Employing epigenetic marks to detect cancer : studies on nasopharyngeal carcinoma and lung cancer

Tumor suppressor genes (TSGs) or oncogenes aberrantly methylated in transcription control regions during early carcinogenesis are potential tools for early detection of cancer. We have identified suitable genes and explored assays based on their methylation status aiming for early detection of cancer: nasopharyngeal carcinoma (NPC) and non-small cell lung cancer (NSCLC). We established and developed further the “multiplex methylation specific-PCR (MMSP)” assay designed to detect the tumor-specific methylation status of several NPC-related genes (paper I). It provided information about the methylation status of multiple genes simultaneously in a single PCR with small amounts of tumor DNA derived from nasopharyngeal swabs. It was shown to be applicable with DNA from as few as 10 cells. The detection rate of NPC from nasopharyngeal swabs was 98%. The false positive rate was zero. We employed the MMSP assay on NPC tumors from two other regions (Morocco and Italy) and compared our results with those on Chinese NPC patients from paper I (paper II). We also did a pilot study using sera from Italian and Chinese NPC. We updated the panel of MMSP markers and modified the assay to improve its applicability to NPC from different geographical locations. We could detect at least any one methylation marker gene in 97% of the EBNA1 positive samples with a specificity of 94%, while the results on sera were less informative than using swabs. We used an established NotI microarray method to identify gene losses (by deletion or methylation) in chromosome 3 of NPC tumors (paper III), This chromosome is known to contain TSGs involved in many cancer types. Ten candidate TSGs were found. Among them, the CpG rich area in the promoter region of Integrin α9 (ITGA9) was confirmed to be hypermethylated in NPC by bisulfite cloned sequencing, bisulfite pyrosequencing and methylation specific PCR. ITGA9 was downregulated in NPC clinical samples and 5-aza-2 ′-deoxycytidine restored the expression of ITGA9 in NPC derived cell lines. The functional role of ITGA9 downregulation in NPC should be explored further. We developed an MMSP assay for analysis of the methylation status of multiple potential TSGs in NSCLC samples (paper IV). Thirty-eight potential TSGs were selected, based on literature search, genome-wide CpG methylation and expression microarrays performed on NSCLC tissues and matched control tissues. After evaluation by methylation specific PCR (MSP) six of these genes were selected for inclusion into the MMSP assay. Subsequently, 70 NSCLC DNA samples with matched controls and 24 non-cancerous DNA samples were screened with this assay. With a cut off of methylation of at least any two of these marker genes 87% of the cancer samples were detected with a specificity of 94%. Early stage I or II NSCLC showed a 100% specificity and 86% sensitivity

Genetic and gene expression analysis of nasopharyngeal carcinoma (NPC)

We examined both the chromosomal copy number changes and differential RNA expression profiles in Nasopharyngeal carcinoma (NPC). Gene expression profiles identified a large number of differentially regulated genes involved in diverse functional processes, while genetic analysis detected extensive genomic abnormalities including large and small, discrete regions of copy number change and loci that exhibit uniparental disomy (UPD). The relationship between chromosomal copy number and level of gene expression were analysed. This revealed that the direct copy number/expression link applies in about 60% of the instances of copy number loss/down-expression and less than 35% of instances of copy number gain/up-expression that were examined. Signalling pathway analysis revealed that numerous components involved in the TGF-β, Wnt/β-catenin and Hedgehog pathways were universally upregulated in NPC tumours, and gene expression pattern of the C666-1 cell line approximated to other NPC tumours, indicating that it is a good tumour model. A preliminary in vitro investigation of signalling pathways revealed that the C666-1 cell line is intact in the activin and Hh signalling pathways but not in the TGF-β pathway. However, the C666-1 cells appear to resist activin-mediated cell growth inhibitio

