Developing a system to simultaneously detect genetic and epigenetic information

Many diseases are associated with changes to the DNA sequence, most notably cancer. Also altered in disease is the way that the DNA is decorated with chemical modifications such as methylation (epigenetic modifications). Being able to extract genetic and epigenetic information using DNA sequencing has revolutionised biomedical research and has led to new ways to diagnose diseases. A particular interest currently is in using genetic and epigenetic characteristics of tumour DNA circulating in the blood or other bodily fluids as a strategy for detecting cancer earlier. However, despite the potential utility of combining genetic and epigenetic information to enhance disease detection, no methods currently exist that can efficiently simultaneously extract this information from the same DNA sequencing data.

Up until now, DNA methylation has predominantly been detected using methods that rely on a process called bisulphite conversion. Bisulphite is a harsh chemical that damages DNA, resulting in decreased sensitivity and a high error rate in the sequencing data. Because it is not known whether any changes in the DNA compared to a reference genome are introduced by bisulphite or real mutations, it is very challenging to simultaneously detect methylation and mutation data using these methods.

Recently, a new bisulphite-free method for detecting DNA methylation called TET-assisted pyridine borane sequencing (TAPS) has been developed by Ludwig Oxford’s Dr Chunxiao Song and Dr Benjamin Schuster-Böckler. This method is both cheaper than bisulphite sequencing and importantly produces data of higher quality, similar to that of standard DNA sequencing.

In this project, funded by an MRC Methodology Research Grant, Dr Benjamin Schuster-Böckler will collaborate with Professor Gerton Lunter (Visiting Professor, Radcliffe Department of Medicine) to develop algorithms that simultaneously detect mutations and DNA methylation from TAPS data.  Experimental data will be provided in collaboration with Ludwig Oxford’s Dr Chunxiao Song and Professor Xin Lu, and Professor Ellie Barnes (Nuffield Department of Medicine). Test data will be used to train machine-learning algorithms to optimise the accuracy of the sequencing method and to establish the best possible experimental parameters for this technique.

The resulting method will greatly increase the utility of the TAPS technique and will make it possible to routinely query a patient’s genetic background, while simultaneously measuring their epigenetic state. This will lead to a much broader understanding of the role of epigenetics in disease and would raise the possibility of using combined genetic and epigenetic information from sequencing data to aid earlier detection of cancer.

Image attribution: Darryl Leja, National Human Genome Research Institute (NHGRI) from Bethesda, MD, USA, CC BY 2.0 https://creativecommons.org/licenses/by/2.0, via Wikimedia Commons

Early Detection of Cancer Month

A summary of Early Detection Month in Oxford, February 2021

New Oxford spin-out Singula Bio launches

Singula Bio is a bold new seed-stage biotechnology company spun out of Oxford University. It aims to become a world leader in developing neoantigen-based individualised cell therapies to use against difficult-to-treat solid malignancies such as ovarian cancer.

This patient-centred approach will pioneer immunological, medical, surgical and computational technologies to generate selective therapies that eliminate cancer, and the ultimate hope is to achieve long-term, high-quality disease-free survival for cancer patients.

Singula Bio was co-founded by Professors Ahmed Ahmed, Enzo Cerundolo and Enda McVeigh from the Nuffield Department of Women’s & Reproductive Health at Oxford University. It is supported by Oxford University Innovation (OUI), the University’s research commercialisation company, and it has secured generous seed-stage investment from IIU Nominees Limited to pursue its goals. Singula Bio is a landmark for OUI as it is the 250th OUI-supported venture to have passed through the office since it opened its doors in 1987.

Motivated by their many patients (and laboratory funding from charities Ovarian Cancer Action and Cancer Research UK) Profs Ahmed and Cerundolo were inspired to improve an individual’s gruelling experience of cancer and to lessen their suffering of other treatments. Together, they have an enormous knowledge in cancer medicine, cancer immunology, cell and molecular biology, and computational biology which has enabled them to design patient-specific cancer cell therapies that harness the power of the patient’s own immune system to fight cancer.

In a tumour, cancer cells carry mutations that appear foreign to a patient’s body and, therefore, their immune system reacts to these mutations. One strong form of an immune reaction is through generating mutation-specific cells called “T cells”.

