ALBERT

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: The International Prognostic Index (IPI) is the most commonly used prognostic model in mantle cell lymphoma. However, the prognostic value of IPI is limited. The recently published Follicular Lymphoma International Prognostic Index (FLIPI) is built on variables (age, stage, lactic dehydrogenase, anemia, and nodal disease) which also are pertinent to mantle cell lymphoma. This study was conducted to evaluate the prognostic value of FLIPI in patients with mantle cell lymphoma. Patients and Methods: A population-based series of 93 patients with mantle cell lymphoma diagnosed in a 7-year period were studied. End points of the study were response to therapy, overall survival, and failure-free survival according to IPI and FLIPI. Results: Applied to the whole series, FLIPI identified 3 risk groups with markedly different outcome with 5-year overall survival rates of 65%, 42%, and 8%, respectively (P 〈 .0001; log-rank 28.13; figure below). Notably, the high-risk group comprised 53% of patients. In contrast, IPI only allocated 16% of cases to the high-risk group and had a lower overall predictive capacity (log-rank 24.8). When both FLIPI and IPI were included in a multivariate analysis, only FLIPI was related to survival. In patients treated with CHOP-based regimens (n = 45) FLIPI also had superior predictive capacity compared to IPI (log-rank, 18.51 versus 11.37), and again only FLIPI retained significance in multivariate analysis. Multivariate analysis of failure-free survival also identified FLIPI, and not IPI, as independently significant. Conclusion: FLIPI is the superior prognostic model as compared to IPI and should be the preferred clinical prognostic index in mantle cell lymphoma. Overall survival according to FLIPI risk groups Overall survival according to FLIPI risk groups

Description: Diffuse large B-cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease. Frequent genetic lesions include alterations of BCL2 and BCL6, as well as aberrations of the RB pathway and the p53 pathway resulting in deregulation of cell cycle progression and apoptosis. MYC translocations and amplifications have been detected in a subset of cases. Recently, MYC was shown to control a major regulatory network in B-cells (Nature Genet. 37; 382–90: 2005). We thus hypothesized that DLBCLs with constitutively active MYC are associated with distinctive genetic and clinical features when compared to cases with wild-type MYC. Thirty seven de novo DLBCLs were analyzed using immunohistochemistry for CD10, bcl6, MUM1, and bcl2 expression, as well as fluorescence in situ hybridization for MYC, BCL2, BCL6, IGH, IGK, and IGL breakpoints and amplifications. The lymphomas were previously extensively characterized for aberrations at the genetic, epigenetic, or protein level of components of the RB pathway (pRb, p16, cyclin D3, cdk4) and the p53 pathway (p53, p14ARF, MDM2, E2F1). Five cases (14%) had MYC alterations (4 breakpoints and 1 amplification). Of the MYC+ DLBCL cases, 2 showed GCB immunophenotype and 3 were of non-GCB type. Perturbation of the p53 pathway components p53, p14ARF, MDM2, and E2F1 were identified in 28%, 16%, 53%, and 20% of the MYC− cases, respectively, and 83% had aberrations of 1 or more p53 pathway component. In contrast, only 1 (20%) of the MYC+ cases had a p53 pathway aberration (MDM2 overexpression; p=0.013). Thus, MYC alterations and p53 pathway aberrations were inversely correlated. RB pathway aberrations were equally frequent among MYC+ (40%) and MYC− (43%) DLBCLs. However, the pattern of aberrations was different. Lack of pRb expression was the only alteration of the RB pathway in the MYC+ DLBCLs, whereas 30 of 31 (97%) MYC− lymphomas had normal pRb expression (p=0.045). p16 alterations (point mutation, deletion, hypermethylation, lack of expression) and/or cyclin D3 overexpression were identified in 12 (38%) MYC− cases but in none of the MYC+ cases (p=0.038). MYC status was also related to cytogenetic BCL2 alterations. Four of 5 (80%) MYC+ DLBCLs showed BCL2 breakpoints or amplification in contrast to 4 of 32 (13%) of MYC− DLBCLs (p=0.005). Furthermore, all MYC+ DLBCLs were bcl2 positive (p=0.05) and MUM1 negative (p=0.022), and the proliferation index was lower in the MYC+ DLBCLs (p=0.038). Clinically, MYC+ DLBCL patients were characterized by advanced stage disease (p=0.037) and high tumor burden (p=0.037) but favorable treatment response (p=0.05). Taken together, our data indicate that DLBCL with MYC alterations constitute a subgroup of DLBCL with distinct clinical and molecular features. The patterns of aberrations of the p53 pathway, the RB pathway, and BCL2 indicate profound differences in the molecular pathogenesis between MYC+ and MYC− DLBCL.

