The impact of transportation time on apoptosis in allogeneic stem cell grafts and the clinical outcome in malignant patients with unrelated donors.

BACKGROUND: The quality of cells in peripheral blood stem cell (PBSC) grafts is important for allogeneic stem cell transplantation outcome. The viability of PBSC grafts may decrease during transportation time between donor and transplant center. We hypothesize that the graft viability based on apoptosis and necrosis in the graft may better reflect graft quality and clinical outcome. METHODS: PBSC graft viability from unrelated donors was analyzed in 91 patients. Viable cells were defined as 7-aminoactinomycin D- and Annexin V-negative. The clinical outcome, including survival, transplant-related mortality and graft-versus-host disease (GvHD), was correlated to graft viability. RESULTS: Grafts transported for 1 day had a median viability of 86.4% (range 63.8 to 98.9%), and grafts transported for 2 days had median viability of 83.2% (range 52.8% to 96.2%) (P = .003). Grafts were divided into two groups based on the median graft viability of 85.1%. Patients who received low viability grafts had lower 1-year survival of 63.7% compared with 88.9% for those who received high viability grafts (P = .007). In the multivariate analysis, transplant-related mortality (TRM) was higher in the low viability group (P = .03), whereas overall survival was not significantly associated with graft viability. The incidence of acute GvHD grade II to IV, chronic GvHD and relapse risk remained comparable between the groups. CONCLUSION: Low graft viability was an independent predictor of 1-year survival and TRM after adjusting for multiple confounders. Better graft quality markers are important for the detection of clinically important variations in the stem cell graft.

Impact of donor-derived CD34 + infused cell dose on outcomes of patients undergoing allo-HCT following reduced intensity regimen for myelofibrosis: a study from the Chronic Malignancies Working Party of the EBMT.

The optimal CD34 + cell dose in the setting of RIC allo-HCT for myelofibrosis (MF) remains unknown. We retrospectively analyzed 657 patients with primary or secondary MF transplanted with use of peripheral blood (PB) stem cells after fludarabine/melphalan or fludarabine/busulfan RIC regimen. Median patient age was 58 (range, 22-76) years. Donors were HLA-identical sibling (MSD) or unrelated (UD). Median follow-up was 46 (2-194) months. Patients transplanted with higher doses of CD34 + cells (>7.0 × 106/kg), had an increased chance of achievement of both neutrophil (hazard ratio (HR), 1.46; P < 0.001) and platelet engraftment (HR, 1.43; P < 0.001). In a model with interaction, for patients transplanted from a MSD, higher CD34 + dose was associated with improved overall survival (HR, 0.63; P = 0.04) and relapse-free survival (HR, 0.61; P = 0.02), lower risk of non-relapse mortality (HR, 0.57; P = 0.04) and higher rate of platelet engraftment. The combined effect of higher cell dose and UD was apparent only for higher neutrophil and platelet recovery rate. We did not document any detrimental effect of high CD34 + dose on transplant outcomes. More bulky splenomegaly was an adverse factor for survival, engraftment and NRM. Our analysis suggests a potential benefit for MF patients undergoing RIC PB-allo-HCT receiving more than 7.0 × 106/kg CD34 + cells.

Granulocyte transfusions could benefit patients with severe oral mucositis after allogeneic hematopoietic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Mucositis is a common complication after allogeneic hematopoietic stem cell transplantation (HSCT), and is caused by a combination of conditioning-induced mucosal damage and severe neutropenia. The symptoms include oral and abdominal pain, inability to swallow food and fluids, and severe diarrhoea. Severe mucositis is associated with increased risk of Graft-versus-Host disease and infection. Granulocyte transfusions (GCX) could be a treatment option, and our objective was to study its feasibility and potential benefits. MATERIAL AND METHODS: This retrospective, single-centre study included 30 patients receiving GCX because of severe oral mucositis after HSCT during 2005-2017. Clinical outcome, response to GCX, change in opiate administration and adverse events were studied. RESULTS: Twenty-seven patients received GCX from donors pre-treated with steroids and G-CSF, and three from donors pre-treated with steroids only. Overall response was 83% (24/29 evaluable patients). Fifteen patients reached a complete response. In 14 of 24 responders, a reduction of the administration of opiate pain relief was seen. In eight patients this reduction was ≥50% of the dose. Adverse events (AEs) were reported in 14 cases, and were mild to moderate, and well manageable with symptomatic treatment. No life-threatening or fatal AEs were recorded. CONCLUSIONS: These results indicate that GCX could be a safe and effective treatment for oral mucositis after HSCT with the potential to reduce the necessity of opiate analgesic treatment in this disorder. No severe AEs were seen in this study, but the risk for severe pulmonary AEs after GCX needs to be considered.

