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Introduction: Despite better therapeutic options and improved survival of diffuse large B-cell lymphoma (DLBCL), 30-40% of the patients still relapse and have dismal prognosis. Recently, the impact of genomic aberrations, allowing lymphoma cells to escape immune recognition on DLBCL pathogenesis has been recognized. However, whether immune related signatures could be used as determinants for treatment outcome has not been rigorously evaluated. Here, our aim was to elucidate the immunologic characteristics of the tumor microenvironment, and associate the findings with outcome in patients with high-risk DLBCL.Patients and methods: We specifically examined gene expression in the tumor microenvironment, using RNA isolated from formalin fixed paraffin-embedded (FFPE) tumor samples from 81 patients with DLBCL. Gene expression analysis was conducted on the NanoString nCounter gene expression platform using a 770-gene PanCancer Immune panel. The predictive impact of the gene expression signatures was assessed using a cohort of 65 high risk DLBCL patients enrolled in the Nordic CHIC trial (Leppä et al., 2016). The findings were validated using exon array data from an independent cohort of 38 high risk DLBCL patients treated in the Nordic CRY-04 trial (Holte et al., 2013; Leivonen et al., 2017a). In both trials, the patients received dose-dense immunochemotherapy (R-CHOEP-14) and systemic central nervous system prophylaxis (HD-Mtx and HD-Ara-C).Results: Unsupervised hierarchical clustering revealed three major clusters of genes with differential expression between the patients. The clusters were enriched for the genes related to focal adhesion and extracellular matrix (ECM) receptor interactions (eg. COL3A1, ITGA, ITGB, VEGFC, VEGFA, PDGFRB, PDGFC, FN1), T/NK-cell signaling (eg. ICAM1, CD3, CD4, CD8 GZMB, ITGB2), and type 1/2 immune responses (eg. IL4, IFNL1, IFNL2, IL13, IL24). Interestingly, type 1/2 immune response signature correlated with survival. In the CHIC cohort, the group of patients (n=10) with high expression of type 1/2 immune response signature genes had a 4-year PFS of 60% in comparison to 91% for those (n=55) with low expression (p=0.021). The corresponding 4 y OS rates were 64% vs 90% (p=ns). The risk of progression was 4.0 fold higher for the patients with high expression of type 1/2 immune response signature genes (95% CI 1.120-14.280, p=0.033). No differences were observed in sex, age, stage, IPI scores, or molecular subgroups between the low and high type 1/2 immune response subgroups. The prognostic impact of the signature was confirmed in the CRY-04 exon array cohort (4-y PFS; 46% vs 80%, p = 0.021, 4-y OS 62% vs 88%; p = 0.014). In the CRY-04 cohort, the risk of progression was 3.6 fold (95% CI 1.13-11.20, p=0.030) and death 4.8 fold (95% CI 1.20-19.26, p=0.027) higher for the group of patients with high expression of the type 1/2 immune response signature genes. In contrast to our data on primary testicular lymphomas (PTLs) (Leivonen et al., 2017b), T/NK cell signature did not correlate with outcome in either of the trial cohorts. Correspondingly, type 1/2 immune signature was not associated with outcome in the cohorts of PTL or DLBCL patients treated with conventional R-CHOP regimen.Conclusion: Our data suggest that immune related signatures exhibit treatment-specific roles in diffuse large B-cell lymphoma. For the high risk DLBCL patients treated with dose-dense immunochemotherapy, high expression of type 1/2 immune response signature genes predicts a poor outcome. A detailed characterization of immune cell composition in the tumor microenvironment and its impact on survival is ongoing.

