The EBMT/EHA CAR-T Cell Handbook

This first open access European CAR-T Handbook, co-promoted by the European Society for Blood and Marrow Transplantation (EBMT) and the European Hematology Association (EHA), covers several aspects of CAR-T cell treatments, including the underlying biology, indications, management of side-effects, access and manufacturing issues. This book, written by leading experts in the field to enhance readers’ knowledge and practice skills, provides an unparalleled overview of the CAR-T cell technology and its application in clinical care, to enhance readers’ knowledge and practice skills

The EBMT/EHA CAR-T Cell Handbook

This first open access European CAR-T Handbook, co-promoted by the European Society for Blood and Marrow Transplantation (EBMT) and the European Hematology Association (EHA), covers several aspects of CAR-T cell treatments, including the underlying biology, indications, management of side-effects, access and manufacturing issues. This book, written by leading experts in the field to enhance readers’ knowledge and practice skills, provides an unparalleled overview of the CAR-T cell technology and its application in clinical care, to enhance readers’ knowledge and practice skills

The EBMT Handbook

This Open Access edition of the European Society for Blood and Marrow Transplantation (EBMT) handbook addresses the latest developments and innovations in hematopoietic stem cell transplantation and cellular therapy. Consisting of 93 chapters, it has been written by 175 leading experts in the field. Discussing all types of stem cell and bone marrow transplantation, including haplo-identical stem cell and cord blood transplantation, it also covers the indications for transplantation, the management of early and late complications as well as the new and rapidly evolving field of cellular therapies. This book provides an unparalleled description of current practices to enhance readers’ knowledge and practice skills

Vincristine-Induced Peripheral Neuropathy: Assessing Preventable Strategies in Paediatric Acute Lymphoblastic Leukaemia

Background: Acute Lymphoblastic Leukaemia is the most common cancer experienced by children with overall survival rates now exceeding 90%. However, most children will experience vincristine-induced peripheral neuropathy (VIPN) during treatment resulting in sensory-motor abnormalities. To date, there are no approved preventative therapeutics or mitigation strategies for VIPN. This body of work set out to: (1) establish a high-throughput and high-content assay with the capacity to identify neuroprotective compounds, (2) test the feasibility of repurposing olesoxime as a neuroprotectant, and (3) compare traditional statistical methods with machine learning models to identify patients at risk of VIPN. Methods: (1) In vitro neuronal cultures were exposed to vincristine to recapitulate the VIPN phenotype and olesoxime assessed as a positive control. The neurotoxicity assay was miniaturised in 384-well microplates with automation steps to reduce manual handling. (2) Olesoxime and vincristine were applied to proliferating malignant cell lines to ensure the efficacy of vincristine was maintained. (3) Machine learning algorithms were developed using data from a local retrospective cohort to predict VIPN. Results: (1) Neurite length was reduced in a dose-responsive manner with vincristine. Assay miniaturisation and automation steps helped facilitate a high-throughput workflow. An optimised multiplexed dye solution enabled image acquisition and neurite quantification. Further, olesoxime was found to protect neurites and deemed suitable as a positive control (2) Cell viability assays confirmed olesoxime did not interfere with vincristine efficacy in leukemia cells. (3) Machine learning algorithms showed equivalency to traditional univariate analysis. The observation of severe class imbalance meant that patients who were least susceptible to VIPN could be identified. Conclusions: This body of work demonstrates the successful development of a neurotoxicity assay suitable for neuroprotectant drug discovery. Olesoxime was found suitable as a positive control in the assay. Further, viability studies indicated that vincristine retains it efficacy with olesoxime, opening the possibility of its use as an adjunctive therapy. Finally, this work developed machine learning models with the capacity to identify patients with VIPN-free survival. The utility of this model may mean that it can be used to stratify patients prospectively in the clinic based on favourable clinical features

Genetic and functional characterisation of Immunoglobulin Heavy Chain Locus-CCAAT enhancer binding protein B-Cell acute lymphoblastic leukaemia

