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Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL) are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin ligase with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiologic KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the bric-a-brac tramtrack broad domain of KLHL6 disrupted its localization and heterodimerization and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL. Significance: Oncogenic BCR signaling sustains DLBCL cells. We discovered that Cullin-3-RING E3 ubiquitin ligase substrate-adapter KLHL6 targets BCR heterodimer (CD79A/CD79B) for ubiquitin-mediated degradation. Recurrent somatic mutations in the KLHL6 gene cause corrupt BCR signaling by disrupting surface BCR homeostasis. Loss of KLHL6 expression and mutant-induced phenotypes associate with targetable disease characteristics in B-cell lymphoma. See related commentary by Leveille et al. See related commentary by Corcoran et al., (©2024 The Authors; Published by the American Association for Cancer Research.)

Post-transplant lymphoproliferative disorders (PTLD) are iatrogenic immune deficiency-associated lymphoid/plasmacytic proliferations developing due to immunosuppression in solid organ or hematopoietic stem cell allograft patients. PTLD are characterized by abnormal proliferation of lymphoid cells and have a heterogeneous clinical behavior. We profiled expression of >700 tumor microenvironment (TME)-related genes in 75 post-transplant aggressive B-cell lymphomas (PTABCL). Epstein-Barr virus (EBV)-positive PT-ABCL clustered together and were enriched for type I interferon pathway and antiviral-response genes. Additionally, a cytotoxicity gene signature associated with EBV-positivity and favorable overall survival (OS) (hazard ratio =0.61; P=0.019). In silico immunophenotyping revealed two subgroups with distinct immune cell compositions. The inflamed subgroup with higher proportions of immune cells had better outcome compared to noninflamed subgroup (median OS >200.0 vs. 15.2 months; P=0.006). In multivariable analysis with EBV status, International Prognostic Index, and rituximab-containing treatment, inflamed TME remained as an independent predictor for favorable outcome. We also compared TME between post-transplant and immunocompetent host diffuse large B-cell lymphomas (n=75) and discovered that the proportions of T cells were lower in PT-diffuse large B-cell lymphomas. In conclusion, we provide a comprehensive phenotypic characterization of PT-ABCL, highlighting the importance of immune cell composition of TME in determining the clinical behavior and prognosis of PT-ABCL.

Marginal zone lymphoma (MZL) is an indolent B-cell malignancy with heterogeneous anatomical and clinical presentation. While MZLs are generally associated with long survival, some patients experience histological transformation to aggressive large B-cell lymphoma. Population-based long-term data on the transformation of MZL is limited. We conducted a nationwide population-based study to estimate the risk of transformation and relative survival in patients diagnosed with MZL in Finland from 1995-2018. We identified a total of 1454 patients with MZL from the Finnish Cancer Registry (FCR). The cumulative incidence of transformation was 4.7% (95% CI, 3.6-6.2) at 10 years. The highest incidence of transformation was observed in the patients with splenic MZL (14.0%; 95% CI, 8.6-22.7). The transformation was associated with a substantially increased risk of death (HR, 5.18; 95% CI, 3.58-7.50). Ten-year relative survival was 79% (95% CI, 73‒83%). Transformation, nodal MZL subtype, and older age at diagnosis were associated with increased excess mortality, whereas patients diagnosed at a later calendar period had a lower excess risk of death. We conclude that transformation resulted in a substantially increased mortality irrespective of MZL subtype compared with the patients without transformation. Our results also suggest a reduction in excess mortality in recent years., (© 2023. The Author(s).)

Inadequate molecular and clinical stratification of the patients with high-risk diffuse large B-cell lymphoma (DLBCL) is a clinical challenge hampering the establishment of personalized therapeutic options. We studied the translational significance of liquid biopsy in a uniformly treated trial cohort. Pretreatment circulating tumor DNA (ctDNA) revealed hidden clinical and biological heterogeneity, and high ctDNA burden determined increased risk of relapse and death independently of conventional risk factors. Genomic dissection of pretreatment ctDNA revealed translationally relevant phenotypic, molecular, and prognostic information that extended beyond diagnostic tissue biopsies. During therapy, chemorefractory lymphomas exhibited diverging ctDNA kinetics, whereas end-of-therapy negativity for minimal residual disease (MRD) characterized cured patients and resolved clinical enigmas, including false residual PET positivity. Furthermore, we discovered fragmentation disparities in the cell-free DNA that characterize lymphoma-derived ctDNA and, as a proof-of-concept for their clinical application, used machine learning to show that end-of-therapy fragmentation patterns predict outcome. Altogether, we have discovered novel molecular determinants in the liquid biopsy that can noninvasively guide treatment decisions., (© 2022 by The American Society of Hematology.)

