Citations

Abstract Purpose: Diffuse large B-cell lymphoma (DLBCL) is molecularly and clinically heterogeneous, and can be subtyped according to genetic alterations, cell-of-origin, or microenvironmental signatures using high-throughput genomic data at the DNA or RNA level. Although high-throughput proteomic profiling has not been available for DLBCL subtyping, MYC/BCL2 protein double expression (DE) is an established prognostic biomarker in DLBCL. The purpose of this study is to reveal the relative prognostic roles of DLBCL genetic, phenotypic, and microenvironmental biomarkers. Experimental Design: We performed targeted next-generation sequencing; IHC for MYC, BCL2, and FN1; and fluorescent multiplex IHC for microenvironmental markers in a large cohort of DLBCL. We performed correlative and prognostic analyses within and across DLBCL genetic subtypes and MYC/BCL2 double expressors. Results: We found that MYC/BCL2 double-high-expression (DhE) had significant adverse prognostic impact within the EZB genetic subtype and LymphGen-unclassified DLBCL cases but not within MCD and ST2 genetic subtypes. Conversely, KMT2D mutations significantly stratified DhE but not non-DhE DLBCL. T-cell infiltration showed favorable prognostic effects within BN2, MCD, and DhE but unfavorable effects within ST2 and LymphGen-unclassified cases. FN1 and PD-1–high expression had significant adverse prognostic effects within multiple DLBCL genetic/phenotypic subgroups. The prognostic effects of DhE and immune biomarkers within DLBCL genetic subtypes were independent although DhE and high Ki-67 were significantly associated with lower T-cell infiltration in LymphGen-unclassified cases. Conclusions: Together, these results demonstrated independent and additive prognostic effects of phenotypic MYC/BCL2 and microenvironment biomarkers and genetic subtyping in DLBCL prognostication, important for improving DLBCL classification and identifying prognostic determinants and therapeutic targets.

Abstract Introduction: Multiple studies have demonstrated that diffuse large B-cell lymphoma (DLBCL) can be divided into subgroups based on their biology. However, these biological subgroups overlap clinically. While R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) remains the standard of care for treating patients with DLBCL, predicting which patients will not benefit from such therapy is important so that alternative therapy or clinical trials can be considered. Most of the studies stratifying patients select biomarkers first, then explore how these biomarkers can stratify patients based on outcome. We explored the potential of using machine learning to first group patients with DLBCL based on survival, then isolating the biomarkers necessary for predicting these survival subgroups. Methods: RNA was extracted from tissue paraffin blocks from 379 R-CHOP treated patients with de novo DLBCL, and from 247 patients with extranodal DLBCL. A targeted hybrid capture RNA panel of 1408 genes was used for next generation sequencing (NGS). Sequencing was performed using an Illumina NextSeq 550 System platform. Ten million reads per sample in a single run were required, and the read length was 2 × 150 bp. An expression profile was generated from the sequencing coverage profile of each individual sample using Cufflinks. A machine learning system was developed to classify patients into four groups based on their overall survival. This machine learning approach based on Naïve Bayesian algorithm was also used to discover the relevant subset of genes with which to classify patients into each of the four survival groups. To eliminate the underflow problem commonly associated with the standard Naïve Bayesian classifiers, we applied Geometric Mean Naïve Bayesian (GMNB) as the classifier to predict the survival group for each patient. Results: Using machine learning, patients were first divided into two groups: short survival (S) and long survival (L). To refine this model, we used the same approach and divided the patients in each group into two subgroups, generating four groups: long survival in the long group (LL), short survival in the long group (LS), long survival in the short group (SL), and short survival in the short group (SS). The hazard ratio for this model was 0.174 (confidence interval: 0.120-0.251), and P-value <0.0001. After defining these four groups, a machine learning algorithm was used to discover the biomarkers from the expression data of the 1408 genes from NGS data. To reduce the effects of noise and avoid overfitting, we employed a 12-step cross validation to obtain a robust measure. For an individual gene, a generalized Naïve Bayesian classifier was constructed on the training of one of the 12 subsets and tested on the other 11 testing subsets. This allowed us to limit the prediction process to 60 genes for each separation step. Using the selected biomarkers, we classified the patients in the original set (379 patients) into LL, LS, SL, and SS groups and then evaluated the survival pattern of these groups. As shown in Fig. 1A, the selected biomarkers predicted survival as expected in the overall survival groups prior to biomarker selection. For additional validation of the system, we used the selected biomarkers to classify a completely new set of 247 samples of patients with extranodal DLBCL. As shown in Fig. 1B, these selected biomarkers successfully predicted the overall survival in this group of patients with an HR of 0.530 (confidence interval: 0.234-1.197, P=0.005). This classification correlated with cell of origin classification, TP53 mutation status, MYC expression, and IRF4 expression. However, in a multivariate analysis, only TP53 mutation was independent in predicting prognosis (P=0.005) and age (below or over 60) (P=0.01) along with the survival grouping (P<0.000001). Conclusions: Using a novel machine learning approach with the expression levels of 180 genes, we developed a model that can reliably stratify patients with DLBCL treated with R-CHOP into four survival subgroups. This model can be used to identify patients who may not respond well to R-CHOP to be considered for alternative therapy and clinical trials. Figure 1 Figure 1. Disclosures Hsi: AbbVie Inc, Eli Lilly: Research Funding. Ferreri: Ospedale San Raffaele srl: Patents & Royalties; BMS: Research Funding; Pfizer: Research Funding; Beigene: Research Funding; Hutchison Medipharma: Research Funding; Amgen: Research Funding; Genmab: Research Funding; ADC Therapeutics: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; PletixaPharm: Membership on an entity's Board of Directors or advisory committees; x Incyte: Membership on an entity's Board of Directors or advisory committees; Adienne: Membership on an entity's Board of Directors or advisory committees. Piris: Millenium/Takeda, EUSA, Jansen, NanoString, Kyowa Kirin, Gilead and Celgene.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Winter: BMS: Other: Husband: Data and Safety Monitoring Board; Actinium Pharma: Consultancy; Janssen: Other: Husband: Consultancy; Agios: Other: Husband: Consultancy; Gilead: Other: Husband: Consultancy; Epizyme: Other: Husband: Data and Safety Monitoring Board; Ariad/Takeda: Other: Husband: Data and Safety Monitoring Board; Merck: Consultancy, Honoraria, Research Funding; Novartis: Other: Husband: Consultancy, Data and Safety Monitoring Board; Karyopharm (Curio Science): Honoraria.