Prof Ahmed, Professor of Gynaecological Oncology at the Nuffield Department of Women’s & Reproductive Health, Oxford University, said:

“A key feature of cancer cells is the preponderance of genetic aberrations in their DNA. These aberrations can make proteins appear foreign to our body’s immune system which then develops immune cells (T cells) to fight cancer cells. Thanks to years of research and technology development we now know how to identify relevant tumour-specific T cells to grow them outside the body and deliver them back to patients to fight cancer cells.”

New Oxford technology assesses cancer patient vulnerability to COVID-19

The web-based COVID risk prediction tool, QCovid, is the product of the latest research to emerge from the QResearch database co-founded by Prof Julia Hippisley-Cox based at the University of Oxford. The analysis of anonymised UK health records of more than 8 million adults using GP records, hospital records including intensive care data, mortality data, Cancer Registry data and COVID-19 testing data from late January 2020 to April 2020 allows clinicians to estimate someone’s  risk of COVID-19 infection as well as the risk of being admitted to hospital with serious illness due to the virus and the potential risk of COVID-19-related death. QCovid takes into account a range of risk factors such as age, gender ethnicity and medical conditions and enables the NHS to make evidenced based decisions when prioritising different patient groups for shielding and COVID-19 vaccination.

The work was commission by England’s Chief Medical Officer Chris Whitty, who involved the team led by clinical epidemiologist, Prof Julia Hippisley-Cox, at the University of Oxford as the group has acquired extensive experience in developing risk prediction tools for a range of diseases, including QCancer for the prediction of having  undiagnosed cancer, which are widely used in the NHS. QCovid has now been validated and published in the British Medical Journal, is accessible to the public at www.qcovid.org and has been adopted by NHS Digital as a way to assess the relative risk of COVID-19 for all members of the population, based on their medical history and other risk factors.

Based on the prediction tool, 1.7 million patients have been added to the shielding list, including many cancer patients. Those within the most ‘vulnerable’ group who are over 70 will have already been invited for vaccination and 820,000 adults between 19 and 69 years will now be prioritised for a vaccination.

CANCER & VULNERABILITY

Although previous studies from the University of Oxford have shown that blood cancer patients are at higher risk of COVID-19, until now no research or model had been published that assessed patients with different types of cancer, including their treatment history and backgrounds. The QCovid algorithm feeding into the prediction tool takes cancer factors such as diagnosis of blood, lung, oral or bone cancers and different cancer therapies into account.

Thus using the QCovid prediction tool, it has a been highlighted that those with blood (acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma) and respiratory (lung, laryngeal, nasopharyngeal and mouth) cancers are at increased COVID-19 risk.

In addition, those undergoing therapy (including recent bone marrow or stem cell transplant, chemotherapy, radiotherapy, immunotherapy or other antibody treatments for cancer and treatments that affect the immune system such as protein kinase inhibitors or PARP inhibitors), have also been identified at higher risk.

Thanks to the QCovid algorithm cancer patients now can be appropriately categorised and prioritised based on their type of cancer, current or previous cancer treatment and other factors such as corresponding health conditions that could make them more vulnerable to COVID-19.

The development of the QCovid model was led by the University of Oxford and involved researchers from Cambridge, Edinburgh, Swansea, Leicester, Nottingham, Liverpool, the London School of Hygiene & Tropical Medicine, Queen’s University Belfast, Queen Mary University of London and University College London. It was funded by the NIHR, NIHR Oxford Biomedical Research Centre, Wellcome Trust (ISSF) and John Fell Fund and supported by EMIS GP practices and the University of Nottingham.

Finding extracellular vesicle biomarkers for oesophageal cancer early detection

Extracellular vesicles (EVs) are entities secreted by cells that can be involved in cell-to-cell communication. They contain messenger proteins and other molecules, which act like ‘instructions’ to recipient cells.  EVs contain proteins both on their inside and outside.

All cells, including cancer cells, release EVs.  EVs from different cell types have slightly different compositions of proteins, which give them the ‘characteristics’ of their parent cell.

Members of the Goberdhan’s lab have previously shown that EVs released by colorectal cancer cells contain different protein when they are subjected to certain types of stress, such as certain nutrient deficiencies.  These ‘switched’ EVs change recipient cell behaviour, for example, increasing cancer cell growth. Researchers can potentially exploit these differences in EV protein composition to define distinct EV sub-populations: a helpful step towards their use as multi-protein biomarkers.

