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Projects using CellBank samples

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Below are summaries of the large number of research studies that have made use of CellBank samples.

A genome wide screen of SNPs to identify susceptibility alleles for childhood acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) is a common cancer of childhood in developed countries. Current evidence indicates it is likely a priori that all risk affected by any exposures will operate in the context of a genetic background of variable susceptibility. The United Kingdom Childhood Cancer Study (UKCCS) has established a unique clinical resource for studying inherited predisposition to ALL. Using DNAs from patients with ALL available through UKCCS we shall conduct a genome-wide association study of tagging SNPs to identify common variants influencing the risk of developing the disease. This information should greatly assist in identifying those at increased risk. In addition associations identified may prove extremely valuable via the functional links they reveal and either endorse current aetiological hypotheses or suggest new ones that merit testing via gene/environment specific hypotheses.

An investigation of the molecular basis for B lineage acute lymphoblastic leukaemia cell migration to extramedullary sites

Acute lymphoblastic leukaemia (ALL) is the commonest childhood cancer. Although massive strides have been made in treating this disease a significant minority of children still suffer disease recurrence (relapse) which may lead on to significant long-term health problems or death. Although leukaemia begins in cells from the bone marrow, most children who relapse have evidence of leukaemia at distant sites round the body, particularly the brain. This study sets out to understand how leukaemic cells might enter the brain and other distant sites and how they survive when they get there. This knowledge may allow identification of new tests and treatments for leukaemia.

Asparaginase-associated pancreatitis during treatment of childhood acute lymphoblastic leukaemia; characteristics and risk factors - a PdL/IBFM phenotype-genotype study

Acute lymphoblastic leukemia (ALL) is the most common cancer in children aged 1-14.9 years, with an incidence of approximately 3.5 cases per 100,000 children in Europe and the U.S. Survival rates have increased to 85% after first line treatment, but many patients are burdened by life-long toxicities. Asparaginase (ASP) is a crucially important antileukaemic drug. Studies have shown that patients receiving prolonged administration of this drug have a superior survival rate, compared to patients who have their therapy truncated due to toxicity. ASP is associated with several toxicities, pancreatitis being one of the most common. Asparaginase Associated Pancreatitis (AAP) has an incidence of 4-10% in contemporary protocols using ASP and is among the most common reasons for truncating ASP treatment. However, risk factors, including genetic predisposition to AAP, are not well known. This study provides deep phenotyping of more than 600 AAP patients based on data from 15 large international childhood ALL groups. Furthermore, we will explore the host genome variants associated with developing AAP. The latter is examined in more than 300 childhood ALL patients. The retrospective AAP phenotype study is the first of its kind in collecting data on a specific toxicity from multiple international childhood ALL groups, i.e. across many treatment protocols. This will provide sufficient power in describing in detail the phenotype of AAP. This large, and unique, dataset will allow exploration of risk factors, long term outcome including risk of chronic pancreatitis, and establish guidelines for ASP re-exposure after an episode of AAP. The Genome Wide Association (GWA) Study investigates ~2,500,000 Single Nucleotide Polymorphisms (SNPs) spread across the genome with emphasis on the exome. We will link individual SNPs as well as biological pathways to the risk of developing AAP. This is done based on SNP analysis of >300 of the AAP cases with available deep phenotype data. This study will provide new knowledge on AAP, enabling a more personalized cancer treatment strategy with ASP. Thereby improving treatment of childhood ALL in patients with high risk of AAP.

Biology of the TEL/ABL (ETV6/ABL)-positive leukaemia

The goal of the project is to collect as many cases of this very rare leukaemia subtype (with TEL/ABL fusion) as possible throughout the world (realistically 10-20) to characterise those genetically and to define in details the biological background of TEL/ABL-psoitive leukaemias. Discovering clinical and biological attributes of the TEL/ABL-positive leukaemia will contribute towards improving the treatment of the patients from this group who have so far faced a particularly unfavourable prognosis.

Cell/Gene therapy for HSCT: Gene edited anti-CD3 chimeric antigen receptor T-cells

Cell therapy is a therapy in which cellular material is introduced into a patient. Generally, intact, living cells are introduced to the patient. For example, T-cells that are engineered to target disease-causing cells, such as cancer cells, may be introduced to a patient. In this way, cancer cells may be destroyed. We have engineered a T-cell that can target and kill other T-cells (which may cause disease), but importantly would not be able to recognise other engineered T-cells.

Characterisation of sub-set of patients

Understanding the genetics of leukaemia has led to improvements in treatment for certain groups of patients. One set of genes, the immunoglobulin genes, are important in lymphoma and multiple myeloma, where they have been studied in detail. A unique group of patients with acute lymphoblastic leukaemia have been identified with abnormalities of these genes. We plan to characterise these abnormalities and relate them to the other genetic changes and the clinical features in ALL compared with other blood cancers. Understanding how these genes function may define a new group of patients in which modified treatment may improve survival.

Characterisation of the gene expression profiles in IGH translocated B-cell precursor acute lymphoblastic leukaemia

Leukaemia is a type of cancer that leads to the uncontrolled accumulation of blood cells. Acute lymphoblastic leukaemia (ALL) is a subtype of leukaemia and is caused by genetic defects in the patient's DNA. How the alterations in our genes lead to the development of leukaemia is not fully understood. We identified genetic defects in patients with ALL that result in the swapping of material between two chromosomes (translocation). We believe that the genes involved in these translocations contribute to the development of leukaemia and propose to study their role. We will assess the similarities and differences that may be present by looking at the levels of gene expression amongst these patients. We will investigate whether we can begin to subgroup these patients according to which signalling pathways may be disrupted. The data generated from this analysis will inform future experiments investigating specific drug combinations that may benefit these patients.

