We performed a prospective phase II study to evaluate clinical safety and outcome in 48 patients with steroid-refractory grade II-IV acute graft-versus-host disease (aGVHD) treated with mesenchymal stromal cells (MSCs). Clinical outcomes were correlated to comprehensive analyses of soluble and cellular biomarkers. Complete resolution (CR) of aGVHD at day 28 (CR-28) occurred in 12 (25%) patients, CR lasting >1 month (CR-B) occurred in 24 (50%) patients. One-year overall survival was significantly improved in CR-28 (75 versus 33%, P=0.020) and CR-B (79 versus 8%, P<0.001) versus non-CR patients. A six soluble biomarker-panel was predictive for mortality (HR 2.924; CI 1.485-5.758) when measured before MSC-administration. Suppression of tumorigenicity 2 (ST2) was only predictive for mortality 2 weeks after but not before MSC-administration (HR 2.389; CI 1.144-4.989). In addition, an increase in immature myeloid dendritic cells associated with decreased mortality (HR 0.554, CI 0.389-0.790). Patients had persisting T-cell responses against defined virus- and leukemia-associated antigens. In conclusion, our data emphasize the need to carefully assess biomarkers in cohorts with homogeneous GVHD treatments. Biomarkers might become an additional valuable component of composite end points for the rapid and efficient testing of novel compounds to decrease lifecycle of clinical testing and improve the success rate of phase II/III trials.
Background:Acute myeloid leukemia (AML) is a hematologic neoplasm characterized by the proliferation of bone marrow myeloblasts. Most of patients achieve remission after intensive chemotherapy, although they still relapse. The immune system play an important role in the pathogenesis of AML, where myeloblasts are be able to switch the immunological synapsis disabling the generation of an anti‐tumor response. In this sense, the presence of a greater number of Th17 lymphocytes has been described in AML.Aims:To elucidate whether the myeloblats modulate the Th17 population and more specifically the two Th17 subpopulations: CCR4+ and CCR4‐.Methods:From February 2015 to March 2018, 20 AML diagnosed patients at the CHN and 20 controls were included. Blood samples were collected at diagnosis from AML patients and in controls. We identified by flow cytometry the populations of T helper cells (Th1, Th2, Th17 CCR4‐CCR6+, Th17 CCR4+CCR6+) with the following monoclonal antibodies: CD3 Horizon‐V450, CCR3 FITC, IFNγRβ PE, CCR4 PE‐Cy7, CD4 PerCP, CCR6 APC, CD45 APC‐H7, CCR5 FITC, CXCR3 APC in a FACSCanto II. Serum level of cytokines were also analysed by CBA Human Th1/Th2/Th17 Cytokine Kit (BD) for IL‐2, IL‐4, IL‐6, IL‐10, TNFα, IFNγ, and IL‐17A. We also study the polimorfism of IL‐17A (rs2275913) and IL‐17F (rs763780) by allele discrimination by real‐time PCR. The SPSS software was used to perform Student́s test or Mann‐Whitney test for unpaired samples between patients and controls, and simple linear regression was used to study the association between levels of cytokines and Th subpopulations in AML patients. P < 0,05 was considered to be statistically significant.Results:Patients and controls characteristics were showed in table 1. Th1 and Th2 cells levels did not differ between AML patients and controls. The mean of Th17 CCR4‐CCR6+ cells was significantly higher in AML patients (192 ± 178/μl) than those in controls (67 ± 38/μl), and the difference was statistically significant (p = 0,027). However, Th17 CCR4+CCR6+ cells levels did not differ between groups. We did not find significant differences in the levels of IL‐2, IL‐4, TNFα and IFNγ citokines. Th17 related cytokines were significantly higher in AML patients than in controls and statistically significant. The mean of cytokines (pg/ml) in AML and in controls was 32,79 ± 86,64 vs 0 (p < 0,01), 3,03 ± 2,65 vs 0,24 ± 0,52 (p < 0,001), 14,37 ± 14,12 vs 2,38 ± 4,17 (p = 0,012), for IL‐6, IL‐10 and IL‐17A respectively. No association was find by simple linear regression between the levels of IL‐6, IL‐10 and IL‐17A, and the Th17 subpopulations. There are also no significant differences between the polymorphisms studied of IL‐17 in patients and controls.imageSummary/Conclusion:Th17 subpopulations could play a role in the pathophysiology of AML. The most relevant subpopulation described is the Th17 CCR4+CCR6+. Protumoral and antitumor properties have been described for this subpopulation and associated cytokines (IL‐6, IL‐10 and IL‐17A). However, our results show an increase in the levels of the Th17 CCR4‐CCR6+ subpopulation and in IL‐6, IL‐10 and IL‐17A in untreated AML patients. The lack of association between Th17 subpopulations and cytokines implies that AML generates a protumoral microenvironment, where Th17 subpopulation and associated cytokines are regardless regulated by the tumor. Specifically, the downregulation of CCR4 antigen expression prevents the cells from migrating and having an antitumor effect and the upregulation of IL‐6, IL‐10 and IL‐17A secretion very likely from Th17 CCR4+CCR6+ could contribute to the progression of the disease.
