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Molecular background delineates outcome of double protein expressor diffuse large B-cell lymphoma

(ABC) DLBCLs, concurrent alterations were infrequent. Clinically, DPE DLBCL de ﬁ ned a prognostic entity, which was independent of the International Prognostic Index (IPI) and cell of origin, and together with the loss of TP53 had a synergistic dismal impact on survival. In the DPE DLBCL, the loss of TP53 was associated with a chemorefractory disease, whereas among the other DLBCLs, no correlation with survival was seen. Importantly, BCL6 translocations identi ﬁ ed non-GCB lymphomas with favorable BN2/C1-like survival independent of IPI and concurrent DPE status. Taken together, our ﬁ ndings de ﬁ ne molecular characteristics of the DPE in DLBCL, and recognize clinically feasible predictors of outcome. Given the emerging taxonomical signi ﬁ cance of BCL2 , BCL6 , MYC , and TP53 , our ﬁ ndings provide further depth and validation to the genomic classi ﬁ cation of DLBCL.


Introduction
The first genomic aberrations that were recognized in B-cell lymphomas were cytogenetic events concerning MYC, 7-9 BCL2, 10,11 and BCL6 12,13 proto-oncogenes. At present, the translational relevance of these translocations is being underlined in the proposed genomic models of DLBCL, where they have subtype dictating roles. 2,3 Moreover, tumors with DLBCL morphology and concurrent MYC and BCL2 or BCL6 translocations are assigned to a separate World Health Organization-defined entity named high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH). 14,15 This distinction is at least clinically justified because HGBL-DT/TH are strongly associated with poor outcomes, 16,17 although the prognosis of HGBL-DH/TH may be less dismal than previously reported. 18 Beyond molecular classification, DLBCLs with concurrent MYC and BCL2 protein expression (double protein expression [DPE]) are also recognized based on their clinically aggressive course. 19,20 Because the concomitant deregulation of MYC and BCL2 can be only partially explained by underlying double hit (DHIT) status, further deregulatory mechanisms are likely to exist. Recently, gene expression profiling studies identified additional DHIT and Burkitt-like GCB DLBCLs with similar clinical behavior as HGBL-DH/TH but no traceable concurrent lesions of MYC and BCL2. 21,22 Some of these lymphomas carry cryptic cytogenetic events that are not detected with routine methodology. 23 However, DPE DLBCLs can originate from either of the gene expression-based molecular subtypes and have remained a biologically poorly characterized entity in the advent of genomic subtyping with unmet clinical need.
Here, we have comprehensively profiled hallmark DLBCL genes, BCL2, BCL6, and MYC and dissected the molecular high-risk contexts that they represent. By integrating protein expression and translocation data with transcription profiles, copy number aberrations, and somatic driver mutations, we discover previously unknown associations between genomic events, gene expression, clinical characteristics, and treatment outcomes. We uncover a novel interplay between TP53 alterations and MYC deregulation in DLBCL, and recognize a subgroup of ultrahigh-risk DPE DLBCLs with concurrent genomic perturbations of TP53. Furthermore, we recognize BCL6 translocation as a major marker for favorable outcome in patients with non-GCB DLBCLs irrespective of concurrent DPE status. We integrate our discoveries with previous findings and recent advances in DLBCL pathogenesis and provide clinically feasible directives that narrow the gap between emerging genomic taxonomy of DLBCL, well-established prognostic markers and routine diagnostic procedures.

Patients and samples
Discovery cohort consisted of 181 patients with primary DLBCL treated according to institutional guidelines with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) or R-CHOP-like immunochemotherapy in Helsinki University Hospital, Finland, from 2002 to 2013.
Patients in this cohort were included in the 1001 DLBCLs study. 1 Patient-matched single nucleotide variations, copy number annotations of driver genes, and RNA sequencing-based COO were determined previously, and the data collected from the supplemental Material.
Validation cohorts comprised of datasets with mutation, transcriptome, and clinical data from 586 1 and 228 3 DLBCL patients. Additional information on the cohorts is provided in the supplemental Material.
The study was approved by the Ethics Committee in Helsinki University Hospital, Finland, the National Authority for Medicolegal Affairs, Finland, and an institutional review board.