Characterisation of the genomic landscape of cytogenetically undefined B-cell precursor acute lymphoblastic leukaemia (BCP-ALL)

In childhood BCP-ALL chromosomal abnormalities are often used to predict prognosis and in risk stratification for treatment; however 25% of cases are cytogenetically undefined, termed 'B-other' BCP-ALL. Clinical heterogeneity clearly exists within this subgroup. We would like to employ high resolution techniques to identify recurrent genomic abnormalities that may form distinct subgroups in 'B-other' BCP-ALL. We will assess the clinical significance of any genomic aberrations found to identify patients that may benefit from modifications to their treatment or novel targeted therapies.

Creation of CEBPE/IGH cell line as a means to understand the role of CEBPE overexpression in acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) has its origins in the early stages of immune B-cell development. Genetic studies of ALL have identified regions of DNA harbouring risk factors that inherited from parent to child, including on chromosome 14 at the gene CEBPE. Some ALL tumour cells also possess a large defect in their DNA on chromosome 14 also involving CEBPE  that occurs spontaneously in immune cells but is not inherited. However, the role of CEBPE in ALL is currently unknown. The objective of this project is to take tumour cells from an ALL patient who possessed such a large DNA defect influencing CEBPE and use this as a model system to investigate how CEBPE promotes cancer development.

Defects in foetal B-lymphoid development and in utero origins of Down syndrome associated childhood acute lymphoblastic leukaemia

There is growing evidence that many childhood leukaemias start to develop in foetal life. Immature blood cells in the foetus, called progenitor cells, may develop abnormalities that make them more susceptible to events after birth that transform them into rapidly proliferating leukaemic cells. This secondary event or ‘second hit’ required to develop childhood leukaemia may happen several months or even years after birth. Children with Down syndrome (DS) have an increased risk of developing acute lymphoblastic leukaemia (ALL) and we believe that this is driven by the presence of an extra copy of chromosome 21 (trisomy 21; T21) in all the cells (including blood progenitor cells). We also know that many children without DS who develop leukaemia have T21 in the leukaemic cells. Therefore I would also like to examine DS fetal BM progenitors to try and understand how T21 may render progenitors more susceptible to leukaemic transformation. Indeed T21 may be one of the ‘first hits’ in childhood leukaemia. The leukaemia that children with DS develop is biologically different from other childhood leukaemias that occur in children without DS; for example infant ALL (ALL developing in a child <1 year of age) is very rare in the DS cohort. We therefore want to compare the properties of fetal DS progenitors with progenitors from DS-ALL and non-DS ALL. We expect DS fetal progenitors to be similar to those from DS-ALL and the characteristics that differentiate them from non-DS ALL may help us understand the origins of other childhood leukaemias, especially infant ALL.

Development of magnetic micro and nanostructures for the application of cell treatment and phenotypic profiling of cell interaction

This project aims to identify a novel signature able to distinguish leukaemic cells from the normal ones. Cancer cells will be specifically targeted with magnetic nanoparticles able to disrupt their membrane, provoking cell death without harming the healthy cells.

Development of pluronic micelle nanocarriers for the treatment of childhood acute lymphoblastic leukaemia

Treatment for children with acute lymphoblastic leukaemia (ALL) is very toxic and can be life threatening, so intensifying current therapies is not a realistic method of improving survival. A naturally occurring drug, Parthenolide, is particularly effective at killing leukaemia cells without harming normal blood cells. Unfortunately, Parthenolide is not soluble in water and so cannot be used to treat children. We will develop new preparations to improve Parthenolide delivery and make it more effective against ALL cells. We will also investigate whether these delivery methods can be used to reduce drug doses to alleviate problems associated with children being over-treated.

Expression of Rho GTPase family members in T-acute lymphoblastic leukaemia

T-cells are white blood cells that are needed to fight infections, but they are also associated with disease. A type of leukaemia arises when T-cells become cancerous in the bone marrow. Like normal T-cells, these leukaemic T-cells can move into the blood and then into tissues, where the leukaemic T-cells can then accumulate. Our research will examine which molecules are different between normal T-cells and leukaemic T-cells, in order to find new therapeutic targets for preventing the spread of leukaemia.

Genetic abnormalities in predicting relapse and post relapse survival in acute lymphoblastic leukaemia

Leukaemia, like other cancers, is a genetic disease. The discovery and characterisation of abnormal genes in the leukaemic cells of children with leukaemia has provided scientists and clinicians with a wealth of information about this disease. Currently many of these abnormal genes help in the diagnosis and treatment of patients. In particular, they can be useful in identifying patients who are at the highest or lowest risk of relapse. However, we do not fully understand the biological or clinical impact of many abnormal genes and there are likely to be abnormal genes that have yet to be discovered. The purpose of this study is to use novel and high-throughput technologies to comprehensively characterise a large cohort of patients for the presence of known abnormal genes and to identify novel abnormal genes. The data produced by this project will be analysed in the context of a modern clinical trial so that the results can be rapidly translated to optimise the treatment and outcome of future children with leukaemia.

Genetic characterisation and therapeutic targeting of paediatric mixed phenotype acute leukaemia

Mixed phenotype acute leukaemia is a rare childhood disease with a poor prognosis (50-60% survival) and no known effective treatment. To date, there have been no studies to identify genetic changes associated with the development of mixed phenotype acute leukaemia and its response to treatment. Such an analysis could reveal previously unrecognised targets for therapy and lay the groundwork for clinical studies of therapeutic agents. We propose to conduct such an analysis by comparing gene expression in samples obtained from patients at the time of leukaemia diagnosis and remission and by comparing genetic alterations with patient clinical characteristics. We will then create an authenticated mouse model of the disease by grafting the human leukaemic cells into mice, in order to screen for and test therapeutic agents. This work will provide a much-needed deeper understanding of the genetic basis of mixed phenotype acute leukaemia and identify treatments that may improve the survival of children with this disease.

