Activated B-cell Subtype Research Articles

Polatuzumab Vedotin in Combination with Immunochemotherapy in Patients with Previously Untreated DLBCL: A Phase 1b/2 Study

Polatuzumab Vedotin in Combination with Immunochemotherapy in Patients with Previously Untreated DLBCL: A Phase 1b/2 Study

BCL2 expression is associated with a poor prognosis independent of cellular origin in primary central nervous system diffuse large B-cell lymphoma.

Primary central nervous system diffuse large B-cell lymphoma (CNS-DLBCL) is a distinct clinicopathological entity with a poor prognosis. Concurrent MYC and BCL2 overexpression predicts inferior prognosis in systemic DLBCL, although their prognostic significance remains unclear in primary CNS-DLBCL. Pretreatment diagnostic biopsy samples were retrospectively evaluated for 79 patients with primary CNS-DLBCL who were treated between January 2001 and December 2017. Histological and immunohistochemical testing were performed to evaluate the patients' statuses for various markers, which were also evaluated for associations with survival outcomes. According to the Hans criteria, 26 patients (32.9%) had the germinal center B-cell subtype and 53 patients (67.1%) had the activated B-cell subtype. Forty-one cases (51.9%) were positive for MYC (expression of ≥ 40%), 33 cases (41.8%) were positive for BCL2 (expression of ≥ 70%), 22 patients (27.8%) were positive for both MYC and BCL2, and 27 patients (34.2%) were negative for both MYC and BCL2. There were no significant differences in survival between the germinal center and activated B-cell subtypes. Furthermore, MYC positivity was not associated with overall survival (p = 0.369) or progression-free survival (PFS) (p = 0.253). However, BCL2 positivity was significantly associated with poor overall survival (p = 0.039) and PFS (p = 0.036). Co-expression of MYC and BCL2 was not associated with survival. Our data suggest that evaluating BCL2 expression may help predict the prognosis in cases of primary CNS-DLBCL.

Abstract CT162: ACCEPT: A phase Ib/II combination of acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for patients with diffuse large B-cell lymphoma (DLBCL)

Abstract Background DLBCL is the most common non-Hodgkin lymphoma (NHL) comprising one third of all adult NHL cases, accounting for an annual incidence in the UK of approximately 5,000 patients. R-CHOP has been accepted as the international standard first-line regimen, with cure rates of around 60-70%. There is a need to further improve efficacy of first line treatment. Chronic and tonic B-cell receptor (BCR) signaling has been identified as a feature of DLBCL. Preclinical models of this lymphoma have demonstrated cytotoxicity through interruption of BCR-signaling. Inhibition of Bruton's tyrosine kinase (BTK), downstream of the BCR has proven active clinical trials in DLBCL, particularly in the activated B-cell subtype. The second generation BTK inhibitor, acalabrutinib, has demonstrated improved target specificity and bioavailability, with potential for better efficacy and tolerability compared to earlier agents. It is appealing to combine this targeted agent with R-CHOP in untreated de novo DLBCL to understand its safety profile and efficacy. Method Patients must be 16 years or older, treatment naive with histologically confirmed DLBCL. All will receive 6 cycles of R-CHOP chemotherapy on a standard 21-day schedule, with the addition of acalabrutinib in cycles 2-6, to allow determination of the subtype by whole transcriptome gene expression profiling during cycle 1. This will be followed by a continuation phase of acalabrutinib only, for 2 cycles of 28 days. The primary objective of the phase I is to establish a recommended phase II dose of acalabrutinib in combination with R-CHOP. The primary objective of the phase II is to assess activity of the combination (by overall response rate) and determine additional safety information. Secondary endpoints include duration of response, PFS and OS and their relation to the cell of origin subtype, and BTK receptor occupancy in peripheral blood mononuclear cells and tumor measured by a drug analogue enzyme-linked immunosorbent assay. The effect of acalabrutinib on antibody-directed cellular cytotoxicity mediated by rituximab will be measured in vitro during treatment. The trial is coordinated by the Cancer Research UK Southampton Clinical Trials Unit. It will recruit up to 24 patients during phase I in a modified classical 6+6 design, and 15 patients during phase II. This is an investigator initiated study that has been granted free access to investigational medicinal product, trial Management and translational study support through a grant from Acerta Pharma B.V. (IST-LY-801) and has endorsement from Cancer Research UK (CRUKDE/16/006). Trial registration: ISRCTN11965217 Progress Phase I of the study opened in April 2017 and has recruited 6 eligible patients to the first cohort at a dose of 100mg acalabrutinib once daily, with a further 4 patients recruited to the second cohort at a dose of 100mg twice daily. Citation Format: Peter W M Johnson, Joshua Caddy, Tom Cumin, Bridgen Merton, Tom Maishman, Angelic Galanopoulou, Gareth Griffiths, Andrew Davies. ACCEPT: A phase Ib/II combination of acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for patients with diffuse large B-cell lymphoma (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT162.

Comparison of the algorithms classifying the ABC and GCB subtypes in diffuse large B-cell lymphoma.

Different immunohistochemical algorithms for the classification of the activated B-cell (ABC) and germinal center B-cell (GCB) subtypes of diffuse large B-cell lymphoma (DLBCL) are applied in different laboratories. In the present study, 127 patients with DLCBL were investigated, all treated with rituximab and cyclophosphamide, hydroxydaunorubicin, oncovin and prednisone (CHOP) or CHOP-like regimens between April 2004 and December 2010. Multi-tumor tissue microarrays were prepared and were tested according to 4 algorithms: Hans; modified Hans; Choi; and modified Choi. For 39 patients, the flow cytometric quantification of CD19 and CD20 antigen expression was performed and the level of expression presented as molecules of equivalent soluble fluorochrome units. The Choi algorithm was demonstrated to be prognostic for OS and classified patients into the GCB subgroup with an HR of 0.91. No difference in the expression of the CD19 antigen between the ABC and GCB groups was observed, but the ABC subtype exhibited a decreased expression of the CD20 antigen compared with the GCB subtype.