Dr Jennifer Allen and Ms Karen Billington from the Goberdhan lab are now applying this concept to the early detection of oesophageal cancer. Barrett’s Oesophagus is a pre-cancerous condition whereby oesophageal cells become damaged. Over time the damage can increase and cancer can develop.

Monitoring patients with Barrett’s Oesophagus is in place to try and identify when cancer has developed, however this is done through invasive and costly endoscopy, which may miss cancer in the very early stages. There is a need to identify when Barrett’s Oesophagus has progressed using less-invasive methods that can be used more regularly, so that cancer can be caught earlier.

Jen and Karen are investigating the potential of using EV proteins as biomarkers, which could be identified though simple blood tests. They are using different types of cells – such as normal oesophagus cells, Barrett’s Oesophagus cells and Oesophageal cancer cells – to compare the proteins found on the EVs released by each of these cell types.

The team is working with Dr Elizabeth Bird-Lieberman, a Gastroenterology Consultant at the JR Hospital, to collect blood samples from patients with Barrett’s Oesophagus, to see if EV information could be extracted and tested through simple blood tests – such as that being developed by Prof Jason Davis.

The aim is to identify a handful of proteins via proteomic analysis, that allows them to differentiate EVs from oesophageal cancer cells. If the protein biomarkers associated with the more cancerous cell lines can be detected in patient blood samples, Barrett’s Oesophagus patients could then be routinely tested for specific EV proteins that indicate the presence of parent cancer cells. This simple test could be carried out much more regularly than endoscopy surveillance and would enable earlier detection and treatment of oesophageal cancer in these patients.

Colorectal cancer cell extracellular vesicles. These two vesicles have become deflated and have the characteristic cup-shaped morphology caused by preparation for electron microscopy. Images generated by Dr John Mason (DPAG) and Dr Errin Johnson (EM Facility Manager, Dunn School).

About the study

The Goberdhan lab members are interested in intracellular signalling and cell communication. Their major focus is on how this goes wrong in cancer and other major human diseases. Specifically, they investigate:

  1. The role of amino acid sensing and stress-induced signalling in regulating cellular growth and intercellular communication involving exosomes.
  2. The regulation of exosome formation and heterogeneity by intracellular signalling pathways and membrane trafficking.
  3. The effect of exosome signalling on recipient cell behaviour and cancer progression, particularly in response to microenvironmental stresses applied to exosome-secreting cells.

 

This study is funded by the CRUK Early Detection Primer Award.

Detecting for multiple cancers in one simple test

Biomarkers – or biological markers – are used in many areas of health and disease as measures of a biological or clinical state. In the context of cancer, identifying biomarkers of early stage cancer is crucial for being able to detect disease earlier and improving the outcomes of patients with cancer. However, biomarkers alone are not sufficient for earlier detection. We also need to develop cost-effective, non-invasive, simple-to-use technologies that can be used in the clinic to detect these biomarkers with high sensitivity, specificity and accuracy.

Professor Jason Davis in the Department of Chemistry at the University of Oxford is working on just that. Professor Davis’ research has focused on developing portable, handheld diagnostic tests that use a range of electroanalytical methods for biomarker detection. This includes recent work on using novel electrochemical impedance-based sensing technology to detect C-reactive protein, a marker of inflammation in the body.

These methods are advantageous for use in diagnostics since they generate results in a few minutes and are more sensitive than other commonly used techniques such as ELISA (enzyme-linked immunosorbent assay). They also do not require the sample to be processed before testing, meaning that a single drop of blood can be analysed directly, without needing further reagents or equipment. Multiple different biomarkers can be analysed simultaneously, potentially allowing multi-cancer blood tests in the future.

To further develop this technology into a clinically implementable assay, five years ago, Osler Diagnostics was spun out of Professor Davis’ lab. The ultimate aim is that this assay could be applied in GP surgeries to test for disease in asymptomatic individuals.

Professor Davis is currently looking at clinical applications within cardiac, cancer and neurological diseases and welcomes interest from researchers who would like to contribute their biomarker ideas and clinical problems.