Genetic definition of the BCR-ABL1-like subgroup in acute lymphoblastic leukaemia

In childhood B-lineage ALL, chromosomal abnormalities are linked to prognosis and are used in risk stratification for treatment. Recently, novel high resolution techniques have uncovered new genetic abnormalities in patients who were previously unclassified. These abnormalities are linked to poor response to treatment, but in some cases they will highlight those patients who may respond to different treatments. We are establishing a screening programme using DNA and RNA approaches to ensure that these patients are accurately identified.

Genetic polymorphism in the PAI-1 gene and risk of osteonecrosis in patients with acute lymphoblastic leaukaemia treated on ALL2003

Some children who are treated for leukaemia develop a side-effect of treatment called osteonecrosis; this affects their bones and in its most severe form can cause very painful joint problems. We are investigating whether some children may have a genetic predisposition to developing this side-effect. If so, we may be able to offer them better, more effective treatment in future.

Genetic regulation of normal and aberrant haematopoiesis

Haematopoiesis is the formation of blood cells. All cellular components of the blood are derived from stem cells that have the ability to become any type of cell in the blood system. Leukaemia is a cancer of the blood and bone marrow, characterised by having too many white blood cells. This is thought to arise when there is a change in the behaviour of genes that normally regulate the development of blood cells. We and others have identified a number of such genes. To understand  exactly what they do, we wish to examine their behaviour in blood stem cells. The umbilical cord blood is a rich source of these stem cells and that is why we wish to obtain this from the cord blood bank. The planned research will increase our understanding of how leukaemia occurs and progresses and to facilitate the development of new anti-leukaemic drugs.

Genetics of acute lymphoblastic leukaemia

Examining DNA from chidren with leukaemia will enable us to identify new risk factors for the disease providing fresh insights into why the disease develops. Furthermore, this information may also show why some children have different outcomes from their disease.

Genomic characterisation of IGH@translocations in B-precursor acute lymphoblastic leukaemia

Understanding the genetics of leukaemia has led to improvements in treatment for certain groups of patients. One set of genes, the immunoglobulin genes, are important in lymphoma and multiple myeloma, where they have been studied in detail. A unique group of patients with acute lymphoblastic leukaemia have been identified with abnormalities of these genes. We plan to characterise these abnormalities using technologies that sequence the patient's genome and allow us to find common abnormalities amongst this group of leukaemia patients. Understanding the genomes of these patients may define a new subgroup in which modified treatment may improve survival.

Genomic landscape of relapsed t(1;19) positive acute lymphoblastic leukaemia

 

Unexpectedly, children that suffer from a quite common subtype of acute lymphoblastic leukaemia that is characterized by a recurrent genomic abnormality (so-called translocation t(1;19)) and in most cases can be cured in the first intent, in the rare event of a disease recurrence cannot be salvaged with the existing chemotherapies. The IntReALL consortium, which coordinates the treatment of ALL at first recurrence, has therefore initiated this project to identify the underlying genomic lesions in this rare subset of patients at need for urgent medical alternatives. The identification of such alternations will provide mechanistic insights about resistance in ALL and hopefully a better rationale for the treatment of this group of patients.

Global methylation patterns in resistant and relapsed childhood acute lymphoblastic leukaemia and effect this may have on resistance

There has been considerable progress in the treatment of childhood leukaemia and in the UK approximately 80% of children with acute lymphoblastic leukaemia (ALL) can be cured with current chemotherapy. Leukaemia is treated with a combination of drugs designed to kill the leukaemia cells. Unfortunately some leukaemia cells are resistant to these drugs. This means that a significant number of children with ALL have disease which is resistant to treatment. One way to overcome this resistance is to study the biology of resistant leukaemia cells and use this information to develop new drugs specifically targeted to overcome the mechanism of resistance. The genetic material in resistant leukaemia cells is known to have some important differences compared to chemotherapy sensitive leukaemia cells. The expression of particular genes (whether they are turned on or off) in leukaemic cells can be related to how the disease responds to chemotherapy. The expression of some genes in leukaemia is controlled by a process called 'DNA methylation.' Our research is investigating the relationship between DNA methylation and the development of resistant disease in childhood acute leukaemia. We know that DNA methylation is a reversible process; therefore understanding how it is involved in resistant disease may lead to the development of new treatments for children with leukaemia in the future.

Identification of genetic variation influencing acute lymphoblastic leukaemia

We have shown that inherited genetic susceptibility contributes to the development of childhood acute lymphoblastic leukaemia. By comparing the genetic makeup of additional patients with ALL we shall identify new genes for the disease. These data will provide fresh insights into how the disease develops and potential novel methods of treatment.

Identification of progression genes in t(8;21) and t(12;22) leukaemias

Leukaemias arise from normal blood cells by a series of genetic changes. We are studying a gene (RUNX1) that is frequently involved in the most common forms of leukaemia. RUNX1 is altered in leukaemia cells by aberrant breakage and rejoining of chromosomes to generate the fusion genes RUNX1-ETO and TEL-RUNX1 in acute myeloid leukaemia and childhood acute lymphoblastic leukaemia respectively. These fusion genes are necessary but not sufficient for leukaemia to develop and we aim to generate new methods to identify other genetic changes that are required for full blown leukaemia to develop. One recent clue from our work is the discovery that RUNX1 and its abnormal counterpart RUNX1-ETO can induce a form of premature ageing (senescence), a natural fail-safe process that must be overcome for cancerous cells to grow out. These observations were made in skin cells and we want to extend our findings to the blood-forming stem cells that are most important in leukaemia

