ddad41c (MD5) Previous issue date: 2011 / Resumo: O câncer da boca é um problema de saúde pública, com grande incidência na população brasileira. O paciente afetado geralmente apresenta-se para o tratamento com a doença em estádios avançados, com conseqüente diminuição das taxas de sobrevida. Diante disso, torna-se imprescindível a detecção do câncer de boca em estádios precoces. A espectroscopia de fluorescência é uma ferramenta diagnóstica não invasiva que pode auxiliar na detecção do câncer em tempo real, com o potencial de fornecer sensibilidade e especificidade semelhantes ao diagnóstico clínico de profissionais experientes. É uma técnica relativamente simples, rápida e acurada que consiste em avaliar a composição bioquímica e a estrutura do tecido pelo espectro de fluorescência emitido por ele, após aplicação de uma fonte de luz. Quando há progressão de um estado normal para um estado alterado, isso é refletido nas características espectrais da fluorescência dos tecidos, podendo ser correlacionada com o exame histopatológico destes tecidos. O objetivo do presente estudo consistiu em discriminar, na mucosa bucal, tecido sadio de neoplásico, por meio de espectroscopia de fluorescência. As avaliações ocorreram nos pacientes do Departamento de Cirurgia de Cabeça e Pescoço e Otorrinolaringologia do Hospital A. C. Camargo e do Laboratório Especial de Laser em Odontologia da USP, São Paulo, Brasil. O estudo obteve aprovação dos Comitês de Ética em Pesquisa das instituições participantes. A amostra consistiu de 150 indivíduos nos quais se realizou a espectroscopia de fluorescência dos quais 55 pacientes eram portadores de carcinoma oral, 30 voluntários com mucosa oral clinicamente normal, 35 pacientes sem lesão oral, com história prévia de neoplasia, que foram submetidos à cirurgia ou à cirurgia e radioterapia, e 30 pacientes com lesão clinicamente detectável e potencialmente maligna. 81 casos (54%) do sexo masculino e a média de idade foi de 62,7 anos. Em relação aos fatores de risco, 76 (50,6%) eram fumantes e 31(20,7%) etilistas. Foram realizadas biópsias e os resultados destas duas metodologias foram comparados, usando o diagnóstico histopatológico como padrão ouro, para identificar características espectrais de cada tipo de lesão. Os espectros foram classificados e comparados com a histopatologia para determinação da eficiência na discriminação diagnóstica empregando-se a fluorescência. A análise consistiu de diversos tipos de processamentos matemáticos dos espectros, sendo as acurácias Bayes 0.513 e 0.618 e J48 0.829 e 0.887 nos comprimentos 532 e 406nm, respectivamente. Observou-se a variabilidade entre as doenças, entre os sítios anatômicos, entre os indivíduos e entre regiões da mesma lesão. Os valores de especificidade e sensibilidade da técnica variam em função do tipo de análise empregada, como na excitação em 406 nm o algoritmo J48+CFS mostrou valores de sensibilidade igual a 88,5% e de especificidade igual a 93,8%, podendo em alguns casos, pode-se constatar o seu potencial de uso como instrumento auxiliar do diagnóstico do câncer de boca / Abstract: Oral cancer is a public health problem with high incidence in the Brazilian population. The affected patient usually presents for clinical investigation, with advanced stage disease, the consequence of this delay is a reduction in survival rates. Given this, it's essential to detect oral cancer in early stages. Fluorescence spectroscopy is a non-invasive diagnostic tool that can aid in cancer detection in real time, with the potential to provide similar sensitivity and specificity for clinical diagnosis of experienced professionals. This technique is relatively simple, fast and accurate which evaluates the biochemical composition and structure of the tissue fluorescence spectrum emitted by it, after application of a light source. When there is a progression from normal state to an altered state, this is reflected in the spectral characteristics of fluorescence of tissues, which may be correlated with the histopathological examination of tissues. The aim of this study was to discriminate, in the oral mucosa, healthy tissue and tumor, through fluorescence spectroscopy. Assessments occurred in patients of the Department of Head and Neck Surgery and Otorhinolaryngology, Hospital A. C. Camargo and the Special Laboratory of Laser in Dentistry, USP, São Paulo, Brazil. The study was approved by the Ethics Research Committee of the participant institutions. The sample consisted of 150 individuals who were submitted to fluorescence spectroscopy, of whom 55 patients had oral carcinoma, 30 volunteers with normal oral mucosa, 35 patients without oral lesions, but with a previous history of malignancy, who underwent surgery or surgery and radiotherapy, and 30 patients with a clinically detectable potentially malignant lesions. 