Chemotherapy-refractory Diffuse Large B-cell Lymphoma Research Articles

Characterization of Hemophagocytic Lymphohistio(HLH）in Relapsed Diffuse Large B-Cell Lymphoma Patients after CAR-T Therapy

Background: Patients with chemotherapy-refractory diffuse large B-cell lymphoma (DLBCL) have poor prognoses. The objective response rate of patients with standard salvage regimens is 26%, while CART therapy have complete remission (CR) rates of 40%- 54% against DLBCL. Half of DLBCL patients relapsed or progressed after chimeric CAR-T cell therapy, but only a few of them were accompanied by HLH. Its clinical characteristics are unclear. This study aimed to investigate the clinical characteristics of these patients. Methods: We retrospectively analyzed the clinical data of 8 patients with relapsed DLBCL lymphoma accompany by HLH after CAR-T therapy between May 2019 and July 2023(NCT02537977 and NCT04036019). We investigate the clinical characteristics of these patients, compare the survival between patients with and without CR after CAR-T therapy, compare the survival between patients with and without CRS after CAR-T therapy. Results: 8 R/R DLBCL patients were enrolled. Median age was 59.5 years (range, 37-69), and 6(75%) patients had non-germinal center B-cell-like (non-GCB) DLBCL, one had GCB DLBCL, one had transformed DLBCL. 2 patients (25.0%) had HLH before CAR-T treatment previously. Among them, 3 patients (37.5%) achieved CR after CART therapy, while 3 patients (37.5%) achieved partial remission. ALL of the patients (100%) relapsed or progressed, and all of them died because of disease progression. With a median follow-up of 7 months, the median overall survival (OS) was 206.0 days (95%CI 106.2-305.8) and progress-free survival (PFS) was 125.0 days (95%CI 40.5-210.0). Particularly，the median time from HLH to death was only 45.0 days (95%CI: 22.8-67.2). 6 patients (75.0%) occurred grade 1-2 CRS, no patient occurred ≥grade 3 CRS, and 2 patients (25.0%) did not occurred any CRS. The median OS was 328.0 days and 142.0 days (95%CI 60.4-223.6) for patients without CRS and with CRS1-2, respectively. There is significantly different in OS for patients with and without CRS ( P=0.03). The median OS was 328.0 days (95%CI 217.6-438.4) and 142.0 days (95%CI 133.4-150.6) for patients with CR and without CR, respectively. The median PFS was 257.0 days (95%CI 103.4-410.6) and 77.0 days (95%CI 8.3-145.7) for patients with CR and without CR. There is significantly different in OS and PFS for patients with CR and other patients ( P=0.01 and P=0.01). Conclusion: Hemophagocytic lymphohistiocytosis (HLH) is a rare and life threatening disorder. The prognosis of patients with relapsed DLBCL lymphoma accompany by HLH after CAR-T therapy is very poor. Patients with CRS during CART therapy may more likely have HLH when DLBCL relapse. The patients without CRS have better survival time than those with CRS during CART therapy. We hypothesis that because both HLH and CRS is inflammatory syndrome resulting from T-cell activation.

Long-Duration Complete Remissions of Diffuse Large B Cell Lymphoma after Anti-CD19 Chimeric Antigen Receptor T Cell Therapy

T cells expressing anti-CD19 chimeric antigen receptors (CARs) can induce complete remissions (CRs) of diffuse large B cell lymphoma (DLBCL). The long-term durability of these remissions is unknown. We administered anti-CD19 CAR Tcells preceded by cyclophosphamide and fludarabine conditioning chemotherapy to patients with relapsed DLBCL. Five of the seven evaluable patients obtained CRs. Four of the five CRs had long-term durability with durations of remission of 56, 51, 44, and 38months; to date, none of these four cases of lymphomas have relapsed. Importantly, CRs continued after recovery of non-malignant polyclonal B cells in three of four patients with long-term complete remissions. In these three patients, recovery of CD19+ polyclonal B cells took place 28, 38, and 28months prior to the last follow-up, and each of these three patients remained in CR at the last follow-up. Non-malignant CD19+ B cell recovery with continuing CRs demonstrated that remissions of DLBCL can continue after the disappearance of functionally effective anti-CD19 CAR Tcell populations. Patients had a low incidence of severe infections despite long periods of B cell depletion and hypogammaglobulinemia. Only one hospitalization for an infection occurred among the four patients with long-term CRs. Anti-CD19 CAR Tcells caused long-term remissions of chemotherapy-refractory DLBCL without substantial chronic toxicities.