The Role of Immune Modulatory MicroRNAs in Tumors

Tumors could evade the control of CD8+ T and/or NK cell-mediated surveillance by distinct immune escape strategies. These include the aberrant expression of HLA class I antigens, coinhibitory or costimulatory molecules, and components of the interferon (IFN) signal transduction pathway. In addition, alterations of the tumor microenvironment could interfere with a proper antitumoral immune response by downregulating or inhibiting the frequency and/or activity of immune effector cells and professional antigen presenting cells. Based on the identification as major mediators of the posttranscriptional silencing of gene expression, microRNAs (miRNAs) have been suggested to play a key role in many biological processes known to be involved in neoplastic transformation. Indeed, miRNA expression is frequently deregulated in many cancer types and could have tumor-suppressive as well as oncogenic potential. This review focused on the characterization of miRNAs, which are involved in the control of the immune surveillance or immune escape of tumors and their use as potential diagnostic and prognostic biomarkers as well as therapeutic targets. Moreover, miRNAs can have dual activities by affecting the neoplastic and immunogenic phenotype of tumors

Proteomic analysis in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a particular type of head and neck cancer with a strong ethnic and enigmatic epidemiology that long puzzles the scientific society. Despite present all over the world, around 80% of all cases are in Asia where it is endemic, followed by low incidence regions in all other continents. Portugal has the second incidence in Europe for women, and third for men (Globocan 2012) among all European countries. Many efforts have been made to clarify and understand this unique geographic and epidemiologic distribution. The onset and evolution of nasopharyngeal carcinoma is a complex multi-stage process and may take a long time to occur. Although the molecular basis remains uncertain, we know that environmental factors, Epstein-Barr virus (EBV) infection and genetic susceptibility are considered to be the three major contributors. Further examination of these genes’ expression in each tumor revealed that around 93 oncogenes are up regulated in each tumor, while the mean number of TSGs down regulated was 109. In Portugal, the works of Souza & Breda have identified important polymorphism markers that may play a role in the onset and NPC development on the Portuguese northern region population. Despite the efforts made, the molecular mechanisms of NPC carcinogenesis and progression remains to be understood. In this regard, current omics methodologies offer a different approach to identify unique miRNAs and proteins that expression signatures associated with the cancer phenotype, reflecting the biological and pathological grade of the disease. Thus, the discovery of useful NPC biomarkers will lead to new diagnostic and prognostic tools. Meanwhile, the treatment of NPC has evolved in the past two decades. Since 2000, intensity modulated radiation therapy (IMRT) has been widely used to treat nasopharyngeal carcinoma. IMRT provides better dose delivery to the target while sparing the surrounding normal tissues while local control reaches 98% at 4 years24-28. At least one prospective randomized controlled trial showed its benefit in salivary protection in HNC. However, despite the excellent local control, 43% of patients will develop distant metastasis before 5 years and die25-26. In Portugal, IMRT is used at the Instituto Português de Oncologia de Lisboa (IPOLFG) since 2009 and represents the current standard of care for HNC RT, including NPC. Until now, several prognostic markers were identified and investigated for screening, and prognostic tools for NPC, have been purposed, particularly in Asia. However, to our knowledge, there are no validated identified markers to predict distant metastasis or outcome. Moreover, the studies exploring this subject have identified a population-based variety of biomarkers stressing an omic translation of NPC ethnic distribution. At the present work, this research explored formalin-fixed paraffin-embedded samples of biopsied nasopharyngeal carcinoma tumors via proteomic analysis. The aim is to describe a tumor profiling from the studied cohort and discover biomarkers to predict distant metastasis. Secondary endpoints are the discovery of biomarkers related to tumor radioresistance and treatment toxicity. Label-free quantitative mass spectrometry was able to identify 12 up-regulated proteins on early-stage primary tumors that were not present on advanced-stage primaries. Moreover, EBV and HSV co-infection was detected. No signs of HPV-related proteins were seen. Although