Higher response rates in patients with severe chronic skin graft-versus-host disease treated with extracorporeal photopheresis.

INTRODUCTION: Different forms of graft-versus-host disease (GVHD) remain a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The prognosis for steroid-refractory chronic GVHD (cGVHD) remains poor. Our aim was to evaluate extracorporeal photopheresis (ECP) treatment in cGVHD patients with different organ involvement to detect subgroups of patients with the best response. MATERIAL AND METHODS: Thirty-four patients who underwent HSCT and developed moderate (n = 7) or severe (n = 27) steroid-refractory or steroid-dependent cGVHD treated with ECP were included in the analysis. A matched cGVHD control patient group untreated with ECP was collected for comparison. RESULTS: Compared to the control group and the stable/progressive disease (SD/PD) patients, individuals with complete/partial remission have higher overall survival and lower transplant-related mortality. Furthermore, patients with complete and partial remission (CR/PR) had significantly higher levels of albumin and platelets after ECP treatment compared to patients with stable or progressive cGVHD (SD/PD). Corticosteroid treatment and other immunosuppressive agents could successfully be tapered in the CR/PR group compared to the SD/PD patients. In this study patients with skin cGVHD are those with the highest rate of CR/PR after ECP treatment. CONCLUSIONS: Our results suggest that ECP treatment is safe and effective for patients with predominantly skin, oral and liver cGVHD.

The importance of graft cell composition in outcome after allogeneic stem cell transplantation in patients with malignant disease.

BACKGROUND: Graft-versus-host disease (GVHD) and relapse remain majobstacles ftreatment success in allogeneic hematopoietic stem cell transplantation (HSCT). In the present study, we evaluated the immune cell profile of the graft to outcome after HSCT. STUDY DESIGN AND METHOD: Flow cytometry data of graft cell subsets [CD34+ , CD3+ , CD19+ , CD4+ , CD8+ , CD3-CD56+ CD16+ , CD4+ CD127low CD25high ] from G-CSF primed peripheral blood stem cell (PBSC) donors was collected retrospectively from 299 patients with hematological malignancies undergoing HSCT between 2006 and 2013. The association to overall survival, transplant-related mortality (TRM), GVHD and probability of relapse was analyzed. Patients outcome from HLA-identical sibling (Sib) (n = 97) and unrelated donors (URD) (n = 202) were analyzed separately as all URD patients received anti-thymocyte globulin (ATG). RESULTS: Five-year overall survival was similar in the two cohorts (68% (Sib) vs 65% (URD)). The relapse incidence was significantly lower in the Sib cohort (24% vs 35%, P = 0.04). Multivariate analysis in the URD group revealed an association between a higher CD8+ dose and less relapse (HR, 0.94; 95%CI, 0.90-0.98; P = 0.006) as well as an association between higher CD34+ dose and both higher TRM (HR, 1.09; 95%CI, 1.02-1.20; P = 0.02) and relapse (HR, 1.09; 95%CI, 1.01-1.17; P = 0.025). The Sib analysis showed an association between a higher graft CD19+ dose and more severe acute GVHD (HR, 1,09; 95%CI, 1.03-1.15; P = 0.003) and TRM (HR, 1.09; 95%CI, 1.01-1.17; P = 0.036). In addition, a higher CD4+ graft content was associated to an increased risk for chronic GVHD (HR, 1.02; 95%CI 1.00-1.04; P = 0.06). CONCLUSION: These data indicate an importance of PBSC dongraft composition in patients with a hematological malignancy.

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 αß T-cells and treated 12 patients post-HSCT suffering from infections and/or poor immune reconstitution. The αß T-cell depleted cell products were characterized by flow cytometry. The median log depletion of αß 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 αß T-cell depleted products as stem cell boosters with encouraging results.

T-cell frequencies of CD8+ γδ and CD27+ γδ cells in the stem cell graft predict the outcome after allogeneic hematopoietic cell transplantation.

