The tumour microenvironment influences outcome in patients with follicular lymphoma (FL), but its impact on transformation is less studied. We investigated the prognostic significance of the tumour microenvironment on transformation and survival in FL patients treated in the rituximab era. We examined diagnostic and transformed biopsies from 52 FL patients using antibodies against CD3, CD4, CD8, CD21 (CR2), CD57 (B3GAT1), CD68, FOXP3, TIA1, PD-1 (PDCD1), PD-L1 (CD274) and PAX5. Results were compared with a second cohort of 40 FL patients without signs of transformation during a minimum of five years observation time. Cell numbers and localization were semi-quantitatively assessed. Better developed CD21+ follicular dendritic cell (FDC) meshworks at diagnosis was a negative prognostic factor for overall survival (OS), progression-free survival (PFS) and time to transformation (TTT) in patients with subsequently transformed FL. Remnants of FDC meshworks at transformation were associated with shorter OS and PFS from transformation. High degrees of intrafollicular CD68+ and PD-L1+ macrophage infiltration, high total area scores and an extrafollicular/diffuse pattern of FOXP3+ T cells and high intrafollicular scores of CD4+ T cells at diagnosis were associated with shorter TTT. Scores of several T-cell subset markers from the combined patient cohorts were predictive for transformation, especially CD4 and CD57.
BackgroundAutologous stem-cell transplantation (ASCT) is a common treatment for lymphoma but it has some mortality.MethodsAll 433 lymphoma patients who underwent ASCT for lymphoma at Karolinska Huddinge 1994–2016 were investigated, including CD34+ cell amounts, medications, infectious and other complications, intensive care, longitudinal laboratory values, and secondary myeloid neoplasia.ResultsThe 100-day non-relapse and overall mortalities were 5.6% and 7.2%. Stem-cell harvests < 5 million CD34+ cells/kg correlated with inferior 100-day and long-term survival. Prior to conditioning (93% BEAM), elevated (both 3–9 and ≥ 10 mg/L) C-reactive protein (CRP) and creatinine, and low albumin (but not higher age) predicted inferior higher 100-day survival. Intravenous antibiotics were given to 97% (22% positive blood cultures) and parenteral nutrition to 89%. After 1 year, 86% had normalized hemoglobin. The 5-year risk for secondary myeloid neoplasia was 4.1%, associated with smaller harvests.ConclusionsBefore starting conditioning, patients should have preferably harvested ≥ 5 million CD34+ cells/kg and normal CRP, albumin, and creatinine. It appears safe to transplant patients ≥ 66 years.Electronic supplementary materialThe online version of this article (10.1186/s40164-019-0131-3) contains supplementary material, which is available to authorized users.
Summary Young patients with diffuse large B‐cell lymphoma (DLBCL) are variably treated with rituximab combined with cyclophosphamide‐doxorubicin‐vincristine‐prednisone (R‐CHOP), CHOP‐etoposide (R‐CHOEP), and anthracycline‐based regimens with the addition of high‐dose cytarabine/methotrexate (R‐HDA/M). Using the nationwide, population‐based Swedish Lymphoma Registry, we evaluated outcome, by treatment and Healthcare Region, in all 751 DLBCL patients aged ≤60 years without central nervous involvement, diagnosed in Sweden between 2007 and 2012. Overall survival was estimated using multivariate Cox analysis. In patients with age‐adjusted international prognostic index (aaIPI) ≥ 2, the 5‐year overall survival (OS) was 70%, 76% and 85% after R‐CHOP, R‐CHOEP and R‐HDA/M, respectively (P = 0·002); the corresponding estimates were 40%, 55%, and 92% in aaIPI = 3 (P = 0·014). There were large therapeutic differences between Sweden's six Healthcare Regions for aaIPI ≥ 2: three were “Moderate” (more R‐CHOP) and three “Intensive” (more R‐CHOEP and R‐HDA/M). Patients with aaIPI ≥ 2 who were treated in the Intensive Regions, showed better OS (P < 0·00005), particularly those with aaIPI = 3 (5‐year OS, 62% vs. 30%; P < 0·00005). There were no regional differences in therapy or survival in patients with aaIPI < 2. We conclude that in younger high‐risk patients, survival appears superior after more intensive therapy than R‐CHOP.