BCL6/BCL2 and MYC Alterations Drawing the Gray Zone between Diffuse Large B-cell Lymphoma and Double, Triple Hit Lymphoma

Diffuse large B-cell lymphoma (DLBCL) Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non- Hodgkin lymphoma that develops from the B-cells in the lymphatic system. Under the microscope, large malignant lymphocytes are seen diffusely throughout the specimen. Diffuse Large B-Cell Lymphoma (DLBCL) accounts for 20– 30% of all lymphoid malignancies. This subtype forms distinct morphological, molecular, and phenotypic variants. Therefore, it is of a great value to classify diffuse large B-cell lymphoma according to gene expression profiling into germinal center B-cell subtype, activated B-cell subtype, and unclassified subtype. The outcomes vary between these subtypes, and the germinal center B-cell subtype has a better outcome with the current standard rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP) immunochemotherapy.

Tonic B-cell receptor signaling in diffuse large B-cell lymphoma

AbstractWe used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO–induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.

Abstract 3939: Evolution and drug resistance of primary and relapse tumors in diffuse large B-cell lymphoma

Abstract Our integrated multi-omics analysis of paired primary and relapse DLBCL tumors provides comprehensive view to tumor evolution, drug resistance and disease heterogeneity. DLBCL is an aggressive systemic disease in which tumors can arise in virtually any part of the body. Activated B-cell (ABC) and germinal center B-cell (GCB)-like subtypes differ molecularly and clinically. Three-year progression-free survival (PFS) in ABC is around 40% vs over 80% in GCB. Whole genome (WGS, 7 patients), transcriptome (total RNA, 7 patients) and methylome sequencing data produced using the Illumina HiSeq platform was complemented with Illumina TruSeq amplicon sequencing covering 102,571 bp of the genome on 788 custom selected targets. Altogether 23 fresh frozen and 28 paraffin embedded tumor samples from 24 patients with an advanced disease and at least one relapse event were included in the study. To investigate the clonal hierarchy of the primary and relapsed tumors we employed dirichlet process clustering using cancer cell fraction (CCF) estimates or variant allele frequencies from somatic point mutations. Divergent evolution was prominent in the GCB subtype whereas in ABC subtype subclonal mutations are rare and only few genomic differences were detected between primary and relapse tumors (p-value=0.047, Pearson's Chi-squared test on 7 patients with WGS data) suggesting pre-treatment drug resistance which is probably triggered by global hypo- or hypermethylation at relapse stage. In the GCB subtype primary and relapse branches have diverged much before diagnosis and subsequently developed independently. Number of emerging primary or relapse specific clonal and subclonal mutations correlate with PSF and overall survival (OS). Mutational signatures were concordant with the inferred evolutionary models in four out of five GCB cases. Contribution of the somatic hypermutation signature was higher in resistant tumors and correlated with time to progression of the disease. We identified 72 relapse tumor related genes including many known B-cell lymphoma genes such as CD79B, IGLL5, PIM1, SGK1. These were used in survival analysis in the CGCI RNA data set derived from 92 diagnostic DLBCL tumors. Patients with subclonal mutations in these genes had shorter PSF and OS (Kaplan-Meier p-values 0.0122 and 0.0071, respectively). To validate and further define our hypothesis we are simulating DLBCL evolution as a discrete time branching process model (Galton-Watson branching processes). The goal is to reconstruct phylogenetic trees from a simulated tumor and explain different types of evolutionary models inferred from the patient data. Despite of remarkable heterogeneity in DLBCL we observed two main evolutionary models which are concordant with disease subtype, patient’s survival, treatment response at diagnosis and relapse, and molecular features of a tumor. Citation Format: Rainer Lehtonen, Amjad Alkodsi, Kaiyang Zhang, Mikko Kivikoski, Emilia Kozlowska, Alejandra Cervera, Annika Pasanen, Harald Holte, Sirpa Leppä, Sampsa Hautaniemi. Evolution and drug resistance of primary and relapse tumors in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3939. doi:10.1158/1538-7445.AM2017-3939

Pan‐lymphoma classification

Introduction: Cell of origin classification of diffuse large B-cell lymphoma (DL-BCL) using gene expression signatures identifies patients with distinct molecular features and survival outcomes. Whilst the identification of activated B-cell (ABC) and germinal center B-cell (GCB) subtypes of DL-BCL is now routinely used in diagnostics, however, a significant proportion of patients display gene expression signatures that are intermediate between classes. Whilst some of these intermediate cases display genuine hallmarks of both ABC or GCB subtypes, leading to an intermediate DL-BCL COO-UNC type, others appear to display distinct molecular signatures. It is possible that that these signatures represent inherent noise in the expression profiling process, or biological noise resulting from, for example, high levels of T-cell infiltration in samples. A more intriguing possibility is that these cases represent intermediate types between DL-BCL and other types of lymphoma. The existence of intermediate molecular signatures raises important questions about which type of treatment is most applicable for these cases. Yet the identification of intermediate molecular signatures is challenging, because existing classification algorithms have tended to focus on a small subset of lymphoma types rather than across the pan-lymphoma spectrum. Moreover, it is not clear what features would be needed to identify the relationship between patients to diverse lymphoma classes. Methods: To address this issue, we have developed a pan-lymphoma classifier using a support vector machine (SVM) with an embedded recursive feature selection (RFS) algorithm. Our training and test dataset consisted of 431 samples, spanning eight different types of lymphoma: ABC (49), GCB (133), COO-UNC (40), Burkitt's (60), Hodgkin's (43), PMBL (35), PBL (39), and Plasmacytoma (32). Gene expression profiles for each sample were obtained using the Illumina DASL platform. We trained the SVM using 10-fold cross-validation with a 70:30 train/test split. On each iteration of the RFS, we removed the single least informative probe. We repeated this until a significant drop in average accuracy of the SVM was reached. Results: Initially training the SVM on 1125 probes, we obtained a pan-lymphoma average classification accuracy of 91%. We then used the SVM-RFS to obtain a list of just 23 probes that gave rise to an average classification accuracy of 86%. This allowed us to develop a between-class distance metric as a score of the relative association of any given sample to a lymphoma class. Retrospective classification of COO-UNC samples showed some share molecular features associated with non-DL-BCL classes. Conclusions: SVM-RFS appears to be a robust approach for identifying classes of lymphoma from underlying molecular phenotypes. A small number of most informative genes provide a separation of cases into subtypes that is in excellent agreement with laboratory diagnoses. Keywords: activated B-cell–like (ABC); GCB lymphoma subtype; gene expression profile (GEP)