Immunohistochemistry and fluorescence in situ hybridization GCB and non-GCB phenotypes were determined from the wholetissue sections as part of routine diagnostics according to the Hans algorithm. 24 Methodological details for BCL2, BCL6, and MYC immunohistochemistry and BCL2, BCL6, and MYC fluorescence in situ hybridization (FISH) are provided in the supplemental Material. In immunohistochemical (IHC) analysis, the cutoff level for BCL2 positivity was set to $50% of the tumor cells being reactive for BCL2 staining (henceforth BCL2 1 ). Qualitative BCL2 overexpression (henceforth BCL2 OE ) was based on staining intensities beyond physiological levels (supplemental Figure 1A). Nuclear staining of MYC in the tumor cells was assessed and cutoff level for positivity was set to $40% (henceforth MYC 1 ). Overexpression of MYC was defined with a cutoff of $70% tumor cell nuclei reactive for MYC staining (henceforth MYC OE ; supplemental Figure 2B). Results from BCL6 stainings were collected from the pathology reports.

Statistical analysis
Clinical data were analyzed using the IBM SPSS Statistics 24.0 software (IBM, Armonk, NY) or in R environment (.v3.4.3) using R package survival (v2.43-3). Differences in categorical variables were assessed with x 2 test. Mutation frequencies were compared using Fisher's exact test. Univariate and multivariate analyses were performed according to the Cox proportional hazard regression model. Survival rates were estimated with the Kaplan-Meier method and the differences compared using the log-rank test. A level of probability P , .05 was considered statistically significant. All comparisons were 2-tailed. Further information on data analyses are included in the supplemental Material.

Patient demographics
Patient demographics of the discovery cohort are described in supplemental Table 1. Based on the Hans algorithm and gene expression profiling, 53% and 45% of the patients were classified as GCB DLBCLs, respectively. Primary therapy was mostly R-CHOP and no treatment decisions were guided by molecular factors. During the median follow-up of 46 months, 32 patients had relapsed and 33 died. Five-year progression-free survival and overall survival (OS) rates were 72% and 75%, respectively.
Molecular context and consequences of BCL2, MYC, and BCL6 deregulation in DLBCL To understand the biological and clinical background and consequences of DPE B-cell lymphomas, we analyzed BCL2, MYC, and BCL6 oncogenes for their structural variations and protein expression and integrated the data with somatic driver mutations, genome-wide transcriptome, and extensive clinical data. The BCL2 locus was targeted by translocations in 24 (16%; BCL2 TL ), copy number gains in 16 (9%), and amplifications in 20 (11%) tumors. BCL2 copy number gains and high-level amplifications displayed similar clinical and biological features and were analyzed as a whole (BCL2 copy number gain/amplification [BCL2 GA ], 20% of the patients [n 5 36]; supplemental Figure 1B-D). As expected, BCL2 TL and BCL2 GA correlated with increased BCL2 gene expression and high BCL2 protein levels ( Figure 1A). Overall, 82 (55%) tumors were BCL2 1 and BCL2 OE was observed in 22 (15%) of the tumors (Table 1; supplemental Table 2). A total of 49 (60%) of the BCL2 1 cases had an underlying structural variation in the BCL2 locus; in all the cases with BCL2 OE , an underlying structural variation was detected ( Figure 1B).
BCL2 TL and BCL2 GA occurred in a mutually exclusive and subtype-specific fashion ( Figure 1C; Table 1). BCL2 TL was almost completely specific to the GCB subtype and clinically associated with advanced stage (Table 1). Differential gene expression (DGE) analysis between BCL2 TL and the other GCB DLBCLs identified only BCL2 itself ( Figure 1D). Similarly, BCL2 GA associated with ABC subtype and the BCL2 GA overexpressed genes included BCL2 and other, mostly 18q located genes, suggesting their involvement in the BCL2 amplicons ( Figure 1E; supplemental Table 3). As expected, the mutational features underlying BCL2 structural variations reflected subtype-specific features (supplemental Figure 1D-E). Taken together, our data suggest that structural variations of BCL2 provide a subtype-specific powerful means to specifically elevate BCL2 expression.
