Factor-mobilized Peripheral Blood Stem Cells Research Articles

Cryopreservation of Growth Factor-Mobilized Peripheral Blood Stem Cells Does Not Compromise Major Outcomes after Allogeneic Hematopoietic Cell Transplantation: A Single-Center Experience

During the COVID-19 pandemic, cryopreservation of allogeneic donor stem cell products ensured availability of products at the start of conditioning for hematopoietic cell transplantation (HCT). Following recommendations from unrelated donor registries, including the National Marrow Donor Program (NMDP), many centers started to cryopreserve related donor peripheral blood stem cell (PBSC) products. Throughout the process, several centers have published outcomes with cryopreserved versus fresh products, some with conflicting results. Even though cryopreservation was initially considered only a temporary measure driven by the pandemic, potential advantages include greater flexibility of transplant timing. However, concern about detrimental effects of cryopreservation including increased risks of graft rejection, relapse and consequently mortality remained. The primary objective was to describe our center's experience comparing outcomes following PBSC transplantation with cryopreserved versus fresh grafts. This was an observational case study with retrospective review comparing cryopreserved grafts (N=213), to a recent historical cohort (controls) using fresh grafts (N=167). In multivariable analyses, the adjusted hazard ratio (fresh vs. cryo) we for overall mortality was HR= 1.20 (95% confidence interval (CI), 0.79-1.82; p=0.40), for non-relapse mortality HR=0.99 (95% CI, 0.55-1.77; p=0.98), and for relapse HR=0.94 (95% CI, 0.60 -1.48; p=0.80). The adjusted hazard ratio for platelet engraftment was HR=1.31 (95% CI, 1.05-1.63; p=.02) and the odds ratio of grades III-IV acute GVHD was OR=1.75 (95% CI 1.01-3.04; p=.05) with fresh compared to cryopreserved grafts. There was no demonstrable difference in the risk of chronic GHVD. Although longer-term follow-up is needed, these data provide preliminary reassurance that in the event of another pandemic or should the logistical need arise in individual patients, cryopreservation of PBSC products constitutes a reasonably safe alternative.

Allogeneic transplantation of bone marrow versus peripheral blood stem cells from HLA-identical relatives in patients with myelodysplastic syndromes and oligoblastic acute myeloid leukemia: a propensity score analysis of a nationwide database.

Bone marrow (BM) and granulocyte colony-stimulating factor-mobilized peripheral blood stem cells (PBSC) are used as grafts from HLA-identical-related donors for adults with myelodysplastic syndrome (MDS). To assess the impact of graft sources on post-transplant outcomes in MDS patients, we conducted a retrospective analysis of a nationwide database. A total of 247 and 280 patients underwent transplantation with BM and PBSC, respectively. The inverse probability of treatment weighting (IPTW) methods revealed that overall survival (OS) was comparable between BM and PBSC (P = .129), but PBSC transplantation was associated with worse graft-versus-host disease (GVHD)-free/relapse-free survival (GRFS) (hazard rate [HR], 1.24; 95% confidence intervals [CIs], 1.00-1.53; P = 0.049) and chronic GVHD-free and relapse-free survival (CRFS) (HR, 1.29; 95% CIs, 1.13-1.73; P = 0.002) than BM transplantation. In the propensity score matched cohort (BM, n = 216; PBSC, n = 216), no significant differences were observed in OS and relapse; 3-year OS rates were 64.7% and 60.0% (P = 0.107), while 3-year relapse rates were 27.1% and 23.5% (P = 0.255) in BM and PBSC, respectively. Three-year GRFS rates (36.6% vs. 29.2%; P = 0.006), CRFS rate (37.7% vs. 32.5%; P = 0.003), and non-relapse mortality rates (13.9% vs. 21.1%; P = 0.020) were better in BM than in PBSC. The present study showed that BM transplantation provides a comparable survival benefit with PBSC transplantation and did not identify an enhanced graft-versus-MDS effect to reduce the incidence of relapse in PBSC transplantation.

HLA-Haploidentical Hematopoietic Cell Transplantation for Treatment of Nonmalignant Diseases Using Nonmyeloablative Conditioning and Post-Transplant Cyclophosphamide.

