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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.

Understanding the nature and timing of leukaemogenic events during the development of childhood acute lymphoblastic leukaemia (ALL) will enable intervention that could prevent ALL in the future. We hypothesised that 9p21 deletion in childhood ALL may unmask predisposing genetic events that would allow us to determine the "nature" of initiating events in childhood ALL; whereas the inclusion, or exclusion, of random �N� nucleotides in normal immunoglobulin gene rearrangements from the developing fetus and the expression of terminal deoxynucleotidyl transferase (TdT) in fetal lymphocytes may allow us to unmask the developmental window during which the first transforming leukaemic event occurs in a pre-leukaemic B cell. The most frequent genetic abnormality in childhood ALL is deletion of chromosome 9p21, with the minimal region of deletion including the CDKN2-locus, making genes at this locus candidates for a predisposing genetic event in ALL. To determine whether genomic imprinting might be involved in ALL at the 9p21 locus we investigated the imprinting status of the candidate genes CDKN2A, CDKN2B and ARF. No evidence for genomic imprinting of ARF was found in this study. Because no expressed polymorphisms could be identified for CDKN2B, and CDKN2A expression was too low in normal tissues, the imprinting status of these genes could not be evaluated. Furthermore investigations in our laboratory have been unable to find genomic imprinting at any of these genes in mice. However, we have shown variation in allelic expression of ARF, which suggests a role for ARF haploinsufficiency in the onset of childhood ALL. A key feature of early human fetal lymphoid development is the absence of random �N� nucleotides between the rearranged V[H], D[H] and J[H] gene segments. The addition of �N� nucleotides at these junctions requires the enzyme terminal deoxynucleotidyl transferase (TdT). TdT is reported to not be expressed during early fetal lymphopoiesis but has been observed by the end of the first trimester, but data are sparse. The reported absence of N nucleotides in the majority of childhood ALLs suggests an early fetal origin. By defining the window-in-time during which TdT-negative B cell development occurs, we will be able to refine the timing of the origin of the B cells that give rise to ALL. Therefore we have sequenced and analysed the V[H]-DJ[H] and D[H]-J[H] junctions from immunoglobulin rearrangements in developing B cells in normal human fetuses aged from 5.1 to 11 weeks gestation. In this study 73 fetal IgH gene rearrangements were amplified from 21 different fetal liver samples. Only eight of the seventy-three rearrangements (11%) analysed in this study had no �N� nucleotides at the N1 (D[H]-J[H]) junction. This finding contrasts with the 24-28% of fetal rearrangements with no �N� nucleotides that have been reported in the literature. Furthermore, �N� nucleotides were shown to be present in the earliest sample, 5.1 weeks gestation. TdT expression was demonstrated by immunohistochemistry at 7.3 weeks and by RT-PCR at 8.3 weeks. B cell development in the fetal liver was detected as early as 6.5 weeks using flow cytometric analysis. Then, IgH gene rearrangements from 99 cases of childhood ALL were analysed. In total, 134 clone-specific IgH gene rearrangements were examined in this study. No association was found between the number of �N� nucleotides from complete and incomplete rearrangements at either the N1 (D[H]-J[H]) or N2 (V[H]-DJ[H]) junctions. Nor was any association observed between ALLs from children [less than or equal to] 3 years of age and those >3 years of age at diagnosis. These findings indicate that ALL IgH rearrangements do not have the paucity of �N� nucleotides that has been previously reported. The findings in this thesis suggest that there is no TdT-negative timepoint during B cell development and that there is no paucity of �N� nucleotides at the N1 junction in either fetal or childhood ALL IgH gene rearrangements.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