Identification of recurrent patterns of gene expression in relapsed ALLR3 patients

Although approximately 85% children with acute lymphoblastic leukaemia are now cured, the disease will return (relapse) in around 15% of children, with only 30-40% surviving. This makes relapsed ALL the fourth most common childhood cancer. As treatments are now given at maximum doses, with toxicity-related deaths matching the number of deaths from disease, it is important that new drugs are identified. Outcomes for relapsed ALL patients could be greatly improved by the characterisation of their DNA to identify possible new drug targets. This would enable better treatment at diagnosis to include the use of targeted therapies, together with conventional chemotherapy, in those patients considered to be high risk. A new relapsed trial (IntReALL) aims to test several novel drugs for relapsed ALL and will be the largest ever clinical trial for ALL, involving 19 countries and more than 1500 children. The trial associated research project will perform key laboratory studies that include techniques to measure very low levels of disease and also monitor levels of proteins that may predict how well a patient will respond to a drug. In addition, relapsed patient DNA will be analysed to identify possible new targets for drugs. In addition, generate of a bank of primary material from IntReALL patients which will be pivotal in the testing of other novel drugs for this disease. Similar applications are underway in other IntReALL partners, which will result in a global laboratory effort to optimise the treatment for relapsed ALL.

Identifying candidate prognostic mutations in relapsed childhood acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) is the commonest childhood cancer. With modern treatment, over 80% of children are cured of this disease. In the remainder, the disease recurs or fails to respond to treatment. In the latter group of children, additional chemotherapy treatment is adminstered and sensitive bone marrow monitoring tests are used to decide which of these children should receive bone marrow transplantation. Using these sensitive tests, children who despite chemotherapy continue to have leukaemia in the bone marrow, are offered bone marrow transplantation. This study proposes to examine the DNA of leukaemic cells from children in the chemotherapy and bone marrow transplant groups to identify specific gene mutations that enable leukaemic cells to resist chemotherapy. Two methods of such testing will be compared. A conventional technique called DHPLC will be compared against a novel technology called 'DNA tagging for pooled populations'. The latter approach is potentially a more streamlined cost-effective technique for the detection of these gene mutations. In the longer-term, these disease-resistance mutations can potentially be tackled using novel targeted therapy that is rapidly becoming available. The findings from this study will be integrated into the forthcoming protocol for treatment of relapse in childhood ALL.

Identifying methylation markers in good risk acute lymphoblastic leukaemia

Acute lymphocblastic leukaemia (ALL) is the most common type of cancer seen in children. Treatment of ALL has improved dramatically in recent times and survival rates are now well above 80%. However, because it is more common than other cancer types in children, it is still one of the main causes of cancer related morbidity and mortality in children. One way in which current treatments can be improved is by identifying patients most likely not to be cured by standard treatment, who can then be given more intensive treatment which can lead to improved survival. However, it is not currently possible to identify all patients at high risk and in a significant number of patients regarded as low risk the disease can come back (known as relapse). Thus new methods are required to allow identification of these patients.

Identifying novel clinically relevant molecular aberrations in pediatric Down syndrome acute lymphoblastic leukemia using next-generation sequencing methods

Previous studies suggest that the genetic landscape of pediatric Down Syndrome Acute Lymphoblastic Leukemia (DS-ALL) patients is dissimilar from that of pediatric ALL patients without Down syndrome and that other genetic lesions may underlie DS-ALL and/or explain the adverse prognosis associated with DS-ALL. We therefore would like to propose to identify genetic alterations in DS-ALL patients with new sequencing techniques, including DNA and RNA sequencing, in order to identify more or other genetic aberrations in DS-ALL patients which may point to new ways to stratify and more effectively treat these patients. Functional studies will address the contribution of aberrant genes or genomic lesions to the survival capacity of leukemic cells.

Identifying the hierarchical lineage of leukaemia initiation in high-hyperdiploid pre-B acute lymphoblastic leukaemia

Acute leukaemias are the most well studied of all cancers mainly due to the ease of access and the relatively limited number of genetic alterations. Investigating monozygotic twins with concordant leukaemia provides a unique opportunity to analyze the early genetic steps of leukaemogenesis. Previous studies by Greaves and his colleagues revealed that several recurrent chromosomal aberrations, associated with specific clinical subtypes of acute lymphoblastic leukaemia (ALL) are present in both co-twins confirming their early (in utero) occurrence and implicating their role in the pathogenesis. However, the stage of the cell maturation process in which these aberrations emerge is poorly understood. Immunoglobulin (IG) and T-cell receptor (TCR) gene arrangements are hallmarks of B- and T-cell development thus can serve as points of reference at determination of the timing of initiation. Very limited data are available in this field with reported studies each including only twin pair or triplet. Our aim is to reveal the IG and TCR gene rearrangements in a group of monozygotic twin children with concordant high-hyperdiploid pre B-ALL and determine in which phase of the B-cell ontogeny the common pre-natal, pre-leukaemic cell occurred. We predict that twin children with concordant pre-B-ALL share some early but not all clonal IG and TCR rearrangements which allows us to determine the cell of origin precisely.

Identifying the T681I gatekeeper mutation in EBF1-PDGFRB Ph-like acute lymphoblastic leukaemia using droplet digital polymerase chain reaction (ddPCR)

Understanding the underlying mechanisms of resistance in Ph-like ALL is critical to find novel therapeutic strategies in the relapse setting. We first focussed on identifying resistance mechanisms in Ph-like ALL patients harboring the EBF1-PDGFRB rearrangement by using well-validated in-vitro saturation mutagenesis screens to predict the spectrum of drug resistant mutations. Our screens suggest that kinase domain (KD) point mutations may represent the primary mechanism of acquired resistance in EBF1-PDGFRB. The most common KD mutation identified was the T681I mutation, which is analogous to BCR-ABL1's T315I gatekeeper mutation. We further hypothesize that these KD mutations may pre-exist at diagnosis at a frequency that cannot otherwise be detected by Sanger, or even next generation sequencing. The primary objective of this study is to determine the presence of the T681I gatekeeper mutation in patients with EBF1-PDGFRB at diagnosis, end of induction, and at relapse by using a droplet digital PCR designed for rare mutation detection.

