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Diffuse large B-cell lymphoma can be classified by gene expression profiling into germinal center and activated B-cell subtypes with different prognoses after rituximab-CHOP. The importance of previously recognized prognostic markers, such as Bcl-2 protein expression and BCL2 gene abnormalities, has been questioned in the new therapeutic era. We analyzed Bcl-2 protein expression, and BCL2 and MYC gene abnormalities by interphase fluorescence in situ hybridization in 327 patients with de novo disease treated with rituximab-CHOP. Isolated BCL2 and MYC rearrangements were not predictive of outcome in our patients as a whole, but only in those with the germinal center subtype of lymphoma. The prognostic relevance of isolated MYC rearrangements was weaker than that of BCL2 isolated translocations, but was probably limited by the rarity of the rearrangements. Seven of eight patients with double hit lymphoma had the germinal center subtype with poor outcome. The germinal center subtype patients with isolated BCL2 translocations had significantly worse outcome than the patients without BCL2 rearrangements (P=0.0002), and their outcome was similar to that of patients with the activated B-cell subtype (P=0.30), but not as bad as the outcome of patients with double hit lymphoma (P

Gene expression profiling (GEP) has stratified diffuse large B-cell lymphoma (DLBCL) into molecular subgroups that correspond to different stages of lymphocyte development - namely germinal center B-cell-like and activated B-cell-like. This classification has prognostic significance, but GEP is expensive and not readily applicable into daily practice, which has lead to immunohistochemical algorithms proposed as a surrogate for GEP analysis. We assembled tissue microarrays from 475 de novo DLBCL patients who were treated with rituximab-CHOP chemotherapy. All cases were successfully profiled by GEP on formalin-fixed, paraffin-embedded tissue samples. Sections were stained with antibodies reactive with CD10, GCET1, FOXP1, MUM1, and BCL6 and cases were classified following a rationale of sequential steps of differentiation of B-cells. Cutoffs for each marker were obtained using receiver operating characteristic curves, obviating the need for any arbitrary method. An algorithm based on the expression of CD10, FOXP1, and BCL6 was developed that had a simpler structure than other recently proposed algorithms and 92.6% concordance with GEP. In multivariate analysis, both the International Prognostic Index and our proposed algorithm were significant independent predictors of progression-free and overall survival. In conclusion, this algorithm effectively predicts prognosis of DLBCL patients matching GEP subgroups in the era of rituximab therapy.

In diffuse large B-cell lymphoma (DLBCL), the clinical and biological significance of concordant and discordant bone marrow (BM) involvement have not been well investigated. We evaluated 712 de novo DLBCL patients with front-line rituximab-containing treatment, including 263 patients with positive and 449 with negative BM status. Compared with negative BM disease, concordant BM adversely impacted overall and progression-free survival, independent of the International Prognostic Index (IPI) and cell-of-origin classification. Once BM is concordantly involved, poor prognosis was not associated with the extent of BM involvement. Conversely, patients with discordant BM showed favorable overall survival similar to stage I-II DLBCL. A BM-adjusted IPI, using three parameters: concordant BM involvement, age >60 years, and performance status >1, improves the risk stratification for DLBCL with positive BM. Intensive immunochemotherapy seemingly rendered survival benefit for patients with concordant BM, as did rituximab maintenance for the discordant BM group. Frequently revealing adverse clinical and molecular characteristics, patients with concordant BM demonstrated gene expression signatures relevant to tumor cell proliferation, migration and immune escape. In conclusion, clinical and biological heterogeneity is seen in DLBCL with positive BM but concordant BM involvement represents a distinct subset with unfavorable gene signatures, high-risk clinicopathologic features and poor prognosis.