The Activated B-Cell Subtype of Diffuse Large B-Cell Lymphoma As Determined By Whole Genome Expression Profiling on Paraffin Embedded Tissue Is Independently Associated with Reduced Overall and Progression Free Survival in the Rituximab Era: Results from the UK NCRI R-CHOP 14 v 21 Phase III Trial

Abstract Introduction: The discovery of 3 distinct molecular subgroups of diffuse large B-cell lymphoma (DLBCL) according to cell-of-origin (COO): germinal centre B-cell (GCB), activated B-cell (ABC) and type III/unclassifiable by gene expression profiling (GEP) (Alizadeh et al, Nature 2000; Rosenwald et al, NEJM 2002) was a key advance in understanding the disease biology. The ABC subtype is associated with inferior survival which has persisted in the rituximab era (Lenz et al, NEJM 2008). Determination of COO by GEP has not been incorporated into routine practice however due to a lack of accessibility and requirement for fresh frozen tissue. Barrans et al (BJH 2012) recently demonstrated that COO could be accurately classified by the Illumina DASL® platform, using RNA extracted from routinely processed FFPE tissue from a population-based cohort of 172 R-CHOP-treated patients, and predicted clinical outcome. The aim of this analysis was to determine GEP-based DLBCL COO using the Illumina DASL® platform, to correlate results with outcome, and to validate this methodology using FFPE tissue samples from patients enrolled in the prospective phase III R-CHOP 14 v 21 trial. Methods: The UK NCRI R-CHOP-14 v 21 trial assessed R-CHOP given 2-weekly versus 3-weekly in 1080 previously untreated DLBCL patients aged ≥18 years and enrolled from 2005-2008. We previously reported that R-CHOP-14 was not superior to R-CHOP-21 for overall survival (OS), progression-free survival (PFS), response rate or safety. COO as determined by the Hans classifier (n=560) was not prognostic for OS (Cunningham et al, Lancet 2013). All patients with sufficient FFPE material remaining were included in this analysis. RNA was extracted and GEP was performed using the Illumina DASL® platform. Cases were classified as ABC, GCB or type III according to the DAC classifier (Care et al, Plos One 2013). Response was assessed using IWG 1999 criteria. PFS and OS were calculated from date of randomisation and analysed using Kaplan-Meier and Cox regression methods. Results: 369 patients had sufficient FFPE material remaining for analysis. COO classification was as follows: ABC 15.2% (n=56), GCB 46.3% (n=171), type III 38.5% (n=142). Baseline characteristics are shown in Table 1. Patients with GCB subtype had a significantly higher incidence of BCL-2 (p<0.001)and double-hit rearrangement (p=0.01) vs. those classified as ABC or type III. The median follow-up was 77.8 months. COO by GEP identified 3 distinct prognostic groups for OS (p=0.04) (Figure 1). 5-year OS for patients classified as ABC, GCB and type III was 60.5% (95% CI: 47.6-73.4), 81.4% (95% CI: 75.5-87.3) and 69.9% (95% CI: 62.1-77.7) respectively. Strong evidence of differences between these groups remained after adjusting for age as a continuous variable, International Prognostic Index (IPI) risk factors (WHO performance status, stage, presence of >1 extranodal site of disease, elevated LDH), sex, bulky disease, B symptoms and trial arm; patients classified as GCB or type III had superior OS versus ABC subtype (HR 0.53, 95% CI: 0.31-0.89; p=0.02) and (HR 0.56, 95% CI: 0.33-0.96; p=0.03) respectively. GCB subtype was also independently associated with superior PFS versus ABC subtype (HR=0.59, 95% CI: 0.37-0.96; p=0.03). The difference in PFS between type III and ABC subtypes did not reach statistical significance, but followed a similar trend (HR=0.65, 95% CI: 0.40-1.07; p=0.09). Results of univariate and multivariate analyses of individual factors and OS are shown in Table 2. Conclusion: Our results demonstrate that the ABC subtype of DLBCL as determined by GEP is independently associated with inferior PFS and OS. Our findings confirm those of Barrans et al (BJH 2012) and serve as a validation cohort for this methodology in the setting of a prospective trial where patients were exclusively R-CHOP-treated. Of note our patient cohort included a high proportion of type III/unclassifiable patients (38.5%) which are being further investigated currently and updated results will be presented at the meeting. Our analysis confirms that GEP-based COO is a significant prognostic biomarker for DLBCL in the rituximab era which can be accurately determined using routinely processed FFPE tissue samples. Table 1 Baseline characteristics Table 1. Baseline characteristics Figure 1. Overall survival by cell-of-origin determined by gene expression profiling group Figure 1. Overall survival by cell-of-origin determined by gene expression profiling group Table 2 Overall survival: univariate and multivariate analyses Table 2. Overall survival: univariate and multivariate analyses Disclosures Cunningham: Medimmune: Research Funding; Merrimack: Research Funding; Celgene: Research Funding; Bayer: Research Funding; Astra-Zeneca: Research Funding; Amgen: Research Funding. Hawkes:Merck Serono: Research Funding; Takeda: Other: travel expenses; BMS: Other: travel expenses, Research Funding. Pocock:Gilead Sciences: Other: Sponsorship to attend the EHA 2016 Meeting; Janssen: Speakers Bureau; Takeda: Honoraria. Ardeshna:Roche: Membership on an entity's Board of Directors or advisory committees, Other: Conference Expenses, Research Funding. Radford:Astra Zeneca: Equity Ownership; GlaxoSmithKline: Equity Ownership; Novartis: Honoraria; Seattle Genetics: Honoraria; Takeda: Honoraria, Research Funding.

