ALBERT

Description: Introduction: MYC/BCL2 double hit lymphoma (DHL), defined as a large B-cell lymphoma with concurrent MYC and BCL2 translocations, is the most common type of DHL. Although multiple studies focused on DHL have been published, several issues regarding impact on prognosis remain controversial including: 1) history of low grade B cell lymphoma; 2) morphology (diffuse large B-cell lymphoma [DLBCL] versus B cell lymphoma unclassifiable with features intermediate between DLBCL and Burkitt lymphoma [BCLU)]; 3) Absence or low expression of MYC or BCL2; 4) MYC translocation partner gene; and especially 5) most effective therapy. The aim of this study was to attempt clarify the prognostic importance of these factors in DHL. Methods: 157 patientsdiagnosed with MYC/BCL2 DHL between 2003 and April 2015 at two institutions were included in this study. MYC and BCL2 gene rearrangement were confirmed by FISH using a MYC breakapart probe and BCL2 and IGH dual color dual fusion probes. BCL6/3q27gene status was tested either by FISH using breakapart probe or by karyotype. MYC partner gene was identified by karyotype. MYC/BCL2 DHL cases were identified if they had rearrangements of MYC and BCL2 but not BCL6. Positive for MYC or BCL2 by immunohistochemistry was defined by 〉40% and 〉50% of lymphoma cells showed positive expression, respectively. Patient survival was analyzed using the Kaplan-Meier method and compared using the log-rank test. Fisher's exact test was used to compare the clinicopathologic features. Statistical analysis was performed using SPSS 23 software. Results: There were 103 men and 54 women with a median age of 61 years (range, 18-87). 110 patients had de novo disease and 47 patients had a history of low-grade B-cell NHL, mostly follicular lymphoma. The clinicopathologic features were similar (P〉0.05) between patients with a history of low-grade B-cell NHL and patients with de novo NHL, and therefore analysis was performed on all 157 DHL cases. Using the 2008 WHO classification, there were 91 DLBCL, 61 BCLU, and 5 composite lymphoma (4 DLBCL + follicular lymphoma and 1 DLBCL + B-lymphoblastic lymphoma). 99% of cases had a germinal center B-cell immunophenotype by the Hans algorithm. MYC expression was observed in 39/47 (83%) and BCL2 in 129/141(91%) of cases. MYC and BCL2 dual expression was present in 34/46(74%) cases. Of the 39 cases assessed, the MYC translocation partner was IGH in 13, IG light chain in 19, and a non-IG gene in 7 cases. 144 patients had complete treatment information: 61 received the R-CHOP regimen initially, 31 R-EPOCH, 29 R-HCVAD, and 23 various other chemotherapy regimens. 39 patients also received stem cell transplant (SCT) including 31 autologous and 8 allogeneic. 62 patients reached complete remission (CR) after initial chemotherapy. The median overall survival was 19 months. In a univariate analysis that evaluated 17 clinicopathologic parameters including those mentioned in introduction, extranodal sites of disease, bone marrow involvement, CNS involvement, advanced stage, and high/high-intermediate International Prognostic Index score were associated with a worse overall survival (OS, P

