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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.

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.

Supplementary Table S1. Clinicopathologic characteristics of patients with de novo DLBCL with dual MYC/TP53 aberrations Supplementary Table S2. Frequency of single or dual abnormal Myc protein, p53 protein, BCL2 protein, MYC gene, and TP53 gene in the studied patients with DLBCL treated with R-CHOP Supplementary Table S3. Prognostic factors by univariate analysis and multivariate analysis in DLBCL Supplementary Table S4. Gene expression signatures identified by comparing DLBCL patients with concurrent MYC-R Mut-TP53 or Mychigh Mut-TP53 alterations (double-positive) with DLBCL patients withSupplementary Table S5. Molecular, genetic and phenotypic status of 8 DLBCL/HGBCL cell lines by targeted next-generation sequencing, fluorescence in situ hybridization (FISH), and immunohistochemistry analysis none or the alterations (double-negative) Supplementary Figure S1. Morphologic and immunophenotypic features of DLBCL with MYC rearrangement (MYC-R) and TP53 mutation (Mut-TP53) dual-alterations and highgrade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) in representative patients. Supplementary Figure S2. Prognostic impact of p53 and Myc protein overexpression as single or double abnormalities in overall DLBCL and GCB/ABC subtypes. Supplementary Figure S3. Heatmap for gene expression signatures of MYC/TP53 dual alterations. Supplementary Figure S4. Representative figures of flow cytometric analysis indicating G2/M phase-cell cycle arrest by NCB057643 treatment for 24 hours in cells with MYC-R or Myc overexpression with Wt-TP53 (OCI-LY19) or Mut-TP53 (GR and TMD8). Supplementary Figure S5. Heatmap for significantly up- or downregulated proteins after INCB057643 treatment (5µM, 24 hours) in OCI-LY19 cells. Supplementary Figure S6. A MDM2 inhibitor DS3032b shows cytotoxic effects selectively in high-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) and wild-type (Wt) TP53. Supplementary Figure S7. Combined DS3032b and INCB057643 treatment has no synergistic cytotoxicity in DLBCL cell lines with MYC aberrations and TP53 mutation (MutTP53). Supplementary Figure S8. INCB057643 treatment (1.25 µM) alone or in combination with ABT-199 (venetoclax, 6.25 mM) for 24 hours induced p21 expression in TMD8 cells, whereas had no effect on p53 (mutant) expression levels.

Supplementary Figures S1 to S3

PDF file - 57KB, Supplemental Table 1. Genes differently expressed in EBV+ DLBCL vs. EBV-negative DLBCL. A. Genes upregulated in EBV+ DLBCL; B. Genes downregulated in EBV+ DLBCL

PDF file - 3781KB, Supplemental Figure 1. Morphologic subtypes of EBV+ DLBCL. A, E, I, and M. The monomorphic subtype is composed of monotonous sheets of large transformed B-cells. B, F, J and N. The polymorphic subtype, canonical large B-cell neoplasm variant contains high density of large neoplastic cells and scattered cells with HRS-like features. C, G, K and O. The polymorphic subtype, Hodgkin-like variant shows lower density of neoplastic cells with HRS-like features. D, H, L and P. The polymorphic subtype, lymphoproliferative disorder-like variant has low density of neoplastic cells without Hodgkin lymphoma-like features. By immunohistochemistry, the monomorphic variant had a GCB phenotype, but all 3 polymorphic variants showed non-GCB phenotype.

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.

The supplementary documents include: (1) Supplementary methods for detection of MYC mutations and rearrangements, assessment of Myc expression, and functional studies. (2) Supplementary Tables S1-S8. Supplementary Table S1. Numbers of MYC mutation present in the coding sequence and the untranslated regions. Supplementary Table S2. Clinical and molecular characteristics of the DLBCL cohort with wild-type or mutated Myc or N11S single nucleotide polymorphism. Supplementary Table S3. List of Myc mutations and corresponding patient survival. Supplementary Table S4. Multivariate survival analysis. Supplementary Table S5. Molecular characteristics of patients with wild-type or mutated MYC untranslated regions. Supplementary Table S6. List of MYC 3Ã,'UTR mutations and corresponding patient survival. Supplementary Table S7. Gene profiling comparisons between wild-type and mutated MYC. Supplementary Table S8. Brief summary of major findings by this study. (3) Legends for Supplementary Figures S1-S5. Supplementary Figure S1. Survival analysis for MYC mutations in DLBCL respective to GCB/ABC subtypes, homo/heterozygosity, and MYC translocations. Supplementary Figure S2. Comparison of Myc levels and gene expression profiles between DLBCL groups. Supplementary Figure S3. Differential expression of genes between the WT-Myc and MUT-Myc groups. Supplementary Figure S4. Comparison of gene expression between the WT-Myc and MUT-Myc groups. Supplementary Figure S5. Differential expression of proteins between the WT-Myc and MUT-Myc groups.