Immunophenotypic and genetic characterisation of leukaemic stem cells in childhood acute myeloid leukaemia

Acute Leukaemia (AL) is the commonest childhood cancer in the UK, accounting for 31% of cases and 30% of deaths (~105/year). AL is divided into Acute Lymphoblastic Leukaemia (ALL) (80% of cases) and Acute Myeloid Leukaemia (AML) (20% of cases). The significant improvements in survivorship in childhood ALL (~85%) have not been mirrored in AML. 10 year survival data from the UK AML MRC12 trial (1995-2002) show: Overall survival ~63% and Relapse Rate (RR) 35%. Thus 35% of children will die of their disease. We aim to study the cellular and molecular structure of malignant clones within paediatric AML. This study will have general implications for understanding the clonal and molecular structures of childhood cancer and the similarities and differences between equivalent structures in adult cancers. Specifically for childhood AML, it will be the first study to identify/characterise in detail the molecular, cellular and functional heterogeneity of tumour propagating populations. From a translational perspective, this work is a platform for follow-on studies to: a) identify chemo-resistant populations in refractory/relapsed disease, which would provide powerful biomarkers; b) therapeutically target chemo-resistant populations.

Investigating the role of genetic variation in risk of childhood acute lymphoblastic leukaemia in Down syndrome

Acute lymphoblastic leukaemia (ALL) is the most common cancer in childhood. Down syndrome (DS) is a genetic disorder caused by an extra chromosome 21, and DS children have a 20-fold increased risk of ALL. Furthermore, children with ALL and DS ("DS-ALL") have increased treatment-related deaths and disease recurrence relative to children without DS. Determining the cause of DS-ALL is, therefore, of paramount importance. The additional chromosome 21 must play a role in increased leukaemia risk, but not every child with DS develops ALL. We predict that additional variations may affect DS-ALL risk. This study is designed to discover why some children with DS contract leukaemia while others do not. We will analyse genetic variation in DS-ALL children compared to DS children without ALL, specifically by investigating single base DNA changes as well as 'copy number variation', that is deleted or duplicated sections of DNA. This will be carried out across the genome, with a special focus on chromosome 21. We will also determine whether genetic variations associated with DS-ALL are also associated with ALL risk in children without DS, in a large set of ALL cases and controls. This will be the first comprehensive investigation of the role of genetics in risk of DS-ALL, and will lead to earlier risk stratification and potentially improved treatment of this disease, as well as shedding light on the causes of ALL in all children.

Investigation of the effects of IKZ1 deletions on expression of Gpr132 in Philadelphia positive acute lymphoblastic leukaemia

Sometimes leukaemia cells have deletions of important genes, and this may affect the way the cells respond to chemotherapy. We are interested in the effects of deletions of a gene called Ikaros, which appears to make leukaemias harder to treat. Following several years’ work in our lab, we now believe that loss of Ikaros causes increased levels of a protein called G2A, which causes the leukaemia cell to divide abnormally. We will test whether the gene which makes the G2A protein is more active in leukaemia cells which don’t have Ikaros. If this is true, it will help us to understand how we might improve the treatment for children with these types of leukaemia.

Investigation of the role of JAK2 mutations in the pathogenesis of acute lymphoblastic leukaemia in children with Down syndrome

Recent advances in our understanding of how leukaemia develops have shown that most cases of childhood leukaemia are initiated by chromosome changes before birth, but require additional mutational alterations to progress to full clinical leukaemia. Several acquired genetic mutations are now known to be present in the leukaemic cells of a significant proportion of Down syndrome (DS) children with ALL. The same genetic aberrations are also found in non-DS ALL, although at a lower incidence. Our aim is to determine which of these genetic events occur early and have the potential to initiate or cause progression of the disease. To do this we are using new technologies to look at individual, single cells for the presence of multiple genetic rearrangements. We are also using neonatal blood spots (Guthrie cards) of DS-ALL children to see whether we can ‘backtrack’ specific genetic abnormalities to birth, several years before they develop leukaemia.

Legacy thiopurine study samples

This study will attempt to validate recent results involving prediction of risk of relapse.

Leukaemogenesis by the CALM-AF10 fusion protein: Development of a NOD-SCID mouse model

Leukaemias carrying CALM-AF10 fusions are associated with a poor outcome on current therapy. Ths project has the potential for unravelling the mechanisms by which this rearrangement causes leukaemia. In addition, this project seeks to identify pathways that may be amenable for targeted treatment, as well as create suitable in vivo models where such therapeutic molecules can be tested.

Lymphocyte development in Down syndrome

Down syndrome is the most common genetic condition in the UK affecting 750 newborn babies every year. Children with Down syndrome have an increased risk of developing acute lymphoblastic leukaemia (ALL) which originates from transformation of the progenitor cells which give rise to B lymphocytes. The sequence of B lymphocyte alterations and the exact events which cause it in Down syndrome remain largely unclear. We are therefore investigating this by studying the effects of genes important for normal B-cell development in healthy children and children with Down syndrome.