Understanding the nature and timing of leukaemogenic events during the development of childhood acute lymphoblastic leukaemia (ALL) will enable intervention that could prevent ALL in the future. We hypothesised that 9p21 deletion in childhood ALL may unmask predisposing genetic events that would allow us to determine the "nature" of initiating events in childhood ALL; whereas the inclusion, or exclusion, of random �N� nucleotides in normal immunoglobulin gene rearrangements from the developing fetus and the expression of terminal deoxynucleotidyl transferase (TdT) in fetal lymphocytes may allow us to unmask the developmental window during which the first transforming leukaemic event occurs in a pre-leukaemic B cell. The most frequent genetic abnormality in childhood ALL is deletion of chromosome 9p21, with the minimal region of deletion including the CDKN2-locus, making genes at this locus candidates for a predisposing genetic event in ALL. To determine whether genomic imprinting might be involved in ALL at the 9p21 locus we investigated the imprinting status of the candidate genes CDKN2A, CDKN2B and ARF. No evidence for genomic imprinting of ARF was found in this study. Because no expressed polymorphisms could be identified for CDKN2B, and CDKN2A expression was too low in normal tissues, the imprinting status of these genes could not be evaluated. Furthermore investigations in our laboratory have been unable to find genomic imprinting at any of these genes in mice. However, we have shown variation in allelic expression of ARF, which suggests a role for ARF haploinsufficiency in the onset of childhood ALL. A key feature of early human fetal lymphoid development is the absence of random �N� nucleotides between the rearranged V[H], D[H] and J[H] gene segments. The addition of �N� nucleotides at these junctions requires the enzyme terminal deoxynucleotidyl transferase (TdT). TdT is reported to not be expressed during early fetal lymphopoiesis but has been observed by the end of the first trimester, but data are sparse. The reported absence of N nucleotides in the majority of childhood ALLs suggests an early fetal origin. By defining the window-in-time during which TdT-negative B cell development occurs, we will be able to refine the timing of the origin of the B cells that give rise to ALL. Therefore we have sequenced and analysed the V[H]-DJ[H] and D[H]-J[H] junctions from immunoglobulin rearrangements in developing B cells in normal human fetuses aged from 5.1 to 11 weeks gestation. In this study 73 fetal IgH gene rearrangements were amplified from 21 different fetal liver samples. Only eight of the seventy-three rearrangements (11%) analysed in this study had no �N� nucleotides at the N1 (D[H]-J[H]) junction. This finding contrasts with the 24-28% of fetal rearrangements with no �N� nucleotides that have been reported in the literature. Furthermore, �N� nucleotides were shown to be present in the earliest sample, 5.1 weeks gestation. TdT expression was demonstrated by immunohistochemistry at 7.3 weeks and by RT-PCR at 8.3 weeks. B cell development in the fetal liver was detected as early as 6.5 weeks using flow cytometric analysis. Then, IgH gene rearrangements from 99 cases of childhood ALL were analysed. In total, 134 clone-specific IgH gene rearrangements were examined in this study. No association was found between the number of �N� nucleotides from complete and incomplete rearrangements at either the N1 (D[H]-J[H]) or N2 (V[H]-DJ[H]) junctions. Nor was any association observed between ALLs from children [less than or equal to] 3 years of age and those >3 years of age at diagnosis. These findings indicate that ALL IgH rearrangements do not have the paucity of �N� nucleotides that has been previously reported. The findings in this thesis suggest that there is no TdT-negative timepoint during B cell development and that there is no paucity of �N� nucleotides at the N1 junction in either fetal or childhood ALL IgH gene rearrangements.

Relapsed ALL remams one of the most common causes of death from disease in children. Broad-range drug resistance is often associated with relapse, although its underlying molecular mechanisms remained poorly understood. The aim of this thesis was to establish an in vivo model using the non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse strain, to facilitate the engraftment, expansion and characterisation of childhood ALL cells, obtained from patients at diagnosis or relapse. Mice were inoculated with leukaemia cells from patients' biopsies and engraftment was monitored by the proportion of human CD45+ cells in the blood. Successful leukaemia engraftment was achieved for 20/20 patient biopsies. Continuous passaging of ten xenografts has also been achieved. Immunophenotypic analysis showed only minor changes in cell surface markers after passage in mice. Leukaemia dissemination in murine bone marrow, liver, spleen and blood was consistent with the human disease. The in vivo responses of ten continuous xenografts to dexamethasone and vincristine, but not methotrexate, significantly correlated with patient outcome (p<0.05). Xenograft sub-lines resistant to vincristine, dexamethasone, methotrexate and cytosine arabinoside were also selected by in vivo drug treatments, although these sublines were not found to be cross resistant to structurally unrelated drugs. Resistance to vincristine, either in in vivo selected sub-lines or inherently resistant xenografts, was not associated with increased activity of drug efflux pumps such as P-gp or MRPl. Class I ?? tubulin levels remained unchanged when compared between vincristine resistant sublines and their parental xenografts. Decreased expression of stathmin and increased polymerised tubulin were observed in vincristine resistant sub-lines, suggesting a possible mechanism of counteracting the depolymerising effects of vincristine. In summary, this study has shown that primary ALL cells engraft efficiently into NOD/SCID mice, and indicates that their response to vincristine and dexamethasone mimics the clinical situation. This model appears to be highly relevant for the study of childhood ALL and will provide the foundation to delineate clinically relevant mechanisms of drug resistance.