From risk indices to reconstitution of immunity : studies of outcome-related factors in patients undergoing allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potent immunotherapeutic procedure which has the possibility to cure otherwise lethal hematological diseases. Its usability in clinical practice is limited by tangible risks of detrimental transplantation-related complications. Consequently, it is of great importance to adjust eligibility criteria and individualize allogeneic HSCT treatment protocols to maximize the chance of a positive outcome for every single patient. The general aim of this thesis has been to study influential outcome-related factors in patients under- going allogeneic HSCT. In the enclosed research papers, specific focus has been directed to improve interpretations of patient comorbidity and hematological indication prior to transplantation (paper I), to optimize stem cell dose (paper II), the choice of graft-versus-host disease (GVHD) prophylaxis (paper III) and to evaluate immune reconstitution after treatment (paper IV). In scientific paper I, we performed a retrospective study of 521 consecutive adult allogeneic HSCT- patients transplanted at the Karolinska University Hospital for hematological malignancy from 2000 to 2012 to compare the predictive capacity of the Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) and the disease risk index (DRI) for transplantation-related mortality (TRM) and overall survival (OS). Both indices could predict OS (with poorer survival in the highest risk groups) but failed to predict differences in TRM. In summary, obtained study data showed that the studied indices should be evaluated according to local data prior to their implementation on individual patients on the single-center level. For patients with pre-existing comorbidities, the use of reduced intensity conditioning regimens (RIC) has made allogeneic HSCT a valid treatment option. In scientific paper II, we sought to determine the optimal hematopoietic stem cell dose (CD34+ cell dose) related to survival in RIC transplantations with peripheral blood stem cell grafts (PBSCT). Using consecutive transplantation registry data from the Center for International Blood and Marrow Transplant Research (CIBMTR), we retrospectively analyzed 1,054 patients with AML or MDS who underwent RIC PBSCT between 2002 and 2011. Grafts from HLA-matched siblings containing < 4 × 106 CD34+ cells/kg recipient and grafts from unrelated donors containing < 6 ×106 CD34+ cells/kg recipient, were associated with higher overall mortality after transplantation. Consequently, CD34+ cell dose in PBSCT grafts should be kept above these respective thresholds if possible. In scientific paper III, we compared the standard GVHD prophylaxis regimen of cyclosporine and methotrexate (CsA/Mtx, n = 106) with a combination of tacrolimus and sirolimus (Tac/Sir, n = 103) in a prospective randomized trial. Based on previous publications on sirolimus, the hypothesis was that Tac/Sir would lead to less acute GVHD and reduced TRM. Analyses of study data did not show any significant differences in incidence of grades II-IV acute GVHD between the groups (CsA/Mtx: 41%, Tac/Sir: 51%), and data for TRM and OS were similar. In conclusion, the two GVHD prophylaxes provided comparable outcomes in patients after matched related or unrelated allogeneic HSCT, but study data indicated differences in toxicity in certain transplantation settings. GVHD prophylaxis and conditioning regimens constitute risk factors for prolonged immuno- deficiency related to impaired transplantation outcomes. In scientific paper IV, we sought to investigate the effects of the two different GVHD prophylaxis protocols (from paper III) on lymphocyte reconstitution and replicative capacity using PCR-derived levels of TREC, KREC and telomere length as proxy markers. Levels of lymphocyte excision circles were not significantly different between the GVHD prophylaxis groups, but patients with TREC or KREC levels above median during study follow-up had reduced TRM and superior OS. In summary, the results and conclusions presented in this thesis may be useful in the continuous endeavor towards a safer and more individualized allogeneic HSCT procedure for future patients. Either as arguments for adjusted transplantation protocols or as basis for future research hypotheses

Individualization of Hematopoietic Stem Cell Transplantation Using Alpha/Beta T-Cell Depletion

Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with several potentially lethal complications. Higher levels of CD3+ T-cells in the graft have been associated with increased risk of graft-versus-host disease (GVHD), but also beneficial graft-versus-leukemia effect and reduced infections. To tackle post-transplant complications, donor lymphocyte infusions have been used but with an increased risk of GVHD. To reduce this risk, we performed depletion of αβ T-cells and treated 12 patients post-HSCT suffering from infections and/or poor immune reconstitution. The αβ T-cell depleted cell products were characterized by flow cytometry. The median log depletion of αβ T-cells was −4.3 and the median yield of γδ T-cells was 73.5%. The median CD34+ cell dose was 4.4 × 106/kg. All 12 patients were alive 3 months after infusion and after 1 year, two patients had died. No infusion-related side effects were reported and no severe acute GVHD (grade III-IV) developed in any patient post-infusion. Overall, 3 months after infusion 11 out of 12 patients had increased levels of platelets and/or granulocytes. In conclusion, we describe the use of αβ T-cell depleted products as stem cell boosters with encouraging results

Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a debilitating complication arising in around half of all patients treated with an allogeneic hematopoietic stem cell transplantation. Even though treatment of severe cGVHD has improved during recent years, it remains one of the main causes of morbidity and mortality in affected patients. Biomarkers in blood that could aid in the diagnosis and classification of cGVHD severity are needed for the development of novel treatment strategies that can alleviate symptoms and reduce the need for painful and sometimes complicated tissue biopsies. Methods that comprehensively profile complex biological systems such as the immune system can reveal unanticipated markers when used with the appropriate methods of data analysis. Here, we used mass cytometry, flow cytometry, enzyme-linked immunosorbent assay, and multiplex assays to systematically profile immune cell populations in 68 patients with varying grades of cGVHD. We identified multiple subpopulations across T, B, and NK-cell lineages that distinguished patients with cGVHD from those without cGVHD and which were associated in varying ways with severity of cGVHD. Specifically, initial flow cytometry demonstrated that patients with more severe cGVHD had lower mucosal-associated T cell frequencies, with a concomitant higher level of CD38 expression on T cells. Mass cytometry could identify unique subpopulations specific for cGVHD severity albeit with some seemingly conflicting results. For instance, patients with severe cGVHD had an increased frequency of activated B cells compared to patients with moderate cGVHD while activated B cells were found at a reduced frequency in patients with mild cGVHD compared to patients without cGVHD. Moreover, results indicate it may be possible to validate mass cytometry results with clinically viable, smaller flow cytometry panels. Finally, no differences in levels of blood soluble markers could be identified, with the exception for the semi-soluble combined marker B-cell activating factor/B cell ratio, which was increased in patients with mild cGVHD compared to patients without cGVHD. These findings suggest that interdependencies between such perturbed subpopulations of cells play a role in cGVHD pathogenesis and can serve as future diagnostic and therapeutic targets

Oral mucositis after tacrolimus/sirolimus or cyclosporine/methotrexate as graft-versus-host disease prophylaxis

Objectives: To determine whether treatment with tacrolimus plus sirolimus (Tac/Sir) as a prophylaxis for graft-versus-host disease worsens severe oral mucositis and delays healing compared to cyclosporine plus methotrexate (CsA/Mtx) following haematopoietic stem cell transplantation. Subjects and methods: The study comprised 141 patients: 73 randomized to receive Tac/Sir and 68 to receive CsA/Mtx. The oral mucositis assessment scale and toxicity grading according to WHO were used to assess the severity, peak and duration of oral mucositis from the day -3 to day 24 post-transplant. Results: Eighty-seven patients developed oral mucositis in the first 24 days post-transplant. No significant difference in oral mucositis severity between the Tac/Sir and CsA/Mtx groups was observed. The peak oral mucositis score occurred on day 10 in both groups. Although oral mucositis scores had returned to baseline in the CsA/Mtx group on day 24 post-transplant, no significant difference compared with the Tac/Sir group was found. Conclusions: The introduction of tacrolimus/sirolimus as a graft-versus-host disease prophylaxis in haematopoietic stem cell transplantation increased neither the incidence nor severity of oral mucositis compared with cyclosporine/methotrexate. Furthermore, oral mucositis healing was not prolonged and followed the same time pattern as cyclosporine/methotrexate

Effect of Graft-versus-Host Disease Prophylaxis Regimens on T and B Cell Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation

Lymphocyte reconstitution is pivotal for successful long-term outcome after allogeneic hematopoietic stem cell transplantation (HSCT), and conditioning regimen and post-transplantation immunosuppression are risk factors for prolonged immunodeficiency. Nevertheless, the effects of different immunosuppressive protocols on lymphocyte output and replicative capacity have not been investigated. Here we assessed T cell receptor excision circles (TREC), kappa-deleting recombination excision circles (KREC), and T cell telomere length (TL) as proxy markers for immune reconstitution in patients in a prospective randomized trial comparing graft-versus-host disease (GVHD) prophylaxis after transplantation (cyclosporine/methotrexate versus tacrolimus/sirolimus; n=200). Results showed that medians of TREC, KREC, and TL were not significantly different between the prophylaxis groups at any assessment time point during follow-up (24 months), but the kinetics of TREC, KREC, and TL were significantly influenced by other transplantation-related factors. Older recipient age, the use of antithymocyte globulin before graft infusion, and use of peripheral blood stem cell grafts were associated with lower TREC levels, whereas acute GVHD transiently affected KREC levels. Patients with lymphocyte excision circle levels above the median at &lt;= 6 months post-transplantation had reduced transplantation-related mortality and superior 5-year overall survival (P&lt;.05). We noticed significant T cell telomere shortening in the patient population as a whole during follow-up. Our results suggest that lymphocyte reconstitution after transplantation is not altered by different immunosuppressive protocols. This study has been registered at ClinicalTrials.gov (identifier: NCT00993343). (C) 2019 American Society for Blood and Marrow Transplantation

A prospective randomized trial comparing cyclosporine/methotrexate and tacrolimus/sirolimus as graft-versus-host disease prophylaxis after allogeneic hematopoietic stem cell transplantation

Improvement of graft-versus-host disease prophylaxis remains an important goal in allogeneic hematopoietic stem cell transplantation. Based on reports of possibly preferential properties of sirolimus, we compared the standard regimen of cyclosporine and methotrexate (n=106) with a combination of tacrolimus and sirolimus (n=103) as graft-versus-host disease prophylaxis after allogeneic hematopoietic stem cell transplantation in a prospective, open, randomized trial. The hypothesis was that the tacrolimus/sirolimus regimen would lead to less acute graft-versus-host disease and reduced transplant-related mortality. There was no significant difference in the cumulative incidence of acute graft-versus-host disease of grades II–IV (41% vs. 51%; P=0.19) or grades III–IV (13% vs. 7%; P=0.09) between the groups. Time to neutrophil engraftment (18 days vs. 17 days; P=0.24) was similar, but time to platelet engraftment was longer in cyclosporine/methotrexate patients (14 vs. 12 days;

DataSheet_1_T cell receptor excision circles are potential predictors of survival in adult allogeneic hematopoietic stem cell transplantation recipients with acute myeloid leukemia.docx

BackgroundLymphocyte neogenesis from primary lymphoid organs is essential for a successful reconstitution of immunity after allogeneic hematopoietic stem cell transplantation (HSCT). This single-center retrospective study aimed to evaluate T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) as surrogate markers for T and B cell recovery, as predictors for transplantation-related outcomes in adult acute myeloid leukemia (AML) patients.MethodsNinety adult patients diagnosed with AML and treated with HSCT between 2010 and 2015 were included in the study. TREC and KREC levels were measured by quantitative PCR at 1, 3, 6, and 12 months after transplantation.ResultsOverall, excision circle levels increased between 3 and 6 months post-HSCT for TREC (p = 0.005) and 1 and 3 months for KREC (p = 0.0007). In a landmark survival analysis at 12 months post-HSCT, TREC levels were associated with superior overall survival (HR: 0.52, 95% CI: 0.34 - 0.81, p = 0.004). The incidence of viral infections within the first 100 days after transplantation was associated with lower TREC levels at 6 months (p = 0.0002). CMV reactivation was likewise associated with lower TREC levels at 6 months (p = 0.02) post-HSCT. KREC levels were not associated with clinical outcomes in statistical analyzes.ConclusionsResults from the present study indicate that TREC measurement could be considered as part of the post-HSCT monitoring to identify AML patients with inferior survival after transplantation. Further prospective studies are warranted to validate these findings.</p