Background: Diffuse large B-cell lymphoma (DLBCL) harbors somatic hypermutation (SHM) in the immunoglobulin heavy chain and light chain variable region genes, IGHV and IGK/LV. Recent studies have revealed that IGV SHM creates neoantigens that activate T-cell responses against B-cell lymphoma. Methods: To determine the clinical relevance of IGV SHM in DLBCL treated with standard immunochemotherapy, we performed next-generation sequencing of the immunoglobulin variable regions and complementarity determining region 3 (CDR3) for 378 patients with de novo DLBCL. The prognostic effects of IGV SHM and ongoing SHM or intra-clonal heterogeneity were analyzed in the training (192 patients), validation (186 patients), and overall DLBCL cohorts. To gain mechanistic insight, we analyzed the predicted IG-derived neoantigens' immunogenicity potential, determined by the major histocompatibility complex-binding affinity and the frequency-of-occurrence of T cell-exposed motifs (TCEMs) in a TCEM repertoire derived from human proteome, microbiome, and pathogen databases. Furthermore, IGV SHM was correlated with molecular characteristics of DLBCL and PD-1/L1 expression in the tumor microenvironment assessed by fluorescent multiplex immunohistochemistry. Results: SHM was commonly found in IGHV and less frequently in IGK/LV. High levels of clonal IGHV SHM (SHMhigh) were associated with prolonged overall survival in DLBCL patients, particularly those without BCL2 or MYC translocation. In contrast, long heavy chain CDR3 length, the presence of IGHV ongoing SHM in DLBCL, and high clonal IGK/LV SHM in germinal center B-cell-like (GCB)-DLBCL were associated with poor prognosis. These prognostic effects were significant in both the training and validation sets. By prediction, the SHMhigh groups harbored more potentially immune-stimulatory neoantigens with high binding affinity and rare TCEMs. PD-1/L1 expression in CD8+ T cells was significantly lower in IGHV SHMhigh than in SHMlow patients with activated B-cell-like DLBCL, whereas PD-1 expression in CD4+ T cells and PD-L1 expression in natural killer cells were higher in IGK/LV SHMhigh than in SHMlow patients with GCB-DLBCL. PD-L1/L2 (9p24.1) amplification was associated with high IGHV SHM and ongoing SHM. Conclusions: These results show for the first time that IGV SHMhigh and ongoing SHM have prognostic effects in DLBCL and potential implications for PD-1/PD-L1 blockade and neoantigen-based immunotherapies.

PD-1/L1 and CTLA-4 blockade immunotherapies have been approved for 13 types of cancers and are being studied in diffuse large B-cell lymphoma (DLBCL), the most common aggressive B-cell lymphoma. However, whether both PD-1 and CTLA-4 checkpoints are active and clinically significant in DLBCL is unknown. Whether PD-1 ligands expressed by tumor cells or by the microenvironment of DLBCL are critical for the PD-1 immune checkpoint is unclear. We performed immunophenotypic profiling for 405 patients with de novo DLBCL using a MultiOmyx immunofluorescence platform and simultaneously quantitated expression/coexpression of 13 immune markers to identify prognostic determinants. In both training and validation cohorts, results demonstrated a central role of the tumor immune microenvironment, and when its functionality was impaired by deficiency in tumor-infiltrating T cells and/or natural killer cells, high PD-1 expression (but not CTLA-4) on CD8+ T cells, or PD-L1 expression on T cells and macrophages, patients had significantly poorer survival after rituximab–CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) immunochemotherapy. In contrast, tumor-cell PD-L2 expression was associated with superior survival, as well as PD-L1+CD20+ cells proximal (indicates interaction) to PD-1+CD8+ T cells in patients with low PD-1+ percentage of CD8+ T cells. Gene-expression profiling results suggested the reversibility of T-cell exhaustion in PD-1+/PD-L1+ patients with unfavorable prognosis and implication of LILRA/B, IDO1, CHI3L1, and SOD2 upregulation in the microenvironment dysfunction with PD-L1 expression. This study comprehensively characterized the DLBCL immune landscape, deciphered the differential roles of various checkpoint components in rituximab–CHOP resistance in DLBCL patients, and suggests targets for PD-1/PD-L1 blockade and combination immunotherapies.

