Page 126 - 2021_09-Haematologica-web

E. Derenzini et al.
Figure 5. The MBN signature identifies prognostically distinct subgroups including activated B cells and a fraction of germinal center B cells/ unclassified diffuse large B-cell lymphoma (DLBCL) enriched in double hit DLBCL cases, providing opportunities for precision therapies. (A) Overall survival (OS) of the germinal center B cells/ unclassified (GCB/U) subset in the discovery cohort (n=146 patients) according to the integration of the targeted gene expression profiling (T-GEP) panel (Lymph2Cx) with the MBN signature (MBN-Sig), distinguishing two risk categories according to the MBN-Sig. (B) Heatmap representing the three informative genes of the MBN-Sig shown as rows and diffuse large B-cell lymphoma (DLBCL) tissue samples shown as columns, in the discovery cohort (n=186 patients). (C) OS of the GCB/U subset in the Sha’s validation cohort (n=340 patients) according to the MBN-Sig, showing superimposable results compared to Figure 4C. (D) Heatmap rep- resenting the three informative genes of the MBN-Sig shown as rows and DLBCL tissue samples shown as columns, in the Sha’s cohort (n=469 patients treated with R-CHOP). (E) Progression-free survival (PFS) of patients treated with R-CHOP versus RB-CHOP in the MBN-Sig high subgroup (Full Sha’s cohort, n=928 patients; MBN- Sig high n=464 patients). (F) OS of patients treated with R-CHOP versus RB-CHOP in the MBN-Sig high subgroup (Full Sha’s cohort, n=928 patients; MBN-Sig high n=464 patients). In all panels the P-value was calculated with the log rank test. NA: not available; DH: double hit; DE: double expressor (based on DEXP_mRNA sta- tus); aaIPI: age adjusted international prognostic index; COO: cell of origin; IPI: international prognostic index); MHG: molecular high grade. RB-CHOP: R-CHOP plus Bortezomib.
(P=0.012) (Figure 5E), which translated in an increased OS rate (P=0.052) (Figure 5F).
Discussion
In this study we applied a customized T-GEP panel (including the Lymph2Cx signature for COO classification and additional genes of potential prognostic and therapeu- tic interest) to two randomized trials34,35 (n=186 patients) performed in the Rituximab era. The aims of this study were the integration of the COO with additional GEP- based variables, and the identification of a gene signature applicable to routine clinical practice, able to refine current prognostication algorithms. The genes of the T-GEP panel were selected considering the relevance of the respective signaling pathways in B-cell lymphomagenesis, but more importantly based on their potential druggability.
Our study confirmed the prognostic value of GEP-based COO determination, which clearly outperformed the IHC-based Hans algorithm (the ABC DLBCL subgroups having a significantly inferior OS in all case series evaluat- ed here) (Online Supplementary Figure S1). The COO retained its prognostic value in patients undergoing ASCT consolidation, suggesting that therapy intensification is not able to overcome the negative prognostic value of the COO. A recursive partitioning analysis integrating COO with MYC/BCL-2 DEXP_mRNA status identified three main subgroups (a low risk non-DEXP_mRNA GCB/U subset and two high-risk groups including DEXP_mRNA GCB/U and ABC-DLBCLs) (Figure 2A). The observation of lower NFKBIA levels in the ABC and DEXP_mRNA GCB/U subgroups (overexpressing MYC and BCL-2 to a similar extent) (Figure 2B) suggests that, despite known biologic differences, these DLBCL subsets could share similar oncogenic dependencies on MYC, BCL-2 and the NF-kB pathway (being NFKBIA a negative regulator of NF-kB signaling). This observations prompted us to design a three-gene prognostic signature integrating MYC, BCL-2 and NFKBIA, which we called the MBN signature. The signature was first tested in our discovery cohort of 186 patients, identifying two subgroups characterized by dif- ferent outcome (Figure 3), and was then applied to three independent datasets (469 patients treated with R-CHOP in the Sha cohort,27 233 patients from the Lenz cohort,36 and 102 patients treated in real-life clinical practice with R-CHOP/R-CHOP-like regimens) confirming its high prognostic significance (total number of tested cases 990). Since the discovery cohort had some unique characteris- tics (such as lack of low aa-IPI cases, a relatively low frac- tion of ABC cases and no uniform first-line treatment), the extensive validation performed in three additional cohorts treated with R-CHOP/R-CHOP-like regimens confirms
that the key findings of the present study are indeed appli- cable to an unselected DLBCL population. Importantly, the MBN signature defined a high-risk group including a significant fraction of ABC cases (in line with data shown in the Online Supplementary Table S3 demonstrating a high- er incidence of MYC/BCL-2 DEXP_mRNA and low NFKBIA expressors in the ABC subgroup), and about 30% of GCB/U cases (Figure 3). Therefore the MBN signature could potentially identify an increased proportion of patients at high risk of treatment failure, compared to standard risk stratifications (COO or DE status). The MBN signature was an independent prognostic predictor, out- performing the MYC/BCL-2-DEXP_mRNA status in multi- variate analyses (Figure 3), thus confirming the added value of the third gene (NFKBIA) for prognostic stratifica- tion. The possible clinical applicability of the MBN signa- ture was tested in the real-life cohort using an RF predic- tion model built on the discovery cohort, providing a reli- able tool for prospective risk stratification (Figure 4). Importantly, the integration of the MBN signature with the COO allowed the identification of two risk categories whithin the GCB/U subset. These findings, which were validated in independent cohorts, could have immediate implications (Figure 5A and C; Online Supplementary Figure S6A to D). Two recently published studies confirmed the heterogeneity of the GCB subgroup and identified gene signatures allowing better risk stratification of this patient subset.26,27 These signatures were able to identify a pro- portion of HG-BCL with DH/TH and a further group lack- ing MYC/BCL-2 re-arrangements but characterized by similar clinical features. However, the fact that these sig- natures are composed by several genes encompassing multiple pathways, could make their successful transla- tion to clinical practice and precision therapy approaches quite challenging.
Our data are in line with these findings confirming that the GCB/U DLBCL subset represents indeed a rather het- erogeneous disease category. The MBN signature could identify the majority of tumors with high-grade molecular features (HG-BCL with DH/TH) in the discovery cohort and Sha’s cohort (Figure 5B and D; Online Supplementary Figure S7A and B). Moreover, by applying the MBN signa- ture to the Sha validation cohort we observed that the MBN-Sig high subroup was significantly enriched in MHG DLBCL cases (Figure 5D; Online Supplementary Figure S7B). Taken together, these data indicate that a sim- ple there-gene signature could efficiently identify high risk GCB/U DLBCL cases. Furthermore, the MBN-signature is based on potentially druggable targets or pathways. For example, NFKBIA (encoding for IkB-α) could be targeted by proteasome inhibitors43-45 and by bromodomain and extraterminal protein (BET) inhibitors, which are able to downregulate MYC while increasing IkB-α levels.46-48 Our
2414
haematologica | 2021; 106(9)