Purpose: Tumor-infiltrating immune cells have prognostic significance and are attractive therapeutic targets. Yet, the clinical significance of their spatial organization and phenotype in diffuse large B-cell lymphoma (DLBCL) is unclear., Experimental Design: We characterized T cells, macrophages, and their spatial interactions by multiplex IHC (mIHC) in 178 patients with DLBCL and correlated the data with patient demographics and survival. We validated the findings on gene expression data from two external DLBCL cohorts comprising 633 patients., Results: Macrophage and T-cell contents divided the samples into T cell-inflamed (60%) and noninflamed (40%) subgroups. The T cell-inflamed lymphoma microenvironment (LME) was also rich in other immune cells, defining immune hot phenotype, which did not as such correlate with outcome. However, when we divided the patients according to T-cell and macrophage contents, LME characterized by high T-cell/low macrophage content or a corresponding gene signature was associated with superior survival [5-year overall survival (OS): 92.3% vs. 74.4%, P = 0.036; 5-year progression-free survival (PFS): 92.6% vs. 69.8%, P = 0.012]. High proportion of PD-L1- and TIM3-expressing CD163- macrophages in the T cell-inflamed LME defined a group of patients with poor outcome [OS: HR = 3.22, 95% confidence interval (CI), 1.63-6.37, Padj = 0.011; PFS: HR = 2.76, 95% CI, 1.44-5.28, Padj = 0.016]. Furthermore, PD-L1 and PD-1 were enriched on macrophages interacting with T cells., Conclusions: Our data demonstrate that the interplay between macrophages and T cells in the DLBCL LME is immune checkpoint dependent and clinically meaningful., (©2021 The Authors; Published by the American Association for Cancer Research.)

The tumor microenvironment (TME) and limited immune surveillance play important roles in lymphoma pathogenesis. Here we aimed to characterize immunological profiles of diffuse large B-cell lymphoma (DLBCL) and predict the outcome in response to immunochemotherapy. We profiled the expression of 730 immune-related genes in tumor tissues of 81 patients with DLBCL utilizing the Nanostring platform, and used multiplex immunohistochemistry to characterize T-cell phenotypes, including cytotoxic T cells (CD8, Granzyme B, OX40, Ki67), T-cell immune checkpoint (CD3, CD4, CD8, PD1, TIM3, LAG3), as well as regulatory T-cells and Th1 effector cells (CD3, CD4, FOXP3, TBET) in 188 patients. We observed a high degree of heterogeneity at the transcriptome level. Correlation matrix analysis identified gene expression signatures with highly correlating genes, the main cluster containing genes for cytolytic factors, immune checkpoint molecules, T cells and macrophages, together named a TME immune cell signature. Immunophenotyping of the distinct cell subsets revealed that a high proportion of immune checkpoint positive T cells translated to unfavorable survival. Together, our results demonstrate that the immunological profile of DLBCL TME is heterogeneous and clinically meaningful. This highlights the potential impact of T-cell immune checkpoint in regulating survival and resistance to immunochemotherapy. (Registered at clinicaltrials.gov identifiers: NCT01502982 and NCT01325194.)

Objectives: Testicular diffuse large B-cell lymphoma (T-DLBCL) is a rare and aggressive extranodal lymphoma. We have previously shown that high content of tumor-infiltrating lymphocytes (TILs) and PD-1 expressing TILs associate with better survival in T-DLBCL. In this study, we have further characterized distinct TIL subtypes and their proportions in association with patient demographics and survival., Methods: We used multiplex immunohistochemistry to characterize TIL phenotypes, including cytotoxic T-cells (CTLs; CD8 + , OX40 + , Granzyme B + , Ki-67 + , LAG-3 + , TIM-3 + , PD-1 + ), CD4 + T-cells (CD3 + , CD4 + , TIM-3 + , LAG-3 + ), regulatory T-cells (Tregs; CD3 + , CD4 + , FoxP3 + ), and T helper 1 cells (Th1; CD3 + , CD4 + , T-bet + ) in 79 T-DLBCLs, and correlated the findings with patient demographics and outcome., Results: We observed a substantial variation in TIL phenotypes between the patients. The most prominent CD8 + TILs were Ki-67 + and TIM-3 + CTLs, whereas the most prominent CD4 + TILs were FoxP3 + Tregs. Despite the overall favorable prognostic impact of high TIL content, we found a subpopulation of T-bet + FoxP3 + Tregs that had a significant adverse impact on survival. Lower content of CTLs with activated or exhausted phenotypes correlated with aggressive clinical features., Conclusions: Our results demonstrate significant variation in TIL phenotypes and emphasize the adverse prognostic impact of Tregs in T-DLBCL., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Understanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. We integrated over 8,000 transcriptomes and 2,000 samples with multilevel genomics of hematological cancers to investigate how immunological features are linked to cancer subtypes, genetic and epigenetic alterations, and patient survival, and validated key findings experimentally. Infiltration of cytotoxic lymphocytes was associated with TP53 and myelodysplasia-related changes in acute myeloid leukemia, and activated B cell-like phenotype and interferon-γ response in lymphoma. CIITA methylation regulating antigen presentation, cancer type-specific immune checkpoints, such as VISTA in myeloid malignancies, and variation in cancer antigen expression further contributed to immune heterogeneity and predicted survival. Our study provides a resource linking immunology with cancer subtypes and genomics in hematological malignancies., Competing Interests: Declaration of Interests S.M. has received honoraria and research funding from Novartis, Pfizer, and Bristol-Myers Squibb, and C.H. received funding from Celgene, Novartis, Oncopeptides, Orion Pharma, and the Innovative Medicines Initiative project HARMONY. The remaining authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Concomitant deregulation of MYC and BCL2 comprises clinically significant, yet poorly characterized biological high-risk feature in diffuse large B-cell lymphoma (DLBCL). To interrogate these lymphomas, we analyzed translocations and protein expression of BCL2, BCL6, and MYC; correlated the findings with comprehensive mutational, transcriptomic, and clinical data in 181 patients with primary DLBCL; and validated the key findings in independent data sets. Structural variations of BCL2 were subtype-specific and specifically increased BCL2 expression. Molecular dissection of MYC deregulation revealed associations with other lymphoma drivers, including loss of TP53, and distinctive gene expression profiles. Double protein expression (DPE) arose from heterogeneous molecular backgrounds that exhibited subtype-dependent patterns. In the germinal center B-cell (GCB) DLBCL, concurrent alterations of MYC and BCL2 loci gave rise to the majority of DPE DLBCLs, whereas among the activated B-cell (ABC) DLBCLs, concurrent alterations were infrequent. Clinically, DPE DLBCL defined a prognostic entity, which was independent of the International Prognostic Index (IPI) and cell of origin, and together with the loss of TP53 had a synergistic dismal impact on survival. In the DPE DLBCL, the loss of TP53 was associated with a chemorefractory disease, whereas among the other DLBCLs, no correlation with survival was seen. Importantly, BCL6 translocations identified non-GCB lymphomas with favorable BN2/C1-like survival independent of IPI and concurrent DPE status. Taken together, our findings define molecular characteristics of the DPE in DLBCL, and recognize clinically feasible predictors of outcome. Given the emerging taxonomical significance of BCL2, BCL6, MYC, and TP53, our findings provide further depth and validation to the genomic classification of DLBCL., (© 2020 by The American Society of Hematology.)