XII. Therapeutic exploitation of autologous T-cell activation in B-cell lymphoma.

XII. Therapeutic exploitation of autologous T-cell activation in B-cell lymphoma.

Anti-CD19 CAR T Cells Administered after Low-Dose Chemotherapy Can Induce Remissions of Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma

We have treated a total of 30 patients with autologous T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19; 22 of 27 evaluable patients obtained either complete remissions (CR) or partial remissions (PR). Ten patients remain in ongoing CRs of 1 to 37 months duration. The CAR was encoded by a gammaretroviral vector and included the variable regions of an anti-CD19 antibody along with CD28 and CD3-zeta moieties. The first 21 patients treated on this protocol have been reported (Kochenderfer et al. Blood 2010, Blood 2012, and Journal of Clinical Oncology 2014). To enhance the activity of the transferred CAR T cells, T-cell infusions in the previously reported patients were preceded by a chemotherapy regimen of high-dose cyclophosphamide (60-120 mg/kg) plus fludarabine. In an attempt to reduce the overall toxicity of our anti-CD19 CAR treatment protocol, we substantially reduced the doses of chemotherapy administered before CAR T-cell infusions. This abstract communicates results from 9 patients with B-cell lymphoma who received a single infusion of 1x106 anti-CD19-CAR-expressing T cells/kg bodyweight preceded by a low-dose chemotherapy regimen consisting of cyclophosphamide 300 mg/m2 and fludarabine 30 mg/m2 (Table). Each chemotherapy agent was administered daily for 3 days. Eight of the 9 treated patients had DLBCL (diffuse large B-cell lymphoma) that was refractory to chemotherapy (chemo-refractory) or that had relapsed less than 1 year after autologous stem cell transplantation (ASCT). Both of these clinical situations carry a grim prognosis, with median overall survivals of only a few months. Despite the very poor prognoses of our patients, one patient with DLBCL obtained a CR and 4 DLBCL patients obtained PRs. In some patients, PRs included resolution of large lymphoma masses. Compared to our previous experience with anti-CD19 CAR T cells preceded by high-dose chemotherapy, toxicity was reduced when CAR T cells were infused after low-dose chemotherapy. None of the 9 patients treated with low-dose chemotherapy and CAR T cells required vasopressor drugs or mechanical ventilation, although some patients did have short-term neurological toxicity. Cytopenias were mild with a mean of only 1.4 days of blood neutrophils<500/microliter. Blood anti-CD19 CAR T-cell levels were assessed in 6 patients with a quantitative PCR assay; we detected CAR+ cells in the blood of all 6 patients. The mean peak absolute number of blood CAR+ T cells was 73 cells/microliter. Six months after infusion, persisting CAR+ T cells were detected in a lymphoma-involved lymph node by flow cytometry. These results demonstrate that anti-CD19 CAR T cells administered after low-dose chemotherapy have significant activity against chemo-refractory DLBCL and could potentially become a standard treatment for aggressive lymphoma.TablePatientAge/GenderMalignancyNumber ofPrior TherapiesClinicalSituationResponse(Duration in Months)166/MDLBCL3Post ASCT relapsePR (7)2*63/FDLBCL2Chemo-refractoryPR (7+)363/MFL7Not chemo-refractoryPR (6+)4*22/MDLBCL6Chemo-refractoryProgression565/MDLBCL4Post ASCT relapsePR (5+)647/MDLBCL2Chemo-refractoryPR (1)728/MDLBCL7Chemo-refractoryProgression862/MDLBCL7Post ASCT relapseCR (1+)954/MDLBCL3Chemo-refractoryProgression*Compassionate exemption was obtained from regulatory agencies to enroll these patients because their poor performance status precluded standard enrollment; M = male; F = female; FL = follicular lymphoma; + indicates ongoing response DisclosuresRosenberg:Kite Pharma: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Imbalance in Alternative Splicing of MCL-1 Inhibits Apoptosis in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) remains eventually fatal in 30-40% of the patients, despite intensive chemotherapy (CHOP) in combination with rituximab. A subgroup of chemotherapy-refractory DLBCL is characterized by high expression levels of both pro- and anti-apoptotic genes, including MCL-1. Alternative splicing of the MCL-1 gene results in a Bcl-2-like anti-apoptotic MCL-1L protein and a BH3-only pro-apoptotic MCL-1S protein. In the present study, we investigated if a switch in alternative splicing of MCL-1 is involved in apoptosis-resistance in primary lymphoma cells of 20 DLBCL patients and 5 DLBCL cell lines.RT-MLPA analysis revealed that MCL-1L and MCL-1S are both expressed in all tested DLBCL samples and DLBCL cell lines, however expression levels varied strongly. An imbalance between the expression levels of MCL-1L and MCL-1S to an anti-apoptotic status was observed in DLBCL patient cells and DLBCL cell lines, especially in activated B-cell like (ABC)-DLBCL, compared to tonsillar germinal center B-cells. MCL-1 mRNA expression was confirmed at protein level using immunohistochemistry and western blot analysis. Co-immunoprecipitation demonstrated that MCL-1L inhibited apoptosis by binding of Bak in MCL-1L positive DLBCL cell lines. Knockdown of MCL-1L with siRNA analysis resulted in induction of apoptosis in both GCB- and ABC-DLBCL cell lines and also in increased sensitivity to the conventional chemotherapeutical drugs etoposide. Downregulation of MCL-1L using flavopiridol induced apoptotic cell death of MCL-1L-positive DLBCL cells with low Bcl-2 expression.In summary, a switch in alternative splicing of MCL-1 occurs in a subgroup of DLBCL leading to an increase in the level of anti-apoptotic MCL-1L that contributes to therapy-resistance. These preclinical data suggest that targeting of MCL-1L might be a therapeutic option for MCL-1L positive DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Ofatumumab Monotherapy for Treatment of Patients with Relapsed/Progressive Diffuse Large B-Cell Lymphoma: Results From a Multicenter Phase II Study

