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The next step in personalised cancer medicine

Researchers at the Botnar Research Centre, University of Oxford have developed technology that facilitates standalone long-read Oxford Nanopore sequencing of single cells. This breakthrough technology has the potential to open new avenues within genomics and enable future discoveries to understand the causes of many human diseases.

The work, in part supported by grants from the UKRI (Innovate UK, EPSRC and MRC), results from a collaboration with researchers from the Department of Chemistry at Oxford University, ATDBio, a world leader in complex oligonucleotide chemistry, and pharmaceutical company BristolMyersSquibbs. The study has been published in this week’s issue of Nature Biotechnology.

“The application of accurate long-read single-cell sequencing will have a transformative effect on the wider single-cell sequencing community, as longer and full-length transcriptomic sequencing allows users to capture more information about the transcriptional and functional state of a cell,” says Assistant Professor Adam Cribbs, senior author of the paper and Group Leader in Systems Biology and Next Generation Sequencing Analysis at the Botnar Research Centre. “This means that we move closer to being able to better understand and diagnose diseases such as cancer”.

Single-cell genomics, the ability to examine all information contained in an individual cell, is a rapidly evolving field and is dominated by droplet-based short-read single-cell sequencing applications. In this approach, cells are encapsulated with barcoded RNA-capture microbeads into droplets within an oil emulsion. Each droplet becomes a discrete reaction vessel, associating a different barcode with each cell’s RNA and a unique molecular identifier (UMI) with each RNA transcript.  Once barcoded, RNA from all cells can be pooled and processed conventionally for next generation sequencing.  During sequencing, both the original RNA sequence and the associated barcode and UMI are determined. Key to measuring abundance of each RNA and correctly associating them with their cell of origin is accurate assignment of the UMIs and barcodes.

Long-read sequencing approaches, such as those of Oxford Nanopore Technologies, are currently revolutionising bulk sequencing approaches. “Long-read single-cell technology has the potential to interrogate not only RNA abundance, but also splice variants, structural variation and chimeric transcripts at the single-cell level. Collectively, the ability to determine these features accurately will improve diagnostics and biological understanding. However, Nanopore sequencing can be inaccurate, which hinders the critical steps of barcode and UMI assignment, making its application to single-cell sequencing challenging,” explains Dr Martin Philpott, first author of the paper and Director of the Next-Generation sequencing facility at the Botnar Research Centre.

To overcome these challenges, the team has developed a new approach called single-cell corrected long-read sequencing (scCOLOR-seq) that identifies and corrects errors in the barcode and UMI sequences, permitting standalone cDNA Nanopore sequencing of single cells. “Each mRNA molecule is tagged with a short sequence which identifies it within a certain droplet,” adds Dr Cribbs. “However, Nanopore long-read sequencing is too error prone to reliably sequence these tags, making it difficult to map the mRNA back to its specific cell. What we’ve been able to do is to develop a practical method for building redundancy into the tag, allowing inaccuracies within the sequencing to be pinpointed, and then correct them. The mRNA can then be linked back to an individual cell.”

The research was developed in collaboration with ATDBio, an Oxford/Southampton based company, created by Professor Tom Brown Sr at the Chemistry Department, University of Oxford. Dr Tom Brown Jnr, Chief Scientific Officer of ATDBio, says, “The new scCOLOR-seq method is the first of many innovations resulting from our collaboration with the Botnar Research Centre team. The collaboration has been one of our most interesting and successful, and we are pleased to see our work recognised in Nature Biotechnology. It’s a great example of how we at ATDBio can apply our expert knowledge of nucleic acid chemistry and complex oligonucleotide synthesis to difficult problems in biology and beyond, together with our corporate and academic partners”.

“This study demonstrates an incredible cross-disciplinary team effort to advance single-cell technologies and is the result of strategic investments into these technologies at our department,” adds Professor Udo Oppermann, Director of Laboratory Sciences at the Botnar Research Centre and co-senior author of the paper. “We will continue our collaborative efforts to develop innovative single-cell approaches and – as demonstrated in the paper- apply this to molecular analyses in primary and secondary bone and other haematological cancers. Our intention is to advance these technologies in personalised medicine approaches such as cancer diagnosis allowing rational clinical decision making.”