Molecular and functional characteristics of leukaemia-initiating cells in MLL-AF4 positive infant acute lymphoblastic leukaemia

There is growing evidence that many childhood leukaemias start to develop in fetal life. Immature blood cells in the fetus may develop abnormalities that make them more susceptible to transform into rapidly proliferating leukaemic cells. One such primary hit is a re-arranged gene called MLL (MLLr). Some MLLr leukaemias develop very early (<12 months of age - infant leukaemia), and these children tend to do poorly even with intensive treatment. They differ from MLLr leukaemias that present later in life by being inherently more aggressive and difficult to treat. I would like to investigate why infant MLLr leukaemia is different biologically and whether this is because of unique fetal cells that undergo the 'first hit'. To do this I shall examine the progenitor cells from fetal bone marrow samples and compare their characteristics and function with that of infant and non-infant MLLr leukaemia cells. This will help us identify unique pathways that initiate the development of infant leukaemia and ultimately design novel therapies to treat this refractory leukaemia.

Molecular characterisation of intrachromosomal amplification of chromosome 21 (iAMP21)

iAMP is arguably the most important molecular prognostic marker for patients with B-lineage acute lymphoblastic leukaemia (ALL) identified in recent years. In 2003, we were the first to define a subset of patients with this abnormality. Since then we have been working towards its full characterisation which will ultimately expand our understanding of the genetic mechanisms driving leukaemogenesis and provide accurate methods of detection. We propose further molecular profiling of these patients to identify recurrent genomic regions. Sophisiticated molecular methods will be used to assess the gene content of aberrant genomic regions. The effect of these changes on gene and protein expression will be investigated and will initiate experiments to assess the potential role of these changes in leukaemogenesis. Retrospective analysis of a large patient cohort will allow correlations with prognosis to be determined.

Molecular characterisation of intrachromosomal amplification of chromosome 21 (iAMP21) - further studies

In childhood acute lymphoblastic leukaemia, chromosomal abnormalities are often used to predict prognosis and in risk stratification for treatment. About 10 years ago, we identified an abnormality known as iAMP21 which was associated with a very high risk of relapse on standard treatment. Now, with intensive therapy, the outcome has improved dramatically. In order to consider less toxic personalised therapy for these patients, we need to understand the way in which this abnormality drives leukaemia. We have made significant progress in this area but data are based on historical samples. With samples from a new cohort of patients using state-of-the-art techniques, we are confident that those genes responsible for the development of leukaemia in these patients will be identified to provide more accurate diagnosis to identify those who require intensive treatment.

Molecular characterisation of the deregulation of CRLF2 in childhood B-cell precursor acute lymphoblastic leukaemia

Overexpression of genes is a frequent and important molecular marker in patients with B-lineage acute lymphoblastic leukaemia. We have identified overexpression of a gene in patients where material is exchanged between the sex chromosomes and chromosome 14, bringing this target gene under the control of a potent enhancing element leading to gene dysregulation. We have identified a deletion of sequence to the target gene which also leads to increased expression. We are in the process of characterising these aberrations, which will ultimately expand our understanding of the genetic mechanisms driving leukaemogenesis and provide accurate methods of detection for what may be a poor-risk genetic abnormality.

MRD monitoring of children with Philadelphia positive acute lymphoblastic leukaemia receiving Dasatinib

A rare subset of childhood acute lymphoblastic leukaemia is characterised by the presence of an aberrant chromosomal translocation. This is called the Philadelphia Chromosome and patients with this subtype have a poor outcome and have been usually transplanted. The translocation results in the increased expression of the ABL gene. The advent of drugs such as Imatinib, which suppress ABL has dramatically improved outcome of these patients. In this trial, we propose to investigate the benefit of an even more potent ABL inhibitor called Dasatinib. We propose to monitor the level of disease using molecular tools that can detect 1 malignant cell in 10,000-100,000 cells. We think almost half will have disease levels lower than this and can be cured with chemotherapy i.e without transplantation.

Novel therapies for TP53 mutated paediatric malignancies

The tumour suppressor gene, TP53 is the guardian of the human genome, providing protection from the development of cancer by correcting any faults that occur in the body during cell division.  The TP53 gene is one of the most frequently inactivated/mutated genes in childhood cancer and is associated with a poor prognosis in many paediatric tumour types, such as leukaemia, neuroblastoma and the brain tumour, medulloblastoma.  Current therapeutic approaches are failing to successfully treat this population of children with TP53-mutant tumours and new approaches are now urgently required.

This project will establish a biologically defined cohort of TP53 mutant tumours across a spectrum of childhood malignancies (acute lymphoid and myeloid leukaemia, lymphoma, neuroblastoma and medulloblastoma). This unique cohort will be utilised firstly to describe the nature and spectrum of TP53 defects and secondly, collaborative matched datasets, investigating chromosomal changes, whole genome aberrations and gene expression levels, will be interrogated to identify any common biology amongst these TP53-mutant tumours.  Importantly these investigations will advance our knowledge and have the potential to identify future therapeutic targets which will ultimately improve outcomes in this difficult to treat group of tumours.

Optimisation of dexamethasone therapy in childhood acute lymphoblastic leukaemia

This study is investigating whether small changes in DNA may alter the way that children with leukaemia process the medicine dexamethasone. We would like to see whether these changes in DNA may mean some children get a higher exposure to dexamethasone, and whether this, in turn, means they experience more side effects.

Pilot project to study the expression of the meiotic regulator PRDM9 by childhood leukaemias

Abnormal numbers and rearrangements of chomosomes are a typical feature of childhood leukaemia and contribute to disease causation; understanding their causes could aid disease treatment and prevention. PRDM9 is a protein important for correct chromosome exchanges during gamete production. Rare forms of PRDM9 are more common among children with leukaemia and there is evidence that it is expressed in non-gamete cells. It is unknown whether PRDM9 is expressed in leukaemia cells, or if it contributes to chromosomal abnormalities causing leukaemia. We will determine which forms of PRDM9 are expressed in leukaemias and whether expression is absent from normal blood cells.