Allogeneic hematopoietic cell transplant (HCT) is often the only curative therapy for patients with nonmalignant diseases; however, many patients do not have an HLA-matched donor. Historically, poor survival has been seen after HLA-haploidentical HCT because of poor immune reconstitution, increased infections, graft-versus-host disease (GVHD), and graft failure. Encouraging results have been reported using a nonmyeloablative T cell-replete HLA-haploidentical transplant approach in patients with hematologic malignancies. Here we report the outcomes of 23 patients with various nonmalignant diseases using a similar approach. Patients received HLA-haploidentical bone marrow (n=17) or granulocyte colony-stimulating factor-mobilized peripheral blood stem cell (n=6) grafts after conditioning with cyclophosphamide 50 mg/kg, fludarabine 150 mg/m2, and 2 or 4 Gy total body irradiation. Postgrafting immunosuppression consisted of cyclophosphamide, mycophenolate mofetil, tacrolimus, ± sirolimus. Median patient age at HCT was 10.8 years. Day 100 transplant-related mortality (TRM) was 0%. Two patients died at later time points, 1 from intracranial hemorrhage/disseminated fungal infection in the setting of graft failure and 1 from infection/GVHD. The estimated probabilities of grades II to IV and III to IV acute GVHD at day 100 and 2-year National Institutes of Health consensus chronic GVHD were 78%, 26%, and 42%, respectively. With a median follow-up of 2.5 years, the 2-year overall and event-free rates of survival were 91% and 78%, respectively. These results are encouraging and demonstrate favorable disease-specific lineage engraftment with low TRM in patients with nonmalignant diseases using nonmyeloablative conditioning followed by T cell-replete HLA-haploidentical grafts. However, additional strategies are needed for GVHD prevention to make this a viable treatment approach for patients with nonmalignant diseases.

GVHD Prevention in 2018: Tried and True or Something New?

Graft-versus-host disease (GVHD) is a major contributor to early and late morbidity and mortality after allogeneic stem cell transplantation. Despite results from a randomized controlled trial demonstrating an increased risk of chronic GVHD with use of growth factor-mobilized peripheral blood stem cells (PBSC) compared with bone marrow, PBSCs are the most widely used graft source in allogeneic transplantation for hematologic neoplasms in the U.S. This lecture will review established, recent, and novel strategies for GVHD prevention in unrelated donor PBSC transplantation and will highlight ongoing clinical research at Fred Hutchinson Cancer Research Center. Clinical trials aimed at defining standard-of-care GVHD prophylaxis after myeloablative and nonmyelablative conditioning will be presented. In addition, novel pharmacologic agents and graft manipulation strategies under investigation will be discussed. (Presented at the 1962nd Meeting, May 12, 2018).

A Study of Human Leukocyte Antigen Mismatched Cellular Therapy (Stem Cell Microtransplantation) in High-Risk Myelodysplastic Syndrome or Transformed Acute Myelogenous Leukemia

The treatment outcomes of myelodysplastic syndrome (MDS) and transformed acute myelogenous leukemia (tAML) remain very unsatisfactory. We designed a combination of human leukocyte antigen (HLA)-mismatched hematopoietic stem cell microtransplantation (MST) with chemotherapy for patients with MDS and tAML and evaluated its effects and toxicity. Patients were between 13 and 79 years old. Patients with MDS (n=21) were given HLA-mismatched MST combined with decitabine and cytarabine; patients with tAML (n=22) were given HLA-mismatched MST combined with decitabine and cytarabine, and also mitoxantrone. Patients in complete remission (CR) also received MST plus decitabine and medium-dose cytarabine chemotherapy without graft-versus-host disease (GVHD) prophylaxis. The overall response rate of the patients with MDS was significantly higher than that of those with tAML (81% vs. 50%; p=.03). The CR rates were 52.4% and 36.4% in the two groups, respectively. There was no difference in the cytogenetic CR rate between the MDS and tAML groups (85.7% vs. 70%, respectively; p=.7). The 24-month overall survival of the patients with MDS was significantly higher than that of the patients with tAML (84.7% and 34.1%, respectively; p=.003). The median recovery times of neutrophils and platelets were, respectively, 14 and 17 days in the patients with MDS, and 16 and 19 days in those with tAML. The treatment-related mortality rates were 4.8% and 18.2%, respectively, in the MDS and tAML groups (p=.34). No GVHD was observed in any patient. Microtransplantation combined with decitabine and chemotherapy may provide a novel, effective, and safe treatment for high-risk MDS and tAML. Microtransplantation (MST) refers to regular chemotherapy combined with granulocyte colony-stimulating factor-mobilized peripheral blood stem cell infusion of human leukocyte antigen-mismatched donor cells without using immunosuppressive agents. It aims to support hematopoietic recovery and perform graft-versus-leukemia (GVL) effects but differs from traditional allogeneic stem cell transplantation because the rate of donor cell chimerism is low and there is and no graft-versus-host disease (GVHD) risk. Thus, a trial was designed to evaluate the safety and efficacy of MST in patients with myelodysplastic syndrome and those with transformed acute myelogenous leukemia. Higher complete remission and cytogenetic complete response rates were observed, and the treatment improved disease progress-free survival, sped hematopoietic recovery, and avoided GVHD.

Negative Impact of Unidirectional Host-versus-Graft Killer Cell Immunoglobulin-like Receptor Ligand Mismatch on Transplantation Outcomes after Unmanipulated Haploidentical Peripheral Blood Stem Cell Transplantation for Acute Myeloid Leukemia.

