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Thesis (M Med(Haematology))-- University of Limpopo (Medunsa Campus), 2008. / Introduction: ALL (Acute Lymphoblastic Leukaemia) is the most common leukaemia in childhood. The two most important features predictive of outcome are age and presenting WBC at diagnosis. NCI risk criteria are applied to all children with precursor B-ALL, dividing them into NCI “high risk” (age < 1 year and ≥ 10 yrs, WBC > 50 x 10 9/ ) and NCI “standard risk” (age ≥ 1 year and < 10 yrs, WBC < 50 x 10 9/ ). Gender, immunophenotyping and genetic studies are other features that have been shown to be associated with outcome. Objectives: To determine the correlation between survival outcome of paediatric patients with ALL and different variables, e.g. biological, haematological, immunophenotypic and cytogenetic features at diagnosis, and to determine the duration of survival of a patient since the diagnosis of ALL, at Dr George Mukhari Hospital. Methods: This study was conducted over the period 2003-2007. Children diagnosed with ALL with ages ranging from 1-12 years, were identified. The hospital and laboratory records were analysed retrospectively. Early prognostic features were identified from patient data. Results: Descriptive statistical measures were used to summarize data. Twenty nine paediatric patients with ALL were identified of which 12 were female and 17 were male. The mean age of patients at diagnosis was 7,2 years. The presenting leucocyte count ranged from 2,5 to 325 x 10 9/ . Cytogenetic studies of three patients were available, all of which were unfavourable prognostic factors. Immunophenotyping revealed ten patients with T-cell ALL, 17 patients with B-cell ALL and two patients whose immunophenotype was unknown as recorded results were not available. Twenty one patients’ survival data were known. The longest duration of survival of a patient was 3,7 years. There were seven patients known to be alive at the end of the study period. Conclusion: The cases reported herein and those described in the literature demonstrate the importance of a careful and multidisciplinary approach in the diagnosis and evaluation of paediatric ALL.

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M.Med.(Haematology), Faculty of Health Sciences, University of the Witwatersrand, 2008 / This study aimed to identify the relevance of these prognostic features in the modern treatment era in South African children. A retrospective analysis of the presentation clinical and laboratory features and treatment outcomes of all children treated for Precursor B cell ALL at the Johannesburg Hospital was performed. Between January 1997 and May 2007, 100 children were reviewed. Clinical features (age, race and gender) emerged as significant prognostic variables. Laboratory features (white cell count and genetic features) lacked significance. Early morphologic response on day 15 identified a subgroup associated with a favourable outcome. However the presence of > 5% blasts was not significantly predictive of relapse or death at this time point. Minimal residual disease (MRD) detection by modified immunoglobulin gene rearrangement and flow cytometry techniques did not improve the predictive value of the morphological assessment. In a low resource setting, the challenge is to design cost effective MRD detection methods to improve the identification of patients at risk for relapse.

Children treated for acute lymphoblastic leukemia (ALL) are immunodeficient and therefore at an increased risk of infection and cancer recurrence. Natural killer (NK) cells are a subset of lymphocytes that are very efficient at combatting infections and cancer; however, children treated for ALL have impaired NK cell number and function. Exercise has the potential to bolster NK cell number and function, at least in healthy children and adults. Limited evidence suggests exercise may also have beneficial effects on NK cells in children treated for cancer. However, these previous exercise immunology studies in children with cancer have yielded low sample sizes. Therefore, the aim of this study was to assess the: 1a) feasibility, 1b) acceptability and 1c) safety of performing an exercise intervention in children with ALL. The secondary objectives were to assess the 2a) effects of acute exercise on NK cell number, function and receptor expression in children receiving maintenance therapy for ALL compared to healthy children, as well as to 2b) assess how the NK response changes over 4 months of therapy, and to 2c) assess the link between physical activity and NK cell number and function at rest in children receiving maintenance therapy for ALL. Children undergoing maintenance therapy for ALL (n=4) were recruited from McMaster Children’s Hospital, and healthy sex and pubertal-status matched children (n=4) were recruited from the Hamilton community. ALL patients completed a total of 3 exercise visits, occurring monthly after their regularly scheduled chemotherapy session. At each exercise visit, children were asked to complete 30 minutes of continuous biking, followed by 1 hour of rest. Blood samples were drawn at rest prior to exercise (PRE), immediately after exercise (POST) and 1 hour into recovery (REC). Healthy children only completed one exercise visit. During recovery, participants were asked to complete a physical activity enjoyment scale (PACES) questionnaire and a structured interview in order to assess exercise acceptability and to gauge participant feedback on study components, respectively. Participants were outfitted with an accelerometer to track physical activity levels between visits. Feasibility was assessed by tracking recruitment statistics, study completion rates and exercise completion rates. Acceptability of accelerometer wear was assessed by tracking accelerometer wear and log rates. Safety was assessed by tracking adverse events. All parameters were reported using descriptive statistics. We approached 22 patients to participate, and 4 children completed the study (100% completion rate) out of a goal of 15. Primary deterrents to participation were that patients and families did not want to extend time spent at the hospital or had time restrictions and that patients were uncomfortable with blood collection methods. Exercise was feasible (94% exercise completion rate), acceptable (4.2 ± 0.38 out of 5 PACES score), and safe. Accelerometer wear rates (61.9% (range 3.7-100.0%)) and log completion rates (69.0% (25.9-100.0)) were moderate. Exercise transiently increased NK cell number and function in healthy children and some children with ALL. There were no patterns in the change of the NK cell response to acute exercise over time. We were unable to assess the link between physical activity and NK cells due to a paucity of data. This study cautiously suggests that exercise is a feasible, acceptable and safe intervention that may increase NK cell number and function in children treated for ALL. / Thesis / Master of Science in Medical Sciences (MSMS) / Children treated for leukemia have weak immune systems, making them more susceptible to developing infections and cancer recurrence. Natural killer cells are a special immune cell that is very effective at combatting cancer and infections; however, children treated for leukemia have very low amounts of natural killer cells and they do not function well. Exercise is a simple way to boost the immune system in healthy adults and children, by increasing the number and function of natural killer cells. We don’t know what effect exercise has on natural killer cells in children with leukemia. Previous studies looking at the effects of exercise on the immune system of children with cancer have not been able to recruit enough children to participate. Therefore, it is also important to investigate why children with cancer may not want to participate in exercise studies looking at immune function. The main goals of this thesis were to assess how likely we are to recruit enough children being treated for leukemia to participate in a study looking at how exercise changes natural killer cells, if our participants enjoyed being part of this study, and how safe exercise is for children being treated for leukemia. We also wanted to learn about how natural killer cells respond to exercise in children being treated for leukemia. We found that most of the children and families that decided not to participate in our study felt they did not have time, and the second most common reason for not participating was because the children experienced anxiety surrounding blood draws for the study. The children that decided to participate in the study enjoyed the exercise and being in the study. We also found that the exercise was safe. Finally, we saw that exercise was able to increase natural killer cell numbers and function in some, but not all, children treated for leukemia. The results of this study suggest that exercise may be a realistic and safe way to improve immune function in some children with leukemia.

