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published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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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.

Thesis (M. Med. Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 36-41).

Thesis (M.Med.Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 36-41). Also available in print.

Thesis (Master Diploma(Medical Technology) -- Cape Technikon, Cape Town, 1990 / Eighty six patients with acute l.eukaemia were studied using morphol.ogical., cytochemical. and immunol.ogical. techniques. The acute l.eukaemias were subdivided using the French-American-British (FAB) cl.assification. The immunophenotyping studies were compared with the morphological classification to assess their contribution to the diagnosis. Acute non-lymphoblastic leukaemia (ANLL) was diagnosed on the basis of morphol.ogy and cytochemical. criteria. In addition this group of patients was studied with antibodies directed against myel.omonocytic antigens. However, no further cl.inical.l.y useful. information was obtained. Patients whose bl.asts did not stain with Sudan black or myel.operoxidase were considered to have acute lymphoblastic leukaemia (ALL). After assessment with monocl.onal. antibodies directed against epitopes expressed on cel.l.s from the l.ymphoid lineage, these patients were subgrouped into non-T-ALL, common-ALL, B-ALL, T-ALL and l.ymphoblastic lymphoma categories. This study confirmed the val.ue of monocl.onal. antibodies for accurately assigning l.ineage to the acute l.eukaemias and particularly in those situations where conventional morphol.ogical. criteria and cytochemical. markers are inconclusive.

