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Improving immunotherapy through epigenetics

Immunotherapy has shown remarkable efficacy against a range of cancers. One approach, termed immune checkpoint blockade therapy, blocks an inhibitory immune receptor called PD-1 to take the brakes off the immune system and allow it to kill cancer cells. However, despite this success, anti-PD-1 therapy is ineffective in the majority of cancer patients.

Research is underway to discover strategies that can overcome tumour resistance to immunotherapy. A promising avenue for further investigation is the manipulation of epigenetic regulators. Epigenetic regulators influence the expression of genes without changing the underlying DNA sequence. They can dampen the response of the immune system and their inhibition has been shown to enhance the response to anti-PD-1 treatment. However, because epigenetic regulators are involved in several aspects of the anti-tumour immune response, inhibiting them can result in potentially opposing effects, with the result of little or no overall benefit.

In this paper published in the journal Cancer Discovery, Professor Yang Shi and his laboratory explore the opposing effects of inhibiting one such epigenetic regulator, LSD1. Using mouse and tumour cell models, they show that when LSD1 is repressed, there is a greater immune cell infiltration into the tumour but this is counteracted by the increased production of a cell regulatory protein called TGF-β that suppresses the ability of these infiltrating immune cells to kill cancer cells.

To tackle these conflicting effects, the team experimentally depleted both LSD1 and TGF-β during anti-PD-1 therapy and demonstrated a significant increase in immune cell infiltration, cytotoxicity and cancer cell killing. This combination treatment led to eradication of these previously resistant tumours and long-lasting protection from tumour re-challenge, making it a promising future strategy for increasing the efficacy of this important class of cancer treatment.

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

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.

New sequencing methods for distinguishing DNA modifications

Chemical modifications made to the DNA base cytosine play an important role in the regulation of gene expression across the genome. Cytosine can be chemically modified in four ways, with 5-methylcytosine (5mC) being the most common. Demethylation of 5mC by the TET family of enzymes results in the stable intermediates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). From what has been discovered so far, these modifications appear to have distinct functions. For example, 5mC is associated with repressed regions of the genome whereas 5hmC is present in active ones. However, to study these modifications further, robust sequencing methods are needed that can detect each of these four modifications specifically.

The traditional gold standard method for detecting DNA methylation is bisulphite sequencing. However, this relies on a harsh chemical treatment that degrades most of the DNA sample and is an indirect detection method, which decreases sequencing quality. Recently, a bisulphite-free method called TAPS has been developed by Ludwig Oxford’s Song lab, which has the advantage of preserving more of the DNA, increasing sensitivity, and directly detecting modified cytosines for improved DNA sequencing quality.

Despite its advantages, TAPS cannot distinguish between the different types of cytosine modifications. Other methods already exist that can do so but these use subtraction, for example, measuring 5mC and subtracting this signal from a combined measure of 5mC and 5hmC to obtain 5hmC levels. In addition to the disadvantages of using bisulphite and/or indirect detection strategies, these subtraction methods also need higher sequencing depths and generate very noisy data that can be difficult to interpret. New subtraction-free methods are therefore needed to specifically, directly and sensitively detect these four cytosine modifications in the genome.

In this paper published in Nature Communications, Dr Yibin Liu from Dr Chunxiao Song’s lab (Ludwig Oxford) and Dr Zhiyuan Hu from Professor Ahmed Ahmed’s lab (Weatherall Institute of Molecular Medicine and Nuffield Department of Women’s and Reproductive Health, University of Oxford) have developed a suite of TAPS-related whole genome sequencing methods for specifically detecting 5mC, 5hmC, 5fC and 5caC. They have named these TAPSβ (for 5mC), chemical-assisted pyridine borane sequencing (CAPS; for 5hmC), pyridine borane sequencing (PS; for 5caC and 5fC) and pyridine borane sequencing for 5caC (PS-c; for 5caC).

With these new methods, the research community is now armed to tackle more of the questions about the distinct and important functions of cytosine modifications in the genome and how their distribution is altered in diseases such as in cancer.

