A phase I prospective, non-randomized trial of autologous dendritic cell-based cryoimmunotherapy in patients with metastatic castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 108) combined with checkpoint inhibitors. Further studies focusing on the biologic indications of antitumor activity and immune system activation could be considered through a phase II trial focusing on treatment responses and immunologic biomarkers.

Impact of previous disease-modifying treatment on safety and efficacy in patients with MS treated with AHSCT.

BACKGROUND: Autologous haematopoietic stem cell transplantation (AHSCT) is a highly effective treatment for multiple sclerosis (MS). The impact of previous long-lasting disease-modifying treatments (DMT) for safety and efficacy of AHSCT is unknown. OBJECTIVE: To explore whether previous DMTs with long-lasting effects on the immune system (anti-CD20 therapy, alemtuzumab and cladribine) affect treatment-related complications, long-term outcome and risk of new MS disease activity in patients treated with AHSCT. METHODS: Retrospective observational study of 104 relapsing remitting patients with MS treated by AHSCT in Sweden and Norway from 2011 to 2021, grouped according to the last DMT used ≤6 months prior to AHSCT. The primary outcomes were early AHSCT-related complications (mortality, neutropenic fever and hospitalisation length), long-term complications (secondary autoimmunity) and proportion of patients with No Evidence of Disease Activity (NEDA-3 status): no new relapses, no MRI activity and no disease progression during the follow-up. RESULTS: The mean follow-up time was 39.5 months (range 1-95). Neutropenic fever was a common AHSCT-related complication affecting 69 (66%) patients. There was no treatment-related mortality. During the follow-up period, 20 patients (19%) were diagnosed with autoimmunity. Occurrence of neutropenic fever, hospitalisation length or secondary autoimmunity did not vary dependent on the last DMT used prior to AHSCT. A total of 84 patients (81%) achieved NEDA-3 status, including all patients (100%) using rituximab, alemtuzumab or cladribine before AHSCT. CONCLUSION: This study provides level 4 evidence that AHSCT in patients previously treated with alemtuzumab, cladribine or rituximab is safe and efficacious.

Peripheral Blood Stem Cell Mobilization in Healthy Donors by Granulocyte Colony-Stimulating Factor Causes Preferential Mobilization of Lymphocyte Subsets.

Background: Allogeneic hematopoietic stem cell transplantation is associated with a high risk of immune-mediated post-transplant complications. Graft depletion of immunocompetent cell subsets is regarded as a possible strategy to reduce this risk without reducing antileukemic immune reactivity. Study design and methods: We investigated the effect of hematopoietic stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) on peripheral blood and stem cell graft levels of various T, B, and NK cell subsets in healthy donors. The results from flow cytometric cell quantification were examined by bioinformatics analyses. Results: The G-CSF-induced mobilization of lymphocytes was a non-random process with preferential mobilization of naïve CD4+ and CD8+ T cells together with T cell receptor αß+ T cells, naïve T regulatory cells, type 1 T regulatory cells, mature and memory B cells, and cytokine-producing NK cells. Analysis of circulating lymphoid cell capacity to release various cytokines (IFNγ, IL10, TGFß, IL4, IL9, IL17, and IL22) showed preferential mobilization of IL10 releasing CD4+ T cells and CD3-19- cells. During G-CSF treatment, the healthy donors formed two subsets with generally strong and weaker mobilization of immunocompetent cells, respectively; hence the donors differed in their G-CSF responsiveness with regard to mobilization of immunocompetent cells. The different responsiveness was not reflected in the graft levels of various immunocompetent cell subsets. Furthermore, differences in donor G-CSF responsiveness were associated with time until platelet engraftment. Finally, strong G-CSF-induced mobilization of various T cell subsets seemed to increase the risk of recipient acute graft versus host disease, and this was independent of the graft T cell levels. Conclusion: Healthy donors differ in their G-CSF responsiveness and preferential mobilization of immunocompetent cells. This difference seems to influence post-transplant recipient outcomes.

The healthy donor profile of immunoregulatory soluble mediators is altered by stem cell mobilization and apheresis.