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.

Supplementary Figure from Genetic Subtyping and Phenotypic Characterization of the Immune Microenvironment and MYC/BCL2 Double Expression Reveal Heterogeneity in Diffuse Large B-cell Lymphoma

Supplementary Data from Genetic Subtyping and Phenotypic Characterization of the Immune Microenvironment and MYC/BCL2 Double Expression Reveal Heterogeneity in Diffuse Large B-cell Lymphoma

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.

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.

Supplementary Figure 1. Morphology and immunophenotype of pSTAT3+ DLBCL. Supplementary Figure 2. Relationship between STAT3 mRNA and pSTAT3 expression and survival analysis based on STAT3 mRNA level. Supplementary Figure 3. Survival analyses based on pSTAT3 expression in DLBCL with MYC/BCL2 double expression and DLBCL without MYC/BCL2 double expression after COO stratification. Supplementary Figure 4. Overall survival (OS) and progression-free survival (PFS) in all DLBCL cases, DLBCL with germinal center B-cell-like phenotype (GCB) and DLBCL with activated B-cell-like phenotype (ABC). Supplementary Figure 5. Survival analyses in 3 and 4 groups based on pSTAT3 expression in lymphoma cells. Supplementary Table 1. Number of cases based on pSTAT3 expression, MYC/BCL2 double expression and COO classification Supplementary Table 2. The numbers and percentages of pSTAT3 positive DLBCL with each cutoff Supplementary Table 3. Clinical characteristics and cell-of-origin differentiation with 30% cutoff for pSTAT3 expression Supplementary Table 4. Distribution of cell-of-origin classification based on 4 groups of pSTAT3 expression in lymphoma cells. Supplementary Table 5. Distribution of cell-of-origin classification based on 3 groups of pSTAT3 expression in lymphoma cells. Supplementary Table 6. Bivariate analyses of pSTAT3 with each variable and pSTAT3.

Recent compelling evidence showed that innate immune effector cells could recognize allogeneic grafts and prime an adaptive immune response. Signal regulatory protein α (SIRPα) is an immunoglobulin superfamily receptor that is expressed on myeloid cells; the interaction between SIRPα and its ubiquitously expressed ligand CD47 elicits an inhibitory signal that suppresses macrophage phagocytic function. Additional studies showed that donor-recipient mismatch in SIRPα variants might activate monocytic allorecognition, possibly as the result of non-self SIRPα-CD47 interaction. However, the frequency of SIRPα variation and its role in hematopoietic stem cell transplantation (HSCT) remains unexplored. We studied 350 patients with acute myeloid leukemia/myelodysplastic syndrome who underwent HLA-matched related HSCT and found that SIRPα allelic mismatches were present in 39% of transplantation pairs. SIRPα variant mismatch was associated with a significantly higher rate of chronic graft-versus-host disease (GVHD; hazard ratio [HR], 1.5; P = .03), especially de novo chronic GVHD (HR, 2.0; P = .01), after adjusting for other predictors. Those with mismatched SIRPα had a lower relapse rate (HR, 0.6; P = .05) and significantly longer relapse-free survival (RFS; HR, 0.6; P = .04). Notably, the effect of SIRPα variant mismatch on relapse protection was most pronounced early after HSCT and in patients who were not in remission at HSCT (cumulative incidence, 73% vs 54%; HR, 0.5; P = .01). These findings show that SIRPα variant mismatch is associated with HSCT outcomes, possibly owing to innate allorecognition. SIRPα variant matching could provide valuable information for donor selection and risk stratification in HSCT.

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.