81 cases (54%) were male and the mean age was of 62.7 years. Regarding risk factors, 76 (50.6%) were smokers and 31 (20.7%) consumed alcohol. Biopsies were performed and the results of these two methods were compared using the histopathologic diagnosis as the gold standard for comparison to the spectral characteristics of each type of lesion. The spectra were classified and compared to histopathology for determining the efficiency in diagnostic discrimination employing fluorescence. The analysis consisted of various types of mathematical processing of spectra, and the accuracies Bayes 0,513 and 0,618 and J48 0,829 and 0,887 in lengths of 532 and 406nm, respectively. It can be observed variability among diseases, among the anatomical sites among individuals and among regions of the same injury. The specificity and sensitivity of the technique varies depending on the type of analysis employed as the excitation at 406 nm the algorithm J48 + CFS showed sensitivity of 88,5% and specificity of 93,8% and may in some cases, one can see its potential use as an aid to diagnosis of oral cancer / Mestrado / Patologia / Mestre em Estomatopatologia

>Magister Scientiae - MSc / Cervical cancer is the leading cause of cancer mortality among black women in South Africa. It is estimated that this disease kills approximately 8 women in South Africa every day. Cervical cancer is caused by the human papillomavirus (HPV) with the most common screening method for cervical cancer being Papanicolaou (Pap) smear, test amongst others. However, less than 20% of South African women go for these tests. There are several reasons why women do not go for these tests but the invasiveness of the test is one of the major causes for the low rate of screening. Lateral flow devices offer medical diagnosis at the point- of-care, allowing for the quick initiation of the appropriate therapeutic response. These tests are more cost-effective for the healthcare delivery industry, and can potentially be used by patients to self-test in the privacy of their homes and allow them to make informed decisions about their health. Therefore, the aim of this study was to use computational methods to identify serum biomarkers for cervical cancer that can be used to develop a point-of-care diagnostic device for cervical cancer. An in silico approach was used to identify genes implicated in the initiation and development of cervical cancer. Several bioinformatics tools were employed to extract a list of genes from publicly available cancer repositories. Multiple gene enrichment analysis tools were employed to analyze the selected candidate genes. Through this pipeline, ~28190 genes were identified from the various databases and were further refined to only 10 genes. The 10 genes were identified as potential cervical cancer biomarkers. A subcellular compartmentalization analysis clustered the proteins encoded by these genes as cell surface, secretory granules and extracellular space/matrix proteins. The selected candidate genes were predicted to be specific for cervical cancer tissue in a cancer tissue specificity meta-analysis study. The expression levels of the candidate genes were compared relative to each other and a graph constructed using gene expression data generated by GeneHub-GEPIS and TiGER databases. Further gene enrichment analysis was performed such as protein-protein interactions, transcription factor analysis, pathway analysis and co-expression analysis, with 9 out of the10 of the candidate genes showing co-expression. A gene expression analysis done on cervical cancer cell lines, other cancer cell lines and normal fibroblast cell line revealed differential expression of the candidate genes. Three candidate genes were significantly expressed in cervical cancer, while the seven remaining genes showed over expression in other cancer types. The study serves as basis for future investigations to diagnosis of cervical cancer, as well as for cancers. Thus, they could also serve as potential drug targets for cancer therapeutics and diagnostics.

