HPV-associated Cancers (CERVIVA)
CERVIVA (www.cerviva.ie) is a multi-investigator collaboration led by Trinity College and encompassing researchers at a range of Irish academic institutions, hospitals and commercial diagnostic or biotechnology companies. Our purpose is to advance high quality peer-reviewed research programmes that provide the best possible information and guidance in the delivery of cancer services to Irish people. Our focus of research is on Human Papillomavirus (HPV) associated cancers, including cervical, head and neck cancers, anal, vaginal and vulval cancers. We work across 4 main thematic areas
- Molecular Epidemiology
- Health Economics
- Health Psychology
- New Technologies
CERVIVA-Vax: Monitoring the impact of HPV vaccination in Ireland
Cervical cancer prevention is changing worldwide. The introduction of primary prevention [vaccination] and changes to screening protocols [HPV testing], raise several challenges for cervical screening programmes, most notably with regard to impact of vaccination on screening as it is currently organised. If vaccination affects women’s likelihood of screening participation, or the performance of screening and diagnostic tests, screening will become less effective. Monitoring the impact of vaccination on screening is, therefore, important.
In Ireland, a national school-based HPV vaccination programme began in 2010. As the vaccine only targets the main HPV types (16/18), it is important that all women, aged 25- 60, continue to have regular cervical smear tests. In 2018, the first set of women who received the vaccine through the school programme (women in the “catch-up” cohort, of whom 19,000 were fully vaccinated), will be eligible to attend for their first CervicalCheck smear.
By following this cohort, CERVIVA-Vax will generate, for the first time, Irish data relating to the early impact of HPV vaccination on cervical screening. CERVIVA-Vax will assess the prevalence of HPV, HPV genotype diversity, abnormal cytology, and histology outcomes in vaccinated girls compared to age-matched unvaccinated woman enrolled in the CERVIVA HPV Primary Screening Study. CERVIVA-Vax will generate, for the first time, HPV prevalence, HPV genotype and molecular biomarker [E6/E7 mRNA, p16ink4a/Ki-67, and methylation markers (CADM1, MAL, miR124)] data on vaccinated women. Finally CERVIVA-Vax will investigate the impact of vaccination on screening uptake.
Changes in screening uptake, HPV infections and detection of cervical abnormalities may have an adverse effect on how well screening works for individual women and the population. By investigating the early impact of HPV vaccination on screening in Ireland, CERVIVA-Vax will be able to inform CervicalCheck, and international screening programmes, of the best cervical screening approach for both vaccinated and unvaccinated women.
HPV Primary Screening Pilot Study: molecular testing of potential triage strategies for HPV-positive
The HPV Primary Screening Pilot will explore the use human papillomavirus (HPV) testing as a first-line cervical screening method. The current method of examining smear test samples is called PAP test where the cells from your smear test sample are looked at under a microscope to check for abnormalities. HPV testing is a slightly different way of examining your smear test sample. It involves a test that looks for the presence of Human Papillomavirus (HPV). HPV is a common virus that is linked with changes in the cells of the cervix (neck of the womb) and cervical cancer. Research has shown that HPV testing has many benefits over the PAP test. One benefit is that it can detect more cervical cell abnormalities than the PAP test can.
The presence of HPV does not always lead to the development of cervical cancer. To ensure HPV testing is effective, we need to find out which HPV positive samples are likely to develop into cancer. To do this, we will look into samples that test positive for HPV in a little more detail. We will do additional tests to look for specific markers that we know are linked to HPV infection. The additional tests will include cytology, HPV 16/18 genotyping, p16INK4a/ki67, and methylation markers. By following women to see what happens over the course of several smear tests we will be able to determine how useful these new approaches to cervical screening are.
HPV and Biomarker Screening [HRB-ICE]
HPV infections play a crucial role in the aetiology of cervical lesions. Various molecular assays have been developed to detect HPV. HPV DNA testing is already being used for the management of cervical disease. While it is generally accepted that HPV DNA testing is more sensitive for detection of cervical disease compared with cytology, it is less specific in particular in younger age groups, largely due to the high prevalence of transient HPV infections. As a consequence, the appropriate framework for HPV testing is paramount to avoid unnecessary testing and follow up. There is a clear need to improve the specificity of HPV DNA testing, and this can be achieved at various levels, including HPV genotyping and viral load assessment. An alternative approach is to triage with some form of secondary biomarkers. Several biomarker options exists for this including (1) measuring HPV E6 and E7 mRNA transcripts, (2) alterations to methylation patterns of several genes, (3) alterations of viral and host genomes (4) detection of cellular proteins overexpressed in HPV infected cells.
Within this project, CERVIVA aims to address the need for additional triage markers for cervical screening through evaluation of a combined approach of HPV DNA, HPV mRNA, p16INK4A/ki67 and a novel panel of 20 mRNA biomarkers developed as part of an FP7 funded programme grant "AutoCast" in a colposcopy controlled study. The data generated from this colposcopy controlled study will support a primary screening algorithm of primary HPV testing followed by biomarker triage.
This will be the first study to evaluate all three biomarker sets in a single population.
Human Papillomavirus (HPV) in Men who have sex with men (MSM); Awareness, Prevention, Surveillance - HPV MAPS Project.
Human Papillomavirus (HPV) is the most common sexually transmitted infection worldwide. Over 90 HPV serotypes have been identified; low risk HPV-types (6 and 11) are associated with genital warts. High risk types (16 and 18) are associated with cervical, oral and anal cancer.
Non-cervical HPV-associated cancers, which individually are relatively rare, now collectively parallel the burden of cervical cancers in developed countries. MSM and in particular HIV+ MSM are disproportionately affected with rates of anal cancer up to 70 times greater than the general population.
The quadrivalent HPV-vaccine (6/11/16/18) prevents persistent HPV infection and diseases associated with vaccine serotypes. In line with other European countries, Ireland has implemented an immunisation programme for girls only since 2008.
Research undertaken by HPV MAPS has identified high prevalence (69%) and persistence (30%) of anal HPV-infection in MSM in Ireland.
Interim analysis of a HPV-awareness study (90 HIV+ MSM) has identified poor knowledge of HPV-infection/associated disease but a high level of HPV-vaccine acceptability.
The purpose of our research is:
- To measure awareness of HPV-infection, disease association and HPV-vaccine acceptability in MSM. This will provide focus for education and prevention interventions.
- Measure functional and immunological efficacy of HPV-vaccine in HIV+ and HIV- MSM <26 years of age. Phase-2 of this study will assess efficacy of HPV-vaccine in a broader group of HIV+ MSM > 26 years of age. This will guide vaccine scheduling recommendations in this patient subgroup.
- To measure prevalence and serotype of HPV-infection/disease at multiple sites (oral and anal) in MSM (vaccine and non-vaccine recipients). Surveillance data will facilitate monitoring of vaccine effectiveness and identify potential replacement strains of HPV.
- To further examine novel bio-pathological mechanisms that may be operational in superficial human epithelial cells facilitating HR-HPV infection and replication. This will further advance understand of HPV related disease.
What influences cervical screening uptake in older women and how can screening programmes translate this knowledge into behaviour changing strategies? A CERVIVA-CervicalCheck co-production project.
Well-organised cervical screening is effective in reducing cervical cancer incidence and mortality. To achieve these benefits, high coverage is essential. In Ireland, the coverage target is 80%. While overall coverage has risen (from 61% to 79%) since the programme started in 2008, it has consistently been lower in older (50-60 years) than younger (25-49 years) women. This distinctive pattern is not seen in other countries with organised programmes and the reasons for it are unknown. Women in their early 50s are at risk of developing cervical cancer and screening at that age substantially reduces cancer risk for the subsequent 30 years. Thus, it is important to attain high screening coverage in older women.
This CERVIVA-CervicalCheck co-production project will generate evidence on the influences on cervical screening participation among older women in Ireland, to inform development and implementation of evidence-based strategies to increase screening coverage in this group.
A mixed-methods design will be used, underpinned by a theoretical framework of behaviour change. Phase 1 will involve in-depth interviews exploring influences on, barriers to, and enablers of, cervical screening participation; older and younger women and women with adequate and inadequate screening histories will be compared. Phase 2 will involve a postal survey of older women, comparing those with adequate and inadequate screening histories, to identify the most important influences on screening participation and inter-relationships between these. Phase 3 will triangulate phase 1 and 2 results and undertake a behavioural analysis to begin to generate evidence-based solutions for how screening participation may be changed.
The findings will be translated by the National Screening Service into strategies to improve participation in older women, thereby optimising effectiveness of cervical screening in Ireland and delivering health gains for individual women and the population. The knowledge gained may also be applicable to other screening programmes in Ireland.
