Double-hit Diffuse Large B-cell Lymphoma Research Articles

Lenalidomide and Obinutuzumab with CHOP for Newly Diagnosed Diffuse Large B-Cell Lymphoma: Phase I Results

Introduction:Diffuse large B-cell Lymphoma (DLBCL) is the most common lymphoid malignancy with ~30K diagnoses each year in the United States. The standard initial therapy for all patients is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) which cures ~65% of patients. Multiple novel agents have shown promising efficacy in the treatment of patients with DLBCL, including lenalidomide (L) and obinutuzumab (O). Obinutuzumab is a novel, humanized, glycoengineered CD20 monoclonal antibody with increased pre-clinical affinity to the FcGRIIIa receptor in comparison to rituximab, and thus may have increased antibody dependent cell-mediated cytotoxicity (ADCC). Lenalidomide is small molecule which is toxic to DLBCL via interferon signaling and immunomodulation. As single agents, L and O achieve overall response rates (ORR) of 28% and 32%, respectively, in patients with relapsed DLBCL (Hernandez-Ilizaliturri, 2011, Morschhauser, 2013). The addition of L to RCHOP may overcome the adverse features of the non-germinal center (non-GCB) subtype of DLBCL. Separate randomized clinical trials are evaluating RCHOP +/- Len in non-GCB DLBCL and RCHOP vs OCHOP in all patients with newly diagnosed DLBCL. We hypothesize the combination of OLCHOP will result in down-regulation of interferon regulatory factor 4, enhanced NK cell-mediated cytotoxicity, and a complete response (CR) rate ≥ to OCHOP (55% in Gather trial, Zelenetz et al, ASH 2013).Methods: Patients with newly diagnosed, CD20+ DLBCL are eligible if they have measurable disease, age ≥18 years, and adequate organ function. In the Phase Ib trial, Obinutuzumab was dosed at 1000mg IV days 1, 8, and 15 during cycle 1, and day 1 on cycles 2-6, CHOP was dosed in standard fashion, and lenalidomide dose was 15mg po daily days 1 - 14 cycles 1 - 6 in the first cohort, and lower dose was to be considered only if we observed DLT in the initial cohort. The Phase Ib trial was designed with a 3+3 design, with dose de-escalation for toxicity. Once the maximum tolerated dose is identified, an additional 44 patients will be enrolled. The primary objectives of this investigator initiated phase Ib/II trial are to determine the maximum tolerated dose and efficacy of LOCHOP. Correlative studies, including minimal disease assessment, cell of origin determination, and immune profiling, are planned on blood and tumor samples.Results: Six patients were enrolled on the Phase Ib trial, with all evaluable for safety and efficacy. The median age was 62.5 years (50-73), with 83% males and a median IPI of 2 (1-3). One patient had double hit DLBCL, and 5 patients were non-GCB DLBCL. No dose limiting toxicities were identified in the six patients treated at L 15mg daily. Toxicities encountered include neutropenia (Grade 3: 50%, Grade 4 33%), thrombocytopenia (grade 3: 17%), and rash (grade 2: 17%). All six patients achieved a complete response, and the Phase II trial has now enrolled 17 patients as of 8/2/16, with accrual projected to complete in December 2016. In patients treated on the Phase II trial, 8 patients have an interim disease assessment with a 100% overall response rate.Conclusions: The combination of Obinutuzumab 1000mg, Lenalidomide 15mg day 1-14, and CHOP is well tolerated in the phase Ib trial, with no dose limiting toxicity encountered, and enrollment is continuing in the Phase II trial. Adverse events do not appear to be different than expected with standard RCHOP. In the patients with at least 1 disease assessment at data cutoff, the efficacy appears promising but additional patients are required. Results from additional patients will be presented at the ASH meeting. DisclosuresWestin:ProNAi: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees. Fayad:Seattle Genetics: Consultancy, Research Funding. Oki:Novartis: Research Funding.

Evaluation of Idelalisib with B-Cell Receptor or Orthogonal Pathway Inhibitors in Diffuse Large B-Cell Lymphoma Cell Lines in Vitro and In Vivo

Background: Idelalisib, a selective oral inhibitor of PI3Kd, is approved for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with follicular lymphoma who have received at least 2 prior therapies. Despite remarkable clinical efficacy, complete responses are rare, highlighting the need to identify more effective therapies, including combinations of novel agents. GS-4059 (ONO-4059) is an investigational next generation Bruton's tyrosine kinase (BTK) inhibitor with improved selectivity compared with ibrutinib. We report here the results of the combination of a PI3Kd inhibitor and GS-4059 in a diffuse large B-cell lymphoma (DLBCL) xenograft model, demonstrating supportive data for our ongoing combination trial in B-cell malignancies (NCT02457598). Additionally, we investigated preclinical orthogonal combination approaches for DLBCL.Methods: Growth inhibition was assessed using CellTiter-Glo Assay after 96 h incubation with idelalisib and GS-4059. CB17-SCID mice were irradiated, implanted subcutaneously with TMD8, and treated BID PO with the PI3Kd inhibitor GS-649443, GS-4059, or coformulated combination when tumors reached 200 mm3. Lysates from tumors or cell cultures were analyzed by Simple Western (Protein Simple). Synergy for antiproliferative effects was assessed using Chalice software (Horizon Discovery, Inc., Lehar et al., Nature Biotech, 2009).Results: Idelalisib and GS-4059 potently inhibited the ABC subtype DLBCL cell line TMD8, which is a B-cell receptor (BCR)-dependent line that exhibits chronic activated B-cell signaling due to mutations in CD79A/CD79B and MYD88 (Kim Y. et al., Hum Pathol, 2014). When a clinically relevant single concentration of idelalisib or GS-4059 was added in combination to a dose responsive effect of the other, a shift in EC50 on cell viability was seen. GS-4059 (50 nM) shifted the EC50 of idelalisib from 141 nM to 5 nM, a 28-fold shift. Idelalisib (1 µM) shifted the EC50 of GS-4059 from 27 nM to 2 nM, a 14-fold shift. Evaluation of downstream signaling pathways implicated in malignant B-cell survival and proliferation showed enhanced inhibition of pAkt S437, pBTK Y223, pErk1/2 T202/Y204, and MYC with a combination of idelalisib and GS-4059, more than either single agent alone. When TMD8 xenografts were treated with a PI3Kd tool compound, GS-649443, GS-4059 or a combination of the 2 inhibitors, a statistically significant decrease in tumor volume was seen as well as tumor regression, when compared with single agent effects (Figure 1A). Evaluation of TMD8 tumor lysates showed strong suppression of pAkt S437, pBTK Y223, pS6RP S235/236, and MYC in tumors treated with both GS-649443 and GS-4059 (Figure 1B). pS6RP S235/236 and MYC, in formalin-fixed paraffin-embedded (FFPE) TMD8 tumors, were profoundly inhibited in tumors treated with combination therapy compared to the monotherapies (Figure 1C). Since the combination of a PI3Kd inhibitor and GS-4059 led to TMD8 tumor regression, an effect correlated to strong down-modulation of MYC, the combination of idelalisib with a bromodomain and extra-terminal (BET) family inhibitor was explored as a potential new orthogonal combination approach for DLBCL. A panel of DLBCL cell lines was evaluated for inhibition of cell viability by idelalisib in combination with GS-5829, a BET inhibitor currently being evaluated in a phase 1 clinical trial. At clinically relevant concentrations, the combination of idelalisib and GS-5829 showed synergistic effects on cell viability in 2 of 6 ABC subtype, 4 of 5 GCB subtype, and 2 of 2 double-hit DLBCL cell. As compared with combination with other agents that inhibit the BCR pathway (GS-4059) or the Bcl-2 pathway (ABT-199), the broadest activity across cell lines was seen with the combination of idelalisib and GS-5829.Conclusion: Idelalisib and GS-4059 demonstrated synergistic inhibition of the TMD8 xenograft with concomitant inhibition of MYC. Screening of other targeted agent combinations in a panel of DLBCL lines revealed broad preclinical activity for the BET inhibitor GS-5829 in combination with idelalisib. This represents a potential orthogonal approach for a new therapeutic strategy for the treatment of B-cell malignancies. [Display omitted] [Display omitted] [Display omitted] DisclosuresMeadows:Gilead Sciences: Employment. Yahiaoui:Gilead Sciences: Employment. Sorensen:Gilead Sciences: Employment. Cui:Gilead Sciences: Employment. Brockett:Gilead Sciences: Employment. Keegan:Gilead Sciences: Employment. Tannheimer:Gilead Sciences: Employment.