Abstract Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities (MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2–targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL-MYC/BCL2-DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC/BCL2/TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. Implications: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg.

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity of B-cell lymphoma. Cell-of-origin (COO) classification of DLBCL is required in routine practice by the World Health Organization classification for biological and therapeutic insights. Genetic subtypes uncovered recently are based on distinct genetic alterations in DLBCL, which are different from the COO subtypes defined by gene expression signatures of normal B cells retained in DLBCL. We hypothesize that classifiers incorporating both genome-wide gene-expression and pathogenetic variables can improve the therapeutic significance of DLBCL classification. To develop such refined classifiers, we performed targeted RNA sequencing (RNA-Seq) with a commercially available next-generation sequencing (NGS) platform in a large cohort of 418 DLBCLs. Genetic and transcriptional data obtained by RNA-Seq in a single run were explored by state-of-the-art artificial intelligence (AI) to develop a NGS-COO classifier for COO assignment and NGS survival models for clinical outcome prediction. The NGS-COO model built through applying AI in the training set was robust, showing high concordance with COO classification by either Affymetrix GeneChip microarray or the NanoString Lymph2Cx assay in 2 validation sets. Although the NGS-COO model was not trained for clinical outcome, the activated B-cell–like compared with the germinal-center B-cell–like subtype had significantly poorer survival. The NGS survival models stratified 30% high-risk patients in the validation set with poor survival as in the training set. These results demonstrate that targeted RNA-Seq coupled with AI deep learning techniques provides reproducible, efficient, and affordable assays for clinical application. The clinical grade assays and NGS models integrating both genetic and transcriptional factors developed in this study may eventually support precision medicine in DLBCL.