Ludwig Oxford and Oxford University welcome Professor Stefan Constantinescu

Oxford’s cancer community is delighted to welcome Professor Stefan Constantinescu, a physician scientist and authority on the signalling pathways and molecular mechanisms of blood cancers, especially myeloproliferative neoplasms, a collection of slow growing blood cancers that can progress to acute malignancies. He is a member of the Ludwig Institute for Cancer Research, Professor of Cell Biology at the Université catholique de Louvain, Director of Research (Honorary) at the Fonds National de la Recherche Scientifique (FRS-FNRS), Belgium and President of the Federation of European Academies of Medicine (FEAM). Constantinescu will spend 25% of his time at the Ludwig Oxford Branch and the remainder of the time at his existing Ludwig laboratory in Brussels.

Constantinescu has received many honors for his work, including membership of the Royal Academy of Medicine of Belgium and the Belgian Government prize for basic medical sciences. He is internationally known for his groundbreaking contributions to our understanding of the mutations and mechanisms that drive myeloproliferative disorders. In a fruitful collaboration with William Vainchenker, he discovered that a mutation (V617F) in a signalling enzyme named Janus kinase 2 (JAK2) occurs in most patients with polycythemia vera, in which red blood cells accumulate abnormally. Constantinescu’s subsequent work demonstrated how this mutation causes disease, leading to the development of novel therapies to treat myeloproliferative disorders and the widespread clinical use of genetic tests to detect the mutation.

Constantinescu has also identified and characterised other common mutations in the thrombopoietin receptor that cause these blood disorders. He has further demonstrated that mutated calreticulins –“chaperone” proteins that otherwise help fold other proteins appropriately—can induce myeloproliferative disorders via abnormal activation of the thrombopoietin receptor, identifying a novel oncogenic mechanism. His discoveries have helped transform the field and continue to open new avenues for the development of targeted therapies.

Constantinescu’s Ludwig Oxford lab will focus on a systematic study of signalling and epigenetic regulation during oncogenesis in chronic myeloid cancers and their progression to the severe condition, secondary acute myeloid leukaemia. Ludwig Oxford’s research programme will be enhanced by Constantinescu’s presence, and his own research programme will benefit from Ludwig Oxford’s expertise in cancer epigenetics, represented by the laboratories of Yang Shi, Chunxiao Song, Skirmantas Kriaucionis and Benjamin Schuster-Böckler.

Read more about the new Constantinescu research group here.

Dr Lennard Lee awarded ACP McElwain Prize for contributions to medical oncology

Dr Lennard Lee, Academic Clinical Lecturer at the University of Oxford, has been awarded the 2020 Association of Cancer Physicians (ACP) McElwain Prize for his contribution to the development of Medical Oncology in the UK.

During the COVID-19 pandemic, Dr Lee’s contributions were best reflected in his creation and implementation of a national prospective observational cohort study of cancer patients during the COVID-19 pandemic, the UK Coronavirus Cancer Monitoring Project (UK CCMP). This was one of the largest global registries and the first to identify that cancer treatments can be safely delivered during a COVID-19 pandemic.

The initial phase of the project has been successfully achieved with the roll out and implementation of the UK CCMP emergency observational response network, as well as studies to determine those in our population who are most at risk – such as blood cancer patients.

Thanks to this project, the UK oncology community now has the tools and the mechanism to learn from each case of COVID-19 in cancer patients and the evidence required to bring about clinical management/service/treatment decision changes to improve the outcomes of cancer patients. The data from this project has helped form the guidelines which have been published from NHS England about the resumption of cancer services and led to the return of near-normal chemotherapy prescribing levels.