Potential of DNA-based blood tests for detecting pancreatic cancer earlier

Pancreatic cancer is sadly a disease with very poor outcomes and only 7.3% of people survive this cancer for 5 years or longer in England (Cancer Research UK). The majority of patients with pancreatic cancer are diagnosed too late for potentially curable treatment to be applied and so there is an urgent need to detect pancreatic cancers earlier with the aim of improving outcomes from this disease.

One strategy for earlier detection is to screen people before they experience any symptoms using a minimally invasive test such as a blood test to look for indicators of pancreatic cancer. Published in the journal Cancers, Dr Shivan Sivakumar (Department of Oncology and Oxford University Hospitals NHS Trust) and colleagues Dr Jedrzej Jaworski (University of Oxford) and Dr Robert Morgan (University of Manchester and Christie NHS Foundation Trust) review the potential of cancer DNA in the blood as an effective and reliable indicator of pancreatic cancer.

DNA from cancer cells can be distinguished from DNA from healthy tissue using either genetic or epigenetic methods (or a combination of both). In the genetic method, cancer can be detected by looking at the DNA sequence, with the presence of cancer-associated DNA sequence changes called mutations or altered fragmentation patterns indicating cancer. In the epigenetic method, chemical modifications to the DNA called methylation are measured that have been shown to change in cancer.

In this review, the authors discuss the potential for DNA-based blood tests for pancreatic cancer earlier detection, the challenges that still need to be overcome and the future perspectives.

Read the full review article on the Cancers journal website.

Pancreatic cancer blood test research in Oxford

In Oxford, we have a couple of research projects underway to study both the genetic and epigenetic methods for detecting pancreatic cancer-derived DNA in the blood.

Dr Siim Pauklin (Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences) is working to identify a pancreatic cancer-specific DNA signature. In the long-term, it is hoped that this can be used as the basis of a simple blood test to detect the presence of pancreatic cancer earlier. This project is funded by the Pancreatic Cancer UK Research Innovation Fund. Read more about Siim’s project here.

Dr Chunxiao Song (Ludwig Institute for Cancer Research) is collaborating with Dr Shivan Sivakumar to apply his TAPS technology to pancreatic cancer. TAPS is a new, more sensitive method for detecting methylation on DNA, which gives it an advantage over other detection methods for measuring the very small levels of circulating tumour DNA in the blood. The team are working to identify patterns of DNA methylation that are specific for pancreatic cancer with the aim of developing this into a diagnostic test. Read more about Chunxiao’s and Shivan’s project on the CRUK Oxford Centre website.

Research projects to detect pancreatic cancer in the blood through non-DNA markers are also in progress in Oxford.

Oxford Cancer alumni’s biotech success

Scenic Biotech was founded in March 2017 as a spin-out of the University of Oxford and the Netherlands Cancer Institute. The company is based on the Cell-seq technology developed by co-founders Sebastian Nijman and Thijn Brummelkamp in their academic labs.

Cell-seq is a large-scale genetic screening platform that allows the identification of genetic modifiers – or disease suppressors – that act to decrease the severity of a disease. These disease-specific genetic modifiers are difficult to identify by more traditional population genetics approaches, especially in the case of rare genetic diseases. By mapping all the genetic modifiers that can influence the severity of a particular disease, Cell-seq unveils a new class of potential drug targets that can be taken forward for drug development.

In a deal worth $375m, Scenic Biotech has recently entered into a strategic collaboration with Genentech, a member of the Roche Group. This will enable discovery, development and commercialisation of novel therapeutics that target genetic modifiers.

Detecting pancreatic cancer through blood tests

Pancreatic ductal adenocarcinoma (PDAC) makes up 95% of all pancreatic cancer cases and has the lowest survival rate, and early diagnostic methods have yet to be developed. As a result, diagnosis often comes at a later stage when treatment options are limited and prognosis is poor.

Diagnosis at this stage often comes from imaging techniques followed by tissue biopsies, which are not appropriate options to use as standardised, early screening methods. New ways to diagnose PDAC at an earlier stage are needed, without the use of invasive procedures.