>Magister Scientiae - MSc / Prostate cancer (PCa) is the second most common form of cancer in men around the world. In many parts of Africa, data on prostate cancer is sparse. This is attributed to poor access to testing and diagnostics. The International Agency for Research on Cancer (GLOBOCAN) estimated that 28,000 deaths occurred as a result of PCa in Africa in 2008, 4500 of which were in South Africa. This figure (28,000) is predicated a rise to 57,000 over the next two decades. Currently, the most commonly used diagnostic tests for PCa are the DRE and PSA tests. The former is highly invasive and both have low specificity and poor sensitivity. Therefore, the need for a less invasive early detection method with the ability to overcome the lack of specificity and sensitivity is required. Biomarkers have recently been identified as a viable option for early detection of disease. Examples of biological indicators for disease are miRNAs. miRNAs are small non-coding RNA molecules which play a key role in controlling gene expression and certain biological processes. Studies have shown that aberrantly expressed miRNAs are a hallmark of several diseases like cancer. miRNA expression has been shown to be associated with tumour development, progression and response to therapy, suggesting their possible use as diagnostic, prognostic and predictive biomarkers. The study aimed to investigate the potential of miRNAs implicated in prostate cancer as putative biomarkers for the disease and evaluating these miRNAs in a panel of prostate as well as several other cancer cell lines using qRT-PCR. An in silico approach was used to identify 13 putative miRNAs implicated in prostate cancer of which 8 were further analysed in a parallel study and 5 in this study. Two publicly available target prediction software were used for target gene prediction of the 5 identified miRNAs. The target genes were subjected to functional analysis using web-based software, DAVID. Functions which were clustered with an enrichment score of 1.3 and greater were considered significant. Targets with gene ontologies linked to “transcription regulation”, “regulation of “apopotosis”, “extracellular region” and “metal ion binding” were considered for further analyses. Protein gene interaction analysis was performed to determine the pathways the target genes are involved in using STRING. Expression profile analysis of the genes in various tissues was also carried out using in silico methods through the TiGER and GeneHub-GEPIS databases. Analysis using DAVID resulted in 9 gene targets for the 5 miRNAs. It was found that miR3 seemed the most promising miRNA for biomarker validation based on the in silico analyses. Its target gene MNT was found to be abundantly expressed in prostate tissue from the TiGER results. The GeneHub-GEPIS results also indicated that the gene’s expression is up-regulated during prostate cancer. The expression levels of the miRNAs analysed using qRT-PCR indicated that miR3 is significantly over-expressed in prostate cancer cells when compared to the other cancer cell lines used in this study, corroborating the results observed from the in silico analyses. Another miRNA with interesting results was miR5. It was predicted to target two genes, YWHAZ and TNFSF13B. In TiGER, both were found to be expressed in prostate tissue. The genes were also found to be up regulated during prostate cancer in GeneHub-GEPIS. The expression level of miR5 in LNCaP was 15.32; it was significantly up-regulated in the cell line using qRT-PCR. However, miR5 was also present in HEPG2-7.06, MCF7-0.79, HT29-1.61 and H157-3.59. Thus, it was concluded it can be used as a biomarker in combination with other miRNAs. The miRNA miR2 was found to target the actin filament protein encoding gene AFAP1. The gene was predicted to be upregulated with a DEU of 33.25 in GeneHuB-GEPIS. The qRT-PCR analysis showed that the expression ratio in LNcaP was 8.79. However, miR2 expression was up-regulated in MCF7-0.85 and HT29-1.09 as well. The expression level of miR1 in BHP1 was found to be 4.85. It can be considered as an indicator for benign prostate hyperplasia. Future work would include investigating the expression of miR3 in a larger panel of cancer cells as well as in patient samples. In addition, analysis of the UTR sequences of the miRNAs targets experimentally to prove that the target genes identified using in silico methods, are indeed regulated by these miRNAs. Furthermore, performing gene “knock-out” studies on the genes that code for the miRNAs to study their roles in prostate cancer development.

No / Dementia is one of the biggest fears in the process of ageing and the most common cause is Alzheimer’s Disease(AD). Topographic disorientation is an early manifestation of AD and threatens activities of their daily lives. Finding solutions are essential in the early diagnosis of dementia if medical treatment and healthcare services to be deployed in time. Recent studies have shown that people with mild cognitive impairment (MCI) may convert to Alzheimer’s disease (AD) over time although not all MCI cases progress to dementia. The diagnosis of MCI is important to allow prompt treatment and disease management before the neurons degenerate to a stage beyond repair. Hence, the ability to obtain a method of identifying MCI is of great importance. This work presents a virtual environment which can be utilized as a quick, easy and friendly tool for early diagnosis of dementia. This tool was developed with an aim to investigate cognitive functioning in a group of healthy elderly and those with MCI. It focuses on the task of following a route, since Topographical Disorientation (TD) is common in AD. The results shows that this novel simulation was able to predict with about 90% overall accuracy using weighting function proposed to discriminate between MCI and healthy elderly.