Frequent Genetic Alterations of PI3K-AKT Pathway and Their Clinical Significance in Germinal Center B-Cell-like Diffuse Large B-Cell Lymphoma

▪Diffuse large B cell lymphoma (DLBCL) comprises two distinct molecular subtypes: germinal center B cell (GCB) subtype and activated B cell (ABC) subtype. The pathogenesis of ABC-DLBCL is characterized by two processes - the activation of NF-KB and a block in terminal B-cell differentiation. However, in GCB-DLBCL only a few biologically relevant pathways have been identified, which has hampered the development of targeted therapies with specific efficacy in this subtype.Recurrent genetic alterations involved in PI3K-AKT signaling pathway have been identified in the patients with solid cancers, and dramatic responses have been observed to PI3K inhibitors in clinical trials. The prevalence and clinical significances of genetic alterations in PI3K-AKT pathway have not been well studied in DLBCL. Previous studies have reported that loss of PTEN expression was observed in almost half of GCB-DLBCL cases but were largely absent in the ABC-DLBCL. In addition, functional studies have recently shown that the inactivation of Gα13 signaling pathway genes may also activate AKT in germinal center driven lymphoma, raising the possibility that additional genes within these pathways are affected in GCB-DLBCL. Herein, we identified genetic alterations involved in the PI3K-AKT pathway and evaluated their clinical impact.We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL uniformly treated with R-CHOP at the BC Cancer Agency. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Mutation status was determined using deep targeted re-sequencing of the coding exons of 61 genes with a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips, and RNAseq. Immunohistochemical staining of phospho-AKT (pAKT) was performed on tissue microarrays (n=332). Cell-of-origin (COO) was assigned by gene expression (Lymph2Cx assay) in 323 cases - 183 GCB, 104 ABC and 36 unclassifiable.GISTIC analysis revealed several COO-specific peaks with copy number changes. Among them, focal 10q23.3 deletion including PTEN was detected in GCB-DLBCL (q-value=1.7e-7), but not in ABC-DLBCL. We also identified two focal amplification peaks in GCB-DLBCL containing microRNAs MIR17HG (13q31.1;q=1.01e-24) and MIR21 (17q23.3; q=1.45e-6), which are known to down-regulate PTEN and result in activation of PI3K-AKT signaling. Furthermore, we detected recurrent INPP4B deletion (4q21.23) in GCB-DLBCL (q= 0.004) only. Of note, the lipid phosphatase INPP4B has been shown to play a role as a tumor suppressor that controls the levels of PI3K lipid products leading to AKT activation and metastasis of some solid cancers. This is consistent with the observation that PTEN and INPP4B deletions were individually associated with increased pAKT protein expression in our cohort (p=0.015 and p<0.0001, respectively).With respect to mutations, GNA13, P2RY8 and ARHGEF1 were more frequently mutated in the GCB than ABC subtype (26% vs 0.6%; p<.0001, 25% vs 7%; p=.0002, and 8% vs 0.5%; p=0.008, respectively). Previously known mutations of PI3KCA and PI3KCB were not recurrently observed in GCB-DLBCL.Based on these genetic data, we found that the PI3K-AKT signaling pathway was more commonly altered in GCB-DLBCL (64% (114/177)) than in ABC-DLBCL (23% (23/98) p<0.0001; Fig1). Consistent with this, pAKT protein expression was significantly higher in GCB-DLBCL than ABC-DLBCL (p<0.0001). The cases with higher pAKT protein expression (defined as the 25% highest pAKT protein expressors) were associated with significantly inferior outcome in GCB-DLBCL (5y-PFS 75% vs 59 %, p= 0.007 and 5y-OS 81% vs 64%, p=0.004; Fig2). At the genetic level PTEN and INPP4B deletions were individually associated with poor outcome in GCB-DLBCL (p=0.01 and p=0.045, respectively), and further, patients whose tumors harbored both genetic alterations had even worse prognosis (p= 0.004, Fig 3).In conclusion, genetic alterations in the PI3K-AKT pathway were frequently observed in GCB-DLBCL and were associated with activation of the pathway and inferior outcomes. These results suggested that AKT and mTOR inhibitors might be beneficial in a greater proportion of GCB-DLBCL than expected. [Display omitted] [Display omitted] DisclosuresSehn:roche/genentech: Consultancy, Honoraria; amgen: Consultancy, Honoraria; seattle genetics: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; TG therapeutics: Consultancy, Honoraria; celgene: Consultancy, Honoraria; lundbeck: Consultancy, Honoraria; janssen: Consultancy, Honoraria. Connors:F Hoffmann-La Roche: Research Funding; Millennium Takeda: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squib: Research Funding; NanoString Technologies: Research Funding. Scott:NanoString Technologies: Patents & Royalties: named inventor on a patent for molecular subtyping of DLBCL that has been licensed to NanoString Technologies.