Building on the success of this work, Lennard is now leading on further studies to understand and better safeguard cancer patients during the pandemic. He has co-launched projects on COVID-19 vaccine efficacy, understanding how this is impacted by cancer treatments, and studies to identify other effective COVID-19 risk reduction interventions.

Lennard Lee said

“I am honoured to receive this award on behalf of all the clinicians and researchers who took part in the UKCCMP. The work by the UK oncology community had global impact, demonstrating that cancer patients can be treated safely during the pandemic. No one should be denied appropriate cancer treatments and it is important that we continue to deliver research excellence in order to protect the vulnerable during this pandemic”.

For more information about the UK CCMP see here.

Two clinical academic research partnerships awarded to Oxford researchers

Dr Karthik Ramasamy and Dr John Jacob are both Oxford clinical researchers that have been awarded a Clinical Academic Research Partnership (CARP) by the MRC. Both have been awarded upwards of £200,000 to fund projects investigating myeloma early detection & brain cancer modelling, respectively. Find out more about the projects being funded with this award below.

Dr Karthik Ramasamy

Earlier diagnosis of the bone marrow cancer myeloma is a high priority for patients since it can both improve survival and allow for better control of symptoms. Every case of myeloma is preceded by a condition called Monoclonal Gammopathy of Undetermined Significance (MGUS) and individuals with MGUS are regularly monitored so that progression to myeloma can be caught early.

While this approach is benefitting some patients, there are two main issues that still need to be overcome to improve earlier myeloma diagnosis:

  1. MGUS is largely symptomless and often undiagnosed, meaning that 80-90% of myeloma patients are diagnosed without first receiving an MGUS diagnosis that would have prompted monitoring for myeloma.
  2. Only 1% of patients with MGUS progress to myeloma every year and the risk of progression is not well defined, placing a large resource burden for monitoring on healthcare providers and creating anxiety for patients.

In this MRC CARP award, Dr Karthik Ramasamy will address both of these challenges. Firstly, working with Professor Kassim Javaid, Professor Daniel Prieto-Alhambra, Dr Constantinos Koshiaris and data from primary care health records, Dr Ramasamy will identify specific clinical signs/symptom clusters associated with MGUS to enable a greater proportion of individuals with MGUS to be diagnosed and monitored. Secondly, additionally collaborating with Dr Ross Sadler, Professor Chris Schofield and Professor James McCullagh, Dr Ramasamy will seek to identify routinely recorded clinical characteristics and additional protein biomarkers that improve prediction of progression from MGUS to myeloma in a cohort of patients undergoing monitoring at Oxford University Hospitals NHS Trust. This latter work is bolstered by a recently awarded CRUK Oxford Centre Development Fund award, which will enable the research team additionally to pilot protein glycosylation analysis in blood samples collected as part of a current study investigating serological markers in plasma cell dyscrasias (BLOOM).

Dr Karthik Ramasamy is Lead Clinician for myeloma and other plasma dyscrasias in Thames Valley Strategic Clinical Network and Divisional Lead of Cancer Research across Thames Valley and South Midlands Research Network.

Learn more about myeloma early detection research in Oxford.

Dr John Jacob

Brain cells that originate in the cerebellum give rise to the most common childhood brain tumour, known as medulloblastoma, which can also affect adults. Although it can be cured, it is often a devastating disease, with common treatment options resulting in an increased risk of adverse side effects such as strokes and seizures.

Like most cancers, the development of new treatment options that improve survival rate and reduce side effects is reliant on researchers establishing models that reflect the human tumours, in order to test the efficacy of new therapeutics before these are tested in patients. Existing models for medulloblastoma have shortcomings making the discovery of new treatment options slow. This is due to:

  1. Growing medulloblastoma cells outside of a patient is hard, due to the change in environmental factors. Growing cells outside the body usually does not accurately reflect the microenvironment that cells are derived from, and so the cells will behave differently and unlike a real tumour. As a result, only a few cell lines from medulloblastoma patients have been successfully grown ex vivo.
  2. Models can be costly, and these include mouse-based models, which pose limitations due to the difference in species
  3. Medullobastoma tumours are genetically different between patients – there is no ‘one size fits all’ model and there is a need to develop more treatment options that target specific genetic subtypes in order to improve survival.