AbstractAbstract 3955 Background:Patients (pts) with relapsed diffuse large B-cell lymphoma (DLBCL) ineligible for autologous stem cell transplant (ASCT) or relapsing after ASCT have a poor prognosis and limited therapeutic options. New treatment options are needed for these pts. Ofatumumab is a human monoclonal antibody that targets a membrane-proximal epitope comprising both the large loop and small loop of CD20, and has shown effective lysis of chemotherapy-refractory DLBCL cells in vitro (Teeling et al. Blood 2004; Cillessen et al. Blood 2007; abstract 2346). We report the first results from an open-label, single-arm, international Phase II trial that evaluated ofatumumab monotherapy in relapsed or progressive DLBCL. Methods:Pts (age ≥18 years) with relapsed or progressive CD20+ DLBCL ineligible for ASCT or with relapsed or progressive disease after ASCT were enrolled between December 2007 and August 2009 (N=81). Relapsed or progressive disease was defined as relapse after a complete remission (CR) or disease progression after partial remission (PR). Pts received 8 weekly infusions of ofatumumab (dose 1, 300 mg; doses 2–8, 1000 mg). The primary endpoint was overall response rate (ORR) evaluated during the 6 months from the start of treatment, as assessed by an Independent Endpoint Review Committee according to the revised response criteria (Cheson et al. J Clin Oncol 2007). Secondary endpoints included duration of response, progression-free survival (PFS), overall survival (OS) and safety. Results:Baseline characteristics are shown in the Table. Pts were heavily pretreated (median 3 prior systemic therapies), and 32% of pts did not respond to their last prior therapy. Nearly all pts (96%) had received prior rituximab-containing treatment; 54% had received 2 or more courses of prior rituximab therapy (Table). Overall, 58% of pts completed all 8 infusions of ofatumumab, 65% received at least 6 infusions, and 81% received at least 4 infusions. The primary reason for treatment discontinuation was disease progression. The ORR (95% CI) was 11% (4–18%), including 3 CRs (4%) and 6 PRs (7%). Of these 9 responding pts, 8 had responded to their last systemic therapy. The median duration of response (95% CI) was 6.9 months (5.3–6.9) and median PFS (95% CI) was 2.5 months (2.3–2.9). Infusion-related events occurred in 59% of pts, primarily occurred during infusion 1 (40% of pts) and infusion 2 (22%), and subsided during subsequent infusions; these events were predominantly grade 1–2 in severity (96% of pts). The most common (>10% of pts) adverse events (AEs; any grade) were diarrhea (17%), fatigue (15%), peripheral edema (15%), neutropenia (14%), abdominal pain (12%), constipation (12%), nausea (12%), pyrexia (11%), anemia (11%) and leukopenia (11%). Of these, ≥ grade 3 events included neutropenia (10%), leukopenia (6%), anemia (5%) and fatigue (1%). Grade 3 or greater thrombocytopenia and febrile neutropenia were reported in 6% and 4% of pts, respectively. The most common infectious events were upper respiratory tract infections (7% of pts), all of which were grade 1–2 events. In total, 13 pts died during the study; 3 pts died during the treatment period and 10 pts died during post-treatment follow up (until 24 months from study start). Deaths were due to disease progression (n=10), sepsis (n=1), circulatory collapse (no further details; n=1) and multi-organ failure (n=1). Conclusions:Single-agent ofatumumab was well tolerated with an ORR of 11% in heavily pretreated pts with relapsed or progressive DLBCL, nearly all of whom had received prior rituximab therapy. Response to last systemic treatment appeared to influence response to ofatumumab in this pt population. Further studies of ofatumumab in combination with chemotherapy in relapsed DLBCL are currently underway. Disclosures:Off Label Use: Ofatumumab is an investigational anti-CD20 monoclonal antibody, currently under development for the treatment of B-cell malignancies (chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Waldenstroms macroglobulinemia and follicular lymphoma) as well as autoimmune diseases (rheumatoid arthritis and multiple sclerosis). Davies:GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees. Padmanabhan:GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gupta:GlaxoSmithKline: Employment. Lin:GlaxoSmithKline: Consultancy, Employment. Davis:GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees. Losic:Genmab A/S: Employment, Equity Ownership. Lisby:Genmab A/S: Employment. Radford:GlaxoSmithKline: Equity Ownership; Genmab: Consultancy, Honoraria.