Pilot study to assess the feasibility of utilising samples from CellBank for whole genome sequencing as part of the 100,000 genome project

Genomics England (GEL) are responsible for delivering the 100,000 genome project on behalf of the Department of Health. This is an NHS transformation programme aimed at developing whole genome sequencing (WGS) capacity for clinical diagnostics. GEL Clinical Interpretation Partnerships (GECIPs) have been set up in order to coordinate sequencing activity with different disease subgroups. The Haematology Oncology GECIP domain covers all activity within this field and includes a childhood leukaemia sub-domain which is led by Professor Moorman. The Haematology Oncology GECIP is proposing to undertake WGS on approximately 4000 patients with the overall goal of deriving new prognostic and predictive biomarkers and ultimately driving the development of new therapies.

Sensitivity of T-acute lymphoblastic leukaemia cells to JAK kinase inhibitors

T-cell acute lymphoblastic leukaemia is an aggressive leukaemia that is most common in children and adolescents. Research over the past 30 years has identified many different oncogenes that are implicated in the development of these leukaemias. It was recently shown that JAK1, JAK3 and IL7R genes are frequently mutated in T-ALL, and preliminary data from studies in cell lines and mouse models has suggested that JAK kinase inhibitors could be used to inhibit these oncoproteins. In this project we will determine whether leukaemia cells with HAK1, JAK3 or IL7R gene mutations are indeed sensitive to JAK kinase inhibitors and which of these JAK kinase inhibitors show the best activity. Based on these data, future clinical trials with JAK kinase inhibitors could be initiated.

Sequential acquisition and clonal architecture of genetic aberrations in paediatric T-acute lymphoblastic leukaemia

This project concentrates on identifying how genetic changes already known to be important in a particular type of childhood leukaemia – T-cell acute lymphoblastic leukaemia (T-ALL) - evolve and in what order. Earlier work from our group has shown that genetic evolution of cancer cells in other types of ALL (B-cell type rather than T-cell type) is non-linear and adopts a branching tree or ‘Darwinian’ evolutionary structure. Understanding of ‘cancer initiating’ events in this disease would allow the development of therapies specific to these early events which are likely to be more successful than therapies targeting events further down the evolving leukaemic branching tree. Our project is summarised in three phases. In phase 1 we will apply techniques we have already used in other subtypes of leukaemia to confirm the ‘branching tree’ model also applies in T cell ALL. We will aim to support our hypothesis that all leukaemic cells within each sample share a large genetic rearrangement within (intra) or between (inter) the chromosomes. These rearrangements are our hypothesised initiating event and are hypothesised to occur in all cells as they happen prior to the ‘branching’ of the tree. In phase 2 we will address a similar question for other genetic changes – subtle changes to the sequence of DNA, the so called ‘genetic code’ – to determine in what order these occur and what their relative importance is as a drug target. In phase 3 we will consider how to apply our new knowledge to the practical screening of diagnostic patient samples and extend it to exploring the ongoing evolution of the leukaemia cell branching tree in relapsed patients and what genetic alterations are important at this stage in the disease. If we have identified a key drug target we will liase within our organisation with the drug development team.

Studies to determine whether molecular markers predict response to therapy and can be used to stratify treatment

The majority of children with acute lymphoblastic leukaemia (ALL) can now be cured of their disease but certain subgroups of patients still have a relatively poor prognosis. One such subgroup is those patients with T-ALL, particularly those that have persistence of detectable disease (Minimal Residual Disease or MRD) at the end of induction therapy. In the last few years mutations have been identified in specific genes of some patients with T-cell ALL that may be associated with response to treatment and long-term outcome. We aim to screen samples from children with T-ALL who have been entered into the current national ALL trial for mutations in 3 genes called NOTCH1, FBXW7 and PTEN that are all involved in the production of T-cells. Presence of mutations will be related to early response to treatment and long-term outcome. We will also ascertain the outcome of patients with mutations in the NOTCH pathway who remain MRD-positive after induction therapy to try and refine the indications for intensification of treatment and thus reduce the number of children subjected to a bone marrow transplant procedure.

The CXCL8-producing T-cell: function in health and carcinogenesis

We have identified a novel type of T-cell - one of the white blood cells that help protect the body from infection - in human neonates. These cells are rarely found in adults but, if present, have markers suggesting they are naïve, newly produced cells from the thymus (the organ from which T-cells emerge). These cells are novel as they make a mediator called CXCL8. T-cell acute lymphoblastic leukaemia cells (T-ALL) arise from transformation of cells as they develop in the thymus. We have looked at a few T-ALL to date and these also seem to make a lot of CXCL8. Previously, it has been shown that T-ALL that make CXC8 seem to have a poor response to therapy. We want to test different T-ALL samples to see if those T-ALL that make CXCL8 have a particular profile and whether by blocking CXCL8 this may have an effect on the ability of the T-ALL to continue to grow.

The prognostic significance of genetic abnormalities in T-cell acute lymphoblastic leukaemia

In childhood B-lineage ALL, chromosomal abnormalities are linked to prognosis and are used in risk stratification for treatment. Novel high resolution techniques have uncovered new genetic abnormalities in T-ALL, particularly those involved in T-cell development, for which the prognostic significance is unclear. We propose to use two recently established techniques of MLPA and targeted next generation sequencing on the cohorts of childhood T-ALL from ALL2003 and ALL97 to conclusively determine whether these new abnormalities are associated with treatment response and evaluate their role in risk stratification of future treatment trials.