Development of a Theory-Based Psychoeducational Intervention for Cervical Cancer Screening
The aim of this project led by our partners at the National Cancer Registry is to develop a theory-based psycho-educational intervention to alleviate the adverse psychological after-effects of colposcopy and related management procedures, such as punch biopsies and LLETZ.
This workpackage is building directly on our work in CERVIVA Phase 1, including our longitudinal quantitative study of psychological impact and health-related quality of life in women attending colposcopy. As part of this workpackage, we conducted a qualitative study involving in-depth interviews with women about their experiences of colposcopy and psychological after-effects. The findings of this study have been written up and submitted for review at a scientific journal. We undertook the first ever systematic review of the adverse psychological after-effects of colposcopy and related procedures.
Currently, we our using the results of the longitudinal quantitative study, qualitative study on women’s experiences of colposcopy and the systematic review to inform the development of an appropriate based psycho-educational intervention to alleviate the adverse psychological after-effects of colposcopy and related procedures.
Optimising cervical screening in the HPV vaccination era [HRB-CARG]
The entry into the cervical screening programme of women who have been vaccinated against HPV will impact on screening effectiveness and, hence, cost-effectiveness. Vaccinated women have a much lower risk of having cervical intra-epithelial neoplasia, and this will affect the performance of the screening test and other investigations. It is likely that new screening protocols will need to be developed (for example, based on primary HPV screening, or less frequent screening, or starting screening at an older age). The challenge for decision-makers and service providers is to decide how screening should be organised for maximum effectiveness and cost-effectiveness in the “HPV vaccination era”.
This workpackage has two main objectives: (1) to ascertain the likely impact of HPV vaccination on current cervical screening protocols and (2) to investigate the effectiveness and cost-effectiveness of alternative screening scenarios in the “HPV vaccination era”.
To achieve this CERVIVA will: in conjunction with key stakeholders : identify a set of plausible and feasible alternative screening scenarios; assess available mathematical and economic models; and identify and parameterise the model most suitable for the Irish setting. This model will then be used to estimate the impact of HPV vaccination on current screening practice, the likely changes over time as more of the screening population are vaccinated, and to make recommendations on the most effective and cost-effective screening options.
Optimal screening and management strategies for cervical cancer [HRB ICE]
The overall goal of health economics is to maximise the health gain achieved by health services within available funding. The application to health economics to cervical screening has the particular goal of finding which screening tests will prevent the most disease at an acceptable cost to the health system. The cost-effectiveness of cervical screening depends in part on the interval between screens. If the interval is very short, such as one or two years then screening may impose high costs with little or no benefit over longer screening intervals. Cervical screening at intervals of three to five years as employed in Ireland is thought to be highly cost-effective. Recent developments in screening technology mean that more advanced tests are now available to detect early stages of cervical disease. These new tests include tests for the DNA and RNA of the human papillomavirus. The health economics research in CERVIVA II is to find which new testing technologies will further enhance the cost-effectiveness of Irish cervical screening services. This involves considering the trade-offs of the likely benefits of new tests that are better able to detect disease against the possible costs of false positives and the costs of the testing technologies themselves.
The CERVIVA new technologies theme is focussed on developing new diagnostic and prognostic tests/technologies for use in cervical screening and cervical cancer pre-cancer diagnostics. We are adopting state-of-the-art approaches and technologies including proteomics, nanotechnology, spectroscopy, imaging, lab-on-a chip, and biochips to develop improved personalised medicine approaches for screening and cervical pre-cancer/cancer diagnostics. We collaborate with national and international partners in this regard through EU FP7, H2020 funded initiatives.
Ongoing work as part of CERVIVA, in partnership with DIT has been developing a novel tool for cervical cancer screening based on Raman spectroscopy. Raman spectroscopy is a powerful tool that can generate a biochemical fingerprint of a sample in a rapid and non-destructive manner. Raman spectroscopy is an optical method based on inelastic light scattering. The sample is illuminated by monochromatic laser light and interactions between the incident photons and molecules in the sample result in scattering of the light. The exact energy required to excite a molecular vibration depends on the masses of the atoms involved in the vibration and the type of chemical bonds between these atoms and may be influenced by molecular structure, molecular interactions and the chemical microenvironment of the molecule. Therefore, the positions, relative intensities and shapes of the bands in a Raman spectrum carry detailed information about the molecular composition of the sample and can be thought of as a biochemical fingerprint. There is convincing evidence from our own and other laboratories that Raman spectroscopy can be used as a diagnostic tool to identify spectral changes in malignant and premalignant cells.
The technology is being developed into ’the molecular Pap test’ which can potentially identify biochemical changes related to CIN and high-risk HPV infection. Raman spectroscopy can differentiate between HPV positive and HPV negative cervical ThinPrep samples with the variation between the samples being attributed to differences in protein, carbohydrate and lipid content.
This programme will conduct the first clinical trial of this technology for cervical screening. The trial is based at the Coombe Women and Infants University Hospital.
Our gynaecological cancer research group works jointly with the Department of Obstetrics and Gynaecology. The group is primarily interested in discovering and validating both diagnostic and prognostic biomarkers for ovarian and other gynaecological cancers. The group also investigates mechanisms of chemoresistance and recurrence in ovarian cancers. Our research is funded by Health Research Board, Emer Casey Foundation, Supporting Ovarian Cancer Knowledge (SOCK), and the Royal City of Dublin Hospital Trust Fund. We also work closely with our industry partners, Fujirebio and Roche.
Gynaecological cancer biobank (DISCOVARY bioresource)
Our group has an extensive biobank [DISCOVARY bioresource] of serum, blood and plasma and tissue from malignant and benign tumours. This biobank underpins all of our translational research and provides important material for investigating tumour markers which can help in early diagnosis and in more targeted treatments. The bioresource is a collaborative effort between St James’s and Trinity College Dublin. This biobank is linked to a dedicated database which provides full clinical information and follow-up data on each patient. This allows us to do studies looking at determinants of survival and treatment response. To date it has recruited over 3000 patients and recruitment is ongoing. A recent epidemiological review of cases from our bioresource reveals our figures are in line with the national averages so we are confident our sample population is representative of the gynaecological cancer cases that present in Ireland.
INNOVATION-the Irish National Network for Ovarian Cancer Collaboration
Our group participates in a nationwide network internationally recognised ovarian cancer clinical and scientific experts. Established in 2014, the aim of INNOVATION is to integrate patient clinical pathways with cutting edge research to improve diagnosis and treatment of ovarian cancer. Further information can be found at http://www.molecularmedicineireland.ie/research/innovation
The role of HE4 in gynaecological cancers
HE4 is a serum biomarker which has both diagnostic and prognostic roles in ovarian cancer. We are currently working with our industry partners to evaluate this biomarker in our centre and assessing the clinical scenarios where it may have potential.
HE4 has also shown utility in endometrial cancer and we are currently investigating this.
Validation of a histological chemotherapy response scoring system following Neoadjuvant Chemotherapy in advanced Ovarian Cancer with correlation to clinical outcomes
There is still considerable controversy over the sequencing of treatment in advanced ovarian cancer. With Primary Debulking Surgery (PDS) forming the cornerstone of treatment for decades, there has now been debate over the possible increasing role of neoadjuvant chemotherapy (NACT) followed by Interval Debulking Surgery (IDS). Optimal cytoreduction at surgery regardless of sequence remains key in optimising overall survival. Recent development of a validated histological chemotherapy response score following IDS for high grade serous ovarian cancer has provided another method of prognostic scoring along with debulking status. We are reviewing our cohort of neoadjuvant chemotherapy and IDS patients and will perform a further independent validation of the Chemotherapy Response Score with assessment of its correlation to clinical outcome.
Validation of chemoresponse markers in ovarian cancer
The group has focussed a lot of work on identification of novel prognostic biomarkers and chemoresponse biomarkers for ovarian cancer. Many biomarkers have been validated and mechanistic functional work is currently in progress. Such markers include TLR4, MyD88, MAD2 and MMP-9.
Towards Clinically Relevant Immunosignatures in Ovarian Cancer (PhD Sara O’Kane, joint student registered UCD) Funded partly by the Emer Casey Foundation
Currently there are no reliable biomarkers for the diagnosis of early ovarian cancer (OC). A lack of specific symptoms of the disease means only 20% of ovarian cancers are diagnosed at an early stage (Stage 1). Autoantibodies are an attractive biomarker entity as they are present in blood and can be adapted into current diagnostic platforms. It is accepted that the complexity of cancer means that a panel of biomarkers will be required as a diagnostic test rather than the more traditional approach of identifying a single biomarker. Autoantibodies associated with ovarian cancer were identified and pathway analysis was performed to determine if autoantibodies can be indicative of pathway dysregulation associated with malignancy. This project is focussed on refining and validating a panel of biomarkers which has the potential to diagnose early ovarian cancer.