MYC locus was mostly altered by translocations (n 5 18; 14% of the cases, MYC TL ), which were enriched in the GCB subtype (Hans algorithm; Table 1). MYC TL s were associated with elevated MYC gene and protein expression ( Figure 2A). Overall, MYC copy number gains were rare (MYC GA , n 5 4), and they did not associate with increased gene expression of MYC or MYC OE (Figure 2A-B). MYC 1 was more common in elderly patients (n 5 46; 63% of cases; Table 1). Of the patients with MYC OE (n 5 16; 11% of cases), 53% harbored MYC TL , whereas the rest of the cases had no detectable structural variation in the MYC locus ( Figure 2B). MYC OE DLBCLs with no structural variants of MYC originated mostly from the non-GCB/ABC subtype, suggesting that additional mechanisms or cryptic translocations could deregulate MYC expression in these lymphomas.
We recognized that MYC TL cooccurred with structural variations of the BCL2 locus ( Figure 2C; 71% of the MYC TL lymphomas, double alteration B-cell lymphomas [DA BCL], see the following section). Besides BCL2 SVs, we recognized cooccurrence between MYC TL and somatic mutations of TP53, CREBBP, and EZH2 ( Figure 2C; false discovery rate [FDR] , 10%). On the protein level, we observed that MYC OE cooccurred with TP53 alterations (supplemental Figure 2B). As further evidence for concurrent loss of TP53 and MYC deregulation, we observed that in the rest of the 1001 DLBCLs cohort and in a separate dataset, TP53-altered DLBCLs were associated with elevated gene expression levels of MYC ( Figure 2D-E). Together, these data suggest that there is an      intricate association between the somatic loss of TP53 and MYC deregulation in DLBCL.
In the DGE analysis, we identified 39 differentially expressed genes between MYC TL and the rest of the DLBCLs ( Figure 2F; supplemental Table 4). MYC TL -associated genes contained CD10 (MME), supporting the enrichment of MYC TL in the GCB phenotype (Table 1). Interestingly, this association was also evident in the DGE analysis of MYC TL among the GCB DLBCLs, suggesting additional enrichment of MYC TL among CD10 1 GCB DLBCLs ( Figure 2G; supplemental Table 5). Meanwhile, MYC TL DLBCLs showed decreased expression of JAK-STAT signaling genes (JAK3, STAT3) and several other genes including EBI3, SEMA7A, BCL2A1, and CFLAR, which were recognized to be downregulated in the double-hit signature described by Ennishi et al. 22 On the protein level, the gene expression profile associated with MYC OE was strongly enriched for biosynthesis and metabolic pathways including nucleotide, amino acid, and ribosome biogenesis, transcription, translation, and telomerase activity (supplemental Figure 2C; supplemental Tables 6 and 7). In fact, the expression of telomerase reverse transcriptase was the most strongly MYC OE -associated gene recognized 25 (supplemental Figure 2C). Genes associated with inflammatory pathways, including cytokine, JAK-STAT, and T-cell receptor signaling pathways were suppressed in MYC OE DLBCLs, suggesting depletion of tumor-infiltrating immune cells in MYC OE lymphomas (supplemental Table 8).

DPE arises from heterogeneous molecular backgrounds that reflect COO-related features
Forty-eight (33%) of the DLBCLs in our cohort were DPEs (BCL2 $50% and MYC $40%). DPE was associated with a clinically highrisk disease (Table 1; high International Prognostic Index [IPI], advanced stage, and older age). DPE lymphomas were derived from either of the molecular subtypes with a slight overrepresentation of ABC/non-GCB DLBCLs (Table 1). Structural aberrations of BCL2 and MYC were detected in 29 (60%) and 15 (32%) of the DPE lymphomas, respectively ( Figure 3A). Apart from these alterations, the genomic drivers underlying DPE lymphomas reflected the established features of the molecular subtypes, and did not strikingly differ from the non-DPE lymphomas ( Figure 3A; supplemental Figure 3).
The DLBCL-like HGBL-DH/TH was detected in 8 (6%) cases from the whole cohort and represented 18% of the DPE lymphoma patients. All the HGBL-DH/TH lymphomas were GCB subtype according to the Hans algorithm, carried BCL2 TL , and were DPE (2 triple-hit lymphomas; Figure 3B; supplemental Table 2). Interestingly, we identified that beyond these canonical DHIT lymphomas, additional cases of the DPE GCB DLBCLs had a combination of MYC TL and BCL2 GA . Together with DHIT, these DA BCLs constituted 58% of the DPE GCBs (Table 1; Figure 3B). Besides MYC and BCL2 alterations, TP53 alterations were associated with DA BCL ( Figure 3B).