Low Expression and Somatic Mutations of the KLHL6 Gene Predict Poor Survival in Patients with Activated B-Cell like Diffuse Large B-Cell Lymphoma

IntroductionDiffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease both clinically and biologically. Based on gene expression profiling, two major molecular subtypes, activated B-cell (ABC)- and germinal center B-cell (GCB)-like DLBCLs, have been recognized. More recently, next generation sequencing has also shed light on the mutational heterogeneity of these lymphomas. While several oncogenes are recurrently mutated and aberrantly expressed in DLBCL, little is known of their functional impact and association with survival.Design and MethodsTo identify mutated oncogenes in DLBCL, we performed whole-exome and RNA sequencing screen for tumor tissue and matched normal DNA from eight high-risk (aaIPI >2) DLBCL patients less than 65 years old. The patients were treated in the Nordic phase II protocol with dose-dense chemoimmunotherapy followed by systemic central nervous system (CNS) prophylaxis.Initial screen was expanded to a Nordic discovery cohort consisting of 38 high-risk DLBCL patients treated in the same protocol. Gene expression was analyzed from the tumor tissue using Affymetrix Human Exon 1.0 ST arrays, quantified with MEAP (Multiple Exon Array Preprocessing, probe annotation version 70) algorithm, and expression of the mutated genes identified. Samples were classified into GCB, ABC and nonclassified subgroups using the Lymphochip gene predictor. At the time of the analysis, median follow-up time was 65 months and predicted 5-year OS 76%. Survival association of the identified mutations and gene expression was validated using RNAseq data from 92 (Cancer Genome Characterization Initiative (CGCI) repository), and oligonucleotide-based microarray expression data from 233 (Lymphoma/Leukemia Molecular Profiling Project, LLMPP) DLBCL patients, respectively. Patients were treated with chemoimmunotherapy in both validation cohorts.ResultsIn our initial screen 922 genes were affected by somatic mutations. The genes included both novel and previously identified lymphoma-related oncogenes, such as MYD88, CD79B and DTX1. We highlight mutations affecting GC marker Kelch-like family member 6 (KLHL6), which is a predicted oncogene and involved in B-cell receptor (BCR) signaling in DLBCL.In the CGCI validation cohort 16% of the patients carried non-silent KLHL6 mutations. According to Kaplan Meier analysis, the patients with KLHL6 mutations had significantly worse survival in comparison to the patients without mutations (5-year OS; 53% vs 84%; p = .010). In multivariate analysis, the prognostic impact of KLHL6 mutations on the survival was independent of IPI (OS; RR, 3.060; 95% CI, 1.233-7.598; p = .016). KLHL6 mutations were equally distributed between the molecular subtypes. However, when the molecular subtypes were analyzed separately, KLHL6 mutations remained predictive for poor survival only in the ABC-like DLBCL (OS; RR, 6.727; 95% CI 1.576-28.714; p= .010).KLHL6 expression was further analyzed in the Nordic discovery cohort. Consistent with its role as a GC marker, KLHL6 expression tended to be higher in the GCB- than the ABC-like DLBCLs. Overall, low KLHL6 expression was associated with poor survival (5-year OS; 93% vs 65%; p = .044). The difference in survival was restricted to the ABC subtype. The prognostic impact of KLHL6 expression in the patients with the ABC-like DLBCL was confirmed in independent LLMPP validation cohort.ConclusionsThe results show that although high KLHL6 expression is characteristic for the GCB DLBCL, mutations and low expression of KLHL6 are clinically more relevant in the ABC subtype pinpointing the patients with the most dismal prognosis. DisclosuresLeppä:CTI Life Sciences: Honoraria; Roche: Honoraria, Other: Travel Expenses, Research Funding; Takeda: Honoraria, Other: Travel expenses; Bayer: Honoraria, Research Funding; Mundipharma: Research Funding; Janssen: Research Funding; Amgen: Research Funding.

Voruciclib Sensitizes High Risk Diffuse Large B Cell Lymphoma to BCL2 Inhibition Mediated Cell Death and Tumor Regression

Diffuse Large B Cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma in adults. Although upfront chemotherapy leads to favorable survival outcomes, relapsed or refractory patients continue to have poor prognosis with limited treatment options. In DLBCL, evasion of apoptosis - a key hallmark of cancer is mediated by functionally redundant BCL family members: BCL2, BCLxL and MCL-1. The BCL2 specific inhibitor venetoclax is approved for treating high-risk CLL, but responses in DLBCL have been limited, potentially due to compensatory upregulation of MCL-1. Currently a well-tolerated drug for inhibition of MCL-1, is unavailable in the lymphoma clinic.Voruciclib, is a novel clinical stage oral CDK inhibitor with potent activity (<10 nM) against CDKs 9, 4, 6 and 1. Multiple mechanisms for downregulation of MCL-1 activity have been described for CDK inhibitors. Arguably best characterized is transcriptional inhibition of MCL-1, a short half-life transcript, via inhibition of transcriptional regulator CDK9. We evaluated MCL-1 expression in the FFPE lymphatic tissues from 33 patients with DLBCL, and found that it was expressed in 52% of cases, of both GC (germinal center) and ABC (activated B-cell)-like type. We therefore investigated whether voruciclib could synergize with venetoclax in pre-clinical models of DLBCL via inhibition of MCL-1. In cell-based assays, exposure of DLBCL cells to voruciclib as a single agent resulted in apoptosis which was preceded by context-dependent downregulation of MCL-1. To further explore the impact of voruciclib on MCL-1 activity and DLBCL viability in vivo, we utilized Presage’s CIVO tumor microinjection technology. CIVO enables investigation of multiple drugs and drug combinations simultaneously in a living tumor facilitating in vivo assessment of anti-tumor drug synergy (Klinghoffer et al. Sci. Transl Med. 2015; Dey et al. PLOS One 2016). Voruciclib was introduced as a single agent or in combination with venetoclax to DLBCL xenografts. Microinjection, resulting in localized tumor exposure to voruciclib, led to MCL-1 downregulation in vivo across multiple models of DLBCL. In contrast, tumor exposure to venetoclax led to MCL-1 upregulation. Co-exposure to voruciclib and venetoclax demonstrated that the ability of voruciclib to downregulate MCL-1 is dominant to the upregulation by venetoclax. Consistent with the hypothesis that MCL-1 compensates for loss of BCL2 function in DLBCL, synergistic cell death was observed when voruciclib was combined with venetoclax. Synergy between voruciclib and venetoclax was observed in vivo in models representing both ABC (RIVA: CI value 0.5) and GC subtypes (NUDHL1 and Toledo: CI values 0.4). Similar activity was noted when venetoclax was combined with A1210477, an investigational MCL-1 inhibitor thereby suggesting MCL-1 downregulation to play a role in the observed synergy between venetoclax and voruciclib. Consistent with these results, preliminary studies on xenografted mice have shown that systemic administration of a sub-efficacious dose of venetoclax in combination with voruciclib led to impediment of tumor growth which was greater than the effect observed with each single agent. Additional systemic studies are ongoing with venetoclax in combination with voruciclib in a panel of DLBCL models to further strengthen this observation. Based on the above findings, a Phase 1b clinical trial has been designed to evaluate the combination of voruciclib and venetoclax in patients with the goal of expediting future treatment options for relapsed/refractory DLBCL. We expect to initiate this trial at multiple centers in early 2017. DisclosuresDey:Presage Biosciences: Employment. Kerwin:Presage Biosciences: Employment. Casalini:Presage Biosciences: Employment. Merrell:Presage Biosciences: Employment. Grenley:Presage Biosciences: Employment. Ditzler:Presage Biosciences: Employment. Dixon:Presage Biosciences: Employment. Burns:Presage Biosciences: Employment. Danilov:ImmunoGen: Consultancy; GIlead Sciences: Research Funding; Astra Zeneca: Research Funding; Pharmacyclics: Consultancy; Takeda: Research Funding; Dava Oncology: Honoraria; Prime Oncology: Honoraria. Klinghoffer:Presage Biosciences: Employment.