// Zijun Y. Xu-Monette 1,* , Meifeng Tu 2,* , Kausar J. Jabbar 1 , Xin Cao 1 , Alexandar Tzankov 3 , Carlo Visco 4 , Qingqing Cai 1 , Santiago Montes-Moreno 5 , Yuji An 1 , Karen Dybkaer 6 , April Chiu 7 , Attilio Orazi 8 , Youli Zu 9 , Govind Bhagat 10 , Kristy L. Richards 11 , Eric D. Hsi 12 , William W.L. Choi 13 , J. Han van Krieken 14 , Jooryung Huh 15 , Maurilio Ponzoni 16 , Andres J.M. Ferreri 16 , Xiaoying Zhao 17 , Michael B. Moller 18 , John P. Farnen 19 , Jane N. Winter 20 , Miguel A. Piris 5 , Roberto N. Miranda 1 , L. Jeffrey Medeiros 1 and Ken H. Young 1,21 1 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Peking University Cancer Hospital and Institute, Beijing, China 3 University Hospital, Basel, Switzerland 4 San Bortolo Hospital, Vicenza, Italy 5 Hospital Universitario Marques de Valdecilla, Santander, Spain 6 Aalborg University Hospital, Aalborg, Denmark 7 Memorial Sloan-Kettering Cancer Center, New York, NY, USA 8 Weill Medical College of Cornell University, New York, NY, USA 9 The Methodist Hospital, Houston, TX, USA 10 Columbia University Medical Center and New York Presbyterian Hospital, New York, NY, USA 11 University of North Carolina School of Medicine, Chapel Hill, NC, USA 12 Cleveland Clinic, Cleveland, OH, USA 13 University of Hong Kong Li Ka Shing Faculty of Medicine, Hong Kong, China 14 Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands 15 Asan Medical Center, Ulsan University College of Medicine, Seoul, Korea 16 San Raffaele H. Scientific Institute, Milan, Italy 17 Zhejiang University School of Medicine, Second University Hospital, Hangzhou, China 18 Odense University Hospital, Odense, Denmark 19 Gundersen Lutheran Health System, La Crosse, WI, USA 20 Feinberg School of Medicine, Northwestern University, Chicago, IL, USA 21 The University of Texas School of Medicine, Graduate School of Biomedical Sciences, Houston, Texas, USA * These authors made equal contributions to this work Correspondence to: Ken H. Young, email: // Keywords : ABC, BCL2, CD5, diffuse large B-cell lymphoma, NF-κB Received : December 29, 2014 Accepted : January 02, 2015 Published : March 08, 2015 Abstract CD5 is a pan-T-cell surface marker and is rarely expressed in diffuse large B-cell lymphoma (DLBCL). Large-scale studies of de novo CD5 + DLBCL are lacking in Western countries. In this study by the DLBCL Rituximab-CHOP Consortium, CD5 was expressed in 5.5% of 879 DLBCL patients from Western countries. CD5 + DLBCL was associated with higher frequencies of >1 ECOG performance status, bone marrow involvement, central nervous system relapse, activated B-cell–like subtype, Bcl-2 overexpression, and STAT3 and NF-κB activation, whereas rarely expressed single-stranded DNA-binding protein 2 (SSBP2), CD30 or had MYC mutations. With standard R-CHOP chemotherapy, CD5 + DLBCL patients had significantly worse overall survival (median, 25.3 months vs . not reached, P < .0001) and progression-free survival (median, 21.3 vs. 85.8 months, P < .0001) than CD5 – DLBCL patients, which was independent of Bcl-2, STAT3, NF-κB and the International Prognostic Index. Interestingly, SSBP2 expression abolished the prognostic significance of CD5 expression, suggesting a tumor-suppressor role of SSBP2 for CD5 signaling. Gene-expression profiling demonstrated that B-cell receptor signaling dysfunction and microenvironment alterations are the important mechanisms underlying the clinical impact of CD5 expression. This study shows the distinctive clinical and biological features of CD5 + DLBCL patients in Western countries and underscores important pathways with therapeutic implications.