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 3038: Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL)

Abstract Background: Molecular studies divide DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal lymphoma (PML). Florescence in situ hybridization (FISH) studies identified another subgroup of DLBCL, classified as DH-DLBCL, with a poor clinical outcome harboring concurrent gene rearrangements of the c-MYC, BCL2 and/or BCL6 proto-oncogenes, resulting in the over-expression of c-Myc, Bcl2 and Bcl6 proteins. Previously, our retrospective review from single institution series revealed that 30 out of 611 DLBCL patients had aberrations in c-MYC and BCL2 or BCL6 by FISH. These patients exhibited inferior response rates (RR) to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS), suggesting that newer therapies are in dire need. DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, resulting in rapid cellular proliferation and resistance to apoptotic stimuli. In ABC-DLBCL, anti-apoptotic factor MCL-1 is implicated in poor prognosis leading to resistance to standard chemotherapy. C-MYC transcriptionally upregulates Mcl1. Translocation of c-MYC in DH-DLBCL may contribute to the aggressive phenotype and chemotherapy resistance via the MCL-1 pathway. We hypothesize that dual inhibition of both anti-apoptotic proteins BCL2 and MCL1 is an effective strategy in inducing lymphoma cell death in DH-DLBCL. Materials & Methods: At the pre-clinical level, we studied 3 novel therapeutic agents targeting BCL2 (ABT-199), c-MYC (JQ-1), and various cell cycle regulatory proteins (p21) and other BCL2 family members affecting ABT-199 activity (irreversible proteasome inhibitor carfilzomib(CFZ)) using DH lymphoma (DHL) cell lines (Val, DOHH-2, ROS-50). DHL cell lines were exposed to ABT-199 (0-10 uM), JQ-1 (0-100 uM) and carfilzomib (CFZ) (0-50 nM) at 24, 48 and 72 hours. Changes in cell viability were evaluated using Presto Blue assay. Subsequently, DHL cells were exposed to doublet combinations of ABT-199, JQ-1 and CFZ for 48 hours. Coefficient of synergy was calculated using CalcuSyn. Results: In vitro, ABT199, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining ABT199 with CFZ and to a lesser degree with JQ-1. Conclusion: ABT199 exhibited strong synergistic activity with CFZ. Dual targeting of BCL2 and c-MYC pathways results in synergistic activity in DHL cell lines. Of interest, this pharmacological interaction could be related to the effects of proteasome inhibition on MCL1 and p21 levels in lymphoma cells, further enhancing the activity of ABT199. Using combination therapy to inhibit c-MYC and the proteasome and in turn decreasing MCL1 will render ABT-199 more effective and be a more potent combination in causing apoptosis and lymphoma cell death. Further pre-clinical work is ongoing. Citation Format: Priyank P. Patel, Alison Zeccola, Juan Gu, Cory Mavis, Sheila N. J. Sait, Vishala Neppalli, Francisco J. Hernandez-Ilizaliturri. Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL). [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 3038.

Abstract 1299: Selinexor, a selective inhibitor of nuclear export (SINE) compound shows enhanced anti-tumor activity when combined with either venetoclax or bendamustine in diffuse large B cell lymphoma (DLBCL) mouse models

Abstract Introduction: Selinexor (sel) is a small molecule inhibitor of CRM1/XPO1, the primary nuclear exporter of over 200 proteins. As such, it affects multiple cellular pathways and has been shown to be broadly synergistic with various drugs in multiple indications. We have previously shown that sel has marked activity in double-hit DLBCL in pre-clinical models and in a small cohort of patients with heavily pre-treated relapsed / refractory double or triple-hit DLBCL. The goal of this study was to test whether combination of sel with either venetoclax (ven), a selective BCL2 inhibitor or bendamustine (benda), a DNA damaging agent, can further enhance the anti-tumor effect of sel in DLBCL. Methods: The effects of sel, benda and ven as single agents and the effects of sel in combination with either benda or ven on cell viability were tested on DLBCL cell lines including Toledo, DoHH2 and SUDHL6 using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots, respectively. Mice were implanted with either Toledo or DoHH2 cells. Toledo inoculated mice were treated with either sel or ven alone or in combination and DoHH2 inoculated mice were treated with sel, ven or benda alone or with sel-benda or sel- ven combinations.%Tumor growth inhibition (%TGI) and overall survival were determined. Xenografts were harvested and analyzed by Immunohistochemestry (IHC). Results: Sel-ven and sel-benda were highly effective both in-vitro and in-vivo. Using MTT assay, we showed that each of the drugs have low IC50 values (nanomolar) and they function synergistically/ additively when combined. In-vivo, in the sel-benda model, treatment with each drug showed%TGI of 37% (sel) and 86% (benda) but in combination%TGI was 107%. Western and IHC analyses showed that sel reduces the expression of key DNA Damage Response (DDR) proteins presumably disabling the cells from over coming the damage induced by benda. In the sel-ven model, in both Toledo and DoHH2 mouse models, individual drugs had little effect on%TGI (Toledo: sel 4%, ABT 28%; DoHH2: sel 37%, ABT 24%) but when combined, treatment showed a synergistic effect (Toledo: 109%; DoHH2: 93%). Moreover, combination treatment of Toledo-derived large xenografts resulted in 30% tumor volume shrinkage that was sustained until the end of the study. Interestingly, BCL2 protein levels were reduced by each drug and to a further extent in the combination-treated group suggesting that the synergistic effect is induced via BCL2. Conclusion: Selinexor is an excellent candidate partner for combination therapies in DLBCL. It shows enhanced antitumor effect with both bendamustine and venetoclax modulating DDR and BCL2 pathway activity, respectively. These data provide rational support for study of sel-ven and sel-benda combination in clinical trials. Citation Format: Sivan Elloul, Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. Selinexor, a selective inhibitor of nuclear export (SINE) compound shows enhanced anti-tumor activity when combined with either venetoclax or bendamustine in diffuse large B cell lymphoma (DLBCL) mouse models. [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 1299.

