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E. Derenzini et al.
Introduction
The biologic complexity of diffuse large B-cell lym- phoma (DLBCL) was first dissected in the early 2000s by gene expression profiling (GEP) studies, which subdivided DLBCL into two groups based on GEP signatures reminis- cent of the respective cell of origin (COO). These studies showed that DLBCL with a gene signature related to acti- vated B lymphocytes (ABC subgroup) had a significantly worse response to anthracycline-based therapies com- pared to those histogenetically related to germinal center B cells (GCB subtype), and were dependent on nuclear fac- tor k-B (NF-kB) signaling.1-3 Since immunohistochemical algorithms failed to reproduce the results of GEP,4-10 the Lymphoma Leukemia Molecular Profiling Project (LLMPP) proposed a targeted GEP (T-GEP) panel (Lymph2Cx) desumed from previous studies on fresh/frozen tissue (FFT).11,12 This assay was applied on the NanoString plat- form to formalin-fixed, paraffin embedded (FFPE) tissue from DLBCL patients treated with R-CHOP,11,12 identify- ing three subgroups: GCB, ABC and unclassified, the latter representing about 15% of all cases and prognostically closer to the GCB.11,13 The reproducibility of this assay was confirmed in several studies.131-5 However, recent results from three independent phase III randomized tri- als16-18 based on COO classification were largely negative. Although these unsatisfactory results could be due to sev- eral reasons, including unexpected toxicities and subopti- mal efficacy of these drugs in vivo, these data also indicate that the clinical development of predictive T-GEP signa- tures able to complement the COO for precision therapy approaches is an urgent unmet need. Besides the COO, current evidence indicates a negative prognostic value of double MYC and BCL-2 protein overexpression deter- mined by immunohistochemistry (IHC).19-21 Furthermore those DLBCL with concurrent MYC and BCL-2 and/or BCL-6 genomic rearrangements are characterized by an even worse prognosis, being now classified as a separate entity, high-grade B-cell lymphoma (HG-BCL) with dou- ble/triple hits (w DH/TH).19,20,22 Recently large genomic studies integrating DNA and RNA sequencing data identi- fied additional DLBCL subgroups beyond the COO and MYC/BCL-2 double expressor (DE) status,23-25 based on the mutational landscape, GEP signatures, copy number changes, and differences in outcome. Furthermore, recent studies identified GEP signatures able to define high-risk populations within the GCB/unclassified (GCB/U) sub- group.26,27 However, given their complexity, large-scale application of these prognostication algorithms could be difficult in daily clinical practice. The aim of this study was the implementation of a simple T-GEP panel able to complement and improve COO-based prognostic strati- fication for routine clinical application. We designed a panel of genes corresponding to those of the Lymph2Cx assay for COO determination plus additional candidates selected because of their potential prognostic and/or therapeutic interest including MYC, BCL-2 and central nodes of NF-kB, Janus kinase (JAK)/signal transducer and activator of transcription (STAT), and phosphatidylinosi- tol-3 kinase (PI3K) signaling.3,28-33 This panel of genes was applied to 186 DLBCL enrolled in two recently reported large Italian trials (DLCL04 and R-HDS0305; clinicaltrials gov. Identifier: NCT00355199 and NCT00499018).34,35 We found that a three-gene signa- ture based on MYC, BCL-2 and NFKBIA (MBN signature), identified a significant fraction of ABC cases and a sub-
group of GCB/U cases (roughly 30%) enriched in HG-BCL w/DH, at increased risk of treatment failure. These data were validated in a real-life cohort and in silico in two large independent series, including one cohort of patients enrolled in the REMoDL-B trial,18,27 where the addition of bortezomib to chemoimmunotherapy provided a signifi- cant advantage for high-risk patients identified by the MBN signature.
Methods
Study design
Patients considered in this study had been enrolled in two prospective randomized phase III clinical trials investigating the role of first line autologous stem cell transplant (ASCT) consolida- tion in intermediate/high-risk DLBCL.34,35 Only cases of DLBCL not-otherwise specified (NOS) (including those originally diag- nosed as DLBCL and nowadays included in the HG-BCL provi- sional category22) were selected for the present study (Figure 1). Patients’ characteristics and study algorithm are summarized in Table 1 and Figure 1.
Results were validated in three independent cohorts, (two in silico validation datasets and one “real-life” cohort): a dataset from Sha and coworkers (n=928 patients: 469 treated with R- CHOP and 459 with R-CHOP plus bortezomib [RB-CHOP]);27 a public dataset from Lenz et al.36 (n=233 patients treated with R- CHOP); a “real-life” cohort including 102 consecutive DLBCL- NOS cases with available FFPE tissue, treated with R-CHOP/R- CHOP-like regimens in Bologna (S.Orsola-Malpighi Hospital), and in Milan (European Institute of Oncology) from 2007 to 2018.
This study was approved by the Institutional Review Boards and Ethics Committees of the participating centers, in accordance with the Declaration of Helsinki.
Procedures
Gene expression was measured on the NanoString nCounter Analysis System (NanoString Technologies, Seattle, USA). The T- GEP panel contains 26 genes: 15 genes for COO subtyping;11 five housekeeping genes (UBXN4, ISY1, R3HDM1, WDR55, TRIM56); and six additional genes (MYC, BCL-2, STAT3, NFKBIA, PTEN, PIK3CA). Besides MYC and BCL-2, the additional genes were selected based on their known functions in key pathways involved in DLBCL lymphomagenesis and potential druggability.
Statistical analysis
Survival data were analyzed retrospectively. We used Kaplan- Meier method37 for overall survival (OS) and progression-free sur- vival (PFS) analyses. Multivariate and univariate analyses were constructed with the Cox proportional hazards regression model. A P-value ≤0.05 was considered statistically significant. Recursive Partitioning Analysis (RPA)38 was applied to classify patients into more homogenous prognostic groups based on survival. All analy- ses were performed using R 3.5.0 software.39 Correlations and dif- ferences in patient characteristics were analyzed with the χ2 and Fisher’s exact test.
Development of the three-gene prognostic signature (MBN signature)
An expression ratio-based test was developed by selecting those genes significantly deregulated in the high risk subgroups identi- fied by the RPA shown in Figure 2A and whose normalized mRNA levels were significantly associated with OS. We defined high and low MYC and BCL-2 expressors based on the median normalized MYC and BCL-2 mRNA levels. The high-risk groups
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haematologica | 2021; 106(9)