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K. Van Roosbroeck et al.
i.e. significantly differentially expressed (SDE, at P val- ues <0.05) in bone marrow samples of patients with CLL at the time of Richter transformation when com- pared to their expression in samples obtained at the time of CLL diagnosis: SDE in group 1b versus group 1a, but not in group 2b versus group 2a (to exclude the time of evolution as a variable) (Figures 1C and 2A,B, Online Supplementary Figure S1 and Online Supplementary Table S2). We excluded miR-34a and miR-17 from the list of Richter-specific SDE miRNA, as these were also signifi- cantly upregulated in the control group of patients with CLL who did not develop RS over the course of follow- up, which was similar to the time to Richter transforma- tion in the RS group. We then extended our cohort to include non-matched samples (Table 1 and Online Supplementary Table S2) and analyzed these with the Firefly custom multiplex miRNA assay as well. We con- sidered the following groups (Figure 1B): group 1a - sam- ples at the time of CLL diagnosis from patients with
A
CLL who later developed RS (n=25); group 1b - samples from patients with CLL at the time of Richter transfor- mation/RS diagnosis (n=9); group 2a - control samples from patients with CLL, at the time of diagnosis, who did not develop RS over a follow-up period equal to or longer than the time between CLL and RS diagnosis of groups 1a and 1b, (n=16); and group 2b - at a time cor- responding to RS transformation in group 1b (n=13). This analysis revealed 15 miRNA that showed signifi- cantly different expression between samples at the time of CLL diagnosis (group 1a) and at the time of RS diag- nosis (group 1b), of which seven are common with the paired analysis (Figure 2A-C and Online Supplementary Figure S2).
Next, we validated these results with a different assay and performed qRT-PCR expression analysis on 25 miRNA in the same extended CLL/RS dataset. This analysis confirmed three differentially expressed miRNA: miR-21 and miR-146b were upregulated and
BC
Figure 1. Workflow of the microRNA screening process and composition of the University of Texas MD Anderson Cancer Center cohort of patients with Richter syn- drome/chronic lymphocytic leukemia. (A) Three-step workflow of the miRNA screening process, consisting of miRNA profiling, array comparative genomic hybridiza- tion (aCGH) analysis and functional analysis. (B) Schematic representation of the extended University of Texas MD Anderson Cancer Center (MDACC) cohort of patients. This cohort consists of patients with Richter syndrome (RS) (group 1), as well as age-, sex- and sample time-matched controls with chronic lymphocytic leukemia (CLL) (group 2). For both groups, samples at CLL diagnosis (group 1a) and at the time of RS diagnosis (group 1b) or a time corresponding to RS diagnosis time in the case of the matched CLL controls (group 2b) are available. Time “t1” between CLL diagnosis and RS diagnosis time is similar for both groups. (C) miR-21, miR-146a, miR-150 and miR-181b are members of the four-miRNA “restricted signature”, while the eight-miRNA “enlarged signature” contains these four miRNA and additionally miR-24, miR-26a, miR-181a and miR-146a. miRNA highlighted in red are upregulated in RS, while miRNA highlighted in green are downregulated. For additional Information, see Online Supplementary Figure S1. IOR: Institute of Oncology Research; DLBCL: diffuse large B-cell lymphoma.
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haematologica | 2019; 104(5)