Background Tipifarnib is a potent and selective inhibitor of the enzyme farnesyltransferase (FT). FT catalyzes post-translational attachment of farnesyl groups required for localization of signaling molecules to the inner cell membrane. CXCL12 is a ligand for CXCR4 that is essential for T cell homing to lymphoid organs and the bone marrow, and for the maintenance of immune cell progenitors. We have previously shown that FT inhibition by tipifarnib downregulates CXCL12 secretion. Herein we report preliminary efficacy, safety and biomarker data from a Phase 2 study of tipifarnib in angioimmunoblastic T-cell lymphoma (AITL) and CXCL12+ peripheral T-cell lymphoma (PTCL) patients (pts). Methods This Phase 2 study (NCT02464228) is a multi-institutional, single-arm, open-label trial initially designed as a two-stage (11+7 pts) design to determine the efficacy, safety and biomarkers of tipifarnib in pts with relapsed/refractory (R/R) PTCL age >/=18 years and a performance status of 0-2. Based on initial findings, the study was amended to include a cohort of AITL (n=12) and PTCL (n=12) pts with the CXCL12 rs2839695 A/A genotype (wt CXCL12 3'UTR cohort). Pts received tipifarnib 300 mg administered orally twice daily on days 1-21 of 28-day treatment cycles until progression of disease (PD) or unacceptable toxicity. The primary endpoint of the study is overall response rate (ORR). Tumor Whole Exon Sequencing (WES) was generated by NGS and gene expression data generated by RNA Seq. Ancillary studies also investigated the prognostic value of CXCL12 expression in pts who received standard of care treatment. Results As of 24 May 2019, 50 PTCL pts (23 AITL, 25 PTCL-NOS, 1 ALK- ALCL, 1 gamma-delta TCL) have been treated with tipifarnib, 19 pts in stages 1 and 2, and 31 pts in the ongoing AITL histology and wt CXCL12 3'UTR cohorts. Median number of prior treatment regimens was 3; 19 pts had a prior stem cell transplant. All pts (n=48 with available safety data) had at least one treatment-emergent adverse event (TEAE); 42 (88%) had at least 1 study drug-related TEAE and 13 (27%) at least 1 drug related SAE. The most frequently observed drug-related TEAEs of Grade >3 occurring in 10% or more of pts were blood and lymphatic system disorders, including neutropenia (40%), thrombocytopenia (33%), leukopenia (25%), anemia and febrile neutropenia (19% each). There have been 14 deaths on study; one related to study drug (lung infection). Of 18 evaluable pts enrolled in Stages 1 and 2 of the trial, 3 partial responses (PR), 2 of them in pts with AITL histology, and 5 best responses of stable disease (SD) were observed. In the AITL cohort (11 evaluable of 16 pts enrolled), a 45% ORR and 73% clinical benefit rate (CBR; 3 CR, 2 PR and 3 SD) was observed. In the wt CXCL12 3'UTR cohort (n=12 evaluable pts), a 42% ORR was observed (3 CR, 2 PR), with 2 of the 3 CRs observed in patients of AITL histology (n=4). A total of 23 AITL subjects were enrolled in the overall study of whom 16 had WES data. A strong association with the activity of tipifarnib was observed in 8 of the 16 (50%) carrying KIR3DL2 gene variants C336R/Q386E: 50% CR rate, 75% ORR, 100% clinical benefit rate. These tumors expressed also very low levels of CXCL5, a ligand for CXCR2, that may mediate resistance to tipifarnib. High Allele Frequency of KIR3DL2 variants predicted CR to tipifarnib treatment (ROC AUC=0.94, p<0.0001) and AITL patients carrying KIR3DL2 gene variants experienced a better outcome with tipifarnib treatment than with prior SOC treatment. While AITL tumors expressed high levels of CXCL12 and responded to tipifarnib, a trend for poor prognosis (22 vs 40 months median OS from diagnosis, HR=1.8, p=0.09) was observed in a series of 50 PTCL subjects treated with SOC therapy. Fifty percent of AITL and 35% of non-AITL samples overexpressed CXCL12. Conclusion The AITL and wt CXCL12 3'UTR cohorts met pre-specified statistical hypotheses supporting proof-of-concept for tipifarnib in PTCL. AITL histology, KIR3DL2 and CXCL12 genotype provided robust tools for the selection/stratification of PTCL subjects treated with tipifarnib. Extended enrollment of AITL patients continues and an update on enrollment and outcomes will be provided at the time of the presentation. Disclosures Sokol: EUSA: Consultancy. Foss:Mallinckrodt: Consultancy; Eisai: Consultancy; Spectrum: Other: fees for non-CME/CE services ; Acrotech: Consultancy; Seattle Genetics: Consultancy, Other: fees for non-CME/CE services ; miRagen: Consultancy. Kim:Donga: Research Funding; Celltrion: Research Funding; J&J: Research Funding; Roche: Research Funding; Kyowa-Kirin: Research Funding; Novartis: Research Funding; Mundipharma: Research Funding. Jacobsen:Kura Oncology: Research Funding. Advani:Kyowa Kirin Pharmaceutical Developments, Inc.: Consultancy; Kura: Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Research Funding; Agensys: Research Funding; Stanford University: Employment, Equity Ownership; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Regeneron: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Celmed: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forty-Seven: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cell Medica, Ltd: Consultancy; Gilead Sciences, Inc./Kite Pharma, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Infinity Pharma: Research Funding; Millennium: Research Funding. Roncero:Kura Oncology: Research Funding. Terol:Janssen: Consultancy, Research Funding; Roche: Consultancy; Gilead: Research Funding; Astra Zeneca: Consultancy; Abbvie: Consultancy. Domingo-Domenech:Bristol-Myers Squibb: Other: Travel expenses; Roche: Other: Travel expenses; Seattle Genetics: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses. Piris:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding. Rodriguez:Kura Oncology: Research Funding. Bolognese:Kura Oncology: Consultancy. Kessler:Kura Oncology: Employment. Mishra:Kura Oncology: Employment. Curry:Kura Oncology: Employment. Kurman:Kura Oncology: Employment. Scholz:Kura Oncology: Employment, Equity Ownership, Patents & Royalties. Gualberto:Kura Oncology: Employment, Equity Ownership, Patents & Royalties.