Mutation in the NPM1 gene occurs in 60% of acute myeloid leukaemia (AML) patients with normal karyotype. NPM1 mutation is potentially a superior minimal residual disease (MRD) marker compared to WT1 gene overexpression by being specific to the malignant clone, although experimental evidence published so far includes very limited numbers of relapsed cases. Also, the stability of the NPM1 mutation has been questioned by reports of the mutation being lost at relapse. In the present study we compared NPM1 mutation and WT1 overexpression as MRD markers in 20 cases of relapsed AML. The 20 patients experienced a total of 28 morphological relapses. Karyotypic evolution was detected in 56% of relapses. All relapses were accompanied by high levels of NPM1 mutation, along with high WT1 mRNA levels, thus demonstrating complete stability of both markers during relapse. Detectable NPM1 mutation following a period of morphological remission was accompanied by a morphological relapse in all cases. In contrast, WT1 expression was detected in 33% of the NPM1 mutation negative samples. This background WT1 expression produced by non-leukaemia cells was highly variable, both between and within patients, and limited the de facto sensitivity of the WT1 expression analysis. The present study therefore provides important experimental evidence demonstrating that NPM1 mutation is superior to WT1 overexpression as marker of MRD in NPM1-mutated AML, even in the presence of extensive karyotypic evolution.

*701 Mutational Profile and Prognostic Significance of TP53 in Diffuse Large B-cell Lymphoma Patients Treated with Rituximab-CHOP: A Report From an International DLBCL Rituximab-CHOP Consortium Program Study Zijun Y. Xu-Monette, Lin Wu, Carlo Visco, Yu Chuan Tai, Alexander Tzankov, Wei-min Liu, Santiago Montes-Moreno, Karen Dybkaer, April Chiu, Attilio Orazi, Youli Zu, Govind Bhagat, Kristy L. Richards, Eric D. Hsi, X. Frank Zhao, William W.L. Choi, Xiaoying Zhao, J. Han van Krieken, Qin Huang, Jooryung Huh, Weiyun Ai, Maurilio Ponzoni, Andres J.M. Ferreri, Fan Zhou, Brad S. Kahl, Jane N. Winter, Wei Xu, Jianyong Li, Ronald S. Go, Yong Li, Miguel A. Piris, Michael B. Moller, Roberto N. Miranda, Lynne V. Abruzzo, L. Jeffrey Medeiros, Ken H. Young Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Roche Molecular Systems, Inc., Pleasanton, CA, USA; San Bortolo Hospital, Vicenza, Italy; University Hospital, Basel, Switzerland; Hospital Universitario Marques de Valdecilla, Santander, Spain; Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark; Memorial Sloan-Kettering Cancer Center, New York, NY, USA; Weill Medical College of Cornell University, New York, NY, USA; The Methodist Hospital, Houston, TX, USA; Columbia University Medical Center and New York Presbyterian Hospital, New York, NY, USA; University of North Carolina School of Medicine, Chapel Hill, NC, USA; Cleveland Clinic, Cleveland, OH, USA; University of Maryland School of Medicine, Baltimore, MD, USA; University of Hong Kong Li Ka Shing Faculty of Medicine, Hong Kong, China; Zhejiang University School of Medicine, Second University Hospital, Hangzhou, China; Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands; City of Hope National Medical Center, Duarte, CA, USA; Asan Medical Center, Ulsan University College of Medicine, Seoul, Korea; University of California San Francisco School of Medicine, San Francisco, CA, USA; San Raffaele H. Scientific Institute, Milan, Italy; Southwest Washington Medical Center, Vancouver, WA, USA; University of Wisconsin Hospital and Clinic, Madison, WI, USA; Feinberg School of Medicine, Northwestern University, Chicago, IL, USA; The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China; Gundersen Lutheran Health System, La Crosse, WI, USA; University of Louisville, Louisville, KY; Odense University Hospital, Odense, Denmark

Differences in genetic origin between nodal and extranodal diffuse large B-cell lymphomas (DLBCL) exist. Using population-based data from the registry of the Danish Lymphoma Group, the present study is the first to analyse clinical implications of nodal versus extranodal presentation of DLBCL. Of 4786 newly diagnosed non-Hodgkin's lymphoma patients in a 16-year period, 1575 (33%) had DLBCL. The annual incidence rate was 2.9 per 100 000; 40% were extranodal. The clinical profile of patients with extranodal DLBCL was different from the nodal DLBCL patients. Extranodal DLBCL was associated with older age and poorer performance score, but also lower tumour burden. In extranodal DLBCL, 51% of the cases were stage I and 36% were stage IV, whereas the patients were relatively equally distributed between the four stages in nodal DLBCL. For stage I patients, extranodal DLBCL was independently associated with poor survival (P = 0.003). In contrast, among stage IV patients those with extranodal DLBCL survived longer (P = 0.009). We conclude that there are important clinical differences between nodal and extranodal DLBCL. The addition of these clinical results to the existing aetiological and genetic data suggests that the distinction between nodal and extranodal DLBCL is not only pathogenetically but also clinically important.