This study explored the influence of mismatched inhibitory killer cell immunoglobulin-like receptor (KIR) ligands on the outcome of haploidentical transplantation using T cell-replete, granulocyte colony-stimulating factor-mobilized peripheral blood stem cells in adult patients with acute myeloid leukemia (AML). Three groups were examined: unidirectional graft-versus-host KIR ligand mismatched (GVH-KIR-MM; n= 33), bidirectional KIR ligand matched (KIR-M; n= 41), and unidirectional host-versus-graft KIR ligand mismatched (HVG-KIR-MM; n= 26). All recipients were treated with the same conditioning regimen (800 cGy total body irradiation, fludarabine, busulfan, and antithymocyte globulin). After a median follow-up of 26 months, the 2-year cumulative incidence of relapse was significantly higher in HVG-KIR-MM (40.3% ± 10.3%) versus others (18.9% ± 4.8%, P= .044). In the standard-risk group, the 2-year disease-free survival (DFS) was significantly lower in HVG-KIR-MM (51.8% ± 11.2%) compared with GVH-KIR-MM (88% ± 8.1%, P= .025). Multivariate analysis showed that HVG-KIR-MM was significantly associated with higher relapse (hazard ratio [HR], 10.7; P= .002) and lower DFS (HR, 3.4; P= .012). Subgroup analysis revealed increased DFS with higher doses of CD3(+)CD8(+) and CD3(-)CD56(+) grafts in GVH-KIR-MM (90.9% ± 8.7%, P= .006); there was no such effect in the other groups. Although our conclusions are limited by the absence of donor KIR genotype data, our study suggests unidirectional KIR ligand incompatibility in the host-versus-graft vector has a detrimental effect on T cell-replete haploidentical transplantation outcomes in adult patients with AML.

Improved survival after transplantation of more donor plasmacytoid dendritic or naïve T cells from unrelated-donor marrow grafts: results from BMTCTN 0201.

To characterize relationships between specific immune cell subsets in bone marrow (BM) or granulocyte colony-stimulating factor-mobilized peripheral blood (PB) stem cells collected from unrelated donors and clinical outcomes of patients undergoing transplantation in BMTCTN 0201. Fresh aliquots of 161 BM and 147 PB stem-cell allografts from North American donors randomly assigned to donate BM or PB stem cells and numbers of transplanted cells were correlated with overall survival (OS), relapse, and graft-versus-host disease (GvHD). Patients with evaluable grafts were similar to all BMTCTN 0201 patients. The numbers of plasmacytoid dendritic cells (pDCs) and naïve T cells (Tns) in BM allografts were independently associated with OS in multivariable analyses including recipient and donor characteristics, such as human leukocyte antigen mismatch, age, and use of antithymocyte globulin. BM recipients of > median number of pDCs, naïve CD8(+) T cells (CD8Tns), or naïve CD4(+) T cells (CD4Tns) had better 3-year OS (pDCs, 56% v 35%; P = .025; CD8Tns, 56% v 37%; P = .012; CD4Tns, 55% v 37%; P = .009). Transplantation of more BM Tns was associated with less grade 3 to 4 acute GvHD but similar rates of relapse. Transplantation of more BM pDCs was associated with fewer deaths resulting from GvHD or from graft rejection. Analysis of PB grafts did not identify a donor cell subset significantly associated with OS, relapse, or GvHD. Donor immune cells in BM but not PB stem-cell grafts were associated with survival after unrelated-donor allogeneic hematopoietic stem-cell transplantation. The biologic activity of donor immune cells in allogeneic transplantation varied between graft sources. Donor grafts with more BM-derived Tns and pDCs favorably regulated post-transplantation immunity in allogeneic hematopoietic stem-cell transplantation.

The Comparison of Allogeneic Stem Cell Transplantation From Family-Mismatched/Haploidentical Donors with Unrelated Donors in Adults with High-Risk Acute Myeloid Leukemia,

