Key Points• High proportions of PD-1 1 and PD-L1 1 leukocytes in the Hodgkin lymphoma microenvironment are associated with inferior outcome.• Expression of PD-L1 and PD-L2 on Hodgkin and Reed-Sternberg cells has no impact on outcome.Immune checkpoint inhibition targeting the programmed death receptor (PD)-1 pathway is a novel treatment approach in relapsed and refractory classical Hodgkin lymphoma (cHL). leukocytes was also associated with inferior OS in a multivariate analysis (HR, 3.46; 95% CI,. This is the first study to show a correlation after multivariate analysis between inferior outcome in cHL and a high proportion of both PD-1 1 and PD-L1 1 leukocytes in the tumor microenvironment.

Background: Aggressive non-Hodgkin lymphoma (aNHL) relapsing after high-dose therapy or, in not transplant-eligible patients, after 1st-line chemotherapy represents an unmet clinical need. Therefore, we aimed at evaluating a salvage combination regimen based on pixantrone, an aza-anthracenadione recently approved in Europe for patients with multiply relapsed aNHL. Etoposide and bendamustine were chosen as companion compounds due to available feasibility data in combination with anthracenadions, and a well-documented efficacy in salvage regimens for relapsed aNHL. Rituximab was added, if the relapse tumor biopsy was CD20+. Aim: The aim of the present analysis was to summarize the preliminary clinical experience with the PREBEN/PEBEN regimen gathered, on a compassionate need basis, at different European sites and representing the platform for a currently ongoing Nordic phase 1/2 trial in relapsed aNHL. Methods: The adopted schedule consisted of pixantrone 50 mg/m2 i.v. day 1+8, etoposide 100 mg i.v. day 1, bendamustine 90 mg i.v. day 1 with or without the addition of rituximab 375 mg/m2 i.v. day 1 (PREBEN/PEBEN). If feasible, each cycle was given at 3-weekly intervals for a maximum of 6 cycles. All patients were assessed for chemosensitivity with PET/CT, already after cycle 1 or 2. G-CSF support was applied and administered according to local practice. Results: A total of 30 heavily pre-treated patients (19 males and 11 females, age range 49-81 yrs; mean N of previous regimens: 3, range 1-7) with aNHL were treated according to the PREBEN/PEBEN schedule. Seventeen had diffuse large B-cell (DLBCL), six transformed indolent (tIND), and seven peripheral T-cell lymphoma (PTCL). All patients had intermediate or high risk IPI prior to start of salvage therapy. Eight patients (27%) had a complete metabolic response (CMR) and seven (23%) a partial one (PMR), resulting in an overall response rate (ORR) of 50%. Among the histological subtypes, the patients with DLBCL, PTCL and tIND had an ORR of 53% (CMR 35%), 57% (CMR 14%), and 33% (CMR in one out of two responders), respectively. Most responses were achieved early (prior to course nr. 4). Response durations ranged between 2 and 23+ months. Among the 17 patients with DLBCL, nine were frail, non transplant-eligible with relapsed disease, six had primary refractory lymphoma progressing through anthracycline-containing 1st line and platinum-containing salvage therapies, and two had relapses occurring after a post-transplant remission period. While most of the relapsed patients with DLBCL responded , i.e. seven (five CMR and two PMR) of the nine (78%) frail relapsed patients and one of the two (50%) patients with post-transplant relapses, only one out six (17%) primary refractory patients exhibited some chemosensitivity. Interestingly, four out of seven PTCL patients achieved a PMR or CMR allowing them to undergo non-myeloablative allogeneic transplant with subsequent sustained response durations. The treatment schedule was feasible and most patients received it on an out-patient basis. The most common grade 3-4 toxicity was of hematological type (mainly neutropenia and thrombocytopenia), occurring in 52% of the patients. Grade 3-4 infections were observed at a frequency of 21%. No septic deaths were recorded. A previously anthracycline exposed, heavily pre-treated 60-year old female PTCL patient developed symptomatic congestive heart failure effectively reversed by angiotensin