The Alzheimer's Disease Life Events study examines whether there is a relationship between life events and Alzheimer's disease (AD). The ADLE study uses a mixed methods approach to answer the central research question:Are life events a risk factor for Alzheimer's disease? The central research question uses the following theory questions to examine:1. Is there a difference between the number of life events between patients and controls, using the Life Events and Difficulties Schedule (LEDS)(Brown and Harris, 1978) as a measurement tool?2. Is there a difference in the way (i.e. positive, neutral and negative) life events are discussed and in the range of emotions expressed when discussing life events between the patients and controls? 3. Are there any differences in the narrative constructions of life events, as interpreted by the Biographic Narrative Interpretive Method (BNIM)(Wengraf, 2001, 2008) between the patient and control groups? 4. Can the differences, between the patient and control groups, in the narratives be developed into a diagnostic marker? 5. Can the Emotion Word Coding (EWC)(Danner et aI., 2000) be used as a diagnostic marker by being applied to text collected from patients and controls over a period of decades? The ADLE study found that the patient group had experienced more life events in comparison with the control group as defined by the LEOS (Brown and Harris, 1978), and that the patient group had experienced more bereavements under the age of 51 years. The evidence supports the association between life events and AD.Even though there were significantly more life events experienced by the patients, the EWC (Danner et aI., 2001) found significantly fewer discussions expressing emotion bythe patients, particularly the negatively described ones. The range of negative and positive words used to describe the life events was significantly fewer too. This implies that the ways the patients express emotions about life events is substantially different from the controls. This finding was mirrored in the thematic field analysis of the BNIM interviews (Wengraf, 2001, 2008), which found differences in the content and structure of the narratives, and the emotional expression in the narratives about life events. A tool has been constructed using the differences between patients and controls to contribute to the early diagnosis of AD. In addition, the ADLE study has contributed to a gap in the knowledge about life events and AD.

Introdução: O carcinoma hepatocelular (CHC) é o câncer primário de fígado mais comum. A cirrose hepática é o principal fator de risco para esse tumor. O rastreamento para o CHC em pacientes com cirrose tem sido recomendado há anos. Acredita-se que a detecção e o tratamento precoce do CHC melhorem a sobrevida dos pacientes. O objetivo deste estudo foi analisar a sobrevida dos pacientes cirróticos com CHC pequeno e identificar fatores preditivos de sobrevida no Brasil. Casuística e Métodos: Entre janeiro de 1998 e dezembro de 2003, 74 pacientes cirróticos com CHC foram avaliados. Eles preenchiam os seguintes critérios: CHC com até três nódulos e no máximo 30 mm de diâmetro cada. Os fatores preditores de sobrevida foram identificados através do método de Kaplan-Meier e o modelo de Cox. Resultados: A média de idade foi de 58 anos (32-77); 71% dos pacientes eram do sexo masculino; 64% tinham hepatite C; 60% eram Child-Pugh A, o valor mediano da pontuação de MELD foi de 11; 79% tinham hipertensão portal. No momento do diagnóstico, 71% tinham uma única lesão; o tamanho do principal tumor era menor que 20 mm em 47%; o valor médio de AFP foi de 131 ng/ml. Três pacientes tinham trombose de veia porta, sugestiva de invasão vascular. Cinqüenta pacientes (67,5%) foram incluídos na lista de transplante hepático, que foi realizado só em quatro pacientes. A ressecção cirúrgica do tumor foi possível em quatro pacientes. Quarenta e oito (64,8%) pacientes receberam tratamento ablativo percutâneo (ablação por radiofreqüência ou injeção percutânea de etanol). Nove pacientes não receberam nenhum tratamento específico para o tumor. A taxa de sobrevida geral foi de 80%; 62%; 41% e 17% em 12, 24, 36 e sessenta meses, respectivamente. O tempo médio de seguimento após o diagnóstico do CHC foi de 23 meses (mediana de 22 meses, variando de um a 86 meses) para todo o grupo. Durante o seguimento, ocorreram 39 óbitos ocorreram relacionados com insuficiência hepática ou progressão do CHC. A análise univariada dos 74 pacientes mostrou que escore MELD maior que 11 (p = 0,016), classificação de Child-Pugh B e C (p = 0,007), AFP > 100 ng/ml (p = 0,006), mais de uma lesão (p = 0,041), diâmetro do tumor > 20 mm (p = 0,009) e presença de invasão vascular (p < 0,0001) foram preditores independentes de sobrevida. A análise de regressão de Cox identificou invasão vascular (RR = 14,60 - IC 95% = 3,3 - 64,56 - p < 0,001) e tamanho do tumor > 20mm (RR = 2,14 - IC 95% = 1,07 - 