Use of Nanotechnology to improve the treatment of ovarian cancer
Ovarian cancer (OC) is the seventh most common female cancer and one of the most devastating diseases affecting the life of many women worldwide. It has become increasingly clear that the effectiveness of the available platinum-based chemotherapies is profoundly inadequate, largely due to the development of acquired resistance in patients with OC. In this study, nanomedicine-based therapeutic approaches are being used to enhance the treatment efficacy with minimal doses of chemotherapeutic drugs against cancers. Nano Diamonds are being coated with drugs and targets such as HE4 used to direct the treatment to the correct cells. The models being used include cell line models and ex vivo explant models.
Outreach and dissemination events are held frequently and often with the associated charities. The clinicians and scientists speak at town hall and public forum events, TV and partake in annual events highlighting the various cancer types; e.g. world ovarian cancer day.
A Public Patient Involvement (PPI) group within the Irish Society of Gynaecological Oncology was established in 2016. A number of workshops have taken place and the group has commenced its first research project. Members of the PPI group were invited as patient representatives on the National Cancer Guidelines committee for diagnosis of ovarian cancer.
Some of the patient charities that work directly with the Trinity Cancer group include:
Ovacare was established in 2011 to improve diagnosis and education of ovarian cancer within Ireland, through sharing global research and best practice, and providing support and advocacy through OvaCare’s dedicated support network. Patient Days take place across the country which provide information and support for ovarian cancer patients and their families. It provides the opportunity to hear from leading clinicians, therapists and researchers specialising in the disease, as well as being a chance to meet fellow patients.
Supporting Ovarian Cancer Knowledge (SOCK) was the brainchild of the late Jane Keating. Following her diagnosis with ovarian cancer, Jane wanted to do something positive as there was limited information available to women about ovarian cancer. SOCK it is a non-profit organisation and is dedicated to raising funds for research into and awareness of ovarian cancer.
The Emer Casey Foundation was established in 2006 following the death of Emer Casey. Emer was the youngest of a family of five daughters and she had just recently qualified as a solicitor at Matheson Solicitors in Dublin when she became ill. She was diagnosed with ovarian/endometrial cancer in February 2006 and she died on June 10th 2006 aged only 28. Since then Emer’s family, friends and work colleagues at Matheson Solicitors have been working on behalf of the Foundation. To date the Foundation has raised in excess of €900,000 and has supported 3 PhD fellowships in Trinity College Dublin.
Lung and Prostate Cancer
Associate Professor Stephen Finn is joint head of the Thoracic Oncology Research Group, head of the Translational Prostate Cancer Group, and co-director of the Cancer Molecular Diagnostics Laboratory (CMD) based at St. James’s Hospital. Dr. Finn’s research has resulted in a Prostate Cancer Foundation (PCF) Young Investigator Award and a co-PI position on a PCF Challenge Award. He is a member of the Irish Prostate Cancer Research Consortium (PCRC) and a steering committee member for Irish Prostate Cancer Outcomes Research (IPCOR). Dr. Finn is a founding member and a lead investigator with the Trans-disciplinary Prostate Cancer Partnership (ToPCaP), and a member of the Australian-Canadian Prostate Cancer Research Alliance. The group have active links with the Harvard School of Public Health, the Dana Farber Cancer Institute, the University of Orebro, Guy’s and St. Thomas’s Hospital and Queensland University of Technology. Additionally, Dr. Finn and his team are associated with the European Thoracic Oncology Platform (ETOP – Lungscape (Steering Committee) , Mesoscape and SPECTAlung (Steering Committee) and the International Association for the Study Lung Cancer (IASLC).
Project: Role of lipids in treatment response and prostate cancer-specific outcomes.
The main objective of this project is to identify tumour biomarkers of dysregulated lipid metabolism that are predictive of treatment response and prostate cancer progression and that can be modified by lifestyle changes. This project bridges epidemiology and molecular biology, linking colleagues in biostatistics, molecular pathology, cancer biology and genetics at Harvard University and Trinity College Dublin.
Project: Discovery and Clinical Implementation of Novel Predictive Biomarkers for Enzalutamide therapy.
This project utilises samples obtained in both the iPROSPECT (Irish PROgramme for Stratified ProstatE Cancer Therapy) and Radium 233/Enzalutamide clinical trial, with the aim of transforming current practice to a more individual and personalised treatment of patients with metastatic prostate cancer in order to improve patient outcomes. Most forms of castration resistant prostate cancer (CRPC) are dependent on the androgen receptor (AR) for survival. While, enzalutamide provides a substantial survival benefit, it is not curative and many patients present with intrinsic and acquired resistance. Although not yet fully understood, resistance can develop through a number of mechanisms, such as AR copy number gain or the generation of splice variants such as AR-V7. The project consists of the following three aims:
- Broad genomic analysis of patient plasma samples using custom made Prostate NGS panels
The data from this aim will allow for (i) an assessment of genomic changes over time within the natural course of disease and treatment, (ii) a comparison of NGS panels (broad vs. targeted mutational screening), and (iii) an evaluation of the feasibility and sensitivity of a liquid based biopsy (plasma) to detect known and novel mutations.
- Broad investigation of AR-V7 in blood samples and circulating tumour cells (CTCs)
The data from this aim will (i) evaluate whether RNA from whole blood is superior to plasma to profile AR status, (ii) determine if baseline AR-V7 may prove useful in predicting patients response to therapy, (iii) identify known and novel drug resistance mutations in a cohort of Irish patients with Prostate Cancer and (iii) assess the utility of CTC numbers as a predictive marker for therapy.
- Investigation of Circular RNAs within AR mutation harbouring cell lines and plasma samples
Circular RNAs (circRNAs) are novel long non coding RNAs, which may play an important role in cancer initiation and progression. Studies have shown that they can act as competitive endogenous RNAs (ceRNAs) or as microRNA (miRNA) sponges. This aim will examine the circRNA/miRNA pathways in prostate cancer progression, and in the development of drug resistance.
Project: Exercise, Prostate Cancer and Circulating Tumour Cells (ExPeCT)
Obesity, known to be associated with a pro-inflammatory, pro-thrombotic humoral milieu, confers a worse prognosis in prostate cancer. Circulating tumour cells (CTCs) are identified in the blood in advanced cancer. Their quantitation provides prognostic information. "Cloaking" of CTCs by adherent platelets impedes natural killer (NK)-cell clearance of CTCs from the circulation, enhancing metastatic spread. NK-cell function in blood and in solid organs is quantitatively and qualitatively reduced in obesity. Platelet cloaking may be enhanced in obesity due to the pro-inflammatory, pro-thrombotic state, and may be a mechanism for worse cancer-specific outcomes in this group. Obesity and its biochemical effects may be influenced by lifestyle changes such as exercise. Physical activity reduces levels of systemic inflammatory mediators and so an aerobic exercise intervention may represent an accessible and cost-effective means of ameliorating the pro-inflammatory effects of obesity. The ExPeCT trial will determine if a prescribed exercise intervention can ameliorate the degree of platelet cloaking in obese and non-obese men with advanced prostate cancer. The specific aims include:
- The enumeration of CTCs and platelet cloaking in all participant samples. The number of CTCs and evidence of platelet cloaking will be compared between the exposed and non-exposed groups and any possible correlations with clinical data noted.
- Project 2 consists of an exercise programme for participants randomised into the exercise arm. Participants in this arm complete six months of prescribed aerobic exercise at a moderate-to-vigorous intensity, which is progressed using percentage heart-rate reserve. CTC number and levels of platelet cloaking will be compared across all three time points, T0, T3 and T6, in both the control and exercise arms and the impact of exercise evaluated.
- Assessment of inflammatory mediators in patient sera and infammatory infliltrate in tumour tissue.
- Determination of a lethality gene signature, and its association with platelet cloaking and obesity.
This project is funded by the World Cancer Research Fund (WCRF).
Project: ‘Identifying long non-coding RNA transcripts associated with resistance to enzalutamide in prostate cancer.’
The key clinical challenge is to identify the mechanisms underpinning enzalutamide resistance so as to guide therapy and improve response rates. Previous assessments have implicated expression of long non-coding RNAs as mediators of enzalutamide resistance. By working with RNA from isogenic parent and enzalutamide resistant cell lines developed in our laboratory, we aim to identify long non-coding RNA transcripts associated with resistance to enzalutamide. This research is supported by a grant from the Pathological Society of Great Britain and Ireland.