converting enzyme inhibitors with normalization of the myocardial ejection fraction. One previously ibritumomab tiuxetan exposed, heavily pretreated patient with tIND developed acute myeloid leukemia with therapy-related cytogenetic features. Conclusions: The PREBEN/PEBEN salvage regimen was feasible in a heavily pre-treated cohort of elderly patients with high-risk aNHL. In individual patients it elicited substantial and durable responses early in the course of therapy. In some younger patients, it proved useful as bridging strategy to a non-myeloablative allogeneic transplant. A phase 1/2 study in relapsed (non-refractory) aNHL was launched in June 2016 (ClinicalTrial.gov identifier: NCT02678299; EudraCT number: 2015-000758-39) and is currently accruing (N=5 pr. Aug 1st, 2016). Disclosures Clausen: Takeda: Research Funding; Novartis: Other: Travel expences; Abbvie: Other: Travel expences. Leppa:Mundipharma: Research Funding; Roche: Honoraria, Other: Travel expenses, Research Funding; Bayer: Research Funding; Janssen: Research Funding; Takeda: Honoraria, Other: Travel expenses; CTI Life Sciences: Honoraria; Amgen: Research Funding; Merck: Other: Travel expenses. Willenbacher:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; European Commision: Research Funding. d'Amore:Servier: Honoraria, Other: Advisory Boards; CTI LIfe Sciences: Honoraria, Other: Advisory Boards.

Background The programmed cell death protein 1 (PD‐1) and its ligand 1 and 2 (PD‐L1/PD‐L2) regulate the immune system, and the checkpoint pathway can be exploited by malignant cells to evade anti‐tumor immune response. Soluble forms (sPD‐1/sPD‐L1/sPD‐L2) exist in the peripheral blood, but their biological and clinical significance is unclear. Method Time‐resolved immunofluorometric assay (TRIFMA) and enzyme‐linked immunosorbent assay (ELISA) were used to measure sPD‐1, sPD‐L1, and sPD‐L2 levels in serum from 131 lymphoma patients and 22 healthy individuals. Results Patients had higher sPD‐1 and sPD‐L2 levels than healthy individuals. In diffuse large B‐cell lymphoma, patients with high International Prognostic Index score had higher sPD‐1 levels and sPD‐L2 levels correlated with subtype according to cell of origin. Compared to other lymphoma types, follicular lymphoma displayed higher sPD‐1 and lower sPD‐L1 levels along with lower ligand/receptor ratios. Conclusion This is the first study to simultaneously characterize pretherapeutic sPD‐1, sPD‐L1, and sPD‐L2 in a variety of lymphoma subtypes. The relation between higher sPD‐1 levels and adverse prognostic factors suggests a possible biological role and potential clinical usefulness of sPD‐1. Moreover, the reverse expression pattern in follicular lymphoma and T‐cell lymphoma/leukemia may reflect biological information relevant for immunotherapy targeting the PD‐1 pathway.

Interaction of checkpoint receptor programmed death 1 (PD-1) with its ligand 1 (PD-L1) downregulates T cell effector functions and thereby leads to tumor immune escape. Here, we aimed to determine the clinical significance of soluble PD-1 (sPD-1) and soluble PD-L1 (sPD-L1) in patients with diffuse large B-cell lymphoma (DLBCL). We included 121 high-risk DLBCL patients treated in the Nordic NLG-LBC-05 trial with dose-dense immunochemotherapy. sPD-1 and sPD-L1 levels were measured from serum samples collected prior to treatment, after three immunochemotherapy courses, and at the end of therapy. sPD-1 and sPD-L1 levels were the highest in pretreatment samples, declining after three courses, and remaining low post-treatment. Pretreatment sPD-1 levels correlated with the quantities of PD1+ T cells in tumor tissue and translated to inferior survival, while no correlation was observed between sPD-L1 levels and outcome. The relative risk of death was 2.9-fold (95% CI 1.12–7.75, p = 0.028) and the risk of progression was 2.8-fold (95% CI 1.16–6.56, p = 0.021) in patients with high pretreatment sPD-1 levels compared to those with low levels. In conclusion, pretreatment sPD-1 level is a predictor of poor outcome after dose-dense immunochemotherapy and may be helpful in further improving molecular risk profiles in DLBCL.