Among the non-GCB DPE lymphomas, the prevalence of DPE was similar between BCL6 TL and non-BCL6 TL non-GCB lymphomas (40% and 43%, respectively), suggesting that non-GCB DPE can arise from both BN2-or MCD-like ABC backgrounds. In comparison with GCB DLBCLs, concurrent alterations of BCL2 and MYC were uncommon (1/26 of the non-GCB DLBCLs). Taken together, concurrent structural variations of BCL2 and MYC are the major drivers of DPE among the GCB subtype, whereas the DPE ABC DLBCLs may arise from cryptic alterations or epigenetically dysregulated loci.
To recognize transcriptome perturbations characteristic for DPE DLBCL, we analyzed the expression levels of gene set exemplars reflecting lymphoma-related biological processes ( Figure 3C). As expected, DPE DLBCLs were associated with expression of gene sets associated with MYC protein expression, including pathways related to biosynthesis, metabolism, and cell cycle ( Figure 3C-D). Furthermore, DPE DLBCLs and especially GCB DA BCLs, were associated with lower expression of genes associated with different tumor-infiltrating immune cells and host response ( Figure 3E-F). Together, our findings suggest that the heterogeneity of DPE DLBCL reflects ABC-and GCB-related differences in genome, transcriptome, and tumor microenvironmental levels.

Clinical behavior of MYC and BCL2 deregulation is dependent on the molecular context
To get an overview of the clinical attributes of the studied molecular characteristics, we performed univariate Cox regression analyses to determine their associations with the risk of death within the whole cohort ( Figure 4A). As previously established, high IPI scores, Hans algorithm-based non-GCB, and gene expression-based ABC subtypes associated with poor OS ( Figure 4A). In the whole study cohort, both BCL2 and MYC protein expression was associated with poor survival, and overexpression of these proteins associated with even less favorable survival ( Figure 4A). As expected, DPE DLBCL was a powerful predictor for poor outcome in the entire cohort ( Figure 4B). This association was also independent of IPI and COO in the multivariate analysis ( Figure 4B, models 1 and 2).
To recognize subtype-dependent outcome predictors, we performed survival analyses stratified according to immunohistochemically defined subtypes ( Figure 4A; for RNAseq-defined subtypes, see supplemental Figure 4A-B). In the GCB DLBCLs, MYC TL translated to poor outcome ( Figure 4A). At the protein level, BCL2 1 or MYC 1 did not associate with survival, whereas BCL2 OE and MYC OE translated to adverse outcome ( Figure 4A). As expected, concurrent deregulation of MYC and BCL2, DPE, HGBL-DH/TH, and DA BCL predicted poor outcome ( Figure 4A). Importantly, DA BCL could recognize additional GCB patients with refractory course that did not meet HGBL-DH/TH criteria ( Figure 4A,C). Of note, all patients with no response to therapy (progressive or stable disease) in the discovery cohort were DA BCLs, and all events recorded in these patients occurred during the first 24 months of follow-up. 18 Similar effect was seen in independent validation set (supplemental Figure 4C). In multivariate analysis with IPI, DPE   In the non-GCB DLBCLs, BCL2 GA had a prominent adverse impact on survival independent of IPI ( Figure 4D, model 1). Interestingly, expression of BCL2 (BCL2 1 or BCL2 OE ) did not recapitulate this effect on survival ( Figure 4A). When BCL2 GA was analyzed among the ABC subtype defined according to the gene expression profile, a trend toward poor survival was seen (supplemental Figure 4B). Meanwhile, MYC OE was identified as a strong predictor of outcome in the patients with non-GCB DLBCL ( Figure 4A). When MYC OE was included in the multivariate analysis with IPI and BCL2 GA , all markers remained independent predictors of poor survival ( Figure 4D, model 2). In addition, DPE status was found to result in a trend toward poor survival among the non-GCB DLBCLs ( Figure 4A).