PhDB-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is a heterogeneous disease in which patient outcome is influenced by genetic lesions. This outcome has improved due to increasingly tailored treatment regimens, selected through risk stratification by use of cytogenetic, copy number alterations (CNAs) classifiers, genomic, and molecular data. Translocations involving the Immunoglobulin Heavy Chain Locus (IGH) comprise 5% of BCP-ALL and lead to overexpression of juxtaposed genes, due to the powerful IGH enhancer elements. Multiple IGH partner genes have been described in BCP-ALL, including five members of the Ccaat Enhancer-Binding Protein (CEBP) transcription factor family. A cohort of 33 IGH-CEBP BCP-ALL patients was identified including 11 IGH-CEBPD, 10 IGH-CEBPA, 8 IGH-CEBPB, 3 IGH-CEBPE and 1 IGH-CEBPG patients, comprising 19% of the IGH cohort, and 1% of ALL as a whole. The patients displayed variation between individual CEBP subgroups, with IGH-CEBPB patients showing higher white blood cell counts (WBC), higher relapse rates, higher number of CNAs and older age than other CEBP patients. The CEBPD subgroup included mostly younger patients, under the age of 10 years, and had the lowest number of CNAs per patient. Deletions of CDKN2A/B were the most commonly occurring CNA followed by intragenic exon 4-7 deletions of IKZF1, which were found exclusively in the IGH-CEBPB and IGH-CEBPD subgroups (p=0.04). A novel intragenic deletion of the tyrosine kinase gene, ABL2, was found in four patients in the cohort, which may represent a deletion unique to this subgroup. This finding in combination with the IKZF1 deletions is suggestive of a BCR-ABL1-like profile. Retroviral expression of the CEBPD gene in CD34+ cells was found to hinder proliferation in transduced cells, potentially through cell cycle arrest via the RB/E2F pathway. RNA sequencing analysis of two IGH-CEBP patients showed very different expression profiles, suggesting two mechanisms of oncogenesis in IGH-CEBP patients: one through inactivation of the CEBP function, leading to deregulation of cell cycle and differentiation control, and another through upregulation of inflammatory factors

The identification of tumour antigens recognized by patients with Duke’s B (Stage II) reactive colorectal cancers using SEREX

A thesis submitted to the Faculty of Creative Arts, Technologies & Science, University of Bedfordshire, in fulfilment of the requirements for the degree of Master of Philosophy.Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in both men and women, posing a serious demographic and economic burden worldwide. In the UK, CRC affects one in every twenty people and it is often detected once well-established and after it has spread beyond the bowel (Stage IIA-C and Stage IIIA-C). A diagnosis at such advanced stages is associated with poor treatment response and survival. However, studies have identified two sub-groups of post-treatment CRC patients – those with good outcome (reactive disease) and those with poor outcome (non-reactive disease). Evidence indicates the presence of an effective immune response differentiates between those patients who respond well to treatment and those who do not. To investigate these underlying mechanisms we used the serological analysis of cDNA recombinant libraries (SEREX) technique to determine which antigens are recognised by patients in each group. Immunoscreening a healthy donor testes cDNA library with sera from three patients with Duke’s B reactive disease led to the identification of five antigens. These were (1) the immunoglobulin heavy constant gamma 3 (G3m marker), IGHG3 gene, located on chromosome 14 at 14q32.33, which encodes IgG3, and was recognised by sera from patients CC005 and CC014; (2) the immunoglobulin heavy constant gamma 2, IGHG2 gene, located on chromosome 14 at 14q32.33 and recognised by CC014 sera; while (3) CYB5R3, (4) RPL37A and (5) SLC34A2 were recognised by CC005, CC014 and CC014 sera respectively. CYB5R3 is a NADH-cytochrome b5 reductase 3 protein which has been shown to be upregulated in lung tissue with a RAS mutation in mice. Ribosomal protein L37a (RPL37A) has previously been shown to be upregulated in astrocytomas and to have a general association with lifetime glioblastoma survival and overall glioblastoma survival. Solute carrier family 34 member 2 (SLC34A2) encodes a protein which acts as a pH-sensitive sodium-dependent phosphate transporter. SLC34A2 has been shown to be upregulated in breast and ovarian cancers and it is suggested that SLC34A2 is involved in the process of carcinogenesis, making it an attractive target in therapeutic strategies and also as a diagnostic biomarker. Although other antigens were found, and their sequences identified, all were unknown and not found in the databases. RT-PCR analysis of the Duke’s B colon cancer cell line SW480 showed consistent expression of BCP-20. Although, expression of SSX2, NY-ESO-1, TSP50, HAGE and RAGE were detected, the data was not easily reproductible. Further optimisation of the PCR conditions and primer pairs would be necessary to confirm these findings. We hope in the future we can disern the role of these antigens in the inflammatory immune responses associated with reactive Dukes’ B colon cancer which would help us better understand the mechanisms which underlie effective anti-tumour responses post-surgery. It may also be that RPL37A is a biomarker for patient survival in colorectal cancer and this would be worthy of further investigation

Helical tomotherapy for total marrow and total skin irradiation : Optimisation, verification, and clinical results