Description: Introduction: ALK+ anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5) resulting in overexpression of NPM-ALK oncoprotein, which activates many oncogenic pathways. The Jab1 (c-Jun activation domain-binding protein-1), initially discovered as a c-Jun co-activator, represents the fifth component of an evolutionary highly conserved 8-subunit protein complex, named COP9 signalosome (CSN). The Jab1/Csn5 gene operates as an oncogene in cancer through multiple mechanisms including cell cycle control via the CDK inhibitor p27. Recent evidence suggests that Jab1/Csn5 is also involved in immune checkpoint regulation through PD-L1 stabilization by inhibition of PD-L1 proteasomal degradation. Recently, we reported that the protein levels of Jab1/Csn5 are the highest in ALCL as compared to other peripheral T-cell lymphomas (PTCL) and significantly correlate with PD-L1 levels in PTCLs (Drakos et al, HemaSphere 2019; (3):p596, EHA abstract). In this study, we hypothesized that NPM-ALK may regulate Jab1/Csn5 expression and thus contributes to cell proliferation as well as PD-L1/PD1 immune checkpoint regulation. Methods: The in vitro system included 4 ALK+ (Karpas 299, DEL, SUPM2, SUDHL1) and 2 ALK- (Mac1, Mac2a) ALCL cell lines as well as murine Ba/F3 parental and Ba/F3 clones stably transfected with NPM-ALK (Ba/F3-NPM-ALK), EEF1G (Ba/F3-EEF1G-ALK) or control plasmid (Ba/F3-MIG). Transient transfections with Jab1/CSN si-RNAs and STAT3 si-RNAs were also performed in ALCL cells. In addition, ALCL cells were treated with ALK (Crizotonib) or STAT3 (XIII) inhibitors. Two animal models were used in the study: 1) in the ex vivo model, Karpas-299 clones stably transfected with Jab1/CSN5 shRNA constructs were generated and injected in both thighs of SCID-beige immunocompromised mice. The Jab1/CSN5 shRNA and the control mice were followed for tumor development and their tumors were measured and analysed by immunohistochemistry. 2) in the patient derived xenograft (PDX) model of ALK+ ALCL, the mice were treated with the ALK inhibitor Ceritinib or control vehicle and were monitored for changes in tumor characteristics over two weeks. Tumor specimens were taken at early time points (24, 48, and 72 hrs) following treatment in order to obtain viable tumor cells for protein analysis. Results: Jab1/Csn5 was substantially upregulated in the Ba/F3-NPM-ALK and Ba/F3-EEF1G-ALK stable clones as compared to paternal or control Ba/F3-MIG clones. Jab1/Csn5 upregulation was associated with high STAT3 activation (Tyr705-phosphorylation) and increased PD-L1 gene expression in this system. Inversely, inhibition of ALK activity was associated with STAT3 de-activation and decreased protein levels of Jab1/Csn5 and PD-L1 in ALK+ ALCL cells. Knocking down STAT3 by siRNA or inhibition of its activity by the XIII inhibitor resulted in decreased levels of Jab1/Csn5 protein in both ALK+ and ALK- ALCL cell lines. The SCID-beige mice that received Jab1/CSN5-shRNA clones showed significant delay in tumor development and longer survival as compared to control mice, which was associated with significantly decreased PD-L1 protein levels and p27 upregulation in the tumor cells (xenografts). Treatment of the PDX mice with Ceritinib, a potent next generation ALK inhibitor, resulted in substantial tumor necrosis after 72 hrs and decreased tumor size at day 7 post-treatment. At earlier time points, de-activation (de-phosphorylation) of ALK kinase and STAT3 was observed in the Ceritinib-treated mice, which was linked to variably lower levels of Jab1/CSN5 and PD-L1 proteins as compared to control mice. Conclusion: Jab1/CSN5 is a novel downstream target of the NPM-ALK oncogenic kinase that regulates its expression, at least in part, through STAT3 activation. The Jab1/CSN5-mediated stabilization of PD-L1 can be efficiently inhibited by Ceritinib in preclinical animal models of ALK+ ALCL. Disclosures Österborg: BeiGene: Research Funding; Janssen: Research Funding; Abbvie: Research Funding; Kancera AB: Research Funding; Gilead: Research Funding. Vega:National Cancer Institute, national Institutes of Health: Other: Grant Funding-R01CA222918.

Description: Background Mantle cell lymphoma (MCL) is an aggressive B cell neoplasm characterized the by t(11;14)(q13;q32)/CCND1-IGH resulting in overexpression of cyclin D1. Although indolent variants are recognized, MCL is generally aggressive and incurable. The MCL International Prognostic Index (MIPI), gene expression-based proliferation signatures, Ki-67 proliferation index, p53 expression, and aggressive histologic features are used for risk stratification, but the prognostic groups defined by each of these markers are clinically heterogeneous, demonstrating that each parameter alone does not fully account for the clinical behavior of these tumors. In this study, we investigated the gene expression profile of 60 cases of MCL and evaluated which genes had expression patterns that correlate with different clinical and histopathological parameters. Design A total of 60 excisional biopsy specimens of MCL were selected for gene expression profiling. Forty (66%) and 16 (26%) cases were untreated and relapse/persistent samples, respectively; in 4 this unknown was unknown. Biopsy sites included lymph nodes (n=38), spleen (n=10), tonsil (n=6) and various other extranodal tissue sites (n=6). The morphologic features were classic in 27 and aggressive (blastoid/pleomorphic) in 33 patients. MIPI was calculated in selected patients with nodal involvement and whose biopsy was an obtained prior to treatment. Each block contained tumor cells representing ≥ 80% of all cells in the biopsy specimen. RNA was extracted from formalin-fixed paraffin-embedded tissue using the Qiagen Allprep FFPE Kit after deparaffinization, according to the manufacturer's instructions. Gene expression was quantified in 200ng of RNA on the Nanostring™ platform. Normalization for RNA loading was performed using the geometric mean of 40 housekeeping genes with a cutoff value 20. Standard QC and data processing were performed using the nSolver™ Analysis Software. Adjusted p-values were used according to the Benjamini-Hochberg procedure. Results Gene expression profiling was different according to the site of involvement of MCL. Compared with nodal involvement, MCL involving spleen showed overexpression of VEGFA (p