BACKGROUND: The clinical presentation of patients with hepatitis C virus (HCV)-positive diffuse large B-cell lymphoma (DLBCL) is different from their HCV-negative counterparts, but the underlying molecular and pathological characteristics are largely under investigated. The virus has a role in lymphomagenesis, as witnessed by the curative potential of antiviral therapy in HCV-related low-grade B-cell lymphomas.METHODS: We performed a case-control study including 44 HCV-positive cases of de novo DLBCL, comparing them with 132 HCV-negative patients as controls (ratio 3 to 1). Cases and controls were matched for age, lactate dehydrogenase level and international prognostic index at presentation. Patients were studied by gene expression profiling for cell-of-origin determination and to perform differential expression analysis between groups, fluorescence in-situ hybridisation and immunohistochemistry for MYC, BCL2 and BCL6, TP53 mutations, and diagnostic specimens reviewed to exclude transformation from low-grade lymphoma.RESULTS: Compared to the HCV-negative controls, patients with HCV-positive de novo DLBCL had differential expression of genes that regulate innate immune response and modulate apoptotic pathways, have higher proliferative index, and lack BCL2 translocations.CONCLUSIONS: HCV-positive DLBCL have distinct molecular and pathological features compared to the HCV-negative counterparts.British Journal of Cancer advance online publication, 26 September 2017; doi:10.1038/bjc.2017.345 www.bjcancer.com.

It has been well established that nuclear factor kappa-B (NF-κB) activation is important for tumor cell growth and survival. RelA/p65 and p50 are the most common NF-kB subunits and involved in the classical NF-kB pathway. However, the prognostic and biological significance of RelA/p65 is equivocal in the field. In this study, we assessed RelA/p65 nuclear expression by immunohistochemistry in 487 patients with de novo diffuse large B-cell lymphoma (DLBCL), and studied the effects of molecular and pharmacological inhibition of NF-kB on cell viability. We found RelA/p65 nuclear expression, without associations with other apparent genetic or phenotypic abnormalities, had unfavorable prognostic impact in patients with stage I/II DLBCL. Gene expression profiling analysis suggested immune dysregulation and antiapoptosis may be relevant for the poorer prognosis associated with p65 hyperactivation in germinal center B-cell-like (GCB) DLBCL and in activated B-cell-like (ABC) DLBCL, respectively. We knocked down individual NF-κB subunits in representative DLBCL cells in vitro, and found targeting p65 was more effective than targeting other NF-κB subunits in inhibiting cell growth and survival. In summary, RelA/p65 nuclear overexpression correlates with significant poor survival in early-stage DLBCL patients, and therapeutic targeting RelA/p65 is effective in inhibiting proliferation and survival of DLBCL with NF-κB hyperactivation.

Contains fulltext : 171804.pdf (Publisher’s version ) (Closed access) CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its clinical and biologic significance in 773 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 231 patients treated with CHOP. We found that CD37 loss (CD37-) in approximately 60% of DLBCL patients showed significantly decreased survival after R-CHOP treatment, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37-, including TP53 mutation, NF-kappaBhigh, Mychigh, phosphorylated STAT3high, survivinhigh, p63-, and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37- status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R-CHOP (M-IPI-R), or IPI plus immunohistochemistry (IHC; IPI+IHC) for CD37, Myc, and Bcl-2, significantly improved risk prediction over IPI alone. Gene expression profiling suggested that decreased CD20 and increased PD-1 levels in CD37- DLBCL, ICOSLG upregulation in CD37+ GCB-DLBCL, and CD37 functions during R-CHOP treatment underlie the pivotal role of CD37 status in clinical outcomes. In conclusion, CD37 is a critical determinant of R-CHOP outcome in DLBCL especially in GCB-DLBCL, representing its importance for optimal rituximab action and sustained immune responses. The combined molecular and clinical prognostic indices, M-IPI-R and IPI+IHC, have remarkable predictive values in R-CHOP-treated DLBCL.

Contains fulltext : 173033.pdf (Publisher’s version ) (Open Access) PRDM1/BLIMP-1, a master regulator of plasma-cell differentiation, is frequently inactivated in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) patients. Little is known about its genetic aberrations and relevant clinical implications. A large series of patients with de novo DLBCL was effectively evaluated for PRDM1/BLIMP-1 deletion, mutation, and protein expression. BLIMP-1 expression was frequently associated with the ABC phenotype and plasmablastic morphologic subtype of DLBCL, yet 63% of the ABC-DLBCL patients were negative for BLIMP-1 protein expression. In these patients, loss of BLIMP-1 was associated with Myc overexpression and decreased expression of p53 pathway molecules. In addition, homozygous PRDM1 deletions and PRDM1 mutations within exons 1 and 2, which encode for domains crucial for transcriptional repression, were found to show a poor prognostic impact in patients with ABC-DLBCL but not in those with germinal center B-cell-like DLBCL (GCB-DLBCL). Gene expression profiling revealed that loss of PRDM1/BLIMP-1 expression correlated with a decreased plasma-cell differentiation signature and upregulation of genes involved in B-cell receptor signaling and tumor-cell proliferation. In conclusion, these results provide novel clinical and biological insight into the tumor-suppressive role of PRDM1/BLIMP-1 in ABC-DLBCL patients and suggest that loss of PRDM1/BLIMP-1 function contributes to the overall poor prognosis of ABC-DLBCL patients.

Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a unique subtype of DLBCL. The impact of rituximab on survival and patterns of treatment failure in PT-DLBCL patient remain controversial. We analyzed the clinical and biological feature of 280 PT-DLBCL cases, 64% of which were treated with rituximab-containing regimens. Although most (95%) patients achieved complete remission, a continuous risk of relapse was observed. Rituximab significantly reduced the cumulative risk of relapse (P=0.022) and improved both progression-free survival (PFS, P=0.012) and overall survival (OS, P=0.027) of PT-DLBCL patients (5-year PFS, 56% vs 36%; 5-year OS, 68% vs 48%). Central nervous system and contralateral testis were the most common sites of relapse, but other extranodal and nodal sites of relapse were also observed. Most cases of PT-DLBCL had a non-germinal center B-cell like (84%) immunophenotype and an activated B-cell like (86%) gene expression profile (GEP) subtype. The distinctive GEP signature of primary testicular lymphoma was relevant to tumor cell proliferation, dysregulated expression of adhesion molecules and immune response, likely accounting for the poor outcome. Accordingly, forkhead box P1 transcription factor (FOXP1) and T-cell leukemia/lymphoma 1 (TCL1) oncogenic activation were confirmed and predicted a significant trend of poor survival. This study provides valuable observations for better understanding of both clinical and biological features in PT-DLBCL patients.

Purpose: MYC is a critical driver oncogene in many cancers, and its deregulation in the forms of translocation and overexpression has been implicated in lymphomagenesis and progression of diffuse large B-cell lymphoma (DLBCL). The MYC mutational profile and its roles in DLBCL are unknown. This study aims to determine the spectrum of MYC mutations in a large group of patients with DLBCL, and to evaluate the clinical significance of MYC mutations in patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) immunochemotherapy. Experimental Design: We identified MYC mutations in 750 patients with DLBCL using Sanger sequencing and evaluated the prognostic significance in 602 R-CHOP–treated patients. Results: The frequency of MYC mutations was 33.3% at the DNA level (mutations in either the coding sequence or the untranslated regions) and 16.1% at the protein level (nonsynonymous mutations). Most of the nonsynonymous mutations correlated with better survival outcomes; in contrast, T58 and F138 mutations (which were associated with MYC rearrangements), as well as several mutations occurred at the 3′ untranslated region, correlated with significantly worse survival outcomes. However, these mutations occurred infrequently (only in approximately 2% of DLBCL). A germline SNP encoding the Myc-N11S variant (observed in 6.5% of the study cohort) was associated with significantly better patient survival, and resulted in reduced tumorigenecity in mouse xenografts. Conclusions: Various types of MYC gene mutations are present in DLBCL and show different impact on Myc function and clinical outcomes. Unlike MYC gene translocations and overexpression, most MYC gene mutations may not have a role in driving lymphomagenesis. Clin Cancer Res; 22(14); 3593–605. ©2016 AACR.