Survival of patients with high-risk diffuse large B-cell lymphoma (DLBCL) is suboptimal, and the risk of central nervous system (CNS) progression is relatively high. We conducted a phase 2 trial in 139 patients aged 18 to 64 years who had primary DLBCL with an age-adjusted International Prognostic Index (aaIPI) score of 2 to 3 or site-specific risk factors for CNS recurrence. The goal was to assess whether a dose-dense immunochemotherapy with early systemic CNS prophylaxis improves the outcome and reduces the incidence of CNS events. Treatment consisted of 2 courses of high-dose methotrexate in combination with biweekly rituximab (R), cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP-14), followed by 4 courses of R-CHOP-14 with etoposide (R-CHOEP) and 1 course of high-dose cytarabine with R. In addition, liposomal cytarabine was administered intrathecally at courses 1, 3, and 5. Coprimary endpoints were failure-free survival and CNS progression rates. Thirty-six (26%) patients experienced treatment failure. Progression occurred in 23 (16%) patients, including three (2.2%) CNS events. At 5 years of median follow-up, failure-free survival, overall survival, and CNS progression rates were 74%, 83%, and 2.3%, respectively. Treatment reduced the risk of progression compared with our previous trial, in which systemic CNS prophylaxis was given after 6 courses of biweekly R-CHOEP (hazard ratio, 0.49; 95% CI, 0.31-0.77; P = .002) and overcame the adverse impact of an aaIPI score of 3 on survival. In addition, outcome of the patients with BCL2/MYC double-hit lymphomas was comparable to the patients without the rearrangements. The results are encouraging, with a low toxic death rate, low number of CNS events, and favorable survival rates. This trial was registered at www.clinicaltrials.gov as #NCT01325194., (© 2020 by The American Society of Hematology.)

Background: The epigenome plays a key role in cancer heterogeneity and drug resistance. Hence, a number of epigenetic inhibitors have been developed and tested in cancers. The major focus of most studies so far has been on the cytotoxic effect of these compounds, and only few have investigated the ability to revert the resistant phenotype in cancer cells. Hence, there is a need for a systematic methodology to unravel the mechanisms behind epigenetic sensitization., Results: We have developed a high-throughput protocol to screen non-simultaneous drug combinations, and used it to investigate the reprogramming potential of epigenetic inhibitors. We demonstrated the effectiveness of our protocol by screening 60 epigenetic compounds on diffuse large B-cell lymphoma (DLBCL) cells. We identified several histone deacetylase (HDAC) and histone methyltransferase (HMT) inhibitors that acted synergistically with doxorubicin and rituximab. These two classes of epigenetic inhibitors achieved sensitization by disrupting DNA repair, cell cycle, and apoptotic signaling. The data used to perform these analyses are easily browsable through our Results Explorer. Additionally, we showed that these inhibitors achieve sensitization at lower doses than those required to induce cytotoxicity., Conclusions: Our drug screening approach provides a systematic framework to test non-simultaneous drug combinations. This methodology identified HDAC and HMT inhibitors as successful sensitizing compounds in treatment-resistant DLBCL. Further investigation into the mechanisms behind successful epigenetic sensitization highlighted DNA repair, cell cycle, and apoptosis as the most dysregulated pathways. Altogether, our method adds supporting evidence in the use of epigenetic inhibitors as sensitizing agents in clinical settings.

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease whose personalized clinical management requires robust molecular stratification. Here, we show that somatic hypermutation (SHM) patterns constitute a marker for DLBCL molecular classification. The activity of SHM mutational processes delineated the cell of origin (COO) in DLBCL. Expression of the herein identified 36 SHM target genes stratified DLBCL into four novel SHM subtypes. In a meta-analysis of patients with DLBCL treated with immunochemotherapy, the SHM subtypes were significantly associated with overall survival (1642 patients) and progression-free survival (795 patients). Multivariate analysis of survival indicated that the prognostic impact of the SHM subtypes is independent from the COO classification and the International Prognostic Index. Furthermore, the SHM subtypes had a distinct clinical outcome within each of the COO subtypes, and strikingly, even within unclassified DLBCL. The genetic landscape of the four SHM subtypes indicated unique associations with driver alterations and oncogenic signaling in DLBCL, which suggests a possibility for therapeutic exploitation. These findings provide a biologically driven classification system in DLBCL with potential clinical applications.