Description: Key Points TP53 3′UTR variations demonstrate prognostic value in DLBCL.

Description: Objective :To evaluate the prognostic significance of RELamplification, expression and c-Rel activation in DLBCL patients and to identify potential mechanisms for impact of c-Rel activation on patient survival. Patients and Design : The study cohort consists 460 de novo DLBCL patients (median follow-up, 46.8 moths) treated with R-CHOP. We assessed the nuclear expression/activation of c-Rel and other NF-κB subunits by immunohistochemistry, REL gene amplification by fluorescence in situ hybridization, and gene expression profiling using Affymetrix GeneChips array. Correlations between expression of nuclear c-Rel, REL mRNA, and expression of TP53, MDM2, MDM4, MYC, BCL2, AKT1, NFKB1, RELA, NFKB2, and RELB, both at the mRNA and protein levels were analyzed using t-tests. The prognostic significance of c-Rel activation, REL mRNA expression, and REL amplification was evaluated in the overall cohort, and different subgroups stratified by COO, status of TP53 mutation (wide type/WT, or mutated/MUT) and expression, Myc, Bcl-2 overexpression, and nuclear expression of other NF-κB subunits. Results :Nuclear c-Rel expression was observed in 29.6% of DLBCL patients and did not correlate with REL mRNA levels (P=0.95) and COO (P=0.77). In contrast, REL mRNA was significantly higher in germinal center B-like (GCB) subtype (P

Description: Background: Primary mediastinal (PMBL), GCB and non-GCB B-cell lymphoma are three major subtypes of DLBCL with distinct clinical outcomes. PMBL and DLBCL of GCB immunophenotype have a higher chance of cure than patients with non-GCB DLBCL when treated with conventional chemotherapy. In the relapsed/refractory setting, there is paucity of data regarding the impact of histological classification of DLBCL on survival following alloSCT in patients who were not eligible for or who have failed a prior autologous SCT (ASCT). Herein, we compare outcomes of alloSCT in these patients. Methods and Patients: We identified 101 de novo DLBCL patients who were treated at our center from January 1, 1998 to December 31, 2011. Seventeen (17%) tumors were PMBL. We determined the cell-of-origin in the remaining patients, based upon the Visco/Young and Choi immunohistochemical algorithms, and classified 62 (61%) as GCB and 22 (22%) as non-GCB tumors. GCB patients were older than non-GCB [median (range): 54 (21-70) vs 48(24-58) years, p=0.003)] and PMBL [26(19-48), P