In modern cancer therapy, radiotherapy (RT) is a vital part of most treatments. Most RT treatments in Sweden are performed using intensity-modulated radiotherapy (IMRT) with fixed or dynamic arc delivery. The dose gradients outside the target are steep, the margins are small, and the treatment delivery is complicated. Complicated treatments increase the requirement for control of the uncertainties in planning and delivery. This requires robust treatments and mitigation of the various uncertainties. In addition, robust planning and rigorous quality assurance (QA) of the patient treatments that takes account of all types of uncertainties are essential. A TomoTherapy device (Accuray Inc., Madison, WI, USA) is an RT device with a linear accelerator mounted on a slip-ring construction, giving it the ability to irradiate while continuously rotating around the patient, very similar to a CT scanner but with megavoltage instead of X-ray energy generation. Since helical tomotherapy can entail long and complicated irradiations, new treatment types targeting RT to large parts of the body have emerged. These new techniques are challenging regarding the optimization and verification of the planning and treatment process. Recurring blood cancers (leukaemias) can be treated with RT and chemotherapy before stem cell transplantation. Traditionally, the patient is irradiated with whole-body irradiation using a conventional linac at a large distance. The treatment can be performed with helical tomotherapy to avoid organs at risks to a greater extent. Another emerging treatment with helical tomotherapy is irradiation of whole-body neoplastic skin lesions, such as mycosis fungoides. These patients were traditionally treated with electron irradiation in different positions, subsequently complemented with x-ray fields in ‘hard-to-reach’ sites. Delivering this treatment with photons is complex but opens the possibility for integrated boost treatments of lesions and simultaneously avoiding OAR and previously treated areas.The clinical follow-up illustrated the potential and usefulness of helical tomotherapy targeting the bone marrow, with more patients surviving without severe complications after one year, than with the previous technique. Further, we showed the possibility to implement irradiation of the entire skin with helical tomotherapy. Overall, we demonstrated the usefulness of helical tomotherapy, and solutions to overcome challenges in implementing large-target techniques

Diversity of cancer stem cells in acute lymphoblastic leukaemia

Phd ThesisFor many cancers research led to controversial results regarding frequency and identity of cancer stem cells, cells that self-renew and are able to reconstitute the full phenotype of the original malignancy. In some malignancies such as acute myeloid leukaemia the hierarchical stem cell model that suggests that only rare and immunophenotypically immature blasts exhibit stem cell characteristics, resembling the normal physiological haematopoietic hierarchy, has been well established. In contrast to that, the stochastic model states that all or at least a substantial proportion of malignant cells has stem cell potential whereby this is supported by extrinsic stimuli. Initially, several studies suggested that acute lymphoblastic leukaemia (ALL) also is organised in a hierarchy. However, using more immunocompromised mouse strains and refined transplantation techniques for in vivo xenotransplantation models, previous findings regarding frequency and restriction of stem cells to very early B-precursor cell stages have been challenged in more recent studies. In order to address the questions of identity and frequency of stem cells in ALL, a robust orthotopic mouse model with the most immunocompromised mouse strain currently available (NOD/scid IL2Rγnull; NSG) was established. Primary diagnostic patient ALLs or blasts harvested from engrafted mouse bone marrow were sorted for B-cell lineage differentiation markers CD10, CD19, CD20 or CD34 to purify candidate stem cell populations of different maturity. All transplanted cell populations, from the most immature CD34+CD19low stage to the already more differentiated stage of CD19+CD20high cells were able to reconstitute the original ALL in mouse bone marrow and followed a typical dissemination pattern with infiltration of the spleen. Furthermore, this more mature CD19+CD20high subpopulation proved self-renewal ability in serial transplantation experiments. To investigate, whether stem cells in ALL are a rare entity or more abundant, unsorted bulk leukaemia blasts were transplanted in limiting dilutions from 1 x 104 to 10 cells per mouse. Cell numbers required for engraftment varied between leukaemias but the leukaemia engrafted in mice when only 10 to 1,000 cells were transplanted. The limiting dilution experiments were repeated with sorted blast populations according to the surface antigens CD10, CD20 and CD34. Stem cell frequencies in all sorted populations were comparable and as few as 10 to 100 cells were sufficient to reconstitute the leukaemia in mice. Stem cells do not seem to be restricted to immature blast populations as in the hierarchical model but a broad spectrum of different blast immunophenotypes display stem cell capabilities. Furthermore, the frequency of stemness among unsorted bulk ALL cells as well as subpopulations of different maturity according to the blast immunophenotype is high and similar. The results from this thesis provide strong evidence for the stochastic cancer stem cell model in B precursor ALL.NHS: The North of England Children’s Cancer Research group (NECCR): Leukaemia and Lymphoma Research: The JGW Paterson foundation