EHA Library - The official digital education library of European Hematology Association (EHA)

In acute lymphoblastic leukemia (ALL), involvement of the central nervous system (CNS) is associated with adverse prognosis. Despite non-detectable CNS involvement in many cases, CNS-directed therapy is indispensible for relapse free survival indicating subclinical CNS manifestation in many patients. However, CNS therapy coincides with increased risk for secondary neoplasms and impaired cognitive skills in ALL survivors.

In this study, we aimed to characterize mechanisms mediating CNS involvement in ALL and to identify targets for CNS leukemia directed treatment.

Primary B-cell precursor (BCP) ALL cells were transplanted onto NOD/SCID mice. At onset of disease related morbidity, human ALL engraftment was analyzed in bone marrow (BM), spleen (S), peripheral blood (PB), and meninges of the recipients by flowcytometry staining for huCD19. Transcriptome profiles of ALL cells isolated from meningeal and BM infiltrates (8 pairs of 8 CNS+ ALLs) were analyzed. Gene expression was validated by qPCR. Proliferation, survival and trans-endothelial migration was analyzed in genetically modified (down-regulation or overexpression) ALL cells and in response to recombinant protein or an antagonizing antibody. In an in vivo model of CNS+ ALL, inhibition of CNS manifestation was evaluated.

At onset of leukemia manifestation in recipients transplanted with BCP ALL, meningeal infiltration was detected along with ALL engraftment in BM, S and PB in a subset of samples (CNS+) in contrast to absent CNS involvement despite full-blown leukemia in BM, S and PB in others (CNS-). In line, magnetic resonance imaging revealed meningeal enhancement in CNS+ recipients.

By expression profiling, the gene coding for vascular endothelial growth factor A (VEGF) was identified to be highly expressed in leukemia cells isolated from CNS as compared to BM-derived cells. Interestingly, VEGF was reported to mediate cell survival, vascular permeability and trans-endothelial cell migration and increased levels of VEGF protein were described in cerebrospinal fluid collected from CNS+ ALL and AML patients.

Differential VEGF expression was confirmed in independent cohorts. VEGF receptor 1 expression was detected on all ALL cells. However, ALL cell proliferation and survival was not affected by VEGF overexpression, down-regulation, or exposure to VEGF or the antagonizing antibody bevacizumab. Further, we addressed the paracrine effect of VEGF on endothelial cells mediating trans-endothelial leukemia migration. Upon VEGF incubation, brain endothelial cells (bEND.3) showed induction of downstream VEGF receptor 2 signaling activity mediating cellular permeability. In a transwell assay, we identified VEGF-dependent trans-endothelial migration of BCP ALL cells (Nalm-6) with significantly increased or decreased migration in the presence of VEGF or bevacizumab through bEND.3 monolayers.

Moreover, we investigated the effect of VEGF inhibition by bevacizumab on CNS leukemia manifestation in 3 patient-derived primografts (4 experiments including 1 replicate) in vivo. Most interestingly, in all 4 experiments anti-VEGF treatment significantly reduced the leukemia load exclusively in the CNS but not in BM, S and PB organ compartments indicating that the CNS+ phenotype is mediated by VEGF and can be controlled by VEGF inhibition.

We identified VEGF as a mediator of trans-endothelial leukemia cell migration and CNS manifestation in ALL. Most importantly, in vivo inhibition of VEGF by bevacizumab significantly decreased involvement of CNS ALL indicating a novel therapeutic strategy to control CNS leukemia.