Long-term survival and probable cure have become norms in acute lymphoblastic leukaemia of childhood. The adverse effects of treatment for leukaemia are diverse and complex. In many cases, treatment effects come to light 1 ong after the end of therapy. These so-ca 11 ed 1 ate effects (which are yet obscure and incompletely understood) have become increasingly important as the number of children surviving leukaemia increases. This thesis describes a comprehensive study of leukaemia survivors attending the Oncology Clinic of the Red Cross War Memorial Children's Hospital. The study sample consisted of all leukaemia survivors in long-term remission, disease free and off treatment up to January 1st, 1984. The study is introduced by a chapter which describes acute lymphoblastic leukaemia and pays particular attention to the effects of the primary disease on organs which may subsequently exhibit late effects of treatment. Treatment of acute lymphoblastic leukaemia is described in some detail and the reasons for current treatment strategies are outlined. Individual modalities of treatment are then discussed with reference to their mechanisms of action and potential for damage to non-neoplastic tissue. The study then examines all systems likely to have been damaged during therapy, in order to achieve a comprehensive impression of the late effects of leukaemia treatment. In each chapter, pertinent literature was reviewed up to January 1987. Growth is a major task of childhood. Many chronic diseases are potential causes of growth failure. A longitudinal retrospective study showed that statured growth in leukaemia survivors was stunted during treatment. Catch-up growth did not occur at the end of treatment, although normal growth velocity was resumed. Adult height was expected to be reduced as a result. In addition to temporary stunting of statured growth, leukaemia survivors showed a progressive increase in weight-for-height during treatment. This trend continued after treatment had ended. These changes in weight and height were peculiar to leukaemia survivors. Control groups of children with solid tumours in long term remission showed less stunting during treatment and had catch-up growth after treatment, except when they had undergone spinal i rradi ati on. Normal endocrine function is a prerequisite for normal growth and development. Although growth hormone responses to insulin-induced hypoglycaemia were frequently and significantly abnormal in survivors of childhood leukaemia, these children grew normally once treatment had stopped. Impaired growth hormone secretion appeared to be a marker of hypothalamic damage caused by leukaemia therapy. Testicular and ovarian function was normal in the absence of irradiation of these organs. Thyroid function was normal in leukaemia survivors although a minority showed evidence of hypothalamic damage in their response to thyrotropin releasing hormone. Normal prolactin levels in children showing other hormonal evidence of hypothalamic damage were thought to indicate the selectivity of damage caused by leukaemia treatment. Adrenal control and function were normal in leukaemia survivors. In the absence of a growth disorder, only thyroid status may need long-term assessment in leukaemia survivors. Intellectual development is a further major task of childhood. A sibling-controlled study of intellectual function indicated an intelligence deficit in children surviving leukaemia and its treatment. This deficit was thought to be the consequence of therapy, since children surviving solid tumours showed no such deficit in comparison with their sibling controls. Survivors of childhood leukaemia also had an increased incidence of visual perceptual difficulty and more school prob 1 ems than survivors of solid tumours, particularly in early primary grades. Intellectual outcome and school performance in leukaemia survivors may be improved by early visual perceptual training. Children surviving acute lymphoblastic leukaemia had significantly more minor motor abnormalities than children surviving solid tumours. Minor motor abnormalities were frequently and significantly associated with abnormalities of the brain visualized by computerized tomography. Neurophysiologic measurement (EEG, VER, BAER) did not contribute to the assessment of neurological outcome and correlated poorly with clinical and CT scan findings. A functional assessment of neurological outcome in leukaemia survivors should include a clinical examination for minor motor dysfunction. Some children manifested other organ-specific damage due to chemotherapy or radiotherapy. These isolated cases are discussed in the form of case reports and literature reviews. Patients have received treatment with cytotoxic drugs in addition to standard leukaemia therapy need to be followed for treatment-specific late effects. The psychological outcome of leukaemia survivors was assessed by means of parent interviews and teacher questionnaires. In terms of a low frequency of behaviour problems reported by these observers, psychosocial adaptation in leukaemia survivors vas surprisingly good. Children surviving solid tumours and healthy school children from the same community (the latter from a literature report) had similar frequencies of behavioural problems. In both leukemic children ana solid tumour control patients, certain patterns of family behaviour ~ere predictive of a poor psychological outcome. It appears that an early family assessment may identify families 'at risk'. If needs to be shewn whether such families would benefit from professional psychological support. In the final chapter a 'functional deficit score' is offered as a measure of overall outcome in terms of late effects of therapy. Patients were rated in five categories (growth, intellectual outcome, neurological status, miscellaneous organ damage and psychosocial adaptation) according to the severity of persistent late effects. Children surviving acute lymphoblastic leukaemia were shown to have been more seriously damaged by their treatment than children surviving solid tumours. The difference in overall damage was the consequence of central nervous system injury. Available evidence indicates that this central nervous system injury is caused by radiotherapy (with or ·thought a synergistic effect with i intrathecal 1 methotrexate) given as central nervous system 'prophylaxis'. With few exceptions, leukaemia survivors in this study had received L400 rads of deep x-ray therapy as cranial irradiation. This dosage has since been reduced world-wide. Current cranial irradiation 'prophylaxis' consists of 1800 rad of megavoltage radiotherapy. Fa 11 ow-up studies of survivor cohorts given such radiotherapy should include the measures embodied in the 'functional deficit score' described above.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer which arises from the malignant transformation of B-cell or T-cell progenitors. Despite recent pioneering improvements in intensified combination chemotherapy, 20% of pediatric and 50% of adult ALL patients present with primary drug-resistant leukemia or develop relapse. Treatment of refractory and relapsed ALL has remained a significant clinical challenge with survival rates following relapse of only 40%, highlighting the need to understand the mechanisms which drive drug resistance and relapse of ALL. Through extensive sequencing analyses of matched diagnostic, remission and relapsed DNA samples from patients with B-precursor ALL (B-ALL) and T-cell ALL (T-ALL) we have identified recurrent relapse-specific gain-of-function mutations in the cytosolic 5'-nucleotidase II (NT5C2) gene in 25% of relapsed T-ALLs and 6% of relapsed B-ALLs. NT5C2 is a highly conserved, ubiquitously expressed enzyme which regulates intracellular purine nucleotide levels by dephosphorylating purine monophosphates. NT5C2 also dephosphorylates key metabolites in the activation of purine analog prodrugs such as 6-mercaptopurine and 6-thioguanine which are routinely used in the treatment of ALL, allowing purine analog nucleosides to be readily exported out of the cell. Here we show that mutant NT5C2 proteins have increased 5’-nucleotidase activity and confer resistance to 6-mercaptopurine and 6-thioguanine chemotherapy when expressed in leukemic cells. Consistently, NT5C2 mutations correlate with early relapse and relapse while under therapy. We present a novel T-ALL conditional inducible knock-in mouse model of the highly recurrent NT5C2 R367Q mutation and show that expression of one Nt5c2 R367Q allele from the endogenous locus in primary T-ALL lymphoblasts induces overt resistance and disease progression under therapy with 6-mercaptopurine in vivo, while surprisingly conferring reduced growth and decreased leukemia initiating activity in the absence of chemotherapy. Metabolically we show that the observed loss of fitness in Nt5c2 R367Q tumors can be explained by a severe depletion of endogenous purine monophosphate metabolites as a result of increased Nt5c2 5’-nucleotidase activity. Consistently, using ultra-sensitive mutation analyses we show that relapse-associated NT5C2 mutations are not detectable at initial disease presentation, indicating that NT5C2-mutant tumor cells are negatively selected by clonal competition in the early stages of disease development and only positively selected under prolonged 6-mercaptopruine chemotherapy which is the backbone treatment for ALL following remission. Our findings present the first known example of chemotherapy resistance and disease progression driven by a tumor clone with decreased leukemia initiating activity, highlighting the intense selective pressure of chemotherapy in the clonal evolution of tumors from diagnosis to relapse. Through extensive biochemical and structural characterizations of recombinant NT5C2 mutant proteins, we have grouped relapse-specific NT5C2 activating mutations into 3 different classes, each conferring unique enzymatic behavior in basal conditions and in response to allosteric activation, and each with unique structural features which mediate increased 5’-nucleotidase activity. Moreover, we identify a novel auto-regulatory switch-off mechanism of the NT5C2 enzyme involving movement of an unstructured flexible loop, and present the first crystal structure view of the NT5C2 C-terminal acidic tail, implicating it as an auto-inhibitory brake to the allosteric activation of the enzyme. The presence of multiple mutational mechanisms of activating such a highly conserved enzyme, especially in light of the inherent loss of fitness to the tumor cells, indicates a strong convergent evolution towards activating NT5C2. This is supported by our discovery that patients can harbor multiple leukemic clones with NT5C2 mutations at relapse. Overall our findings highlight NT5C2 as a major driver of drug resistance and relapse of ALL and pinpoint metabolic susceptibilities which could be exploited therapeutically to target NT5C2-mutant tumors in the future. Our in-depth structural and enzymatic knowledge of mutant NT5C2 proteins will serve as an essential tool in the rational drug development of novel NT5C2 inhibitors with increased specificity and selectivity for mutant NT5C2, while our novel Nt5c2 R367Q knock-in mouse model will serve as a platform for the pre-clinical testing of both NT5C2 inhibitors and alternative compounds selective for Nt5c2-mutant leukemias which can be used for prevention and treatment of relapsed ALL.

Thesis (Ph. D.)--University of Florida, 2006. / Title from title page of source document. Document formatted into pages; contains 227 pages. Includes vita. Includes bibliographical references.