Purpose: Sinonasal tract diffuse large B-cell lymphoma (SNT-DLBCL), a rare extranodal lymphoma, is not well characterized. We performed a population-based study to determine cell-of-origin, clinical presentation and impact of rituximab (R) and central nervous system (CNS) directed chemotherapy on survival., Patients and Methods: Patients with SNT-DLBCL were identified from pathology databases. Clinical information was collected and outcomes between different treatment modalities evaluated., Results: Thirty-two percent of the patients had germinal centre B-cell phenotype. Forty-six patients were treated with curative intent using CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or CHOP-like chemotherapy, 21 (46%) before and 25 (54%) in the R-era. Additionally, 24 (52%) received CNS-directed chemotherapy. Addition of R to chemotherapy reduced the risk of progression (RR = 0.368, 95% CI 0.138-0.976, P = 0.045) and death (RR = 0.245, 95% CI 0.068-0.883, P = 0.032), and translated into better survival (5-year PFS, 67% vs 38%, P = 0.037; 5-year OS, 81% vs 48%, P = 0.020). CNS-directed chemotherapy reduced the risk of progression (RR = 0.404, 95% CI 0.159-1.029, P = 0.057) and death (RR = 0.298, 95% CI 0.093-0.950, P = 0.041), and translated into favorable survival (5-year PFS, 67% vs 32%, P = 0.050; 5-year OS 82% vs 43%, P = 0.030)., Conclusion: Patients with SNT-DLBCL benefit from rituximab and CNS-directed chemotherapy., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: Ibrutinib has shown activity in non-germinal center B-cell diffuse large B-cell lymphoma (DLBCL). This double-blind phase III study evaluated ibrutinib and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in untreated non-germinal center B-cell DLBCL., Patients and Methods: Patients were randomly assigned at a one-to-one ratio to ibrutinib (560 mg per day orally) plus R-CHOP or placebo plus R-CHOP. The primary end point was event-free survival (EFS) in the intent-to-treat (ITT) population and the activated B-cell (ABC) DLBCL subgroup. Secondary end points included progression-free survival (PFS), overall survival (OS), and safety., Results: A total of 838 patients were randomly assigned to ibrutinib plus R-CHOP (n = 419) or placebo plus R-CHOP (n = 419). Median age was 62.0 years; 75.9% of evaluable patients had ABC subtype disease, and baseline characteristics were balanced. Ibrutinib plus R-CHOP did not improve EFS in the ITT (hazard ratio [HR], 0.934) or ABC (HR, 0.949) population. A preplanned analysis showed a significant interaction between treatment and age. In patients age younger than 60 years, ibrutinib plus R-CHOP improved EFS (HR, 0.579), PFS (HR, 0.556), and OS (HR, 0.330) and slightly increased serious adverse events (35.7% v 28.6%), but the proportion of patients receiving at least six cycles of R-CHOP was similar between treatment arms (92.9% v 93.0%). In patients age 60 years or older, ibrutinib plus R-CHOP worsened EFS, PFS, and OS, increased serious adverse events (63.4% v 38.2%), and decreased the proportion of patients receiving at least six cycles of R-CHOP (73.7% v 88.8%)., Conclusion: The study did not meet its primary end point in the ITT or ABC population. However, in patients age younger than 60 years, ibrutinib plus R-CHOP improved EFS, PFS, and OS with manageable safety. In patients age 60 years or older, ibrutinib plus R-CHOP was associated with increased toxicity, leading to compromised R-CHOP administration and worse outcomes. Further investigation is warranted.

Primary testicular lymphoma is a rare and aggressive lymphoid malignancy, most often representing diffuse large B-cell lymphoma histologically. Tumor-associated macrophages and tumor-infiltrating lymphocytes have been associated with survival in diffuse large B-cell lymphoma, but their prognostic impact in primary testicular lymphoma is unknown. Here, we aimed to identify macrophages, their immunophenotypes and association with lymphocytes, and translate the findings into survival of patients with primary testicular lymphoma. We collected clinical data and tumor tissue from 74 primary testicular lymphoma patients, and used multiplex immunohistochemistry and digital image analysis to examine macrophage markers (CD68, CD163, and c-Maf), T-cell markers (CD3, CD4, and CD8), B-cell marker (CD20), and three checkpoint molecules (PD-L1, PD-L2, and PD-1). We demonstrate that a large proportion of macrophages (median 41%, range 0.08-99%) and lymphoma cells (median 34%, range 0.1-100%) express PD-L1. The quantity of PD-L1 + CD68 + macrophages correlates positively with the amount of PD-1 + lymphocytes, and a high proportion of either PD-L1 + CD68 + macrophages or PD-1 + CD4 + and PD-1 + CD8 + T cells translates into favorable survival. In contrast, the number of PD-L1 + lymphoma cells or PD-L1 - macrophages do not associate with outcome. In multivariate analyses with IPI, PD-L1 + CD68 + macrophage and PD-1 + lymphocyte contents remain as independent prognostic factors for survival. In conclusion, high PD-L1 + CD68 + macrophage and PD-1 + lymphocyte contents predict favorable survival in patients with primary testicular lymphoma. The findings implicate that the tumor microenvironment and PD-1 - PD-L1 pathway have a significant role in regulating treatment outcome. They also bring new insights to the targeted thera py of primary testicular lymphoma., (Copyright© 2018 Ferrata Storti Foundation.)