MYC translocation and/or BCL 2 protein expression are associated with poor prognosis in diffuse large B-cell lymphoma.

Genomic alterations and protein expression levels have been established as prognostic factors for survival in patients with diffuse large B‐cell lymphoma (DLBCL). In particular, double‐hit DLBCL (DHL), which exhibits translocations in MYC and BCL2 and/or BCL6, is known to be associated with a poor prognosis. However, the clinical significance of gene alterations and protein expression levels for MYC, B‐cell lymphoma (BCL)2, and BCL6 are unclear. In this study, we analyzed 61 adult patients diagnosed with DLBCL without DHL, who were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone, or similar regimens. There were no differences in the distribution of MYC expression rates among the different MYC gene statuses. In log–rank tests, MYC translocation was a prognostic factor for overall survival (OS; P = 0.011), whereas BCL2 and BCL6 translocation were not prognostic indicators (P = 0.999 and P = 0.925, respectively). Although the expression levels of MYC and BCL6 were not significantly associated with OS, the expression of BCL2 was a prognostic factor for OS (P = 0.027). Furthermore, copy number gains in the MYC, BCL2, and BCL6 genes did not affect OS. MYC translocation (hazard ratio, 4.769; range, 1.518–14.98; P = 0.007) and BCL2 protein expression (hazard ratio, 3.072; range, 1.002–9.413; P = 0.049) were independent prognostic factors for survival in multivariate analyses. In conclusion, MYC translocation and BCL2 expression may need to be investigated at the initial diagnosis to predict prognosis in patients with DLBCL.

Using the pathology report in initial treatment decisions for diffuse large B-cell lymphoma in the era of precision medicine: reports from the 57th American Society of Hematology annual meeting

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma in the Western world,and is potentially curable with standard R-CHOP chemoimmunotherapy. We are now in an era that the heterogeneity of DLBCL is defined genetically and molecularly,and rational subset-specific therapeutic targets are guiding clinical trials.Primary mediastinal DLBCL is a unique clinicopathologic entity, and alternatives to R-CHOP may confer superior outcome. Rearrangement of the myc oncogene occurs in 10 % of patients with DLBCL, and confers a very poor prognosis with standard R-CHOP, particularly when there is concomitant rearrangement of bcl-2, a condition referred to as double-hit DLBCL. A larger subset of DLBCL demonstrates overexpression of both myc and bcl-2 by immunohistochemistry. Analyze the source of cells by gene expression profile, immunohistochemistry algorithms,or a novel Lymph2Cx platform,provides prognostic information, and guides therapeutic decisions in both relapsed and de novo disease. This article reviews latest research presented at the 57th American Society of Hematology (ASH) annual meeting on the definition of specific subsets of DLBCL and selection of subtype-specific treatment,including novel approaches under investigation. Understanding these key features of the pathology report, and limitations of these assays defining subsets of DLBCL, allows for a precision medicine approach to this disease. Key words: Lymphoma, large B-cell, diffuse; Pathology; Precision medicine; American Society of Hematology annual meeting

Pre-Clinical Investigation of Targeted Therapies for Double Hit Diffuse Large B-Cell Lymphoma (DH-DLBCL)