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: Abstract 1548 Introduction: The introduction of Rituximab as supplement to chemotherapy has significantly improved outcome in diffuse large B-cell lymphoma (DLBCL). Still, a fraction of patients are resistant or relapse shortly after treatment. Improved stratification of patients with DLBCL for standard immunochemotherapy or alternative treatment strategies is therefore urgently needed. Although DLBCL profiling based on mRNA expression may be helpful, this has not proven clinically efficient, and the prognostic value of immunohistochemical algorithms is controversial. In addition, novel therapeutic options are essential since the current alternative treatment modalities are often not curative. MicroRNAs (miRs) are particularly attractive molecules for clinical use since they are well conserved in formalin fixed paraffin embedded (FFPE) tissue, and novel data imply that they may be targeted directly in the patients. Materials and methods: RNA was extracted from diagnostic biopsies from DLBCL patients (n=97) treated uniformly with immunochemotherapy (R-CHOP n=80 or R-CHOEP n=17). GCB/non-GCB profiling was done by immunohistochemistry according to the Hans classification. MiR profiles were generated using Affymetrix microRNA version 1.0 arrays. Data analyses were performed using R/biocondutor and the webtool “SignS” that uses parallel computing for finding survival related genes and signatures from gene-expression datasets. Survival analysis was performed in R using the survival package. Univariate analysis was performed by comparing Kaplan-Meier survival estimates using Log-rank test. Cox proportional hazards regression model was used for multivariate analysis. Results and discussion: The median follow-up time for all patients was 3.4 years. The estimated 3-year over all survival probability was 82.8% (95% CI: 75.4%-90.9%). No difference in survivability was observed between the R-CHOP and the R-CHOEP treated cohort (P=0.145). High IPI (〉 2) was significantly associated with inferior overall survival (OS, P = 0.038), but not progression free survival (PFS, P = 0.083). Univariate analysis showed that in this cohort the Hans classification was not prognostic (P=0.73; (52 GBC and 37 non-GCB subtypes; 8 NA)). Seven miRs were differentially regulated between GCB and non-GCB using a cutoff of P〈 0.05. Five miRs were upregulated in non-GCB lymphomas: miR-625, miR-222, miR-221, miR-155 and miR-503, two were downregulated (miR-181a, miR-181b). For survival analysis, we initially applied a multivariate approach (Robust likelihood-based survival modeling, RBsurv), which identified a subset of miRs that significantly associates with poor survival. These include one upregulated miR, and four down regulated miRs. In order to obtain cross validated survival estimates, we applied three different algorithms; FCSM(SignS), TGD(SignS) and GLMboost(SignS) to the same sample set. These combined bioinformatic models identified a total of 17 deregulated miRs that significantly associate with survival. Among these, 9 are predicted by more that one algorithm, and interestingly, all 4 models identify a novel upregulated and potential oncogenic miR in patients treated by immunochemotherapy. When the cross-validated predictors were combined into a unified robust “miR-survival-predictor”, the performance is as good as, or even better, than the IPI. In addition, our model is a superior predictor of survival than the GCB/non-GCB classification according to Hans. Our data are currently being validated in a test set of 60 patients, and functional studies of the novel putative oncomiR are ongoing. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Disruption of miR34a, -b, and -c has been implicated in lymphomagenesis, and in CLL it has been suggested that miR34a expression is a surrogate marker for TP53 disruption associated with a poor prognosis. P53 is a transcription factor for the miR34s, and overexpression of miR34s in p53 deficient cells can reinstate p53 functions (Chang et al, Mol Cell 26, 2007, He et al, Nature 447, 2007). However, in cellular senescence miR34a may be activated independently of p53 (Christoffersen et al, Cell Death Differ17, 2010). A recent multicenter study shows that in diffuse large B-cell lymphoma (DLBCL), TP53 disruption is still a negative prognostic factor for survival after the implementation of Rituximab (Xu-Monette et al, Blood 19, 2012). Information on the role of the miR34s in normal CD19+ B-cells (PBL-B), activated B-cells, and de novo diffuse large B-cell lymphoma (DLBCL) is limited. Aims Given that MIR34A and MIR34B/C locate to regions of allelic loss in DLBCL (1p36.23 and 11q23.1, respectively), and the importance of the miR34 targets in DLBCL pathogenesis, we investigated a large panel of newly diagnosed cases of DLBCLs for MIR34A and MIR34B/C promoter methylation, TP53 mutational status, clinical presentation patterns, and outcome. Methods MIR34A/B/C promotor methylation was performed by MsMCA and bisulfite sequencing. Histone modifcations at the MIR34A/B/C promotor was examined by ChIP RT-qPCR. Expression of miR34s was performed using miRCURY LNA™ Universal RT-qPCR. The coding sequences and splice sites of exons 5-9 of the TP53gene were scanned for mutations by PCR and denaturing gradient gel electrophoresis (DGGE). Differences in clinical characteristics using the one-way Anova, the Pearson chi-square, or Fisher’s exact tests. Overall survival was estimated using the Kaplan-Meier method and log-rank test. For assessment of independent predictors of survival a multivariate Cox regression hazard model with backward stepwise (likelihood ratio) entry was applied. Effects not meeting a p-value 〈 0.05 were removed from the model. Results We show that only miR34a-5p is expressed in PBL-B, and significantly induced in activated B-cells (P=0.017) and reactive lymph nodes (P