Liquid biopsies are becoming a more popular option to fill this demand. Taking a blood sample is minimally invasive, quick, and can tell us a lot of information about a person from their cfDNA (cell free DNA). cfDNA is released from cells and circulates in the blood, containing information about the cell they come from.

Methylation on cfDNA often appears in cancer patients, making it an effective biomarker that can be used to diagnose the presence of cancer with high accuracy and specificity about the cancer (such as location). The concept has many applications, including in the earlier diagnosis of PDAC.

The identification of these biomarkers in blood is often limited to the technology used, with DNA being damaged by the harsh chemicals that are used in the processing. The recent development of TAPS technology at the University of Oxford has helped to overcome this, using a bisulphate-free method, and making it a perfect method for PDAC biomarker identification.

DPhil students Paulina Siejka-Zielinska and Felix Jackson and Postdoctoral Researcher Jingfei Chang from Dr Chunxiao Song’s lab in collaboration with Dr Shivan Sivakumar (consultant medical oncologist) have been investigating TAPS as a method to identify PDAC biomarkers. Using blood samples from PDAC patients and healthy individuals, they are applying TAPS technology to prove that it can be used to accurately detect pancreatic cancer biomarkers in cfDNA.

Preliminary results from this study suggest that cfDNA methylation can be used for the identification of PDAC, as well as being able to accurately distinguish between pancreatic cancer and other pancreatic disorders that effect the DNA, such as pancreatitis.

If this is the case, then the results from this study will make for solid grounds for the application of TAPS in the earlier screening for pancreatic cancer.

About the Song Lab

The Song Lab combine various chemical biology and genome technologies to develop novel tools to analyse the epigenome. The lab apply these tools to two main research areas: the use of epigenetic modifications in circulating cell-free DNA from the blood for non-invasive disease diagnostics including early detection of cancer, and understanding the contribution of epigenetic heterogeneity in cancer development.

Most recently, the TAPS technology developed at the Song Lab has led to the creation of the start up Base Genomics, which has been launched to set a new gold standard in DNA methylation detection using this TAPS technology. Base Genomics will initially focus on developing a blood test for early-stage cancer and minimal residual disease. You can read more about it here.

$410 million buy out for Oxford cancer detection technology

Biotechnology company, Base Genomics, launched in June 2020 based on Oxford’s Dr Chunxiao Song’s innovative TET-assisted pyridine borane sequencing (TAPS) technology. This week, Base Genomics was bought out by Exact Sciences for $410 million.

TAPS is a new method for measuring DNA methylation, a chemical modification on cytosine bases. DNA methylation is frequently altered in cancer and these altered DNA methylation levels are preserved in the small amounts of DNA that are released into the blood from cancer cells. With its enhanced sensitivity over the standard methodology for measuring DNA methylation, TAPS has great potential as the basis for a multi-cancer blood test.

“This acquisition by Exact Sciences will enable us to accelerate the clinical and commercial development of Base Genomics and unlock a new era for early cancer detection. This is a big step forwards”

says Base Genomics co-founder and chemistry lead, Dr Yibin Liu, who co-invented the technology while a post-doc at the Ludwig Institute for Cancer Research, Oxford Branch.

Exact Sciences will continue to build on the Base Genomics team in Oxford, creating a world-leading research centre for early stage cancer detection.

“I am thrilled that the TAPS technology developed in my lab has received this level of investment. We can now proceed much more rapidly to fully leverage the power of this technology for cancer detection and patient benefit”

says Dr Chunxiao Song, Assistant Member of the Ludwig Institute for Cancer Research, Oxford Branch and Base Genomics co-founder.

Chunxiao Song’s research has received funding from the Ludwig Institute for Cancer Research, Cancer Research UK and the NIHR Oxford Biomedical Research Centre. TAPS is continuing to be developed in Chunxiao’s lab, for example it was recently adapted for long-read sequencing, to further its application to other fields of biomedical research.

The developmental origins of resistant infant leukaemia

The Roy and Milne labs are investigating the developmental origins of infant leukaemia and its influence on the biology of the disease