Background: Molecular studies divide DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal lymphoma (PML). Florescence in situ hybridization (FISH) studies identified another subgroup of DLBCL, classified as DH-DLBCL, with a poor clinical outcome harboring concurrent gene rearrangements of the c-MYC, BCL2 and/or BCL6 proto-oncogenes, resulting in the over-expression of c-Myc, Bcl2 and Bcl6 proteins. DH-DLBCL has inferior response rates (RR) to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS). DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, resulting in rapid cellular proliferation and resistance to apoptotic stimuli. In ABC-DLBCL, anti-apoptotic factor MCL-1 is implicated in poor prognosis leading to resistance to standard chemotherapy. C-MYC transcriptionally upregulates Mcl1. Translocation of c-MYC in DH-DLBCL may contribute to the aggressive phenotype and chemotherapy resistance via the MCL-1 pathway. We hypothesize that dual inhibition of both anti-apoptotic proteins BCL2 and MCL1 is an effective strategy in inducing lymphoma cell death in DH-DLBCL.Materials & Methods: At the pre-clinical level, we studied novel therapeutic strategies targeting key-regulatory pathways abnormally enhanced by the dual BCL2/BCL6 and c-MYC gene rearrangements or amplification using DH lymphoma cell lines (Val, DOHH-2, ROS-50). We evaluated the therapeutic value of three novel agents targeting BCL2 (ABT199), c-MYC (JQ-1), and cell cycle regulatory proteins (p21) and other BCL2 family members affecting ABT199 activity (carfilzomib (CFZ), an irreversible proteasome inhibitor) in a panel of DH-DLBCL cell lines. We retrospectively evaluated our single institution outcome experience over the last 15 years in DH-DLBCL patients. Using the lymphoma translational database that includes 611 DLBCL patients, we identified 30 patients (M=17/F=13) with aberrations in c-MYC and BCL2 or BCL6 by FISH. Demographic characteristics and clinical data were collected and analyzed.Results: In vitro, ABT199, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining ABT199 with CFZ and to a lesser degree with JQ-1. Subsequently, we studied the outcome of DHL patients treated at our institute. Mean age at diagnosis was 63 years, mostly Caucasians (N=28). Using the Han's algorithm, 21 of the DH-DLBCL were GCB, 8 were non-GCB, and 1 unclassifiable. 22 patients had rearrangement of c-MYC and BCL2/ or BCL-6 by FISH and 8 had gain/amplification/aneuploidy at these loci. At diagnosis, 96% (n=29) patients presented with stage III/IV, 67% (n=20) with High-intermediate/High IPI score, and 77% (n=23) had extra nodal disease. Front-line chemo-immunotherapy included 1) standard doxorubicin-containing regimens: R-CHOP (N=11) or R-EPOCH (N=8), or 2) intensified regimens: R-DHAC (N=3) and R+HyperCVAD/R+HDMTXCytarabine (N=8). Prophylaxis IT chemotherapy was administered to 21 (70%) patients. The complete remission (CR) rate was 67% (n=20) and 37% (n=11) patients underwent HDC-ASCS (N=9)/allo-BMT (N=2) in first (N=9) or second-remission (N=2). The use of intensified regimens was associated with a non-statistically significant improvement is PFS and OS. Similarly, an improvement in OS was observed among patients receiving HDC-ASCT/allo-BMT as consolidation. As previously noted, CNS prophylaxis was associated with an improvement in OS. Mcl-1 and other Bcl-2 family members expression levels are been determined by IHC. These results highlight the poor clinical outcome of this patient population and the need for alternative therapeutic options.Conclusion: Our data suggests that ABT199 exhibited strong synergistic activity with CFZ. Dual targeting of BCL2 and c-MYC pathways results in synergistic activity in DHL cell lines. Of interest, this pharmacological interaction could be related to the effects of proteasome inhibition on MCL1 and p21 levels in lymphoma cells, further enhancing the activity of ABT199. Using combination therapy to inhibit c-MYC and the proteasome and in turn decreasing MCL1 will render ABT-199 more effective and be a more potent combination in causing apoptosis and lymphoma cell death. Further pre-clinical work is ongoing. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Evaluation of the Oral Proteasome Inhibitor Ixazomib in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL), the most common human lymphoma, comprises a genetically and clinically diverse group of aggressive B cell non-Hodgkin lymphomas (NHL-B) among a small group of important human cancers increasing in incidence in the US over the last four decades.Current frontline DLBCL therapy although fairly successful (~70-80% remission rate with frontline R-CHOP chemo-immunotherapy), frequently relapses (~40% within 2-3yrs.) often with refractory (r/r) DLBCL leaving only very poor salvage therapy options (<20% PR/CR) and short survival. Of note, 2-3% of DLBCL patients with rearrangements of MYC and either BCL2 or BCL6 gene, so called double-hit lymphoma (DHL), are associated with an even more dismal outcome. Investigation of novel therapeutic approaches for refractory DLBCL is still in progress. Ixazomib (MLN9708), an investigational, orally bioavailable, small-molecule, reversible inhibitor of the 20S proteasome, was pre-clinically evaluated in refractory DLBCL models. We initially screened the activity of ixazomib in 28 DLBCL cell lines using viability assays with increasing doses of ixazomib (0-1000 nM) for 72 hours. Exposure of ixazomib resulted in a dose-dependent inhibition of cell viability with IC50s ranging from as low as 20 nM up to 200 nM. When we compared the IC50 values of ixazomib in DLBCL cell lines with other proteasome inhibitors such as bortezomib (BZ) and carfilzomib (CFZ), the IC50s of ixazomib (average 120 nM) is 16-fold higher than BZ (average 7.4 nM) and 8.8-fold higher than CFZ (average 13.5 nM). We have also evaluated the activity of ixazomib in primary cells from patients with refractory DLBCL (7 cases). The IC50 values of ixazomib in primary refractory DLBCL ranges from 40 nM to 200 nM. Similar drug concentrations of ixazomib were tested on normal peripheral blood mononuclear cells, but showed no significant cellular toxicity. The inhibition effect of ixazomib was noticeable in two representative DLBCL cell lines, MZ (GCB subtype) and RC (double hit-DLBCL) with IC50 well below 50 nM. Thus, we selected these cell line to test whether ixazomib can induce apoptosis. Annexin V assays showed that ixazomib effectively induces apoptosis in both MZ and RC cells in a dose- and time-dependent manner. To assess the functional significance of ixazomib-induced cell growth inhibition and apoptosis in DLBCL, proteomic approach by reverse phase protein array (RPPA) was used. RC and MZ cells were treated with ixazomib (IC75) for 12 hrs and 24 hrs, and protein extracts were analyzed by a customized RPPA with 285 validated antibodies. The profiles for RC and MZ after ixazomib treatment were quite different in that few common proteins get down/up-regulated. This could be due to the fact that RC is a double hit (BCL-2 x Myc) DLBCL cell line and utilizes different growth/survival pathways than MZ cells, particularly activation of protein synthesis pathway (IGF-PI3K-PDK1-ATK-mTOR-S6K-S6) and the growth/survival pathway (Fibronectin-FAK-MEK1-p38-ELK1). Ixazomib treatment significantly down-regulated these two pathways in RC cells. Several common proteins that were significantly up-regulated after 24 hrs of ixazomib treatment in both RC and MZ cells are the apoptotic related proteins (Caspase 3, cleaved caspase 7, and cleaved PARP) and the DNA damage related proteins (H2AX, Histone H-3, DM-Histone-H3, and HM-K9-Histone-H3). These results suggest that DNA damage could be one of ixazomib's mechanisms of action. In conclusion, our results indicate that ixazomib is an active proteasome inhibitor in refractory DLBCL cells, and warrants further investigation as a potential therapy for treatment of therapy-resistant DLBCL. DisclosuresPham:Amgen, Onyx, Millennium: Research Funding.

Integrative Analysis of Diffuse Large B Cell Lymphoma Mutational Landscape: A Lysa Study