Abstract 4164Since August 2008, we have been continuously doing allogeneic stem cell transplantation (SCT) from family-mismatched/haploidentical donors (FMT) in high-risk acute myeloid leukemia (AML) lacking of HLA-identical siblings, well-matched unrelated donors (WM-UD), and partially-matched unrelated donors (PM-UD), and a pilot study about the encouraging outcomes of FMT using unmanipulated donor cells and less aggressive conditioning regimen without T-cell depletion were reported at last ASH (2010;116:Abstract 3533). We herein investigated the clinical outcomes of FMT in high-risk AML compared to SCT from WM-UD and PM-UD in order to weigh up the pros and cons of FMT.Between August 2008 to December 2010, 69 adult patients with high-risk AML underwent allogeneic SCT from WM-UD (n=33), PM-UD (n=13), and FMT (n=23). Conditioning regimen of FMT consisted of total body irradiation (800 cGy), fludarabine (150 mg/m2/day), busulfex (6.4 mg/kg/day), and ATG (thymoglobulin, Genzyme, 5.0 mg/kg). Unmanipulated granulocyte colony stimulating factor-mobilized peripheral blood stem cells (PBSCs) were used for FMT. Patients (n=46) transplanted from WM-UD and PM-UD received myeloablative (n=23, 70%) or reduced-intensity conditioning (n=14, 30%), and bone marrow (n=11, 23%) or PBSCs (n=36, 77%). Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus and short course of methotrexate, which was identical for the three donor groups.The type of AML was de novo (n=59, 86%) and secondary (n=10, 14%). The majority of patients (n=66, 95%) had intermediate (n=52, 75%) or unfavorable (n=14, 20%) cytogenetic and molecular features based on criteria of 2011 NCCN guidelines. Three patients with favorable cytogenetics were second complete remission (n=2) or had persistent disease (n=1) at transplantation. All but one patients (99%) and 49 donors (71%) were cytomegalovirus (CMV)-seropositive. Offspring (n=12), mothers (n=7), and siblings (n=4) were donors for FMT. There was no significant difference in patients and transplant characteristics, such as age and pre-transplant disease status, among the three donor groups.All patients were successfully engrafted. The median time to neutrophil (>0.5^109/L) and platelet (>20^109/L) recovery were 12 days (range, 10–21) and 13 days (range, 8–60) for WM-UR, 12 days (range, 9–22) and 13 days (range, 9–25) for PM-UR, and 11 days (range, 10–17) and 12 days (range, 8–40) for FMT, respectively. There was no significant difference in the neutrophil (P=0.370) and platelet (P=0.487) recovery times between groups. With 24 months (range, 6–34) of median follow-up for surviving patients, overall survival (OS), disease-free survival (DFS), relapse, and non-relapse mortality were 82.7%, 82.9%, 9.4%, and 7.6% for WM-UD, 61.5%, 61.5%, 30.8%, and 7.7% for PM-UD, and 68.9%, 70.0%, 17.9%, and 12.1% for FMT. Multivariate analyses revealed that PM-UD had significantly inferior OS (HR 3.68, 95% CI 1.04–13.1) and DFS (HR 3.1, 95% CI 0.90–10.73), and higher relapse rate (HR 6.09, 95% CI 1.28–29.01) than WM-UD, whereas no significant difference in FMT [OS (HR 2.08, 95% CI 0.59–7.30), DFS (HR 1.95, 95% CI 0.60–6.40), and relapse (HR 1.19, 95% CI 0.55–2.57)]. The occurrence of acute GVHD (aGVHD) with grade II-IV (WM-UD vs. PM-UD vs. FMT; 48.5% vs. 38.5% vs. 52.2%; P = 0.65) and chronic GVHD (cGVHD; WM-UD vs. PM-UD vs. FMT; 51.9% vs. 38.5% vs. 47.8%; P = 0.25) were not significantly different in three donor groups. CMV reactivation was more common in FMT (91.3%, P=0.047) than WM-UD (66.7%) and PM-UD (61.5%), but the occurrence of CMV disease was not different (WM-UD vs. PM-UD vs. FMT; 6.1% vs. 7.7% vs. 17.4%; P=0.390). Causes of death were the recurrence of leukemia (n=3) and refractory cGVHD (n=1) for WM-UD, the recurrence of leukemia (n=4) and aGVHD combined with CMV enteritis (n=1) for PM-UD, and the recurrence of leukemia (n=4) and aGVHD combined with CMV enteritis (n=1), and CMV enteritis combined with veno-occlusive disease (n=1) for FMT.We demonstrate that survival outcomes of FMT using a Korean-adapted protocol are comparable to SCT from WM-UD in contrast to PM-UD showing inferior outcomes than WM-UD. Our data suggest that FMT may be a better option than PM-UD for high-risk AML in the absence of WM-UD as well as HLA-identical sibling donors if complemented by more efficient CMV prophylactic measures, which needs to be validated with more large scale prospective trials in the future. Disclosures:No relevant conflicts of interest to declare.