the clinical outcomes of early and advanced primary tumors were identical, this can be attributed to the known effectiveness of intensive chemoradiation. Moreover, the difference in tumor profiling may reflect that those earlystage tumors could benefit from de-escalation protocols. We were able to detect the presence of a pool of 10 proteins related to distant metastases. Among them, the significant presence of interferon and tyrosine kinase proteins generates the hypothesis that they may represent therapeutic targets. Validation is needed.O carcinoma da nasofaringe (NPC) é um tipo particular de cancro da cabeça e pescoço com um epidemiologia enigmática e forte cariz étnico que há muito intriga a comunidade científica. Apesar de presente em todo o mundo, 805 dos casos encontram-se na Ásia, onde é endémico, seguido de regiões de media e baixa incidência em todos os outros continentes. Portugal tem a segunda incidência em mulheres e a terceira em homens dentre todos os países da Europa (Globocan 2012). Muitos esforços foram realizados para esclarecer e compreender esta distribuição epidemiológica e geográfica. A instalação e evolução do NPC é um processo com múltiplas fases e pode demorar um longo período de tempo para ocorrer. Apesar da base molecular da sua origem permanecer incerta, sabe-se que factores ambientais, a infecção pelo vírus Epstein-Barr e a susceptibilidade genética do hospedeiro são os três maiores contribuidores. Análise aprofundada da expressão genética revelou que cerca de 93 oncogenes estão sobre regulados enquanto que o número médio de genes supressores de tumor down regulated é de cerca de 109. Em Portugal, o trabalho de Souza & Breda identificaram um importante marcador de polimorfismo que pode estar relacionado com a instalação do NPC na população portuguesa na região norte. À despeito dos esforços realizados, o mecanismo molecular da carcinogênese do NPC e sua progressão permanecem por ser esclarecidos. A este respeito, as actuais tecnologias de ômica oferecem uma abordagem diferente capaz de identificar miRNAs e proteínas únicos que expressam assinatura com um fenótipo do cancro, refletindo o grau biológico e patológico da doença. Assim, a descoberta de biomarcadores úteis levará a novas ferramentas prognósticas. Ao mesmo tempo, o tratamento do NPC evoluiu consideravelmente nas últimas duas décadas. Desde 2000, a radioterapia com intensidade modulada do feixe (IMRT) tem sido amplamente utilizada para o tratamento do carcinoma da nasofaringe. IMRT fornece melhor entrega da dose ao alvo enquanto poupa os tecidos adjacentes sadios ao mesmo tempo que o controlo local alcança 98% aos 4 anos (Lee et al, 2002). Pelo menos uma meta-análise demonstrou o benefício na capacidade de proteção salivar em carcinomas da cabeça e pescoço. Entretanto, apesar do excelente controlo local, até 43% dos doentes vão morrer devido às metástases à distância antes dos 5 anos. Em Portugal, IMRT é utilizada no Instituto Português de Oncologia de Lisboa (IPOLFG) desde 2009 e representa o actual estado da arte no tratamento da maioria dos carcinomas da cabeça e pescoço incluindo NPC. Até o momento, diversos marcadores prognósticos de NPC foram propostos, particularmente na Ásia. Contudo, até o momento não são utilizados marcadores validades para predizer metástases ou evolução do doente. Estudos avaliando esta questão identificaram uma população variada de biomarcadores distintos a outras regiões geográficas, ressaltando a diversidade étnica da população dos doentes com NPC. Neste trabalho, esta pesquisa explorou amostras de carcinoma da nasofaringe provenientes de biopsias fixadas em formalina e embebidas em parafina através de análise proteómica. O objectivo é descrever o perfil tumoral da coorte estudada e descobrir biomarcadores capazes de predizer metástases à distância. Objectivos secundários é a descoberta de biomarcadores relacionados ao tumor que determinem radioresistência ou toxicidade ao tratamento. Espectroscopia de massa por ressonância magnética foi capaz de identificar 12 proteínas sobre expressadas nas em tumores primários iniciais que não estão presentes em tumores primários avançados. Além disso, foi detectada uma coinfecção EBV e HSV sem sinais de proteínas relacionadas ao HPV. Apesar dos resultados clínicos terem sido idênticos em ambos os grupos, isto pode ser explicado pela efectividade e intensidade do tratamento combinado. Além disso, a diferença em perfil tumorais podem refletir um perfil de doentes que poderia se beneficiar de protocolos de desintensificação. Esta pesquisa foi capaz de detectar a presença de 10 proteínas relacionadas aos doentes com metástases à distância. Entre elas, a presença de interferon e inibidores da tirosina cinase geram a hipótese de que podem representar potenciais alvos terapêuticos