The International Prognostic Index (IPI) is widely used for risk stratification of patients with diffuse large B-cell lymphoma (DLBCL). However, even among patients with low-risk disease, according to the IPI a substantial proportion of patients ultimately succumb to their disease. Using mature population-based data from the Danish Lymphoma Group, we analyzed if prognostic clinical pretreatment factors could be identified in patients with low-risk DLBCL. One hundred seventy-seven patients, all with a prognostic profile as favorable as possible according to the IPI and treated with anthracycline-based combination chemotherapy (92%) or loco-regional radiotherapy/surgery (8%) with curative intent were included. The median age was 50 years and 170 achieved complete remission. The median follow-up time was 11 years. Twenty-six patients relapsed, with a median time to relapse of 12.1 months. Overall survival at 5 years and 10 years was 85% and 75%, respectively. Stage II was associated with poor response to treatment (P=0.044). In a multivariate analysis, Stage II (P=0.001) and age50 years (P=0.043) were independently associated with poor outcome. Patients without these adverse factors had an excellent prognosis, with a survival at 5 and 15 years of 90% and 80%, respectively. In contrast, patients with both adverse factors had poor outcome, with survival at 5 and 15 years of 70% and 29%, respectively (P0.001). The present data suggest that risk stratification of DLBCL patients with a favorable IPI score can be improved by the simple use of two clinical pretreatment factors.

The International Prognostic Index (IPI) is widely used as a predictive model in diffuse large B-cell lymphoma (DLBCL) patients of all ages and stages. To determine the optimal IPI-based prognostic system at the time of diagnosis in younger patients with limited-stage DLBCL, the authors evaluated the age-adjusted IPI and the recently proposed stage-adjusted IPI, and constructed an IPI-based model adjusted for both age and stage.From the population-based LYFO registry of the Danish Lymphoma Group, 233 patients not older than 60 years with Stage I-II DLBCL treated with anthracycline-based chemotherapy with or without involved-field irradiation were identified. The primary endpoint of analysis was overall survival.At the end of the observation period, 151 patients were alive with a median follow-up time of 8.3 years. All the variables in the age-adjusted and the stage-adjusted IPI had major prognostic significance (Por = 0.0001). Log-rank analyses of survival showed highly significant differences between the subgroups in each model (P0.0001); however, the stage-adjusted IPI was more powerful (chi-square test = 44.99) than the age-adjusted IPI (chi-square test = 36.27). By using the median age of the cohort (50 years) as a cutoff point, age was found to be a strong prognostic factor (P = 0.0036). An age-/stage-adjusted IPI was constructed, the predictive power of which was equal to the stage-adjusted IPI (chi-square test = 44.16) but with different distribution of patients between the risk groups, making the proposed model better suited to identify poor-risk patients.The data from the current study suggest that the proposed age-/stage-adjusted IPI is the superior IPI-based model in predicting outcome in younger patients with localized DLBCL.

Mutations of the p53 tumor suppressor gene have been associated with a poor clinical outcome in some series of diffuse large B-cell lymphoma (DLBCL). However, conflicting results have been reported in other studies. The purpose of this study was to analyze the p53 mutations in DLBCL from twelve centers, and to correlate the structural profiles of the mutations with clinical outcome. The p53 mutations were identified in 102 of 477 cases of DLBCL for a frequency of 21.4%. These included 92 missense mutations, 5 nonsense mutations, 4 deletions, and 1 insertion. The presence of any p53 mutation correlated with poor overall survival (OS; P=0.002). Sixty-two of 102 cases (61%) had mutations in the DNA binding domains of the p53 gene, and the mutations in the DNA-binding domains were found to be the most important predictor of poor OS (P

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