Intermediate-Risk Acute Lymphoblastic Leukemia (ALL) Patients with and without Relapse Differentially Depend on Survival Signals From Microenvironment
Authors: Rhein Peter , Mitlohner Rita , Kirschner-Schwabe Renate , Hagemeier Christian , Cario Gunnar , Stanulla Martin , Schrappe Martin , Fichtner Iduna , Ratei Richard , Ludwig Wolf-Dieter , Karawajew Leonid 
Published: October 2019

Abstract
Abstract 752
Risk stratification in the childhood ALL-BFM clinical trial has been based on molecular/cytogenetic markers and the in vivo response to treatment (blast reduction in peripheral blood after prednisone prephase, bone marrow clearance after induction treatment and minimal residual disease (MRD) after induction and induction consolidation). These criteria classify about 50% of ALL cases into standard-risk (SR) and high-risk (HR) groups. The intermediate-risk (IR) ALL group, showing moderate levels of MRD, provides the majority of relapses, but still lacks specific prognostic markers which could distinguish between those patients subsequently relapse and those remaining relapse-free (IR+r and IR-r). We established a bank of NOD/SCID ALL xenografts representing IR+r and IR-r subgroups (12 vs 13 patients) and characterized the samples in a broad series of functional assays. There was no difference in the sensitivity to drugs (dexamethasone, daunorubicin, L-asparaginase and vincristine) between the IR+r and IR-r groups, however, the rate of spontaneous apoptosis in vitro was significantly higher in the IR+r group than in the IR-r group (69 +/− 7% vs 39 +/− 6%, p=0.02). Given that in the presence of stromal cells the cell death level has been similar in both IR groups (9 +/− 5% vs 7 +/− 7%, p=0.8), these observations suggest that the IR+r ALL is more dependent on survival signals from microenvironment than IR-r ALL. In the NOD/SCID engraftment model, leukemic cells from IR+r patients engrafted more rapidly than cells from IR-r patients (93 +/− 12 days vs 170 +/− 24 days, p=0.006), pointing to a better in vivo cooperation between leukemia cells and microenvironment in the IR+r cases. In order to identify the underlying molecular pathways, gene expression changes induced in the presence and absence of stroma have been investigated at genome-wide level (Human Gene 1.0 ST Affymetrix array). Of the apoptosis-regulating genes, a caspase inhibitior from the IAP family, BIRC3, was found to be up-regulated significantly higher in the IR-r than in the IR+r group (3.7 +/− 1.1 vs 1.7 +/− 0.1-fold up-regulation, p=0.016). The adhesion molecule VCAM1 has been generally up-regulated by the co-incubation with stroma, with a tendency to a higher up-regulation in the IR-r (4.4 +/− 0.7-fold) than in the IR+r (2.7 +/− 1.0-fold) group. We further speculated that interaction with microenvironment is a mutual process which also implicates gene expression changes of the stroma. To this end, mouse stromal cells (MS5 cell line) were incubated with a series of ALL samples (n=3), purified by MACS sorting and investigated using Mouse Gene 1.0 ST Affymetrix arrays. The list of genes whose expression increased by at least 2-fold in all 3 cases (n=10), included the macrophage inflammatory peptide-1alpha (MIP1A/CCL3), a chemokine known to regulate chemotaxis of lymphocytes. Our data suggest that the cooperation of ALL with microenvironment may provide a promising approach to molecularly discriminate ALL cases with relapse and to identify potential relapse-associated targets within the IR-ALL group.

Disclosures:
No relevant conflicts of interest to declare.