Description: Abstract 1730 Poster Board I-756 The sonic hedgehog (SHH) pathway, associated with proliferative stem cell niches of many organs, is frequently deregulated in diverse cancers. We have previously shown that the SHH pathway augments the survival of tumor cells by inducing antiapoptotic molecules including bcl-2 and provides resistance to a number of conventional cancer therapies. Aberrant activation of the SHH pathway has been associated with activation and maintenance of lymphoid malignancies. Additionally, our recent data indicates that p63, a p53 family member and an important marker of stem cells has multiple interacting nodes with the SHH pathway. Based on these observations, we hypothesized i) that there is crosstalk between the SHH and p53/p63 networks in cells and, ii) inhibition of the SHH pathway with simultaneous activation of the p53 pathway would result in increased cell death in cancer cell lines of lymphoid origin. Using SDS-PAGE followed by immunoblotting and RT-PCR we surveyed a panel of 18 leukemia/lymphoma cell lines for components of the SHH pathway and the p53/p63/p73 network. Robust SHH pathway expression was observed in 15 of the 18 cell lines examined. Interestingly, in p53 deficient cell lines there was an increase in p63/p73 expression as compared to cell lines with wild type (WT) p53. A set of the previously analyzed diffuse large B- cell lymphoma (DLBCL) cell lines were selected and were representative of both p53 deficient and WT cell lines and also the activated B-like DLBCL (ABC) and germinal center B-like DLBCL (GCB) subgroups. These cells were treated with cyclopamine, an inhibitor of the SHH pathway and/or nutlin, an HDM2 antagonist. Cell viability (MTS assay) was measured using both compounds at various drug concentrations and time points. In addition we also investigated the effects of the drugs individually and in combination on components of the p53/p63 and SHH axes and their targets. Our findings suggest that treatment of p53 (WT) cell lines with a combination of nutlin and cyclopamine results in reduced cell survival than treatment with either drug alone and at lower drug concentrations and that the p53 status of the cell line may be more important in determining therapeutic response to the selected compounds. In addition the p53/p63 pathway may have a novel role in regulation of specific components of the SHH pathway in cells of lymphoid origin. In conclusion, these observations provide proof of concept that a combinatorial therapeutic approach, targeting both the p53 and SHH axes would provide a more robust and favorable response in large B cell lymphomas. Acknowledgments This work was supported in part by the The Mehta Family Foundation Disclosures No relevant conflicts of interest to declare.

Description: Key Points A subset of DHL patients may be cured, and some patients may benefit from intensive induction. Further investigations into the roles of SCT and novel agents are needed.

Description: Context and objective Approximately 5-10% of diffuse large B-cell lymphoma (DLBCL) patients carry MYC gene translocations (MYC-translocation+) with a poor prognosis after standard chemotherapy. MYC-translocation+ DLBCL patients carrying BCL2 translocations (MYC+/BCL2+ double-hit lymphoma) have a worse survival. The efficacy of adjuvant radiotherapy in this setting is unknown. The purpose of this study is to evaluate the efficacy of radiotherapy as a part of the therapeutic regimen for patients with MYC-translocation+ DLBCL. Patients and methods From the International DLBCL R-CHOP consortium program, we selected 581 patients with de novo DLBCL treated with standard R-CHOP immunochemotherapy (diagnosed and treated from 2000 to 2010). We excluded patients with transformed DLBCL, primary mediastinal, cutaneous, testicular or central nervous system large B-cell lymphomas, patients with HIV infection, and patients not treated with R-CHOP. The median follow-up was 54.9 months. Fluorescence in situ hybridization assessing MYC was performed for all the patients (n=581) and results were correlated with available clinical data to identify clinicopathologic features associated with MYC translocation, and to evaluate the prognostic significance of MYC translocations regarding overall survival (OS, from the time of diagnosis to death from any cause) and progression-free survival (PFS, from the time of diagnosis to relapse or death from any cause). In the MYC-translocation+ DLBCL group, 38 patients received chemotherapy alone and 21 patients received chemotherapy with adjuvant radiation therapy. The clinicopathologic features and survival of MYC-translocation+ DLBCL patients treated with (n=21) and without radiotherapy (n=38) after immunochemotherapy were compared to in order to evaluate the radiotherapy efficacy and other confounding factors. Results MYC translocations were detected in 59 DLBCL patients (10.2%). Patients with MYC-translocation+ DLBCL more often had bulky tumors, involvement of multiple extranodal sites, and poorer OS (hazard ratio [HR]: 2.0, 95% confidence interval [CI]: 1.20 - 3.35, P= .0083) and PFS (HR: 1.96, 95% CI: 1.22 - 3.13, P= .0005) independent of the International Prognostic Index score. Poor survival was primarily attributable to patients with MYC+/BCL2+ double-hit DLBCL who were predominantly of germinal center B-cell subtype. Among MYC-translocation+ DLBCL patients, a better survival was achieved in patients who received radiotherapy (for OS, HR: .32, 95% CI: .15 - .71, P= .0049; for PFS, HR: .35, 95% CI: .17 - .73, P= .0043). Conversely, radiotherapy abolished the adverse impact of MYC translocations. In addition, radiotherapy was associated with better survival in the subset of patients with MYC+/BCL2+ double-hit lymphoma (P = .017 for OS, and P = .05 for PFS). However, owing to the common use of radiotherapy as consolidation therapy, the favorable prognoses in the group of patients who received radiotherapy could also be attributed to limited-stage disease and more frequent complete remission (CR) after first-line treatment and therefore these factors confound interpretation of the data. To address these issues, we evaluated radiotherapy efficacy in separate patient groups: patients who achieved CR, non-CR (PR/SD/PD) patients, and patient with stage I/II, or stage III/IV disease. The efficacy of radiotherapy appeared more apparent in patients with advanced disease who did not achieve CR after first-line chemotherapy. Multivariate analysis after adjustment for stage and IPI score validated that radiotherapy significantly improved OS (HR: .28, 95% CI: .10 - .81, P= .018) and PFS (HR: .32, 95% CI: .13 - .80, P= .015) of MYC-translocation+ DLBCL patients. Conclusions The presence of MYC translocations in DLBCL is an important biomarker that facilitates prognostic prediction and treatment stratification independent of the IPI score. For chemoresistant MYC-translocation+ DLBCL patients, radiotherapy seems to be an effective adjuvant regimen likely due to the higher frequency of extranodal involvement and bulky disease in MYC-translocation+ DLBCL patients. Our results provide a rationale for larger scale studies to assess the potential role of radiotherapy in the management of MYC-translocation+ DLBCL patients, particularly patients with MYC+/BCL2+double-hit DLBCL. Disclosures: Winter: Millenium: Research Funding; Novartis : Research Funding; Pfizer (Wyeth): Research Funding; Seattle Genetics: Research Funding; Spectrum: Research Funding; Janssen (Pharmacyclics): Research Funding; Spectrum (Allos): Consultancy; Sanofi Aventis: Consultancy; Tgen: Consultancy; AMBIT Biosciences (Spouse): Research Funding; Celgene (Spouse): DSMB, DSMB Other, Research Funding; Ariad Pharmaceuticals (Spouse): Research Funding; Novartis (Spouse): Consultancy, Research Funding; Amgen (Spouse): Consultancy, Research Funding; Astellas (Spouse): Research Funding; Caremark/CVS: Consultancy; Pfizer (Spouse): Consultancy; Sanofi Aventis (Spouse): DSMB, DSMB Other; Bristol Myers Squibb (Spouse): DSMB, DSMB Other; UptoDate, Inc.(Spouse): Patents & Royalties.