The impact of intra-graft T cells on the clinical outcome after allogeneic hematopoietic cell transplantation has been investigated. Most previous studies have focused on the role of αß cells while γδ cells have received less attention. It has been an open question whether γδ cells are beneficial or not for patient outcome, especially with regards to graft versus host disease. In this study, graft composition of γδ cell subsets was analyzed and correlated to clinical outcome in 105 recipients who underwent allogeneic hematopoietic cell transplantation between 2013 and 2016. We demonstrate for the first time that grafts containing higher T-cell proportions of CD8+γδ cells were associated with increased cumulative incidence of acute graft versus host disease grade II-III (50% vs 22.6%; P = 0.008). Additionally, graft T-cell frequency of CD27+γδ cells was inversely correlated with relapse (P = 0.006) and CMV reactivation (P = 0.05). We conclude that clinical outcome after allogeneic hematopoietic cell transplantation is influenced by the proportions of distinct γδ cell subsets in the stem cell graft. We also provide evidence that CD8+γδ cells are potentially alloreactive and may play a role in acute graft versus host disease. This study illustrates the importance of better understanding of the role of distinct subsets of γδ cells in allogeneic hematopoietic cell transplantation.

Complete Remission with Reduction of High-Risk Clones following Haploidentical NK-Cell Therapy against MDS and AML.

Purpose: To evaluate the safety, efficacy, and immunobiological correlates of allogeneic NK-cell-based therapy in primary chemotherapy-refractory or relapsed high-risk myelodysplastic syndrome (MDS), secondary AML (MDS/AML), and de novo AML patients.Experimental Design: Sixteen patients received fludarabine/cyclophosphamide conditioning combined with total lymphoid irradiation followed by adoptive immunotherapy with IL2-activated haploidentical NK cells.Results: NK-cell infusions were well-tolerated, with only transient adverse events observed in the 16 patients. Six patients achieved objective responses with complete remission (CR), marrow CR, or partial remission (PR). Five patients proceeded to allogeneic hematopoietic stem cell transplantation (HSCT). Three patients are still free from disease >3 years after treatment. All evaluable patients with objective responses (5/5 evaluable) had detectable donor NK cells at days 7/14 following infusion and displayed reduction of tumor cell clones, some of which carried poor prognosis mutations. Residual lin-CD34+CD123+CD45RA+ blast cells in responders had increased total HLA class I and HLA-E expression. Responding patients displayed less pronounced activation of CD8+ T cells and lower levels of inflammatory cytokines following NK-cell infusion. Intriguingly, despite omission of systemic IL2, all patients displayed increased frequencies of activated Ki-67+CD127-FoxP3+CD25hiCD4+ Treg cells of recipient origin following NK-cell therapy.Conclusions: Overall, this study suggests that high-risk MDS is responsive to NK-cell therapy and supports the use of haploidentical NK-cell infusions as a bridge to HSCT in refractory patients. Objective clinical responses and reduction of high-risk clones were associated with detectable donor-derived NK cells, immunoediting of residual blast cells, and less pronounced host immune activation. Clin Cancer Res; 24(8); 1834-44. ©2018 AACR.

Quality of the hematopoietic stem cell graft affects the clinical outcome of allogeneic stem cell transplantation.

BACKGROUND: In approximately two-thirds of patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) no suitable related donor can be identified but an unrelated HLA-matched donor can be found through international donor registries. HSCT grafts from unrelated donors are commonly collected at distant sites. Therefore, graft storage and transportation becomes crucial in the HSCT process. We aimed to study the impact of graft quality on clinical outcome and identify factors affecting graft quality. STUDY DESIGN AND METHODS: We investigated the influence of graft quality on the clinical outcome in 144 HSCT patients. Graft quality was assessed by determining the viability (7-aminoactinomycin D [7AAD]) on a frozen-thawed sample from the peripheral blood stem cell (PBSC) graft. RESULTS: Patients receiving PBSCs with inferior quality (i.e., viability < 64% in the frozen-thawed sample) more frequently developed acute graft-versus-host disease (aGVHD) Grades I to IV than patients receiving grafts with better quality (p = 0.025). The transplant-related mortality (TRM) was higher in the group receiving grafts with lower viability (p = 0.03). The viability of the frozen-thawed samples was highly variable (median, 64%; range, 24%-96%). No correlation could be observed when comparing the viability in newly arrived PBSC grafts to frozen-thawed vials. Grafts with white blood cell (WBC) count of more than 300 × 10(9) /L had lower viability than those with lower WBC counts (p < 0.001). CONCLUSION: Graft quality affects clinical outcome. Patients receiving grafts with inferior quality had more aGVHD and higher TRM. There is a need for better analyses for assessing graft quality in routine HSCT care; analysis using 7AAD on fresh PBSC grafts is not sufficient.