Despite better therapeutic options and improved survival of diffuse large B-cell lymphoma (DLBCL), 30-40% of the patients experience relapse or have primary refractory disease with a dismal prognosis. To identify biological correlates for treatment resistance, we profiled microRNAs (miRNAs) of matched primary and relapsed DLBCL by next-generation sequencing. Altogether 492 miRNAs were expressed in the DLBCL samples. Thirteen miRNAs showed significant differential expression between primary and relapse specimen pairs. Integration of the differentially expressed miRNAs with matched mRNA expression profiles identified highly anti-correlated, putative targets, which were significantly enriched in cancer-associated pathways, including phosphatidylinositol (PI)), mitogen-activated protein kinase (MAPK), and B-cell receptor (BCR) signaling. Expression data suggested activation of these pathways during disease progression, and functional analyses validated that miR-370-3p, miR-381-3p, and miR-409-3p downregulate genes on the PI, MAPK, and BCR signaling pathways, and enhance chemosensitivity of DLBCL cells in vitro. High expression of selected target genes, that is, PIP5K1 and IMPA1, was found to be associated with poor survival in two independent cohorts of chemoimmunotherapy-treated patients (n = 92 and n = 233). Taken together, our results demonstrate that differentially expressed miRNAs contribute to disease progression by regulating key cell survival pathways and by mediating chemosensitivity, thus representing potential novel therapeutic targets.

Purpose We compared the efficacy of ofatumumab (O) versus rituximab (R) in combination with cisplatin, cytarabine, and dexamethasone (DHAP) salvage treatment, followed by autologous stem-cell transplantation (ASCT) in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). Patients and Methods Patients with CD20 + DLBCL age ≥ 18 years who had experienced their first relapse or who were refractory to first-line R-CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone)-like treatment were randomly assigned between three cycles of R-DHAP or O-DHAP. Either O 1,000 mg or R 375 mg/m 2 was administered for a total of four infusions (days 1 and 8 of cycle 1; day 1 of cycles 2 and 3 of DHAP). Patients who experienced a response after two cycles of treatment received the third cycle, followed by high-dose therapy and ASCT. Primary end point was progression-free survival (PFS), with failure to achieve a response after cycle 2 included as an event. Results Between March 2010 and December 2013, 447 patients were randomly assigned. Median age was 57 years (range, 18 to 83 years); 17% were age ≥ 65 years; 63% had stage III and IV disease; 71% did not achieve complete response (CR) or experience response for < 1 year on first-line R-CHOP. Response rate for O-DHAP was 38% (CR, 15%) versus 42% (CR, 22%) for R-DHAP. ASCT on protocol was completed by 74 patients (33%) in the O arm and 83 patients (37%) in the R arm. PFS, event-free survival, and overall survival were not significantly different between O-DHAP versus R-DHAP: PFS at 2 years was 24% versus 26% (hazard ratio [HR], 1.12; 95% CI, 0.89 to 1.42; P = .33); event-free survival at 2 years was 16% versus 18% (HR, 1.10; P = .35); and overall survival at 2 years was 41% versus 38% (HR, 0.90; P = .38). Positron emission tomography negativity before ASCT was highly predictive for superior outcome. Conclusion No difference in efficacy was found between O-DHAP and R-DHAP as salvage treatment of relapsed or refractory DLBCL.

Purpose: To compare disease-free survival (DFS) after maintenance therapy with the selective protein kinase C β (PKCβ) inhibitor, enzastaurin, versus placebo in patients with diffuse large B-cell lymphoma (DLBCL) in complete remission and with a high risk of relapse after first-line therapy., Patients and Methods: This multicenter, phase III, randomized, double-blind, placebo-controlled trial enrolled patients who were at high risk of recurrence after rituximab-cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Patients (N = 758) with stage II bulky or stage III to IV DLBCL, three or more International Prognostic Index risk factors at diagnosis, and a complete response or unconfirmed complete response after 6 to 8 cycles of R-CHOP were assigned 2:1 to receive oral enzastaurin 500 mg daily or placebo for 3 years or until disease progression or unacceptable toxicity. Primary end point was DFS 3 years after the last patient entered treatment. Correlative analyses of biomarkers, including cell of origin by immunohistochemistry and PKCβ expression, with efficacy outcomes were exploratory objectives., Results: After a median follow-up of 48 months, DFS hazard ratio for enzastaurin versus placebo was 0.92 (95% CI, 0.689 to 1.216; two-sided log-rank P = .541; 4-year DFS, 70% v 71%, respectively). Independent of treatment, no significant associations were observed between PKCβ protein expression or cell of origin and DFS or overall survival., Conclusion: Enzastaurin did not significantly improve DFS in patients with high-risk DLBCL after achieving complete response to R-CHOP. Achievement of a complete response may have abrogated the prognostic significance of cell of origin by immunohistochemistry., (© 2016 by American Society of Clinical Oncology.)