In this CARP award, Dr John Jacob (Nuffield Department of Clinical Neurosciences) aims to investigate a specific genetic subtype of medulloblastoma (known as sonic hedgehog medulloblastoma) – and test if the presence of the tumour microenvironment, which consists of non-cancerous cerebellar tissue, is necessary to better simulate the typical tumour growth conditions.

Working alongside Associate Professor Esther Becker (Nuffield Department of Clinical Neurosciences) and Dr Benjamin Schuster-Boeckler (Big Data Institute & Oxford Ludwig Cancer Institute), the team hope to recreate the in vivo tumour microenvironment more accurately compared to existing models. The Becker group previously established a methodology to grow human cerebellar neurons from human induced pluripotent stem cells (hiPSC). By using hiPSCs to form miniature cerebellar structures, termed cerebellar organoids, the tumour microenvironment can be recreated in a dish.

The team hope to overcome the existing modelling difficulties by growing medulloblastoma cells on these organoids. Dr Jacob, aided by the computational biology expertise of Dr Schuster-Boeckler will then investigate the growth of individual tumour cells, in a more physiological context. The resulting outcomes could mean the development of a new model to test patient-specific therapies on, in order to assess their toxicities and efficacy more accurately.

Dr John Jacob is a consultant neurologist who is interested in better understanding the genetic complexity of cancer and what it means for personalised treatment. Through this award and collaboration, Dr Jacob hopes to improve the success of therapy development and ultimately improve the repertoire of therapies of this cancer.

The Medical Research Council Clinical Academic Research Partnership scheme allows NHS consultants with a PhD or MD to participate in collaborative high-quality research partnerships with established leading biomedical researchers.

 

New clinical prediction tools for myeloma

Myeloma is a cancer of the bone marrow that caused 117,077 deaths worldwide in 2020 (International Agency for Research on Cancer). Earlier diagnosis improves the rate of survival but unfortunately, delays in myeloma diagnosis are common and result in poorer patient outcomes.

One of the reasons for the diagnostic delay is that myeloma symptoms are non-specific and relatively common in people without cancer. For example, back pain is associated with myeloma yet there are many other non-myeloma causes of this symptom. Additional measures are therefore needed to highlight the possibility of myeloma in patients where GPs do not originally suspect this disease.

GPs frequently order simple laboratory tests, such as the full blood count, to investigate patients presenting with non-specific symptoms. Previous work by Dr Constantinos Koshiaris, Dr Jason Oke, Dr Brian Nicholson and colleagues from Oxford’s Nuffield Department of Primary Care Health Sciences and the University of Exeter identified certain abnormalities in blood test results that indicate a higher risk of myeloma, such as low haemoglobin which can be observed up to 2 years before a myeloma diagnosis.

In this paper published recently in the British Journal of General Practice, the Oxford researchers have developed new clinical prediction models for myeloma that incorporate both symptoms and blood test results. Using the Clinical Practice Research Datalink (GOLD version), a primary care database containing electronic health records for more than 11 million patients in the UK, the team identified the most common symptoms and full blood count results recorded for patients with myeloma. The most predictive of these were included in the models they developed and the new tools were validated against a set of test data. Decisions made using their prediction models resulted in fewer false positives and more true positives when compared to single tests or symptoms alone.

By identifying patients at highest risk of myeloma in primary care, these new prediction rules have the potential to reduce diagnostic delays by a substantial amount. Further research is now needed to understand more about the feasibility and implementation of this tool in the primary care setting and the impact it will have on the diagnostic pathway and patient outcomes.

Early Detection Award for research into the clinical application of single cell genomics

Myelodysplastic syndromes (MDS) are a group of blood cancers in which the bone marrow fails to make normal levels of blood cells. MDS can be broadly classified into two major groups: high-risk MDS, in which patients progress to acute myeloid leukaemia with a very poor survival rate; and low-risk MDS, in which the disease is less aggressive but patients still suffer from a huge burden of symptoms, often the result of anaemia.