4,2 - p = 0,030) como preditores independentes de pior sobrevida. O tratamento do CHC esteve relacionado com melhor sobrevida. Conclusão: A identificação de CHC pequeno com até 20 mm de diâmetro está relacionada com melhores taxas de sobrevida. Por outro lado, a presença de invasão vascular, apesar do tamanho pequeno das lesões, é um fator associado a péssimo prognóstico. / Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Liver cirrhosis is the major risk factor for this tumor. Screening for HCC in patients with cirrhosis has been recommended, in the belief that detection and treatment of early HCC improves patient survival. The aims of this study were to analyze the overall survival of small HCC in cirrhotic patients and identify independent predictors of survival, in Brazil. Methods: Between January 1998 and December 2003, seventy-four cirrhotic patients with hepatocellular carcinoma were evaluated satisfying the following criteria: HCC of 30 mm or smaller and a maximum of three lesions. Predictors of survival were identified using the Kaplan-Meier and the Cox model. Results: Mean age was 58 years-old (32-77), 71% of patients was male, 64% had hepatitis C, 60% were Child-Pugh A, mean MELD score was 11 and 79% had portal hypertension. At the time of diagnosis, 71% had one tumor, the size of the main tumor was smaller than 20 mm in 47%, mean AFP level was 131 ng/ml. Three patients had portal vein thrombosis, suggesting vascular invasion. Fifty patients (67.5%) were included in the liver transplant list, but it was performed in only four patients. Tumor resection was possible in four patients. Forty-eight (64.8%) patients received percutaneous treatment (radiofrequency ablation or percutaneous ethanol injection). Nine patients did not receive any cancer treatment. The overall survival rates were 80%, 62%, 41% and 17% at 12, 24, 36 and 60 months, respectively. The mean length of follow-up after HCC diagnosis was 23 months (median 22 months, range 1-86 months) for the entire group. During follow-up a total of 39 deaths related to liver failure or HCC progression occurred. Univariate analysis of the 74 patients showed that MELD score greater than 11 (p = 0.016), Child-Pugh classification (p = 0.007), AFP > 100 ng/ml (p = 0.006), more than one lesion (p = 0.041), tumor diameter > 20 mm (p = 0.009) and presence of vascular invasion (p < 0.0001) were significant predictors of survival. Cox regression analysis identified vascular invasion (RR = 14.60 - IC 95% = 3.3 - 64.56 - p < 0.001) and tumor size > 20mm (RR = 2.14 - IC 95% = 1.07 - 4.2 - p = 0.030) as independent predictors of decreased survival. Treatment of HCC was related to increased overall survival. Conclusion: Identification of small tumors of up to 20 mm diameter is related to increase survival. Nevertheless, vascular invasion, in spite of the small diameter of the lesions, is a factor associated with dismal prognosis.

Early diagnosis represents an effective way to improve patient prognosis in cancer. New opportunities for cancer diagnosis and screening may arise from identification of cancer-specific epigenetic alterations in the cell-free circulating DNA (cirDNA). This study investigated biomarkers at the level of DNA methylation in the plasma cirDNA of individuals affected with prostate cancer or colorectal cancer. A methylation-sensitive restriction enzyme-based method was used to enrich methylated DNA fractions, which were interrogated on CpG island and human genome tiling microarrays. A number of genes and non-coding loci exhibited differential methylation between prostate cancer patients and controls. The candidate loci identified from these microarray experiments underwent verification by bisulfite modification coupled with pyrosequencing. Our results suggest that microarray-based studies of DNA methylation in the cirDNA can be a promising avenue for the identification of epigenetic biomarkers in cancer.

Early diagnosis represents an effective way to improve patient prognosis in cancer. New opportunities for cancer diagnosis and screening may arise from identification of cancer-specific epigenetic alterations in the cell-free circulating DNA (cirDNA). This study investigated biomarkers at the level of DNA methylation in the plasma cirDNA of individuals affected with prostate cancer or colorectal cancer. A methylation-sensitive restriction enzyme-based method was used to enrich methylated DNA fractions, which were interrogated on CpG island and human genome tiling microarrays. A number of genes and non-coding loci exhibited differential methylation between prostate cancer patients and controls. The candidate loci identified from these microarray experiments underwent verification by bisulfite modification coupled with pyrosequencing. Our results suggest that microarray-based studies of DNA methylation in the cirDNA can be a promising avenue for the identification of epigenetic biomarkers in cancer.