We evaluated the predictive value of interim positon emission tomography (I-PET) after one course of chemoimmunotherapy in patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL). One hundred and twelve patients with DLBCL were enrolled. All patients had PET/computed tomography (CT) scans performed after one course of chemotherapy (PET-1). I-PET scans were categorized according to International Harmonization Project criteria (IHP), Deauville 5-point scale (D 5PS) with scores 1-3 considered negative (D 5PS > 3) and D 5PS with scores 1-4 considered negative (D 5PS = 5). Ratios of tumor maximum standardized uptake value (SUVmax) to liver SUVmax were also analyzed. We found no difference in progression-free survival (PFS) between PET-negative and PET-positive patients according to IHP and D 5PS > 3. The 2-year PFS using D 5PS = 5 was 50.9% in the PET-positive group and 84.8% in the PET-negative group (p = 0.002). A tumor/liver SUVmax cut-off of 3.1 to distinguish D 5PS scores of 4 and 5 provided the best prognostic value. PET after one course of chemotherapy was not able to safely discriminate PET-positive and PET-negative patients in different prognostic groups.

Translocations affecting both MYC and BCL2 are associated with a poor prognosis in diffuse large B-cell lymphomas. We have examined genetic aberrations combined with analyses of protein expression of respective gene products. Fluorescence in situ hybridization (FISH) for translocations of BCL2 and MYC and del17p13 was performed. Immunohistochemistry analyses included BCL2, MYC and TP53 protein expression. Sixty-seven patients with high-risk DLBCL participating in a prospective multicenter study were included. Six patients with simultaneous translocations of BCL2 and MYC had impaired overall (OS) (p = 0.009) and progression-free survival (PFS) (p = 0.009). Six patients with high coexpression of MYC and BCL2 proteins also had impaired OS (p = 0.004) and PFS (p = 0.002). Combining these groups identified nine patients with impaired OS (p = 0.004) and PFS (p = 0.005). Sixteen patients had double-hit translocation or protein expression and/or del17p13 and/or high TP53. This combined endpoint was associated with impaired OS (p = 0.008) and PFS (p = 0.036), and identified 70% of all deaths.

The prognostic impact of the tumor microenvironment in diffuse large B-cell lymphoma has not been systematically assessed. We analyzed mRNA and antigen expression of monocytes, macrophages, lymphocytes, dendritic and natural killer cells in pretreatment tumor samples of patients with high-risk diffuse large B-cell lymphoma using gene expression microarray and immunohistochemistry. The patients were treated in a Nordic phase II study with dose-dense chemoimmunotherapy and central nervous system prophylaxis. Of the studied markers for non-malignant inflammatory cells, CD68 expression and CD68(+) macrophage counts correlated with favorable outcome. Five-year progression-free survival rates were 83% and 43% for the patients with high and low CD68 mRNA levels, respectively (P=0.007), while overall survival rates were 83% and 64%, respectively (P=ns). The patients with high CD68(+) macrophage counts had better 5-year progression-free survival (74% versus 40%; P=0.003) and overall survival (90% versus 60%; P=0.009) than the patients with low macrophage counts. Low CD68(+) macrophage count retained its prognostic impact on overall survival with age-adjusted International Prognostic Index [RR=5.0 (95% CI 1.024-19.088); P=0.017]. The findings were validated in three independent cohorts of patients treated with chemoimmunotherapy. In contrast, in patients treated with chemotherapy, high CD68(+) macrophage count was associated with poor progression-free survival (40% versus 72%; P=0.021) and overall survival (39% versus 72%; P=0.015). Together, the data suggest that macrophages exhibit a dual, treatment-specific role in diffuse large B-cell lymphoma. For the patients treated with chemoimmunotherapy, high pretreatment CD68 mRNA levels and CD68(+) macrophage numbers predict a favorable outcome., (Copyright© Ferrata Storti Foundation.)

Background: Despite improved survival for the patients with diffuse large B-cell lymphoma (DLBCL), the prognosis after relapse is poor. The aim was to identify molecular events that contribute to relapse and treatment resistance in DLBCL., Methods: We analysed 51 prospectively collected pretreatment tumour samples from clinically high risk patients treated in a Nordic phase II study with dose-dense chemoimmunotherapy and central nervous system prophylaxis with high resolution array comparative genomic hybridization (aCGH) and gene expression microarrays. Major finding was validated at the protein level immunohistochemically in a trial specific tissue microarray series of 70, and in an independent validation series of 146 patients., Results: We identified 31 genes whose expression changes were strongly associated with copy number aberrations. In addition, gains of chromosomes 2p15 and 18q12.2 were associated with unfavourable survival. The 2p15 aberration harboured COMMD1 gene, whose expression had a significant adverse prognostic impact on survival. Immunohistochemical analysis of COMMD1 expression in two series confirmed the association of COMMD1 expression with poor prognosis., Conclusion: COMMD1 is a potential novel prognostic factor in DLBCLs. The results highlight the value of integrated comprehensive analysis to identify prognostic markers and genetic driver events not previously implicated in DLBCL., Trial Registration: ClinicalTrials.gov NCT01502982.

Diffuse large cell B-cell lymphoma (DLBCL) is the most common lymphoma in the western world. The most common symptom is lymphadenopathy. General symptoms may include a decrease in performance status, loss of weight, fever and night sweats. DLBCL is treated with a combination of anthracyclin-based chemotherapy and the CD20 antibody rituximab. 70% of the patients can be cured with the combination therapy. To date, clinical risk factors remain as the cornerstone of treatment decisions and prognosis. Biological prognostic factors and targeted therapies are, however, under active research and it is likely that patients will in the near future be treated according to risk-adapted and biomarker-driven therapies.