Abstract Background Tipifarnib is a potent and selective inhibitor of the enzyme farnesyltransferase (FT). FT catalyzes post-translational attachment of farnesyl groups required for localization of signaling molecules to the inner cell membrane. CXCL12 is a chemokine that is essential for T cell homing to lymphoid organs and the bone marrow, and for the maintenance of immune cell progenitors. Herein we report preliminary efficacy, safety and biomarker data from a Phase 2 study of tipifarnib in angioimmunoblastic T-cell lymphoma (AITL) and CXCL12+ peripheral T-cell lymphoma (PTCL) patients (pts). Methods This Phase 2 study is a multi-institutional, single-arm, open-label trial initially designed as a two-stage (11+7 pts) cohort to determine the efficacy, safety and biomarkers of tipifarnib in pts with relapsed/refractory (R/R) PTCL, age >/=18 years and a performance status of 0-2. Based on initial findings, the study was amended to include a cohort of AITL (n=12) and PTCL (n=12) pts with the CXCL12 rs2839695 A/A genotype (CXCL12+ cohort). Pts received tipifarnib 300 mg administered orally twice daily on days 1-21 of 28-day treatment cycles until progression of disease (PD) or unacceptable toxicity. The primary endpoint of the study is overall response rate (ORR). Ancillary studies are also ongoing to investigate the prognostic value of CXCL12 expression in patients who received standard of care treatment. Results As of 25 July 2018, 34 PTCL pts (13 AITL, 1 ALK- ALCL, 20 PTCL-NOS) have been treated with tipifarnib, 19 pts in stages 1 and 2, and 15 pts in the ongoing AITL histology and CXCL12 cohorts. Median number of prior treatment regimens was 3. The most common treatment-related adverse events (AE) (grade ≥ 3) are hematological, including neutropenia (50%), thrombocytopenia (43%), leukopenia (33%), febrile neutropenia (27%), and anemia (20%). Skin and subcutaneous tissue disorders were reported in 9 pts, 6 of them with AITL histology. One pt with AITL experienced an episode of possible Stevens Johnson Syndrome that resolved with dose discontinuation and did not recur upon re-challenge at one dose level reduction. Of 18 evaluable patients enrolled in Stages 1 and 2 of the trial, 3 partial responses (PR), 2 of them in pts with AITL histology, and 4 best responses of stable disease (SD) were observed. Pre-treatment tumor tissue CXCL12 expression correlated with favorable pt outcomes. In the AITL cohort (10 evaluable pts), 1 PR and 1 SD have been observed so far, with 5 pts pending cycle 2 response evaluation. In the CXCL12+ cohort (n=3 evaluable pts), 1 SD has been observed, with 2 pts pending cycle 2 response evaluation. Plasma levels of CXCL12 decreased over time with tipifarnib treatment. Expression of CXCL12 mRNA and other biomarkers in pre-treatment biopsies of pts in the AITL and CXCL12+ cohorts are being evaluated using RT-PCR assays. In addition, the prognostic value of CXCL12 is being investigated in approximately 100 diagnostic specimens of PTCL pts who received standard therapy. Preliminary data suggest that CXCL12 overexpression is observed in approximately 25% of PTCL and negatively affects pt survival. Conclusion Preliminary activity of tipifarnib was observed in PTCL pts, particularly in those with tumors of AITL histology and high CXCL12 expression and enrollment continues. Disclosures Witzig: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sokol:Mallinckrodt Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy; Spectrum Pharmaceuticals: Consultancy. Jacobsen:Merck: Consultancy; Seattle Genetics: Consultancy. Kim:Takeda: Research Funding; J&J: Research Funding; Mundipharma: Research Funding; Roche: Research Funding; Novartis: Research Funding; Kyowa-Kirin: Research Funding; Celltrion: Research Funding. Foss:Miragen: Consultancy, Speakers Bureau; Spectrum: Consultancy; Seattle genetics: Consultancy; Mallinkrodt: Consultancy. Advani:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kura: Research Funding; Merck: Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board, Research Funding; Kyowa: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Regeneron: Research Funding; Janssen: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Cell Medica: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Astra Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Autolus: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board, Research Funding; Gilead/Kite: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millenium: Research Funding; Agensys: Research Funding; Celgene: Research Funding; Forty Seven: Research Funding; Infinity: Research Funding. Marin Niebla:Amgen: Other: Medical education of Staff, Speakers Bureau; Roche: Consultancy, Other: Medical education of Staff, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Other: Medical education of Staff, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Medical education of Staff, Speakers Bureau. Piris:Kura: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Curry:Kura Oncology: Employment, Equity Ownership. Gualberto:Kura Oncology: Employment, Equity Ownership.