Project: Developing A Rationale For Gene Silencing and Epigenetic Targeted Therapies For Stem Cell Populations In Advanced Prostate Cancer
The aim of this project is to investigate the anti-cancer efficacy of cationic solid lipid nanoparticles as an siRNA carrier system for use in the treatment of prostate cancer. It will also investigate the anti-cancer effect of the simultaneous administration of this complex with a pre-clinical lysine demethylase. Cell line models will include established prostate cancer cell lines, Enzalutamide resistant clones and their respective cancer stem cell subpopulations.
Ms Oner is funded by the ‘Research fellowship program for PhD students’ from TUBITAK (The Scientific and Technological Research Council of Turkey).
Project: Optimisation and characterisation of assays to identify Met exon 14 skipping in formalin fixed, paraffin embedded (FFPE) non-small cell lung cancer (NSCLC) samples.
The hepatocyte growth factor (HGF) receptor (MET) is frequently altered in non-small cell lung cancer (NSCLC). Tumours with a specific type of MET mutation – Met exon 14 skipping - have been shown in clinical trials to be more sensitive to MET inhibitors (e.g. Crizotinib) when compared to other mutation types. The prevalence of MetEx14 skipping has been shown to be highest in sarcomatoid carcinoma of the lung. We aim to optimise and characterise a number of different laboratory assays to accurately identify Met exon 14 skipping in FFPE NSCLC samples. These assays include a one-step RT-PCR end-point PCR assay, an RNA in situ hybridization (RISH) technique using a specific BaseScope™ Assay and next-generation sequencing.
Project: A retrospective cohort study of PD-L1 by RNA in-situ hybridisation (RISH) as a potentially superior companion biomarker for immune checkpoint inhibitors in non-small cell lung cancer (NSCLC).
The aim of this project is to explore a new scientific technique called RNA in-situ hybridisation (RISH) RNAScope, which will be used to detect PD-L1; a biomarker expressed in a percentage of patients diagnosed with non-small cell lung cancer (NSCLC). Digital pathology and image analysis will also be used to give a more accurate interpretation of PD-L1 staining if compatible with RISH. Currently immunohistochemistry (IHC) is the gold standarsd method for detecting PD-L1 positivity in patients with NSCLC, however reading IHC stained slides for PD-L1 is very difficult and has proven to be subjective amongst pathologists. Pemprolizumab immunotherapy (Keytruda) is only administered to patients who have a tumour expressing 50% or greater positivity for PD-L1 by IHC, however in a study carried out in 2015, a group of patients who tested negative for PD-L1 actually responded to the drug. Another study showed that the sensitivity of RISH for detecting PD-L1 was greater than IHC, which begs to question, is it possible to lower the threshold with this new assay and will digital pathology give a more accurate interpretation of positive PD-L1 tumour cells.
Project: Enhancing Detection of Resistance Mutations in EGFR Targeted Therapy for Non Small Cell Lung Carcinoma
The aim of the study is to identify sensitivity of EGFR mutation status in retrospective and prospective patients with EGFR-activating mutation to de novo and acquired T790M mutations in cf-DNA in patients with EGFR-activating mutations. One factor that may impede uptake of cf-DNA testing in a National Testing environment is that most blood samples collected in a hospital setting will use standard EDTA vacutainer tubes. In essence these are non-optimal for EGFR mutation testing using cf-DNA, as they require processing within 4 hours in order to protect sample integrity. For this reason we are conducting a comparator study, which compares standard EDTA vacutainer tubes versus specific Cell-Free DNA Collection Tubes. The aim of this comparator study is to monitor for effective detection of EGFR mutation status following storage of the blood samples held in specific Cell-Free DNA Collection Tubes over an extended period of time. In non-small cell lung carcinoma, the importance of circulating tumour cell detection and molecular characterisation is emerging. These cells detach from the primary tumour and move to a new site via the bloodstream to cause tumour growth. They are valuable biomarkers for disease diagnosis and progression as well as response to treatment; however, they have not progressed to the clinical mainstream for NSCLC. We aim to use the presence or absence of CTCs as an additional quality control for the assessment of plasma mutations. The hypothesis is that when CTCs are present there is a higher chance of ctDNA containing tumour DNA content
Reclassification of THyroid cancer - Implications for patient Management and well-being (RyTHM)
A project employing Next Generation Sequencing (NGS) in Fine Needle Aspiration and Liquid Biopsies of Thyroid to determine the implications for patient management and wellbeing.
In 2015, changes to the guidelines for treating patients with thyroid disease were published and in 2016 an international panel of pathologists and clinicians reclassified a subtype of thyroid cancer to reflect that it is noninvasive and has a low risk for recurrence.
These changes will affect the way the disease is viewed and managed by caregivers and patients. It may eliminate the psychological impact of receiving a cancer diagnosis, while impacting on psychological wellbeing as a result ongoing monitoring and active surveillance.
This project aspires to mitigate the financial burden on service provision and psychological burden of watchful waiting on the patient by augmenting pathology assessment with molecular diagnostics to better classify thyroid lesions and accurately monitor ongoing disease or progression.
Biological Characteristics of Oesophago-Gastric Tumours and their Clinical Correlates
Malignancies of the lower oesophagus and oesophago-gastric junction are increasing in prevalence. Worldwide, squamous cell carcinoma is the most common form of oesophageal cancer, however in the western world the incidence of adenocarcinoma has been increasing in recent years, most commonly seen in the lower oesophagus and the junction between oesophagus and stomach. These tumours, currently treated with a combination of surgery, chemotherapy and radiotherapy, at present have a 5 year survival rate of <20%. Our study seeks to investigate the biological nature of these tumours as they de-differentiate, becoming more high grade and aggressive.
Current research suggests that epithelial-mesenchymal transition (EMT), involving de-differentiation of epithelial cells back to a multi-potent state, may in some circumstances lead to the development of cancer stem cells. This is evidenced by the fact that the reprogramming factors which are involved in EMT have also been shown to play an oncogenic role in many organ systems.
Cancer cells may be present within the same tumour in various stages of de-differentiation, a phenomenon known as intra-tumour heterogeneity. Cancer stem cells function as one such biologically unique subpopulation of cells within a tumour which self-renew indefinitely, and are often insensitive to current cancer treatments.
We seek to investigate the areas within lower oesophagus and oesophago-gastric junction tumours which have the greatest degree of de-differentiation, identified based on histological characteristics, in addition to location at the pushing/infiltrative edge of the tumour. We postulate that this will allow us to examine the role of stem cells in tumour development. Accordingly, we aim to correlate these biological findings with patient outcome: specifically, response to therapy and disease-free survival.
Resection specimens of patients who underwent surgery with curative intent for lower oesophageal/oesophago-gastric junction tumours will be examined, specifically looking at the role of cancer stem cells in this process. For this we will use immunohistochemistry, and functional genomics analysis of micro RNAs and messenger RNAs. These histological and molecular characteristics will then be correlated with tumour response to treatment and overall survival outcomes for these patients.
Additionally, we will also seek to determine if there is an association between levels of specific micro RNAs in patients’ pre-operative serum samples and in their final resection specimens. Potential links between pre-operative clinically measurable parameters and aggressive tumour biology may thus be elucidated.
Head and Neck Cancer (non-thyroid)
ECHO (Epidemiology of HPV infection in Oral cancer in Ireland)
Human papillomavirus (HPV) is the single most important aetiological agent in the pathogenesis of cervical cancer and pre-cancer. It is increasingly recognised that HPV is causally implicated in other cancers, including head and neck cancer. The incidence of head and neck cancer, and more specifically oropharyngeal cancer, has been increasing over the last decade. An average of 411 cases are now diagnosed annually in Ireland. Oropharyngeal carcinoma is increasing in younger age groups and among females, who are less likely to have strong tobacco or alcohol histories, suggesting the importance of HPV in these cohorts. In the US, approximately 35% of oropharyngeal tumours are HPV-positive. The prevalence of HPV in other head and neck cancers – notably those of the oral cavity and larynx – is less firmly established. In addition, it is unclear whether the survival advantage associated with HPV-positivity in some clinical series and trials persists at the population-level.
In this project, CERVIVA will extend its scope into HPV associated cancers beyond the cervix, by generating the first data on HPV prevalence and genotype distribution, and associations between HPV status and outcome, in head and neck cancers in Ireland.
Tumour metastasis is the pivotal contributory factor in determining prognosis for cancer patients. Haematogenous dissemination of circulating tumour cells [CTCs] from the primary tumour is essential for establishment of metastases at secondary sites. A key goal in cancer research is to understand the mechanism centrally underlying metastasis. Our group has a large research programme in this area and are working in partnership with Becton Dickinson in an Enterprise Ireland funded Innovation Partnership to “Decipher the most clinically and biologically relevant circulating tumour cells [CTCs] in cancer metastasis”.