BACKGROUND: Peripheral blood stem cells from healthy donors mobilized by granulocyte colony-stimulating factor (G-CSF) and thereafter harvested by leukapheresis are commonly used for allogeneic stem cell transplantation. METHODS: Plasma levels of 38 soluble mediators (cytokines, soluble adhesion molecules, proteases, protease inhibitors) were analyzed in samples derived from healthy stem cell donors before G-CSF treatment and after 4 days, both immediately before and after leukapheresis. RESULTS: Donors could be classified into two main subsets based on their plasma mediator profile before G-CSF treatment. Seventeen of 36 detectable mediators were significantly altered by G-CSF; generally an increase in mediator levels was seen, including pro-inflammatory cytokines, soluble adhesion molecules and proteases. Several leukocyte- and platelet-released mediators were increased during apheresis. Both plasma and graft mediator profiles were thus altered and showed correlations to graft concentrations of leukocytes and platelets; these concentrations were influenced by the apheresis device used. Finally, the mediator profile of the allotransplant recipients was altered by graft infusion, and based on their day +1 post-transplantation plasma profile our recipients could be divided into two major subsets that differed in overall survival. DISCUSSION: G-CSF alters the short-term plasma mediator profile of healthy stem cell donors. These effects together with the leukocyte and platelet levels in the graft determine the mediator profile of the stem cell grafts. Graft infusion also alters the systemic mediator profile of the recipients, but further studies are required to clarify whether such graft-induced alterations have a prognostic impact.

Granulocyte colony-stimulating factor alters the systemic metabolomic profile in healthy donors.

INTRODUCTION: Peripheral blood stem cells mobilized by granulocyte colony-stimulating factor (G-CSF) from healthy donors are commonly used for allogeneic stem cell transplantation. The effect of G-CSF administration on global serum metabolite profiles has not been investigated before. OBJECTIVES: This study aims to examine the systemic metabolomic profiles prior to and following administration of G-CSF in healthy adults. METHODS: Blood samples were collected from 15 healthy stem cell donors prior to and after administration of G-CSF 10 µg/kg/day for 4 days. Using a non-targeted metabolomics approach, metabolite levels in serum were determined using ultrahigh performance liquid chromatography-tandem mass spectrometry and gas chromatography/mass spectrometry. RESULTS: Comparison of the metabolite profiles of donors before and after G-CSF treatment revealed 239 metabolites that were significantly altered. The major changes of the metabolite profiles following G-CSF administration included alteration of several fatty acids, including increased levels of several medium and long-chain fatty acids, as well as polyunsaturated fatty acids; while there were lower levels of other lipid metabolites such as phospholipids, lysolipids, sphingolipids. Furthermore, there were significantly lower levels of several amino acids and/or their metabolites, including several amino acids with known immunoregulatory functions (methionine, tryptophan, valine). Lastly, the levels of several nucleotides and nucleotide metabolites (guanosine, adenosine, inosine) were also decreased after G-CSF administration, while methylated products were increased. Some of these altered products/metabolites may potentially have angioregulatory effects whereas others may suggest altered intracellular epigenetic regulation. CONCLUSION: Our results show that G-CSF treatment alters biochemical serum profiles, in particular amino acid, lipid and nucleotide metabolism. Additional studies are needed to further evaluate the relevance of these changes in healthy donors.

Immunomodulation Induced by Stem Cell Mobilization and Harvesting in Healthy Donors: Increased Systemic Osteopontin Levels after Treatment with Granulocyte Colony-Stimulating Factor.

Peripheral blood stem cells from healthy donors mobilized by granulocyte colony-stimulating factor (G-CSF) and harvested by leukapheresis are commonly used for allogeneic stem cell transplantation. The frequency of severe graft versus host disease is similar for patients receiving peripheral blood and bone marrow allografts, even though the blood grafts contain more T cells, indicating mobilization-related immunoregulatory effects. The regulatory phosphoprotein osteopontin was quantified in plasma samples from healthy donors before G-CSF treatment, after four days of treatment immediately before and after leukapheresis, and 18-24 h after apheresis. Myeloma patients received chemotherapy, combined with G-CSF, for stem cell mobilization and plasma samples were prepared immediately before, immediately after, and 18-24 h after leukapheresis. G-CSF treatment of healthy stem cell donors increased plasma osteopontin levels, and a further increase was seen immediately after leukapheresis. The pre-apheresis levels were also increased in myeloma patients compared to healthy individuals. Finally, in vivo G-CSF exposure did not alter T cell expression of osteopontin ligand CD44, and in vitro osteopontin exposure induced only small increases in anti-CD3- and anti-CD28-stimulated T cell proliferation. G-CSF treatment, followed by leukapheresis, can increase systemic osteopontin levels, and this effect may contribute to the immunomodulatory effects of G-CSF treatment.