The p53 protein is a primary mediator of apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has categorised a wild type p53 gene in the majority of childhood acute lymphoblastic leukaemia (ALL) cases, in which instance the p53 protein functions as a modulator of chemotherapy-induced cell death. In contrast, certain p53-induced proteins, such as p21WAF1, can act in an anti-apoptotic manner, and bestow resistance to chemotherapy. Previous studies of the p53 pathway in ALL have utilised cell lines and primary material. In this study a model of ALL was utilised that had previously been developed from a heterogeneous panel of patient biopsies established as xenografts in immune-deficient mice, and are adaptable for short term in vitro culture. A wild-type p53 protein response to etoposide and nutlin-3 exposure was a feature of the whole ALL xenograft panel, irrespective of clinical characteristics and disease biology. While a range of p53 target genes were induced in B-cell precursor (BCP)-ALL and T-ALL xenografts after etoposide exposure, there was negligible induction of p21WAF1 in T- ALL samples. Further work with the histone deacetylase inhibitor vorinostat facilitated p53-independent induction of p21WAF1 in BCP-ALL samples, yet failed to induce p21WAF1 in T- ALL. An association was observed between reduced p21WAF1 expression in the T-ALL samples and decreased histone H3 acetylation in the p21WAF1 promoter together with increased cytosine methylation in the first exon/intron of the p21WAF1 gene. These results suggest that p21WAF1 in T-ALL cells is subject to epigenetic modifications that cause transcriptional silencing. Defective induction of p21WAF1 in T-ALL xenografts was associated with increased sensitivity to the death-inducing effects of drugs, phosphatidylserine (PS) externalisation and caspase-3/-7 activity after drug exposure, indicating that p21WAF1 may exert an anti-apoptotic activity. As proof of principle, p21WAF1 was silenced in Nalm-6 cells by micro-RNA transduction and these cells exhibited increased sensitivity and rapid PS externalisation after drug exposure. A combination of a p21WAF1 inhibitory agent and vorinostat gave some pharmacological evidence to suggest that p21WAF1 inhibition could enhance drug efficacy. Overall, these investigations provide insight into the epigenetic regulation of p21WAF1 and demonstrate an anti-apoptotic role for p21WAF1 in childhood ALL cells.

Interest in cellular immunotherapy has increased with the recognition of the pivotal role that dendritic cells (DC) play in the adaptive immune system. The preparation of DC to present tumour antigens and subsequent induction of tumour specific T cells have been widely documented. This thesis studied the ability of cord blood (CB) stem cells to differentiate into functional CD34+DC, followed by the optimisation of electroporation of RNA into these cells. Total RNA derived from a leukaemic cell line and a primary human leukaemic sample was electroporated into CD34+DC DC and we were able to generate anti-leukaemic cytotoxic T lymphocytes (CTL). The CTL specifically targeted leukaemia but not normal cells. While the in vitro data showed promising results of the CTL specificity, a NOD-SCID model of human ALL was established to allow the CTL to be tested in vivo. We established a reproducible model of human ALL in NOD-SCID mouse using four primary human ALL samples. The adoptively transferred anti-leukaemic CTL into the ALL bearing NOD-SCID mice showed that ALL engraftment was significantly delayed. However, the addition of total RNA loaded CD34+DC DC did not enhance the in vivo CTL effect. Lastly, by dissecting the CTL response, we found that the polyclonal CTL were targeting survivin, HM1.24 and CT-7 antigens. The CTL clones generated from these polyclonal CTL showed high specificity for leukaemia but not normal cells. In conclusion, these preliminary data support the use of total RNA electroporated CD34+DC as a means of inducing anti-leukaemic CTL, and have demonstrated the efficacy of the CTL in a NOD-SCID model of ALL. This study has also provided insight into the polyclonal CTL response and future studies will likely continue along this path.