IntroductionDiffuse Large B Cell Lymphoma (DLBCL) is the most common lymphoid malignancy, accounting for 30-40% of all Non Hodgkin Lymphomas. Gene expression profiling (GEP) has identified three main subtypes of DLBCL: Germinal Center B-cell like (GCB), Activated B-Cell like (ABC) and Primary Mediastinal B-cell Lymphoma (PMBL). Recently, Next Generation Sequencing (NGS) has enabled a more detailed characterization of DLBCL mutational profiles. Conventional techniques such as immunohistochemistry (IHC) and FISH are also widely used to describe DLBCL. However, no study has yet performed an integrative analysis of the mutational, gene expression, IHC and FISH profiles of DLBCL, in order to provide a comprehensive view of this disease.Methods215 patients with de novo DLBCL in the prospective, multicenter and randomized LNH-03B clinical trials led by the LYmphoma Study Association (LYSA) were included in this study. Microarray-based GEP identified 81 ABC, 83 GCB, 18 PMBL and 33 other. Mutational profiles of patients' tumor DNA were established using Lymphopanel NGS, designed to identify mutations in 34 genes important for lymphomagenesis. For each recurrently mutated gene, we applied ROMER (Ritchie, Nucleic Acids Res, 2015) to perform gene set enrichment analysis on differential expression profiles of mutant and wild-type patients, using a multifactorial model accounting for subtype. The gene sets were obtained from the MSIGDB Hallmarks (Subramanian A, PNAS, 2005) and Signaturedb (Schaffer, Immunol Rev, 2006) collections. When possible, IHC was performed for IgM (n=150), MYC (n=140), BCL2 (n=148), BCL6 (n=146), CD10 (n=152), FOXP1 (n=147) and MUM1 (n=152); FISH was performed for MYC (n=131), BCL2 (n=133) and BCL6 (n=131).ResultsAs expected, EZH2 mutations were significantly associated with upregulation of GCB gene expression (p<10-3), as well as downregulation of bivalent genes (p<10-2), H3K27me3 targets (p<10-2) and GSK343 upregulated genes (p=0.02) (Beguelin et al, Cancer Cell, 2013). IHC and FISH data further cemented EZH2 mutations' link to GCB subtype, and particularly the t(14;18)-positive subset (CD10+: OR=3.9 and p=0.01, MUM1-: OR=0.12 and p<10-3, BCL2+: OR=8.1 and p=0.04, BCL2 rearranged: OR=6.1 and p=0.04). BCL2 and CREBBP mutations were also linked to GCB subtype (CD10+ and MUM1-), and BCL2 mutations correlated with double-hit GCB DLBCL (Myc+: OR=6.6 and p<10-2, MYC rearranged: OR=7.6 and p=0.03, BCL2 rearranged: OR=20 and p<10-3).An association between BCL6 translocations and ABC subtype was confirmed, via a correlation with ABC-enriched CD79B mutations (p=0.02), although interestingly not with MYD88 mutations. MYD88 mutations were correlated with an upregulation of genes involved in proliferation or repressed by PRDM1 (FDR=0.04 each), as well as with an upregulation of genes involved in checkpoint controls, such as E2F targets and genes involved in DNA repair (FDR=0.03 each). All MYD88 mutants expressed FOXP1 in IHC (p<10-3) and MYD88 mutations were also correlated with IgM IHC positivity (OR=3.3 and p<10-2). TNFAIP3 mutations, also involved in constitutive NFkB activation, were associated with an upregulation of genes regulated by NFkB in response to TNF (FDR=0.02), as well as with an upregulation of KRAS-activated genes (FDR<10-2).PMBL-enriched mutations in our cohort were frequently associated with IgM and FoxP1 negative IHC, as expected (WHO 2008 and Roschewski, Nat Rev Clin Onc). XPO1 and ITPKB mutations were correlated with JAK-STAT pathway activation in the total cohort, including upregulation of interferon-inducible genes for both gene mutations (FDR=0.02 and FDR=0.08 respectively) and upregulation of BCL6-repressed genes for XPO1 mutations only (FDR=0.02). Interestingly, CD58 mutations were significantly correlated with upregulation of Nfkb pathway target genes (FDR=0.06), perhaps due to their negative impact on CD2 activation and ROS production inhibition.ConclusionThe results herein provide steps toward a comprehensive, multi-level overview of DLBCL. We highlight differential gene set expression linked to gene mutation status, as well as driver translocation-associated mutational profiles. By using an integrative analysis approach, this study broadens our understanding of DLBCL subtypes' diverse genetic backgrounds. DisclosuresBriere:St. Louis Hospital, Paris, France: Employment. Salles:Celgene Corporation; Roche: Speakers Bureau; Calistoga Pharmaceuticals, Inc.; Celgene Corporation; Genentech, Inc.; Janssen Pharmaceutica Products, L.P.; Roche: Consultancy; Celgene Corporation; Roche and Gilead Sciences: Research Funding.

Aggressive B cell Lymphoma: Optimal Therapy for MYC-positive, Double-Hit, and Triple-Hit DLBCL.

Approximately 10% of cases of diffuse large B cell lymphoma (DLBCL) harbor a MYC rearrangement and this has been associated with an inferior outcome following standard therapy across many different studies. Double-hit and triple-hit lymphomas harbor concurrent rearrangements of MYC and BCL2 and/or BCL6 and are also associated with a very aggressive course and poor clinical outcome. It is unclear and there is lack of consensus on how these diseases should be approached therapeutically. They are characterized typically by high tumor proliferation and likely require Burkitt lymphoma-type strategies and several retrospective studies suggest that more intensive approaches than rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) may be beneficial. One challenge in this respect is that most patients with these diseases are older than 60 years and generally have poor tolerability of regimens typically used in Burkitt lymphoma. Dose-adjusted EPOCH-R is an alternative effective immunochemotherapy platform for DLBCL and is effective in Burkitt lymphoma, and retrospective studies suggest that it is effective and feasible in patients with DLBCL that harbors a MYC rearrangement with or without a BCL-2 translocation (double-hit). A multicenter study of this approach in MYC-rearranged DLBCL is ongoing and preliminary results are very encouraging. There is a lack of consensus on the role of consolidation stem cell transplantation in patients who achieve a good response to initial therapy but at this point in time, no (retrospective) studies have demonstrated any benefit. These diseases are also associated with a high rate of CNS involvement and progression and checking for cerebrospinal fluid by cytology and flow cytometry at initial diagnosis should be considered. In summary, based on retrospective data and preliminary prospective data (as more mature data is awaited), while Burkitt-type regimens may be feasible in young patients, DA-EPOCH-R is a reasonable approach for patients with these diseases, particularly for those over the age of 60 years. CNS prophylaxis is a reasonable consideration (depending on clinical characteristics) given that the rate of CNS progression is high. Many exciting new small molecule inhibitors such as BCL-2 and MYC inhibitors are in development and are exciting for these diseases and attempts are underway to combine them with effective immunochemotherapy platforms.