Effect of Total Nucleated and CD34(+) Cell Dose on Outcome after Allogeneic Hematopoietic Stem Cell Transplantation.

During more recent years only few studies have analyzed the effect of total nucleated cell (TNC) and CD34(+) cell dose in allogeneic hematopoietic stem cell transplantation (HSCT). A single-center analysis included 544 patients, 227 with a sibling donor and 317 with an unrelated donor. Most patients (n = 292) were treated with myeloablative conditioning, whereas the remaining patients (n = 252) received reduced-intensity conditioning. Bone marrow (BM) (n = 121) and peripheral blood stem cell (PBSC) grafts (n = 423) were analyzed separately. Median TNC and CD34(+) cell dose was 3.2 × 10(8)/kg versus 11.6 × 10(8)/kg in BM and 3.9 × 10(6)/kg versus 8.1 × 10(6)/kg in PBSC. In the BM group we found a higher TNC and CD34(+) cell dose was associated with a faster neutrophil engraftment (P < .001 and P = .02). In the PBSC group we found patients given a very high (≥11 × 10(6)/kg) CD34(+) cell dose had decreased rates of survival (P = .001) and increased relapse (P = .02). A high CD34(+) cell dose correlated with faster platelet engraftment (P < .01). In HSCT using PBSCs, the CD34(+) cell doses should be kept below 11 × 10(6)/kg but over 2.5 × 10(6)/kg.

Analysis of donor and recipient ABO incompatibility and antibody-associated complications after allogeneic stem cell transplantation with reduced-intensity conditioning.

Allogeneic hematopoietic stem cell transplantation (HSCT) can be performed across the ABO blood group barrier. The impact of ABO incompatibility on clinical outcome is controversial. A retrospective analysis of 310 patients who underwent HSCT with reduced-intensity conditioning between 1998 and 2011 was performed to investigate the frequency and clinical implications of anti-RBC antibodies in passenger lymphocyte syndrome (PLS) after minor ABO mismatch (mm), persistent or recurring recipient type ABO antibodies (PRABO) after major ABO mm HSCT, and autoimmune hemolytic anemia (AIHA). Transplantation characteristics and clinical outcome were analyzed by univariate and multivariate analysis for groups with or without anti-RBC antibodies. ABO blood group incompatibility did not affect clinical outcome despite an increased requirement of blood transfusion. Twelve patients with AIHA, 6 patients with PLS, and 12 patients with PRABO post-HSCT were identified. AIHA did not affect overall survival (OS) or transplant-related mortality (TRM), but patients with AIHA had a lower incidence of grades II to IV acute graft-versus-host disease (P = .05). OS in the PLS group was 0% compared with 61% in the whole group receiving minor ABO mm transplants (P < .001). Comparing PRABO patients with those receiving a major ABO mm HSCT, the OS was 17% versus 73% (P = .002) and TRM was 50% versus 21% (P = .03). At our center, PLS after minor ABO mm and PRABO antibodies after major ABO mm HSCT are significant risk factors for decreased OS and TRM. Our results suggest that occurrence of unexpected ABO antibodies after HSCT warrant a wider investigation individual to find the underlying cause.

Rapid salvage treatment with virus-specific T cells for therapy-resistant disease.

BACKGROUND: Viral infections are major complications after allogeneic hematopoietic stem cell transplantation (HSCT). During posttransplant immunosuppression the regular T-cell control is compromised. Even if treatment strategies against infections caused by herpes viruses such as cytomegalovirus, Epstein-Barr virus, and adenovirus have improved, the mortality rate is still considerable. If primary antiviral therapy fails or cannot be tolerated, adoptive therapy with virus-specific cytotoxic T cells (CTL) can be utilized. METHODS: In this study, we used virus-specific CTLs to treat 8 patients suffering from severe viral infections after allogeneic HSCT. Using positive selection with HLA multimers and magnetic beads, we isolated CTLs from both frozen donor material as well as third-party donors within hours. RESULTS: At 90 days after CTL infusions 7 out of 8 patients were still living. CTLs infused from third-party donors were detected in 5 of 6 patients up to 76 days after infusion. No graft-versus-host disease associated with CTL infusions was observed. CONCLUSIONS: Our separation approach offers a rapid alternative for adoptive CTL therapy if primary antiviral treatment strategies fail. Because no prolonged expansion steps are needed, this method may be used for early treatment of patients suffering from life-threatening infectious complications.

Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study.

BACKGROUND: Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS: A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 µg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS: We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION: Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING: European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.

Improved survival after allogeneic hematopoietic stem cell transplantation in recent years. A single-center study.

We analyzed the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) over the past 2 decades. Between 1992 and 2009, 953 patients were treated with HSCT, mainly for a hematologic malignancy. They were divided according to 4 different time periods of treatment: 1992 to 1995, 1996 to 2000, 2001 to 2005, and 2006 to 2009. Over the years, many factors have changed considerably regarding patient age, diagnosis, disease stage, type of donor, stem cell source, genomic HLA typing, cell dose, type of conditioning, treatment of infections, use of granulocyte-colony stimulating factor (G-CSF), use of mesenchymal stem cells, use of cytotoxic T cells, and home care. When we compared the last period (2006-2009) with earlier periods, we found slower neutrophil engraftment, a higher incidence of acute graft-versus-host disease (aGVHD) of grades II-IV, and less chronic GVHD (cGHVD). The incidence of relapse was unchanged over the 4 periods (22%-25%). Overall survival (OS) and transplant-related mortality (TRM) improved significantly in the more recent periods, with the best results during the last period (2006-2009) and a 100-day TRM of 5.5%. This improvement was also apparent in a multivariate analysis. When correcting for differences between the 4 groups, the hazard ratio for mortality in the last period was 0.59 (95% confidence interval [CI]: 0.44-0.79; P < .001) and for TRM it was 0.63 (CI: 0.43-0.92; P = .02). This study shows that the combined efforts to improve outcome after HSCT have been very effective. Even though we now treat older patients with more advanced disease and use more alternative HLA nonidentical donors, OS and TRM have improved. The problem of relapse still has to be remedied. Thus, several different developments together have resulted in significantly lower TRM and improved survival after HSCT over the last few years.

Persistence of human parvovirus B19 in multipotent mesenchymal stromal cells expressing the erythrocyte P antigen: implications for transplantation.

Multipotent mesenchymal stromal cells (MSCs) are used to improve the outcome of hematopoietic stem cell transplantation (HCST) and in regenerative medicine. MSCs may harbor persistent viruses that may compromise their clinical benefit, however. Retrospectively screened, 1 of 20 MSCs from healthy donors contained parvovirus B19 (B19) DNA. MSCs express the B19 receptor (P antigen/globoside) and a co-receptor (Ku 80) and can transmit B19 to bone marrow cells in vitro, suggesting that the virus can persist in the marrow stroma of healthy individuals. Two patients undergoing HSCT received the B19-positive MSCs as treatment for graft-versus-host disease; neither developed viremia nor symptomatic B19 infection. These findings demonstrate for the first time that persistent B19 in MSCs can infect hematopoietic stem cells and underscore the importance of monitoring B19 transmission by MSC products.

Major ABO blood group mismatch increases the risk for graft failure after unrelated donor hematopoietic stem cell transplantation.

Two hundred twenty-four patients with leukemia transplanted with an unrelated donor between 1991 and 2003 at the Karolinska University Hospital were analyzed according to association between graft failure and ABO, RhD, MNSs, and Kidd blood group antigen compatibility. Median age was 29 years (range: 0-55). Conditioning consisted of total-body irradiation or busulfan-based myeloablative conditioning. A bone marrow graft was given to 152 patients, and 72 patients received peripheral blood stem cells. Most patients received graft-versus-host disease prophylaxis with cyclosporine and MTX. Graft failure (GF) was seen in 6 (2.7%) patients. In the multivariate analysis major ABO mismatch (odds ratio [OR] 14.9, 95% confidence interval [CI] 2.01-110, P = .008) and HLA-allele mismatch (6.42, 1.19-34.8, P = .03) was significantly associated to GF. In patients with and without major ABO mismatch the incidence of GF was 7.5% and 0.6% (P = .02), respectively. Using an ABO major mismatched graft increases the risk for GF after unrelated donor hematopoietic stem cell transplantation.