Description: Abstract 949 Introduction: Diffuse large B cell lymphoma (DLBCL) has a highly variable outcome, and individual risk assessment is largely based on clinical features. Gene expression profiling (GEP) stratifies patients into those with germinal center B-cell (GCB) and activated B-cell subtype (ABC) subtype with different prognoses. These groups have been shown to predict prognosis in patients treated with CHOP or R-CHOP. Conversely, the role of other recognized prognostic markers, such as BCL2 gene abnormalities or Bcl2 expression has been questioned in the new therapeutic era. Materials and Methods: In 438 patients treated with R-CHOP for de novo DLBCL, we analyzed the tumors by immunohistochemistry for Bcl2 protein expression and by interphase fluorescence in situ hybridization (FISH) for BCL2 translocation and other abnormalities. All cases were successfully studied by GEP. The cutoff for Bcl2 protein expression, 60%, used as prognostic factor was determined using receiver operating characteristic curves. Progression-free survival (PFS) and overall survival (OS) were assessed. Results: The t(14;18)(q32;q21) was detected in 82 cases (18.7%) and BCL2 gains occurred in 63 cases (14.3%). Both t(14;18) and BCL2 gains strongly correlated with higher levels of Bcl2 protein expression (p

Description: Purpose: Prognosis of lymphoma patients is usually estimated at the time of diagnosis, and the estimates are guided by the International Prognostic Index (IPI). However, survival rates calculated at the time of diagnosis may not be predictive of outcome for patients who have survived 1 or more years. For these patients conditional survival is more informative as only those patients who have already survived a period of time after treatment are considered. Conditional survival data have not been reported for lymphoma patients and the impact of the IPI on conditional survival is not known. Patients and Methods: Conditional survival was estimated for 1,209 patients with diffuse large B-cell lymphoma from the population-based LYFO-registry of the Danish Lymphoma Group. The patients were diagnosed between 1983 and 1998 with a median follow-up time of 7.8 years, and all patients were treated with curative intent. The Kaplan-Meier method was used to estimate conditional survival at 0–5 years after diagnosis. Results: The probability of surviving 5 years increased with each year survived for the first 3 years after diagnosis (from 43% to 70%) after which the increase was minimal (Figure 1). The median survival increased from 38 months for all patients to between 108 and 120 months conditioned on survival for at least 3 to 5 years. The distribution of patients in the IPI risk groups changed significantly over time. However this effect was only seen in the first 5 years. At diagnosis, 28% of the patients were allocated to the high-intermediate and high-risk groups, but only 11% of patients who survived 5 years were from these risk groups (P 〈 .0001). Risk group allocation did not change the following 5 years (P = .7747). At the time of diagnosis overall survival differed greatly between the IPI risk groups (P 〈 .0001; figure 2). However, 5 years after diagnosis the rates were similar (P = .0586). Furthermore, the prognostic impact of the individual clinical variables of the IPI (age, stage, performance score, LDH, and extranodal disease) was also significant at diagnosis, but 2 years after diagnosis only age had prognostic impact. Multivariate analysis (including the IPI and the individual IPI variables) of patients who survived ≥3 years identified only age as a prognostic factor (relative risk 4.1; P 〈 .001). Conclusion: For patients with diffuse large B-cell lymphoma who have survived more than 1 year after diagnosis, the conditional survival probability provides more accurate prognostic information than the conventional survival rate estimated from the time of diagnosis. Furthermore, age is the most important factor for long-term survival, whereas the remaining IPI factors are not informative. Figure Figure Figure Figure

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.