Characterisation of circulating tumour cells in ovarian and breast cancer
Given that CTCs are essentially the ‘liquid phase’ of the cancer metastatic cascade, an understanding of their biology, survival characteristics and dynamic interaction with components of the blood and vascular system are essential to increasing the knowledge base of metastasis. However, many issues still remain in relation to: detection, characterisation, biological activity and disease forming activity of these cells. CTCs can exist in many forms including singlets, doublets and clusters, clusters are thought to represent more aggressive disease. This is currently being interrogated using a novel CTCID capture mechanism.
Understanding how Platelet-Cancer Cell Interactions aid Survival in the Circulation and Drive Metastasis in Ovarian Cancer
CTCs are tumour cells shed from a primary tumour and carried through the bloodstream, which is a necessary step for the establishment of metastases at secondary sites. To unlock the full clinical potential of CTCs what is needed is a means to identifying diagnostically /prognostically relevant forms, i.e. those cells with the highest propensity to form metastatic tumours. To achieve this, we must understand the molecular mechanisms driving the establishment, the survival and the invasive capabilities of CTCs. Initial work focused on the interaction with ovarian cancer cells, however, they have demonstrated that there is universal, potent and dynamic interaction between platelets and cancer cells, which aids survival and drives a pro-metastatic phenotype in cancer cells. This work has led to the identification of gene panel that are hypothesised to be potential significant effector molecules of the platelet’s pro-metastatic drive; including Plasminogen activator inhibitor type 1 (PAI1) and Monocyte chemotactic protein 1 (MCP1) and this is being investigated as part of this project.
Understanding the role of the platelet-cancer cell interaction in immune surveillance
In addition, to examining the effect of platelets on cancer cells, the research group has extended this work to study how this interaction influences immune surveillance. Understanding how CTCs survive in the vasculature by evading natural immune responses is central to establishing which CTCs are biologically/clinically relevant to metastasis. The immune system has a natural response to tumours, initially involving cells of the innate immune system, including NK cells and macrophages. This response however, can fail, facilitating cancer cell survival and disease progression. Our work has focussed on establishing if platelets, through their direct interaction with cancer cells, can evoke this evasion of the natural immune response. Our work on NK cells, which are major components of the anti-tumour innate immune response, has conclusively demonstrated that the activity of NK cells is significantly diminished by platelet cloaking of cancer cells. We have demonstrated that platelet cloaking significantly decreases NK cell activity towards cancer cells via the modulation of the NK cell tumour recognition systems, NKG2D-MICA/MICB and CD96/CD226-CD155/CD112. In addition, we have direct evidence that platelet cloaked cancer cells specifically modulate the phenotype of macrophages. Macrophages have a crucial role in regulating immune responses to tumours, but their function also becomes hijacked by tumour-derived signals to support cancer progression. These hijacked macrophages, known as tumour associated macrophages [TAMs], adopt an anti-inflammatory, pro-tumour phenotype and have been implicated in the processes of invasion and intra/extravasation, ultimately supporting metastasis. We have now demonstrated that platelet cloaking enhances the immunomodulatory effect of macrophages on cancer cells, promoting the development of an anti-inflammatory, pro-metastatic phenotype in macrophages. Our work is now focussing on how this system can be manipulated to improve the treatment of metastatic disease.
Metastasis, Resilience and Educating Platelets
This research is involved with dissecting those molecular features that make some cancers resilient to metastasis to confirm biomarkers that are protective. We will build on our existing bodies of work which have detailed the molecular taxonomy of thyroid cancer and the role of circulating tumour cells in the metastatic cascade. We have demonstrated that platelets cloak circulating tumour cells offering protection during the process of metastasis, but further that the cloaking process is mediated by von Willebrand Factor (vWF). We are examining if there are different functional consequences in the effect of vWF derived from de-novo expression in the cancer cell compared with that derived from endothelial cells. We are investigating the potential to target and modify de-novo production of vWF in metastatic cells as a potential therapeutic intervention.
Platelets are equipped with RNA processing machineries, such as pre-mRNA splicing, pre-miRNA processing, and mRNA translation. Since platelets are devoid of a nucleus, most RNA transcripts in platelets are derived from megakaryocytes during thrombocytogenesis. However, platelets can also ingest RNA molecules during circulation and/or interaction with other cell types, and it is now emerging that platelet RNA biomarker signatures can be altered in the presence of cancer.
Beyond the roles in regulating haemostasis and coagulation, an emerging body of evidence indicates that platelets serve much more comprehensive functions in various diseases. The sheer number of blood circulating platelets provides a remarkable membrane system. This biologically active surface is the major source and translator of cross-talk between platelets and other cells allowing them to be “educated” by different cell encounters through a tumour-platelet axis.
This project will address the fundamental questions underpinning lethal metastases and focussed on the interplay between tumour cells, vWF and tumour educated platelets including:
- how metastatic disease is established,
- how vWF mediates platelet cloaking,
- how platelets are active drivers of metastasis
- how particular phenotypes confer resilience to metastasis
- how progression of disseminated cancer might be interrupted.
The project applies a novel strategy to elucidate the intersecting roles of vWF and platelets in driving metastasis (or offering protection from it), and offers the potential to devise novel therapeutic approaches to abrogate the lethal consequences of metastatic disease.
Sepsis and Health Care Associated Infection
PROSPeR will reduce mortality, morbidity, hospital length of stay as well as the costs of testing and treatment for sepsis and HCAI patients by addressing the fundamental deficiencies of existing practice. Contemporary Sepsis care consisting of timely appropriate antimicrobial therapy and protocol driven supportive care, has failed to significantly reduce mortality in the last 2 decades. Mortality in patients with sepsis is greater when complicated by secondary Health Care Associated Infection (HCAI). Current HCAI control measures comprise a series of protocol driven physical interventions designed to limit cross infection with multi-resistant organisms. They are not individualised and are only partially effective. There is currently no reliable Sepsis predictive biomarkers. Therefore an urgent unmet medical need for predictive biomarkers for patients with sepsis who develop HCAI exists.
We hypothesise that HCAI and Sepsis are clinical manifestations of an underlying immune compromise; a failure of innate immune antigen presenting mechanisms and concomitant impaired T cell activation. This programme will develop a laboratory pipeline to allow for rapid diagnosis, optimal isolation of relevant T cell populations from patients’ blood samples, followed by integrated analysis of gene expression and metabolomic metrics.
Cancer Stem Cells
Towards Clinical Targeting of Cancer Stem Cell Therapy-Resistance
It is widely believed that clinical targeting of tumour-initiating, treatment-resistant ‘Cancer Stem Cells’ (CSCs) can dramatically reduce the mortality rates associated with malignancy, particularly in patients with therapy-resistance disease. Before this can be achieved, the therapy-resistant properties of CSCs must be understood. Historically it has been believed that therapy-resistance was a universal property of CSCs, a paradigm that informed most clinical-targeting strategies, but had never been tested. We recently demonstrated, for the first time, that therapy-resistance is a property of specific CSCs within the tumour and not a universal property. Furthermore, we demonstrated that CSCs displaying resistance to one type of chemotherapy drug can be sensitive to another chemotherapy drug. Mechanistically, therapy-resistant CSCs were shown to express several mechanisms to efflux chemotherapy drugs and repair chemotherapy-induced DNA damage, mechanisms that were naïve and further upregulated by prolonged chemotherapy (Figure 1). Having described similar findings in two very different malignancies [ovary (Ffrench et al 2017) and lung (MacDonagh et al 2017)], it seems likely that similar observations will be reported in other malignancies and be translated towards improved CSC-targeting in the clinic. The importance of this work is reflected in its publication in the prestigious Nature journal ‘Cell Death & Disease’ (Ffrench et al 2017). In addition, our review discussing the clinical implications of these findings is published in their ‘Cancer Stem Cell Special Series’ by the highly topical journal ‘Molecular Cancer’ (Gasch et al 2017). We propose an advancement to CSC Theory, where tumours contain specific populations of therapy-resistant CSCs (CSCRES), which must be identified and targeted specifically in different patient groups within different malignancies (Figure 2). Our on-going research aims to identify and target CSCRES populations within therapy-resistant malignancy.