BCL6 TL was found to associate with excellent survival in the patients with non-GCB and ABC DLBCL ( Figure 4A,E; supplemental Figure 4B). The association was independent of IPI and could be validated in an independent cohort ( Figure 4E, model 1; supplemental Figure 4D). The outcomes of the BCL6 TL non-GCB/ ABC DLBCLs were comparable with the non-HGBL-DH/TH GCB DLBCLs (5-year OS; 93% vs 85%, respectively). Interestingly, among the non-GCB DLBCLs with no BCL6 TL , DPE translated to poor survival (supplemental Figure 4E). Conversely, BCL6 TL associated with good outcome among the non-GCB DPE lymphomas (supplemental Figure 4F-G)  Overall survival  Finally, because we recognized that DPE status was associated with poor survival independent of COO and IPI scores in the whole cohort, we extended our stratified approach to DPE DLBCLs ( Figure 5A). When the impact of IPI or molecular subgroups on outcome was assessed in the patients with DPE DLBCLs, no significant differences in survival were found ( Figure 5A). Neither did we did observe differences in survival between the HGBL-DH/TH or DA lymphomas and other DPE DLBCLs ( Figure 5A). However, MYC OE was associated with poor survival in the DPE DLBCLs ( Figure 5A). Given the cooccurrence of TP53 alterations and MYC deregulation in our cohort, we then examined the effect of TP53 alterations on the outcome in DPE DLBCL (10/48 patients). The association of TP53 alterations with poor outcome was restricted solely to the DPE phenotype and was not seen in the patients with non-DPE DLBCL or in the whole cohort ( Figure 5B-C; supplemental Figure 5A). In the DPE DLBCL, point mutations and copy number losses of TP53 showed similar clinical behavior, suggesting that loss of TP53 in the context of DPE identifies a subset of ultrahighrisk lymphomas with treatment refractory clinical course (supplemental Figure 5B). In separate validation cohorts of 586 1 and 228 3 patients with double expression defined according to gene expression, a similar synergistic effect on survival was seen ( Figure 5D-E).

Discussion
Recently identified genetic signatures beyond the COO-based lymphoma classification have not only provided new insights to the pathogenesis of DLBCL but also established a platform for clinical interventions. Meantime, however, the biological background of a clinically relevant prognostic entity, DPE B-cell lymphoma, has remained poorly described. Here, we have comprehensively characterized BCL2, BCL6, and MYC oncogenes and dissected the molecular and clinical background of well-recognized HGBL-DH/TH and DPE entities that they establish. Our results elucidate the molecular underpinnings of high-risk lymphoma and provide additional evidence for many recent findings. Furthermore, our approach elucidates interactions between different prognostic factors and identifies relevant biomarkers.
Deregulation of BCL2 expression is attributed to the subtypespecific structural variations of the BCL2 locus, which underlie BCL2 overexpression and often DPE. We highlight BCL2 GA for its clinical and translational relevance. First, BCL2 GA was associated with dismal survival within the non-GCB subtype, 26  was not recapitulated by BCL2 immunohistochemistry among the mostly BCL2 1 non-GCB lymphomas. Second, the genomic background of BCL2 GA ABC DLBCLs displayed similarities with the C5-cluster characteristics identified by Chapuy et al, 2 further suggesting that BCL2 GA is a feasible marker for bona fide ABC and C5-like DLBCLs among the heterogenous non-GCB entity. Third, among the GCB subtype, additional HGBL-DH/TH-like treatmentrefractory lymphomas with poor outcomes can be identified when "functional DHIT" of BCL2 GA and MYC TL is considered. 28 Together, our results justify copy number analysis and guide interpretation of BCL2 amplifications in clinical practice to aid risk stratification and molecular phenotyping upon diagnosis.
Interestingly, we did not identify any association between BCL6 TL and elevated gene expression or BCL6 immunoreactivity. Yet, of clinical relevance, BCL6 TL recognized one-quarter of non-GCB DLBCLs with excellent survival, although 40% of these lymphomas were also DPEs. Our results support rationale for profiling BCL6 translocations in non-GCB DLBCLs to recognize lymphomas with a favorable BN2 3 and C1 2 -like clinical behavior. So far, the interpretation has been that these non-GCB DLBCLs resemble marginal zone B-cell lymphomas genetically, and are likely to have COO within the non-GCB/ABC lymphomas different from their MCD/C5-like counterparts. Importantly, our results clarify that the favorable outcome of these lymphomas is irrespective of DPE and BCL2 positivity.