Primary mediastinal large B-cell lymphoma (PMBCL) is a subtype of diffuse large B-cell lymphoma (DLBCL) accounting for 2% to 4% of all non-Hodgkin lymphomas. We report a family of 3 siblings with PMBCL and their cousin with extranodal DLBCL. The histopathological characteristics of lymphomas of all 4 patients are similar, implying post-germinal center differentiation and growth deregulation by other mechanisms than BCL2-mediated inhibition of apoptosis and suggesting a shared biological background. We aimed to identify the genetic defect underlying lymphoma susceptibility in this family using exome sequencing and linkage analysis. The only variant segregating in all 4 patients and not reported in genetic databases was 5533C>A (His1845Asn) in the MLL gene. To our knowledge, this is the first time when familial clustering of PMBCL is reported. Although we propose MLL as a candidate predisposition gene for this condition, this finding needs to be validated in additional cases.

Objectives: To determine whether serum vascular endothelial growth factor (s-VEGF) levels and VEGF gene expression in tumor tissue predict survival of diffuse large B-cell lymphoma (DLBCL) patients treated with chemoimmunotherapy., Methods: VEGF levels were measured in serum samples from 102 patients <65 yrs with high-risk DLBCL using a quantitative sandwich enzyme immunoassay technique. Exon array data set of tumor tissues from 32 patients was concurrently used to determine VEGF-A exon and gene expression. All patients were treated in a Nordic phase II study with six dose-dense chemoimmunotherapy courses followed by systemic central nervous system prophylaxis., Results: After a median follow-up time of 40 months, 3-yr progression-free survival (PFS) was inferior in patients with high s-VEGF levels compared to those with low levels (59% vs. 83%, P = 0.005). The relative risk of progression or relapse was 3.1-fold (95% confidence interval 1.34-6.91, P = 0.008). The predictive capacity of s-VEGF levels on PFS was most pronounced in the DLBCLs of non-germinal center subtype. In contrast to serum data, VEGF mRNA expression in the lymphoma tissue did not predict outcome, and no correlation was found between s-VEGF levels and lymphoma VEGF expression., Conclusion: Pretreatment s-VEGF level is a predictor of PFS after chemoimmunotherapy and may help to further stratify high-risk DLBCL patients into low- and high-risk groups., (© 2012 John Wiley & Sons A/S.)

Rituximab, a CD20-specific antibody, is used with chemotherapy as a treatment for diffuse large B cell lymphoma (DLBCL). Although many patients benefit from the addition of rituximab to chemotherapy, a favourable response is not achieved in approximately 30% of cases. This sets a prerequisite to better understand the response and resistance mechanisms of rituximab. To do so, we analyzed the gene expression profiles of one rituximab unresponsive and two responsive DLBCL cell lines. In the responsive cells, rituximab affected the expression of genes related to apoptosis, lymphocyte signaling and cytokine response. Our data show rituximab-response to be associated with gene expression in classical signaling cascades involved in cell growth and differentiation, such as previously identified MAPK and completely novel Wnt and TGF-β pathways. Furthermore, our findings support earlier observations that rituximab can induce direct apoptosis and suggest the cell of origin to be associated with the cellular outcome. After validation of cellular results, we used a cohort of 233 R-CHOP treated DLBCL patients and found several of the most differentiating genes to have impact on survival. Together, the results provide an advanced picture of the CD20 mediated signaling of DLBCL cells and may provide new targets in future treatment protocols.

We have previously shown that expression of CD40 has a favorable prognostic impact in diffuse large B-cell lymphoma (DLBCL) after anthracycline-based chemotherapy. Here we examined the prognostic value of immunohistochemically defined CD40 expression in 95 patients with DLBCL treated with both anthracycline-based chemotherapy and rituximab. Using a 10% cut-off level, 77% of the patients had CD40-positive tumors and showed a superior overall survival (p = 0.02 log-rank, hazard ratio 0.35, 95% CI 0.14-0.88, p = 0.03 Cox regression). When adjusted for International Prognostic Index in multivariate analysis, CD40 was not an independent prognostic factor (hazard ratio 0.39, 95% CI 0.15-1.04, p = 0.06 Cox regression). However, even after the introduction of immunochemotherapy, CD40 has a potential prognostic impact in DLBCL. Additional and larger studies are necessary, regarding the immunohistochemical robustness of CD40 and the biological mechanisms that contribute to the superior prognosis in CD40-expressing DLBCL.

Development of targeted agents for the treatment of diffuse large B-cell lymphoma includes clinical evaluation of enzastaurin, an agent that suppresses signaling through protein kinase C-beta and AKT pathways. To determine whether protein kinase C-beta expression has prognostic significance for diffuse large B-cell lymphoma patients treated with immunochemotherapy, we analyzed the expression of protein kinase C-beta II, BCL-2 and cell of origin immunohistochemically from pretreatment samples of 95 diffuse large B-cell lymphoma patients. All patients received rituximab with CHOP or CHOEP. According to Kaplan-Meier analyses, overall survival at 3 years was better among the patients with low than high protein kinase C-beta II protein levels (94 vs 76%, P=0.036). The prognostic value of protein kinase C-beta II expression on survival was seen in the patients with low and high International Prognostic Index risk groups, and in all molecular entities. Gene expression data from an independent set of 233 diffuse large B-cell lymphoma patients treated with a combination of rituximab and CHOP-like chemotherapy was analyzed in comparison. Accordingly, a better 3-year overall survival was observed among the subgroup with low protein kinase C-beta II mRNA levels (84 vs 68%, P=0.005). In multivariate analysis with cell of origin, protein kinase C-beta II mRNA expression remained as an independent predictor for overall survival. Together, the data show that protein kinase C-beta II expression has prognostic significance in diffuse large B-cell lymphoma patients treated with immunochemotherapy.