MYC-IG rearrangements are negative predictors of survival in DLBCL patients treated with immunochemotherapy: a GELA/LYSA study

AbstractDiffuse large B-cell lymphoma (DLBCL) with MYC rearrangement (MYC-R) carries an unfavorable outcome. We explored the prognostic value of the MYC translocation partner gene in a series of MYC-R de novo DLBCL patients enrolled in first-line prospective clinical trials (Groupe d'Etudes des Lymphomes de l'Adulte/Lymphoma Study Association) and treated with rituximab-anthracycline–based chemotherapy. A total of 774 DLBCL cases characterized for cell of origin by the Hans classifier were analyzed using fluorescence in situ hybridization with BCL2, BCL6, MYC, immunoglobulin (IG)K, and IGL break-apart and IGH/MYC, IGK/MYC, and IGL/MYC fusion probes. MYC-R was observed in 51/574 (8.9%) evaluable DLBCL cases. MYC-R cases were predominantly of the germinal center B-cell–like subtype 37/51 (74%) with no distinctive morphologic and phenotypic features. Nineteen cases were MYC single-hit and 32 cases were MYC double-hit (MYC plus BCL2 and/or BCL6) DLBCL. MYC translocation partner was an IG gene in 24 cases (MYC-IG) and a non-IG gene (MYC-non-IG) in 26 of 50 evaluable cases. Noteworthy, MYC-IG patients had shorter overall survival (OS) (P = .0002) compared with MYC-negative patients, whereas no survival difference was observed between MYC-non-IG and MYC-negative patients. In multivariate analyses, MYC-IG predicted poor progression-free survival (P = .0051) and OS (P = .0006) independently from the International Prognostic Index and the Hans classifier. In conclusion, we show in this prospective randomized trial that the adverse prognostic impact of MYC-R is correlated to the MYC-IG translocation partner gene in DLBCL patients treated with immunochemotherapy. These results may have an important impact on the clinical management of DLBCL patients with MYC-R who should be routinely characterized according to MYC partner gene. These trials are individually registered at www.clinicaltrials.gov as #NCT00144807, #NCT01087424, #NCT00169143, #NCT00144755, #NCT00140660, #NCT00140595, and #NCT00135499.

DH DLBCL with Myc/bcl-6 rearrangement and rare cutaneous presentation- a case report

Diffuse Large B Cell Lymphoma (DLBCL) is the most common non Hodgkin Lymphoma type accounting for 25% of the cases. Double Hit (DH) DLBCL is an aggressive form in which MYC rearrangement is associated with either BCL2 or BCL6 rearrangement. Cutaneous lesion is a rare initial presentation of DHL.

Abstract 2578: Synthetic lethal approaches to aurora inhibition in aggressive B-cell non-Hodgkin lymphoma

Abstract We explored synthetic lethality in the context of aurora inhibition with novel combination therapies for aggressive B-NHL. Aurora A and B are serine/threonine kinases and when amplified dysregulate the mitotic phase of the cell cycle. Pre-clinical and clinical evidence demonstrate that aurora inhibition has therapeutic benefit in aggressive B-NHL. Alisertib, an aurora inhibitor has single agent activity in B-NHL mouse xenograft tumors and clinical trials. Pre-clinical studies demonstrate Bruton's tyrosine kinase (BTK) is persistently expressed despite alisertib therapy. TMAs for DLBCL (S0313, S0515, N = 60) and MCL (S0213, S0601, N = 45) were generated to evaluate aurora A, B and BTK expression by IHC. We developed B-NHL cell lines resistant to alisertib to evaluate mechanisms of acute and chronic resistance and knock down of aurora A and B to evaluate Myc, Bcl2 and BTK expression. Mouse B-NHL xenograft models were conducted to demonstrate synthetic lethality of aurora plus BTK inhibition. We demonstrate synergistic inhibition of cell proliferation associated with apoptosis, particularly in ABC and double hit DLBCL and mantle cell lymphoma (MCL) cells. Ibrutinib completely inhibited BTK and AKT activity at 3.0μM without affecting BTK or AKT protein level in B-NHL cells that co-over-express Myc and Bcl-2. Aurora A and B knockdown show compromised oncogene expression. IHC indicated in DLBCL and MCL aurora B to be differentially over-expressed versus aurora A and BTK. Alisertib plus ibrutinib was highly synergistic in inhibiting cell proliferation and inducing apoptosis in B-NHL cells. Moreover, this effect was amplified with the addition of rituximab. Acute and chronic resistance to alisertib was due to ERK1/2 activation. Significantly greater apoptosis was induced in the triplet combination (alisertib + ibrutinib + rituximab) compared to doublet combinations (ibrutinib + rituximab or alisertib + ibrutinib or alisertib + rituximab). Mouse xenograft models of MCL (Granta-519) and double hit DLBCL (U2932) showed ibrutinib alone had no anti-B-NHL activity. Ibrutinib + alisertib lead to tumor growth inhibition (TGI) of ∼50% in MCL and ∼30% in DLBCL (p = 0.005). Interestingly, ibrutinib + rituximab lead to a TGI of ∼60% (p = 0.005) in both B-NHL mouse models. In contrast, triplet therapy with alisertib plus ibrutinib plus rituximab demonstrated a statistically significant tumor TGI of ∼70-90% (p = 0.005) in both B-NHL mouse models with an associated enhanced overall survival (&amp;gt;70 days). Harvested tumors at the end of treatment(s) demonstrated target inhibition and increased apoptosis with triple therapy. Inhibition of BTK plus rituximab in the presence of aurora inhibition is synthetic lethal in B-NHL and warrant clinical trial evaluation. Citation Format: Daruka Mahadevan, Carla Morales, Laurence Cooke, Manjari Pandey, Catherine Spier, Wenqing Qi. Synthetic lethal approaches to aurora inhibition in aggressive B-cell non-Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2578. doi:10.1158/1538-7445.AM2015-2578

Abstract LB-062: XPO1 is a rational target for double and triple-hit aggressive B-cell lymphomas