Description: Introduction: Aggressive MCL has a poor prognosis with a 21-40% complete remission (CR) after CHOP and a duration of response of only 10-16 months. More intense therapy could improve these statistics. Rituximab is effective in MCL and has minimal toxicity. Methods: A prospective phase II trial of R-HCVAD (considered to be one cycle) alternating every 21 days with R- M/A (considered to be another cycle) as described earlier (Ann Oncol. 13, suppl 2, 2002 #24). Prophylaxis with mesna, calcium leucovorin, prednisone eyedrops, G-CSF, antibacterial, antifungal, and antiviral therapy. CBC with differential and platelet counts X 2-3/week. Re-staging every 2 cycles including upper and lower endoscopies. Patients in complete remission (CR) after 6 courses of a planned 6-8 cycles were not offered consolidation with stem cell transplant. Post-treatment evaluation was performed every 3 months for 1 yr, every 4 months for 2 yrs, every 6 months for 2 years, then annually. Results: Of 100 patients registered, one was ineligible and two decided to not receive the treatment after registration, leaving 97 evaluable for analysis of response, survival and toxicity. An analysis of response after the first 6 cycles shows an 87% CR/CRu rate. With a median follow up of 40 months, the 3-year FFS and overall survival (OS) were 67% and 81%, respectively. Adverse factors for FFS were: Grade 4 hematologic toxicity was significant. Five patients died during treatment of sepsis (3), pulmonary hemorrhage (1), and unknown cause (1). Four patients developed myelodysplasia/acute myelogenous leukemia after treatment and while in CR and three have died, for a total of 8 deaths in the study (8%). Conclusion: R-HCVAD alternating with R-M/A without stem cell transplant is an effective regimen for treatment of aggressive untreated MCL, specially for patients ≤ 65 years old. Toxicity is as expected for an intense regimen. This encouraging data warrants continued follow-up and comparison with existing/new therapies in future trials.