Monotherapy with the anti-CD20 monoclonal antibody rituximab can induce complete responses (CR) in patients with follicular lymphoma (FL). Resting FcRγIII+ (CD16+) natural killer (NK) cells respond strongly to rituximab-coated target cells in vitro. Yet, the contribution of NK cells in the therapeutic effect in vivo remains unknown. Here, we followed the NK cell repertoire dynamics in the lymph node and systemically during rituximab monotherapy in patients with FL. At baseline, NK cells in the tumor lymph node had a naïve phenotype albeit they were more differentiated than NK cells derived from control tonsils as determined by the frequency of CD56dim NK cells and the expression of killer cell immunoglobulin-like receptors (KIR), CD57 and CD16. Rituximab therapy induced a rapid drop in NK cell numbers coinciding with a relative increase in the frequency of Ki67+ NK cells both in the lymph node and peripheral blood. The Ki67+ NK cells had slightly increased expression of CD16, CD57 and higher levels of granzyme A and perforin. The in vivo activation of NK cells was paralleled by a temporary loss of in vitro functionality, primarily manifested as decreased IFNγ production in response to rituximab-coated targets. However, patients with pre-existing NKG2C+ adaptive NK cell subsets showed less Ki67 upregulation and were refractory to the loss of functionality. These data reveal variable imprints of rituximab monotherapy on the NK cell repertoire, which may depend on pre-existing repertoire diversity.
Diffuse large B-cell lymphoma (DLBCL) incidence rises with increasing age. Rituximab-anthracycline-based regimens offer a potential cure but also risks of adverse events, especially in the elderly. Using Swedish registers, we conducted a nationwide, population-based study of DLBCL in the very elderly. We obtained information on clinical characteristics, residence, comorbidity, therapy and survival for the 1194 patients aged ≥80 years diagnosed in Sweden 2007-2014. To address selection bias, we also investigated treatment differences between Sweden's Healthcare Regions and whether there were survival differences between the Regions. The 2-year overall and relative survivals were better in patients aged ≥80 years given treatment with curative intent (54%; 64%) than low-intensity (26%; 33%), or palliative treatment (6%; 7%). The fraction of patients treated with curative intent varied between the Healthcare Regions (45-76%). Survival was significantly inferior in Regions with few patients treated with curative intent (multivariable hazard ratio 1.3, 95% confidence interval 1.1-1.6). When treatment intensity and Regions competed, Regions were no longer independent, suggesting that Regional survival differences are due to therapeutic differences. Furthermore, we found that the age-adjusted International Prognostic Index was independently associated with survival. We conclude that patients aged ≥80 years with DLBCL appear to benefit from rituximab-anthracycline-based treatment given with curative intent.