Abstract Mutation and constitutive expression of MYC and BCL2 and/or BCL6 (a.k.a. double and triple-hit lymphomas) defines subsets of diffuse large B-cell lymphoma (DLBCL) pts with particularly poor outcome. Almost 60% of pts with BCL2 and MYC translocations die within six months of diagnosis due to chemorefractory disease, a prognosis that cannot be overcome with intense chemotherapy. A further hindrance to patient survival is that these double and triple-hit lymphomas are frequently found in the elderly who have limited tolerability to aggressive chemotherapeutic regimens. To identify chemoresistant double and triple-hit DLBCL, we screened 38 DLBCL cell lines for mutations in BCL2, MYC and BCL6 by FISH and response to chemotherapy administration in vitro. We found 3 triple-hit and 2 double-hit DLBCL cell lines. Previous work in our laboratory showed that the nuclear export protein, XPO1, is a critical regulator of MYC, BCL2 and BCL6 mRNA transport in DLBCL. We therefore analyzed XPO1 amplification (by PCR) and expression (by immunoblot) in these cell lines and found that all 5 cells expressed XPO1 at higher levels than centroblasts and that, in at least 2 of them, this was result of gene amplification. Inhibition of XPO1 with selinexor, a Selective Inhibitor or Nuclear Export (SINE) compound, increased nuclear localization of MYC, BCL2 and BCL6 transcripts and consequently decreased their protein expression. Moreover, selinexor administration induced chemosensitization in doxorubicin-resistant DLBCL cells through decreasing DNA damage repair mechanisms as demonstrated by comet assays. To determine the potential extent of DLBCL patients that could benefit from such a treatment, we first analyzed 110 DLBCL patient samples by immunohistochemistry and found that XPO1 expression was increased in 85 cases compared to normal tonsils. Within the cohort analyzed, 6 patients had double or triple hit lymphoma (by FISH) and all overexpressed XPO1. Since this population has higher incidence of chemorefractory disease, we decided to develop a pre-clinical model of this disease using primary patient samples. We developed two patient-derived xenograft (PDX) models representing a double and a triple-hit lymphoma. In both cases XPO1 was also amplified. We compared the molecular and pathological characteristics of 12 generations of PDXs with the original pt sample, and found that protein expression and mutations of these genes were stable. We therefore administered selinexor alone, CHOP alone (cyclophosphamide, vincristine, doxorubicin and dexamethasone) and the combination of selinexor with CHOP to these PDXs. We found that selinexor significantly decreased tumor growth, compared to vehicle or CHOP alone. Moreover, combinatorial treatment indicated that selinexor was able to revert the chemorefractoriness of these DLBCLs, without inducing toxicity by clinical chemistry or pathologic examination. Analysis of selinexor vs. vehicle treated PDXs indicated a decrease in protein levels of MYC, BCL6, BCL2 as well as DNA damage and checkpoint regulators such as CHK1. In summary, selinexor has potent anti-proliferative effects in double/triple-hit DLBCL and can be safely combined with CHOP to enhance cancer cell death. These data present a new therapeutic approach for pts with double/triple hit lymphomas, and provide rational support for the study of selinexor/CHOP combination in clinical trials. Citation Format: Rossella Marullo, ShaoNing Yang, Tami Rashal, Yosef Landesman, Robert Carlson, Sharon Shacham, Leandro C. Cerchietti. XPO1 is a rational target for double and triple-hit aggressive B-cell lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-062. doi:10.1158/1538-7445.AM2015-LB-062

ABC, GCB, and Double-Hit Diffuse Large B-Cell Lymphoma: Does Subtype Make a Difference in Therapy Selection?

Personalized therapy for the treatment of patients with cancer is rapidly approaching and is an achievable goal in the near future. A substantial number of novel targets have been developed into therapeutic agents. There is a substantial variability to antitumor activity by novel therapeutics because of the unique heterogeneity and biology that exists both between and within lymphoma subtypes. Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL). Approximately 40% of patients have refractory disease or disease that will relapse after an initial response, and the majority of patients with relapsed DLBCL will succumb to the disease. There are two major biologically distinct molecular subtypes of DLBCL: germinal center B-cell (GCB) and activated B-cell (ABC). ABC DLBCL is associated with substantially worse outcomes when treated with standard chemoimmunotherapy. In addition to GCB and ABC subtypes, double-hit lymphomas (approximately 5% to 10% of patients) and double-expressor lymphomas, which overexpress MYC and BCL2 protein, are aggressive DLBCLs and are also associated with a poor prognosis. Double-hit lymphomas have concurrent chromosomal rearrangements of MYC plus BCL2 (or less likely, BCL6). Advances in molecular characterization techniques and the development of novel agents targeting specific subtypes of DLBCL have provided a foundation for personalized therapy of DLBCL based on molecular subtype. A number of early clinical trials evaluating combinations of novel targeted agents with standard chemotherapy (R-CHOP) have been completed and have demonstrated the feasibility of this approach with encouraging efficacy. As such, molecular classification of DLBCL is not only important for prognostication, but moves to center stage for personalization of therapy for DLBCL.

Double hit diffuse large B-cell lymphomas: diagnostic and therapeutic challenges.

Although diffuse large B-cell lymphoma (DLBCL) is curable with standard chemoimmunotherapy, over 30% of patients with advanced stage disease experience refractory disease or progression. Recent studies suggest that rearrangement of the myc oncogene occurs in approximately 10% of patients with DLBCL, and confers a very poor prognosis, particularly when there is concomitant rearrangement of bcl-2, a condition referred to as "double hit DLBCL". Using immunohistochemistry, up to 30% of patients have evidence of increased expression of myc, which occurs in both activated B-cell and germinal center type DLBCL. When bcl-2 is also positive by immunohistochemistry, prognosis is also poor. There are no randomized studies guiding treatment for patients with double hit DLBCL, but new datasets are emerging suggesting a possible role for dose-adjusted EPOCH infusional chemotherapy with rituximab. This review will conclude with a survey of novel agents which may be rationally incorporated into chemotherapy platforms for this high risk subset of DLBCL.

Dysregulated CXCR4 expression promotes lymphoma cell survival and independently predicts disease progression in germinal center B-cell-like diffuse large B-cell lymphoma.

Abnormal expression of the chemokine receptor CXCR4 plays an essential role in tumor cell dissemination and disease progression. However, the significance of CXCR4 overexpression in de novo diffuse large B cell lymphoma (DLBCL) is unknown. In 743 patients with de novo diffuse large B cell lymphoma (DLBCL) who received standard Rituximab-CHOP immunochemotherapy, we assessed the expression of CXCR4 and dissected its prognostic significance in various DLBCL subsets. Our results showed that CXCR4+ patients was associated with male, bulky tumor, high Ki-67 index, activated B-cell-like (ABC) subtype, and Myc, Bcl-2 or p53 overexpression. Moreover, CXCR4+ was an independent factor predicting poorer progression-free survival in germinal-center B-cell-like (GCB)-DLBCL, but not in ABC-DLBCL; and in patients with an IPI of ≤2, but not in those with an IPI>2. The lack of prognostic significance of CXCR4 in ABC-DLBCL was likely due to the activation of p53 tumor suppressor attenuating CXCR4 signaling. Furthermore, concurrent CXCR4+ and BCL2 translocation showed dismal outcomes resembling but independent of MYC/BCL2 double-hit DLBCL. Gene expression profiling suggested that alterations in the tumor microenvironment and immune responses, increased tumor proliferation and survival, and the dissemination of CXCR4+ tumor cells to distant organs or tissues were underlying molecular mechanisms responsible for the CXCR4+ associated poor prognosis.