Molecular targeted therapies for diffuse large B‐cell lymphoma based on apoptosis profiles

Diffuse large B-cell lymphoma (DLBCL) is the most common type of adult non-Hodgkin lymphoma and is treated with chemotherapy in combination with rituximab. Despite this aggressive therapy, the disease is fatal in 30-40% of patients. Inhibition of the apoptosis signalling pathways is strongly related to response to chemotherapy and eventual clinical outcome. In order to survive, lymphoma cells depend on disruption of the apoptosis pathway by mutations in apoptosis inducing genes or by continuous expression of anti-apoptotic proteins. The development of molecules targeting these apoptosis inhibitors provides a very promising opportunity to specifically target tumour cells without toxicity to non-malignant cells in DLBCL patients. Sensitivity for most of these antagonists can be predicted based on biological markers, suggesting the possibility of pre-defining patients who will most likely benefit from these targeted therapies. Experimental therapies aimed at restoring the upstream apoptosis pathway or targeting apoptosis inhibitors are currently being tested in clinical trials and are expected to be effective particularly in chemotherapy-refractory DLBCL, providing hope for patients who are refractory to current therapies.

9221 A subset of chemotherapy-refractory diffuse large B-cell lymphomas is characterized by constitutive upstream activation of the intrinsic apoptosis pathway

9221 A subset of chemotherapy-refractory diffuse large B-cell lymphomas is characterized by constitutive upstream activation of the intrinsic apoptosis pathway

T-Cell Response in B-Cell Lymphomas: Favorable or Unfavorable?

To the Editors: In a recent issue of Clinical Cancer Research, Chang and colleagues reported that in patients with diffuse large B-cell lymphoma, the presence of an intense cytotoxic T-cell infiltrate was associated with a favorable clinical outcome (1). These observations are in contrast with our previous reports, and by other reports, which show that the presence of many activated cytotoxic T-cells in primary nodal diffuse large B-cell lymphomas is in fact a strong International Prognostic Index–independent indicator for an unfavorable clinical outcome (2, 3). We recently confirmed this association by microarray expression analysis, which also identified a group of patients with primary nodal diffuse large B-cell lymphoma with a very poor clinical outcome that was characterized by high expression levels of cytotoxic T-cell effector molecules including granzyme B and perforin (4). The possible causes for these conflicting data are not clear but could result from differences in the methods used for quantification of the T cells or by selection of analyzed areas of tumor specimens.It is unclear why Chang et al. did not cite these previous articles. Chang et al. conclude that enhancing this putative antitumor T-cell immune response is a possible target for diffuse large B-cell lymphoma immunotherapy (1). Based on our data, and the data reported by Hasselblom et al. (3), we think that this is unlikely. Apparently, in cases with many activated CTLs, these T cells are also incapable of effectively killing the lymphoma cells. Hasselblom et al. showed that this is probably not caused by the presence of Foxp3 immunosuppressive T cells (3). We have previously suggested that lymphoma cells might escape from CTL-induced apoptosis not only by interfering with proper antigen presentation but also by disruption of the intracellular apoptosis pathway (2). This latter mechanism could also explain the poor response to the apoptosis-inducing chemotherapy. Thus, from this point of view, not enhancing the antilymphoma T-cell response, but rather, restoring apoptosis sensitivity will be a promising new therapy for patients with chemotherapy-refractory diffuse large B-cell lymphoma. Recently, we have shown that this is indeed a highly promising approach (5).No potential conflicts of interest were disclosed.