Correlation of Toll-like Receptor (TLR) and PD-1 Pathways in Central Nervous System Lymphoma: A Clinicopathological Study on Outcomes

Introduction:Most patients with central nervous system lymphoma (CNSL) have an inferior response to therapy compared with systemic diffuse large B-cell lymphoma (DLBCL), in part possibly because 95% of CNSL are of activated B-cell (ABC) phenotype. Recent studies have revealed the genetic basis of pathways active in CNSL. Activated anti-apoptotic NFκB and B-cell receptor pathways are key to sustained cancer cell survival. Myeloid differentiation primary-response protein 88 (MYD88) is an adaptor protein that upon stimulation of TLR, mediates downstream activation of the NFκB pathway. The objective of this study is to examine the association of MYD88 mutation and PD-1/PD-L1 expression with clinical outcome and the overlap between PD-1/ PD-L1 expression and MYD88 L265P mutation in CNSL.Methods:Twenty-two patients with CNSL on whom complete treatment records and tissue were available were included in our study following IRB approval. On a corresponding archived formalin-fixed, paraffin-embedded tissue block from each case, 10 5-um unstained slides for immunohistochemistry and a 10-50 um tissue curl for MYD88 DNA analysis by PCR were prepared. PAX5, CD3, CD68, PD-1 and PD-L1 immunostains were performed and evaluated by light microscopy by two of the authors blinded to the clinical data, including one hematopathologist. Extracted DNA was analyzed using allele-specific PCR designed to detect the common MYD88 gene mutation c.794T>C (p.L265P). DLBCL immunophenotype data, based on Hans algorithm was extracted from the pathology reports. Comparisons between two groups were analyzed using Chi-square and Student t tests. Kaplan Meier method was used to calculate progression-free survival (PFS) and overall survival (OS). Log-rank test was used to determine the differences in survival. Pearson and Spearman’s rho coefficients were calculated for correlation analysis. Statistical analysis was performed using SPSS.22 software.Results:The median age at diagnosis was 67 years (range 28-81). Thirteen patients were male (59%). Twelve patients had ECOG performance status of > 1. Median LDH at diagnosis was 190 U/L. Two patients with CNSL had HIV. Two patients had received prior solid-organ transplant. Seventeen patients received high-dose methotrexate-based therapy, and 9 received radiation. In terms of histopathology, 19 (86%) were of ABC subtype, all were CD20 positive, and 4 were EBV-positive, including both post-transplant cases. The median PFS of all patients was 6.52 months, and the OS was 12 months. Tumor content of the tissue sections was estimated from H&E and PAX-5 stains and showed 30-100% tumor areas, barring 3 cases with <20%. Tumor infiltrating lymphocyte (TIL) content (CD3) was estimated as low (50%), moderate (36%) or high (14%). Tumor associated macrophage (TAM) infiltration (CD68) was estimated as 32% high, 59% moderate and 9% low. 32% of tumors had either PD-L1 or PD-1 expression; all 4 EBV-associated tumors were positive for PD-L1. In 68% of cases, TAMs were positive for PD-L1. TILs co-expressed PD-1 in all cases. MYD88 data was available for 21 patients and 15/21 (71%) were positive. Twenty patients were included in the survival analysis. There was no significant association of MYD88 mutational status or tumor PD-1/PD-L1 expression with PFS or OS. While no correlation was found between the tumor PD1/PD-L1 status and the degree of TAM infiltration, TIL infiltration or the baseline lymphocyte count, there was a negative correlation between MYD88 mutation and tumor PD-1/ PD-L1 (p=0.004;r2=-0.623).Conclusion: The high prevalence of tumor PD-1/PD-L1 and MYD88 gene mutation are similar to previous reports, however, we found an inverse correlation between MYD88 mutation status and tumor PD-1/PD-L1 expression, not previously reported. This could be due to one pathway being dominant in the individual tumor or to down regulation of checkpoint molecules mediated by MYD88 and needs further exploration. The presence of either PD-1/PD-L1 or MYD88 mutation is associated with a poor prognosis. This study further supports the rational for trials with TLR/NFkB pathway inhibitors and checkpoint inhibitors that penetrate the blood brain barrier.We acknowledge the Vanderbilt Translational Pathology Shared Resource (TPSR) supported by NCI/NIH Cancer Center Support Grant 2P30 CA068485-14 and the Vanderbilt Mouse Metabolic Phenotyping Center Grant 5U24DK059637-13. DisclosuresReddy:KITE: Membership on an entity's Board of Directors or advisory committees; INFINITY: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; GILEAD: Membership on an entity's Board of Directors or advisory committees.