MYC translocation partners are diverse, and a significant proportion of noncanonical MYC TL are missed even with routine widespread break-apart FISH-based approaches. 29 Recent seminal works have applied nonconventional next-generation sequencing-based techniques to detect translocation events that are not likely to recapitulate the diversity of MYC TL s, which were reported at lower frequencies than expected (8% 2 and not reported 3 vs 14% of the cases observed here). Indeed, both groups proposed highly concordant but MYC agnostic genomic classification models. Chapuy et al reported a genetic subgroup (C2) characterized by loss of TP53 that was associated with a dismal clinical course 30,31 and aneuploidy genotype suggesting chromosomal instability. 32 Although supported by recent findings by Wright et al, 33 the role of TP53-driven subtype remains under debate. 34 We found that TP53 mutations cooccur with MYC TL and further recognized a global association between MYC deregulation and loss of TP53. The referred-to pivotal studies were not designed to pursue the comprehensive characterization of the molecular background of DPE lymphoma. According to our findings, DPE lymphoma arises from heterogenous molecular and genomic backgrounds. In GCB DLBCL, the majority of the DPE lymphomas carry concomitant structural lesions of BCL2 and MYC (DA BCL), whereas in ABC/non-GCB DLBCLs, concurrent lesions are virtually absent, and DPE can arise from both BN2-/C1-like or MCD-/C5-like backgrounds. We speculate that in ABC/non-GCB DLBCL, deregulation on epigenetic, transcriptional, or posttranslational levels affect aberrant MYC protein expression. In DPE DLBCLs, transcriptomic features reflect MYC protein expression-related pathways and a less abundant immune cell infiltration that is especially characteristic for GCB DA BCLs.
Clinical characteristics of DPE DLBCL patients have also remained poorly characterized. Our findings associate DPE with a clinically high-risk disease at diagnosis. Importantly, despite the underlying potentially confounding associations with risk factors, DPE status has independent prognostic effect on survival. Although DPE and HGBL-DH/TH patients have been suggested to benefit from more intensive treatment regimens, such an effect has neither been studied nor demonstrated in post hoc analysis of the phase 3 Cancer and Leukemia Group B 50303 trial, 35 where DPE lymphoma patients were heavily underrepresented (16% of the Cancer and Leukemia Group B cohort vs 33% observed here, with the same cutoff). Furthermore, given the recognized positive correlation between DPE and age in our study, implementing more aggressive therapeutic approaches for these patients has limitations. Further clinical studies are needed on phosphatidylinositol 3-kinase or BCL2 inhibition for DPE and other BCL2deregulated lymphomas. 36 Other novel approaches including antibody-drug conjugates 37 or immunotherapies with T-cell engaging bispecific antibodies 38 and chimeric antigen receptor T cells 39,40 could provide much-needed new therapies for these patients.
Taken together, we have comprehensively characterized and clarified the clinical and molecular framework for DPE lymphomas. According to our findings, the heterogenous pathogenesis of the DPE reflects COO features, and the unfavorable impact on survival is dependent on the molecular context of the lymphoma. We highlight TP53 alterations as major prognostic markers for poor outcome among DPE entity, whereas BN2/C1-like DLBCLs with BCL6 TL s have favorable outcomes regardless of DPE. Our results provide rationale and update interpretation guidelines for FISH-and IHC-based profiling of primary DLBCL upon diagnosis. The data are of translational importance and narrow the gap between recent genome-wide findings 2,3 and clinically relevant diagnostic and riskevaluation measures.  Conflict-of-interest disclosure: S.L. reports consultancy and research funding (not related to this study) from Janssen-Cilag; consultancy, honoraria, and research funding (not related to this study) from Roche; consultancy, honoraria, and research funding (not related to this study) from Takeda; consultancy and honoraria from Novartis; research funding (not related to this study) from Bayer; consultancy and research funding (not related to this study) from Celgene; and consultancy from Gilead. The remaining authors declare no competing financial interests.