Comparative Analysis of Gene Expression Profiles between Diagnosis and Relapse of Childhood Acute Lymphoblastic Leukemia.
Authors: Szczepanski Tomasz , de Ridder Dick , van der Velden Vincent H.J. , Schonewille Tom , van Wering Elisabeth , van Dongen Jacques J.M. , Staal Frank J.T. 
Published: October 2019

Abstract
Approximately 30% of childhood acute lymphoblastic leukemia (ALL) patients relapse, which is the most frequent adverse event in this otherwise curable disease. The mechanisms of relapse are largely unknown. Earlier studies indicated that some relapses might originate from subclones with many different biological features compared to the original ALL clones at diagnosis. Therefore, we aimed at detailed comparison of gene expression profiles between diagnosis and relapse of childhood ALL. The study group consisted of 41 children, 27 diagnosed with B-cell precursor ALL (BCP-ALL) and 14 with T-cell precursor ALL (T-ALL). All samples obtained at diagnosis and relapse were subjected to purification using CliniMACS system and enriched to more than 95% of blasts in each sample. RNA isolation and gene expression profiling were performed according to standard procedures using Affymetrix HG-U133+2 set GeneChip arrays (Affymetrix). The samples were also screened at the RNA level for the most common genetic aberrations occurring in ALL such as t(9;22), t(4;11), t(12;21) and TAL1 deletion. The studies at the DNA level involved detailed comparison of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements between diagnosis and relapse to assess clonal evolution. GeneChip array data were quantile normalized and background was removed using robust multichip analysis. Significance Analysis of Microarrays (SAM) and t-test were applied to find differentially expressed probe sets between diagnosis and relapse using both the paired and unpaired criterion. The p values < 0.05 were considered significant. The paired SAM analysis revealed 388 significantly differentially expressed (SDE) probe sets for BCP-ALL and 10 SDE probe sets for T-ALL. The differences in expression levels were relatively low, generally not exceeding two-fold. SDE gene sets revealed in our study were mainly different from previously published data, which is most probably due to more stringent purification procedures. Using Ingenuity Systems the SDE genes in BCP-ALL could be significantly linked to several networks involved in cell cycle, DNA replication, recombination, and repair, cellular assembly and organization, cellular growth, proliferation and cancer. There were no significant differences in gene expression profiling in smaller immunophenotypic and cytogenetic ALL subgroups as well as in relation to remission duration (early vs. late relapse). Several SDE genes were found when comparing the ALL with stable Ig/TCR configuration and with some clonal evolution (22 probes for T-ALL and 8 probes for BCP-ALL). In conclusion, discrete differences of gene expression profiles between diagnosis and relapse of childhood ALL indicate heterogeneous origin of relapse. Many relapses represent the simple outgrowth of the original clone, while in other cases many different (leukemia-related) relapse mechanisms might be involved.