Allogeneic donor peripheral blood “stem cell” apheresis: prospective comparison of two apheresis systems

Granulocyte-colony-stimulating factor-mobilized peripheral blood stem cells, collected by white blood cell apheresis, are used for more than 80% of allogeneic and most autologous hematopoietic stem cell transplantations. Optimal donor and recipient outcomes require maximized stem cell collection efficiency and minimized non-target cell contamination. Therefore, improved apheresis technology is desirable. The safety and feasibility of apheresis collections with the novel, electronics-assisted apheresis system Spectra Optia v.5.0 (CaridianBCT) were recently demonstrated. An unpublished optimization trial had furthermore determined that different settings than manufacturer-installed default might result in improved apheresis yields. The first prospective comparison of allogeneic peripheral blood stem cell apheresis with the Spectra Optia versus the COBE Spectra (CaridianBCT) mononuclear cell (MNC) in a routine clinical setting is reported here; "optimized" machine settings were used. Assessed variables included collection efficiency, product characteristics, donor outcomes, and frequency of operator interventions. Outcomes were additionally compared with historical data from the Spectra Optia in default mode. The mean CD34+ cell collection efficiency CE1 was 7.9% greater with the Spectra Optia than with the COBE Spectra MNC. Variability of outcomes was equally great. Reduced platelet (PLT) attrition necessitated 90% fewer autologous PLT reinfusions. Spectra Optia products contained 50% fewer red blood cells, but 50% more granulocytic lineage cells. Less operator input was required, although 26% of Spectra Optia apheresis procedures required triggering of the first chamber flush. Apheresis yield and collection efficiency were also markedly greater than in default-mode Spectra Optia collections. Using optimized machine settings, peripheral blood stem cell apheresis outcomes with the automated apheresis system Spectra Optia exceed results with the COBE Spectra MNC or the Spectra Optia in the default mode.

Genome-wide Profiling in AML Patients Relapsing after Allogeneic Hematopoietic Cell Transplantation

Molecular pathogenesis of relapse after allogeneic hematopoietic cell transplantation is poorly understood. Data regarding relapse mechanisms after transplantation is scarcely available. We investigated genomic aberrations (GAs) in 21 patients undergoing related and unrelated HLA-matched transplantation in leukemic blasts before transplant and at relapse after transplantation. We found a higher number of GAs after transplantation, suggesting increased genomic instability during relapse. Two of 21 patients showed a large homozygous region spanning the whole HLA-locus on chromosome 6p in the relapse sample. In both patients sequence-based HLA typing of the blasts revealed a loss of the patient-specific allele at the mismatched locus leading to homozygosity for the HLA haplotype shared by the patient and the donor. In addition, GAs were found in critical regions such as 12p13, 13q12.2, and 17p13. Our results suggest that escape from immunologic surveillance may be a relevant mechanism of relapse after transplantation in patients with GAs on chromosome 6p. A combination of continuous immunologic pressure mediated by donor T cells and clonal evolution of myeloid leukemia may result in acquired GAs after transplantation.

Factors influencing engraftment in HLA-haploidentical/mismatch related transplantation with combined granulocyte-colony stimulating factor-mobilized peripheral blood and bone marrow for patients with leukemia

Unmanipulated HLA-haploidentical/mismatch related transplantation with combined granulocyte-colony stimulating factor-mobilized peripheral blood stem cells (G-PBSCs) and granulocyte-colony stimulating factor-mobilized bone marrow (G-BM) has been used as an alternative transplantation strategy for patients without an HLA-matched donor. In this transplantation setting, factors associated with hematopoietic recovery have not been defined completely. The aim of this study was to investigate the factors influencing the engraftment in this transplantation setting for patients with leukemia. The study group comprised 104 patients with leukemia who underwent transplantation at a single institution between 2005 and 2008. Factors correlating with neutrophil and platelet engraftment post-transplantation were analyzed retrospectively. All patients achieved an absolute neutrophil count of 500/μL with a mean time of 13.6 days (range 8–20 days) and a platelet count over 20 × 10 9/L with a mean time of 20.2 days (range 16–26 days). In univariate analysis, donor and age were associated with increased risk of neutrophil engraftment, but their significance was lost upon multivariate analysis. The sex, age, donor, CD34 + cell dose, conditioning regimen, mismatched locus, ABO mismatched and diagnosis have no effect on platelet engraftment. Our results suggest that it is an ideal approach to treat patients with leukemia with HLA-haploidentical/mismatched related transplantation with combined G-PBSCs and G-BM for a high level of stem cells without delayed engraftment.

Peripheral blood stem cell mobilization: new regimens, new cells, where do we stand

