Upregulated Cholesterol Biosynthesis Is a Biomarker for Isolated Central Nervous System Relapse in Acute Lymphoblastic Leukaemia

Background:

Acute lymphoblastic leukaemia (ALL) is the commonest childhood cancer. In early trials CNS relapse was a common occurrence, and the introduction of CNS-directed therapy in the 1960s-70s resulted in the largest single improvement in outcome for childhood ALL. Today, despite universal intensive CNS-directed therapy - with significant associated toxicity - the CNS is involved in around 50% of ALL relapses, with 50% of these being isolated CNS (iCNS) relapse. Whilst many factors appear to increase risk of CNS relapse, few are specific for CNS relapse. Discovery of biomarkers for CNS relapse would allow increased therapy for children at high risk, and potentially less CNS-directed therapy for those at low risk.

Despite the clinical significance of CNS infiltration, relatively little is known about the biological differences between systemic and CNS ALL. In the CNS, leukaemic cells form plaques adherent to the leptomeninges, bathed in low-nutrient, low-oxygen CSF. It was hypothesised that leukaemic cells adapt metabolically to this nutritionally poor CNS microenvironment, and these adaptations may be targetable as specific biomarkers for CNS relapse.

Findings:

Human precursor-B ALL cell lines (SEM (t(4;11) and REH (t(12;21)) were purified from the CNS and spleen after xenotransplantation into immunodeficient (NSG) mice, and transcriptomic analysis was carried out using RNASeq. Gene Set Enrichment Analysis (GSEA), showed upregulation of the cholesterol biosynthesis pathway in the CNS compared with systemic ALL (adj. p<0.0001). This finding was confirmed in publicly available microarray data of human ALL cells retrieved from CNS and bone marrow samples (GEO dataset GSE60926; p=006). To further validate this finding, metabolic analysis of xenografted SEM cells was performed using HPLC-mass spectrometry, showing an increase in intracellular abundance of cholesterol-precursor mevalonate (p=0.003) in SEM cells in the CNS compared with spleen.

To confirm lack of cholesterol as a key metabolic challenge for ALL cells in the CNS, total cholesterol was quantified in CSF and plasma from normal human controls and NSG mice, with and without leukaemia. To investigate the effect of leukaemia on CSF cholesterol abundance in humans, CSF from patients at diagnosis (i.e. with untreated ALL) and during maintenance therapy was analysed. In control samples from humans, there was approximately 1000-fold lower total cholesterol in CSF compared to plasma (in murine CSF there was approx. 500-fold lower cholesterol). In addition, plasma cholesterol was around 33% lower in mice xenografted with SEM cells (p=0.02), with a trend towards a similar reduction in CSF cholesterol (p=0.08). In humans there was an approx. 2-fold increase in CSF cholesterol between samples taken at diagnosis compared with samples taken during maintenance chemotherapy (i.e. CSF cholesterol was lower in children with untreated ALL, p=0.03).

Having shown upregulated cholesterol biosynthesis to be an adaptation to the CNS microenvironment, the utility of finding this signature in the bone marrow of children at diagnosis as a biomarker of CNS relapse was assessed. Publicly available data from GEO dataset GSE11877/NCI TARGET program was analysed for association between upregulated cholesterol biosynthesis and CNS relapse. Upregulation of 2 or more genes in the cholesterol biosynthetic pathway, determined using a z-score approach, (n=44 upregulated, n=163 not upregulated) was associated with a 3-fold increased risk of isolated CNS but not bone marrow relapse (28.5% absolute risk of iCNS relapse compared with 9.5% after 14 years follow up, p=0.0007). This increase in CNS relapse risk remained after multivariate analysis. Early validation using data from the NOPHO ALL2008 trial of all risk groups (n=163) showed that both children with iCNS relapse had upregulation of cholesterol synthesis by these criteria. Further validation in additional independent cohorts is ongoing.

Conclusions:

These findings suggest that cholesterol biosynthesis is a key metabolic pathway for childhood ALL cells infiltrating the CNS. This is in keeping with very low levels of cholesterol detected in the CSF, particularly in the presence of leukaemia. Upregulated cholesterol synthesis genes in diagnostic bone marrow are associated with increased risk of CNS relapse in high risk ALL, with supporting evidence of a similar association in standard-risk ALL.