About the researchers

The Davis Group runs an interdisciplinary research programme within the Department of Chemistry that develops and applies methods for the fabrication of advanced functional interfaces, and are actively engaged in the development of molecular detection, diagnostic, theranostic, and imaging methodologies.

Understanding how inherited and acquired mutations interact to affect cancer

There are two types of genetic variation that affect cancer. So-called somatic variation results from changes (mutations) in a person’s DNA that are acquired during their lifetime in individual parts of the body. These mutations only occur in some cells in the body and are often the result of damage with age or by carcinogens such as sunlight, smoking and some infections. By contrast, germline variation is inherited and so occurs in every cell in the body since birth. An example of germline variation is inheriting a mutation in the BRCA1 gene, which is associated with increased risk of breast cancer in families with these mutations.

Many research studies have investigated the separate effects of somatic and germline variation on cancer risk, progression and response to therapy. However, these studies generate an incomplete picture. For example, designing bespoke therapies to target cancer cells containing specific somatic mutations has had variable success, perhaps in part due to differing underlying germline variation between individuals. To make further progress, we need to learn more about whether germline and somatic variants interact to affect cancer. This is the question that Dr Ping Zhang asked as a post-doc in Dr Gareth Bond’s lab when it was at the Oxford Branch of the Ludwig Institute for Cancer Research.

In this paper published recently in the journal Cancer Research, the Ludwig Oxford researchers worked with colleagues at several other institutions to investigate the interplay between germline and somatic variants affecting the activity of the p53 tumour suppressor protein. p53 is a key protector against the development of cancers and somatic mutations in the gene coding for p53 are found in over half of all human cancers. Perturbation of p53 activity also influences cancer progression and drug response.

In this study, the team discovered evidence that germline cancer-risk p53 pathway mutations cooperate with somatic p53 gene mutations to alter cancer risk, progression and response to therapy, and can be used to identify novel, more effective therapies. With this increased understanding, this work has the potential to guide further discovery of future anti-cancer drug targets and novel combination therapies for enhancing precision medicine.

This work was funded by the CRUK Oxford Centre Development Fund along with the Ludwig Institute for Cancer Research, and the Nuffield Department of Medicine.

Reprogramming tumour cells using an antimalarial drug

Cancers metabolise a large amount of oxygen in order to create the energy needed to divide, grow and spread rapidly. This results in oxygen-starved, or ‘hypoxic’, environments around tumour cells.

This proves problematic as hypoxic tumours behave more aggressively and are more resistant to most treatments, especially radiotherapy. Radiotherapy relies on oxygen to attack cancer cells, and previous studies have shown that three-times higher doses of radiation are needed to destroy tumours in hypoxic environments, compared to those in oxygen rich environments.

Researchers from the University of Oxford and Oxford University Hospitals Trust have investigated the potential for the commonly used anti-malarial and pneumonia drug Atovaquone to improve lung tumour receptiveness to cancer treatments such as chemotherapy and radiotherapy.

The ATOM study, published today in Clinical Cancer Research, administered Atovaquone to patients with non-small cell lung cancer before the surgical removal of their tumours. Using state-of-the-art scans to measure tumour hypoxia, this study found that tumours had 55% less hypoxic volumes than those who didn’t receive the drug.

Following genetic analysis, it was shown that Atovaquone successfully disrupted the metabolic pathways of the tumour that are involved in the consumption of oxygen to create energy for tumour cells. The drug successfully reprogrammed the tumour cell metabolism so that more oxygen was present around the tumour, making it more susceptible to treatments.

Atovaquone is a highly-promising clinical drug, as it is already in wide circulation in the prevention and treatment of malaria. As an FDA-approved drug that is cheap and has hardly any side effects, it could be quickly adopted into clinical use if shown to improve the impact of cancer treatments such as radiation therapy. Laboratory experiments have shown its effect is not lung cancer-specific and it is likely to decrease the hypoxic environment of many types of tumour and so may improve treatment outcomes for many different cancer patients

Professor Geoff Higgins, Consultant Oncologist at the Oxford University Hospitals Trust and lead researcher of this University of Oxford project said;

“Although radiotherapy is already an extremely effective treatment, there is scope to further increase its ability to cure patients. Reprogramming a cancer cell to make it more receptive is one way of doing this.