Granulocyte colony-stimulating factor-mobilized peripheral blood stem cells are widely used to reconstitute hematopoiesis; however, preclinical and clinical studies show that improvements to this mobilization can be achieved. We discuss the development of new mobilizing regimens and evaluation of new findings on mobilized stem cell populations that may improve the utility and convenience of peripheral blood stem cell transplant. Chemokines and their receptors regulate leukocyte trafficking, and altering chemokine signaling pathways mobilizes stem cells. In recent trials, combination use of the chemokine (C-X-C motif) receptor 4 antagonist AMD3100 and granulocyte colony-stimulating factor mobilized more CD34 cells in fewer days than granulocyte colony-stimulating factor alone and allowed more patients to proceed to autotransplant. In preclinical studies the chemokine GRObeta synergizes with granulocyte colony-stimulating factor and when used alone or with granulocyte colony-stimulating factor mobilizes more primitive hematopoietic stem cells with less apoptosis, higher integrin activation, lower CD26 expression and enhanced marrow homing compared with granulocyte colony-stimulating factor. Hematopoietic stem cells mobilized by GRObeta or AMD3100 demonstrate superior engraftment and contribution to chimerism in primary and secondary transplant studies in mice, and peripheral blood stem cells mobilized by AMD3100 and granulocyte colony-stimulating factor in patients demonstrate enhanced engraftment capabilities in immunodeficient mice. Alternate regimens differentially mobilize stem cell populations with unique intrinsic properties with the potential to expand the utility of hematopoietic transplantation. Continued mechanistic evaluation will be critical to our understanding of mechanisms of mobilization and their use in regenerative medicine.

Late stent thrombosis after transcoronary transplantation of granulocyte-colony stimulating factor-mobilized peripheral blood stem cells following primary percutaneous intervention for acute myocardial infarction

Transcoronary transplantation of autologous stem cells (ASC) can improve cardiac function after myocardial infarction. For ASC-propagation, granulocyte-colony stimulating factor (G-CSF) is widely used. However, a clinical long-term safety profile of combining G-CSF therapy and transcoronary ASC-transplantation does not exist. We report on three cases of late stent thrombosis in a cohort of 24 patients who had undergone intracoronary infusion of G-CSF-mobilized ASC after primary stenting for acute myocardial infarction.

A high proportion of donor CD4+ T cells expressing the lymph node-homing chemokine receptor CCR7 increases incidence and severity of acute graft-versus-host disease in patients undergoing allogeneic stem cell transplantation for hematological malignancy

CC-chemokine receptor 7 (CCR7), a chemokine receptor required for transmigration into lymphoid organs, is only expressed by naive and central memory T cells. T cells with a capacity of homing into lymphoid organs can initiate acute graft-versus-host disease (GVHD) in mice and respond vigorously in vitro to alloantigens in humans, but their impact on clinical outcomes is unknown. We evaluated prospectively the distribution of naive, central memory and CCR7neg memory T-cell subsets in 39 bone marrow and 23 granulocyte colony-stimulating factor-mobilized peripheral blood stem cell allografts and investigated their impact on patient outcomes. Ranges of the relative proportions of CCR7+ cells within CD4+ and CD8+ T-cell populations were broad, but did not differ between the two sources of allografts. By multivariate analysis, high percentage of donor-derived CD4+CCR7+ T cells (>73.5%) significantly correlated with incidence, earliness of onset and severity of acute GVHD, conferring the highest adjusted hazard ratio (HR=3.9; 95% confidence interval 1.4-10.8; P=0.008) without interfering in other clinical events, especially chronic GVHD and relapse. Determination of the percentage of CD4+CCR7+ T cells in the graft provides a predictive indicator of acute GVHD. Partial depletion of this subset may reduce the risk of acute GVHD while preserving immunotherapeutic effects.

139 INVITED Non-myeloablative transplantation in solid tumors-results in other solid tumors

We previously described a nonmyeloablative hematopoietic stem cell transplantation regimen in dog leukocyte antigen (DLA)-identical littermate recipients consisting of low-dose total body irradiation (TBI) before and mycophenolate mofetil (MMF)/cyclosporine (CSP) given after transplant to control both graft-vs-host and residual host-vs-graft reactions. In this study, we sought to develop a reduced-intensity regimen to achieve engraftment across major histocompatibility complex barriers in DLA-haploidentical littermate recipients.We tested a regimen of 450-cGy TBI with or without postgrafting MMF/CSP for 28 and 35 days, respectively, and with the administration of monoclonal antibody (mAb) S5 (anti-CD44), at a dose of 0.2 mg/kg/day from days −7 through −2, prior to receiving TBI.One of six dogs conditioned with 450-cGy TBI alone achieved engraftment of granulocyte colony-stimulating factor-mobilized peripheral blood stem cells. Three of six dogs achieved sustained donor cell engraftment using 450-cGy TBI and posttransplantation MMF/CSP. None of three dogs given mAb S5 followed by 450-cGy TBI showed signs of donor cell engraftment. However, when S5 mAb pretreatment was added to 450-cGy TBI and postgrafting MMF/CSP, 10 of 12 dogs achieved sustained engraftment (p = 0.008 or 0.007 vs 450-cGy alone or to S5 + 450-cGy TBI without MMF/CSP, respectively), with only three dogs developing severe graft-vs-host disease on this short regimen of immunosuppression.These results show that engraftment across a DLA haplotype-mismatched barrier can be achieved after reduced-intensity conditioning when mAb S5 directed at CD44 is added to this regimen.