Inhibition of the Intrinsic Apoptosis Pathway Downstream of Caspase-9 Activation Causes Chemotherapy Resistance in Diffuse Large B-Cell Lymphoma

Inhibition of the apoptosis cascade is an important cause of therapy resistance in diffuse large B-cell lymphomas (DLBCL). In this study, we investigated possible mechanisms and expression levels of apoptosis-related genes in the apoptosis pathway that may be responsible for differences in chemotherapy sensitivity between DLBCL patients. Twenty-eight DLBCL patient samples were investigated for their expression levels of apoptosis-related genes using reverse transcription-multiplex ligation-dependent probe amplification analysis. Functional analysis of the intrinsic, caspase-9-mediated pathway was done using fluorescence-activated cell sorting analysis, Western blot analysis, and immunohistochemistry. Two DLBCL groups were identified: one with low expression levels of both proapoptotic and antiapoptotic genes and one group with high expression levels of these genes. DLBCL with high expression levels of proapoptotic and antiapoptotic genes frequently seemed to be refractory to clinical chemotherapy. Functional analysis in these latter DLBCL samples and DLBCL cell lines with comparable expression profiles revealed high levels of spontaneous caspase-9 activity without induction of apoptosis, indicating disruption of the apoptosis pathway downstream of caspase-9 activation. This disruption of the apoptosis pathway could be restored using a small-molecule XIAP antagonist. We conclude that the intrinsic, caspase-9-mediated apoptosis pathway is constitutively activated in part of chemotherapy-refractory DLBCL with concomitant downstream inhibition of the convergence apoptosis pathway and that inhibition of XIAP might be an alternative therapy for chemotherapy-refractory DLBCL.

Genes and Proteins Associated with the Tumor Microenvironment Are Differentially Expressed in Cured Versus Primary Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma.

Background: The aim of this study was to identify possible differences in gene expression profiles (GEP) between two clinically very different patient populations, cured versus primary chemotherapy-refractory diffuse large B-cell lymphoma (DLBCL).Material and methods: Tumor samples from 37 patients with de novo DLBCL, stage II-IV, either in continuous primary complete remission (n=24) or with progressive disease during primary treatment (n=13) were selected from a larger study cohort and examined with respect to GEP using spotted 55K oligonucleotide arrays. Formalin fixed paraffin-embedded tissue for immunohistochemical analysis was available in 33 of 37 cases, and was used for confirmation on protein level.Results: The top 86 genes with the greatest statistical ability of discriminating the two patient groups were chosen for further analysis. Seventy-nine were over-expressed in the cured cohort, many of them coding for proteins expressed in the tumor microenvironment: CD68, several proteolytic enzymes and proteins involved in remodelling of extra cellular matrix and inflammation. Furthermore, MHC class I molecules, CD3δ, NK transcript 4, and ICAM-1 were overexpressed indicating an enhanced immunological reaction. Immunohistochemistry showed that the frequency of cells expressing CD68, defining the macrophage population, was higher in the cured cohort and that expression of lysozyme, cathepsin D, UPAR, and ICAM-1, was mainly seen within the reactive cells. Tumor infiltrating CD8+ T-cells were more frequent within the cured cohort, corresponding to the increased MHC class I expression seen within this group.Conclusions: In DLBCL, genes coding for antigens present in the tumor microenvironment are differentially expressed in patients with cured vs. chemotherapy-refractory disease. Our findings suggest that a reactive microenvironment, including tumor infiltrating T-cells and macrophages, may have impact on outcome of chemotherapy in DLBCL.