Building Cancer Stem Cell Hierarchies
It is now known that different populations of CSC are organised as hierarchical networks within the heterogeneous tumour. Successful clinical targeting of specific CSC populations requires elucidation and characterisation of these CSC Networks. To facilitate this, we have developed a pipeline through which different CSC populations can be identified, and their relationship to one another elucidated, which is described in detail in our Ffrench et al 2014 review. This data allows us to model CSC Networks within a tumour. Once elucidated, we demonstrated that chemotherapy results in a dramatic alteration of the original CSC Network, where therapy-sensitive CSCs are eliminated and CSCRES thrive. As we discuss in our recent review (Gasch et al 2017), this indicates and approach where the pre- and post-therapy CSC Network must be targeted differently for improved patient outcome. Our on-going research aims to build novel CSC Networks in other malignancies and inquiries for collaboration with other research groups are welcome.
Collaborative Cancer Stem Cell Research
CSC biology is of high interest to many cancer researchers, and is facilitated by our specialist knowledge and experience. We regularly collaborate with research teams from within TCD, Ireland, and around the World. To date, we have published and have active collaborations in the areas of ovarian, lung, breast, oesophageal and prostate cancers. Our research group can provide general input into stem cell and CSC biology, particularly the identification of stem cell or CSC molecular signatures within collaborators data. More specifically, our group can provide assistance with validation of putative cell types as validated CSCs via an in vivo tumour-generation xenograft model. Our group is a world-leading authority on the use of single-cell analysis for ‘CSC Discovery’, which involves marker- (ALDH, CD44 etc.) and stem cell biology- (spheroid-formation, holoclone, hoechst dye efflux assays etc.) based screening. CSC Networks are elucidated though our ‘Single Cell Asymmetric Division’ (SCAD) assay. These approaches are detailed in our Ffrench et al 2014 review and collaborations with researchers are welcome.
Controlling Differentiation of Human Pluripotency
Pluripotency, the potential of certain stem cells to produce cells representative of all three germ layers via differentiation, is a highly-topical research area in which we have many years of experience. In recent years, many stem cell researchers have focused on the ‘primed’ states through which pluripotent stem cells appear to transition en route to differentiation down specific lineages. We recently demonstrated that key inflammation regulator ‘MyD88’ (Myeloid Differentiation Response Gene 88) plays a key role in the differentiation of human pluripotent embryonal carcinoma CSCs. Our study demonstrates that high MyD88 expression is required for maintenance of the pluripotent state, while its loss transitions cells to a new, ‘Primed Self-Renewal’ state that readily responds to differentiation morphogen all-trans retinoic acid (RA). Mechanistically, loss of MyD88 facilitates a change in the profile of protein secreted by the cell into the niche, a profile that is sufficient to differentiate pluripotent CSCs, and facilitate upregulation of RA-signalling. We propose that regulators such as MyD88 act as ‘Differentiation Gatekeepers’, which we demonstrate can be targeted to facilitate ‘Forced-Differentiation’ as a ‘Differentiation Therapy’ strategy for cancer treatment. The importance of this work, is indicated by its publication in the prestigious Nature journal ‘Cell Death and Differentiation’ (Sulaiman et al 2017). Our on-going research aims to further elucidate the MyD88 mechanism and identify specific Differentiation Gatekeepers in other malignancies.
Next Generation Sequencing
Ion Torrent™ Next-Generation Sequencing (NGS) Technology
Ion Torrent™ technology directly translates chemically encoded information (A, C, G, T) into digital information (0, 1) on a semiconductor chip. This approach marries simple chemistry to proprietary semiconductor technology; it's Watson meets Moore. The result is a sequencing technology that is simpler, faster, more cost effective and scalable than any other technology available. The semiconductor has transformed every industry it's touched. Just as the microprocessor enabled desktop computing to displace the mainframe, semiconductor technology will inevitably democratize sequencing, putting it within the reach of any lab or clinic.
In nature, when a nucleotide is incorporated into a strand of DNA by a polymerase, a hydrogen ion is released as a byproduct. Here’s how the technology is used to call a base: If a nucleotide, for example a C, is added to a DNA template and is then incorporated into a strand of DNA, a hydrogen ion will be released. The charge from that ion will change the pH of the solution, which can be detected by our proprietary ion sensor. Our sequencer—essentially the world's smallest solid-state pH meter—will call the base, going directly from chemical information to digital information. The Ion Personal Genome Machine™ (PGM™) sequencer then sequentially floods the chip with one nucleotide after another. If the next nucleotide that floods the chip is not a match, no voltage change will be recorded and no base will be called.
The semiconductor will inevitably transform the life sciences, just as it has transformed every other industry it has touched. By creating a direct connection between chemical and digital information, Ion Torrent technology will democratize research, providing a fast, simple, scalable sequencing solution that every lab can afford. Eventually, Ion Torrent technology will also be able to provide diagnostics that are less expensive and more reliable, improving human health around the world.
- Targeted DNA/RNA sequencing
Sequencing individual genes, gene regions, or sets of genes is a common targeted sequencing approach used in cancer and inherited disease research to screen known or discover novel germline and/or somatic mutations in focused areas of the genome. Targeted RNA sequencing is a highly affordable solution for following up on transcriptome studies, or it can be used to rapidly focus in on pathway- or disease-related content. With very low amounts of input total RNA (as low as 500 pg of unfixed RNA or 5 ng of RNA isolated from FFPE samples), expression of a handful, hundreds or even thousands of RNA targets can be assayed in a simple NGS-based workflow enabling the monitoring of messenger RNA (mRNA), non-coding RNA (ncRNA), small RNA, and gene fusions.
- Exome sequencing
Targeted sequencing of the exome employs enrichment strategies that target coding exons. The exome encompasses approximately 1% of the genome, yet contains approximately 85% of disease-causing mutations. For genetic researchers trying to unravel the causes of over 6,800 rare diseases, exome sequencing enables the identification of single-nucleotide variants (SNVs), small insertions or deletions (indels), and rare de novo mutations that explain the heritability of complex diseases.
- Whole Genome Sequencing (WGS)
WGS entails sequencing the entire genome and comparing that to a reference genome in order to detect the full range of genetic variation such as SNPs, insertions, deletions, inversions, complex rearrangements, and copy number variation.
- Small RNA Sequencing
The Ion PGM provides sequencing-based analysis of all known and novel small RNA transcripts, such as microRNA (miRNA), short interfering RNA (siRNA), and piwi-interacting RNA (piRNA), in a strand-specific, hypothesis-free fashion.
- Epigenetic Sequencing
As CpG DNA methylation is not preserved during DNA amplification, studying methylation requires that the DNA be treated to either preserve or select for methylation status prior to DNA sequencing. Techniques used to prepare DNA for methylation analysis include: the selection of methylated DNA by affinity enrichment, enzymatic compartmentalization using methylation sensitive enzymes, or chemical treatment of the DNA using bisulfite.
Proteins bound to DNA can impact the expression of genes, as well as the accessibility and packaging of DNA. To study these interactions, protein–DNA complexes can be isolated by chromatin immunoprecipitation (ChIP) and then characterized by sequencing the associated DNA. This approach is called ChIP - Seq.
The OncoNetwork consortium is comprised of twelve -translational cancer research institutes with many years of experience in adopting the latest molecular techniques for sensitive and accurate detection of targetable mutations in solid tumours.
The consortium has worked collaboratively to develop and validate a series of targeted mutation detection kits including a Solid Tumour Fusion Transcript kit. This represents a key solution designed to aid clinical-decision making and enable highly accurate and reliable multiplexed sequencing of formalin-fixed, paraffin embedded (FFPE) tumor samples. The next-generation sequencing (NGS) approach enables the detection of rearrangements involving the ALK, ROS-1, RET, and NTRK1 genes from as little as 10ng of RNA. The target content, focusing on lung cancer, but potentially applicable to other solid tumors.
Marked for in vitro diagnostic use in the European Union, the Oncomine Solid Tumour Fusion Transcript kit for, was verified with leading clinical researchers from the OncoNetwork Consortium spanning 10 different countries and with years of experience in implementing NGS technologies in clinical laboratories.
Genetic rearrangements that cause fusion transcripts are an important and expanding class of actionable biomarkers in cancer. Current approaches to detect numerous variations in a clinical laboratory rely on the use of multiple tests, each designed to detect a different genetic rearrangement. When used in a sequential manner with other required pathology tests, there is a significant risk of the tumor sample being consumed before an actionable variant is uncovered. The new kit overcomes this hurdle with its low RNA sample input requirement of FFPE tissue (10ng extracted nucleic acid per reaction), ultimately providing actionable information to a greater number of patients and beneficial insight that may help guide treatment.