The pretransplantation serum cytokine profile in allogeneic stem cell recipients differs from healthy individuals, and various profiles are associated with different risks of posttransplantation complications.

Cytokines play a key role in regulation of normal and malignant hematopoiesis, angiogenesis, and inflammation. Serum levels of several cytokines are altered in patients with hematologic malignancies, and pretransplant cytokine levels seem to have a prognostic impact in patients treated with allogeneic stem cell transplantation. However, the cytokine system constitutes an interacting functional network, and it may therefore be more relevant to look at serum cytokine profiles rather than the serum levels of single cytokines in allotransplanted patients. We therefore investigated the pretransplantation serum levels of 35 cytokines in a group of 44 consecutive allogeneic stem cell transplantation patients, mainly with a primary diagnosis of acute leukemia. Serum samples were collected before the start of myeloablative conditioning therapy when all patients were in complete hematologic remission. Unsupervised hierarchical clustering analysis identified three major patient groups/subsets. These groups differed especially in the levels of hepatocyte growth factor and granulocyte-colony stimulating factor, and one of the groups was characterized by low early treatment-related morbidity and high levels of hepatocyte growth factor and granulocyte-colony stimulating factor. The degree of weight gain/fluid retention after conditioning therapy did not differ between the patient subsets, but fluid retention showed a significant correlation with pretransplantation serum levels of basic fibroblast growth factor. We conclude that the pretransplantation serum cytokine profile shows a considerable variation even between patients in complete hematologic remission and is associated with clinicopathologic features.

Future perspectives: should Th17 cells be considered as a possible therapeutic target in acute myeloid leukemia patients receiving allogeneic stem cell transplantation?

Th17 cells seem to promote proinflammatory effects, and their development seems to depend on intracellular signaling initiated by IL1ß, supported by IL6 and IL23 and mediated by STAT3 and RORC2. Even though primary human AML cells may affect Th17 development through their constitutive cytokine release, the levels of circulating Th17 cells in older patients with untreated AML do not differ from healthy controls and show only minor variations during and following conventional intensive chemotherapy. IL17-A is the signature cytokine of Th17 cells, but in vitro studies have failed to demonstrate a direct antileukemic effect of IL17 on primary human AML cells for most patient samples. However, several observations suggest that Th17 cells mediate antileukemic effects through other mechanisms and are important in allogeneic stem cell transplantation. Firstly, genetic variants in IL23/Th17 pathway have a prognostic impact with regard to both development of GVHD and posttransplant infections. Secondly, circulating IL17-secreting cells are detected during early posttransplant pancytopenia, and their ability to release IL17 is associated with later GVHD. Thirdly, a high number of Th17 cells in allogeneic stem cell grafts are associated with later acute GVHD, levels of circulating Th17 cells are increased at the onset of acute GVHD, and these levels normalize during treatment. In the present article, we review previous studies of Th17 cells in AML and in the development of GVHD, possible therapeutic strategies and available therapeutic tools for targeting of Th17 cells.

The chemokine system in allogeneic stem-cell transplantation: a possible therapeutic target?

Further improvements in allogeneic stem-cell transplantation will probably depend on a better balance between immunosuppression to control graft-versus-host disease and immunological reconstitution sufficient to ensure engraftment, reduction of infection-related mortality and maintenance of post-transplant antileukemic immune reactivity. The chemokine network is an important part of the immune system, and, in addition, CXCL12/CXCR4 seem to be essential for granulocyte colony-stimulating factor-induced stem-cell mobilization. Partial ex vivo graft T-cell depletion based on the expression of specific chemokine receptors involved in T-cell recruitment to graft-versus-host disease target organs may also become a future therapeutic strategy; an alternative approach could be pharmacological inhibition (single-receptor inhibitors or dual-receptor inhibitors) in vivo of specific chemokine receptors involved in this T-cell recruitment. Future clinical studies should therefore be based on a better characterization of various immunocompetent cells, including their chemokine receptor profile, both in the allografts and during post-transplant reconstitution.

Is there a scientific basis for a recommended standardization of collection and cryopreservation of peripheral blood stem cell grafts?