Background Checkpoint proteins regulate the immune system and their down-regulation of effector mechanisms is exploited by malignant cells to evade antitumor immune response. Membrane bound programmed death 1 protein (PD-1) and its ligands 1 and 2 (PD-L1 and PD-L2) are pivotal members of the immune checkpoint family. The significance of the PD-1 pathway have been demonstrated in a variety of malignant diseases and suppressing the pathway with targeting antibodies can re-establish antitumor immunity. Soluble forms of the proteins (sPD-1, sPD-L1 and sPD-L2) are detectable in the peripheral blood, but their biological and clinical significance are still largely unknown. To our knowledge, this is the first study to simultaneously characterize sPD-1, sPD-L1 and sPD-L2 in different types of lymphoma. Aims The aim of the present study was to measure the pre-therapeutic sPD-1, sPD-L1 and sPD-L2 levels in different lymphoid malignancies and correlate them with clinico-pathological features and outcome. Methods We established and validated a Time Resolved Immunoflourometric assay (TRIFMA) to determine levels of sPD-1 and sPD-L2. Soluble PD-L1 levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) applied according to manufacturer's instructions. In total, archival serum samples from 102 patients were analysed in duplicate for each protein. This single-institution study population consisted of patients with chronic lymphocytic leukemia (CLL; n=42), diffuse large B cell lymphoma (DLBCL; n=33), follicular lymphoma (FL; 15) and Hodgkin lymphoma (HL; n=12). In addition, 22 and 17 healthy controls were included for analysis of sPD-1/sPD-L1 and sPD-L2, respectively. Information on clinical parameters, response to treatment and outcome was obtained from medical records. Measured levels were compared between patient subgroups and controls using a non-parametric test (Mann-Whitney). P-values below 0.05 were considered statistically significant. Results Soluble PD-1 levels were higher in patients, taken as one group, compared to controls (p=0.0002). When analyzed according to lymphoma type, patients with DLBCL, CLL and FL still had significantly elevated sPD-1 levels as compared to controls (p=0.0089, p=0.0008 and p<0.0001, respectively). Highest sPD-1 levels were observed in FL and these were also significantly higher than in any of the other lymphoma subtypes analyzed. Soluble PD-L1 levels were similar in patients, taken as one group, and controls. Interestingly, patients with FL had lower sPD-L1 levels than patients from all the other lymphoma subtypes and it was the only histology whose sPD-L1 levels differed significantly (lower values) from healthy controls (p=0.0017, see table 1). Moreover, we identified a reverse expression pattern of sPD-1/sPD-L1 in FL as compared to other lymphoma subtypes, as FL had significantly higher sPD-1 and lower sPD-L1 than all other remaining patients (see table 1). Soluble PD-L2 levels were generally higher than those of sPD-1 and sPD-L1, and the highest values were found in healthy controls. When analyzed according to histological subtype, patients with CLL, DLBCL and FL had all significantly lower sPD-L2 levels than healthy controls (see table 1). Interestingly, the sPD-1, sPD-L1 and sPD-L2 levels measured in HL patients did not differ significantly from controls, however, this may be due to limited sample size. Conclusion We simultaneously characterized sPD-1, sPD-L1 and sPD-L2 in selected lymphomas subtypes and found varying levels across the investigated lymphoma entities. In particular, we identified a reverse expression pattern of sPD-1 and sPD-L1 in FL compared to other lymphoid malignancies. We were the first to also characterize sPD-L2 levels in the same patients and found its levels to be generally high throughout lymphoma entities, but highest in healthy controls. A correlation analysis with outcome-related parameters is ongoing. Disclosures No relevant conflicts of interest to declare.