* The OncoNetwork Consortium members include these researchers:
Pierre Laurent-Puig1, Cecily Vaughn2, Ludovic Lacroix3, Marjolijn Ligtenberg4, Bastiaan Tops4, Christoph Noppen5, Henriette Kurth5, Nicola Normanno6, Aldo Scarpa7, Ian Cree8, Orla Sheils9, Harriet Feilotter10, José Carlos Machado11, Jose Costa11, Kazuto Nishio12
1 Université Paris Descartes, Paris, France
2 ARUP- Institute for Clinical and Experimental Pathology, Utah, USA
3 Institut Gustave Roussy, Paris, France
4 Radboud University Nijmegen Medical Centre, The Netherlands
5 VIOLLIER AG, Basel, Switzerland
6 Centro Ricerche Oncologiche Mercogliano, Avellino, Italy
7 ARC-NET University of Verona, Italy
8 Warwick University Medical School, United Kingdom
9 Trinity College Dublin, Ireland
10 Queen’s University, Ontario Canada
11 IPATIMUP Medical Faculty of Porto, Portugal
12 Faculty of Medicine, Kinki University, Osaka, Japan
Cancer Stem Cell
In recent years the cancer stem cell (CSC) theory of tumorigenesis has received vastly increased attention. Our department is home to the only pure Cancer Stemness research group in Ireland. It is now generally accepted that most, if not all, cancers are driven by a population of stem-cell like cancer cells. These CSCs can self-renew and differentiate to generate tumors in vivo, thrive in the hypoxic environment of tumors and are resistant to current therapeutics. Thus CSC-targeting is an area through which the efficiency of cancer treatment may be improved.
The primary characteristic defining CSCs is their differentiation capacity. Our group investigates the relationship between the differentiation potential of CSC populations and tumour grade, metastasis, recurrence and chemoresistance. We employ an embryonal carcinoma model of cancer stemness, which we have shown is highly relevant to ovarian cancer (Gallagher et al 2009: d’Adhemar et al 2010: Figures 1 and 2). The group’s primary aim is development of a therapeutically relevant method or targeting CSCs without harming non-CSCs within the body.
Michael Gallagher (B.Sc, PhD) trained in the Stem Cell department at the Genome Institute of Singapore and is primarily interested in transcriptional and post-transcriptional networks that regulate early CSC differentiation. To date we have characterised gene and miRNA expression and modeled translation at a whole genome level, generating substantial lists of putative gene and miRNA targets. This data is used as a platform for functional analysis of the effects of gene and miRNA targeting on CSC differentiation, tumor generation and chemoresistance.
Postgraduate Students and Projects
Salah Elbaruni (B.Sc, M.Sc):Functional analysis of TGF-beta pathway genes on differentiation on ovarian cancer stem cells. TGF-beta signaling was identified by array analysis of CSCs and Salah has characterised this pathway in CSCs and is currently functionally analyzing the role of TGF-beta signaling in CSC differentiation.
Yvonne Salley (B.Sc, M.Sc):Generation of stem cell-like cells from prostate cancer. Yvonne’s work is part of the Cancer Research Ireland (CRI) funded Prostate Cancer Research Consortium and involves generation and characterization of stem cell-like holoclones from prostate cancer cells.
Sebastian Vencken (B.Sc):Functional analysis of miRNAs involved in differentiation of ovarian cancer stem cells. This CRI funded project assesses the necessity of miRNAs identified in our data set of the differentiation of CSCs.
Aoife Cooke (B.Sc):The role of TLR/MyD88 signaling in ovarian cancer stem cell differentiation and chemoresistance in normal, hypoxic conditions. This CRI funded project exploits our identification of a TLR/MyD88 switch in CSC differentiation. Aoife is fully characterizing the role of TLR4/MyD88 signalling in CSC differentiation and chemoresistance in normal and hypoxic conditions.
Brendan Ffrench (B.Sc):Isolation and characterization of novel cancer stem cell populations from ovarian cancer. This project if funded by The Emer Casey Foundation and will establish a protocol for the isolation of multiple CSC populations from chemoresistant and chemosensitive primary and recurrent ovarian cancers.
Charles d’Adhemar (M.D):TLR4/MyD88 expression in ovarian cancer samples. Charles has completed a comprehensive analysis of TLR4/MyD88 expression in ovarian tumour samples, associating them with tumor grade (d’Adhemar et al 2010).
Aoife Canney (M.D):Assessment of EMT in ovarian cancer. Tumor cells are believed to travel to secondary sites and establish secondary malignancies via a process termed epithelial-mesenchymal transition (EMT). Aoife is characterizing this process in ovarian CSCs.
For more information please contact Michael Gallagher: firstname.lastname@example.org
Gallagher MF, ElBaruni S et al 2009: Regulation of microRNA biosynthesis and expression in 2102Ep embryonal carcinoma stem cells is mirrored in ovarian serous adenocarcinoma patients. Journal of ovarian research, 2: 19.
Heffron CCBB, Gallagher MF et al 2007: Global mRNA analysis to determine a transcriptome profile of cancer stemness in a mouse model. Anticancer research, 27:1319-1324.
D’Adhemar C, Gallagher MF et al 2010: Functional Expression Analysis of TLR-4 and MyD88 in Epithelial Ovarian Neoplasia. In prep
European Union Funded Programmes
Dr. Cara Martin/ Prof John O’Leary
Dr. Helen Keegan
Cervical cancer is the second most common cancer in women worldwide and remains a major cause of morbidity and mortality. Human papillomaviruses (HPVs) are implicated in over 99% of cervical cancers. HPV contributes to neoplastic progression through the interaction of two of its oncogenes E6 and E7 which interfere with critical cell cycle regulatory processes. Nucleic assays such as multiplex PCR, nucleic acid sequence based amplification (NASBA), DNA sequencing and microarrays are time-consuming, labour intensive and complex investigations when performed isolation. Minaturisation of these assays into lab-on-a-chip diagnostic platforms presents huge potential for the integration of multiple molecular assays into easy-to-use, flexible, point of care diagnostic systems, with improved power to diagnose accurately and represents a major step forward in cervical cancer screening and diagnostics. Such platforms have the potential to be applied to other diagnostic systems including other cancers, biosafety platforms and food technology. The department has collaborated on the European funded projects:- Microactive and Autocast.
The "MicroActive" project is a specific targeted research project under the EU 6th IST Framework Programme. The project started in December 2005 and closed in December 2008.
During the MicroActive project a microfluidic device for molecular diagnostics intended for use in the doctors’ office was developed. A platform for nucleic acid extraction and Human papillomavirus (HPV) E6 and E7 transforming oncogene expression was developed. “Proof of principle” was established on cervical clinical specimens. Microfluidics and biotechnology formed the basis for the development of two disposable biochips, one for nucleic acid purification and the other for HPV amplification. The project was coordinated by SINTEF (Norway) with Institut fur Mikrotechnik Mainz (Germany), IMTEK (Germany), BioFluidix (Germany), NorChip (Norway) and Coombe Women and Infants University Hospital (Ireland) affiliated with University of Dublin, Trinity College, as project partners. Results from the project are now available through the project web-site http://www.sintef.no/microactive and through the publishable final activity report
Baier T, et. al.,"Hands-free sample preparation platform for nucleic acid analysis", Lab Chip, 2009, DOI: 10.1039/b910421f
Keegan H, et al,"Comparison of HPV detection technologies: Hybrid capture 2, PreTech HPV Proofer and analysis of HPV viral load in HPV16, HPV18, and HPV33 E6/E7 mRNA positive specimens"Journal of Virological Methods(2009)
The “AutoCast” (Automated Cancer Screening based on Real-time PCR) project, is a collaborative EU 7th framework funded project under the “Health” work programme commencing in August 2008 for 3 years.
In this project, we will develop a novel low cost automated real time PCR/probe technology, in a microarray biochip format with corresponding automated detection instrumentation for use as a rapid “point of care” diagnostic device for cervical cancer screening in both a clinical and laboratory setting. A multiplex real-time amplification assay will be coupled to detection on an array platform for the detection of human papillomaviruses and human biomarker genes in a new combinatorial approach to cervical screening. The project is coordinated by GenoID (Hungary) with Fraunhofer Institut (Germany), Austrian Research Centers (Austria), Biofluidix (Germany), Jenoptik (Germany), IMTEK, (University of Freiburg, Germany) and University of Dublin, Trinity College affiliated with Coombe Women and Infants University Hospital (Dublin, Ireland). Further information is available on this project at the following website: http://www.genoid.net/index.php/autocast/
The Department also has collaborations with other European partners in the areas of microfluidic devices and diagnostics for neonatal sepsis, ovarian cancer, prostate cancer and in novel cell sorting techniques.