High-dose chemotherapy followed by autologous stem cell transplantation has been used extensively during the last two decades in the treatment of hematologic malignancies. The vast majority of recent transplantations have been performed using mobilized peripheral blood stem cells, because they have become the preferred source of hematopoietic cells rather than bone marrow stem cells. The mobilization is achieved by growth factors, eventually combined with chemotherapy, and the cells are then harvested and cryopreserved until reinfusion. Despite extensive use for many years, few attempts have been made to standardize the various steps in mobilization, harvesting and cryopreservation. Furthermore, the autografts only represent relative stem cell enrichment and contain a wide range of more mature hematopoietic and immunocompetent cells; the potential clinical importance of these normal cells is largely unknown and represents an additional non-standardized factor in this treatment. We have reviewed the various methodologic approaches for stem cell mobilization, collection and cryopreservation of autografts with a special focus on the cryopreservation procedures, immunocompetent cells in the graft, and cytokine content of the graft supernatant. We conclude that the factors/aspects mentioned above should be standardized in future clinical studies of autotransplantation for human hematologic malignancies. Alternatively, detailed methodologic descriptions should be required when the results are published. Standardization of autograft preparation and cryopreservation will be achieved if/when transplantation units assess and adopt new standards based not only on the technology but, more importantly, on the quality of evidence and data related to that technology/methodology.

Self-treatment of an ABO-incompatible blood transfusion.

Unfortunately, transfusion errors continue to pose a major clinical problem. In practice there is a remarkable lack of understanding of the reasons behind transfusion medicine, a worrying disregard for protocol and an offhand attitude to bedside checking. Clearly transfusion medicine education, as often said, must be more extensively incorporated into the core curriculum of medical, nursing and laboratory staff. In this immunological case report, following accurate pre-transfusion procedures and routine laboratory testing, we highlight, once more, that it would be better for the patient to rely on proper bedside checking rather than on their own immune system in order to avoid serious side effects related to transfusions. Albeight this case we had "a happy ending", we must always bear in mind that one cannot compromise on safety and the quality care of patients.

Evaluation of the persistence and characteristics of indeterminate reactivity against hepatitis C virus in blood donors.

BACKGROUND: A substantial number of individuals are excluded from blood donation due to indeterminate results in screening tests for hepatitis C virus antibody (anti-HCV). Disclosure of the characteristics of the indeterminate serologic pattern could optimize the testing and the management and care of blood donors. STUDY DESIGN AND METHODS: Ninety-two former blood donors deferred from blood donation due to consistent reactivity in anti-HCV enzyme immunoassay and indeterminate HCV recombinant immunoblot assay (RIBA) results were retested for anti-HCV to examine the extent of disappearance of reactivity. In addition, they were screened for selected viral, immunologic, and inflammatory variables to detect possible causes of the test reactivity pattern. RESULTS: After a median observation time of 75 months, 66 of 92 individuals had persistent indeterminate HCV RIBA results. Reactivity against the nonstructural NS5 antigen was the most frequent finding. A significant association was detected both between indeterminate reactivity in HCV RIBA and against the NS5 antigen independently and detectable antibody against adenovirus. Novel indeterminate reactivity showed increased prevalence during autumn and winter months. CONCLUSION: Indeterminate HCV RIBA reactivity in blood donors persists over years. Increased prevalence during autumn and winter and association to detection of adenovirus antibody indicate that viral infection and cross-reactivity are etiologic factors in indeterminate HCV RIBA reactivity. Further prospective studies are needed to confirm the results.

[Blood transfusion and pretransfusion testing in patients with warm autoimmune haemolytic anaemia]. / Blodtransfusjon og pretransfusjonsutgreiing ved autoimmun hemolytisk anemi av varmetype.

BACKGROUND: In warm autoimmune haemolytic anaemia, patients have red cell autoantibodies that complicate the serological compatibility testing before transfusion. Different autoadsorption techniques are utilised for more extensive pretransfusion testing. We reviewed the literature on blood transfusion in these patients. We also present our experience with autoadsorption by papain and polyethylene glycol methods. MATERIAL AND METHODS: Blood samples from 13 patients with warm autoantibodies were analysed in parallel. After autoadsorption, antibody screening and identification tests were performed to detect possible red cell alloantibodies. If the serological testing is inadequate, clinically important alloantibodies can be ignored before transfusion, which can lead to haemolytic transfusion reactions. RESULTS: Autoantibodies were completely adsorbed in six of the 13 patients. In five, alloantibodies were detected. In three patients, different results were obtained with the two different techniques. INTERPRETATION: Restrictive transfusion practice in warm autoimmune haemolytic anaemia is recommended. Adequate pretransfusion testing in a specialised laboratory is required before transfusion. Autoadsorption with polyethylene glycol and papain can supplement each other in pretransfusion testing.