Chemotherapy-Refractory Diffuse Large B-Cell Lymphomas (DLBCL) Are Effectively Killed by Ofatumumab-Induced Complement-Mediated Cytoxicity.

The combination of chimeric CD20 monoclonal antibody (mAb) rituximab (R) with chemotherapy (CHOP) has significantly improved clinical outcome in patients with diffuse large B-cell lymphoma (DLBCL). However, the efficacy of R-CHOP is variable and DLBCL remains fatal in 30–40% of the patients. We recently developed the novel fully human CD20 mAb, ofatumumab (2F2) that is currently in clinical development for CLL and follicular lymphoma (phase III) and rheumatoid arthritis (phase II). In this study, we compared ofatumumab with rituximab for their ability to induce complement dependent cytotoxicity (CDC) of isolated lymphoma cells of chemotherapy-refractory DLBCL patients. Human type II CD20 mAb 11B8 does not induce CDC and was used as a control. Ofatumumab and rituximab induced CDC of all DLBCL samples tested, including the DLBCL cell lines SUDHL4, SUDHL5 and HT and lymphoma cells derived from ten chemotherapy-refractory DLBCL patients. Ofatumumab was significantly more effective in inducing CDC in nine out of ten DLBCL tumor samples when compared to rituximab (p=0.001, Two-way-Anova). The lethal doses (LD50) for ofatumumab (0.1 ± 2.8 μg/ml) was significantly lower when compared to the LD50 for rituximab (6.4 ± 4.9 μg/ml, p=0.04, Mann-Whitney test). The control CD20 mAb 11B8 showed no CDC induction of DLBCL cells. Sensitivity of DLBCL patient cells to ofatumumab- and rituximab-induced CDC negatively correlated with expression of complement defense molecule CD59, but not with expression of CD46, CD55, as determined by immunohistochemistry or apoptosis inhibitors Bcl-2 and XIAP. Functional inhibition of CD55 and CD59 using blocking mAb demonstrated that ofatumumab-induced CDC of DLBCL tumor cells was less sensitive to expression of these complement defense molecules than rituximab-induced CDC. We conclude that chemotherapy-refractory DLBCL cases are sensitive to CD20 mAb induced CDC with ofatumumab being the most effective mAb, especially in patients expressing high levels of CD59. Based on our results ofatumumab should be considered as a valuable therapy option for chemotherapy-refractory DLBCL patients.

Human soluble TRAIL/Apo2L induces apoptosis in a subpopulation of chemotherapy refractory nodal diffuse large B‐cell lymphomas, determined by a highly sensitive in vitro apoptosis assay

Resistance to chemotherapy in therapy-refractory diffuse large B-cell lymphomas (DLBCL) is related to inhibition of the intrinsic apoptosis pathway. Human soluble tumour necrosis factor (TNF)-related apoptosis-inducing ligand (hsTRAIL/Apo2L) induces apoptosis via the alternative, death-receptor mediated apoptosis pathway and might be an effective alternative form of therapy for these lymphomas. This study investigated whether hsTRAIL/Apo2L could actually induce apoptosis in isolated lymphoma cells of DLBCL biopsies of patients with chemotherapy-refractory DLBCL. Twelve out of a total of 22 DLBCL samples were sensitive to hsTRAIL/Apo2L. These sensitive lymphomas included seven clinically chemotherapy-refractory lymphomas. Furthermore, hsTRAIL/Apo2L induced apoptosis in DLBCL cells and in B-cell lines that showed high expression levels of inhibitors of the intrinsic apoptosis pathway: Bcl-2 and/or X-linked inhibitor of apoptosis (XIAP). hsTRAIL/Apo2L-sensitive lymphoma cells showed expression of the TRAIL receptors R1 and/or R2 and absence of R3 and R4. We conclude that hsTRAIL/Apo2L induced apoptosis in a subpopulation of chemotherapy-refractory nodal DLBCL and that disruption of the intrinsic apoptosis-mediated pathway and expression of Bcl-2 and XIAP did not confer resistance to hsTRAIL/Apo2L-induced apoptosis in DLBCL. Thus, based on our results, further exploration of hsTRAIL/Apo2L as an alternative treatment for patients with chemotherapy-refractory DLBCL should be considered.