Bone Marrow-Derived Cell Mobilization with G-CSF and GM-CSF in Patients with Acute Myocardial Infarction: A Pilot Study on Feasibility, Safety and Efficacy.

Introduction. Intracoronary or intramyocardiac injection of bone marrow stem cells or growth factor-mobilized peripheral blood stem cells (PBSC) may contribute to myocardial regeneration and neovascularization of the ischemic myocardium either in animal models or in human. This effect is probably due to a selective colonization of the ischemic area by PBSC. Very few studies were done in the acute setting of ischemic heart disease, with controversial results. Aims of this pilot study were: i) to verify feasibility and safety of PBSC mobilization in patients with acute myocardial infarction (AMI); ii) to monitor clinical effects of PBSC mobilization in terms of global and segmental myocardial perfusion and function.Patients and methods. Eight male patients (mean age: 51,7±5,6 years) were enrolled. All were treated with a primary percutaneous transluminal coronary angioplasty (PTCA) for an anterior (5 patients) or an inferior (3 patients) AMI. The mobilization regimen consisted of G-CSF 5 μg/kg/12h from day 1 to day 3 and GM-CSF 2.5 μg/kg/24h from day 1 to day 5 (starting within 24 hours from PTCA). Since transient severe hypotension was noticed in the first three patients, the protocol was amended by reducing and tapering GM-CSF administration (2.5 μg/kg/24h at days 3, 5, 7). All patients underwent coronary angiography, intracoronary doppler flow study, echocardiography, and nuclear Thallium scan before treatment and at 6 months apart.Results. WBC and PBSC peaked during the 3th day of mobilization. Mean WBC and PBSC peaks were 34960± 10794 leukocytes/μL and 29.71± 30.8 CD34+/μL. Five in hospital adverse events were recorded: severe hypotension in 3 patients (as previously mentioned), atrial fybrillation in 1 patient, recurrent ischemia in 1 patients. No death was observed. At present 7 patients completed a 6-months follow-up evaluation: target lesion revascularization rate was 14,3% (1 patient) and target vessel revascularization rate was 42,8% (3 patients). Angiographic mean ejection fraction increased from 49,8±11,9 to57,1±8,9 (p=NS), and mean coronary flow reserve raised from 1,63±0,42 to 2,45±0,36 (p=0,005). Perfusion improvement was observed by nuclear study in 66% of patients.Conclusions. We conclude that: a) cytokine-induced stem cell mobilization is feasible in AMI patients with adequate GM-CSF administration; b) myocardial perfusion appears to improve within the first six months of follow-up; c) the 3 cases of progressive coronary disease give a concern about a potential negative effect of hematopoietic cytokines (particularly GM-CSF) on silent coronary lesions. However, PBSC mobilization using G-CSF only is worthwhile of further investigation.

Acute graft-versus-host disease and steroid treatment impair CD11c + and CD123 + dendritic cell reconstitution after allogeneic peripheral blood stem cell transplantation

Human dendritic cells (DC) comprise 2 subsets—plasmacytoid CD123 + and myeloid CD11c + DC—that may have distinct roles in the regulation of immunity after allogeneic hematopoietic stem cell transplantation. In this study, we analyzed the kinetics of CD123 + DC and CD11c + DC reconstitution in 31 patients who underwent transplantation with allogeneic granulocyte colony-stimulating factor-mobilized peripheral blood (PB) stem cells from HLA-identical sibling donors after myeloablative conditioning. Lineage marker-negative HLA-DR + CD11c + CD11c + DC and lineage marker-negative HLA-DR + CD123 + CD123 + DC, as well as monocytes and lymphoid subsets, were enumerated in donor grafts and in the PB of patients at various time points after transplantation. Reconstitution of both CD11c + DC and CD123 + DC to normal levels occurred within 6 to 12 months and was not affected by the diagnosis, preparatory regimen, or graft composition. However, PB CD11c + DC and CD123 + DC counts were significantly reduced in patients with acute GVHD grade II to IV (at 1 and 3 months) and grade I (at 1 month). Patients with chronic GVHD instead showed reduced CD123 + DC counts only 6 months after transplantation. Moreover, treatment with steroids (>0.1 mg/kg) was significantly associated with reduced PB CD11c + DC and CD123 + DC counts at all time points after transplantation. In multivariate analysis, only acute GVHD affected DC reconstitution early after transplantation. These results will prompt new studies addressing whether DC reconstitution correlates with immunity against infectious agents or with graft-versus-tumor reactions after PB stem cell allotransplantation.

In vitro expansion of human basophils by interleukin-3 from granulocyte colony-stimulating factor-mobilized peripheral blood stem cells.