“I’m delighted that the results from the study are so positive so that we can take the next step towards repurposing a well-established drug as a new, effective anti-cancer treatment.”

Professor Higgins’s team are now investigating the potential of this drug further in a study called the ARCADIAN trial. In this study they hope to demonstrate the safety of using Atovaquone in combination with chemotherapy and radiotherapy, before assessing whether combining this drug with such treatments improves survival of patients with lung cancer.

This study was led by Geoff Higgins, Professor at the Department of Oncology, University of Oxford, and Consultant Clinical Oncologist at the Oxford University Hospitals Trust. It was funded by the Howat Foundation, who support translational research for the benefit of cancer patients.

Studying viral genetics to aid liver cancer early detection

Chronic Hepatitis C virus (HCV) infection causes liver damage and is a significant risk factor for liver cancer. There are now cures available for chronic HCV infection and the World Health Organisation has set a target to eliminate HCV by 2030. However, although curing HCV reduces the risk of liver cancer, individuals with a history of chronic HCV infection remain at higher risk.

There are multiple types of HCV that differ in their genetic sequences. Previous research has established that not all HCV genotypes present the same level of risk for liver cancer. The next step is to discover which particular viral genetic motifs are most associated with liver cancer so the HCV-infected individuals who are at the highest risk of liver cancer can be identified. This will enable more targeted surveillance to detect liver cancer earlier when treatment is more likely to be successful.

Professor Ellie Barnes and Dr Azim Ansari (Nuffield Department of Medicine) have been awarded funding as part of a wider Wellcome Trust Collaborative Award led by Professor Graham Foster (Queen Mary’s University, London) to study anti-viral drug resistance and long-term effects of HCV in Pakistan. HCV infection is highly prevalent in Pakistan with up to 20% of the population infected in hotspot regions.

A cohort of ~500 individuals with HCV-associated liver cancer will be recruited and samples will be collected for viral whole genome sequencing. The Oxford team will then analyse these sequences, comparing to people with HCV infection but not cancer, to identify any genetic patterns that are linked to cancer.

This work complements the recently launched Cancer Research UK-funded DeLIVER programme which, among other features, will study host and viral genetics in a cohort of individuals with HCV and liver cancer in the UK.

Christina Ye awarded CRUK pre-doctoral fellowship

Immune related toxicity is a common side effect of treatment with Immune Checkpoint Blockers for cancer – but the degree to which the development of these side effects is related to overall oncological outcome is unclear. As an Academic Foundation Programme Trainee within OUCAGS (https://www.oucags.ox.ac.uk), I had a four month block of time to work in a lab to gain experience of research. I worked with Dr Benjamin Fairfax’s group in the WIMM/Department of Oncology to explore the relationship between immune toxicity and clinical outcomes. Working with other members of the group, and Dr Anna-Olsson Brown in Liverpool, we found that patients who developed immune related toxicity appeared to have better long-term clinical outcomes including overall survival. Indeed, we found the development of toxicity was a key predictor of the cancer responding to treatment. This work is currently in-press in the British Journal of Cancer.

This period of time in the lab stimulated my interest in research and helped in my decision to apply for an Academic Clinical Fellowship in Dermatology. I was successful in this and I have a further nine months of protected research built into my training this year, which I again plan to spend working in Ben’s group. As a trainee dermatologist I am particularly intrigued by the rash patients frequently develop when they first receive immunotherapy. There is evidence to suggest that another side effect of immunotherapy, colitis, is secondary to the activation of resident memory T cells. Conversely, when you look at the gene expression in CD8 T cells after treatment with checkpoint blockers you can see up-regulation of genes involved in skin trafficking. I will explore whether this rash is indicative of T cell trafficking to the skin, or activation of resident memory T cells, or something completely separate.

Cancer Research UK awards biannual pre-doctoral research bursaries, aimed at providing ‘short term funding to allow clinicians and other health professionals to get involved in research projects early in their career’. Ben encouraged me to apply to this CRUK scheme to further explore the mechanistic basis of the rash in immunotherapy and I am very grateful to have been awarded this funding. Personally I am hoping to gain training in immunological techniques and bioinformatic analysis during this period, and I hope the results we generate will provide further insights into the cells cancer immunotherapy affects and how it works.