Contains fulltext : 152029.pdf (Publisher’s version ) (Closed access) MYC dysregulation, including MYC gene rearrangement and Myc protein overexpression, is of increasing clinical importance in diffuse large B-cell lymphoma (DLBCL). However, the roles of MYC and the relative importance of rearrangement vs overexpression remain to be refined. Gaining knowledge about the tumor biology associated with MYC dysregulation is important to understand the roles of MYC and MYC-associated biology in lymphomagenesis. In this study, we determined MYC rearrangement status (n=344) and Myc expression (n=535) in a well-characterized DLBCL cohort, individually assessed the clinical and pathobiological features of patients with MYC rearrangement and Myc protein overexpression, and analyzed the prognosis and gene expression profiling signatures associated with these MYC abnormalities in germinal center B-cell-like and activated B-cell-like DLBCL. Our results showed that the prognostic importance of MYC rearrangement vs Myc overexpression is significantly different in germinal center B-cell-like vs activated B-cell-like DLBCL. In germinal center B-cell-like DLBCL, MYC-rearranged germinal center B-cell-like DLBCL patients with Myc overexpression significantly contributed to the clinical, biological, and prognostic characteristics of the overall Myc-overexpressing germinal center B-cell-like DLBCL group. In contrast, in activated B-cell-like DLBCL, the occurrence, clinical and biological features, and prognosis of Myc overexpression were independent of MYC rearrangement. High Myc levels and Myc-independent mechanisms, either tumor cell intrinsic or related to tumor microenvironment, conferred significantly worse survival to MYC-rearranged germinal center B-cell-like DLBCL patients, even among Myc(high)Bcl-2(high) DLBCL patients. This study provides new insight into the tumor biology and prognostic effects associated with MYC dysregulation and suggest that detection of both MYC translocations and evaluation of Myc and Bcl-2 expression is necessary to predict the prognosis of DLBCL patients.

Purpose: Epstein–Barr virus–positive (EBV+) diffuse large B-cell lymphoma (DLBCL) of the elderly is a variant of DLBCL with worse outcome that occurs most often in East-Asian countries and is uncommon in the Western hemisphere. We studied the largest cohort of EBV+ DLBCL, independent of age, treated with rituximab combined with CHOP (R-CHOP) in developed Western countries. Experimental design: A large cohort (n = 732) of patients with DLBCL treated with R-CHOP chemotherapy is included from the multicenter consortium. This study group has been studied for expression of different biomarkers by immunohistochemistry, genetic abnormalities by FISH and mutation analysis, genomic information by gene expression profiling (GEP), and gene set enrichment analysis (GSEA). Results: Twenty-eight patients (4.0%) were positive for EBV with a median age of 60.5 years. No clinical characteristics distinguished patients with EBV+ DLBCL from patients with EBV-negative (EBV−) DLBCL. Genetic aberrations were rarely seen. NF-κB p50, phosphorylated STAT-3, and CD30 were more commonly expressed in EBV+ DLBCLs (P < 0.05). Significant differences in survival were not observed in patients with EBV+ DLBCL versus EBV− DLBCL. However, CD30 expression combined with EBV conferred an inferior outcome. GEP showed a unique expression signature in EBV+ DLBCL. GSEA revealed enhanced activity of the NF-κB and JAK/STAT pathways independent of molecular subtype. Conclusions: The clinical characteristics of patients with EBV+ versus EBV− DLBCL are similar and EBV infection does not predict a worse outcome. EBV+ DLBCL, however, has a unique genetic signature. CD30 expression is more common in EBV+ DLBCL and, consistent CD30 and EBV is associated with an adverse outcome. Clin Cancer Res; 20(9); 2338–49. ©2014 AACR.

Purpose: Activated signal transducer and activator of transcription 3 (STAT3) regulates tumor growth, invasion, cell proliferation, angiogenesis, immune response, and survival. Data regarding expression of phosphorylated (activated) STAT3 in diffuse large B-cell lymphoma (DLBCL) and the impact of phosphorylated STAT3 (pSTAT3) on prognosis are limited. Experimental Design: We evaluated expression of pSTAT3 in de novo DLBCL using immunohistochemistry, gene expression profiling (GEP), and gene set enrichment analysis (GSEA). Results were analyzed in correlation with cell-of-origin (COO), critical lymphoma biomarkers, and genetic translocations. Results: pSTAT3 expression was observed in 16% of DLBCL and was associated with advanced stage, multiple extranodal sites of involvement, activated B-cell–like (ABC) subtype, MYC expression, and MYC/BCL2 expression. Expression of pSTAT3 predicted inferior overall survival (OS) and progression-free survival (PFS) in patients with de novo DLBCL. When DLBCL cases were stratified according to COO or MYC expression, pSTAT3 expression did not predict inferior outcome, respectively. Multivariate analysis showed that the prognostic predictability of pSTAT3 expression was due to its association with the ABC subtype, MYC expression, and adverse clinical features. GEP demonstrated upregulation of genes, which can potentiate function of STAT3. GSEA showed the JAK–STAT pathway to be enriched in pSTAT3+ DLBCL. Conclusions: The results of this study provide a rationale for the ongoing successful clinical trials targeting the JAK–STAT pathway in DLBCL. Clin Cancer Res; 20(19); 5113–23. ©2014 AACR.