Introduction Cannabinoid receptors type 1 (CB1) and type 2 (CB2) are tentative treatment-targets in cancer. They are activated by endogenous cannabinoids and by plant cannabinoids such as tetrahydrocannabinol (THC). CB2 is expressed in normal and malignant lymphocytes while CB1 expression is low in normal lymphocytes but high in mantle cell lymphomas and half of cases of chronic lymphocytic leukemia (CLL). Agonists to CB1 and CB2 induce cell death of CB1 or CB2 expressing lymphoid cell lines (Gustafsson, K. et al. Int J Cancer 2008). CB1 and CB2 regulate tissue localization and homing of leukocytes (Muppidi JR, et al. J Exp Med. 2011; Wasik et al., 2014, Oncoscience). We here report the effects of Sativex, which contains a whole-plant mixture of Cannabis sativa with exact proportions of THC and the partial CB1-antagonist cannabidiol (CBD), on patients with indolent B-cell lymphoma. Methods Patients, 18-80 years, with a leukemic indolent B-cell lymphoma without treatment indication, were given a single administration of Sativex, as an oral mucosal spray. The cannabis compound was given at 9 AM and patients were sampled at 0, 2, 4, 6, 24 and 168 hours. They were also sampled at a prior non-treatment day at the same hours of the day to compensate for any circadian rhythms of blood leukocytes. Blood samples were analyzed using blood chemistry and flow cytometry to quantify lymphoma and non-malignant cells. Apoptosis was measured by caspase-3 activation. CB1 and CB2 mRNA levels were quantified using qPCR in enriched lymphoma cells. Results 23 patients were included (Table 1). Maximum tolerated dose was determined to be 7 actuations, containing 18.9mg THC and 17.5mg CBD. This dose was given to 15 patients. Side effects were mostly grade 1 and manageable (Table 2) and all patients could return home at 3 PM. At every time point on the treatment day, there was a significant decrease in lymphocyte counts compared with 0 hours (2 hours, P = 0.004; 4 hours, P < 0.001; 6 hours, P = 0.007), with nadir usually at 4 hours after drug administration (median nadir 0.85 relative to baseline). On the control day, lymphocytes decreased significantly at 4 hours (P = 0.031) and 6 hours (P = 0.026) (median nadir of 0.93 compared to baseline). Changes in clonal B cells were the same as in lymphocytes. The larger median nadir on treatment day was not due to increased cell-death as measured by activated caspase 3. In the non-clonal B-cells, there was no circadian variation during the control day, but a decrease after treatment was detectable (2 hours: P = 0.01; 4 hours: P = 0.034; 6 hours: P = 0.031). T-cells showed no circadian changes and decreased after treatment (4 hours, P = 0.06; 6 hours, P = 0.009). For NK cells, the pattern, regardless of administration of cannabinoids, was a decrease at 6 hours (6 hours no drug, P = 0.051; 6 hours with drug P = 0.013). A week after administration of the cannabis compound, all non-malignant lymphocytes had returned to baseline levels, but the clonal B cells had significantly increased (P = 0.011). Neutrophils increased significantly after treatment (4 hours, P = 0.007; 6 hours, P = 0.005) whereas platelets decreased at 2 hours (P = 0.003). CB2 mRNA was expressed in all lymphomas and 17/23 lymphomas expressed CB1 mRNA. There was no correlation between baseline levels of CB2, CB1 or plasma concentrations of THC and CBD to nadir of lymphocytes (all P > 0.4). The cannabis compound reduced lymphocyte levels both in CB1-positive and CB1-negative lymphoma (CB1+, P = 0.028; CB1-, P = 0.013). Conclusion This study demonstrates that it is safe to administrate a single dose of Sativex to elderly patients with indolent B-cell lymphoma with regards to adverse events. We show that the cannabis compound quickly reduces lymphoma cell numbers in peripheral blood. There was no evidence of activation of caspase 3; this suggests that the reduction of lymphoma cells in blood might be due to redistribution from blood rather than apoptosis. We have also detected an apparent circadian rhythm of the peripheral numbers of malignant lymphocytes. Our findings suggest that the drug might promote homing of lymphoma cells from blood into secondary lymphoid organs where they receive pro-survival signals. Therefore, this cannabinoid compound should be used with caution in patients with indolent leukemic lymphomas. Further studies are needed to dissect the signaling pathways affected by cannabinoids in B-cell lymphoma. Disclosures Wahlin: Roche and Gilead: Consultancy. OffLabel Disclosure: Sativex is an oromucosal spray containing whole plant Cannabis sativa. In Europe is is registred for use against spasticity caused by multiple sclerosis.
