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A 3-gene signature in DLBCL
included the ABC and double expressor GCB/unclassified (GCB/U) DLBCL (hereafter defined as DEXP_mRNA); the low risk group was constituted by the non-DEXP_mRNA GCB/U subset. Since the expression levels of MYC and BCL-2 on one hand and NFKBIA on the other hand had opposing patterns being inversely associated with OS (with higher MYC/BCL-2 and lower NFKBIA levels associated with worse outcome), we combined the expres- sion levels of the three genes in a synthetic predictor called MBN- signature (MBN-Sig) and defined as:
MBN-Sig= (MYC + BCL-2)/NFKBIA
Detailed information on study cohorts (Online Supplementary Table S1), T-GEP procedures with list of genes and target sequences, fluorescence in situ hybridization (FISH), IHC methods and antibodies (Online Supplementary Table S2), and random forest (RF) classifier are described in the Online Supplementary Appendix.
Results
Univariate analyses and a decision-tree classification model integrating the cell of origin and MYC/BCL-2 status
Given their established clinical relevance, we first inves- tigated the prognostic significance of T-GEP-based COO classification and MYC/BCL-2 status in the R-HDS0305 and DLCL04 trials34,35 (discovery cohort). Patient’s charac- teristics are summarized in Table 1. In line with previous findings11,12 COO classification by T-GEP clearly outper- formed the immunohistochemical Hans algorhitm for sur- vival prediction and retained its prognostic significance in the presence or absence of ASCT consolidation (Online Supplementary Figure S1A to D). In order to investigate the prognostic impact of concurrent overexpression of MYC and BCL-2, we defined high and low expressors based on
Figure 1. Study algorithm. On the left, the discovery cohort is represented; 224 diffuse large B-cell lymphoma (DLBCL) patients enrolled in the DLCL04 (n= 130) and R-HDS0305 (n= 94) trials with available formalin-fixed, paraffin embedded (FFPE) tissue were initially considered in this analysis. Targeted gene expression profiling (T- GEP) success rate was 92.4% (n=207), with 17 cases not yelding enough high- quality mRNA to undergo successful GEP assessment. Only cases originally diagnosed as DLBCL non-otherwise specified (NOS) were considered. Therefore 21 cases classified in differ- ent DLBCL categories were excluded; 99 NOS-DLBCL FFPE patient samples from the DLCL04 trial and 87 samples from the R-HDS0305 trial were finally included in this study. On the right, the three validation cohorts: a cohort of 928 patients from Sha and coworkers27 (469 treated with R-CHOP; 459 with RB- CHOP), a public gene expression dataset (Affymetrix Human Genome U133 Plus 2.0 Array), GSE10846, (https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE1 0846), including 233 patients treated with R-CHOP regimen (Lenz et al. 2008)36; an additional validation cohort including 102 consecutive DLBCL NOS cases with available FFPE tissue, treat- ed with R-CHOP/R-CHOP-like regimens. RB-CHOP: R-CHOP plus bortezomib.
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