Intense Cytarabine-Based Chemo-Immunotherapy, Central Nervous System (CNS) Prophylaxis and Early High-Dose Chemotherapy and Autologous Stem Cell Support (HDC-ASCS) in First Remission Are Associated with an Improved Clinical Outcome in Double-Hit (DHL)/Triple-Hit (THL) Diffuse Large B-Cell Lymphoma (DLBCL)

Based on molecular studies, DLBCL is now further divided in three subtypes with distinct pathogenesis and clinical outcomes. Fluorescence in situ hybridization (FISH) studies identified a subgroup of DLBCL with a poor clinical outcome harboring concurrent gene rearrangements of the MYC, BCL2 and/or BCL6 proto-oncogenes leading to the over-expression of c-Myc, Bcl-2 and Bcl-6, inferior response rates to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS). This group of patients is now categorized as double-hit (DHL) or triple-hit (THL) DLBCL. Immunohistochemistry (IHC) studies suggested that DLBCL patients with over-expression of Bcl-2 and c-Myc proteins exhibit a similar clinical course than those patients with DHL/THL. Alternative transcriptional (i.e. gene amplification or chromosomal gain) or post-translational regulatory mechanisms (yet to be defined) may be responsible for the over-expression of c-Myc, Bcl-2, or Bcl-6 in some patients with DHL/THL phenotype. The appropriate therapy for DHL/THL remains to be defined, but retrospective studies had demonstrated that at standard doses of R+CHOP are suboptimal. In order to study the impact of more aggressive therapeutic approaches in the management of DHL/THL, we retrospectively evaluated our single institution outcome experience over the last 14 years. Using the lymphoma translational database that includes 611 DLBCL patients, we identified 24 patients (M=13/F=11) (4%) with FISH-confirmed DHL/THL DLBCL. Demographic characteristics, clinical data, treatment history in the front line (including the use of CNS prophylaxis, high-dose chemotherapy and autologous stem cell support [HDC-ASCS] or allogeneic bone marrow transplant [allo-BMT]) were collected. In addition, response rate, PFS and OS were calculated. Since the first case-reports were published, we observed a steady increase in the number of DHL/THL patients at our Institution (7 vs. 18 cases before or after 2011) which represents an increasing medical awareness of this new clinical entity. The mean age at diagnosis was 62 years (25 to 85 years), most of them Caucasians (N=22). The median Ki67 proliferation index was 90%. Using the Han’s algorithm, 11 of the DHL/THL were categorized as germinal center B-cell like (GCB), 6 patients as non-GCB, and 7 patients could not be classified. By FISH studies, 58% were DHL (involvement of MYC and BCL2 [N=22] or BCL6 [N=10]) and 42% THL (involvement of MYC, BCL2 and BCL6, N=10). Gene rearrangements involving MYC, BCL2, or BCL6 were observed in 18 patients and MYC, BCL2 or BCL6 gain in 6. Clinically, 95% of the patients presented with stage III/IV, 67% with High-intermediate/High IPI score, and 79% of the patients had extranodal disease. Front-line chemo-immunotherapy included 1) standard doxorubicin-containing regimens: R+CHOP (N=10) or R-EPOCH (N=7), or 2) intensified regimens: R-DHAC (N=2) and R+HyperCVAD/R+HDMTXCytarabine (N=4). Prophylaxis IT chemotherapy was administered to 16 (67%) patients. The complete remission (CR) rate was 62.5% and 29% of the patients underwent HDC-ASCS (N=4)/allo-BMT (N=2) in first (N=2) or second-remission (N=4). In this group of patients, the use of intensified regimens was associated with a non-statistically significant improvement is OS when compared to R+CHOP/R+DA-EPOCH (OS at 3 years of 85.7% vs. 58%). Similarly, an improvement in OS was observed among patients receiving HDC-ASCT/allo-BMT as consolidation (71% vs. 60%, P=0.27). Of interest, CNS prophylaxis was associated with an improvement in OS at 3 years (81% vs. 37%, P=0.032). No differences in clinical outcomes were observed between DHL/THL harboring gene-rearrangements vs. gene gain. In summary, our single institution experience suggests that DHL/THL are increasingly being recognized as more aggressive sub-types of DLBCL with a dismal outcome with conventional therapeutic approaches. A high-index of suspicion should be raised and testing for DHL/THL in those DLBCL presenting with advance stage, multiple extra-nodal site of disease, and an elevated Ki67 index should be done. The accurate identification of DHL/THL patients is necessary, as they appear to benefit from rituximab-based intensified cytarabine-containing regimens, CNS prophylaxis and HDC-ASCT/allo-BMT consolidation. DisclosuresNo relevant conflicts of interest to declare.

BCL-2 and c-MYC Rearrangements in Leukemic Phase of Diffuse Large B Cell Lymphoma Predicts Central Nervous System Involvement

IntroductionDiffuse large B-cell lymphomas (DLBCL) are the most common lymphoid neoplasms but rarely with leukemic phase. Few studies had described the clinical characteristics of DLBCL with leukemic phase and found that these patients had high incidence of centre nervous system (CNS) involvement but with no knowledge of the reason.MethodsWe reviewed the clinical characteristics of 40 DLBCL patients in leukemic phase during 2001 and 2012. The DNA probes were LSI BCL2 and MYC Dual Color, Break Apart Rearrangement Probe were usedto detect the abnormalities of 18q21 and 8q24 by FISH.We also used LSI IGH/BCL2 Dual Color, Dual Fusion Translocation Probe and LSI IGH/MYC/CEP 8 Tri-Color Dual Fusion Probesto detect the partner fusion genes of BCL2 or MYC.ResultsThe median age of these 40 patients was 46 years(range, 15-75) with 19 men patients. The median WBC was 12.32×109/l(range, 1.2-66.57)with a median 35.74% (range, 1–90) of circulating lymphomatous cells identified by flow cytometry.14 of these 40 patients (35%)had CNS involvement.There were gender differences between CNS involvement or not. Ten of the fourteen patients (71.4%) with CNS involvement were male, while in non CNS involvement group only 9 of 26 (34.6%) cases were male (p=0.046).57.1% patients with CNS involvement had IPI scores 4-5, which was higher than those without CNS involvement (11.5%, p=0.001).All of these 14 patients had elevated LDH was and was significantly higher than those without (61.5%, p=0.007).There were 14 patients (33.5%) had the BCL2 or MYC aberrationrespectively and nine of them simultaneously had both of c-MYC and BCL2 rearrangement, which we defined as double-hit DLBCL patients here. No IGH/BCL2 fusion gene was detected and only one patient with IGH/MYC fusion with c-MYC rearrangement. Eight of the nine double-hit patients had CNS involvement, while only six of the other 31 patients with either BCL2 or MYC or none rearrangements had CNS involvement (88.9% vs. 19.4%; p＜0.001). By logistic regression analysis, we found that double-hit with BCL2 and c-MYC rearrangement was the only one independent factor correlated with CNS involvement with relative risk 15.3 (95% confidence interval, 1.4-171.1).ConclusionDLBCL patients in leukemic phase had higher incidence of CNS involvement and double-hit with BCL2 and c-MYC gene rearrangements might contribute to CNS involvement in this population. DisclosuresNo relevant conflicts of interest to declare.