A number of studies support the belief that human basophils play an important role in allergic inflammation. The exact mechanism of basophil activation at the site of allergic inflammation, however, has not been well understood, mainly due to their low number in blood and difficulty in obtaining a sufficient number of highly purified basophils for investigation. The purpose of this study is to expand human basophils in vitro with high yield and purity by culturing peripheral blood stem cells (PBSCs). We collected PBSC-rich mononuclear cells containing CD34+ cells (0.15-4.9%) by leukapheresis from patients with malignant lymphoma and lung cancer during haematopoietic recovery after chemotherapy plus granulocyte colony-stimulating factor-induced mobilization. PBSC-rich mononuclear cells were cultured in the presence of IL-3. When PBSC-rich mononuclear cells containing more than 1% of CD34+ cells were cultured, 20.0-83.3% of the cells, mostly with a yield of >10%, were metachromatic cells after 3 weeks of culture. These cells resembled mature peripheral blood basophils morphologically when examined by light and electron microscopy. Flow cytometric analysis showed that they expressed both FcepsilonRI and FcgammaRII. FcepsilonRI cross-linking resulted in intracellular calcium mobilization, histamine release and synthesis of cysteinyl leukotrienes. The intracellular histamine content and the release of these chemical mediators triggered by anti-IgE antibodies were comparable to those of peripheral blood basophils. These findings suggest that PBSC-derived basophils expanded in vitro are morphologically and functionally mature and will be a useful tool for the analysis of basophil functions.

Dog leukocyte antigen-haploidentical stem cell allografts after anti-CD44 therapy and reduced-intensity conditioning in a preclinical canine model.

We previously described a nonmyeloablative hematopoietic stem cell transplantation regimen in dog leukocyte antigen (DLA)-identical littermate recipients consisting of low-dose total body irradiation (TBI) before and mycophenolate mofetil (MMF)/cyclosporine (CSP) given after transplant to control both graft-vs-host and residual host-vs-graft reactions. In this study, we sought to develop a reduced-intensity regimen to achieve engraftment across major histocompatibility complex barriers in DLA-haploidentical littermate recipients. We tested a regimen of 450-cGy TBI with or without postgrafting MMF/CSP for 28 and 35 days, respectively, and with the administration of monoclonal antibody (mAb) S5 (anti-CD44), at a dose of 0.2 mg/kg/day from days -7 through -2, prior to receiving TBI. One of six dogs conditioned with 450-cGy TBI alone achieved engraftment of granulocyte colony-stimulating factor-mobilized peripheral blood stem cells. Three of six dogs achieved sustained donor cell engraftment using 450-cGy TBI and posttransplantation MMF/CSP. None of three dogs given mAb S5 followed by 450-cGy TBI showed signs of donor cell engraftment. However, when S5 mAb pretreatment was added to 450-cGy TBI and postgrafting MMF/CSP, 10 of 12 dogs achieved sustained engraftment (p = 0.008 or 0.007 vs 450-cGy alone or to S5 + 450-cGy TBI without MMF/CSP, respectively), with only three dogs developing severe graft-vs-host disease on this short regimen of immunosuppression. These results show that engraftment across a DLA haplotype-mismatched barrier can be achieved after reduced-intensity conditioning when mAb S5 directed at CD44 is added to this regimen.

Impaired T and NK cell response of bone marrow and peripheral blood stem cell products to interleukin (IL)-2

The function of steady-state and interleukin (IL)-2-co-cultured mononuclear cells differs significantly between bone marrow (BM) products, growth factor-mobilized peripheral blood stem cell (PSC) products and normal peripheral blood mononuclear cells (PBMC). The natural killer (NK) cell activity and T cell proliferative response of PSC products from non-Hodgkin’s lymphoma (NHL) patients are significantly higher than that of BM products and similar to normal PBMC. However, following a five-day co-culture with IL-2 (100 IU/ml), the NK activity of PSC, PBMC, and BM products (lytic units) was increased 176-, 40-, and 14-fold, respectively, compared to that observed prior to IL-2 culture. In contrast, lymphokine activated killer (LAK) cytotoxicity prior to IL-2 culture was low in PSC and BM products and normal PBMC, but was significantly increased in PSC products and PBMC following IL-2 co-culture. The proliferative response of PSC and BM products to the T cell mitogen phytohemagglutinin (PHA) was significantly lower than that observed with normal PBMC; however, PSC had a significantly higher response than cells from BM products. Similar patterns of T cell PHA mitogenic response were observed after IL-2 co-culture. In addition, the IL-2 mitogenic responses of IL-2-co-cultured PSC and BM products were also significantly lower than that observed with PBMC co-cultured with IL-2. The IL-2 mitogenic response of PBMC was also significantly increased compared to prior to IL-2 co-culture; whereas, the IL-2 mitogenic responses from PSC and BM cells were not. In summary, co-culture with IL-2 can increase the NK and LAK cell cytotoxicity of PSC and BM products from NHL patients, but IL-2 co-culture does not improve T cell function within either BM or PSC products.