The prognostic significance of IZF1, other B-cell differentiation gene abnormalities and related genetic changes in childhood B cell precursor acute lymphoblastic leukaemia (BCP-ALL)

 

Chromosomal abnormalities are linked to prognosis and are used in risk stratification for treatment of childhood ALL. Novel high resolution techniques have uncovered new genetic abnormalities particularly those involved in B-cell development, for which the prognostic significance is unclear. We propose to use two recently established techniques of MLPA and customised genomic array analysis on the cohorts of childhood ALL from ALL2003 and ALL97to conclusively determine whether these new abnormalities are associated with treatment response and evaluate their role in risk stratification of future treatment trials.

The role of CD33 and anti-CD33 therapy in childhood acute myeloid leukaemia

Relapse is the commonest cause of death in childhood acute myeloid leukaemia (AML). Multiple courses of chemotherapy is the mainstay of treatment but a ceiling of benefit has been reached and toxicity is significant. Relapse is due to the persistence of cells that grow slowly and can evade death by conventional chemotherapy, called leukaemic stem cells. Novel treatments that can target these cells are required. The aim of our project is to increase understanding of a protein called CD33 and novel agents that target CD33. This protein is present on the surface of AML cells and the leukaemia stem cells. We aim to identify new targets that will work in combination with the current anti-CD33 treatments available to create novel combination therapies.

The role of miRNAs in developing haematopoetic cells and infant leukaemia

Cancer development is in many cases caused by a dysregulation of gene expression due to the aberrant action of transcription factors, which are proteins responsible for the correct expression of genes. Recently, a further level of gene regulation has been described following the discovery of small RNA molecules known as microRNAs (miRNAs) which act as repressors of gene expression. Subsequent work has demonstrated that miRNAs are also key players in cancer development and progression, including cancers of the blood system. A number of leukaemias occurring in infants are known to have a pre-natal origin; however their precise cellular source and mechanism of emergence remain undefined. The aim of this project is to establish the repertoire of miRNAs expressed in cells of the developing blood system and in paediatric leukaemia samples. The function of miRNAs will then be further studied to uncover their role in normal blood development and how these processes become dysregulated in cancer development. The results obtained from these studies are likely to shed light on leukaemia pathogenesis and may highlight novel strategies for therapeutic interventions.

The use of phenotypic high content screening for the predictin of clinical response to chemotherapy in paediatric acute myeloid leukaemia (AML)

While 80% of all children's cancers are curable, Acute Myeloid Leukaemia is one of the exceptions with only 60% of children responding to standard chemotherapy for this disease. The challenge is to provide help to the doctors to treat the 40% of children who progress to second line therapy. With so many anti-cancer drugs now available, it would be highly desirable if there was a way to determine which ones in advance might be most effective for a particular child. Imagen Therapeutics has a test which may be able to address this challenge. However, the first step to exploring whether our test will work is to see if it can predict, in a set of banked AML samples, the clinical outcome of the children concerned. If it can, then this would be strong evidence that the assay can be used to guide future second and third line therapy in paediatric AML.

Tracking the juvenile myelomonocytic leukaemia stem cell

This research project will study a rare and fatal form of childhood blood cancer that is called Juvenile Myelomonocytic Leukaemia (JMML). This type of blood cancer usually presents within the first 2 years of life and it carries very poor prognosis. To date, the only available treatment is transplantation of haematopoietic cells from a healthy donor to the patient (stem cell transplant). Unfortunately, JMML is very difficult to treat and even after treatment, the disease often returns. The aim of this project is to identify the exact types of cells where the disease originates and study their characteristics in great detail. We believe that these cells are also most likely to be responsible for the high rate of disease relapse and the disappointing response of JMML to conventional leukaemia therapy. We hope that understanding the pathways which have led to the abnormal growth and development of the disease will help to develop much better treatment targeted to the abnormal cells responsible for generating and propagating JMML. Furthermore, with this project we aim to use the latest technologies available in order to detect all the genetic abnormalities that can lead to the disease development; and understand how the different genetic alterations affect the natural course of the disease. This will enable doctors in the future to identify more effectively the high risk patients and apply the most appropriate treatments for each patient, with the overall aim to improve the outcome of the children with this disease. Lastly, our detailed genetic analysis will enable us to identify patients that carry inherited mutations and provide the families of these children with the appropriate follow up for the family members that are likely to be affected.

Transcriptional regulation of LMO2 in T-acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia is a cancer of the white blood cells and is the commonest cancer to affect children. Although cure rates for some subtypes of leukaemia have improved rapidly over recent decades, these advances are not shared by all sub-types. T-acute lymphoblastic leukaemia is one such subtype and has been shown to be associated with mis-regulation of genes associated with the control of transcription, the first step on the way to producing active proteins. LMO2 is misregulated in over 60% of patients with T-acute lymphoblastic leukaemia and the cause for this is not currently clear. By using state of the art DNA chip technology, only available since the completion of the genome project, I have identified sequences of DNA that are mis-regulated in T-acute lymphoblastic leukaemia cell lines and, if they are also active in patient samples, we will have gained greater understanding of how this gene is inappropriately switched on in this disease, which may suggest ways to identify possible new and more targeted treatments.

Whole genome sequencing of genetically defined subtypes of childhood acute lymphoblastic leukaemia

The cancer cells of patients with leukaemia carry genetic abnormalities which are the drivers of the disease. Some genetic abnormalities are associated with an excellent response to standard treatment whilst others indicate that the patient will ultimately relapse. This project focusses on a rare subtype of childhood leukaemia which is associated with an extremely poor outcome. We will use state of the art technology to sequence (“read”) the entire genome (“genetic code”) of patients with this poor risk abnormality and compare it to the genome of patients with a related abnormality that has a good prognosis. The results of this study will help us to understand why different patients response differently to the same treatment. It will also help in the development of novel therapies for patients with a poor prognosis.