Mechanisms of central nervous system disease in childhood acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) is the commonest childhood malignancy. Once a universally fatal disease, modern therapy has achieved excellent outcome and the majority of children achieve long term cure. Yet relapse remains a challenge. One of the major hurdles in achieving complete cure is the relapse of ALL at extramedullary sites such as the central nervous system (CNS). Despite significant advances in understanding leukaemia biology, most predictors of leukaemic behaviour are accurate for the bone marrow disease only. The precise timing, frequency and properties of CNS infiltrating leukaemic cells are not elucidated. Therefore, the broad aim of this thesis is to develop a better understanding of the mechanisms of leukaemic entry and infiltration patterns of leukaemic cells in the CNS. In order to address the frequency and pattern of CNS infiltration, a xenograft model using primary leukaemic cells from children with B-cell precursor (BCP) ALL in NOD/Scid IL2Rγ null (NSG) mice was established. The majority of samples from children with and without overt CNS disease were able to infiltrate the CNS in NSG mice. CNS infiltration was seen in mice engrafted with small numbers of cells and distinct immunophenotypic subpopulations. The leukaemic samples followed a distinct and reproducible pattern of CNS infiltration with leukaemic infiltrates in the meninges while sparing the CNS parenchyma. To investigate whether a distinct set of leukocyte trafficking molecules provided tissue specificity for entering the CNS, leukaemic cells retrieved from the bone marrow and the CNS were assessed for expression levels of selected chemokine receptors and P-selectin glycoprotein ligand-1 (PSGL1). Additionally, chemotaxis assays were utilized to investigate the function of the chemokine receptor CXCR4. Despite surface expression of chemokine receptors on leukaemic cells and presence of chemokines in the CNS, no evidence for positive selection of a high-expressing subpopulation was seen. Overall, it appears that, unlike the bone marrow, chemokine receptors do not direct leukaemic cell trafficking to the CNS. To investigate the published observation that interleukin-15 (IL-15) expression in leukaemic samples correlates with the risk of CNS disease, the effects of IL-15 stimulation on BCP-ALL cells were assessed. IL-15 and IL-15 receptor subunits were noted to be expressed at mRNA level in samples from BCP-ALL patient and cell lines. Exogenous IL-15 stimulated leukaemic cell proliferation and upregulated genes associated with migration and invasion in SD1 cells. A higher proliferative advantage was observed at low-serum conditions which mimics conditions found in the CNS. Therefore a plausible mechanistic link was established for the association of CNS disease with high IL-15 expression levels. In cerebrospinal fluid (CSF) samples from patients, quantitative PCR (qPCR) was utilized to detect submicroscopic levels of CNS disease. In approximately 40% patients, qPCR using patient specific primers tested positive for the presence of leukaemic DNA. Therefore this test is much more sensitive than conventional diagnostic techniques which only detect CNS disease in 2-5% of patients. CSF supernatants were also tested to assess whether the levels of chemokines could be used to diagnose patients with qPCR positive disease. Although differences in the levels of chemokines between qPCR positive and negative patients were noted, the values are not sufficiently discriminatory to be clinically useful. In conclusion, CNS entry appears to be a much more frequent property of leukaemic cell than previously appreciated. Leukocyte trafficking molecules do not appear to play an instructive role in the CNS entry and therefore, it is unlikely that expression levels of leukocyte trafficking molecules or the levels of chemokines in the CSF will be useful biomarkers of CNS disease. In addition, CNS disease appears to be present at diagnosis in at least 40% of patients. Therefore, attempts at blocking leukaemic entry into the CNS are unlikely to be therapeutically useful. Instead, analysing and targeting factors that allow long-term survival of leukaemic cells in the CNS may be a better strategy to eradicate CNS disease and prevent leukaemic relapse.