Objective: For more than two decades, CHOP (and later R-CHOP) has been the standard frontline treatment for diffuse large b-cell lymphoma (DLBCL). However, anthracycline-based chemotherapy regimens increase risk of cardiotoxicity with congestive heart failure (HF) being the best documented cardiovascular complication of anthracycline use. However, cardiotoxicity has also been associated with other chemotherapeutic drugs. In this nationwide cohort study, we compared the incidence of development of HF in patients with DLBCL or follicular lymphoma (FL) treated with or without anthracyclines. Methods: DLBCL and FL patients > 16 years both receiving anthracycline in the form of R-CHOP/CHOEP or non-anthracycline chemotherapy regiments were identified in the Danish National Lymphoma Registry (LYFO) from 2000-2012. Anthracycline exposed patients were required to receive ≥3 cycles of R-CHOP or R-CHOEP upfront. The non-anthracycline exposed patients consisted of patients receiving non-anthracycline containing chemotherapy (such as CVP, CEOP and Bendamustine). Exclusion criteria were i) treatment with chemo therapy regimens more intensive than R-CHOP/CHOEP, ii) up-front high dose autologous stem cell transplantation and iii) the following heart comorbidities prior to the beginning of the study: HF (ICD-10 code: “I50”), Ischemic Heart Disease (ICD-10 code: “I20-I25”), Arrhythmic Heart Disease (ICD-10 code: “I48-I49”) and Cardiomyopathy (ICD-10 code: “I42”). Information on heart comorbidities in our study population were retrieved from the Danish National Patient Registry. Patient follow-up started from 30 days after ended chemotherapy to minimize confounding by early HF on the planned number of R-CHOP treatments. Follow-up ended when HF occurred, on the day of emigration from Denmark, day of death or the end of the study on 31th. December 2014, whichever came first. Univariate and multivariable cox regression analyses were performed to assess the association between cumulative anthracycline treatments and HF (adjusted analysis included gender, age, Ann Arbor stadium, radiotherapy, comorbidities and type of lymphoma). Cumulative R-CHOP treatments were stratified into groups of “3-5”, “6” and “>6” treatments. Cumulative incidence curves were computed to illustrate risk of HF over time according to number of treatments. Results: The study included 2,696 patients; 2,007 received anthracycline and 689 received non-anthracycline containing chemotherapy. In total, 1979 patients were diagnosed with DLBCL and 717 with follicular lymphoma. Anthracycline users were slightly younger (median age 64.03 vs 65.2 years (p-value 6 cycles, the cumulative risks were 4.01 %, 7.86% and 9.33% (fig 1). Compared to patients receiving non-anthracycline containing chemotherapy (reference), patients receiving anthracycline containing chemotherapy had a higher risk of developing HF during follow-up with adjusted hazard ratio (HR) of 3.73 (95% CI 1.38-10.09) for patients receiving 3-5 cycles, 4.38 (95% CI 1.83-10.48) for patients receiving 6 cycles, and 8.22 (95% CI 3.50-19.31) for patients receiving >6 cycles of anthracycline treatment. Conclusion: This present study confirms that inclusion of anthracyclines in a multi-agent chemotherapy regimen is responsible for the increased risk of HF observed after treatment for lymphoma and that the rate of HF is correlated to the number of treatment cycles. Patients treated without anthracyclines had a negligible risk of HF supporting the safety of these regimens in patients considered at substantial risk of cardiotoxicity.

Background: Survival of patients with high-risk diffuse large B-cell lymphoma (DLBCL) is suboptimal, and the risk of central nervous system (CNS) progression relatively high. We investigated the efficacy of dose-dense chemoimmunotherapy and systemic CNS prophylaxis in two completed Nordic trials including patients less than 65 years with high-risk DLBCL. We combined individual patient data from these studies to compare clinical outcome and prognostic factors in patients treated with CNS prophylaxis given in the beginning (CHIC) vs at the end (CRY-04) of therapy. Patients and methods: Inclusion criteria were age 18-65 years, primary DLBCL or grade 3 follicular lymphoma without signs of CNS involvement, WHO performance score 0-3, age adjusted International Prognostic Index (aaIPI 2-3) and/or involvement of anatomical sites associated with an increased risk for CNS recurrence (e.g. testis, facial sinuses, orbita). In CRY-04, six courses of R-CHOEP14 were followed by HD-Mtx and HD-Ara-C. In CHIC, treatment consisted of two courses of HD-Mtx in combination with R-CHOP14, followed by four courses of R-CHOEP14 and one course of R-HD-AraC. In addition, liposomal AraC was administered intrathecally at courses 1, 3 and 5. Primary end points were failure free survival (FFS; disease progression, discontinuation of protocolled therapy due to toxicity, death from any cause) at three years and CNS progression rate at 1,5 years. Secondary endpoints included progression free survival (PFS; disease progression or death from any cause) and overall survival (OS) at three years. Results: Among 303 patients enrolled in the trials (CRY-04, n=160 and CHIC, n=143), 295 (CRY-04, n=154 and CHIC, n=139) met inclusion criteria and were evaluable for baseline characteristics and primary endpoints. Median age (54 and 56 years, p=0.222), male/female ratio, stage, and aaIPI scores were comparable in the two cohorts. Three-year FFS was 63 % in CRY-04 and 77% in CHIC (p=0.018) after a median follow-up of 5 and 3 years, respectively. Cumulative incidence rates of CNS progression were 5.0% and 2.4% (p=0.22), and three-year OS 80% and 86% (p=0.508), respectively. Treatment in the CHIC reduced the risk of systemic progression (aaIPI adjusted RR=0.484, 95%CI 0.300-0.782, p=0.003). PFS benefit with CHIC vs CRY-04 was observed across pre-specified subgroups, and particularly in patients < 60 years old (p=0.007), with low proliferation index (Ki67 expression