Abstract 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.

Abstract Abstract 2638 We recently demonstrated that over 30% of cases with splenic marginal-zone lymphoma (SMZL) express distinctive immunoglobulin (IG) receptors that utilize a single polymorphic variant of the IGHV1-2 gene (IGHV1-2*04) and also exhibit restricted antigen-binding site motifs and precise targeting of somatic hypermutation (SHM). On these grounds, we proposed the existence of molecular subtypes of SMZL defined by immunogenetic analysis of the IG receptors with implications for selection by specific (super) antigenic element(s) in the development of at least a major subset of SMZL. In order to gain insight as to whether antigen involvement is relevant only prior to the malignant transformation or if it continues to affect the SMZL clone itself leading to intraclonal diversification (ID) through ongoing SHM, we conducted a large-scale subcloning study of rearranged IG heavy variable genes, in a total of 471 subcloned sequences from 22 SMZL cases. The analysis was intentionally biased towards cases expressing IGHV1-2*04 receptors that exhibit a series of distinctive immunogenetic features, including biased usage of the IGHD3-3 and IGHD3-10 genes, unusually long heavy complementarity-determining region 3 (VH CDR3) and minimally/borderline mutated status (identity to the germline in the range of 97–99.6%). Hence, the study group included 16 IGHV1-2*04 cases and 6 cases utilizing other IGHV genes. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and a median of 21 (9–46) colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences from the same sample. Overall, 15/22 cases (68%) carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, of which 12 utilized the IGHV1-2*04 gene whereas the remaining 3 utilized other IGHV genes. The high frequency of ID within the IGHV1-2*04 group, ranging from limited to (often) pronounced, is noteworthy in view of the generally low level of SHM among IGHV1-2*04 receptors. Detailed analysis of the distribution and molecular features of CMs revealed: (i) restricted ID patterns, in the sense of identical mutations in certain VH positions among subclones of different cases; (ii) “hotspots” of ID, i.e. particular codons exhibiting intense ongoing mutational activity (especially notable in this respect was codon 39 in VH FR2); (iii) a predominance of conservative amino acid changes, characterized by somatically introduced amino acid belonging to the same biochemical category as the mutating amino acid; and (iv) limited diversification within VH CDR3. Additionally, in 7/12 IGHV1-2*04 cases with CMs, identified ID patterns delineate distinct “clusters” of subcloned sequences with unique as well as shared mutations and closely similar if not identical VH CDR3s (including identical VH CDR3 length), pointing to “branching” of the malignant clone into distinct subclones, perhaps able to evolve along related yet distinct pathways. In conclusion, our study indicates that the SHM mechanism may continuously operate in certain subsets of SMZL, especially those expressing IGHV1-2*04 receptors. Although the precise timing of interactions with antigen(s) and their functional implications for SMZL evolution will likely remain difficult to define accurately, the results reported here suggest a role for persistent antigenic stimulation, at least for certain immunogenetically defined subsets of SMZL. Disclosures: Mollejo: Red Temática de Investigación Cooperativa en Cancer (RETICC): Research Funding; Asociación Española contra el Cancer: Research Funding.

Abstract 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.