279 - Manganese Porphyrin, MnTE-2-PyP5+, Enhances Chemotherapeutic Response in Hematological Malignancies

The prognosis for multiple myeloma (MM) and the activated B-cell subtype of diffuse large B-cell lymphoma (ABC DLBCL) are poor. Gene expression profiling studies have revealed that nuclear factor kappa-B (NF-kB), a critical survival protein, is constitutively active in MM and ABC DLBCL, and contributes to the poor prognosis. Use of agents that target NF-kB have the potential to improve patient outcome. Our laboratory, previously demonstrated that Mn(III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP5+), a redox cycling agent, acts as a pro-oxidant to enhance glucocorticoid-induced apoptosis in WEHI7.2 murine lymphoma cells. An increase in H2O2 and the presence of GSH are critical for the porphyrin’s ability to cause this effect. Alone, MnTE-2-PyP5+ was neither able to increase oxidative stress nor induce apoptosis. In the presence of dexamethasone, a synthetic glucocorticoid, MnTE-2-PyP5+ redox cycles with GSH, glutathionylates the p65 NF-kB subunit, and inhibits NF-kB activity in WEHI7.2 cells. Collectively these studies suggest that MnTE-2-PyP5+ is likely to enhance apoptosis in oxidized environments or in combination with other agents that increase H2O2 levels and in cells that overexpress NF-kB. In this study we found that MnTE-2-PyP5+ synergized with the drugs that are standard of care for DLBCL (cyclophosphamide, doxorubicin, vincristine and glucocorticoids) to enhance cell death in DLBCL human cell lines. The ABC DLBCL cells were also sensitive to MnTE-2-PyP5+ alone. In addition, DLBCL cells with increased C-MYC and BCL-2, which models a DLBCL patient population that also has a poor prognosis, were sensitive to MnTE-2-PyP5+ alone. In MM cells, MnTE-2-PyP5+ synergized with glucocorticoids and bortezomib to enhance cell death. Both drugs increase H2O2 and are used for the treatment of MM. These findings suggest that the addition of MnTE-2-PyP5+ to the standard therapy for DLBCL and MM may overcome NF-kB-mediated survival, enhance cell death, and ultimately improve patient survival.

Control of translational activation by PIM kinase in activated B-cell diffuse large B-cell lymphoma confers sensitivity to inhibition by PIM447.

The PIM family kinases promote growth and survival of tumor cells and are expressed in a wide variety of human cancers. Their potential as therapeutic targets, however, is complicated by overlapping activities with multiple other pathways and remains poorly defined in most clinical scenarios. Here we explore activity of the new pan-PIM inhibitor PIM447 in a variety of lymphoid-derived tumors. We find strong activity in cell lines derived from the activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL). Sensitive lines show lost activation of the mTORC1 signaling complex and subsequent lost activation of cap-dependent protein translation. In addition, we characterize recurrent PIM1 protein-coding mutations found in DLBCL clinical samples and find most preserve the wild-type protein's ability to protect cells from apoptosis but do not bypass activity of PIM447. Pan-PIM inhibition therefore may have an important role to play in the therapy of selected ABC-DLBCL cases.

Evolution of frontline treatment of diffuse large B-cell lymphoma: a brief review and recent update.

Various strategies have been implemented to improve the outcomes of diffuse large B-cell lymphoma (DLBCL). In recent years, remarkable advances have been achieved, based on the discovery of cell-of-origin in DLBCL and on more effective targeted agents. This commentary will summarize recent updates on the evolution of frontline therapies for DLBCL, focusing on the upcoming promising frontline chemotherapy platforms and on activated B-cell subtype DLBCL and double-hit DLBCL.

Abstract 1762: Body mass index shows etiologic heterogeneity for risk of diffuse large B-cell lymphoma (DLBCL) subtype defined by cell-of-origin

Abstract Background. DLBCL is the most common non-Hodgkin lymphoma (NHL) subtype in western countries and is known to be clinically heterogeneous. Gene expression profiling has identified two major, biologically distinctive DLBCL subtypes on the basis of their cell-of-origin (COO): the germinal center B-cell (GCB) - characterized by BCL2 rearrangement and C-REL amplification, and activated B-cell (ABC) - characterized by constitutive activation of the NF-kB pathway. Recently, the InterLymph Subtypes Project identified medical history, lifestyle, and family history risk factors for DLBCL using a large pooled analysis of case-control studies. In this study we evaluated these same risk factors for etiologic heterogeneity as defined by DLBCL COO. Methods. For this analysis we used a clinic-based study of newly diagnosed NHL cases and frequency matched controls, enrolled from 2002-2012, with 474 DLBCL cases and 2203 controls. COO was determined clinically using the Hans algorithm, which is based on immunohistochemistry markers using formalin-fixed, paraffin-embedded tumor tissue. Amongst DLBCL cases 107 were ABC, 207 were GCB, and 160 were missing subtype (tissue unavailable). Risk factor data, including body mass index (BMI), smoking, alcohol use, any allergy, asthma, blood transfusion, diabetes, rheumatoid arthritis, sun exposure, lived on a farm and family history of hematologic malignancy, were collected from self-administered questionnaires. Polytomous logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals for ABC and GCB subtypes, adjusted for age and sex. Results. The mean age of cases was 60.4 years with 53% male; the mean age of controls was 61.6 years and 53% male. We identified a strong positive association for BMI with risk of ABC but not GBC DLBCL (p-heterogeneity 0.025). Compared to BMI 18.5-24.9 kg/m2, those with a BMI of 25.0-29.9 (OR = 1.65; 0.93-2.93) or 30+ (OR = 2.61; 1.48-4.62) were at increased risk of ABC DLBCL. There was no association for a BMI of 25.0-29.9 (OR = 0.91; 0.63-1.31) or 30+ (OR = 1.21; 0.83-1.76) with GCB DLBCL. There was no evidence of significant heterogeneity between ABC and GCB DLBCL for the other risk factors that we evaluated. Further adjustment of the BMI association for educational level, smoking, and alcohol only slightly attenuated these findings. Conclusions. We observed a strong positive association of BMI with ABC but not GCB DLBCL. In contrast, other evaluated risk factors showed no heterogeneity by COO. While BMI has not been clearly associated with NHL overall, there has been evidence for an association with DLBCL. The etiologic heterogeneity we observed for ABC DLBCL is notable as BMI is also a strong risk factor for multiple myeloma, another post-germinal center B-cell malignancy. While requiring replication, our findings suggest BMI may influence B-cell neoplasia through effect on post-germinal center pathways. Citation Format: Matthew K. Breitenstein, Megan M. O’Byrne, Andrew L. Feldman, Carrie A. Thompson, William R. Macon, Grzegorz S. Nowakowski, Stephen M. Ansell, Susan L. Slager, Thomas M. Habermann, James R. Cerhan. Body mass index shows etiologic heterogeneity for risk of diffuse large B-cell lymphoma (DLBCL) subtype defined by cell-of-origin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1762.