Contains fulltext : 118936.pdf (Publisher’s version ) (Open Access) Diffuse large B-cell lymphoma can be classified by gene expression profiling into germinal center and activated B-cell subtypes with different prognoses after rituximab-CHOP. The importance of previously recognized prognostic markers, such as Bcl-2 protein expression and BCL2 gene abnormalities, has been questioned in the new therapeutic era. We analyzed Bcl-2 protein expression, and BCL2 and MYC gene abnormalities by interphase fluorescence in situ hybridization in 327 patients with de novo disease treated with rituximab-CHOP. Isolated BCL2 and MYC rearrangements were not predictive of outcome in our patients as a whole, but only in those with the germinal center subtype of lymphoma. The prognostic relevance of isolated MYC rearrangements was weaker than that of BCL2 isolated translocations, but was probably limited by the rarity of the rearrangements. Seven of eight patients with double hit lymphoma had the germinal center subtype with poor outcome. The germinal center subtype patients with isolated BCL2 translocations had significantly worse outcome than the patients without BCL2 rearrangements (P=0.0002), and their outcome was similar to that of patients with the activated B-cell subtype (P=0.30), but not as bad as the outcome of patients with double hit lymphoma (P

CD30, originally identified as a cell-surface marker of Reed-Sternberg and Hodgkin cells of classical Hodgkin lymphoma, is also expressed by several types of non-Hodgkin lymphoma, including a subset of diffuse large B-cell lymphoma (DLBCL). However, the prognostic and biological importance of CD30 expression in DLBCL is unknown. Here we report that CD30 expression is a favorable prognostic factor in a cohort of 903 de novo DLBCL patients. CD30 was expressed in ∼14% of DLBCL patients. Patients with CD30(+) DLBCL had superior 5-year overall survival (CD30(+), 79% vs CD30(-), 59%; P = .001) and progression-free survival (P = .003). The favorable outcome of CD30 expression was maintained in both the germinal center B-cell and activated B-cell subtypes. Gene expression profiling revealed the upregulation of genes encoding negative regulators of nuclear factor κB activation and lymphocyte survival, and downregulation of genes encoding B-cell receptor signaling and proliferation, as well as prominent cytokine and stromal signatures in CD30(+) DLBCL patients, suggesting a distinct molecular basis for its favorable outcome. Given the superior prognostic value, unique gene expression signature, and significant value of CD30 as a therapeutic target for brentuximab vedotin in ongoing successful clinical trials, it seems appropriate to consider CD30(+) DLBCL as a distinct subgroup of DLBCL.

MDM2 is a key negative regulator of the tumor suppressor p53, however, the prognostic significance of MDM2 overexpression in diffuse large B-cell lymphoma (DLBCL) has not been defined convincingly. In a p53-genetically defined large cohort of de novo DLBCL patients treated with R-CHOP chemotherapy, we assessed MDM2 and p53 expression by immunohistochemistry (n=478), MDM2 gene amplification by fluorescence in situ hybridization (n=364), and a single nucleotide polymorphism in the MDM2 promoter, SNP309, by SNP genotyping assay (n=108). Our results show that MDM2 overexpression, unlike p53 overexpression, is not a significant prognostic factor in overall DLBCL. Both MDM2 and p53 overexpression does not predict for an adverse clinical outcome in patients with wild-type p53, but predicts for significantly poorer survival in patients with mutated p53. Variable p53 activities may ultimately determine the survival differences as suggested by the gene expression profiling analysis. MDM2 amplification was observed in 3 of 364 (0.8%) patients with high MDM2 expression. Presence of SNP309 did not correlate with MDM2 expression and survival. This study indicates that evaluation of MDM2 and p53 expression correlating with TP53 genetic status is essential to assess their prognostic significance, and important for designing therapeutic strategies that target the MDM2-p53 interaction. MDM2 is a key negative regulator of the tumor suppressor p53, however, the prognostic significance of MDM2 overexpression in diffuse large B-cell lymphoma (DLBCL) has not been defined convincingly. In a p53-genetically defined large cohort of de novo DLBCL patients treated with R-CHOP chemotherapy, we assessed MDM2 and p53 expression by immunohistochemistry (n=478), MDM2 gene amplification by fluorescence in situ hybridization (n=364), and a single nucleotide polymorphism in the MDM2 promoter, SNP309, by SNP genotyping assay (n=108). Our results show that MDM2 overexpression, unlike p53 overexpression, is not a significant prognostic factor in overall DLBCL. Both MDM2 and p53 overexpression does not predict for an adverse clinical outcome in patients with wild-type p53, but predicts for significantly poorer survival in patients with mutated p53. Variable p53 activities may ultimately determine the survival differences as suggested by the gene expression profiling analysis. MDM2 amplification was observed in 3 of 364 (0.8%) patients with high MDM2 expression. Presence of SNP309 did not correlate with MDM2 expression and survival. This study indicates that evaluation of MDM2 and p53 expression correlating with TP53 genetic status is essential to assess their prognostic significance, and important for designing therapeutic strategies that target the MDM2-p53 interaction.