Ovarian cancer is the leading cause of death from gynaecological malignancy in the western world. About 200,000 cases of ovarian cancer occur worldwide each year with over 350 new cases in Ireland. The majority of ovarian cancer patients present in advanced stages (III or IV). This is because the symptoms of ovarian cancer are not recognised immediately and sensitive screening tests are not currently available. Treatment usually involves surgery followed by chemotherapy. While many women respond well initially, a worrying number of women will face a recurrence of disease, often with the tumour becoming less amenable to chemotherapy over time.
The DISCOVARY consortium was formed to specifically address issues in relation to ovarian and endometrial cancer diagnostics and prognostics and supports the development of translational research in Ireland. The consortium is built on ongoing collaborations between gynaecologists, oncologists, pathologists, scientists and bioinformaticians throughout Ireland, representing the following institutions, Obstetrics and Gynaecology, Histopathology, IMM, TCD; The Conway Institute, UCD; Biomedical Diagnostics Institute, DCU; Centre for Cancer Research and Cell Biology, Queens University Belfast and the following hospitals; St James’s, The Coombe Women and Infants University Hospital, The Mater, St Vincents University Hospital, Belfast City, University College Hospital Galway and University College Hospital, Cork. The consortium has the backing of ICORG and the Irish Cancer Society. The consortium has been fortunate to receive funding from the Emer Casey Foundation. Family and friends of Emer established the foundation following her untimely death from ovarian/uterine cancer in June 2006
Various projects are being carried out under the umbrella of the DISCOVARY consortium and are detailed below.
Identification and validation of novel serum biomarkers for ovarian cancer (Emer Casey PhD Fellow Mairead Murphy)
This project involves screening bloods from ovarian cancer patients in an attempt to identify novel markers, which can be used to diagnose ovarian cancer at an early stage, to discriminate between malignant and benign disease and to diagnose recurrent disease.
Developing novel therapeutic approaches in chemoresistant ovarian cancer patients and investigation of the role of hypoxia in ovarian cancer chemoresistance using a novel lab-on-a-chip device (Emer Casey PhD Fellow Lynda McEvoy in collaboration with Biomedical Diagnostics Institute, Dublin City University)
Understanding the biological mechanisms underlying chemoresistance and recurrence of ovarian cancer is an important goal. This will involve establishing the role of previously identified genes and miRNAs in the pathogenesis of ovarian cancer and recurrent ovarian cancer. A possible cause of chemoresistance is the microenvironment of the tumour, which is characterised by areas of poor oxygen supply known as hypoxia. The results of various experimental studies suggest that tumour hypoxia plays an important role in the development of typical features of malignancy such as invasiveness, metastatic potential, and resistance to treatment. An understanding of the key elements and control of the hypoxia pathway could reduce unnecessary chemotherapy treatment and toxicity, prolong survival and lead to more effective therapy for ovarian cancer. This project is also directed at employing a lab-on-a-chip device to test tumour cells for their response to chemotherapy prior to treatment. Our overall objective is to identify panels of markers that would define if an ovarian tumour will respond or be resistant to chemotherapy or if it will recur.
Hsa-miR-141 and hsa-miR-223 are central to Ovarian Serous Carcinoma Pathogenesis through Regulation of JAG1 and SMARCD1 proteins (Lisa Keogh)
The group has examined microRNA expression profiles with a view to distinguishing ovarian and primary peritoneal serous carcinoma histotypes. We are focussing our attention on specific gene regulators in this project. We believe they become over-expressed when the cancer genes are switched off. In this project we hope to conclusively prove this phenomenon using cells grown in culture. We will then test a large series of ovarian cancers to monitor levels of regulator molecules. To confirm our findings we will also examine a series of ovarian cancers for specific cancer promoting genes and their protein products.
miRNA Profiling in Ovarian Cancer Serum (Ream Langhe)
There is emerging research about the role of microRNAs in a variety of pathologic conditions; including both solid and hematologic malignancies. miRNA profiling has allowed the identification of signatures associated with diagnosis, prognosis and response to treatment of tumours. miRNAs have not been extensively studied in ovarian cancer and we propose to examine expression in serum of ovarian cancer patients. We intend to profile miRNAs in malignant, recurrent, borderline and benign disease and compare this to age matched controls. Functional studies will also be performed to establish how the miRNAs work
Cell cycle regulatory proteins and their potential role in early diagnosis, treatment and prognosis of borderline and invasive ovarian cancer (Alhadi Araibi)
Borderline tumours are a well delineated and clearly defined group of epithelial ovarian tumours. Their potential for progression is currently unknown. They can also recur early and in an aggressive manner. Tumorigenesis is a result of aberrant decisions in cell fate. Cells can improperly differentiate, resulting in tumour progression, or uncontrollably proliferate, resulting in tumour growth. MCMs, CDC6, CDt1, Geminin, P16 and NuF2 are some of the cell cycle regulatory proteins that have been extensively assessed and found to be differentially expressed in various tumours including breast, uterine cervix, colorectal and prostatic cancers. Their level of expression and significance in ovarian cancer remains to be elucidated in this project.
Pro-inflammatory pathways and differentiation in ovarian epithelial cancer – a model of cancer stem cells, hypoxia and chemoresistance (Charles d'Adhemar)
Toll-like-receptors (TLR’s) act as an immune pattern recognition system. Once stimulated they produce a pro-inflammatory response involving activation of MyD88 and NFkB molecules with the elaboration of cytokines, chemokines and growth factors. From a cancer cell perspective, such an environment forms an ideal paradigm for malignant progression. This project aims to investigate the significance of pro-inflammatory pathways in epithelial ovarian cancers to define whether manipulation of hypoxic and inflammatory conditions may result in a therapeutically relevant reduction of proliferation, as well as increased differentiation of ovarian malignancy towards less aggressive phenotypes.
The Irish Cervical Screening Research Consortium
The Irish Cervical Screening Research Consortium "Cerviva" was established in 2005 to perform high quality research into cervical screening options, offer support to the Irish Cervical Screening Programme and improve the quality of cervical screening in Ireland. The consortium is comprised of scientists, clinicians, epidemiologists, health economists and has significant hospital, academic, biotechnology and national agency support.
Cerviva seeks to introduce major innovations in the cervical screening process through the introduction of automation, ancillary viral testing for use in management triage and the development of objective biomarkers and biosensors for use as prognostic indicators of disease outcome.
There are eight individual component projects underway within CERVIVA. These include:
- Introduction of automated cytology: a cost base analysis of automated screening compared with manual screening approaches
- Introduction of a virtual slide based external quality assessment (EQA) system in Irish cytopathology.
- Population based survey of the attitudes of women to cervical screening and HPV oncogenic testing.
- Establishment of a cervical screening and Human Papillomavirus Virus out reach education programme.
- HPV DNA and mRNA testing in the Irish cervical screening population.
- Screening HIV positive women for HPV RNA transforming genes as a role in the prevention of cervical cancer in this population.
- Examination of the effect of introducing a HPV vaccine in the Irish population: a longitudinal analysis of risk.
- Development of cervical biochips/ biosensors
A publicly accessible website www.cerviva.ie has been established. This website is regularly update with news items relating to the projects with a list of publications and presentations to date and contains links to national and international cancer agencies and research groups.
- National Centre for Sensor Research, Dublin City University (DCU)
- Kevin Street College of Technology (DIT)
- Dept of Obstetrics and Gynaecology Royal College of Surgeons in Ireland (RCSI)
- Dept of Histopathology, Trinity College Dublin (TCD)
- Dept of Pathology, University College Cork (UCC)
- Dept of Pathology, University College Galway (UCG)
- Dept of Pathology, University College Dublin (UCD)
- Free University of Amsterdam
- University of Oslo
- Dept of Pathology, Coombe Women's Hospital, Dublin (Coombe)
- Dept of Cytopathology, St. James's Hospital Dublin (St James)
- Dept of Obstetrics and Gynaecology, St. James's Hospital Dublin (St James)
- Cancer Molecular Diagnostics, St. James's Hospital, Dublin (St James)
- Dept of Cytopathology, St.Luke's Hospital Dublin (St Luke's)
- Dept of Cytopathology, University Hospital Galway (UHG)
- Dept of Cytopathology, Beaumont Hospital, Dublin (Beaumont)
- Dept of Pathology, The National Maternity Hospital, Dublin (NMH)
- Dept of Pathology, The Rotunda Hospital, Dublin (Rotunda)
- Dept of Cytopathology, Cork University Hospital, Cork (CUH)
- Affymetrix, UK
- Celera Diagnostics / Applied Biosystems, UK
- Cytyc, USA
- Tripath, USA
- Dakocytomation, Denmark
- Digene Corporation, USA
- Norchip AS, Norway
- Roche Diagnostics, UK
- Biotrin, Ireland
- SlidePath, Dublin (a DCU campus company)