There are a number of exciting new targeted treatment options for low-risk MDS. However, these do not work in all patients and, particularly given the high economic cost of newer treatments, current biomarkers are not sufficiently predictive of treatment response. There is a need to more precisely categorise MDS to predict the disease trajectory and the response to therapy so that the most effective treatment can be given to each patient.

Large investments in sequencing technology in clinical laboratory services are enabling precision medicine in certain cancers and revolutionising patient care. Dr Onima Chowdhury, MRC Clinical Academic Research Fellow and Consultant Haematologist (MRC Weatherall Institute of Molecular Medicine and Oxford University Hospitals) is working with Professor Adam Mead, Dr Supat Thongjuea and Dr Lynn Quek at the MRC WIMM to explore the use of single-cell genomics in the clinical diagnosis and management of MDS. Funded by a Cancer Research UK Early Detection and Diagnosis Primer Award, the team will seek to develop a simple, clinically applicable processing and analysis pipeline, as well as identifying biomarkers that correlate and can perhaps supersede current diagnostic modalities.

Long-term, the team hope that this approach will be able to improve outcomes of patients through improved diagnosis, risk prediction and targeted treatment in MDS and other haematological malignancies.

Prof Andi Roy receives new award for immune-cell research

Co-funded by Cancer Research UK and Children with Cancer UK, Andi is one of 5 to receive £1 million each to investigate children’s and young people’s cancers.

Detecting myeloma earlier

Several research projects are underway in Oxford focusing on different points in the clinical care pathway to improve myeloma early detection.

Funding boost for OxPLoreD early detection study

OxPLoreD is an observational cohort study sponsored by Johnson and Johnson that will recruit 1650 patients from across the UK with pre-cancerous lymphoproliferative disorders. These conditions include monoclonal B-cell lymphocytosis and monoclonal gammopathy of unknown significance that put individuals at higher risk of developing the blood cancers chronic lymphocytic leukaemia and multiple myeloma respectively.

The aim of the study is to look for new ways to find and treat blood cancer sooner by identifying clinical, genomic and immunological predictive markers of progression from these pre-cancerous conditions to malignant disease. The study will also explore the possibility of a future early intervention trial for the subgroup of patients at highest risk of progression.

OxPLoreD is one of the seven clinical trials that have received an £8m funding boost from UK Research and Innovation (UKRI) and will work in partnership with Genomics England. The funding will speed up the adoption of whole genome sequencing in the study of cancer. Genetic analysis is a critical tool that can allow clinicians to select the most appropriate treatments for each patient. In the OxPLoreD study, genetic analysis might be able to identify individuals at highest risk of disease progression that would benefit from earlier treatment. In the longer term this may also enable the identification of those people who would benefit from certain types of treatment.

Alison Cave, UKRI challenge director says:

“Research tells us that one-in-two people in the UK population will get cancer. That stark statistic shows just how important it is for us to seek new treatments. The use of genetic analysis opens new possibilities in our drive to beat cancer. The projects for which we have announced funding today are exciting pointers to future diagnosis and precision treatments”

The funding has been delivered through UKRI’s Industrial Strategy Challenge Fund’s £210m data to early diagnosis and precision medicine (DEDPM) programme. The challenge aims to combine research data and evidence from the NHS to create new and improved ways of identifying disease and treatment pathways.

Prof. Sir Mark Caulfield, Chief Scientist at Genomics England says:

“The 100,000 Genomes Project, Genomics England has analysed the genomes of over 17,000 cancer participants and this suggests that up to half have revealed mutations of potential clinical significance. The DEDPM programme is a major opportunity to expand the application of whole genome sequencing into clinical trials involving cancer where support from the ISCF is likely to deliver significant clinical benefit”

For more information about the other trials funded by this scheme, see the UK Research and Innovation announcement.