Epstein - Barr Virus Expression and Correlation with the Immunohistochemical Profile of Hodgkin Lymphoma: A Five Year Study

INTRODUCTION : Hodgkin lymphoma constitutes around 1% of all cancers worldwide and about 12% of lymphomas. It is characterized by heterogeneous cellularity comprising of small quantity of specific neoplastic cells, the Reed Sternberg cells. There are many evidences based on Epidemiology, molecular studies and immunologic assays which link Hodgkin lymphoma to EBV infection. Histochemical assays are often used for localizing EBV nucleic acid as well as the protein in the malignant Reed Sternberg /Hodgkin cell. EBV virus expresses latent membrane protein which can be detected immunohistochemically or by EBER, which is the viral RNA detected by in situ hybridization. AIM AND OBJECTIVES : To analyze the expression pattern of Epstein Barr Virus (EBV) Latent Membrane Protein 1(LMP1) and Ki-67 in various histologic subtypes of Hodgkin lymphoma and to compare the expression of LMP1 with CD15 and CD30 expression of HRS cells in Hodgkin lymphoma. METHODS : Blocks of 34 cases of Hodgkin lymphoma diagnosed between January 2011 to December 2015 were retrieved. Immunohistochemical staining for LMP1 and Ki-67 was done and it was correlated with parameters such as histopathological type, sex, age and stage of the disease. RESULTS : LMP1 positivity was seen in 22 out of 34 cases studied. Maximum EBV expression was seen in mixed cellularity subtype (50%). There was no significant gender predilection in EBV expression.CD15, CD30 and LMP1 were positive in 13 cases. There was a positive correlation between EBV expression and Ki-67 proliferative index with a high statistical significance (p value < .01) High Ki-67 index was more commonly seen in EBV associated Hodgkin lymphoma. CONCLUSION : Hence our study justifies the role of EBV in the oncogenesis of Hodgkin lymphoma. More elaborate and extensive studies are warranted to further emphasize this theory

Mesenchymal stem cell expressing TRAIL as targeted therapy against sensitised tumour

Tapping into the ability of engineered mesenchymal stem cells (MSCs) to mobilise into the tumour has expanded the scope of cancer treatment. Engineered MSCs expressing tumour necrosis factor (TNF)-related apoptosis inducing ligand (MSC-TRAIL) could serve as a platform for an efficient and targeted form of therapy. However, the presence of cancer stem cells (CSCs) that are resistant to TRAIL and apoptosis may represent a challenge for effective treatment. Nonetheless, with the discovery of small molecular inhibitors that could target CSCs and tumour signalling pathways, a higher efficacy of MSC-TRAIL mediated tumour inhibition can be achieved. This might pave the way for a more effective form of combined therapy, which leads to a better treatment outcome. In this review, we first discuss the tumour-homing capacity of MSCs, its effect in tumour tropism, the different approach behind genetically-engineered MSCs, and the efficacy and safety of each agent delivered by these MSCs. Then, we focus on how sensitisation of CSCs and tumours using small molecular inhibitors can increase the effect of these cells to either TRAIL or MSC-TRAIL mediated inhibition. In the conclusion, we address a few questions and safety concerns regarding the utilization of engineered MSCs for future treatment in patients