Gene expression profiling studies initially enabled diffuse large B-cell lymphoma to be divided into germinal center and activated B-cell-like subtypes, which define high- and low-risk patient groups when treated with chemotherapy. Attempts to reproduce the prognostic classification immunohistochemically have, however, provided inconsistent results. The aim of this study was to determine whether modified immunohistochemical classification of cell of origin focusing on activated B-cell-like markers could be used to predict the outcome of immunochemotherapy-treated diffuse large B-cell lymphoma patients. The expression of CD10, Bcl-6, MUM1/IRF4, Bcl-2, and FOXP1 was determined immunohistochemically from 88 samples of diffuse large B-cell lymphoma patients treated uniformly with R-CHOP. When the modified classification using MUM1/IRF4 and FOXP1 positivities as activated B-cell-like markers was applied to distinguish the patients between the activated B-cell-like and other diffuse large B-cell lymphoma subtypes, a significantly worse outcome was seen for the patients with the activated B-cell-like phenotype (3-year failure-free survival 63 vs 82%, P=0.048, overall survival 69 vs 85%, P=0.110). Similarly, according to the Muris algorithm, the group 2 patients representing Bcl-2-positive post-germinal center patients showed an inferior outcome in comparison to the group 1 patients (failure-free survival 59 vs 81%, P=0.041, overall survival 67 vs 82%, P=0.159). In contrast, when the classification of the same cohort was performed according to the Hans algorithm, no significant difference in survival was observed between the germinal center and non-germinal center patients. In conclusion, the data suggest that both the modified activated B-cell-like and Muris classifications define the non-germinal center phenotype as an adverse risk factor in R-CHOP-treated diffuse large B-cell lymphoma patients.

Objectives: Non-germinal center (non-GC) phenotype, high level expression of the transcription factor forkhead box protein P1 (FOXP1) and anti-apoptotic protein Bcl-2 have been identified as unfavorable prognostic factors for diffuse large B-cell lymphoma (DLBCL) patients treated with chemotherapy. Our aim was to re-evaluate the prognostic impact of these biologic factors on the survival of the patients treated with immunochemotherapy., Methods: Expression of Bcl-2 and FOXP1, and cell of origin based on the Hans algorithm were determined immunohistochemically from samples of 117 de novo DLBCL patients treated with R-CHOP and R-CHOEP regimens, and correlated with clinical data., Results: Consistent with our previous studies, no significant difference in 2-yr survival rates between the GC- and non-GC phenotypes was found. Both FOXP1 and Bcl-2 expression were associated with the non-GC phenotype. For all patients, no prognostic impact of FOXP1 positivity on survival was observed. However, Bcl-2 negative patients had a better survival as compared to Bcl-2 positive patients [failure free survival (FFS) 97% vs. 71%, P = 0.001 and overall survival (OS) 97% vs. 82%, P = 0.034]. When Bcl-2 related survival was analyzed in the GC- and non-GC subgroups, a significant prognostic effect of Bcl-2 on FFS was seen only in the non-GC group of patients (positive 65% vs. negative 100%, P = 0.011). A trend for the difference in OS was also observed (positive 84% vs. negative 100%, P = 0.082)., Conclusions: The data demonstrate that expression of Bcl-2 and FOXP1 is associated with the non-GC phenotype, but only Bcl-2 expression continues to be of prognostic significance in DLBCL patients treated with immunochemotherapy.

Germinal center (GC) and non-GC phenotypes are predictors of outcome in diffuse large B-cell lymphoma (DLBCL) and can be used to stratify chemotherapy-treated patients into low- and high-risk groups. To determine how combination of rituximab with chemotherapy influences GC-associated clinical outcome, GC and non-GC phenotypes were identified immunohistochemically from samples of 90 de novo DLBCL patients treated with rituximab in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-like regimen (immunochemotherapy). One hundred and four patients previously treated with chemotherapy served as a control group. Consistent with previous studies, chemotherapy-treated patients with immunohistochemically defined GC phenotype displayed a significantly better overall (OS) and failure-free survival (FFS) than the non-GC group (OS, 70% vs 47%, P = .012; FFS, 59% vs 30%, P = .001). In contrast, immunohistochemically defined GC phenotype did not predict outcome in immunochemotherapy-treated patients (OS, 77% vs 76%, P = ns; FFS, 68% vs 63%, P = ns). In comparison, International Prognostic Index (IPI) could separate the high-risk patients from low- and intermediate-risk groups (OS, 84% vs 63%, P = .030; FFS, 79% vs 52%, P = .028). We conclude that rituximab in combination with chemotherapy seems to eliminate the prognostic value of immunohistochemically defined GC- and non-GC phenotypes in DLBCL.