Contains fulltext : 118768.pdf (Publisher’s version ) (Closed access) Diffuse large B-cell lymphoma (DLBCL) is stratified into prognostically favorable germinal center B-cell (GCB)-like and unfavorable activated B-cell (ABC)-like subtypes based on gene expression signatures. In this study, we analyzed 893 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). We show that MYC/BCL2 protein coexpression occurred significantly more commonly in the ABC subtype. Patients with the ABC or GCB subtype of DLBCL had similar prognoses with MYC/BCL2 coexpression and without MYC/BCL2 coexpression. Consistent with the notion that the prognostic difference between the 2 subtypes is attributable to MYC/BCL2 coexpression, there is no difference in gene expression signatures between the 2 subtypes in the absence of MYC/BCL2 coexpression. DLBCL with MYC/BCL2 coexpression demonstrated a signature of marked downregulation of genes encoding extracellular matrix proteins, those involving matrix deposition/remodeling and cell adhesion, and upregulation of proliferation-associated genes. We conclude that MYC/BCL2 coexpression in DLBCL is associated with an aggressive clinical course, is more common in the ABC subtype, and contributes to the overall inferior prognosis of patients with ABC-DLBCL. In conclusion, the data suggest that MYC/BCL2 coexpression, rather than cell-of-origin classification, is a better predictor of prognosis in patients with DLBCL treated with R-CHOP.

BackgroundHigh levels of cyclin E (CCNE) are accompanied by shorter survival in cyclophosphamide, hydroxydaunorubicin, oncovin and prednisone (CHOP)-treated diffuse large B-cell lymphomas (DLBCL), independent of the international prognostic index (IPI). Data on the prognostic role of CCNE in the ‘rituximab (R)-era’ are lacking.MethodsTo test reproducibility and applicability of observations from the ‘pre-R era’ to the ‘R era’, we examined the prognostic role of CCNE expression by immunohistochemistry in 1579 DLBCL on tissue microarrays (TMA); 339 patients were treated by CHOP and 635 by R-CHOP.Results1209 samples (77%) were evaluable; failures were due to missing TMA punches and fixation artefacts. Mean expression of CCNE was 13% (0–85%); applying a cut-off of >16%, 382 DLBCL (31%) were positive. CCNE did not correlate with any of the known variables (IPI, primary site, cell of origin, proliferation, and BCL2- or C-MYC rearrangements). We were able to reproduce data suggesting an IPI- and response to therapy independent, negative prognostic impact of CCNE in CHOP-treated DLBCL patients: CCNE-positive cases had a median survival of 16 months compared with 57 months in negative ones (p=0.012). In R-CHOP-treated patients the prognostic impact of CCNE was abrogated and only IPI, cell of origin and response to therapy had a prognostic significance.ConclusionsAddition of R to CHOP overcomes the negative prognostic impact of CCNE in DLBCL. Thus, R not only prolongs survival in DLBCL but also serves a cautionary note that prognostic factors should not be transferred into the ‘R era’ without proper scientific studies.