miR-205: A Potential Biomedicine for Cancer Therapy

microRNAs (miRNAs) are a class of small non-coding RNAs that regulate the expression of their target mRNAs post transcriptionally. miRNAs are known to regulate not just a gene but the whole gene network (signaling pathways). Accumulating evidence(s) suggests that miRNAs can work either as oncogenes or tumor suppressors, but some miRNAs have a dual nature since they can act as both. miRNA 205 (miR-205) is one such highly conserved miRNA that can act as both, oncomiRNA and tumor suppressor. However, most reports confirm its emerging role as a tumor suppressor in many cancers. This review focuses on the downregulated expression of miR-205 and discusses its dysregulation in breast, prostate, skin, liver, gliomas, pancreatic, colorectal and renal cancers. This review also confers its role in tumor initiation, progression, cell proliferation, epithelial to mesenchymal transition, and tumor metastasis. Restoration of miR-205 makes cells more sensitive to drug treatments and mitigates drug resistance. Additionally, the importance of miR-205 in chemosensitization and its utilization as potential biomedicine and nanotherapy is described. Together, this review research article sheds a light on its application as a diagnostic and therapeutic marker, and as a biomedicine in cancer

Multidimensional Approach to Understanding Head and Neck Cancer

Head and neck is the sixth most common cancer type worldwide, with the highest incidence observed in South and Southeast Asia. Comprised of a heterogeneous group of malignancies, head and neck cancers (HNC) span several anatomical subsites, including the: oral cavity, oropharynx, hypopharynx, nasopharynx, larynx, salivary glands, paranasal sinuses and nasal cavity. While ~90% of these neoplasms are morphologically characterized as squamous cell carcinoma, etiological differences across cancer subsite, along with geographic attributes that influence the variable global incidence and mortality trends, make HNC a therapeutically challenging and behaviorally variable disease. The goal of this dissertation is to improve the current state of knowledge surrounding HNC through the use of descriptive and molecular epidemiological methods. To do so, the first aim utilized several sources of cancer registry data to assess trends in head and neck, and separately nasopharyngeal, carcinoma to better understand how temporal changes in environmental and behavioral risk factors, as well as advances in clinical practices, influence cancer onset and survival. Culminating in four manuscripts, the first two focused on nasopharyngeal carcinoma (NPC); findings revealed Asian/Pacific Islanders living in the U.S., a non-endemic region, to have comparable rates of NPC incidence as those in endemic Thailand. Interestingly, the Epstein-Barr virus (EBV) related differentiated non-keratinizing subtype was shown to be increasing across nearly all gender and race groups in the U.S., while survival rates appeared to vary. The remaining studies highlighted the role of decreasing tobacco, alcohol and betel quid use, three major risk factors in HNC development, on decreasing laryngeal and oral cavity cancer rates across the U.S. and Southeast Asia. Nevertheless, as the world’s largest tobacco manufacturer and consumer of ~30% of the world’s cigarettes, Chinese males in Shanghai experienced a 9.1% annual increase in laryngeal cancer between 1998 and 2002. The reductions in tobacco use have elucidated the role of human papilloma virus (HPV) as an important causal factor in oropharyngeal carcinoma (OPC). To date, this etiological shift has almost exclusively been studied and proposed to effect Western, more developed countries. Using descriptive epidemiology in aim one to inform molecular analyses in aim two, the results of these studies challenged previous assumptions. Having undergone rapid socioeconomic change, cancer has superseded infectious disease as the leading cause of mortality in Thailand. In accordance with economic growth, changes in cultural norms have allowed for an etiological transition that has led to an increase in oral HPV infections, and consequently OPC incidence. Although far from the 80% prevalence observed in the U.S., OPC HPV rates in Thailand have increased from 16% in 2012 to 26% in 2017—expecting to exceed 50% by 2030. Despite medical advances in HNC treatment, 5-year relative survival has remained disappointingly stable at 50-60% over the past few decades. Recent studies have shown the presence of tumor-infiltrating lymphocytes to be an important prognostic tool in guiding HNC treatment. For the last aim, dietary factors, which have previously been shown to be important in HNC risk, progression and prognosis, were assessed and shown to significantly influence tumor immune response and subsequently survival. Overall, the research presented in this dissertation not only provides a better understanding of HNC on a global scale, but also offers a basis for future studies to aid in elucidating etiology and improving treatment and prevention for this highly disfiguring and deadly disease.PHDEnvironmental Health SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155216/1/argirion_1.pd