Background: Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with a high rate of relapses after therapy. Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with varied outcome. For both diseases there is a need for new therapies. Cannabinoid receptors (CBs), which are overexpressed in most cases of MCL and CLL compared to normal B cells (Islam et al., 2003; Gustafsson et al., 2008; Freund et al., 2016) are promising novel therapeutic targets. CBs are membrane-bound receptors that convey signals from the microenvironment to the cells. There are two types of CBs: CB1 and CB2. CB1 is suggested to be involved in retention and/or egress of MCL cells from the tissue to the blood circulation (Wasik et al., 2014). CB2 is expressed by normal B-cells where it regulates positioning and retention of cells in tissue (Pereira et al., 2009; Basu et al., 2011; Muppidi et al., 2011) and in pre-B-cell acute lymphoblastic leukemia, involved in the energy metabolism (Chan et al., 2017). The retention/egress of the B-cell lymphoma cells is mainly regulated by chemokine receptors and adhesion molecules. The chemokine receptor CXCR4 is one of the most highly expressed chemokine receptors in MCL and CLL. 2-arachidonoylglycerol (2-AG, CB1/CB2 endogenous ligand) and CXCL12 (CXCR4 ligand) are synthetized and secreted by stromal cells in the bone marrow (Kose et al., 2018; Burger and Gribben, 2014). The endocannabinoids levels in cancer are suggested to have a role in cancer progression (Sailler et al., 2014) while CXCL12 is already a candidate target for therapy using a CXCR4 inhibitor AMD3100. Aim: To investigate a possible crosstalk between CBs and CXCR4 in MCL and CLL cells. Methods: Patients with newly diagnosed MCL (n=8) or CLL (n=25) gave informed consent to participate in the study. Lymphoma cells were enriched by negative selection. Fifteen primary lymphoma samples and the JeKo MCL cell line were subjected to chemotaxis towards CXCL12 and/or 2-AG. CXCR4 membrane expression was assessed by flow cytometry. Selective CB1 and CB2 antagonists were used to investigate the underlying mechanisms. CB1, CB2 and CXCR4 encoding genes levels were measured by qPCR and normalized to B cells from tonsil. Results and Conclusion: 2-AG induced chemotaxis in 11/15 MCL and CLL samples. In JeKo, 2-AG-induced migration was blocked by a CB2 antagonist, suggesting that signaling via CB2 is involved. When the primary cells were subjected to migration towards CXCL12, two patterns of chemotaxis were observed. The first pattern was seen in 7/15 samples that migrated towards CXCL12. In these samples, the migration was inhibited when 2-AG was combined with CXCL12. The second type of response was observed in 8/15 samples, those samples did not migrate towards CXCL12 but chemotaxis was enhanced by combining 2-AG and CXCL12. MCL and CLL samples expressed variable mRNA levels of CB1 (RFI range: 0.0-204) and CB2 (RFI range: 0.8-14.3) and all expressed CXCR4 at mRNA (RFI range: 0.1-215.8) and protein (MFI range: 1278-19301) levels that did not differ neither between the two diseases nor between the two migratory groups. When all 15 samples were combined, CB1 mRNA levels, but not CB2 mRNA, correlated to the chemotaxis towards CXCL12 (Spearman correlation coefficient = 0.626; p=0.01). In contrast, CB2 mRNA levels, but not CB1, correlated to chemotaxis towards 2-AG (Spearman correlation coefficient = 0.532; p=0.04), which is in agreement with the effects observed in JeKo. Furthermore, CB1 and CB2 mRNA levels correlated to chemotaxis towards the combination of CXCL12 and 2-AG both (for CB1 mRNA: Spearman correlation coefficient= 0.588; p=0.02 and for CB2 mRNA: 0.589; p=0.02). Neither CXCL12-induced CXCR4 receptor internalization, nor recycling was influenced by 2-AG incubation. Our findings indicate a novel pathway regulating chemotaxis of MCL and CLL implicating a cross-talk between CBs and CXCR4. The fact that the capacity to internalize CXCR4 remained intact after incubation with 2-AG suggests that the reduced CXCL12-mediated migration when 2-AG was combined could be due to an impaired downstream signaling in lymphoma cells. Importance: Lymphoma cells residing in the tissue receive pro-survival stimuli and are protected from chemotherapy by signals from the microenvironment. A better understanding of how lymphoma cell migration and tissue retention are regulated can be a step towards more efficient therapies. Disclosures Wahlin: Gilead: Consultancy, Honoraria, Research Funding; Roche: Research Funding.
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