Treatment Type Determines Gene Expression Changes from Pretreatment (PTx) to Relapse (Rel) in Acute Promyelocytic Leukemia (APL).
Authors: Cui Qingping , Kim Haesook , Zhou Da-Cheng , Wiernik Peter H. , Tallman Martin S. , Gallagher Robert E. 
Published: October 2019

The molecular changes responsible for the recurrence of APL after achieving complete remission are largely unknown. The Phase III trial E2491 provided a venue for investigating whether treatment type had an effect on the changes in gene expression that occur from PTx to Rel, since ¼ of patients were randomized to receive only chemotherapy (C-set patients), while ¾ of patients additionally received all-trans retinoic acid (ATRA) either during the induction and/or maintenance phases of treatment (A-set patients). High-quality, ethanol-stored RNA was available from matched PTx and Rel samples with ≥85% blasts from 3 C-set and 3 A-set patients, who had the following characteristics:

Patient-Set PTx WBC FAB Class P-Rα Type PTx Mutations Rel Mutations Days on ATRA Days off ATRA 1-C 68.6 M3 V None Flt3ITD & D835 0 N/A 2-C 2.4 M3 S Flt3ITD & D835 Flt3ITD 0 N/A 3-C 0.4 M3v S Flt3ITD & D835 Flt3ITD 0 N/A 4-A 77.4 M3v V Flt3ITD PML-RARα 142 Maintenance 0 5-A 3.0 M3 L - - 365 Maintenance 344 6-A 10.9 M3 V PML PML 19 Induction 410

Gene expression analysis utilized the Affymetrix Human Genome U-133 Plus 2.0 chip. Of 54,613 gene probe sets (gps), 6220 gps for named genes with a mean value >100 for all samples and a ≥100 absolute difference between PTx and Rel values were selected for study. By unsupervised hierarchical cluster analysis, the overall samples segregated into 2 major groups, PTx or Rel with single exceptions. The PTx samples co-segregated into 2 groups according to the co-incident parameters of high WBC count and V-form PML-RARα (Patients 1,4,6 vs 2,3,5). Most notably, the Rel samples segregated by treatment type, C-set and A-set. In order to identify the gps most significantly contributory to differences between the C- and A-set gene expression profiles, we made two linked analyses. First, 575 gps were selected based on the criteria of an ≥1.5-fold difference, p <0.05, between the average expression level of each gps in the 3 relapse samples of the A-set vs the C-set. Second, we identified 92/575 genes with a statistically-significant fold-change (p ≤0.05, paired T-test) from PTx to Rel. After editing out redundant gps, 21 unique gene transcripts (ugt) were upregulated (Up) and 54 ugt were downregulated (Dn) from PTx to Rel. There was a high degree of concordance between the fold-difference values in the Rel samples and the fold-change values from PTx to Rel. Remarkably, for 19/21 Up ugt and 49/54 Dn ugt, the selection was based on greater change in A-set than C-set samples. Prominent among the selected A-set ugt were those affecting DNA repair/replication (Up: NPN; Dn: RAD17, RPA1, PARP1, MCM3) and signal transduction (Up: STAT3, IRAK3, LIMK2, GRB10; Dn, STS-1, CSNK2A1, PAK2, OPN3). These results indicate that the addition of ATRA to chemotherapy-based treatment of APL (all patients received 2 courses of consolidation chemotherapy) had a major impact on the gene expression profile at relapse, which further suggests that ATRA treatment made an important and unique contribution to the molecular progression process leading to relapse.