A New Therapeutic Era in GCB and ABC Diffuse Large B-cell Lymphoma Molecular Subtypes: A Cell of Origin-Driven Review.

In the past 15 years, advances in molecular biology have exposed the genetic and physiopathologic heterogeneity of diffuse large B-cell lymphoma (DLBCL). Subsets of patients have been identified in which current chemoimmunotherapies may not be as efficacious, such as the activated B-cell subtype (ABC). In this review, we present an in-depth study of the differences between the two main DLBCL subsets (germinal center B cell [GCB] and ABC), focusing specifically on their different genetic features, active tumoral pathways, and pathologic features. We also discuss the bridges that have been built from the bench to the forefront of patient care through translational research, including the use of immunohistochemistry versus gene profiling to categorize patients with DLBCL and current clinical trial data pertaining to new possible targeted therapies for patients with these two subtypes of DLBCL. We hope that clinicians use this review as a tool to better understand the complexity of the two more prevalent DLBCL subtypes seen in the day to day practice and update their knowledge in both current and upcoming novel treatment options that can potentially change the outcomes of this population.

CARMA1- and MyD88-dependent activation of Jun/ATF-type AP-1 complexes is a hallmark of ABC diffuse large B-cell lymphomas

AbstractA hallmark of the diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) type, a molecular subtype characterized by adverse outcome, is constitutive activation of the transcription factor nuclear factor–κB (NF-κB), which controls expression of genes promoting cellular survival and proliferation. Much less, however, is known about the role of the transcription factor activator protein-1 (AP-1) in ABC DLBCL. Here, we show that AP-1, like NF-κB, was controlled by constitutive activation of the B-cell receptor signaling component caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) and/or the Toll-like receptor signaling component myeloid differentiation primary response gene 88 (MyD88) in ABC DLBCL cell lines. In contrast to germinal center (GC) B-cell (GCB) DLBCL, ABC DLBCL cell lines expressed high levels of the AP-1 family members c-Jun, JunB, and JunD, which formed heterodimeric complexes with the AP-1 family members activating transcription factor (ATF) 2, ATF3, and ATF7. Inhibition of these complexes by a dominant-negative approach led to impaired growth of a majority of ABC DLBCL cell lines. Individual silencing of c-Jun, ATF2, or ATF3 decreased cellular survival and revealed c-Jun/ATF2-dependent control of ATF3 expression. As a consequence, ATF3 expression was much higher in ABC vs GCB DLBCL cell lines. Samples derived from DLBCL patients showed a clear trend toward high and nuclear ATF3 expression in nodal DLBCL of the non-GC or ABC subtype. These findings identify the activation of AP-1 complexes of the Jun/ATF-type as an important element controlling the growth of ABC DLBCL.

Optimal Management of Double-Hit Lymphoma.

Although the majority of patients with diffuse large B-cell lymphoma (DLBCL) can be cured by using standard immunochemotherapy, a sizeable proportion of patients cannot. Therefore, it is critical to be able to identify these patients up-front by looking at various clinical and biologic characteristics. DLBCL is a heterogeneous disease with clinical, morphologic, and genetically distinct subtypes, and it is important to understand how these various factors impact treatment outcome as a prerequisite for the development of effective and specific treatment strategies for clinically and molecularly defined subtypes.Whereas the role of clinical characteristics, such as those comprising the International Prognostic Index score, in predicting outcome has been understood for some time, many recent studies demonstrate a prognostic role for individual tumor biomarkers. Biomarkers are thus becoming more andmore of an important consideration in first-line optimal therapy selection. Most DLBCL cases can be divided into one of two molecular subtypes—a germinal center B-cell subtype or an activated B-cell (ABC) subtype—on the basis of mechanisms of oncogenic activation.Outcomes for patients with the ABC subtype are significantly inferior after standard therapy than are outcomes for those with the germinal center B-cell subtype; therefore, there is a need todevelop novel strategies for theABC subtype. It has beenhelpful that anumberof recently developed agents have shown selective activity within the ABC subtype, and randomized clinical trials to test someof these agents in the up-front setting are in progress. Role of MYC and BCL2 Whereas discovering the cell of origin has been helpful in the development of therapeutic agents, there has been much interest and focus in the literature on the roles ofMYC and BCL2 in DLBCL. Several retrospective studies have investigated these roles, and it has been shown that after standard CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or R-CHOP(rituximabplusCHOP) therapy, cases that harbor a MYC rearrangement, which is usually detected by fluorescent in situ hybridization, or both aMYC and a BCL2 rearrangement (double-hit lymphomas [DHLs]) have inferior outcomes. In patients with DHL, BCL2 is typically the translocation partner withMYC but it can sometimes be BCL6, or there can be three rearrangements with BCL2 and BCL6 (triple-hit lymphoma). In addition, by using immunohistochemistry to assess expression ofMYCandBCL2 inDLBCL,various groups have demonstrated that approximately 20% to 25% of newly diagnosed patients with DLBCL have high levels of MYC and BCL2 protein expression, without translocations in the majority of cases. These cases have been called double-expresser lymphomas, and patients do not respond well to treatment with standard R-CHOP therapy. An important question, therefore, is how should these patients be managed?