An Analysis Of Mechanisms Of Central Nervous System Infiltration In Acute Lymphoblastic Leukaemia Using Primary Cells Xenografted Into Immunodeficient Mice

Despite great advances in the treatment of paediatric acute lymphoblastic leukaemia (ALL), disease in the central nervous system (CNS) continues to pose challenges. Current diagnostic tests are insensitive and risk factors for CNS relapse are poorly understood. This results in all children receiving intensive CNS-directed therapy which may be associated with acute and/or chronic neurotoxicity. A better understanding of the mechanisms of CNS engraftment is a necessary pre-requisite for diagnostic and therapeutic advances. To investigate if primary cells infiltrate the CNS, and whether this property resides in any particular sub-clonal compartment we undertook a comprehensive analysis of the CNS engrafting potential of primary ALL cells in immunodeficient NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice. In addition, we used this model to investigate whether chemokine receptor expression drives CNS infiltration in pre-B ALL, as previously shown for T-ALL.

CNS engraftment was seen in 18/23 pre-B ALL samples (78%). A consistent pattern of engraftment was observed with plaques of disease in the leptomeninges, relative sparing of the ventricles and complete absence of parenchymal infiltration. This implies ALL transit across the blood-CSF barrier rather than the blood-brain barrier – an important distinction, as leucocytes utilise distinct physiological trafficking mechanisms to cross these two barriers. To examine the frequency of cells with CNS-engrafting potential, intra-femoral transfer of 10 to 10,000 cells was performed. CNS engraftment was seen with as few as 10 initial cells. In addition, the ability to engraft the CNS was not restricted to any particular immunophenotypic compartment and was seen in CD19⁺CD10^high, CD19⁺CD10^low, CD19⁺CD20^low, CD19⁺CD20^high, CD19⁺CD34^high, CD19⁺CD34^low fractions. These data suggest that CNS engraftment potential is present at high-frequency in the bulk leukaemic population at diagnosis. To examine candidate trafficking molecules governing CNS entry, we investigated chemokine receptor expression on pre-B ALL blasts using quantitative PCR and flow cytometry. The chemokine receptors CXCR3, CXCR4 and CXCR7 were expressed by pre-B ALL but were not up-regulated in cells retrieved from the CNS compared to the bone marrow (BM).

In conclusion, in xenograft models of pre-B ALL, CNS engrafting potential is present at high frequency in patient diagnostic BM specimens and does not appear to be restricted to sub-clonal compartments. These findings have important implications for the design of risk-adapted CNS therapy. Firstly, our studies indicate that CNS entry is a common property of leukaemic blasts and therefore the current dogma of CNS-directed therapy for all patients appears to have a rational scientific basis. Secondly, it is unlikely that chemokine receptor expression profiling will be a useful biomarker for CNS disease in pre-B ALL. Finally, identifying factors that facilitate long-term survival of cells in the CNS (which may also enhance long-term survival in the bone marrow) may be a better therapeutic strategy than attempts to block cell entry.