3046 Introduction For numerous malignant and non-malignant hematological diseases allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy. However, a major complication is the acute graft versus host disease (aGVHD), which is life-threatening and substantially reduces efficacy of HSCT. In particular, the outcome for patients with severe steroid-resistant aGVHD is very poor. Therefore, it remains important to search for new therapeutic strategies for the treatment of aGVHD. Objective Feasibility of the generation mesenchymal stroma cells (MSCs) expanded with human plasma and platelet lysate (hPPL) was tested as well as the feasibility and safety of the application of MSCs in patients with steroid-refractory extensive aGVHD. Method In an open-label, non-randomized prospective phase I/II study MSCs were extracted from the bone marrow of healthy volunteers, expanded with human plasma and platelet lysate (hPPL), and stored. Patients with steroid-refractory extensive aGVHD were treated with ∼2×106/kg MSC. Response rate, transplantation-related deaths, and other adverse events were assessed for up to 12 months after the last infusion of the cells. Results Between January 2009 and December 2010, 18 patients were treated, 5 children and 13 adults, median age was 32.5 years (range 1.3–65.9). Organ involvement of the aGVHD was 67% skin, 83% gastro-intestinal and 28% liver. Overall grade was II for 4 (22%), III for 13 (72%), and IV for 1(6%) patients. 1 patient received one infusion, all other patients received two or more infusions. No patient had side-effects during or immediately after infusions of the MSC. Median follow-up was 5.5 months (range 0.33–12). Complete overall response was observed in 11 (61%) patients after a median of 65 days (range 10–184 days). The overall survival was significant better (p <0.001) compared to non-responders. Of the 11 patients who reached a CR, 8 relapsed median 59 days (1–244) after reaching CR. Three children with subsequently limited cGVHD, allo immune lung and auto-immune cytopenia and 5 adults with all relapse GVHD of the gut, median 98 days (35–302 days). However, GVHD of the gut was then sensitive for the treatment with steroids. Overall, 7 patients died, 4 due to progression of aGVHD, 1 patient due to abdominal bleeding and 2 due to sepsis. Conclusion Generation and infusion of MSCs in steroid-resistant aGVHD grade II- IV is a feasible, safe and very effective. In addition, also patients who initially responded to MSCs but develop later a relapse of aGVHD during tapering or cessation of immunosuppressive drugs become again sensitive to the treatment with steroids. Disclosures: No relevant conflicts of interest to declare.
736 Introduction Despite multiple improvements in the last decade in the field of HSCT, acute graft versus host disease (aGVHD) remains a life-threatening complication and reduces substantially efficacy of HSCT. In particular, the outcome of patients with severe steroid-resistant aGVHD is very poor. Therefore, it remains important to search for new therapeutic strategies for the treatment of aGVHD. Objective Feasibility of the generation and efficacy of mesenchymal stroma cells (MSCs) generated with fetal calf serum (FCS) has been suggested recently. However, FCS is a putative source of prions and virus transmission. Therefore, the feasibility of the generation of MSCs expanded with human plasma and platelet lysate (hPPL) was tested as well as the feasibility and safety of the application of hPPL-MSCs in patients with steroid-refractory aGVHD. Furthermore multiple immunological changes after infusion of MSC were characterized, in vitro. However, truly active mechanisms in human are poorly understood as well as whether infusion of MSC selectively impairs GVHD-inducing immune cells or also anti-virus and anti-leukemia reactive T-cells. Therefore, phenotypical and functional changes in immunological cell types and cytokine levels were investigated. Method In an open-label, non-randomized prospective phase I/II study MSCs were extracted from the bone marrow of healthy volunteers, expanded with hPPL, and stored. Patients with steroid-refractory aGVHD grade II to IV were treated with hPPL-MSC. 50 patients were included and received up to four infusions. Response rate, transplantation-related deaths, and other adverse events were assessed for up to 12 months after the last infusion of the cells. In addition, a comprehensive phenotypical and functional analysis was performed with PBMCs and serum isolated from all patients before, during, and after infusion of MSC. Results Between January 2009 and July 2012, 50 patients were included, 2 patients dropped out, 5 patients are so far incompletely documented. Thus 43 patients were so far available for analysis, 6 children and 37 adults. Median age was 51,5 years (1.3–65.9). Organs involved in aGVHD were the skin (56%), the gastro-intestinal tract (86%) and the liver (33%). Overall grade was II for 11 (26%), III for 28 (65%), and IV for 3 (7%) patients. Mean number of infusion were 3 (1–4). No severe side effects were observed. Median follow-up was 4.06 months (range 0.43–12). Complete overall response was observed in 24 patients (56%) after a median of 53 days (range 3–116 days). The overall survival was significantly better in responders when compared to non-responders (p <0.001). Most patients who relapsed with GVHD of the gut were again sensitive to steroids, except one patient who then responded well to a second cycle of MSCs. Immunological monitoring suggests that anti-viral and anti-leukemia reactive T-cells are well preserved in all patients who responded to MSC treatment. In addition, we identified biomarkers which associate even 2 weeks after MSC infusion with a complete resolution of GVHD, which occurred usually after months. Conclusion Generation and infusion of hPPL-MSCs in steroid-resistant aGVHD grade II- IV is feasible, safe and appears to be effective. In addition, also patients who initially responded to hPPL-MSCs but develop later a relapse of aGVHD during tapering or cessation of immunosuppressive drugs become again sensitive to the treatment with steroids. Infusion of MSC did not impair anti-virus and anti-leukemia reactive T-cells. Identified biomarkers predict very early a usually late clinical resolution of GVHD, thus might be useful to early guide clinical decisions. Disclosures: No relevant conflicts of interest to declare.
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