Contains fulltext : 185425.pdf (Publisher’s version ) (Closed access) We identified multiple single nucleotide variants (SNVs) in the TP53 3' untranslated region (3'UTR) in tumor specimens from 244 patients with diffuse large B-cell lymphoma (DLBCL). Patients carrying a wild-type TP53 coding sequence (CDS) and 1 or more 3'UTR SNVs had a better 5-year survival rate than patients carrying a wild-type CDS and the reference 3'UTR, yet there is no statistically significance difference in overall survival (OS). In contrast, 3'UTR variation predicted poorer OS for patients with a mutant TP53 CDS. We then sequenced TP53 3'UTR in 247 additional DLBCL patients as a validation set. Altogether, we identified 187 novel SNVs; 36 occurred at least twice. Most of the newly identified 3'UTR SNVs were located at sites that are complementary to seed sequences of microRNAs (miRNAs) that are predicted or experimentally known to target TP53. Three SNVs disrupt the seed match between miR-125b and the TP53 3'UTR, thereby impeding suppression of p53 by this miRNA. In addition, a germline SNV (rs78378222) located in the TP53 polyadenylation signal resulted in downregulation of both p53 messenger RNA and protein levels and reduction of cellular apoptosis. This study is the first to demonstrate the prognostic value of the TP53 3'UTR in cancer.

Contains fulltext : 108767.pdf (Publisher’s version ) (Closed access) TP53 mutation is an independent marker of poor prognosis in patients with diffuse large B-cell lymphoma (DLBCL) treated with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (CHOP) therapy. However, its prognostic value in the rituximab immunochemotherapy era remains undefined. In the present study of a large cohort of DLBCL patients treated with rituximab plus CHOP (R-CHOP), we show that those with TP53 mutations had worse overall and progression-free survival compared with those without. Unlike earlier studies of patients treated with CHOP, TP53 mutation has predictive value for R-CHOP-treated patients with either the germinal center B-cell or activated B-cell DLBCL subtypes. Furthermore, we identified the loop-sheet-helix and L3 motifs in the DNA-binding domain to be the most critical structures for maintaining p53 function. In contrast, TP53 deletion and loss of heterozygosity did not confer worse survival. If gene mutation data are not available, immunohistochemical analysis showing > 50% cells expressing p53 protein is a useful surrogate and was able to stratify patients with significantly different prognoses. We conclude that assessment of TP53 mutation status is important for stratifying R-CHOP-treated patients into distinct prognostic subsets and has significant value in the design of future therapeutic strategies.

Gene expression profiling (GEP) has stratified diffuse large B-cell lymphoma (DLBCL) into molecular subgroups that correspond to different stages of lymphocyte development-namely germinal center B-cell-like and activated B-cell-like. This classification has prognostic significance, but GEP is expensive and not readily applicable into daily practice, which has lead to immunohistochemical algorithms proposed as a surrogate for GEP analysis. We assembled tissue microarrays from 475 de novo DLBCL patients who were treated with rituximab-CHOP chemotherapy. All cases were successfully profiled by GEP on formalin-fixed, paraffin-embedded tissue samples. Sections were stained with antibodies reactive with CD10, GCET1, FOXP1, MUM1, and BCL6 and cases were classified following a rationale of sequential steps of differentiation of B-cells. Cutoffs for each marker were obtained using receiver operating characteristic curves, obviating the need for any arbitrary method. An algorithm based on the expression of CD10, FOXP1, and BCL6 was developed that had a simpler structure than other recently proposed algorithms and 92.6% concordance with GEP. In multivariate analysis, both the International Prognostic Index and our proposed algorithm were significant independent predictors of progression-free and overall survival. In conclusion, this algorithm effectively predicts prognosis of DLBCL patients matching GEP subgroups in the era of rituximab therapy.Leukemia accepted article preview online, 22 March 2012; doi:10.1038/leu.2012.83.

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 (p60%) tumors had significantly inferior PFS in the GCB subgroup (p=0.03), but not in the ABC subgroup (p=0.54). Multivariate analysis revealed that the presence of the t(14;18), but not Bcl2 protein expression, was independent of the International Prognostic Index in predicting outcome of our patients. Conclusions: Patients with the GCB subtype and t(14;18) exhibit a significantly worse prognosis than patients without t(14;18) when treated with R-CHOP. The assessment of t(14;18) by FISH approach not only functions as a valuable prognosticator for individual risk estimation in GCB-DLBCL patients in addition to the established parameters, but also provides valuable result for therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.